近藤 征史

There has been growing concern about the potential threat of hormone-disrupting chemicals like bisphenol A to various aspects of animal and human health. We studied the effects of bisphenol A on the Cl- secretion in human airway epithelial Calu-3 cells. Pretreatment with bisphenol A (IC50 = 60 muM, for 30 min) prevented isoproterenol (10 nM)-generated short-circuit current (I-sc) more potently than 17beta-estradiol or tamoxifen (IC50 = 1 mM). 5'-Nitro-2-(3-phenylpropylamino) benzoate-sensitive apical conductance potentiated by isoproterenol was not affected by the pretreatment with either of these estrogenic compounds. The effects of bisphenol A were simulated in I-sc responses to forskolin (10 muM) and 8-bromo-cAMP (1 mM). Nystatin permeabilization of Calu-3 monolayers revealed that bisphenol A attenuated 8-bromo-cAMP-induced basolateral K+ current, which is sensitive to clotrimazole (30 muM) and insensitive to charybdotoxin (100 nM), without affecting the apical Cl- current. Bisphenol A, but neither 17beta-estradiol nor tamoxifen, interrupted the charybdotoxin-sensitive component of I-sc stimulated by 1-ethyl-2-benzimidazolinone (1-EBIO; 500 muM). The inhibitory effects of bisphenol A on these Cl- secretory stimuli were remarkable when applied to the apical rather than the basolateral membrane. Alternatively, long-term incubation of bisphenol A (1 muM; 12-72 h) had no discernible effect on isoproterenol- and 1-EBIO-induced Cl- secretion. These findings indicate that short- term exposure to bisphenol A attenuates transepithelial Cl- secretion through inhibition of both cAMP- and Ca2+-activated K+ channels on the basolateral membrane, interacting from the cytosolic surface in Calu-3 cells.

A group of candidate tumor suppressor genes (designated CACNA2D2, PL6, 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2, and HYAL1) has been identified in a 120-kb critical tumor homozygous deletion region (found in lung and breast cancers) of human chromosome 3p21.3. We studied the effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2, and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells by recombinant adenovirus-mediated gene transfer in vitro and in vivo. We found that forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p21.3 120-kb region-deficient (homozygous) H1299 and A549 cells but not in the 3p21.3 120-kb region-heterozygous H358 and the normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, Ad-NPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 tumor xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, our results, coupled with other studies demonstrating a tumor suppressor role for the RASSSF1A isoform, suggest that multiple contiguous genes in the 3p21.3 120-kb chromosomal region may exhibit tumor suppressor activity in vitro and in vivo.

1. Recently, a patch formulation of tulobuterol, a β-adrenoceptor (AR) agonist, has been developed using a transdermal delivery system. The present study was designed to determine whether β-AR function and asthma control were affected by the sustained-released β-AR agonist. 2. Tulobuterol (2 mg) was applied daily for 8 weeks to seven patients with bronchial asthma in whom the morning dip in the peak expiratory flow (PEF) rate developed even though inhaled glucocorticoids were being taken. After treatment with tulobuterol, the early morning reduction in PEF was suppressed and PEF values were increased from 367 ± 35 to 439 ± 38 L/min (P < 0.05). The rescue use of inhaled β-AR agonists was decreased from 6.9 ± 2.0 to 1.0 ± 0.7 puffs/week (P < 0.01). Symptom scores also decreased from 8.3 ± 3.4 to 2.1 ± 1.4 score/week (P < 0.01). 3. Next, we sought to examine the effects of exposure to tulobuterol on β-AR function in guinea-pig tracheal smooth muscle. After exposure of tissues to tulobuterol (0.01-10 μmol/L) for 45 min, the inhibitory effects of tulobuterol on methacholine-induced contractions were attenuated in a concentration-dependent manner. However, the inhibitory effects of tulobuterol were not affected after exposure to 0.01 μmol/L tulobuterol (a concentration greater than serum levels in clinical use). In contrast, the inhibitory effects of procaterol were not affected after exposure to tulobuterol under the same experimental conditions. 4. These results indicate that the combination of sustained-released tulobuterol with inhaled glucocorticoid therapy is beneficial to patients with bronchial asthma who suffer from symptoms induced by the morning dip in PEF. Moreover, chronic exposure to lower concentrations of tulobuterol does not lead to desensitization of β-AR in airway smooth muscle.

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them into lung cancer NCl-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30-40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. We found that lung cancers (n = 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of several lung cancer lines 30-90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5' region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. These results are consistent with SEMA3B functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.

Recently we identified FUS1 as a candidate tumor suppressor gene (TSG) in the 120 kb 3p21.3 critical region contained in nested lung and breast cancer homozygous deletions. Mutation of FUS1 is infrequent in lung cancers which we have confirmed in 40 other primary lung cancers. In addition, we found no evidence for FUS1 promoter region methylation. Because haplo-insufficiency or low expression of Fus1 may play a role in lung tumorigenesis, we tested the effect of exogenously induced overexpression of Fus1 protein and found 60-80% inhibition of colony formation for non-small cell lung cancer lines NCI-H1299 (showing allele loss for FUS1) and NCI-H322 (containing only a mutated FUS1 allele) in vitro. By contrast, a similar level of expression of a tumor-acquired mutant form of FUS1 protein did not significantly suppress colony formation. Also, induced expression of Fus1 under the control of an Ecdysone regulated promoter decreased colony formation 75%, increased the doubling time twofold, and arrested H1299 cells in G1. In conclusion, our data are consistent with the hypothesis that FUS1 may function as a 3p21.3 TSG, warranting further studies of its function in the pathogenesis of human cancers.

Background: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. Methods: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell Lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures, We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. Ail statistical tests were two-sided. Results: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 63% of breast cancer lines, in 30% of primary NSCLCs, and in 39% of primary breast tumors but in none of the nonmalignant lung tissues, RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046), Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. Conclusion: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MS-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences, BS contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI(-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

We used overlapping and nested homozygous deletions, contig building, genomic sequencing, and physical and transcript mapping to further define a similar to 630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor genes (TSGs) on chromosome 3p21.3, This location was identified through somatic genetic mapping in tumors, cancer cell lines, and premalignant lesions of the lung and breast, including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize, and annotate a set of 25 genes that likely constitute the complete set of protein-coding genes residing in this similar to 630-kb sequence. A subset of 19 of these genes was found within the deleted overlap region of similar to 370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal similar to 120-kb segment and 11 genes lying in the distal similar to 250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Four penes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (CACNA2D2/alpha2 delta -2, SEMA3B [formerly SEMA(V) BLU, and HYAL1] or small cell lung cancer (SEMA3B, BLU, and HYAL1) cell lines. We found six of the genes to have two or more amino acid sequence-altering mutations including BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, the putative lung cancer TSG in this location may either be inactivated by tumor-acquired promoter hypermethylation or belong to the novel class of haploinsufficient genes that predispose to cancer in a hemizygous (+/-) state hut do not show a second mutation in the remaining wild-type allele in the tumor. We discuss the data in the context of novel and classic cancer gene models as applied to lung carcinogenesis. Further functional testing of the critical genes by gene transfer and gene disruption strategies should permit the identification of the putative lung cancer TSG(s), LUCA. Analysis of the similar to 630-kb sequence also provides an opportunity to probe and understand the genomic structure, evolution; and functional organization of this relatively gene-rich region.

Smad family members are essential intracellular signaling components of the transforming growth factor-beta (TGF-β) superfamily involved in a range of biological activities. Two highly homologous molecules, Smad2 and Smad3, have so far been identified as receptor-activated Smads for TGF-β signaling and have become the focus of intensive studies. However, no definite differences in regulation or function have been established between these TGF-β signaling molecules. In the present study, we show that the expression of Smad3, but not its dose relative, Smad2, is down-regulated by TGF-β mediated signals themselves in human lung epithelial cells. This down-regulation of Smad3 by TGF-β treatment did not appear to result from shortening of the half-life of Smad3 mRNA. Constitutive expression of Smad3 in the presence of TGF-β induced apoptotic cell death, with an adverse effect on the cell growth of human lung epithelial cells. Apoptotic cell death could also be induced by forced expression of Smad2 in the presence of TGF-β, but less efficiently than by that of Smad3. These findings clearly define the distinctions between Smad2 and Smad3 for the first time in that a qualitative difference was observed with regard to the regulation of their expression in response to TGF-β, while Smad2 and Smad3 appeared to have quantitatively different capabilities regarding the induction of apoptotic cell death in human lung epithelial cells.

We examined 61 lung cancer cases to determine whether alterations of p73, a novel monoallelically expressed p53-like molecule, may be involved in the pathogenesis of lung cancer, Allelic loss at the p73 locus at 1p36.33 mas observed in 42% (11 of 26 informative eases), and squamous cell carcinoma tended to carry this lesion most frequently, Somatic mutations in the p73 gene itself, however were not detected, despite our extensive search, We found interindividual difference in the allelic expression of p73 in normal lung, as well as intertissue variance, even within the same individual, hut preferential loss or the expressed allele appeared to be an unlikely mechanism for p73 inactivation, This study, consequently, suggests the presence of an as yet unidentified tumor suppressor gene or genes within the subtelomeric region of 1p, warranting further studies aimed at its isolation.

In previous reports, we described that DPC4/Smad4 and Smad2 are mutated in a fraction of human lung cancers and suggested possible roles of the downstream mediators of transforming growth factor-beta (TGF-beta)-elicited signals in the pathogenesis of this most common cancer. In the present study, we investigated whether another downstream mediator, human TGF-beta-activated kinase I (hTAKI), also is altered in lung cancer. For this purpose, the hTAKI gene was cloned with the aid of an expression sequence tag database search and cDNA library screening, and hTAKI was found to be expressed ubiquitously in 2 distinct isoforms regulated in a tissue-specific manner in fetal and adult normal tissues. Interestingly, hTAKI was assigned to the chromosome region 6q14-21, which is deleted frequently in various human malignancies, including lung cancer. Despite our extensive search for alterations in 39 lung cancer specimens as well as in 16 lung cancer cell lines, somatic mutations of hTAKI were not identified, indicating that hTAKI itself is not a frequent target for genetic alterations in lung cancer. (C) 1998 Wiley-Liss, Inc.

Accumulating evidence suggests that the p53 gene is a good target for molecular epidemiological studies. We previously reported an association between the presence of p53 mutations and lifetime cigarette consumption. Although over 675 p53 mutations have been reported in lung cancers in the literature thus far, very little is known about the nature of such changes in lung cancers in the absence of a smoking background. In the present study, we therefore analysed 69 non-small-cell lung cancer specimens from individuals without any history of active smoking and identified p53 mutations in 26% of the cases. Statistical analysis of the present cohort of non-smokers also showed absence of significant relationship between p53 mutations and age, sex, histological type or disease stage. Comparison of mutational spectra between the present results in non-smokers and previously reported mutations in smokers clearly demonstrated G:C to T:A transversions to be significantly less frequent in non-smokers than in smokers (OR 5.35, 95% CI 1.77 - 16.12). Interestingly, G:C to C:G and G:C to A:T mutations were also observed in tumours of non-smokers at similar frequencies to G:C to T:A mutations, suggesting that these mutations can occur relatively frequently in the absence of active smoking. This study is, to our knowledge, the largest so far analysing a well-defined cohort of non-smokers in a single laboratory.

Novel human epithelial cell lines retaining characteristic features of normal peripheral airway cells were established by transfecting the SV40 large T antigen gene into primary in vitro outgrowths from normal peripheral lung specimens. These lines, designated as HPL1A to HPL1E, showed the polygonal shapes typical of epithelial cells and expressed cytokeratin in abundance. Ultrastructural examination revealed the presence of microvilli, multivesicular bodies, and multilamellar body-like structures that are characteristic of type II pneumocytes, but expression of CC10 transcripts, a highly specific marker for Clara cells, was also observed. Response to transforming growth factor β, epidermal growth factor (EGF), and hepatocyte growth factor, all of which are thought to be important growth-regulatory molecules for cellular proliferation and developmental processes of peripheral lung, was apparent. In the HPL1A case, markedly altered cell morphology and cytoskeletal organization, potent inhibition of cell growth, and increased expression of an extracellular matrix protein were noted with transforming growth factor β. Interestingly, both EGF and hepatocyte growth factor stimulated anchorage-dependent growth, whereas only EGF could sustain anchorage-independent proliferation. The HPL1 lines are, to our knowledge, the first series of stable epithelial lines of human peripheral lung to be described. They should be valuable for investigating various aspects of growth regulation and oncogenic processes, including the mechanisms of acquisition of anchorage independence and the interrelationships of genetic changes identified previously in lung cancers. In addition, the HPL1 lines may also prove useful for development of in vitro models for other human lung disorders as well as to elucidate the mechanisms of peripheral lung differentiation.

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.