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Cell-intrinsic effects such as induction of apoptosis and/or inhibition of cell proliferation have been proposed as the major antitumor responses to histone deacetylase inhibitors (HDACi). These compounds can also mediate immune-modulatory effects that may contribute to their anticancer effects. However, HDACi can also induce anti-inflammatory, and potentially immunosuppressive, outcomes. We therefore sought to clarify the role of the immune system in mediating the efficacy of HDACi in a physiologic setting, using preclinical, syngeneic murine models of hematologic malignancies and solid tumors. We showed an intact immune system was required for the robust anticancer effects of the HDACi vorinostat and panobinostat against a colon adenocarcinoma and two aggressive models of leukemia/lymphoma. Importantly, although HDACi-treated immunocompromised mice bearing established lymphoma succumbed to disease significantly earlier than tumor bearing, HDACi-treated wild-type (WT) mice, treatment with the conventional chemotherapeutic etoposide equivalently enhanced the survival of both strains. IFN-γ and tumor cell signaling through IFN-γR were particularly important for the anticancer effects of HDACi, and vorinostat and IFN-γ acted in concert to enhance the immunogenicity of tumor cells. Furthermore, we show that a combination of vorinostat with α-galactosylceramide (α-GalCer), an IFN-γ-inducing agent, was significantly more potent against established lymphoma than vorinostat treatment alone. Intriguingly, B cells, but not natural killer cells or CD8(+) T cells, were implicated as effectors of the vorinostat antitumor immune response. Together, our data suggest HDACi are immunostimulatory during cancer treatment and that combinatorial therapeutic regimes with immunotherapies should be considered in the clinic., (©2013 AACR.)

Around 20% of patients with acute lymphoblastic leukemia are Philadelphia chromosome positive (Ph-positive acute lymphoblastic leukemia) and express the Bcr/Abl tyrosine kinase. Treatment with the tyrosine kinase inhibitor Imatinib is currently standard for chronic myelogenous leukemia, which is also caused by Bcr/Abl. However, Imatinib has shown limited efficacy for treating Ph-positive acute lymphoblastic leukemia. In our study, we have investigated the effect of Imatinib therapy on murine P190 Bcr/Abl lymphoblastic leukemia cells. Three of four cultures were very sensitive to treatment with 5 mumol/L Imatinib. Significant cell death also initially occurred when the same cultures were treated in the presence of stromal support. However, after 6 days, remaining cells started to proliferate vigorously. The Bcr/Abl tyrosine kinase present in the cells that were now able to multiply in the presence of 5 mumol/L Imatinib was still inhibited by the drug. In concordance with this, the Abl ATP-binding pocket domain of Bcr/Abl in the resistant cells did not contain point mutations which would make the protein Imatinib resistant. The effect of stroma in selecting Imatinib-resistant lymphoblasts did not require direct cell-cell contact. SDF-1alpha could substitute for the presence of stromal cells. Our results show that stroma selects Imatinib-resistant Bcr/Abl P190 lymphoblasts that are less dependent on Bcr/Abl tyrosine kinase activity. Therefore, therapy for Ph-positive acute lymphoblastic leukemia, aimed at interfering with the protective effect of stroma in combination with Imatinib, could be of benefit for the eradication of the leukemic cells.

Photodynamic therapy (PDT) with endogenous protoporphyrin IX derived from 5-aminolevulinic acid or its derivatives has been established for treatments of several premalignancies and malignancies; however, the mechanism of the modality is not fully elucidated. The mitochondrial permeability transition pore consists mainly of the mitochondrial outer membrane voltage-dependent anion channel and the peripheral benzodiazepine receptor (PBR) and the mitochondrial inner membrane adenine nucleotide translocator (ANT). These mitochondrial proteins are responsible for the permeability transition that leads to apoptosis. In the present study, the human leukemia cell line, Reh, was treated with PDT using hexaminolevulinate (HAL). More than 80% of apoptotic Reh cells were found after HAL-mediated PDT (HAL-PDT) with high-molecular-weight (50 kbp) DNA fragmentation. Addition of PK11195 or Ro5-4864, two ligands of PBR, during HAL-PDT significantly inhibited the apoptotic effect. Bongkrekic acid, a ligand for ANT, also reduced the PDT effect. Although the mitochondrial transmembrane potential collapsed, neither cytosolic translocation of mitochondrial cytochrome c nor activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase were found. However, nuclear translocation of mitochondrial apoptosis-inducing factor (AIF) was shown by both immunoblotting and immunocytochemistry. Because AIF is the sole one among all proapoptotic factors involved in caspase-dependent and caspase-independent pathways that induces the high-molecular-weight DNA fragmentation, we conclude that HAL-PDT specifically targets PBR, leading to apoptosis of the Reh cells through nuclear translocation of mitochondrial AIF. This study suggests PBR as a possible novel therapeutic target for HAL-based PDT of cancer.

Genistein (GEN) is an isoflavone known to inhibit both tyrosine protein kinases and DNA topoisomerase II. The effects of GEN on cell proliferation and cell cycle kinetics of human myelogenous leukemia HL-60 and lymphocytic leukemia MOLT-4 cell cultures were studied, and the data were compared to results obtained with normal human lymphocytes stimulated to proliferate with phytohemagglutinin. GEN concentrations greater than 50 micrograms/ml (185 microM) were cytotoxic to HL-60 and MOLT-4 cells following exposure for 24 h; in HL-60 cell cultures, a population of cells with decreased DNA content and nuclear fragmentation characteristic of apoptosis was observed within 8 h. The 50% inhibition concentration after 24 h of exposure for HL-60 and MOLT-4 cells was 8.5 and 13.0 micrograms/ml, respectively. Normal proliferating lymphocytes survived a 24-h exposure of up to 200 micrograms/ml GEN. Short-term (4-8 h) exposures of MOLT-4 or HL-60 cells to 5-20 micrograms/ml GEN resulted in a suppression of cell progression through S or through both S and G2 phases, respectively, while equivalent treatment had no effect on proliferating lymphocytes. A stathmokinetic experiment using MOLT-4 cells revealed that as little as 5 micrograms/ml GEN suppressed cell exit from S to G2 phase by 40%, with a terminal point of action at or near the S-G2 border. Cell progression through the very early portion of G1 phase (G1A, characterized by postmitotic chromatin decondensation) was also suppressed by approximately 40%, whereas cell advancement through the remainder of the G1 phase was not markedly affected. Longer (24 h) exposure of proliferating lymphocytes to 20 micrograms/ml GEN led to an S-phase arrest, while similar treatment of leukemic cells caused cell arrest in G2 phase and an increase in the number of cells entering the cycle at higher DNA ploidy. The mitogen-induced transition of lymphocytes from G0 to G1 phase was extremely sensitive to inhibition by GEN; the 50% inhibition concentration was 1.6 micrograms/ml. The chemotherapeutic value of GEN may be due to the fact that, in terms of cytotoxicity, this agent is more active against proliferating leukemic cells than against normal proliferating lymphocytes. The sensitivity of the G0 to G1 transition in normal lymphocyte cultures and the suppressive effect of GEN on the G1A exit in MOLT-4 cells both suggest that protein kinases involved in chromatin decondensation may be a target of this drug. In light of the observation that lymphocyte stimulation is sensitive to the presence of GEN, the drug is expected to be a strong immunosuppressant.

Exposure of exponentially growing human promyelocytic of lymphocytic leukemic cells to the putative DNA topoisomerase II inhibitor fostriecin (FST), at a concentration of 1 microM, results in the suppression of their rate of progression through the S and G2 phases of the cell cycle. At concentrations between 5 microM and 0.5 mM, FST triggers endonucleolytic DNA degradation in human promyelocytic leukemia cells, resulting in apoptotic cell death; this effect is not selective for any particular phase of the cell cycle. Little or no apoptotic cell death is observed in lymphocytic leukemic cells at any FST concentration. Because FST, unlike other inhibitors of topoisomerase II, such as teniposide (TN) or amsacrine (m-AMSA), does not stabilize cleavable DNA-topoisomerase complexes, the observed differences between the effects of FST versus TN or m-AMSA on the cell cycle may provide clues regarding the role of such complexes in the kinetic effects of these inhibitors. The present results, therefore, are compared with our earlier data on the effects of TN and m-AMSA on the same cells. The only observed difference is the loss of cell cycle phase-specific triggering of DNA degradation by FST in human promyelocytic leukemia cells, compared to the S phase-specific effects of TN and m-AMSA. Therefore, stabilization of the DNA-topoisomerase cleavable complexes may be essential in the selectivity of cell kill during S phase. However, it appears that the presence of stabilized complexes is not essential to the suppression of cell progression through S or G2 or the induction of apoptotis or necrosis, in general, by topoisomerase II inhibitors.

The coordinated gold compound, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-triethylphosphine-gold (auranofin) was found to be effective in increasing the life span of C57BL x DBA/2 F1 mice inoculated with the lymphocytic leukemia P388. A number of dose schedules were used, the lowest dose being 6 mg/kg every fourth day and the highest dose being 6.0 mg/kg twice daily for 9 days; the lowest and highest doses produced treated versus control ratios of 140 and 220%, respectively. All treatment groups achieved the minimum treated versus control ratio of 125%. Animal weights remained stable at twice-daily and high-dose-daily regimens. Increased life span and weight changes were both found to correlate with drug concentration and/or dose frequency.

Amino acid utilization was evaluated in seven children with acute lymphocytic leukemia treated with succinylated Acinetobacter glutaminase-asparaginase. All patients received food p.o. ad libitum and glucose-electrolyte solutions i.v.; four patients received an i.v. amino acid supplement (1.5 g/kg/day). Although all patients were in negative energy balance, there was a significant linear regression between nitrogen balance and nitrogen intake during Days 1 to 7 and Days 8 to 14 of the study. The slope of the regression line, reflecting exogenous nitrogen utilization, was not significantly different from that found in healthy young men ingesting adequate or subadequate energy intakes. The Y-intercept (-210 mg/kg/day) indicated an obligatory nitrogen loss that was much greater than normal. Most of the nitrogen loss was due to urinary excretion. Ammonia and urea accounted for 77 to 91% of the urine nitrogen. Urinary glutamate accounted for 4 to 10% of this loss. Urine protein excretion was abnormally high in each of the patients, ranging from 987 to 3440 mg/day. Urine excretion of N-acetyl-beta-glucosaminidase and beta 2-microglobulin was also abnormally high, despite normal blood urea nitrogen and serum creatinine, suggesting that these children had renal tubular dysfunction. The antileukemic effect of succinylated Acinetobacter glutaminase-asparaginase did not appear to be altered by amino acid supplementation. These data indicate that amino acid supplementation can improve nutritional status in patients treated with succinylated Acinetobacter glutaminase-asparaginase.

The pharmacodynamic parameters of 1-beta-D-arabinofuranosylcytosine (ara-C) in patient plasma and its active anabolite 1-beta-D-arabinofuranosylcytosine-5-triphosphate (ara-CTP) in circulating and bone marrow blast cells were studied in 20 pediatric patients with acute leukemia. ara-C (3 g/m2) was administered as a short-term infusion over 3 h every 12 h for a total of eight doses. The peak plasma concentration of ara-C ranged from 0.02 to 5.6 microM after the first dose of ara-C. The area under the concentration-time curve (AUC) of ara-C in plasma ranged from 302 to 20,298 microMh after the first dose of ara-C. The half-life of elimination (t1/2,el) of ara-C from plasma was 2.4 +/- 1.5 h in three patients with acute nonlymphoblastic leukemia (ANLL) and 4.78 +/- 4.1 h in 9 patients with acute lymphoblastic leukemia (ALL). The intracellular peak concentration of ara-CTP in circulating blast cells averaged 432.2 +/- 14.5 microM and 544.3 +/- 330 microM in patients with ANLL and ALL, respectively. The elimination kinetics of ara-CTP was monoexponential with t1/2,el of 3.30 +/- 0.8 h and 6.9 +/- 2.8 h in patients with ANLL and ALL. DNA synthetic capacity (DSC) of the blast cells was inhibited to between 24 and 64% of control after the first dose of ara-C and it declined further to between 1 and 32% after four doses of ara-C. The AUC of ara-CTP in leukemic cells ranged from 1,073 to 14,751 microMh and it was not related to the AUC of ara-C in plasma. The pharmacodynamic parameters of ara-CTP in circulating blast cells were more homogeneous in patients with ANLL than in patients with ALL. Four of six patients (67%) with ANLL and six of 14 patients (43%) with ALL achieved either complete remission or partial remission with high dose ara-C. We conclude that treatment of pediatric patients with leukemia in relapse with high dose ara-C is tolerable and moderately successful. Inhibition of DSC is positively correlated with the probability of having zero nadir peripheral blast cells. In turn there is a trend for a zero nadir peripheral blast cell count to be related to achievement of a response to therapy. This latter result is consistent with the results of larger studies in adults with leukemia.

Using a sensitive radioimmunoassay, concentrations of vincristine and its products were determined in cerebrospinal fluid (CSF) and corresponding blood samples from four patients with lymphoma and two patients with leukemia who had received i.v. bolus injection of 2 mg vincristine. Serial samples of CSF were withdrawn from a CSF reservoir in two patients during a 24-hr period following injection. The first time point after injection at which measurable levels of vincristine were detected in the CSF was 30 min; the concentrations were within the lower range of sensitivity of the assay and were 20- to 30-fold lower than were corresponding serum samples. Despite a prolonged terminal half-life of vincristine in the serum (14.4 to 37.5 hr) following i.v. bolus injection, concentrations of vincristine in the CSF ranged between undetectable within the limits of the assay and 1.1 X 10(-9) M during the 24-hr period of observation. The highest CSF concentration of vincristine (2.6 X 10(-9) M) has been observed in a patient receiving cranial irradiation for active meningeal lymphoma. No measurable levels of vincristine or its products were detected in spot samples of CSF from three patients. Penetration of vincristine and its products into the CSF of humans after i.v. bolus injection appears to be very poor and may account for the uncommon occurrence of central neurotoxicity following its clinical use.

Patients in remission with advanced acute lymphatic leukemia were randomly assigned to receive chemotherapy alone or chemotherapy plus immunotherapy with a Burkitt lymphoma tissue culture cell line (RAJI). Remission duration in both groups were identical. Complement-dependent cytotoxic antibody was seen in 5 of 8 immunized patients and 0 of 8 controls. This antibody reacted with RAJI and both allogeneic and autologous acute leukemia cells. Antibody titers began to rise after 2 months, peaked at 4 months, and then declined prior to relapse in all patients. The time course of the increase in mixed-leukocyte culture response to RAJI was similar to immunized patients. An increased in vitro response to phytohemagglutinin was seen during drug administration in all patients. Although no clinical benefit was seen in this small number of patients with the RAJI injections, these in vitro responses are encouraging and new immunization schedules will be investigated.

Cultured human T-cell leukemia lymphocytes have enhanced sensitivity to growth inhibition by deoxyadenosine. We have used flow cytometry to investigate the mechanism of deoxyadenosine toxicity in cultured T-leukemic cells. Comparative studies on deoxyadenosine-resistant Epstein-Barr virus-transformed B-lymphocyte cell lines were also performed. After exposure of T-cells to low concentrations of deoxyadenosine (3 microM), in the presence of an adenosine deaminase inhibitor (erythro-9-[3-(2-hydroxynonyl)]adenosine), accumulation of cells of cells with a G1 DNA content was demonstrated. In contrast, B-cell lines showed a similar degree of growth inhibition after exposure to 200 to 400 microM deoxyadenosine but were blocked in S phase. The T-cell G1 block was associated with a rise in the deoxyadenosine triphosphate pool, and both these phenomena were prevented by the addition of deoxycytidine. The biochemical mechanism of this G1 block induced by deoxyadenosine in T-cells is not understood.

The ability of methotrexate (MTX) to pass from the blood into the interstitial space and seminiferous tubule of the rat was investigated using testicular micropuncture. MTX was administered to anesthetized adult Wister rats via a femoral vein cannula. Constant plasma levels of MTX were achieved by giving a priming dose followed by a constant infusion of 1, 10, or 100 mg/kg/hr with 6 to 27 rats studied at each dose. Blood (via a jugular vein cannula), testicular interstitial fluid, and seminiferous tubule fluid (via direct micropuncture) were periodically sampled during the 4 hr of drug infusion. Under steady-state conditions, when compared to corresponding plasma values, MTX levels were 2- to 4-fold lower in the testicular interstitial fluid and 18- to 50-fold lower in the seminiferous tubule. These results indicate that, in the rat, a significant blood-testis barrier to MTX exists at the tubular but probably not at the capillary-interstitial level. If these results can be extrapolated to humans, they do not provide a pharmacological explantation for the frequent occurrence of leukemic relapse in the interstitium of the testes in boys with acute lymphocytic leukemia.

A patient with refractory T-cell acute lymphoblastic leukemia was treated with eight courses of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), over a 5-month period. After developing resistance to dCF, he responded to treatment with the combination of dCF and 9-beta-D-arabinofuranosyladenine (ara-A). We monitored the levels in plasma and urine of adenosine, 2'-deoxyadenosine, and ara-A as well as the accumulation of their nucleotide derivatives in erythrocytes and circulating lymphoblasts. We also monitored the activities of adenosine deaminase and S-adenosylhomocysteine (AdoHcy) hydrolase and the concentrations of AdoHcy and S-adenosylmethionine in lymphoblasts. Production of 2'-deoxyadenosine was related to both the duration of dCF infusion and the magnitude of cytolysis that occurred during treatment: much more 2'-deoxyadenosine was produced by dCF infusion when disease was active than by the same infusion given during remission. Resistance to dCF was associated with a decrease of greater than 90% in the amount of deoxyadenosine 5'-triphosphate accumulated by circulating lymphoblasts. Infusion of dCF resulted in increases of up to 20-fold in the concentration of AdoHcy in circulating lymphoblasts, causing a decrease in the S-adenosylmethionine:AdoHcy ratio (the "methylation index") from a pretreatment value of greater than 40:1 to less than 4:1. This ratio decreased to 2.5:1 during combined treatment with dCF and ara-A, which caused nearly complete inactivation of lymphoblast AdoHcy hydrolase. Decline in the methylation index was accompanied by inhibition of the methylation of newly synthesized lymphoblast RNA. Impaired ability to catabolize AdoHcy may have contributed to the cytolytic responses to dCF and ara-A, as well as to hepatic and central nervous system toxicity associated with their combined use.

The effects of six clinically active drugs were tested against a ttansplantable leukemia in inbred strain 2 guinea pigs. Cytoxan and 6-mercaptopurine were found to elicit a therqeutic response against this leukemia based on complete tumor regression of the established tumor as well as a substantial increase in survival time. Animals dying in the untreated control and drug-treated groups revealed typical generalized lymphoblastic leukemia. However, only Cytoxan-treated animals that had relapsed exhibited central nervous system involvement originating from the arachnoid membrane. A tow-drug combination of Cytoxan and 1-(2-chloroethyl)-3(trans-4-methylcyclohexyl)-1-nitrosourea was found not only to prevent meningeal leukemia development but also to result in "curing" all animals from their leukemia. This observation was based on a complete clinical, hematological, and histopathological "remission" period up to 176 days. The administration of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea alone was observed not only to control the systemic leukemia but also to prevent central nervous system involvement. No relapses occurred after the first "remission" period was achieved in the groups of animals that received 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea.

Illudins are low molecular weight natural products which were previously evaluated as anticancer drugs using rodent tumor models. In the present studies, we used in vitro cultures of human cancer cells to reevaluate their potential as anticancer agents. Using continuous exposure, Illudins S and M were cytotoxic to human leukemia cells at concentrations of 6-100 nM, but dihydroilludin M was 3 orders of magnitude less toxic, thus identifying a ketone site as a structural feature critical for cytotoxicity. Cytokinetic studies showed that illudin S caused a complete block at the G1-S phase interface of the cell cycle. Kinetics of inhibition of radiolabeled thymidine, uridine, and leucine incorporation suggested a primary effect on DNA synthesis. In colony and liquid culture assays, cell killing was time dependent but near maximal with a 2-h exposure. Myeloid and T-lymphocyte leukemia cells were most sensitive (50% inhibitory concentration, 6-11 nM), but B-cell leukemia/lymphoma, melanoma, and ovarian carcinoma cells were at least 10 times more resistant. Bone marrow granulocyte/macrophage progenitors showed intermediate sensitivity. Illudin S was equally effective against CEM T-lymphocyte leukemia cells expressing the multidrug resistance phenotype associated with Mr 180,000 glycoprotein and the parental cell line. CEM cells resistant to doxorubicin, epipodophyllotoxins, and 1-beta-D-arabinofuranosylcytosine showed only a 2-fold increased resistance to illudin S. Illudins are novel and potent cytotoxins which may be preferentially active against human myeloid and T-cell leukemias, including cells resistant to more conventional chemotherapeutic agents. The present studies illustrate the breadth of information which can be obtained on a new agent using present in vitro screening procedures and human cells.

A 9-year-old white boy developed a fatal primary Epstein-Barr virus (EBV) infection while receiving chemotherapy for acute lymphoblastic leukemia in remission. Histopathological findings at the height of the proliferative phase of the illness were compatible with a virally induced hemophagocytic syndrome. The infection spontaneously converted to complete aplasia of the bone marrow and lymph nodes. Serological studies disclosed that the patient had no antibodies to EBV prior to the infection, but during the acute phase he showed a spectrum and titers of antibodies to EBV-specific antigens characteristic of a current primary EBV infection. A lymph node biopsy obtained 5 weeks after onset revealed Epstein-Barr nuclear antigen in approximately 50% of the cells. The boy's condition deteriorated rapidly, with disseminated candidiasis resulting in cardiorespiratory failure and death. Lymph nodes obtained at autopsy no longer contained Epstein-Barr nuclear antigen-positive cells.

A rapid propidium iodide staining method was used for analysis of single-cell suspensions of bone marrow and tumor biopsies by flow microfluorometry. With this technique, information on the proliferative status of target tissues can be obtained within 10 min of sample removal. DNA histograms and labeling index of sequential bone marrow biopsies from a patient with Stage IV diffuse lymphocytic leukemia and treated with 1-beta-D-arabinofuranosylcytosine infusion showed pronounced reduction in the percentage of cycling cells. In contrast, sequential tumor biopsies from a melanoma patient on methotrexate-citrovorum factor rescue therapy showed no changes. In sequential bone marrow biopsies of 3 patients on high-dose methotrexate-citrovorum factor rescue, initial accumulation of cells in G1-S (Day 1) was followed by a significant proliferative response (Days 4 to 7) and return to pretherapy values. In contrast, no recovery similar to that of the bone marrow was seen in tumor cells.

Between June 1977 and December 1979, 72 evaluable patients with childhood non-T-cell acute lymphoblastic leukemia were induced into complete remission using vincristine, prednisone, and doxorubicin. All received asparaginase consolidation and central nervous system prophylaxis with cranial irradiation and intrathecal methotrexate. All patients then received prolonged intensification with vincristine, prednisone, and doxorubicin, and half of them were randomized to receive weekly high-dose asparaginase. Continuation therapy was with vincristine, prednisone, methotrexate, and 6-mercaptopurine. After a median follow-up of 57 months, there were four remission deaths and 25 relapses. Central nervous system relapse was the first event in 4% of patients. There were fewer treatment failures in the asparaginase-treated group [2-sided, p = 0.04 (0.07 controlling for standard and high-risk groups)]. Asparaginase toxicity occurred in six patients (8%) and was self-limited, but it precluded further use of the drug in those patients. The major toxicity of this treatment program was drug-induced cardiomyopathy which occurred in 10 patients (14%) and was fatal in three of them. In summary, we conclude that the intensive use of high-dose asparaginase has an important role in the treatment of children with acute lymphoblastic leukemia. The morbidity of multiple doses of doxorubicin outweighed its antileukemic advantage in standard-risk patients.

An investigation was undertaken of the mechanism of resistance to nitrogen mustard (HN2) and other alkylating agents, with particular emphasis on the interaction between cross-resistance and drug transport mechanisms in L5178Y lymphoblasts. Dose-survival curves demonstrated that the D0 for HN2-sensitive cells (L5178Y) treated with HN2 in vitro was 9.79 ng/ml and the D0 for HN2-resistant cells (L5178Y/HN2) was 181.11 ng/ml; thus, sensitive cells were 18.5-fold more responsive than were resistant cells and the difference was highly significant (p less than 0.001). A similar evaluation of 5 additional alkylating agents, including chlorambucil, melphalan, 1,3-bis(2-chloroethyl)-l-nitrosourea, Mitomycin C, and 2,3,5-tris(ethyleneimino)-1,4-benzoquinone, revealed that L5178Y/HN2 cells were also cross-resistant, in part, to each of these compounds. Furthermore, the degree of cross-resistance was remarkably similar; for each drug, dose-survival studies showed that HN2-resistant cells were approximately 2- to 3-fold more resistant to therapy than were sensitive cells. L5178Y/HN2 cells were also cross-resistant to cyclophosphamide in vivo; after treatment with cyclophosphamide, DBA/2 female mice that were given inoculations of L5178Y cells, but not those given transplants of L5178Y/HN2 cells, showed a significant prolongation of survival time (p less than 0.01). Transport of HN2, hydrolyzed derivative of HN2 and choline by L5178Y lymphoblasts in vitro was not competitively inhibited by any of the other alkylating agents, suggesting that transport of these compounds was by an independent mechanism. These findings suggest that the mechanism whereby L5178Y/HN2 cells are cross-resistant to other alkylating agents may involve nontransport factors and that these other drugs may bypass a major portion of HN2 resistance by using independent transport systems.

Mercaptopurine (MP) is a purine antimetabolite widely used for remission maintenance in the therapy of acute lymphoblastic leukemia. In order to study the biochemical parameters affecting MP activity, leukemic cells were obtained from ten patients with acute lymphoblastic leukemia at the time of diagnosis and from the same patients at the time of their initial marrow relapse. Hypoxanthine phosphoribosyltransferase (HPRT), the enzyme that converts MP to its active, nucleotide metabolite, thioinosine monophosphate; alkaline phosphatase, the primary catabolic enzyme of thioinosine monophosphate; and 5-phosphoribosyl-1-pyrophosphate (PRPP), the cellular ribose-phosphate donor essential for MP activation, were all measured within the patients' leukemic cells. There was marked interpatient variability in the three biochemical parameters studied with a greater than 10-fold range in alkaline phosphatase activity and an approximately 100-fold range in HPRT activity and PRPP levels. Four patients developed changes in biochemical parameters that influence MP activity at the time of relapse. In three of the four patients, alterations in more than one of these three biochemical parameters were noted. Three of four patients had a greater than 50% decrease in intracellular HPRT activity, four of four had a greater than 50% decrease in intracellular PRPP, and two of four had a greater than 9-fold increase in intracellular alkaline phosphatase activity at relapse. Two of four patients demonstrated changes in all three parameters at relapse in the directions that could have resulted in decreased MP sensitivity (i.e., decreased HPRT, decreased PRPP, and increased alkaline phosphatase). There was no correlation between pretreatment values of HPRT, PRPP, and alkaline phosphatase and remission duration. These results indicate that: (a) there is marked variation in HPRT, PRPP, and alkaline phosphatase in patients with acute lymphoblastic leukemia and b) following MP-containing maintenance chemotherapy, some patients develop biochemical changes that may result in decreased sensitivity to MP.

The RNA content of intact cells and isolated nuclei of normal human lymphocytes and mononuclear cell populations (containing at least 50% blasts) from patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) was measured by flow cytometry based on metachromatic red luminescence of acridine orange-stained cells. Relative nuclear RNA (n-RNA) and cellular RNA (c-RNA) content was estimated in relation to luminescence of RNase-treated nuclei, which served as a standard. The mean values (+/- SE) of n-RNA were 22.6 +/- 3.2, 25.8 +/- 3.2, and 51.5 +/- 6.1 arbitrary units for normal lymphocytes, ALL, and ANLL cell populations, respectively. The mean values for c-RNA were 51.3 +/- 5.2, 71.9 +/- 11.3, and 128 +/- 13.4 for the same cell populations, respectively. The differences between normal lymphocytes or ALL and ANLL cell populations were statistically significant (t-test, with respect to both n- and c-RNA), while differences between normal and ALL populations were not. The proportions of n-RNA versus c-RNA were similar within all three types of cell populations. The intercellular variabilities with respect to n- and c-RNA among the G0/1 cell populations of all three types of cells were characterized by the coefficient of variation (CV) of the mean RNA and the third moment about the mean (MOM3). CV and MOM3 of c-RNA were significantly different between all three types of cell populations, whereas CV and MOM3 of n-RNA showed significant differences between control and leukemic cells. Thus, mean RNA, CV, and MOM3 of RNA on the cellular and nuclear levels of G0/1 cells discriminate normal lymphocytes, ALL lymphoblasts, and ANLL blast cells from each other fully on statistically significant levels.

L-Asparaginase was added to vincristine and prednisone for induction of first remission in 815 children with acute lymphocytic or acute undifferentiated leukemia. This combination resulted in an overall remission rate of 93%. The addition of L-asparaginse to the standard induction regimen using prednisone and vincristine did not significantly increase the morbidity or mortality rate during the induction period. The most common side effect was transient L-asparaginase-induced hyperglycemia. The safe administration of L-asparaginase i.m. and the dose efficacy of 6000 I.U./sq m were confirmed. For these reasons, L-asparaginase should be combined with vincristine and prednisone for the initial induction of children with acute lymphocytic or acute undifferentiated leukemia.

A batch elution method with hydroxylapatite was developed to assay DNA damage by a set of antitumor anthracycline derivatives and was standardized with respect to the kinetics of unwinding, size of the alkaline unwinding unit, and fidelity of selective elution of single and double-stranded DNA. The method was applied to a study of a set of 10 antitumor anthracycline derivatives which inhibit growth of CCRF-CEM human leukemia cells over a range of potencies exceeding four orders of magnitude. The derivatives, including Adriamycin, daunorubicin, and carminomycin, vary in structure at C-4 and C-13, with substitutions at C-14 and N and stereochemical differences at C-4'. In a static model (fixed drug concentrations and incubation times), the potency [1/ID37 (concentration of agent that inhibits cell growth by 37%)] of nine of the ten derivatives may be expressed as functions of DNA damage (n), inhibition of thymidine incorporation (l), and drug retention (r): ID37 = Ka(r/l . n)kb, with a coefficient of correlation of greater than 0.99. A kinetic model with 4-demethoxydaunorubicin (varying concentrations and incubation times) was also described. Following initial uptake and a period of rapid loss after cells are washed free of excess drug, the change in agent concentration in the cells follows first-order kinetics. The cell index (cell number after 50 hr in drug-free growth medium/cell number after initial 2-hr exposure with drug) may be expressed linearly in terms of the kinetics of drug loss (coefficient of correlation, greater than 0.98), or as functions of 1/n (coefficient of correlation, greater than 0.958), 1/l . n (coefficient of correlation, greater than 0.963, or r/l . n (coefficient of correlation, greater than 0.963). These studies may be used to define a class of similarly acting anthracycline agents and to give some insight into the mechanism of action of the agents that fall within the class.

A patient with acute lymphoblastic leukemia refractory to conventional combination chemotherapy schedules was treated with high-dose 1-beta-D-arabinofuranosylcytosine (ara-C) (8 doses of 3 g/m2). The patient achieved complete remission but relapsed 6 weeks later. Two cell lines, PER-145 and PER-163, were established from bone marrow samples obtained before and after treatment, respectively. Both cells represent common acute lymphoblastic leukemia cells (CALLA+, HLA-DR+, sIg-, cIg-). Exposure of the two cell lines to ara-C in vitro revealed that the primary line PER-145 is susceptible to ara-C, while cell line PER-163 is more than 1000-fold more resistant (based on 50% inhibitory doses for growth in culture). Moreover, it was observed that ara-C concentrations from 1 to 33 micrograms/ml resulted in the stimulation of this cell line. Five weeks after his relapse the patient was given another high-dose ara-C course during which the blasts increased by a factor of 10, thus showing a response in vivo similar to that of the PER-163 cells in vitro. This pair of human acute lymphoblastic leukemia cells provides a unique opportunity to investigate the mechanisms underlying the acquired resistance and proliferative response to ara-C.

A combination of mitoxantrone, vincristine, and prednisone was used to treat 19 patients with acute lymphocytic leukemia. Of these, 12 were patients with acute lymphoblastic leukemia (ALL) (9 in first relapse and 5 primarily refractory to standard induction therapy with daunorubicin, vincristine, and prednisone), 2 had a phenotypic ALL relapse after an initial diagnosis of acute myelocytic leukemia and 5 had terminal deoxynucleotidyl transferase positive blastic phase chronic myelogenous leukemia (BCML). Eight patients with ALL (and of these, four with primarily anthracycline resistant disease), and two with BCML achieved complete remission. Five patients died in induction (three ALL from sepsis and two BCML from bleeding), and five had progressive disease. Median duration of response was 5 months, with two primarily refractory ALL patients remaining in continuing complete remission at 28 and 31 months. Treatment was well tolerated, with minimal nausea and vomiting, and oral mucositis. Posttreatment transient hepatic dysfunction was seen in 80% of patients. Mitoxantrone, vincristine, and prednisone are an active combination for the treatment of relapsed or refractory ALL and of terminal deoxynucleotidyl transferase positive BCML. The finding that four of five primarily refractory ALL patients were induced in complete remission supports the contention that mitoxantrone and anthracyclines are not cross-resistant.

2'-Deoxycoformycin (2'-dCF), a tight-binding inhibitor of adenosine deaminase, was administered to 26 pediatric patients with acute lymphoblastic leukemia in a Phase I study. Doses ranged from 0.25 to 1.0 mg/kg given i.v. for 3 consecutive days. Common toxicity included nausea, vomiting, diarrhea, hepatocellular enzyme elevations, and conjunctivitis. Lymphopenia occurred in all patients. The most serious adverse effects were acute tubular necrosis and central nervous system toxicity, which appeared to be dose related. In addition, two patients given the 0.75-mg/kg dose developed severe hepatic toxicity, although this could not be ascribed definitively to 2'-dCF. Antitumor activity was observed in eight patients, two of whom experienced a complete remission. Inhibition of lymphoblast adenosine deaminase activity was noted in the majority of cases and was observed at all doses. Antileukemic activity occurred at doses of 2'-dCF which were not associated with limiting toxicities. These results suggest that 2'-dCF is active against acute lymphoblastic leukemia and that a starting dose of 0.5 mg/kg/day be utilized in Phase II studies.

Four patients with refractory chronic lymphocytic leukemia were treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and initially received 4 mg/m2 i.v. weekly. Clinical responses to therapy varied: Patient A had a minimal response; whereas Patient D showed an 85% decrease in lymphocyte count at 2 wk; and Patients B and C had intermediate responses. The pretreatment mononuclear cell adenosine deaminase activities, which ranged from 1.6 to 44.6 nmol adenosine/h/10(6) cells, decreased to approximately 1 nmol adenosine /h/10(6) cells 24 h following 2'-deoxycoformycin, and increased to 15 to 50% of the pretreatment activity prior to the second drug treatment. The clinical response to 2'-deoxycoformycin was unrelated to the pre- or posttreatment adenosine deaminase activities or to the rate of return of enzyme activities following treatment. The plasma deoxyadenosine levels and the leukemic cell dATP concentrations rose slightly with therapy, but there was no correlation between the magnitude of increase and clinical response. No significant levels of DNA strand breaks were observed in the leukemic cells following treatment, although the NAD levels decreased slightly in two patients. When peripheral mononuclear cells from the patients and two controls were incubated in vitro for 24 h with 2'-deoxycoformycin and increasing concentrations of deoxyadenosine, a concentration-dependent increase in dATP and decrease in NAD were observed in both the patients and normals. The normal cells, and cells from two patients, developed a significant number of DNA strand breaks. However, there was no relationship between the formation of DNA breaks and the degree of accumulation of dATP or depletion of NAD, or between any of these changes and subsequent clinical responses to 2'-deoxycoformycin. Based on this study, it appears that the antitumor activity of 2'-deoxycoformycin in chronic lymphocytic leukemia is unrelated to the induction of DNA strand breaks or to changes in the levels of dATP or NAD in the leukemic cells.

L-Asparaginase, in the dose of greater than or equal to 6000 IU/sq m three times weekly, was demonstrated to be an effective agent in reinduction of remissions in childhood leukemia. Four hundred thirteen children with acute lymphocytic leukemia were treated with L-asparaginase. Doses i.m. ranged from 300 to 12,000 IU/sq m. None of the patients had received prior asparaginase therapy. 6-Mercaptopurine was given p.o. concurrently. All of the patients had experienced several previous relapses, and their disease was not responsive to 6-mercaptopurine. L-Asparaginase was found to be effective in reinducing remissions at the following rates: 9.5% for 300 IU/sq m; 35.1% for 3,000 IU/sq m; 53.5% for 6,000 IU/sq m; and 62.5% for 12,000 IU/sq m. The drug was given three times weekly for four weeks. Hypersensitivity reactions occurred in 6.5% of patients.

Neocarzinostatin (NCS) is an acidic protein (molecular weight, 10,700) isolated from Streptomyces carzinostaticus that has antitumor activity both in model rodent systems and in humans. In vitro it inhibits the growth of a human lymphoblastic leukemic cell line (CCRF-CEM) at a very low concentration (the amount of drug that causes a 50% inhibition of growth compared to control cultures as extrapolated from a dose-response curve (ID50), 2.4 X 10(-9) M). We covalently coupled NCS to the N-hydroxysuccinimide ester of agarose and obtained a product that, by a variety of biochemical and immunological criteria, has been demonstrated to be devoid of any free or loosely bound NCS. Agarose-bound NCS, which is unable to enter cells because of its size, retains a significant amount of inhibitory activity (ID50, 6 to 15 X 10(-9) M) and is also capable of inhibiting tritiated deoxythymidine incorporation into CCRF-CEM cells. Since agarose-bound NCS cannot enter mammalian cells, the above findings indicate that NCS is able to exert its toxic effects by binding to or reacting with receptors on the cell membrane.

Methotrexate (MTX)-resistant sublines of malignant human cells were selected in vitro by stepwise increase in drug concentration in the medium. By this procedure a subline of Burkitt's lymphoma cells (RAJI) was made 290-fold resistant (RAJI/MTX-R), T-cell leukemia cells (CCRF-CEM) were obtained 210-fold resistant (CEM/MTX-R), and 3 MTX-resistant human osteosarcoma lines were selected: TE-85/MTX-R (19-fold resistant; relative to wild-type); MG-63/MTX-R (8-fold resistant); and SAOS-2/MTX-R (200-fold resistant). We also studied a B-cell lymphoblastoid line, WI-L2/m4, that was 13,000-fold resistant. Assay of cellular dihydrofolate reductase (DHFR) showed the following pattern of activity in resistant cell lines, relative to parental cell activity: RAJI/MTX-R, 550-fold increased; CEM/MTX-R, unchanged; TE-85/MTX-R, 4-fold increased; MG-63/MTX-R, 6-fold increased; SAOS-2/MTX-R, unchanged; and WI-L2/m4, 110-fold increased. Measurement of MTX membrane transport showed decreased uptake in CEM/MTX-R and SAOS-2/MTX-R, relative to parental cell lines. The other DHFR-overproducing cells all gave normal initial MTX uptake rates but increased total uptake. The DHFR-overproducing lines all had significant cross-resistance to both metoprine and trimetrexate; the two lines with defective MTX transport were not cross-resistant, and the CEM/MTX-R cells showed collateral sensitivity to these agents. Only minor cross-resistance to homofolic acid was found in all MTX-resistant lines. The highly MTX-resistant RAJI/MTX-R and WI-L2/m4 cells showed minor cross-resistance to the dual inhibitor of thymidylate synthetase and DHFR, CB3717 (5- and 15-fold, respectively). These studies demonstrated that, depending upon the mechanism of resistance, MTX-resistant human tumor cells may be effectively killed by antifolates with different routes of uptake into cells, or with a different enzyme target. Thus, there are at least three functionally distinct classes of folate antagonist with antitumor activity.

In leukocyte population freshly isolated from the blood of 26 patients with acute leukemia, we have measured several parameters including glucocorticoid receptors, nucleoside incorporation, percentage of cells in S phase, and steroid-induced cell lysis. In addition, in some cases, the short-term response to steroid therapy was determined. Although, in all the patients studied, leukocytes were found to contain glucocorticoid receptors, we failed to demonstrate any correlation between the level of binding sites and the in vitro or in vivo response to glucocorticoids. This absence of correlation could be in part explained by the marked heterogeneity of the steroid response demonstrated in leukocyte subpopulations. It appears, however, that the degree of steroid action in vitro as well as the extent of spontaneous and dexamethasone-induced cell death may be related to the number of cells in the S phase of the cell cycle.

Glucocorticoid receptors were studied in leukemic cells from 14 newly diagnosed adults with acute lymphoblastic leukemia. Receptors were found in all cases; total receptor levels ranged from 1,348 to 18,697 sites per cel (median, 7,553). To determine the utility of glucocorticoid receptor levels for predicting response to glucocorticoid therapy, lymphoblasts were studied for receptors on nine occasions, and the patients were then treated with dexamethasone as a single agent. Response to glucocorticoid therapy appeared to correlate with lymphoblast receptor level. Five of six patients with greater than 4,500 total receptor sites per cell responded; all three patients with less than 4,500 receptors failed to respond. Glucocorticoid receptor level did not correlate with response to combination chemotherapy, duration of remission, or survival. Glucocorticoid receptors were studied at the initial presentation and following relapse in five patients. In two patients, the receptor level did not change following therapy; both patients at diagnosis had leukemias resistant to glucocorticoids. Three patients at relapse developed leukemias with lower receptor levels; these patients had leukemias sensitive to glucocorticoids at diagnosis. Our data suggest that the glucocorticoid receptor levels of lymphoblasts may be useful in predicting response to glucocorticoid therapy in adult acute lymphoblastic leukemia. They also suggest that, following relapse, cases initially sensitive to glucocorticoid may develop resistance by selection of cells with fewer receptors.

Between 1972 and 1974, Childrens Cancer Study Group enrolled 724 children with newly diagnosed acute lymphoblastic leukemia on a single randomized clinical trial. Study CCG-101 was designed to test four types of presymptomatic central nervous system and sanctuary therapies consisting of (a) 2400-rad craniospinal radiation therapy (RT) plus 1200-rad extended-field RT, which included the liver, spleen, kidneys, lower abdomen, and gonads; (b) 2400-rad craniospinal RT; (c) 2400-rad cranial RT plus intrathecal methotrexate (i.t. MTX); and (d) i.t. MTX alone. Patients all received a 28-day induction course of vincristine, prednisone, and L-asparaginase and were maintained subsequently on a regimen consisting of daily 6-mercaptopurine, weekly MTX, and monthly pulses of vincristine and prednisone. Patients treated with six doses of i.t. MTX alone had a significantly higher incidence of central nervous system relapse than did patients treated with 2400-rad craniospinal RT plus 1200-rad abdominal RT, 2400-rad craniospinal RT, or 2400-rad cranial RT plus i.t. MTX. There was no significant differences in marrow remission duration or survival of the treatment groups. There appears to be a benefit with regard to length of bone marrow remission and survival for patients with initial white blood counts greater than or equal to 20,000/cu mm treated with cranial RT plus i.t. MTX. The majority of the patients remaining on study have now discontinued maintenance therapy. The 8-year overall estimated survival rate on this study is 56%, and the disease-free survival rate is 52%.

Glucorticoid receptors were studied in various populations of normal human peripheral blood lymphocytes and leukemic lymphoblasts. Normal lymphocytes contain low levels of glucocorticoid receptor (approximately 2,500 sites/cell) which are identical in T- and non-T-fractions. Phytohemagglutinin treatment increases levels about 3-fold. Leukemic lymphoblasts contain larger numbers of receptor sites. Presence of receptor is correlated with in vitro sensitivitiy to glucocorticoids and in vivo response to therapy. Quantity of receptor is also correlated with complete remission duration independently of leukemic cell type (T or null), initial WBC, or age of patient. Quantitative determination of glucocorticoid receptor levels in acute lymphoblastic leukemia may be of value both as an independent prognostic variable and in suggesting which patients should receive glucocorticoid therapy.

A pharmacokinetic study of methotrexate levels in blood and cerebrospinal fluid was performed in 42 patients who received one or more courses of high-dose methotrexate at 500 mg/sq m infused i.v. over a 24-hr period. Methotrexate level in the lumbar cerebrospinal fluid reached 1.2 x 10(-7) M at 0.5 hr and remained constant at that level for the 1st 24 hr. Similar methotrexate levels were noted in the ventricular fluid obtained through an Ommaya device on three patients with brain tumors treated with high doses of methotrexate. Preliminary clinical results using high-dose methotrexate combined with simultaneous intrathecal methotrexate in 23 children with newly diagnosed acute lymphocytic leukemia indicate that this treatment program is safe to administer and to date appears effective in the prevention of central nervous system leukemia.

Cultured human T-lymphoblastoid cell lines are more sensitive than B-cell lines to 2'-deoxyadenosine in the presence of 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase. This difference is related to the greater efficiency with which T-lymphoblasts accumulate cytotoxic levels of dATP derived from the adenosine deaminase substrate 2'-deoxyadenosine (dAdo). Previous work has shown that differences in dATP accumulation by cultured T- and B-lymphoblastoid cell lines cannot be explained by large differences in the levels of dAdo-phosphorylating or dAdo nucleotide (dAXP)-degrading activities in cytoplasmic extracts of these cells, although it has been proposed that intact B-cell lines may catabolize intracellular dAXP more rapidly than do T-cell lines. To further examine the determinants of dAdo sensitivity in T- and B-lymphoblasts, we have studied dAdo and dAXP metabolism in the human T- and B-cell lines CEM and WI-L2 and in hybrids generated by fusion of these cell lines. The hybrid nature of the fusion products was established by nutritional studies and by analyses of cellular surface antigens, DNA content, and enzymatic activities. We found that WI-L2 X CEM hybrids and another T X B hybrid derived from fusion of the SB human B-cell line with CEM were 30- to 40-fold less sensitive to dAdo and about 10-fold less sensitive to the dAdo analogue 9-beta-D-arabinofuranosyladenine than was CEM, or about as resistant as were their B-cell parental lines. Our studies confirm that CEM avidly accumulates dAXP from dAdo but does not catabolize intracellular dAXP. In contrast, WI-L2, SB, and WI-L2 X CEM and SB X CEM hybrids rapidly degraded intracellular dAXP, which limited their ability to undergo dAXP pool expansion. Expression of dAXP catabolic activity in T X B hybrids behaved as a dominant mechanism, conferring resistance to dAdo- and dAdo-related nucleosides to T X B hybrids. It has been postulated that cell fusion may play a role in the progression of tumors and contribute to diversity among the cells that compose clonal tumors. We have speculated that fusion of a malignant T-lymphoblast with an activated B-cell might be a mechanism for the evolution of drug resistance in acute T-cell leukemia.

Fludarabine phosphate (NSC 312878), an adenosine deaminase resistant analogue of 9-beta-D-arabinofuranosyladenine, has entered clinical trials. Eleven patients with acute leukemia in relapse received 14 courses of fludarabine phosphate as a 5-day continuous infusion administered at doses of 40 to 100 mg/m2/day. Toxicity was characterized by uniform myelosuppression, as well as occasional nausea, vomiting, and hepatotoxicity. Three episodes of metabolic acidosis and lactic acidemia were noted. In addition, three patients suffered neurotoxicity. Two of these three patients had a severe neurotoxicity syndrome characterized by blindness, encephalopathy, and coma. Neither patient recovered neurological function. Neuropathological findings at autopsy were characterized by a diffuse, necrotizing leukoencephalopathy which was most severe in the occipital lobes. The medullary pyramids and posterior columns were also severely affected. This sporadic fatal neurotoxicity was observed only at doses greater than 40 mg/m2/day. The maximum tolerated dose for a 5-day infusion of fludarabine phosphate is thus 40 mg/m2/day.

Terminal deoxynucleotidyl transferase activity and cell surface markers were measured in peripheral lymphoid cells from 27 children with acute lymphoblastic leukemia in various phases of their disease. Lymphoblasts from untreated patients had smooth surface ultrastructure but heterogeneous surface receptors. Greater than 60% of lymphoblasts from 4 to 7 untreated patients formed rosettes with sheep red blood cells. Transferase activity was variable, ranging from 8 to 210 units/10(8) blasts, but it was consistently elevated at diagnosis and in relapse. Transferase levels did not correlate with the presence of lymphoblast surface receptors. During induction therapy transferase activity decreased rapidly, but it remained elevated in peripheral lymphoid cells even when blasts were not detectable in peripheral blood smears. Patients in remission had normal surface receptors and undetectable or minimally elevated levels of transferase. Terminal transferase activity may be a sensitive biochemical marker for a primitive cell population and may be important in the evaluation of therapeutic effectiveness in acute lymphoblastic leukemia.

We examined the ability of verapamil, a Ca2+ channel blocker, to overcome Vinca alkaloid and multiple drug resistance in our CEM/VLB100 and CEM/DOX human leukemic lymphoblasts. Compared with the parent CCRF-CEM cells, CEM/VLB100 cells are approximately equal to 200- to 800-fold resistant to vinblastine and express cross-resistance to vincristine, doxorubicin, and other "natural product" drugs, as determined by comparing 50% inhibitory concentrations in a 48-h growth inhibition assay. Verapamil (10 microM) decreased the 50% inhibitory concentrations for Vinca alkaloids in CEM/VLB100 cells by approximately equal to 75- to 85-fold but caused only slight (approximately equal to 2- to 5-fold) decreases in 50% inhibitory concentrations for anthracyclines, epipodophyllotoxins, and other tubulin-binding drugs (colchicine and podophyllotoxin). Qualitatively similar results were obtained with doxorubicin-resistant cells, termed CEM/DOX; verapamil caused a 19-fold increase in doxorubicin toxicity but 67- and 3500-fold increases in the toxicities of vinblastine and vincristine, respectively. These results indicate that the effect of verapamil is relatively greater for Vinca alkaloids, with less pronounced effects for the other natural product drugs against which these cells express multiple drug resistance. In flow cytometric studies, individually nontoxic or minimally toxic concentrations of vinblastine plus verapamil caused measurable accumulation in the G2 + M phase as early as 4 h after the drug combination was added to cultures of CEM/VLB100 cells; this finding correlated with a comparable increase in the number of cells in mitosis and measurable decreases in the total number of cells. Since similar effects on cell cycle distribution, percentage of cells in mitosis, and cell number were seen when CEM/VLB100 cells were treated with toxic concentrations of vinblastine alone, we conclude that the primary toxicity of the vinblastine-verapamil combination stems from the alkaloid. Further, the rapid lytic effect of the drug combination was associated with cellular vacuolization. The vacuoles did not stain for lipids, and increases in their number were evident within 2 to 4 h after drug treatment. We suggest that verapamil enhances Vinca alkaloid cytotoxicity by altering "cryptic" cytotoxic targets, possibly related to these vacuoles, through some as yet undefined membrane effect.