Anikó Venetianer

THE FUNCTION OF HEAT-SHOCK PROTEINS IN STRESS TOLERANCE

We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid‐resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid‐sensitive Faza 967 cells from which clone 2 was derived. We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance. Our experiments show that clone 2 cells were not protected from the killing effect of heat by pretreatment with sodium arsenite, whereas Faza 967 cells were. These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells. Stable heat‐resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat‐inducibility of hsp68. These results suggest that several hsps are needed to protect the critical biological processes at high temperature. The heat‐resistant hepatoma cells also became resistant to several anticancer drugs. The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P‐glycoprotein. Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P‐gp may participate in protection against environmental stress such as heat.

Antiproliferative and Apoptosis‐Inducing Effects of Hemin in Hepatoma Cells

Abstract: Hemin is an extremely versatile molecule that may have cytotoxic or cytoprotective effects on certain cells. We investigated the effect of hemin on the growth of hepatoma cells, including the multidrug‐resistant ones. Searching for new tools that interfere with the growth of hepatomas is an important area of clinical research. Cell viability and proliferation of drug‐sensitive and multidrug‐resistant hepatoma cell lines was determined using the trypan‐blue exclusion test XTT/PMS and colony‐forming ability assays. Apoptosis was assessed by confocal microscopy and DNA ladder assay. Hemin inhibited the proliferation and induced apoptosis in both drug‐sensitive and multidrug‐resistant hepatoma cells overexpressing functional P‐glycoprotein. zVAD‐fmk inhibited the hemin‐induced decrease in cell viability, pointing to a role of caspases in hemin‐induced apoptosis. The antiproliferative and apoptosis‐inducing effects of hemin might be considered in the design of treatment for patients with hepatoma.

Overexpression of P-glycoprotein in heat-and/or drug-resistant hepatoma variants

We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68.Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 μg/ml colchicine (termed ashighly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stableheat-resistant variants of clone 2 were also isolated, which showed increaseddrug resistance to several drugs, i.e. they becamemoderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heatresistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against enviromental stress such as heat.

Isolation and characterization of L-cell variants with altered sensitivity to glucocorticoids.

Stable glucocorticoid-resistant receptor-containing (R+)variants of L cells have been obtained and characterized. Five clones of such cells were obtained after chemical mutagenesis of the A9HT cell line. These clones and an additional, spontaneously resistant subclone of the LB82 cell line all contain glucocorticoid receptors which appear normal with regard to quantity, affinity for steroids, and in vivo localization in subcellular compartments. Somatic cell hybrids were formed between two of these R+-resistant clones, a previously isolated R−-resistant clone, and three different sensitive L cell parents. In no case was steroid resistance dominant. Thus, these resistant L cells appear to be candidates for complementation analysis. The R+-resistant clones may be hindered in as yet undefined steps of glucocorticoid action.

Biotransformation of 17β-hydroxy-11β-(4-dimethylaminophenyl)17α-1-propynyl-estra-4,9-dien-3-one (RU486) in rat hepatoma variants

Abstract Metabolism of the synthetic steroid 17β-hydroxy-11β-(4-dimethylaminophenyl)17α-1-propynl-estra-4,9-dien-3-one (RU486) occurs in the dedifferentiated S-H56-125 variant of Reuber hepatoma. Considering that rat liver cytochrome P450 (P450) monooxygenases are engaged in different oxidative steps of the metabolism of RU486, the influence of several prototype P450 inducers was investigated. The data obtained by treating H56 and S-H56-125 hepatoma cells with different P450 inducers (dexamethasone (DEX), benzathracene, phenobarbital) or with a specific P450 inhibitor, troleandomycin, led us to conclude that CYP3A is involved in the hydroxylation of RU486. This form is induced by DEX independently of the availability of the canonical glucocorticoid receptor.

Effect of phenobarbital on the glucocorticoid receptor in rat hepatoma cells

Phenobarbital is a potent inducer of several liver-specific genes such as those encoding detoxication enzymes, including cytochromes P450. However, the mechanisms of action of the barbiturate are poorly understood. Since both, phenobarbital and glucocorticoids, are capable of inducing the same cytochrome P450 species, we asked whether the glucocorticoid receptor could participate to the phenobarbital induced responses. The results presented here show that phenobarbital was able to induce a two-fold increase in the affinity of the glucocorticoid receptor for the binding of dexamethasone, as well as a 30% increase of the receptor number in Reuber rat hepatoma cells of the Fao line. These effects may have a biological significance since they were paralleled by an enhancement of the dexamethasone-induced tyrosine aminotransferase activity, a glucocorticoid inducible function in rat hepatoma cells and in rat liver. To our knowledge, phenobarbital is the first compound shown to be able to induce, in intact cells, an increase in the affinity of the glucocorticoid receptor for the binding of its ligand.

mRNA Levels of Related Abcb Genes Change Opposite to Each Other upon Histone Deacetylase Inhibition in Drug-Resistant Rat Hepatoma Cells

The multidrug-resistant phenotype of tumor cells is acquired via an increased capability of drug efflux by ABC transporters and causes serious problems in cancer treatment. With the aim to uncover whether changes induced by epigenetic mechanisms in the expression level of drug transporter genes correlates with changes in the drug resistance phenotypes of resistant cells, we studied the expression of drug transporters in rat hepatoma cell lines. We found that of the three major rat ABC transporter genes Abcb1a, Abcb1b and Abcc1 the activity of only Abcb1b increased significantly in colchicine-selected, drug-resistant cells. Increased transporter expression in drug-resistant cells results primarily from transcriptional activation. A change in histone modification at the regulatory regions of the chromosomally adjacent Abcb1a and Abcb1b genes differentially affects the levels of corresponding mRNAs. Transcriptional up- and down-regulation accompany an increase in acetylation levels of histone H3 lysine 9 at the promoter regions of Abcb1b and Abcb1a, respectively. Drug efflux activity, however, does not follow tightly the transcriptional activity of drug transporter genes in hepatoma cells. Our results point out the need for careful analysis of cause-and-effect relationships between changes in histone modification, drug transporter expression and drug resistance phenotypes.

Changes in the expression of differentiated functions during long-term cultivation of rat hepatoma cells

The stability of the expression of six differentiated functions was examined during long-term cultivation of rat hepatoma cells. Faza 967 cell line—a clonal descendant of the Reuber H35 hepatoma—is characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenetic enzymes; secretion of serum albumin; and the presence of liver isozymes of alcohol dehydrogenase (ADH-L), aldolase (aldolase-B) and five isozymes of lactate dehydrogenase (LDH). During the 3-year-long cultivation of Faza 967 cells TAT specific activity, inducibility, and albumin production were reduced drastically whereas the expression of the three liver-specific isozymes examined was maintained. The majority of Faza 967 cells were able to perform gluconeogenesis after 3 years of continuous cultivation. Our results show that long-term cultivation of hepatoma cells may change the expression of certain liver-specific functions independently of the expression of other differentiated functions.

Sensitivity to glucocorticoids of lymph node cells stimulated in vivo by oxazolone.

Sensitization of B10.A mice with oxazolone results in delayed‐type hypersensitivity (DTH) and a two‐to three‐fold increase in the weight of regional lymph nodes by the third day. Hydrocortisone treatment of the animals 3 days after sensitization suppresses the manifestation of DTH on the tenth day hut not on the seventieth day and significantly decreases the weight of lymph nodes both in control and oxazolone‐sensitized mice. The regional lymph node cells of oxazolone‐stimulated mice were, like the controls, sensitive to dexamethasone in vitro, as judged by viability and thymidine incorporation. Dexamethasone binding capacity of the regional lymph node cells 72 h after oxazolone sensitization increased. However, the dissociation constant for dexamethasone, the binding specificity, and the nuclear transfer of the hormone were not altered. Since high doses of hydrocortisone given.1 days after sensitization were unable to erase permanently the DTH lo oxazolone, it seems very likely that cells responsible for the immunological memory survive the high dose of hydrocortisone.

Genetic analysis of dexamethasone resistance in L cells by somatic cell hybridization

Stable dexamethasone resistant and receptor-containing (R+) variants of L cells have been characterized by somatic cell hybridization. Neither of the variants had a clearly dominant phenotype in hybrids with dexamethasone-sensitive fibroblast lines, i.e. the resistance of the variants was not due to transdominant factors. Somatic cell hybrids formed between one of the R+-resistant clones and an independent resistant fibroblast cell line showed complementation--the hybrid clones were as sensitive to the steroid as the sensitive parental lines. Complementation, however, disappeared after continued culture of the clones. The return of the dexamethasone-sensitive phenotype was not always linked with similar changes in the responsiveness to another steroid, e.g. progesterone. Our clones can be considered to be resistant variants, designated death-less (d-), where the cells are defective in a non-receptor component involved in the hormone response. The fact that complementation can occur indicates the existence of at least two such steps in the pathway.