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Results for “human mammary epithelial cells (HMEC)”

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12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

YAP1 Dysfunction Promotes Molecular Properties Linked to Breast Cancer Susceptibility

YAP1 is a cotranscription factor that promotes malignant and stem cell properties in cancer. We previously found that YAP1 dysregulation is associated with aging in human mammary epithelia. With increased age, YAP1 expression changes in luminal epithelial cells, the prospective breast cancer cell of origin. Because age is a significant risk factor for breast cancer, we tested whether YAP1 dysregulation acted early in cancer progression by conferring cellular states associated with increased cancer susceptibility. In this study, we find that with increased age and genetic risk for developing cancer, human breast tissues showed significantly increased YAP1 expression, and cultured primary human mammary epithelial cells (HMEC) showed significantly increased expression of both YAP1 and its transcriptional targets. Increased YAP1 expression in cultured HMEC induced gene expression changes associated with increased cancer susceptibility, such as genes associated with stem cell states, increased telomerase activity, breast cancer progression, and increased age and genetic breast cancer risk. Furthermore, overexpression of YAP1 in post-stasis HMEC—finite lifespan cells that have bypassed a retinoblastoma-mediated senescence barrier—promoted properties related to increased growth potential. We found that YAP1 dysregulation in finite epithelial cells allows for access to gene programs and functions that are typically thought to be restricted to stem cells. We hypothesize that YAP1 acts early in breast cancer progression, long before the development of a tumor, to impose cancer-susceptible molecular states.

YAP1↗
NASA NTRS2002

Irradiated HMEC from A-T Heterozygous Breast Tissue

Women who are heterozygous for ataxia-telangiectasia (A-T) carry a single defective ATM gene in chromosome 11 q22-23, and have been statistically determined with high significance within a defined database to be approximately 5-fold more susceptible for developing breast cancer than their noma1 counterpart. Breast cancer susceptibility of these A-T heterozygotes has been hypothesized to include consequence of response to damage caused by low levels of ionizing radiation. Prophylactic mastectomy specimens were donated by a 41 year-old obligate A-T heterozygote who was located prior to her elective surgery through an existing pedigree. Harvest of that breast tissue provided an isolate of long-term growth human mammary epithelial cells (HMEC), designated WH612/3. An isolate of presumed normal long-term growth HMEC, designated 48R, was obtained from Dr. Martha Stampfer (Lawrence Berkeley Laboratory, University of California), and the A-T heterozygous HMEC were transformed with E6 and E7 oncogenes of human papilloma virus Type-16 in the laboratory of Dr. Ray White (Hunt- Cancer Institute, University of Utah) for use in this study. The objective of this study is to study the expression of end points that may bear on cancer outcome following irradiation of HMEC. Specific end points are cell survival, cell cycle, p53 expression, and apoptosis. Survival curves, immunostaining, and flow cytometery are used to examine these end points. Radiation-induced cell killing shows less shoulder development in the survival curve for WH61U3 compared to 48R HMEC, suggesting less repair of damage in the former HMEC. Additional information is included in the original extended abstract.

Richmond, Robert↗
DOE OSTI2020

Methods for immortalization of epithelial cells

Methods for inducing non-clonal immortalization of normal epithelial cells by directly targeting the two main senescence barriers encountered by cultured epithelial cells. In finite lifespan pre-stasis human mammary epithelial cells (HMEC), the stress-associated stasis barrier was bypassed, and in post-stasis HMEC, the replicative senescence barrier, a consequence of critically shortened telomeres, was bypassed. Early passage non-clonal immortalized lines exhibited normal karyotypes. Methods of efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation and immortalization during human carcinoma progression, methods for screening for toxic and environmental effect on progression, and the development of therapeutics targeting the process of immortalization.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Review↗
NASA NTRS2003

Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia

Cultures of human mammary epithelial cells (HMECs) contain a subpopulation of variant cells with the capacity to propagate beyond an in vitro proliferation barrier. These variant HMECs, which contain hypermethylated and silenced p16(INK4a) (p16) promoters, eventually accumulate multiple chromosomal changes, many of which are similar to those detected in premalignant and malignant lesions of breast cancer. To determine the origin of these variant HMECs in culture, we used Luria-Delbruck fluctuation analysis and found that variant HMECs exist within the population before the proliferation barrier, thereby raising the possibility that variant HMECs exist in vivo before cultivation. To test this hypothesis, we examined mammary tissue from normal women for evidence of p16 promoter hypermethylation. Here we show that epithelial cells with methylation of p16 promoter sequences occur in focal patches of histologically normal mammary tissue of a substantial fraction of healthy, cancer-free women.

NASA Discipline Radiation Health↗
NASA NTRS2003

Ionizing radiation induces heritable disruption of epithelial cell interactions

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

Non-NASA Center↗
NASA NTRS2001

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

NASA Discipline Radiation Health↗
NASA NTRS2001

Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

Richmond, Robert C.↗
NASA NTRS2003

Radiation Dose-effects on Cell Cycle, Apoptosis, and Marker Expression of Ataxia Telangiectasia-Heterozygous Human Breast Epithelial Cells

Ataxia-telangiectasia (A-T) is a radiation-sensitive genetic condition. AT-heterozygous human mammary epithelial cells (HMEC) were irradiated using a Cs137 source in order to compare cell cycle, apoptosis, and marker expression responses across 3 radiation doses. No differences in cell cycle and apoptosis were found with any of the radiation doses used (30, 60, and 90 rads) compared with the unirradiated control (0 rad). At the same doses, however, differences were found in marker expression, such as keratin 18 (kl8), keratin 14 (k14), insulin-like growth factor I receptor (IGF-IR), and connexin 43 (cx43). This may indicate that radiation sensitivity in the heterozygous state may be initiated through signal transduction responses.

Cruz, A.↗
NASA NTRS2004

Biodosimetry as a New Paradigm for Determination of Radiation Risks and Risk-Mitigation in Astronauts Exposed to Space Radiation

Predicting risk of cancer in astronauts exposed to space radiation is challenging partly because uncertainties of absorption of dose and the processing of dose-related damage at the cellular level degrade the confidence of predicting the expression of cancer. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the macromolecular profiles related to malignant transformation in cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported, This is the demonstration of two dose-responsive field-effects of enhanced protein-expression. In one case, expression of keratin 18 (K18) in cultures of human mammary epithelial cells (HMEC) irradiated with cesium-137 gamma-rays is enhanced following exposure of log phase cells to relatively low doses of 30 to 90 cGy. K18 has been reported by a marker for tumor staging and for apoptosis. In the second case, expression of connexin 43 (Cx43) is increased in irradiated stationary phase cultures of HMEC, indicating enhanced formation of gap junctions. Gap junctions have been reported to be involved in bystander effects following irradiation. It is a biodosimeter for assessing radiogenic damage. It is suggested further that such biomolecular dosimetry may introduce a new paradigm for assessing cancer risk and risk-mitigation in individuals, a requirement for managing radiation health in astronauts during extended missions in space. This new paradigm is built upon the statistical power provided by the use of functional genomics and proteomics represented in combined gene- and protein-expression assays.

Richmond, Robert↗
DOE OSTI2021

Early growth response 2 (EGR2) is a novel regulator of the senescence programme

Abstract Senescence, a state of stable growth arrest, plays an important role in ageing and age‐related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up‐regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down‐regulates p16 levels and increases the pool of p16− p21− ‘reversed’ cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2001

Differentiation and Genomic Instability in a Human Mammary Cell Model

Harvest of prophylactic mastectomy specimens from an obligate heterozygote for ataxia-telangiectasia provided autologous fibroblasts as well epithelial cells (HMEC). The routine availability of these autologous cells has provided an opportunity to study cell-cell interactions in coculture and monoculture, and in 3-dimensional cultures grown in the NASA rotating bioreactor. HMEC and stromal fibroblasts grown in 2-dimensional monoculture were both observed to produce extracellular matrix. Similar matrix was encountered in 3-dimensional cultures containing HMEC. Metaphases were analyzed. For stromal fibroblasts, genomic aberrations were found in 18% of metaphase spreads. For HMEC, aberrations were greater such that a majority were found to be abnormal. The level of genomic instability determined for these noncancerous cells in 2-dimensional monoculture should be useful for generating a human cell model that can correlate the effects of differentiation in 3-dimensional coculture on the level of genomic instability.

Richmond, R.↗