Sp17 gene expression in myeloma cells is regulated by promoter methylation.
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.
Wang
Z
Z
Division of Hematology and Oncology, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
Zhang
Y
Y
Ramsahoye
B
B
Bowen
D
D
Lim
S H
SH
D000954:Antigens, Surface; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008565:Membrane Proteins; D009101:Multiple Myeloma / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000954:Antigens, Surface; D000964:Antimetabolites, Antineoplastic; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C090172:SPA17 protein, human; D015500:Chloramphenicol O-Acetyltransferase; D001374:Azacitidine