Bernard W Futscher

PMID: 16799634;Abstract:

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2′- deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer. © 2007 Nature Publishing Group All rights reserved.

PMID: 10709100;Abstract:

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue- restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5- aza-2'-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer. (C) 2000 Wiley-Liss, Inc.

PMID: 19716837;PMCID: PMC2783766;Abstract:

Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortal to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, and alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome. © 2009 Elsevier Inc. All rights reserved.

PMID: 14580325;Abstract:

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme with tumor suppressor activity; however, the molecular mechanisms of MnSOD antitumor effects remain unclear. We hypothesized that MnSOD activity in cancer cells might cause downstream changes in the expression of other tumor suppressor genes. To determine whether maspin, a tumor suppressor gene that inhibits breast cancer cell invasion and metastasis, might be a target of MnSOD, we forced MnSOD expression in several human breast and prostate cancer cell lines by adenovirus-mediated gene transfer and measured maspin mRNA expression. Forced expression of MnSOD caused maspin mRNA to accumulate in a dose-dependent manner in both human breast and prostate cancer cells. Normal p53 was not necessary to mediate the effect of MnSOD because MnSOD up-regulated maspin in cells that harbor wild-type p53 and in cells that harbor mutant p53. Moreover, the effects of MnSOD on maspin were not due to demethylation of the maspin promoter. Analyses of maspin promoter activity, transcriptional run-on, and mRNA stability showed that maspin mRNA stability was the major mechanism for maspin up-regulation by MnSOD. Our findings identify a mechanism underlying MnSOD antitumor effects and provide evidence to support MnSOD as a genetic therapy in the treatment of human breast and prostate cancers.

PMID: 1515367;Abstract:

Genomic and gene-specific DNA interstrand cross-links produced by nitrogen mustard (HN2) were measured in the human tumor cell line Colo320HSR. Following exposures that produced greater than or equal to 1 log cell kill, it was found that HN2-induced DNA interstrand cross-links were produced and processed in a heterogeneous fashion within the genome. Cross-links were detected in the amplified, overexpressed c-myc oncogene, whereas in the weakly expressed N-ras gene and the nontranscribed, high copy number alpha-satellite sequence (of chromosome 20), cross-links were not detected. The cross-links in the c-myc oncogene disappeared more rapidly than total genomic cross-links. These results suggest that HN2-induced DNA interstrand cross-links are produced and processed in the genome in a nonrandom fashion.