Identification of structural DNA variations in human cell cultures after long-term passage.

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8-12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.

Influence of proteasome inhibitor bortezomib on the expression of multidrug resistance genes and Akt kinase activity.

The goal of this work was to study the mechanisms of ABC family transport proteins' regulation by a new-generation antitumor drug - the proteasome inhibitor bortezomib (Velcade). ABC transporters determine the multidrug resistance of tumor cells (MDR). We confirmed our previously discovered observation that bortezomib affects the expression of genes involved in the formation of MDR (ABCB1 gene, also known as MDR1, and ABCC1-MRP1), reducing the amount of their mRNA. This effect was found to depend on Akt kinase activity: the Akt activity inhibitor Ly 294002 increased the amount of MRP1 mRNA in KB 8-5 cells. It was also shown that bortezomib increased the amount of Akt kinase phosphorylated form in cell lines of malignant cells KB 8-5 and K 562/i-S9 that overexpressed ABCB1 transporter (Pgp), and did not affect the amount of activated Akt in the corresponding wild-type cells. When exposed to bortezomib, selection of resistant to it cell variants was much faster for a Pgp-overexpressing cell population (compared to wild-type cells). It is shown that bortezomib affects the amount of MRP1 gene mRNA, relocating the multifunctional protein YB-1, dependent on Akt activity, from cytoplasm to nuclei of MCF-7 breast cancer cells. The data indicate that the transcriptional activity of YB-1 might be one of the mechanisms that determine the effect of bortezomib on the amount of MRP1 gene mRNA.

Influence of exogenous RAR alpha gene on MDR1 expression and P-glycoprotein function in human and rodent cell lines.

The goal of our study was to obtain direct evidence of co-ordinated regulation of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) and differentiation in tumour cells and to study some signalling pathways involved in joint regulation of these two cell phenotypes. The sublines of human melanoma (mS) and hepatoma (human HepG2 and rat McA RH 7777) cell lines were obtained by retroviral infection of the wild-type cells with the cDNA of the human retinoic acid receptor alpha (RAR alpha). The resulting sublines stably overexpressed exogenous RAR alpha gene. The infectants became more differentiated than the parental cells as determined by a decrease in the synthesis of the embryo-specific alpha-fetoprotein in HepG2 and McA RH 7777 hepatoma cells and by an increase in melanin synthesis in mS cells. The differentiation of human cells was accompanied by an increase in the amounts of MDR1 mRNA but not by an increase in P-gp activity as a drug transporter, in contrast, in the rat RAR alpha overexpressing cells P-gp functional activity was elevated. Treatment with cytotoxic drug (colchicine) or retinoic acid (RA) resulted in a slight increase in P-gp activity in the parental and RAR alpha-infected melanoma cells, whereas the increase in P-gp function in the infected hepatoma cells (both human and rat) was very prominent. Thus, we provide new evidence that cell differentiation caused by the overexpression of the gene participating in the differentiation programme leads to overexpression of MDR1 gene and drug resistance and that this effect is tissue and species specific. These data imply that the activation of the RA-controlled signalling pathway up-regulates MDR1 gene expression.

Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function.

We compared the influence of exogenous N-ras oncogene and treatment with PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.

Influence of exogenous ras and p53 on P-glycoprotein function in immortalized rodent fibroblasts.

The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)