No abstract

Hepatocyte growth factor (HGF) and its receptor, the product of c-MET proto-oncogene, are highly expressed in both fetal and adult lung, though their physiologic functions in the lung are largely unknown. In the present study, we examined whether alveolar type II cells in the lung are the target of HGF and whether HGF has any effects on growth of these cells. The alveolar epithelial type II cells were isolated from the lungs of adult male Sprague-Dawley rats by elastase digestion, and the cells were used to determine whether they express HGF and c-MET mRNAs and whether recombinant HGF has any effect on their DNA synthesis in primary culture. The effects were further compared with those induced by epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), transforming growth factor-alpha (TGF-alpha), and transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis and in situ hybridization revealed that type II cells express c-MET mRNA but not HGF mRNA. HGF stimulated [3H]thymidine incorporation into type II cells in primary cultures. An increase was also seen in labeling index as determined by nuclear immunostaining of bromodeoxyuridine-incorporated DNA. While aFGF (200 ng/ml) exerted an effect comparable to HGF (25 ng/ml) on DNA synthesis in type II cells, EGF (20 ng/ml) and TGF-alpha (100 ng/ml) had lesser effects. TGF-beta 1, a potent inhibitor of epithelial cell proliferation, at 0.25 to 2 ng/ml, did not inhibit HGF-induced [3H]thymidine incorporation into type II cells. The results indicate that HGF exerts its effects on type II cells as a potent mitogen by a paracrine mode of action.

KW‐2189 has been selected as a lead compound for clinical trial among duocarmycin derivatives with structural similarity to CC‐1065, a cyclopropylpyrroloindole. The purpose of this study was to examine the DNA‐binding potency and the mechanisms of cytotoxicity of KW‐2189. In order to analyze DNA‐binding activity of KW‐2189, plasmid pBR322 was treated with KW‐2189 with or without pretreatment with carboxyl esterase, which we demonstrated to be an activating enzyme, and the products were examined by agarose gel electrophoresis and restriction enzyme analysis. Cytotoxic activity was examined by exposing a human small cell lung cancer cell line, NCI‐H69 to KW‐2189 with or without carboxyl esterase. Alkaline elution was performed to examine whether KW‐2189 induces DNA strand breaks. DNA treated with KW‐2189 and carboxyl esterase migrated faster than KW‐2189‐treated DNA, which migrated at the same rate as untreated DNA. In addition DNA treated with esterase‐activated KW‐2189 was protected from digestion by some restriction enzymes. KW‐2189 showed concentration‐ and time‐dependent growth inhibitory effect with IC50 values (drug concentration required for 50% growth inhibition) of 58 nM (96 h) to 1900 nM (1 h) in H69 cells. The IC50 values of 4‐h exposure of H69 to KW‐2189 with 0, 26, 130, 650 mU/ml carboxyl esterase were 460, 120, 30, and 7 nM, respectively. Time‐dependent enhancement of cytotoxicity by carboxyl esterase was also observed. KW‐2189 induced DNA strand breaks in H69 cells in a concentration‐dependent manner around the IC50 value. We conclude that 1) KW‐2189 is activated by carboxyl esterase to its active form(s), 2) activated KW‐2189 has a stronger DNA‐binding activity and cytotoxicity than KW‐2189, 3) DNA cleavage is one of the major mechanisms of KW‐2189‐mediated cytotoxicity.

No abstract

In an attempt to develop the most effective cytokine gene therapy, we transfected mouse interleukin(IL)-2, mouse IL-4, and human IL-6 cDNAs into mouse melanoma cells, B16F10. Transfection with IL-4 cDNA decreased the tumorigenicity of B16F10 most strongly. We investigated whether gene therapy with IL-4-transfected B16F10 cells was possible. Flow-cytometric analysis showed that major histocompatibility complex class I and II expression in B16F10 and IL-4-cDNA-transfected B16F10 (B16F10-IL4) cells did not differ. Doubling times of B16F10 and B16F10-IL4 were 20.1 and 21.1 h respectively. The growth of B16F10 cells was retarded if C57BL/6 mice were inoculated with B16F10-IL4 at the contralateral sides. When 5 x 10(5) B16F10 cells were transplanted subcutaneously into the flanks of C57BL/6 mice, they all developed a tumor mass, whereas no tumor masses formed in those transplanted with B16F10-IL4 cells within 60 days. No nude, severe combined immunodeficient or beige mice were able to reject parental B16F10 or B16F10-IL4 cells, although, B16F10-IL4 tumor growth in all these immunodeficient mice was slower than that of B16F10. Therefore, we concluded that T and natural killer cells are necessary for rejection of B16F10-IL4 tumor cells.