No abstract

The mechanisms underlying sperm protein 17 (Sp17) gene expression in myeloma cells remained unclear. Using reverse transcription -polymerase chain reaction (RT -PCR), Sp17 transcripts were detected in ARK-B, ARP-1, RPMI-8226 and KMS-11 but not in H929, IM-9, MM1-R and U266 cells. Using a panel of primer pairs in methylation-sensitive PCR to amplify overlapping gene segments, our screening studies showed that the HpaII sites at À359 and À350 are involved in the regulation of Sp17 gene expression. To confirm the differences in methylation status between Sp17-positive and Sp17-negative cell lines, KMS-11 cells (Sp17-positive) and IM-9 cells (Sp17-negative) were subjected to the more accurate method of bisulphite conversion. KMS-11 cells were more hypomethylated at these HpaII sites of exon 1 compared to IM-9 cells, indicating the association of hypomethylated promoter with Sp17 gene expression. In addition, the level of methylation at other CpG sites within the promoter sequence was also higher in IM-9 than KMS-11. Exon 1 was cloned into a reporter vector, pCAT*3 Enhancer. Chloramphenicol acetyl transferase ( Even though Sp17 shows a restricted normal tissue expression, the mechanisms regulating Sp17 aberrant expression in myeloma cells are not known. We therefore set out to determine the mechanisms of the Sp17 gene expression since such information may provide the opportunity to manipulate the gene for therapeutic purposes. Changes in gene methylation occur frequently in cells that have undergone malignant transformation. These changes are thought to be responsible for the aberrant expression of the genes encoding some CT antigens. Since Sp17 is a CT antigen, we hypothesised that Sp17 gene expression might be controlled by methylation of the promoter sequence so that the gene is silenced by DNA methylation. To test this hypothesis, we have investigated Sp17 gene expression in eight MM cell lines and