Leukemia (2000) 14, 1444–1450  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu Increased sensitivity of multidrug-resistant myeloid leukemia cell lines to lovastatin L Maksumova, K Ohnishi, F Muratkhodjaev, W Zhang, L Pan, A Takeshita and R Ohno

Department of Medicine III, Hamamatsu University School of Medicine, 3600, Handa-cho, Hamamatsu, 431-3192, Japan

Lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibitor of neoplastic cell proliferation and a powerful reportedly inhibits proliferation and induces apoptosis of tumor inducer of apoptosis and cell cycle alterations (G0/G1 cell cells with MDR-1 coded P-glycoprotein (Pgp) expression. In 8–11 this study we investigated the sensitivity to lovastatin of eight cycle phase block) in various tumor cell lines. Moreover, myeloid leukemia cell lines: K562, NOMO-1, NB4 and its retinoic lovastatin has been reported to preferentially target multidrug- acid (RA) resistant subline NB4/RA, and their multidrug-resist- resistant neoplastic cells in myeloma,12 breast carcinoma13 ant (MDR) sublines: K562/ADR, NOMO-1/ADR, NB4/MDR and and neuroblastoma14 compared with their parental drug-sensi- NB4/RA/MDR. MTT and apoptosis assays revealed that K- tive cells in vitro. As for leukemia cell lines, a human multi- 562/ADR, NOMO-1/ADR and NB4/RA/MDR were more sensitive drug-resistant myeloid leukemia cell line, HL-60/VCR, was to lovastatin than their parental cell lines, while NB4/MDR 15 showed the same level of sensitivity as parental NB4 cells, recently shown to be equally sensitive to lovastatin, suggest- which already were very sensitive to lovastatin. Significant ing that the increased sensitivity to lovastatin observed by elevation of transcript levels of HMG-CoA reductase was some investigators was not a general phenomenon for drug- observed by semiquantitative RT-PCR analysis in more than resistant neoplastic cells expressing P-glycoprotein. three lovastatin-sensitive MDR sublines, but not in NB4/MDR The present study was therefore undertaken to further compared with the parental cell lines. HMG-CoA reductase address the relationships between Pgp expression and mRNA levels were up-regulated more than two-fold by the exposure to lovastatin in all of the parental non-Pgp-expressing sensitivity to lovastatin in other multidrug-resistant myeloid cell lines. In NB4/MDR, HMG-CoA reductase mRNA level was leukemia cell lines. elevated to a similar extent as in parental NB4, whereas in three other MDR sublines which showed preferential sensitivity to lovastatin, their HMG-CoA reductase mRNA levels were not sig- Materials and methods nificantly elevated after 24- and 48-h treatment with lovastatin. These results indicate a connection between drug resistance and regulation of the mevalonate pathway, and further Reagents strengthen the clinical possibility that drug resistant leukemias would be susceptible to treatment with lovastatin. Leukemia Lovastatin was generously provided by Merck Research Lab- (2000) 14, 1444–1450. oratories, Rahway, NJ, USA. To convert the inactive form of Keywords: lovastatin; HMG-CoA reductase; myeloid leukemia lovastatin to the active lactone form, the drug was dissolved cells; P-glycoprotein in ethanol, heated at 50°C in 0.1 N NaOH, neutralized with HCl, and stored unfiltered as a 4 mg/ml stock solution at 4°C for 2 months. DL-mevalonic acid lactone, MTT (3-(4,5-dime- Introduction thylthiazol-2-yl)-2, 5-diphenyltetrazolium-bromide), and pro- pidium iodide were obtained from Sigma-Aldrich, Tokyo, Hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) Japan. is a key enzyme in the mevalonate pathway, from which thou- sands of vital molecules are derived including cholesterol, which is involved in membrane structure and steroid pro- Cell lines duction, ubiquinone and heme-␣, which are associated with the mitochondrial respiratory chain, and dolichol, which is A human chronic myelogeneous leukemia cell line, K-562, required for glycoprotein synthesis.1 Recent studies have was obtained from the Japanese Cancer Research Resources identified over 40 proteins with mevalonate-derived prenyl Bank and its adriamycin-resistant subline, K-562/ADR, was groups attached post-translationally.2 These proteins include kindly provided by Dr T Tsuruo (University of Tokyo, Tokyo, p21 ras proteins, lamin A and lamin B which may play essen- Japan).16 A human myelomonocytic leukemia cell line, tial roles in the progression through the cell cycle3–5 as well NOMO-1, and its adriamycin-resistant subline, NOMO- as several Mr 20 000–25 000 proteins which may regulate 1/ADR, were kindly provided by Dr M Ogura (Aichi Cancer critical functions involved in cellular growth control.2 Thus, Center, Nagoya, Japan) and Dr M Tanimoto (Nagoya Univer- physiological regulation of HMG-CoA reductase is strongly sity, Nagoya, Japan).17 A human promyelocytic leukemia cell believed to be essential for the maintenance of cellular line, NB4, was kindly provided by Dr M Lanotte (Hospital homeostasis. Saint-Louis, Paris, France)18 and its retinoic acid-resistant sub- A number of highly selective inhibitors of the mevalonate line, NB4/RA, was kindly provided by Dr K Kitamura pathway have been developed to decrease elevated plasma (Nagoya University).19 cholesterol.6 These agents have also been considered as NB4/MDR and NB4/RA/MDR sublines were established by potential antitumor agents because of their ability to inhibit transduction of NB4 and NB4/RA with HaMDR retrovirus and the growth of cells dependent on de novo synthesis of choles- subsequent selection of the transduced cells with 4 ng/ml terol.7 Lovastatin, one of the specific competitive inhibitors of vincristine as reported previously.20 All cell lines were HMG-CoA reductase, has been demonstrated to be a strong cultured in RPMI 1640, supplemented with 1% penicillin/streptomycin, 1 mmol/l L-glutamine and 10% heat- inactivated fetal calf serum (FCS, Gibco BRL, Auckland, New ° Correspondence: L Maksumova; Fax: 81 53 434 2910 Zealand), at 37 Cin5%CO2 humidified atmosphere. Adria- Received 23 February 2000; accepted 21 April 2000 mycin (400 nM) was added every 2 weeks to the culture MDR leukemia cell lines and lovastatin L Maksumova et al 1445 medium of K-562/ADR and NOMO/ADR, and vincristine Results (4 ng/ml) was added to the complete medium of NB4/MDR and NB4/RA/MDR cell lines. Cells were grown for 7 days in MDR-1 gene expression and membrane Pgp the absence of the drugs prior to use in specific experiments. distribution

Since drug-resistant cell lines used have come from different Flow cytometric analysis of apoptosis and sources, and methods by which they become drug resistant P-glycoprotein are variable, all cell lines were examined for MDR-1 gene expression and Pgp distribution before starting experiments Apoptosis was identified and quantified by flow cytometry with lovastatin. As shown in Figure 1, K-562/ADR, with propidium iodide (PI) staining.21 P-glycoprotein was ana- NOMO/ADR, NB4/MDR and NB/RA/MDR cells highly lyzed by a flow cytometric assay using biotinylated MRK16 expressed mRNA of MDR-1 gene, while their parental mAb (b-MRK16) (Kyowa, Sunto-Gun, Japan) and streptavidin- counterpart showed low or undetectable levels of the gene. RED670 conjugate (SA-RED 670) (Gibco BRL, Gaithersburg, The membrane distribution of Pgp in resistant sublines varied MD, USA) as described previously.22 Briefly, cells were first from 57.2% in NB4/MDR up to 99.6% in K-562/ADR incubated in 0.5% Polyglobin N (Miles, West Haven, NJ, (Figure 2), whereas membrane Pgp was not detected in any of USA) at room temperature to block non-specific binding, and the parental drug-sensitive cells (data not shown). then incubated with either 1 ␮g of b-MRK16 or 1 ␮g subclass- matched biotinylated IgG2a mAb (negative control) for 30 min on ice. After washing twice in PBS, the cell pellet was resus- The viability and apoptosis of leukemia cell lines in pended and incubated with 1 ␮g of SA-RED670 conjugate for response to lovastatin 30 min on ice. The cells were then washed and resuspended in PBS containing 2% of FCS for flow cytometric assay. The cytotoxic effects of lovastatin on eight leukemia cell lines were evaluated by MTT assay. Cells were treated with lovasta- tin (0.1 to 100 ␮M) for 48 h. The growth of all cell lines was DNA fragmentation assay inhibited by lovastatin in a time- and dose-dependent manner. Simultaneous addition of mevalonate (300 ␮M) completely Apoptosis was further confirmed by the detection of DNA prevented the cell growth inhibition, indicating that lovastatin fragmentation described previously.23 specifically depleted mevalonate in the cells. The concen- trations of lovastatin to induce a 50% decline in MTT activity

(MTT50) are shown in Figure 3. NB4 cells were apparently the Cell viability most sensitive to lovastatin, and a significant decrease of MTT was observed at a concentration as low as 2 ␮M, which was Cell viability was measured by MTT assay as described by around 40- and 10-fold lower than those for K-562 and Mosmann24 with slight modification. After incubation for NOMO-1 cells, respectively (P Ͻ 0.01). NB4/RA was signifi- 48 h, 10 ␮l (5 mg/ml) MTT was added to each well, and plates cantly more resistant to lovastatin than NB4 cells. Multidrug- ° were incubated at 37 C for 4 h. Acid-isopropanol (0.04 N HCl resistant sublines showed two- to 2.5-fold lower MTT50 values in isopropanol) (100 ␮l) was added to each well and mixed than those of their parental cell lines except for NB4/MDR, by pipetting to dissolve the reduced MTT formazans, and rela- the parental cell line of which already showed very high sensi- tive cell viability was measured by ELISA reader at dual wave- tivity to lovastatin. When DNA from lovastatin-treated cells length of 550 and 600 nm with consequent processing of the was analyzed on agarose gel, typical apoptotic DNA lad- data using Delta Soft 4.0B software. dering was observed (Figure 4). Flow cytometric analysis of lovastatin-treated cell lines typically showed pre-G1 peaks due to nuclear fragmentation. As shown in Figure 5, lovastatin mRNA analysis induced accumulation of pre-G1 peaks to a greater extent in

Total RNA was isolated with Tri-Reagent (MRC, Cincinnati, OH, USA). mRNA expression was assessed by reverse transcriptase PCR (RT-PCR). For the RT reaction, RNA (10 ␮g) was primed with random hexamer and reverse transcribed with 2.5 ␮l Superscript MMLV reverse transcriptase (Gibco BRL). The cDNA generated was amplified by using primers for GAPDH (glyceraldehyde-phosphate-dehydrogenase) des- cribed in Ref. 25; set primers for HMG-CoA reductase reported in Ref. 14 and primers for MDR-1 described in Ref. 26. PCR mixture (50 ␮l) contained 1 ␮l of the RT reaction, 20 pmol of each dNTP (0.2 mmol/l) and 2.5 U of Gold- AmplyTag polymerase (Perkin Elmer, Branchburg, NJ, USA). The PCR reaction protocol for GAPDH and MDR-1 was as follows: 94°C for 1 min, 55°C for 1 min and 72°C for 1.5 min; for HMG-CoA reductase: 94°C for 1.5 min, 60°C for 1.5 min Figure 1 Expression of MDR-1 mRNA. Total RNA was purified and 72°C for 2 min for 30 cycles. The expected PCR products: from leukemia cell lines and subjected to semiquantitative RT-PCR analysis with oligonucleotide primers specific for MDR-1. After 24 898 bp (GAPDH), 243 bp (MDR) and 525 bp (HMG-CoA cycles (shown to be at the linear phase of amplification), PCR products reductase), were size fractionated on to agarose gel and were electrophoresed on to a 2% agarose gel and stained with ethid- stained with ethidium bromide. ium bromide.

Leukemia MDR leukemia cell lines and lovastatin L Maksumova et al 1446

Figure 2 P-gp expression in multidrug-resistant leukemia sublines. P-gp was analyzed by flow cytometry. Solid lines show the result from samples incubated with b-IgG2a (negative control), and dotted lines show those with b-MRK16. The mean fluorescence intensities are specific in the Figure.

Figure 3 Cytotoxic effects of lovastatin on myeloid leukemia cell lines by MTT assay. The concentrations of lovastation required to ach- Figure 4 DNA fragmentation in leukemia cell lines following lova- ␮ ieve a decline in MTT activity by 50% (MTT50) in leukemia cell lines statin treatment for 48 h. The lanes in the gel were loaded with 5 l are shown in the Figure. *P Ͻ 0.01 by the Wilcoxon test. (1 ␮g) of the genomic DNA.

multidrug-resistant sublines than parental cell lines. The result was consistent with the findings of the MTT assay. Thus, multi- Before the treatment with lovastatin, all cell lines expressed drug-resistant sublines were for the most part more sensitive detectable levels of HMG-CoA reductase mRNA, but multi- to lovastatin than their parental cell lines in terms of cytotoxic- drug-resistant sublines displayed significantly higher levels of ity and apoptosis. mRNA than their parental cell lines except for NB4/MDR (Figure 6). Following the lovastatin treatment, HMG-CoA Modulation of HMG-CoA reductase by lovastatin in reductase mRNA levels were increased by more than two-fold leukemia cell lines in all parental cell lines, whereas in multidrug-resistant sub- lines, which showed higher sensitivity to lovastatin and higher The effects of lovastatin on HMG-CoA reductase mRNA levels mRNA expression in basal conditions, the HMG-CoA were next evaluated by semiquantitative RT-PCR analysis. reductase mRNA levels were not further up-regulated except

Leukemia MDR leukemia cell lines and lovastatin L Maksumova et al 1447

Figure 5 Flow cytometric analysis of leukemia cells exposed to lovastatin for 48 h. The percentages of cells in the pre-G1 (apoptotic) fraction are shown in the upper left corner of the histograms.

for NB4/MDR, in which the low pre-treatment mRNA was of sensitivity to lovastatin of parental Pgp-negative leukemia increased to a similar extent as in its parental NB4 cell line cells was NB4ϾNOMO-1ϾNB4/RAϾK562. The human acute by the lovastatin treatment (Figure 6). promyelocytic leukemia cell line, NB4, was very sensitive to ␮ lovastatin, with MTT50 of as low as 2 M. Of note, in the phase I study of lovastatin as an anti-cancer drug, doses up to Discussion 25 mg/kg/day were well tolerated and yielded peak serum lev- els in the range of 0.1 to 3.95 ␮M.28 Besides, the effects of In this study we investigated the sensitivity to lovastatin of lovastatin on normal marrow progenitors and hematopoiesis various leukemia cell lines and their MDR Pgp-expressing were reportedly negligible.29,30 Therefore, our data further sublines in terms of growth inhibition and apoptosis. We used strengthen the idea that lovastatin might have a potential two sublines which had been selected as resistant clones by therapeutic role in the treatment of acute promyelocytic leu- step-wise administration of adriamycin (K-562/ADR and kemia.11 In contrast, the human chronic myelogeneous leuke- NOMO-1/ADR) and two sublines which had been transduced mia cell line, K562, displayed low sensitivity to lovastatin. The with a MDR-1 gene vector (NB4/MDR and NB4/RA/MDR). susceptibility of cells to cytotoxic and apoptotic signals Neither MDR-related protein nor lung-resistant protein was appears to be regulated by complex cellular factors involved detectable in the above drug resistant cell lines.27 The order in the cell death pathway.31 Many apoptosis-suppressing pro-

Leukemia MDR leukemia cell lines and lovastatin L Maksumova et al 1448

Figure 6 Expresssion of HMG-CoA reductase mRNA. (a) Total RNA was purified from lovastatin-treated and -untreated leukemia cell lines and subjected to semiquantitative RT-PCR analysis with oligonucleotide primers specific for HMG-CoA reductase (upper panel) and for GAPDH (control gene). After 28 cycles (shown to be at the linear phase of amplification), PCR products were electrophoresed on to a 2% agarose gel and stained with ethidium bromide. (b) Densitometric analysis of the HMG-CoA reductase mRNA levels in leukemia cell lines: *P Ͻ 0.01 by the Wilcoxon test.

teins have been implicated in relative resistance and suscepti- K-562 cells, the finding that K-562 cells were most resistant

bility to antitumor treatment. For instance, Bcl-XL and BCR- to lovastatin in our study is not surprising. ABL32,33 are shown to be highly expressed in K-562 cells.34,35 The mechanisms whereby lovastatin suppresses cell growth

Moreover, a strong association between Bcl-XL and Pgp and triggers apoptosis are unclear at present, although either expression has been found in leukemic cell lines.36,37 In view ras-dependent or ras-independent pathways have been dis- of the wide expression of apoptosis-protecting molecules in cussed.38 Moreover, the mechanism by which some multi-

Leukemia MDR leukemia cell lines and lovastatin L Maksumova et al 1449 drug-resistant neoplastic cells develop preferential sensitivity References to lovastatin also remains to be elucidated. It has been shown 1 Golstein JL, Brown MS. Regulation of the mevalonate pathway. that neoplastic cells require high level of cholesterol biosynth- Nature 1990; 343: 425–430. 39 esis. Pgp plays a role in cholesterol biosynthesis by trans- 2 Schmidt RA, Schneider CJ, Glomset JA . Evidence of post-trans- porting of sterol precursors from plasma membrane to endo- lational incorporation of a product of mevalonic acid into Swiss plasmic reticulum.40,41 Thus, it has been speculated that Pgp 3T3 cell proteins. J Biol Chem 1984; 259: 10175–10180. may down-regulate de novo cholesterol synthesis by a feed- 3 Gibbs JB. Ras C-terminal processing enzymes – new drug targets? back mechanism via the mevalonate pathway.15 Of note, Cell 1991; 65: 1–4. 4 Lutz RJ, Trujillo MA, Denham KS, Wenger L, Sinensky M. Nucleo- cholesterol level was found to increase by as much as 50% plasmic localization of prelamin A: implications for prenylation- in some multidrug resistant cells in comparison with their dependent lamin A assembly into the nuclear lamina. Proc Natl parental cells.42,43 Present study showed that Pgp-expressing Acad Sci USA 1992; 89: 3000–3004. leukemia cell lines K-562/ADR, NOMO-1/ADR and 5 Wolda SL, Glomset JA. Evidence for modification of lamin B by NB4/RA/MDR were more sensitive to lovastatin than their par- a product of mevalonic acid. J Biol Chem 1988; 263: 5997–6000. ental cell lines in terms of inhibition of proliferation and 6 Endo A. The discovery and development of HMG-CoA reductase inhibitors. J Lipid Res 1992; 33: 1569–1582. induction of apoptosis. However, despite the high expression 7 Larsson O. HMG-CoA reductase inhibitors: role in normal and of MDR-1 mRNA and Pgp on membrane, the MDR-1 gene- malignant cells. Crit Rev Oncol Hematol 1996; 22: 197–212. transduced variant of NB4, NB4/MDR, was not more sensitive 8 Perez-Sala D, Collado-Escobar D, Mollinedo F. Intracellular alkal- to lovastatin, showing equal sensitivity to lovastatin as their ization suppresses lovastatin-induced apoptosis in HL-60 cells parental cells. The discrepancy between NB4/MDR and through the inactivation of a pH-dependent endonuclease. J Biol NB4/RA/MDR could probably be explained by the different Chem 1995; 270: 6235–6242. 9 Jones KD, Couldwell WT, Hinton DR, Su Y, He S, Anker L, Law HMG-CoA reductase mRNA levels. We found that HMG-CoA RE. Lovastatin induces growth inhibition and apoptosis in human reductase transcript levels were significantly different between malignant glioma cells. Biochem Biophys Res Commun 1994; parental cells and multidrug-resistant sublines except for 205: 1681–1687. NB4/MDR. Treatment with lovastatin did not lead to further 10 Marcelli M, Cunningham GR, Haidacher SJ, Padayatty SJ, Sturgis significant increase in HMG-CoA reductase mRNA in drug L, Kagan C, Denner L. Caspase-7 is activated during lovastatin- resistant variants, but did in drug sensitive parental cells, sug- induced apoptosis of the prostate cancer cell line LNCaP. Cancer Res 1998; 58: 76–83. gesting some inability of drug resistant counterparts to com- 11 Dimitroulakos J, Nohynek D, Backway KL, Hedley DW, Yeger H, pensate for loss of enzyme function. The result that NB4 and Freedman MH, Minden MD, Penn LZ. Increased sensitivity of NB4/MDR cells showed equal sensitivity to lovastatin can acute myeloid leukemias to lovastatin-induced apoptosis: a poten- probably be explained by the equal expression of HMG-CoA tial therapeutic approach. Blood 1999; 93: 1308–1318. reductase in these two cell lines (Figure 6) and thus by the 12 Holmberg M, Sandberg C, Nygren P, Larsson R. Effects of lovasta- equal compensatory resources to overcome lovastatin- tin on a human myeloma cell line: increased sensitivity of a multi- drug-resistant subline that expresses the 170 kDa P-glycoprotein. induced mevalonate blockade. Otherwise the equal sensitivity Anticancer Drugs 1994; 5: 598–600. of NB4 and NB4/MDR cells could suggest that other mech- 13 Diamond AM, Jaffe D, Murray JL, Safa AR, Samuels BL, Hatfield anisms besides Pgp and the transcript level of HMG-CoA DL. Lovastatin effects on human breast carcinoma cells: differen- reductase may be involved in the complexity of the sensitivity tial toxicity on an adriamycin-resistant derivative and influence on phenomenon to lovastatin of NB4 cell line which was already selenocysteine tRNAs. Biochem Mol Biol Int 1996; 38: 345–355. very sensitive to lovastatin before MDR-1 transfection. Inter- 14 Dimitroulakos J, Yeger H. HMG-CoA reductase mediates the bio- logical effects of retinoic acid on human neuroblastoma cells: lov- estingly, that the RA-resistant subline of NB4, NB4/RA (Pgp astatin specifically targets P-glycoprotein expressing cells. Nature negative), was significantly less sensitive to lovastatin than Med 1996; 2: 326–333. NB4 cells, supports recent findings that retinoic acid respon- 15 Hunakova L, Sedlak J, Sulikova M, Chovancova J, Duraj J, Chorv- siveness is associated with increased sensitivity to lovasta- ath B. Human multidrug-resistant (MRP, p190) myeloid leukemia tin.11,14 On the other hand, NB4/RA cells when made MDR HL-60/ADR cells in vitro: resistance to the mevalonate pathway resistant show a significantly higher expression of HMG-CoA inhibitor lovastatin. Neoplasma 1997; 44: 366–369. 16 Tsuruo T, Saito H, Kawabata H, Ohhara T, Hamada H, Utakoji T. reductase than their parental counterparts and undergo induc- Characteristics of resistance to adryamicin in human myelogenous tion of profound apoptosis. HMG-CoA reductase is regulated leukemia K-562 resistant to adryamicin and in isolated clones. Jpn in a complex manner,1 further investigations are required to J Cancer Res 1986; 77: 682–692. analyze the translational processes and activity of HMG-CoA 17 Hamaguchi M, Morishita Y, Takahashi I, Mitinori O, Takamatsu reductase in multidrug-resistant and drug sensitive leukemia J, Saito H. FDP D-dimer induces the secretion of interleukin-1, cells, and to elucidate the mechanisms underlying differential urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in human promonocytic leukemia cell line. Blood cytotoxic responses to lovastatin. 1991; 77: 94–100. The present study indicates that Pgp plays a certain regulat- 18 Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, ory role in lovastatin sensitivity of neoplastic cells due to a Berger R . NB4, a maturation inducible cell line with t(15;17) connection between multidrug resistance and regulation of marker isolated from a human acute promyelocytic leukemia mevalonate pathway, and suggest the clinical possibility that (M3). Blood 1991; 77: 1080–1086. drug resistant leukemias would be susceptible to treatment 19 Kitamura K, Kiyoi H, Yoshida H, Saito H, Ohno R, Naoe T. Mutant AF-2 domain of PML-RARa in retinoic acid-resistant NB4 cells: with lovastatin. differentiation induced by RA is triggered directly through PML- RARa and its down-regulation in acute promyelocytic leukemia. Acknowledgements Leukemia 1997; 11: 1950–1956. 20 Sugimoto Y, Sato S, Toukahara S, Suzuki M, Okochi E, Gottesman We thank Dr Y Sugimoto (Cancer Chemotherapy Center, MM, Pastan I, Tsuruo T. Coexpression of multidrug resistance gene Japanese Foundation for Cancer Research, Tokyo, Japan) for (MDR-1) and herpes simplex virus thymidine kinase gene in a bici- stronic retroviral vector Ha-MDR-IRES-TK allows selective killing the generous assistance in transduction of MDR-1 gene vector of MDR-1-transduced human tumors transplanted in nude mice. into NB4 and NB4/RA cells. We also thank Dr Quang-Kim Cancer Gene Ther 1997; 4: 51–58. Tran for his helpful review of the manuscript. 21 Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C.

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Leukemia