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Keywords:

  • all-trans retinoic acid ;
  • multidrug resistance;
  • P-glycoprotein;
  • acute promyelocytic leukaemia

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDMENTS
  7. References

We analysed the relationship between all-trans retinoic acid (ATRA) resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL). There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA-transduced NB4 subline and between ATRA-resistant NB4 cells (NB4/RA) and an MDR1 cDNA-transduced NB4/RA subline. PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA-treated cell lines. Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA-induced complete remission.

Several mechanisms of all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukaemia (APL) have been proposed, most of which, however, were not well elucidated. Some investigators have highlighted the role of P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in retinoid resistance ( Su et al, 1994 ; Kizaki et al, 1996 ). P-gp is an energy-dependent efflux pump that generally decreases intracellular drug accumulation. Therefore, decreased intracellular accumulation of retinoid may occur in retinoid-resistant APL cells. Here, we have tried to clarify the relationship between P-gp-associated MDR and ATRA resistance.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDMENTS
  7. References

An APL cell line, NB4, was kindly provided by Dr Lanotte (Hospital Saint-Louis, Paris, France). The ATRA-resistant subline, NB4/RA, was obtained after culturing NB4 with ATRA (Sigma Chemical, St Louis, MO, USA) ( Kitamura et al, 1997 ). NB4/MDR and NB4/RA/MDR were established by the transduction of NB4 and NB4/RA, respectively, with HaMDR retrovirus, as reported previously ( Sugimoto et al, 1997 ). K562 and NOMO-1, and their adriamycin-resistant sublines, K562/ADR and NOMO-1/ADR ( Takeshita et al, 1995 ), were also used.

After informed consent, APL cells were obtained from 10 patients with APL and isolated over Ficoll-Paque (Pharmacia, Uppsala, Sweden). Five were newly diagnosed, and five were relapsed patients who had experienced difficulty in achieving second complete remission (CR) with ATRA therapy.

Reverse transcription PCR (RT–PCR) assay for MDR1, MDR-related protein (MRP) and lung-resistant protein (LRP) expression was performed as reported previously ( Takeshita et al, 1995 ). A total of 25–40 cycles was performed.

P-gp, MRP and LRP were stained by immunofluorescence using MRK16, anti-MRP and anti-LRP monoclonal antibodies (Nitirei, Tokyo), respectively, as described previously ( Takeshita et al, 1995 ; den Boer et al, 1997 ). Ten thousands events were counted using the EPICS Elite flow cytometer (Coulter, Hialeah, FL, USA).

Intracellular accumulation of rhodamine-123 (Rh123) was analysed by preincubation of cells (1 × 106/ml) in the presence or absence of 2 μmol/l PSC833 (Novartis Pharma, Basle, Switzerland) for 1 h and by further incubation in the presence of 200 ng/ml Rh123 for 1 h. Ten thousands events were counted using the EPICS Elite.

Intracellular accumulation of ATRA was determined by ATRA's characteristic emission curve, which was excited by a 325 nm line of ultraviolet (UV), as reported previously ( Kahan, 1967). After preincubation at 37°C in the presence or absence of 2 μmol/l PSC833 for 1 h, cells were incubated with 1 μmol/l ATRA at 37°C for 6 h. Then, 10 000 events were counted immediately using the EPICS Elite. The degree of dissociation (%) between the fluorescence intensity (FI) of cells that reacted with or without ATRA was analysed by the channel-by-channel subtraction method.

Myeloid differentiation was evaluated by cell cycle distribution, cell morphology, the nitroblue tetrazolium (NBT) reduction activity and the expression of CD11b after incubation with ATRA (10−7 mol/l) in the presence or absence of 2 μmol/l PSC833 for 3–5 days as described previously ( Kitamura et al, 1997 ).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDMENTS
  7. References

NB4/MDR, NB4/RA/MDR, K562/ADR and NOMO-1/ADR cells expressed MDR1 mRNA by RT–PCR. Neither MRP mRNA nor LRP mRNA was detectable in NB4, NB4/MDR, NB4/RA and NB4/RA/MDR. These results were confirmed by flow cytometric analysis.

Intracellular ATRA accumulation in NB4 cells was increased in a time- and dose-dependent manner. There was no difference in the intracellular concentration of ATRA between NB4 and NB4/MDR cells and between NB4/RA and NB4/RA/MDR cells (Fig 1). Similar results were observed between K562 and K562/ADR cells and between NOMO-1 and NOMO-1/ADR cells. PSC833 did not increase the intracellular ATRA accumulation in these cells.

image

Figure 1. /ADR cells. The bar graphs are the means of five experiments. Statistical analyses of the intracellular ATRA accumulations in each cell line in the presence and absence of PSC833 or those between two different cell lines were performed using the Student's t-test. P-values are included in the figure.

Download figure to PowerPoint

After 5 days of culture with ATRA, there was no difference in the apoptosis determined by cell cycle distribution between NB4 and NB4/MDR (57·6% vs. 53·6%), and between NB4/RA and NB4/RA/MDR (23·9% vs. 24·3%). PSC 833 did not increase the apoptosis.

After 5 days of culture with ATRA, there was no difference in the amounts of CD11b, the NBT reduction activity and the morphology between NB4 and NB4/MDR cells, and between NB4/RA and NB4/RA/MDR cells ( 1 Table I); PSC833 did not affect these parameters for differentiation in these cells.

Table 1. Table I. NBT reduction activity and the amount of CD11b after incubation with ATRA in the presence or absence of PSC833 for 5 days. * Cells were cultured without ATRA and PSC833.Thumbnail image of

MDR1 mRNA and P-gp were not detectable in three out of five relapsed patients. Intracellular ATRA accumulation of APL cells from relapsed cases was not less than that of the cases at studied diagnosis (26·6 ± 2·8% vs. 21·9 ± 3·3%). PSC833 did not increase the intracellular ATRA accumulation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDMENTS
  7. References

This is the first report on the analysis of intracellular ATRA accumulation in intact APL cells. Dermine et al (1993 ) analysed the intracellular ATRA in NB4 and its ATRA-resistant subline by high-performance liquid chromatography (HPLC) and reported that the ATRA-resistant cells contained more ATRA than original cells, but this was not significant. As ATRA is chemically unstable to light, heat and oxidation ( Hashimoto et al, 1995 ), HPLC may not be suitable for quantifying it precisely. Therefore, using a multilaser-equipped cytometer, we determined intracellular ATRA accumulation by its emission characteristics excited by UV and demonstrated that the intracellular ATRA accumulation was not affected by P-gp. We probably detected only intracellular free ATRA, because the mRNA levels of cytoplasmic retinoic acid-binding proteins (CRABP) were not different between NB4 and NB4/RA (data not shown). We also demonstrated the ineffectiveness of an MDR modifier, PSC833, on ATRA resistance, which supported a limited role for P-gp in ATRA resistance.

Kizaki et al (1996 ) and Su et al (1994 ) explained ATRA resistance in part by the P-gp expression in HL60 cells. In these two studies, HL60 cells might have simultaneously acquired both P-gp-associated MDR and P-gp-dissociated ATRA resistance in the culture, or the mechanism of acquired ATRA resistance might be fundamentally different between NB4 and HL60 cells, because HL60 was established from FAB-M2 acute myeloid leukaemia without PML/RARα.

We reported previously that CR was achieved by ATRA alone in 16 (80%) out of 20 post-chemotherapy relapsed/refractory APL ( Ohno et al, 1993 ). The rate was similar to that (89%) of newly diagnosed APL treated with ATRA alone or in combination with chemotherapy ( Asou et al, 1998 ). In addition, recent studies have shown significantly lower P-gp expression in APL, even in relapsed or resistant APL, than in other types of AML ( Takeshita et al, 1996 ). These reports and our present study suggest that P-gp plays a limited role in ATRA resistance in APL cells.

ACKNOWLEDMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDMENTS
  7. References

We would like to express our sincere gratitude to Ms Yoshimi Suzuki, Ms Noriko Anma, Ms Naoko Sugiura and Ms Keiko Natsume for technical assistance, and to Dr Kiyoshi Shibata (Equipment Centre, Hamamatsu University School of Medicine) for his co-operation. This study was partly supported by grants-in-aid from the Ministry of Health and Welfare (no. 9-2) and from the Ministry of Education and Culture (no. 10670939).

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDMENTS
  7. References
  • 1
    Asou, N., Adachi, K., Tamura, J., Kanamaru, S., Kageyama, S., Hiraoka, A., Omoto, E., Akiyama, H., Tsubaki, K., Saito, K., Kuriyama, K., Oh, H., Kitano, K., Miyawaki, S., Takeyama, K., Yamada, O., Nishikawa, K., Takahashi, M., Matsuda, S., Ohtake, S., Suzushima, H., Emi, N., Ohno, R.. (1998) Analysis of prognostic factors in newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Journal of Clinical Oncology, 16, 78 85.
  • 2
    Den Boer, M.L., Zwaan, C.M., Pieters, R., Kazemier, K.M., Rottier, M.M.A., Flens, M.J., Scheper, R.J., Veerman A.J.P. (1997) Optimal immunocytochemical and flow cytometric detection of P-gp, MRP and LRP in childhood acute lymphoblastic leukemia. Leukemia, 11, 1078 1085.
  • 3
    Dermine, S., Grignani, F., Clerici, M., Nervi, C., Sozzi, G., Talamo, G.P., Marcheshi, E., Formelli, F., Parmiani, G., Pelicci, P.G., Gambacorti-Passerrini C. (1993) Occurrence of resistance to retinoic acid in the acute promyelocytic leukemia cell line NB4 is associated with altered expression of the PML/RARα protein. Blood, 82, 1573 1577.
  • 4
    Hashimoto, Y., Kagechika, H., Kawachi, E., Fukazawa, H., Saito, G., Shudo K. (1995) Correlation of differentiation-inducing activity of retinoids on human leukemia cell lines HL60 and NB4. Journal of Cancer Research and Clinical Oncology, 121, 696 698.
  • 5
    Kahan J. (1967) The fluorescence properties of vitamin A and their changes during photodecomposition. Acta Chemica Scandinavica, 21, 2515 2524.
  • 6
    Kitamura, K., Kiyoi, H., Yoshida, H., Saito, H., Ohno, R., Naoe T. (1997) Mutant AF-2 domain of PML-RARα in retinoic acid-resistant NB4 cells: differentiation induced by RA is triggered directly through PML-RARα and its down-regulation in acute promyelocytic leukemia. Leukemia, 11, 1950 1956.
  • 7
    Kizaki, M., Ueno, H., Yamazoe, Y., Shimada, M., Takeyama, N., Muto, A., Matsushita, H., Nakajima, H., Morikawa, M., Koeffler, H.P., Ikeda Y. (1996) Mechanisms of retinoid resistance in leukemic cells: possible role of cytochrome P450 and P-glycoprotein. Blood, 87, 725 733.
  • 8
    Ohno, R., Yoshida, H., Fukutani, H., Naoe, T., Ohshima, T., Kyo, T., Endoh, N., Fujimoto, T., Kobayashi, T., Hiraoka, A., Mizoguchi, H., Kodera, Y., Suzuki, H., Hirano, M., Akiyama, H., Aoki, N., Shindo, H., Yokomaku, S.. (1993) Multi-institutional study of all-trans retinoic acid as differentiation therapy of refractory acute promyelocytic leukemia. Leukemia, 7, 1722 1727.
  • 9
    Su, G.M.I., Davey, M.W., Davey, R.A., Kidman A. (1994) Development of extended multidrug resistance in HL60 promyelocytic leukaemia cells. British Journal of Haematology, 88, 566 574.
  • 10
    Sugimoto, Y., Sato, S., Tsukahara, S., Suzuki, M., Okuchi, E., Gottesman, M.M., Pastan, I., Tsuruo T. (1997) Coexpression of multidrug resistance gene (MDR1) and herpes simplex virus thymidine kinase gene in a bicistronic retroviral vector Ha-MDR-IRES-TK allows selective killing of MDR1-transduced human tumors transplanted in nude mice. Cancer Gene Therapy, 4, 51 58.
  • 11
    Takeshita, A., Shinjo, K., Ohnishi, K., Ohno R. (1995) New flow cytometric method for detection of minimally expressed multidrug resistance P-glycoprotein on normal and acute leukemia cells using biotinylated MRK16 and streptavidin-RED670 conjugate. Japanese Journal of Cancer Research, 86, 607 615.
  • 12
    Takeshita, A., Shinjo, K., Ohnishi, K., Ohno R. (1996) Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells. British Journal of Haematology, 93, 18 21.