Yoshihiro Wakazono

Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor κB (NF-κB) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes

We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells.

In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro

Abstract: We recently showed that recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) maintained the viability of human neutrophils in incubation for up to 72 hours. However, it is not known whether rhG‐CSF can enhance neutrophil survival in in vivo situations. To clarify this issue, we investigated neutrophil survival in vitro following in vivo injection of rhG‐CSF. Neutrophils were obtained from 4 pediatric patients with malignancies and healthy adult volunteers before and after rhG‐CSF administration. Neutrophils obtained before rhG‐CSF treatment started to undergo apoptosis after 24 h of incubation. In contrast, the survival of neutrophils drawn after rhG‐CSF administration increased by approximately 24 h. Concomitantly, the appearance of typical ladder‐like DNA fragmentation was delayed. Such an increase in neutrophil survival was inhibited by coincubation with either H 7 (10 μmol/1) or H 8 (20 μmol/1), which worked as protein kinase C inhibitors. Although our study did not measure neutrophil survival in vivo directly, it provides us with further evidence that rhG‐CSF may function to prolong neutrophil life expectancy in vivo.

Induction of apoptosis in childhood acute leukemia by chemotherapeutic agents: Failure to detect evidence of apoptosis in vivo

Abstract:  This study is designed to investigate whether apoptosis occurs in vivo in pediatric patients with acute leukemia during induction therapy. When patients with common acute lymphoblastic leukemia (cALL) and acute myeloblastic leukemia (AML) were treated with prednisolone (60 mg/m2/day, p.o. or i.v.) and etoposide (150 mg/m2/day, i.v.), respectively, the blast cell counts fell to below 30% and 5%, respectively, in 1 week. However, during this cytoreduction phase, neither morphologically apoptotic cells nor fragmentation of DNA derived from peripheral blast cells were detected at any preparations. On the other hand, cALL but not AML cells spontaneously undergo apoptosis following their culture in vitro. The addition of autologous serum instead of fetal calf serum substantially prevented apoptosis from occurring spontaneously in cALL cells. When cALL and AML cells freshly obtained from patients before therapy were treated in vitro with 10 μmol/1 prednisolone and 20 μg/ml etoposide, respectively, these cells underwent apoptosis within 6 hours, as determined by a morphological and DNA fragmentation assay. These in vivo and in vitro findings suggest that, although anticancer drugs may induce apoptosis in vivo, these apoptotic cells cannot be detected due to their rapid removal from the circulation.

ASSOCIATION OF HIGH MOLECULAR WEIGHT DNA FRAGMENTATION WITH APOPTOTIC OR NON-APOPTOTIC CELL DEATH INDUCED BY CALCIUM IONOPHORE

Calcium ionophore (A23187)‐induced high molecular weight (HMW) and internucleosomal DNA fragmentation were investigated in human leukemia cell lines. An apoptosis‐sensitive cell line, HL‐60, showed HMW, internucleosomal DNA fragmentation and morphological changes of apoptosis by A23187. MOLT‐4, which is resistant to apoptosis, exhibited only HMW DNA fragmentation and died of necrosis under the same conditions. Autodigestion experiments suggested the endonucleolytic activity to cause HMW fragmentation in the cytoplasm of both cell lines. The activity was more dependent on Mg2+ than Ca2+ in HL‐60, whereas it was Ca2+‐dependent in MOLT‐4. These results suggest that HMW DNA fragmentation is not specific to apoptosis.

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine-guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.

Somatic mutations at T-cell antigen receptor and glycophorin a loci in pediatric leukemia pateints following chemotherapy: comparison with HPRT locus mutation

Frequencies of somatic mutations in pediatric patients with leukemia were evaluated following intensive treatment at three different loci: the hypoxanthine-guanine phosphoribosyl transferase (HPRT), T-cell antigen receptor (TCR), and glycophorin A (GPA) gene. Thirty-two children with acute lymphoblastic leukemia (ALL), nine children with acute myelogenous leukemia (AML), and 20 age-matched healthy controls were included in the study of mutant frequencies (Mfs) at the HPRT and TCR loci. Among these patients and controls, individuals with heterozygous MN blood type, i.e., 14 children with ALL, three children with AML, and nine healthy controls, served for the further assessment of variant frequency (Vf) at the GPA locus. In ALL patients, geometric mean Mfs and Vfs at these loci were significantly higher than in healthy controls. The high Mf value at the HPRT locus persisted for up to 8 years after the end of chemotherapy. On the other hand, the Mf values at the TCR locus and Vf values at the GPA locus declined gradually with time. In AML patients, on the other hand, the geometric mean Mf only at the TCR locus was significantly higher than in the controls, albeit to a lesser degree than in ALL patients. These data suggest that anti-cancer therapy induces somatic mutations at various loci and that ALL patients are more susceptible to mutagenic intervention than are AML patients.

Normal mutation frequencies of somatic cells in patients receiving growth hormone therapy

The number of reported cases of malignancy developing in growth hormone (GH) users worldwide has increased to more than 40. However, the causal relationship between GH administration and the occurrence of malignancies is still uncertain. We investigated somatic cell mutation frequencies (Mfs) or variant frequency (Vf) at three gene loci in patients with pituitary dwarfism receiving GH therapy to clarify the genetic effect of GH. Eighty-eight patients receiving GH therapy for at least 3 months and 42 age-matched healthy controls were studied. Mfs at hypoxanthineguanine phosphoribosyltransferase (HPRT) and T-cell receptor (TCR) loci in GH users were not significantly higher than in the controls. Although a few patients seemed to have a slightly increased Vf at the glycophorin A (GPA) locus, the difference was not statistically significant. In addition, there was no tendency for the Mfs (Vf) at these loci to increase with the duration of the GH therapy. These data seem to exclude the possibility that GH induces genetic instability in patients with pituitary dwarfism who are receiving GH therapy.

Augmentation by aphidicolin of 1-β-d-arabinofuranosylcytosine-induced c-jun and NF-κB activation in a human myeloid leukemia cell line: Correlation with apoptosis

Abstract 1-β- d -arabinofuranosylcytosine (ara-C) (2 μM) can induce apoptosis in a human myeloid leukemia cell line, U937, after 4 h of incubation. Pretreatment of cells with aphidicolin (2 μM) augments ara-C-induced apoptosis, since it was first observed at 0.4 μM ara-C and became more intense at 2 and 10 μM. Although aphidicolin itself had a marginal effect on c- jun expression, it significantly augmented ara-C induced c- jun upregulation by shortening the lag time and lowering ara-C concentrations necessary for the induction of detectable c- jun transcripts. Aphidicolin and ara-C acted synergistically to increase NF-κB DNA binding activity as determined by an electrophoretic mobility shift assay. Expression of c- myc was slightly increased through the DNA degradative phase, and was then downregulated. Thus, the activation of NF-κB and c- jun expression seems to be well correlated with the potentiation by aphidicolin of ara-C-induced apoptosis.

Different effect of thymidine kinase loss on TTP pools; comparison among human leukemia cell lines

Thymidine kinase (TK)-deficient cells were established from six human leukemia cell lines to evaluate the role of TK in maintaining intracellular TTP pools. The residual TK activities in mutant cells were less than 3% of those of wild-type strains, except for a B-lymphoid cell line, Ball-1 (8.7%). In a promyelocytic leukemia cell line (HL-60), a splenic B cell line (WI-L2) and Ball-1, a mutational loss of TK resulted in a decrease of TTP pools by 80%, 33% and 54%, respectively. On the other hand, in the T cell lines, Molt-3, Molt-4 and CEM, TTP did not show any significant differences between parent and TK-deficient cells. TK-deficient HL-60 cells had, however, comparable levels of dATP, dGTP and dCTP with wild-type cells. An analysis of growth characteristics showed that the decrease of TTP was not due to the change of the cell cycle distribution. These results indicate that TK plays a different role in maintaining TTP pools among human leukemia cell lines.

Domperidone with ORT in the treatment of pediatric acute gastroenteritis in Japan: A multicenter, randomized controlled trial

Domperidone is an antiemetic that is often prescribed for children with acute gastroenteritis in Japan. In this study, the authors assessed the efficacy of domperidone prescription in combination with oral rehydration treatment (ORT) in the treatment of vomiting during acute gastroenteritis in children during the early period. They performed a prospective multicenter randomized trial in Japan. Patients received either ORT or ORT and domperidone prescription. The primary outcome was the proportion of patients who had vomiting during the first 2 hours after randomization. A total of 56 children were eligible; 24 received ORT alone, and 32 received ORT and prescribed domperidone suppository. Results showed that 27.3% of children in the ORT group vomited as compared with 20.7% of children in the ORT and domperidone group (P = .41). In this study, it appears that domperidone in combination with ORT in the treatment of acute gastroenteritis does not reduce vomiting in the early period.