Abstract
BackgroundPTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.ResultsUsing a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.ConclusionsWe show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.
Highlights
Phosphatase and tensin homologue (PTEN) is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers
The regions examined for methylation, the position of these regions relative to the PTEN and KLLN genes, and the type of assay used are described in Figure 1 and Table 1
Using combined bisulphite restriction analysis (COBRA) we found that the KLLN gene body, as well as the KLLN and PTEN transcription start sites (TSS) were completely unmethylated in all 36 cancer cell lines analysed (Figure 2)
Summary
PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. Mutation of the PTEN tumour suppressor gene occurs in various sporadic cancers, including 38% of Promoter CpG island hypermethylation, which can result in the transcriptional silencing of gene expression, is an alternative mechanism of gene inactivation. These challenges arise due to the fact that the PTENP1 pseudogene ( known as PTEN2), shares 97.8% sequence identity with the PTEN mRNA sequence, and 91% identity with a 921 bp region of the PTEN CpG island. Most previous studies have chosen to analyse PTEN expression using immunohistochemistry [12,13], PTEN-specific RT-PCR assays that utilise sequence variations between the PTEN and PTENP1 genes have been described [14]
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