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Moro-López M, Farré R, Otero J, Sunyer R. Trusting the forces of our cell lines. Cells Dev 2024; 179:203931. [PMID: 38852676 DOI: 10.1016/j.cdev.2024.203931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Revised: 05/03/2024] [Accepted: 06/04/2024] [Indexed: 06/11/2024]
Abstract
Cells isolated from their native tissues and cultured in vitro face different selection pressures than those cultured in vivo. These pressures induce a profound transformation that reshapes the cell, alters its genome, and transforms the way it senses and generates forces. In this perspective, we focus on the evidence that cells cultured on conventional polystyrene substrates display a fundamentally different mechanobiology than their in vivo counterparts. We explore the role of adhesion reinforcement in this transformation and to what extent it is reversible. We argue that this mechanoadaptation is often understood as a mechanical memory. We propose some strategies to mitigate the effects of on-plastic culture on mechanobiology, such as organoid-inspired protocols or mechanical priming. While isolating cells from their native tissues and culturing them on artificial substrates has revolutionized biomedical research, it has also transformed cellular forces. Only by understanding and controlling them, we can improve their truthfulness and validity.
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Affiliation(s)
- Marina Moro-López
- Unit of Biophysics and Bioengineering, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain
| | - Ramon Farré
- Unit of Biophysics and Bioengineering, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBER-RES), Barcelona, Spain; Institut Investigacions Biomèdiques August Pi Sunyer (IDIBAPS), Barcelona, Spain
| | - Jorge Otero
- Unit of Biophysics and Bioengineering, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBER-RES), Barcelona, Spain; Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, Barcelona, Spain
| | - Raimon Sunyer
- Unit of Biophysics and Bioengineering, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain; Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Bioingeniería (CIBER-BBN), Barcelona, Spain.
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2
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Induced Pluripotent Stem Cells to Model Juvenile Myelomonocytic Leukemia: New Perspectives for Preclinical Research. Cells 2021; 10:cells10092335. [PMID: 34571984 PMCID: PMC8465353 DOI: 10.3390/cells10092335] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Revised: 08/24/2021] [Accepted: 08/31/2021] [Indexed: 11/16/2022] Open
Abstract
Juvenile myelomonocytic leukemia (JMML) is a malignant myeloproliferative disorder arising in infants and young children. The origin of this neoplasm is attributed to an early deregulation of the Ras signaling pathway in multipotent hematopoietic stem/progenitor cells. Since JMML is notoriously refractory to conventional cytostatic therapy, allogeneic hematopoietic stem cell transplantation remains the mainstay of curative therapy for most cases. However, alternative therapeutic approaches with small epigenetic molecules have recently entered the stage and show surprising efficacy at least in specific subsets of patients. Hence, the establishment of preclinical models to test novel agents is a priority. Induced pluripotent stem cells (IPSCs) offer an opportunity to imitate JMML ex vivo, after attempts to generate immortalized cell lines from primary JMML material have largely failed in the past. Several research groups have previously generated patient-derived JMML IPSCs and successfully differentiated these into myeloid cells with extensive phenotypic similarities to primary JMML cells. With infinite self-renewal and the capability to differentiate into multiple cell types, JMML IPSCs are a promising resource to advance the development of treatment modalities targeting specific vulnerabilities. This review discusses current reprogramming techniques for JMML stem/progenitor cells, related clinical applications, and the challenges involved.
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Abstract
Abstract We have considered viruses and their contribution to breast cancer. Mouse mammary tumour virus The prevalence of mouse mammary tumour virus (MMTV) is 15-fold higher in human breast cancer than in normal and benign human breast tissue controls. Saliva is the most plausible means of transmission. MMTV has been identified in dogs, cats, monkeys, mice and rats. The causal mechanisms include insertional oncogenesis and mutations in the protective enzyme ABOBEC3B. Human papilloma virus The prevalence of high risk human papilloma viruses (HPV) is frequently six fold higher in breast cancer than in normal and benign breast tissue controls. Women who develop HPV associated cervical cancer are at higher than normal risk of developing HPV associated breast cancer. Koilocytes have been identified in breast cancers which is an indication of HPV oncogenicity. The causal mechanisms of HPVs in breast cancer appear to differ from cervical cancer. Sexual activity is the most common form of HPV transmission. HPVs are probably transmitted from the cervix to the breast by circulating extra cellular vesicles. Epstein Barr virus The prevalence of Epstein Barr virus (EBV) is five fold higher in breast cancer than in normal and benign breast tissue controls. EBV is mostly transmitted from person to person via saliva. EBV infection predisposes breast epithelial cells to malignant transformation through activation of HER2/HER3 signalling cascades. EBV EBNA genes contribute to tumour growth and metastasis and have the ability to affect the mesenchymal transition of cells. Bovine leukemia virus Bovine leukemia virus (BLV) infects beef and dairy cattle and leads to various cancers. The prevalence of BLV is double in human breast cancers compared to controls. Breast cancer is more prevalent in red meat eating and cow’s milk consuming populations. BLV may be transmitted to humans from cattle by the consumption of red meat and cow’s milk. Conclusion The evidence that MMTV, high risk HPVs and EBVs have causal roles in human breast cancer is compelling. The evidence with respect to BLV is more limited but it is likely to also have a causal role in human breast cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s13027-021-00366-3.
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Affiliation(s)
- James S Lawson
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
| | - Wendy K Glenn
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
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4
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Fresques T, Zirbes A, Shalabi S, Samson S, Preto S, Stampfer MR, LaBarge MA. Breast Tissue Biology Expands the Possibilities for Prevention of Age-Related Breast Cancers. Front Cell Dev Biol 2019; 7:174. [PMID: 31555644 PMCID: PMC6722426 DOI: 10.3389/fcell.2019.00174] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2019] [Accepted: 08/12/2019] [Indexed: 12/24/2022] Open
Abstract
Preventing breast cancer before it is able to form is an ideal way to stop breast cancer. However, there are limited existing options for prevention of breast cancer. Changes in the breast tissue resulting from the aging process contribute to breast cancer susceptibility and progression and may therefore provide promising targets for prevention. Here, we describe new potential targets, immortalization and inflammaging, that may be useful for prevention of age-related breast cancers. We also summarize existing studies of warfarin and metformin, current drugs used for non-cancerous diseases, that also may be repurposed for breast cancer prevention.
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Affiliation(s)
- Tara Fresques
- Department of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, United States
| | - Arrianna Zirbes
- Department of Population Sciences, Beckman Research Institute at City of Hope, Duarte, CA, United States.,Center for Cancer and Aging Research, Beckman Research Institute at City of Hope, Duarte, CA, United States
| | - Sundus Shalabi
- Department of Population Sciences, Beckman Research Institute at City of Hope, Duarte, CA, United States.,Center for Cancer and Aging Research, Beckman Research Institute at City of Hope, Duarte, CA, United States.,Medical Research Center, Al-Quds University, Jerusalem, Palestine
| | - Susan Samson
- Breast Science Advocacy Core, Breast Oncology Program, University of California, San Francisco, San Francisco, CA, United States
| | | | - Martha R Stampfer
- Department of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, United States
| | - Mark A LaBarge
- Department of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.,Department of Population Sciences, Beckman Research Institute at City of Hope, Duarte, CA, United States.,Center for Cancer and Aging Research, Beckman Research Institute at City of Hope, Duarte, CA, United States
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5
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Abbadie C, Pluquet O, Pourtier A. Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses? Cell Mol Life Sci 2017; 74:4471-4509. [PMID: 28707011 PMCID: PMC11107641 DOI: 10.1007/s00018-017-2587-9] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2017] [Revised: 06/27/2017] [Accepted: 07/06/2017] [Indexed: 01/01/2023]
Abstract
Senescence is a cell state occurring in vitro and in vivo after successive replication cycles and/or upon exposition to various stressors. It is characterized by a strong cell cycle arrest associated with several molecular, metabolic and morphologic changes. The accumulation of senescent cells in tissues and organs with time plays a role in organismal aging and in several age-associated disorders and pathologies. Moreover, several therapeutic interventions are able to prematurely induce senescence. It is, therefore, tremendously important to characterize in-depth, the mechanisms by which senescence is induced, as well as the precise properties of senescent cells. For historical reasons, senescence is often studied with fibroblast models. Other cell types, however, much more relevant regarding the structure and function of vital organs and/or regarding pathologies, are regrettably often neglected. In this article, we will clarify what is known on senescence of epithelial cells and highlight what distinguishes it from, and what makes it like, replicative senescence of fibroblasts taken as a standard.
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Affiliation(s)
- Corinne Abbadie
- Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, 59000, Lille, France.
| | - Olivier Pluquet
- Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, 59000, Lille, France
| | - Albin Pourtier
- Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, 59000, Lille, France
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6
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Garbe JC, Vrba L, Sputova K, Fuchs L, Novak P, Brothman AR, Jackson M, Chin K, LaBarge MA, Watts G, Futscher BW, Stampfer MR. Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations. Cell Cycle 2015; 13:3423-35. [PMID: 25485586 PMCID: PMC4613853 DOI: 10.4161/15384101.2014.954456] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16INK4; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional “passenger” errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.
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Key Words
- BaP, benzo(a)pyrene
- CT, cholera toxin
- DDR, DNA damage response
- DMR, differentially methylated regions
- HMEC, human mammary epithelial cells
- OIS, oncogene-induced senescence
- PD, population doublings
- RB, retinoblastoma protein
- TTS, transcription start site
- X, oxytocin
- c-Myc
- carcinogenesis
- genomic instability
- human mammary epithelial cells
- immortalization
- p, passage
- p16INK4a
- p16sh, shRNA to p16INK4A
- senescence
- telomerase
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Affiliation(s)
- James C Garbe
- a Life Sciences Division ; Lawrence Berkeley National Laboratory ; Berkeley , CA USA
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Van Doorslaer K, Burk RD. Association between hTERT activation by HPV E6 proteins and oncogenic risk. Virology 2012; 433:216-9. [PMID: 22925336 PMCID: PMC3449093 DOI: 10.1016/j.virol.2012.08.006] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2012] [Revised: 06/11/2012] [Accepted: 08/03/2012] [Indexed: 01/16/2023]
Abstract
Expression of activated telomerase and subversion of the p16/pRb pathway is sufficient and essential for the in vitro immortalization of primary keratinocytes. Most cancers-including cervical carcinoma-over-express hTERT, the catalytic domain of the telomerase complex. Only a limited set of viruses within the Alphapapillomavirus genus are oncogenic. The viral functions responsible for this distinction are not well understood. The human papillomavirus type 16 E6 protein activates the hTERT promoter. We used a luciferase-based assay to test the ability of 29 viral types, representing all current species within the Alphapapillomavirus genus, to activate the hTERT promoter. We show that oncogenic types specifically activate the hTERT promoter, while non-oncogenic types do not. Statistical analysis supports the notion that activation of the hTERT promoter is uniquely associated with oncogenic types, independent of evolutionary relationships. This finding begins to shed light on the viral phenotypes correlated with oncogenic potential.
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Affiliation(s)
- Koenraad Van Doorslaer
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA
| | - Robert D. Burk
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA
- Department of Pediatrics, Epidemiology and Population Health; and Obstetrics & Gynecology and Women’s Health, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA
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8
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Bazarov AV, Lee WJ, Bazarov I, Bosire M, Hines WC, Stankovich B, Chicas A, Lowe SW, Yaswen P. The specific role of pRb in p16 (INK4A) -mediated arrest of normal and malignant human breast cells. Cell Cycle 2012; 11:1008-13. [PMID: 22333593 DOI: 10.4161/cc.11.5.19492] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G 1 was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.
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Affiliation(s)
- Alexey V Bazarov
- Department of Medicine, University of California at San Francisco, San Francisco, CA, USA
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9
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Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K, Feiler HS, Wyrobek AJ, Stampfer MR. Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. Cancer Res 2009; 69:7557-68. [PMID: 19773443 DOI: 10.1158/0008-5472.can-09-0270] [Citation(s) in RCA: 132] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Normal human epithelial cells in culture have generally shown a limited proliferative potential of approximately 10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence-associated beta-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.
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Affiliation(s)
- James C Garbe
- Life Sciences Divisions, Lawrence Berkeley National Laboratory, Berkeley, California, USA
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10
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Fridman AL, Tainsky MA. Critical pathways in cellular senescence and immortalization revealed by gene expression profiling. Oncogene 2008; 27:5975-87. [PMID: 18711403 DOI: 10.1038/onc.2008.213] [Citation(s) in RCA: 256] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Bypassing cellular senescence and becoming immortal is a prerequisite step in the tumorigenic transformation of a cell. It has long been known that loss of a key tumor suppressor gene, such as p53, is necessary, but not sufficient, for spontaneous cellular immortalization. Therefore, there must be additional mutations and/or epigenetic alterations required for immortalization to occur. Early work on these processes included somatic cell genetic studies to estimate the number of senescence genes, and microcell-mediated transfer of chromosomes into immortalized cells to identify putative senescence-inducing genetic loci. These principal studies laid the foundation for the field of senescence/immortalization, but were labor intensive and the results were somewhat limited. The advent of gene expression profiling and bioinformatics analysis greatly facilitated the identification of genes and pathways that regulate cellular senescence/immortalization. In this review, we present the findings of several gene expression profiling studies and supporting functional data, where available. We identified universal genes regulating senescence/immortalization and found that the key regulator genes represented six pathways: the cell cycle pRB/p53, cytoskeletal, interferon-related, insulin growth factor-related, MAP kinase and oxidative stress pathway. The identification of the genes and pathways regulating senescence/immortalization could provide novel molecular targets for the treatment and/or prevention of cancer.
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Affiliation(s)
- A L Fridman
- Department of Pathology, Program in Molecular Biology and Genetics, Barbara Ann Karmanos Cancer Institute, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA
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11
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Halldorsson S, Asgrimsson V, Axelsson I, Gudmundsson GH, Steinarsdottir M, Baldursson O, Gudjonsson T. Differentiation potential of a basal epithelial cell line established from human bronchial explant. In Vitro Cell Dev Biol Anim 2007; 43:283-9. [PMID: 17876679 DOI: 10.1007/s11626-007-9050-4] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2007] [Accepted: 06/14/2007] [Indexed: 11/30/2022]
Abstract
Due to the cellular complexity of the airway epithelium, it is important to carefully define bronchial cell lines that capture the phenotypic traits of a particular cell type. We describe the characterization of a human bronchial epithelial cell line, VA10. It was established by transfection of primary bronchial epithelial cells with retroviral constructs containing the E6 and E7 oncogenes from HPV16. The cell line has been cultured for over 2 yr, a total of 60 passages. Although prolonged culture resulted in increased chromosomal instability, no major phenotypic drift in marker expression was observed. The cells expressed cytokeratins 5, 13, 14, and 17 suggesting a basal-like phenotype. This was further supported by the expression of alpha6beta4 integrins and the basal cell-associated transcription factor p63. The VA10 cell line generated high transepithelial electrical resistance in suspended and air-liquid interface culture, indicating functionally active tight junction (TJ) complexes. Immunocytochemistry showed the typical reticular structures of occludin and TJ-associated F-actin. VA10 produced pseudostratified layer in air-liquid interface culture with expression of p63 restricted to the basal layer. Furthermore, VA10 produced round colonies when cultured in laminin-rich reconstituted basement membrane, and immunostaining of claudin-1 and the basolateral marker beta4 integrin revealed colonies that generated polarization as expected in vivo. These data indicate that VA10 epithelia have the potential to model the bronchial epithelium in vivo and may be useful to study epithelial regeneration and repair and the effect of chemicals and potential drug candidates on TJ molecules in airway epithelia.
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Chamulitrat W, Huber A, Riedel HD, Stremmel W. Nox1 Induces Differentiation Resistance in Immortalized Human Keratinocytes Generating Cells that Express Simple Epithelial Keratins. J Invest Dermatol 2007; 127:2171-83. [PMID: 17460729 DOI: 10.1038/sj.jid.5700843] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
We have shown that superoxide radical-generating NADPH oxidase 1 (Nox1) is increased in intermediate human transformed cells. It was unknown whether Nox1 overexpression could accelerate early transformation steps. We demonstrated that Nox1 rendered human immortalized (GM16) keratinocytes resistant against Ca(2+)/serum-induced differentiation. Nox1-transfected cells produced fast dividing resistant cells within 7-10 days after DMEM exposure. Progenitor lines (or Nox1 lines) were reproducibly generated from Nox1-transfected cells, while no lines were obtained from control transfections. From several attempts to generate control cells, one resistant population was obtained from untransfected GM16 cells after a 6-week DMEM exposure. Prolonged passaging of the control line could induce Nox1. Compared with the control line, Nox1 lines showed greater expression of Nox1, Rac1, p47phox, p67phox, NOXO1, and NOXA1 with concomitant increased superoxide generation. All five Nox1 lines contained varying amounts of E-cadherin, involucrin, vimentin, and K8/K18, while the control line did not. Since vimentin and K8/K18 are associated with malignant progression in different types of human epithelial tumors, our data demonstrate that Nox1 accelerated neoplastic-like progression by inducing generation of progenitor cells. Our data also emphasize the importance of Nox1 in inducing resistance against differentiation-induced cell death, suggesting a contribution of Nox1 and its oxidants during early stage of cell transformation.
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Affiliation(s)
- Walee Chamulitrat
- Department of Internal Medicine IV, University of Heidelberg, Heidelberg, Germany.
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13
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Beliveau A, Bassett E, Lo AT, Garbe J, Rubio MA, Bissell MJ, Campisi J, Yaswen P. p53-dependent integration of telomere and growth factor deprivation signals. Proc Natl Acad Sci U S A 2007; 104:4431-6. [PMID: 17360541 PMCID: PMC1838618 DOI: 10.1073/pnas.0700260104] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Ectopically expressed hTERT enables p16(INK4A)(-) human mammary epithelial cells to proliferate in the absence of growth factors, a finding that has led to the hypothesis that hTERT has growth regulatory properties independent of its role in telomere maintenance. We now show that telomerase can alter the growth properties of cells indirectly through its role in telomere maintenance, without altering growth stimulatory pathways. We find that telomere dysfunction, indicated by 53BP1/phosphorylated histone H2AX foci at chromosome ends, is present in robustly proliferating human mammary epithelial cells long before senescence. These foci correlate with increased levels of active p53. Ectopic expression of hTERT reduces the number of foci and the level of active p53, thereby decreasing sensitivity to growth factor depletion, which independently activates p53. The continuous presence of hTERT is not necessary for this effect, indicating that telomere maintenance, rather than the presence of the enzyme itself, is responsible for the increased ability to proliferate in the absence of growth factors. Our findings provide a previously unrecognized mechanistic explanation for the observation that ectopically expressed hTERT conveys growth advantages to cells, without having to postulate nontelomeric functions for the enzyme.
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Affiliation(s)
- Alain Beliveau
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
| | - Ekaterina Bassett
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
| | - Alvin T. Lo
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
| | - James Garbe
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
| | - Miguel A. Rubio
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
| | - Mina J. Bissell
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
| | - Judith Campisi
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
- Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945
| | - Paul Yaswen
- *Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
- To whom correspondence should be addressed at:
Lawrence Berkeley National Laboratory, Mailstop 977R225A, 1 Cyclotron Road, Berkeley, CA 94720. E-mail:
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Li Y, Pan J, Li JL, Lee JH, Tunkey C, Saraf K, Garbe JC, Whitley MZ, Jelinsky SA, Stampfer MR, Haney SA. Transcriptional changes associated with breast cancer occur as normal human mammary epithelial cells overcome senescence barriers and become immortalized. Mol Cancer 2007; 6:7. [PMID: 17233903 PMCID: PMC1784108 DOI: 10.1186/1476-4598-6-7] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2006] [Accepted: 01/18/2007] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND Human mammary epithelial cells (HMEC) overcome two well-characterized genetic and epigenetic barriers as they progress from primary cells to fully immortalized cell lines in vitro. Finite lifespan HMEC overcome an Rb-mediated stress-associated senescence barrier (stasis), and a stringent, telomere-length dependent, barrier (agonescence or crisis, depending on p53 status). HMEC that have overcome the second senescence barrier are immortalized. METHODS We have characterized pre-stasis, post-selection (post-stasis, with p16 silenced), and fully immortalized HMEC by transcription profiling and RT-PCR. Four pre-stasis and seven post-selection HMEC samples, along with 10 representatives of fully immortalized breast epithelial cell lines, were profiled using Affymetrix U133A/B chips and compared using both supervised and unsupervised clustering. Datasets were validated by RT-PCR for a select set of genes. Quantitative immunofluorescence was used to assess changes in transcriptional regulators associated with the gene expression changes. RESULTS The most dramatic and uniform changes we observed were in a set of about 30 genes that are characterized as a "cancer proliferation cluster," which includes genes expressed during mitosis (CDC2, CDC25, MCM2, PLK1) and following DNA damage. The increased expression of these genes was particularly concordant in the fully immortalized lines. Additional changes were observed in IFN-regulated genes in some post-selection and fully immortalized cultures. Nuclear localization was observed for several transcriptional regulators associated with expression of these genes in post-selection and immortalized HMEC, including Rb, Myc, BRCA1, HDAC3 and SP1. CONCLUSION Gene expression profiles and cytological changes in related transcriptional regulators indicate that immortalized HMEC resemble non-invasive breast cancers, such as ductal and lobular carcinomas in situ, and are strikingly distinct from finite-lifespan HMEC, particularly with regard to genes involved in proliferation, cell cycle regulation, chromosome structure and the DNA damage response. The comparison of HMEC profiles with lines harboring oncogenic changes (e.g. overexpression of Her-2neu, loss of p53 expression) identifies genes involved in tissue remodeling as well as proinflamatory cytokines and S100 proteins. Studies on carcinogenesis using immortalized cell lines as starting points or "normal" controls need to account for the significant pre-existing genetic and epigenetic changes inherent in such lines before results can be broadly interpreted.
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Affiliation(s)
- Yizheng Li
- Section of Bioinformatics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Jing Pan
- Applied Genomics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Jian-Liang Li
- Section of Bioinformatics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Jee Hyung Lee
- Applied Genomics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Chris Tunkey
- Molecular Profiling and Biomarker Discovery, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Katie Saraf
- Molecular Profiling and Biomarker Discovery, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - James C Garbe
- Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
| | - Maryann Z Whitley
- Section of Bioinformatics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Scott A Jelinsky
- Molecular Profiling and Biomarker Discovery, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
| | - Martha R Stampfer
- Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
| | - Steven A Haney
- Applied Genomics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA
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15
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Christman SA, Kong BW, Landry MM, Kim H, Foster DN. Modulation of p53 expression and its role in the conversion to a fully immortalized chicken embryo fibroblast line. FEBS Lett 2005; 579:6705-15. [PMID: 16313905 DOI: 10.1016/j.febslet.2005.10.066] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2005] [Revised: 10/06/2005] [Accepted: 10/27/2005] [Indexed: 12/30/2022]
Abstract
We have established a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (SC-1) that has been in continuous culture for more than three years. This is only the second report of a spontaneously immortalized reverse transcriptase (RT)-negative chicken cell line. The SC-1 cells emerged from crisis (at about passage 29-31) with a slower growth rate than primary cells. Passage 50 SC-1 cells expressed similar levels of p53 mRNA, but slightly lower levels of p53 protein than passage 6 CEF cells. By passage 120, p53 mRNA levels were significantly decreased in the SC-1 cells, while protein levels were slightly increased compared to passage 6 CEF cells. However, functional analysis of p53 revealed reduced activity in later passage SC-1 cells. Other p53-related genes including p21WAF1, p27Kip1, MDM-2, and the p16INK4a alternate reading frame (ARF) sequence showed similar patterns of differential mRNA expression. Levels of p15INK4b mRNA and protein were dramatically decreased in SC-1 cells, suggesting that the Rb pathway also has been compromised. Telomerase expression was undetectable in SC-1 cells. Fluorescence-activated cell sorting analysis showed that SC-1 and primary cells contained a similar proportion of G0/G1 phase cells, unlike the only other spontaneously immortalized chicken cell line (DF-1). The present study suggests that alterations in the p53 and Rb pathways cause fluctuations in expression levels of important cell-cycle regulatory genes during crucial transition periods as the SC-1 spontaneously immortalized chicken fibroblast cells progress toward becoming a fully committed cell line.
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Affiliation(s)
- Shelly A Christman
- Department of Animal Science, University of Minnesota, 495 AnSci/VetMed, 1988 Fitch Ave., St. Paul, MN 55108, USA
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16
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Gewin L, Myers H, Kiyono T, Galloway DA. Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex. Genes Dev 2004; 18:2269-82. [PMID: 15371341 PMCID: PMC517520 DOI: 10.1101/gad.1214704] [Citation(s) in RCA: 185] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The critical immortalizing activity of the human papillomavirus (HPV) type-16 E6 oncoprotein is to induce expression of hTERT, the catalytic and rate-limiting subunit of telomerase. Additionally, E6 binds to a cellular protein called E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for proteasome-dependent degradation. Although telomerase induction and p53 degradation are separable and distinct functions of E6, binding of E6 to E6-AP strongly correlated with the induction of hTERT. Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction. A yeast two-hybrid screen to find new targets of the E6/E6-AP E3 ubiquitin ligase complex identified NFX1. Two isoforms of NFX1 were found: NFX1-123, which coactivated with c-Myc at the hTERT promoter, and NFX1-91, which repressed the hTERT promoter. NFX1-91 was highly ubiquitinated and destabilized in epithelial cells expressing E6. Furthermore, knockdown of NFX1-91 by shRNA resulted in derepression of the endogenous hTERT promoter and elevated levels of telomerase activity. We propose that the induction of telomerase by the HPV-16 E6/E6-AP complex involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.
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Affiliation(s)
- Lindy Gewin
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
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17
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Petersen OW, Gudjonsson T, Villadsen R, Bissell MJ, Rønnov-Jessen L. Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia. Cell Prolif 2003; 36 Suppl 1:33-44. [PMID: 14521514 PMCID: PMC2933221 DOI: 10.1046/j.1365-2184.36.s.1.4.x] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value of knowing the cellular origin of individual tumours is clear and should aid in designing effective therapies. To do this, however, we need strategies aimed at defining the nature of stem and progenitor cell populations in the normal breast. In this review, we will discuss our technical approach for delineating the origin of the epithelial cell types. A major step forward was the purification of each cell type by the application of immunomagnetic cell sorting based on expression of lineage-specific surface antigens. We then developed chemically defined media that could support either the luminal epithelial or the myoepithelial cell phenotype in primary cultures. Having succeeded in continuous propagation presumably without loss of markers, we could show that a subset of the luminal epithelial cells could convert to myoepithelial cells, signifying the possible existence of a progenitor cell population. By combining the information on marker expression and in situ localization with immunomagnetic sorting and subsequent immortalization, we have identified and isolated a cytokeratin 19-positive suprabasal putative precursor cell in the luminal epithelial compartment and established representative cell lines. This suprabasal-derived epithelial cell line is able to generate both itself and differentiated luminal epithelial and myoepithelial cells, and in addition, is able to form elaborate terminal duct lobular unit (TDLU)-like structures within a reconstituted basement membrane. As more than 90% of breast cancers arise in TDLUs and more than 90% are also cytokeratin 19-positive, we suggest that this cell population contains a breast-cancer progenitor.
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Affiliation(s)
- Ole William Petersen
- Structural Cell Biology Unit, Institute of Medical Anatomy, The Panum Institute, Copenhagen, Denmark.
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18
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Stampfer MR, Garbe J, Nijjar T, Wigington D, Swisshelm K, Yaswen P. Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines. Oncogene 2003; 22:5238-51. [PMID: 12917625 DOI: 10.1038/sj.onc.1206667] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We describe novel effects of p53 loss on immortal transformation, based upon comparison of immortally transformed human mammary epithelial cell (HMEC) lines lacking functional p53 with closely related p53(+) lines. Our previous studies of p53(+) immortal HMEC lines indicated that overcoming the stringent replicative senescence step associated with critically short telomeres (agonescence), produced indefinite lifespan lines that maintained growth without immediately expressing telomerase activity. These telomerase(-) 'conditionally immortal' HMEC underwent an additional step, termed conversion, to become fully immortal telomerase(+) lines with uniform good growth. The very gradual conversion process was associated with slow heterogeneous growth and high expression of the cyclin-dependent kinase inhibitor p57(Kip2). We now show that p53 suppresses telomerase activity and is necessary for the p57 expression in early passage p53(+) conditionally immortal HMEC lines, and that p53(-/-) lines exhibit telomerase reactivation and attain full immortality much more rapidly. A p53-inhibiting genetic suppressor element introduced into early passages of a conditionally immortal telomerase(-) p53(+) HMEC line led to rapid induction of hTERT mRNA, expression of telomerase activity, loss of p57 expression, and quick attainment of uniform good growth. These studies indicate that derangements in p53 function may impact malignant progression through direct effects on the conversion process, a potentially rate-limiting step in HMEC acquisition of uniform unlimited growth potential. These studies also provide evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function.
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Affiliation(s)
- Martha R Stampfer
- Lawrence Berkeley National Laboratory, Life Sciences Division, Berkeley, CA 94720, USA.
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19
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Abstract
Human epithelial cells encounter two senescence barriers that enforce a limited proliferative potential. A first barrier is mediated by the retinoblastoma protein, and can be overcome by multiple types of errors, many of which are observed in human cancers. A second, extremely stringent telomere-dependent barrier, is a consequence of repression of telomerase activity. Although relieved by ectopic hTERT expression, the nature of the errors required to overcome this latter barrier during in vivo carcinogenesis have not yet been defined. Attainment of immortality and telomerase reactivation are crucial to human carcinoma development; the derangements responsible for attainment of immortality may be rate-limiting and permissive for further progression to malignancy.
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20
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Petersen OW, Nielsen HL, Gudjonsson T, Villadsen R, Rank F, Niebuhr E, Bissell MJ, Rønnov-Jessen L. Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. THE AMERICAN JOURNAL OF PATHOLOGY 2003; 162:391-402. [PMID: 12547698 PMCID: PMC1851146 DOI: 10.1016/s0002-9440(10)63834-5] [Citation(s) in RCA: 217] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
A breast carcinoma biopsy showed cytochemical evidence of epithelial mesenchymal transition and an alpha-smooth muscle actin-positive stromal reaction. To study the lineage, and the nature of the cells in the stromal reaction, we derived a novel cell line, HBFL-1, from the explanted biopsy. HBFL-1 cells are immortal and exhibit a shared non-random X-chromosome inactivation pattern with the epithelial tumor of origin. Yet they closely resemble normal, finite-life-span fibroblasts by morphology, lack of tumor formation in nude mice, marker expression profile, protein pattern using two-dimensional gel electrophoresis and the ability to undergo myofibroblast conversion. HBFL-1 interacts reciprocally with tumor cells in collagen gel to induce activation of MMP2, leading to tumor-like behavior of epithelial colonies. In vivo, HBFL-1 cells resembled normal-derived myofibroblasts and conferred a significant 3.5- to 7-fold increase in MCF-7 tumor size in nude mice. However, that they were indeed not normal fibroblasts was revealed by residual keratin expression and formation of epithelial microfoci in a reconstituted basement membrane and in nude mice. We conclude that breast cancer can generate its own nonmalignant stroma and that one function for this is that of a reciprocal interaction with epithelial tumor cells to facilitate tumor growth.
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Affiliation(s)
- Ole William Petersen
- Structural Cell Biology Unit, Department of Medical Anatomy, Section A, the Panum Institute, Copenhagen, Denmark.
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21
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Santos JH, Hunakova L, Chen Y, Bortner C, Van Houten B. Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death. J Biol Chem 2003; 278:1728-34. [PMID: 12424245 DOI: 10.1074/jbc.m208752200] [Citation(s) in RCA: 161] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In order to understand the molecular events following oxidative stress, which lead to persistence of lesions in the mtDNA, experiments were performed on normal human fibroblast (NHF) expressing human telomerase reverse transcriptase (hTERT). The formation and repair of H(2)O(2)-induced DNA lesions were examined using quantitative PCR. It was found that NHF hTERTs show extensive mtDNA damage ( approximately 4 lesions/10 kb) after exposure to 200 microm H(2)O(2), which is partially repaired during a recovery period of 6 h. At the same time, the nDNA seemed to be completely resistant to damage. Cell sorting experiments revealed persistent mtDNA damage at 24 h only in the fraction of cells with low mitochondrial membrane potential (Delta Psi m). Further analysis also showed increased production of H(2)O(2) by these cells, which subsequently undergo apoptosis. This work supports a hypothesis for a feed-forward cascade of reactive oxygen species generation and mtDNA damage and also suggested a possible mechanism for persistence of lesions in the mtDNA involving a drop in Delta Psi m, compromised protein import, secondary reactive oxygen species generation, and loss of repair capacity.
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Affiliation(s)
- Janine Hertzog Santos
- Laboratory of Molecular Genetics and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA
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22
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Spänkuch-Schmitt B, Bereiter-Hahn J, Kaufmann M, Strebhardt K. Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. J Natl Cancer Inst 2002; 94:1863-77. [PMID: 12488480 DOI: 10.1093/jnci/94.24.1863] [Citation(s) in RCA: 259] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference. METHODS Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells) were transfected with small interfering (si) RNAs targeted against the human PLK1 or lamin genes. Northern and western blot analyses were used to examine PLK1 gene expression in transfected cancer cells and normal cells (human mammary epithelial cells [HMECs]). The phenotype, proliferation, and cell cycle distribution of cells transfected with siRNAs were also monitored by fluorescence microscopy and fluorescence-activated cell sorting analysis. RESULTS All cancer cell lines transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. siRNA4, which had the strongest inhibitory effect, reduced PLK1 mRNA in MCF-7 cells by 70% and PLK1 protein in MCF-7 cells by 95% 24 hours after transfection. Cell proliferation was reduced by between 66% and 99% 48 hours after transfection, and apoptosis was increased from 1%-5% to 13%-50% in transfected cells. Transfected SW-480 cells were mitotically arrested, and their centrosomes had lost the ability to nucleate microtubules. HMECs took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did not inhibit their proliferation. CONCLUSIONS PLK1 function appears to be essential for centrosome-mediated microtubule events and, consequently, for spindle assembly. siRNAs targeted against human PLK1 may be valuable tools as antiproliferative agents that display activity against a broad spectrum of neoplastic cells at very low doses.
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Affiliation(s)
- Birgit Spänkuch-Schmitt
- Department of Obstetrics and Gynecology, School of Medicine, J. W. Goethe-University, Frankfurt, Germany
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23
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Shen ZY, Xu LY, Chen MH, Shen J, Cai WJ, Zeng Y. Progressive transformation of immortalized esophageal epithelial cells. World J Gastroenterol 2002; 8:976-81. [PMID: 12439909 PMCID: PMC4656402 DOI: 10.3748/wjg.v8.i6.976] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the progressive transformation of immortal cells of human fetal esophageal epithelium induced by human papillomavirus, and to examine biological criteria of sequential passage of cells, including cellular phenotype, proliferative rate, telomerase, chromosome and tumorigenicity.
METHODS: The SHEE cell series consisted of immortalized embryonic esophageal epithelium which was in malignant transformation when cultivated over sixty passages without co-carcinogens. Cells of the 10th, 31st, 60th and 85th passages were present in progressive development after being transfected with HPV. Cells were cultivated in a culture flask and 24-hole cultural plates. Progressive changes of morphology, cell growth, contact-inhibition, and anchorage-dependent growth characteristics were examined by phase contrast microscopy. The cell proliferation rate was assayed by flow cytometry. The modal number of chromosomes was analyzed. HPV18E6E7 was detected by Western blot methods and activities of telomerase were analyzed by TRAP. Tumorigenicity of cells was detected with soft agar plates cultivated and with tumor formation in SCID mice.
RESULTS: In morphological examination the 10th passage cells were in good differentiation, the 60th and 85th passages cells were in relatively poor differentiation, and the 31st passage cells had two distinct differentiations. The characteristics of the 85th and 60th passage cells were weakened at contact-inhibition and anchorage-dependent growth. Karyotypes of four stages of cells belonged to hyperdiploid or hypotriploid, and bimodal distribution of chromosomes appeared in the 31st and 60th passage cells. All of these characteristics combined with a increasing trend. The activities of telomerase were expressed in the latter three passages. Four fourths of SCID mice in the 85th passage cells and one fourth of SCID mice in the 60th passage cells developed tumors, but the cells in the 10th and 31st passage displayed no tumor formation.
CONCLUSION: In continual cultivation of fetal esophageal epithelial cells with transduction of HPV18E6E7, cells from the 10th to the 85th passage were changed gradually from preimmortal, immortal, precancerous to malignantly transformed stages. All of these changes were in a dynamic progressive process. The establishment of a continuous line of esophageal epithelium may provide a in vitro model of carcinogenesis induced by HPV.
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Affiliation(s)
- Zhong-Ying Shen
- Department of Tumor Pathology, Medical College of Shantou University, Guandong Province, China.
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24
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Yaswen P, Stampfer MR. Molecular changes accompanying senescence and immortalization of cultured human mammary epithelial cells. Int J Biochem Cell Biol 2002; 34:1382-94. [PMID: 12200033 DOI: 10.1016/s1357-2725(02)00047-x] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Limits on the proliferative potential of cultured normal human cells may be consequences of pathways that exist to suppress tumorigenicity. Human mammary epithelial cells (HMEC) employ several mechanisms to prevent unlimited growth. One mechanism may be activated by stress, and is associated with upregulated expression of p16(INK4a). In serum-free medium, some HMEC arise spontaneously which do not express p16. These "post-selection" HMEC are capable of long-term proliferation, but ultimately cease growth when their telomeres become very short. As they approach a growth plateau, termed agonescence, post-selection HMEC populations accumulate chromosome abnormalities. In contrast to the crisis exhibited by cells lacking functional p53, agonescent cells can be maintained as viable cultures. Although transduction of hTERT, the catalytic subunit of telomerase, into post-selection cells can, by itself, efficiently produce immortality and avoid agonescence, the errors that produce telomerase reactivation during carcinogenesis are not known. The block to endogenous telomerase reactivation in HMEC is extremely stringent. However, if one predisposing error is present, the probability greatly increases that additional error(s) required for immortalization may be generated by genomic instability encountered during agonescence. In p53(+) HMEC immortalized after chemical carcinogen exposure, the events involved in overcoming agonescence can be temporally separated from activation of telomerase. We have used the term "conversion" to describe the gradual process that leads to telomerase activation, telomere length stabilization, decreased p57 (KIP2) expression, and increased ability to grow uniformly well in the presence or absence of TGF beta. In the presence of active p53, conversion may represent a rate-limiting step in immortal transformation.
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Affiliation(s)
- Paul Yaswen
- Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop 70A-1118, Berkeley, CA 94720, USA.
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25
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Olsen CL, Gardie B, Yaswen P, Stampfer MR. Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion. Oncogene 2002; 21:6328-39. [PMID: 12214273 DOI: 10.1038/sj.onc.1205780] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2002] [Revised: 06/06/2002] [Accepted: 06/18/2002] [Indexed: 11/08/2022]
Abstract
Using an estrogen-inducible retroviral system, we demonstrate that oncogenic Raf-1 induces growth arrest and morphological changes in finite lifespan human mammary epithelial cells (HMEC). This arrest does not rely on expression of the cyclin-dependent kinase inhibitor (CKI) p16(INK4a), nor on changes in expression of the CKIs p21(Cip1), p14(ARF), p27(Kip1) or p57(Kip2). The Raf-induced arrest is independent of viral oncogene mediated inactivation of p53 and pRB, or c-myc overexpression. Flow cytometric analysis demonstrates that cells arrest in both G1 and G2. The Raf-induced arrest is mitigated or eliminated in some immortally transformed HMEC. Immortal HMEC that have both overcome replicative senescence and undergone the recently described conversion process maintain growth in the presence of transduced oncogenic Raf-1; they also gain EGF-independent growth and a low frequency of anchorage-independent growth. However, HMEC that have overcome replicative senescence but have not undergone conversion and HMEC immortalized by transduction with the catalytic subunit of telomerase, hTERT, remain severely growth arrested. These results indicate that the molecular mechanisms responsible for the Raf-1-induced growth arrest may vary among different finite lifespan cell types, and that in HMEC, this mechanism is altered during the conversion process, rather than as a direct consequence of overcoming senescence or expressing hTERT.
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Affiliation(s)
- Catherine L Olsen
- Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA
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26
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Vitali L, Yakisich JS, Vita MF, Fernandez A, Settembrini L, Siden A, Cruz M, Carminatti H, Casas O, Idoyaga Vargas V. Roscovitine inhibits ongoing DNA synthesis in human cervical cancer. Cancer Lett 2002; 180:7-12. [PMID: 11911964 DOI: 10.1016/s0304-3835(01)00827-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The effect of roscovitine, a purine analogue and cyclin dependent kinase inhibitor, on DNA synthesis rate in tissue mini-units obtained from human cervical cancers was investigated. Roscovitine (100 microM) gave a DNA synthesis rate inhibition by 61% (P<0.0001; range 23-93%) within 30 min of incubation. This inhibitory effect was concentration-dependent. The results suggest that the inhibition of tumor DNA synthesis rate is due to a direct effect on the DNA synthesis machinery via presently unknown mechanisms. In addition, the potential application of CDKs inhibitors as preventive agents is discussed.
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Affiliation(s)
- Liliana Vitali
- Instituto de Investigaciones Bioquimicas 'Fundacion Campomar', FCEyN, UBA, Patricias Argentinas 435 (C. P. 1405), Buenos Aires, Argentina
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27
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Gudjonsson T, Villadsen R, Nielsen HL, Rønnov-Jessen L, Bissell MJ, Petersen OW. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. Genes Dev 2002; 16:693-706. [PMID: 11914275 PMCID: PMC155359 DOI: 10.1101/gad.952602] [Citation(s) in RCA: 277] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC(+)) and epithelial-specific antigen (ESA(+)) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC(-)/ESA(+)). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC(+)/ESA(+) epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC(-)/ESA(+) epithelial cell line was able to generate itself as well as MUC(+)/ESA(+) epithelial cells and Thy-1(+)/alpha-smooth muscle actin(+) (ASMA(+)) myoepithelial cells. The MUC(-)/ESA(+) epithelial cell line further differed from the MUC(+)/ESA(+) epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC(+)/ESA(+) epithelial cell line formed acinus-like spheres. In contrast, the MUC(-)/ESA(+) epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC(-)/ESA(+) epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast.
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Affiliation(s)
- Thorarinn Gudjonsson
- Structural Cell Biology Unit, Institute of Medical Anatomy, The Panum Institute, DK-2200 Copenhagen N, Denmark
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28
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Toouli CD, Huschtscha LI, Neumann AA, Noble JR, Colgin LM, Hukku B, Reddel RR. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene 2002; 21:128-39. [PMID: 11791183 DOI: 10.1038/sj.onc.1205014] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2001] [Revised: 09/29/2001] [Accepted: 10/01/2001] [Indexed: 12/17/2022]
Abstract
We directly compared two methods of immortalizing human mammary epithelial cells (HMECs). Cells were transfected with an expression plasmid either for hTERT, the catalytic subunit of telomerase, or for the simian virus 40 (SV40) early region genes. Under standard culture conditions, HMECs were not immortalized by hTERT unless they had spontaneously ceased expression of the p16(INK4a) tumor suppressor gene. Untransfected HMECs had low levels of telomerase expression, and immortalization by both methods was associated with an increase in telomerase activity and prevention of telomere shortening. SV40-induced immortalization was accompanied by aberrant differentiation, loss of DNA damage response, karyotypic instability and, in some cases, tumorigenicity. hTERT-immortalized cells had fewer karyotypic changes, but had intact DNA damage responses, and features of normal differentiation. Although SV40-immortalized cells are useful for studies of carcinogenesis, hTERT-immortalized cells retain more properties of normal cells.
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Affiliation(s)
- Christian D Toouli
- Children's Medical Research Institute, 214 Hawkesbury Road, Westmead 2145, Australia
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29
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Tsao SW, Wong N, Wang X, Liu Y, Wan TS, Fung LF, Lancaster WD, Gregoire L, Wong YC. Nonrandom chromosomal imbalances in human ovarian surface epithelial cells immortalized by HPV16-E6E7 viral oncogenes. CANCER GENETICS AND CYTOGENETICS 2001; 130:141-9. [PMID: 11675135 DOI: 10.1016/s0165-4608(01)00473-3] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
We had previously immortalized human ovarian surface epithelial (HOSE) cells using HPV16E6E7 ORFs. In order to identify crucial genetic events involved during cell immortalization, the genomic profile of immortalization of five HOSE cell lines was analyzed by comparative genomic hybridization. Our results showed that chromosomal imbalance was common in HOSE cells after immortalization. The common chromosomal imbalances identified in immortal HOSE cells are: +19q13.1 (5/5 lines), -13q12 approximately qter (4/5 lines), +5q15 approximately q33 (3/5 lines), +20q11.2 approximately q13.2 (3/5 lines) and -22q11.2 approximately qter (3/5 lines). Other chromosomal imbalances, which were detected in two of the five immortal HOSE cell lines, included gains on chromosome 1 and 11q12 approximately q13, and losses on 2p, 4q, 8p, 10p and 11q14 approximately qter. The chromosomal imbalances observed in HOSE cells before immortalization include -8pter approximately p11.2, -11q23 approximately qter, -13q12 approximately qter and +19 which may represent early genetic events during cell immortalization. The genomic profile was examined in one HOSE cell line (HOSE 6-3) at various stages of immortalization. The genomic profiles of HOSE 6-3 cells after crisis were largely stable. A few additional chromosomal imbalances were detected in the immortalized HOSE cells after an extensive culture period including +11pter approximately q23, -15q23 approximately qter, and +17q12 approximately qter. Identification of nonrandom chromosomal imbalance in immortalized HOSE cells may facilitate the identification of specific chromosomes harboring genes involved in the immortalization of human ovarian surface epithelial cells.
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MESH Headings
- Cell Line
- Cell Transformation, Viral
- Cells, Cultured
- Chromosome Aberrations
- Chromosomes, Human, Pair 13
- Chromosomes, Human, Pair 19
- Chromosomes, Human, Pair 20
- Chromosomes, Human, Pair 22
- Chromosomes, Human, Pair 5
- Epithelial Cells/pathology
- Female
- Genetic Techniques
- Humans
- Nucleic Acid Hybridization
- Oncogene Proteins, Viral/genetics
- Open Reading Frames
- Ovarian Neoplasms/genetics
- Ovary/pathology
- Papillomavirus E7 Proteins
- Repressor Proteins
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Affiliation(s)
- S W Tsao
- Department of Anatomy, Faculty of Medicine, University of Hong Kong, SAR, Hong Kong, China.
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30
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Gewin L, Galloway DA. E box-dependent activation of telomerase by human papillomavirus type 16 E6 does not require induction of c-myc. J Virol 2001; 75:7198-201. [PMID: 11435602 PMCID: PMC114450 DOI: 10.1128/jvi.75.15.7198-7201.2001] [Citation(s) in RCA: 153] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human papillomavirus type 16 (HPV-16) E6 activates telomerase specifically in epithelial cells. The oncogene c-myc has also been shown to activate telomerase in several cell types. Here we show that while both HPV-16 E6 and c-myc require intact E boxes to transactivate the hTERT promoter, E6 does not induce hTERT transcription simply by inducing expression of c-myc. Moreover, hTERT transactivation by HPV-16 E6 correlates with its ability to bind the cellular E6-associated protein (E6AP), suggesting that E6 and E6AP may target a regulator of hTERT expression.
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Affiliation(s)
- L Gewin
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
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31
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Stampfer MR, Garbe J, Levine G, Lichtsteiner S, Vasserot AP, Yaswen P. Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells. Proc Natl Acad Sci U S A 2001; 98:4498-503. [PMID: 11287649 PMCID: PMC31863 DOI: 10.1073/pnas.071483998] [Citation(s) in RCA: 101] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Failures to arrest growth in response to senescence or transforming growth factor beta (TGF-beta) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16(INK4A) resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-beta. The ability to maintain growth in TGF-beta was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-beta resistance may explain our previously described gain of TGF-beta resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-beta resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.
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Affiliation(s)
- M R Stampfer
- Lawrence Berkeley National Laboratory, Life Sciences Division, Berkeley, CA 94720, USA.
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32
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Yaswen P, Stampfer MR. Epigenetic changes accompanying human mammary epithelial cell immortalization. J Mammary Gland Biol Neoplasia 2001; 6:223-34. [PMID: 11501582 DOI: 10.1023/a:1011364925259] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Acquisition of immortality may be an early and crucial step in malignant progression. We hypothesize that acquisition of unlimited growth potential in individual human mammary epithelial cells (HMEC) requires inactivation of several distinct negative growth constraints as well as reactivation of a mechanism to maintain telomeres on chromosomes. Some of the heritable changes that occur during HMEC immortalization, i.e., loss of expression of cyclin dependent kinase inhibitors p16INK4a and p57KIP2, loss of TGFbeta-mediated growth inhibition, and derepression of telomerase, appear to occur without identifiable mutations in the genes and pathways involved. The absence of mutations, combined with the fact that the changes are often incremental over several cell generations even in clonal populations indicates that some changes associated with immortalization can be epigenetic. We have used the term "conversion" to describe the gradual epigenetic process in chemical carcinogen-immortalized HMEC that leads to activation of telomerase, stabilization of telomere length, and ability to grow uniformly well in the presence or absence of TGFbeta. Characterization of the epigenetic mechanisms involved in immortalization may uncover additional factors that drive tumor progression, and that may be responsive to novel forms of intervention.
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Affiliation(s)
- P Yaswen
- Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
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33
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Stampfer MR, Yaswen P. Immortal transformation and telomerase reactivation of human mammary epithelial cells in culture. TELOMERASE, AGING AND DISEASE 2001. [DOI: 10.1016/s1566-3124(01)08006-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
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34
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Pillai MR, Nair MK. Development of a condemned mucosa syndrome and pathogenesis of human papillomavirus-associated upper aerodigestive tract and uterine cervical tumors. Exp Mol Pathol 2000; 69:233-41. [PMID: 11115364 DOI: 10.1006/exmp.2000.2335] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
High-risk human papillomaviruses (HPVs) have been shown to be involved in the pathogenesis of many squamous carcinomas, particularly those of the uterine cervix. A number of random studies have also reported association of high-risk HPV subtypes with cancers of the oral cavity, larynx, hypopharynx, and esophagus. The roles of other molecular factors involved during HPV infection in these tumors still remain unclear. Recent findings from our laboratories have suggested possible mechanisms associated with HPV-mediated carcinogenesis. Both p53 mutation-dependent and mutation-independent pathways may be associated with HPV-mediated carcinogenesis, the former mainly in upper aerodigestive tract tumors (UADT) and the latter in cervical tumors. In cervical tumors, inactivation of the p53 tumor suppressor protein by the E6 gene product of high-risk HPVs and mutation of the p53 gene in UADT is associated with alterations in the apoptotic regulatory bcl-2 and bax genes, leading to downregulation of programmed cell death (PCD) and increased cell proliferation. HPV infection is also associated with increased tissue angiogenesis and activation of telomerase. Altered kinetics of telomere fragments is evident in HPV-infected tissue. We therefore believe that the combined manifestations of all these factors may contribute to development of a "condemned mucosa syndrome" facilitating development UADT and cervical cancers. A distinct step in the pathogenesis of both types of tumors may only be in the mode of p53 inactivation, whereas all other events appear to be strongly correlated to the presence of HPV. The development and validation of such a molecular model has significant clinical priority. It can be used to identify target populations or individuals for intervention, to monitor effects of intervention, and to determine which individuals or groups are at increased risk of developing cancer.
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Affiliation(s)
- M R Pillai
- Division of Laboratory Medicine, Regional Cancer Centre, Thiruvananthapuram. India
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35
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Sandhu C, Donovan J, Bhattacharya N, Stampfer M, Worland P, Slingerland J. Reduction of Cdc25A contributes to cyclin E1-Cdk2 inhibition at senescence in human mammary epithelial cells. Oncogene 2000; 19:5314-23. [PMID: 11103932 DOI: 10.1038/sj.onc.1203908] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of 'self-selection', and as a consequence exhibit diminished p16INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in fibroblast, p21Cip1 was not increased at senescence in HMECs. There was no increase in p27Kip1 levels nor in KIP association with targets cdks. While p15INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15INK4B. The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase.
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Affiliation(s)
- C Sandhu
- Cancer Research, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ontario, Canada
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36
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Abstract
Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS) already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene, and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies of in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the known in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TGFbeta. We propose that overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.
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Affiliation(s)
- M R Stampfer
- Lawrence Berkeley National Laboratory, University of California, Life Sciences Division, Berkeley 94720, USA.
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37
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Therrien JP, Drouin R, Baril C, Drobetsky EA. Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair. Proc Natl Acad Sci U S A 1999; 96:15038-43. [PMID: 10611334 PMCID: PMC24769 DOI: 10.1073/pnas.96.26.15038] [Citation(s) in RCA: 68] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li-Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G(1) arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li-Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-jun loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair.
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Affiliation(s)
- J P Therrien
- Division of Pathology, Department of Medical Biology, Faculty of Medicine, Laval University, Quebec, Canada
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