Shin-ichi Kaiho

Synthetic analogues of vitamin D3 with an oxygen atom in the side chain skeleton A trial of the development of vitamin D compounds which exhibit potent differentiation‐inducing activity without inducing hypercalcemia

Four analogues of vitamin D3 with an oxygen atom in the side chain skeleton were synthesized to determine whether their differentiation‐inducing activity could be separated structurally from their activity to induce hypercalcemia. The order of the in vitro potency to reduce nitroblue tetrazolium in human myeloid leukemia cells (HL‐60) was 22‐oxa‐1α25‐(OH)2D3 > 1α,25‐(OH)2D3 > 20‐oxa‐1α,25‐(OH)2D3≒22‐oxa‐1α‐(OH) D3 > 1α‐(OH)D3 > 20‐oxa‐1α‐(OH)D3. 22‐Oxa‐1α,25‐(OH)2D3, was also about 10‐times more potent than 1α,25‐(OH)2D3 in suppressing proliferation and inducing differentiation of mouse myelomonocytic leukemia cells (WEHI‐3), but the former was much weaker than the latter in inducing the release of 45Ca from prelabeled fetal mouse calvaria. These results suggest that the differentiation‐inducing activity of vitamin D compounds can be separated structurally from their activity to induce hypercalcemia.

Sensitive assay systems for detection of hemoglobin with 2,7-diaminofluorene: Histochemistry and colorimetry for erythrodifferentiation

Sensitive and rapid assays, colorimetry and histochemistry, for hemoglobin in erythroid cells are established. The assays are based on pseudoperoxidase activity of hemoglobin using 2,7-diaminofluorene as a hydrogen donor for the peroxidase, instead of benzidine which is widely benzidine which is widely used for the detection of small amounts of hemoglobin but which is a potent carcinogen and has been banned from laboratory use. In the presence of hydrogen peroxide, hemoglobin catalyzes the formation of a blue compound (fluorene blue), which has a broad absorption band between 500 and 690 nm with a peak at 610 nm, from 2,7-diaminofluorene. The reagent is safe to use in the laboratory. The methods could be applied to the detection of hemoglobin in Friend erythroleukemia cells induced to cell differentiation along the erythroid pathway by dimethyl sulfoxide.

Reorganization of microfilaments in macrophages after LPS stimulation.

Lipopolysaccharide (LPS), a potent activating substance of macrophages, induced the reorganization of microfilaments in macrophages obtained from C3H/HeN mice. At 1 min after LPS addition, a slight disassembly of actin was observed. At 2 to 4 min, there was a gradual assembly; then, at 5 and 6 min, a subsequent rapid disassembly occurred. We employed two methods to observe this process. One was the RITC-phalloidin staining of actin filaments and the other was the extraction of monomeric actin and unstable actin filaments with Triton X-100 solution. The results obtained by the two methods were basically in agreement. Nevertheless, there was a discrepancy between the results from the two methods, concerning the ratio of assembly and disassembly. The RITC-phalloidin staining was more sensitive in detecting actin assembly and less sensitive in detecting the disassembly than the extraction with Triton X-100 solution was. This difference suggests that some of the unstable filaments, which were extracted with Triton X-100 solution and fixed with formalin, were formed during the LPS-induced reorganization process. This reversible actin assembly could not be observed in the LPS-nonresponder, C3H/HeJ mouse macrophages. We concluded that the observed process could be attributed to LPS-signal triggering pathways subsequent to LPS binding and that a necessary component to initiate effective LPS-signaling, which is probably deficient in C3H/HeJ mice, is involved in this reorganization process of LPS-stimulated macrophages.

Association of CD44 with OTS-8 in tumor vascular endothelial cells

Endothelial cells in solid tumors play an important role in tumor growth, invasion and metastasis through angiogenesis. We have recently cloned two tumor vascular antigens from isolated rat tumor vascular endothelial cells (TEC). One is CD44, a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. The other is OTS-8, a marker for osteoblast into osteocyte transition and type I alveolar epithelial cells termed as E11 antigen and RTI40, respectively. To test for a possible interaction between the two antigens on endothelial cells in tumor angiogenesis, we examined in vivo association of CD44 with OTS-8 using lysates of isolated rat TEC and COS-7 cells cotransfected with CD44 and OTS-8 expression plasmids. The association was detected by direct co-immunoprecipitation of the two types of cells lysed with digitonin, whereas the detection was lost when lysed with Nonidet P-40. To confirm this association, intact COS-7 cells cotransfected were reacted with homobifunctional N-hydroxysuccinimide ester crosslinking reagents. Immunoblot analysis showed a crosslinked CD44/OTS-8 protein complex of 120 kDa, suggesting the proximity of the two proteins. These findings provide evidence of a weak physical association between CD44 and OTS-8 in TEC, and suggest that OTS-8 may alter the mode of endothelial cell growth and/or migration induced by CD44 in tumor angiogenesis.

Gonadotropins Stimulate Growth of MCF-7 Human Breast Cancer Cells by Promoting Intracellular Conversion of Adrenal Androgens to Estrogens

Estrogen receptor (ER)-positive breast cancers initially respond well to estrogen ablation treatment but finally acquire refractoriness, the phenomenon that is a major clinical problem. Because some breast cancers synthesize estradiol (E2) and E2 synthesis is regulated by gonadotropins in normal ovaries, and because circulating gonadotropins are elevated in postmenopausal women and during estrogen ablation treatment, we hypothesized that gonadotropins might modulate estrogen synthesis/metabolism in breast cancer tissue as well. To test this possibility, MCF-7 cells were treated with dehydroepiandrosterone (DHEA) or human chorionic gonadotropin (hCG; ∼LH), each alone or in combination. Cell growth (3-day treatment) was assayed by the MTT method and estrogen synthesis (24-hour treatment) was measured using the ERE-luciferase reporter system. First, MCF-7 cell growth was stimulated by DHEA in a concentration-dependent manner with a maximal effect at 10–4M. Although hCG alone did not have a significant proliferative effect, hCG significantly and dose dependently stimulated MCF-7 cell growth in the presence of a submaximal concentration of DHEA (10–7 M). This stimulatory effect of DHEA and hCG was blocked by a pure antiestrogen ICI182,780 and an aromatase inhibitor, arimidex. Using MCF-7 cells transfected with the ERE-luciferase reporter system, hCG treatment was shown to increase ERE-mediated transcription. These results indicate that MCF-7 cells intrinsically converted DHEA into E2 upon hCG stimulation, then grew their own cells DHEA- and hCG-dependently. We conclude that gonadotropins can act on breast cancer cells and accelerate conversion of DHEA into estrogens, thereby stimulating growth of estrogen-dependent tumor cells. This phenomenon, at least in part, could explain: (1) an increased tissue concentration of E2 in postmenopausal breast cancer; (2) acquisition of hormone refractoriness during estrogen ablation treatment, and (3) the effectiveness of GnRH antagonist/superagonist in some postmenopausal breast cancer patients.

Effective Cancer Targeting Using an Anti‐tumor Tissue Vascular Endotheliumspecific Monoclonal Antibody (TES‐23)

Immunoconjugate targeting of solid tumors has not been routinely successful because the endothelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an antitumor tissue endothelium‐specific monoclonal antibody (TES‐23). TES‐23, an IgG1 monoclonal antibody raised against rat KMT‐17 fibrosarcoma‐derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES‐23‐NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti‐tumor effects against rat KMT‐17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of 125I‐labeled TES‐23 into BALB/c mice bearing Meth‐A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES‐23 was greater than that of the control IgG. In the present study, we report the anti‐tumor effects of this monoclonal antibody in mice bearing Meth‐A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES‐23 might recognize these antigens.

Molecular cloning and characterization of antigens expressed on rat tumor vascular endothelial cells.

We have previously prepared monoclonal antibodies (MAbs) against tumor vasculature using cultured rat tumor endothelial cells (TECs) isolated from solid KMT‐17 tumors and identified 40 and 80 kDa antigens recognized by TES‐17 and TES‐23 MAbs, respectively. To clarify the nature of antigens on tumor vasculature, molecular cloning was conducted by screening the rat TEC cDNA library. Two antigens were detected: a 40 kDa OTS‐8 antigen, which had been defined as a differentiation marker for osteoblastic lineage, and an 80 kDa CD44H antigen. Northern blots showed that OTS‐8 mRNA was expressed exclusively in the lung, in addition to TECs, while CD44H mRNA was detected in the lung, intestine, spleen, thymus and peripheral blood cells, in addition to TECs. Immuno‐histochemistry of KMT‐17 tumors revealed that OTS‐8 and CD44 were expressed on sprouting TECs. In addition, TES‐23 MAb stained TECs of tubular vessels as well as sprouting TECs, but anti‐rat CD44 MAbs stained only sprouting TECs. PCR showed that CD44 cDNA with a splice in exon 6 (CD44ex6) was present in rat TECs at low levels. Our results indicate that OTS‐8 and CD44 are expressed on rat sprouting TECs and that TES‐23 MAb recognizes CD44H on sprouting TECs as well as an unknown epitope on TECs of tubular vessels that could not be recognized by anti‐rat CD44 MAbs. Int. J. Cancer 86:799–805, 2000.

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats : Involvement of intravascular thrombosis and fibrinogenesis

We have reported that immunization of rat tumor‐derived endothelial cells (TEC) isolated from KMT‐17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES‐23, one of these MAbs, recognizes a naturally occurring 80‐kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of 125I‐labeled TES‐23 in rats bearing KMT‐17 solid tumors. We also examined the effect of treatment using unconjugated TES‐23 on tumor growth and histo‐pathological changes in tumor tissues. Biodistribution studies showed localization of TES‐23 into tumor tissues 60 min after intravenous injection. TES‐23 suppressed significantly the growth of KMT‐17 solid tumors following administration for 5 days. Histo‐pathological examination showed that TES‐23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES‐23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors. Int. J. Cancer 82:853–859, 1999.

Inhibition of Friend cell erythrodifferentiation by modification of membrane phospholipid composition by choline analogues.

Dimethylsulfoxide-stimulated Friend leukemia cell erythrodifferentiation was inhibited by choline analogues such as N-monomethylethanolamine and N,N-dimethylethanolamine. Phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine were then accumulated in the cell membranes. N-Monomethylethanolamine also inhibited Friend leukemia cell erythrodifferentiation stimulated by hexamethylene bisacetamide and N-methylacetamide, but did not inhibit differentiation induced by sodium butyrate. This inhibitory effect of N-monomethylethanolamine was partially abrogated by spermine.

Effect of immuno-modulator Lobenzarit-2Na (CCA) on cytoskeleton - II. Effect on response of microfilament in LPS-stimulated macrophages.

We reported in a preceding paper that Lobenzarit-2Na (CCA) affected RNA polymerase-I activity in nuclear suspension prepared from rat hepatocytes. This effect of CCA on RNA polymerase-I was thought to be the secondary effect, that is, CCA primarily modified the reactivity of cytoskeletal components around the nuclei.Previously it was also reported that CCA inhibited the IL-1 production by LPS-stimulated macrophages. So we studied whether CCA could modify the cytoskeletal re-organization process in LPS-stimulated macrophages or not.Results reported here show that microfilament system in macrophages changes its configulation in a biphasic manner within 10 min after LPS stimulation. CCA reduced the magnitude of this configurational change without affecting the timing of process.Because CCA could be transported into macrophages through some kind of carrier in plasma membrane, this effect of CCA on microfilament system was likely to be the direct action in cytoplasm.