BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-α occupancy in the IKKB promoter and inhibits NF-κB activity in antiestrogen-resistant human breast cancer cells.

PURPOSE: We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity ("BRCA1-mimetics"), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 ("NSC35446.HCl"), also inhibited the growth of antiestrogen-resistant LCC9 tumor xenografts. The purpose of this study was to investigate the down-stream effects of NSC35446.HCl and its mechanism of action. METHODS: Here, we studied antiestrogen-resistant (LCC9, T47DCO, MCF-7/RR, LY2), ERα-negative (MDA-MB-231, HCC1806, MDA-MB-468), and antiestrogen-sensitive (MCF-7) cell lines. Techniques utilized include RNA-seq, qRT-PCR, cell growth analysis, cell-cycle analysis, Western blotting, luciferase reporter assays, TUNEL assays, in silico analysis of the IKKB gene, and ChIP assays. RESULTS: SC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen-resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ERα-negative breast cancer cell lines. IKKB (IKKß, IKBKB), an upstream activator of NF-κB, was identified as a BRCA1-mimetic-regulated gene based on an RNA-seq analysis. NSC35446.HCl inhibited IKKB, IKKA, and IKKG/NEMO mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB activity and expression of NF-κB target genes. In silico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ERα was recruited to the ERE-like full-site and five of the nine half-sites and that ERα recruitment was inhibited by NSC35446.HCl in LCC9 and T47DCO cells. CONCLUSIONS: These studies identify functional EREs in the IKKB promoter and identify IKKB as an ERα and NSC35446.HCl-regulated gene, and they suggest that NF-κB and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.

DIM (3,3'-diindolylmethane) confers protection against ionizing radiation by a unique mechanism.

DIM (3,3'-diindolylmethane), a small molecule compound, is a proposed cancer preventive agent that can be safely administered to humans in repeated doses. We report that administration of DIM in a multidose schedule protected rodents against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing was initiated before or up to 24 h after radiation. Physiologic submicromolar concentrations of DIM protected cultured cells against radiation by a unique mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress. Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced ATM signaling and NF-κB activation. DIM also caused activation of ATM in rodent tissues. Activation of ATM by DIM may be due, in part, to inhibition of protein phosphatase 2A, an upstream regulator of ATM. In contrast, DIM did not protect human breast cancer xenograft tumors against radiation under the conditions tested. In tumors, ATM was constitutively phosphorylated and was not further stimulated by radiation and/or DIM. Our findings suggest that DIM is a potent radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like response and NF-κB survival signaling.

B-cell translocation gene 2 (BTG2) stimulates cellular antioxidant defenses through the antioxidant transcription factor NFE2L2 in human mammary epithelial cells.

The B-cell translocation gene 2, BTG2, a member of the BTG/TOB (B-cell translocation gene/transducers of ErbB2) gene family, has been implicated in cell cycle regulation, normal development, and possibly tumor suppression. Previously, it was shown that BTG2 expression is lost or down-regulated in human breast cancers. We now report that BTG2 protects human mammary epithelial cells from oxidative stress due to hydrogen peroxide and other oxidants. BTG2 protection against oxidative stress is BRCA1-independent but requires the antioxidant transcription factor NFE2L2 and is associated with up-regulation of the expression of antioxidant enzymes, including catalase and superoxide dismutases 1 and 2. BTG2 stimulation of antioxidant gene expression is also NFE2L2-dependent. We further demonstrate that BTG2 is a binding partner for NFE2L2 and increases its transcriptional activity. In addition, BTG2 is detectable at the antioxidant response element (ARE) of several NFE2L2-responsive genes. Finally, we show that the ability of BTG2 to associate with NFE2L2, to protect cells against oxidative stress, and to stimulate antioxidant gene expression requires box B, a short highly conserved amino acid motif characteristic of BTG2/TOB family proteins, but does not require boxes A or C. These findings suggest a novel role for BTG2 as a co-activator for NFE2L2 in up-regulating cellular antioxidant defenses.

Novel DNA damage checkpoint in mitosis: Mitotic DNA damage induces re-replication without cell division in various cancer cells.

DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair.

Transcriptional regulation of the base excision repair pathway by BRCA1.

Inactivation of the breast cancer susceptibility gene BRCA1 plays a significant role in the development of a subset of breast cancers, although the major tumor suppressor function of this gene remains unclear. Previously, we showed that BRCA1 induces antioxidant-response gene expression and protects cells against oxidative stress. We now report that BRCA1 stimulates the base excision repair pathway, a major mechanism for the repair of oxidized DNA, by stimulating the activity of key base excision repair (BER) enzymes, including 8-oxoguanine DNA glycosylase (OGG1), the DNA glycosylase NTH1, and the apurinic endonuclease redox factor 1/apurinic endonuclease 1 (REF1/APE1), in human breast carcinoma cells. The increase in BER enzyme activity appears to be due, primarily, to an increase in enzyme expression. The ability of BRCA1 to stimulate the expression of the three BER enzymes and to enhance NTH1 promoter activity was dependent upon the octamer-binding transcription factor OCT1. Finally, we found that OGG1, NTH1, and REF1/APE1 each contribute to the BRCA1 protection against oxidative stress due to hydrogen peroxide and that hydrogen peroxide stimulates the expression of BRCA1 and the three BER enzymes. These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage.

BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity.

The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ((20)LxxLL(24) and (92)LxxLL(96)), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant (20)LxxLL(24) motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant (92)LxxLL(96) motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact (92)LxxLL(96) motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

BRCA1 localization to the telomere and its loss from the telomere in response to DNA damage.

BRCA1, a tumor suppressor, participates in DNA damage signaling and repair. Previously, we showed that BRCA1 overexpression caused inhibition of telomerase activity and telomere shortening in breast and prostate cancer cells. We now report that BRCA1 knockdown causes increased telomerase reverse transcriptase expression, telomerase activity, and telomere length; but studies utilizing a combination of BRCA1 and telomerase reverse transcriptase small interfering RNAs suggest that BRCA1 also regulates telomere length independently of telomerase. Using telomeric chromatin immunoprecipitation assays, we detected BRCA1 at the telomere and demonstrated time-dependent loss of BRCA1 from the telomere following DNA damage. Further studies suggest that BRCA1 interacts with TRF1 and TRF2 in a DNA-dependent manner and that some of the nuclear BRCA1 colocalizes with TRF1/2. Our findings further suggest that Rad50 is required to localize BRCA1 at the telomere and that the association of BRCA1 with Rad50 does not require DNA. Finally, we found that BRCA1 regulates the length of the 3' G-rich overhang in a manner that is dependent upon Rad50. Our findings suggest that BRCA1 is recruited to the telomere in a Rad50-dependent manner and that BRCA1 may regulate telomere length and stability, in part through its presence at the telomere.

Scatter factor protects tumor cells against apoptosis caused by TRAIL.

Scatter factor (SF) and its receptor c-Met are overexpressed in various tumor types, and their expression often correlates with a poor prognosis. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a proposed tumor-specific chemotherapy agent, but its clinical usage is limited by acquisition of TRAIL resistance by tumors. The goals of this study were to determine whether and how SF protects tumor cells against TRAIL and whether SF-induced TRAIL resistance could be reversed. We used MTT assays, trypan blue dye exclusion assays, apoptosis assays, RNA interference, luciferase reporter assays, immunoprecipitation/western blotting, and other cell biological techniques to study SF protection of cultured human tumor cells against TRAIL. SF conferred resistance to TRAIL in various human prostate carcinoma and breast carcinoma cell lines. SF inhibited TRAIL-induced caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and cell death. SF protection against TRAIL required c-Akt; but unlike protection against adriamycin, it did not require Src signaling or the classical pathway of nuclear factor-kappaB activation. Protection against TRAIL was blocked by knockdown of X-linked inhibitor of apoptosis or FLICE-inhibitor protein (FLIP) (a component of the death-inducing signaling complex). We found that c-Met physically associates with several TRAIL receptors and SF regulates their protein stability. Protection against TRAIL was blocked by a novel small molecule inhibitor of c-Met (PHA665752) and by an inhibitor of cyclooxygenase 2. In conclusion, these findings elucidate potential mechanisms of TRAIL resistance in tumors that overexpress the SF/c-Met and identify possible means of reversing this resistance.

Transcription-dependent epidermal growth factor receptor activation by hepatocyte growth factor.

The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-alpha and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr(845) and Tyr(1068) increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-alpha (~5-fold) and HB-EGF (~23-fold) expression. Tyr(845) and Tyr(1068) phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G(2)-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics.

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells.

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4',5-trimethoxy-3'-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.

Camptothecin and Fas receptor agonists synergistically induce medulloblastoma cell death: ROS-dependent mechanisms.

Medulloblastoma, a common malignant pediatric brain tumor, is highly resistant to death receptor-mediated apoptosis despite death receptor expression by tumor cells. Developing new strategies to overcome this resistance to death receptor activation could positively impact therapeutic outcomes. We explored the modulation of death receptor-induced medulloblastoma cell death by the topoisomerase I inhibitor camptothecin (CPT). CPT significantly increased the human medulloblastoma DAOY cell death response to agonistic anti-Fas antibody (CH-11). Cell death after CPT, CH-11, and CPT+CH-11 treatment was 9, 7, and 33%, respectively. Isobologram analysis showed that CH-11 and CPT act synergistically to induce cell death in DAOY cells. A similar pattern of synergism between CPT and CH-11 was found in ONS-76 medulloblastoma cells. Synergistic cell death was found to be predominantly apoptotic involving both extrinsic and intrinsic pathways as evidenced by annexin V staining, cleavage of caspases (3, 8, and 9), Bid and PARP, and cytoprotection by caspase inhibitors. Flow cytometric analyses showed that expression of cell surface Fas or Fas ligand did not change with drug treatment. Western blot analyses showed that the combination of CH-11+CPT induced a significant decrease in XIAP levels. Furthermore, reactive oxygen species, especially O2, were elevated after CPT treatment, and even more so by the CH-11+CPT treatment. The antioxidants glutathione and N-acetyl-cysteine prevented cell death induced by CPT+CH-11. Moreover, the mitochondrial respiratory chain complex I inhibitor rotenone potentiated CH-11-induced apoptosis in DAOY cells. Taken together, these findings show that CPT synergizes with Fas activation to induce medulloblastoma apoptosis through a mechanism involving reactive oxygen species and oxidative stress pathways.

Ribotoxic stress sensitizes glioblastoma cells to death receptor induced apoptosis: requirements for c-Jun NH2-terminal kinase and Bim.

A prominent feature of glioblastoma is its resistance to death receptor-mediated apoptosis. In this study, we explored the possibility of modulating death receptor-induced cell death with the c-Jun-NH2-terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and inducing "ribotoxic stress." We found that anisomycin and death receptor ligand anti-Fas antibody CH-11 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induce apoptosis in multiple human glioblastoma cell lines. For example, in U87 cells, anisomycin reduced the IC50 of CH-11 by more than 20-fold (from 500 to 25 ng/mL). Cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P<0.001), respectively. Anisomycin and TRAIL were found to be similarly synergistic in glioblastoma cells maintained as tumor xenografts. The potentiation of death receptor-dependent cell death by anisomycin was specific because emetine, another ribosome inhibitor that does not induce ribotoxic stress or activate JNK, did not have a similar effect. Synergistic cell death was predominantly apoptotic involving both extrinsic and intrinsic pathways. Expression of Fas, FasL, FLIP, and Fas-associated death domain (FADD) was not changed following treatment with anisomycin+CH-11. JNK was activated 10- to 22-fold by anisomycin+CH-11 in U87 cells. Inhibiting JNK activation with pharmacologic inhibitors of JNKK and JNK or with dominant negative mitogen-activated protein kinase (MAPK) kinase kinase 2 (MEKK2) significantly prevented cell death induced by the combination of anisomycin+CH-11. We further found that anisomycin+CH-11 up-regulated the proapoptotic protein Bim by approximately 14-fold. Simultaneously inhibiting Bim expression and JNK activation additively desensitized U87 cells to anisomycin+CH-11. These findings show that anisomycin-induced ribotoxic stress sensitizes glioblastoma cells to death receptor-induced apoptosis via a specific mechanism requiring both JNK activation and Bim induction.

Growth factor signaling pathways modulate BRCA1 repression of estrogen receptor-alpha activity.

The breast cancer susceptibility gene BRCA1 is mutated in about one half of all hereditary breast cancer cases, and its expression is frequently decreased in sporadic cancers. Previously, we demonstrated a functional interaction between the BRCA1 and estrogen receptor-alpha (ER-alpha) proteins that causes inhibition of ER-alpha signaling. Here, we examined the role of growth factor signaling pathways in modulating this interaction. We found that underexpression of BRCA1 caused ligand-independent activation of ER-alpha that was mediated through phosphatidylinositol-3 kinase (PI3K)/c-Akt signaling. BRCA1 underexpression also enhanced estrogen-inducible ER-alpha activity in a PI3K/Akt-dependent manner. Exogenous c-Akt conferred estrogen-independent ER-alpha activation and rescued the BRCA1 repression of estrogen-stimulated ER-alpha activity. BRCA1 knockdown stimulated c-Akt activity, in part, by inhibiting the activity of protein phosphatase 2A, an enzyme that dephosphorylates Akt. ERs with point mutations of several growth factor-targeted serine residues (S167A, S118A, and S118/167A) were resistant to repression by BRCA1, although the single point mutant receptors still associated with the BRCA1 protein. The enhanced ER-alpha activity attributable to BRCA1 knockdown was dependent, in part, on serine residues 167 and 118 of ER-alpha. BRCA1 knockdown caused an increase in ER-alpha phosphorylation on serine-167 (but not serine-118 or serine-104/106) that was dependent on PI3K/Akt signaling and was mimicked by pharmacologic inhibition of protein phosphatase 2A. These findings suggest that BRCA1 regulates Akt signaling and the PI3K/Akt pathway modulates the ability of BRCA1 to repress ER-alpha, in part through serine phosphorylation events in the activation function-1 domain of ER-alpha.

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer.

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.

BRCA1 inhibition of telomerase activity in cultured cells.

Telomerase, an enzyme that maintains telomere length, plays major roles in cellular immortalization and cancer progression. We found that an exogenous BRCA1 gene strongly inhibited telomerase enzymatic activity in human prostate and breast cancer cell lines and caused telomere shortening in cell lines expressing wild-type BRCA1 (wtBRCA1) but not a tumor-associated mutant BRCA1 (T300G). wtBRCA1 inhibited the expression of the catalytic subunit (telomerase reverse transcriptase [TERT]) but had no effect on the expression of a subset of other components of the telomerase holoenzyme or on the expression of c-Myc, a transcriptional activator of TERT. However, endogenous BRCA1 associated and partially colocalized with c-Myc; exogenous wtBRCA1 strongly suppressed TERT promoter activity in various cell lines. The TERT inhibition was due, in part, to suppression of c-Myc E-box-mediated transcriptional activity. Suppression of TERT promoter and c-Myc activity required the amino terminus of BRCA1 but not the carboxyl terminus. Finally, endogenous BRCA1 and c-Myc were detected on transfected mouse and human TERT promoter segments in vivo. We postulate that inhibition of telomerase may contribute to the BRCA1 tumor suppressor activity.

The breast cancer susceptibility gene BRCA1 regulates progesterone receptor signaling in mammary epithelial cells.

The progesterone receptor (PR) plays roles in normal mammary development and breast cancer formation, where it may exert both stimulatory and inhibitory actions. Previously, the breast cancer susceptibility gene product BRCA1 was found to interact with and inhibit the transcriptional activity of estrogen receptor-alpha. In this study, we found that exogenous wild-type BRCA1 inhibited the activity of the PR in transient transfection assays utilizing a mouse mammary tumor virus-Luc reporter. Wild-type BRCA1 inhibited the activity of endogenous PR in human breast cancer cells (T47D and MCF-7) and inhibited the activity of exogenous PR-A, PR-B, and [PR-A plus PR-B] isoforms. On the other hand, knockdown of endogenous BRCA1 using small interfering RNA enhanced the progesterone-stimulated activity of the PR by about 4-fold. We documented an in vivo association of the endogenous BRCA1 with PR isoforms A and B and a direct in vitro interaction between BRCA1 and PR, which was partially mapped. Whereas down-regulation of the coactivator p300 contributes to the BRCA1-mediated repression of estrogen receptor-alpha, this mechanism does not contribute to inhibition of PR activity, because exogenous p300 did not rescue the BRCA1 repression of PR activity. The BRCA1-PR interaction has functional consequences. Thus, we showed that BRCA1 inhibits the expression of various endogenous progesterone-responsive genes and inhibits progesterone-stimulated proliferation of T47D cells. Finally, exogenous progesterone caused an exaggerated proliferative response in the mammary glands of mice harboring a mammary-targeted conditional deletion of the full-length isoform of Brca1. These findings suggest that BRCA1 regulates the activity of progesterone, a major hormone of pregnancy that may also participate in mammary carcinogenesis.

Sensitization of glioma cells to Fas-dependent apoptosis by chemotherapy-induced oxidative stress.

A prominent feature of glioblastoma is its resistance to death from Fas pathway activation. In this study, we explored the modulation of Fas-induced glioblastoma death with chemotherapeutic agents. Camptothecin significantly increased the glioblastoma cell death response to Fas receptor activation regardless of p53 status. Sublethal concentrations of camptothecin reduced the IC50 of agonistic anti-Fas antibody (CH-11) 10-fold, from 500 to 50 ng/mL, in human U87 glioblastoma cells (p53 wild-type). Cell viability in response to camptothecin, CH-11 alone, and the combination of camptothecin + CH-11 was found to be 84%, 85%, and 47% (P < 0.001), respectively. A similar pattern of relative cytotoxicity was found in U373 cells (p53 mutant). We further examined the pathways and mechanisms involved in this apparent synergistic cytotoxic response. Cell death was found to be predominantly apoptotic involving both extrinsic and intrinsic pathways as evidenced by annexin V staining, cleavage of caspases (3, 8, and 9), increased caspase activities, Smac release, and cytoprotection by caspase inhibitors. Expression of Fas-associated death domain, and not Fas, Fas ligand, or caspase proteins, increased following cell treatment with camptothecin + CH-11. Camptothecin treatment enhanced c-jun-NH2-kinase activation in response to CH-11, but inhibition of c-jun-NH2-kinase did not prevent cell death induced by the combination treatment. Reactive oxygen species, especially H2O2, were elevated following camptothecin treatment; and H2O2 enhanced cell death induced by CH-11. The antioxidants glutathione and N-acetyl-cysteine prevented cell death induced by camptothecin + CH-11. These findings show that camptothecin synergizes with Fas activation to induce glioblastoma apoptosis via a mechanism involving reactive oxygen species and oxidative stress pathways.

Cyclin D1 antagonizes BRCA1 repression of estrogen receptor alpha activity.

The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor alpha (ERalpha)-dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERalpha binding. Cyclin D1 and BRCA1 were both capable of binding ERalpha in a common region of the ERalpha hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERalpha and for rescue of BRCA1-mediated ERalpha transcriptional repression. In chromatin immunoprecipitation assays, 17beta-estradiol (E2) enhanced ERalpha and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERalpha recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERalpha recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERalpha recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERalpha repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.

Role of NF-kappaB signaling in hepatocyte growth factor/scatter factor-mediated cell protection.

The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIalpha inhibitor). We investigated the role of nuclear factor kappa B (NF-kappaB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin-Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-kappaB associated with the transient loss of the inhibitory subunit IkappaB-alpha. Exposure to HGF/SF caused the activation of an NF-kappaB luciferase reporter that was blocked or attenuated by the expression of a mutant 'super-repressor' IkappaB-alpha. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-kappaB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-kappaB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IkappaB-alpha super-repressor. The ability of HGF/SF to activate NF-kappaB signaling was dependent on c-Akt --> Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3'-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-kappaB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-kappaB activity. HGF/SF induced the expression of several known NF-kappaB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-kappaB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-kappaB through a c-Akt --> Pak1 signaling pathway that is also dependent on Src, and that NF-kappaB contributes to HGF/SF-mediated protection against ADR.

Structural determinants of the BRCA1 : estrogen receptor interaction.

Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-alpha), resulting in the inhibition of estradiol (E2)-stimulated ER-alpha transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1-302) and the ligand-binding domain/activation function-2 (LBD/AF-2) region (within aa 282-420) of ER-alpha. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-alpha interaction. We found that the N-terminal RING domain (aa 20-64) is not required for the BRCA1 : ER-alpha interaction. We identified two separate contact points for ER-alpha, one within aa 1-100 and the other within aa 100-200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 N-terminus, we found that aa 67-100 and 101-133 are required for the interaction with ER-alpha, but that aa 1-67 and 134-302 are dispensible. Previously, we showed that BRCA1 aa 1-302 does not inhibit E2-stimulated ER-alpha transcriptional activity but does bind to ER-alpha and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1-100 and BRCA1 aa 101-200 (but not aa 201-300) each inhibited ER-alpha activity, although not as efficiently as full-length BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86-95) impaired the ability of both truncated (aa 1-100) and full-length (aa 1-1863) BRCA1 proteins to interact with and/or repress ER-alpha activity. Based on these findings, a partial BRCA1 : ER-alpha three-dimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-alpha interaction are discussed.