Yukio Satow

c-kit point mutation of extracellular domain in patients with myeloproliferative disorders.

Summary. c‐kit is a tyrosine kinase receptor whose ligand is stem cell factor (SCF). Gene alteration of the c‐kit extracellular domain was analysed by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) in 25 patients with myeloproliferative disorders (MPD). In the N‐terminal part of the domain, mobility shifts indicating sequence alteration were detected in three of the patients, two primary myelofibrosis (PMF) and one chronic myelogenous leukaemia (CML). The subsequent sequencing revealed the same point mutations at codon 52 causing amino acid substitution (Asp → Asn). To our knowledge this is the first report with a c‐kit point mutation found in human fresh tumour cells.

Platelet‐derived growth factor expression in accelerated and blastic phase of chronic myelogenous leukaemia with myelofibrosis

Summary Myelofibrosis is sometimes associated with accelerated and blastic phase of chronic myelogenous leukaemia (CML). In order to investigate the role of platelet derived growth factor (PDGF) in this pathogenesis, expression and production of PDGF was studied in the blast cells from 11 patients. Five patients had myelofibrosis with myeloid blasts, while six patients did not show fibrosis, including three with myeloid blasts and three with lymphoid blasts. PDGF‐A chain transcript was expressed in most of the patients. On the other hand, PDGF‐B chain transcript was detected in all of the five patients with myeloid blasts and with fibrosis, in one of the three patients with myeloid blasts and without fibrosis, and in none of the three lymphoid crisis patients without fibrosis. In the patients with myeloid blasts and with fibrosis, PDGF protein. PDGF‐AB and/or PDGF‐BB, was found to be secreted from blast cells. In addition, the PDGF activity in the culture of myeloid blasts from two patients with fibrosis was also growth stimulatory for human marrow fibroblasts. These results suggest that expression and secretion of PDGF‐AB or PDGF‐BB in blast cells play an important role in the pathogenesis of marrow fibrosis associated with accelerated and blastic phase of CML.

No Alteration in DNA Topoisomerase I Gene Related to CPT‐11 Resistance in Human Lung Cancer

Mutations and reduced expression of DNA topoisomerase I (topo I) gene have been shown to be important in the acquisition of resistance to camptothecin and its analogues in vitro, but their significance has not been verified in vivo. We investigated possible topo I gene mutations and topo I mRNA expression levels in 127 samples obtained from 56 patients with lung tumors, including patients who had developed clinical resistance to topo I inhibitors. No mutations were detected in any of the samples examined and expression levels did not differ significantly between clinically resistant cases and others. However, the topo I mRNA expression level was significantly higher in small cell lung carcinomas than in non‐small cell lung carcinomas (P<0.05). These results suggest that topo I mRNA levels may affect CPT‐11 sensitivity in human lung cancer. However, topo I gene mutations and reduced topo I mRNA expression may not be the main mechanism of clinically acquired resistance to camptothecin analogues in vivo.

Platelet Derived Growth Factor Expression, Myelofibrosis and Chronic Myelogenous Leukemia

CML is often associated with myelofibrosis, and fibrosis in the accelerated phase is one of the diagnostic criteria for this accelerated phase. In this review, the mechanism of myelofibrosis associated with CML is discussed with emphasis on the cell origin of the production and release of platelet derived growth factor (PDGF) and its interaction with marrow fibroblasts. In the initial stage of myelofibrosis in chronic phase CML, atypical small megakaryocytes might leak PDGF, possibly PDGF-AB, together with other growth factors. As the clinical phase of the disease progresses to accelerated or blastic phase, a larger quantity of PDGF-AB or PDGF-BB might be secreted from blastic cells with myeloid phenotype. In addition some fibroblasts may be attracted by the PDGF and proliferate, and deposit collagen as well as fibronectin in the bone marrow stroma.

Decreased L-selectin expression in CD34-positive cells from patients with chronic myelocytic leukaemia

Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA‐5, VLA‐4, LFA‐1, ICAM‐1, L‐selectin and c‐kit) on bone marrow CD34++ cells from 16 CML patients by three‐colour flow cytometry. The mean percentage of cells expressing L‐selectin in the CD34++CD38+  ∼  ++ fraction from untreated CML patients was significantly lower, and that in the CD34++CD38− fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon‐α (IFN‐α), the mean percentage of the cells expressing L‐selectin in the CD34++CD38− fraction from three patients with a low percentage of Ph1(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph1(+) cells. In addition, L‐selectin expression rate was inversely correlated to the percentage of Ph1(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34++ fraction except LFA‐1. These data suggest that decreased L‐selectin expression in CML CD34++ cells reflects one of the features of malignant CML progenitors.

Influence of Paternal 252Cf Neutron Exposure on Abnormal Sperm, Embryonal Lethality, and Liver Tumorigenesis in the F1 Offspring of Mice

Experiments were conducted to determine whether neutron‐induced genetic damage in parental germline cells can lead to the development of cancer in the offspring. Seven‐week‐old C3H male mice were irradiated with 252Cf neutrons at a dose of 0, 50, 100, or 200 cGy. Two weeks or 3 months after irradiation, the male mice were mated with virgin 9‐week‐old C57BL females. Two weeks after irradiation, the irradiated male mice showed an increased incidence of sperm abnormalities, which led to embryo lethalities in a dose‐dependent manner when they were mated with unirradiated female mice. Furthermore, liver tumors in male offspring of male mice in the 50 cGy group were significantly increased in 19 of 44 (43.2%) animals, in clear contrast to the unirradiated group (1 of 31; 3.2%) (P < 0.01). In the 100 cGy group, 6 of 39 (15%) mice had lesions. At 3 months after irradiation abnormal sperm and embryonal lethality were not significantly increased. The incidences of liver tumors in male offspring from the 50 cGy, 100 cGy and 200 cGy groups were 6 of 20 (30%), 5 of 22 (23%) and 1 of 19 (5%), respectively, which are not significantly increased compared with the control. It is concluded that increased hepatic tumor risk in the F1 generation may be caused by genetic transmission of hepatoma‐associated trait(s) induced by 252Cf neutron irradiation.

Leukemic Transformation of Primary Myelofibrosis: Immunophenotype, Genotype and Growth Characteristics of Blast Cells

Blast cells from six patients with leukemic transformation of primary myelofibrosis (PMF) were studied by morphology, immunophenotype and genotype as well as response to hematopoietic growth factors. The majority of the patients showed granulocytic or granulo-monocytic blasts, and only one had T lymphoid-monocytic blasts. None of the patients showed rearrangement of Ig or TCR genes, or the existence of the bcr-abl fused gene. A prominent growth response to GM-CSF and IL-3 was evident in all of the patients examined in liquid as well as semisolid cultures. The response to G-CSF was observed in four of the six patients in suspension culture, and in two of three patients in the clonogenic assay. Stem cell factor (SCF) was a weak growth stimulant, however the combination of this factor with GM-CSF or IL-3 was synergistically stimulatory. These results suggest that leukemic transformation of PMF occurs mainly at the level of myeloid stem cell, and that GM-CSF, IL-3, G-CSF and SCF are major growth factors for the blast cells in these cases.

Effect of Tritiated Water on Female Germ Cells: Mouse Oocyte Killing and RBE

The relative biological effectiveness (RBE) of tritiated water (HTO) was investigated using mouse immature oocytes. Juvenile mice, having immature oocytes that are highly sensitive to radiation killing, were exposed to HTO, 137Cs gamma rays, 60Co gamma rays, or 252Cf fission neutrons. The 137Cs gamma rays were used to simulate the changing dose rate (but not the radiation quality) received from HTO (see section 2). On the 14th day after birth, female ICR strain mice were injected once in the abdominal cavity with HTO at levels of 1.70, 3.40, 6.81, or 10.21 MBq/10g body weight, and their ovaries were removed 2 weeks after the injection. (The cumulative doses are equivalent to 0.039, 0.077, 0.159 and 0.246 Gy, respectively.) The survival rate of oocytes was determined by light microscopy inspection of serial sections, and the results of the different exposures were compared. The number of surviving oocytes decreased exponentially with increasing dose of HTO or 137Cs, and RBEs of HTO compared to 137Cs gamma rays ranged from 1.1 to 3.5. The relative effectiveness of the different types of radiations was compared, and the results of the comparison, in order of increasing effectiveness, was 137Cs, 60Co, HTO, and 252Cf. The RBE of 252Cf compared to 60Co gamma rays ranged from 1.6 to 3.5.

Chemotactic activities of peripheral blood polymorphonuclear leukocytes and peritoneal exudate polymorphonuclear leukocytes in MRL mice

Chemotactic responsiveness to fMet-Leu-Phe in concentrations of 10(-8) to 10(-4) M in the Boyden chamber was compared between peritoneal exudate cells (PEC) and polymorphonuclear leukocytes (PMN) isolated from peripheral blood, between MRL/Mp-+/+ (MRL-+/+) and MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice, and between young (6-9 week old) and aged (16-24 week old) mice. Chemotactic responsiveness of PEC did not differ between MRL-+/+ and MRL-lpr/lpr, and young and aged mice. While, PMN showed greater chemotaxis in aged MRL-+/+ mice than that in aged MRL-lpr/lpr mice. These results suggest that chemotactic responsiveness of PMN differ from that of PEC which is assumed to be preactivated by an inflammatory agent injected into the peritoneal cavity to elicit cells. Less responsiveness of PMN to the bacterial origin peptide might relate to the autoimmune disease of this murine model.

Teratogenicity of Tritium Water to Rat Embryos

Abstract In the purpose to learn the teratogenic effects of tritium, tritiated water of 50, 75, 100, 125 and 150mCi was injected into the abdominal cavity of pregnant rats individually on days 7, 8, 9, 10 and 11 of pregnancy, respectively, and the anomalies that developed were studied on day 18 of pregnancy. The anomalies occurred at the highest frequency in the cases administered with 100mCi on day 9 of pregnancy, the anomalies combined accounting for 81.3 % of the implanted embryos (100% of the surviving embryos). Among them, cardiovascular anomalies accounted for 68.8 % of the implanted embryos (84.6 % of the surviving embryos). The LD50 was 132mCi in cases administered on day 9 of pregnancy.