Progesterone receptor B gene inactivation and CpG hypermethylation in human uterine endometrial cancer

The expressions of two isoforms of human progesterone receptor (PR) are und er the control of the two different promoters. Recent studies revealed diff erences between these isoforms, PRA and PRB, in their expression and functi on in endometrial cells. Aberrant methylation of normally unmethylated CpG islands has been associated with inactivation of several genes in human can cers. In this study, we investigated the methylation status and the express ion of the two different PR isoforms, PRA and PRB, in uterine endometrial c arcinoma (UEC) using methylation-specific PCR (MSP), reverse transcription- PCR (RT-PCR), the 5' rapid amplification of cDNA ends method (5'RACE), and immunohistochemical staining, The results of RT-PCR and 5'RACE suggest that only PRB is inactivated, although PRA is activated in all UEC cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, restored PRB expression in all cell lines, suggesting that inactivation of this gene is through methylation. By MSP and direct DNA sequencing, PRB was methylated, whereas PRA was unmethylated in all of the cell lines. To determine the met hylation status of PRB in UEC patients, we investigated 83 cancerous and 33 normal samples. Sixty-two of 83 cancer samples had only methylated alleles of PRB, although all cancer samples had only unmethylated PRB alleles. Sev enty-one of 83 cancer samples were negative for PRB expression. All 62 canc er samples that had only methylated PRB alleles were negative for PRB expre ssion. No significant changes were observed in PRA methylation status or im munohistochemistry positivity in normal and cancer samples. To determine wh ether de novo methylation of PRB occurred in UEC patients, we studied 32 pa irs of cancer and normal samples from the same patient. Twenty of 32 cancer samples had only methylated PRB alleles, although all 32 normal samples ha d only unmethylated PRB alleles. The loss of unmethylated alleles was well correlated with negativity in immunohistochemical staining for PRB. This is the first report of the selective methylation and the subsequent silencing of PRB in uterine endometrial cancer.