Elk-1, an ets related gene codes for at least two splice variants Elk-1, which regulates c-fos transcription and DElk-1, both of which function as transcriptional activators. To investigate the role of Elk-1 and DElk-1 proteins in apoptosis; we have developed rat ®broblast cell lines and human breast cancer cell lines expressing Elk-1 and DElk-1. The expression of Elk-1 and DElk-1 proteins in the Elk-1/DElk-1 transfectants were analysed by immuno¯uorescence, immunohistochemistry, and Western blot analysis. The Elk-1 unlike DElk-1 transfectants showed a shortened and¯attened morphology compared to the parental cells. We have found that calcium ionophore treatment of Rat-1 Elk-1, MCF-7 Elk-1, Rat-1 DElk-1 and MCF-7 DElk-1 transfectants resulted in programmed cell death. These results indicate that constitutive expression of Elk-1 and DElk-1 proteins triggers apoptosis in Rat-1 ®broblasts and breast cancer cells when treated with calcium ionophore.Keywords: Elk-1; DElk-1; apoptosis; calcium ionophore; breast cancer; MCF-7; Rat-1The Elk-1 gene belongs to the ets family of ternary complex factors (TCFs), i.e. Elk-1, SAP1, and NET/ ERP/SAP2/Elk-3 (Rao et al., 1989;Hipskind et al., 1991;Giovane et al., 1994;Lopez et al., 1994;Dalton and Treisman, 1992;Price et al., 1995;Nozaki et al., 1996). The Elk-1 gene codes for at least two alternately spliced products Elk-1 (Rao et al., 1989) and DElk-1 (Rao and Reddy, 1993) which function as transcriptional activators (Rao and Reddy, 1992;Bhattacharya et al., 1993), are substrates for MAP kinases Marias et al., 1993;Hill et al., 1993) and JNK protein kinases (Gupta et al., 1996;Whitmarsh et al., 1995). As mentioned earlier, the Elk-1 protein is a TCF which in association with serum response factor (SRF) forms a ternary complex on the serum response element (SRE) of the c-fos promoter and regulates cfos transcription (Hipskind et al., 1991). The TCF's which includes Elk-1 have three domains with similar sequences and functions. The ets domain mediates DNA binding, the SRF interaction domain interacts with SRF to form a ternary complex with the c-fos SRE and the C-terminal domain activates transcription upon phosphorylation by MAP kinases (Rao et al., 1989;Rao and Reddy, 1992;Dalton and Treisman, 1992;Janknecht et al., 1993Janknecht et al., , 1994Marias et al., 1993;Giovane et al., 1994;Hipskind et al., 1994;Kortenjann et al., 1994;Lopez et al., 1994;Hill et al., 1995;Price et al., 1995;Whitmarsh et al., 1995) and JNK kinases (Gupta et al., 1996). Thus Elk-1 represents a key link between signal transduction and induction of gene transcription.The Gag-Myb-Ets fusion protein, identi®ed in the avian acute leukemia virus E26 was shown to inhibit apoptosis and induce erythroid di erentiation in hematopoietic cells (Athanasiou et al., 1996). Similarly the Ets-1 proto-oncogene was shown to be required for the normal survival and activation of B and T cells while an Ets-1 splice variant was shown to induce apoptosis in human colon cancer cells indicating a role in apoptosis (Bories et al....

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear–cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.

Elk-1, a c-Fos protooncogene regulator, which belongs to the ETS-domain family of transcriptional factors, plays an important role in the induction of immediate early gene expression in response to a variety of extracellular signals. In this study, we demonstrate for the ®rst time the in vitro and in vivo interaction of Elk-1 with BRCA1 splice variants BRCA1a and BRCA1b using GST-pull down assays, co-imunoprecipitations/Western blot analysis of cell extracts from breast cancer cells and mammalian two-hybrid assays. We have localized the BRCA1 interaction domain of Elk-1 protein to the conserved ETS domain, a motif involved in DNA binding and protein ± protein interactions. We also observed binding of BRCA1 proteins to other ETS-domain transcription factors SAP1, ETS-1, ERG-2 and Fli-1 but not to Elk-1 splice variant DElk-1 and c-Fos protooncogene. Both BRCA1a and BRCA1b splice variants function as growth suppressors of human breast cancer cells. Interestingly, our studies reveal that although both Elk-1 and SAP-1 are highly homologous members of a subfamily of ETS domain proteins called ternary complex factors, it is only Elk-1 but not SAP-1 that can augment the growth suppressive function of BRCA1a/1b proteins in breast cancer cells. Thus Elk-1 could be a potential downstream target of BRCA1 in its growth control pathway. Furthermore, we have observed inhibition of c-Fos promoter activity in BRCA1a transfected stable breast cancer cells and over expression of BRCA1a/1b attenuates MEK-induced SRE activation in vivo. These results demonstrate for the ®rst time a link between the growth suppressive function of BRCA1a/1b proteins and signal transduction pathway involving Elk-1 protein. All these results taken together suggest that one of the mechanisms by which BRCA1a/1b proteins function as growth/tumor suppressors is through inhibition of the expression of Elk-1 target genes like c-Fos.

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