The sequence of K-ras intron 2, which has been associated with lung tumor susceptibility in inbred mouse strains, was analyzed in susceptible strain GR and in resistant strains PT and UT. In the latter case, the intron had a tandem repeat of a 37-bp sequence with variant GC of its two single-nucleotide polymorphisms (SNPs), as earlier reported for resistant strains AKR, C57BL/c, and C3H/A. Strain GR did not differ in intron structure from susceptible strains A/He and ICR, having one copy of the 37-bp sequence with SNP variant CA. By gel retardation assay, a DNA probe corresponding to "susceptible" allele CA of K-ras region 278-307 formed an additional complex with nuclear proteins extracted from the lungs, as compared with probes corresponding to the "resistant" GC and "intermediate" CC alleles. With specific antibodies, the protein binding to the susceptible allele was identified as transcription factor GATA-6. Reverse transcription with subsequent multiplex PCR did not reveal a significant difference in K-ras expression for susceptible and resistant strains. The results suggest that SNPs of K-ras intron 2 do not affect the level of K-ras expression but do control the binding of GATA-6, which plays an important role in lung differentiation.

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed a molecular defect in at least one allele in 51 (98%) tumors.

Multiple endocrine neoplasia (MEN) unites a group of syndromes caused by tumors or hyperplasia of several endocrine glands. We examined and treated patients (father and daughter) with Sipple disease, or type II MEN; the diagnosis was confirmed at Institute of Clinical Endocrinology (Moscow). The aim of our report was to supplement the clinical picture of the disease. Clinical examinations showed thickened corneal nerves, the dry eye syndrome, and abnormal refraction.

A number of genetic syndromes and cancer are caused by chromosomal microstructural rearrangements. It is sometimes impossible to detect this pathology microscopically. A complex of molecular methods makes it possible to detect subchromosomal deletions, point mutations, and functional anomalies on the respective genes, which cause a disease. Achievements of comprehensive analysis have been demonstrated in cases of some well-known diseases, the Prader-Willi syndrome, Engelmann's syndrome, the Martin-Bell syndrome, hereditary multiple exostotic chondrodysplasia, and retinoblastoma. DNA-diagnosis protocols are the result of this study.

The epigenetic (i.e. heritable states that are mediated by changes in DNA other than nucleotide sequence) mechanisms of regulation of gene expression have been recently the focus of intensive studies. Genomic imprinting refers to the epigenetic gene marking that results in monoallelic expression. The epigenetic mechanism of imprinting is based on the gamete-specific methylation of some mammalian genes, which restricts their expression on one of the parental chromosomes. The imprinted genes control fetal and placental development, cell proliferation and adult behavior. Changes in the normal imprinting patterns give rise to numerous genetic diseases, including cancer. Examining the molecular processes that mediate these methylation genome changes will give use a great insight into the mechanisms of regulation of gene activity and into the etiology of some human genetic diseases.

The identification and characterization of genes either induced or repressed in human brain tumors are important in understanding the mechanism of tumor initiation and progression, stages of malignancy, in developing new approaches to the diagnosis and treatment of tumors. In this paper, differential hybridization of arrayed human fetal brain and postnatal brain and postnatal brain cDNA libraries revealed differences in the rate of hybridization signals with total cDNA probes of the human brain and glioblastoma multiforme for more than 150 cDNA clones. Sixteen nucleotide sequences with changed contents in tumors were identified by repeated differential hybridization of the cDNA clones selected by primary screening with the same total cDNA probes of the human brain and glioblastoma multiforme and by Northern-hybridization of RNA samples from the human and glial tumors. The results of an analysis of the increased expression of the gene encoding apolipoprotein E. DNA-binding protein B, mitochondrial (16S and 12S) and cytoplasmic 28S rRNAs, Alu-containing transcripts, inactivation of cullin 1 gene and potential tumor suppressor gene TSC-22 are described.

Ploidometry of prostatic cell nuclei at different stages of carcinogenesis was made in 80 patients using 8 mcm histological sections stained by Felgen. Ploidy of 1332 nuclei of prostatic epithelial cells and 396 lymphocytes (tissue ploidy standard) was studied on computer image analyser "Imager-CG (computer program version Avtan-San). The study covered interphasic nuclei of the cells of the germinative zones of normal and hyperplastic structures, areas of intraepithelial neoplasia and adenocarcinoma with various disdifferentiation degree. Significant differences in ploidy and proliferative activity of prostatic cells at different carcinogenesis stages were revealed. Ploidometry data may help to take decision in differential histopathological diagnosis of benign and malignant tumors of the prostate.

Expression of DNA sequences, related to MMTV env gene, in peripheral blood lymphocytes, which was strictly specific for human mammary carcinoma, has been previously reported. These sequences (homologous to env gene site coding for MMTV gp52 envelope antigen) expressed in T cells can play the key role in virus infection transmission and propagation. In order to elucidate the possible routes of env MMTV-homologous sequences expression, we tried to induced it in donot T lymphocytes by various methods: hormone and virus treatment (related genome "saving" at the expense of the added virus envelope), T cell culturing with conA, interferon-2, and 5-azacytidine. RT-PCR with primers specific for the gp52-coding area of MMTV env gene showed expression of env-homologous sequences in donor T cells cultured in medium with 5-azacytidine. Indirect immunofluorescence with monospecific serum to MMTV gp52 detected gp52 analogous genes only in cultures with 5-azacytidine but not other agents. We therefore suggested that MMTV env-homologous sequences in donors are situated in the methylated promoter zone. Expression of these sequences in T cells, specific for human mammary carcinoma, can be due to demethylation of the promoter and induction of env-homologous sequences to the level of translation of gp52 analogous antigens or by initial location of some of the expressed sequences in the demethylated zone of the genome.

Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.

We showed that transposon P(GUS.p53.259H), mapped to chromosome 3 and carrying a dominant mutation p53(259)H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts- in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53(259)H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P(GUS.p53.259H) transposon itself. The effect of the P(GUS.p53.259H) appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo(alpha)pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P(GUS.p53.259H) effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts- clones induced by P(GUS.p53.259H) was established.

A chimeric p53 cDNA was constructed so that the fragment coding for 39 residues of the chicken p53 tetramerization domain replaced the corresponding region of human p53. The chimeric cDNA substantially inhibited the colony-forming ability of transfected human and mouse cells, suggesting a suppressory potential for its product. The chimeric p53 activated promoters containing p53-responsive elements. In contrast to wild-type human p53, the chimeric p53 remained capable of transcription activation in the presence of dominant-negative mutant p53-His175. This makes the chimeric p53 a convenient model for elaborating gene therapy protocols for tumors with dominant-negative p53 forms. The chimeric p53 may be used to study the role of transdominance of p53 mutants in carcinogenesis and the interactions of p53 with related transcription factors (p73, p63).

The spectrum and frequencies of RB1 structural defects were studied in tumors and peripheral blood lymphocytes of patients with various forms of retinoblastoma. Single strand conformation polymorphism (SSCP) and heteroduplex (HA) analyses, along with direct sequencing, revealed 47 mutations, including 24 new ones. Of these, 42.5% were nonsense mutations, 15% were missense mutations, 15% affected splicing sites, and 27.5% were frameshifts resulting from microdeletions or microinsertions. Six polymorphisms were found, including three new ones located in the coding region. Microsatellite analysis with markers Rbint2, Rbint20, D13S262, and D13S284 revealed a loss of heterozygosity for at least one marker in 71% tumors.

Female transgenic FVB mice transfected with the mammary erbB-2/neu oncogene were injected 0.1 ml 0.9% solution of sodium chloride (control), 1 meg Vilon peptide (Lys-Glu) or Epitalon peptide (Ala-Glu-Asp-Glu), s.c., 5 days in succession once a month, beginning from the age of 2 months. The characteristics of mammary tumor induction in the control and experimental groups did not differ until the age of 9 months. Later on, Epitalon-treated mice revealed distinct inhibition of carcinogenesis. One tumor per animal was detected in 7% (control), 4% (Vilon) and 16% (Epitalon) (p < 0.05). Two or more tumors per animal were in 75%, 95% and 56%, respectively (p < 0.05). Largest diameter of mammary adenocarcinoma in the Epitalon group was smaller than in controls by 33% (p < 0.05). Although the number of mice with metastases to the lung in all three groups was practically identical, their incidence in the Vilon group was 2.6 times higher than in Epitalon-treated animals (p < 0.05). Largest diameter of metastasis in the Epitalon group was the smallest, too. Our data point to inhibition of mammary carcinogenesis by Epitalon in transgenic erbB-2/neu mice.

The proportion of BRCA1/2 genes varies widely among populations. We investigated 52 cases of breast cancer: 24 cases of familial breast cancer, 22--early-onset (till 40 years) and 6--bilateral cancer. The frequency of carriers of mutations among familial cancer cases was 16%, among cases of disease till 40 years--9%. 538insC BRCA1 was the most common genetic mutation among hereditary cases of breast cancer.

Hybridization with cDNA arrays was used to obtain expression profiles of 214 protein-tyrosine kinase, protein-tyrosine phosphatase, dual-specific phosphatase, and other genes for kidney carcinomas (KC) and normal kidney tissues of 34 patients and for seven carcinoma cell lines. Computer analysis revealed three clusters of genes coexpressed in KC. A proliferating-cell gene cluster included MET, VIM, MYC, TOP2A, PCNA, etc. A neoangiogenesis and blood-cell gene cluster included LCK, HCK, FGR, MMP9, CSFR1, VEGF, FLT1, and KDR. A cluster corresponding to normal, differentiated kidney cells included ERBB2 (HER2) for receptor protein-tyrosine kinase, several phosphatase genes (PTPRE, PTPRB, DUSP9), and EGF. The results suggested that MET, DUSP9, PCNA, TOP2A, and VIM may serve as diagnostic and prognostic markers in KC. Tubulin and topoisomerase II were assumed to be promising targets for cell proliferation inhibitors in KC.

Hamster tumor cell lines obtained with the Rous sarcoma virus and characterized by a high metastatic activity in vitro were transfected with the gene for C2+/calmodulin-dependent serine-threonine death-associated protein kinase (DAPk). Expression of DAPk in tumor cells dramatically reduced their survival in the blood of syngenic animals and their ability to produce metastases, but did not affect their tumorigenicity or the primary tumor growth. The DAPk-induced change in the metastatic phenotype was not accompanied by substantial changes in production and phosphorylation of v-Src or focal adhesion proteins (focal adhesion kinase and paxilline). The resulting system of transfected cells with a modulated metastatic potential provide a convenient model to study the molecular mechanisms of tumor progression at various steps.

The paper presents current data on theepigenetic regulation of gene expression in malignant tumors. Methylation of CpG islands in the gene promoter regions not accompanied by DNA structural changes leads to gene inactivation. The analytical methods of DNA methylation are described in detail. Methylation of certain genes in different tumor types are examplified. Since methylation is an early marker of carcinogenesis, there is an opportunity of elaborating of prospective diagnostic protocols.