Inactivation of p53 Function in Cultured Human Mammary Epithelial Cells Turns the Telomere-Length Dependent Senescence Barrier from Agonescence into Crisis

Garbe, James C.; Holst, Charles R.; Bassett, Ekaterina; Tlsty, Thea D.; Stampfer, Martha R.

doi:10.4161/cc.6.15.4519

Cited by 62 publications

(96 citation statements)

References 57 publications

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“…However, both the MYC/RAS-HMEC and shp53/MYC/RAS-HMEC cultures formed significantly fewer colonies at later passages and, despite their capacity for AIG, eventually stopped proliferating. These data support the contention that telomerase activation is a key rate-limiting step in malignant progression, and further analysis is currently underway (26).…”

Section: Resultssupporting

confidence: 69%

TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

Cipriano¹,

Kan²,

Graham³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation, serves as an important tumor-suppressive barrier. We show here that finite lifespan human mammary epithelial cells (HMEC) undergo a p16/ RB-and p53-independent OIS in response to oncogenic RAS that requires TGF-β signaling. Suppression of TGF-β signaling by expression of a dominant-negative TGF-β type II receptor, use of a TGF-β type I receptor inhibitor, or ectopic expression of MYC permitted continued proliferation upon RAS expression. Surprisingly, unlike fibroblasts, shRNA-mediated knockdown of ATM or CHK2 was unable to prevent RAS-mediated OIS, arguing that the DNA damage response is not required for OIS in HMEC. Abrogation of TGF-β signaling not only allowed HMEC lacking p53 to tolerate oncogenic RAS but also conferred the capacity for anchorage-independent growth. Thus, the OIS engaged after dysregulated RAS expression provides an early barrier to malignant progression and is mediated by TGF-β receptor activation in HMEC. Understanding the mechanisms that initiate and maintain OIS in epithelial cells may provide a foundation for future therapies aimed at reengaging this proliferative barrier as a cancer therapy. (ii) constitutive growth signaling, (iii) unlimited replication potential, and (iv) invasive potential (1). Early studies using normal mouse cells indicated that a limited set of genetic manipulations could confer neoplastic potential (2). However, normal human cells have been more difficult to transform to malignancy, indicative of their more stringent tumor-suppressive pathways. Extensive study of cultured human mammary epithelial cells (HMEC) has identified two senescence barriers. One involves the stress-associated induction of the cyclin-dependent kinase inhibitor p16 before attaining critically short telomeres. This stasis barrier can be overcome by inhibiting p16, allowing continued proliferation, which results in agonescence, a proliferative barrier mediated by telomere depletion (3). Additionally, the ability of dysregulated oncogenic signaling to induce senescence in human cells has implicated oncogene-induced senescence (OIS) as an important tumor-suppressive barrier. A number of recent studies have demonstrated the physiological relevance of OIS in human tumorigenesis and in vivo tumor mouse models (4). Additionally, the presence of senescent cells in benign but not advanced tumors argues that OIS serves as an early tumorsuppressive barrier that needs to be dismantled for full oncogenic progression (4). In human fibroblasts, OIS could be bypassed by disabling p16 or molecular components of the DNA damage response (DDR), including ATM, CHK2, or p53, before RAS, MOS, or STAT5 overexpression (5-9). However, OIS in HMEC has been shown to be independent of p53 and the p16-RB pathway after oncogenic RAF-1 expression (10). The contrasting responses between epithelial and fibroblast cells argue that the signaling networks responsible for OIS have tissue specificity. Indeed, fibrob...

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Section: Resultssupporting

confidence: 69%

TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

Cipriano¹,

Kan²,

Graham³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…[13][14][15][16] Polyploidy associated with senescence is generally assessed by flow cytometry which gives no information as to mode of mitotic cycling. 2,[17][18][19] The special type of endopolyploidy had shown production of genome reduced cells (e.g., 4n/8C to 2n/2C) with chromosomal abnormalities occurring immediately before (i.e., pre-senescence) the change to flat cells-only. Thus, re-appearance of this polyploid reductional division in reverted cells would firstly, show persistence of the process in senescence, and secondly, the reverted cells might be a source for polyploidaneuploidization.…”

Section: Introductionmentioning

confidence: 99%

“…1 Recently, senescence arrest-barriers from activated cell cycle checkpoint controls were molecularly defined for human mammary epithelial cells and comparisons were made to events for senescent fibroblast cells. 2 The major issue in replicative senescence is the state of the telomeres which for human cells, becomes progressively shorter in young to old cell growths. [2][3][4][5] These happenings give rise to chromosomal instability (CIN) which solicits a senescence proliferation arrest.…”

Section: Introductionmentioning

confidence: 99%

“…2 The major issue in replicative senescence is the state of the telomeres which for human cells, becomes progressively shorter in young to old cell growths. [2][3][4][5] These happenings give rise to chromosomal instability (CIN) which solicits a senescence proliferation arrest. Replicative senescence is therefore, considered as an antitumor mechanism with possibilities in therapy.…”

Section: Introductionmentioning

confidence: 99%

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Genetic stability of senescence reverted cells: Genome reduction division of polyploidy cells, aneuploidy and neoplasia

Walen

2008

Cell Cycle

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Cell senescence from exhausted cell expansions to cells with short, dysfunctional telomeres is considered to be a non-replicative, irreversible state with possibility in tumor therapy. This leads to questions of senescence-stability which in genomic-probe, manipulated senescent flat cells resulted in reversions to mitotic cells. Additionally, rarer mitoses were present spontaneously in months, old, live flat cell cultures. These latter senescence-escaped cells were analyzed by cytogenetics to determine their origin from either stable G 0 /G 1 diploid and/or from unstable endopolyploid cells. Endo-polyploidization in senescence is associated with re-replication of genomically damaged G 2 /M cells. One source for genomic damage is senescence-specific occurrence of heterochromatization. It causes gluing of chromosomes together with consequent malsegregations in mitosis which was a feature of the present reverted cells. In addition endo-polyploidy cycled with the characteristic presence of diplochromosomes (i.e., pairs of sister chromosomes) undergoing two consecutive bipolar divisions into genome reduced cells. Both diploid and tetraploid, aneuploid cells were also present as reverted cells. For in vitro cell senescence reversion to mitotic cells is therefore, concluded to be associated with occurrence of genomic instability. These results are discussed with reference to a meiotic-like somatic cell division of cycling endopolyploidy and as a possible mechanism of aneuploidization in tumor development. The extracellular matrix is evaluated in regard to a role as a protective shield against nuclear budding-offs (karyoplasts) from the flat cells to form mitotically-capable reverted cells.

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“…Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation [4][5][6][7][8][9][10][11][12][13][14][15]16 . The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic 5,15 .…”

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confidence: 99%

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

LaBarge¹,

Garbe²,

Stampfer³

2013

JoVE

Self Cite

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Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells [1][2] . Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells 3 . The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using lowstress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor) 4-5

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Inactivation of p53 Function in Cultured Human Mammary Epithelial Cells Turns the Telomere-Length Dependent Senescence Barrier from Agonescence into Crisis

Cited by 62 publications

References 57 publications

TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

Genetic stability of senescence reverted cells: Genome reduction division of polyploidy cells, aneuploidy and neoplasia

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

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