Toshiyuki Itaya

In vivo antitumor activity of herbimycin A, a tyrosine kinase inhibitor, targeted against BCR/ABL oncoprotein in mice bearing BCR/ABL-transfected cells

Herbimycin A, a benzoquinoid ansamycin antibiotic, has been shown to reverse the oncogenic phenotype of p60v-src transformed cells because of the inhibition of src protein tyrosine kinase. We previously demonstrated that herbimycin A displayed antitumor activity on the in vitro growth of Philadelphia chromosome-positive leukemia cells and BCR/ABL-transfected murine hematopoietic FDC-P2 cells through the inhibition of BCR/ABL protein tyrosine kinase. In this study, the transformed FDC-P2 cells were demonstrated to be tumorigenic in syngeneic DBA/2 mice. The intraperitoneal (i.p.) injection of the transformed tumor cells into DBA/2 mice induced infiltrations of abdominal organs, and then all of the mice died within time periods proportional to the cell numbers of inoculation. In mice that received an i.p. inoculation with greater than 1 x 10(5) cells, in vivo administration of herbimycin A by i.p. injection inhibited tumor formation and significantly prolonged survival time, and further, in mice inoculated with 1 x 10(4) cells, herbimycin A completely suppressed the in vivo growth of transformant FDC-P2 cells and brought about a complete remission. The present study revealed the in vivo efficacy of herbimycin A in mice bearing BCR/ABL-transfected cells.

Establishment and Characterization of a New Ph1-Positive Chronic Myeloid Leukemia Cell Line MC3 with Trilineage Phenotype and an Altered p53 Gene

A new Ph1-positive leukemic cell line (MC3) expressing the P210bcr/abl oncoprotein was established from a patient with CML in blast crisis. The MC3 cells showed the trilineage phenotype of myeloid, lymphoid (CD19) and megakaryocytoid lineages, and had a proliferative response to rhIL-1 and rhIL-3 in the serum-free culture. These results and the expression of CD34 indicated that the MC3 cells have characteristics of hematopoietic progenitor cells. Recently, it has been documented that alterations of the p53 gene in leukemic cells are frequently detected during the blast crisis of CML. The MC3 cells contained the altered p53 gene. In addition, the original leukemic cells showed the point-mutational activation of the N-ras gene and an additional chromosomal abnormality inv(3q), but the MC3 cells contained no such abnormalities, indicating that not all of the original leukemic cells had these abnormalities. Thus, the MC3 cell line may provide several insights into investigations of the blast crisis in CML as well as hematopoietic progenitor cells.

XENOGENIZATION OF TUMOR CELLS BY TRANSFECTION WITH PLASMID CONTAINING env GENE OF FRIEND LEUKEMIA VIRUS

A rat hepatocellular carcinoma cell line (cKDH‐8 cl‐11) showed decreased tumorigenicity after transfection with an envelope gene derived from a Friend leukemia virus (FV‐env gene). FV‐env gene product was found by indirect immunofluorescence staining to be expressed on the cell surface of the FV‐env gene‐transfected cells. The FV‐env‐transfected cells (FV‐env cKDH‐8), however, grew well in X‐irradiated immunosuppressed rats, indicating that the reduction in tumorigenicity of the transfected cells is based on immunological reaction in the host. The rats which rejected FV‐env cKDH‐8 cells showed resistance to rechallenge with the parent cKDH‐8 cl‐11 tumor cells. These results suggest that the FV‐env gene product may elicit antitumor immunity against FV‐env cKDH‐8 cells in a host with a resultant reduction in the tumorigenicity of these cells.

Effect of Herbimycin A, an inhibitor of tyrosine kinase, on protein tyrosine kinase activity and phosphotyrosyl proteins of Ph1-positive leukemia cells

Herbimycin A, a benzoquinonoid anasamycin antibiotic, preferentially inhibited the in vitro growth of Ph1-positive leukemia cell lines. On the other hand, genistein, which was developed as an inhibitor of receptor-type tyrosine kinase, and other protein kinase inhibitors showed no selective inhibition of Ph1-positive leukemia cell growth. Herbimycin A also displayed an abrogative effect on the transformation of murine hematopoietic cells by transfection with a bcr/abl oncoprotein-expressing retroviral vector. The antitumor action of herbimycin A on Ph1-positive leukemia cells is related to an inhibition of activity of bcr/abl protein tyrosine kinase and a subsequent reduction of the constitutive phosphotyrosyl proteins, however, the antibiotic has no effect on the expression of bcr/abl mRNA and oncoprotein. Therefore, herbimycin A may provide an important insight into the oncogenic action of bcr/abl oncoprotein and the future development of oncoprotein-targeted therapeutic agents.

The activity of suppressor cells in the spleen of murine bone marrow chimeras

An intrasplenic injection (i.s.) of BALB/c bone marrow cells induces a higher survival rate than an intravenous injection (i.v.) in irradiated C3H/He recipients. Coculture experiments revealed the presence of alloantigen-specific and nonspecific suppressor cells in the spleens of mice injected i.s. and i.v. Suppressor activity decreased 50-60 days after bone marrow transplantation in i.v. chimeras, while there was no decrease in i.s. chimeras. In vitro suppressor activity was correlated with in vivo activity. Histopathological changes in the liver were examined. A total of 28% of the i.v. chimeras showed severe changes compared with 6% of the i.s. chimeras. The spleen indices of i.v. and i.s. chimeras were compared. Although there was no statistically significant difference between the spleen indices of i.v. chimeras and those of i.s. chimeras, spleen indices of i.v. chimeras tended to be higher than those of i.s. chimeras. These results show that suppressor cells in i.s. chimeras appear to inhibit graft-versus-host reactions more efficiently. Furthermore, an adoptive transfer assay showed that suppressor cells detected in i.s. chimeras were effective in vivo. We therefore suggest that suppressor cells detected in vitro correlate with in vivo activity and may play some role in the induction and maintenance of transplantation tolerance.

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

SummaryRat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.

Administration of rhG-CSF Increases Complete Remission Rates After CHOP and ProMACE/CytaBOM for Non-Hodghn's Lymphoma: A Pilot Study

To evaluate the clinical effects of the administration of recombinant human granulocyte-stimulating factor (rhG-CSF) post chemotherapy for patients with advanced-staged intermediate-grade or high-grade non-Hodgkins malignant lymphoma (NHL), we conducted this multicenter study and compared the responses between both the regimens, CHOP as a first-generation chemotherapy and ProMACE/CytaBOM as a third-generation chemotherapy, when combined with the rhG-CSF administration. In this multicenter study, where forty patients were registered, patients in both the CHOP and ProMACE/CytaBOM groups were treated with the original regimen designs without the necessity of reducing drug dosages when combined with the administration of rhG-CSF. The administration of rhG-CSF post both of the cytotoxic therapies brought about much higher rates of complete remission in both the groups (CHOP, 75 percent; ProMACE/CytaBOM, 75 percent), as compared with those of the previous study without the rhG-CSF administration. Regarding res...