Differences in the epigenetic regulation of MT-3 gene expression between parental and Cd+2 or As+3 transformed human urothelial cells

Abstract

Background: Studies have shown that metallothionein 3 (MT-3) is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2) or arsenite (As+3).The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine.

Results: The histone deacetylase inhibitor MS-275 induced MT-3 mRNA expression in both parental UROtsa cells and their transformed counterparts. The demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC) had no effect on MT-3 mRNA expression. ChIP analysis showed that metal-responsive transformation factor-1 (MTF-1) binding to metal response elements (MRE) elements of the MT-3 promoter was restricted in parental UROtsa cells, but MTF-1 binding to the MREs was unrestricted in the transformed cell lines. Histone modifications at acetyl H4, trimethyl H3K4, trimethyl H3K27, and trimethyl H3K9 were compared between the parental and transformed cell lines in the presence and absence of MS-275. The pattern of histone modifications suggested that the MT-3 promoter in the Cd+2 and As+3 transformed cells has gained bivalent chromatin structure, having elements of being "transcriptionally repressed" and "transcription ready", when compared to parental cells. An analysis of MT-3 staining in urinary cytologies showed that a subset of both active and non-active patients with urothelial cancer shed positive cells in their urine, but that control patients only rarely shed MT-3 positive cells.

Conclusion: The MT-3 gene is silenced in non-transformed urothelial cells by a mechanism involving histone modification of the MT-3 promoter. In contrast, transformation of the urothelial cells with either Cd+2 or As+3 modified the chromatin of the MT-3 promoter to a bivalent state of promoter readiness. Urinary cytology for MT-3 positive cells would not improve the diagnosis of urothelial cancer, but might have potential as a biomarker for tumor progression.

Figure 1

Expression of MT-3 mRNA in UROtsa parent, Cd⁺² and As⁺³ transformed cell lines. UROtsa parent and the transformed cell lines were seeded at a 1:10 ratio in the presence of MS-275 till they reached confluency and the expression of MT-3 was determined by RT-PCR analysis. A. Real time PCR analysis of MT-3 in UROtsa parent cells. B. Real time PCR analysis of MT-3 in Cd⁺² transformed UROtsa cells. C. Real time PCR analysis of MT-3 in As⁺³ transformed UROtsa cells. The expression of MT-3 was normalized to that of β-actin. The determinations were performed in triplicates and the results shown are the mean ± SE. * Statistically significant compared to untreated control cells. D. Ethidium bromide stained gel showing the expression of MT-3 in the UROtsa cell lines by semiquantitativePCR.

Figure 2

Expression of MT-3 in parent, Cd⁺² and As⁺³ transformed cells after removal of MS-275. Cells were cultured with 10 μM MS-275 until they reached confluency after which the drug was removed and the cells were allowed to recover for 24 h following which RNA was extracted from the recovered cells. A. RT-PCR analysis of MT-3 expression in UROtsa parent cells. B. RT-PCR analysis of MT-3 expression in Cd⁺² transformed UROtsa cells. C. RT-PCR analysis of MT-3 expression in As⁺³ transformed UROtsa cells. Graphs represent real time RT-PCR data whereas ethidium bromide stained gels show the semiquantitative PCR analysis data. The expression of MT-3 was normalized to that of β-actin. The determinations were performed in triplicates and the results shown are the mean ± SE.*Statistically significant compared to untreated control cells.

Figure 3

Effect of Zn⁺² on MT-3 levels in the parent and transformed UROtsa cell lines. UROtsa parent and the transformed cell lines were seeded at a 1:10 ratio in the presence of MS-275 until they reached confluency, following which the cells were allowed to recover for 24 h without the drug. The cells were then exposed to 100 μM zinc for 24 h and RNA was extracted. A. Real time RT-PCR analysis of MT-3 expression in UROtsa parent cells. B. Real time RT-PCR analysis of MT-3 expression in Cd⁺² transformed UROtsa cells. C. Real time RT-PCR analysis of MT-3 expression in As⁺³ transformed UROtsa cells. The expression of MT-3 was normalized to that of β-actin. The determinations were performed in triplicates and the results shown are the mean ± SE.*Statistically significant compared to untreated control cells. D. Ethidium bromide stained gel showing the expression of MT-3 in the UROtsa cell lines using semiquantitativePCR.

Figure 4

Schematic illustration of the MT-3 promoter. The distributions of the MREs and the designated amplified regions are indicated.

Figure 5

MT-3 promoter (region 2) associated histone modifications in the cell lines after treatment with MS-275. A-D. ChIP-qPCR of acetyl H4, trimethy H3K4, trimethyl H3K9 and trimethyl H3K27 at the MT-3 promoter using primers for region 2. The amplification value of the immunoprecipitated DNA was normalized to percentage of input (non-precipitated DNA). The determinations were performed in triplicates and the results shown are the mean ± SE.*Statistically significant compared to untreated control cells. E. Ethidium bromide stained gel showing the amplification of the histone modifications using semiquantitative PCR.

Figure 6

MT-3 promoter (region 1) associated histone modifications in the cell lines after treatment with MS-275. A-D. ChIP-qPCR of acetyl H4, trimethy H3K4, trimethyl H3K9 and trimethyl H3K27 at the MT-3 promoter using primers for region 1. The amplification value of the immunoprecipitated DNA was normalized to percentage of input (non-precipitated DNA). The determinations were performed in triplicates and the results shown are the mean ± SE.*Statistically significant compared to untreated control cells. E. Ethidium bromide stained gel showing the amplification of the histone modifications using semiquantitative PCR.

Figure 7

Recruitment of MTF-1 to the MREs of the MT-3 promoter after treatment with MS-275. UROtsa parent and transformed cells were seeded at a 1:10 ratio and were grown in the presence of 10 μM MS-275 until they reached confluency. They were then exposed to 100 μM Zn⁺² for 4 h and the chromatin was immunoprecipitated with the MTF-1 antibody. PCR was performed with primers specific to each region as specified in Table 2 and Figure 4 to assess the level of precipitated sequences. A. ChIP-q PCR analysis of MTF-1 recruitment to MRE a and MREb. B. ChIP-q PCR analysis of MTF-1 recruitment to MRE-c. C. ChIP-q PCR analysis of MTF-1 recruitment to MREe, MREf and MREg. The amplification value of the immunoprecipitated DNA was normalized to percentage of input (non-precipitated DNA). The determinations were performed in triplicates and the results shown are the mean ± SE.*Statistically significant compared to untreated control cells. Graphs represent real time PCR data whereas ethidium bromide stained gels represent semiquantitative PCR data.

Figure 8

Kaplan Meier plot demonstrating prolonged survival of MT-3 negative bladder cancer. The survival distributions between MT-3 positive (solid line) and MT-3 negative (dotted line) bladder cancer patients. The data shows a statistically significant difference (Log Rank 5.067, p = 0.024; Tarone-Ware 5.071; p = 0.024).