Tetsuro Okabe

Treatment of Myelodysplastic Syndromes with Recombinant Human Granulocyte Colony-Stimulating Factor: A Preliminary Report

PURPOSE The expansion of an abnormal hemopoietic stem cell line is responsible for the myelodysplastic syndromes, which are characterized by pancytopenias, often resulting in lethal infections. Cloned granulocyte colony-stimulating factor (G-CSF) was recently shown to enhance the growth and differentiation of normal granulocyte progenitor cells in vitro. The aim of our study was to examine the effects of recombinant human G-CSF in patients with myelodysplastic syndromes. PATIENTS AND METHODS Four patients with myelodysplastic syndromes and one patient with smoldering acute myelogenous leukemia following the occurrence of a myelodysplastic syndrome received recombinant human G-CSF by intravenous infusion for six days. Patients received different dosage levels (50 to 1,600 micrograms/m2). RESULTS A response was seen in all patients, with an increase in both immature myeloid cells in the bone marrow and mature granulocytes in the peripheral blood. The dose levels that could stimulate granulocytopoiesis differed among patients. CONCLUSION These results suggest that, at least in some cases of myelodysplastic syndromes, granulocytopenia can be improved by G-CSF, although it still remains to be determined whether the increase in the number of granulocytes is due to the differentiation and maturation of the myelodysplastic clone or restoration of a residual normal clone.

Granulocytosis and colony-stimulating activity (CSA) produced by a human squamous cell carcinoma.

A patient with a squamous cell carcinoma accompanied by a marked granulocytosis (100,000/mm3) of unknown origin was examined for Colony‐Stimulating Activity (CSA). The pleural fluid and the tumor extract revealed high CSA. The floating cells in the pleural fluid were successfully transplanted into nude mice as a localized tumor with cyst formation. The tumor invariably caused a marked granulocytosis (100,000–300,000/mm3) with induction of a conspicuous splenic granulopoiesis in the transplanted mice. High CSA were demonstrated in their cystic fluid as well. Media conditioned by the primary cultures of these tumor cells revealed the same CSA, demonstrating the direct production of CSA by the tumor itself. These results indicate the presence of human CSA producing tumor and that such a tumor may in part account for a marked granulocytosis of unknown origin observed in some patients with cancer. Cancer 43:605–610, 1979.

Androgen Receptor-Dependent Activation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells: Role of Phosphatidylinositol 3-Kinase/Akt Pathway

The mechanisms of testosterone-induced vasodilatation are not fully understood. This study investigated the effect of testosterone on nitric oxide (NO) synthesis and its molecular mechanism using human aortic endothelial cells (HAEC). Testosterone at physiological concentrations (1-100 nm) induced a rapid (15-30 min) increase in NO production, which was associated with phosphorylation and activation of endothelial NO synthase (eNOS). Then, the involvement of the androgen receptor (AR), which is abundantly expressed in HAEC, was examined. The effect of testosterone on eNOS activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA. In contrast, testosterone-induced eNOS phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERalpha small interference RNA. 5alpha-Dihydrotestosterone, a nonaromatizable androgen, also stimulated eNOS phosphorylation. Next, the signaling cascade that leads to eNOS phosphorylation was explored. Testosterone stimulated rapid phosphorylation of Akt in a time- and dose-dependent manner, with maximal response at 15-60 min. The rapid phosphorylation of eNOS or NO production induced by testosterone was inhibited by Akt inhibitor SH-5 or by phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p85alpha subunit of PI3-kinase. In conclusion, testosterone rapidly induces NO production via AR-dependent activation of eNOS in HAEC. Activation of PI3-kinase/Akt signaling and the direct interaction of AR with p85alpha are involved, at least in part, in eNOS phosphorylation.

Production of bone-resorbing activity and colony-stimulating activity in vivo and in vitro by a human squamous cell carcinoma associated with hypercalcemia and leukocytosis.

A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.

Isolation and characterization of vitamin-A-storing lung cells

Vitamin A-storing cells have been shown to be distributed among various organs and tissues, including the lungs. In order to investigate this unique type of cell, the in vitro isolation has been carried out from rat lungs. Lungs were perfused with EGTA and collagenase solution in situ, and were digested with trypsin-collagenase solution at 60-min intervals for 2 h. Then, the cell suspensions obtained were incubated at 37 degrees C in F-10 medium supplemented with 10% fetal bovine serum (FBS) for 72 h. Non-adherent cells were then removed by vigorous washing with medium, and the resultant cell monolayer was harvested with trypsin to remove the contaminating macrophages. These cell fractions were shown to contain more than 96% of vitamin A-storing cells, judged by electron and fluorescence microscopic examinations. The cells grown in vitro retained well the overall morphology characteristic of the vitamin A-storing cells found in lung tissues. The isolated cells grew well in vitro and the growth was inhibited by D-valine or cis-hydroxyproline. The progeny of the cells still contained vitamin A lipid droplets after several transfer generations. Characteristic networks of fibronectin were also demonstrated around the cells. These results have shown that vitamin A-storing cells in the lung was successfully isolated from rat lungs and the cells possessed fibroblast-like characters storing vitamin A in small lipid droplets.

Establishment and characterization of a carcinoembryonic antigen (CEA)‐producing cell line from a human carcinoma of the exocrine pancreas

A human carcinoembryonic antigen (CEA)‐producing cell line, T3M‐4, has been established from explant cultures of a primary human pancreatic exocrine adenocarcinoma transplanted into nude mice. The tumor had metastasized in the patient. The tumor obtained from metastatic lymph nodes was the initial source for implantation in athymic nude mice. In the primary culture, host fibroblasts were eliminated by the use of the antiserum raised against nude mouse cells. T3M‐4 cells have been continuously propagated in vitro during the past 26 months. The cells grew in a monolayered sheet with about 31 hours of population doubling time. The cells exhibited epithelial morphologic features resembling the structure of the original tumor, and they showed tumor takes when inoculated into athymic nude mice. Xenografts established from the cell line have retained a similar histology to the original tumor on serial transplantation. Chromosomal analysis revealed the cell line to be a human aneuploid one with a hyperdiploid mode. T3M‐4 cells possess the characteristic function of CEA secretion in vitro in culture and in vivo in nude mice bearing the tumors produced by inoculation with the cultured cells. In view of these characteristics, T3M‐4 cell line represents a new human pancreatic exocrine adenocarcinoma cell line that produces CEA.

1α,25-Dihydroxy vitamin D3 (calcitriol) stimulates proliferation of human circulating monocytes in vitro

Previous studies demonstrated that human circulating monocytes can proliferate in vitro when incubated with lectin‐induced factor(s) from lymphocytes [(1985) Biochem. Biophys. Res. Commun., in press]. This study shows that human monocytes were induced to proliferate when incubated with 1α,25‐dihydroxy vitamin D3 (calcitriol) at physiological concentrations. The optimal dose was about 10 nM. Proliferative activity was examined both by measuring the [su3H]thymidine incorporation and by counting cell nuclei. Among other derivatives of vitamin D3, 1α,24 R‐dihydroxyvitamin D3 and 1α,24R,25‐trihydroxyvitamin D3 stimulated mitotic activity of monocytes. Addition of both calcitriol and lectin‐stimulated lymphocyte‐conditioned medium to the monocyte culture had an additional effect on the mitotic activity of monocytes.

Ginsenoside Rb1 Prevents MPP+-Induced Apoptosis in PC12 Cells by Stimulating Estrogen Receptors with Consequent Activation of ERK1/2, Akt and Inhibition of SAPK/JNK, p38 MAPK

Ginsenoside Rb1 shows neuroprotective effects in various neurons, including dopaminergic cells. However, the precise mechanisms of action are uncertain. In this paper, we examine whether Rb1 has a neuroprotective effect on MPP+-induced apoptosis and attempt to clarify the signaling pathway in PC12 cells. Apoptosis of PC12 cells was determined by DNA fragmentation assay, the activation of caspase-3, or by the inactivation of Bcl-xL. Rb1 inhibited MPP+-induced caspase-3 activation and DNA fragmentation and activated Bcl-xL in MPP+-treated PC12 cells. These antiapoptotic effect was abrogated in PC12 cells transfected with estrogen receptor siRNA. Levels of DNA fragmentation were increased by wortmannin or PD 98059, while they were decreased by SB 203580 or SP 600125 in MPP+-treated PC12 cells. Rb1 increased phosphorylation levels of ERK1/2 or Akt in MPP+-treated PC12 cells, while it reduced phosphorylated p38 or SAPK/JNK. The increased phosphorylation of ERK/1/2 or Akt by Rb1 was abrogated by estrogen receptor siRNA. Rb1-induced inhibition of SAPK/JNK or p38 phosphorylation was also abolished by estrogen receptor siRNA. These results suggest that ginsenoside Rb1 protects PC12 cells from caspase-3-dependent apoptosis through stimulation of estrogen receptor with consequent activation of ERK1/2 and Akt and inhibition of SAPK/JNK and p38.

Activation of the c-K-ras oncogene in a human pancreas carcinoma

The human pancreas carcinoma cell line T3M-4 contains activated c-Kirsten (K)-ras oncogene detectable by the DNA-mediated gene transfer technique using NIH/3T3 cells. DNA fragments containing coding lesions have been cloned, and nucleotide sequence analysis suggests that the T3M-4 oncogene has been activated by a single nucleotide transition from A to C in the second exon, which results in the substitution of histidine for glutamine in coden 61 of the predicted amino acid sequence. The quantity analysis of c-K-ras oncogene in the DNA and RNA of T3M-4 cells revealed that the c-K-ras gene was amplified and overexpressed in T3M-4 cells. These findings indicate that the T3M-4 c-K-ras oncogene is activated by different mutational events.

A platelet factor that stimulates the proliferation of vascular endothelial cells

The effects of platelet factors on the growth of cultured porcine aortic endothelial cells were studied. Human platelet lysate stimulated the incorporation of [3H] thymidine into DNA. Gel chromatography on Sephadex G-75 revealed at least two peaks of activity on endothelial cells, the major peak being at an apparent molecular weight of 20,000. This activity was heat-labile and trypsin-sensitive, and did not stimulate the growth of fibro-blasts.