Affiliations of authors: W. E. Achanzar, E. M. Brambila, M. P. Waalkes, Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, NC; B. A. Diwan, Intramural Research Support Program, SAIC-Frederick, NCI-Frederick, Frederick, MD; M. M. Webber, Departments of Medicine and Zoology, Michigan State University, East Lansing, MI.
Correspondence to: Michael P. Waalkes, Ph.D., Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, MD F009, 111 Alexander Dr., Research Triangle Park, NC 27709 (e-mail: waalkes{at}niehs.nih.gov).
ABSTRACT
Although several epidemiologic studies show an association between arsenic exposure and prostate cancer, it is still unknown whether human prostate epithelial cells are directly susceptible to arsenic-induced transformation. This study was designed to determine whether the nontumorigenic human prostate epithelial cell line RWPE-1 could be malignantly transformed in vitro by arsenite. RWPE-1 cells were continuously exposed to 5 µM arsenite and monitored for signs of transformation, assessed as changes in matrix metalloproteinase-9 levels. After 29 weeks of exposure, the arsenite-exposed RWPE-1 cells (referred to as CAsE-PE) showed a marked increase in matrix metalloproteinase-9 secretion, a common finding in prostate malignancies. Malignant transformation was confirmed when CAsE-PE cells produced aggressive undifferentiated malignant epithelial tumors in nude mice. The tumors stained positive for human prostate-specific antigen, confirming their origin. These results are the first report of arsenite-induced malignant transformation of a human epithelial cell line and provide an important in vitro model for studying the mechanisms underlying arsenic-induced carcinogenesis in humans.
Development and culture of the RWPE-1 cell line has been described previously (12,13). RWPE-1 cells were derived from normal human prostate epithelium, were immortalized with human papillomavirus 18, and are nontumorigenic (12,13). For up to 29 weeks, cells were cultured continuously in keratinocyte serum-free medium supplemented with 50 µg/mL bovine pituitary extract, 5 ng/mL epidermal growth factor, antibiotics and 5 µM as sodium arsenite. Parallel cultures maintained in arsenite-free medium served as passage-matched controls. Cells were passaged weekly, with new cultures seeded with 1 x 106 cells in 75-cm2 flasks. Previous studies (14,15) revealed that increased secretion of matrix metalloproteinase-9 (MMP-9) is associated with Ras-induced or cadmium-induced malignant transformation of RWPE-1 cells. In addition, increased MMP-9 secretion occurs in prostate tumors and primary cultures derived from prostatic cancers and is associated with aggressive malignancies (16,17). Thus, we periodically assayed the long-term arsenite-exposed RWPE-1 cells (referred to as CAsE-PE cells) for MMP-9 activity by zymography as described (15).
Compared with MMP-9 activity from passage-matched RWPE-1 cells, no discernible increases in MMP-9 activity from CAsE-PE cells were detected until after 29 weeks of continuous arsenite exposure. At this time, CAsE-PE cells had a 2.2-fold (95% confidence interval [CI] = 2.0 to 2.5) increase in MMP-9 activity compared with passage-matched control RWPE-1 cells (Fig. 1). Because the observed increase in MMP-9 activity suggested that arsenic-induced malignant transformation had occurred, the CAsE-PE cells and passage-matched RWPE-1 cells were inoculated into the renal capsules of male nude mice (NCr-nu) to test their ability to form tumors. Male NCr-nu mice were obtained at 8 weeks of age from the NCI-Frederick Animal Production Area breeding colony and were housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC) accredited facility under conditions that met or exceeded recommendations outlined in the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication No. 8623, 2985). Within 10 weeks, five of five mice inoculated with the CAsE-PE cells developed tumors, whereas none of five mice inoculated with the control RWPE-1 cells developed tumors, a statistically significant difference (P = .008, two-sided Fishers exact test). After staining sections of the tumors with hematoxylin and eosin, histologic examination of the tumors revealed them to be undifferentiated epithelial tumors (Fig. 2, A
), which is consistent with the majority of epithelial prostate tumors and with previous reports of tumors formed by malignantly transformed prostate epithelial cells (12,15,18). The CAsE-PE-derived tumors were highly aggressive and invaded the surrounding capsular muscle and adjacent normal renal parenchyme (Fig. 2, A
). The invasive nature of the tumors is consistent with the increased MMP-9 secretion observed in CAsE-PE cells in vitro because MMP-9 appears to facilitate prostate tumor invasion and metastasis (16). By immunohistochemical analysis, tumor sections stained strongly positive with an antibody specific for human prostate-specific antigen (Fig. 2, B
), confirming that the tumors were of human prostatic origin.
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Understanding the mechanisms involved in arsenic-induced carcinogenesis has been hampered by the lack of suitable animal models. Inorganic arsenic is recognized as a human carcinogen even though, in the absence of treatment with other agents, arsenic-induced tumors have been difficult to produce in rodents (7,2123). Consequently, mechanistic studies rely heavily on experiments performed using in vitro systems, the validity of which is always a critical issue. Our laboratory has previously demonstrated arsenite-induced malignant transformation of rat liver epithelial cells (8), and has identified several important molecular events associated with arsenite-induced transformation (911). More importantly, several of these changes correspond with alterations found in the livers of humans exposed long-term to inorganic arsenic (24). Nevertheless, there are differences between humans and rodents with respect to susceptibility to arsenic-induced tumors, highlighting the need for human cell-based in vitro models of arsenic-induced carcinogenesis. The development of a model such as the one described herein will allow further analysis of the molecular events associated with inorganic arsenic-induced carcinogenesis in a human system. This will permit additional validation of results obtained in rodent cells and potentially provide clues to understanding why humans are apparently more sensitive than rodents to the carcinogenic effects of inorganic arsenic.
NOTES
This project was funded in part by contract NO-1-CO-12400 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
The authors are grateful to Dr. Jerrold M. Ward for assistance with the pathological analysis. The authors also thank Drs. Jie Liu and Dr. Hua Chen for their critical evaluation of the manuscript.
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Manuscript received May 23, 2002; revised September 19, 2002; accepted October 4, 2002.
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