Mechanism of induction of transforming growth factor-beta type II receptor gene expression by v-Src in murine myeloid cells.

Transforming growth factor (TGF)-beta1 plays an important role during hematopoiesis. Previously, we had shown that the growth of a v-Src-transformed myeloid cell line was markedly more inhibited by TGF-beta treatment when compared with the wild-type myeloid cell line. To investigate the increased growth sensitivity of the v-Src-transformed myeloid cell line, 32D-src, to TGF-beta, we examined expression of the TGF-beta type II receptor (TGF-beta RII) gene in myeloid cell lines. Northem blot analysis showed that expression of approximately 8- and 6-kb species of TGF-beta RII transcripts was markedly increased in the 32D-src cell line. The expression of the TGF-beta RII promoter linked to a reporter gene was increased 23-fold by v-Src. DNA transfection and electrophoretic mobility shift assay revealed that v-Src induces TGF-beta RII promoter activity through an AP1/ATF2-like sequence (-219 to -172), ETS binding sites (+1 to +36), and the inverted CCAAT box (-81 to -77). Novel DNA-protein complexes with ETS binding sites are significantly increased in v-src-transformed cell lines compared with the control cell line. These results suggest that v-Src induces activity of the TGF-beta RII promoter through multiple elements by inducing expression of nuclear proteins interacting with these elements.

The p120-v-Abl protein interacts with E2F-1 and regulates E2F-1 transcriptional activity.

The E2F family of transcription factors regulates cell cycle progression, and deregulated expression of E2F-1 can lead to neoplastic transformation. In myeloid cells, introduction and expression of the Abelson leukemia virus causes growth factor independence. Here, the p120 v-Abl protein activates E2F-1-mediated transcription through a physical interaction with the E2F-1 transcription factor. BCR-Abl and c-Abl also stimulate E2F-1-mediated transcription. Our results suggest a new mechanism by which v-Abl leads to factor-independent myeloid cell proliferation: the activation of E2F-1-mediated transcription.

Vitamin E succinate induces Fas-mediated apoptosis in estrogen receptor-negative human breast cancer cells.

Vitamin E succinate (VES), a derivative of the fat-soluble vitamin D-alpha-tocopherol (vitamin E), inhibited growth and induced apoptotic cell death of estrogen receptor-negative human breast cancer cells. VES-induced apoptosis in MDA-MB-231 and SKBR-3 cells occurred through a Fas pathway. Total protein levels of the Fas receptor (Fas; APO-1/CD-95) and the Fas ligand (Fas-L) were increased following VES treatment. In addition, VES increased cell surface Fas expression. Fas-neutralizing antibodies and Fas-L antisense oligonucleotides blocked VES-induced apoptosis. The presence of Fas-L antisense oligonucleotides also completely blocked the VES-mediated increase in Fas-L protein expression. These data indicate a role for Fas signaling in VES-mediated apoptotic cell death of human breast cancer cells. These findings also suggest that VES may be of clinical use in the treatment of aggressive human breast cancers, particularly those that are refractory to antiestrogen therapy.

p120-v-Abl expression overcomes TGF-beta1 negative regulation of c-myc transcription but not cell growth.

Transformation of interleukin-3 dependent (IL-3) 32D-123 myeloid cells by p120-v-Abl produced the factor-independent 32D-abl cell line. In 32D-abl cells, myc expression was found to be significantly higher than in the parental cells and was correlated with increased E2F-1 protein expression and DNA binding ability. Surprisingly, in 32D-abl cells, TGF-beta1, a potent G1/S inhibitor of 32D-123 and 32D-abl cell growth, increased E2F transactivation as shown by increased c-myc promoter-CAT and GAL4-E2F-1 activity. In addition, TGF-beta1 was also found to increase E2F-1 protein levels but had no effect on steady-state retinoblastoma (RB) protein levels or phosphorylation state. In the absence of TGF-beta1, transient expression of RB in v-Abl expressing cells resulted in decreased c-myc transcription, inhibition of GAL4-E2F-1 driven transactivation and inhibition of cellular proliferation. RB and v-Abl were found to physically associate in vivo and in vitro via v-Abl's ATP binding region. In summary, these studies established that in myeloid cells: (1) v-Abl binds RB resulting in increased E2F-1-driven c-myc transcription, and (2) an alternative pathway exists for TGF-beta1-mediated growth inhibition of v-Abl-transformed cells, in which increased rather than decreased E2F-mediated c-myc transcription is observed.

Nuclear localization of v-Abl leads to complex formation with cyclic AMP response element (CRE)-binding protein and transactivation through CRE motifs.

Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies identify the localization of the oncogenic form of the abl gene product encoded by the Abelson murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with the transcriptional regulator cyclic AMP response element-binding protein (CREB). We have mapped the specific domains within each of the proteins responsible for this interaction. We have shown that complex formation is a prerequisite for transcriptional potentiation of CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the activation of promoters containing an intact CRE. These observations identify a novel function for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor.