BAD Overexpression Research Articles

Mitochondrial apoptotic priming is a key determinant of cell fate upon p53 restoration

Reactivation of p53 in established tumors typically results in one of two cell fates, cell cycle arrest or apoptosis, but it remains unclear how this cell fate is determined. We hypothesized that high mitochondrial priming prior to p53 reactivation would lead to apoptosis, while low priming would lead to survival and cell cycle arrest. Using a panel of Kras-driven, p53 restorable cell lines derived from genetically engineered mouse models of lung adenocarcinoma and sarcoma (both of which undergo cell cycle arrest upon p53 restoration), as well as lymphoma (which instead undergo apoptosis), we show that the level of mitochondrial apoptotic priming is a critical determinant of p53 reactivation outcome. Cells with high initial priming (e.g., lymphomas) lacked sufficient reserve antiapoptotic capacity and underwent apoptosis after p53 restoration. Forced BCL-2 or BCL-XL expression reduced priming and resulted in survival and cell cycle arrest. Cells with low initial priming (e.g., lung adenocarcinoma and sarcoma) survived and proceeded to arrest in the cell cycle. When primed by inhibition of their antiapoptotic proteins using genetic (BCL-2 or BCL-XL deletion or BAD overexpression) or pharmacologic (navitoclax) means, apoptosis resulted upon p53 restoration in vitro and in vivo. These data demonstrate that mitochondrial apoptotic priming is a key determining factor of cell fate upon p53 activation. Moreover, it is possible to enforce apoptotic cell fate following p53 activation in less primed cells using p53-independent drugs that increase apoptotic priming, including BH3 mimetic drugs.

Survivin Regulates Bad Gene Expression by Binding to Its Promoter and Modulates Cell Cycle and Apoptosis in Esophageal Carcinoma Cell.

Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an antiapoptotic and a proapoptotic protein, respectively, survivin and Bad play an important role in carcinogenesis of the most human cancers including EC. However, the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression, and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. A Bad overexpression experiment was conducted to confirm the biological effect on knockdown of survivin via modulating Bad expression. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues (88%, 35/40) was remarkably high, compared with the distal nontumor tissues (48%, 19/40, p < 0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal nontumor tissues (95%, 38/40, p < 0.01). Overexpression of survivin decreases Bad mRNA and protein expression and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression and induces cell cycle arrest and apoptosis. Bad overexpression inducing apoptosis of esophageal carcinoma cell shows the similar apoptotic effect with survivin knockdown. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.

Combination treatment of TRAIL, DFMO and radiation for malignant glioma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancer. Another promising cancer therapy is difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which is oraly administered and well tolerated. Nevertheless, many types of cancer, including gliomas, have exhibited resistance to TRAIL-induced apoptosis and similarly the potency of DFMO should be enhanced to optimize therapeutic efficacy. In this study we sought to determine whether DFMO, in combination with TRAIL and radiation, could result in an enhanced anti-glioma effect in vitro. We investigated the effect of DFMO, TRAIL and radiation in various combinations on a panel of glioblastoma cell lines (A172, T98G, D54, U251MG). Viability and proliferation of the cells were examined with trypan blue exclusion assay, crystal violet and xCELLigence system. Apoptosis (Annexin-PI), cell cycle and activation of caspase-8 were tested with flow cytometry. BAD protein levels were determined by Western blot analysis. DFMO induced BAD overexpression. Combination treatment with DFMO, TRAIL and radiation significantly reduced cell viability in all cell lines tested. Increased induction of cell death and cell cycle arrest was confirmed with flow cytometry in A172 and D54 cell lines, while enhanced activation of annexin and caspase-8 was revealed in U251MG and T98G cells. The treatment of glioblastoma cell lines with combination of DFMO, TRAIL and radiation showed an enhanced effect. This combination treatment may represent a novel strategy for targeting glioblastoma.

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

Specific epiblast loss and hypoblast impairment in cattle embryos sensitized to survival signalling by ubiquitous overexpression of the proapoptotic gene BAD.

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.

BAD overexpression inhibits cell growth and induces apoptosis via mitochondrial-dependent pathway in non-small cell lung cancer

BackgroundThe pro-apoptotic Bcl-2 protein BAD initiated apoptosis in human cells and has been identified as a prognostic marker in non-small cell lung cancer (NSCLC). In this study, we aimed to explore the functions of BAD in NSCLC.MethodsOverexpression of BAD was performed by transfecting different NSCLC cell lines with wild-type BAD. Cell proliferation, cell cycle, apoptosis, and invasion were characterized in vitro. Tumorigenicity was analyzed in vivo. Western blot was performed to determine the effects of BAD overexpression on the Bcl-2 family proteins and apoptosis-related proteins.ResultsOverexpression of BAD significantly inhibited cell proliferation in H1299, H292, and SPC-A1 but not in SK-MES-1 and H460 cell lines in vitro. BAD overexpression also reduced the tumorigenicity of H1299/SPC-A1 cell in vivo. However, no appreciable effects on cell cycle distribution and invasion were observed in all these cell lines. BAD overexpression also induced apoptosis in all cell types, in which process expression of mitochondrial cytochrom c (cyto-c) and caspase 3 were increased, whereas Bcl-xl, Bcl-2, Bax and caspase 8 expressions did not changed. These findings indicated that a mitochondrial pathway, in which process cyto-c was released from mitochondrial to activate caspase 3, was involved in BAD overexpression-mediated apoptosis.ConclusionsOur data suggested that increased expression of BAD enhance apoptosis and has negative influence on cell proliferation and tumor growth in NSCLC. Bad is a new potential target for tumor interventions.

Abstract 3762: Second generation PIM inhibitors exhibit improved activity in solid tumor models

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) includes constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphomas, leukemias, multiple myeloma, prostate and bladder cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, and promoting tumor cell survival and proliferation. In the HEK-293T cell line, enhanced PIM kinase substrate BAD phosphorylation (pBAD) was observed following PIM and BAD overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, and more effectively by second generation PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-µM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the UM-UC-3 human bladder cancer cell line. PIM1 and PIM2 overexpression models were established in the human prostate cancer cell line 22RV-1 and the non-tumorigenic mouse NIH-3T3 cell background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In vivo xenograft studies using both PIM overexpression models and a clinically relevant solid tumor model facilitated identification of a lead candidate with demonstrated efficacy and favorable toxicity. IND-enabling studies with a lead candidate are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3762. doi:1538-7445.AM2012-3762

The Bcl-2 Homology Domain 3 (BH3) Mimetic ABT-737 Reveals the Dynamic Regulation of Bad, a Proapoptotic Protein of the Bcl-2 Family, by Bcl-xL

The proteins of the B-cell lymphoma 2 (Bcl-2) family are important regulators of apoptosis under normal and pathological conditions. Chemical compounds that block the antiapoptotic proteins of this family have been introduced, such as 4-[4-[(4'-Chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide (ABT-737), a BH3-mimetic that neutralizes Bcl-2 and Bcl-xL. In this study, we used ABT-737 to explore the dynamic regulation of Bcl-2 proteins in living cells of different origins. Using ABT-737 as well as RNA interference or the application of growth factors, we examined the impact of the functional availability of the antiapoptotic proteins Bcl-2 and Bcl-2-extra large (Bcl-xL) on the Bcl-2 network. We report that ABT-737 increases the expression of Bcl-2-associated death promoter (Bad), a proapoptotic partner of the proteins Bcl-2 and Bcl-xL. Our observations indicate that Bad overexpression induced by ABT-737 results from the control of its normally rapid protein turnover, leading to the stabilization of this protein. We demonstrate the relevance of Bad post-translational regulation by Bcl-xL to the physiological setting using RNA interference against Bcl-xL as well as the application of epidermal growth factor, a growth factor that promotes the dissociation of Bad from Bcl-xL. Our results highlight a new facet of the mode of action of the antiapoptotic proteins Bcl-2 and Bcl-xL consisting of the regulation of the stability of the protein Bad. Finally, our results shed light on the mode of action of ABT-737, currently the best characterized inhibitor of the antiapoptotic proteins of the Bcl-2 family, and bear important implications regarding its use as an anticancer drug.

Abstract 1046: BAD is a multifunctional protein in breast cancer cells

Abstract BAD is a multifunctional protein in breast cancer cells R. Fernando1, M. Cekanova2, J. Woraratphoka2, M. Sukhthankar2, J. Bahn2, S. Dharmawardhana3, N. Siriwardana2, N. Moustaid-Moussa2, A. Strom4, S. Baek2, Q. Wong5, M. Zou5, R. Donnell2, J. Wimalasena2 1NCI, Bethesda, MD; 2University of Tennessee, Knoxville, TN; 3University of Puerto Rico Medical Sciences, San Juan, PR; 4University of Houston, Houston, TX; 5University of Oklahoma Medical Center, Oklahoma City, OK. BAD is a typical BH3 protein and, like many other BCL-2 family proteins, regulates apoptosis. However, several recent studies suggest that BAD has non-apoptotic roles. Previously, we had shown that BAD inhibits the MCF7 cell cycle via abrogation of cyclin D1 synthesis (JBC 282, 28864, 2007). Using tetracycline-regulated stable BAD overexpression in MCF7 cells, we now report that BAD regulates a wide variety of functions: since BAD binds to c-Jun (JBC, 2007), we investigated the ability of BAD to regulate the AP1 reporter activity. BAD inhibited MEK and activated RAS (V12, S35) regulated AP1 activity, but not the increase due to AKT. BAD overexpression blocked ERK activation by MEK1, but not by active AKT. Wildtype BAD inhibited estrogen- or serum-induced activation of c-Jun, JNK, and ERK, but not p38 MAPK. However, the S112/S136 double mutant BAD had no inhibitory effects, suggesting a requirement for phosphorylation. We then explored if BAD can regulate signaling pathways and found that it can inhibit beta catenin mRNA and protein expression. Several signaling proteins, including total AKT, active GSK 3 beta, and apoptosis-related soluble FasL, TNF alpha, TRAIL, EGFR, and RBM5, which may regulate FasL were significantly (p&amp;lt;0.01) regulated by BAD. BAD decreased invasion of MCF7 cells through Matrigel (p&amp;lt;0.05) and decreased cellular VEGF (p50% (p&amp;lt;0.01). Surprisingly, BAD decreased protein expression of PPAR gamma, active AMPK, LKB1, and active acetylCoA carboxylase (all at p&amp;lt;0.01). We analyzed the expression of several markers in breast cancer specimens: phospho (p)-BAD, BAD and cyclin D1 using tissue microarrays containing normal and neoplastic tissue. In addition, we checked the expression levels of ERK-1, phospho (p)-ERK1, AKT, and phospho-AKT in surgical pathology blocks with normal and neoplastic breast epithelial cells. As expected, cytoplasmic p-BAD expression was significantly and positively correlated with nuclear BAD, nuclear p-BAD, as well as nuclear cyclin D1. Our data showed that cytoplasmic cyclin D1 was positively correlated with cytoplasmic ERK-1, and cytoplasmic cyclin D1 was negatively correlated with nuclear AKT expression. Nuclear BAD and AKT were negatively correlated with nuclear cyclin D1 and nuclear ER-beta. Cytoplasmic expression of p-ERK1 was negatively correlated with nuclear AKT and positively correlated with nuclear cyclin D1, and nuclear ER-beta. Taken together, these results suggest that BAD may be a multifunctional protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1046.

Hepatocyte Growth Factor Promotes Cell Survival by Phosphorylation of BAD in Gastric Cancer Cells

Hepatocyte growth factor (HGF) is one of the survival factors with a potent ability to promote cell survival by inhibiting apoptosis. However, the mechanism by which HGF inhibits apoptosis is not completely understood. To explore the genes associated with stomach cancer cell survival by HGF, we used cDNA microarray technology and selected 26 genes up- or downregulated in NUGC-3 cells during HGF treatment. Among them, BAD was confirmed to be upregulated at the RNA and protein levels by HGF treatment. We investigated the effect of BAD induced by HGF on cell survival. HGF treatment inhibited apoptosis induced by BAD overexpression and enhanced BAD phosphorylation. Pretreatment of NUGC-3 cells with PI3K inhibitors, LY 294002, decreased HGF-induced BAD phosphorylation on Ser136 whereas an MEK inhibitor, PD 98059, decreased BAD phosphorylation on Ser112. In conclusion, increases in BAD levels as well as BAD phosphoryation by HGF might contribute to HGF-mediated cell survival in NUGC-3 cells.

Breast Cancer Cell Proliferation Is Inhibited by BAD: REGULATION OF CYCLIN D1

Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.

Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1

The anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X(L) is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X(L) is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X(L) abolishes the Bcl-X(L)-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X(L), antagonizes autophagy inhibition by Bcl-2/Bcl-X(L) and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X(L).

Overexpression of BAD preferentially augments anoikis.

BAD is a BH3-only protein, and its proapoptotic activity is negatively regulated by serine phosphorylation. Here, we show that overexpression of BAD preferentially augments anchorage loss-induced apoptosis (anoikis). Gene transfer-mediated BAD overexpression alone did not induce apoptosis in attached MDCK cells but strongly augmented apoptosis when cells were cultured in suspension. In contrast, overexpression of another BH3-only protein, BID, displayed much lower augmentation of anoikis, suggesting a preferential contribution of BAD to anoikis. During suspension culture, unphosphorylated BAD was gradually increased and targeted to the mitochondria. Cotransfection of BAD with constitutively active Akt cDNA strongly inhibited this change. In contrast, the increase of unphosphorylated BAD was not significantly inhibited by several phosphatase inhibitors or cotransfection with a dominant negative calcineurin cDNA, implying that the increase may be mainly due to a decrease of serine kinase activity, such as that of Akt. Similar results were observed in COS-7 cells, suggesting that BAD overexpression can increase sensitivity of anchorage-dependent cancer cells to anoikis. Thus, we propose that BAD can serve as a valuable gene therapeutic molecule to inhibit carcinoma progression.

Bad Overexpression Sensitizes NIH/3T3 Cells to Undergo Apoptosis Which Involves Caspase Activation and ERK Inactivation

The effect of Bad overexpression on apoptosis was demonstrated by a mouse Bad transgene stably expressed in NIH/3T3 cells. The cells overexpressing Bad treated with either serum starvation or ceramide showed apoptotic characteristics evident at 18 and 8 h, respectively. Whether serum deprivation and ceramide utilize a common death pathway requires further investigation. The time for the first apoptosis detection was shortened to 2 h and was prominent at 4 h, while above that time cells were maintained under serum-depleted conditions in the presence of ceramide (40 μM). Further investigation revealed that the activity of caspase-3 (CPP32) was elevated after ceramide treatment in Bad-transfected cells compared to that of the cells without Bad transfection, indicating the involvement of caspase cascade. Furthermore, the Bad-transfected cells showed reduced phosphorylation of extracellular signal-regulated kinase (ERK). Taken together, we hypothesize that Bad-overexpressing NIH/3T3 cells in the presence of ceramide undergo apoptosis by activating caspase cascade. Simultaneously, the cell survival pathway was blocked possibly by inactivation of the MAPK pathway such as the down-regulation of ERK.

Bad can act as a key regulator of T cell apoptosis and T cell development.

Bad is a distant relative of Bcl-2 and acts to promote cell death. Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis. We generated bad transgenic mice to study the action of upregulated Bad expression on T cell apoptosis. The T cells from these mice are highly sensitive to apoptotic stimuli, including anti-CD95. The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed. We show that the proapoptotic function of Bad in primary T cells is regulated by Akt kinase and that Bad overexpression enhances both cell cycle progression and interleukin 2 production after T cell activation. These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.

Expression and function of a proapoptotic Bcl-2 family member Bcl-XL/Bcl-2-associated death promoter (BAD) in rat ovary.

Bcl-2-related anti- and proapoptotic proteins are important in the decision step of the intracellular death program upstream from the caspase proteases. Targeted overexpression of Bcl-2 in ovarian somatic cells of transgenic mice leads to decreased apoptosis of granulosa cells and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation. The ability of exogenous Bcl-2 proteins to promote follicle cell survival suggests that the transgene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellular apoptosis process in favor of cell survival. We used the yeast two-hybrid system to search for ovarian Bcl-2 interacting proteins. The screening of an ovarian fusion complementary DNA library yielded several clones encoding for the death agonist Bcl-XL/Bcl-2-associated death promoter (BAD). Dimerization of Bcl-2-related proteins mediated by the consensus Bcl-2 homology (BH) domains is essential for their apoptosis-regulating function. Consistent with these observations, yeast two-hybrid assays indicated that the interaction of Bcl-2 with BAD is dependent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis showed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hybridization analysis indicated BAD mRNA expression in granulosa cells of different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 days and did not alter during the developmentally occurring apoptosis found about postnatal day 18 when the first group of early antral follicles were formed. Similarly, BAD mRNA levels did not change during follicle atresia induced by estrogen withdrawal in immature rats. To study the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell line. Overexpression of BAD induced apoptosis in both cell types, and the effect of BAD was reversed by a membrane-permeable caspase inhibitor, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Bcl-2-interacting protein in the ovary, and BAD mRNA is constitutively expressed in granulosa cells, suggesting that BAD is an essential part of the ovarian cell death process. Because BAD overexpression in granulosa cells leads to apoptosis, future studies on ovarian BAD binding proteins and hormonal regulation of the interactions among different Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.