Masatoshi Seki

Radiation-Induced Myeloid Leukemia in C3H/He Mice and the Effect of Prednisolone Acetate on Leukemogenesis

We found that the incidence of spontaneous myeloid leukemia in C3H/He male mice was less than 1%, but it could be increased considerably by total-body X irradiation. The induction of myeloid leukemia was seen to increase after doses from 0.47 Gy (3%) to 2.84 Gy (23.9%), and then decrease after a dose of 4.73 Gy (13.6%). The administration of prednisolone acetate (synthesized glucocorticoid) after irradiation resulted in a significant increase in the incidence of myeloid leukemia from 23.9 to 38.5% after a dose of 2.84 Gy; however, corticosterone, a glucocorticoid secreted by cells, did not have such an enhancing effect.

Exacerbating factors of radiation-induced myeloid leukemogenesis

The spontaneous incidence of myeloid leukemia in female mice was slightly higher than in male mice, whereas the radiation-induced incidence was significantly lower than in male mice. We also examined whether the incidence of myeloid leukemia was related to inflammatory response. Mice had a piece of cellulose acetate membrane inserted into the peritoneal cavity to cause inflammation. This did not affect the incidence of myeloid leukemia in unirradiated mice at all, but in 2.84 Gy irradiated mice the incidence (35.9% in male, 26.0% in female mice) increased significantly compared with irradiated-only mice (23.9% and 12.0%, respectively). From these results, the physiological fluctuation of humoral factors by means of inflammatory response is considered to increase the development of radiation-induced myeloid leukemia.

ROLE OF THE RETICULAR CELLS DURING MATURATION PROCESS OF THE ERYTHROBLAST

Through electron microscopic observations of the normal mouse spleen, the following findings were obtained concerning denucleation mechanism of the erythroblast:

Radiation-Induced Neoplastic Transformation of C3H10T1/2 Cells Is Suppressed by Ascorbic Acid

X-ray induced transformation of C3H10T1/2 cells was suppressed in a concentration-dependent manner by administration of ascorbic acid after irradiation (0.1-20 micrograms/ml for the first week) in the culture medium. The dose-response curve was shifted about 60% downward and was slightly steeper in the presence of ascorbic acid (5 micrograms/ml for the first week) than in its absence. The 1-week treatment procedure revealed that cells initiated by radiation remained susceptible to ascorbic acid until the time of morphological phenotype expression. The neoplastically transformed phenotype expressed after incubation for 8 weeks could no longer be suppressed by ascorbic acid even after culture transfer. Similarly, the neoplastically transformed phenotype suppressed for 8 weeks by ascorbic acid treatment was not subsequently expressed in the absence of ascorbic acid. On the basis of the oxygen-detoxifying nature of ascorbic acid, we postulated that expression of the neoplastically transformed phenotype is promoted by reactive oxygen species and peroxy radicals generated in cells during the whole assay period. The data may be useful as a guide for chemopreventive efforts against radiation carcinogenesis.

ROLE OF THE RETICULAR CELLS DURING MATURATION PROCESS OF THE ERYTHROBLAST REPORT III. THE FATE OF PHAGOCYTIZED NUCLEUS.

In the previous reports259 26), it was described that the erythroblasts in the pulp cord of normal mouse spleen formed the erythroblastic islet of BES.SIS~* 4. 5) and denucleation of erythroblasts was induced by the phagocytic activity of reticular cells. The phagocytized nucleus is digested in the cytoplasm of reticular cells. This phenomenon has been hitherto known as “ingestion of extruded normoblastic nuclei 15)”, and it was also reported previously from this laboratoryzs). This paper deals with the description of the detailed ingestive process as revealed by electron microscopical and histochemical studies.

Myeloproliferative disorder due to abnormal production of hematopoietic stimulators.

A new kind of myeloproliferative disorder (L-8313) has been discovered. It was transplantable into syngeneic mice with spleen cells. The mice showed hepato-splenomegaly with a marked leukocytosis and anemia 3 weeks after transplantation of L-8313 cells. The number of GM-CFU and CFU-S per spleen increased to more than 40 times normal. The results of chromosomal and PGK analysis demonstrated that these increased stem cells were of host origin. Both the culture medium of the spleen cells and the serum from L-8313 bearing mice showed high levels of IL-3, BPA and CSF. Consequently, hematopoietic cells of the host mice underwent remarkable proliferation in response to these stimulating factors when L-8313 cells were transplanted. We also have been successful in establishing an in-vitro cell line and have maintained it for over one year. The phenotype of L-8313 cells was Thy 1.2 positive. Some L-8313 cells showed a positive acid phosphatase reaction but the cytochemical character of myeloid lineage was not observed. Therefore, L-8313 is considered to be a T-cell derived hematopoietic regulatory cell neoplasm with the ability to produce several hematopoietic stimulating factors.

Improvement of anemia in W/Wv mice by recombinant human erythropoietin (rHuEPO) mediated through epo receptors with lowered affinity

We studied the effects of recombinant human erythropoietin (rHuEPO) on anemic W/WV mice which manifested severe anemia accompanied by mutation of the W gene encoding tyrosine kinase type receptor (c-kit gene) of bone marrow hematopoietic cells. Nine-week-old male W/WV mice or normal littermates (+/+) were used. Since serum EPO concentration in W/WV mice increased in proportion to severity of anemia, EPO production in the kidneys of these animals was found to be regulated normally. Hematocrit in +/+ mice increased and a maximal response was also obtained with 2,000 IU/kg of rHuEPO. On the other hand, hematocrit in W/WV mice increased in a dose-responsive manner by administration with 2,000 and 10,000 IU/kg, showing different responses to rHuEPO in these two types of mice. The responsiveness of W/WV mice to rHuEPO was low in terms of increases in erythroblastic precursor cells (CFU-E), and immature cells in the bone marrow. Scatchard analysis of the specific binding of 125I-rHuEPO against bone marrow cells revealed that the different responsiveness to rHuEPO between W/WV and +/+ mice may be correlated with differences in affinity of EPO receptor of bone marrow cells in these mice. From these results, a high dose of rHuEPO is capable of improving the anemia in W/WV mice that had EPO receptors with lowered affinity, indicating the possible effectiveness of rHuEPO in anemic patients with EPO receptor abnormality.

Granulocytic Leukemia, Mouse

The spleen is greatly swollen; its capsule is tightly stretched, and its surface is smooth. Its weight is increased several times (Table 11). The cut surface of the spleen may be dark red, yellow-white, or mottled by both colors. Rarely, it has a gray-green tone (chloroleukemia). The bone marrow is pale yellow and translucent. The liver is usually pale yellow and enlarged. Numerous small white branched patterns corresponding to Glisson’s capsule can be observed on its surface. The lymph nodes are white and moderately swollen (greenish in cases of chloroleukemia). Hemorrhages in the lung or other tissues are sometimes encountered.

Production of interleukin-3 from a T-cell neoplasm

We have established a new T-cell line (CL-8313) that produces interleukin-3 from the spleen cells of mice with a radiation-induced myeloproliferative disorder. IL-3 activity was detected at an extremely high level in the culture medium of the CL-8313 cell line in the absence of any exogenous stimulator. A large amount of IL-3 transcript also was demonstrated in CL-8313 cells. BPA- and CSF-activity was detected at a high level in the culture medium of the CL-8313 cell line. Southern blot analysis of high molecular weight DNA from the CL-8313 cells showed they had unique rearrangement of the antigen receptor beta chain gene. Therefore, we concluded that CL-8313 cells belonged to T-lineage lymphocytes and constitutively produced a high level of IL-3.

Effects of recombinant human granulocyte colony-stimulating factor on the growth potential of two murine myeloid leukemias

We investigated the in vitro and in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the proliferation of two murine leukemic cell lines. The rhG-CSF stimulated leukemic colony formation of the promyelocytic leukemic cell line L-8801 in methylcellulose culture and increased the number of L-8801 cells in liquid culture. However, rhG-CSF treatment prolonged the median survival period of mice implanted with L-8801 cells and the emergence of the leukemic blast cells in peripheral blood. Meanwhile, rhG-CSF had no influence on that of the megakaryoblastic leukemic cells L-8057 and failed to prolong the median survival period of L-8057 leukemic mice. Receptor binding analysis revealed that L-8801 cells expressed a G-CSF receptor (Kd=125 pM, 479 binding sites/cell) and L-8057 cells had no G-CSF receptors. Then, we examined the growth potential of these cells. The median survival period was longer for mice implanted with L-8801 cells cultured with rhG-CSF for 72 h in vitro than for cells grown without rhG-CSF. Furthermore, the median survival period of mice implanted with spleen cells from L-8801 leukemic mice treated with rhG-CSF was prolonged compared with those from leukemic mice without rhG-CSF. In contrast, there was no effect of rhG-CSF on the growth potential of the spleen from L-8057 leukemic mice. The results of our present study demonstrate that rhG-CSF reduced the growth of L-8801 leukemic cells in vitro and in vivo mediated through G-CSF receptors, thereby suppressing the development of leukemia.