SPAN-Xb expression in myeloma cells is dependent on promoter hypomethylation and can be upregulated pharmacologically.
SPAN-Xb is a novel cancer-testis antigen in multiple myeloma (MM). In this study, we determined the mechanisms regulating SPAN-Xb expression in MM. SPAN-Xb promoter sequence was first cloned into the CAT-reporter vector to determine the role of promoter methylation in the regulation of gene expression. Tumor cells were treated with 5-azacytidine and a panel of cytokines were used to determine their ability to induce SPAN-Xb expression. Bisulfite conversion with sequence analysis was applied to a panel of tumor cells and normal tissues to correlate the CpG dinucleotide hypomethylation and SPAN-Xb expression. We found that SPAN-Xb promoter function could be silenced by methylation. 5-Azacytidine induced promoter hypomethylation and resulted in SPAN-Xb expression, at both the transcript and protein levels. Hypomethylation of the CpG dinucleotides at positions -310, -307, -299 and -221 within the SPAN-Xb promoter strongly predict for SPAN-Xb expression. Both IL-7 and GM-CSF were also able to upregulate the expression of SPAN-Xb in myeloma cells, but only after the promoter sequence has been hypomethylated. Our results provide the first evidence showing the role of promoter methylation in the primary regulation of SPAN-Xb and the ability of IL-7 and GM-CSF to further enhance SPAN-Xb gene and protein expression in myeloma cells.
Wang
Zhiqing
Z
Division of Hematology and Oncology, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
Zhang
Jian
J
Zhang
Yana
Y
Lim
Seah H
SH
D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D020014:K562 Cells; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D007376:Interleukin-2; D015851:Interleukin-7; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C414403:SPANXA1 protein, human; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015500:Chloramphenicol O-Acetyltransferase; D001374:Azacitidine