Seiji Koishi

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.

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.

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.

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.

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.

Analysis of mutations at the DNA repair genes in acute childhood leukaemia

Deficiency in DNA repair capability is considered to be responsible for oncogenesis. Hereditary and sporadic cancers in various tissues have been reported to have mutations at the DNA repair genes. In this study we analysed two excision repair genes (ERCC1 and XPCC) and two mismatch repair genes (hMSH2 and hMTH1) in the leukaemic blasts of newly diagnosed paediatric patients by use of reverse transcriptase (RT)–polymerase chain reaction (PCR). Analysis of the leukaemic blasts from 15 patients demonstrated no alterations at the XPCC, hMSH2 or hMTH1 genes. Blasts from one patient with acute mixed lineage leukaemia revealed an abnormally migrated product of the ERCC1 gene by RT‐PCR. His leukaemic blasts showed a reduced expression of ERCC1 protein by Western blotting. Since bone marrow cells at remission showed only normally migrated product, the ERCC1 gene mutation was considered to be specific for the leukaemic blasts. This is the first report describing a mutation at the ERCC1 gene in acute childhood leukaemia.

Inhibition of superoxide production and chemotaxis by methotrexate in neutrophils primed by TNF-α or LPS

Abstract: We have demonstrated recently that methotrexate (MTX) inhibits superoxide generation and chemotaxis induced by N‐formylmethionyl‐leucyl‐phenylalanine (fMLP) in neutrophils primed by granulocyte colony‐stimulating factor (G–CSF). To extend these observations, we examined the in vitro effect of MTX on fMLP‐stimulated superoxide generation and chemotaxis in neutrophils primed by either tumor necrosis factor α (TNF‐α) or bacterial lipopolysaccharide (LPS). MTX inhibited superoxide generation and chemotaxis more efficiently in TNF‐α‐or LPS‐primed neutrophils than in unprimed neutrophils. When either hypoxanthine or guanosine was added to the culture medium, the effects of MTX were partially counteracted. Furthermore, MTX caused a significant inhibition of both superoxide production induced by phorbol 12‐myristate‐13‐acetate and chemotaxis induced by interleukin 8 in G‐CSF‐primed neutrophils. These results may support the hypothesis that neutrophils primed by different stimuli are more susceptible to the inhibitory effects of MTX on superoxide generation and chemotaxis irrespective of chemoattractants. Such an effect can be partly attributed to the perturbation of purine nucleotide biosynthesis.

Myelodysplastic syndrome presenting as third malignancy after non-Hodgkin's lymphoma and osteosarcoma

Abstract The patient was initially diagnosed as having non-Hodgkins lymphoma and was cured following treatment with prednisolone, vincristine, daunorubicin, l-asparaginase, and cyclophosphamide. Seven years and two months later, he developed osteosarcoma in his right femur. He received chemotherapy consisting of methotrexate, carboplatin, etoposide, and ifosfamide and again obtained remission. After 2 years and 7 months, however, he was found to have pancytopenia with morphological abnormalities in the erythroid and myeloid series. Diagnosis of myelodysplastic syndrome (MDS) was made. Cytogenetic analysis of bone marrow cells revealed -5 and -7, which is typical for secondary MDS. This is a rare case of third malignancy presumably caused by alkylating agents.

Constitutive endonuclease to induce high molecular weight or internucleosomal DNA fragmentation in freshly isolated leukemia cells

Using an autodigestion method, we investigated endogenous endonuclease(s) in leukemia cells freshly obtained from pediatric patients with various types of leukemia. Endonucleolytic activity was found to cause both high molecular weight and internucleosomal DNA fragmentation at a neutral pH in whole cell lysates of all common acute lymphoblastic leukemia (cALL) blasts, which was Mg2+-dependent and Ca2+-independent. Whole lysates from most acute myeloblastic leukemia (AML) cells possessed similar endonuclease activity, but both Mg2+ and Ca2+ were required for the activity. Our results suggest that leukemia cells of different lineages have distinct constitutive endonucleases, which may play a role in the occurrence of apoptosis in these cells.