COMPARISON OF CELL CYCLE DURATION IN THE MONOLAYER CELL LINE OF HEPATOMA ZAJDELA AND ITS SUBLINES 3H AND 9C CULTIVATED IN VITRO.

We studied proliferative features of cells in monolayer line of rat hepatoma Zajdela (the original, parent line) and in the sublines 3H and 9C cloned from different types of the colonies of the parental line. These sublines also differed by cytomorphometric parameters, by the types of colonies formed at recloning of these cells in vitro, and by tumorogenicity at transplantation to a rat. Using a time-lapse video of native living cells, we analyzed the cell cycle duration (CCD) and its relationship to a cell shape. Direct measurement of the CCD (a time period from mitosis to mitosis) was performed in individual cells of non-synchronized cultures. Average value of CCD in the parent Zajdela line appeared to be 14.6 ± 0.2 hours, that was higher (at P < 0.05) than in 3H and 9C sublines (13.9 ± 0.2 and 13.5 ± 0.3 hours, respectively). The analysis of CCD distribution histogram showed that all three lines contained a common population of cells with CCD close to 14 hours. Besides, the parent cell line had about 1/3 of cells with a higher CCD (16.7 ± 0.2 hours) while the subline 9C had, on the contrary, 1/3 of cells with a lower CCD (12.6 ± 0.1 hours). In addition, the parameters of cell area, coefficient of cell spreading and coefficient of cell polarization showed the highest correlation to CCD in cells of subline 3H, which are primarily fibroblast-shaped cells (P < 0.01) : the larger the cell area, the longer the CCD; the more flattened or polarized the cell is, the shorter its CCD. In the parental cell line and the subline 9C, both consisting of preferably epithelium-shaped cells, the correlation between CCD and cell shape was less pronounced and showed the opposite direction, that may be explained by a difference in the origin of the cell lines. When considering the differences of CCD in the pairs of daughter cells, we introduce the concept of «the coefficient of symmetry of a cell division¼. The lower its value, the greater the similarity of CCD in a pair of daughter cells. Possible connection of the cell parameters studied in vitro to the tumorigenicity of these cells is discussed.

USING OF CELL LINE HepG2 FOR ASSESSMENT OF TOXIC AND METABOLIC EFECTS OF ANTIPSYCHOTIC DRUGS.

In order to study in vitro the toxic and metabolic effects of antipsychotic drugs (AP) on the cells of hepatic origin we used human hepatoblastoma cell line HepG2. We cultured HepG2 cells in the presence of two AP of the first and second generations (haloperidol and olanzapine, respectively) adding them to the culture medium in concentrations that may at the therapeutic use of AP take place in liver and other tissues of a high lipid content. In the process of cultivation, we detected several products of carbohydrate and lipid metabolism, measured activity of four hepatocellular enzymes in the culture medium, and estimated cell viability/proliferation in the MTS-test. We observed that both AP performed a toxic effect on HepG2 cells, the effect was manifested by a decrease in cell viability/proliferation and an increase in alkaline phosphatase activity in the culture medium. The toxic effect of olanzapine was less pronounced in comparison to haloperidol. According to the data from literature, AP upregulate the expression of lipogenesis genes in the cells of central nervous system, adipose tissue and liver, that might lead to hyperlipidemia. However, we observe in our experiments no increase in the levels of total cholesterol, of cholesterol in lipoproteins of high and low density, of triglycerides in the culture medium containing haloperidol or olanzapine. That observation may have been due to the fact that both AP, which are cationic amphiphiles, are capable to inhibit intracellular traffic of lipids. We also found no effects of haloperidol and olanzapine on the activity of aspartate aminotransferase and gamma-glutamyltransferase, while both AP did reduce the alanine aminotransferase activity. Our work proves that HepG2 cells can be helpful as an in vitro model to obtain new data on metabolic effects of drugs on the cells of hepatic origin and to assess the risk of a drug hepatotoxicity in preclinical studies.

[Spontaneous cytotoxicity treated with anti-tumor drugs of splenocytes of rats on a model of hepatoma cell monolayer cultures].

A purpose of this research is the study of spontaneous cytotoxic activity of effector cells (EC) of the innate immunity of animals against target cells (TC) of cultured hepatoma. There are established differences in the cytotoxic potential of freshly non-activated mouse and rat splenocytes: TC exhibit resistance to splenocytes of mice C3HA and are exposed to active dose-dependent killing under the influence of splenocytes outbred rats. There were revealed two mechanisms of killing of clip-target by splenocytes of rats--secretory variant (Zajdel hepatoma) and the path of classical apoptosis (hepatomas HTC, MH-22a and BWTG3). Single intraperitoneal administration of anticancer drugs cyclophosphamide (CP) and 5-fluorouracil (5-FU) to animals inhibits secretory pathway, whereas the activity of the apoptotic mechanism is enhanced after administration of CF to animals and is unchanged under the action of 5-FU.

Antioxidative activity of high density lipoproteins in vivo.

The present study consists of experimental and clinical investigations. It was shown that a single intravenous injection of a large dose of human HDL3 (200 mg protein) to rabbits with induced hypercholesterolemia (plasma cholesterol 500-700 mg/dl) was accompanied by a significant elevation of plasma HDL and led to a decrease (P < 0.05) of conjugated dienes and trienes by 20-30% after 6 h. Conjugated dienes remained stable for 24 h after HDL administration. In the clinical investigations a weak but statistically significant negative correlation (r = 0.262; P = 0.006) between HDL cholesterol and the content of conjugated dienes in the plasma of a total group of healthy subjects and patients with coronary heart disease (CHD) was found. The data allowed us to conclude that, in addition to other antioxidative systems, HDL also take part in the protection of plasma lipids from peroxidation.