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Counter, Christopher M.

Overview:

The Counter lab studies the molecular mechanisms underlying the evolution of normal cells into cancer. The lab is divided into two major areas studying key features of human cancers.

Immortalization: We have shown that the ability of cancer cells to keep dividing, or become immortal, is a fundamental aspect of tumorigenesis, and is due to elongation of telomeres. Current efforts focus on the molecular biology of telomere-binding proteins in regulating telomere length.

Proliferation: The ability of tumor cells to proliferate inappropriately is a hallmark of cancer. One gene that plays a key role in this process is the oncogene Ras. We have shown that Ras exerts its oncogenic signals through different proteins at different phases of cancer. Current studies focus on how these different pathways promote cancer and how to inhibit their activity.

Positions:

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Assistant Professor in Radiation Oncology

Radiation Oncology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 1990

B.S. — McMaster University

Ph.D. 1996

Ph.D. — McMaster University

News:

Grants:

Translational Research in Surgical Oncology

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
January 01, 2002
End Date
August 31, 2021

The role of dietary copper in melanoma

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 25, 2015
End Date
August 31, 2020

Genetics Training Grant

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 1979
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Pharmacology Industry Internships for Ph.D. Students

Administered By
Pharmacology & Cancer Biology
AwardedBy
American Society for Pharmacology and Experimental Therapeutics
Role
Participating Faculty Member
Start Date
January 01, 2017
End Date
December 31, 2019

Viral Oncology Training Grant

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1980
End Date
June 30, 2019

RalA signal transduction

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2002
End Date
March 31, 2019

Dynamic requirements of Ras signaling during cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2006
End Date
December 31, 2018

Duke University Program in Environmental Health

Administered By
Environmental Sciences and Policy
AwardedBy
National Institute of Environmental Health Sciences
Role
Mentor
Start Date
July 01, 2013
End Date
June 30, 2018

The role of KRAS codon bias in tumorigenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2016
End Date
February 28, 2018

Center for Molecular & Cellular Studies of Ped Disease

Administered By
Pediatrics
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
April 11, 2003
End Date
November 30, 2017

Training Program in Developmental and Stem Cell Biology

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2001
End Date
October 31, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

Leveraging copper chelation as targeted therapy for BRAF-driven thyroid cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
Triangle Community Foundation
Role
Principal Investigator
Start Date
June 01, 2015
End Date
June 01, 2017

Defining RAS isoform- and mutation-specific roles in oncogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
June 22, 2016
End Date
May 31, 2017

Defining RAS isoform- and mutation-specific roles in oncogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
June 22, 2016
End Date
May 31, 2017

Defining RAS isoform- and mutation-specific roles in oncogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
June 22, 2016
End Date
May 31, 2017

The effect of oncogenic Ras signaling strength on cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2013
End Date
March 31, 2017

A Platform for Real-time Drug Profiling of Patient-Derived Melanomas

Administered By
Surgery
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2014
End Date
July 31, 2016

Transfusion Medicine and Hematology

Administered By
Medicine, Hematology
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
July 01, 1975
End Date
June 30, 2016

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Examining infection-mediated metastasis with single-cell resolution

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
March 01, 2014
End Date
February 29, 2016

Pleiotrophin, a paracrine regulator of hematopoietic stem cell fate

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of California - Los Angeles
Role
Principal Investigator
Start Date
May 01, 2014
End Date
December 31, 2015

Copper reduction as a novel therapy in BRAF-mutant positive cancers

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 20, 2013
End Date
July 01, 2015

Reducing dietary copper for the treatment of BRaf mutation-positive melanoma

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2013
End Date
June 30, 2015

JUN PROTEINS IN EPIDERMAL HOMEOSTASIS AND NEOPLASIA

Administered By
Dermatology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
August 09, 2010
End Date
May 31, 2015

Instrumentation for Quantitative Phosphoproteomics and Acetylomics

Administered By
Duke Center for Genomic and Computational Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2014
End Date
May 14, 2015

Evaluating the impact of KRas codon bias on pancreatic cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2012
End Date
March 31, 2014

The role of RalA in pancreatic tumorigenesis

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2008
End Date
July 19, 2013

Small molecular weight eNOS inhibitors for the treatment of pancreatic cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2011
End Date
March 31, 2013

Preclinical evaluation of NOS inhibitors for the treatment of pancreatic cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 17, 2009
End Date
July 31, 2011

NF-kB, CDK4 and JNK in Epidermal Growth Regulation

Administered By
Dermatology
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
April 05, 2005
End Date
March 31, 2010

The Molecular Basis of Telomerase Function & Regulation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 23, 1999
End Date
November 30, 2009

Developing inhibitors of RalA function for the treatment of pancreatic cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 12, 2007
End Date
February 28, 2009

Targeting Functional Domains in Telomerase

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2002
End Date
April 30, 2004
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Awards:

John J. Abel Award in Pharmacology. American Society for Pharmacology and Experimental Therapeutics.

Type
National
Awarded By
American Society for Pharmacology and Experimental Therapeutics
Date
January 01, 2006

Publications:

Isoform-Specific Effects of Wild-Type Ras Genes on Carcinogen-Induced Lung Tumorigenesis in Mice.

The gene KRAS is commonly mutated in lung cancer to encode a constitutively active and oncogenic protein that is well established to initiate and maintain lung tumorigenesis. However, the remaining wild-type KRAS protein, or the other family members HRAS and NRAS, can still be activated in the presence of oncogenic KRAS. Moreover, loss of any one of these three genes has been shown to increase the sensitivity of mice to the carcinogen urethane, which induces Kras mutation-positive early lung lesions. To determine the contribution of progressively disrupting Hras and Nras genes on urethane lung tumorigenesis, mice with different combinations of wild-type and null alleles of Hras and Nras were exposed with urethane and tumor burden was assessed. As previously reported, loss of one allele of Hras increased the sensitivity of mice to this carcinogen, and this effect was further exacerbated by the loss of the second Hras allele. However, loss of one or both alleles of Nras failed to alter tumor burden, either in the absence or presence of Hras, after exposure to urethane. Additionally, no obvious difference between lung lesions in mice with wild-type versus null alleles was detected, suggesting that wild-type Ras proteins may exert a tumor suppressive effects at the time of initiation, although other interpretations are certainly possible. In summary, these data suggest that in some genetic backgrounds inactivation of different wild-type Ras genes can have different effects on urethane-induced lung tumorigenesis.

Authors
Weyandt, JD; Carney, JM; Pavlisko, EN; Xu, M; Counter, CM
MLA Citation
Weyandt, JD, Carney, JM, Pavlisko, EN, Xu, M, and Counter, CM. "Isoform-Specific Effects of Wild-Type Ras Genes on Carcinogen-Induced Lung Tumorigenesis in Mice." PloS one 11.12 (January 2016): e0167205-.
PMID
27911940
Source
epmc
Published In
PloS one
Volume
11
Issue
12
Publish Date
2016
Start Page
e0167205
DOI
10.1371/journal.pone.0167205

Utility of telomerase-potl fusion protein in vascular tissue engineering

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called potl-TERT, is a chimeric fusion between the DNA binding protein potl and TERT. Because potl-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of potl-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using potl-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of potl-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs. Copyright © 2010 Cognizant Comm. Corp.

Authors
Petersen, TH; Hitchcock, T; Muto, A; Calle, EA; Zhao, L; Gong, Z; Gui, L; Dardik, A; Bowles, DE; Counter, CM; Niklason, LE
MLA Citation
Petersen, TH, Hitchcock, T, Muto, A, Calle, EA, Zhao, L, Gong, Z, Gui, L, Dardik, A, Bowles, DE, Counter, CM, and Niklason, LE. "Utility of telomerase-potl fusion protein in vascular tissue engineering." Cell Transplantation 19.1 (April 14, 2010): 78-87.
Source
scopus
Published In
Cell Transplantation
Volume
19
Issue
1
Publish Date
2010
Start Page
78
End Page
87
DOI
10.3727/096368909X478650

Sec5 and Exo84 foster oncogenic ras-mediated tumorigenesis.

The genes encoding the Ras family of small GTPases are mutated to yield constitutively active GTP-bound oncogenic proteins in one third of all human cancers. Oncogenic Ras binds to and activates a number of proteins that promote tumorigenic phenotypes, including the family of Ral guanine nucleotide exchange factors (RalGEF). Activated RalGEFs convert the Ral family of small GTPases, composed of RalA and RalB, from an inactive GDP-bound state to an active GTP-bound state. As both RalA and RalB have been implicated in a variety of tumorigenic phenotypes, we sought to determine which proteins downstream of Rals promote transformation and tumorigenesis. Here, we report that shRNA-mediated knockdown of the Ral effector proteins Sec5 and Exo84, but less so in the case of RalBP1, reduced oncogenic RalGEF-mediated transformation and oncogenic Ras-driven tumorigenic growth of human cells. These results suggest that Rals promote oncogenic Ras-mediated tumorigenesis through, at least in part, Sec5 and Exo84.

Authors
Issaq, SH; Lim, K-H; Counter, CM
MLA Citation
Issaq, SH, Lim, K-H, and Counter, CM. "Sec5 and Exo84 foster oncogenic ras-mediated tumorigenesis." Mol Cancer Res 8.2 (February 2010): 223-231.
PMID
20145037
Source
pubmed
Published In
Molecular cancer research : MCR
Volume
8
Issue
2
Publish Date
2010
Start Page
223
End Page
231
DOI
10.1158/1541-7786.MCR-09-0189

Aurora-A phosphorylates, activates, and relocalizes the small GTPase RalA.

The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA.

Authors
Lim, K-H; Brady, DC; Kashatus, DF; Ancrile, BB; Der, CJ; Cox, AD; Counter, CM
MLA Citation
Lim, K-H, Brady, DC, Kashatus, DF, Ancrile, BB, Der, CJ, Cox, AD, and Counter, CM. "Aurora-A phosphorylates, activates, and relocalizes the small GTPase RalA." Mol Cell Biol 30.2 (January 2010): 508-523.
PMID
19901077
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
30
Issue
2
Publish Date
2010
Start Page
508
End Page
523
DOI
10.1128/MCB.00916-08

Utility of telomerase-pot1 fusion protein in vascular tissue engineering.

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

Authors
Petersen, TH; Hitchcock, T; Muto, A; Calle, EA; Zhao, L; Gong, Z; Gui, L; Dardik, A; Bowles, DE; Counter, CM; Niklason, LE
MLA Citation
Petersen, TH, Hitchcock, T, Muto, A, Calle, EA, Zhao, L, Gong, Z, Gui, L, Dardik, A, Bowles, DE, Counter, CM, and Niklason, LE. "Utility of telomerase-pot1 fusion protein in vascular tissue engineering." Cell Transplant 19.1 (2010): 79-87.
Website
http://hdl.handle.net/10161/4616
PMID
19878625
Source
pubmed
Published In
Cell Transplantation
Volume
19
Issue
1
Publish Date
2010
Start Page
79
End Page
87
DOI
10.3727/096368909X478650

Utility of telomerase-pot1 fusion protein in vascular tissue engineering.

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

Authors
Petersen, TH; Hitchcock, T; Muto, A; Calle, EA; Zhao, L; Gong, Z; Gui, L; Dardik, A; Bowles, DE; Counter, CM; Niklason, LE
MLA Citation
Petersen, TH, Hitchcock, T, Muto, A, Calle, EA, Zhao, L, Gong, Z, Gui, L, Dardik, A, Bowles, DE, Counter, CM, and Niklason, LE. "Utility of telomerase-pot1 fusion protein in vascular tissue engineering." Cell transplantation 19.1 (2010): 79-87.
Source
scival
Published In
Cell Transplantation
Volume
19
Issue
1
Publish Date
2010
Start Page
79
End Page
87
DOI
10.3727/096368909X478650

ELR+ CXC chemokines and oncogenic Ras-mediated tumorigenesis.

The small GTPase Ras is mutated to remain in the active oncogenic state in one-third of human cancers, thereby promoting tumorigenesis. It has recently come to light that one consequence of oncogenic Ras signaling is secretion of cytokines vascular endothelial growth factor (VEGF), interleukin 6 (IL6), hCXCL1 (Gro-alpha) and hCXCL8 (IL8). As the latter two belong to the ELR+ Cys-X-Cys (CXC) chemokine family, we investigated whether the entire family of ELR+ CXC chemokines plays a role in oncogenic Ras-mediated tumorigenesis. We now demonstrate that oncogenic Ras induced the expression and secretion of the ELR+ CXC chemokine family in different tumorigenic human cells and that these chemokines are elevated in tumor specimens. Moreover, genetic ablation of the common receptor for these chemokines, mCXCR2, reduced oncogenic Ras-driven tumorigenesis in mice. Taken together, we suggest that oncogenic Ras induces the secretion of the ELR+ CXC chemokine family to promote tumorigenesis. This chemokine signature may identify the presence of Ras activation in cancer and perhaps even serve as targets for oncogenic Ras-driven tumor cells.

Authors
O'Hayer, KM; Brady, DC; Counter, CM
MLA Citation
O'Hayer, KM, Brady, DC, and Counter, CM. "ELR+ CXC chemokines and oncogenic Ras-mediated tumorigenesis." Carcinogenesis 30.11 (November 2009): 1841-1847.
PMID
19805574
Source
pubmed
Published In
Carcinogenesis
Volume
30
Issue
11
Publish Date
2009
Start Page
1841
End Page
1847
DOI
10.1093/carcin/bgp198

POT1 association with TRF2 regulates telomere length.

Deleting the OB folds encoding the telomeric single-stranded DNA (ssDNA)-binding activity of the human telomeric protein POT1 induces significant telomere elongation, suggesting that at least one critical aspect of the regulation of telomere length is disrupted by this POT1(DeltaOB) mutant protein. POT1 is known to associate with two proteins through the protein interaction domain retained in POT1(DeltaOB)-the telomeric double-stranded DNA-binding protein TRF2 and the telomere-associated protein TPP1. We report that introducing a mutation that reduces association of POT1 with TRF2, but not a mutation that reduces the association with TPP1, abrogates the ability of POT1(DeltaOB) to promote telomere elongation. Mechanistically, expression of POT1(DeltaOB) reduced the association of TRF2 with POT1, RAP1, and TIN2; however, of these proteins, only ectopic expression of POT1 suppressed the telomere elongation induced by POT1(DeltaOB). Lastly, replacing endogenous POT1 with a full-length POT1 mutant defective in the association with TRF2 induced telomere elongation. Thus, we conclude that the association of POT1 with both ssDNA and TRF2 is critical for telomere length homeostasis.

Authors
Kendellen, MF; Barrientos, KS; Counter, CM
MLA Citation
Kendellen, MF, Barrientos, KS, and Counter, CM. "POT1 association with TRF2 regulates telomere length." Mol Cell Biol 29.20 (October 2009): 5611-5619.
PMID
19651898
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
29
Issue
20
Publish Date
2009
Start Page
5611
End Page
5619
DOI
10.1128/MCB.00286-09

Defining the cooperative genetic changes that temporally drive alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of RMS known as alveolar (aRMS) have a 5-year survival rate of <30%. aRMS tissues exhibit a number of genetic changes, including loss-of-function of the p53 and Rb tumor suppressor pathways, amplification of MYCN, stabilization of telomeres, and most characteristically, reciprocal translocation of loci involving the PAX and FKHR genes, generating the PAX7-FKHR or PAX3-FKHR fusion proteins. We previously showed that PAX3-FKHR expression in primary human myoblasts, cells that can give rise to RMS, cooperated with loss of p16INK4A to promote extended proliferation. To better understand the genetic events required for aRMS formation, we then stepwise converted these cells to their transformed counterpart. PAX3-FKHR, the catalytic unit of telomerase hTERT, and MycN, in cooperation with down-regulation of p16INK4A/p14ARF expression, were necessary and sufficient to convert normal human myoblasts into tumorigenic cells that gave rise to aRMS tumors. However, the order of expression of these transgenes was critical, as only those cells expressing PAX3-FKHR early could form tumors. We therefore suggest that the translocation of PAX3 to FKHR drives proliferation of myoblasts, and a selection for loss of p16INK4A/p14ARF. These early steps, coupled with MycN amplification and telomere stabilization, then drive the cells to a fully tumorigenic state.

Authors
Naini, S; Etheridge, KT; Adam, SJ; Qualman, SJ; Bentley, RC; Counter, CM; Linardic, CM
MLA Citation
Naini, S, Etheridge, KT, Adam, SJ, Qualman, SJ, Bentley, RC, Counter, CM, and Linardic, CM. "Defining the cooperative genetic changes that temporally drive alveolar rhabdomyosarcoma." Cancer Res 68.23 (December 1, 2008): 9583-9588.
PMID
19047133
Source
pubmed
Published In
Cancer Research
Volume
68
Issue
23
Publish Date
2008
Start Page
9583
End Page
9588
DOI
10.1158/0008-5472.CAN-07-6178

The cytoplasmic deacetylase HDAC6 is required for efficient oncogenic tumorigenesis.

Histone deacetylase inhibitors (HDACI) are promising antitumor agents. Although transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanism and targets by which HDACIs achieve their antitumor effects remain poorly understood. It is not known whether any of the HDAC members support robust tumor growth. In this report, we show that HDAC6, a cytoplasmic-localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. We found that HDAC6 expression is induced upon oncogenic Ras transformation. Fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several cancer cell lines reduces anchorage-independent growth and the ability to form tumors in mice. The loss of anchorage-independent growth is associated with increased anoikis and defects in AKT and extracellular signal-regulated kinase activation upon loss of adhesion. Lastly, HDAC6-null mice are more resistant to chemical carcinogen-induced skin tumors. Our results provide the first experimental evidence that a specific HDAC member is required for efficient oncogenic transformation and indicate that HDAC6 is an important component underlying the antitumor effects of HDACIs.

Authors
Lee, Y-S; Lim, K-H; Guo, X; Kawaguchi, Y; Gao, Y; Barrientos, T; Ordentlich, P; Wang, X-F; Counter, CM; Yao, T-P
MLA Citation
Lee, Y-S, Lim, K-H, Guo, X, Kawaguchi, Y, Gao, Y, Barrientos, T, Ordentlich, P, Wang, X-F, Counter, CM, and Yao, T-P. "The cytoplasmic deacetylase HDAC6 is required for efficient oncogenic tumorigenesis." Cancer Res 68.18 (September 15, 2008): 7561-7569.
PMID
18794144
Source
pubmed
Published In
Cancer Research
Volume
68
Issue
18
Publish Date
2008
Start Page
7561
End Page
7569
DOI
10.1158/0008-5472.CAN-08-0188

Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells.

Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference-mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme.

Authors
Tomlinson, RL; Abreu, EB; Ziegler, T; Ly, H; Counter, CM; Terns, RM; Terns, MP
MLA Citation
Tomlinson, RL, Abreu, EB, Ziegler, T, Ly, H, Counter, CM, Terns, RM, and Terns, MP. "Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells." Mol Biol Cell 19.9 (September 2008): 3793-3800.
PMID
18562689
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
19
Issue
9
Publish Date
2008
Start Page
3793
End Page
3800
DOI
10.1091/mbc.E08-02-0184

Distinct functions of POT1 at telomeres.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.

Authors
Barrientos, KS; Kendellen, MF; Freibaum, BD; Armbruster, BN; Etheridge, KT; Counter, CM
MLA Citation
Barrientos, KS, Kendellen, MF, Freibaum, BD, Armbruster, BN, Etheridge, KT, and Counter, CM. "Distinct functions of POT1 at telomeres." Mol Cell Biol 28.17 (September 2008): 5251-5264.
Website
http://hdl.handle.net/10161/1343
PMID
18519588
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
28
Issue
17
Publish Date
2008
Start Page
5251
End Page
5264
DOI
10.1128/MCB.00048-08

The protein hSnm1B is stabilized when bound to the telomere-binding protein TRF2.

hSnm1B is member of the SNM family of exonucleases involved in DNA processing and is known to be localized to telomeres via binding to the telomere-binding protein TRF2. Here we demonstrate that the C terminus of hSnm1B facilitates the concentration of hSnm1B on telomeres by promoting ubiquitin-mediated degradation of hSnm1B that is not localized to telomeres, as well as by blocking protein degradation and fostering localization to telomeres via binding of TRF2. Finally, a mutant of hSnm1B stabilized independently of exogenous TRF2-induced cell death. Taken together, we speculate that sequestering hSnm1B at telomeres by a combination of stabilizing the protein when bound to telomeres and degrading it when not bound to telomeres may be a means to prevent potentially lethal effects of unregulated hSnm1B activity.

Authors
Freibaum, BD; Counter, CM
MLA Citation
Freibaum, BD, and Counter, CM. "The protein hSnm1B is stabilized when bound to the telomere-binding protein TRF2." J Biol Chem 283.35 (August 29, 2008): 23671-23676.
PMID
18593705
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
35
Publish Date
2008
Start Page
23671
End Page
23676
DOI
10.1074/jbc.M800388200

Tumour maintenance is mediated by eNOS.

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.

Authors
Lim, K-H; Ancrile, BB; Kashatus, DF; Counter, CM
MLA Citation
Lim, K-H, Ancrile, BB, Kashatus, DF, and Counter, CM. "Tumour maintenance is mediated by eNOS." Nature 452.7187 (April 3, 2008): 646-649.
PMID
18344980
Source
pubmed
Published In
Nature
Volume
452
Issue
7187
Publish Date
2008
Start Page
646
End Page
649
DOI
10.1038/nature06778

Tethering telomeric double- and single-stranded DNA-binding proteins inhibits telomere elongation.

Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3' single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening.

Authors
Etheridge, KT; Compton, SA; Barrientos, KS; Ozgur, S; Griffith, JD; Counter, CM
MLA Citation
Etheridge, KT, Compton, SA, Barrientos, KS, Ozgur, S, Griffith, JD, and Counter, CM. "Tethering telomeric double- and single-stranded DNA-binding proteins inhibits telomere elongation." J Biol Chem 283.11 (March 14, 2008): 6935-6941.
PMID
18178559
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
11
Publish Date
2008
Start Page
6935
End Page
6941
DOI
10.1074/jbc.M708711200

Oncogenic ras-induced expression of cytokines: a new target of anti-cancer therapeutics.

The Ras family of small guanosine triphosphatases normally transmit signals from cell surface receptors to the interior of the cell. Stimulation of cell surface receptors leads to the activation of guanine exchange factors, which, in turn, convert Ras from an inactive GDP-bound state to an active GTP-bound state. However, in one third of human cancers, RAS is mutated and remains in the constitutively active GTP-bound state. In this oncogenic state, RAS activates a constellation of signaling that is known to promote tumorigenesis. One consequence of this oncogenic RAS signal in cancer cells is the upregulation of the cytokines interleukin (IL)-6, IL-8, and chemokine growth-regulated oncogene 1 (GRO-1). We review the evidence supporting a role for these cytokines in oncogenic RAS-driven solid tumors.

Authors
Ancrile, BB; O'Hayer, KM; Counter, CM
MLA Citation
Ancrile, BB, O'Hayer, KM, and Counter, CM. "Oncogenic ras-induced expression of cytokines: a new target of anti-cancer therapeutics." Mol Interv 8.1 (February 2008): 22-27. (Review)
PMID
18332481
Source
pubmed
Published In
Molecular interventions
Volume
8
Issue
1
Publish Date
2008
Start Page
22
End Page
27
DOI
10.1124/mi.8.1.6

Genetic modeling of Ras-induced human rhabdomyosarcoma.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Historically, rhabdomyosarcoma has been studied by the manipulation of human cell lines derived from primary rhabdomyosarcoma tumor tissue adapted to grow in culture. Recently, mouse models have been added to the arsenal of tools to study this disease in vivo. However, given the emerging understanding of the genetic variability and mutability of human tumor-derived cell lines, and the existing differences between human and murine tumorigenesis, we sought to uniformly dissect the genetic events required to generate rhabdomyosarcoma from primary human skeletal muscle precursors. To this end, primary human skeletal muscle cells were transformed with defined genetic elements to corrupt the p53, Rb, Myc, telomerase, and Ras pathways, resulting in cells that, when assayed as subcutaneous xenografts in immunocompromised mice, formed tumors indistinguishable at the immunohistochemical level from the embryonal histologic variant of rhabdomyosarcoma. This chapter will discuss the techniques used to transform primary human skeletal muscle cells, the assays used to verify expression of the ectopically expressed genetic elements, and the methods used to evaluate the tumorigenic capacity of the resulting cell lines.

Authors
Linardic, CM; Counter, CM
MLA Citation
Linardic, CM, and Counter, CM. "Genetic modeling of Ras-induced human rhabdomyosarcoma." Methods Enzymol 438 (2008): 419-427.
PMID
18413264
Source
pubmed
Published In
Methods in Enzymology
Volume
438
Publish Date
2008
Start Page
419
End Page
427
DOI
10.1016/S0076-6879(07)38028-2

DNA-based animal models of human disease: From genotype to phenotype

Biomedical research utilizes animal models to elucidate human disease processes at the cellular and molecular level and for the development of new therapies. Traditionally, mammalian models have been limited to the mouse, primarily because of well characterized genetic lines and the ability to manipulate the genome to directly test hypotheses regarding causal mutations and disease phenotypes. The emerging availability of genome sequences of other mammals (bovine, canine, equine, feline, and porcine) now permits utilization of the mammal in which the phenotype best approximates the human condition. Equally important is the use of somatic cell nuclear cloning (SCNT) coupled with targeted germline manipulation to create animals to resolve the molecular mechanisms of the disease state. Our efforts have focused on the pig, which has emerged as an important biomedical mammalian model due to its closer physiology to humans. The utility of porcine genetically-defined tumour, cardiovascular and neurological disease models is described.

Authors
Schook, LB; Kuzmuk, K; Adam, S; Rund, L; Chen, K; Rogatcheva, M; Mazur, M; Pollock, C; Counter, C
MLA Citation
Schook, LB, Kuzmuk, K, Adam, S, Rund, L, Chen, K, Rogatcheva, M, Mazur, M, Pollock, C, and Counter, C. "DNA-based animal models of human disease: From genotype to phenotype." Developments in Biologicals 132 (2008): 15-25.
PMID
18817282
Source
scival
Published In
Developments in biologicals
Volume
132
Publish Date
2008
Start Page
15
End Page
25

A method to generate genetically defined tumors in pigs.

As a biomedical model, pigs offer many advantages and hence have been utilized extensively for toxicology, Crohn's disease, diabetes, and organ transplantation, as well as many other research areas. However, the advantages of porcine models, particularly its large size and similarity to humans, were not exploited previously to any large degree for cancer research. One reason for this lack of porcine cancer models was the inability to induce cancer in pigs genetically. This chapter describes a rapid, reproducible, and genetically malleable method to induce large tumors in pigs.

Authors
Adam, SJ; Counter, CM
MLA Citation
Adam, SJ, and Counter, CM. "A method to generate genetically defined tumors in pigs." Methods Enzymol 439 (2008): 39-51.
PMID
18374155
Source
pubmed
Published In
Methods in Enzymology
Volume
439
Publish Date
2008
Start Page
39
End Page
51
DOI
10.1016/S0076-6879(07)00404-1

Characterization of the porcine ATM gene: towards the generation of a novel non-murine animal model for Ataxia-Telangiectasia.

Ataxia-Telangiectasia (A-T) is a genetic disorder causing cerebellar degeneration, immune deficiency, cancer predisposition, chromosomal instability and radiation sensitivity. Among the mutations responsible for A-T, 85% represent truncating mutations that result in the production of shorter, highly unstable forms of ATM (AT-mutated) protein leading to a null ATM phenotype. Several ATM-deficient mice have been created however none reflects the extent of neurological degeneration observed in humans. In an attempt to identify an alternative animal model, we have characterized the porcine ortholog of ATM (pATM). When compared to the human ATM (hATM), the pATM showed a high level of homology in the coding region, particularly in the regions coding for functional domains, and had extensive alternative splicing of the 5'UTR, characteristic for the human ATM mRNA. Six different 5'UTRs resulting from alternative splicing of the first three exons were identified. The porcine 5'UTRs varied in size, had multiple ATG codons and different secondary structures, supporting the possibility of complex transcriptional regulation. Three of the six transcripts demonstrated alternative splicing of exon 3, the first putative coding exon, altering the translation start and giving rise to a putative protein lacking the N-terminus substrate binding domain (82-89 aa) involved in activation of human p53 and BRCA1 pathways. Real time-PCR analysis revealed variable expression levels of total ATM transcripts in individual tissues. Although each splice variant was ubiquitously expressed among the tissues, differences in the relative abundances of specific 5'UTRs were observed. The extensive alternative splicing of the pATM gene resembles the complex splicing observed in the hATM and could provide insights for differences observed between mice and humans with regards to the onset of A-T. Thus, the pig may provide a more relevant clinical model of A-T.

Authors
Rogatcheva, MB; Fritz, KL; Rund, LA; Pollock, CB; Beever, JE; Counter, CM; Schook, LB
MLA Citation
Rogatcheva, MB, Fritz, KL, Rund, LA, Pollock, CB, Beever, JE, Counter, CM, and Schook, LB. "Characterization of the porcine ATM gene: towards the generation of a novel non-murine animal model for Ataxia-Telangiectasia." Gene 405.1-2 (December 15, 2007): 27-35.
PMID
17933474
Source
pubmed
Published In
Gene
Volume
405
Issue
1-2
Publish Date
2007
Start Page
27
End Page
35
DOI
10.1016/j.gene.2007.08.014

From bread to bedside: What budding yeast has taught us about the immortalization of cancer cells

The budding yeast Saccharomyces cerevisiae is a formidable model system indeed. With the entire genome sequenced, unparalleled genetic malleability, and an eukaryotic background, this system is virtually beyond compare for studying the multitude of biological pathways that are conserved amongst eukaryotes. Importantly with regards to human cancer, many of the cellular processes of the mammalian cell can be found, admittedly in a stripped-down version, in yeast. In this regard, yeasts are fertile ground for elucidating mechanisms and identifying the key players in cellular processes. This invaluable information can then act as a guide for the cancer cell biologist attempting to navigate the analogous pathway in the far more complex and elaborate system of the mammalian cell. One example of where yeast has been used in this regard is in understanding how cancer cells acquire the ability to divide indefinitely. Here we highlight the enormous contributions made by studies performed in the model system of S. cerevisiae to our understanding of this tumourigenic process. © 2007 Springer.

Authors
Banik, SSR; Counter, CM
MLA Citation
Banik, SSR, and Counter, CM. "From bread to bedside: What budding yeast has taught us about the immortalization of cancer cells." (December 1, 2007): 123-139. (Chapter)
Source
scopus
Publish Date
2007
Start Page
123
End Page
139
DOI
10.1007/978-1-4020-5963-6_5

Oncogenic Ras-induced secretion of IL6 is required for tumorigenesis.

Ras is mutated to remain in the active oncogenic state in many cancers. As Ras has proven difficult to target therapeutically, we searched for secreted, druggable proteins induced by Ras that are required for tumorigenesis. We found that Ras induces the secretion of cytokine IL6 in different cell types, and that knockdown of IL6, genetic ablation of the IL6 gene, or treatment with a neutralizing IL6 antibody retard Ras-driven tumorigenesis. IL6 appears to act in a paracrine fashion to promote angiogenesis and tumor growth. Inhibiting IL6 may therefore have therapeutic utility for treatment of cancers characterized by oncogenic Ras mutations.

Authors
Ancrile, B; Lim, K-H; Counter, CM
MLA Citation
Ancrile, B, Lim, K-H, and Counter, CM. "Oncogenic Ras-induced secretion of IL6 is required for tumorigenesis." Genes Dev 21.14 (July 15, 2007): 1714-1719.
PMID
17639077
Source
pubmed
Published In
Genes & development
Volume
21
Issue
14
Publish Date
2007
Start Page
1714
End Page
1719
DOI
10.1101/gad.1549407

The PAX3-FKHR fusion gene of rhabdomyosarcoma cooperates with loss of p16INK4A to promote bypass of cellular senescence.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of <30%. ARMS is characterized by a chromosomal translocation generating the PAX3-FKHR fusion gene. However, ectopic expression of PAX3-FKHR often induces inhibition of cell proliferation, or cell death, when expressed in nonmuscle cells. This prompted us to explore the effect of expressing PAX3-FKHR in more relevant cells, specifically primary human skeletal muscle cells because these cells can be converted to a tumorigenic state that mimics rhabdomyosarcoma. PAX3-FKHR expression promoted both fetal and postnatal primary human skeletal muscle cell precursors to bypass the senescence growth arrest checkpoint. This bypass was accompanied by epigenetic DNA methylation of the p16(INK4A) promoter and correspondingly a loss of expression of this tumor suppressor. Knockdown of p16(INK4A) cooperated with PAX3-FKHR to drive proliferation past senescence, whereas reintroduction of wild-type p16(INK4A) in post-senescent cells caused growth arrest. Thus, PAX3-FKHR acts in concert with loss of p16(INK4A) to promote inappropriate proliferation of skeletal muscle cells. This association between PAX3-FKHR expression and p16(INK4A) loss was seen in human ARMS tumor tissue, as both human rhabdomyosarcoma cell lines and tissue microarrays showed a trend toward down-regulation of p16(INK4A) protein in alveolar subsets. We surmise that the generation of the PAX3-FKHR fusion protein may require loss of p16(INK4A) to promote malignant proliferation of skeletal muscle cells as an early step in ARMS tumorigenesis.

Authors
Linardic, CM; Naini, S; Herndon, JE; Kesserwan, C; Qualman, SJ; Counter, CM
MLA Citation
Linardic, CM, Naini, S, Herndon, JE, Kesserwan, C, Qualman, SJ, and Counter, CM. "The PAX3-FKHR fusion gene of rhabdomyosarcoma cooperates with loss of p16INK4A to promote bypass of cellular senescence." Cancer Res 67.14 (July 15, 2007): 6691-6699.
PMID
17638879
Source
pubmed
Published In
Cancer Research
Volume
67
Issue
14
Publish Date
2007
Start Page
6691
End Page
6699
DOI
10.1158/0008-5472.CAN-06-3210

Genetic induction of tumorigenesis in swine.

The transition from basic to clinical cancer research for a number of experimental therapeutics is hampered by the lack of a genetically malleable, large animal model. To this end, we genetically engineered primary porcine cells to be tumorigenic by expression of proteins known to perturb pathways commonly corrupted in human cancer. Akin to human cells, these porcine cells were quite resistant to transformation, requiring multiple genetic changes. Moreover, the transformed porcine cells produced tumors when returned to the isogenic host animal. The ability to now rapidly and reproducibly genetically induce tumors of sizes similar to those treated clinically in a large mammal similar to humans in many respects will provide a robust cancer model for preclinical studies dependent on generating large tumors.

Authors
Adam, SJ; Rund, LA; Kuzmuk, KN; Zachary, JF; Schook, LB; Counter, CM
MLA Citation
Adam, SJ, Rund, LA, Kuzmuk, KN, Zachary, JF, Schook, LB, and Counter, CM. "Genetic induction of tumorigenesis in swine." Oncogene 26.7 (February 15, 2007): 1038-1045.
PMID
16964292
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
26
Issue
7
Publish Date
2007
Start Page
1038
End Page
1045
DOI
10.1038/sj.onc.1209892

Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells.

BACKGROUND: The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. RESULTS: We now show that inhibition of RalA but not RalB expression universally reduced the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. CONCLUSIONS: RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.

Authors
Lim, K-H; O'Hayer, K; Adam, SJ; Kendall, SD; Campbell, PM; Der, CJ; Counter, CM
MLA Citation
Lim, K-H, O'Hayer, K, Adam, SJ, Kendall, SD, Campbell, PM, Der, CJ, and Counter, CM. "Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells." Curr Biol 16.24 (December 19, 2006): 2385-2394.
PMID
17174914
Source
pubmed
Published In
Current Biology
Volume
16
Issue
24
Publish Date
2006
Start Page
2385
End Page
2394
DOI
10.1016/j.cub.2006.10.023

hSnm1B is a novel telomere-associated protein.

Artemis, a member of the beta-CASP family, has been implicated in the regulation of both telomere stability and length. Prompted by this, we examined whether the other two putative DNA-binding members of this family, hSnm1A and hSnm1B, may associate with telomeres. hSnm1A was found to not interact with the telomere. Conversely, hSnm1B was found to associate with telomeres in vivo by both immunofluorescence and chromatin immunoprecipitation. Furthermore, the C terminus of hSnm1B was shown to interact with the TRF homology domain of TRF2 indicating that hSnm1B is likely recruited to the telomere via interaction with the double-stranded telomere-binding protein TRF2.

Authors
Freibaum, BD; Counter, CM
MLA Citation
Freibaum, BD, and Counter, CM. "hSnm1B is a novel telomere-associated protein." J Biol Chem 281.22 (June 2, 2006): 15033-15036.
PMID
16606622
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
22
Publish Date
2006
Start Page
15033
End Page
15036
DOI
10.1074/jbc.C600038200

Genetically engineered human cancer models utilizing mammalian transgene expression.

Cancer models are vital to cancer biology research, and multiple cancer models are currently available that utilize either murine or human cells, each with particular strengths and weaknesses. The ability to transform primary human cells into tumors through the expression of specific transgenes offers many advantages as a cancer model, including genetic malleability and the ability to transform specific cell types. Until recently, the conversion of primary human cells into tumors through transgene expression required the use of viral genetic elements, which unfortunately adds uncertainty regarding which cancer pathways are affected and how they are affected. In recent years multiple reports have described the transformation of primary human cells into tumors using only mammalian transgenes. This review focuses on these five cancer models, comparing the different cell types which were transformed into tumors and which transgenes were expressed, as well as the cancer pathways affected in the disparate models. These genetically-engineered human cancer models offer a valuable tool to complement existing cancer models and further cancer research.

Authors
Kendall, SD; Adam, SJ; Counter, CM
MLA Citation
Kendall, SD, Adam, SJ, and Counter, CM. "Genetically engineered human cancer models utilizing mammalian transgene expression." Cell Cycle 5.10 (May 2006): 1074-1079. (Review)
PMID
16687931
Source
pubmed
Published In
Cell Cycle
Volume
5
Issue
10
Publish Date
2006
Start Page
1074
End Page
1079
DOI
10.4161/cc.5.10.2734

A genetically defined normal human somatic cell system to study ras oncogenesis in vivo and in vitro.

Transgenic mice, cultured murine cells, and human cancer cell lines have widely been used to study Ras oncogenesis. Although extremely valuable systems, they could not be used to study Ras function in genetically defined human cells. In this regard, Ras is required for tumor formation in normal human somatic cells expressing SV-40 T/t antigens, which inactivate the tumor suppressors p53 and Rb and activate the oncogene c-Myc, and hTERT, the catalytic subunit of telomerase. Such a system allows not only the general requirements of Ras to be dissected in matched cells from different organisms or tissues but also the individual pathways required for tumor growth to be defined in human cells. This review will detail the methods of creating stable T/t Ag, TERT, Ras-expressing cell lines, as well as commonly used techniques of soft agar and xenograft tumor formation.

Authors
O'Hayer, KM; Counter, CM
MLA Citation
O'Hayer, KM, and Counter, CM. "A genetically defined normal human somatic cell system to study ras oncogenesis in vivo and in vitro." Methods Enzymol 407 (2006): 637-647. (Review)
PMID
16757358
Source
pubmed
Published In
Methods in Enzymology
Volume
407
Publish Date
2006
Start Page
637
End Page
647
DOI
10.1016/S0076-6879(05)07050-3

Use of retrovirus expression of interfering RNA to determine the contribution of activated K-Ras and ras effector expression to human tumor cell growth.

Cancer is a multistep genetic process that includes mutational activation of oncogenes and inactivation of tumor suppressor genes. The Ras oncogenes are the most frequently mutated oncogenes in human cancers (30%), with a high frequency associated with cancers of the lung, colon, and pancreas. Mutational activation of Ras is commonly an early event in the development of these cancers. Thus, whether mutated Ras is required for tumor maintenance and what aspects of the complex malignant phenotype might be promoted by mutated Ras are issues that remain unresolved for these and other human cancers. The recent development of interfering RNA to selectively impair expression of mutated Ras provides a powerful approach to begin to resolve these issues. In this chapter, we describe the use of retrovirus-based RNA interference approaches to study the functions of Ras and Ras effectors (Raf, RalA, RalB, and Tiam1) in the growth of pancreatic carcinoma and other human tumor cell lines. Finally, we also compare the use of constitutive and inducible shRNA expression vectors for analyses of mutant Ras function.

Authors
Baines, AT; Lim, K-H; Shields, JM; Lambert, JM; Counter, CM; Der, CJ; Cox, AD
MLA Citation
Baines, AT, Lim, K-H, Shields, JM, Lambert, JM, Counter, CM, Der, CJ, and Cox, AD. "Use of retrovirus expression of interfering RNA to determine the contribution of activated K-Ras and ras effector expression to human tumor cell growth." Methods Enzymol 407 (2006): 556-574.
PMID
16757353
Source
pubmed
Published In
Methods in Enzymology
Volume
407
Publish Date
2006
Start Page
556
End Page
574
DOI
10.1016/S0076-6879(05)07045-X

A network of genetic events sufficient to convert normal human cells to a tumorigenic state.

Although great progress has been made at identifying and characterizing individual genes involved in cancer, less is known about how the combination of such genes collaborate to form tumors in humans. To this end, we sought to genetically recreate tumorigenesis in normal human cells using genes altered in human cancer. We now show that expression of mammalian proteins that inactivate the tumor suppressors Rb and p53 in conjunction with the oncoproteins Ras and Myc and the telomerase subunit hTERT is sufficient to drive a number of normal human somatic cells to a tumorigenic fate. This provides a blueprint of the events that lead to human cancer, allowing different cancers to be genetically modeled from normal human cells.

Authors
Kendall, SD; Linardic, CM; Adam, SJ; Counter, CM
MLA Citation
Kendall, SD, Linardic, CM, Adam, SJ, and Counter, CM. "A network of genetic events sufficient to convert normal human cells to a tumorigenic state." Cancer Res 65.21 (November 1, 2005): 9824-9828.
PMID
16267004
Source
pubmed
Published In
Cancer Research
Volume
65
Issue
21
Publish Date
2005
Start Page
9824
End Page
9828
DOI
10.1158/0008-5472.CAN-05-1543

Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance.

While tumors become addicted to oncogenes like Ras, the microenvironment in which tumor cells reside changes during tumorigenesis; the cells are surrounded initially by normal tissue and later by tumor tissue. Hence, we asked if Ras exerts its oncogenic effects through the same set of effectors during different stages of tumorigenesis. We now show in human cells that the Ras effector pathways MAPK, RalGEF, and PI3K are required to initiate tumor growth. Conversely, activation of the PI3K/AKT pathway replaced Ras once tumors formed, although other effectors were still activated independently of Ras, presumably by factors provided upon the establishment of a tumor microenvironment. Thus, as tumorigenesis progresses the addiction of cancers to their initiating oncogene is reduced to, at least in the case of Ras, the PI3K/AKT pathway.

Authors
Lim, K-H; Counter, CM
MLA Citation
Lim, K-H, and Counter, CM. "Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance." Cancer Cell 8.5 (November 2005): 381-392.
PMID
16286246
Source
pubmed
Published In
Cancer Cell
Volume
8
Issue
5
Publish Date
2005
Start Page
381
End Page
392
DOI
10.1016/j.ccr.2005.10.014

Blood vessels engineered from human cells.

Tissue engineering has made considerable progress in the past decade, but advances have stopped short of clinical application for most tissues. We postulated that an obstacle in engineering human tissues is the limited replicative capacity of adult somatic cells. To test this hypothesis, the effectiveness of telomerase expression to extend cellular lifespan was assessed in a model of human vascular tissue engineering. Telomerase expression in vascular cells isolated from elderly patients enabled the successful culture of engineered autologous blood vessels. Engineered vessels may one day provide a source of bypass conduit for patients with atherosclerotic disease.

Authors
Poh, M; Boyer, M; Solan, A; Dahl, SLM; Pedrotty, D; Banik, SSR; McKee, JA; Klinger, RY; Counter, CM; Niklason, LE
MLA Citation
Poh, M, Boyer, M, Solan, A, Dahl, SLM, Pedrotty, D, Banik, SSR, McKee, JA, Klinger, RY, Counter, CM, and Niklason, LE. "Blood vessels engineered from human cells." Lancet 365.9477 (June 18, 2005): 2122-2124.
PMID
15964449
Source
pubmed
Published In
The Lancet
Volume
365
Issue
9477
Publish Date
2005
Start Page
2122
End Page
2124
DOI
10.1016/S0140-6736(05)66735-9

Genetic modeling of human rhabdomyosarcoma.

Rhabdomyosarcoma, a malignancy showing features of skeletal muscle differentiation, is the most common soft tissue sarcoma of childhood. The identification of distinct clinical presentation patterns, histologic tumor types, and risk groups suggests that rhabdomyosarcoma is a collection of highly related sarcomas rather than a single entity. In an effort to understand this seemingly heterogeneous malignancy, we constructed a genetically defined but malleable model of rhabdomyosarcoma by converting less differentiated human skeletal muscle cell precursors (SkMC) and committed human skeletal muscle myoblasts (HSMM) into their malignant counterparts by targeting pathways altered in rhabdomyosarcoma. Whereas the two cell types were both tumorigenic, SkMCs gave rise to highly heterogeneous tumors occasionally displaying features of rhabdomyosarcoma, whereas HSMMs formed rhabdomyosarcoma-like tumors with an embryonal morphology, capable of invasion and metastasis. Thus, despite introducing the same panel of genetic changes, altering the skeletal muscle cell of origin led to different tumor morphologies, suggesting that cell of origin may dictate rhabdomyosarcoma tumor histology. The ability to now genetically induce human rhabdomyosarcoma-like tumors provides a representative model to dissect the molecular mechanisms underlying this cancer.

Authors
Linardic, CM; Downie, DL; Qualman, S; Bentley, RC; Counter, CM
MLA Citation
Linardic, CM, Downie, DL, Qualman, S, Bentley, RC, and Counter, CM. "Genetic modeling of human rhabdomyosarcoma." Cancer Res 65.11 (June 1, 2005): 4490-4495.
PMID
15930263
Source
pubmed
Published In
Cancer Research
Volume
65
Issue
11
Publish Date
2005
Start Page
4490
End Page
4495
DOI
10.1158/0008-5472.CAN-04-3194

Activation of RalA is critical for Ras-induced tumorigenesis of human cells.

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

Authors
Lim, K-H; Baines, AT; Fiordalisi, JJ; Shipitsin, M; Feig, LA; Cox, AD; Der, CJ; Counter, CM
MLA Citation
Lim, K-H, Baines, AT, Fiordalisi, JJ, Shipitsin, M, Feig, LA, Cox, AD, Der, CJ, and Counter, CM. "Activation of RalA is critical for Ras-induced tumorigenesis of human cells." Cancer Cell 7.6 (June 2005): 533-545.
PMID
15950903
Source
pubmed
Published In
Cancer Cell
Volume
7
Issue
6
Publish Date
2005
Start Page
533
End Page
545
DOI
10.1016/j.ccr.2005.04.030

Blood vessels engineered from human cells [3] (multiple letters)

Authors
Kassem, M; Burns, J; Abd-Allah, B; Torensma, R; Niklason, LE; Counter, CM
MLA Citation
Kassem, M, Burns, J, Abd-Allah, B, Torensma, R, Niklason, LE, and Counter, CM. "Blood vessels engineered from human cells [3] (multiple letters)." Lancet 366.9489 (2005): 891-893.
PMID
16154007
Source
scival
Published In
Lancet
Volume
366
Issue
9489
Publish Date
2005
Start Page
891
End Page
893
DOI
10.1016/S0140-6736(05)67311-4

Loss of hPot1 function leads to telomere instability and a cut-like phenotype.

The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2. Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation. However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death. Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans. Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability. In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation. However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation. We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.

Authors
Veldman, T; Etheridge, KT; Counter, CM
MLA Citation
Veldman, T, Etheridge, KT, and Counter, CM. "Loss of hPot1 function leads to telomere instability and a cut-like phenotype." Curr Biol 14.24 (December 29, 2004): 2264-2270.
PMID
15620654
Source
pubmed
Published In
Current Biology
Volume
14
Issue
24
Publish Date
2004
Start Page
2264
End Page
2270
DOI
10.1016/j.cub.2004.12.031

Characterization of interactions between PinX1 and human telomerase subunits hTERT and hTR.

The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activity in vitro, less is known about how this activity is regulated in vivo to ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase function in vivo is PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show that in vitro PinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activity in vivo by binding to the assembled hTERT.hTR complex.

Authors
Banik, SSR; Counter, CM
MLA Citation
Banik, SSR, and Counter, CM. "Characterization of interactions between PinX1 and human telomerase subunits hTERT and hTR." J Biol Chem 279.50 (December 10, 2004): 51745-51748.
PMID
15381700
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
50
Publish Date
2004
Start Page
51745
End Page
51748
DOI
10.1074/jbc.M408131200

Genomics and clinical medicine: rationale for creating and effectively evaluating animal models.

Because resolving human complex diseases is difficult, appropriate biomedical models must be developed and validated. In the past, researchers have studied diseases either by characterizing a human clinical disease and choosing the most appropriate animal model, or by characterizing a naturally occurring or induced mutant animal and identifying which human disease it best resembled. Although there has been a great deal of progress through the use of these methods, such models have intrinsic faults that limit their relevance to clinical medicine. The recent advent of techniques in molecular biology, genomics, transgenesis, and cloning furnishes investigators with the ability to study vertebrates (e.g., pigs, cows, chickens, dogs) with greater precision and utilize them as model organisms. Comparative and functional genomics and proteomics provide effective approaches for identifying the genetic and environmental factors responsible for complex diseases and in the development of prevention and treatment strategies and therapeutics. By identifying and studying homologous genes across species, researchers are able to accurately translate and apply experimental data from animal experiments to humans. This review supports the hypothesis that associated enabling technologies can be used to create, de novo, appropriate animal models that recapitulate the human clinical manifestation. Comparative and functional genomic and proteomic techniques can then be used to identify gene and protein functions and the interactions responsible for disease phenotypes, which aids in the development of prevention and treatment strategies.

Authors
Swanson, KS; Mazur, MJ; Vashisht, K; Rund, LA; Beever, JE; Counter, CM; Schook, LB
MLA Citation
Swanson, KS, Mazur, MJ, Vashisht, K, Rund, LA, Beever, JE, Counter, CM, and Schook, LB. "Genomics and clinical medicine: rationale for creating and effectively evaluating animal models." Exp Biol Med (Maywood) 229.9 (October 2004): 866-875. (Review)
PMID
15388881
Source
pubmed
Published In
Experimental biology and medicine (Maywood, N.J.)
Volume
229
Issue
9
Publish Date
2004
Start Page
866
End Page
875

Leveling the playing field.

Using a genetically malleable model system that allows direct comparisons between human and mouse cells, Weinberg and colleagues lay to rest any doubt that murine cells are more easily transformed than human cells, and additionally, find that there may be cell type constraints on transformation of human cells.

Authors
Lim, K-H; Counter, CM
MLA Citation
Lim, K-H, and Counter, CM. "Leveling the playing field." Mol Cell 15.4 (August 27, 2004): 491-492.
PMID
15327763
Source
pubmed
Published In
Molecular Cell
Volume
15
Issue
4
Publish Date
2004
Start Page
491
End Page
492
DOI
10.1016/j.molcel.2004.08.014

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.

Authors
Yeh, E; Cunningham, M; Arnold, H; Chasse, D; Monteith, T; Ivaldi, G; Hahn, WC; Stukenberg, PT; Shenolikar, S; Uchida, T; Counter, CM; Nevins, JR; Means, AR; Sears, R
MLA Citation
Yeh, E, Cunningham, M, Arnold, H, Chasse, D, Monteith, T, Ivaldi, G, Hahn, WC, Stukenberg, PT, Shenolikar, S, Uchida, T, Counter, CM, Nevins, JR, Means, AR, and Sears, R. "A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells." Nat Cell Biol 6.4 (April 2004): 308-318.
PMID
15048125
Source
pubmed
Published In
Nature Cell Biology
Volume
6
Issue
4
Publish Date
2004
Start Page
308
End Page
318
DOI
10.1038/ncb1110

Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1.

The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.

Authors
Armbruster, BN; Linardic, CM; Veldman, T; Bansal, NP; Downie, DL; Counter, CM
MLA Citation
Armbruster, BN, Linardic, CM, Veldman, T, Bansal, NP, Downie, DL, and Counter, CM. "Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1." Mol Cell Biol 24.8 (April 2004): 3552-3561.
PMID
15060173
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
24
Issue
8
Publish Date
2004
Start Page
3552
End Page
3561

Creating porcine biomedical models through recombineering.

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to 'forward genetics', in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the 'reverse genetics' approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a 'genetic model' organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide 'clones' of genetically modified animals.

Authors
Rogatcheva, MM; Rund, LA; Swanson, KS; Marron, BM; Beever, JE; Counter, CM; Schook, LB
MLA Citation
Rogatcheva, MM, Rund, LA, Swanson, KS, Marron, BM, Beever, JE, Counter, CM, and Schook, LB. "Creating porcine biomedical models through recombineering." Comp Funct Genomics 5.3 (2004): 262-267.
PMID
18629152
Source
pubmed
Published In
Comparative and Functional Genomics (Hindawi)
Volume
5
Issue
3
Publish Date
2004
Start Page
262
End Page
267
DOI
10.1002/cfg.404

In vivo regulation of hTERT expression and telomerase activity by androgen.

PURPOSE: The most effective therapy for metastatic prostate cancer is androgen deprivation. Genes activated directly or possibly even indirectly by this steroid hormone represent potential targets for anticancer therapy. One such gene may be hTERT, which encodes the catalytic subunit of telomerase. In prostate cancer cells telomerase is activated, permitting sustained proliferation. Therefore, we tested whether hTERT gene expression is modulated in prostate cancer cells in vitro and in vivo by androgens. MATERIALS AND METHODS: Transcriptional activation of hTERT during androgen stimulation was assayed by luciferase assays using the hTERT promoter fused to the luciferase gene and by reverse transcriptase-polymerase chain reaction to detect endogenous hTERT mRNA in LNCaP cells. hTERT mRNA levels and telomerase activity were also measured in CWR22 prostate cancer cells implanted in mice that were subsequently castrated and left untreated or administered androgen. RESULTS: We report that the endogenous hTERT promoter is activated during the administration of androgen to androgen sensitive LNCaP prostate cancer cells. However, this effect was indirect since an hTERT promoter construct was not activated by androgens and transcription of the endogenous gene was not stimulated early enough in cultured cells to be considered a direct target of this steroid hormone. Importantly in an in vivo model of human prostate cancer androgen deprivation led to a decrease in hTERT expression, followed by a decrease in telomerase activity, which was reversed by a single administration of androgen. CONCLUSIONS: The hTERT gene is regulated in human prostate cancer cells in vivo by androgens.

Authors
Guo, C; Armbruster, BN; Price, DT; Counter, CM
MLA Citation
Guo, C, Armbruster, BN, Price, DT, and Counter, CM. "In vivo regulation of hTERT expression and telomerase activity by androgen." J Urol 170.2 Pt 1 (August 2003): 615-618.
PMID
12853842
Source
pubmed
Published In
The Journal of Urology
Volume
170
Issue
2 Pt 1
Publish Date
2003
Start Page
615
End Page
618
DOI
10.1097/01.ju.0000074653.22766.c8

Human arteries engineered in vitro.

There is a pressing need to develop methods to engineer small-calibre arteries for bypass surgery. We hypothesized that the rate-limiting step that has thwarted previous attempts to engineer such vessels from non-neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non-neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

Authors
McKee, JA; Banik, SSR; Boyer, MJ; Hamad, NM; Lawson, JH; Niklason, LE; Counter, CM
MLA Citation
McKee, JA, Banik, SSR, Boyer, MJ, Hamad, NM, Lawson, JH, Niklason, LE, and Counter, CM. "Human arteries engineered in vitro." EMBO Rep 4.6 (June 2003): 633-638.
PMID
12776184
Source
pubmed
Published In
EMBO Reports
Volume
4
Issue
6
Publish Date
2003
Start Page
633
End Page
638
DOI
10.1038/sj.embor.embor847

Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model.

We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitro phenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis.

Authors
Rich, JN; Shi, Q; Hjelmeland, M; Cummings, TJ; Kuan, C-T; Bigner, DD; Counter, CM; Wang, X-F
MLA Citation
Rich, JN, Shi, Q, Hjelmeland, M, Cummings, TJ, Kuan, C-T, Bigner, DD, Counter, CM, and Wang, X-F. "Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model." J Biol Chem 278.18 (May 2, 2003): 15951-15957.
PMID
12590137
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
18
Publish Date
2003
Start Page
15951
End Page
15957
DOI
10.1074/jbc.M211498200

Putative telomere-recruiting domain in the catalytic subunit of human telomerase.

Telomerase, the enzyme that elongates telomeres, is essential to maintain telomere length and to immortalize most cancer cells. However, little is known about the regulation of this enzyme in higher eukaryotes. We previously described a domain in the hTERT telomerase catalytic subunit that is essential for telomere elongation and cell immortalization in vivo but dispensable for catalytic activity in vitro. Here, we show that fusions of hTERT containing different mutations in this domain to the telomere binding protein hTRF2 redirected the mutated hTERT to telomeres and rescued its in vivo functions. We suggest that this domain posttranscriptionally regulates telomerase function by targeting the enzyme to telomeres.

Authors
Armbruster, BN; Etheridge, KT; Broccoli, D; Counter, CM
MLA Citation
Armbruster, BN, Etheridge, KT, Broccoli, D, and Counter, CM. "Putative telomere-recruiting domain in the catalytic subunit of human telomerase." Mol Cell Biol 23.9 (May 2003): 3237-3246.
PMID
12697823
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
23
Issue
9
Publish Date
2003
Start Page
3237
End Page
3246

Telomere shortening in cultured autografts of patients with burns.

In extensive third-degree burns, donor sites for conventional split thickness skin grafts are limited. In such cases, cultured epithelial (keratinocyte) grafts are prepared from small samples of the patient's own skin and expanded in tissue culture, a process that may incur very many cell divisions. Telomeres shorten with each cell division, and are markers of cellular proliferative history. We therefore measured telomere length in healed cultured epithelial autografts from four patients with burns, and noted that their telomeres were shorter than those in non-cultured skin from the same individuals, and than those in skin of healthy donors older than 80 years. Such great loss of telomeric DNA suggests that engrafted cells might have a shortened lifespan, which could have negative repercussions on the long-term viability of these grafts.

Authors
Counter, CM; Press, W; Compton, CC
MLA Citation
Counter, CM, Press, W, and Compton, CC. "Telomere shortening in cultured autografts of patients with burns." Lancet 361.9366 (April 19, 2003): 1345-1346.
PMID
12711471
Source
pubmed
Published In
The Lancet
Volume
361
Issue
9366
Publish Date
2003
Start Page
1345
End Page
1346
DOI
10.1016/S0140-6736(03)13042-5

Mutational analysis defines a minimum level of telomerase activity required for tumourigenic growth of human cells.

A hallmark of cancer cells is the ability to proliferate indefinitely. This acquisition of an immortal lifespan usually requires the activation of telomerase, the enzyme that elongates telomeres. Human telomerase is minimally composed of the reverse transcriptase subunit hTERT, and the RNA subunit hTR. While hTR is ubiquitously expressed in human cells, the hTERT subunit is generally transcriptionally repressed in most normal somatic cells, but is illegitimately activated to restore telomerase activity in cancer cells. Indeed, in the thousands of different human tumours assayed, 85% were scored positive for telomerase activity. However, the levels of telomerase activity detected in tumour samples can vary substantially and even some normal somatic cells have been found to have low levels of enzyme activity. As the functional significance of low levels of telomerase activity is unclear, we investigated whether there is a minimum level of telomerase activity required for tumourigenesis. Using mutants of hTERT that induce varying levels of telomerase activity, we show that there does indeed exist a threshold of activity required for the processes of immortalization, transformation and tumourigenesis. Thus, low levels of activity detected in certain somatic cells would not be expected to contribute to tumourigenesis, nor does the mere detection of telomerase in cancer cells necessarily signify an immortal lifespan.

Authors
Hamad, NM; Banik, SSR; Counter, CM
MLA Citation
Hamad, NM, Banik, SSR, and Counter, CM. "Mutational analysis defines a minimum level of telomerase activity required for tumourigenic growth of human cells." Oncogene 21.46 (October 10, 2002): 7121-7125.
PMID
12370834
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
21
Issue
46
Publish Date
2002
Start Page
7121
End Page
7125
DOI
10.1038/sj.onc.1205860

C-terminal regions of the human telomerase catalytic subunit essential for in vivo enzyme activity.

Most human cancer cells are thought to acquire the ability to divide beyond the capacity of normal somatic cells through illegitimately activating the gene hTERT, which encodes the catalytic subunit of telomerase. While telomerase reverse transcriptase (TERT) is conserved in most eukaryotes, mounting evidence suggests that the C terminus of the human protein may have functions unique to higher eukaryotes. To search for domains responsible for such functions, we assayed a panel of tandem substitution mutations encompassing this region of human TERT for in vitro and in vivo functionality. We found four clusters of mutations that inactivated the biochemical and biological functions of telomerase, separated by mutations that had little or no effect on enzyme activity. We also identified a region where mutations generate catalytically active but biologically inert proteins. This C-terminal region that dissociates activities of telomerase (C-DAT) does not appear to be involved in nuclear localization or protein multimerization. Instead, it appears that the C-DAT region is involved in a step of in vivo telomere synthesis after the assembly of a catalytically active enzyme. Intriguingly, all of the described regions reside in a portion of TERT that is dispensable for cellular viability in yeast, arguing for a divergent role of the C terminus in higher eukaryotes.

Authors
Banik, SSR; Guo, C; Smith, AC; Margolis, SS; Richardson, DA; Tirado, CA; Counter, CM
MLA Citation
Banik, SSR, Guo, C, Smith, AC, Margolis, SS, Richardson, DA, Tirado, CA, and Counter, CM. "C-terminal regions of the human telomerase catalytic subunit essential for in vivo enzyme activity." Mol Cell Biol 22.17 (September 2002): 6234-6246.
PMID
12167716
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
17
Publish Date
2002
Start Page
6234
End Page
6246

Distinct requirements for Ras oncogenesis in human versus mouse cells.

The spectrum of tumors associated with oncogenic Ras in humans often differs from those in mice either treated with carcinogens or engineered to sporadically express oncogenic Ras, suggesting that the mechanism of Ras transformation may be different in humans. Ras stimulates primarily three main classes of effector proteins, Rafs, PI3-kinase, and RalGEFs, with Raf generally being the most potent at transforming murine cells. Using oncogenic Ras mutants that activate single effectors as well as constitutively active effectors, we find that the RalGEF, and not the Raf or PI3-kinase pathway, is sufficient for Ras transformation in human cells. Thus, oncogenic Ras may transform murine and human cells by distinct mechanisms, and the RalGEF pathway--previously deemed to play a secondary role in Ras transformation--could represent a new target for anti-cancer therapy.

Authors
Hamad, NM; Elconin, JH; Karnoub, AE; Bai, W; Rich, JN; Abraham, RT; Der, CJ; Counter, CM
MLA Citation
Hamad, NM, Elconin, JH, Karnoub, AE, Bai, W, Rich, JN, Abraham, RT, Der, CJ, and Counter, CM. "Distinct requirements for Ras oncogenesis in human versus mouse cells." Genes Dev 16.16 (August 15, 2002): 2045-2057. (Review)
PMID
12183360
Source
pubmed
Published In
Genes & development
Volume
16
Issue
16
Publish Date
2002
Start Page
2045
End Page
2057
DOI
10.1101/gad.993902

The nucleolar localization domain of the catalytic subunit of human telomerase.

Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTR-binding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTR-binding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.

Authors
Etheridge, KT; Banik, SSR; Armbruster, BN; Zhu, Y; Terns, RM; Terns, MP; Counter, CM
MLA Citation
Etheridge, KT, Banik, SSR, Armbruster, BN, Zhu, Y, Terns, RM, Terns, MP, and Counter, CM. "The nucleolar localization domain of the catalytic subunit of human telomerase." J Biol Chem 277.27 (July 5, 2002): 24764-24770.
PMID
11956201
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
27
Publish Date
2002
Start Page
24764
End Page
24770
DOI
10.1074/jbc.M201227200

Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression.

Human postnatal bone marrow stromal stem cells (BMSSCs) have a limited life-span and progressively lose their stem cell properties during ex vivo expansion. Here we report that ectopic expression of human telomerase reverse transcriptase (hTERT) in BMSSCs extended their life-span and maintained their osteogenic potential. In xenogenic transplants, hTERT-expressing BMSSCs (BMSSC-Ts) generated more bone tissue, with a mineralized lamellar bone structure and associated marrow, than did control BMSSCs. The enhanced bone-forming ability of BMSSC-Ts was correlated with a higher and sustained expression of the early pre-osteogenic stem cell marker STRO-1, indicating that telomerase expression helped to maintain the osteogenic stem cell pool during ex vivo expansion. These results show that telomerase expression can overcome critical technical barriers to the ex vivo expansion of BMSSCs, and suggest that telomerase therapy may be a useful strategy for bone regeneration and repair.

Authors
Shi, S; Gronthos, S; Chen, S; Reddi, A; Counter, CM; Robey, PG; Wang, C-Y
MLA Citation
Shi, S, Gronthos, S, Chen, S, Reddi, A, Counter, CM, Robey, PG, and Wang, C-Y. "Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression." Nat Biotechnol 20.6 (June 2002): 587-591.
PMID
12042862
Source
pubmed
Published In
Nature Biotechnology
Volume
20
Issue
6
Publish Date
2002
Start Page
587
End Page
591
DOI
10.1038/nbt0602-587

N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo.

Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits. However, catalytic activity alone is insufficient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerase-negative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation. We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution. Additionally, we discovered a novel domain, DAT, that "dissociates activities of telomerase," where mutations left the enzyme catalytically active, but was unable to function in vivo. Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.

Authors
Armbruster, BN; Banik, SS; Guo, C; Smith, AC; Counter, CM
MLA Citation
Armbruster, BN, Banik, SS, Guo, C, Smith, AC, and Counter, CM. "N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo." Mol Cell Biol 21.22 (November 2001): 7775-7786.
PMID
11604512
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
21
Issue
22
Publish Date
2001
Start Page
7775
End Page
7786
DOI
10.1128/MCB.21.22.7775-7786.2001

Inhibition of telomerase is related to the life span and tumorigenicity of human prostate cancer cells.

PURPOSE: Telomerase, the enzyme that catalyzes the elongation of telomeres, is illegitimately activated in the majority of cancers, including that of the prostate, where it may greatly extend the life span of malignant cells. The inhibition of telomerase by molecular intervention has been shown to lead eventually to cell death in several tumor or in vitro immortalized cell lines and in 1 case prevent tumor growth in vivo. Therefore, we tested whether a similar strategy may be used to limit the tumorigenic potential of late stage prostate cancer cells. MATERIALS AND METHODS: PC-3, LNCaP and DU-145 human prostate cancer cells were infected with a retrovirus encoding a dominant-negative version of the catalytic subunit of telomerase (DN-hTERT). Subclones or polyclonal populations were assayed for DN-hTERT expression, telomerase activity, telomere length, cell life span and in most cases tumorigenicity in nude mice. RESULTS: DN-hTERT expression levels directly correlated with cell life span and tumorigenic growth. PC-3 cells expressing high levels of DN-hTERT died rapidly and failed to form tumors in nude mice, whereas cells expressing the lowest levels proliferated the longest and generated tumors that later spontaneously regressed. Similarly the inhibition of telomerase activity in LNCaP cells was greater than in DU-145 cells and correspondingly LNCaP cells had a shorter life span. CONCLUSIONS: DN-hTERT expression limits the life span and tumorigenic potential of human prostate cancer cells, although the onset of these effects appears to be dictated by the expression level of DN-hTERT. Therefore, telomerase represents an attractive target for potentially managing prostate cancer. Nevertheless, effective means of inhibiting the enzyme may be required for a therapeutically useful outcome.

Authors
Guo, C; Geverd, D; Liao, R; Hamad, N; Counter, CM; Price, DT
MLA Citation
Guo, C, Geverd, D, Liao, R, Hamad, N, Counter, CM, and Price, DT. "Inhibition of telomerase is related to the life span and tumorigenicity of human prostate cancer cells." J Urol 166.2 (August 2001): 694-698.
PMID
11458119
Source
pubmed
Published In
The Journal of Urology
Volume
166
Issue
2
Publish Date
2001
Start Page
694
End Page
698

A genetically tractable model of human glioma formation.

Gliomas remain one of the deadliest forms of cancer. Improved therapeutics will require a better understanding of the molecular nature of these tumors. We, therefore, mimicked the most common genetic changes found in grade III-IV gliomas, disruption of the p53 and RB pathways and activation of telomere maintenance and independence from growth factors, through the ectopic expression of the SV40 T/t-Ag oncogene, an oncogenic form of H-ras (H-ras(V12G)), and the human telomerase catalytic subunit hTERT in normal human astrocytes. The resulting cells displayed many of the hallmarks of grade III-IV gliomas, including greatly expanded life span and growth in soft agar and, most importantly, were tumorigenic with pathology consistent with grade III-IV neuroectodermal tumors in mice. This model system will, for the first time, allow the biological significance of selected genetic alterations to be studied in human gliomas.

Authors
Rich, JN; Guo, C; McLendon, RE; Bigner, DD; Wang, XF; Counter, CM
MLA Citation
Rich, JN, Guo, C, McLendon, RE, Bigner, DD, Wang, XF, and Counter, CM. "A genetically tractable model of human glioma formation." Cancer Res 61.9 (May 1, 2001): 3556-3560.
PMID
11325817
Source
pubmed
Published In
Cancer Research
Volume
61
Issue
9
Publish Date
2001
Start Page
3556
End Page
3560

The MEK pathway is required for stimulation of p21(WAF1/CIP1) by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta)can induce the cyclin-dependent kinase inhibitors p21 and p15 in a variety of cell types. We have shown previously that Smad3 is required for the growth inhibitory activity of TGF-beta, whereas overexpression of Smads is not sufficient to activate the expression of p21 in HaCaT cells. These data suggest that an additional signaling pathway may be involved in stimulating p21 in HaCaT cells. Given the recent finding that the mitogen-activated protein kinase (MAPK) pathway can cause p21 induction and arrest cells, we examined the involvement of this pathway for p21 and p15 induction by TGF-beta. We found that TGF-beta can regulate the MAPK pathway, leading to the increased transactivation ability of transcription factor Elk. Constitutively active components in the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway significantly decrease p21 induction by TGF-beta. Both constitutively active MEK and inhibitors for MEK have no effect on Smad activity, including DNA binding, localization, and interaction with coactivator p300/CBP. These findings suggest that the MAPK pathway may be an independent pathway that is involved in p21 and p15 induction by TGF-beta.

Authors
Hu, PP; Shen, X; Huang, D; Liu, Y; Counter, C; Wang, XF
MLA Citation
Hu, PP, Shen, X, Huang, D, Liu, Y, Counter, C, and Wang, XF. "The MEK pathway is required for stimulation of p21(WAF1/CIP1) by transforming growth factor-beta." J Biol Chem 274.50 (December 10, 1999): 35381-35387.
PMID
10585406
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
50
Publish Date
1999
Start Page
35381
End Page
35387

Creation of human tumour cells with defined genetic elements.

During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

Authors
Hahn, WC; Counter, CM; Lundberg, AS; Beijersbergen, RL; Brooks, MW; Weinberg, RA
MLA Citation
Hahn, WC, Counter, CM, Lundberg, AS, Beijersbergen, RL, Brooks, MW, and Weinberg, RA. "Creation of human tumour cells with defined genetic elements." Nature 400.6743 (July 29, 1999): 464-468.
PMID
10440377
Source
pubmed
Published In
Nature
Volume
400
Issue
6743
Publish Date
1999
Start Page
464
End Page
468
DOI
10.1038/22780

Characterization of the repeat-tract instability and mutator phenotypes conferred by a Tn3 insertion in RFC1, the large subunit of the yeast clamp loader

The RFC1 gene encodes the large subunit of the yeast clamp loader (RFC) that is a component of eukaryotic DNA polymerase holoenzymes. We identified a mutant allele of RFC1 (rfc1::Tn3) from a large collection of Saccharomyces cerevisiae mutants that were inviable when present in a rad52 null mutation background. Analysis of rfc1::Tn3 strains indicated that they displayed both a mutator and repeat-tract instability phenotype. Strains bearing this allele were characterized in combination with mismatch repair (msh2Δ, pms1Δ), double-strand break repair (rad52), and DNA replication (pol3-01, pol30-52, rth1Δ/rad27Δ) mutations in both forward mutation and repeat-tract instability assays. This analysis indicated that the rfc1::Tn3 allele displays synthetic lethality with pol30, pol3, and rad27 mutations. Measurement of forward mutation frequencies in msh2Δ rfc1:Tn3 and pms1Δ rfc1:Tn3 strains indicated that the rfc1::Tn3 mutant displayed a mutation frequency that appeared nearly multiplicative with the mutation frequency exhibited by mismatch-repair mutants. In repeat-tract instability assays, however, the rfc1::Tn3 mutant displayed a tract instability phenotype that appeared epistatic to the phenotype displayed by mismatch-repair mutants. From these data we propose that defects in clamp loader function result in DNA replication errors, a subset of which are acted upon by the mismatch- repair system.

Authors
Xie, Y; Counter, C; Alani, E
MLA Citation
Xie, Y, Counter, C, and Alani, E. "Characterization of the repeat-tract instability and mutator phenotypes conferred by a Tn3 insertion in RFC1, the large subunit of the yeast clamp loader." Genetics 151.2 (1999): 499-509.
PMID
9927446
Source
scival
Published In
Genetics
Volume
151
Issue
2
Publish Date
1999
Start Page
499
End Page
509

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization.

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Authors
Counter, CM; Hahn, WC; Wei, W; Caddle, SD; Beijersbergen, RL; Lansdorp, PM; Sedivy, JM; Weinberg, RA
MLA Citation
Counter, CM, Hahn, WC, Wei, W, Caddle, SD, Beijersbergen, RL, Lansdorp, PM, Sedivy, JM, and Weinberg, RA. "Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization." Proc Natl Acad Sci U S A 95.25 (December 8, 1998): 14723-14728.
PMID
9843956
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
25
Publish Date
1998
Start Page
14723
End Page
14728

Expression of TERT in early premalignant lesions and a subset of cells in normal tissues.

Activation of telomerase, the enzyme that synthesizes the telomere ends of linear chromosomes, has been implicated in human cell immortalization and cancer cell pathogenesis. Enzyme activity is undetectable in most normal cells and tissues, but present in immortal cells and cancer tissues. While expression of TERC, the RNA component of telomerase, is widespread, the restricted expression pattern of TERT, the telomerase catalytic subunit gene, is correlated with telomerase activity, and its ectopic expression in telomerase-negative cells is sufficient to reconstitute telomerase activity and extend cellular lifespan. We have used in situ hybridization to study TERT expression at the single-cell level in normal tissues and in various stages of tumour progression. In normal tissues, including some that are known to be telomerase-negative, TERT mRNA was present in specific subsets of cells thought to have long-term proliferative capacity. This included mitotically inactive breast lobular epithelium in addition to some actively regenerating cells such as the stratum basale of the skin. TERT expression appeared early during tumorigenesis in vivo, beginning with early pre-invasive changes in human breast and colon tissues and increasing gradually during progression, both in the amount of TERT mRNA present within individual cells and in the number of expressing cells within a neoplastic lesion. The physiological expression of TERT within normal epithelial cells that retain proliferative potential and its presence at the earliest stages of tumorigenesis have implications for the regulation of telomerase expression and for the identification of cells that may be targets for malignant transformation.

Authors
Kolquist, KA; Ellisen, LW; Counter, CM; Meyerson, M; Tan, LK; Weinberg, RA; Haber, DA; Gerald, WL
MLA Citation
Kolquist, KA, Ellisen, LW, Counter, CM, Meyerson, M, Tan, LK, Weinberg, RA, Haber, DA, and Gerald, WL. "Expression of TERT in early premalignant lesions and a subset of cells in normal tissues." Nat Genet 19.2 (June 1998): 182-186.
PMID
9620778
Source
pubmed
Published In
Nature Genetics
Volume
19
Issue
2
Publish Date
1998
Start Page
182
End Page
186
DOI
10.1038/554

Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase.

The expression of telomerase, the enzyme that synthesizes telomeric DNA de novo, is suppressed in normal somatic human cells but is reactivated during tumorigenesis. This reactivation appears to arrest the normal loss of telomeric DNA incurred as human cells divide. Since continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may represent an important step in the acquisition of the cell immortalization which occurs during tumor progression. The telomerase holoenzyme is composed of both RNA and protein subunits. In humans, mRNA expression of hTERT (hEST2), the candidate telomerase catalytic subunit gene, appears to parallel the levels of telomerase enzyme activity, suggesting that induction of hTERT is necessary and perhaps sufficient for expression of telomerase activity in tumor cells. To test this model directly, we ectopically expressed an epitope-tagged version of hTERT in telomerase-negative cells and show that telomerase activity was induced to levels comparable to those seen in immortal telomerase-positive cells and that the expressed hTERT protein was physically associated with the cellular telomerase activity. We conclude that synthesis of the hTERT telomerase subunit represents the rate-limiting determinant of telomerase activity in these cells and that this protein, once expressed, becomes part of the functional telomerase holoenzyme.

Authors
Counter, CM; Meyerson, M; Eaton, EN; Ellisen, LW; Caddle, SD; Haber, DA; Weinberg, RA
MLA Citation
Counter, CM, Meyerson, M, Eaton, EN, Ellisen, LW, Caddle, SD, Haber, DA, and Weinberg, RA. "Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase." Oncogene 16.9 (March 5, 1998): 1217-1222.
PMID
9528864
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
16
Issue
9
Publish Date
1998
Start Page
1217
End Page
1222
DOI
10.1038/sj.onc.1201882

hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization.

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.

Authors
Meyerson, M; Counter, CM; Eaton, EN; Ellisen, LW; Steiner, P; Caddle, SD; Ziaugra, L; Beijersbergen, RL; Davidoff, MJ; Liu, Q; Bacchetti, S; Haber, DA; Weinberg, RA
MLA Citation
Meyerson, M, Counter, CM, Eaton, EN, Ellisen, LW, Steiner, P, Caddle, SD, Ziaugra, L, Beijersbergen, RL, Davidoff, MJ, Liu, Q, Bacchetti, S, Haber, DA, and Weinberg, RA. "hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization." Cell 90.4 (August 22, 1997): 785-795.
PMID
9288757
Source
pubmed
Published In
Cell
Volume
90
Issue
4
Publish Date
1997
Start Page
785
End Page
795

The catalytic subunit of yeast telomerase.

Telomerase is an RNA-directed DNA polymerase, composed of RNA and protein subunits, that replicates the telomere ends of linear eukaryotic chromosomes. Using a genetic strategy described here, we identify the product of the EST2 gene, Est2p, as a subunit of telomerase in the yeast Saccharomyces cerevisiae. Est2p is required for enzyme catalysis, as mutations in EST2 were found to result in the absence of telomerase activity. Immunochemical experiments show that Est2p is an integral subunit of the telomerase enzyme. Critical catalytic residues present in RNA-directed DNA polymerases are conserved in Est2p; mutation of one such residue abolishes telomerase activity, suggesting a direct catalytic role for Est2p.

Authors
Counter, CM; Meyerson, M; Eaton, EN; Weinberg, RA
MLA Citation
Counter, CM, Meyerson, M, Eaton, EN, and Weinberg, RA. "The catalytic subunit of yeast telomerase." Proc Natl Acad Sci U S A 94.17 (August 19, 1997): 9202-9207.
PMID
9256460
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
17
Publish Date
1997
Start Page
9202
End Page
9207

The roles of telomeres and telomerase in cell life span.

Telomeres cap and protect the ends of chromosomes from degradation and illegitimate recombination. The termini of a linear template cannot, however, be completely replicated by conventional DNA-dependent DNA polymerases, and thus in the absence of a mechanisms to counter this effect, telomeres of eukaryotic cells shorten every round of DNA replication. In humans and possibly other higher eukaryotes, telomere shortening may have been adopted to limit the life span of somatic cells. Human somatic cells have a finite proliferative capacity and enter a viable growth arrested state called senescence. Life span appears to be governed by cell division, not time. The regular loss of telomeric DNA could therefore serve as a mitotic clock in the senescence programme, counting cell divisions. In most eukaryotic organisms, however, telomere shortening can be countered by the de novo addition of telomeric repeats by the enzyme telomerase. Cells which are "immortal' such as the human germ line or tumour cell lines, established mouse cells, yeast and ciliates, all maintain a stable telomere length through the action of telomerase. Abolition of telomerase activity in such cells nevertheless results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Therefore, loss of terminal DNA sequences may limit cell life span by two mechanisms: by acting as a mitotic clock and by denuding chromosomes of protective telomeric DNA necessary for cell viability.

Authors
Counter, CM
MLA Citation
Counter, CM. "The roles of telomeres and telomerase in cell life span." Mutat Res 366.1 (October 1996): 45-63. (Review)
PMID
8921986
Source
pubmed
Published In
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume
366
Issue
1
Publish Date
1996
Start Page
45
End Page
63

Telomerase activity in normal leukocytes and in hematologic malignancies.

Telomeres are essential for function and stability of eukaryotic chromosomes. In the absence of telomerase, the enzyme that synthesizes telomeric DNA, telomeres shorten with cell division, a process thought to contribute to cell senescence and the proliferative crisis of transformed cells. We reported telomere stabilization concomitant with detection of telomerase activity in cells immortalized in vitro and in ovarian carcinoma cells, and suggested that telomerase is essential for unlimited cell proliferation. We have now examined the temporal pattern of telomerase expression in selected hematologic malignancies. We found that, unlike other somatic tissues, peripheral, cord blood, and bone marrow leukocytes from normal donors expressed low levels of telomerase activity. In leukocytes from chronic lymphocytic leukemia (CLL) patients, activity was lower than in controls in early disease, and comparable with controls in late disease. Relative to bone marrow, telomerase activity was enhanced in myelodysplastic syndrome (MDS) and more significantly so in acute myeloid leukemia (AML). Regardless of telomerase levels, telomeres shortened with progression of the diseases. Our results suggest that early CLL and MDS cells lack an efficient mechanism of telomere maintenance and that telomerase is activated late in the progression of these cancers, presumably when critical telomere loss generates selective pressure for cell immortality.

Authors
Counter, CM; Gupta, J; Harley, CB; Leber, B; Bacchetti, S
MLA Citation
Counter, CM, Gupta, J, Harley, CB, Leber, B, and Bacchetti, S. "Telomerase activity in normal leukocytes and in hematologic malignancies." Blood 85.9 (May 1, 1995): 2315-2320.
PMID
7727765
Source
pubmed
Published In
Blood
Volume
85
Issue
9
Publish Date
1995
Start Page
2315
End Page
2320

Telomeres and telomerase in human cancer. (Review)

Telomerase has recently come into the limelight as one of the most prevalent tumour markers, due to its nearly ubiquitous presence in malignant tissues and absence from most somatic tissues. The essential role of telomeres in unlimited cell proliferation and that of the enzyme in telomere maintenance have suggested that telomerase inhibitors may be effective in cancer therapy. We provide here a compendium and an evaluation of the available data relating to this hypothesis.

Authors
Bacchetti, S; Counter, CM
MLA Citation
Bacchetti, S, and Counter, CM. "Telomeres and telomerase in human cancer. (Review)." International Journal of Oncology 7.3 (1995): 423-432.
PMID
21552856
Source
scival
Published In
International Journal of Oncology
Volume
7
Issue
3
Publish Date
1995
Start Page
423
End Page
432

Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes.

We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.

Authors
Counter, CM; Botelho, FM; Wang, P; Harley, CB; Bacchetti, S
MLA Citation
Counter, CM, Botelho, FM, Wang, P, Harley, CB, and Bacchetti, S. "Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes." J Virol 68.5 (May 1994): 3410-3414.
PMID
8151802
Source
pubmed
Published In
Journal of virology
Volume
68
Issue
5
Publish Date
1994
Start Page
3410
End Page
3414

Telomerase activity in human ovarian carcinoma.

Telomeres fulfill the dual function of protecting eukaryotic chromosomes from illegitimate recombination and degradation and may aid in chromosome attachment to the nuclear membrane. We have previously shown that telomerase, the enzyme which synthesizes telomeric DNA, is not detected in normal somatic cells and that telomeres shorten with replicative age. In cells immortalized in vitro, activation of telomerase apparently stabilizes telomere length, preventing a critical destabilization of chromosomes, and cell proliferation continues even when telomeres are short. In vivo, telomeres of most tumors are shorter than telomeres of control tissues, suggesting an analogous role for the enzyme. To assess the relevance of telomerase and telomere stability in the development and progression of tumors, we have measured enzyme activity and telomere length in metastatic cells of epithelial ovarian carcinoma. We report that extremely short telomeres are maintained in these cells and that tumor cells, but not isogenic nonmalignant cells, express telomerase. Our findings suggest that progression of malignancy is ultimately dependent upon activation of telomerase and that telomerase inhibitors may be effective antitumor drugs.

Authors
Counter, CM; Hirte, HW; Bacchetti, S; Harley, CB
MLA Citation
Counter, CM, Hirte, HW, Bacchetti, S, and Harley, CB. "Telomerase activity in human ovarian carcinoma." Proc Natl Acad Sci U S A 91.8 (April 12, 1994): 2900-2904.
PMID
8159676
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
91
Issue
8
Publish Date
1994
Start Page
2900
End Page
2904

Telomerase, cell immortality, and cancer.

Authors
Harley, CB; Kim, NW; Prowse, KR; Weinrich, SL; Hirsch, KS; West, MD; Bacchetti, S; Hirte, HW; Counter, CM; Greider, CW
MLA Citation
Harley, CB, Kim, NW, Prowse, KR, Weinrich, SL, Hirsch, KS, West, MD, Bacchetti, S, Hirte, HW, Counter, CM, and Greider, CW. "Telomerase, cell immortality, and cancer." Cold Spring Harb Symp Quant Biol 59 (1994): 307-315. (Review)
PMID
7587082
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
59
Publish Date
1994
Start Page
307
End Page
315

The telomere hypothesis of cellular aging.

Authors
Harley, CB; Vaziri, H; Counter, CM; Allsopp, RC
MLA Citation
Harley, CB, Vaziri, H, Counter, CM, and Allsopp, RC. "The telomere hypothesis of cellular aging." Exp Gerontol 27.4 (July 1992): 375-382.
PMID
1459213
Source
pubmed
Published In
Experimental Gerontology
Volume
27
Issue
4
Publish Date
1992
Start Page
375
End Page
382

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Authors
Counter, CM; Avilion, AA; LeFeuvre, CE; Stewart, NG; Greider, CW; Harley, CB; Bacchetti, S
MLA Citation
Counter, CM, Avilion, AA, LeFeuvre, CE, Stewart, NG, Greider, CW, Harley, CB, and Bacchetti, S. "Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity." EMBO J 11.5 (May 1992): 1921-1929.
PMID
1582420
Source
pubmed
Published In
EMBO Journal
Volume
11
Issue
5
Publish Date
1992
Start Page
1921
End Page
1929
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