Alterations of (CA)n DNA repeats and tumor suppressor genes in human gastric cancer.

We have examined whether alterations of simple (CA)n DNA repeats, as observed in human colon cancers, occur during human gastric carcinogenesis and whether such alterations reflect genomic instability that could lead to other genetic changes. A total of 22 gastric cancer samples were analyzed: 15 well or moderately differentiated adenocarcinomas, 6 signet-ring cell carcinomas, and 1 poorly differentiated adenocarcinoma. When (CA)n repeat sequences were examined at 10 loci, one adenocarcinoma showed a loss of repeat sequences at five loci, three adenocarcinomas gained a repeat at one locus, and one adenocarcinoma had new, repeated sequences at five loci. Three samples showed mutations in the p53 gene, two in exon 5 (both GC to AT transition at a CpG dinucleotide) and one in exon 7 (AT to GC transition). Only one sample with a p53 mutation also showed altered (CA)n repeats. A putative tumor suppressor gene, connexin 32, was not altered as assessed by single-strand conformation polymorphism analysis. These results suggest that genomic instability revealed by (CA)n repeat changes does not seem to contribute to induction of point mutations in p53 or connexin 32 genes but may participate in loss of heterozygosity at APC/MCC loci. The results are consistent with the hypothesis that different mechanisms are involved in the gain and loss of (CA)n repeats.

Some biochemical mechanisms underlying the impairment of T and B cell immunity in C3HA mice during hepatoma growth.

In thymocytes of C3HA mice carrying the transplantable and ortoaminoazotoluene induced hepatomas at the time of their intense growth a drastic decrease in adenosine deaminase activity set in and 3-4-fold augmentation of intracellular concentration of dATP and dGTP, potential inhibitors of ribonucleoside diphosphate reductase was observed, leading to the reduction of the DNA synthesis. The latter event was evidenced by a suppressed 14C-thymidine incorporation into thymocytes DNA in vitro, decreased thymidine kinase activity, intracellular dTTP and depletion of dCTP pools. Only in the terminal period of hepatocarcinogenesis (12 months) a 4-fold increase in the corticosterone serum concentration was observed. As for the mice carrying transplantable 22a hepatoma, serum hormone levels augmented 4-fold as early as 24 h after tumor implantation and thereafter kept increased two fold. An elevated activity of terminal deoxynucleotidyl transferase in mouse thymocytes has been shown to be characteristic of the late periods of tumor growth reflecting the arrest of the immature cortical thymocyte differentiation. By the time hepatomas emerged in the course of hepatocarcinogenesis in spleen T and B lymphocytes a significant drop in the activity of adenosine deaminase (3-4-fold) and purine nucleoside phosphorylase (2-8-fold) was noted--the events directly correlated with the weakening of cell immune functions. The disorders described were accompanied by the accumulation of dGTP in spleen T lymphocytes, dATP in B lymphocytes and inhibition of DNA synthesis, predominantly in T lymphocytes. In the latter instance the pool of dCTP was found to be depleted. In spleen T and B lymphocytes of mice carrying solid 22a hepatoma when the peak of its growth was reached (day 5) the rate of DNA synthesis dropped. Later on (from day 8 to the animal death), however, in spite of the suppression of immune function and the decrease in adenosine deaminase activity a drastic stimulation of DNA synthesis in spleen T and B lymphocytes was observed. The increase in spleen T suppressor activity in the course of intense growth of the both types of hepatomas coincided in the time with the stimulation of the CTP-dependent thymidine kinase isoenzyme activity in total T lymphocyte population of the same organ.

[Methodological approaches to the study of tumor and body tissue metabolism in vitro and in vivo]. / Metodologicheskie podkhody k issledovaniiu metabolizma opukholei i tkanei organizma in vitro i in vivo.

The idea is put forward that in vitro measurements of the potential activity even of the key enzymes are not sufficient for correct evaluation of the rate of complex enzymic processes in vivo. The rate of the in vivo formation of final (not intermediate) products from the corresponding precursors is thought to be the most adequate criterion for it. The idea is substantiated by our studies on the glycolysis enzymes and RNA pyrimidine nucleotide synthesis. Such an approach permits gaining in particular, exact information on both features of the RNA synthesis in experimental tumours and mechanisms underlying a successful competition of the tumour and host tissues for RNA precursors. The latter phenomenon is one of manifestations of the tumour capacity to act as a trap for nitrogen.

[Genetic defects as markers of tumor development]. / Geneticheskie defekty kak markery opukholevogo rosta.

Carcinogenesis is long-term multistep accumulation of defects of genes responsible for cell division, DNA repair, and apoptosis. The functions of these genes are known both for norm and for pathologies caused by their damage and resulting in "asocial" cell behavior. Owing to the recent progress in studying the mechanisms of carcinogenesis, some genetic defects may be considered from the applied point of view (as tumor markers rather than as pathogenetic factors) and employed in diagnostics. Thus detection of mutant alleles in biological fluids (e.g., beyond the tumor) suggests higher risk of carcinogenesis. Genetic defects are a new class of tumor markers and have a substantial diagnostic potential. In contrast to known protein markers (alpha-fetoprotein, etc.) used in clinical practice, DNA markers are oncospecific (as these are in direct cause-and-effect relationships with carcinogenesis) and universal (as there is not a single tumor cell without a genetic defect). Analysis of DNA markers may be employed not only in diagnostics or tumor growth monitoring (assessment of treatment efficiency, early detection of recurrence or metastasis), but also (prospectively) in screening (tumor detection at the presymptomatic stage, identification of high-risk groups). Theoretical grounds, prospects, problems, and methods of this new field are considered.

[Analysis of DNA excreted from the body as an approach to diagnosis and monitoring tumor growth]. / Analiz ékskretiruemoi iz organizma DNK kak podkhod k vyiavleniiu rastushchei v organizme opukholi.

Proceeding from their early data showing that some portion of DNA originating from apoptotic cells can enter the blood stream and pass through the renal barrier by preserving its template capabilities, the authors analyzed urine DNA from 29 patients with colorectal cancer. PCR was used to compare DNA samples from the normal mucosa surrounding the tumor and from the urine collected just prior to surgery. Six microsatellite loci were studied with oligonucleotide primers. The following results were obtained: i) 3 cases showed differences in one of the studied loci in normal and tissue DNA; ii) some patients displayed changes in urine DNA microsatellite loci, namely: disappearance of some alleles (loss of heterozygocity) and appearance of new ones; iii) there were no differences in microsatellite patterns of lymphocytic DNA (taken as a control) and urine DNA in healthy donors. The findings are discussed in view of current concepts of tumor clonal heterogeneity and interpreted as a promising approach to diagnosing and monitoring tumor growth.

[Combination of immunodepression and disorders in nucleic acid metabolism of lymphoid tissue as a manifestation of a paraneoplastic syndrome]. / Sopriazhennost' immunodepresii i narushenii nukleinovogo obmena limfoidnoi tkani kak proiavlenie paraneoplasticheskogo sindroma.

Several physiological, biochemical, and molecular biological approaches to the study of factors determining immunodepression in tumor-bearing animals are considered. Cancer cells release substances of nucleic and peptide nature that suppress the functional activity of macrophages and lymphocytes and stimulate cell proliferation in organs and tissues of the host. Suppressor T cells capable of inhibiting the function of helper T cells and impairing the differentiation of killer T cells are activated. The suppression of T- and B-cell-mediated immunity in the tumor host involves disturbances of nucleic acid metabolism in those cells as well as hypersecretion of glucocorticoids. The impairments of lymphocyte proliferation and differentiation that result in reduced immune responsiveness are attributable to drastic alterations in the metabolism of purine and pyrimidine nucleotides and to the damage sustained by the lymphocyte's DNA.

[Biochemical and functional characteristics of thymus and spleen lymphocytes in C3HA mice during the growth of hepatoma 22 and after immunization with sheep erythrocytes]. / Biokhimicheskaia i funktsional'naia kharakteristika limfotsitov timusa i selezenki myshei C3HA pri roste gepatomy 22 i posle immunizatsii éritrotsitami barana.

Activities of key enzymes of purine metabolism [adenosine deaminase (AD); purine nucleoside phosphorylase (PNP); 5'-nucleotidase] were studied; changes in DNA content, nucleus ploidity in thymocytes, T- and B-lymphocytes in the C3HA mouse spleen during solid 22 hepatoma growth and after the immunization were monitored. Immunological properties of lymphocytes were also investigated measuring antibody formation and the reaction of blasttransformation in response to phytohemagglutinin, concanavalin A and lipopolysaccharide. Within the first 48 hrs after the tumor implantation and immunization certain nonspecific biochemical mechanisms of lymphocytes activation (elevated AD activity, decreased activity of 5'-nucleotidase, augmented intracellular DNA levels, polyploidity) were revealed. As the solid 22 hepatoma reached the maximum growth rate specific alterations in the activities of the purine metabolism key enzymes were observed reflecting the response of thymus and spleen lymphocytes to the presence of the malignant tumor.

[Changes in purine metabolism and in the immune response in the thymic and splenic lymphocytes of C3HA mice during chemical hepatocarcinogenesis]. / Izmeneniia purinovogo obmena i immunnogo otveta v limfotsitakh timusa i selezenki myshei C3HA pri khimicheskom gepatokantserogeneze.

The activities of adenosine deaminase, purine nucleoside phosphorylase, membrane 5'-nucleotidase and DNA content in thymus and spleen lymphocytes as well as the immune function of T and B lymphocytes of the spleen of C3HA mice with o-AAT-induced hepatomas were studied 1, 3 weeks and 3, 8, 12 months after o-AAT treatment was instituted. In the early stages of the hepatocarcinogenesis (up to 3 months), the elevation of the activity of all the enzymes and DNA content in thymocytes and T and B lymphocytes was observed. These changes coincided with the enhancement of the immune responses that manifested in the increased content of PFC, EA-RFC and high response to PHA and Con A. In the late stages, the decreased activities of purine nucleosides and nucleotide metabolizing enzymes correlated with disturbances of lymphocyte differentiation and lowering of the host immune response.

[Change in the strength of DNA-protein binding in T- and B-lymphocytes of the spleen of C3HA mice during chemical hepatocarcinogenesis]. / Izmenenie prochnosti sviazi DNK-belok v T- i B-limfotsitakh selezenki myshei linii C3HA pri khimicheskom gepatokantserogeneze.

The tightness of DNA-protein binding in the nuclei of mouse spleen T- and B-lymphocytes was assessed, using nucleoprotein celite chromatography, and changes in the number of T- and B-suppressors in the course of o-AAT-induced chemical hepatocarcinogenesis were studied. Attenuation of DNA-protein bonds in T-lymphocytes at the early stages (up to 3 months) was observed, and by the time of hepatoma formation (8 months) about 50% of T-lymphocyte DNA was loosely bound to proteins, which is a typical feature of quiescent cells. In B-lymphocytes attenuation of DNA-protein interaction was only observed by the 8th month of carcinogenesis. By the time of hepatoma formation the number of T-suppressors in mouse spleen increased 2.8-fold, while the number of B-suppressors in lymph nodes remained unchanged.

[Changes in the lymphoid cells of DNA and purine nucleotide synthesis and sensitivity to glucocorticoids associated with impairment of differentiation and immune function during tumor growth in mice. Thymocytes]. / Izmeneniia v limfoidnykh kletkakh sinteza DNK, purinovykh nukleotidov i chuvstvitel'nosti k gliukokortikoidam, sopriazhennye s narusheniem differentsirovki i immunnoi funktsii u myshei v protsesse opukholevogo rosta. Timotsity.

Some biochemical mechanisms underlying the impairments of cellular immunity were studied in C3Ha mice in the course of growth of transplantable and induced (ortoaminoazotoluol) solid hepatomas. During intensive hepatoma growth, the adenosine deaminase activity in host thymocytes was shown to be drastically (6 times) reduced, resulting in the elevation of dATP and dGTP concentrations (6- and 7-fold, respectively), the potential inhibitors of ribonucleoside diphosphate reductase. Consequently, the rate of DNA synthesis was reduced as can be evidenced by the decrease of (a) thymidine kinase activity, (b) 14C-thymidine incorporation into DNA, and (c) dTTP and dCTP pools. By the terminal period of hepatoma growth (both transplantable and induced one), the serum corticosterone content increased 3- and 8-fold, respectively. At the same time, specific binding of [3H]triamsinolone acetonide by thymocytes was augmented and the activity of terminal deoxynucleotidyl transferase increased the latter alterations, which can be regarded as a reflection (including other parameters mentioned) of the arrest of T-lymphocyte differentiation at the level of immature cortex thymocytes.

[Changes in DNA and purine nucleotide synthesis in lymphoid cells and sensitivity to glucocorticoids associated with the impairment of differentiation and immune function in mice during tumor growth. Spleen T- and B-lymphocytes]. / Izmeneniia v limfoidnykh kletkakh sinteza DNK, purinovykh nukleotidov i chuvstvitel'nosti k gliukokortikoidam, sopriazhennye s narusheniem differentsirovki i immunnoi funktsii u myshei v protsesse opukholevogo rosta. T- i B-limfotsity selezenki.

Biochemical impairments in spleen immunocompetent cells (T- and B-lymphocytes) were revealed in host (C3HA mice) of transplantable and ortoaminoazotoluol-induced hepatomas in the course of their growth. As soon as hepatoma emerged (chemical carcinogenesis), the activity of adenosine deaminase and purine nucleoside phosphorylase in T- and B-lymphocytes were found to be reduced 2-6 and 7-10-fold, respectively in parallel with the impairment of their immune system. These alterations were accompanied by the increase in concentrations of dGTP in T-lymphocytes (5.4-fold) and of dATP in B-lymphocytes (4-fold) as well as with the inhibition of DNA synthesis, predominantly in T-lymphocytes. In both T- and B-lymphocytes, the dCTP pool was decreased. In the spleen, T- and B-lymphocytes of mice carrying transplantable 22 hepatoma 22 by the moment of its maximal growth (5th day), the DNA synthesis was inhibited as revealed by the reduction of (a) thymidine kinase activity, (b) rate of the labeled thymidine incorporation into DNA, and (c) intracellular dTTP and dCTP concentrations. In latter periods (from 8th day up to the moment of death), drastic stimulation of DNA synthesis in spleen T- and B-lymphocytes was observed irrespective of the impairments in the immune function and the decrease of the adenosine deaminase activity. In the course of growth of both transplantable and induced solid hepatomas in host spleen T- lymphocytes, the activity of the CTP-dependent thymidine kinase isoenzyme increased, coinciding in time with the activation of antigen-specific T-suppressors in the same organ.

[Characteristics of the mechanism of action of complex copper (II) compounds with alpha-amino acids]. / Nekotorye osobennosti mekhanizma deistviia kompleksnykh soedinenii nedi (II) s al'fa-aminokislotami.

There was no direct inhibition of DNA synthesis in ascites hepatoma 22A cells after intraperitoneal injection of single doses of copper (II) complexes with amino acids into tumor-bearing C3HA mice. Meanwhile cis-dichlorodiamine platinum (II) (DDP) as well as sarcolysine showed such inhibition. Copper (II) complexes with alpha-amino acids displayed as significant superoxide dismutase-like activity at concentrations corresponding to therapeutic doses of these compounds. The complexes of copper (II) combined with DDP give an additive antitumor effect in solid tumors of mice.

[Change in activity of enzymes for purine metabolism and RNA and DNA biosynthesis in macrophages, reflecting impairment of their functions in neoplastic growth]. / Izmenenie aktivnosti fermentov purinovogo obmena i biosinteza RNK i DNK v makrofagakh, otrazhaiushchie narushenie ikh funktsii pri zlokachestvennom roste.

The changes in the biochemical parameters of peritoneal macrophages and their coupling to the secretory and phagocytic functions in CH3A mice during the growth of the reinoculated solid hepatoma 22a were studied. The DNA and RNA synthesis during the active tumour growth was more intense than that in resident macrophages. The activity of uridine kinase increased up to 156.0 +/- 12.0 nmol/hour/10(8) but was absent in resident macrophages. This was accompanied by a 7.2-fold increase of interleukin-1 synthesis as determined by the [3H]thymidine incorporation into thymocyte DNA in response to concanavalin A administration to C3H mice. Similar changes were observed in peptone-stimulated macrophages. A specific feature of macrophages from tumour-bearing mice was the impairment of activity of purine exchange enzymes and the efficiency of phagocytosis that were unobserved in peptone-stimulated macrophages. The activity of adenosine deaminase and purine nucleoside phosphorylase was inhibited as a result of their preincubation with zymosan, a phagocytosis-stimulating agent. This was accompanied by a significant decrease of the first chemiluminescence peak resulting from disturbances in Fc-reception. Macrophages of tumour-bearing animals possessed an increased 2.2-fold activity of membrane-bound AMP 5'-nucleotidase concomitant with the lack or decrease of the amplitude of the second chemiluminescence peak reflecting the disturbances in digestion resulting from phagocytosis.

[Opposite effect of p53 on nucleotide metabolizing enzyme activity in Rat1 cells and their sublines, transformed by N-RAS or v-mos oncogenes]. / Protivopolozhnye éffekty p53 na aktivnost' fermentov nukleinovogo obmena v kletkakh Rat1 i ikh subliniiakh, transformirovannykh onkogenami N-RAS ili v-mos.

The effects of exogenous human p53 and its various mutants (Ala-141, His-175, His-194, Trp-248, His-273) on two key enzymes of purine uptake, adenosine deaminase (AD) and hypoxanthine phosphoribosyl transferase (HPRT), has been studied in Rat 1 immortalized fibroblasts and their sublines transformed by N-RAS or v-mos oncogenes. Introduction into Rat1 cells of both wild type (wt) and mutant p53 produced a 2- to 7.5-fold increase in the AD activity, p53 mutants having a stronger effect than p53wt. In contrast, the HPRT activity decreased 8- to 10-fold in cells containing exogenous p53wt, while p53 mutants partly lost their ability to inhibit HPRT. Transformation of Rat1 by ras or mos oncogenes was also accompanied by an increase in the AD activity (4-5-fold and 1.5-2-fold, respectively) as well as by suppression of HPRT (20-fold and 2-fold, respectively). However, simultaneous expression of exogenous p53 and ras or p53 and mos produced opposite effects, i.e., a dramatic decrease in the AD activity and complete (p53wt, His-273) or partial (His-175, Trp-248) restoration of the HPRT activity. Possible functional significance and mechanisms of AD and HPRT regulation by p53 as well as the role of modifications of activity of nucleotide synthesis enzymes in the cooperative effect of predominant oncogenes and mutant p53 oncogenes in tumour transformation are discussed.

[The pool of free purine and pyrimidine nucleosides and bases in the thymocytes, and splenic T- and B-lymphocytes of C3HA mice during the growth of solid hepatoma 22a]. / Analiz pula svobodnykh purinovykh i pirimidinovykh nukleozidov i osnovanii v timotsitakh, T- i B-limfotsitakh selezenki myshei C3HA v protsesse rosta solidnoi gepatomy 22a.

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.

Detection of mutant k-ras sequences in the urine of cancer patients.

DNA fragments from apoptotic cells crossing the renal barrier retain their matrix functions, which allows PCR identification of mutant sequences in excreted DNA. We investigated the possibility of detecting k-ras mutations in urinary DNA of tumor patients (colon cancer). In some patients with k-ras codon 12 mutations in tumor cell DNA the same changes were detected in the urinary DNA. The possibility of using this approach for early diagnosis and monitoring of tumors is discussed.