Sp17 gene expression in myeloma cells is regulated by promoter methylation

Br J Cancer. 2004 Oct 18;91(8):1597-603. doi: 10.1038/sj.bjc.6602160.

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 -359 and -350 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 (CAT) activity was restored in cells transfected with the recombinant plasmid, indicating the promoter function of exon 1. Exposure of Sp17-negative cell lines to the hypomethylating agent, 5-azacytidine, resulted in the upregulation of Sp17 gene expression. Our results therefore provide evidence for the regulation of Sp17 gene expression by promoter methylation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Surface
  • Antimetabolites, Antineoplastic / pharmacology
  • Azacitidine / pharmacology
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Chloramphenicol O-Acetyltransferase / metabolism
  • CpG Islands
  • DNA Methylation*
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Multiple Myeloma / genetics*
  • Multiple Myeloma / metabolism
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Neoplasm / genetics
  • RNA, Neoplasm / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription, Genetic
  • Tumor Cells, Cultured

Substances

  • Antigens, Surface
  • Antimetabolites, Antineoplastic
  • Carrier Proteins
  • RNA, Messenger
  • RNA, Neoplasm
  • SPA17 protein, human
  • Chloramphenicol O-Acetyltransferase
  • Azacitidine