Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins

Abstract

BRCA1 is a tumor suppressor gene that is mutated in families with breast and ovarian cancer. Several BRCA1 splice variants are found in different tissues, but their subcellular localization and functions are poorly understood at the moment. We previously described BRCA1 splice variant BRCA1a to induce apoptosis and function as a tumor suppressor of triple negative breast, ovarian and prostate cancers. In this study we have analyzed the function of BRCA1 isoforms (BRCA1a and BRCA1b) and compared them to the wild-type BRCA1 protein using several criteria like studying expression in normal and tumor cells by RNase protection assays, subcellular localization/fractionation by immunofluorescence microscopy and Western blot analysis, transcription regulation of biological relevant proteins and growth suppression in breast cancer cells. We are demonstrating for the first time that ectopically expressed GFP-tagged BRCA1, BRCA1a, and BRCA1b proteins are localized to the mitochondria, repress ELK-1 transcriptional activity and possess antiproliferative activity on breast cancer cells. These results suggest that the exon 9, 10, and 11 sequences (aa 263-1365) which contain two nuclear localization signals, p53, Rb, c-Myc, gamma-tubulin, Stat, Rad51, Rad50 binding domains, angiopoietin-1 repression domain are not absolutely required for mitochondrial localization and growth suppressor function of these proteins. Since mitochondrial dysfunction is a hallmark of cancer, we can speculate that the mitochondrial localization of BRCA1 proteins may be functionally significant in regulating both the mitochondrial DNA damage as well as apoptotic activity of BRCA1 proteins and mislocalization causes cancer. J. Cell. Physiol. 219: 634-641, 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1

Schematic representation of the BRCA1, BRCA1a, and BRCA1b isoforms (Not drawn to scale).

Figure 2

Western blot analysis of BRCA1a and BRCA1b expression in Cos-1 cells. Cos-1 cells were transfected with 12 μg of CMVFlag BRCA1a/1b and CMVFlag vector. The whole cell lysates (∼75 μg) were resolved on a 10 % SDS-PAGE. The blot was probed with an anti-FLAG antibody as described previously.

Figure 3

(a) Analysis of expression BRCA1, BRCA1a and BRCA1b transcripts by RNAase protection assay. RNAase protection assay was carried out on ∼ 30 micrograms of total RNA as described by us previously. Lane1, HBL100; Lane 2, Normal human mammary gland; Lane3, MCF7; Lane 4, CAL-51; Lane 5, SKOV3; Lane 6, NIHOVCAR3; Lane 7, Hela; Lane 8, HL60; Lane 9, A431; Lane 10, COLO320; Lane 11, MOLT-4; Lane 12, K562; Lane 13, COS cells transfected with vector CMV; Lane 14, COS cells transfected with BRCA1a expression plasmid. Protected 200bp (corresponding to BRCA1) and 118bp (corresponding to BRCA1a and BRCAlb) is shown by arrows (b) Represents actin control. (c) Schematic representation of antisense RNA probe used for RNAase protection assay and also the expected fragments from BRCA1, BRCA1a/1b transcripts are shown.

Figure 3

(a) Analysis of expression BRCA1, BRCA1a and BRCA1b transcripts by RNAase protection assay. RNAase protection assay was carried out on ∼ 30 micrograms of total RNA as described by us previously. Lane1, HBL100; Lane 2, Normal human mammary gland; Lane3, MCF7; Lane 4, CAL-51; Lane 5, SKOV3; Lane 6, NIHOVCAR3; Lane 7, Hela; Lane 8, HL60; Lane 9, A431; Lane 10, COLO320; Lane 11, MOLT-4; Lane 12, K562; Lane 13, COS cells transfected with vector CMV; Lane 14, COS cells transfected with BRCA1a expression plasmid. Protected 200bp (corresponding to BRCA1) and 118bp (corresponding to BRCA1a and BRCAlb) is shown by arrows (b) Represents actin control. (c) Schematic representation of antisense RNA probe used for RNAase protection assay and also the expected fragments from BRCA1, BRCA1a/1b transcripts are shown.

Figure 4

Effect of BRCA1a and BRCA1b on GAL-4-ELK-1 transcriptional activation. (a) MCF7 (b) HLR-ELK-1 cells were plated at a density of 6×105 cells per well in 6-well plates. Cells were cotransfected with 1 μg each of GAL-4-Elk-1, MEK, E1bLuc and pCMV βgal. Cells were also transfected with various concentrations of BRCA1a/BRCA1b, BRCA1a/BRCA1b Y1853 ter mutants and pcDNA3 vector using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. DNA was held constant at 5 μg using Gem3. Forty-eight hours after transfections, cells were harvested and assayed for luciferase activity using a luciferase assay system (Promega). Transfections were normalized for β-galactosidase activity. Values represent relative luciferase activity as percent of pcDNA3 vector control.

Figure 5

a Mitochondrial localization of BRCA1, BRCA1a, and BRCCA1b. The GFP tagged BRCA1, BRCA1a, or BRCA1b was transiently expressed in MCF-7. The tagged proteins were detected by immunostaining with anti-GFP antibody. MitoTracker staining and DAPI staining are shown in parallel. BRCA1, BRCA1a and BRCA1b proteins are shown to co-localize with the Mitotracker (red color) which specifically stains the mitochondria. (b) BRCA1 Localization and Expression in COS1 and HeLa Cells. Cells were transfected with pCDNA3-BRCA1 or pFLAG-BRCA1 using Fugene 6 (Roche). Mitochondria, cytoplasmic and nuclear extracts were prepared 48 hours after transfection using a mitochondrial isolation kit for cultured cells (Pierce). Extracts were also prepared from HeLa cells. BRCA1 was detected by Western blot analysis using BRCA1 (D-20; Santa Cruz) or BRCA1 (Ab-1; Calbiochem) Cytochrome c was detected using cytochrome-c (H-104; Santa Cruz). M: mitochondria; C: cytoplasm; N: nucleus. The column labeled Hela represents the endogenous BRCA1 isolated from Hela cells and the columns labeled pCDNA-BRCA1 and pFLAG-BRCA1 refers to the COS-1 transfected BRCA1 proteins. (c) BRCA1a and BRCA1b Localization and Expression in COS1 Cells. Cells were transfected with pEGFP-C1-BRCA1a/1b using Fugene 6 (Roche). Mitochondria, cytoplasmic and nuclear extracts were prepared 48 hours after transfection using a mitochondrial isolation kit for cultured cells (Pierce). BRCA1 was detected by Western blot analysis using Anti-GFP (Zymed) or BRCA1 (Ab-1; Calbiochem Cytochrome c was detected using cytochrome c (H-104; Santa Cruz). M: mitochondria; C: cytoplasm; N: nucleus.

Figure 5

a Mitochondrial localization of BRCA1, BRCA1a, and BRCCA1b. The GFP tagged BRCA1, BRCA1a, or BRCA1b was transiently expressed in MCF-7. The tagged proteins were detected by immunostaining with anti-GFP antibody. MitoTracker staining and DAPI staining are shown in parallel. BRCA1, BRCA1a and BRCA1b proteins are shown to co-localize with the Mitotracker (red color) which specifically stains the mitochondria. (b) BRCA1 Localization and Expression in COS1 and HeLa Cells. Cells were transfected with pCDNA3-BRCA1 or pFLAG-BRCA1 using Fugene 6 (Roche). Mitochondria, cytoplasmic and nuclear extracts were prepared 48 hours after transfection using a mitochondrial isolation kit for cultured cells (Pierce). Extracts were also prepared from HeLa cells. BRCA1 was detected by Western blot analysis using BRCA1 (D-20; Santa Cruz) or BRCA1 (Ab-1; Calbiochem) Cytochrome c was detected using cytochrome-c (H-104; Santa Cruz). M: mitochondria; C: cytoplasm; N: nucleus. The column labeled Hela represents the endogenous BRCA1 isolated from Hela cells and the columns labeled pCDNA-BRCA1 and pFLAG-BRCA1 refers to the COS-1 transfected BRCA1 proteins. (c) BRCA1a and BRCA1b Localization and Expression in COS1 Cells. Cells were transfected with pEGFP-C1-BRCA1a/1b using Fugene 6 (Roche). Mitochondria, cytoplasmic and nuclear extracts were prepared 48 hours after transfection using a mitochondrial isolation kit for cultured cells (Pierce). BRCA1 was detected by Western blot analysis using Anti-GFP (Zymed) or BRCA1 (Ab-1; Calbiochem Cytochrome c was detected using cytochrome c (H-104; Santa Cruz). M: mitochondria; C: cytoplasm; N: nucleus.

Figure 5

a Mitochondrial localization of BRCA1, BRCA1a, and BRCCA1b. The GFP tagged BRCA1, BRCA1a, or BRCA1b was transiently expressed in MCF-7. The tagged proteins were detected by immunostaining with anti-GFP antibody. MitoTracker staining and DAPI staining are shown in parallel. BRCA1, BRCA1a and BRCA1b proteins are shown to co-localize with the Mitotracker (red color) which specifically stains the mitochondria. (b) BRCA1 Localization and Expression in COS1 and HeLa Cells. Cells were transfected with pCDNA3-BRCA1 or pFLAG-BRCA1 using Fugene 6 (Roche). Mitochondria, cytoplasmic and nuclear extracts were prepared 48 hours after transfection using a mitochondrial isolation kit for cultured cells (Pierce). Extracts were also prepared from HeLa cells. BRCA1 was detected by Western blot analysis using BRCA1 (D-20; Santa Cruz) or BRCA1 (Ab-1; Calbiochem) Cytochrome c was detected using cytochrome-c (H-104; Santa Cruz). M: mitochondria; C: cytoplasm; N: nucleus. The column labeled Hela represents the endogenous BRCA1 isolated from Hela cells and the columns labeled pCDNA-BRCA1 and pFLAG-BRCA1 refers to the COS-1 transfected BRCA1 proteins. (c) BRCA1a and BRCA1b Localization and Expression in COS1 Cells. Cells were transfected with pEGFP-C1-BRCA1a/1b using Fugene 6 (Roche). Mitochondria, cytoplasmic and nuclear extracts were prepared 48 hours after transfection using a mitochondrial isolation kit for cultured cells (Pierce). BRCA1 was detected by Western blot analysis using Anti-GFP (Zymed) or BRCA1 (Ab-1; Calbiochem Cytochrome c was detected using cytochrome c (H-104; Santa Cruz). M: mitochondria; C: cytoplasm; N: nucleus.

Figure 6

Effect of BRCA1, BRCA1a and BRCA1b on the growth of MCF7 breast cancer cells. Cells were co-transfected with pcDNA3 or pcDNA BRCA1, BRCA1b, BRCA1a and selected with G418 and the resulting colonies were stained and counted. The number of colonies obtained by pcDNA3 clone was considered as 100%. Each experiment was repeated at least three times and the bars shown represent standard deviation.