Transport proteins of the ABC family and multidrug resistance of tumor cells.

Some new data concerning the role of transport proteins of the ABC family in multidrug resistance (MDR) of human tumor cells, and problems connected with regulation of these proteins are considered. MDR is a complex phenomenon that may be caused simultaneously by several mechanisms functioning in one and the same cell. Among them there may be the alterations of activity of several transport proteins. Activation of these proteins may be associated with alterations of activities of different cell protective systems and of the signal transduction pathways involved in regulation of proliferation, differentiation, and apoptosis. Clinical significance of multifactor MDR is discussed.

Role of P-glycoprotein in evolution of populations of chronic myeloid leukemia cells treated with imatinib.

Imatinib mesylate (imatinib) is a new generation preparation that is now successfully used for treatment of cancer, particularly for chemotherapy of chronic myeloid leukemia (CML). Imatinib inhibits the activity of chimeric kinase BCR-ABL, which is responsible for the development of CML. The goal of this study was to investigate the role of a multidrug resistance protein, P-glycoprotein (Pgp), in the evolution of CML treated with imatinib. We demonstrate here that although imatinib is a substrate for Pgp, cultured CML cells (strain K562/i-S9), overexpressing active Pgp, do not exhibit imatinib resistance. Studies of CML patients in the accelerated phase have shown variations in the number of Pgp-positive cells (Pgp+) among individual patients treated with imatinib. During treatment of patients with imatinib for 6-12 months, the number of Pgp-positive cells significantly increased in most patients. The high number of Pgp+ cells remained in patients at least for 4.5 years and correlated with active Rhodamine 123 (Rh123) efflux. Such correlation was not found in the group of imatinib-resistant patients examined 35-60 months after onset of imatinib therapy: cells from the imatinib-resistant patients exhibited efficient Rh123 efflux irrespectively of Pgp expression. We also compared the mode of Rh123 efflux by cells from CML patients who underwent imatinib treatment for 6-24 months and the responsiveness of patients to this therapy. There were significant differences in survival of patients depending on the absence or the presence of Rh123 efflux. In addition to Pgp, patients' cells expressed other transport proteins of the ABC family. Our data suggest that treatment with imatinib causes selection of leukemic stem cells characterized by expression of Pgp and other ABC transporters.

Development of drug resistance in the population of colon cancer cells under the effect of multifunctional protein YB-1.

The effects of YB-1 gene on the expression level of P-glycoprotein and drug resistance of tumor cells were studied in cultured HCT116 colon cancer cells. Transitory transfection of chimeric YB-1/GFP gene rendered HCT116 cells a selective advantage in a medium with vinblastine, which caused translocation of the chimeric protein into cell nuclei. This was paralleled by an increase in the expression of P-glycoprotein (multiple drug resistance protein).

Intracellular localization and content of YB-1 protein in multidrug resistant tumor cells.

The multifunctional mammalian protein YB-1 is a member of the large DNA- and RNA-binding protein family with an evolutionarily ancient cold-shock domain. YB-1 is involved in multiple DNA- and mRNA-dependent events and regulates gene expression at various levels. It can be found both in the nucleus and the cytoplasm. Bound to DNA in the cell nucleus, YB-1 functions as a transcription factor interacting with inverted CCAAT-box (Y-box) in promoters and enhancers of multiple genes. In particular, YB-1 regulates activity of the multidrug resistance (MDR) genes MDR1 and LRP. In tumors, YB-1 has been suggested to be an early and global marker of MDR. In this study, we compared amounts of YB-1 mRNAs and intracellular localization of YB-1 protein in six pairs of drug sensitive and drug resistant sublines of diverse tumors. We have shown that neither great increase in the level of YB-1 mRNA nor substantial increase in the number of cells with nuclear localization of YB-1 are obligatory traits of drug resistant tumor cell populations. However, the cells with highest amounts of YB-1 mRNA also demonstrated increased quantities of MDR1, MRP1, BCRP, and LRP mRNAs encoding different MDR proteins. Transfection of two different populations of drug-sensitive cells with YB-1 cDNA led to increase in the amount of YB-1 mRNA. The quantities of MRP1 and LRP mRNAs increased in both populations. Introduction of YB-1 small hairpin RNA (shRNA) resulted in decreased amounts of YB-1 mRNA, as well as MRP1, LRP, and MDR1 mRNAs (in three different cell lines). Our data suggest that although YB-1 regulates several MDR genes, it could not be regarded as a global marker of already formed drug resistant tumor cell populations. It is most likely that at the first steps of MDR development YB-1 activity is necessary for propagation of resistant cell populations rather than for maintenance of drug resistance.

Studies of some mechanisms of drug resistance in chronic myeloid leukemia (CML).

CML is the myeloproliferative disorder connected with the specific chromosome translocation (9;22) and occurrence of the fusion gene/protein BCR-ABL. BCR-ABL protein is believed to inhibit apoptosis and to cause drug resistance. We investigated the correlation of two different forms of BCR-ABL mRNA in 94 pts with their overall survival. It was found that b2a2 (but not b2a3) mRNA expression correlates with longer survival of patients treated with chemotherapy. We did not find an influence of different types of BCR/ABL mRNA on the survival of pts treated with interferon-alpha. FAS/APO-1 antigen was expressed by the cells of 34% of the pts in CML blast crisis (BC) and directly correlated with the the expression of CD34, CD13 and CD14 differentiation antigens. FAS/APO-1 non-expression correlated with higher rate of remissions in BC. We investigated P-glyco-protein (Pgp) expression and functional activity in 40 BC CML pts. 2-fold shorter survival was found in the pts with Pgp expression. Pgp expression strongly correlated with CD13 antigen. Consecutive studies of pts in BC CML show that Pgp expressing cells often do not multiply in the course of BC CML. We postulate that Pgp may be regarded as differentiation marker of the cells and the unfavorable prognostic factor in BC CML.

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.

Alterations of melanin synthesis in human melanoma cells selected in vitro for multidrug resistance.

Previous data showing the correlation of multidrug resistance (MDR) and differentiation in tumor cell populations (Melloni et al. 1988; Stavrovskaya et al. 1990) suggest that: 1) isolation of MDR cells by cytostatic drugs leads to the selection of more differentiated cell variants and 2) in more differentiated cell variants the activity of MDR-related P-glycoprotein (Pgp) is more prominent than in less differentiated cells. Here we used human melanoma cell line mS and two variants selected from mS population: a) MDR variant of mS selected by colchicine (mS-0.5) and b) mS-trRAR/2--variant obtained by introduction of expressing retinoic acid receptor RAR-alpha cDNA into mS cell. The differentiation status, expression of MDR1 gene and Pgp functioning were compared in wild-type cells and mS variants. Electron microscopic examination of melanosomes showed that the mS-0.5 subline comprised more differentiating cells in the population than parental mS cultures and that these cells were at later stages of melanogenesis. The increase in the degree of differentiation in mS-0.5 population coincided with MDR1 gene overexpression, occurrence of Pgp molecules on the cell membrane and acceleration of Pgp-mediated Rhodamine 123 (Rh123) efflux. mS-trRAR/2, proved to be more differentiated than mS cells. The MDR1 mRNA level and Rh123 efflux were not elevated in mS-trRAR/2 cells, however, retinoic acid (RA) treatment increased both the degree of differentiation and Rh123 efflux in mS-trRAR/2 to a greater extent than in mS cultures. Thus, the data obtained in this study are in favor of the suppositions mentioned above. The mechanisms of coordinated alterations of differentiation and Pgp activity in MDR cells are discussed.

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)

Early steps of the P-glycoprotein expression in cell cultures studied with vital fluorochrome.

This study shows that flow cytometry analysis of the rate of fluorochrome Rh123 efflux may be used for detection of cells at initial steps of P-glycoprotein expression and of minor subpopulations of multidrug-resistant (MDR) variants in human cell lines. This method also evaluates the fraction of low-level MDR cells among peripheral blood leukocytes of patients with chronic myeloid leukemia. The alterations in Pgp function were revealed in rat hepatoma cells after short treatment with colchicine.

Clonal analysis of the ability of tumor cells to form colonies in semi-solid medium.

Near-diploid mouse tumor cells CAK.25 AG(r) are characterized by low cloning efficiency in semi-solid medium:: about 10-5 per cell plated in methylcellulose. The properties of cells that have formed colonies in methylcellulose were compared with those of the parental population. Nine of 12 subclones of CAK-25 AG(r) picked from methylcellulose retained parental cloning efficiency when retested for growth, in semi-solid medium. Thus most colonies in methylcellulose were not formed by variant cells different from the parental cell population. Cloning efficiency of CAK-25 AG(r) and of its subclones on the solid substratum was high. All six sub-subclones randomly isolated from the solid substratum had the ability to form colonies in methylcellulose at a frequency no lower than that of the parental clone. Thus, most cells of the population had not lost the ability to form colonies in methylcellulose. The results suggest that the probability of a cell's initiating a colony in semi-solid medium is heritable and identical for most cells of the line under investigation.

Cross-resistance of transformed mouse cells to some drugs.

The Colcemid-resistant L--53 cell strain was examined for cross-resistance to metaphase inhibotors (Vincristine, Vinblastine, estradiol-17beta), an antitumor antibiotic (Rubomycin C) and an alkylating agent (Lycurim), compared with the Colcemid-sensitive L cells. The L-53 cells proved to be resistant besides colchicine to Vincristine, Vinblastine and estradiol-17beta concerning their antimitotic effect. The comparison of the viability of L and L-53 cells in the presence of Rubomycin C and Lycurim showed a resistance of the L-53 cells to Rubomycin C, while the effect of Lycurim was the same on both cell lines. The chromosome-mutagenic action of Lycurim was also equal on both cell lines.