Departments of Pathology and * Surgery, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202
Received December 9, 2003; accepted January 23, 2004
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ABSTRACT |
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Key Words: cadmium; arsenite; arsenic; bladder cancer; UROtsa; cell culture.
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INTRODUCTION |
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Despite the epidemiological base to support a role for both Cd+2 and As+3 in the development of bladder cancer, it is unknown if either pollutant can directly cause the malignant transformation of human urothelial cells. Therefore, the goal of this study was to determine if Cd+2 or As+3 are able to effect the malignant transformation of human urothelial cells. The strategy employed was to expose the nontumorigenic urothelial cell line, UROtsa, to long-term in vitro exposure to Cd+2 and As+3, the endpoints being the ability of the cells to form colonies in soft agar and tumors when heterotransplanted into nude mice. The UROtsa cell line was derived from normal human urothelium lining the ureter and was immortalized using the SV-40 large T-antigen (Petzoldt et al., 1994, 1995
). These cells, when grown on serum-containing growth medium, remain undifferentiated with features of basal epithelial cells, are immortal, grow as a contact-inhibited monolayer, do not form colonies in soft agar, and do not form tumors in nude mice. The UROtsa cells have also been adapted to grow in a serum-free growth medium (Rossi et al., 2001
). Under serum-free conditions, the cells have enhanced differentiation, displaying a stratified morphology consistent with the structural features associated with the intermediate layers of the urothelium. The cells grown in serum-free medium retained the properties of immortality, contact inhibition, and nontumorigenicity, as noted for UROtsa cells when grown on serum-containing growth medium. The two distinct growth conditions allowed expansion of the above strategy to include the question of whether malignant transformation by either Cd+2 or As+3 might be influenced by the medium in which they were grown or the differentiated state of the cells.
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MATERIALS AND METHODS |
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Cell growth.
Growth curves of the malignantly transformed cells were obtained using the methylthiazoletetrazolium (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay following a 1:20 subculture of the cells (Denizot and Lang, 1986).
Growth in soft agar.
Before testing for tumor growth in nude mice, all cultures were tested for their ability to form colonies in soft agar using a slight modification of the procedure described by San and coworkers (San et al., 1979). Briefly, 60-mm diameter dishes were prepared with a 5-ml underlay of 0.5% agar in DMEM containing 5% fetal calf serum. On top of the underlayer we placed either 2 x 104 or 2 x 105 cells in 1.5 ml of 0.25% agar in DMEM containing 5% fetal calf serum. The dishes were incubated at 37°C in a 5% CO2: 95% air atmosphere inside humidified plastic containers to prevent evaporation. Cultures were examined microscopically 24 h after plating, to confirm an absence of large clumps of cells, and thereafter at 7, 14, and 21 days after plating.
Tumorigenicity in nude mice.
To test for malignant transformation, the respective cultures, at passage numbers that showed colony formation in soft agar, were each inoculated, sc, at a dose of 1 x 106 cells in the dorsal thoracic midline of 10 nude (NCr-nu/nu) mice. Tumor formation and growth were assessed weekly. All mice were sacrificed by 10 weeks after injection or when clinical conditions dictated euthanasia. Tumor samples were paraffin-embedded, sectioned, stained with H&E, and analyzed by light microscopy.
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RESULTS |
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The UROtsa cells grown on both serum-containing and serum-free growth media did not survive continuous exposure to 4.0 and 8.0 µM As+3, or to 5.0 and 9.0 µM Cd+2. These cultures displayed greater than 99% cell death by 30 days of exposure and 30 additional days of feeding every 3 days did not result in the formation of surviving colonies of cells. The response of the UROtsa cells grown on both serum-containing and serum-free growth media to 1.0 µM of As+3 or Cd+2 was qualitatively similar to each other. Light microscopic examination revealed no cell death and only a slight increase in the granular appearance of the cell monolayers over the initial 30 days of exposure. Between 30 and 48 days of exposure, over 95% of the cells in all four experimental groups died and detached from the culture growth surface. For each group, cell death occurred within 96 h of the first signs of toxicity. Toxicity was characterized first by a very granular appearance of the cytoplasm of cells, followed by rounding of the cells and eventual detachment from the growth surface. Cell death in all four groups was extensive and no organized groups of cells were left attached to the growth surface. The four experimental groups were continued on a 3-day feeding schedule with growth medium containing 1.0 µM As+3 or Cd+2. Continued microscopic examination disclosed that within 15 to 30 days all four experimental groups had multiple clones of proliferating cells. These cells were allowed to attain confluency and were subcultured at a 1:4 ratio. The resulting cultures had growth rates very similar to the parental UROtsa cells and were subcultured 4 to 8 additional times without incident. Over the next several subcultures, each of the four experimental groups again experienced cell toxicity, which accounted for over 95% cell death. However, in each instance, multiple clones developed quickly and the respective cultures rapidly regained confluency. Upon subculture, the cells from all four groups were noted to have growth rates far in excess of the parental UROtsa cells and were thereafter subcultured at a 1:10 (serum-free) or 1:20 (serum-containing) ratios on a weekly schedule. These cultures were expanded for 8 additional passages to assure no further toxicity and stocks of the cells were preserved in liquid nitrogen.
Light microscopic examination of these cultures showed that all retained an epithelial morphology regardless of growth medium composition or of whether exposure was to Cd+2 or As+3 (Figs. 1A- 1F). This examination also revealed that there were only minor differences in morphology between the cells grown on serum-containing medium when exposed to Cd+2 or As+3, or when each were compared to the parental UROtsa cells. There was a significant difference in morphology for the cells grown on serum-free growth medium. The UROtsa cells treated with Cd+2 or As+3 both lost most of their ability to stratify upon reaching confluence when compared to the parental UROtsa cells. An analysis of cell growth revealed that the UROtsa cells exposed to 1.0 µM As+3 or Cd+2 had higher growth rates compared to the parental UROtsa cells (Figs. 2A and 2B). For cells transformed using serum-containing growth medium; the doubling times of the As+3-exposed cells was 22.1 h, that of the Cd+2-exposed cells 26.6 h, and that of unexposed control cells 43.1 h (Fig. 2A). This difference was similar for cells transformed using serum-free growth medium; the doubling times of the As+3-exposed cells being 34.6 h, that of the Cd+2-exposed cells 36.6 h, and that of unexposed control cells 56.1 h (Fig. 2B). At the time of storage, the cell lines were given new designations: URO-ASSC, for cells treated with As+3 on serum-containing growth medium; URO-ASSF, for cells treated with As+3 on serum-free growth medium; URO-CDSC, for cells treated with Cd+2 on serum-containing growth medium; and, URO-CDSF, for cells treated with Cd+2 on serum-free growth medium. The cells from these stocks were assessed for growth in soft agar and formation of tumors in nude mice.
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The four cell lines were each injected into10 nude mice at an inoculum of 1 x 106 cells per mouse. For the mice injected with the URO-ASSC cells, 9 of 10 mice formed tumors, with the tumors being visible between 2 and 4 weeks following inoculation. Tissue was harvested for histology at an approximate size of 0.5 to 1.5 cm in diameter (between 4 and 9 weeks after inoculation). For the mice injected with the URO-CDSC cells, 9 of 10 mice formed tumors, with the tumors being visible between 3 and 5 weeks of inoculation. Tissue was harvested for histology at an approximate size of 0.5 and 1.5 cm in diameter (between 5 and 10 weeks following inoculation). For the mice injected with the URO-ASSF cells, 5 of 8 mice formed tumors (2 mice died of unknown cause), with the tumors being visible between 5 and 7 weeks of inoculation. Tissue was harvested for histology at an approximate size of 0.5 and 1.0 cm in diameter (10 weeks following inoculation). For the mice injected with the URO-CDSF cells, 7 of 10 mice formed tumors, with the tumors being visible between 4 and 6 weeks from inoculation. Tissue was harvested for histology at an approximate size of 0.5 to 1.0 cm in diameter (6 to 10 weeks following inoculation).
Histology of Nude Mouse Tumors Produced by Cd+2-Transformed Cells
The tumors formed by the URO-CDSC cells were composed of infiltrating masses and nests of moderately differentiated cells (Fig. 3A) with stratification of the malignant epithelial phenotype from the exterior to the central portions of the neoplastic masses. Within the tumor masses, the basal (more peripherally located) cells were small and compact and phenotypically similar to invasive transitional cell carcinoma. Toward the center of the tumor masses, larger eosinophilic cells were present that had a lower nuclear/cytoplasmic ratio with some features of a squamous phenotype. These more differentiated cells had abundant eosinophilic cytoplasm containing swirls of filamentous material, but no squamous "pearls" or desmosomes (Fig. 3B). The nuclei were somewhat smaller and compact with condensed chromatin and occasional small nucleoli. Areas of necrosis were rare even within the largest tumor masses, but when present, there was prominent calcification admixed with irregular, loose eosinophilic laminations within the centrally located necrotic areas (Fig. 3C). The tumors formed by the URO-CDSF cells were smaller in the nude mice and were composed of tight nests and whorls of moderately differentiated cells more closely resembling the invasive urothelial phenotype (Fig. 3D). There was very little tendency for central necrosis or calcification within the centers of the tumor masses even in the largest tumor masses. Although there were focal areas within the tumor nests that demonstrated centrally located differentiation (Fig. 3E) toward a squamous phenotype, this was not as prominently seen as in the URO-CDSC tumors, and the cytoplasmic filaments and swirls were rare in the URO-CDSF tumors. There was a slight increase in nuclear pleomorphism as evidenced by vesicular nuclei, nuclear lobulations and occasional nucleoli (Figs. 3E and 3F).
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DISCUSSION |
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A unique finding in the present study was the morphology of the tumors produced by the heterotransplantation of the arsenite-transformed UROtsa cells into nude mice. In the general patient population, the overwhelming majority of bladder cancers are transitional cell carcinomas with little or no evidence of squamous differentiation. In contrast, the tumor heterotransplants formed by the arsenite-transformed UROtsa cells displayed dominant features of squamous differentiation. These features included: concentrically laminated deposits resembling keratin "pearls," granules resembling the keratohyaline seen in the granular layer of the skin epidermis; and cells with prominent intercellular connections similar to the granulosa or spinous cells within the skin. The significant question that this finding poses is whether the squamous differentiation is an artifact of the model system or if it is a real feature to be expected in bladder cancers that arise from urothelial cells experiencing a long-term, substantial exposure to arsenite. The best answer to this question would be a study detailing the type of bladder cancers produced by patients in areas where there are high concentrations of arsenic in the drinking water; however, to the authors' knowledge no such study currently exists. While an artifact cannot be ruled out, there is indirect evidence to suggest that the feature of squamous differentiation is a legitimate property of the urothelium transformed by long-term exposure to arsenite. The most compelling evidence is that a hallmark of high level environmental arsenic exposure is hyperkeratosis and skin cancer (Kitchin, 2001; Rossman et al. 2001
; Steinmaus et al. 2000
). As such, it would not be surprising if the urothelial cells of the bladder expressed a squamous phenotype as these epithelial cells underwent malignant transformation by an agent known to produce hyperkeratosis in the epithelial cells of the skin. Specificity for arsenite is also suggested by the finding that malignant transformation of the UROtsa cells with Cd+2-exposure resulted in tumor heterotransplants, with only modest evidence of squamous differentiation. If the squamous differentiation were an artifactual property inherent in the UROtsa cells, it would be expected that a similar treatment protocol with Cd+2 would have produced similar results regarding squamous differentiation of the heterotransplants; however, only very rare profiles of squamous change were detected in the Cd+2-induced tumors. That the squamous differentiation was not a rare one-time event is suggested by the finding that As+3-exposed UROtsa cells grown on serum-containing and serum-free media both produced tumors with squamous differentiation when heterotransplanted into nude mice. The present results suggest that bladder tumors diagnosed in areas where high concentrations of arsenic exist in the drinking water should be examined carefully for features of squamous differentiation. It is possible that squamous differentiation in urothelial tumors could be a biomarker for instances where arsenic had a significant role in neoplastic transformation.
There has been only one other study that has had success in malignantly transforming human epithelial cells with arsenite (Achanzar et al. 2002). These studies utilized an immortalized epithelial cell culture, RWPE-1, derived from the prostate glanda site not associated with keratinocyte differentiation. In this study, the RWPE-1 cells were malignantly transformed when exposed to 5 µM sodium arsenite for 29 weeks. The resulting cultures formed epithelial tumors when heterotransplanted into nude mice. These tumors were positive for the production of prostate-specific antigen, but displayed no features of a squamous phenotype. There were several interesting differences between the studies in the RWPE-1 and UROtsa cells. The RWPE-1 cells were exposed to 5 µM As+3 for 29 weeks, a concentration which caused no notable death to the cells. The malignantly transformed cells were identified by the formation of foci of cells that had lost contact inhibition of growth. These cells, when heterotransplanted into nude mice formed invasive tumors as noted by invasion of the underlying muscle layer. In contrast, the UROtsa cells experienced over 95% cell death when treated with a 5-fold less concentration of As+3, and the malignantly transformed cells arose from clones that survived this initial As+3-induced toxicity. Cultures arising from these clones had highly elevated growth rates but did not form foci of cells indicative of a loss of contact inhibition. These cells, when heterotransplanted into nude mice, formed tumors that did not invade the underlying muscle layer of the mouse. Thus, the UROtsa cells were more sensitive to As+3 toxicity than were the RWPE-1 cells, but the malignantly transformed RWPE-1 cells formed a more aggressive tumor.
To the authors' knowledge, the present study is also the first to demonstrate the Cd+2-induced malignant transformation of human urothelial cells. This is a significant finding since Cd+2 is classified as a known human carcinogen with the potential for implication as a bladder carcinogen; although the data is much less extensive than that linking As+3 as a bladder carcinogen (IARC, 1993; Siemiatycki et al., 1994
; Waalkes, 2000
). The present malignant transformation of human urothelial cells is compelling evidence that Cd+2 has the potential to be a human bladder carcinogen. Furthermore, the model system developed should provide a valuable set of human cell lines to study the development and progression of cadmium-induced bladder cancer. The tumor heterotransplants produced by the Cd+2-transformed cells were epithelial in character and had features consistent with those expected of an undifferentiated transitional cell carcinoma of the bladder. An additional significant finding was evidence that the level of Cd+2 required to malignantly transform urothelial cells is quite low when compared to the prostate, an organ where there is substantial evidence for a role of Cd+2 as a prostate carcinogen (Goering et al. 1995
; Waalkes et al. 1997
). The concentration of cadmium in the prostate of people with no known occupational exposure to cadmium is between 11 and 28 µM (Achanzar et al. 2001
; Elinder, 1985
). In studies demonstrating the cadmium-induced malignant transformation of human prostate epithelial cells (RWPE-1 cells), it was shown that 10 µM Cd+2 induced malignant cell transformation following 8 weeks of continuous exposure to the metal (Achanzar et al. 2001
). This level of Cd+2 exposure was not noted to be lethal to the cells. In comparison, the UROtsa cells were malignantly transformed at a 10-fold lower Cd+2 concentration and this concentration produced significant cytotoxicity to the cells. This evidence indirectly suggests that human bladder urothelium might be susceptible to relatively low concentrations of Cd+2.
In summary, these studies provide evidence that both cadmium and arsenite have the potential to be human bladder carcinogens.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, 501 N. Columbia Road, Grand Forks, ND 58202-9037. Fax: (701) 777-3108. E-mail: ssomji{at}medicine.nodak.edu
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