Insoo Bae

BRCA1 Induces Antioxidant Gene Expression and Resistance to Oxidative Stress

Mutations of the breast cancer susceptibility gene 1 (BRCA1), a tumor suppressor, confer an increased risk for breast, ovarian, and prostate cancers. To investigate the function of the BRCA1 gene, we performed DNA microarray and confirmatory reverse transcription-PCR analyses to identify BRCA1-regulated gene expression changes. We found that BRCA1 up-regulates the expression of multiple genes involved in the cytoprotective antioxidant response, including glutathione S-transferases, oxidoreductases, and other antioxidant genes. Consistent with these findings, BRCA1 overexpression conferred resistance while BRCA1 deficiency conferred sensitivity to several different oxidizing agents (hydrogen peroxide and paraquat). In addition, in the setting of oxidative stress (due to hydrogen peroxide), BRCA1 shifted the cellular redox balance to a higher ratio of reduced to oxidized glutathione. Finally, BRCA1 stimulated antioxidant response element-driven transcriptional activity and enhanced the activity of the antioxidant response transcription factor nuclear factor erythroid-derived 2 like 2 [also called NRF2 (NFE2L2)]. The ability of BRCA1 to stimulate antioxidant response element-dependent transcription and to protect cells against oxidative stress was attenuated by inhibition of nuclear factor erythroid-derived 2 like 2. These findings suggest a novel function for BRCA1, i.e., to protect cells against oxidative stress. This function would be consistent with the postulated role of BRCA1 as a caretaker gene in preserving genomic integrity.

CR6 : A third member in the MyD118 and Gadd45 gene family which functions in negative growth control

MyD118 and Gadd45 are two related genes which encode for proteins that play important roles in negative growth control, including both growth suppression and apoptosis. A strategy was employed to clone new members of the MyD118 and Gadd45 family of genes. Based on alignment of the deduced amino acid sequences, one cDNA clone was found to encode for the murine homologue of human CR6, originally cloned as an IL-2 immediate-early response gene. The murine and human CR6 proteins were observed to be 97% identical, indicating that CR6 is an evolutionarily conserved protein. Analysis of CR6 expression during hematopoietic cell development associated with growth arrest and apoptotic cell death, upon exposure of hematopoietic cells to a variety of growth arrest and apoptotic stimuli, and in a variety of murine tissues, has revealed that CR6 expression differs significantly from the expression of the related MyD118 and Gadd45 genes. Nevertheless, CR6, like MyD118 and Gadd45, suppressed colony formation of human lung carcinoma H1299 cells. These data suggest that CR6 plays similar, but not identical, roles to MyD118 and Gadd45 in negative control of cell growth.

BRCA1 Regulates Gene Expression for Orderly Mitotic Progression

Germline mutations of the BRCA1 confer an increased risk for breast cancer, ovarian cancer, and several other tumor types, including prostate cancer. In sporadic (non-hereditary) breast and ovarian cancers, BRCA1 expression is frequently decreased or absent, implicating BRCA1 in the etiology of these cancers. To study the contribution of BRCA1 to sporadic cancers, we tested the effect of knocking down endogenous BRCA1 on the gene expression profiles of human prostate (DU-145) and breast (MCF-7) cancer cell lines. DNA microarray and independent confirmatory RNA analyses revealed that treatment with BRCA1 small interfering (si) RNA caused decreased expression of genes involved in cell cycle regulation and in DNA replication and repair. In particular, BRCA1-siRNA caused down-regulation of multiple genes implicated in the mitotic spindle checkpoint (eg., BUB1, BUB1B, HEC, STK6, and BIRC5), chromosome segregation (eg., ESPL1, NEK2, PTTG1, and multiple kinesins and kinesin-like proteins), centrosome function (eg., ASPM), cytokinesis (eg., PRC1, PLK, MPHOSPH1, and KNSL2), and the transition into and progression through mitosis (eg., B-type cyclins, CDC2, and CDC20). Consistent with these findings, cells treated with BRCA1-siRNA showed attenuation of the mitotic spindle checkpoint, demonstrated by a failure of cells to arrest in metaphase following treatment with nocodazole, an agent that activates the spindle checkpoint. On the other hand, BRCA1-siRNA did not attenuated the ionizing radiation-inducible G2 checkpoint or the G2 decatenation checkpoint. Finally, BRCA1 knockdown caused or promoted the accumulation of binucleated and multinucleated cells, suggesting a defect in the coordination of cytokinesis and karyokinesis; and BRCA1-siRNA caused centrosome amplification. These findings suggest that BRCA1 transcriptionally regulates gene expression for orderly mitotic progession.

Induction of BCL2 family member MCL1 as an early response to DNA damage

When ML-1 human myeloid leukemia cells are exposed to DNA damaging agents, they exhibit dramatic changes in the expression of a variety of gene products. This includes an increase in p53 (wild-type), a decrease in BCL2, a p53-dependent increase in the BCL2 family member BAX, and increases in Growth Arrest and DNA Damage-inducible (GADD) genes such as GADD45; these changes occur as early events in a sequence that culminates in DNA damage-induced apoptosis. DNA damaging agents have now been tested for effects on expression of another BCL2 family member, MCL1, a gene expressed during ML-1 cell differentiation. Expression of MCL1 was found to increase upon exposure of ML-1 cells to various types of DNA damaging agents, including ionizing radiation, ultraviolet radiation, and alkylating drugs. The increase in MCL1 occurred rapidly and was transient, levels of the MCL1 mRNA being elevated within 4u2009h and having returned to near baseline within 24u2009h. An increase in the Mcl1 protein was also seen, with the maximal increase occurring at an intermediate dose of IR (5 Gray) and lesser increases occurring at either lower or higher doses. The increase in expression of MCL1 was further studied using a panel of human cell lines that includes cells containing or not containing alterations in p53 as well as cells sensitive or insensitive to the apoptosis-inducing effects of DNA damage. The DNA damage-induced increase in MCL1 mRNA did not depend upon p53 as it was seen in cells lacking functional p53. However, the increase did depend upon susceptibility to apoptosis as it was not seen in cells insensitive to apoptosis-induction by DNA damaging agents. These findings demonstrate that cytotoxic DNA damage causes an increase in the expression of MCL1 along with increases in GADD45 and BAX and a decrease in BCL2. Furthermore, while the increase in GADD45 is seen both in cells that undergo growth arrest and in cells that undergo apoptosis in response to DNA damage, alterations in the profile of expression of BCL2 family members occur exclusively in cells that undergo the apoptotic response, with some family members increasing through p53-dependent (BAX) and others through p53-independent (MCL1) pathways. Overall, expression MCL1 can increase during the induction of cell death as well as during the induction of differentiation.

Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1α/2α is mediated through chelation of iron

Hypoxia‐inducible factor‐1 alpha (HIF‐1α) is the regulatory subunit of the heterodimeric transcription factor HIF‐1 that is the key regulator of cellular response to low oxygen tension. Under normoxic conditions, HIF‐1α is continuously degraded by the ubiquitin‐proteasome pathway through pVHL (von Hippel–Lindau tumor suppressor protein). Under hypoxic conditions, HIF‐1α is stabilized and induces the transcription of HIF‐1 target genes. Quercetin, a flavonoid with anti‐oxidant, anti‐inflammatory, and kinase modulating properties, has been found to induce HIF‐1α accumulation and VEGF secretion in normoxia. In this study, the molecular mechanisms of quercetin‐mediated HIF‐1α accumulation were investigated. Previous studies have shown that, in addition to being induced by hypoxia, HIF‐1α can be induced through the phosphatidylinositol 3‐kinase (PI3K)/Akt and p53 signaling pathways. But our study revealed, through p53 mutant‐type as well as p53 null cell lines, that neither the PI3K/Akt nor the p53 signaling pathway is required for quercetin‐induced HIF‐1α accumulation. And we observed that HIF‐1α accumulated by quercetin is not ubiquitinated and the interaction of HIF‐1α with pVHL is reduced, compared with HIF‐1α accumulated by the proteasome inhibitor MG132. The use of quercetins analogues showed that only quercetin and galangin induce HIF‐1/2α accumulation and this effect is completely reversed by additional iron ions. This is because quercetin and galangin are able to chelate cellular iron ions that are cofactors of HIF‐1/2α proline hydroxylase (PHD). These data suggest that quercetin inhibits the ubiquitination of HIF‐1/2α in normoxia by hindering PHD through chelating iron ions. J. Cell. Biochem. 103: 1989–1998, 2007.

Nuclear factor (erythroid-derived 2)-like 2 regulates drug resistance in pancreatic cancer cells.

Objective: To investigate the molecular basis of drug resistance in pancreatic cancer. Methods: The expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels in pancreatic cancer tissues and cell lines was analyzed. Clinical relevance between Nrf2 activation and drug resistance was demonstrated by measuring cell viability after Nrf2 and adenosine 5&vprime;-triphosphate-binding cassette, subfamily G member 2 (ABCG2) regulation by overexpression or knock-down of these genes. Activity of ABCG2 was measured by Hoechst 33342 staining. Results: Abnormally elevated Nrf2 protein levels were observed in pancreatic cancer tissues and cell lines relative to normal pancreatic tissues. Increasing Nrf2 protein levels either by overexpression of exogenous Nrf2 or by activating endogenous Nrf2 resulted in increased drug resistance. Conversely, a reduction in endogenous Nrf2 protein levels or inactivation of endogenous Nrf2 resulted in decreased drug resistance. These changes in drug resistance or sensitivity were also positively correlated to the expression levels of Nrf2 downstream genes. Similarly, the expression of ABCG2 was correlated with drug resistance. Conclusions: Because the intrinsic drug resistance of pancreatic cancers is, in part, due to abnormally elevated Nrf2 protein levels, further research on regulating Nrf2 activity may result in the development of novel pancreatic cancer therapies.Abbreviations: Nrf2 - nuclear factor (erythroid-derived 2)-like 2, ABCG2 - adenosine 5&vprime;-triphosphate-binding cassette, subfamily G member 2, MRP - multidrug resistance protein

BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

A recent study of breast cancer patients with and without BRCA1 gene mutations found significantly lower levels of VEGF in serum from patients with BRCA1 mutations (Tarnowski, B., Chudecka-Glaz, A., Gorski, B., and Rzepka-Gorska, I. (2004) Breast Cancer Res. Treat. 88, 287–288). Here, we describe a possible mechanistic explanation for this correlation. Because hypoxia in tumors stimulates VEGF expression and secretion we hypothesized that altered BRCA1 protein levels in breast tumors could affect hypoxia-stimulated VEGF promoter activity. This possibility was tested in cells transfected with various combinations of expression plasmids for BRCA1, BRCA1 specific inhibitory RNAs (BRCA1-siRNAs), HIF-1α, and a VEGF promoter-reporter and then incubated in normoxia (21%, O2) or hypoxia (0.1%, O2). As predicted, increased BRCA1 levels enhanced both hypoxia-stimulated VEGF promoter activity and the amounts of VEGF mRNA, as determined by semiquantitative RT-PCR and quantitative real time PCR. Using the ChIP assay, we discovered that BRCA1 could be recruited to the endogenous human VEGF promoter along with HIF-1α following hypoxia. An interaction between BRCA1 and HIF-1α was found in human breast cancer cells. We also found that hypoxia-stimulated VEGF promoter activity and secretion was reduced in cells containing reduced amounts of endogenous BRCA1 protein (obtained by transfecting with BRCA1 siRNAs). A mechanistic explanation for these effects is provided by our finding a reduced half-life and reduced accumulation of HIF-1α in hypoxic cells transfected with BRCA1-siRNAs and that proteasome inhibitors blocked these effects of BRCA1-siRNAs. Thus, our results suggest that normal amounts of BRCA1 function in hypoxia to regulate HIF-1α stability, probably by interacting with HIF-1α.

Pretreatment of indole-3-carbinol augments TRAIL-induced apoptosis in a prostate cancer cell line, LNCaP

Prostate cancer is one of the most common cancers in men and is the second leading cause of cancer‐related deaths in the USA. Many anti‐tumor agents against prostate cancer cells have been developed, but their unacceptable systemic toxicity to normal tissues frequently limits their usage in clinics. Several previous studies have demonstrated that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) can induce cell death in a variety of transformed cells including prostate cancer cells, but not normal cells. Indole‐3‐carbinol (I3C), a phytochemical that is produced in fruits and vegetables, may play an important role in the prevention of many types of cancer, including hormone‐related ones such as breast and prostate cancer. In this study, we examined the potential sensitizing effects of I3C on TRAIL‐mediated apoptosis in a prostate cancer cell line, LNCaP. When LNCaP cells were incubated with I3C (either 30 or 90 μM) for 24 h and then treated with TRAIL (100 ng/ml), enhanced TRAIL‐mediated apoptosis was observed. The enhanced apoptosis measured by poly(ADP‐ribose) polymerase and caspase 3 cleavage. We also observed that loss of cell viability after treatment with I3C/TRAIL is greater compared with I3C and TRAIL alone. To determine the molecular mechanisms involved in the enhanced apoptosis, we examined the expression of two TRAIL death receptors (DR4 and DR5) and two TRAIL decoy receptors (DcR1 and DcR2). We found that treatment with I3C induced DR4 and DR5 expression at both transcriptional and translational levels. These findings suggest that I3C may be an effective sensitizer of TRAIL treatment against TRAIL‐resistant prostate cancer cell lines such as LNCaP.

BRCA1 modulates xenobiotic stress-inducible gene expression by interacting with ARNT in human breast cancer cells.

Previously, we have reported that BRCA1 regulates the expression of various classes of genes, including genes involved in xenobiotic stress responses (Bae, I., Fan, S., Meng, Q., Rih, J. K., Kim, H. J., Kang, H. J., Xu, J., Goldberg, I. D., Jaiswal, A. K., and Rosen, E. M. (2004) Cancer Res. 64, 7893–7909). In the present study, we have investigated the effects of BRCA1 on xenobiotic stress-inducible gene expression. In response to aryl hydrocarbon receptor (AhR) ligands, cytoplasmic AhR becomes activated and then translocates to the nucleus where it forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT). Subsequently, the AhR·ARNT complex binds to the enhancer or promoter of genes containing a xenobiotic stress-responsive element and regulates the expression of multiple target genes including cytochrome P450 subfamily polypeptide 1 (CYP1A1). In this study, we have found that endogenous and overexpressed exogenous wild-type BRCA1 affect xenobiotic stress-induced CYP1A1 gene expression. Using a standard chromatin immunoprecipitation assay, we have demonstrated that BRCA1 is recruited to the promoter regions of CYP1A1 and CYP1B1 along with ARNT and/or AhR following xenobiotic exposure. Our findings suggest that BRCA1 may be physiologically important for mounting a normal response to xenobiotic insults and that it may function as a coactivator for ARNT activity. Using immunoprecipitation, Western blotting, and glutathione S-transferase capture assays, a xenobiotic-independent interaction between BRCA1 and ARNT has been identified, although it is not yet known whether this is a direct or indirect interaction. We have also found that the inducibility of CYP1A1 and CYP1B1 transcripts following xenobiotic stress was significantly attenuated in BRCA1 knockdown cells. This reduced inducibility is associated with an altered stability of ARNT and was almost completely reversed in cells transfected with an ARNT expression vector. Finally, we have found that xenobiotic (TCDD) treatments of breast cancer cells containing reduced levels of BRCA1 cause the transcription factor ARNT to become unstable.

CR6-interacting factor 1 (Crif1) regulates NF-E2-related factor-2 (Nrf2) protein stability by proteasome-mediated degradation

Free radicals generated by oxidative stress cause damage that can contribute to numerous chronic diseases. Mammalian cells respond to this damage by increased transcription of cytoprotective phase II genes, which are regulated by NRF2. Previously, it has been shown that NRF2 protein levels increase after oxidative stress because its negative regulator, KEAP1, loses its ability to bind NRF2 and cause its proteasome-mediated degradation during oxidative stress. Here, we show that CRIF1, a protein previously known as cell cycle regulator and transcription cofactor, is also able to negatively regulate NRF2 protein stability. However, in contrast to KEAP1, which regulates NRF2 stability only under normal reducing conditions, CRIF1 regulates NRF2 stability and its target gene expression under both reducing and oxidative stress conditions. Thus, CRIF1-NRF2 interactions and their consequences are redox-independent. In addition, we found that CRIF1, unlike KEAP1 (which only interacts with N-terminal region of NRF2), physically interacts with both N- and C-terminal regions of NRF2 and promotes NRF2 ubiquitination and subsequent proteasome-mediated NRF2 protein degradation.