[Arf6, RalA and BIRC5 protein expression in non small cell lung cancer].

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.

[Down-regulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1 and HYAL2 genes in non-small cell lung cancer].

Chromosomal and genome abnormalities of 3p are frequent events in many epithelial tumours, including lung cancer. Several critical regions with high frequency of hemi--and homozygous deletions in tumours were detected on 3p and more then 20 different cancer-related genes were identified in 3p21.3 locus. Real-time PCR was used to measure mRNA level of tumour-suppressor genes and candidates in 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1 and HYAL2 in basic types of non-small cell lung cancer (NSCLC)--squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). Significant (from 2 to 100 times) and frequent (from 44 to 100%) mRNA level decrease was shown in NSCLC. Level and frequency of mRNA decrease for all genes depended on histological type of NSCLC. Down-regulation of RASSF1A and ITGA9 was associated significantly with AC progression, the same tendency was found for genes RBSP3/CTDSPL, NPRL2/G21, HYAL1 and HYAL2. On the contrary, down-regulation of all genes in SCC was not associated with clinical stages, tumor cells differentiation and metastases in lymph nodes. Significant decrease of RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1 and HYAL2 mRNA levels (on average, 5-13 times) with high frequency (83-100%) was already shown at the first stage of SCC. Simultaneous decrease of all six genes mRNA level was found in the same tumor samples and was not depended on their localization on 3p21.3 and functions of the proteins. Spearman's correlation coefficient r(s) was from 0.63 to 0.91, P < 0.001. Co-regulation of gene pairs ITGA9 and HYAL2, HYAL1 and HYAL2, which mediate cell-cell adhesion and cell-matrix interaction, was suggested based on the obtained data. It was shown that genetic and epigenetic mechanisms were important for down-regulation of RBSP3/CTDSPL and ITGA9 genes. These results supported the hypothesis on simultaneous inactivation of cluster cancer-related genes in extended 3p21.3 locus during development and progression of lung cancer and other epithelial tumors. Significant and frequent decrease of mRNA level of six genes in SCC could be important for development of specific biomarker sets for early SCC diagnosis and new therapeutic approaches/strategies for NSCLC.

[Suppression of WIFI transcript and protein in non-small cell lung carcinomas].

Changes in WIFI expression, an extracellular inhibitor of Wnt pathway, in non-small cell lung carcinomas were analyzed. Frequent (67% cases) suppression of WIFI transcript in non-small cell lung carcinomas were found. Our results, together with previously published data, suggest that inhibition of WIFI expression often occurs in squamous cell carcinomas and is less typical of adenocarcinomas. It was also found that a decrease in the WIFI transcript in tumors is parallel to concomitant suppression of the WIFI protein level. Our results provide further evidence that the WIFI suppression is a frequent event in the lung carcinogenesis, which might lead to disregulation of Wnt signaling pathway and contribute to tumor progression.

[c-Met and HGF expression in non-small-cell lung carcinomas].

Alterations in the c-Met/HGF system are frequently observed in various types of tumors and can directly influence tumor progression. In particular, the c-Met/HGF system can influence progression of lung tumors. The altered c-Met and HGF expression occurs in non-small-cell lung carcinomas. In this work we analyzed changes in c-Met and HGF expression in non-small-cell lung carcinomas by comparing expression levels with those in adjacent non-malignant tissues using semi-quantitative PCR. The c-Met transcript was detected in 50% of squamous cell carcinoma samples with an elevated level observed in 2 out of 25 cases (8%). HGF expression was detected only in two squamous cell carcinomas. At the same time, the c-Met transcript was observed in all 5 studied adenocarcinoma samples with an increased level compared to adjacent non- malignant tissue in 3 cases. HGF transcript was found in 2 adenocarcinoma samples. Therefore, c-Met rather than HGF transcript is frequently observed in non-small-cell lung carcinomas, especially in adenocarcinomas. According to the results of other studies, the c-Met transcript can serve as an indicator of the aggressive behavior and progression of non-small-cell lung carcinomas.

[Transcription TIMP3, DAPk1 and AKR1B10 genes in squamous cell lung cancer].

Lung cancer is one of the most frequent neoplasia in the Russia, the United States and Europe. This cancer is associated with functional activity changes of many genes. In the present study TIMP3, DAPK1 and AKR1B10 genes transcription analysis of squamous cell lung cancer specimens was carried out using reverse transcription-PCR. Substantial increasing of AKR1B10 transcription level is revealed in 80% tumor samples. TIMP3 and DAPK1 transcription level is considerably decreased in 76 and 72% tumor specimens, accordingly. These results may point out that all three genes are important for squamous cell lung cancer tumorogenesis while AKR1B10 is potential oncogene whereas TIMP3 and DAPK1 are potential tumor suppressor genes. We suggest that revealed substantial transcription level-changes of investigated genes may be used for oncodiagnostics.

[Analysis of polymorphic variants of gene GIPC1 CGG repeats in healthy individuals and in patients with breast cancer and non-small cell lung cancer].

The GIPC1 gene product promotes clustering of some transmembrane receptors, including those involved in carcinogenesis, and protects them against ubiquitin-dependent degradation. The 5' untranslated region of GIPC1 contains a polymorphic trinucleotide CGG repeat, which has not been characterized earlier. In the present study, we have carried out comparative analysis of the allele and genotype frequencies of this repeat in 129 samples of breast cancer (BC), 58 samples of non-small cell lung cancer (NSCLC), and 215 samples of healthy donors. The CGG repeat in the 5' untranslated GIPC1 gene region was shown to be highly polymorphic and represented by at least eight alleles. Alleles CGG10-13 were major, occurring at frequencies of 22, 41, 27, and 9%, respectively; the total frequency of the remaining alleles was approximately 1%. Heterozygosity of the CGG repeat was 0.70. Allele CGG12 was shown to be associated with high risk of developing NSCLC (alpha = 0.05).

[Abnormal methylation of p16/CDKN2A AND p14/ARF genes GpG Islands in non-small cell lung cancer and in acute lymphoblastic leukemia]. / Sravnitel'nyi analiz anomal'nogo metilirovaniia CpG-ostrovkov, raspolozhennykh v promotornykh oblastiakh genov p16/CDKN2A i p14/ARF, pri nemelkokletochnom rake legkogo i ostrom limfoblastnom leikoze.

Multiplex methylation-sensitive PCR and methylation-specific PCR were employed in studying the methylation of CpG islands in the p16/CDKN2A and p14/ARF promoter and the first exon regions in non-small cell lung cancer (54 samples) and acute B-cell lymphoblastic leukemia (61 samples). Differences in methylation were detected between types of neoplasia as well as between CpG islands studied within the same types of tumors. High level of the p16/CDKN2A first exon CpC island methylation was revealed in non-small cell lung cancer (68%) and in acute B-cell lymphoblastic leukemia (55%) and the CpG island of p14/ARF first exon was nonmethylated in these types of tumors. The methylation of CpG-rich fragments of genes p16/CDKN2A and p14/ARF promoters was analysed. As was found out, CpG islands located in 5' areas of one and the same gene can differ in methylation frequencies. The comparison of sensitivity between methylation-specific PCR and methylation-sensitive PCR used in the methylations studies was carried out.

[Molecular markers of various stages of nonsmall cell lung cancer development]. / Molekuliarnye markery razlichnykh stadii razvitiia nemelkokletochnogo raka legkogo.

Studies performed in the recent decade in the Laboratory of the Regulation of Cell and Virus Oncogenes associated structural and functional defects of several oncogenes and tumor suppressor genes with various stages of non-small-cell lung cancer. High risk of lung cancer was established for carriers of rare alleles of the Hras1 minisatellite, the hypermethylated p16INK4A promoter, and microsatellite defects in chromosome regions 3p12, 3p14.2, 3p22-24, 3p21, 3p25, 9p21, and 17p13. Analysis of the Hras1 minisatellite and microsatellites located in two minimal deletion overlap regions of 1p36 was shown to allow a more reliable prognosis in lung cancer. The results testified again that panels of molecular markers are useful for individual risk assessment, early and differential diagnosis, and prognosis in cancer.

[Papilloma virus infection in pretumor and tumor masses of the larynx]. / Papillomavirusnaia infektsiia v predopukholevykh i opukholevykh obrazovaniiakh gortani.

There has been recent evidence suggesting that papilloma virus infection is of no small importance in the pathogenesis of tumors of the head and neck. This study has involved a screening of the mucosa having laryngeal pretumors and tumors for papilloma virus infection by polymerase chain reaction and immunohistochemistry. Specimens were studied in 130 patients. All the patients with laryngeal papillomatosis were found to have human papilloma virus (HPV) types 6 and 11. HPV-18 was detected in 6 (13%) of 48 cases of laryngeal cancer and in 4 (9%) of 46 cases of laryngeal mucosal precancer. Mixed HPV infection of 2-3 different types was revealed in 6 (20%) of 30 HPV-positive patients. Such a wide survey of laryngeal pretumors and tumors in the Russian population for virus infection has been conducted for the first time.

[Development of chronic obstructive pulmonary disease correlates with mini- and microsatellite locus instability]. / Razvitie khronicheskikh obstruktivnykh boleznei legkikh korreliruet s nestabili'nost'iu mini- i mikrosatellitnykh lokusov.

Allele distribution at a highly polymorphic minisatellite adjacent to the c-Hras1 gene as well as deletions of microsatellite markers, D3S966, D3S1298, D9S171, and a microsatellite within p53 gene, were examined in bronchial epithelium specimens obtained from 53 chronic obstructive pulmonary disease (COPD) patients and healthy donors. A higher frequency of rare Hras1 minisatellite alleles in COPD patients than in the individuals without pulmonary pathology (6.6% versus 2.2%; P < 0.05) was shown. This difference was most pronounced in the group of ten COPD patients with idiopathic pulmonary fibrosis. Three of these patients had rare Hras1 minisatellite allele (P < 0.02 in comparison with healthy controls). Alterations in at least one of the microsatellite markers (deletions or microsatellite instability) were detected in bronchial epithelium samples obtained from: 4 of 10 COPD patients with pneumofibrosis (40%); 15 of 43 COPD patients (34.9%) without pneumofibrosis; and 8 of 20 tobacco smokers (40%) without pulmonary pathology. These defects were not observed in the analogous samples obtained from healthy nonsmoking individuals. No statistically significant differences were revealed between COPD patients and healthy smokers upon comparison of both the total number of molecular defects and the number of defects in the individual chromosomal loci. The total number of molecular defects revealed in bronchial epithelium samples from the individuals of two groups examined correlated with the intensity of exposure to tobacco smoke carcinogens (r = 0.28; P < 0.05). These findings suggest that rare alleles at the Hras1 locus may be associated with hereditary predisposition to COPD and the development of pneumofibrosis, while mutations in microsatellite markers result from exposure to tobacco smoke carcinogens and are not associated with the appearance of these pathologies.

[Profile of methylation of certain tumor growth suppressing genes in non-small cell lung cancer]. / Profil' metilirovaniia nekotorykh genov-supressorov opukholevogo rosta pri nemelkokletochnom rake legkogo.

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5' regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk-Rb-E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.

[Allelotyping the Hras1 minisatellite: formation of carcinogenic risk groups and predicting the course of non-small cell lung cancer]. / Allelotipirovanie minisatellita Hras1: formirovanie grupp kantserogennogo riska i prognozirovanie techeniia nemelkokletochnogo raka legkogo.

PCR-based typing of Hras1 minisatellite alleles was carried out in 226 non-small cell lung cancer (NSCLC) patients and 207 unaffected controls. Application of this method permitted detection of four common (a1 to a4) and 25 other alleles, differing from any common allele by one or more repeat units. Depending on their frequency in control group, these alleles were defined as intermediate or rare (the frequency over 0.5% or less than 0.5%, respectively). It was established that the frequency of rare alleles in the group of NSCLC patients (7.1%) was statistically significantly higher than in healthy individuals (2.2%, p = 0.002), while the difference in the distribution of common and intermediate alleles between the compared groups was not statistically significant. In addition, rare Hras1 alleles were more frequent (p = 0.02) among nonsmoking patients compared to the patients subjected to of tobacco carcinogens. The presence of "heavy" (a3-a4) alleles was associated with an increased risk of low-differentiated and/or actively metastasizing tumors and also with the risk of lung cancer in the patients under 50 years of age (p < 0.05). These data indicate that an approach including application of modern highly sensitive techniques of Hras1 allele typing in combination with preliminary examination of healthy control population can be employed for identifying carcinogenic risk groups as well as for prognosis of the NSCLC clinical course.

[Increased risk of occurrence of pulmonary adenocarcinoma in man is associated with the presence of rare alleles of the Hras1 minisatellite]. / Povyshennyi risk vozniknoveniia adenokartsinom legkogo u cheloveka assotsiiruetsia s nalichiem redkikh allelei minisatellita Hras1.

Data on allelotyping of minisatellite sequence within the Hras1 protooncogene locus in 60 patients with lung adenocarcinoma (LAC) are presented. Allele distribution was analyzed with respect to the effect of tobacco smoke carcinogens (smoking factor). Results were compared with the analogous data obtained for patients with squamous-cell-carcinoma and for individuals without cancer. In contrast to the patients with squamous cell-carcinoma, the frequency of the Hras1 locus rare alleles in patients with lung-adenocarcinoma was higher than in individuals without cancer. More striking difference between the latter groups was demonstrated for nonsmoking patients (17.6% versus 2.7% of healthy individuals, P = 0.0005). In smoking LAC patients, higher frequencies of the common a4 allele (2.5 kb in size under the MspI/HpaII digestion) were found. Our findings point to the combined effect of endogenous and exogenous factors on the development of lung adenocarcinomas in humans. In this work, we discuss the possible mechanism of association between the rare minisatellite alleles and the predisposition to lung cancer.

[Restriction polymorphism of the proto-oncogene c-Ha-ras-1 in patients with multiple primary malignant neoplasms and non-small-cell lung cancer]. / Restriktsionnyi polimorfizm protoonkogena c-Ha-ras-1 u patsientov s pervichno-mnozhestvennymi zlokachestvennymi novoobrazovaniiami i nemelkokletochnkym rakom legkogo.

Restriction fragment length polymorphism in the human c-Ha-ras-1 locus, associated with a minisatellite sequence, was examined in 45 multiple primary cancer (MPC) patients, 56 patients with squamous cell lung cancer (SCLC), 21 patients with lung adenocarcinoma (LAC), and 53 individuals having no oncopathology. Southern analysis of cellular DNA revealed the presence of 4 common alleles (with collective allele frequency close to 94% in the control group) and a set of rare alleles. Allele a3, (2.1 kb in size under MspI/HpaII digestion) was shown to be more frequent in the MPC than in the control group. The same tendency was observed in the patients with highly differentiated cell lung cancer. An increased frequency of the a4 allele (2.5 kb under MspI/HpaII digestion) was observed in the patients with adenocarcinomas as well as in the patients with metastases and low levels of tumor tissue differentiation. The elevated frequencies of a3 in the MPC group and of a4 in the LAC patients did not correlate with increased risk of the cancers mentioned above but was associated with type of tumor progression. Previously, it was reported that the mini-satellite sequence within the c-Ha-ras-1 locus possesses enhancer activity. Our data indirectly confirm the hypothesis that the efficiency of minisatellite modulator activity is associated with fragment size.

[Synthesis and distribution of oncoproteins in tumor tissue]. / Sintez i raspredelenie onkobelkov v opukholevykh tkaniakh.

Expression of myc, fos, src, ras and sis oncoproteins was studied in biopsy material of tumors, metastases and "normal" surrounding tissues from patients with different histological types of stomach and lung cancer, melanoma and other malignancy using immunoblotting. Besides, the immunohistochemical distribution of these oncoproteins under lung cancer and precancer conditions was analysed. The oncoproteins expression was significantly higher in cancer as compared with precancer and "normal' surrounding tissues. C-myc and c-fos gene products were detected in all the malignant tissues irrespectively to histogenesis of tumors, while the level of c-myc expression was rather high. The high level of c-fos expression was observed in stomach carcinomas and at early stages of lung tumor progression. C-src and c-sis genes expression varied in tumors of different histogenesis. C-src proteins were found in 60% of lung cancer but it was practically absent in stomach carcinomas and in melanomas. C-sis gene product was observed in some melanomas and lung carcinomas. Ras gene can be activated at early stages of tumor progression of stomach carcinomas and lung adenocarcinomas and at later stages of tumor progression in melanomas and small-cell lung carcinomas. Thus, there are some correlations between oncoprotein expression and tumor tissue histogenesis and progression.