Arabinosylguanine-induced apoptosis of T-lymphoblastic cells: incorporation into DNA is a necessary step.

9-Beta-D-Arabinosylguanine (ara-G) is a recently introduced and effective treatment for T-cell acute lymphoblastic leukemia, but how ara-G and ara-G triphosphate (ara-GTP) kill cells is not known. We hypothesized that, in cycling T-lymphoblastoid cells, ara-G may act directly by incorporation into DNA, which may lead to apoptosis. Hence, blocking the incorporation of ara-G monophosphate (ara-GMP) into DNA may prevent apoptosis. To test this hypothesis, we performed experiments in a T-lymphoblastic leukemia cell line (CCRF-CEM) after synchronization with a double aphidicolin block. Intracellular accumulation of ara-GTP was neither cell cycle dependent nor affected by aphidicolin (53 +/- 5 microM/h without aphidicolin, 50 +/- 5 microM/h with aphidicolin). Cells at the G1-S boundary accumulated 75 +/- 7 microM ara-GTP with minimal incorporation into DNA (5 +/- 2 pmol ara-GMP/mg DNA) and had little biochemical or morphological evidence of apoptosis. In marked contrast, cells in S phase had significantly more ara-G incorporated into DNA (24 +/- 4 pmol ara-GMP/mg DNA), although the cytosolic concentration of ara-GTP (85 +/- 7 microM) was similar to that in the G1-enriched population. In the S-phase cells, there was a corresponding increase in apoptosis (measured as high molecular weight DNA fragmentation and morphological changes), and the incorporation of ara-GTP into DNA resulted in a >95% inhibition of DNA synthesis. There was a direct linear relationship between the number of cells in S phase and both the total number of ara-GMP molecules in DNA and the inhibition of DNA synthesis. Blocking of ara-GTP incorporation into S-phase DNA abolished biochemical and morphological features of apoptosis, even in the presence of cytotoxic level of intracellular ara-GTP. Taken together, these data demonstrate that the incorporation of ara-GTP into DNA is the critical event that mediates the induction of apoptosis in CCRF-CEM cells.     

Metabolic basis of arabinonucleoside selectivity for human leukemic T- and B-lymphoblasts

Purine analogues are potentially useful agents for selective chemotherapy of lymphoproliferative diseases. We compared the toxic effects of various arabinonucleosides against eight human T- and B-lymphoblastoid lines. The arabinosides of cytosine (ara-C), 2-fluoroadenine (F-ara-A), adenine (ara-A) and guanine (ara-G) all inhibited the growth of T-lymphoblasts at concentrations below 2 microM. Only ara-G showed strong selectivity for T-cells, as indicated by a 15- to 250-fold greater toxicity toward T-cell lines than B-cell lines. To investigate the biochemical basis for ara-G selectivity, we compared the metabolism of the arabinonucleosides in CCRF-CEM (T-) versus PF-2S (B-) lymphoblasts. Comparison of arabinonucleoside triphosphate accumulation indicated differences favoring selective ara-GTP formation in T-cells. In contrast, ara-C, ara-A, and F-ara-A formed almost corresponding amounts of their triphosphates in both cell types. Triphosphate accumulation correlated directly with inhibition of DNA synthesis in CCRF-CEM and PF-2S cells. PF-2S cells accumulated less than 20% ara-GTP from the nucleoside than did CCRF-CEM cells. Nucleoside kinase measurements showed no significant differences in arabinonucleoside phosphorylation that could account for the preferential ara-GTP accumulation in T-cells. After removal of arabinonucleoside-containing medium, ara-GTP levels in PF-2S cells declined with a half-life of 49 min whereas, in CCRF-CEM cells, the level of analogue triphosphate remained unchanged. Furthermore, the half-life of ara-CTP, ara-ATP, and F-ara-ATP in the B-cells was 3- to 5-fold longer than that of ara-GTP. These results indicate that ara-G is more selective than other known arabinonucleosides; such selectivity warrants further assessment of the therapeutic potential of this agent against T-cell malignancies and other lymphoid disorders.     

Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5'-nucleotidase mRNA measurement in cell lines and in patients with leukemia.

The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.     

Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. ³H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas ³H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine–guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.     

Modulation of arabinosylnucleoside metabolism by arabinosylnucleotides in human leukemia cells

Previous studies have indicated that deoxycytidine kinase (dCK) is requisite and rate limiting in the phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on the pathway to their respective cytotoxic 5'-triphosphates. In K562 cells, the rate of triphosphate accumulation was maximal during incubation with 10 microM ara-C (35 microM/h) and 300 microM F-ara-A (102 microM/h). Under these conditions, accumulation of cellular ara-CTP plateaued at about 110 microM after 3 h, whereas in separate cultures, F-ara-ATP continued to accumulate at a linear rate to cellular concentrations greater than 500 microM after 5 h. Other laboratories have demonstrated that dCK activity in cell-free extracts was inhibited by ara-CTP. To determine whether ara-CTP exhibited the same activity in whole cells, K562 cells were preincubated with ara-C to accumulate 110 microM ara-CTP. After washing into medium containing F-ara-A, the rate of F-ara-ATP accumulation was significantly decreased (37 microM/h). However, cells loaded with F-ara-ATP exhibited an increased rate of ara-CTP accumulation (110 microM/h) that resulted in cellular ara-CTP concentrations in excess of 400 microM after 5 h. This stimulation was proportional to the cellular concentration of F-ara-ATP, achieving a maximum effect between 75 and 100 microM. Phosphorylation of ara-C by cell-free extracts supplemented with physiological levels of ribo- and deoxyribonucleoside 5'-triphosphates was stimulated by addition of F-ara-ATP. The decreased rate of accumulation of products of dCK in intact cells containing 110 microM ara-CTP suggests that this active triphosphate may limit its own synthesis and phosphorylation of other substrates. In contrast, stimulation of the accumulation of ara-CTP in cells containing F-ara-ATP suggests new possibilities for the design of combination chemotherapy regimens.     

Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

Background

We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug) and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies.

Methods

Three immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of < 0.05 considered significant.

Results

The multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P < 0.05) and lowered dCK protein (24% to 56%, P < 0.05) without affecting CDA protein. However, paclitaxel increased both dCK (10% to 50%) and CDA (75% to 153%) activity (P < 0.05). Paclitaxel caused substantial declines in the accumulation of the deaminated and phosphorylated metabolites in H520 cells (P < 0.05); the metabolites were not measurable in the remaining two cell lines. The ratio of dCK to CDA mRNA levels corresponded to the combination index (CI) estimated for sequential paclitaxel-gemcitabine.

Conclusion

In summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the selection of an appropriate anticancer drug regimen.     

Evaluation of the combination of nelarabine and fludarabine in leukemias: clinical response, pharmacokinetics, and pharmacodynamics in leukemia cells.

PURPOSE: A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate. PATIENTS AND METHODS: Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days. RESULTS: Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27). CONCLUSION: Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.     

Hexadecylphosphocholine inhibits inositol phosphate formation and protein kinase C activity.

Hexadecylphosphocholine (HePC) inhibits protein kinase C (PKC) from NIH3T3 cells in cell-free extracts with a 50% inhibitory concentration of about 7 microM. Inhibition is competitive with regard to phosphatidylserine with a Ki of 0.59 microM. In order to determine whether HePC affects PKC in intact cells, the bombesin or tetradecanoylphorbolacetate-induced, PKC-mediated activation of the Na+/H(+)-antiporter was determined. It is demonstrated that HePC causes a drastic inhibition of this enzyme indicating a similar sensitivity of PKC to HePC in intact cells compared to cell-free extracts. In addition to the effects on PKC, treatment of NIH3T3 cells with HePC depresses the bombesin-induced formation of inositol 1,4,5-trisphosphate and the concomitant mobilization of intracellular Ca2+. Dose-response curves for the inhibition of inositol 1,4,5-trisphosphate formation and Ca2+ mobilization reveal 50% inhibitory concentrations of 2 or 5 microM, respectively. Polyphosphorylated phosphoinositides accumulate in HePC-treated cells indicating that the depression of inositol 1,4,5-trisphosphate generation is not caused by an inhibition of phosphoinositide kinases. Addition of bombesin to HePC-treated cells in the presence of LiCl revealed no evidence for an accelerated rate of inositol 1,4,5-trisphosphate turnover by the phospholipid analogue. It is concluded that HePC inhibits phosphoinositidase C in intact cells. The data strongly suggest that the growth-inhibitory effect of HePC is at least in part explained by the interference with mitogenic signal transduction.     

Transport and metabolism of 9-beta-D-arabinofuranosylguanine in a human T-lymphoblastoid cell line: nitrobenzylthioinosine-sensitive and -insensitive influx

Nitrobenzylthioinosine (NBMPR), dipyridamole, and dilazep, potent inhibitors of nucleoside transport, were found to be ineffective in preventing 9-beta-D-arabinofuranosylguanine (ara-G)-induced inhibition of MOLT 4 and CCRF CEM cell growth, ara-G (2.0 microM) was metabolized to 9-beta-D-arabinofuranosylguanine 5'-triphosphate in MOLT 4 cells, and the levels of this metabolite were not affected by the presence of 5.0 microM NBMPR in the incubation medium. Permeation of the MOLT 4 cell membrane by ara-G occurred primarily by means of the NBMPR-sensitive nucleoside transport system. However, a residual transport component accounting for 10-20% of the total transport activity was demonstrated in the presence of NBMPR. This component was inhibited by adenine and hypoxanthine but not by dilazep, dipyridamole, or other nucleosides. In contrast, inhibitors of nucleoside transport readily reversed the cytotoxic effect of 7-deazaadenosine (tubercidin) in both MOLT 4 and CCRF CEM cells. The levels of tubercidin 5'-triphosphate formed from 2.0 microM tubercidin in MOLT 4 cells were reduced by 80% in the presence of 5.0 microM NBMPR. The influx of tubercidin into MOLT 4 cells was found to occur primarily by means of the NBMPR-sensitive nucleoside transport system. This same system mediated the transport of ara-G into human erythrocytes.     

Immunohistochemical and genetic evaluation of deoxycytidine kinase in pancreatic cancer: relationship to molecular mechanisms of gemcitabine resistance and survival.

Gemcitabine is considered the standard first-line therapy for patients with advanced pancreatic cancer. More recent strategies have focused on improving the efficacy of gemcitabine by either improving the method of delivery or by combining gemcitabine with other non-cross-resistant chemotherapy agents or with small-molecule drugs. However, the clinical benefits, response rates, and duration of responses have been modest. Deoxycytidine kinase (dCK) is the rate-limiting enzyme involved in the metabolism of gemcitabine. The expression of dCK has been postulated to be correlative of gemcitabine resistance. We determined the relationship of dCK immunohistochemical protein expression and/or genetic status of dCK in a panel of human pancreatic cancer tissues and pancreatic cancer cell lines and determined the relationship of these variables to the clinical outcome of patients treated with gemcitabine. We report that dCK protein expression is expressed in the majority of pancreatic cancers analyzed (40 of 44 cases, 91%) and showed a range of labeling intensities ranging from 1+ (labeling weaker in intensity than normal lymphocytes present in same section) to 3+ (labeling greater in intensity than normal lymphocytes present in same section). When labeling intensity was compared with survival, low dCK expression (1+ labeling) was correlated with both overall survival (P < 0.009) and progression-free survival following gemcitabine treatment (P < 0.04). Low dCK labeling intensity was also significantly correlated with patient age (70.3 +/- 8.1 versus 59.8 +/- 7.4 years; P < 0.0006), suggesting that age-related methylation of the dCK gene may account in part for the observed differences. Sequencing of the entire dCK coding sequence in 17 cell lines and 9 patients' cancer tissues with disease progression while on gemcitabine did not identify any mutations, suggesting that genetic alterations of dCK are not a common mechanism of resistance to gemcitabine for this tumor type. Moreover, dCK labeling showed similar patterns and intensities of labeling among matched pretreatment and post-treatment tissues. In summary, pretreatment levels of dCK protein are most correlated with overall survival following gemcitabine treatment and are stable even after resistance to gemcitabine is clinically documented.     

Treatment of normal and malignant cells with nucleoside analogues and etoposide enhances deoxycytidine kinase activity

Deoxycytidine kinase (dCK), one of the rate-limiting enzymes in the intracellular metabolism of many antileukaemic drugs, was shown to be stimulated after treatment of human tonsillar lymphocytes by 2-chloro-2'-deoxyadenosine (cladribine, CdA) (Sasvári-Székely, et al., Biochem Pharmacol 1998, 56, 1175-1179). Here we present a comparative study of different normal and malignant cells in respect to the activation of dCK by CdA. G-phase lymphocytes showed a higher sensitivity for dCK stimulation than S-phase cells. Normal and leukaemic peripheral blood mononuclear cells, as well as the promyelocytic cell line HL60 responded to CdA treatment by a 2-5-fold increase in activity of dCK. However, no significant stimulation was detected either in CCRF-CEM T-lymphoblastoid cells, or in K562 myeloid cells. Thymidine kinase (TK) activity was not stimulated in any cases. Treatment of these cells with several other analogues beside CdA, such as 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), 2-fluoro-1-beta-D-arabinosyladenine (Fludarabine, FaraA) and 1-beta-D-arabinosylcytosine (cytarabine, araC) gave similar results to CdA treatment. Enhancement of dCK activity could also be achieved with the topoisomerase II inhibitor, etoposide. In contrast, 2-chloro-riboadenosine (CrA) had no effect on the dCK at concentrations of 10 microM or less, while dCyd and 5-aza-dCyd caused slight inhibition. These results indicate that treatment of cells with several inhibitors of DNA synthesis potentiates the dCK activity. The drugs widely differ in their stimulatory effect on dCK, and there are also 'responsive' and 'non-responsive' cells with respect to dCK activation. Thus, enhancement of the dCK activity by specific drugs in 'responsive' cells might give a rationale for combination chemotherapy.     

Growth inhibition and modulation of kinase pathways of small cell lung cancer cell lines by the novel tyrosine kinase inhibitor STI 571

Small cell lung cancer (SCLC) is an aggressive cancer characterized by several autocrine growth mechanisms including stem cell factor and its receptor c-Kit. In order to arrive at potentially new and novel therapy for SCLC, we have investigated the effects of the tyrosine kinase inhibitor, STI 571, on SCLC cell lines. It has been previously reported that STI 571 does not only inhibit cellular Abl tyrosine kinase activity but also the PDGF receptor and c-Kit tyrosine kinases at similar concentrations (approximately 0.1 microM). There is no expression of the PDGF-receptor, and the Abl kinase is not activated by SCLC, but over 70% of SCLC contain the c-Kit receptor. Utilizing this preliminary data, we have determined that three (NCI-H69, NCI-H146 and NCI-H209) of five (including NCI-H82 and NCI-H249) SCLC cell lines had detectable c-Kit receptors and were inhibited in growth and viability at concentrations 1 - 5 microM of STI 571 after 48 h of treatment. The SCLC cell lines, NCI-H69, NCI-H146 and NCI-H209, showed a dose-response (tested between 0.1 - 10 microM) inhibition of tyrosine phosphorylation of c-Kit as well as in vitro kinase activity (at 5 microM) of c-Kit in response to STI 571. STI 571 inhibited cell motility, as assessed by time-lapsed video microscopy, within 6 h of STI 571 treatment (5 microM). STI 571 also decreased intracellular levels of reactive oxygen species (ROS) by at least 60%, at a concentration (5 microM) that also inhibited cell growth. Cell cycle analysis of STI 571 responsive cells showed that cells were generally slowed in G2/M phase, but there was no arrest at G1/S. A downstream phosphorylation target of c-Kit, Akt, was not phosphorylated in response to stem cell factor in the presence of STI 571. These data imply that STI 571 inhibits growth of SCLC cells through a mechanism that involves inactivation of the tyrosine kinase c-Kit. The effectiveness of STI 571 in this study suggests this drug may be useful in a clinical trial, for patients with SCLC. Oncogene (2000) 19, 3521 - 3528     

Sensitization of ara-C-resistant lymphoma cells by a pronucleotide analogue

Adequate intracellular concentrations of ara-CMP, the monophosphorylated derivative of ara-C, are essential for its cytotoxicity. The critical step for ara-CMP formation is intracellular phosphorylation of ara-C by deoxycytidine kinase (dCK). A common nucleoside resistance mechanism is mutation affecting the expression or the specificity of dCK. We describe the ability of a tert-butyl S-acyl-thioethyl (SATE) derivative of ara-CMP (UA911) to circumvent ara-C resistance in a dCK-deficient human follicular lymphoma cell line (RL-G). The RL-G cell line was produced by continuous exposure to gemcitabine and displayed low dCK mRNA and protein expression that conferred resistance both to ara-C (2,250-fold) and to gemcitabine (2,092-fold). RL-G cells were able to take up the UA911 pronucleotide by diffusion and metabolize it to the corresponding ara-CMP and ara-CTP nucleotides, exhibiting a 199-fold reduction in resistance ratios, and a similar cell cycle arrest to the parental RL-7 cells. Exposures to 10, 50 or 100 microM concentrations of UA911 produced 160 +/- 7, 269 +/- 8 and 318 +/- 62 pmol ara-CTP/mg protein in RL-7 cells, and 100 +/- 12, 168 +/- 10 and 217 +/- 39 pmol ara-CTP/mg protein in RL-G cells, respectively. Exposure of RL-G cells to underivatized, radiolabeled ara-C produced no detectable amounts of the active triphosphate metabolites. We conclude that the UA911 pronucleotide is capable of overcoming dCK-mediated resistance. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the intracellular delivery of ara-CMP to dCK-deficient cells.     

Inhibition of cell proliferation, protein kinase C, and phorbol ester-induced fos expression by the dihydropyridine derivative B859-35.

The dihydropyridine derivative B859-35 inhibits phospholipid- and calcium-dependent protein kinase C (PKC) in cell-free extracts from NIH3T3 cells. Inhibition is competitive with regard to phosphatidylserine. At 1 microM phosphatidylserine, half-maximal inhibition (IC50) is obtained at approximately 2.5 microM B859-35. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-dependent activation of the Na+/H+ antiporter was used to determine whether the enzyme is also affected in intact cells. The activity of the antiporter was monitored by following the dimethylamiloride-sensitive cytosolic alkalinization. It is demonstrated that B859-35 depresses the TPA-induced alkalinization with an IC50 of 5 microM, indicating that PKC in intact cells and the enzyme in cell-free extracts are equally sensitive to the drug. TPA-induced expression of the c-fos gene was used as an additional marker for intracellular PKC activity. Activation of c-fos expression was determined by measuring chloramphenicol acetyltransferase (CAT) activity in cells transfected with a c-fosCAT construct in which the CAT gene is expressed under the control of the endogenous human c-fos promoter. The studies revealed that 2.5 microM B859-35, a concentration equivalent to the IC50 in cell-free extracts, significantly depresses TPA-induced c-fosCAT expression. B859-35 inhibited cellular proliferation of NIH3T3 cells with an IC50 of approximately 5 microM. This is close to the IC50 for the anti-PKC activity of B859-35. It is suggested that the inhibition of PKC contributes to the growth inhibition following exposure to B859-35.     

Novel leukemic cell lines resistant to clofarabine by mechanisms of decreased active metabolite and increased antiapoptosis

Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA‐resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20‐fold and 80‐fold more CAFdA‐resistant than HL‐60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL‐60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL‐60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4‐h incubation with 10 μM CAFdA was 20 pmol/10⁷cells in HL/CAFdA20 and 3 pmol/10⁷cells in HL/CAFdA80 compared with 63 pmol/10⁷cells in HL‐60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA‐induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL‐60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.     

The tyrosine kinase inhibitors imatinib and AG957 reverse multidrug resistance in a chronic myelogenous leukemia cell line

The K562 cell line derived from a chronic myelogenous leukemia (CML) patient exhibits ATP-dependent exclusion of the multidrug resistance (MDR)-type drugs. The protein tyrosine kinases inhibitors, imatinib mesylate and AG957 allowed for increased doxorubicin and calcein-AM accumulation in these cells. Maximal modulation was achieved at 3 and 10 microM imatinib and AG957, respectively. This imatinib concentration is comparable to the plasma steady state levels observed in patients. Although the increase in cellular accumulation followed a time course similar to apoptotic manifestations induced by these drugs, the two phenomena seem independent. There was no correlation between the levels of MDR reversal and apoptosis in clones derived from the K562 cell line. Moreover, whereas protein kinase inhibitors induced apoptosis in only a fraction of the cells, the MDR reversal occurred in all of them. Inhibition of apoptosis by a non-specific inhibitor of caspases was not associated with MDR reversal. The consequence of these findings is that combination of tyrosine kinase inhibitors with antileukemic drugs is likely to have the added beneficial effect of allowing MDR-type drugs better access to cells.     

SRC kinase and mitogen-activated protein kinases in the progression from normal to malignant endometrium.

PURPOSE: The purpose of this research was to determine whether a correlation exists between the levels of activated mitogen-activated protein kinase (MAPK) and Src kinases and the progression from normal to malignant endometrium. EXPERIMENTAL DESIGN: We measured total and phosphorylated levels for extracellular signal-regulated kinase 1/2, p38, stress-activated protein kinase/c-Jun NH(2)-terminal kinase, and Src kinases from 33 frozen endometrial adenocarcinomas and 38 benign endometrial specimens by quantitation of signals from Western blots using antibodies against these kinases. RESULTS: Elevated phospho-extracellular signal-regulated kinase 1/2 (150 +/- 40 versus 46 +/- 7; P = 0.03), phospho-Src (28 +/- 5 versus 4 +/- 1), and phospho-p38 (131 +/- 16 versus 27 +/- 7; P < 0.001) was detected in benign versus malignant endometrium when the Western blot signal of activated kinase was normalized to total kinase levels and beta actin. A modest increase in active c-Jun NH(2)-terminal kinase was detected in carcinoma versus benign specimens (51 +/- 13 versus 43 +/- 10; P = 0.8). Expression of total kinases (normalized to beta-actin) was higher in carcinoma versus benign specimens, respectively (extracellular signal-regulated kinase 1/2, 9 +/- 2 versus 0.7 +/- 0.1; Src, 7 +/- 2 versus 0.4 +/- 0.1; stress-activated protein kinase c-Jun NH(2)-terminal kinase, 2 +/- 0.4 versus 0.2 +/- 0.02; P < 0.001; and p38, 1 +/- 0.2 versus 0.4 +/- 0.1; P < 0.01). Immunohistochemistry for active and total Src kinases and MAPKs detected positive staining in epithelial and stroma cells. CONCLUSIONS: These data demonstrated that, in contrast with breast cancer, the progression from normal to malignant endometrium is not associated with activation of MAPK and Src kinases. Elevation of these active kinases in benign endometrium may contribute to endometrial resistance to the antiestrogen action of tamoxifen.