Innovation and Partnerships Office

Long-term Growth of Finite Life Span Normal Human Epithelial Cells IB-2077

APPLICATIONS OF TECHNOLOGY:

  • Examine carcinogenesis, aging, expression of genes, proteins and miRNA, signaling pathways, epigenetics, and genomic stability
  • Develop quantitative assays for potentially pro or anti carcinogenic or aging agents, or toxic agents
  • Generate transformed HMEC lines

ADVANTAGES:

  • Enables the creation of large standardized batches of normal HMEC (60 PD)
  • Elucidates a model that appears to closely replicate in vivo carcinogenesis
  • May provide for easier generation of immortalized HMEC lines

ABSTRACT:

Martha Stampfer and colleagues at Berkeley Lab have developed a system for consistently achieving as many as 60 population doublings (PD) in a cell culture of finite lifespan human mammary epithelial cells (HMEC).   Previous methods developed by Stampfer and currently distributed commercially achieve nearly equivalent PD, but these cell types (post-selection) have already overcome one of the barriers to immortality and contain properties not representative of cells most prevalent in normal human breast.   Conventional culturing methods used in most laboratories for normal HMEC typically yield 30 PD of active growth at best.

Berkeley Lab’s recent success in culturing cells is the result of a data-supported model of the proliferation barriers encountered by HMEC that offers new perspectives on malignant progression. The paradigm, developed by Stampfer’s team, predicts that HMEC encounter two mechanistically distinct proliferation barriers. The first, stasis, is a consequence of various stresses and appears to be telomere length independent. The second is an extremely stringent barrier imposed by critically shortened telomeres, producing telomere dysfunction and genomic instability. This characterization of proliferation barriers and senescence encountered by cultured HMEC is consistent with in vivo studies of human carcinoma progression.

Based on this model, the Berkeley Lab researchers developed a medium consisting of a ratio of two previously developed media that increases the culturing of pre-stasis HMEC to approximately 45 PD. The addition of an anti-stress growth factor to the medium in primary or secondary passage aids the cells in postponing the onset of the stress barrier and can increase the PD to approximately 60.

Besides enabling the generation of large standardized HMEC batches and related assays, the Berkeley Lab media and method may also provide for easier generation of transformed HMEC lines from pre-stasis HMEC populations rather than through the commercially available post-selection HMEC that have already overcome the stasis barrier.

STATUS:

  • Published US Patent Application 2010-0022000 available at www.uspto.gov. Available for licensing or collaborative research.

FOR MORE INFORMATION:

Researcher’s website: http://hmec.lbl.gov/mindex.html

Referenced Publication:

Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K, Feiler HS, Wyrobek AJ, Stampfer MR (2009). 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.

Other method references:

LaBarge MA, Garbe JC, Stampfer MR (2013). Processing of human reduction mammoplasty and mastectomy tissues for cell culture. J Visualized Experimentation 71: e50011.

Stampfer MR, LaBarge MA, Garbe JC (2013). An Integrated Human Mammary Epithelial Cell Culture System for Studying Carcinogenesis and Aging , in: Cell and Molecular Biology of Breast Cancer, ed. H. Schatten, Springer, NY pp323-361.

Papers that have used this new methodology:

Bloushtain-Qimron N, Yao, J, Snyder, EL, Shiptisin, M, Campbell, LL, Mani, SA, Hu, M, Chen, H, Ustyansky, V, Antosiewicz, JE, Argani, P, Halushka, MK, Thomson, JA, Pharoah, P, Porgador, A, Sukumar, S, Parsons, R, Richardson, AL, Stampfer, MR, Gelman, RS, Nikolskaya, T, Nikolsky, Y, Polyak, K (2008). Cell type-specific DNA methylation patterns in the human breast, Proc Nat Acad Sci USA 105:14076-81.

LaBarge MA, Nelson CM, Villadsen R, Fridrikdottir A, Ruth JR, Stampfer MM, Petersen OW, and Bissell MJ (2009). Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments. Integr Biol:1, 70-79.

Novak, P, Jensen, TJ, Garbe, JC, Stampfer, MR, Futscher, BW (2009). Step-wise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization, Cancer Res 69:5251-58.

Vrba, L, Jensen, TJ, Garbe, JC, Heimark, RL, Cress, AE, Dickinson, S, Stampfer, MR, Futscher, BW (2010). DNA Methylation Control of Normal Cell-Type Specific Expression of miR-200c, PLoS One 5(1): e8697.

Lapuk, A, Marr, H, Jakkula, L, Pedro, H, Bhattacharya, S, Purdom, E, Hu, Z, Simpson, L, Pachter, L, Durinck, S, Wang, N, Parvin, B, Fontenay, G, Speed, T, Garbe, J, Stampfer, M, Bayandorian, H, Dorton, S, Clark, T, Schweitzer, A, Wyrobek, A, Feiler, H, Spellman, P, Conboy, J, Gray, J (2010). Exon-level microarray analyses identify alternative splicing programs in breast cancer. Mol Can Res 8; 961–74.

Bishop CL, Bergin AH, Fessart D, Borgdorff V, Hatzimasoura E, Garbe JC, Stampfer MR, Koh J, Beach DH (2010). Primary cilium dependent and independent Hedgehog signaling inhibits p16INK4A. Mol Cell 40, 533–547.

Cipriano R, Kan CE, Graham J, Danielpour D, Stampfer M, Jackson MW (2011). TGF-ß signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells. Proc Nat Acad Sci USA 108:8668-73.

Chanson L, Brownfield D, Garbe JC, Kuhn I, Stampfer MR, Bissell MJ, LaBarge MA (2011). Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells, Proc Nat Acad Sci USA, 108:3264-9.

Sherman, MY, Meng, L, Stampfer, M, Gabai, VL, Yaglom, JA. Oncogenes induce senescence with incomplete growth arrest and suppress the DNA damage response. Aging Cell 10:949-961, 2011.

Vrba L, Garbe JC, Stampfer MR, Futscher BW (2011). Epigenetic regulation of normal human mammary cell type specific miRNAs. Genom Res 21:2026-2037.

Garbe JC, Pepin F, Pelissier F, Sputova K, Fridriksdottir A, Guo DE, Villadsen R, Park M, Petersen OW, Barowsky A, Stampfer MR, LaBarge MA (2012). Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia. Cancer Res 72:3687-701.

Novak P, Stampfer MR, Munoz-Rodriguez JL, Garbe JC, Ehrich M , Futscher BW Jensen TJ (2012). Cell-Type Specific DNA Methylation Patterns Define Human Breast Cellular Identity. PLoS One 8(2): e53776.

Vrba L, Muñoz-Rodríguez JL, Stampfer MR, Futscher BW (2013). miRNA Gene Promoters Are Frequent Targets of Aberrant DNA Methylation in Human Breast Cancer. PLoS One 7(12): e52299.

Junk, DJ, Cipriano, R, Stampfer, MR, Jackson, MW (2013). Constitutive CCND1/CDK2 Activity Substitutes for p53 Loss, or MYC or Oncogenic RAS Expression in the Transformation of Human Mammary Epithelial Cells. PLoS One 8(2): e53776.

Garcia, H, Miecznikowski, JC, Safina, A, Commane, M, Ruusulehto, A, Kilpinen, S, Leach, RW, Attwood, K, Li, Y, Degan, S, Omilian, AR, Guryanova, O, Papantonopoulou, O, Wang, J, Buck, M, Liu, S, Morrison, C, Gurova, KV (2013). Facilitates Chromatin Transcription Complex Is an ‘‘Accelerator’’ of Tumor Transformation and Potential Marker and Target of Aggressive Cancers Cell Rep 4, 159–73.

Sputova, K, Garbe, JC,Pelissier, FA, Chang, E, Stampfer, MR, Labarge, MA (2013). Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length. Genome Integr 4:4.

Stovall, DB, Wan, M, Miller, LD, Cao,P, Maglic, D, Zhang Q, Stampfer, MR, Liu, W, Xu, J, Sui, G (2013). The Regulation of SOX7 and its Tumor Suppressive Role in Breast Cancer. Am J Path 183:1645.

Overhoff, MG, Garbe, JC, Koh, J, Stampfer, MR, Beach, DH, Bishop, CL (2014). Cellular senescence mediated by p16INK4A-coupled miRNA pathways. Nuc Acid Res 42:1606-18.

Pelissier, FA, Garbe, JC, Ananthanarayanan, B, Miyano, M, Lin, C, Jokela, T, Kumar, S, Stampfer, MR, Lorens, JB, LaBarge, LA (2014). Age-related dysfunction in mechano-transduction impairs differentiation of human mammary epithelial progenitors. Cell Rep 7:1926-39.

Garbe, JC, Vrba, L, Sputova, K, Fuchs, L, Novak, P, Brothman, AR, Jackson, M, Chin, K, LaBarge, MA, Watts, G, Futscher, BW, Stampfer, MR (2014). Immortalization of Normal Human Mammary Epithelial Cells in Two Steps by Direct Targeting of Senescence Barriers Does Not Require Gross Genomic Alterations. Cell Cycle, 13:3423-35.

Pathiraja, TN, Kaushik, KN, Thakkar, N, Jiang, S, Stratton, S, Liu, Z, Gagea, M, Xi, S, Shah, PK, Phan, L, Lee, M-H, Andersen, J, Stampfer, MR, Barton, MC. TRIM24 Links Glucose Metabolism with Transformation of Human Mammary Epithelial Cells. Oncogene, in press.

REFERENCE NUMBER: IB- 2077

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