Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Comparative and Experimental Medicine

Major Professor

Jay Wimalasena

Committee Members

Roger Carroll, Hwa-Chain Robert Wang, Hildegard Schuller, Ana Kitazono


BAD is a Central Player in Cell Death and Cell Cycle Regulation in Breast Cancer Cells

The estrogen, 17-b estradiol (E2) can stimulate proliferation or induce differentiation or death depending on the cell context. In MCF 7 cells, an estrogen receptor (ER) positive human breast cancer cell line, E2 has been shown to stimulate cell proliferation and may have an anti-apoptotic effect. E2 rapidly activates mitogen activated protein kinases (MAPKs) in mammalian cells in an ER dependent fashion. The mechanisms underlying and the physiological significance of the signaling through MAPK remain to be determined. This signal pathway may represent a potential mechanism by which E2 regulates cell proliferation, growth as well as apoptosis. Recent observation of Raf-1 kinase (which relays signals from cell surface receptor tyrosine kinase to MAPKs) activation by E2 in MCF 7 cells explained a possible link between cell surface and intracellular components of the signaling pathway. PI-3 kinase and its down stream member protein kinase B (PKB/Akt) have been shown to be strongly involved in mitogenic and anti-apoptotic signaling in MCF 7 cells.

Chemical and biological inhibition of PI-3 kinase diminished E2 supported cell survival and concomitant Akt, MAPK (ERK) and its down stream target p90RSK activities. On the other hand, interference of endogenous Ras with a dominant negative construct abolished signal mechanisms involving ERK as well as Akt. These observations suggested that Ras/ERK and PI-3 Kinase pathways could operate as separate entities or cross talk and cooperate to enhance survival or impede apoptosis in MCF 7 cells in the presence of E2.

BCL2 family proteins play critical decision-making roles during apoptosis. BH3 domain only, pro-apoptotic member, BAD has been intensively investigated for its contribution in regulation of apoptosis at the mitochondrial level. Post-translational phosphorylation modulates BAD’s function and, most survival factors impose inhibitory phosphorylations on BAD via active Akt and MAPK pathways.

E2 rapidly induces Akt and ERK signaling pathways resulting in phosphorylation of BAD on two serine residues, and abolishes apoptosis in response to a variety of stimuli in MCF-7 cells. Therefore, we hypothesized that E2 targets BAD for its anti-apoptotic effect. Data presented clearly show that E2 effects are at least partly mediated via Ras originated ERK/p90RSK and PI-3 kinase/Akt pathways that add inhibitory phosphate groups onto corresponding serine 75 and 99 residues of BAD there by inactivating its pro-apoptotic tendencies. We demonstrate the significance of BAD and its functional serine residues in E2 mediated anti-apoptosis with studies conducted with anti-sense mRNA and phosphorylation site mutants of BAD. Over-expression of wild type or phosphorylation site mutant of BAD did not enhance the basal cell death, which was remarkably lessened by the inhibition of endogenous BAD. E2 could rescue cells from TNFa induced death in control cultures and in wild type BAD transfected cells but not in phosphorylation site mutant BAD expressing cells. This observation agrees with previous studies that demonstrate the importance of BAD’s phosphorylation sites in growth factor mediated cell protection. This study shows for the first time that E2 is an anti-apoptotic agent in breast cancer cells whose response is mediated by non-genomic cytoplasmic signal networks that converge on BAD in turn regulating the mitochondrial membrane potential change.

When transiently over-expressed, wild type or dual phosphorylation site mutant of BAD did not induce apoptosis, as observed in the first part of this study, but the rate of growth of these cells was remarkably low compared to the untransfected or control vector transfected cells. This suggested that the presence of extra amount of BAD could reduce the growth of MCF-7 cells. Similar phenomenon have been reported with the overexpression of BCL2 in malignant as well as normal epithelial cells. When over expressed, BCL2 arrests cell cycle in G0/G1 phase and this effect appears to depend on the amino terminal BH4 domain. Interestingly in a variety of epithelial cell types, endogenous BCL2 was shown to localize to the chromosomes of mitotic nuclei. This pattern of BCL2 expression is considered to be indicative of its special role in cell proliferation.

We hypothesized that BAD also exhibits a cell cycle related function in breast cancer epithelial cells, which was tested in the second part of this study. We evaluated the distribution pattern of nuclear BAD in several breast cancer cell lines. BAD overexpression lessened cyclin D1protein levels that agreeably correlate with the G1-S transition block observed. Cyclin D1 regulation by BAD was assayed at the transcriptional level using a luciferase reporter under the control of the cyclin D1 promoter. In asynchronous cells and in synchronous and E2 treated cells, BAD over-expression significantly reduced the cyclin D1 reporter activity. Overexpression of a dual phosphorylation site mutant of BAD did not alter the cyclin D1 expression pattern or the cell cycle progression suggesting an involvement of these serine residues in BAD’s nuclear functions. Furthermore, the BH3 domain deletion mutant and the S91A mutant, both of which carry intact S75 and S99 residues, exerted inhibitory effects similar to that of wild type BAD.

Wild type BAD overexpression as well as BAD depletion from nuclear extracts diminished the labeled AP-1/TRE DNA binding with the nuclear factors. This demonstrates that BAD is capable of binding and sequestering some components from the nuclear extracts which bind with AP-1/TRE element. Collectively our data suggest that in addition to pro-apoptotic functions, BAD regulates the cell cycle at least partly via AP-1/TRE element. These functions appear to depend on its S75 and S99 phosphorylation status that are also the targets of pro-survival mechanisms. Further, these results also suggest that the AP-1 transcription regulation by BAD is kept at its minimal level in normal cells as a result of the fine balance of its phosphorylation state.

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