Search

A number of human immunodeficiency virus type-1 (HIV) positive subjects are also opiate abusers. These individuals are at high risk to develop neurological complications. However, little is still known about the molecular mechanism(s) linking opiates and HIV neurotoxicity. To learn more, we exposed rat neuronal/glial cultures prepared from different brain areas to opiate agonists and HIV envelope glycoproteins gp120IIIB or BaL. These strains bind to CXCR4 and CCR5 chemokine receptors, respectively, and promote neuronal death. Morphine did not synergize the toxic effect of gp120IIIB but inhibited the cytotoxic property of gp120BaL. This effect was blocked by naloxone and reproduced by the mu opioid receptor agonist DAMGO. To examine the potential mechanism(s) of neuroprotection, we determined the effect of morphine on the release of chemokines CCL5 and CXCL12 in neurons, astrocytes, and microglia cultures. CCL5 has been shown to prevent gp120BaL neurotoxicity while CXCL12 decreases neuronal survival. Morphine elicited a time-dependent release of CCL5 but failed to affect the release of CXCL12. This effect was observed only in primary cultures of astrocytes. To examine the role of endogenous CCL5 in the neuroprotective activity of morphine, mixed cerebellar neurons/glial cells were immunoneutralized against CCL5 prior to morphine and gp120 treatment. In these cells the neuroprotective effect of opiate agonists was blocked. Our data suggest that morphine may exhibit a neuroprotective activity against M-tropic gp120 through the release of CCL5 from astrocytes., ((c) 2010 Wiley-Liss, Inc.)

The cytotoxic effect of 1-beta-D-arabinofuranosyl cytosine (araC) depends on the intracellular phosphorylation into its active compound araCTP, on the degree of degradation of araCTP and on the incorporation of araCTP into DNA. Deoxycytidine triphosphate (dCTP) inhibits the phosphorylation of araC (by feedback inhibition of the enzyme deoxycytidine kinase) and the incorporation of araCTP into DNA (by competition for DNA polymerase). In a T-lymphoblastic cell line, we studied whether the cytotoxicity of araC (2 nM-50 microM) could be enhanced by decreasing the concentration of dCTP, using the nucleoside-analogue cyclopentenyl cytosine (CPEC), an inhibitor of the enzyme CTP synthetase. Preincubation of the cells with CPEC (100-1,600 nM) for 2 hr increased the concentrations of araCMP 1.6-9.5-fold, which was significant for each concentration of CPEC used. The concentration of araCDP remained low, whereas the concentration of araCTP changed depending on the concentration of araC used. With 2-15 microm of araC and a preincubation with 400 nM of CPEC, the araCTP concentration increased by 4-15% (not significant), and the total amount of araC nucleotides increased significantly by 21-45%. When using a concentration of araC of 2 nM after a preincubation with CPEC of 100 nM, the concentration of araCMP increased by 60% (p = 0.015), whereas that of araCTP decreased by 10% (p = 0.008). This was compensated by an increase of 41% (p = 0.005) of araCTP incorporation into DNA, which represented 43% of all araC metabolites. Moreover, by performing pulse/chase experiments with 400 nM of CPEC and 2 microM of araC, the retention of cytosolic araCTP and the incorporated amount of araCTP into DNA were increased by CPEC. The modulation by CPEC of araC metabolism was accompanied by a synergistic increase of araC-induced apoptosis and by an additive effect on the araC-induced growth inhibition., (Copyright 2002 Wiley-Liss, Inc.)

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

Drug resistance is one of the major reasons for failure of chemotherapy of acute leukemia with cytosine arabinoside (ARA-C). In order to overcome this problem we have investigated the interaction of ARA-C with 3-deazauridine (3-DU) against HL-60 myeloid leukemic cells. 3-DU is an interesting agent to use in combination with ARA-C, since drug-resistant cells that are deficient in deoxycytidine kinase are very sensitive to this uridine analogue. We have observed that for both short and long drug exposure there was a potent synergistic interaction between ARA-C and 3-DU with respect to their cytotoxic effects on HL-60 leukemic cells. This synergy could be explained by an increased cellular uptake of ARA-C to ARA-CTP by the leukemic cells in the presence of 3-DU, due to the reduction in the pool of dCTP produced by this latter analogue. Since dCTP is a potent feedback inhibitor of the phosphorylation of ARA-C by deoxycytidine kinase, the reduction in the dCTP produced by 3-DU results in an increased rate of phosphorylation of the arabinosyl analogue. Our results suggest that ARA-C and 3-DU may be an interesting drug combination to circumvent drug resistance in the chemotherapy of acute leukemia.

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) "suicide" technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara-C increased from 10(-8) to 10(-5) M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara-C even while the cells are in active DNA synthesis.

The antiviral activities of antileukemic drugs 1-beta-D-arabinofuranosylcytosine (Cytarabine; Ara-C), 2,2'-anhydro-1-beta-D-arabinofuranosylcytosine (Ancitabine; Cyclo-C), and N4-behenoyl-1-beta-D-arabinofuranosylcytosine (Enocitabine; BH-AC) were evaluated in vitro against human cytomegalovirus (HCMV) in comparison with those of five other antiviral drugs. Both Ara-C and Cyclo-C showed the strongest inhibitory effect to HCMV. BH-AC inhibited the replication of HCMV and depicted almost as the same dose-response curve as Ganciclovir (DHPG). In the presence of Ara-C, Cyclo-C, or BH-AC, triphosphate forms of the nucleoside analogs were detected in the HCMV-infected cells, and synthesis of HCMV DNA was strongly suppressed. Thus, Ara-C, Cyclo-C, and BH-AC were not only antileukemic, but also antiviral in vitro. However, Ara-C and Cyclo-C may not be suitable as anti-HCMV agents, because they are cytotoxic or excreted rapidly in the urine in vivo [Van Voris, 1984; Hirayama et al., 1974]. Because of lower toxicity and longer retention in vivo, BH-AC may be expected as an anti-HCMV agent in patients with leukemia, in addition to serving as an antileukemic drug.

Concentrations of ara-CTP in leukemic cells isolated from CSF and of ara-C in lumbar CSF were measured following intraventricular ara-C administration in two girls with refractory meningeal leukemia. CSF samples were collected with a permanent intrathecal-lumbar catheter. In contrast to the comparatively short retention of ara-C in the CSF (t1/2 1.8 to 2.9 hours), there was a high accumulation and an extremely long retention of ara-CTP in the leukemic cells (t1/2 8.1 to 36 hours). The patients included in this study had an ara-C-resistant disease. No obvious relationship was seen between concentrations of ara-C in the CSF and of ara-CTP in the leukemic cells. Similar studies were performed after simultaneous intraventricular administration of hydrocortison and ara-C. Hydrocortison did not increase ara-CTP retention in the leukemic cells, nor did it effect CSF pleocytosis.

Eight pediatric patients with acute lymphoblastic leukemia (ALL) were treated with intermediate-dose cytosine arabinoside (ID-AraC, 1 g/m2) in combination with adriamycin except one patient. Of the eight patients, four refractory to the initial induction therapy and one in bone marrow relapse gained complete remission with two to three cycles of this therapy. Four of the five patients have been in continuous remission for 4 to 24 months with the maintenance therapy of monthly administration of ID-AraC. One patient in central nervous system (CNS) relapse has continued in remission from CNS leukemia after two cycles of the therapy. Side effects of ID-AraC and adriamycin were generally mild to moderate and tolerable in all children. These results suggest that the use of ID-AraC and adriamycin might prove effective in the treatment of ALL refractory to other regimens.

Therapeutic efficacy of anticancer drugs against malignant diseases is limited because of the selection and regrowth of drug resistant cells. This problem is compounded by the development of cross-resistance to other useful chemotherapeutic agents which severely limits treatment alternatives. Development of approaches to overcome and/or circumvent drug resistance will depend on the precise understanding of the mechanism(s) of resistance not only at the target tumor cell level (in vitro) but also in vivo. The data on L1210 sensitive to araC transplanted s.c. demonstrated that although target cells are highly sensitive to araC, the lack of in vivo response of solid L1210 cells (s.c.) is primarily due to limited drug delivery to the target. Approaches that would modify these processes should improve on the therapeutic selectivity of this agent. In contrast, in cells where the mechanism of resistance is deletion of a key anabolic enzyme (e.g. CdR kinase), these cells are collaterally sensitive in vitro and in vivo to DAUR. This type of resistance, however, could not be overcome by liposome encapsulation of araC or araCTP. With respect to AM resistance, the concentration of VRP for in vitro reversal of resistance of P-388/AM was difficult to achieve in vivo without significant host toxicity. Continuous infusion of VRP, however, demonstrated that at the maximally tolerated doses of VRP it was not possible to achieve any significant therapeutic effects against P-388/AM. The use of liposomes or modulation by VRP does not appear to be a fruitful approach in overcoming AM resistance, especially in cells which possess a high degree of resistance. Metabolic modulation of FU by dLCF appears to be a promising area of investigation and deserves further investigation concerning quantitation of the intracellular pools of folate and ways to modulate them.

We have demonstrated that Ara-C incorporates in leukemic cell DNA and that the extent of this incorporation correlates significantly with loss of clonogenic survival. This relationship between formation of (Ara-C)DNA and cytotoxicity is also obtained in the presence of modulating agents such as thymidine. Further, the (Ara-C)DNA is structurally abnormal and undergoes strand scission upon exposure to alkali. We have also demonstrated that the incorporated Ara-C residue serves as a poor primer terminus for further chain elongation and thereby results in inhibition of DNA synthesis. These findings provide new insights into the mechanism of action of the most effective agent in the treatment of acute myelogenous leukemia. These findings have also been extended to demonstrate that Ara-C results in the induction of leukemic cell differentiation as well as lethal cellular events.

Cytosine arabinoside (Ara-C) is a very potent inhibitor of DNA synthesis in mammalian cells. This inhibition does not appear to be due to the direct inhibition of DNA polymerase by Ara-CTP (triphosphate of Ara-C) because it is a very weak competitive inhibitor of this enzyme. The inhibition of DNA synthesis appears to be due to the incorporation of Ara-C into DNA producing chain termination of polydeoxynucleotide elongation.

The effect of thymidine (dThd) and hydroxyurea (HU) on the cellular metabolism of 1-beta-D-arabinofuranosylcytosine (Ara-C) was investigated in the human promyelocytic cell line HL-60. Both dThd and HU increased the cellular uptake and rate of formation of Ara-CTP. Measurement of ribo- and deoxyribonucleotide triphosphate pools implicated a reduction of the dCTP as the mechanism of this effect. dThd and HU had opposite effects on the incorporation of Ara-C into DNA per unit time, but both enhanced the incorporation of Ara-C per unit of newly synthesized DNA. In a Phase I trial Ara-C was given by continuous infusion for five days at 100 mg/m2, and HU by mouth every six hours with dose escalation from 0.375 to 1.78 g/m2 every six hours. Myelosuppression was the dose-limiting toxicity; the major nonhematologic toxicity was skin rash. To date responses have been observed in chronic myelogenous leukemia in blast crisis and diffuse histiocytic lymphoma.

Plasma level Ara-C and Ara-U in vivo and intracellular Ara-CTP pools in vivo and in vitro were measured using high-performance liquid chromatography and radioimmunoassay. Plasma Ara-C during High Dose therapy was found in two phases; one, a peak at t1/2 of approximately 5-8 minutes, the other approaching that of Continuous Infusion with a t1/2 of about 6 to 8 hours. There appears to be no relationship between peak levels of plasma Ara-C and intracellular Ara-CTP formed during High Dose therapy. Intracellular Ara-CTP pools were found to be higher in peripheral blood than in bone marrow during High Dose treatment and were also higher in bone marrow of patients treated with High Dose rather than conventional dose Ara-C. In vitro experiments with various concentrations of Ara-C on patient cells prior to treatment suggest that patients may benefit from High Dose therapy when an increase in intracellular Ara-CTP occurred with higher extracellular concentrations of Ara-C. In patients with metabolically sensitive cells containing the necessary enzymes to activate Ara-C to Ara-CTP, where resistance is due to limited drug transport, High Dose therapy may prove beneficial. Conversely, in patients with no increase in Ara-CTP pools in vitro due to a depletion of activating enzymes, High Dose treatment may not be warranted. Therefore, it may be possible to determine patients most likely to benefit from High Dose chemotherapy by measuring pre-treatment in vitro intracellular Ara-CTP.

The cytotoxicity of many antitumor agents exhibits cell-cycle phase specificity. Using a human lymphoma cell line synchronized by thymidine block, we have investigated the differential uptake of radioactive 1-beta-D-arabinofuranosylcytosine (ara-C) and prednisolone in various phases of the cycle. Uptake of ara-C is highest during early S and declines steadily throughout the rest of the cycle. In comparison, prednisolone demonstrates no significant age-dependent difference in uptake although its cytotoxicity is cell cycle sensitive.

Pharmacological data are currently available for a number of antineoplastic agents which have shown clinical activity in advanced breast carcincoma. Preclinical data reveal a relationship between therapeutic response and certain pharmacokinetic parameters such as time of effective cytotoxic exposure (Teff) and the product of concentration with time (Cxt). We have attempted to apply human pharmacologic data to get estimates of these parameters for 6 active agents in breast cancer, to relate them to response rates, and to suggest a method for estimating the role of individual drugs in a multidrug combination. The response rates for 6 single agents were obtained from literature review and related to estimates of Teff and Cxt. The Cxt-response relations for single drugs were linear for cyclophosphamide, 5-fluorouracil, and thiotepa; exponential for vincristine; and relatively flat for methotrexate and cytoxine arabinoside. Most Teff values for the active single agents clustered about 15 hr/dose. From the graphs of response rate vs Cxt, the individual contribution of each agent in a combination study was estimated to arrive at a predicted response rate. The predicted response rates for the combination studies correlated with the actual response rates determined in the clinical study, for 6 of 6 nonrandomized studies and for 12 of 14 randomized studies analyzed. In 2 studies, deviations from the predicted response rate were attributed to differences in study design or analysis. There was no correlation between Teff and predicted response rate. Analyses of pharmacokinetic data may be useful to simulate combination chemotherapy studies to predict the effectiveness of clinical trials in breast cancer. Since the pharmacologic data were not obtained for any of the agents in the actual clinical trials done, we can only speculate on the usefulness of this method. We would encourage the prospective collection of this data in future clinical trails.

HeLa cell mitochondria were allowed to incorporate 3H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.

Tumor cells were isolated from malignant effusions of three patients with disseminated solid tumors of different origin. Intracellular accumulation of nondiffusible cytosine arabinoside (ara-C) nucleotides was used to measure phosphorylation. Mouse leukemia L 1210 and L 1210/CA, and ara-C-resistant subline, were used as reference cells. Phosphorylation activity was similar in the cells from all three solid tumors and showed a linear increase with drug concentrations of 0.1--100 micograms/ml. In contrast to activity in L 1210 cells, the in vitro activity was not saturable at drug levels up to 100 micrograms/ml. Ara-C inhibited the incorporation of thymidine into DNA 84%--90% in the solid tumor cells at a concentration of 10 micrograms/ml. Higher drug concentrations did not result in further inhibition. In one patient, DNA synthesis of tumor cells isolated before and after intraperitoneal instillation of 1,000 mg ara-C was measured. The in vivo inhibition was found to correspond to the in vitro data. Solid tumor cells isolated from malignant effusion have no greatly reduced capacity for cellular formation of ara-C/nucleotides, but higher drug levels than achieved with conventional therapy are necessary for sufficient ara-C nucleotide synthesis.