Lois A. Annab

BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor

Mutational inactivation of BRCA1 confers a cumulative lifetime risk of breast and ovarian cancers. However, the underlying basis for the tissue-restricted tumor-suppressive properties of BRCA1 remains poorly defined. Here we show that BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor α (ERα), a principal determinant of the growth, differentiation, and normal functional status of breasts and ovaries. In Brca1-null mouse embryo fibroblasts and BRCA1-deficient human ovarian cancer cells, ERα exhibited ligand-independent transcriptional activity that was not observed in Brca1-proficient cells. Ectopic expression in Brca1-deficient cells of wild-type BRCA1, but not clinically validated BRCA1 missense mutants, restored ligand-independent repression of ERα in a manner dependent upon apparent histone deacetylase activity. In estrogen-dependent human breast cancer cells, chromatin immunoprecipitation analysis revealed the association of BRCA1 with ERα at endogenous estrogen-response elements before, but not after estrogen stimulation. Collectively, these results reveal BRCA1 to be a ligand-reversible barrier to transcriptional activation by unliganded promoter-bound ERα and suggest a possible mechanism by which functional inactivation of BRCA1 could promote tumorigenesis through inappropriate hormonal regulation of mammary and ovarian epithelial cell proliferation.

Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage

Telomerase is often re‐activated in human cancers and is widely used to immortalize cells in culture. In addition to the maintenance of telomeres, telomerase has been implicated in cell proliferation, genomic instability and apoptosis. Here we show that human telomerase reverse transcriptase (hTERT) is targeted to the mitochondria by an N‐terminal leader sequence, and that mitochondrial extracts contain telomerase activity. In seven different human cell lines, mitochondrial telomerase increases hydrogen‐peroxide‐mediated mitochondrial DNA damage. hTERT expression did not alter the rate of hydrogen peroxide breakdown or endogenous cellular levels. Because the damaging effects of hydrogen peroxide are mediated by divalent metal ions (Fenton chemistry), we examined the levels of bioavailable metals. In all cases, higher levels of chelatable metals were found in hTERT‐expressing cells. These results suggest that mitochondrial telomerase sensitizes cells to oxidative stress, which can lead to apoptotic cell death, and imply a novel function of telomerase in mitochondrial DNA transactions.

Estrogen upregulation of BRCA1 expression with no effect on localization

Alterations in the expression of the breast and ovarian cancer susceptibility gene BRCA1 may contribute to the development of mammary and ovarian neoplasia. The sex‐steroid estrogen modulates cell proliferation of normal and neoplastic breast and ovarian epithelial cells, but the role of estrogen regulation on the expression of BRCA1 remains to be defined. In this study, estrogen‐regulated BRCA1 expression was examined in breast and ovarian cancer cells. Estrogen stimulated the proliferation of estrogen receptor (ER)–positive breast MCF‐7, C7‐MCF‐7, and ovarian BG‐1 cells as well as the expression of the estrogen‐inducible pS2 gene. This was concomitant with upregulation of BRCA1 mRNA (2.5‐ to 5.0‐fold) and a 3‐ to 10‐fold induction of BRCA1 protein (230 kDa). Cell fractionation studies localized the BRCA1 protein to the nucleus in both unstimulated and estrogen‐stimulated cells. The antiestrogen ICI‐182780 inhibited estrogen‐induced cell proliferation, BRCA1 mRNA induction, and BRCA1 protein expression in ER‐positive cells. Conversely, estrogen did not influence expression of BRCA1 in HBL‐100 cells that lacked the estrogen receptor, although the constitutive levels of BRCA1 mRNA (but not protein) in these cells were 5‐ to 30‐fold higher than in other breast and ovarian cancer cells. Secretion of the BRCA1 protein into the cell medium did not account for the discrepancy between the mRNA and protein levels in HBL‐100 cells. Proliferation of HBL‐100 cells was not affected by either estrogen or ICI‐182780. Taken together, these data support a role for the steroid estrogen and the involvement of the estrogen receptor pathway in the modulation of expression of BRCA1. We therefore propose that stimulation of cell proliferation may be a prerequisite for upregulation of BRCA1 in breast and ovarian cancer cells. Mol. Carcinog. 22:102–109, 1998.

Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG‐1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild‐type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1‐blocked ER‐positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1‐blocked ER‐positive BG‐1 ovarian cancer cells. For all three drugs, BRCA1‐blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (ΔΨm), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1‐blocked and control cells. However, apoptosis induction was greater in BRCA1‐blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 μM in the control cells and 2.68 μM for the BRCA1‐blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild‐type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1‐mutated ovarian cancer patients.

Evidence for two senescence loci on human chromosome I

Microcell‐mediated introduction of a neo‐tagged human chromosome I (HC‐1‐neo) into several immortal cell lines has previously been shown to induce growth arrest and phenotypic changes indicative of replicative senescence. Somatic cell hybridization studies have localized senescence activity for immortal hamster 10W‐2 cells to a cytogenetically defined region between 1q23 and the q terminus. Previous microcell‐mediated chromosome transfer experiments showed that a chromosome 1 with an interstitial q‐arm deletion (del‐1q) lacks senescence inducing activity for several immortal human cell lines that are sensitive to an intact HC‐1‐neo. In contrast, our studies reveal that the del‐1q chromosome retains activity for 10W‐2 cells, indicating that there are at least two senescence genes on human chromosome 1. Sequence‐tagged site (STS) content analysis revealed that the q arm of the del‐1q chromosome has an interstitial deletion of approximately 63 centimorgans (cM), between the proximal STS marker D1S534 and distal marker D1S412, approximately 1q12 to 1q31. This deletion analysis provides a candidate region for one of the senescence genes on 1q. In addition, because this deletion region extends distally beyond 1q23, it localizes the region containing a second senescence gene to approximately 1q31‐qter, between D1S422 and the q terminus. STS content analysis of a panel of 11 10W‐2 microcell hybrid clones that escaped senescence identified 2 common regions of loss of 1q material below the distal breakpoint of del‐1q. One region is flanked by markers D1S459 and ACTN2, and the second lies between markers W1‐4683 and D1S1609, indicating that the distal 1q senescence gene(s) localizes within 1q42‐43. Genes Chromosom Cancer 16:55–63 (1996).

Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast.

The BRCA1 gene, mutations of which contribute significantly to hereditary breast cancer, was not identified in the existing YAC and BAC libraries. The gene is now available only as a set of overlapping fragments that form a contig. In this work we describe direct isolation of a genomic copy of BRCA1 from human DNA by transformation-associated recombination (TAR) cloning. Despite the presence of multiple repeats, most of the primary BRCA1 YAC isolates did not contain detectable deletions and could be stably propagated in a host strain with conditional RAD52. Similar to other circular YACs, approximately 90kb BRCA1 YACs were efficiently and accurately retrofitted into bacterial artificial chromosomes (BACs) with the Neo(R) mammalian selectable marker and transferred as circular BAC/YACs in E. coli cells. The BRCA1 BAC/YAC DNAs were isolated from bacterial cells and were used to transfect mouse cells using the Neo(R) gene as selectable marker. Western blot analysis of transfectants showed that BRCA1 YACs isolated by a TAR cloning contained a functional gene. The advantage of this expression vector is that the expression of BRCA1 is generated from its own regulatory elements and does not require additional promoter elements that may result in overexpression of the protein. In contrast to the results with cDNA expression vectors, the level of BRCA1 expression from this TAR vector is stable, does not induce cell death, maintains serum regulation, and approximates the level of endogenously expressed BRCA1 in human cells. The entire isolation procedure of BRCA1 described in this paper can be accomplished in approximately 10 days and can be applied to isolation of gene from clinical material. We propose that the opportunity to directly isolate normal and mutant forms of BRCA1 will greatly facilitate analysis of the gene and its contribution to breast cancer.

Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line

Statement of findingsWe tested the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as the inhibition of ovarian cell proliferation. Introduction of BRCA1 antisense retroviral constructs into BG-1 estrogen-dependent ovarian adenocarcinoma cells resulted in reduced BRCA1 expression. BRCA1 antisense pooled populations and derived subclones were able to proliferate in monolayer culture without estrogen, whereas control cells began to die after 10 days of estrogen deprivation. In addition, both populations and subclones of BRCA1 antisense infected cells demonstrated a growth advantage in monolayer culture in the presence of estrogen and were able to proliferate in monolayer culture without estrogen, while control cells did not. Furthermore, clonal studies demonstrated that reduced levels of BRCA1 protein correlated with growth in soft agar and greater tumor formation in nude mice in the absence of estrogen. These data suggest that reduction of BRCA1 protein in BG-1 ovarian adenocarcinoma cells may have an effect on cell survival during estrogen deprivation both in vitro and in vivo.

Differential responses to retinoic acid and endocrine disruptor compounds of subpopulations within human embryonic stem cell lines

The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). The large cell population responds to retinoic acid differentiation with greater than a 50% reduction in cell number and loss of Oct-4 expression, whereas the number of the small cell population does not change and Oct-4 protein expression is maintained. In addition, four estrogenic compounds altered SSEA-3 expression differentially between the two cell subpopulations changing their ratios relative to each other. Both populations express stem cell markers Oct-4, Nanog, Tra-1-60, Tra-1-80 and SSEA-4, but express low levels of differentiation markers common to the three germ layers. Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). We propose the differential response to RA in these populations is due to differential gene expression of Notch signaling members, CoupTF1 and CoupTF2, chromatin remodeling and histone modifying genes that render the small population resistant to RA differentiation. The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy.

Epigenetic Enzymes, Age, and Ancestry Regulate the Efficiency of Human iPSC Reprogramming

Epigenetic enzymes regulate higher‐order chromatin architecture and cell‐type specific gene expression. The ATPase BRG1 and the SWI/SNF chromatin remodeling complex are epigenetic enzymes that regulate chromatin accessibility during steady and transitional cell states. Experiments in mice show that the loss of BRG1 inhibits cellular reprogramming, while studies using human cells demonstrate that the overexpression of BRG1 enhances reprogramming. We hypothesized that the variation of SWI/SNF subunit expression in the human population would contribute to variability in the efficiency of induced pluripotent stem cells (iPSC) reprogramming. To examine the impact of an individuals sex, ancestry, and age on iPSC reprogramming, we created a novel sex and ancestry balanced cohort of 240 iPSC lines derived from human dermal fibroblasts (DF) from 80 heathy donors. We methodically assessed the reprogramming efficiency of each DF line and then quantified the individual and demographic‐specific variations in SWI/SNF chromatin remodeling proteins and mRNA expression. We identified BRG1, BAF155, and BAF60a expression as strongly correlating with iPSC reprogramming efficiency. Additionally, we discovered that high efficiency iPSC reprograming is negatively correlated with donor age, positively correlated with African American descent, and uncorrelated with donor sex. These results show the variations in chromatin remodeling protein expression have a strong impact on iPSC reprogramming. Additionally, our cohort is unique in its large size, diversity, and focus on healthy donors. Consequently, this cohort can be a vital tool for researchers seeking to validate observational results from human population studies and perform detailed mechanistic studies in a controlled cell culture environment. Stem Cells 2018;36:1697–1708

Combination of functional genomics approaches and microarray analyses to define candidate senescence genes

Combination of functional genomics approaches and microarray analyses to define candidate senescence genes