Ikuya Usami

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.

Role of Protein Tyrosine Phosphorylation in Etoposide-Induced Apoptosis and NF-κB Activation

Abstract When a human myeloid cell line, U937, was incubated with etoposide (10 μg/mL), morphologically apoptotic cells first appeared at 3 hr and increased with time to 50% at 6 hr. Pretreatment of U937 cells for 30 min with a potent tyrosine kinase inhibitor, herbimycin A (10 μM), significantly suppressed the appearance of apoptotic morphological changes. Concomitantly, herbimycin A pretreatment prevented both high molecular weight and internucleosomal DNA fragmentation induced by etoposide. Two major bands at 30 and 66 kDa with enhanced tyrosine phosphorylation inhibited by herbimycin A were detectable after 30 min of incubation with etoposide. In addition, herbimycin A prevented etoposide-induced NF-κB activation. The expressions of Bcl-2 and Bax, on the other hand, were not affected by herbimycin A pretreatment. Herbimycin A was also found to inhibit 1-β- d -arabinofuranosylcytosine-induced apoptotic changes and NF-κB activation. These results suggest that activation of tyrosine kinase(s) may play an important role in apoptotic processes induced by a variety of anti-cancer drugs.

Prevention of etoposide-induced apoptosis by proteasome inhibitors in a human leukemic cell line but not in fresh acute leukemia blasts: A differential role of nf-κb activation

Recent research indicates that the proteasome is one of the non-caspase proteases involved in apoptotic signaling pathways. Nuclear factor-kappaB (NF-kappaB) activation, one of the key factors in apoptosis, can be prevented through abrogation of IkappaBalpha degradation by proteasome inhibition. We have investigated the effects of the proteasome inhibitors carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal (MG132) and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL) on apoptosis and NF-kappaB activation induced by etoposide, using a human leukemia cell line (U937) and leukemia blasts freshly isolated from patients with acute leukemia. Pretreatment of U937 cells with MG132 or LLnL inhibited etoposide-induced morphological apoptosis and caspase-3 activation. Furthermore, MG132 or LLnL prevented NF-kappaB activation and IkappaBalpha degradation, but not IkappaBalpha phosphorylation at Ser32. Other inhibitors of NF-kappaB activation, including pyrrrolidine dithiocarbamate (an antioxidant) and the peptide SN50 (an inhibitor of translocation of activated NF-kappaB into the nucleus), also attenuated etoposide-induced apoptosis. In leukemia blasts, although proteasome inhibitors suppressed NF-kappaB activation induced by etoposide, they were unable to prevent morphological apoptosis. Moreover, proteasome inhibitors by themselves caused apoptosis in leukemia blasts at the concentrations employed in this study. These results suggest that the role that NF-kappaB plays in apoptosis induced by etoposide in a human leukemia cell line may be different from the role it plays in freshly isolated leukemia blasts.

Outcome of childhood acute lymphoblastic leukemia with induction failure treated by the Japan Association of Childhood Leukemia study (JACLS) ALL F-protocol.

Children with acute lymphoblastic leukemia (ALL) who fail to achieve complete remission (CR) after induction therapy (induction failure: IF) have a poor prognosis; however, there have been few prospective studies in patients with IF.

Evaluation of mutant frequencies at the hprt and the T-cell receptor loci in pediatric cancer patients before treatment

Mutant frequencies (Mfs) at the two genetic loci, the hypoxanthine phosphoribosyl transferase (hprt) gene and the T-cell receptor (TCR) gene were evaluated in pediatric cancer patients before starting chemotherapy or radiotherapy. The study population consisted of 27 patients with various solid tumors (mean age +/- SD; 5.5 +/- 5.1 years, range; 0.2-14.5 years), 5 patients with acute leukemia (10.3 +/- 6.1, 1.3-17.0 years), and 26 healthy controls (11.6 +/- 4.0, 4.4-22.2 years). Although the age distributions were different, the mean Mf values of the hprt and the TCR loci were comparable among these three groups. On an individual basis taking the age factor into consideration, the hprt-Mfs of 3 patients with solid tumors, i.e., two patients with Hodgkins disease and one patient with Askin tumor, were found to be well above the 95% confidence limit. There were no patients with a TCR-Mf exceeding the 95% confidence limit. These data suggest the possibility that some patients with solid tumors may be predisposed to mutational susceptibility before treatment. The assay of the hprt-Mf appears more sensitive than the TCR-Mf assay in distinguishing these 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.

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.

Treatment of children with congenital heart disease and growth retardation with recombinant human growth hormone

Seven prepubertal short children with congenital heart disease were treated with recombinant human growth hormone (GH). Although complete surgical correction was performed for their heart disease at least 2 years before the start of GH therapy, improvement in growth was less than expected in these children. They received 0.5 IU kg−1 week−1 of GH daily for 2 years or more. The growth rate increased from a mean of 4.3 cm year−1 before treatment to a mean of 7.8 cm year−1 in the first year and to a mean of 6.3 cm year−1 in the second year of treatment. Their mean standardized height improved from −3.41 ± 0.78 to −2.54 ± 0.62 after 2 years. The mean height age difference minus the bone age difference became positive in these children. We conclude that recombinant GH increases the growth rate in children with congenital heart disease and prepubertal growth retardation.

Kawasaki disease with lymphadenopathy and fever as sole initial manifestations

Aim:  Initial presentation with only cervical lymphadenopathy and fever is one of the pitfalls in the diagnosis of Kawasaki disease (KD). As the number of such patients is small, their clinical features have remained uncertain. The purpose of the present study is to characterise the features of such KD patients, especially in comparison with those of patients with common onset.

Methotrexate inhibits superoxide production and chemotaxis in neutrophils activated by granulocyte colony-stimulating factor

Treatment of circulating human neutrophils with recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) for 30 min augmented superoxide generation and chemotaxis induced by N‐formylmethionyl‐leucyl‐phenylalanine (fMLP) in a dose dependent manner. When neutrophils were treated with 1 μM of methotrexate (MTX) for 60 min after incubation with rhG‐CSF (10 ng/ml), the effects of rhG‐CSF on superoxide generation and chemotaxis were inhibited by approximately 49 and 29%, respectively. Although inhibitory effects of MTX were also seen in neutrophils not pretreated with rhG‐CSF, the degree of inhibition was much less. The addition of either hypoxanthine or guanosine at a concentration of 100 μM to the culture medium significantly attenuated the effects of MTX. However, in neutrophils obtained from a patient with Lesch‐Nyhan syndrome, which lacked hypoxanthine‐guanine phosphoribosyl transferase activity, neither hypoxanthine nor guanosine had any rescue effect. These results suggest that MTX inhibits superoxide generation and chemotaxis in rhG‐CSF‐activated neutrophils, at least in part, by disturbing purine nucleotide biosynthesis.