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Title: Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

Abstract

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1more » expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular transformation.« less

Authors:
;  [1];  [2];  [1]
  1. National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States)
  2. Molecular Toxicology and Informatics Group, Biomolecular Screening Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Morrisville, NC 27560 (United States)
Publication Date:
OSTI Identifier:
22465791
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 286; Journal Issue: 3; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ARSENIC; CADMIUM; CARCINOGENS; DNA; GENES; METHYLATION; NEOPLASMS; PHENOTYPE; PROSTATE

Citation Formats

Pelch, Katherine E., Tokar, Erik J., Merrick, B. Alex, and Waalkes, Michael P., E-mail: waalkes@niehs.nih.gov. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium. United States: N. p., 2015. Web. doi:10.1016/J.TAAP.2015.04.011.
Pelch, Katherine E., Tokar, Erik J., Merrick, B. Alex, & Waalkes, Michael P., E-mail: waalkes@niehs.nih.gov. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium. United States. doi:10.1016/J.TAAP.2015.04.011.
Pelch, Katherine E., Tokar, Erik J., Merrick, B. Alex, and Waalkes, Michael P., E-mail: waalkes@niehs.nih.gov. 2015. "Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium". United States. doi:10.1016/J.TAAP.2015.04.011.
@article{osti_22465791,
title = {Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium},
author = {Pelch, Katherine E. and Tokar, Erik J. and Merrick, B. Alex and Waalkes, Michael P., E-mail: waalkes@niehs.nih.gov},
abstractNote = {Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular transformation.},
doi = {10.1016/J.TAAP.2015.04.011},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 286,
place = {United States},
year = 2015,
month = 8
}
  • Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchialmore » epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro-angiogenic effect. • EMT in arsenic-transformed cells activates β-catenin. • β-Catenin activation increases VEGF expression in arsenic-transformed cells. • β-Catenin activation is likely independent of canonical Wnt signaling. • EMT in arsenic-transformed cells promotes angiogenesis via β-catenin–VEGF pathway.« less
  • Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less
  • Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomousmore » growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a valuable model for arsenic-induced lung cancer.« less
  • In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferativemore » capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.« less
  • Chronic exposure to arsenic can generate reactive oxidative species, which can induce certain proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8). TNF-α, IL-6 and IL-8 have been shown to be involved in the development and progression of various cancers, including bladder cancer. This study aimed to investigate the joint effect of the polymorphism of TNF-α − 308 G/A, IL-6 − 174 G/C, IL-8 − 251 T/A and urinary arsenic profiles on urothelial carcinoma (UC) risk. This study evaluated 300 pathologically-confirmed cases of UC and 594 cancer-free controls. Urinary arsenic species were detected using high-performance liquidmore » chromatography-linked hydride generator and atomic absorption spectrometry. The polymorphism of TNF-α − 308 G/A, IL-6 − 174 G/C and IL-8 − 251 T/A was determined using polymerase chain reaction-restriction fragment length polymorphism. The joint effects on UC risk were estimated by odds ratios and 95% confidence intervals using unconditional logistic regression. We found that the TNF-α − 308 A/A and IL-8 − 251 T/T polymorphisms were significantly associated with UC. Moreover, significant dose–response joint effect of TNF-α − 308 A/A or IL-8 − 251 T/T genotypes and arsenic methylation indices were seen to affect UC risk. The present results also showed a significant increase in UC risk in subjects with the IL-8 − 251 T/T genotype for each SD increase in urinary total arsenic and MMA%. In contrast, a significant decrease in UC risk was found in subjects who carried the IL-8 − 251 T/T genotype for each SD increase in DMA%. - Highlights: • Joint effect of the TNF-α -308 A/A genotype and urinary total arsenic affected UC. • Joint effect of the IL-8 -251 T/T genotype and urinary total arsenic affected UC. • Urinary total arsenic level, TNF-α -308 A/A and IL-8 -251 T/T genotype affected UC.« less