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 replicative potential of hepatocytes and liver stem cells]. / Replikativnyi potentsial gepatotsitov i stvolovye kletki pecheni

The cellular basis of liver growth is reviewed from overall recent and previous data. According to the present-day ideas, the adult mammalian liver contains at least two cellular populations with many properties similar to the stem cells of renewing tissues that provide for the liver postnatal growth and parenchyma regeneration under various conditions. According to the present nomenclature, the differentiated parenchyma cells--hepatocytes--are a unipotent committed population of stem cells. In addition, there is a system of nonparenchymal multipotent stem cells or oval cells in the liver. Certain key models of liver growth, regeneration, and repopulation that contributed to development of these notions are considered. The recent data are discussed in the context of yet unclear cellular mechanisms providing for the tremendous replicative potential of hepatocytes, the role of polyploidy in the growth effects, and the sources of malignant transformation in the liver.

[Damage to the genome, p53 and cell polyploidization in ontogeny]. / Povrezhdenie genoma, p53 i poliploidizatsiia kletok v ontogeneze.

The protein p53 is a universal tumor suppressor in humans and negative regulator of cell proliferation, which is induced in cells in response to the damage of DNA and controls arrest of the cell cycle at specific checkpoints. A recently discovered function of gene p53 is the maintenance of the diploid state of the genome and a block of the formation of the population of cycling polyploids. This review examines models of disturbed control of the cell cycle due to loss, inactivation, or hyperexpression of gene p53 or its effector gene p21 in transgenic animals, i.e., homozygous mutant mice (p53-/-), (ERCC-1-/-), as well as in genetically defective cell lines in vitro. We discuss mechanisms responsible for the transition from the diploid to the polyploid state owing to the disturbed control of cell cycle by p53 and arising during the ontogenetic polyploidization of cells in normal animals.

[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 prospects for the development and use in ecological research of a cytogenetic method of analysing micronuclei in hepatocytes]. / Perspektivy razrabotki i primeneniia v ékologicheskikh issledovaniiakh tsitogeneticheskogo metoda analiza mikroiader v gepatotsitakh.

The article deals with peculiarities of hepatocyte proliferation which determine the fields of uses for a micronuclear test in hepatocytes and first of all for ecological biomonitoring of genotoxic pollution. Theoretical problems are considered that substantiate applied practices for performing the micronuclear test. Among them there are methods of stimulating the mitotic activity (partial resection of liver, necrogenic hepatotoxins, chemical inductors of adaptive growth); effects of cellular kinetics (volume of proliferative pool, blockade of premitotic period) on quantitative criteria of the method. The capacity of hepatocyte population to preserve and accumulate injuries of genetic material is 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.

[Complete change of the cell composition of the liver parenchyma following exposure to dipin and partial resection]. / Polnaia smena kletochnogo sostava parenkhimy pecheni posle vozdeistviia dipina i chastichnoi rezektsii.

The phenomenon of total replacement of preexisting and damaged hepatocytes in mice were demonstrated by the method of autoradiography. Adult mice were injected an alkylating drug Dipin 2 h prior to partial hepatectomy and then proliferating cells were labelled by means of multiple injections of 14C-thymidine. Dipin in combination with mitotic stimulation induced multiple mitotic aberrations in proliferating hepatocytes resulting in degeneration, death and then elimination of prelabelled liver cells. New parenchymal tissue originated from non-labelled preneoplastic nodules. These hepatocyte nodules grew in size, propagated and 8-10 months later completely replaced the preexisting hepatocytes.

[Cytogenetic study of the effect of gossypol on mouse germ cells]. / Tsitogeneticheskoe issledovanie deistviia gossipola na muzhskie polovye kletki myshei.

The clastogenic and mutagenic activities of a new antifertility and antitumor agent gossypol were studied in the mouse male germ cells. Results of the present work indicate that at the doses 125 and 250 mg/kg the drug does not significantly increase frequencies of the micronuclei in the early spermatids and sperm head abnormalities. Hence, genotoxic influence can not be proposed as responsible for the antifertility effect of gossypol.

[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.