Decreased expression of KAI1/CD82 metastasis suppressor gene is associated with loss of heterozygosity in melanoma cell lines.
KAI1/CD82, a metastasis suppressor gene of prostate cancer, is located on the human chromosome 11p11.2. Down-regulation of KAI1/CD82 during tumor progression and metastasis has been reported in several cancers, but the mechanism of this down-regulation remains unknown. The relationship between down-regulation of KAI1/CD82 mRNA expression and KAI1/CD82 gene alterations in human melanoma cell lines were investigated. The promoter methylation status was examined after a 331-bp GC-rich fragment of the promoter region was amplified in G361, SK-MEL-24 and SK-MEL-28 cell lines treated with bisulfite. In order to detect methylated CpGs in all three cell lines, 331-bp fragments were sequenced. To examine the restoration of KAI1/CD82 mRNA and protein expression, the cells were exposed to methylase inhibitor, 5-aza-2'-deoxycytidine (5-AzaC). Bisulfite-sequencing data showed no methylation in G361 and SK-MEL-24 cells, and slight methylation in SK-MEL-28 cells at CpG sites 23-26 in the promoter. Real-time PCR and flow cytometry analysis showed that 5-AzaC-treated cells restored KAI1/CD82 mRNA and protein expression in SK-MEL-24 and SK-MEL-28 cells, compared to the controls. The restoration of KAI1/CD82 mRNA and protein expression detected no significant difference between SK-MEL-24 and SK-MEL-28 cells. This means that 5-AzaC did not affect the methylated cells only. Loss of heterozygosity (LOH) at polymorphic microsatellite loci on the human chromosome 11 in the human melanoma cells was also examined. Microsatellite analysis showed LOH at D11S1344 in SK-MEL-24 and SK-MEL-28 cells, and G361 showed allelic imbalance. In conclusion, this study suggests that down-regulation of KAI1/CD82 mRNA expression in human melanoma cell lines is related to LOH or allelic imbalance, but not to methylation of the KAI1/CD82 gene region.
Kim
Young Il
YI
East-West Medical Research Institute, Kyunghee University, College of Medicine, Seoul 130-702, Korea.
Shin
Min-Kyung
MK
Lee
Jin-Woo
JW
Chung
Joo-Ho
JH
Lee
Mu-Hyoung
MH
D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D015536:Down-Regulation; D005434:Flow Cytometry; D015870:Gene Expression; D016147:Genes, Tumor Suppressor; D006801:Humans; D051861:Kangai-1 Protein / Q000096:biosynthesis* / Q000235:genetics*; D019656:Loss of Heterozygosity; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
C497688:CD82 protein, human; D051861:Kangai-1 Protein; D012333:RNA, Messenger