Toshiki Ohkubo

Sequence-dependent antitumor effect of VP-16 and 1-β-d-arabinofuranosylcytosine in L1210 ascites tumor

The sequence-dependence of the antitumor effect of etoposide (VP-16) and 1-beta-D-arabinofuranosylcytosine (ara-C) was investigated against the L1210 ascites tumor in BDF1 mice. Treatment with VP16 (7.5 or 15 mg/kg) and ara-C (25 or 500 mg/kg) was administered intraperitoneally on days 1, 4 and 7 after tumor inoculation. Six hour pretreatment with 15 mg/kg VP16 followed by 500 mg/kg ara-C yielded a 100% cure rate, but only a 20% cure rate was obtained with the reverse sequence. Simultaneous administration of 15 mg/kg of VP-16 and 500 mg/kg ara-C interacted synergistically, producing a 70% cure rate. In contrast with the results obtained with VP-16 and 500 mg/kg ara-C, simultaneous administration of 25 mg/kg ara-C neither antagonized nor potentiated the antitumor effect of VP-16. Twenty-five mg/kg ara-C was too low to produce any antitumor effect with VP-16 in simultaneous administration. At every dose investigated, pretreatment with VP-16 followed by ara-C was the most effective antitumor schedule in L1210 leukemia. This sequence of drug administration did not cause greater toxicity as measured by weight loss or toxic death.

Decreased DNA polymerase sensitivity to 1-β-d-arabinofuranosylcytosine 5′-Triphosphate in P388 murine leukemic cells resistant to vincristine☆

P388 murine leukemic cells 28.6-fold resistant to vincristine were cross-resistant to doxorubicin and etoposide. Although the intracellular ara-CTP in P388 murine leukemic cells resistant to vincristine (P388/VCR) was significantly higher than that in the parent cells, the cytotoxic effect of ara-C was not significantly different between the parent and resistant cells. Therefore, we investigated the DNA synthesis in P388/VCR and its parent cell line using the permeable cell system. The DNA synthesis in P388/VCR cells was less sensitive to ara-CTP and dTTP than that in P388 parent cells. These results suggested that the characteristics of DNA polymerase in P388/VCR cells might change when the cells developed multiple drug resistance.

ENHANCED INCORPORATION OF 1-BETA -D-ARABINOFURANOSYLCYTOSINE BY PRETREATMENT WITH ETOPOSIDE

We examined the effects of timing of administration of etoposide on cytosine arabinoside (ara-C) incorporation into DNA in L1210 ascites tumor. At 1 hr after injection of ara-C, 3-hr and 6-hr pretreatments with 15 mg/kg of etoposide increased ara-C incorporation to more than 200% as compared to that of ara-C given alone. Simultaneous administration of etoposide, however, decreased ara-C incorporation to 33% of that of ara-C alone. These results might explain the previously reported sequence dependency of the antitumor effect of etoposide and ara-C.

Intracellular dCTP/ARA-CTP Ratio and the Cytotoxic Effect of ARA-C

In this study, the relationship between the dCTP/ara-CTP ratio and the cytotoxic effect of cytosine arabinoside (ara-C) was investigated. Intracellular levels of dNTPs and ara-CTP were analyzed by high-performance liquid chromatography. Although doubling time and intracellular dCTP levels were different in each of the cells, there was a consistent relation between the intracellular dCTP/ara-CTP ratio and the cytotoxic concentration of ara-C. These data suggest that the intracellular dCTP/ara-CTP ratio is one of the important factors for considering the cytotoxic action of ara-C.

Heterogeneous Effects of G-CSF and GM-CSF on Cell Growth and Ara-C Cytotoxicity in Childhood Leukemias Which Express Myeloid Markers

It is uncertain if acute lymphoblastic leukemia (ALL) cells expressing myeloid makers can respond to granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). We investigated the effects of G-CSF (0.01 microgram/ml) and GM-CSF (0.01 microgram/ml) on [3H]thymidine (TdR) uptake, and the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in leukemia cells from 17 pediatric patients. ALL cells without myeloid markers did not respond to G-CSF or GM-CSF. On the other hand, these cytokines enhanced the [3H]TdR uptake and cell growth, not only of AML cells but also of ALL cells expressing myeloid antigens. However, G-CSF and GM-CSF did not always enhance the growth inhibitory effect of the cell cycle specific drug ara-C when the cells were co-cultured with the drug. There was no relationship between cell growth and the amount of [3H]TdR incorporation or the intracellular ara-CTP level. These results indicate the heterogeneous effects of G-CSF and GM-CSF on cell growth and ara-C sensitivity in childhood leukemia cells.

Effects of UFT (Mixed Compound of Tegafur and Uracil) on Cell Kinetics and Inhibition of Thymidylate Synthase in L1210 Ascites Tumor

Previous work in our laboratory showed that UFT (mixed compound of tegafur and uracil, molar ratio 1:4, respectively) caused the prolonged reduction of dTTP in L1210 leukemia cells in comparison with 5-fluorouracil (5-FU). The purpose of this study was to assess the effect of UFT on cell cycle distribution and thymidylate synthase activity of a leukemia cell line as compared with 5-FU. UFT and 5-FU were orally given to BDF1 mice bearing L1210 ascites tumor on day 3 after the tumor inoculation. Cell cycle distribution patterns at 24 hr after the drug administration showed a higher percentage of S phase in tumor cells treated with UFT than in those treated with 5-FU. Until 6 hr after the oral administration of the drugs, UFT inhibited the incorporation of [3H] deoxyuridine into DNA more long than 5-FU did. These results indicated that UFT has longer and stronger inhibitory effects on DNA replication than 5-FU in vivo under the employed experimental conditions (i.e., low and single doses of these fluorinated pyrimidines).

Effects of 5-fluorouracil and a combination of tegafur and uracil (UFT) on nucleotide metabolism in L1210 ascites tumor.

Changes in the deoxyribonucleotide pools following a single oral administration of 13 mg/kg of 5-fluorouracil (5-FU) or of 64.8 mg/kg of UFT (a mixed compound of tegafur and uracil) were investigated. We compared their pharmacodynamics and effects on nucleotide metabolism in L1210 ascites tumor on day 3 after intraperitoneal tumor inoculation. The intracellular dTTP pool decreased to half the control level 1-6 hr after the administration of 5-FU. The dTTP pools rapidly recovered after 6 hr. In contrast, UFT kept the intracellular dTTP level to 1/3 to 1/2 of the control level for 24 hr. Either drug elevated the intracellular dATP pools, but decreased dCTP pools. UFT influenced the intracellular dATP and dCTP levels longer than 5-FU. Orally administered UFT seemed to exert a longer and more potent inhibitory effect on thymidylate synthetase than equimolar 5-FU. In view of these results, we suggest that UFT could be a more potent chemotherapeutic drug than 5-FU in oral administration.

Membrane transport of 1-β-d-arabinofuranosylcytosine and accumulation of 1-β-d-arabinofuranosylcytosine 5′-triphosphate in P388 murine leukemic cells resistant to vincristine☆

The membrane transport of ara-C and intracellular ara-CTP accumulation were investigated in P388 murine leukemic cells resistant to vincristine (P388/VCR) and its parent cell line. The transport of ara-C in P388/VCR cell line was a 1.4-fold increase at 30 sec compared to that in P388 parent cell line (P less than 0.01). The increase of the transport of ara-C in P388/VCR cell line, however, was not completely abolished by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBTI) to the level in parent cell line. Scatchard analyses revealed that the resistant cells had significantly less NBTI binding sites than the parent cells had. These results suggested that the changes in ara-C transport in P388/VCR cells were due, in part, to increase of NBTI-insensitive transport sites in the membrane. The measurement of the intracellular ara-CTP concentration by high-performance liquid chromatography revealed that the intracellular ara-CTP level in P388/VCR cells was also significantly higher than that in parent cells (1.4-fold, P less than 0.01). As the transport of ara-C is rate limiting at a concentration of 1 microM in the both cell lines, we concluded that the accumulation of ara-CTP in P388/VCR cells might have partially resulted from the enhancement of the ara-C transport.

Incorporation ofN 4-behenoyl-1-β-d-arabinofuranosylcytosine into DNA as 1-β-d-arabinofuranosylcytosine

BHAC is a newly synthesized lipophilic derivative of ara-C. To clarify its pharmacological mode of action, P388 murine leukemic cells were incubated with two different types of14C-labeled BHAC, [cytosine-2-14C]BHAC and [acyl-1-14C]BHAC, and DNA was extracted with phenol. The phenol-extracted DNA was then hydrolyzed by nuclease P1 and analyzed with high-performance liquid chromatography (HPLC). The radioactivity of DNA, from the cells incubated with [cytosine-2-14C]BHAC, was detected as ara-CMP. But the radioactivity of DNA, from the cells incubated with [acyl-1-14C]BHAC, was hardly detected. On the other hand, the main radioactivity of the acid soluble fraction was determined as ara-CTP. On the basis of our results, BHAC is not phosphorylated directly to produce N4-behenoly-ara-CTP, but is mainly converted to ara-C which subsequently produces ara-CTP, the active metabolite of the drug, and which is then incorporated into DNA.

Deoxyribonucleoside triphosphate pools and ara-ctp levels in P388 murine leukemic cells treated with 1-B-D-Arabinofuranosylcytosine-5′ -stearylphosphate which is a newly synthesized derivative OF 1-B-D-arabinofuranosylcytosine

Abstract1-B-D-arabinofuranosylcytosine-573′-stearylphosphate (C18PCA), which is an Ara-CMP ester and one of the most promising orally effective anti-leukemic drugs, is a newly synthesized derivative of Ara-C. The antitumor effect of C18PCA and Ara-C was investigated against the P388 ascites tumor in BDF1 mice. Treatment with C18PCA (100 mg kg-1, orally) and Ara-C (40 mg kg-1, subcutaneously) was administered on days 1, 3 and 5 after tumor inoculation. The percentage increase in lifespans of the mice treated with C18PCA or Ara-C were 84.4% and 53.9%, respectively. The determination of the plasma Ara-C concentration revealed that the plasma concentration of Ara-C was retained much longer in mice which orally received C18PCA than in those which received Ara-C. By using high-performance liquid chromatography, it was revealed that the deoxyribonucleo-side triphosphate pools increased gradually but Ara-CTP concentration once increased, then decreased rapidly when Ara-C was administered subcutaneously. On the other hand, both the intracellular deoxyribonucleoside triphosphate (dNTP) pools and Ara-CTP level increased gradually after oral administration of C18PCA. We concluded that these longer-term biochemical effects, even if the plasma concentration of Ara-C and Ara-CTP level were low, might be correlated with antitumor effects of C18PCA.