PMC:13916 / 29290-37420 JSONTXT

{"project":"Colil","denotations":[{"id":"T33","span":{"begin":4603,"end":4605},"obj":"9296497"},{"id":"T34","span":{"begin":2200,"end":2202},"obj":"9361806"},{"id":"T35","span":{"begin":5083,"end":5085},"obj":"8674108"},{"id":"T36","span":{"begin":3651,"end":3652},"obj":"9218874"},{"id":"T37","span":{"begin":3653,"end":3654},"obj":"9010238"},{"id":"T38","span":{"begin":3655,"end":3657},"obj":"9121124"},{"id":"T39","span":{"begin":3658,"end":3660},"obj":"8917547"},{"id":"T40","span":{"begin":3661,"end":3663},"obj":"9008167"},{"id":"T41","span":{"begin":3664,"end":3666},"obj":"9296497"},{"id":"T42","span":{"begin":4380,"end":4382},"obj":"8589721"},{"id":"T43","span":{"begin":2777,"end":2779},"obj":"9121124"},{"id":"T44","span":{"begin":2780,"end":2782},"obj":"9467943"},{"id":"T45","span":{"begin":2900,"end":2902},"obj":"9241077"},{"id":"T46","span":{"begin":6830,"end":6832},"obj":"9155518"},{"id":"T47","span":{"begin":2451,"end":2452},"obj":"9010238"},{"id":"T48","span":{"begin":7187,"end":7189},"obj":"9699523"},{"id":"T49","span":{"begin":7190,"end":7192},"obj":"9639385"},{"id":"T50","span":{"begin":7193,"end":7195},"obj":"7795653"},{"id":"T51","span":{"begin":6365,"end":6367},"obj":"9212093"},{"id":"T52","span":{"begin":1119,"end":1121},"obj":"2910432"},{"id":"T53","span":{"begin":5086,"end":5088},"obj":"8563759"},{"id":"T54","span":{"begin":5089,"end":5091},"obj":"9171368"},{"id":"T55","span":{"begin":4081,"end":4083},"obj":"8637708"},{"id":"T56","span":{"begin":5802,"end":5804},"obj":"8700535"},{"id":"T57","span":{"begin":2897,"end":2899},"obj":"9221812"},{"id":"T58","span":{"begin":2203,"end":2205},"obj":"9002648"},{"id":"T59","span":{"begin":2206,"end":2208},"obj":"9074768"},{"id":"T60","span":{"begin":6874,"end":6876},"obj":"7878464"},{"id":"T61","span":{"begin":2194,"end":2196},"obj":"9135581"},{"id":"T62","span":{"begin":2197,"end":2199},"obj":"7654326"},{"id":"T63","span":{"begin":7184,"end":7186},"obj":"9988281"},{"id":"T64","span":{"begin":6360,"end":6361},"obj":"9267023"},{"id":"T65","span":{"begin":6362,"end":6364},"obj":"9008167"},{"id":"T66","span":{"begin":2903,"end":2905},"obj":"9155518"},{"id":"T67","span":{"begin":6106,"end":6108},"obj":"9582019"},{"id":"T68","span":{"begin":5369,"end":5371},"obj":"9662340"},{"id":"T69","span":{"begin":3152,"end":3154},"obj":"9149021"},{"id":"T70","span":{"begin":7460,"end":7462},"obj":"9269993"},{"id":"T71","span":{"begin":5171,"end":5173},"obj":"8700535"},{"id":"T72","span":{"begin":2536,"end":2537},"obj":"9010238"},{"id":"T73","span":{"begin":2538,"end":2540},"obj":"9121124"},{"id":"T74","span":{"begin":2007,"end":2009},"obj":"8780885"},{"id":"T75","span":{"begin":4193,"end":4195},"obj":"8589721"},{"id":"T76","span":{"begin":4944,"end":4946},"obj":"9121124"},{"id":"T77","span":{"begin":4947,"end":4949},"obj":"9008167"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Discussion\nThe present studies show that reduction of BRCA1 levels, using an antisense retroviral vector in the estrogen dependent BG-1 ovarian carcinoma cell line, may aid in confirmation of the hypothesis that BRCA1 functions as a tumor suppressor gene by playing a pivotal role in the balance between cell death and cell proliferation. BRCA1 RNA and protein levels were successfully reduced in pooled and isolated subclones of antisense-infected populations of BG-1 cells. Decreased BRCA1 levels appeared to affect the ability of BG-1 cells to arrest growth or die in the absence of estrogenic growth-inducing conditions. We found that BRCA1 antisense cells, both as pooled populations and individual subclones, also exhibited enhanced growth in monolayer culture on plastic in the presence of estrogen compared with control vector-infected colonies. All BRCA1 antisense subclones were able to proliferate as well as exhibit a decreased death rate in estrogen-deprived media, whereas parental and control subclones failed to grow. Death after estrogen withdrawal has been shown in previous studies using BG-1 cells [13,35]. BRCA1 antisense subclones demonstrated other traits associated with a tumorigenic phenotype, such as the ability to grow in soft agar independent of estrogen, whereas control clones could only form colonies with the addition of estrogen. In ovariectomized nude mice, a BRCA1 antisense clone (AS-4) was examined for tumorigenicity compared with a control clone (NEO). The AS-4 clone formed a greater number of and larger tumors than NEO in the absence of estrogen, and in general formed tumors faster in the presence of estrogen. The main conclusion from these studies is that BG-1 clones with reduced levels of BRCA1 protein have a survival advantage over controls in the absence of estrogen both in vitro and in vivo.\nThe response of BRCA1 mRNA and protein levels to mitogens and hormones in vitro suggests that BRCA1 may play a role in regulation of cell growth or maintenance [21]. During estrous, many hormones and growth factors interact in a complex manner as survival factors and inducers of cell proliferation, which are then balanced with growth inhibitors [36,37,38,39,40]. The mechanism by which BRCA1 can regulate or influence these processes has not yet been identified. It has been shown that BRCA1 is induced as a result of the mitogenic activity of the estrogen receptor in estrogen receptor-positive cells [9,10]. Direct estrogen stimulation is not required for BRCA1 transcription, however [9,41]. In support of this, BRCA1 expression has been shown to occur in the mouse ovary (granulosa and thecal cells of small and medium follicles) independent of hormonal status, and even in ovaries from estrogen receptor -/- deficient mice [41,42]. In contrast, the tumors from patients with BRCA1 mutations appear to have downregulation of estrogen receptors [43,44,45]. Previous experiments in our laboratory showed that another hormone, progesterone, could also cause a modest increase of BRCA1 mRNA in BG-1 cells after 24 h exposure without an increase in growth (unpublished data). Progesterone has been found [46] to inhibit cell proliferation and induce apoptosis significantly in two ovarian carcinoma cell lines. Thus, although BRCA1 may not be regulated directly by hormones, the BRCA1 gene product may be involved in the regulation of hormone response pathways, and the present results may demonstrate that loss of BRCA1 may result in loss of inhibitory control of these mitogenic pathways.\nBRCA1 transcription is regulated with the cell cycle, and highest levels correlate with the G1/S-phase boundary [5,9,41,47,48,49]. The present studies show that reduction of BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in vitro and in vivo, suggesting that BRCA1 may normally be acting as a growth inhibitor. Similar to our findings with ovarian carcinoma cells, accelerated growth, anchorage independence and tumorigenicity is associated with BRCA1 antisense introduction into mouse NIH3T3 cells [50]. In addition, increased proliferation of mammary cells is induced with antisense oligonucleotides to BRCA1 [51]. Conversely, introduction of full-length BRCA1 by retrovirus-mediated gene transfer inhibited growth of breast and ovarian cancer cell lines in both in vitro and in vivo experiments [51], and transfection of BRCA1 into colon cancer cells inhibited new DNA synthesis by 50% in addition to inhibition of S-phase progression, possibly through direct transactivation of the cell cycle inhibitor p21 WAF1/CIP1 [49]. \nIn multicellular mammalian organisms, a balance between cell proliferation and cell death is extremely important for the maintenance of normal healthy tissues. This is especially important during early embryonic development as well as in the development and function of adult tissues such as the gonadal cells (ie ovarian and testes) [41,48]. For example, BRCA1 expression is critical during development, as evidenced by the embryonic lethality in transgenic knockout mice [29,30,52]. Alternatively, overexpression of BRCA1 may activate apoptosis or cell death [14]. Human prostate cells with an introduced wild-type BRCA1 cDNA demonstrated a threefold to sixfold increase in chemosensitivity, as well as an increased susceptibility to drug-induced apoptosis [53]. We found that clones with even moderately reduced levels of BRCA1 protein appeared to be relatively resistant to death due to estrogen deprivation. Previous studies in our laboratory showed that response of parental BG-1 cells and antisense clones to gamma radiation were consistent with a p53 wildtype phenotype, indicating that loss of estrogen dependence is probably not due to a p53 mutation (unpublished data). Shao et al [14] demonstrated that BRCA1 transfected into mouse 3T3 fibroblasts resulted in increased programmed cell death. In support of this hypothesis, it has been shown that p53 and BRCA1 can form stable complexes, and can coactivate p21 and bax genes, which may lead to the activation of the apoptosis pathway [15]. The present data, which show that cells with a reduction in BRCA1 have a survival advantage in conditions where control cells fail to thrive, also supports this hypothesis.\nLike p53, BRCA1 has also been implicated in DNA damage and repair pathways [7,48,54]. According to this model, cells without normal BRCA1 activity may accumulate genetic alterations as a result of failure to arrest and repair DNA damage or self-destruct, thereby leading to genomic instability and neoplastic progression. It may not be coincidental that BRCA1-mutant breast cancers are preferentially linked to a 'specific' histopathologic pattern that includes a high S-phase fraction of cells, aneuploidy, and hormone receptor-negative status [45]. In addition, it has been demonstrated [20] that hormone-dependent tumors such as breast and ovarian cancers have a decreased ability to undergo apoptosis. Although mutations in this gene are uncommon in sporadic breast and ovarian tumors, BRCA1 expression levels and protein levels have been found to be reduced in sporadic human breast carcinomas [16,17,18,19]. Other mechanisms that involve gene regulation may allow for decreased expression of BRCA1 in sporadic tumors. Hypermethylation has been observed in some sporadic breast tumors in the promoter region of BRCA1, which may account for decreased BRCA1 transcription [55]. Low BRCA1 levels found in sporadic cancers may play an important role in tumorigenesis. The present data suggest that diminished levels of BRCA1 not only accelerate proliferation in the BG-1 ovarian carcinoma cell line, but appear to alter tumorigenesis. The exact mechanism may be unknown, but decreased BRCA1 levels appear to affect the ability to arrest growth or die in the absence of estrogenic growth-inducing conditions. We propose that the loss or reduction of BRCA1 may predispose a cell population to neoplastic transformation by altering the balance between cell death and proliferation/survival, rendering it more sensitive to secondary genetic changes."}