Loss of mismatch repair activity in simian virus 40 large T antigen-immortalized BPH-1 human prostatic epithelial cell line

Area of Science:

• Molecular Biology
• Cell Biology
• Cancer Research

Background:

• Simian virus 40 large T antigen (SVLTAg) is known to immortalize cells, but the underlying mechanisms remain incompletely understood.
• DNA mismatch repair (MMR) is crucial for maintaining genomic stability by correcting errors during DNA replication.

Purpose of the Study:

• To investigate the role of DNA mismatch repair (MMR) activity in SVLTAg-induced cellular immortalization.
• To analyze MMR activity and the expression of MMR genes in the SVLTAg-immortalized human prostate epithelial cell line BPH-1.

Main Methods:

• Analysis of MMR activity in BPH-1 cells using bacteriophage M13mp2 for mispair repair assays.
• Reverse-transcription polymerase chain reaction (RT-PCR) to assess the expression levels of MMR genes (hMLH1, hPMS1, hPMS2, hMSH2, hMSH3, hMSH6).
• Demethylation treatment with 5-aza-2'-deoxycytidine to investigate the role of hypermethylation.
• Western blotting and exon analyses to examine hMLH1 gene status.

Main Results:

• BPH-1 cells exhibited deficiency in repairing G:T, A:C, and G:G mispairs.
• Low expression levels of MMR genes hMSH3, hMSH6, and hPMS1 were observed in BPH-1 cells compared to normal prostate tissues.
• Demethylation did not restore the expression of downregulated MMR genes, ruling out hypermethylation as the cause.
• The hMLH1 gene, despite being expressed, was found to be mutated and/or deleted in BPH-1 cells.

Conclusions:

• SVLTAg-induced immortalization of human prostate epithelial cells is associated with significant deficiencies in DNA mismatch repair (MMR).
• The observed MMR deficiency in BPH-1 cells is linked to the low expression of hMSH3, hMSH6, and hPMS1, and mutation/deletion of hMLH1.
• These findings suggest that impaired MMR activity plays a critical role in the immortalization process mediated by SVLTAg.