Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation
Abstract
Aberrations affecting the tumor suppressor gene p16INK4a have been described for a variety of tumors. In breast cancer, approximately 50% of tumors show low or lack p16 expression. While evidence provided by some studies has implicated a possible role for p16 in normal replicative senescence, other studies have suggested that the Rb, pathway through which p16 functions, may not be involved in senescence control. Previously we observed that all immortal lines derived from normal mammary epithelium which were analysed for p16 displayed inactivation of this gene through distinct mechanisms, supporting p16 inactivation as a possible necessary event in escape from senescence. To further clarify this issue, we have analysed p16 expression in a panel of normal finite lifespan human mammary epithelial cells (HMEC) from initial propagation through growth arrest, using media which confer different replicative capacity. Approximately 10-25-fold increase in p16 expression was observed for all normal HMEC with initial onset of a senescence phenotype following 15-25 population doublings in culture. These cells also displayed expression of the senescence associated beta-galactosidase. Interestingly, HMEC with additional long term replicative capacity (approximately 80 population doublings) arose from these growth arrested cultures, showing lack of p16 expression. This extended growth capacity appears to be associated with a methylation phenomenon since treatment of these cells with the methylation inhibitor 5-aza-2-deoxycytidine resulted in growth arrest concurrent with reacquisition of p16 expression and senescence associated beta-galactosidase. Analysis of p21waf1 expression revealed no change in expression during growth in vitro. These results support p16INK4a as the 9p senescence gene and suggest a role for p16 loss in the escape from initial onset of senescence and in acquisition of an extended life span of human mammary epithelial cells.
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Primary human fibroblasts and epithelial cells express increasing levels of p16 during culture.
“Primary human fibroblasts and epithelial cells also express increasing levels of p16 during culture (Reznikoff et al., 1996; Brenner et al., 1998; Rheinwald et al., 2002; Benanti and Galloway, 2004).”
From Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors (2005) · cites this paper as Brenner et al., 1998
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“In these postselection MECs, the p16Ink4a gene is silenced, but the cells do not immortalize spontaneously, and they undergo replicative senescence though they have 30-40 additional doublings compared to preselection MECs (Brenner et al., 1998; Wong et al., 1999).”
p16INK4a inactivation is not required to immortalize human mammary epithelial cells
Herbert, B., Wright, W. E., & Shay, J. W. (2002) · Oncogene · doi:10.1038/sj.onc.1205902 · Cited by 86 · see citing works
“HMECs grown on feeder layers did not exhibit an early growth slow down while HMECs grown on plastic showed a typical plateau phase associated with ‘self-selection’ (Figure 1, Romanov et al., 2001; Kiyono et al., 1998; Brenner et al., 1998; Huschtscha et al., 1998; Ramirez et al., 2001).”
BackgroundInfluentialReplicative senescence revisited.
Marcotte, R., & Wang, E. (2002) · The Journals of Gerontology Series A: Biological Sciences and Medical Sciences · doi:10.1093/gerona/57.7.b257 · Cited by 86 · see citing works
“Moreover, epithelial cells that display increased proliferative potential following an initial onset of senescent phenotype after 20–25 population doublings lack p16 INK4a expression (25).”
BackgroundDNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium
Plachot, C., & Lelièvre, S. A. (2004) · Experimental Cell Research · doi:10.1016/j.yexcr.2004.04.024 · Cited by 86 · see citing works
Genetic and Epigenetic Changes in Mammary Epithelial Cells May Mimic Early Events in Carcinogenesis
Tlsty, T. D., Crawford, Y., Holst, C. R., Fordyce, C., Zhang, J., McDermott, K. M., Kozakiewicz, K., & Gauthier, M. L. (2004) · Journal of Mammary Gland Biology and Neoplasia · doi:10.1023/b:jomg.0000048773.95897.5f · Cited by 84 · see citing works
“Similar to previous studies in human skin fibroblasts and HMEC (1,5,6,11,12), the epithelial cell populations undergo a limited number of population doublings prior to entering a proliferative plateau (Fig.”
Hinshelwood, R. A., Huschtscha, L. I., Melki, J. R., Stirzaker, C., Abdipranoto, A., Vissel, B., Ravasi, T., Wells, C. A., Hume, D. A., Reddel, R. R., et al. (2007) · Cancer Research · doi:10.1158/0008-5472.can-07-1284 · Cited by 81 · see citing works
Immortalization of human small airway epithelial cells by ectopic expression of telomerase
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“This activity is shared with other tumor suppressor genes, for example, p16INK4a (38, 39) and p21/Waf/Sdi1, initially uncovered as a clone involved in the senescence of human diploid fibroblasts (40).”
BackgroundEpithelial cell senescence: an adaptive response to pre-carcinogenic stresses?
Abbadie, C., Pluquet, O., & Pourtier, A. (2017) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-017-2587-9 · Cited by 74 · see citing works
Stampfer, M. R., Garbe, J. C., Nijjar, T., Wigington, D., Swisshelm, K. L., & Yaswen, P. (2003) · Oncogene · doi:10.1038/sj.onc.1206667 · Cited by 74 · see citing works
“Postselection HMEC cease growth with a mean TRF of B5 kb, and expression of SA-bgal (Stampfer et al., 1997; Brenner et al., 1998; Romanov et al., 2001).”
BackgroundSelf-citationCellular Plasticity: A Route to Senescence Exit and Tumorigenesis
Blander, H. D., Morel, A., Senaratne, A. P., Ouzounova, M., & Puisieux, A. (2021) · Cancers · doi:10.3390/cancers13184561 · Cited by 74 · see citing works
“It has been estimated that about one in 104 epithelial cells spontaneously escapes from stasis and reenters the cell cycle to give rise to new clones [50,54,55].”
BackgroundDoes pH 6 β-galactosidase activity indicate cell senescence?
Krishna, D. R., Sperker, B., Fritz, P., & Klotz, U. E. (1999) · Mechanisms of Ageing and Development · doi:10.1016/s0047-6374(99)00031-7 · Cited by 73 · see citing works
The thorny path linking cellular senescence to organismal aging
Patil, C. K., Mian, S. I., & Campisi, J. (2005) · Mechanisms of Ageing and Development · doi:10.1016/j.mad.2005.08.001 · Cited by 73 · see citing works
“Should a single culture condition be used, or should conditions be optimized for each cell type—or each species? And what is optimal? For example, p16 induction can limit the proliferation of human epithelial cells in standard culture and in vivo (Reznikoff et al., 1996; Brenner et al., 1998; Erickson et al., 1998; Di…”
Hinshelwood, R. A., Melki, J. R., Huschtscha, L. I., Paul, C. L., Song, J. Z., Stirzaker, C., Reddel, R. R., & Clark, S. J. (2009) · Human Molecular Genetics · doi:10.1093/hmg/ddp251 · Cited by 72 · see citing works
Nuclear Accumulation of Globular Actin as a Cellular Senescence Marker
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“Many tumors contain immortal cells or cells with an extended replicative life span (8, 9).”
BackgroundProcessing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture
LaBarge, M. A., Garbe, J. C., & Stampfer, M. R. (2013) · Journal of Visualized Experiments · doi:10.3791/50011 · Cited by 74 · see citing works
Self-citationHuman epithelial cell immortalization as a step in carcinogenesis
Stampfer, M. R., & Yaswen, P. (2003) · Cancer Letters · doi:10.1016/s0304-3835(02)00707-3 · Cited by 70 · see citing works
Self-citationPremature aging/senescence in cancer cells facing therapy: good or bad?
Calvo, L., Ghadaouia, S., Martinez, A., & Rodier, F. (2015) · Biogerontology · doi:10.1007/s10522-015-9593-9 · Cited by 71 · see citing works
Pampalona, J., Soler, D., Genescà, A., & Tusell, L. (2009) · Mutation research. Fundamental and molecular mechanisms of mutagenesis · doi:10.1016/j.mrfmmm.2009.10.001 · Cited by 69 · see citing works
Garbe, J. C., Vrba, L., Sputova, K., Fuchs, L., Novák, P., Brothman, A. R., Jackson, M. W., Chin, K., LaBarge, M. A., Watts, G. S., et al. (2014) · Cell Cycle · doi:10.4161/15384101.2014.954456 · Cited by 67 · see citing works
Self-citationImportance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer
Lemaire, M., Chabot, G. G., Raynal, N. J. ‐., Momparler, L. F., Hurtubise, A., Bernstein, M. L., & Momparler, R. L. (2008) · BMC Cancer · doi:10.1186/1471-2407-8-128 · Cited by 67 · see citing works
“PCR amplifications were performed using HotStar Taq Polymerase (Qiagen) and specific primers spanning different exons for human 18S ribosomal RNA (18S) (Gen Bank accession number X03205) [8], p57 [8] and p16 [25].”
MethodMyocyte death and renewal: modern concepts of cardiac cellular homeostasis
Ellison, G. M., Torella, D., Karakikes, I., et al. (2007) · Nature Clinical Practice Cardiovascular Medicine · doi:10.1038/ncpcardio0773 · Cited by 67 · see citing works
- Authors
- Brenner, A. J., Stampfer, M. R., & Aldaz, C. M.
- Affiliations
- The University of Texas MD Anderson Cancer Center (US)
- Lawrence Berkeley National Laboratory (US)
- Venue
- Oncogene
- Published
- 1998-07-16
- Volume
- 17
- Issue
- 2
- Pages
- 199-205
- ISSN
- 0950-9232 (ISSN-L)
- 1476-5594
- OpenAlex
- W1979165906
- Cited by
- 272
- References
- 0
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 3 · 2023: 10 · 2022: 3 · 2021: 5 · 2020: 6 · 2019: 10 · 2018: 5 · 2017: 8 · 2016: 9 · 2015: 8 · 2014: 4 · 2013: 12 · 2012: 9
- FWCI
- 9.80
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Oncogene (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute (CA-24844)
- Topics
- Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Keywords
- p16INK4a, replicative senescence, mammary epithelial cells, senescence-associated β-galactosidase, DNA methylation, decitabine, p21, Rb pathway, tumor suppressor inactivation, cell immortalization, growth arrest, breast cancer
- MeSH terms
- beta-Galactosidase, Breast, Epithelial Cells, Female, Humans, Promoter Regions, Genetic, Cyclins, Cellular Senescence, DNA Methylation, Cyclin-Dependent Kinase Inhibitor p16, Cyclin-Dependent Kinase Inhibitor p21
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