Frequent Abnormalities Of MTAP Gene In BERP35T-2 Cells And Chinese Non-small Cell Lung Cancers

5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadenosine: ortho- phosphate methylthioribosyltransferase, EC 24.2.28) plays a key role in purine and polyamine metabolism and in the regulation of transmethylation reactions. MTAP is abundant in normal cells but deficient in many kinds of tumors and tumor-derived cell lines. MTAP is located on human chromosome 9p21, where tumor suppressor genes such as pi6, p!4ARF and pi5 also map. It is believed that MTAP is frequently codeleted with pi6.By 2-Dimension Electrophoresis analysis, MTAP was observed to be down-regulated in the BERP35 cells originated from BEP2D cells irradiated by a single dose of high-energy a particles. To investigate the association of down-regulation of MTAP gene with malignancy of cells passaged from BERP35, total proteins from BEP2D cells and BERP35T-2 derived from BERP35, which shows further malignancy, were extracted and analyzed by 2-D electrophoresis. MTAP was found to be expressed at very low level in BERP35T-2 cells somewhat similar to BERP35.Northern Blot and Western Blot were performed respectively to confirm differential expression of MTAP gene between BEP2D and BERP35T-2. MTAP was found to be expressed 5 times lower at mRNA level as well as protein level in BERP35T-2 than in BEP2D.Genomic DNA was extracted from cell lines BEP2D, BERP35T-2 and 44cases of Chinese non-small cell lung cancers. No homozygous deletions and mutations were detected in BERP35T-2 cells, and no methylation was detected in the CpG island of the promoter of MTAP gene by Methylation-Specific PCR. Three STSs, D9S171, D9S162 and RH99034, flanking MTAP gene region, were analyzed by PCR amplification, and LOH (loss of heterozygosity) was found at RH99034 locus.Genomic alterations were investigated in 44 samples. Homozygous deletions of MTAP were detected in 4 of 44 NSCLC samples. No Mutation was found in all cases by SSCP. The CpG island in the promoter of MTAP gene was methylated in 3 out of 44 cases detected by MSP. Seven microsatellite loci, D9S162, D9S1749, D9S916, RH99034, D9S1748, D9S1752 and D9S977, distributing from 9p21 to 9p22, were selected to investigate frequency of LOH in 44 NSCLC samples. LOH frequencies of the seven STSs markers were 13.8%(5/36), 56.2%(18/32), 54.2%(17/33), 70.3% (19/27), 57.1%(16/28), 30.8%(8/26) and 13.3%(4/30), respectively. 26 of 42 informative samples showed at least one locus lost.15 fresh samples of the 44 cases were analyzed for MTAP mRNA and protein expression by RT-PCR and Western Blot. Down-regulation or loss of MTAP expression was found in 11 cases, showing consistency with the alterations occurring at genomic level. Furthermore, RT-PCR was performed to observe the differences between MTAP gene and tumor suppressor genes pi6, p!4 and pi5. Unexpectedly, it was found that pi6 and pi4 were expressed at higher level in 3 of 7 tumor samples respectively than in matched normal tissues, while pi5 mRNA was deficient in 5 tumor samples.The results indicate that down-regulation of MTAP in BERP35T-2 cell line caused by LOH at RH99034 may contribute to the elevating malignancy of BERP35T-2. LOH occurred at 9p21 genomic region in Chinese NSCLC may contribute more to deficiency or down-regulation of MTAP than homozygous deletions and methylations of the only CpG island in the promoter region.