Haruyo Hirota

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.

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.

Analysis of hprt gene mutation following anti-cancer treatment in pediatric patients with acute leukemia

We evaluated the genotoxic effect of cancer therapy on somatic cell mutation by isolating 6-thioguanine-resistant mutants in peripheral lymphocytes. The study population comprised 45 children with acute lymphoblastic leukemia (ALL), 13 children with acute myelogenous leukemia (AML) and 28 age-matched healthy controls. The geometric mean mutant frequency for ALL patients was 7.8 x 10(-6), which was significantly higher than that for AML patients (1.7 x 10(-6)) or for healthy controls (1.1 x 10(-6)). Fifteen patients with ALL showed a high mutant frequency above 10 x 10(-6), although 10 of them had completed their treatment at least 24 months earlier. Moreover, repeated measurements of mutant frequency at intervals of 12 or more months revealed that the values were very stable. Structural hypoxanthine-guanine phosphoribosyl transferase (hprt) gene alterations, as determined by Southern blot analysis, were seen in 23% (12/52) of mutant clones derived from ALL patients, but not in those from the controls. These results suggest that intensive anti-cancer therapy of children may produce persistent somatic mutations, which could be related to the appearance of second neoplasms.

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.

Biomarkers in long survivors of pediatric acute lymphoblastic leukemia patients: late effects of cancer chemotherapy.

In order to elucidate the late effects of cancer chemotherapy, mutant frequencies (Mfs) at the hypoxanthine phosphoribosyl transferase (hprt) locus were evaluated in pediatric patients with early pre-B acute lymphoblastic leukemia (ALL). Hprt-Mfs were measured at least 2 years after completion of chemotherapy. Ten out of 15 patients were found to have hprt-Mfs exceeding the 99% confidence limits as calculated from observations of healthy controls. Although there was some intraindividual variation, serial measurements of hprt-Mfs with intervals of more than 6 months revealed that hprt-Mfs were fairly stable. Patients with high Mfs tended to have sibling clones as detected by clonality analysis using the T-cell receptor (TCR) rearrangement pattern, but clonality did not have a major effect on the Mfs. On the other hand, Mfs at the TCR locus and sister chromatid exchange frequency were within the normal range in all patients. These data suggest that chemotherapy can cause persistent genotoxicity in vivo in a subset of pediatric ALL patients and that the hprt-Mf is a useful method for measuring such an effect.

Somatic cell mutation frequency at the HPRT, T-cell antigen receptor and glycophorin A loci in Cockayne syndrome

Skin fibroblasts of patients with Cockayne syndrome (CS) are hypersensitive to the lethal or mutagenic effect of ultraviolet light, which may cause genetic instability. Up to now, however, no systematic study of in vivo somatic cell mutation in CS cells has been reported. This article describes our investigation of the mutation frequencies (Mfs) at three different loci, i.e. hypoxanthine-guanine phosphoribosyl transferase (HPRT), T-cell antigen receptor (TCR) and glycophorin A (GPA), in six patients with CS. Mfs at the HPRT and TCR loci were found to be within the normal range as determined in age-matched controls. In the GPA locus of two patients, there was a slight increase, but it was much smaller than that reported in other DNA repair deficient syndromes. The frequency of spontaneous HPRT mutation in Epstein-Barr virus transformed B-lymphoblastoid cells derived from CS patients was similar to that in cells from normal children. The molecular characterization of the representative HPRT mutant T cell clones from CS patients did not show any structural alterations. These results may explain, at least in part, why CS is not associated with predisposition to cancer.

Aphidicolin potentiates apoptosis induced by arabinosyl nucleotides in human myeloid leukemia cell lines

We investigated the effect of aphidicolin, an inhibitor of DNA polymerase alpha and delta, on the induction of apoptosis by arabinosyl nucleosides in a human promyelocytic leukemia cell line, HL-60. Pretreatment of HL-60 cells with aphidicolin (2 microM) significantly increased the number of morphologically apoptotic cells induced by 1-beta-D arabinofuranosylcytosine (ara-C) during 4 hr of incubation. This is consistent with the appearance of DNA fragmentation as determined quantitatively by diphenylamine or by agarose gel electrophoresis. The inhibition of cell growth on day 3 after drug exposure was correlated with the degree of apoptosis: Such synergistic interaction between aphidicolin and ara-C has also been observed in other human myeloid leukemia cell lines, U937 and KG-1. In addition, the induction of apoptosis by 9-beta-D arabinofuranosyladenine or 9-beta-D arabinofuranosylguanine is augmented by aphidicolin.