Bernard W Futscher

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.

PMID: 8062263;Abstract:

The multidrug resistance-associated protein (MRP) gene is a member of the ATP-binding cassette transporter gene superfamily and may be partially responsible for clinical drug resistance. Reverse transcriptase-polymerase chain reaction was used to measure MRP mRNA in normal hematopoietic cells from bone marrow and peripheral blood as well as patients with high risk acute myelocytic leukemia and multiple myeloma. All normal peripheral blood cells, regardless of cell lineage (CD4, CD8, CD14, CD15, CD19, CD56), expressed a similar basal level of MRP mRNA. Specimens from bone marrow containing mixed lineages also expressed a similar basal level of MRP expression. In patients with acute myelocytic leukemia, 10 of 12 (83%) of the specimens had detectable MRP mRNA, but the level of expression was similar to that of normal blood cells and low compared to a cell line known to overexpress MRP (H69/AR). All myeloma patients (12 of 12) had detectable MRP mRNA expression at levels comparable to normal peripheral blood and bone marrow cells. We conclude that MRP is commonly expressed in normal hematopoietic cells as well as certain hematopoietic malignancies. The therapeutic relevance of MRP expression is unknown, but these studies emphasize the importance of measuring MRP expression in normal cells as a point of reference and comparison for detection in malignant cells. We also recommend obtaining sequential specimens from patients, which may reveal an increased expression of MRP from baseline as the disease progresses and becomes resistant.

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).