Keratin 6 expression correlates to areas of squamous differentiation in multiple independent isolates of As⁺³‐induced bladder cancer

Abstract

This laboratory has shown that arsenite (As+3) exposure can cause the malignant transformation of the UROtsa human urothelial cell line. This single isolate formed subcutaneous tumors with a histology similar to human urothelial cell carcinoma. The tumors also displayed areas of squamous differentiation of the urothelial cells, an infrequent, but known component of human bladder cancer. In the present study, five additional independent isolates of As+3 -transformed urothelial cells were isolated and each were shown to produce subcutaneous urothelial cell tumors with a characteristic histology very similar to those described in the initial report. That there were underlying phenotypic differences in the 6 independent isolates was demonstrated when they were assessed for their ability to form tumors within the peritoneal cavity. It was shown that two isolates could form hundreds of small peritoneal tumor nodules, one isolate a moderate number of tumor nodules, and three isolates no or only one tumor nodule. The peritoneal tumors were also characterized for their degree of squamous differentiation of the urothelial cells and, while areas of squamous differentiation could be found, such differentiation was substantially reduced compared to subcutaneous tumors. Immunostaining for keratin 6 was tested as a potential marker for malignant urothelial cells that had undergone squamous differentiation. Keratin 6 was shown to consistently stain only cells having some evidence of squamous differentiation. Keratin 16 was shown to follow the staining pattern of keratin 6. The isolates and tumor heterotransplants were all examined for keratin 6, 16 and 17 mRNA and protein expression.

Comparison of expression patterns of keratin 6, 7, 16, 17, and 19 within multiple independent isolates of As+3- and Cd+2-induced bladder cancer

“…Tumor tissue for analysis of keratin expression Tumor tissue for the analysis of keratin mRNA and protein expression was obtained from pre-existing specimens of subcutaneous and peritoneal tumors produced in immuno-compromised mice (NCr-nu/nu) from the seven isolates of Cd +2 and six isolates of As +3 -transformed UROtsa cells as described previously (Cao et al 2010;Somji et al 2010). …”

“…The isolation and growth of the seven isolates of the Cd +2 -transformed UROtsa cells and six isolates of the As +3 -transformed UROtsa cells have been described previously (Cao et al 2010;Sens et al 2004;Somji et al 2010). They were grown and maintained using identical conditions.…”

“…Real-time analysis of keratin mRNA expression The expression of keratin 6, 7, 16, 17, and 19 were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) using commercially available primers from Qiagen (Valencia, CA) as described previously (Cao et al 2010;Somji et al 2010). Amplification was monitored by SYBR Green fluorescence.…”

“…The laboratory has also examined the expression of keratin 6 and its binding partner in the series of As +3 -transformed cell lines and keratin 7 in the Cd +2 -transformed cell lines (Somji et al 2010;Cao et al 2010). In the series of As +3 -derived tumors, immunostaining for keratin 6 showed that immunoreactivity was confined to areas of squamous differentiation in all the subcutaneous tumors.…”

“…The histology of subcutaneous tumor heterotransplants produced by these original isolates of Cd +2 -and As +3 -transformed UROtsa cells displayed histologic features of human urothelial carcinoma with areas of squamous differentiation. The laboratory subsequently generated six additional Cd +2 -transformed cell lines and five additional As +3 -transformed cell lines and each were shown to produce subcutaneous tumor heterotransplants with a histology of human urothelial carcinoma with areas of squamous differentiation (Somji et al 2010;Cao et al 2010). In addition to consistently producing a tumor with urothelial cancer histology, each series of Cd +2 -and As +3 -transformed cell lines were also shown to display features consistent with tumor heterogeneity.…”