Monomethylarsonous acid induces transformation of human bladder cells

Abstract

Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMA(III)) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 muM MMA(III) for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMA(III), which had a 40-50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMA(III) exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMA(III) for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMA(III). These observations are the first demonstration of MMA(III)-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMA(III) may be carcinogenic in human tissues.

Fig. 1

Metabolism of inorganic arsenic in mammals.

Fig. 2

Cytotoxicity of MMA^III to UROtsa cells. Cells were treated with 0.5–10 μM MMA^III for 24, 48, and 72 h. Values represent means ± SEM. Assay results are portrayed as percentage value of untreated samples (n = 3). Significant changes in viability were identified with ANOVA followed by Bonferroni’s multiple comparisons test. P < 0.05 was considered significant and marked by asterisk(s) *(24 h), **(48 h), and ***(72 h).

Fig. 3

Evaluation via confocal microscopy of UROtsa cells before, during, and after transformation with MMA^III. Morphology of untreated UROtsa cells (A). Morphology of URO-MSC cells treated with 0.05 μM MMA^III for 12 (B), 24 (C), and 52 (D) weeks. Magnification used for photographs is ×200.

Fig. 4

Anchorage-independent growth of URO-MSC cells. Light microscopy of colony growth 14 days after plating for negative control, UROtsa, positive control, URO-ASSC, URO-MSC24, and URO-MSC52 cells (A). Colonies were photographed at ×200. (B). A single plane of agar was selected, and colonies were manually counted in twenty randomly selected microscope fields. Bars represent mean ± SEM (n = 3). However, error bars are too small to visualize. Data were further analyzed with Student’s t test to identify significant changes in the ability to form colonies in soft agar. Statistically significant values (P < 0.05) are marked with an asterisk (*).

Fig. 5

Tumorigenicity of URO-MSC cells in SCID mice. Tumor volumes were measured two times per week. Data are tumor volume (mm³) per time (days). Bars represent mean ± SEM (n = 4). However, error bars in UROtsa and URO-MSC24 tumors are too small to visualize. Significant changes in tumor volume were identified with ANOVA followed by Bonferroni’s multiple comparisons test. P < 0.05 was considered significant. URO-ASC tumors are statistically different in tumor volume from UROtsa 30 days after injection. URO-MSC52 tumors are significantly different from UROtsa 40 days after injection.

Fig. 6

Ki-67 staining of tumor tissue from UROtsa (A) and URO-MSC52 (B) cells. Representative photograph of Ki-67-stained tissue sections demonstrates the increased proliferation found in URO-MSC52 tumors. Magnification used for photographs is ×400.