[Igf-1 and its isoform expression in hepatic cell tumors and the surrounding tissue in mice liver carcinogenesis induced by diethylnitrozamine].

The expression of the Igf-1 gene in mice liver at different stages of development of hepatocellular carcinoma induced by diethylnitrozamine--from the initial diffuse tissue dysplasia and nodular hyperplasia to the development of multiple adenomas and carcinoma--has been analyzed. It was marked that the level of Igf-1 expression in all liver neoplasms decreased; it increased only in the liver tissue surrounding the carcinoma. The dependence of Igf-1 expression on inflammatory processes accompanying tumor growth was analyzed on the model of acute liver damage by diethylnitrozamine. It was established that the level of Igf-1 expression in liver tissue under acute damage in sexually mature mice was the same as in the control group. By the means of semiquantitative evaluation of the products of two Igf-1 splice isoforms--locally active (Mgf) and circulating (Igf- 1v4)--it has been shown that the amount of mRNA of both isoforms in hepatocellular carcinoma was lower, and in tissue surrounding the tumor higher, than in the samples of the control group. At the same time, the proportion of transcripts of isoforms was stable.

Changes in the Proteasome Pool during Malignant Transformation of Mouse Liver Cells.

Multiple forms of proteasomes regulate cellular processes by destroying proteins or forming the peptides involved in those processes. Various pathologies, including carcinogenesis, are related to changes in the functioning of the proteasome forms. In this study, we looked at the changes in the pool of liver proteasomes during nodular regenerative hyperplasia and formation of adenoma and hepatocellular carcinoma in mice treated with Dipin, followed by partial liver resection. The relative content of various proteasome forms was determined using Western blot analysis. The chymotrypsin-like activity of proteasomes was assessed from the hydrolysis of the commercial Suc-LLVY-AMC substrate. It was found that changes in the proteasome pool appeared already during the formation of diffuse nodules, the changes being the increased expression of the X(ß5) constitutive subunit and the LMP7(ß5i) and LMP2(ß1i) immune subunits, accompanied by the increase of the total proteasome pool and the decrease in the chymotrypsin-like activity. These changes were more pronounced in hepatocellular carcinoma. The content of the total proteasome pool and the LMP2(ß1i) immune subunit and the chymotrypsin-like activity in adenoma were intermediate compared to those in the samples of liver with diffuse nodules and carcinoma. In addition, the level of the Rpt6 subunit present in the 19S proteasome activator was increased in carcinoma. Our results indicate that nodular regenerative hyperplasia and adenomatosis may be stages preceding carcinogenesis. We also conclude that there is a need to find signalling pathways that change the expression of various proteasome subunits during carcinogenesis. The 19S proteasome activator, which is overexpressed in malignant tumours, can be a promising target for the development of new anticancer drugs.

[Cytogenetic aberrations in the cells of liver and spermatogenetic epithelium in senescence accelerated SAMP1 and SAMR1 mice]. / Tsitogeneticheskie aberratsii v kletkakh pecheni i spermatogennogo épiteliia u uskorenno stareiushchikh myshei linii SAMP1 i SAMR1.

It was shown that during ontogenesis, the mice prone to (SAMP1) and resistant against accelerates senescence did not differ substantially in the frequency of cytogenetic aberrations in the hepatocytes and spermatogenic cells (spermatozoa and circular spermatids). These data suggest that in the mice of both lines, the processes of appearance, development, and functioning of complex biological systems, such as liver and testis, take place against the background of high genetic instability. The role of genetic instability in senescence is discussed.

[The organizational characteristics of the nucleolar region in the diploid and polyploid nuclei of neoplastic hepatocytes during dipin-induced carcinogenesis]. / Osobennosti organizatsii iadryshkovogo apparata v diploidnykh i poliploidnykh iadrakh novoobrazovannykh gepatotsitov pri dipinovom kantserogeneze.

The use of the number of nucleoli as a criterion allowed us to identify significant differences in the organization of nucleoli in newly formed hepatocytes under the conditions of dipine-induced carcinogenesis 8-10 weeks after its initiation, as compared with normal liver cells of adult mice. The number of nucleoli in tetraploid new hepatocytes was equal to that in the diploid nuclei of normal liver (on average 3.5 and 3.6, respectively) and the number in the new octaploid nuclei was equal to that in tetraploid (6.2 and 6.4, respectively). A higher extent of association of nucleolar organizers under the conditions of endoreproduction or the association of homologous chromosomes in G1 period of the cell cycle is discussed as a possible mechanism responsible for the decrease in the number of nucleoli.

[The mutagenic and modifying properties of emoxipin studied by micronucleus assay in liver cells]. / Izuchenie mutagennykh i modifitsiruiushchikh svoistv émoksipina s pomoshch'iu analiza mikroiader v kletkakh pecheni.

Emoxypin is a medicinal drug from the group of 3-oxypyridines. We studied the capacity of emoxypin to affect the spontaneous level of micronuclear aberrations in the hepatocytes (to decrease or increase it by exerting a mutagenic effect) using the micronucleus test, as well as the capacity to modulate (enhance or weaken) the effects of nitrosomethylurea and X-irradiation. The results obtained do not suggest cytogenetic activity of emoxypin. The nature of "spontaneous" micronuclear aberrations in the liver are discussed, as well as the causes of their age-related increase and adequacy of this model to search for antimutagens.

An improved method of mouse liver micronucleus analysis: an application to age-related genetic alteration and polyploidy study.

The performance of a micronucleus test in liver cells in vivo requires two laborious procedures: stimulation of hepatocytes to division and dissociation of liver tissue into a single-cell suspension. We propose the method of inhalation treatment of mice with carbon tetrachloride to induce cell proliferation and alkaline dissociation of previously fixed tissue. The micronucleus incidence and ploidy classes in terms of cytophotometric DNA content were determined in liver of mice of three age groups (around 2.5, 5.0 and 7.0 months old) after CCl4 treatment or partial hepatectomy. The data obtained show that both methods give the same results. The fraction of micronucleated hepatocytes was 0.69% at the age of 2.5 months; it increased to 8.5% and then to 13.5% at 5.0 and 7.0 months respectively. Simultaneously, the ploidy classes changed both with the aging of the animal and after induced liver regeneration. The percentage distribution of micronucleated cells by ploidy class showed that cells carrying micronuclei were the higher ploidies rather than the population in general. Since polyploid cells contain multiple molecular targets for genetic damage, the micronucleation index per genome unit was estimated. Then the real rate of accumulation of both intrinsic endogenous (and probably the exogenously induced) preclastogenic genetic alterations in hepatocytes during the adulthood of mice was evaluated to be 0.03% per diploid genome per day. This seems to be the first description of the phenomenon of liver cell aging in terms of micronuclear aberrations.

[The cytogenetic monitoring of the southern Aral Sea area. An evaluation of the genotoxic activity of the water]. / Tsitogeneticheskii monitoring Iuzhnogo Priaral'ia. Otsenka genotoksicheskoi aktivnosti vody.

The work gives the data indicating the ability of water sampled from different sources of South Priaralje to induce chromosomal abnormalities in somatic and sexual cells of mammals. It has been shown also that the studied water samples can exert modifying effects on the clastogenic activity of supermutagen nitrosomethylurea. Season-dependent fluctuations of level of spontaneous and induced mutations have been revealed in three tissue systems (liver, cerebellum, and spermatogenic epithelium).

[The assessment of the level of genetic damages accumulated with age and induced in liver cells by the micronucleus production]. / Otsenka urovnia nakoplennykh s vozrastom i indutsirovannykh geneticheskikh povrezhdenii v kletkakh pecheni po produktsii mikroiader.

Latent genetic disturbances in aging liver cells can be registered during interphase by the appearance of micronuclei resulting from certain chromosomal aberrations. Micronuclei were also detected in postmitotic hepatocytes of mouse liver regenerating after partial resection of CCl4 poisoning. In 1.5- and 2-month-old mice, the proportion of micronuclei-containing cells was on average 0.59 and 0.89%, respectively. At the age of 4 and 7 months, the proportion of aberrant cells in hepatocyte population, including cells containing multiple micronuclei, increased to 5.93 and 11.7%, respectively. In order to evaluate parameters used to characterize "spontaneous" aging, experiments were performed in which genetic disturbances were induced by x-irradiation or treatment with dipin, an alkylating agent (individually or in combination); the effect was determined one and two months after the treatment. The yield of micronuclei under the conditions of a mild treatment (irradiation at a dose of 0.7 and 1.4 Gr or dipin at a dose of 30 mg/kg body weight) was similar to that observed during aging. The possible reasons for the increased (as compared to the published data) rate of genetic disturbances in arbitrary intact animals are discussed.

[The dependence of the cytokinetic effects of alkylating antitumor preparations on the phase of the hepatocyte cell cycle in regenerating liver]. / Zavisimost' tsitokineticheskikh éffektov alkiliruiushchikh protivoopukholevykh preparatov ot fazy kletochnogo tsikla gepatotsitov regeneriruiushchei pecheni.

Effects of alkylating antitumor drugs on resting (G0 phase of cell cycle) and proliferating (G1, S, G2 and M phases) hepatocytes were studied in regenerating mouse liver. Cell cycle kinetics (fraction of labeled mitoses, labeling and mitotic indices) were determined by 3H-thymidine autoradiography. Dipin and fotrin as a DNA-damaging agents attack mainly resting (G0) and proliferating (G1) cells. Effect of the damage results in the inhibition of DNA synthesis and G2 phase arrest in the following mitotic cycle. An alkylating drug phopurin as well as ara-C both suppress the mitotic progression in proliferating hepatocytes and do not influence the resting cells.

Functional control of hepatocyte proliferation. Comparison with the temporal control of cardiomyocyte proliferation.

A 4.1-fold difference in liver weight, a 2.5 fold difference in hepatocyte number, and a 1.5-fold difference in mean ploidy were seen in mice on the day of weaning (day 21) reared four (fast-growing group) or sixteen (slow-growing group) to a litter. These differences in proliferation parameters were eliminated in adult mice that had similar liver weights. The kinetics of the changes were not time dependent, i.e. under temporal control, but corresponded to liver-weight kinetics. The definitive number of hepatocytes was established by 3 weeks of age in the fast-growing mice and by 3 months in the slow-growing ones. In contrast to the hepatocytes, multiplication of heart myocytes of the same mice was inhibited by 3-4 days after birth and polyploidization ceased about 3 weeks after birth in mice from both fast- and slow-growing litters, and a stable (approximately 40%) difference in cardiac myocyte number and ploidy persisted throughout the entire period of ontogenesis. It is concluded that hepatocyte proliferation is under functional control whereas cardiac myocyte proliferation is time dependent. Postmitotic hypertrophy of the cell cytoplasm is functionally dependent in both heart and liver. In slow-growing mice, an increase in heart weight occurs only as a result of cytoplasmic hypertrophy, whereas in the liver, cell number and ploidy are both involved.

[Analysis of the postnatal growth of the mouse liver by estimating the hepatocyte count, their weight and ploidy]. / Analiz postnatal'nogo rosta pecheni myshi na osnove ucheta chisla gepatotsitov, ikh massy i ploidnosti.

Changes in the total number of hepatocytes, their distribution by the ploidy classes, as well as changes in the protein content of the cells were studied in 0.5-6 month old mice. The data obtained made it possible to estimate quantitatively the contribution of different growth components: increase in cell number, hypertrophy and polyploidization of cells, to the total increase of the liver mass. From 2 weeks to 1 month, the liver mass is increased via polyploidization (by 70%) and hypertrophy (by 30%). From 1 to 2 months, the liver mass increases due to hyperplasia (by 65%) and polyploidization (35%). After 2 months, the liver growth is practically terminated. The calculated equivalent mass of the liver, i. e. derivative of all three growth components, coincides fairly well with the factual changes in the liver mass.

Genome multiplication in cardiomyocytes of fast- and slow-growing mice.

The number of myocytes and the percentage of cells with a high degree of ploidy increased in the heart ventricles of fast-growing mice compared with slow-growing ones. The mean incidence of octa- and hexadecaploid (by summary DNA content) myocytes was 7% in the slow-growing and 23% in the fast-growing, weaned mice. In these groups, the total myocyte number varied by 20%. There were 43% more myocyte genomes in the heart ventricles of the fast-growing mice than in those of the slow-growing mice. The same differences in cell number and ploidy persist in 90-day-old mice in spite of feeding ad libitum after weaning.

[Changes in the protein mass and ploidy level of the myocytes in the mouse heart]. / Izmeneniia massy belka i urovnia ploidnosti miotsitov v serdtse myshei.

Neonatal mice were grown until 3 weeks of age at a rate of four or sixteen per litter (groups I and II, respectively). The group I animals were characterized by accelerated growth. At day 21 of postnatal development their body and heart weights were three times greater than those of the group II animals. The mean protein content in cardiomyocytes of the group I animals increased faster, correlating with the increase of the heart weight. The fast-growing mice showed an increase in the number of polyploid cardiomyocytes (polynucleate cells mainly), amounting to 15-16% as compared with 2-4% seen in normal and slow-growing animals.

[Measurement of the absolute cell count in the heart and liver. The quantitative preservation of proteins and DNA in isolated cells]. / Izmerenie absoliutnogo chisla kletok v serdtse i pecheni. Kolichestvennoe sokhranenie belkov i DNK v izolirovannykh kletkakh.

Some improvement has been made of the Belov et al. (1975) method of alkaline dissociation of tissues. No cell loss occurs during cell suspension preparation or other procedures. The improved method was shown to be suited for determination of absolute numbers of cardiomyocytes and hepatocytes. Comparison of the living and dissociated hepatocytes revealed preservation of the total cell dry weight and DNA content (classes of ploidy) in the cells treated with formol and concentrated alkali. The method is recommended for studying the cell number, weight, ploidy and mitoses, and for DNA autoradiography.