Methylation of CpG Islands Was Involved in the Down-Regulation of DAZAP2 in Multiple Meyloma Cells.

Our previous studies showed that DAZAP2 (deleted in azoospermia associated protein 2) was the most profoundly downregulated gene in bone marrow mononuclear cells from multiple meyloma (MM) patients. The results implicated a role for DAZAP2 as a potential tumor suppressor involved in the origin and development of MM. To further understand the molecular mechanism underlying this deregulation, we analyzed epigenetic changes (i.e., DNA methylation) associated with DAZAP2 in MM, given that aberrant promoter methylation contributes to tumorigenesis through inducing transcriptional suppression and tumor suppressor inactivation. Bioinformatics analysis showed that there are three CpG islands in the DAZAP2 promoter region. The fragments of DAZAP2 promoter region with different length were separately PCR-amplified and inserted into the luciferase reporter, pGL2-Basic vector. The reporter constructs were transfected into COS-7 cells and their luciferase activities measured. The results showed that the fragment containing the third CpG island was the most active among the constructs tested. When this fragment was treated with M. Sss I methylase, its luciferase activity was almost lost. On the other hand, after 5-aza-2′-deoxycytidine treatment, the expression of DAZAP2 was restored in KM3 cells, an MM cell line associated with DAZAP2 downregulation. To extend these observations, bone marrow samples from MM patients and normal controls were collected and genomic DNA, RNA and proteins were extracted from each sample for RT-PCR, MSP (methylation-specific PCR) and Western blot analyses. The results were in general agreement with that reported previously, demonstrating that the protein and mRNA of DAZAP2, while detectable in 100% normal controls, were undetectable in 62.5% MM patients. The MSP data further corroborated with RT-PCR and Western blot analysis (94%), which is characterized by a negative correlation between promoter methylation and downregulation of DAZAP2 in multiple myeloma. These results indicate that promoter hypermethylation may play an important role in downregulation of DAZAP2 gene expression in multiple myeloma.