BRCA1 Zinc RING Finger Domain Disruption Alters Caspase Response in Ovarian Surface Epithelial Cells

Background The frequently occurring 185delAG mutation occurs in the amino-terminal zinc RING domain of the breast and ovarian cancer susceptibility gene, BRCA1. We sought to determine differential cell viability and apoptotic response of human ovarian surface epithelial cells with and without the 185delAG mutation. Results BRCA1wt and BRCA1+ cells were treated with staurosporine. Cell proliferation assays showed BRCA1wt cells grew to a greater extent compared to BRCA1+ cells. Trypan blue exclusion assays confirmed this observation. Western immunoblot analysis revealed that caspase 3 levels were higher after staurosporine treatment in BRCA1+ cells than in wild type cells, while full length DNA Fragmentation Factor 45 levels were lower in BRCA1+ cells. While there was no significant difference in levels of excision repair cross complementing protein1 (ERCC1) with BRCA1 status, BRCA1+ cells demonstrated cleavage of polyribose ADP polymerase (PARP) before wild type cells. Conclusions Disruption of the BRCA1 RING domain caused altered cell viability and caspase-dependent apoptotic response after chemotoxic stress.


Background
The breast and ovarian cancer susceptibility gene, BRCA1, is located at 17q21, and encodes a 1863 amino acid protein. Mutations in this gene account for 60% of hereditary ovarian cancers [1]. Loss of heterozygosity in this gene occurs in 30-70% of sporadic ovarian carcinomas [2]. Species homology studies have shown that while the entire 22 exon gene is poorly conserved, the terminal ends maintain over an 80% homology between rat, human and mouse [3]. BRCA1 has long been known to function in DNA repair. Studies have shown BRCA1 is upregulated in cells treated by DNA damaging agents such as cisplatinum [4]. BRCA1 has been shown to interact with DNA repair proteins such as Rad50 and Rad51, the tumor suppressor genes RB and BRCA2, transcriptional factors (RNA pol II, histone deacetylase complex, ctIP) as well as influence numerous cyclins and cyclin dependent kinases contributing to cell cycle regulation [5][6][7][8][9][10][11][12]. More recently, BRCA1 has been shown to influence apoptosis in a p53 independent manner [13]. This apoptotic response involved the c-jun kinase (JNK) pathway, though the details of this mechanism remain unclear [14].
The highly acidic carboxy-terminal (BRCT) region of BRCA1 has been suggested to play a role in transactivation [11]. BRCT interacts with BRCA2, Rad51, other tumor suppressing elements, as well as numerous transcription factors, such as RNA helicase A and STAT1 [15,16]. Recently, it has been discovered that truncation of this region resulted in suppression of apoptosis following proapoptotic stimuli [17]. Further, these studies also suggested that the BRCT region facilitates apoptotic functions within the caspase pathway.
The amino terminal (BRNT) of BRCA1 contains a highly conserved zinc binding or RING finger domain also involved in multiple functions within the cell. Molecular modeling has shown that this domain contains two zinc finger-like motifs connected through linking C 3 HC 4 regions [18]. Naturally occurring splice variants of the gene suggest at least two transcription initiation points above and below the coding region for the RING domain [19]. Truncation studies have shown that the RING domain may function in direct protein binding of ER-α, ATF1, and BARD1, a ubiquitin ligase [20][21][22]. While zinc RING domains are common motifs in several protein families such as oncoproteins and regulatory proteins, the actual function of the domain differs among these proteins. For example, inhibitors of apoptosis proteins, (IAPs), contain one to three tandem baculovirus inverted repeat (BIR) domains as well as a carboxy terminal RING domain. Previous studies have shown this RING domain essential in the anti-apoptotic function of some IAPs [23].
The most common mode a cell uses to undergo apoptosis is the cysteine-aspartate specific protease (caspase) pathway. This proteolytic cascade may be triggered by a wide variety of stimuli and employs numerous initiation routes within the cell. While there is extensive crosstalk between the caspases, the two most common initiator pathways are the Fas/Fas ligand pathway, involving caspase 8 and caspase 10, and the mitochondrial pathway, triggering caspase 9 [24,25]. Caspase 3, a pivotal downstream protease, functions in virtually every caspase pathway and serves as an executioner in the cells by cleavage of downstream targets which lead to irreversible chromosomal degradation. Perhaps the most prominent caspase 3 substrate is DNA Fragmentation Factor 45 (DFF45), an inhibitor of caspase-activated DNase [26]. Following caspase 3mediated cleavage, DFF45 releases DFF40, the DNase responsible for DNA fragmentation into the characteristic apoptotic DNA ladder. Caspase 3 also deactivates vital DNA repair enzymes such as poly ribose ADP polymerase (PARP) [27]. Cleavage of PARP has been regarded as a hallmark of caspase-dependent apoptosis [28].
No study to date has explored the possible involvement of the BRCA1 amino-terminal RING domain in caspase-me-diated apoptosis. Therefore, ovarian surface epithelial cell lines with and without the 185delAG BRCA1 mutation were used to ascertain whether the RING domain of the amino-terminal affected apoptosis. This mutation, common among families with hereditary ovarian cancer, is a frameshift mutation occurring at the beginning of the C 3 HC 4 region of exon 2 that essentially interrupts RING domain function [19,29,30]. The results showed that disruption of the BRCA1 amino-terminal RING domain altered caspase 3 activation and subsequent DFF45 and PARP cleavage, resulting in accelerated STS-induced apoptosis.

Results
Loss of BRCA1 expression resulted in increased cell death when exposed to 1 µM staurosporine treatment SV-40 large T antigen transfected ovarian surface epithelial cell lines from women with and without an amino-terminal BRCA1 mutation were employed to ascertain the function of the amino-terminal RING domain in apoptosis [31]. To confirm BRCA1 status in these cell lines, whole cell lysates were western blotted using a monoclonal anti-BRCA1 antibody against the amino-terminal ( Figure 1). Using the MCF7 breast carcinoma cell line as a positive control, MCC5 cells expressed the full length 216 kDa BRCA1 protein and were confirmed BRCA1wt. In contrast, the HIO3261-77 cells were found to have significantly reduced levels of full length BRCA1 than the wild type cell line, confirming the mutated amino-terminal BRCA1 (BRCA1+) in these cells. Due to the high molecular weight of BRCA1, actin could not be used as a loading control. Thus, the membranes were stained with 7% Amido black with the protein front used as a loading control.
Having confirmed the BRCA1 status of these cell lines, cell viability was then assayed under cytotoxic stress. Cells were treated with 1 µM STS for 3 h and subjected to MTS    p<.001 C assay at 0, 24, and 48 h (Figure 2A). Results were reported as percent growth of respective untreated cells allowed to grow in serum containing media. BRCA1wt cells grew 17% greater at 24 h and 8% greater at 48 h than BRCA1+ cells. This difference proved to be statistically significant (p < .03 and p < .01, respectively). BRCA1wt cells appeared to recover at 72 h while BRCA1+ cells continued to decline in growth (data not shown).
To confirm that the difference in cell viability was due to alterations in survival response after STS treatment and not an intrinsic property of the individual cell lines, growth of both cell lines was examined by MTS assay under the same conditions in the absence of STS ( Figure 2B). Linear regression analysis of cell growth revealed that the slopes of the BRCA1wt cells, (0.12) and that of the BRCA1+ cells, (0.14) were essentially the same.
To ensure the survival difference after STS treatment seen between the BRCA1wt and BRCA1+ cells was associated with cell death, a trypan blue exclusion assay was conducted ( Figure 2C). Cells were plated in triplicate and both adherent and suspended cell populations were assayed with results reported as percent of total population dead. No appreciable difference was observed in the amount of death between the cell lines at 0, 1, or 3 h (1%, 3.5%, and 2.5%, respectively). There was, however, a significantly higher percentage of dead cells in the BRCA1+ cell line at 24 h (12.8%; p < .027) and 48 h (24.6%; p < .001).

Amino-terminal BRCA1 mutation was associated with elevated caspase 3 activation following STS treatment
To investigate the role of amino-terminal of BRCA1 in apoptosis, the effect of STS was examined on elements of the caspase pathway ( Figure 3). First, to determine whether a mutation in amino-terminal RING domain of BRCA1 preferentially targeted either the mitochondrial [25] or Fas/Fas ligand apoptotic pathway [24], levels of the respective initiator caspases 9 and 8 were determined. Both cell lines produced activated caspases 8 and 9 by 1 h after treatment with equivalent levels at 3 h.
Next, levels of the executioner caspase 3 were examined. Once more, both cell lines produced activated caspase 3 by 1 h after treatment. However, BRCA1+ cells showed significantly more active caspase 3 by 3 h after treatment than the wild type. To quantify the difference in caspase activation, the immunoblots were scanned and analyzed via ImagQuant densitometry (Table 1). Densitometric analysis revealed that although BRCA1+ cells initially had lower levels of caspase 3, after 3 h STS treatment, caspase 3 levels were 72% higher. Levels of STS-induced caspase 7, a structural and functional homolog of caspase 3 [32] were also evaluated ( Figure 3). Procaspase 7 began to be cleaved at 1 h of treatment and was completely processed by 3 h of treatment in both BRCA1 wt and BRCA1+ cells. Although caspase 7 plays a subsidiary role in DNA fragmentation and apoptosis morphology [33], densitometric analysis illustrated that BRCA1+ cells contained substantially reduced levels of procaspase 7 in untreated cells (39% of that seen in BRCA1wt), and during initial STS treatment (31% of that seen in BRCA1wt at 0.5 h).
To determine whether elevated levels of cleaved caspase 3 resulted in increased cleavage of caspase 3 substrates, DFF45 cleavage was studied (Figure 3). Degradation of full length DFF45 was used to indicate caspase 3 activity. In both cell lines, DFF45 began to significantly degrade by 1 h after treatment. In BRCA1wt cells, the levels of full length DFF45 were 95% of control at 0.5 h, 40% of control at 1 h, and 22% of control at 1.5 h. In contrast, in BRCA1+ cells, full length DFF45 was only 71% of control at 0.5 h, 16% of control at 1 h, and 10% of control at 1.5 h (Table 1).

Amino-terminal BRCA1 mutation caused increased degradation of caspase-linked DNA repair proteins
To ascertain whether increased caspase 3 activity in BRCA1+ cells could also affect DNA repair pathways, the DNA repair enzymes PARP, a known substrate of caspase  3, [27] and ERCC1, a repair protein not dependent on caspase 3 [35] were examined ( Figure 4). Interestingly, cleavage and inactivation of PARP was noted only at 3 h after STS treatment in BRCA1wt cells. In contrast, accelerated cleavage and inactivation of PARP was seen in BRCA1+ cells as early as 1 h after STS treatment. Levels of ERCC1 were not significantly different between BRCA1wt and BRCA1+ cells. With BRCA1's extensive connection to DNA repair already established [35], we also examined whether an amino-terminal BRCA1 mutation reduced cellular survival by enhanced caspase 3-dependent cleavage and subsequent inactivation of the caspase 3-dependent DNA repair enzyme PARP. As with DFF45, PARP cleavage was significantly enhanced in BRCA1+ cells suggesting that decreased DNA repair capacity in BRCA1+ cells may, in part, be due to premature inactivation of PARP. This pattern was not seen with ERCC1 and may be due to the choice of apoptotic stimulus used. While the exact mechanism remains unclear, STS has been shown to initiate caspase driven apoptosis in a manner distinct from chemotherapeutic agents such as cisplatinum, etoposide, and tamoxifen, which directly cause DNA damage and tend to favor ERCC1 activation [37,38].

Discussion
Previous studies have shown that truncation of the highly acidic carboxy-region BRCT resulted in resistance to caspase induced apoptosis [17]. Further, this failure of apoptosis was traced specifically to caspase 8 and the Fas/FasL pathway. In contrast, our data showed that the 185delAG mutation, affecting the amino-terminal domain of BRCA1, conferred an increased apoptotic response with no caspase pathway preferentially selected. Instead, this amino-terminal mutation favored elevated caspase 3 levels that subsequently facilitated enhanced apoptosis by inactivating the caspase-dependent DNA repair proteins PARP and by inactivating DFF45, an inhibitor of the caspase-dependent DNase, DFF40. The nature of the 185delAG frameshift makes it difficult to determine if the apoptotic alterations seen are directly caused by the mutation or by downstream effects of it and certainly deserves further scrutiny. In this way, carboxy-and amino-terminal BRCA1 mutations may target two distinct anti-apoptotic pathways. Therefore, disruptions in either or both BRCA1 terminals effect apoptotic response. In summary, our findings suggest a possible novel mechanism by which the amino-terminal of BRCA1 suppresses apoptosis and facilitates DNA repair in human ovarian surface epithelial cells.

Conclusions
The 185delAG mutation in the BRCA1 gene disrupts the zinc linker region of the amino-terminal RING domain. Disruption of this domain triggered an elevated caspase 3dependent apoptotic response and affected downstream proteins such as DFF45 and PARP.

Induction of Apoptosis and Cell Viability Assessment
Cells were grown in 100 mm tissue culture disks until confluent. Cultures were treated with 1 µM staurosporine (STS) (Sigma) in serum containing medium until collected. Control samples were rinsed in DPBS, drained, and fresh medium was added.
Cell growth was determined by the MTS (Promega, Madison, WI) colorimetric assay following STS treatment. The assay was performed in 96 well microtiter places according to manufacturer's instructions and is based on soluble formazan production by dehydrogenase enzymes found in metabolically active cells. Samples were seeded in six wells per time point at 2.5 × 10 3 cells per well. Absorbance was determined at 490 nm using a Dynex MRX plate reader (Dynex Technologies, Chantilly, VA) and the results expressed as the mean absorbance of triplicate experiments ± SE.

SDS-PAGE and Western Blot Analysis
In order to observe changes at the onset of apoptosis, only adherent cell populations were trypsinized, pelleted 5 minutes at 500 × g, and lysed in ice cold lysis buffer (10 mM Tris-HCl (pH 7. Polyclonal antibodies to caspase 3, caspase 9, and PARP were purchased from Cell Signaling (Beverly, MA), a monoclonal antibody to caspase 7 and a polyclonal antibody to DFF45 were obtained from BD Transduction (San Diego, CA). Polyclonal anti-caspase 9, and monoclonal actin antibodies were purchased from Alexis (San Diego, CA) and Sigma, respectively. Membranes were incubated and developed according to the Enhanced Chemiluminescent Protocol, according to manufacturer's instructions (Amersham). After initial blotting, membranes were reprobed for actin to ensure even loading.
BRCA1 status of the cell lines used in this study was confirmed via western immunoblotting. Cells were mixed with equal volume of 2X loading buffer, vortexed and boiled for 5 minutes. One hundred thousand cells were separated by 5% SDS-PAGE, transferred by wet-transfer to PVDF membrane, and blotted as described above using monoclonal antibody specific for the N-terminus of BRCA1 (Oncogene Boston, MA). After blotting, the PVDF membrane was stained with 2% amido black in 7% gla-

Figure 4
Effect of amino-terminal BRCA1 mutation on DNA repair protein expression. BRCA1wt and BRCA1+ cells were treated and lysed as in Figure 3 and probed for cleaved PARP (86 kDa) and ERCC1 (36 kDa). Membranes were then probed for actin for loading consistency. Images are representative of at least 3 independent treatments blotted twice.
C .5 1 1.5 3 C . 5  cial acetic acid and the protein fronts of all lanes were compared for loading accuracy.

Statistical Analysis
Samples for MTS and trypan blue exclusion assays were performed in triplicate and the data subjected to the Student's paired t-test analysis for determination of statistical significance between BRCA1+ and BRCA1wt samples. Two-tailed results are reported as P values within the corresponding figures.