Human Mammary Epithelial Cells Senescence Barriers Shown by Long-term Culture of Normal Molecular Distinctions between Stasis and Telomere Attrition

Normal human epithelial cells in culture have generally shown a limited proliferative potential of f10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclindependent 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 B-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. [Cancer Res 2009;69(19):7557–68]

[1]  M. Stampfer,et al.  Stepwise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization. , 2009, Cancer research.

[2]  G. Saretzki,et al.  Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress , 2008, Journal of Cell Science.

[3]  M. Stampfer.  Cholera toxin stimulation of human mammary epithelial cells in culture , 1982, In Vitro.

[4]  G. Saretzki,et al.  DNA damage in telomeres and mitochondria during cellular senescence: is there a connection? , 2007, Nucleic acids research.

[5]  J. Campisi,et al.  Cellular senescence: when bad things happen to good cells , 2007, Nature Reviews Molecular Cell Biology.

[6]  James D Iglehart,et al.  Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes. , 2007, Cancer cell.

[7]  T. Tlsty,et al.  Inactivation of p53 Function in Cultured Human Mammary Epithelial Cells Turns the Telomere-Length Dependent Senescence Barrier from Agonescence into Crisis , 2007, Cell cycle.

[8]  Mina J. Bissell,et al.  Evidence for a stem cell hierarchy in the adult human breast , 2007, The Journal of cell biology.

[9]  M. Stampfer,et al.  Growth of normal human mammary cells in culture , 1980, In Vitro.

[10]  M. Stampfer,et al.  Solar flair. , 2003, Molecular Cancer.

[11]  Jianmin Zhang,et al.  p16INK4a modulates p53 in primary human mammary epithelial cells. , 2006, Cancer research.

[12]  Jianmin Zhang,et al.  p16INK4a Prevents Centrosome Dysfunction and Genomic Instability in Primary Cells , 2006, PLoS biology.

[13]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[14]  K. Chin,et al.  In situ analyses of genome instability in breast cancer , 2004, Nature Genetics.

[15]  Cynthia A Afshari,et al.  Histologically normal human mammary epithelia with silenced p16(INK4a) overexpress COX-2, promoting a premalignant program. , 2004, Cancer cell.

[16]  Gordon K Smyth,et al.  Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2004, Statistical applications in genetics and molecular biology.

[17]  J. Barrett,et al.  Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks , 2004, Nature Cell Biology.

[18]  L. Haupt,et al.  Loss of Chromosomal Integrity in Human Mammary Epithelial Cells Subsequent to Escape from Senescence , 2001, Journal of Mammary Gland Biology and Neoplasia.

[19]  M. Francolini,et al.  Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains , 2003, Oncogene.

[20]  P. Yaswen,et al.  Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines , 2003, Oncogene.

[21]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[22]  P. Yaswen,et al.  Human epithelial cell immortalization as a step in carcinogenesis. , 2003, Cancer letters.

[23]  Stanley N Cohen,et al.  Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  B. Gilchrest,et al.  Evidence that exposure of the telomere 3′ overhang sequence induces senescence , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Yaswen,et al.  Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion , 2002, Oncogene.

[26]  P. Yaswen,et al.  Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P. Cassoni,et al.  Editorial: the oxytocin/oxytocin receptor system-expect the unexpected. , 2001, Endocrinology.

[28]  J. Shay,et al.  Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. , 2001, Genes & development.

[29]  Thea D. Tlsty,et al.  Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes , 2001, Nature.

[30]  A. Sapino,et al.  Biological relevance of oxytocin and oxytocin receptors in cancer cells and primary tumors. , 2001, Annals of oncology : official journal of the European Society for Medical Oncology.

[31]  E. Verspohl,et al.  Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells , 2001, International journal of experimental diabetes research.

[32]  P. Yaswen,et al.  Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells , 1999, Oncogene.

[33]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  A. Brenner,et al.  Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation , 1998, Oncogene.

[35]  P. Yaswen,et al.  Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells. , 1997, Molecular biology of the cell.

[36]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[37]  A. Sapino,et al.  Oxytocin enhances myoepithelial cell differentiation and proliferation in the mouse mammary gland. , 1993, Endocrinology.

[38]  P. Yaswen,et al.  Culture systems for study of human mammary epithelial cell proliferation, differentiation and transformation. , 1993, Cancer surveys.

[39]  C B Harley,et al.  Telomere length predicts replicative capacity of human fibroblasts. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  E. Lane,et al.  Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. , 1989, Journal of cell science.

[41]  M. Stampfer,et al.  Human mammary epithelial cells in culture: differentiation and transformation. , 1988, Cancer treatment and research.

[42]  I. Jolliffe.  Principal Component Analysis , 2005 .

[43]  M. Stampfer,et al.  Factors influencing benzo[a]pyrene metabolism in human mammary epithelial cells in culture. , 1985, Carcinogenesis.

[44]  Martha R. Stampfer,et al.  Isolation and growth of human mammary epithelial cells , 1985 .

[45]  M. Stampfer,et al.  Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[46]  S. L. Hammond,et al.  Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract. , 1984, Proceedings of the National Academy of Sciences of the United States of America.