Delayed cytotoxicity of 6-mercaptopurine is compatible with mitotic death caused by DNA damage due to incorporation of 6-thioguanine into DNA as 6-thioguanine nucleotide.

Many protocol studies have shown that low dose 6-mercaptopurine (6MP) in maintenance chemotherapy for childhood acute lymphoblastic leukemia (ALL) can be utilized to cure the disease. Mitotic or reproductive cell death has been recognized after G2 arrest when cells are treated with antitumor agents. The precise mechanism of mode of action of 6MP still remains unclear. We found delayed cytotoxic effect of 6MP in P388 murine leukemic cells. Morphological study showed that 6MP induced delayed death was characterized by an enlargement of cell size and multinucleated nuclei. Agarose gel electrophoresis of fragmented DNA from cells treated with 6MP showed the typical ladder pattern. These findings were compatible with mitotic death. Our results make us hypothesize that the delayed cytotoxicity of 6MP is one of the drug induced mitotic deaths caused by DNA damage due to incorporation of 6-thioguanine (6TG) into DNA as thioguanine nucleotide (TGN). Mitotic death may be a mechanism for killing the cycling cells from residual leukemic cells in G0 or long G1 phases in the treatment of childhood ALL.

Enhanced cytotoxic interaction between 5-fluorouracil and 4-hydroperoxycyclophosphamide against L1210 murine leukemic cells: applicability to ex vivo purging.

Eradication of contaminated tumor cells in bone marrow is a matter of utmost concern in the setting of autologous bone marrow transplantation. 4-Hydroperoxycyclophosphamide (4-HC) is often used for ex vivo chemical purging of contaminated tumor cells in bone marrow. The marrow from patients pretreated with 5-fluorouracil (5-FU) is enriched with multifactor-responsive high proliferative potential colony-forming cells. To develop an efficient ex vivo chemical purging system, we evaluated interaction between 4-HC and 5-FU. We investigated the antitumor effect of cyclophosphamide, a mother compound of 4-HC, and 5-FU against L1210 ascites tumor in B6D2F1 mice. The median lifespan of the mice treated with 4-HC or 5-FU alone was 8 and 12 days, respectively. The combination of both drugs significantly extended the median lifespan to 18.5 days. The median effect plot analysis indicated a synergistic cytotoxic interaction between 5-FU and 4-HC in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl terazolium bromide (MTT) assay. Clonogenic assay also showed that combination of 4-HC and 5-FU significantly reduced L1210 leukemic colonies to 20% of untreated control. Bone marrow cells from the mice treated with 5-FU at 150 mg/kg body weight was resistant to 4-HC at concentrations as high as 0.2 microgram/ml, which was more than 70% inhibitory concentration for colony formation in L1210 leukemic cells. Findings suggest that sequential treatment with in vivo 5-FU followed by ex vivo 4-HC could selectively enhance antitumor effects of 4-HC in tumor cells remaining in bone marrow.

Aclarubicin inhibits etoposide induced apoptosis through inhibition of RNA synthesis in P388 murine leukemic cells.

It has been reported that aclarubicin inhibits etoposide (VP-16) induced cytotoxicity in human lung cancer cell lines (1, 2). However, it still remains unclear how aclarubicin (ACR) inhibits etoposide-induced cytotoxicity. We report here that the combination of ACR and VP-16 showed antagonistic cytotoxic effect in P388 murine leukemic cells. DNA unwinding assay showed that 1000 ng/ml ACR significantly reduced VP-16 induced early DNA double strand(ds) breaks compared to that of VP-16 alone at a concentration of 10 microM. However, ACR did not inhibit VP-16 induced early DNA double strand breaks at a concentration of 100 ng/ml, a clinically achievable concentration. Furthermore, DNA repair occurred within two hours after removing VP-16 even if ACR was co-cultured at concentrations of 100 and 1000 ng/ml. DNA agarose gel electrophoresis and detection of sub-G1 fraction by flowcytometer showed that 100 ng/ml of ACR inhibited VP-16 induced DNA ladder formation and formation of sub-G1 fraction. Radioactive precursor incorporation studies showed that VP-16 inhibited DNA synthesis rather than RNA synthesis. On the other hand, ACR selectively inhibited RNA synthesis at a concentration of 100 ng/ml. The VP-16 induced increment of [3H]-L-leucine uptake was canceled by addition of 100 ng/ml of ACR. These data suggest that ACR inhibited VP-16 induced apoptosis by the inhibition of RNA synthesis along with protein synthesis, but not early DNA double strand breaks and DNA repair at a concentration of 100 ng/ml in P388 murine leukemic cells.

FK506 inhibits anti-IgM antibody-induced apoptosis and 17 kD endonuclease activity in the human B-cell line, MBC-1, established from Burkitt's lymphoma.

The endonuclease which causes antibody-induced apoptotic cell death in B cells is not completely understood. We previously established a B-cell line (MBC-1) from a patient with Burkitt's lymphoma at the leukaemic stage which demonstrated the typical morphology and internucleosomal DNA fragmentation of apoptotic cell death when treated with anti-IgM antibody. FK506, an immunosuppressive agent and calcineurin inhibitor, partially rescued the anti-IgM antibody-induced cell death in these MBC-1 cells. DNA SDS-PAGE nuclease activity assay demonstrated that a 17 kD protein exhibited endonuclease activity. Active gel assay showed nuclease activity in the cellular nuclear extract not treated with anti-IgM antibody. This nuclease activity was inhibited by FK506 at concentrations of 10-200 ng/ml in the active gel assay. These results raise the possibility that the 17 kD endonuclease is one of the nuclear members of the immunophilin family, which may function as an endogenous endonuclease in MBC-1 cells.

Combined oral administration of etoposide and arabinofuranosylcytosine-5'-stearylphosphate enhances the antitumor effect against P388 ascites tumors.

We investigated the antitumor effect of oral administration of etoposide and arabinofuranosylcytosine-5'-stearylphosphate (C18PCA) against P388 ascites tumors in B6D2F1 mice. Etoposide (25 mg/kg) and C18PCA (5 mg/kg) were given orally on days 1-5 after tumor inoculation. The median life span of the mice treated with etoposide or C18PCA alone was 19.5 and 18 days, respectively. The combination of both drugs significantly extended the median life span to 33 days. To clarify this enhancement of the increase in median life span, we examined intracellular deoxyribonucleoside triphosphate (dNTP) pools, cell-cycle distribution, DNA fragmentation, and the time course of the plasma drug concentration. Etoposide had no effect on intracellular dNTP pools in this experimental system, whereas treatment of cells with C18PCA or with the combination of both drugs resulted in a significant increase in dTTP pools to values ranging from 1.8- to 2.0-fold higher than the control levels. There was a significant increase in cells in the S + G2/M phase when cells had been treated with both etoposide and C18PCA. Agarose-gel electrophoresis of the extracted DNA revealed that C18PCA enhanced the fragmentation of DNA, with a length of about 180 bp being induced by etoposide. The plasma peak levels of etoposide (1000 nM) and ara-C (50 nM) were observed at 20 and 30 min after the simultaneous administration of both drugs, respectively. The plasma etoposide level gradually decreased to 10% of the peak level at 240 min after administration. On the other hand, the plasma concentration of ara-C was maintained at above 20 nM at 240 min. These observations suggest that C18PCA and etoposide act on P388 murine leukemic cells by accumulating cells in the S + G2/M phase. Even if the plasma concentration of ara-C is low, the repair of DNA damage by etoposide may be hindered in the presence of ara-C following an increase in DNA fragmentation.

[Pharmacokinetic and clinical evaluations of flomoxef in neonates].

To evaluate pharmacokinetics and clinical efficacy of flomoxef (6315-S, FMOX) in neonates, FMOX was administered to 21 neonates. With 20 mg/kg and 40 mg/kg of intravenous drip-infusion of FMOX 60 minutes, half lives (T 1/2's) was 64.9 minutes and 130.3 minutes, respectively, and when 20 mg/kg of FMOX was infused intravenously to 2 cases, half lives were 70.8 minutes and 110.1 minutes, respectively. When 45-100 mg/kg of FMOX was administered to 17 neonates with infections (pneumonia 8, sepsis 1, sepsis suspected 2, intrauterine infection 2, urinary tract infection 2, omphalitis 2), the efficacy rate was 88.2% (15 of 17). No adverse reactions were observed clinically in the 21 neonates. Transient elevation of eosinophilia was observed in 1 case and transient elevation of S-GOT and S-GPT 1 in another. These results suggest that FMOX is an effective and safe antibiotic to use in neonates.

Incidence of herpes zoster in infancy in Japan.

A search of the literature has revealed only 12 reports of herpes zoster during infancy in Japan. Of these, seven cases were thought to be mother-child infections where the mother had been infected with varicella and transmitted the VZV to the fetus, producing postnatal herpes zoster. The earliest case of maternal varicella infection occurred at 14 weeks of gestation. In the remaining 6 cases infection took place between the 17th and 32nd week of gestation. Postnatal onset of herpes zoster in the infants occurred within three months in two cases and at seven to eight months in the other four cases. In two cases it was unclear whether it was a mother-child infection. In three cases no clinical manifestations of maternal VZV infection were observed, but in these cases during early infancy the patients might have acquired asymptomatic infection.

[Pharmacokinetics and clinical studies on aztreonam in neonates].

Pharmacokinetic and clinical studies on aztreonam (AZT) were performed in neonates. Serum concentrations and urinary excretion of AZT were determined in 12 neonates with ages between 0 and 7 days (birth weights were between 1,260 and 3,500 g) upon intravenous injection or 1 hour drip intravenous infusion of AZT at 20 mg/kg. Serum concentrations of AZT at 1 hour after i.v. administration were 54.0 +/- 12.5 micrograms/ml, and half-lives were 6.01 +/- 0.70 hours. Serum concentrations of AZT reached their peaks at the end of drip infusion with levels of 42.1 +/- 17.6 micrograms/ml in the d.i.v. group and half-lives were 6.40 +/- 1.88 hours. Urinary recovery rates in the first 12 hours after administration were 28.5 +/- 6.4% for the i.v. group and 32.3 +/- 13.9% for the d.i.v. group. AZT was administered to 12 neonatal patients (2 cases of sepsis, 2 cases of suspected sepsis, 3 cases of pneumonia, 2 cases of urinary tract infection and 3 cases for prophylaxis), and clinical effectiveness, bacteriological efficacy and adverse reactions were evaluated. Clinical efficacies in 9 cases except 3 cases with prophylactic use were excellent in 1 case, good in 5 cases, fair in 1 case, poor in 1 case and unknown in 1 case, thus the efficacy rate was 75%. Bacteriological effects in 3 strains with Gram-negative bacilli were eradicated in 2 strains and unchanged in 1 strain, hence the bacteriological eradication rate was 66.7%. Increased GOT and GPT were observed in 1 cases as abnormal laboratory test results, but the abnormality was not serious.(ABSTRACT TRUNCATED AT 250 WORDS)