Influence of RAR α gene on MDR1 expression and P-glycoprotein function in human leukemic cells
© Stromskaya et al; licensee BioMed Central Ltd. 2005
Received: 22 October 2004
Accepted: 24 May 2005
Published: 24 May 2005
Multidrug resistance (MDR) phenotype of malignant cells is the major problem in the chemotherapy of neoplasia. The treatment of leukemia with retinoids is aimed on the induction of leukemic cells differentiation. However the interconnections between retinoid regulated differentiation of leukemic cells and regulation of MDR remains unclear.
Four lines of cultured leukemic cells of diverse types of differentiation were infected with RAR α gene and stable transfectants were isolated. We investigated the differentiation of these cells as well as the expression of RAR α and MDR1 genes and P-glycoprotein (Pgp, MDR protein) functional activity in these cells.
All RAR α transfected sublines demonstrated the increase in the quantity of RAR α mRNA. All these sublines became more differentiated. Intrinsic activity of MDR1 gene (but not Pgp functional activity) was increased in one of the transfectants. All-trans-retinoic acid (ATRA) induced Pgp activity in two of three infectants to a larger extent than in parental cells.
The data show that RARα regulates MDR1/ Pgp activity in human leukemic cells, in the first place, Pgp activity induced by ATRA. These results show that RARα overexpression in leukemic cells could result in MDR.
Multidrug resistance (MDR) phenotype of malignant cells is the major problem in the chemotherapy of neoplasia. P-glycoprotein (Pgp) activity is recognised to be one of the major mechanisms responsible for MDR. Pgp transports many structurally diverse compounds across the cell membrane and confers the MDR phenotype in tumor cells . A number of signaling pathways participate in the regulation of MDR1 gene expression and the activity of its product, Pgp . Some of these signaling pathways could participate in coordinated regulation of MDR1/ Pgp activity, cell proliferation and cell differentiation. It was shown that retinoic acid (RA) can modulate MDR1 gene expression [3–5]. Retinoids are known to be involved into the regulation of the cell growth, differentiation and apoptosis. In the last decade retinoids became implicated into the treatment of leukemia and some solid tumors . This approach changed the focus of the haematological diseases treatment from the cytotoxicity of the anti-cancer drugs to the reversal of arrested maturation of leukemic cells. Retinoids act via two families of receptors (RARs – RARα, RARβ, RARγ) and RXRs (RXRα, RXRβ, RXRγ). There is the evidence that RARα is the crucial receptor mediating the biological effects during retinoid signaling in some cells . Cell differentiation caused by the stable overexpression of receptor RARα was shown to result in constitutive over expression of MDR1 gene in some cultured cells of solid tumors . However the interconnections between RA/RARα regulated differentiation of leukemic cells and regulation of MDR1/Pgp activity remains unclear. In some leukemic cells RA did not influence MDR1 and/or Pgp activity, while in the others it either augmented or reduced MDR1/Pgp expression [5, 8]. The aim of this study is to investigate if effects of all-trans-retinoic acid (ATRA) on MDR1/Pgp activity in leukemic cells are connected with RAR α expression and with the leukemic cell differentiation. We isolated sublines of cultured leukemic cells characterized by the stable RAR α overexpression and investigated the constitutive and ATRA induced MDR1/Pgp activity in these cells. Our data show that various RAR α transformed leukemic cell lines acquired more differentiated phenotype. Constitutive level of MDR1 gene expression increased in one of RAR α overexpressing cell sublines. RAR α overexpression did not influence Pgp functional activity while Pgp activity induced by ATRA was elevated in all infectants studied. This shows that the main effect of RAR α in the cells studied is its influence on the induced functional activity of Pgp.
Cell lines and culture
Lines of cultured leukemic cells used in the study: H9 cells (acute human T-cell leukemia) , KG-1 cell line (cells of acute myelogenous leukemia) , K562 cell line (cells obtained from the patient in blast crisis of chronic myeloid leukemia) , NB4 (acute promyelocytic leukemia) .
Cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco, USA), 2 mM L-glutamine, 50 μg/ml gentamycin at 37°C in a fully humidified atmosphere of 95% air and 5% CO2. All the derived cell lines described in this paper were obtained by retroviral infection and selection with the appropriate antibiotic. ATRA (all-trans-retinoic acid, Sigma, USA) was added to the culture medium at seeding or 24 hours after seeding (see Legends to Figures).
Expression vector and retrovilal infection
The PA317/LRARSN retroviral vector-producing cell line was used. All the procedure was described earlier . In brief, the vector used contains a cDNA fragment harbouring the complete coding sequence of the RAR α gene driven by the Moloney murine leukemia virus long-terminal repeat as well as the SV40 early promoter-driving neomycin phosphotransferase gene (neo) as a selectable marker . The cells (4 × 105 per 25-cm2 flask) were seeded 24 h before infection. Conditioned medium from a retrovirus-producing cell line was filtered through a 0.45-μm membrane (Millipore, USA), diluted 1:1 with medium, containing 1% serum and 8 μg /ml Polybrene and added to the cells for 24 h at 37°C, 5% CO2. Further selection were carried out by culturing cells in medium supplemented with 400 μg/ml G418 (Gibco, USA) for at least 21 days. The medium was changed twice a week. The pool of G418-resistant cells was resuspended in culture medium and progressively expanded.
Assay of cell growth, apoptosis and differentiation
Cells were seeded into 24-well plates (1 × 104 cell per well) and the cell number was counted at days 1, 3, 5 and 8 after seeding. The apoptosis in the populations of the parental and RARα infected cell lines was performed using the standard procedure . Cells were collected 24 h after seeding, washed with PBS, and fixed in 70% ethanol overnight at 4°C. Fixed cells were suspended in citric buffer and stained with propidium iodide (5 mcg/ml) in PBS for 1 hour at 4°C. DNA content was subsequently measured by FACScan (Becton Dickinson, USA).
The immunophenotype of the cells was evaluated as previously described . Surface expression of the following antigens was determined: CD3, CD5, CD7, CD8, CD11b, CD13, CD15, CD33, CD34, HAE3 and HAE9. In brief, cells were incubated with phycoerythrin-labelled monoclonal mouse antibodies for 20 min at 4°C (Becton, Dickinson), washed with RPMI 1640 medium and analyzed with a flow cytometer (Becton Dickinson).
Analysis of rhodamine 123 (Rh123) efflux by the cells
The technique used in the study was described in . Cells were loaded with 5 μg/ml Rh123 (Sigma) for 10 min at 37°C, washed twice with cold PBS, pH 7.2, and incubated for 30 min in dye-free medium at 37°C. After the completion of incubation, cell were washed twice with cold PBS. Cell fluorescence was measured on a flow cytometer FACScan (Becton Dickinson, USA). Each measurement counted 5000 events. Non-viable cells were gated out of the analysis on the basis of side scatter.
RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of RARα and MDR1 genes expression
Specific gene primers used for RT-PCR
Influence of RARα gene overexpression on cell differentiation, proliferation and spontaneous apoptosis
Influence of RARα overexpression on MDR1 gene activity
ATRA (5 μM applied for 48 h) increased MDR1 gene expression in all examined cell lines either in parental or RARα transfected cells (Fig. 6). In H9/RAR cells effect of ATRA on MDR1 expression was significantly greater in comparison with parental cells. In other ATRA treated RARα transformed cell sublines MDR1 expression was undistinguishable from ATRA treated parental cells (Fig 6).
Influence of RARα gene transformation on Rh123efflux by the cells
Influence of RARα transformation on the intrinsic and induced expression of MDR1 gene
Rh123 efflux (mean fluorescence intensity)
The treatment of leukemia with retinoids is aimed on the induction of leukemic cells differentiation. The question is: are there interconnections between RA/RARα regulated differentiation of leukemic cells and MDR1/Pgp activity? In this study we have isolated more differentiated variants of the cultured leukemic cells by the introduction into the cells of RARα gene encoding one of RA receptors. All RARα transformed leukemic cell populations were characterized by the higher RARα gene expression in comparison with the parental cells. All RARα transformed leukemic cell populations became more differentiated. This was demonstrated by the studies of the differentiation markers, by the increase in the number of cells dying by spontaneous apoptosis and by the decrease of the proliferation rates of most RARα transfected cell sublines. Thus, RARα overexpression could result in the increase of the differentiation of various leukemic cell populations.
We compared MDR1 gene expression and Pgp functional activity tested by Rh123 retention in parental and RARα transformed cells. The results are summarized in the Table 2. Increased constitutive (uninduced) expression of MDR1 gene was found in one of four cell lines after RARα transformation (H9/RAR, Table 2, Fig. 6). In the previous experiments with melanoma and hepatoblastoma human cells we have shown that constitutive expression of MDR1 gene was increased after RARα transfection in both RARα transformed cell sublines . Thus interconnections between regulation of the basal MDR1 and RARα activities could exist both in the cells of solid tumors and in the leukemic cells. Our data suggest, that in the cell populations of solid tumors RARα overexpression could be accompanied by constitutive MDR1 over-expression more often than in the cells of hematopoietic malignancies.
Our study did not reveal the occurrence of the functional Pgp in leukemic cells studied after RARα transformation. In H9/RAR cells elevation of the constitutive MDR1 expression did not lead to the increase in Rh123 efflux (Fig. 7A, Table 2). Some studies also have described discrepancies between Pgp (protein) or MDR1 mRNA expression and Pgp function in leukemic cells [18, 19]. These discrepancies could occur for a variety of reasons. Anyway, our data show that increase in the differentiation of leukemic cell populations induced by RARα overexpression did not result in the elevation of constitutive Pgp functional activity. In our previous study we found that RARα overexpression did not change Pgp functional activity in two RARα transformed sublines of human cells (melanoma and hepatoblastoma) but did change it in the rat cells . It seems that exogeneous RARα in the cells of human malignancies does not influence basal Pgp functional activity.
In KG-1/RAR characterized by the increased differentiaion (Table 1) we had not found increase in the constitutive MDR1 expression and Pgp functional activity decreased (Fig. 7C, Table 2). It is known that blood stem cells and early progenitors expressing CD34 antigen also express high levels of functionally active Pgp . Maturation of these cells is accompanied by the decrease in Pgp expression and even more rapid decrease in Pgp functional activity . It may be suggested that alterations of Pgp function in KG-1/RAR are connected with the differentiation of these cells.
The situation with Pgp functional activity induced by ATRA in the cells studied differs from the situation with constitutive activity of this protein. In all three RARα transfected cells ATRA had induced Pgp fuctional activity (Fig. 8. Table 2). Moreover, in two RARα transformed sublines (H9/RAR and KG-1/RAR) ATRA activated Pgp, while in the parental cells it had either no effect (H9) or activated Pgp to a lesser extent (KG-1) (Table 2). These data suggest that RARα participate in the control of induced, but not in constitutive Pgp functional activity in leukemic cells.
The regulation of MDR1 gene transcription and Pgp functional activities are the complex processes [1, 2]. The studies of these processes are underway. Our data show that RARα gene overexpression could influence the induced Pgp functional activity in leukemic cells, i.e. could participate in the occurrence of multidrug resistance in the populations of these malignant cells. It seems that this influence could depend on the cell context.
This work was supported by grants 04-04-48613a and 02-04-48200 from the Russian Foundation for Basic Research.
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