Recent Advances in the Studies of Molecular Mechanisms Regulating Multidrug Resistance in Cancer Cells.

Here we present new approaches to better understanding multidrug resistance (MDR) development in cancer cells, such as identification of components of a complex process of MDR evolution. Recent advances in the studies of MDR are discussed: 1) chemotherapy agents might be involved in the selection of cancer stem cells resulting in the elevated drug resistance and enhanced tumorigenicity; 2) cell-cell interactions have a great effect on the MDR emergence and evolution; 3) mechanotransduction is an important signaling mechanism in cell-cell interactions; 4) proteins of the ABC transporter family which are often involved in MDR might be transferred between cells via microvesicles (epigenetic MDR regulation); 5) proteins providing cell-to-cell transfer of functional P-glycoprotein (MDR1 protein) via microvesicles have been investigated; 6) P-glycoprotein may serve to regulate apoptosis, as well as transcription and translation of target genes/proteins. Although proving once again that MDR is a complex multi-faceted process, these data open new approaches to overcoming it.

Connection between Proliferation Rate and Temozolomide Sensitivity of Primary Glioblastoma Cell Culture and Expression of YB-1 and LRP/MVP.

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.

Problems of Glioblastoma Multiforme Drug Resistance.

Glioblastoma multiforme (GBL) is the most common and aggressive brain neoplasm. A standard therapeutic approach for GBL involves combination therapy consisting of surgery, radiotherapy, and chemotherapy. The latter is based on temozolomide (TMZ). However, even by applying such a radical treatment strategy, the mean patient survival time is only 14.6 months. Here we review the molecular mechanisms underlying the resistance of GBL cells to TMZ including genetic and epigenetic mechanisms. Present data regarding a role for genes and proteins MGMT, IDH1/2, YB-1, MELK, MVP/LRP, MDR1 (ABCB1), and genes encoding other ABC transporters as well as Akt3 kinase in developing resistance of GBL to TMZ are discussed. Some epigenetic regulators of resistance to TMZ such as microRNA and EZH2 are reviewed.

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.

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.

Cellular mechanisms of multidrug resistance of tumor cells.

Multidrug resistance (MDR) is the protection of a tumor cell population against numerous drugs differing in chemical structure and mechanisms of influence on the cells. MDR is one of the major causes of failures of chemotherapy of human malignancies. Recent studies show that the molecular mechanisms of MDR are numerous. Cellular drug resistance is mediated by different mechanisms operating at different steps of the cytotoxic action of the drug from a decrease of drug accumulation in the cell to the abrogation of apoptosis induced by the chemical substance. Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating. The most investigated mechanisms with known clinical significance are: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of the glutathione detoxification system; c) alterations of the genes and the proteins involved into the control of apoptosis (especially p53 and Bcl-2).

Golgi complex is brefeldin A resistant in multidrug resistant cells.

The multidrug resistance (MDR) is one of the main reasons for chemotherapeutic failures in cancer patients. The overexpression of mdr1 gene product, P-glycoprotein (Pgp), leads to the appearance of resistant tumor cells. In the previous paper (Erokhina, 1997) we have demonstrated that the first stages of Pgp-mediated MDR are accompanied by the reorganization of cytoskeleton elements and the vacuolar system. These data were true for two independently isolated sublines of Syrian hamster embryo fibroblasts transformed by Raus sarcoma virus. In this study, we continued the investigation of the properties of the vacuolar system in Pgp-expressing cells. Brefeldin A (BFA), which is not a Pgp substrate, affects different elements of the vacuolar system and blocks vesicular transport. Our data demonstrate that BFA has different effects on parental and resistant cells. In parental cells, the Golgi apparatus and vesicular transport are sensitive to BFA, while in resistant sublines, BFA affects the vesicular transport but not the Golgi apparatus structure. We discuss the existence of similar and different BFA targets in parental and resistant cells and their role in the evolution of multidrug resistance mechanisms.

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.

The role of phosphatidylinositol 3-kinase in the regulation of cell response to steroid hormones.

Phosphatidylinositol 3-kinase (PI-3 kinase) has been implicated in the regulation of many cellular processes, including growth and transformation. We describe the effect of glucocorticoids on cell growth, phosphoinositide formation and PI-3 kinase activity in Rous sarcoma virus-transformed hamster fibroblasts (HET-SR). Using a prolonged dexamethasone treatment of HET-SR cells we have selected a new glucocorticoid receptor-positive cell subline, HET-SR(h), that was resistant to growth inhibitory action of dexamethasone and/or non-hormonal drugs (vinblastine, adriamycin) and was characterized by higher levels of phosphoinositide formation and increased PI-3 kinase activity. Study of the short-term hormone action has shown that both dexamethasone-sensitive and -resistant sublines responded to hormone by a decrease in phospholipid turnover rate. At the same time, in both cell lines activation of PI-3 kinase after dexamethasone addition was revealed. Dexamethasone-dependent activation of PI-3 kinase was more significant and maintained for a longer period in HET-SR(h) cells than in parent HET-SR cells. Finally, by transfecting p110*, a constitutively active catalytic subunit of PI-3 kinase, into hormone-sensitive HET-SR cells, we have found a marked increase in cell resistance to growth inhibitory dexamethasone action. These results suggest that PI-3 kinase may serve as one of the factors providing cell resistance to cytostatic drugs.

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.

Induction of P-glycoprotein functional activity and multidrug resistance by a soluble factor(s) produced by some mammalian cells.

In the previous study we have found that Djungarian hamster fibroblasts with high levels of multidrug resistance (MDR) (colchicine-resistance index RI of 1000 to 42000) produce soluble factor(s) communicating MDR to the drug-sensitive cells of the same species by elevating the functional activity of P-glycoprotein (Pgp). Here we have shown that these cells can influence human tumor cells in the same fashion. Rat hepatoma McA RH7777 cells and their colchicine-resistant derivatives are shown to produce a factor with similar effects (induction of MDR and Pgp functional activity in the drug-sensitive cells). These effects seem to depend on the drug resistance level of the donor cells. Our results show that induction of the Pgp-mediated MDR is not species-specific and the tumor cells with intrinsic MDR (arising from the tissue with a high level of Pgp expression) can produce a factor(s) communicating this type of drug resistance to the sensitive cells.

Studies of P-glycoprotein in chronic myelogenous leukaemia patients: expression, activity and correlations with CD34 antigen.

Over-expression of the P-glycoprotein (Pgp), transmembrane drug efflux pump, has been shown to cause multidrug resistance of tumour cells (MDR). To investigate the clinical significance of Pgp expression for chronic myeloid leukaemia (CML) diagnosis and monitoring we have studied 38 CML patients in various phases of the disease (chronic phase, CP; accelerated phase, AP; blast crisis, BC). Anti-Pgp monoclonal antibody UIC2 and FACScan analysis were used. Pgp functional activity was investigated by evaluation of verapamil influence upon rhodamine 123 efflux from the cells. Correlations between Pgp and CD34 expression were investigated. In CP, Pgp-expressing cells were found in 2/14 patients; in one of them Pgp proved to be non-functional. There were few Pgp-expressing cells in AP cases. The group of BC patients consisted of cases resistant to chemotherapy. This gave us the opportunity to consider whether drug resistance of BC CML patients is preferentially connected with Pgp-mediated MDR. 11/22 BC patients had 20% or more of Pgp-expressing blasts in the peripheral blood. In all four Pgp+ BC cases studied for Pgp activity this protein was functional. Only 4/22 BC patients demonstrated large (40% or more) fractions of Pgp+ blasts. Moreover, sequential studies of 11 BC CML patients during treatment revealed an increase in the number of Pgp-expressing cells in only two cases. This suggests that Pgp+ cells did not often accumulate in BC CML patients due to chemotherapy and are the cause of drug resistance in only a few cases. A positive correlation between Pgp and CD34 expression was found (r = 0.69; P = 0.0004). 3/22 BC CML patients had large fractions of both Pgp+ and CD34+ blasts in their peripheral blood. The BC CML patients with this immunophenotype of blast cells may represent a subtype of BC CML resistant to treatment due to Pgp overexpression.

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.

Colchicine-resistance and enhancement of P-glycoprotein activity after co-cultivation of drug-sensitive cells with multidrug resistant variants.

The role of cellular interactions in the resistance of Djungurian hamster cells to colchicine (CH) and in the efficiency of P-glycoprotein function was studied. Mixtures of CH-resistant and CH-sensitive cells as well as control unmixed cells were propagated for 3 days and the sensitivity of the cells to CH was measured by colony forming assay. Identification of individual subpopulations was possible due to genetic marker (6TG-resistance). The data show that the survival of CH-sensitive cells in CH-supplemented medium increased after co-cultivation with CH-resistant counterparts. To measure Pgp activity the fluorescent dye RH123 and FACScan analysis were used. Pgp-mediated RH123 efflux increased after co-cultivation of CH-sensitive and CH-resistant cells.

Partial restoration of the actin cytoskeleton in transformed Syrian hamster fibroblasts selected for low levels of 'typical' multidrug resistance.

Two independent colchicine (CLC)-resistant sublines of Rous sarcoma virus-transformed Syrian hamster fibroblasts were isolated. Each subline represented variants with 11- and 12.4-fold resistance, respectively, their 23- and 23.7-fold resistant descendants, as well as variants cultured in CLC-free medium for 10 months without loss of resistance. All variants demonstrated 'typical' multidrug resistance. The parental cells contained actin in dispersed form, as determined by rhodamine-phalloidin staining. In contrast, already in 11- and 12.4-fold resistant sublines up to 30% of cells demonstrated restored stress fibers. Cultivation in CLC-free medium leads to the accumulation of cells with a partially restored actin cytoskeleton. Putative mechanisms of up-regulation of stress fiber assembly in cells with P-glycoprotein-mediated multidrug resistance are discussed.

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.

Multinucleation-induced improvement of the spreading of transformed cells on the substratum.

Multinucleation of various cultured cells was produced by polyethylene glycol-induced fusion or by cytochalasin-induced block of mitosis. It was found that multinucleation induced by both methods considerably improved deficient spreading of all the tested transformed fibroblastic lines; average substratum area occupied by one cell and divided per number of nuclei was 2.0-2.5 times larger for multinucleated cells than for mononucleated ones. Improved spreading was accompanied by increased area of lamellar cytoplasm, increased number of focal contacts, and, in certain lines, by the appearance of actin bundles; numerous microtubules and intermediate filaments radiated from perinuclear zones into the lamellas of multinucleated cells. The number of cell-associated fibronectin fibrils was not increased by multinucleation. Cycloheximide did not prevent the improvement of spreading, suggesting that this effect was not due to any alterations of protein synthesis. Colcemid considerably decreased the effect of multinucleation but did not abolish it completely. It is suggested that increase of spreading is due to multinucleation-associated alterations of quantitative interrelationships between various cell components. One of these alterations is probably increased density of microtubules per unit length of outer cell edge.