Bernard W Futscher

PMID: 9929513;Abstract:

N-Acetyltransferases (NATs) play an important role in the biotransformation of a wide variety of arylamine drugs and carcinogens. Two genes (NAT1, NAT2) have been identified and allelic variation in NAT2 has been associated with arylamine toxicity in adults. Little information has been reported on expression of NAT genes during embryonic and fetal development although substrate specific NAT activity has been detected. The current study investigated the expression of NAT1 and NAT2 in mice pre- and postnatally. RNA was isolated from maternal liver, embryonic tissue at gestational days (GD) 10, 15, and 18, or neonates at neonatal day 3. Reverse transcription-polymerase chain reaction was performed using primers designed to amplify portions of either the NAT1 or the NAT2 gene. NAT1 and NAT2 mRNAs were detected in the embryo/placental complex at GD 10 and in GD 15 and 18 embryos. NAT2 but not NAT1 was expressed in GD 18 and neonatal day 3 hepatic tissue. These data demonstrate the differential expression of NAT genes in the mouse embryo and suggest a potential role for NAT in development.

PMID: 16457875;Abstract:

Objective.: Maspin expression is often deregulated in human cancer cells compared to their normal cells due to loss of epigenetic control. In contrast to normal human ovarian surface epithelial (HOSE) cells, ovarian carcinoma cells display a gain of maspin mRNA expression. The objective of this study was to determine whether gain of maspin expression in ovarian cancer is governed by epigenetic mechanisms. Methods.: We examined the cytosine methylation and chromatin accessibility status of the maspin promoter in normal HOSE cells and ovarian carcinoma cells with real-time RT-PCR, sodium bisulfite genomic sequencing, and chromatin accessibility assays. 5-Aza-2′-deoxycytidine (5-aza-dC) was used to induce demethylation of the maspin promoter. Ad p53 was used to induce transient overexpression of wild-type p53. Results.: Normal HOSE cells were maspin-negative in association with methylation of the maspin promoter. In the maspin-positive ovarian cancer cell lines, the maspin promoter was unmethylated. Increased maspin expression in ovarian carcinoma cells was accompanied by a more accessible chromatin structure in the maspin promoter. In the maspin-negative ovarian cancer cell line A222, maspin could be induced following 5-aza-dC treatment or by forced overexpression of p53. Conclusions.: These results suggest that changes in cytosine methylation and chromatin accessibility play an important role in maspin expression in human ovarian carcinoma. Deregulation of maspin expression in ovarian cancer is due to loss of epigenetic control as has been shown in other cancers. This observation provides further evidence of the strict epigenetic control of the maspin gene. © 2005 Elsevier Inc. All rights reserved.

PMID: 17121431;Abstract:

Polyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 μM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs. ©Adenine Press (2006).

PMID: 21684831;PMCID: PMC3230447;Abstract:

Background: Chronic arsenic exposure is a worldwide health problem. How arsenic exposure promotes a variety of diseases is poorly understood, and specific relationships between experimental and human exposures are not established. We propose phenotypic anchoring as a means to unify experimental observations and disease outcomes. Objectives: We examined the use of phenotypic anchors to translate experimental data to human pathology and investigated research needs for which phenotypic anchors need to be developed. Methods: During a workshop, we discussed experimental systems investigating arsenic dose/exposure and phenotypic expression relationships and human disease responses to chronic arsenic exposure and identified knowledge gaps. In a literature review, we identified areas where data exist to support phenotypic anchoring of experimental results to pathologies from specific human exposures. Discussion: Disease outcome is likely dependent on cell-type-specific responses and interaction with individual genetics, other toxicants, and infectious agents. Potential phenotypic anchors include target tissue dosimetry, gene expression and epigenetic profiles, and tissue biomarkers. Conclusions: Translation to human populations requires more extensive profiling of human samples along with high-quality dosimetry. Anchoring results by gene expression and epigenetic profiling has great promise for data unification. Genetic predisposition of individuals affects disease outcome. Interactions with infectious agents, particularly viruses, may explain some species-specific differences between human pathologies and experimental animal pathologies. Invertebrate systems amenable to genetic manipulation offer potential for elaborating impacts of specific biochemical pathways. Anchoring experimental results to specific human exposures will accelerate understanding of mechanisms of arsenic-induced human disease.

PMID: 2104539;Abstract:

This study was initiated to determine if DNA-damaging chemotherapeutic agents can suppress the expression of oncogenes. The effects of three structurally related bifunctional alkylating agents on the steady state mRNA levels of c-myc, c-fos, N-ras, and β-actin in the human colon carcinoma cell line Colo320HSR were examined. Colo320HSR has an amplified c-myc oncogene, which is highly overexpressed, and is assumed to be one of the transforming genes of this cell line. Two concentrations of mechlorethamine, L-phenylalanine mustard, and 4-hydroperoxycyclophosphamide, which produced 1 or 3 log cell kills were used to examine the effects of drug exposure on the expression of specific genes. Steady state mRNA levels were measured by Northern blot analysis. Following a 1-h drug exposure, RNA was isolated from cells at 0, 6, 12, and 24 h following drug removal. The agents used produced changes in the expression of specific genes, and all three did so in a similar fashion. Immediately following drug removal, the steady state expression of c-myc in treated cells was increased 2- to 3-fold compared to control. At 6 and 12 h following drug removal, c-myc levels were depressed 2.5- to 5-fold. By 24 h, c-myc expression approached, but remained below, control levels. Immediately following drug removal, c-fos levels were increased 3- to 4-fold, and from 6 to 24 h following drug removal, c-fos levels gradually returned to, or fell below low basal levels. During the 24-h time course, drug treatment had little or no effect on the steady state levels of N-ras or β-actin. These data support the hypothesis that alkylating agents may suppress the expression of specific transforming genes.