Ранее в линиях клеток мелкоклеточного (SCLC) и немелкоклеточного (NSCLC) рака легкого наблюдали метилирование CpG-островка в области гена SEMA3B (3p21.31). Согласно данным NCBI (Build 36) эта область соответствует первому интрону гена. В представленной работе изучено метилирование двух CpG-островков гена SEMA3B – промоторного и интронного – при светлоклеточном почечноклеточном раке (RCC). С применением метилспецифичной полимеразной цепной реакции и бисульфитного секвенирования впервые показано метилирование с высокой частотой промоторного CpG-островка гена SEMA3B при RCC – 56% (34/61) и интронного – 35% (17/48). Установлена достоверная обратная корреляция между снижением содержания мРНК этого гена при RCC и метилированием промоторного CpG-островка (P < 0.05 по Фишеру). В случае интронного островка такая корреляция не выявлена. Этот результат позволяет предположить, что метилирование промоторного CpG-островка участвует в инактивации гена-супрессора SEMA3B при RCC.

Представлена новая технология сравнительной геномной гибридизации на NotI-микрочипах (международный патент Каролинского института WO02/086163). Методика основана на проведении сравнительной гибридизации NotI-обогащенных проб из геномной ДНК опухолевой и нормальной тканей с помощью принципиально новых NotI-микрочипов, на которых иммобилизованы NotI-связующие клоны. Проанализирован 181 NotI-связующий геномный локус из хромосомы 3 человека в 200 образцах злокачественных опухолей различных органов: почек, легких, молочной железы, яичников, шейки матки, предстательной железы. Наиболее часто (более чем в 30% случаев) изменения – делеции и метилирование – идентифицированы в NotI-сайтах, расположенных в локусах MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4, что указывает на их возможную связь с развитием заболевания. Метилирование этих геномных локусов подтверждено с помощью метил-специфичной полимеразной цепной реакции и бисульфитного секвенирования. Полученные результаты указывают на перспективность использования разработанной методики для решении ряда проблем онкогеномики.

Earlier, methylation of a CpG island in the SEMA3B gene (3p21.31) was observed in cell lines of small-cell and non-small-cell lung carcinoma. According to NCBI (Build 36), that island belonged to intron 1 of the gene. Our study concerns the methylation of two CpG islands, promoter and intronic, in the SEMA3B gene in patients with clear cell renal cell carcinoma (RCC). Methylation-specific PCR and bisulfite sequencing revealed a high frequency of methylation in the promoter CpG island (34/61, 56%) and somewhat lower, in the intronic (17/48, 35%). A significant inverse correlation was found between the SEMA3B mRNA level and methylation of the promoter CpG island in RCC (P < 0.05 according to Fisher's exact test). The intronic island showed no such correlation. Thus, we suggest that the methylation of the promoter CpG island contributes to the inactivation of the SEMA3B suppressor gene in RCC tissue.

New comparative genome hybridization technology on NotI-microarrays is presented (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-probes from tumor and normal genomic DNA with the principle of new DNA NotI-microarrays. Using this method 181 NotI linking loci from human chromosome 3 were analyzed in 200 malignant tumor samples from different organs: kidney, lung, breast, ovary, cervical, prostate. Most frequently (more than in 30%) aberrations--deletions, methylation,--were identified in NotI-sites located in MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4 genes, that suggests they probably are involved in cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results demonstrate perspective of using this method to solve some oncogenomic problems.

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.

The investigation of the cancer-associated structural and epigenetic changes in cell genome is a major approach for understanding mechanisms of cancerogenesis. To investigate these genome changes, novel technique of microarrays comprising NotI-linking genome clones was developed. Twenty eight samples from patients with cervical cancer were analyzed using NotI microarrays of human chromosome 3. Deletions, amplifications and methylation were detected for 109 out of 182 NotI clones with different frequency. Notably, 17 NotI-linking clones showed genomic changes in more than 35% of tumor samples investigated, which suggests involvement of genes associated with these clones in development of cervical cancer.

Ген RASSF1A (3p21.31) относится к группе генов-супрессоров опухолевого роста. Изучен уровень метилирования СрО-островка в промоторном районе этого гена в первичных опухолях почек, молочной железы и яичников в московской популяции (172 случая). Использован метод ПЦР, специфичной к метилированному аллелю (МСП), а также анализ с помощью метилчувствительных рестрикционных эндонуклеаз и последующей ПЦР (МЧРА). Результаты этих методов с высокой достоверностью (согласно ранговой корреляции Спирмана, P < 10 -6) согласуются между собой и с ранее полученными данными бисульфитного секвенирования. По данным МСП и МЧРА частота метилирования промоторного района гена RASSF1A составила 86% (25/29) и 94% (50/53) при почечноклеточном раке, 64% (18/28) и 78% (32/41) при раке молочной железы и 59% (17/29) и 73% (33/45) в эпителиальных опухолях яичников соответственно. Применение набора метилчувствительных рестриктаз (HpaII, HhaI, BshI236I, AciI) повысило чувствительность анализа и позволило изучить статус метилирования 18 СрО-пар промоторного района гена RASSF1A. На основании этих данных удалось оценить плотность метилирования СрО-островка, которая достигала 72% в опухолях почки, 63% при раке молочной железы и 58% при раке яичников (за 100% принято произведение исследованных 18 СрО-пар на число образцов, в которых выявлено метилирование). Метилирование промоторного района гена RASSF1A (11-35%) наблюдали и в образцах ДНК из гистологически нормальной ткани, прилежащей к опухоли, но оно полностью отсутствовало в этих же участках ДНК из крови здоровых доноров (0/15). Частота метилирования СрО-островка в опухолях не зависела от стадии, степени анаплазии и наличия метастазов. С другой стороны, эпигенетическую модификацию гена RASSF1A в опухолях наблюдали существенно чаще, чем геми- и гомозиготные делеции в области этого гена. Согласно этим наблюдениям, метилирование промоторного района гена RASSF1A относится к ранним событиям в онкогенезе и представляет один из главных путей инактивации этого гена в эпителиальных опухолях.

Methylation of the promoter CpG-islands of the candidate tumor suppressor gene RASSF1A (3p21.31) was studied in primary tumors of kidney, breast and ovary (172 cases). Methylation-specific PCR (MSP) and methyl-sensitive restriction endonuclease digestion followed by PCR (MSRA) were applied. Statistically significant correlation (P << 10(-6)) was shown for the results of the MSP and MSRA, and the data of bisulfite sequencing reported earlier. The frequency of RASSF1A methylation according to MSP and MSRA was 86% (25/29) and 94% (50/53) in renal cell carcinoma (RCC) and 64% (18/28) and 78% (32/41)--in breast carcinoma (BC) samples, and 59% (17/29) and 73% (33/45) in ovarian epithelial tumors (OET), respectively. The use of several methyl-sensitive restriction enzymes (HpaII, HhaI, Bsh12361, AciI) enhanced the sensitivity of MSRA and allowed to analyze methylation status of 18 CpG-pairs in the RASSF1A CpG-island. Density of methylation of the RASSF1A CpG-island was 72% (644/900) in RCC, 63% (361/576) in BC, and 58% (346/594) in OET samples (18 CpG-pairs multiplied to the number of samples shown methylation were assumed as 100%). The RASSF1A gene methylation was also observed in samples of morphologically normal tissues adjacent to corresponding tumors (11-35%), but it was not detected in blood DNAs of healthy donors (0/15). The RASSF1A methylation frequency did not show significant correlation to tumor stage, grade and metastases (P = 0.3-1.0). The RASSF1A gene methylation was observed more frequently than other investigated aberrations--hemi- and homozygous deletions inside or around this gene. These observations are consistent with the hypothesis that the RASSF1A gene methylation is an early event in the carcinogenesis and one of the dominant ways of its inactivation.

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL (3p26-p25), the region of homozygous deletions AP20 (3p22-p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).

The nucleotide sequence of 300 NotI clones with short human genomic DNA inserts was determined. More that 70% clones corresponded to GC-rich genomic regions with clustered NotI sites. Presumably, at least 10% of the NotI sites form clusters in the human genome. Inserts from 33 clones proved highly homologous to sequenced human genes and cDNAs. NotI clusters were assumed to be associated with genes involved in regulating cell proliferation and differentiation.

A new DNA fragment from the human chromosome 3 was studied. Fluorescence in situ hybridization showed that this fragment is located mainly in the unstable regions of the chromosome. The known repeated elements in the fragment were rearranged, it contained matrix attachment regions (MAR) and numerous sequences known to be associated with recombination sites. More than 60% of the fragment was composed of nonrandomly distributed direct repeats. The distribution of direct repeats suggested their origin from unequal recombination of homologous chromosomal fragments. The DNA structure and distribution of direct repeats are supposed to be related with the propensity to recombination within and between the chromosomes.