4.BIOLOGY DIVISION
Isolation of a Noble Lung Cancer Suppressor Gene,MYO18B
Loss of heterozygosity on chromosome 22q has been detected in approximately 60% of advanced non-small cell lung carcinoma (NSCLC) as well as small cell lung carcinoma (SCLC), suggesting the presence of a tumor suppressor gene on 22q that is involved in lung cancer progression. Here, we isolated a myosin family gene, MYO18B, located at chromosome 22q12.1 and found that it is frequently deleted, mutated, and hypermethylated in lung cancers (78). Somatic MYO18B mutations were detected in 19% (14/75) of lung cancer cell lines and 13% (6/46) of primary lung cancers of both SCLC and NSCLC types. MYO18B expression was reduced in 88% (30/34) of NSCLC and 47% (8/17) of SCLC cell lines. Its expression was restored by treatment with 5-aza-2*-deoxycytidine in 11/14 cell lines with reduced MYO18B expression, and the promoter CpG island of the MYO18B gene was methylated in 17% (8/47) of lung cancer cell lines and 35% (14/40) of primary lung cancers. Furthermore, restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth. These results indicate that the MYO18B gene is a strong candidate for a novel tumor suppressor gene whose inactivation is involved in lung cancer progression.
Genetic and Epigenetic Alterations of Tumor Suppressor Genes
Association of RASSF1 methylation with the clinicopathological features, allelic imbalance at 3p21.3, p53 mutations, and K-ras mutations was examined in stage I lung adenocarcinomas (79). RASSF1 methylation was detected in 35/110 (32%) adenocarcinomas, and correlated with adverse survival. The correlation between RASSF1 methylation and allelic imbalance at 3p21.3 was significant, whereas the correlation between RASSF1 methylation and p53 mutations was borderline. However, there was no correlation or inverse correlation between RASSF1 hyper-methylation and K-ras mutations. These results indicate that the epigenetic inactivation of RASSF1 plays an important role in the progression of lung adenocarcinoma, and that RASSF1 methylation is a useful molecular marker for the prognosis of patients with stage I lung adenocarcinoma. Among six osteosarcoma cell lines, both the p16INK4a and p14ARF genes were homozygously deleted in two cell lines and hypermethylated in one cell line (80). Two of the candidate tumor suppressor genes, ATF-2 and ICAT, were not inactivated in lung cancer and colon cancer, respectively (81,82). The Met832 variant of the E-cadherin gene was detected as a possible germline mutation among 3 gastric cancer patients in a case of familial gastric cancer (83). A simple method, Gene Specific-Primer Extension Preamplification (GS-PEP), to increase the copy number of genomic DNA fragments for a gene of interest prior to PCR amplification, was developed for the mutational analysis of various genes in cancer cells using small amounts and/or low quality of DNA (84).
Molecular Mechanism of Chromosome 9p21 Deletions in Lymphoid Leukemia
To understand the molecular pathways underlying 9p21 deletions, which inactivate the p16INK4a and p15ARF tumor suppressor genes, in lymphoid leukemia, breakpoints were cloned from 15 lymphoid leukemia cell lines (85). The sequences of breakpoint junction indicated that the majority of deletions were mediated by illegitimate V(D)J recombination. Moreover, those sequences have V(D)J recombination potentials. These results indicate that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, is a major cause of 9p21 deletions in lymphoid leukemia.
Genetic Polymorphisms and Lung Cancer Susceptibility
Male smokers possessing the wild type CYP2A6 gene had high risk for tobacco-induced lung squamous cell carcinoma and small cell carcinoma (86). It was revealed that NQO1-Pro/Pro and GSTT1-null genotypes are risk factors for lung adenocarcinoma (87). It was also revealed that the human PAS1 locus is located in the vicinity of D12S1034 and a genetic variation at this locus is involved in susceptibility to lung adenocarcinoma (88).
Functional Analysis of Cancer-related Genes
The EVI1 gene was expressed in a megakaryocyte cell lineage and enforced expression of this gene in UT-7/GM cells induced megakaryocyte differentiation (89). Thus, it was suggested that the EVI1 gene is involved in the progression of megakaryocytic differentiation and dysmegakaryocytopoiesis in the 3q21q26 syndrome could be partly due to the differ-entiation capacity of leukemia cells and/or megakaryocytes by constitutive expression of the EVI1 gene.
Since genetic polymorphisms of DNA repair genes associated with the activities and expression levels of their products may modulate cancer susceptibility of individuals, the effect of IVS1+5G/C SNP in the MYH gene on the difference in the expression levels of its products was investigated (90). This SNP resulted in reduced translation efficiency of its transcripts. This was the fourth case of SNPs that cause alterations in translation initiation sites and translation efficiencies in human cells.
Establishment of High- and Low-metastatic Human Cancer Cell Lines
Relevant animal models for metastasis of lung cancer and osteosarcoma cell lines are needed to understand the biology and to develop the treatment modality of cancer metastasis. Since the HuO9 osteosarcoma cell line was metastasized to the lung after intravenous injection, two sublines with high metastatic potential to the lung were established from the parental cells by in vivo selection. These sublines were useful for studies on the treatment of pulmonary metastatic osteosarcoma (91). High-and low-metastatic sublines were also isolated from the A549 human lung adenocarcinoma cell line (92).