Sperm protein 17 (Sp17) is a spermatozoa protein that we have previously identified to be aberrantly expressed in myeloma cells. Using a combination of RT-PCR and Northern blot analysis, we demonstrated that Sp17 showed a very restricted normal tissue expression, being detected only in normal testis, suggesting that Sp17 is a novel Cancer-Testis antigen in multiple myeloma. We subsequently showed that Sp17 expression in myeloma cell lines was regulated through promoter methylation and could be upregulated by demethylating agent such as 5-azacytidine. In the present study, we have used a combination of real time PCR and immunohistochemistry on a large panel of normal tissues to determine the pattern of differential expression of Sp17 in normal tissues and in myeloma cells. We also investigated, using restriction digest/PCR, whether or not any differential expression of Sp17 in these normal tissues is also regulated by promoter methylation.
Using real time PCR, we found that Sp17 transcripts could be detected in some normal tissues. However, the levels of expression were less than 2% of those in normal testis. In contrast, Sp17+ myeloma cells expressed 3–18% of normal testis levels of Sp17 transcripts. This results, therefore, are similar to those obtained by real time PCR in some other CT antigens in which levels of <3% were detected in some normal tissues. Immunohistochemical evaluation using two Sp17 murine monoclonal antibodies, each directed at a non-overlapping B-cell epitope, showed Sp17 protein to be detected only in testis and not any other normal tissues. Specificity of binding of the antibodies to testis was also confirmed in competitive binding assays. These combined results therefore point to a differential expression of Sp17 in normal tissues and over-expression (compared to some normal tissues) of Sp17 in some myeloma cells.
We next determined if the differential expression of Sp17 in normal tissue was in fact regulated through promoted methylation. We previously identified that the HpaII sites at -359 and −350 of the Sp17 gene were involved in the regulation of Sp17 gene expression. Demethylation at these sites resulted in Sp17 gene expression. We have, therefore, used HpaII digest/PCR across these restriction sites in six normal tissues, i.e. kidney, blood, pancreas, skeletal muscle, spleen and testis. Normal testis consistently failed to produce any PCR products when amplified across the HpaII sites after restriction digest, suggesting that these HpaII sites were demethylated. In contrast, the corresponding HpaII sites in kidney, blood, pancreas, skeletal muscles and spleen consistently produced PCR products of the expected size, indicating methylation and insensitivity to HpaII restriction digest of the gene sequence within the Sp17 promoter. Control amplification across another gene segments in all normal tissues were positive.
These results therefore provide evidence that the myeloma CT antigen, Sp17, exhibit a differential normal tissue expression that is regulated through promoter methylation.