Yoko Hosoya

Altered expression of several genes in highly metastatic subpopulations of a human pulmonary adenocarcinoma cell line

Non-small cell lung cancer is associated with approximately 85% mortality due to its high metastatic potential. Therapeutic efforts have failed to produce a significant improvement in prognosis. In this situation, a better understanding of the key factors of metastasis may be useful for designing new molecular targets of therapy. In order to identify these factors, we compared the expression profiles of two subpopulations of an adenocarcinoma cell line with a high metastatic potential, PC9/f9 and PC9/f14, with the parent cell line, PC9, using a cDNA array. The expression of 15 genes was found to be significantly enhanced or reduced in the highly metastatic subpopulations. The expression of matrix metalloproteinase-2 (MMP-2), plasminogen activator inhibitor-1 (PAI-1) and interleukin-1 (IL-1 alpha) were upregulated in the highly metastatic subpopulations, while the expression of carcinoembryonic antigen (CEA), caspase-5, Fas ligand, Prk/FNK, cyclin E, cyclin B1, Ki-67, proliferating cell nuclear antigen (PCNA), Smad4, macrophage proinflammatory human chemokine-3 alpha (MIP-3 alpha)/LARC, Met and CD44 were downregulated. Data from the literature suggest that the altered expression of MMP-2, PAI-1, IL-1 alpha, CEA, caspase-5, Fas ligand, Prk/FNK and Smad4 promotes the highly metastatic phenotype. The differential expression of these genes was confirmed by Northern blot analysis, standard reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. This analysis in subpopulations of a lung cancer cell line indicated that the highly metastatic potential of lung cancer may be induced not by an alteration in the expression of a single gene, but by the accumulation of alterations in the expression of several genes involved in extracellular matrix (ECM) adhesion disruption, ECM degradation, escape from apoptosis, and resistance to transforming growth factor-beta(1) (TGF-beta(1)). Strategies for inhibiting metastasis of pulmonary adenocarcinoma should be designed accordingly.

Microsatellite instability and loss of heterozygosity on chromosome 10 in rat mammary tumors induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine

Microsatellite instability (MI) and loss of heterozygosity (LOH) were examined in mammary tumors induced in Sprague‐Dawley × F344 F1 female rats by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). Examination of 62 microsatellite loci revealed MI in nine of 15 (60%) PhIP‐induced mammary tumors, and five of these MI‐positive tumors had mutations in more than one microsatellite locus. In contrast, two of 12 (17%) 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced mammary tumors were MI positive but had mutations at only one locus each. Further, by using 37 polymorphic markers specific LOH was observed in four of 15 PhIP‐induced mammary tumors on distal parts of rat chromosome 10, which is homologous to human chromosome 17q, with no background level of LOH. Similarly, DMBA‐induced mammary tumors showed specific LOH on the same region of chromosome 10. These data suggest that mismatch‐repair deficiency and loss of chromosome 10 are involved in carcinogenesis of PhIP‐induced rat mammary tumors.

A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity.

The lacI gene has been used as a target gene in various mutation assays. We modified single strand conformation polymorphism (SSCP) analysis by introducing restriction digestion to detect mutations in the gene rapidly, and determined the sensitivity of the method. The entire coding sequence and partial promoter region of the lacI gene were amplified by the polymerase chain reaction with [alpha-32P]dCTP in a 1247 base pair fragment, digested into eight restriction fragments, and analyzed by SSCP. The sensitivity of the method was assessed using 160 phages with lacI mutations, which were selected by assay of expression of beta-galactosidase after their infection into E. coli. Of the 160 mutants, 146 (91.3%) showed shifted bands in the first condition of SSCP analysis (without glycerol, 20 degrees C). The remaining 14 mutants were analyzed in a second condition (with 5% glycerol, 20 degrees C), and eight of them showed shifted bands (cumulatively 96.3% of the 160 mutants). The remaining six mutants were analyzed in a third condition (with 5% glycerol, 10 degrees C), and all of them showed shifted bands (cumulatively 100%). Sequencing of the restriction fragments with mobility shifts in the 160 mutants revealed 108 kinds of mutations, 100 (92.6%) being detected in the first condition, seven (cumulatively 99.1%) in the second condition, and one (cumulatively 100%) in the third condition. This method greatly reduced the time to identify lacI mutations, and allowed the detection of multiple mutations in one lacI mutant. The results also show that in general PCR-SSCP analysis is very sensitive when test fragments are shorter than about 250 base pairs and electrophoresis is performed under at least two conditions.

Alteration of the PTEN/MMAC1 gene locus in primary lung cancer with distant metastasis

The PTEN/MMAC1 gene located at 10q23, has been proposed to be a tumor suppressor gene. To determine the involvement of alteration of the PTEN/MMAC1 gene in carcinogenesis and the progression of primary lung cancers, we analyzed tumor samples of primary and distant metastatic sites and normal lung tissue samples of 30 patients with advanced lung cancer with distant metastasis. The tissues were analyzed for allelic deletion and mutational inactivation of PTEN/MMAC1 by loss of heterozygosity (LOH) analysis, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and direct sequence analysis. LOH of the PTEN/MMAC1 locus was common in each histologic type of primary lung cancer. In this study, the overall allelic deletion rate was 33.3% (7/21). Allelic loss at the primary site and that at the metastatic site of each patient, were identical; in most cases, it seemed that the allelic loss had occurred before metastasis. Sequence analysis of the PTEN/MMAC1 gene revealed a G to C substitution located 8 bp upstream of the coding region of exon 1 and which seems to be a polymorphism, in 4 of the 30 cases. Somatic mutations of the PTEN/MMAC1 gene were not identified in any of the tumors at the primary and metastatic sites. These data indicate that point mutations in the PTEN/MMAC1 gene are probably not an important factor in tumorigenesis and the progression of a major subset of lung cancers. Due to frequent allelic loss at the PTEN/MMAC1 locus occurring at a stage earlier than the metastatic process, alternative mechanisms in which the remaining allele is inactivated such as methylation or homozygous deletion of a small region of the gene that can not be detected by the usual analysis, or alteration of other important tumor suppressor genes lying close to the PTEN/MMAC1 gene on 10q23, may be involved in the tumorigenesis of lung cancers of all histologic subtypes.

Increased expression of the LGALS3 (galectin 3) gene in human non-small-cell lung cancer.

Patients with lung cancer have a poor prognosis because of the high metastatic potential of the neoplasm. Therefore, identifying new molecular targets for anti‐metastatic therapy is very important. To identify novel key factors of tumor metastasis in lung cancer, we established the gene expression profiles of two adenocarcinoma cell line variants, PC9/f9 and PC9/f14, by use of genome‐wide human cDNA microarray analysis and comparing these profiles with that of the parental cell line, PC9. The PC9/f9 and PC9/f14 cell lines were selected for analysis because of their high metastatic potential. We identified five genes in the highly metastatic cell lines that showed a significantly enhanced or reduced expression and that had not been reported to be involved in metastasis of lung cancer. One of the overexpressed genes that was identified encoded the β‐galactoside–binding protein LGALS3 (Galectin 3). LGALS3 has been reported to be overexpressed in a variety of human cancers, but not in lung cancer, and to be involved in tumor metastasis. We examined the expression of LGALS3 by use of real‐time quantitative reverse transcription–polymerase chain reaction in 38 lung cancer cell lines and in tumor tissue obtained by thoracoscopic biopsy. A population (10/30) of the non–small‐cell lung cancers examined was found to overexpress the LGALS3 gene at levels three times higher than those of normal epithelial cells. In contrast, all small‐cell lung cancers either failed to express the gene or expressed it at a very low level. The mean of the relative expression of the LGALS3 gene in non–small‐cell lung cancer (3.065 ± 3.976) was significantly higher than those of small‐cell lung cancer (0.02 ± 0.03) (P < 0.025). This is the first report of alterations of LGALS3 gene expression in lung cancer. These results, together with the previous reports on Galectin 3 function, suggest that Galectin 3 may play a role in the process of metastasis in non–small‐cell lung cancer that overexpresses Galectin 3, but not in small‐cell cancer. Accordingly, LGALS3 may be a phenotypic marker that excludes small‐cell lung cancer and may represent a novel target molecule in non–small‐cell lung cancer therapy.

The promoter region of the human BUBR1 gene and its expression analysis in lung cancer

Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers.

Reduced transcription of the RB2/p130 gene in human lung cancer

Reduced expression of the retinoblastoma gene (RB)2/p130 protein, as well as mutation of exons 19, 20, 21, and 22 of the same gene, has been reported in primary lung cancer. However, it has been suggested by other investigators that mutational inactivation and loss of the RB2/p130 gene and protein, respectively, are rare events in lung cancer. In order to determine the contribution and mechanisms of RB2/p130 gene inactivation to lung cancer development and progression, we quantified RB2/p130 mRNA expression levels in a range of human lung cancer cell lines (n = 13) by real‐time reverse transcription (RT)‐polymerase chain reaction (PCR) analysis. In comparison to normal lung tissue, reduced transcription of the RB2/p130 gene was found in all small cell lung cancer cell lines examined, along with six out of the eight nonsmall cell lung cancers tested, most of which had inactivation of RB/p16 pathway. On the basis of Western blot analysis, the expression of RB2/p130 protein was consistent with RNA expression levels in all lung cancer cell lines examined. In addition, the mutational status of the RB2/p130 gene (specifically, exons 19, 20, 21, and 22) was determined in 30 primary lung cancers (from patients with distant metastasis) and 30 lung cancer cell lines by PCR‐single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing. There was no evidence of somatic mutations within the RB2/p130 gene in the 60 lung cancer samples (both cell lines and tumors) assessed, including the 11 lung cancer cell lines that displayed reduced expression of the gene. Furthermore, hypermethylation of the RB2/p130 promoter was not found in any of the above‐mentioned 11 cell lines, as determined by a DNA methylation assay, combined bisulfite restriction analysis (COBRA). The results of the present study suggest that the reduced RB2/p130 expression seen in lung cancer may be in part transcriptionally mediated, albeit not likely via a mechanism involving hypermethylation of the RB2/p130 promoter. The observed reduction in RB2/p130 gene expression may be due to histone deacetylation, altered mRNA stability, and/or other forms of transcriptional regulation.

Reduced transcription of the Smad4 gene during pulmonary carcinogenesis in idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) have an increased risk of developing lung cancer. To identify key molecules involved in malignant transformation in IPF, we analyzed the expression profiles of lung and lung tumor tissue from patients with lung cancer and IPF (lung cancer/IPF) using cDNA arrays and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Reduced expression of the Smad4 gene was identified in all eight tumor samples from the lung cancer/IPF patients using real-time RT-PCR. Expression levels of Smad4 were significantly lower in tumors from lung cancer/IPF patients than in those from lung cancer patients without IPF or in lung cancer cell lines (p<0.01). Mutational analysis of TGF-β type II receptor and Smad4 was performed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The methylation status of the Smad4 promoter was analyzed using methylation-specific PCR with subsequent sequence analysis. No mutations were detected in the eight tumor samples, but hypermethylated regions were detected in the Smad4 promoter in two of the eight tumors with reduced Smad4 expression. Promoter reporter assays showed that the activity of the Smad4 promoter containing the sequence of the methylated region was significantly stronger than that of the Smad4 promoter with a deleted methylated region (p<0.002). Our findings indicate that the loss of the growth inhibitory response to TGF-β signaling may be crucial in pulmonary carcinogensis or in the progression of lung cancer in IPF patients in whom TGF-β is overexpressed; hypermethylation of the Smad4 promoter region may be one mechanism by which this occurs. These findings are useful for the development of preventive measures or treatment for lung cancer patients with IPF.

Mutation analysis of the gene encoding the human mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) in human cell lines resistant to growth inhibition by transforming growth factor β1 (TGF-β1)

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is involved in activating the transforming growth factor beta(1) (TGF-beta(1)), an inhibitor of the cell proliferation, and limiting the insulin-like growth factor 2 mediated-growth stimulation. The M6P/IGF2R gene has been reported to be mutated and deleted in various cancers, and is a candidate tumor suppressor gene. We studied the genomic structure of the M6P/IGF2R gene and designed the intron primers to detect mutations in the M6P/IGF2R gene of genomic DNA samples. The M6P/IGF2R gene consists of 48 exons. The previously reported 23 mutations of the M6P/IGF2R gene in human cancers, liver, breast, and gastrointestinal tumors, are located in five exons, exon 27, 28, 31, 40, 48. Using the intron primers designed in this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing, we performed an initial analysis of the complete coding sequences of the M6P/IGF2R gene in 21 human cell lines resistant to growth inhibition by TGF-beta(1). An adenine-to-guanine transition, resulting in an asparagine-to-serine amino acid substitution, was found in one lung adenocarcinoma cell line at exon 40 where the mutation has been previously reported in human cancers. This is the first report of a mutation of the M6P/IGF2R gene in lung tumor. These results indicated that the mutation in M6P/IGF2R may be involved in human lung cancinogenesis.