Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine and to determine whether there are differences in resistance mechanisms between troxacitabine, gemcitabine, and cytarabine in human leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell line was highly sensitive to the antiproliferative effects of troxacitabine, gemcitabine, and cytarabine with inhibition of proliferation by 50% observed at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase (dCK)-deficient variant (CEM/dCK(-)) was resistant to all three drugs. In contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhibited high levels of resistance to cytarabine (1150-fold) and gemcitabine (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis of troxacitabine transportability by the five molecularly characterized human nucleoside transporters [human equilibrative nucleoside transporters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, hCNT2, and hCNT3] revealed that short- and long-term uptake of 10-30 microM [(3)H]troxacitabine was low and unaffected by the presence of either nucleoside transport inhibitors or high concentrations of nonradioactive troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that deficiencies in nucleoside transport were unlikely to impart resistance to troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145(R); 6300-fold) that exhibited reduced uptake of troxacitabine was cross-resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK activity toward deoxycytidine in DU145(R) cell lysates was <20% of that in DU145 cell lysates, and no activity was detected toward troxacitabine. Sequence analysis of cDNAs encoding dCK revealed a mutation of a highly conserved amino acid (Trp(92)-->Leu) in DU145(R) dCK, providing a possible explanation for the reduced phosphorylation of troxacitabine in DU145(R) lysates. Reduced deamination of deoxycytidine was also observed in DU145(R) relative to DU145 cells, and this may have contributed to the overall resistance phenotype. These results, which demonstrated a different resistance profile for troxacitabine, gemcitabine, and cytarabine, suggest that troxacitabine may have an advantage over gemcitabine and cytarabine in human malignancies that lack or have low nucleoside transport activities.     

Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB.

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.     

On the pharmacokinetics of 2-chloro-2'-deoxyadenosine in humans.

The antitumoral effect of 2-chloro-2'-deoxyadenosine (CdA) in the treatment of lymphoproliferative diseases in general and of hairy cell leukemia in particular has recently been demonstrated. Detailed information on the pharmacokinetics of CdA, however, is lacking. The pharmacokinetics of CdA after 2- and 24-h infusions of 0.14 mg/kg was described in 12 patients with lymphoproliferative diseases using a newly developed high-performance liquid chromatography method. The plasma concentration data from individual patients were fitted to a two-compartment model with alpha- and beta-half-lives of 35 +/- 12 (mean +/- SD) min and 6.7 +/- 2.5 h, respectively. The volume of distribution was 9.2 +/- 5.4 liters/kg. The steady-state concentration of CdA during the 24-h infusion was 22.5 +/- 11.1 nM. The areas under the time versus concentration curves were 552 +/- 258 and 588 +/- 185 nM x h, respectively, for the 24- and 2-h infusions. The interindividual variability of the determinants of the plasma pharmacokinetics of CdA was small (the coefficients of variation were between 0.22 and 0.58). At 6.3 +/- 1.5 h after the start of the 2-h infusion, the concentration of CdA was the same as the steady-state concentration during the 24-h infusion. When the mean plasma concentrations of the 12 patients were fitted to a 3-compartment model, the half-lives of the alpha-, beta-, and tau-phases were 8 min, 1 h 6 min, and 6.3 h, respectively. The long terminal half-life of CdA after 2-h infusion supports the use of intermittent infusions.     

Potentiation of antitumor effects of cyclophosphamide derivatives in B-chronic lymphocytic leukemia cells by 2-chloro-2'-deoxyadenosine.

Because 2-chloro-2'-deoxyadenosine (CdA) is active in B-chronic lymphocytic leukemia (B-CLL), and may interfere with DNA repair, we investigated the potentiating effect of CdA on the cytotoxicity induced in vitro in B-CLL lymphocytes by cyclophosphamide (CP) derivatives, which induce DNA damage by DNA cross-linking. Exposure to CdA at clinically achievable concentrations for 2 h, followed by mafosfamide (MAF) or 4-hydroxycyclophosphamide (4HC) for 22 h, resulted in synergistic cytotoxicity in the majority of B-CLL samples tested. Synergy between CdA and MAF was observed in cell samples of sensitive/untreated patients, as well as in cells of resistant/pretreated patients, particularly at the highest concentrations of MAF. In the cells treated with CdA and MAF, we observed loss in ATP and hallmarks of apoptosis, as evidenced by cellular morphology and high molecular weight DNA fragmentation. The synergy could be explained neither by an influence of MAF on the phosphorylation of CdA, nor by an increase in the incorporation of CdA into DNA in the presence of MAF. The in vitro synergy between CdA and CP derivatives provides a rationale for the use of this association in B-CLL patients.     

Effects of cytotoxicity of 2-chloro-2'-deoxyadenosine and 2-bromo-2'-deoxyadenosine on cell growth, clonogenicity, DNA synthesis, and cell cycle kinetics

The cytotoxic effects of the adenosine deaminase resistant analogues 2-bromo-2'-deoxyadenosine (2-BrdAdo) and 2-chloro-2'-deoxyadenosine (2-CldAdo) have been compared with those of deoxyadenosine (dAdo). Like 2-CldAdo, 2-BrdAdo is highly effective in inhibiting the growth of many T-lymphoblastoid, B-lymphoblastoid, and myeloid cell lines in culture. Concentrations required to inhibit growth of CCRF-CEM human T-lymphoblastoid cells by 50% (IC50) are: 2-CldAdo, 0.045 microM; 2-BrdAdo, 0.068 microM; dAdo, 0.9 microM in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine. Like dAdo, 2-BrdAdo causes a much greater decrease in DNA synthesis than in RNA and protein synthesis. For each of the nucleosides the concentration required to cause 50% inhibition of DNA synthesis (as measured by thymidine incorporation) in an 18-h exposure is very similar to the IC50 for growth and to the concentration required to decrease viability (clonogenicity) over 18 h by 50% (EC50). A fraction of CCRF-CEM cells (approximately equal to 30%) is resistant to killing by exposure to 2-BrdAdo or 2-CldAdo for 4 h at concentrations 100 times the EC50, but 3% of cells are resistant to exposure for 4 h to a concentration of dAdo 3 times the EC50. Each of the three nucleosides causes accumulation of cells in S phase, the accumulation becoming more marked with longer periods of exposure and with higher concentrations of nucleoside. During exposures for 18-24 h at a concentration of nucleoside near the EC50 most cells accumulate in S, with most in early S, whereas exposure to concentrations greater than EC95 accumulates cells at the G1/S border. This suggests that loss of viability is associated with a blockade of some process specifically occurring at the initiation of S phase. At an optimum dosage schedule, 2-BrdAdo and 2-CldAdo have similar therapeutic effects against L1210 in vivo, both producing over 99% cell kill, but the optimum dosage of 2-CldAdo is lower.     

Second primary tumors and immune phenomena after fludarabine or 2-chloro-2'-deoxyadenosine treatment.

The purine nucleoside analogs fludarabine and 2-chloro-2'-deoxyadenosine display substantial activity in the treatment of various chronic lymphoproliferative disorders. Their major toxicities are primarily immunosuppression and myelosuppression. The profound influence of these drugs on the immune system has raised questions as to the emergence of secondary neoplasms or auto-immune disorders after their use. Based on a literature review and on personal observations, this article reviews the potential clinical importance of these concerns.     

Biochemical mechanisms of drug resistance. XII. Uridine kinases from mouse leukemic cells sensitive and resistant to 5-azacytidine

Partially purified uridine kinases from 5-azacytidine-sensitive and -resistant leukemic cells of AKR mice were separated on a Sephadex G-200 column. The elution profile of both enzymes was similar although specific activity of uridine kinase from 5-azacytidine-resistant cells was more than 50% lower than that isolated from the parental cell line. The apparent Km constants with respect to the concentration of phosphate donor and acceptor were identical for both uridine kinases, while V_max for enzymes from the resistant line were significantly decreased. The enzymes under study were tested for their sensitivity to SH-blocking agents—p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide—and to urea, trypsin and cytidine 5′-triphosphate. Inactivation following p-chloromercuribenzoate affected the activity of uridine kinase from sensitive cells more markedly than that of uridine kinase from resistant ones. When the feedback effect of cytidine 5′-triphosphate was examined, the results indicated, in both cases, a non-competitive inhibition with respect to phosphate acceptor, K_i constant being the same (0.079 mM). Furthermore, v_i/v_o ratios for the intact and trypsin-digested uridine kinases from both cell lines plotted against varying amounts of cytidine 5′-triphosphate yielded a single curve in each instance. The significance of these findings is discussed.     

Imatinib resistance in multidrug-resistant K562 human leukemic cells

The multidrug resistance phenotype (MDR) is one of the major causes of failure in cancer chemotherapy and it is associated with the over-expression of P-glycoprotein (P-gp or MDR1) in tumor cell membranes. A constitutive NF-kappaB activity has been observed in several haematological malignancies and this is associated with its anti-apoptotic role. In the present work, the relationship between NF-kappaB and MDR phenotype was evaluated in wild type K562 human leukemic cells (K562-WT) and in its vincristine-resistant counterpart, K562-Vinc cells. These data showed that K562-Vinc cells, which express an active P-gp, exhibited MDR phenotype. The resistant indexes (IC(50)(K562-Vinc)/IC(50)(K562-WT)) for structurally unrelated drugs like imatinib, doxorubicin and colchicine were 8.0+/-0.3, 2.8+/-0.4 and 44.8+/-8.8, respectively. The imatinib resistance was reversed by P-gp blockade suggesting the involvement of P-gp in imatinib transport. We observed that NF-kappaB was constitutively activated in both cell lines but in a lesser extent in K562-Vinc. The inhibition of NF-kappaB with BAY 11-7082 increased the cytotoxicity of imatinib in K562-Vinc cells but not in K562-WT. Further, the co-administration of imatinib and BAY 11-7082 sensitized multidrug-resistant K562 cells to cell death as detected by increased percentage of annexin V positive cells. The induced cell death in K562-Vinc cells was associated with activation of caspases 9 and 3. Finally, we provide data showing that BAY 11-7082 down-regulates the expression of P-gp suggesting that the activity of NF-kappaB could be functionally associated to this protein in K562 cells. Our results indicate that the vincristine-resistant K562 cells which developed MDR phenotype, exhibited resistance to imatinib associated with a functional P-gp over-expression. This resistance could be partially overcome by the inhibition of NF-kappaB pathway.     

The effects of acetaldophosphamide, a novel stable aldophosphamide analogue, on normal human and leukemic progenitor cells in vitro: implications for use in bone marrow purging

Acetaldophosphamide (A-ALD), a novel in vitro active and stable derivative of aldophosphamide, kills human bone marrow-derived granulocyte-macrophage colony-forming cells (GM-CFC) independent of the cell cycle. The surviving fraction of GM-CFC is an exponential function of the drug concentration and time of exposure. Variation of marrow light-density cell concentration between 2 x 10(6) and 10 x 10(6)/ml does not significantly influence its GM-CFC toxicity. Marrow depleted of GM-CFC by A-ALD subsequently generates GM-CFC when grown in suspension cultures. During the early period after treatment with A-ALD the number of surviving GM-CFC (size of surviving GM-CFC compartment) does influence the speed of the GM-CFC repopulation in suspension cultures. The importance of the number of surviving GM-CFCs for the growth and maintenance of GM-CFC population in such suspension cultures diminishes with time. No significant differences are observed after 2 wk, indicating that the ancestor stem cell population and its regenerative potential responsible for in vitro hematopoiesis have not been significantly affected by the drug treatment. A-ALD-treated progenitor cells retain their ability to integrate with the previously established marrow stromal cell layer and generate GM-CFC within this layer to an extent comparable to that of untreated marrow cells. The effect of A-ALD on human hematopoiesis is comparable to that of 4-hydroperoxycyclophosphamide. Its advantage over 4-hydroperoxycyclophosphamide is a greater stability in vitro. It has sparing effect on GM-CFC ancestor cells. Its toxicity to myeloid leukemia cell line (KBM-3)-derived clonogeneic cells is higher than to the GM-CFC. It is similar in doxorubicin-sensitive (KBM-3) and -resistant (KBM-3/DOX) leukemic cells. Thus, A-ALD appears to be a promising drug for in vitro purging of bone marrow cells.     

Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate

Trimetrexate (TMQ) is a lipophilic antifolate shown to have antitumor activity in humans. TMQ-resistant sublines of the MOLT-3 human acute lymphoblastic leukemia cell line were developed and were designated as MOLT-3/TMQ200, MOLT-3/TMQ800, and MOLT-3/TMQ2500 based on degrees of resistance to TMQ. The TMQ resistance was accompanied by 5- to 7-fold increases in dihydrofolate reductase activity and markedly reduced cellular TMQ accumulation. Methotrexate accumulation was not impaired in TMQ-resistant cells. TMQ retention (efflux) was unchanged in these TMQ-resistant cells. Verapamil enhanced the TMQ accumulation in the resistant cells to the level seen in the parent cells but had no effects on the TMQ retention. These sublines were cross-resistant not only to methotrexate but also to vincristine, doxorubicin, daunorubicin, and mitoxantrone. There was no cross-resistance to bleomycin or cisplatin. Resistance to vincristine, doxorubicin, daunorubicin, and mitoxantrone was reversed by verapamil. TMQ resistance was only minimally reversed by verapamil and methotrexate resistance not affected at all. Both cellular accumulation and retention of vincristine and daunorubicin in the TMQ-resistant cells were markedly decreased. Verapamil enhanced both accumulation and retention of the drug. Plasma membrane fractions of the TMQ-resistant cells analyzed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue revealed the presence of a distinct band with a molecular weight of 170,000. Immunoblot analysis with 125I-labeled monoclonal antibody raised against P-glycoprotein of multidrug-resistant Chinese hamster ovary cells (C219) cross-reacted with the Mr 170,000 protein of the TMQ-resistant cells. These results show that the TMQ-resistant cells displayed not only decreased TMQ uptake and increased dihydrofolate reductase but also characteristics associated with a classical multidrug-resistant phenotype. Multidrug resistance includes lipophilic antifolate.     

Differentiation and the multiple drug resistance phenotype in human leukemic cells

A common feature of mammalian cell lines selected for multiple drug resistance is the overexpression of a high-molecular-weight surface membrane glycoprotein(s). While its amount has been shown to be related to the degree of resistance of such cells, its function in this phenomenon remains obscure. Because there are some biochemical and functional similarities between drug-resistant cells and differentiated cells, we asked if resistance-associated glycoproteins were also associated with cellular differentiation. Using three monoclonal antibodies against antigens known to be associated with differentiation and three monoclonal antibodies that distinguish our multiple drug-resistant human leukemic CEM/VLB100 cells from their drug-sensitive counterparts, we found that the resistant cells were neither altered in their apparent state of differentiation nor were they altered in their ability to respond to a differentiation stimulus with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. We did find, however, that one antibody that recognizes the resistance-associated glycoprotein, Mr 180,000 glycoprotein (gp180), was only minimally altered in amount bound after treatment with the phorbol ester, but two other resistance-associated glycoproteins, Mr 155,000 glycoprotein (gp155) and, to a lesser extent, Mr 130,000 glycoprotein (gp130), were reduced in expression after this treatment. We suggest that the function of the previously described "marker" glycoprotein associated with multiple drug resistance remains unknown, but that the expression of two other resistance-associated glycoproteins also appears to be related to cellular differentiation or maturation in these cells.     

On the bioavailability of oral and subcutaneous 2-chloro-2'-deoxyadenosine in humans: alternative routes of administration.

PURPOSE: The antimetabolite 2-chloro-2'-deoxyadenosine (CdA) is a promising alternative to alkylating agents for the treatment of lymphoproliferative disorders. Its use, however, is hampered by the need for intravenous (IV) administration. The aim of the present study was to determine the bioavailability of subcutaneously (SC) and orally administered CdA, and to establish an oral dose of CdA that could supersede IV administration. PATIENTS AND METHODS: A previously developed high-performance liquid chromatography method was used for the determination of plasma CdA concentrations in 13 patients. Ten patients were treated on alternate days with 0.14 mg/kg/d CdA as a 2-hour IV infusion or by a SC injection. Three of these patients were also given 0.14 mg/kg CdA orally in enteric-coated capsules. Ten patients were administered CdA orally that was dissolved in phosphate-buffered saline (PBS) after treatment with 20 mg omeprazole 1 and 6 hours before the administration of CdA. RESULTS: The bioavailability of SC CdA was 102% +/- 28% (mean +/- SD), and the bioavailability of CdA administered in enteric-coated capsules was 19%, 24%, and 60%. In the three patients who were given 0.14 mg/kg orally dissolved in PBS, the bioavailability was 48% +/- 8%, whereas in the seven patients who received 0.28 mg/kg, the bioavailability was 55% +/- 17%. In the 10 patients who were treated with the CdA solution orally, the coefficients of variation of the areas under the curve (AUCs) after oral and IV administration were similar. Thus, oral administration did not add to the interindividual variability. CONCLUSIONS: We conclude that orally administered CdA can supersede IV infusion if the dose is doubled. SC administration gives a high peak concentration of short duration with an AUC identical to that of IV infusion. Thus, SC injection can also be used as an alternative to IV infusion.     

Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases.

Cytotoxicity of Adriamycin on human colon adenocarcinoma cell lines was investigated. Concentrations of Adriamycin producing 50% inhibition were very similar in HT29, Sw480, Sw620, and Sw1116 cells, whereas Caco-2 cells were relatively insensitive. As compared to the Sw1116 cell line, Caco-2 cells were also insensitive to mitoxantrone. Sensitivity to cisplatin, 5-fluorouracil, or ethacrynic acid was comparable in both cell lines. To find the mechanism for this mitoxantrone and Adriamycin resistance, several potential Adriamycin-detoxifying systems were characterized and quantified in both Sw1116 and Caco-2 cells. No dramatic differences in glutathione content and expression of both selenium dependent- and independent glutathione peroxidase, UDP-glucuronyltransferase, and cytochrome P-450 were found. However, highly significant differences in glutathione S-transferase activity were present, the expression of both class pi and class alpha glutathione S-transferases being much higher in the Caco-2 cell line. In addition, a slightly higher content of P-170 glycoprotein was present in the Caco-2 cells. These findings suggest that glutathione S-transferases, and to a lesser extent the P-170 glycoprotein, may be involved in mitoxantrone and Adriamycin resistance of Caco-2 colon carcinoma cells.     

M2, a novel myelomonocytic cell surface antigen and its distribution on leukemic cells

The selectivity of a novel myelomonocytic cell surface antigen, designated M2, has been assessed in a series of 208 leukemias. The M2 antigen is defined by a monoclonal antibody (VIM-2) of the IgM class. Its expression within the normal hemopoietic system is restricted to myelomonocytic cells. Lymphocytes, erythrocytes, thrombocytes and their morphologically recognizable precursors are negative. Sixty of the 66 acute myeloblastic leukemias (= 91%) and 28 of the 30 myeloid blast crises of CML patients (= 93%) were M2-positive. As expected from our findings with normal myeloid cells, the myeloid cells found in stable phase of CML were also in all instances, M2-positive. Quite in contrast, lymphoid cells from patients with B-CLL, T-CLL, prolymphocytic leukemia, hairy-cell leukemia, lymphoblastic lymphoma, Sézary syndrome, from CML patients in lymphoid blast crisis and from the majority of patients with ALL, were completely M2-negative. Also negative were the blast cells of patients with acute megakaryoblastic leukemia and acute erythroleukemia. A direct comparison of M2 expression with the display of the 3-fucosyl-N-acetyllactosamine determinant, the structure recognized by most of the anti-myeloid monoclonal antibodies reported so far, shows that more AMLs are M2-positive and the proportion of M2-positive blast cells in individual AML samples is higher.     

Molecular and biochemical mechanisms of fludarabine and cladribine resistance in a human promyelocytic cell line

2F-Adenine arabinoside (fludarabine, Fara-A) and 2-chloro-2'-deoxyadenosine (cladribine, CdA) are nucleoside analogues with antineoplastic activity in vitro and in vivo. Lack of clinical resistance between CdA and Fara-A has been demonstrated in patients with chronic lymphocytic leukemia (G. Juliusson et al., N. Engl. J. Med., 327: 1056-1061, 1992). To clarify the differences in mechanism of resistance to CdA and Fara-A in vitro, we developed two stable, resistant cell lines, HL60/CdA and HL60/ Fara-A, by exposure to increasing concentrations of analogues over a period of 8 months. Resistant cells tolerated >8,000 and 5-fold higher concentrations of CdA and Fara-A, respectively. The specific activity of the nucleoside phosphorylating enzyme (using deoxycytidine as substrate) in cell extracts from HL60/CdA and HL60/Fara-A mutants was about 10 and 60%, respectively, compared with the parental cell line. Western blot analysis using a polyclonal antibody showed no detectable deoxycytidine kinase (dCK) protein in CdA-resistant cells, whereas in Fara-A-resistant cells, it was at the same level as in the parental cells. The mitochondrial enzyme deoxyguanosine kinase was not altered in resistant cell lines. The HL60/CdA cells showed cross-resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, Fara-A, arabinofuranosyl cytosine, difluorodeoxyguanosine, and difluorodeoxycytidine toxicity, most likely because of the decreased phosphorylation of these analogues by dCK. Using real-time quantitative PCR, the mRNA levels of dCK and cytosolic 5'-nucleotidase (5'-NT), a major nucleoside dephosphorylating enzyme, were measured. It was shown that the dCK mRNA levels in both CdA- and Fara-A resistant cells were decreased in parallel with the activity. The expression of 5'-NT mRNA was not significantly elevated in CdA- and Fara-A resistant cells, as compared with the parental cells. Ribonucleotide reductase maintains a balanced supply of deoxynucleotide triphosphate pools in the cell and may also be a major cellular target for CdA and Fara-A nucleotides. Except for the deoxycytidine triphosphate level, the intracellular deoxynucleotide triphosphate pools were significantly higher in Fara-A-resistant cells compared with the parental cell line. This might be a consequence of mutation or altered regulation of ribonucleotide reductase activity and may explain the 2-5-fold cross-resistance to several nucleoside analogues observed with HL60/Fara-A cells. It is likely that the resistance for CdA was mainly attributable to a dCK deficiency, and Fara-A-resistant cells might have another contributing factor to the resistance beyond the dCK deficiency.     

Membrane lipid modification and sensitivity of leukemic cells to the thioether lipid analogue BM 41.440.

Since the ether lipid anticancer drugs are membrane targeted, we examined the effect of membrane lipid structural alteration on their cytotoxicity. Enrichment with docosahexaenoic acid increased the sensitivity to the thioether lipid BM 41.440, compared to control cells enriched with oleic acid. The effect was dependent upon drug concentration, time, and the extent of cellular fatty acid enrichment. Other polyunsaturated fatty acids had a similar effect, which was proportional to the degree of unsaturation of the molecule inserted. Depletion of cellular glutathione with buthionine sulfoximine increased the sensitivity to ether lipid, but prooxidants such as Fe2+ and antioxidants such as vitamin E had little effect. The addition of serum to the incubation medium markedly diminished the cytotoxicity of ether lipids for cells modified with both docosahexaenoic acid and oleic acid, probably due to binding of the drug to serum components. The toxicity of another ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, was not affected appreciably by membrane alteration. Drug uptake studies with a radiolabeled BM 41.440 analogue, 1-[3H]hexadecylthio-2-ethyl-rac- glycero-3-phosphocholine, demonstrated no difference in transport at early time points and no difference in accumulation up to 60 min. We conclude that increases in cellular and/or membrane fatty acid polyunsaturation heighten the cytotoxic effect of a membrane-active ether lipid. The effect is not due to a change in drug transport or accumulation. It may be related to a change in oxidative events. These observations provide further confirmation of the membrane being the target of ether lipid action, using biochemical rather than morphological techniques. Most importantly, this observation offers a potential innovative approach to therapy.     

Methylation analysis of the human multidrug resistance 1 gene in normal and leukemic hematopoietic cells.

Expression of the human multidrug resistance 1 gene (MDR1), which encodes the P-glycoprotein transmembrane efflux pump, has been associated with treatment failure of some leukemias, primarily acute myeloid leukemia (AML). To elucidate the epigenetic events associated with overexpression of MDR1 in AML, we analyzed the methylation status of a 2000 bp region within the MDR1 locus using a bisulphite genomic sequencing technique. A CpG-rich domain, approximately 1 kb in size, encompasses the promoter region, exon I, and intron I. This domain was found to be relatively unmethylated in five out of six primary and cultured human hematopoietic cells, as well as five out of six AML patient samples, independent of the MDR1 phenotype. The data suggest that the methylation status of the CpG-rich domain does not act as a 'switch' to regulate expression of the MDR1 gene. In addition, we found an upstream Alu repeat sequence to be unmethylated in three out of five cultured hematopoietic cell lines, both MDR1 expressing and non-expressing. However, analysis of primary CD8-positive T cells and AML patient samples revealed dense methylation of this region which is consistent with methylation of Alu repeat sequences observed in somatic tissues.     

Two distinct molecular mechanisms underlying cytarabine resistance in human leukemic cells

To understand the mechanism of cellular resistance to the nucleoside analogue cytarabine (1-beta-D-arabinofuranosylcytosine, AraC), two resistant derivatives of the human leukemic line CCRF-CEM were obtained by stepwise selection in different concentrations of AraC. CEM/4xAraC cells showed low AraC resistance, whereas CEM/20xAraC cells showed high resistance. Both cell lines showed similar patterns of cross-resistance to multiple cytotoxic nucleoside analogues, with the exception that CEM/20xAraC cells remained sensitive to 5-fluorouridine and 2-deoxy-5-fluorouridine. Both cell lines were sensitive to 5-fluorouracil and to a variety of natural product drugs. Although both CEM/4xAraC and CEM/20xAraC cells displayed reduced intracellular accumulation of [(3)H]AraC, only CEM/4xAraC cells showed reduced uptake of [(3)H]uridine, which was used to assess nucleoside transport activities. Genes encoding proteins known to be involved in nucleoside transport, efflux, and metabolism were analyzed for the presence of mutations in the two cell lines. In CEM/4xAraC cells, independent mutations were identified at each allele of human equilibrative nucleoside transporter 1 (hENT1; SLC29A1), one corresponding to a single-nucleotide change in exon 4, the other being a complex intronic mutation disrupting splicing of exon 13. In contrast to CEM/20xAraC cells, CEM/4xAraC cells did not bind the hENT1/SLC29A1 ligand nitrobenzylmercaptopurine ribonucleoside and lacked detectable hENT1/SLC29A1 protein. In CEM/20xAraC cells, independent intronic mutations impairing splicing of exons 2 and 3 were found at each allele of the deoxycytidine kinase gene. These studies point to at least two distinct mechanisms of AraC resistance in leukemic cells.     

Mechanisms involved in the development of adriamycin resistance in human leukemic cells

We have developed three adriamycin (ADR)-resistant K562 sublines with different degrees of resistance. These sublines show a decreased accumulation and an increased efflux of ADR in proportion to the degree of resistance. Two membrane proteins (mol. wt 170,000 and 230,000) reactive with monoclonal antibody against P-glycoprotein were highly expressed in both the K562/ADR200 and the K562/ADR500 subline. Less resistant K562/ADR80 cells contained only small amounts of mol. wt 230,000 protein. Thus, the level of P-glycoprotein expression was not proportionate to the degree of ADR efflux. Verapamil treatment could not completely reverse ADR resistance. No significant change of glutathione-s-transferase activity nor in the level of DNA topoisomerase II was detected in resistant sublines. In our sublines it seems that P-glycoprotein is one of the mechanisms for resistance, but additional mechanisms may be involved.