The long-term objective of my program since 1976 has been to develop and characterize an experimentally tractable human mammary epithelial cell (HMEC) culture system for use in a wide variety of studies on normal human cell biology and carcinogenesis. Our aim has been to understand normal HMEC growth control processes, and to determine how these processes may be altered during immortal and malignant transformation, and during aging. Our assumption has been that clear understanding of what constitutes abnormal processes is dependent upon understanding what constitutes normal healthy biology. Our desire to facilitate widespread use of human epithelial cells for molecular and cellular biology studies has led us to develop an HMEC system that is relatively easy to use, can provide large quantities of standardized cell populations, and is well-characterized. Ongoing collaborative studies assessing the effects of cell-cell and cell-ECM components aim to improve the ability of this in vitro culture system to accurately reflect in vivo biology.
To address our goals, we have generated an integrated HMEC culture system containing:
1) Normal finite lifespan HMEC (really normal; most commercially available “normal” HMEC are highly aberrant post-stasis cells; see website)
a. The normal HMEC have long-term growth, ~30-60 population doublings.
b. Cells are available from women aged 16-91, allowing study of aging effects.
c. Multiple lineages (myoepithelial, luminal, progenitor) are present, allowing study of lineage differentiation.
2) Aberrant finite lifespan HMEC
a. Normal HMEC were exposed to a variety of oncogenic agents/genomic changes reflective of known in vivo breast cancer etiology (e.g., chemical carcinogens, stress, p53 or p16 inactivation).
b. Exposed cultures produced aberrant cells that bypassed or overcame a stress-associated senescence barrier (stasis) to become aberrant post-stasis cultures.
c. A variety of distinct post-stasis phenotypes were generated, reflecting different potential types of in vivo pathways of malignant transformation.
3) Immortally transformed lines isogenic to normal and aberrant finite HMEC
a. Exposed cultures produced rare clonal lines possessing genomic errors and a variety of distinct phenotypes, including both basal and luminal.
b. Non-clonal lines without gross genomic errors have been derived by direct targeting of tumor-suppressor barriers.
c. Since non-malignant immortal lines have overcome the major tumor suppressor barriers, they can be readily transformed to malignancy following exposure to specific oncogenes.
Detailed information on the derivation, characterization, and methods for growth of these cells, as well as information on how other labs may obtain these cells, can be found on my website: http://hmec.lbl.gov.
Our laboratory’s long-term emphasis on extensive development and characterization of one human epithelial cell type model system has enabled us to gain a unique overview of human cellular growth, aging, and transformation. Our studies led us to produce a new model of the tumor-suppressive senescence barriers encountered by cultured normal finite lifespan HMEC as they grow, senesce, overcome senescence barriers, and gain immortality and malignancy (Garbe et al. 2007, 2009, Stampfer et al. 2013). These ongoing studies have indicated that most normal cultured HMEC cease proliferation due to a stress-associated senescence barrier (stasis) mediated by the retinoblastoma protein, rather than telomere attrition. Normal HMEC prior to this barrier (pre-stasis) display significant biological differences compared to finite post-stasis HMEC. Immortality derives from overcoming the replicative senescence barrier (due to telomere attrition), and requires reactivation of telomerase activity; telomerase activity also confers resistance to oncogene-induced senescence. Both pre- and post-stasis finite HMEC show many significant differences compared to non-malignant immortally transformed lines, whose molecular phenotype more closely resembles tumor-derived lines than finite cultures. Significantly, our in vitro transformation model is consistent with the molecular changes observed during malignant transformation in vivo; as such, it makes possible examination of factors that may enhance or inhibit carcinogenesis at different stages of progression.
Our current methods for culture of normal pre-stasis HMEC support extensive proliferative capacity, and make available sufficient normal cells for most experimental purposes. These HMEC cultures can now be used to better understand normal HMEC biology, and along with their isogenic transformed derivatives, allow novel experimentation on the mechanisms responsible for malignant progression.
Pelissier, FA, Garbe, JC, Ananthanarayanan, B, Miyano, M, Lin, C, Jokela, T, Kumar, S, Stampfer, MR, Lorens, JB, LaBarge, LA. Age-related dysfunction in mechano-transduction impairs differentiation of human mammary epithelial progenitors. Cell Reports, in press.
Overhoff, MG, Garbe, JC, Koh, J, Stampfer, MR, Beach, DH, Bishop, CL. Cellular senescence mediated by p16INK4A-coupled miRNA pathways. Nuc Acid Res 42:1606-18, 2014.
Stampfer MR , LaBarge MA, Garbe JC. An integrated human mammary epithelial cell culture system for studying carcinogenesis andaAging, in Cell and Molecular Biology of Breast Cancer, ed. H. Schatten, Springer, NY pp323-361, 2013.
LaBarge MA, Garbe JC, Stampfer MR. Processing of human reduction mammoplasty and mastectomy tissues for cell culture. J Visualized Experimentation 71: e50011, 2013.
Vrba L, Muñoz-Rodríguez JL, Stampfer MR, Futscher BW. miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer. PLoS One 7(12): e52299, 2013.
Sputova, K, Garbe, JC,Pelissier, FA, Chang, E, Stampfer, MR, Labarge, MA. Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length. Genome Integr 4:4 2013.
Garbe JC, Pepin F, Pelissier F, Sputova K, Fridriksdottir A, Guo DE, Villadsen R, Park M, Petersen OW, Barowsky A, Stampfer MR, LaBarge MA. Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia. Cancer Res 72:3687-701, 2012.
Novak P, Stampfer MR, Munoz-Rodriguez JL, Garbe JC, Ehrich M , Futscher BW Jensen TJ. Cell-type specific DNA methylation patterns define human breast cellular identity. PLoS One 8(2): e53776, 2012
Vrba L, Garbe JC, Stampfer MR, Futscher BW. Epigenetic regulation of normal human mammary cell type specific miRNAs. Genom Res 21:2026-2037, 2011.
Vrba, L, Jensen, TJ, Garbe, JC, Heimark, RL, Cress, AE, Dickinson, S, Stampfer, MR, Futscher, BW. DNA methylation control of normal cell-type specific expression of miR-200c. PLoS One 5(1): e8697, 2010.
Bishop CL, Bergin AH, Fessart D, Borgdorff V, Hatzimasoura E, Garbe JC, Stampfer MR, Koh J, Beach DH. Primary cilium dependent and independent Hedgehog signaling inhibits p16INK4A. Mol Cell 40, 533–547, 2010.
Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K, Feiler HS, Wyrobek AJ, Stampfer MR, Molecular distinctions between the stasis and telomere attrition senescence barriers demonstrated by long-term culture of normal human mammary epithelial cells. Cancer Res 69:7557-68, 2009.
Novak, P, Jensen, TJ, Garbe, JC, Stampfer, MR, Futscher, BW, Step-wise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization. Cancer Res 69:5251-58, 2009.
Garbe, J, Holst, CR, Bassett, E, Tlsty, T, Stampfer, MR. Inactivation of p53 function in cultured human mammary epithelial cells turns the telomere-length dependent senescence barrier from agonescence into crisis. Cell Cycle 6: 1927-1936, 2007.
Li Y, Pan J, Li J-L, Lee J-H, Tunkey C, Saraf K, Garbe J, Jelinsky S, Stampfer MR, Haney, SA, Transcriptional changes associated with breast cancer occur as normal human mammary epithelial cells overcome senescence barriers and become immortalized. Mol Can 6: 7-24 2007.
Stampfer, M, 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 22: 5238-5251, 2003.
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 21: 6328-6339 2002.
Romanov, SR, Kozakiewicz, K, Holst, CR, Stampfer, MR, Haupt, LM, Tlsty, TD, Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409: 633-637, 2001.
Stampfer, MR, Garbe, J, Levine, G, Lichtsteiner, S, Vasserot, AP, Yaswen, P, hTERT expression can induce resistance to TGFß growth inhibition in p16INK4A(-) human mammary epithelial cells. Proc Natl Acad Sci (USA) 98: 4498-4503, 2001.
Brenner, AJ, Stampfer, MR, Aldaz, M, Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with inactivation. Oncogene 17: 199-205, 1998.
Stampfer, MR, Bodnar, A, Garbe, J, Wong, M, Pan, A, Villeponteau, B, Yaswen, P, Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells. Mol Biol Cell 8: 2391-2405, 1997.
Stampfer, MR, Yaswen, P, Alhadeff, M, Hosoda, J. TGFß induction of extracellular matrix associated proteins in normal and transformed human mammary epithelial cells in culture is independent of growth effects. J Cell Physiology 155: 210-221, 1993.