近藤 征史

The insulin-like growth factor 2 (IGF2) gene is regulated in a complex manner, involving developmentally regulated use of four different promoters as well as transcriptional repression of the maternal allele due to genomic imprinting. It has been well documented that the liver is an exceptional organ in which overall transcription from the four IGF2 promoters is markedly imbalanced towards preferential paternal expression only in fetal life, this being relaxed during the postnatal period, resulting in biallelic expression thereafter. We previously reported a marked allelic-expression imbalance in the overall transcription of IGF2 in hepatocellular carcinoma (HCC), leading to preferential expression nonrandomly from the paternal allele. The study presented here, using 18 HCC specimens taken directly from patients, showed that this molecular change often reflects promoter switching from the adult P1 promoter to the fetal PZ, P3, and P4 promoters. Interestingly, however, we found that restoration of allele-specific expression of the P1 promoter nonrandomly from the paternal allele was also frequent in HCC, suggesting retention of an imprint for paternal expression from the P1 promoter of IGF2 in adult normal liver and altered availability of its modifying factor or factors in HCC. Further studies of the molecular mechanisms involved in the fluctuation of promoter usage and genomic imprinting of IGF2 are warranted to gain an insight into the biology of the liver in terms of development and oncogenesis. (C) 1997 Wiley-Liss, Inc.

Chromosome 3p is frequently deleted in various cancers including examples in the lung. A novel gene, termed FHIT, was recently isolated from the fragile site at 3p14.2, with aberrant transcripts being reported in lung cancer tumor specimens. To avoid overlooking tumor-specific altered transcripts due to contaminating normal cells in primary tumors, FHIT alterations were examined in 41 lung cancer cell lines in the present study. Lack of detectable expression or exclusive expression of aberrantly spliced transcripts, often accompanied by intragenic homozygous deletions, were observed in 7 of 24 non-small cell lung cancers (29%) but in 0 of 17 small cell lung cancers (0%). Extensive reverse transcription-PCR-single-strand conformation polymorphism analysis revealed polymorphisms and alternative splicing but failed to identify point mutations. These results suggest distinct mechanisms for FHIT alterations in lung tumorigenesis and that further studies of this interesting gene are warranted.

The chromosome region 18q21 is frequently deleted in lung cancers, Recent identification of JV18-1 at this locus led us to examine whether or not it might also be altered in lung cancers, as is the case for the closely related DPC4 tumor suppressor gene. A missense somatic mutation and a 9-bp in-frame deletion were detected in the highly conserved region of JV18-1 among 57 lung cancer specimens taken directly from patients, The total alterations in JV18-1 and DPC4, however, are not sufficient to account for all 18q21 deletions in lung cancers, These findings suggest that although JV18-1 and DPC4 may play roles in a limited fraction of lung cancers, another tumor suppressor gene may also exist in this chromosome region.

Accumulating evidence suggests that altered DNA methylation may play a role in the oncogenesis of human neoplasms, including lung cancer, The presence of aberrant hypermethylations at 3p, 9p, lip, and 17p, which are known to be hot spots for allele loss in lung cancers, is suggested to be a reflection of the existence of tumor suppressor genes in these chromosomal regions, In the present study, we investigated the methylation status of the Rb locus at 13q14 as well as that of the bcl-2 locus at 18q21 in 134 lung cancer specimens, representing all major histological subtypes, As a result, 18q21 was identified to be the fifth chromosomal region affected by frequent tumor-specific aberrant hypermethylation in lung cancers, The occurrence of aberrant hypermethylation at the bcl-2 locus at 18q21 was restricted to non-small cell lung cancers, and among non-small cell lung cancers, such epigenetic aberrations were observed most frequently in adenocarcinomas without any association with bcl-2 expression, Interestingly, allelic loss at the bcl-2 locus was also seen in 40% (7 of 17 informative cases) of adenocarcinomas; this frequency was also the highest among values for the various histological subtypes of lung cancers, These results suggest that aberrant hypermethylation at the bcl-2 locus may be a reflection of a putative tumor suppressor gene residing at 18q21, and aberrant hypermethylation might play a role in its inactivation, In contrast, altered methylation status of the Rb locus appears to be quite rare in lung cancers, if present at all.

Genomic imprinting is defined as a gamete-specific modification causing differential expression of the two alleles of a gene in somatic cells. While insulin-like growth factor 2 (IGF2) and H19 both at 11p15 are imprinted in normal lung, we observed frequent loss of imprinting (LOI) of the IGF2 and H19 genes in the lung cancer. While genomic imprinting is thought to play an important role in embryonal development and possibly in the development of certain embryonal tumors such as Wilms' tumor, these results suggest that altered imprinting may also play a role in the oncogenesis of this common cancer of adults, lung cancer.

It has been well documented that the liver is an exceptional organ in which the monoallelic expression of insulin-like growth factor 2 (IGF2) due to genomic imprinting is relaxed during the postnatal period, resulting in biallelic expression thereafter. In the present study, changes in the status of genomic imprinting were examined in 15 hepatocellular carcinomas (HCCs) as well as in 29 liver biopsies of chronic hepatitis or liver cirrhosis without clinical evidence of HCC, following screening for heterozygotes with an ApaI polymorphism in IGF2 in 34 HCCs and 80 such non-HCC cases. Extreme allelic-expression imbalance, leading to restoration of monoallelic IGF2 expression, was observed in 15 (100%) of 15 informative HCCs for the polymorphism with this monoallelic IGF2 expression appearing to be non-random from the paternal allele. Interestingly, the same allelic-expression imbalance was also present in a significant fraction of noncancerous liver specimens of patients with underlying disease known to be associated with HCC development. In contrast, the status of genomic imprinting of H19, another gene closely mapped at 11p15 under opposite imprinting, was strictly maintained in seven (100%) of seven cases informative for an RsaI polymorphism of H19. Together with the previous reports on altered genomic imprinting of IGF2 and H19 in embryonal lesions such as Wilms tumors as well as in lung cancers, the results suggest that perturbations of imprinting status occur as locus and tumor-type specific events in the development of human cancers.

Genomic imprinting at 11p15 is suggested to play a role in certain pediatric tumors such as Wilms' tumor, based on the findings of selective maternal loss of this chromosomal region. Although the allele loss at 11p15 is also frequent in a number of cancers of adults including lung, breast, and bladder cancers, possible involvement of genomic imprinting in these tumors has not been investigated extensively. p57KIP2, a newly described member of the p21 cyclin-dependent kinase (CDK) inhibitor family which is thought to negatively regulate the cell cylce at the G1 checkpoint, has been mapped to 11p15. In the present study, we searched for somatic p57KIP2 mutations in lung cancer, but failed to find such alterations. Interestingly, however, we found that the p57KIP2 gene is imprinted with maternal expression and that the maternal alleles had been selectively lost in 11 of 13 (85%) lung cancer cases carrying 11p15 deletions, this being a significant bias (P=0.01). These data provide the first evidence that genomic imprinting may play a role in the oncogenesis of not only rare pediatric tumors but also this common cancer of adults, suggesting that the imprinted p57KIP2 CDK inhibitor gene is a potential target for maternally biased 11p15 deletions.

The antibacterial activity of a new quinolone drug for oral use, balofloxacin (BLFX), against fresh clinical isolates (between November 1992 and March 1993) was investigated. Sensitivities of Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae to BLFX were distributed in the 0.1 ~1.56 μg/ml range, and the activity was more potent than that of ofloxacin (OFLX), by 2~4 times, and those of norfloxacin (NFLX) and enoxacin (ENX), by 4~8 times. BLFX exhibited activity against Haemophilus influenzae which was 4 times stronger than those of OFLX and ENX. Although the activity of BLFX against Klebsiella pneumoniae was inferior to those of OFLX and ENX, by 2~4 times, it was equivalent to that of NFLX. The clinical effects of BLFX on respiratory infectious diseases were investigated in 14 patients. The clinical efficacy was excellent in 4 and effective in 7, all of whom showed improvement in infectious symptoms. In 5 patients with pneumonia, including one with mycoplasma pneumonia and 2 with acute bronchitis particularly, the symptoms were markedly improved. Ten pyogenic bacterial strains were isolated from 10 patients, and all were eradicated in the 7 patients in whom bacteriological evaluation was possible. Mild loss of apetite and elevated S-GPT and BUN were observed, but no specific problematic side effects appeared. This new quinolone drug, for oral use, was considered to be a safe and useful in chemotherapy for respiratory infectious diseases.

Accumulating evidence suggests that deregulation of the insulin-like growth factor II (IGF2) and H19 genes at 11p15, due to loss of imprinting (LOI), plays a role in the oncogenesis of Wilms' tumors. We previously reported LOI of IGF2 in carcinomas of the lung, a common cancer of adults. We show here that LOI of H19 is also a frequent event in lung cancer development, and correlated with hypomethylation of the promoter region. Furthermore, the present study also revealed that overexpression of H19 is often associated with LOI of H19 in lung cancers retaining both parental alleles, showing a contrast to LOI in Wilms' tumors. Interestingly, in contrast to frequent LOI of the imprinted genes at 11p15, LOI was not observed for the remaining gene known to be imprinted in man, SNRPN at 15q12.

Frequent occurrence of 11p deletions has been reported for diverse types of human cancers including lung cancers and Wilms' tumors. In contrast to the well documented identification of preferential retention of the paternal allele in Wihms' tumors, no data have hitherto been available for cancers of adults. Taking advantage of the paternal allele-specific expression of IGF2 in the normal lung, we examined 79 lung cancer cases to investigate allelic loss at 11p15 and the parental origin of the retained alleles. While 11 of 36 (31%) informative lung cancer cases exhibited 11p15 deletions, only seven of these (64%) retained the paternal allele in tumors (P = 0.274), showing a contrast to the strong paternal bias in childhood tumors. Our strategy eliminates the need for parental DNAs for the determination and should be applicable to other adulthood tumors carrying 11P deletions such as breast and bladder cancers.