Kheem S. Bisht

SIRT3 Is a Mitochondria-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress

The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.

Thioredoxin Reductase as a Potential Molecular Target for Anticancer Agents That Induce Oxidative Stress

Redox-sensitive signaling factors regulate multiple cellular processes, including proliferation, cell cycle, and prosurvival signaling cascades, suggesting their potential as molecular targets for anticancer agents. It is logical to set constraints that a molecular target should meet at least one of the following criteria: (1) inhibition of prosurvival signaling pathways; (2) inhibition of cell cycle progression; or (3) enhancement of the cytotoxic effects of anticancer agents. Therefore, we hypothesized that thioredoxin reductase 1 (TR), a component of several redox-regulated pathways, might represent a potential molecular target candidate in response to agents that induce oxidative stress. To address this issue, permanent cell lines overexpressing either the wild-type (pCXN2-myc-TR-wt) or a Cys-Ser mutant (pCXN2-myc-mTR) TR gene were used, as were parental HeLa cells treated with 1-methyl-1-propyl-2-imidazolyl disulfide (IV-2), a pharmacologic inhibitor of TR. Cells were exposed to the oxidative stressors, H2O2 and ionizing radiation (IR), and analyzed for changes in signal transduction, cell cycle, and cytotoxicity. Analysis of HeLa cells overexpressing the pCXN2-myc-TR-wt gene showed increased basal activity of nuclear factor κB (NFκB) and activator protein (AP-1), whereas HeLa cells expressing a pCXN2-myc-mTR gene and HeLa cells treated with IV-2 were unable to induce NFκB or AP-1 activity following H2O2 or IR exposure. Fluorescence-activated cell sorting analysis showed a marked accumulation of pCXN2-myc-mTR cells in the late G1 phase, whereas pCXN2-myc-TR-wt cells showed a decreased G1 subpopulation. Chemical inhibition of TR with IV-2 also completely inhibited cellular proliferation at concentrations between 10 and 25 μmol/L, resulting in a G1 phase cell cycle arrest consistent with the results from cells expressing the pCXN2-myc-mTR gene. Following exposure to H2O2 and IR, pCXN2-myc-mTR– and IV-2–treated cells were significantly more sensitive to oxidative stress-induced cytotoxicity as measured by clonogenic survival assays. Finally, IV-2–treated cells showed increased tumor cell death when treated with H2O2 and IR. These results identify TR as a potential target to enhance the cytotoxic effects of agents that induce oxidative stress, including IR.

Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA)

Abstract Vijayalaxmi, Pickard, W. F., Bisht, K. S., Leal, B. Z., Meltz, M. L., Roti Roti, J. L., Straube, W. L. and Moros, E. G. Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA). Freshly collected peripheral blood samples from four healthy human volunteers were diluted with RPMI 1640 tissue culture medium and exposed in sterile T-75 tissue culture flasks in vitro for 24 h to 835.62 MHz radiofrequency (RF) radiation, a frequency employed for customer-to-base station transmission of cellular telephone communications. An analog signal was used, and the access technology was frequency division multiple access (FDMA, continuous wave). A nominal net forward power of 68 W was used, and the nominal power density at the center of the exposure flask was 860 W/m2. The mean specific absorption rate in the exposure flask was 4.4 or 5.0 W/kg. Aliquots of diluted blood that were sham-exposed or exposed in vitro to an acute dose of 1.50 Gy of γ radiation were used as negative or positive controls. Immediately after the exposures, the lymphocytes were stimulated with a mitogen, phytohemagglutinin, and cultured for 48 or 72 h to determine the extent of genetic damage, as assessed from the frequencies of chromosomal aberrations and micronuclei. The extent of alteration in the kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to mitotic indices, incidence of exchange aberrations, excess fragments, binucleate cells, and micronuclei. In contrast, the response of the lymphocytes exposed to γ radiation was significantly different from both RF-radiation- and sham-exposed cells for all of these indices. Thus, under the experimental conditions tested, there is no evidence for the induction of chromosomal aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 835.62 MHz RF radiation at SARs of 4.4 or 5.0 W/kg.

Radiosensitizing and anti-proliferative effects of resveratrol in two human cervical tumor cell lines

Resveratrol is a polyphenol isolated from the skins of grapes that has been shown to significantly alter the cellular physiology of tumor cells, as well as block the process of initiation and progression. At least one mechanism for the intracellular actions of resveratrol involves the suppression of prostaglandin (PG) biosynthesis. The involvement of PGs and other eicosanoids in the development of human cancer is well established. PGs are synthesized from arachidonic acid via the cyclooxygenase pathway and have multiple physiological and pathological functions. In addition, evidence has arisen suggesting that PGs may be implicated in the cytotoxic and/or cytoprotective response of tumor cells to ionizing radiation (IR). As such, we hypothesized that tumor cells may exhibit changes in the cellular response to IR following exposure to resveratrol, a naturally occurring compound that inhibits cyclooxygenase-1 (COX-1) activity. Thus, clonogenic cell survival assays were performed using irradiated HeLa and SiHa cells pretreated with resveratrol prior to IR exposure, and resulted in enhanced tumor cell killing by IR in a dose-dependent manner. Further analysis of COX-1 inhibition indicated that resveratrol pretreatment: (1), inhibited cell division as assayed by growth curves; and (2), induced an early S phase cell cycle checkpoint arrest, as demonstrated by fluorescence-activated cell sorting, as well as bromodeoxyuridine pulse-chase analysis. These results suggest that resveratrol alters both cell cycle progression and the cytotoxic response to IR in two cervical tumor cell lines.

Chromosome Damage and Micronucleus Formation in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (847.74 MHz, CDMA)

Abstract Vijayalaxmi, Bisht, K. S., Pickard, W. F., Meltz, M. L., Roti Roti, J. L. and Moros, E. G. Chromosome Damage and Micronucleus Formation in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (847.74 MHz, CDMA). Radiat. Res. 156, 430–432 (2001). Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m2 (95 mW/cm2). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of γ radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 ± 0.3°C. Immediately after the exposures, lymphocytes were cultured at 37 ± 1°C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of γ-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.

BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression

ABSTRACT Chromatin status is characterized in part by covalent posttranslational modifications of histones that regulate chromatin dynamics and direct gene expression. BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein that is thought to play a role in chromatin organization and gene expression. BORIS is a cancer-germ line gene; these are genes normally present in male germ cells (testis) that are also expressed in cancer cell lines as well as primary tumors. This work identifies SET1A, an H3K4 methyltransferase, and BAT3, a cochaperone recruiter, as binding partners for BORIS, and these proteins bind to the upstream promoter regions of two well-characterized procarcinogenic genes, Myc and BRCA1. RNA interference (RNAi) knockdown of BAT3, as well as SET1A, decreased Myc and BRCA1 gene expression but did not affect the binding properties of BORIS, but RNAi knockdown of BORIS prevented the assembly of BAT3 and SET1A at the Myc and BRCA1 promoters. Finally, chromatin analysis suggested that BORIS and BAT3 exert their effects on gene expression by recruiting proteins such as SET1A that are linked to changes in H3K4 dimethylation. Thus, we propose that BORIS acts as a platform upon which BAT3 and SET1A assemble and exert effects upon chromatin structure and gene expression.

Measurement of DNA Damage in Mammalian Cells Exposed In Vitro to Radiofrequency Fields at SARs of 3–5 W/kg

Abstract Li, L., Bisht, K. S., LaGroye, I., Zhang, P., Straube, W. L., Moros, E. G. and Roti Roti, J. L. Measurement of DNA Damage in Mammalian Cells Exposed In Vitro to Radiofrequency Fields at SARs of 3–5 W/kg. Radiat. Res. 156, 328–332 (2001). In the present study, we determined whether exposure of mammalian cells to 3.2–5.1 W/kg specific absorption rate (SAR) radiofrequency fields could induce DNA damage in murine C3H 10T½ fibroblasts. Cell cultures were exposed to 847.74 MHz code-division multiple access (CDMA) and 835.62 frequency-division multiple access (FDMA) modulated radiations in radial transmission line (RTL) irradiators in which the temperature was regulated to 37.0 ± 0.3°C. Using the alkaline comet assay to measure DNA damage, we found no statistically significant differences in either comet moment or comet length between sham-exposed cells and those exposed for 2, 4 or 24 h to CDMA or FDMA radiations in either exponentially growing or plateau-phase cells. Further, a 4-h incubation after the 2-h exposure resulted in no significant changes in comet moment or comet length. Our results show that exposure of cultured C3H 10T½ cells at 37°C CDMA or FDMA at SAR values of up to 5.1 W/kg did not induce measurable DNA damage.

DNMT1 as a Molecular Target in a Multimodality-Resistant Phenotype in Tumor Cells

We have previously shown that hydrogen peroxide–resistant permanent (OC-14) cells are resistant to the cytotoxicity of several exogenous oxidative and anticancer agents including H2O2, etoposide, and cisplatin; and we refer to this process as an oxidative multimodality-resistant phenotype (MMRP). Furthermore, OC-14 cells contain increased activator protein 1 activity, and inhibition of activator protein 1 reversed the MMRP. In this study, we show that permanent Rat-1 cell lines genetically altered to overexpress c-Fos also displayed a similar MMRP to H2O2, etoposide, and cisplatin as OC-14 cells. Gene expression analysis of the OC-14 cells and c-Fos–overexpressing cells showed increased DNMT1 expression. Where OC-14 and c-Fos–overexpressing cells were exposed to 5-aza-2′-deoxycytidine, which inhibits DNMT activity, a significant but incomplete reversal of the MMRP was observed. Thus, it seems logical to suggest that DNMT1 might be at least one target in the MMRP. Rat-1 cells genetically altered to overexpress DNMT1 were also shown to be resistant to the cytotoxicity of H2O2, etoposide, and cisplatin. Finally, somatic HCT116 knockout cells that do not express either DNMT1 (DNMT1−/−) or DNMT3B (DNMT3B−/−) were shown to be more sensitive to the cytotoxicity of H2O2, etoposide, and cisplatin compared with control HCT116 cells. This work is the first example of a role for the epigenome in tumor cell resistance to the cytotoxicity of exogenous oxidative (H2O2) or systemic (etoposide and cisplatin) agents and highlights a potential role for DNMT1 as a potential molecular target in cancer therapy. (Mol Cancer Res 2008;6(2):243–9)

CTCFL/BORIS is a methylation-independent DNA-binding protein that preferentially binds to the paternal H19 differentially methylated region

The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele.

DNA methyltransferase 1 and 3B activate BAG-1 expression via recruitment of CTCFL/BORIS and modulation of promoter histone methylation

In a previous genomic analysis, using somatic methyltransferase (DNMT) knockout cells, we showed that hypomethylation decreased the expression of as many genes as were observed to increase, suggesting a previously unknown mechanism for epigenetic regulation. To address this idea, the expression of the BAG family genes was used as a model. These genes were used because their expression was decreased in DNMT1(-/-), DNMT3B(-/-), and double knockout cells and increased in DNMT1-overexpressing and DNMT3B-overexpressing cells. Chromatin immunoprecipitation analysis of the BAG-1 promoter in DNMT1-overexpressing or DNMT3B-overexpressing cells showed a permissive dimethyl-H3-K4/dimethyl-H3-K9 chromatin status associated with DNA-binding of CTCFL/BORIS, as well as increased BAG-1 expression. In contrast, a nonpermissive dimethyl-H3-K4/dimethyl-H3-K9 chromatin status was associated with CTCF DNA-binding and decreased BAG-1 expression in the single and double DNMT knockout cells. BORIS short hairpin RNA knockdown decreased both promoter DNA-binding, as well as BAG-1 expression, and changed the dimethyl-H3-K4/dimethyl-H3-K9 ratio to that characteristic of a nonpermissive chromatin state. These results suggest that DNMT1 and DNMT3B regulate BAG-1 expression via insulator protein DNA-binding and chromatin dynamics by regulating histone dimethylation.