Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P

Inhibitory effects of heparin plus cortisone acetate on endothelial cell growth both in cultures and in tumor masses.

A combination of heparin and cortisone acetate significantly inhibited both embryonic angiogenesis and the tumor growth of Lewis lung carcinoma (3LL) transplanted into C57BL/6 mice, although each of these agents used alone affected neither angiogenesis nor tumor growth. On the other hand, this combination neither decreased the number of metastatic foci in the lung nor prolonged the survival time of mice with 3LL. All tumor-bearing mice died of hemothorax due to pulmonary metastases. Cortisone acetate by itself increased metastasis, and addition of heparin did not affect accelerated metastasis. Because an antiangiogenic activity appears independent of metastasis acceleration by cortisone acetate, the use of steroids other than cortisone acetate having no metastasis-promotion effect should be required for an antiangiogenic tumor therapy in the presence of heparin. Heparin plus cortisone acetate prevented the DNA synthesis of cultured vascular endothelial cells but not that of cultured 3LL cells. Additionally, oral administration of this combination decreased the [3H]thymidine labeling of endothelial cells of tumor blood vessels prior to the suppression of tumor growth. The specific inhibition of the growth of endothelial cells by heparin plus cortisone acetate was revealed in both the in vitro and the in vivo tests.

The primary structure of rat rig/ribosomal protein S15 gene.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.

Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (homologue to human CD38) from islets of Langerhans.

We report the cloning and cDNA sequence of rat CD38, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase. Rat CD38 is composed of 303 amino acids and shares a high degree of homology with human and mouse CD38. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.

Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF120918.

This study was aimed at characterizing the role of BCRP/MXR/ABCP (BCRP) in resistance of the human ovarian tumor cell lines T8 and MX3 to camptothecins more extensively and investigating whether resistance can be reversed by inhibiting BCRP by GF120918. Camptothecins studied were topotecan, CPT-11, and its active metabolite SN-38, 9-aminocamptothecin, and the novel experimental camptothecins NX211, DX8951f, and BNP1350. Notably, DX8951f and BNP1350 appeared to be very poor substrates for BCRP, with much lower resistance factors observed both in T8 and MX3 cells than observed for the other camptothecins tested. In the presence of a nontoxic dose level of GF120918, the intracellular accumulation of topotecan in the T8 and MX3 cells was completely restored to the intracellular levels observed in the sensitive IGROV1 parental cell line. This resulted in almost complete reversal of drug resistance to topotecan and to most of the other topoisomerase I drugs tested in the T8 cell line and to complete reversal in the MX3 cells. However, coincubation of DX8951f or BNP1350 with GF120918 did not affect the cytotoxicity of either of these drugs significantly. From the combined data, we conclude that the affinities of topoisomerase I drugs for BCRP are, in decreasing order: SN-38 > topotecan > 9-aminocamptothecin approximately CPT-11 > NX211 > DX8951f > BNP1350. Furthermore, GF120918 appears to be a potent reversal agent of BCRP-mediated resistance to camptothecins, with almost complete reversal noted at 100 nM. Potential BCRP-mediated resistance to topoisomerase I inhibitors can also be avoided by using the BCRP-insensitive drugs DX8951f or BNP1350. This observation may have important clinical implications for future development of novel camptothecins.

Human gene encoding CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase): organization, nucleotide sequence and alternative splicing.

We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as a second messenger for glucose-induced insulin secretion [Takasawa et al. (1993a) Science 259, 370-373] and that CD38 has both ADP-ribosyl cyclase (ADRC) and cADPR hydrolase activities [Takasawa et al. (1993b) J. Biol. Chem. 268, 26052-26054]. In this study, we determined the structure of the human CD38 gene, and showed that two mRNA forms originated by alternative splicing from the CD38 gene. The human CD38 gene consists of 8 exons that extend more than 77 kb on the human genome. Exon 1 encoded the 5'-untranslated region of the mRNA, the N-terminal end of CD38 and the putative transmembrane domain, and exon 2-8 encoded the remainder of CD38: the exon-intron organization of the human CD38 gene is similar to that of the Aplysia ADRC gene [Nata et al. (1995) Gene 158, 213-218]. This structural conservation between human and Aplysia genes suggests that both genes may have evolved from a common ancestral gene.

The structure of the Aplysia kurodai gene encoding ADP-ribosyl cyclase, a second-messenger enzyme.

The complete nucleotide (nt) sequences of the cDNA and gene encoding the marine mollusk Aplysia kurodai (Ak) ADP-ribosyl cyclase (ADRC) which synthesizes cyclic ADP-ribose (cADP-ribose), a second messenger for Ca2+ mobilization from endoplasmic reticulum, were determined. Ak ADRC consists of 258 amino acids (aa) (29 kDa). It shares 86% aa sequence homology with that from A. californica, and 31-32% homology with the human, rat and mouse cluster of differentiation 38 (CD38) that has both ADRC and cADP-ribose hydrolase activities. The Ak ADRC-encoding gene (ADRC) spans approx. 7 kb and contains eight exons and seven introns. The transcription start point (tsp) determined by primer extension analysis and S1 mapping is 28 bp downstream from the TATA box. This gene is expressed specifically in the ovotestis, although the mammalian CD38-encoding gene is expressed in many kinds of tissues and cells. The 5'-flanking region contains several consensus sequences responsible for the germ-cell-specific expression of the mouse zona pellucida 3 (ZP3) and Drosophila melanogaster chorion genes. The existence of the consensus sequences located at nt -1649, -1161, -234 and -90 may account for the ovotestis-specific expression of the Ak ADRC gene.

Structural determination of Saccharomyces cerevisiae rig gene and identification of its product as ribosomal protein S21.

rig was originally isolated from a rat insulinoma-derived cDNA library. The 145 amino acid sequence of the rig protein is invariant in mammalian cDNAs. In this paper, we have isolated the cDNA and genomic clones for yeast (Saccharomyces cerevisiae) rig, determined their nucleotide sequences, and identified the gene product. The gene and the mRNA encode a basic protein of 142 amino acids which has 61.3% amino acid identity with mammalian rig protein. On two-dimensional gel electrophoresis, the in vitro transcription/translation product of yeast rig cDNA co-migrated with yeast ribosomal protein S21. These results led to the conclusion that yeast rig ribosomal protein S21 and to the determination of the previously unknown primary structure of yeast S21 protein. Unlike most ribosomal protein genes of S. cerevisiae, the gene exists as a single copy in a haploid set of the yeast genome and has no intron, locating at chromosome VII or XV.

Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit.

We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29-37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa laminin binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecular mass of 33,000 which lacked typical signal sequences, N-linked glycosylation sites and putative transmembrane domains. These results indicate that the cDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.

New aspects of the physiological significance of NAD, poly ADP-ribose and cyclic ADP-ribose.

Cyclic ADP-ribose is generated from NAD+ in glucose-stimulated beta-cells by CD38. Cyclic ADP-ribose mobilizes Ca2+ from the endoplasmic reticulum to secrete insulin. The amino acid residues of Cys-119 and Cys-201 in CD38 are essential for the synthesis and hydrolysis of cyclic ADP-ribose.

Potent and broad antitumor effects of DX-8951f, a water-soluble camptothecin derivative, against various human tumors xenografted in nude mice.

PURPOSE: We have previously reported that DX-8951f, a water-soluble and nonprodrug camptothecin (CPT) derivative, exhibits both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The purpose of this study was to evaluate the therapeutic efficacy of DX-8951f against human tumor xenografts in nude mice and to compare its activity with those of CPT-11 and other current CPT derivatives. METHODS: The antitumor activity of DX-8951f against xenografts of several different types of human tumors was determined in nude mice using a schedule in which DX-8951f was administered intravenously every 4th day for a total of four injections. RESULTS: Against both gastric adenocarcinoma SC-6 and its CPT-11-resistant variant, SC-6/CPT-11, DX-8951f demonstrated superior antitumor activity and antitumor activity over a broader range of doses than did CPT-11, SK&F104864 (hycamtin, topotecan) and GG-211 (GI147211). DX-8951f at 75 mg/kg was effective (growth inhibition rate IR > or = 58%) against 15 of 16 lines of human cancers examined (6 colon cancers, 5 lung cancers, 2 breast cancers, 1 renal cancer and the above 2 gastric cancers), and exhibited excellent antitumor activity (IR > or = 80%) against 14 of these lines. CPT-11 exhibited antitumor activity with IR values of 58% and higher against 11 lines and IR values of 80% and higher against only eight of the same 16 human tumors. DX-8951f was effective in inhibiting the growth of an SN-38-resistant tumor and some P-glycoprotein-expressing tumors, but CPT-11 was not. CONCLUSIONS: DX-8951f exhibited potent antitumor activity against various types of human tumor xenografts. Its in vivo antitumor effects were superior to those of current camptothecin analogs against certain tumors.

Systemic administration of a synthetic lipid A derivative, DT-5461a, reduces tumor blood flow through endogenous TNF production in hepatic cancer model of VX2 carcinoma in rabbits.

Intravenous administration of DT-5461a, synthetic low-toxicity lipid A derivative, significantly inhibited the growth of VX2 tumor transplanted in the liver of rabbits. DT-5461a induced high levels of TNF activity in tumor tissue from 30 to 60 min after the administration, while no TNF activity was detected in the adjacent nontumorous liver tissue. Simultaneous measurement of microcirculatory blood flow in both tumorous and nontumorous regions in the liver by laser doppler velocimetory revealed that intravenous administration of DT-5461a caused a significant blood flow reduction in tumor region, but not in nontumorous counterparts. Tumor blood flow was significantly reduced by 40 to 60% at 30 to 90 min after the DT-5461a administration as compared with preadministration value. In contrast, local administration of human recombinant TNF alpha through the hepatic artery induced blood flow reduction not only in tumor region but also in nontumorous liver tissue. These results suggest that systemic administration of DT-5461a induced selective tumor microcirculatory blood flow reduction via local endogenous TNF production.

DT-5461a, an antitumor synthetic lipid a analog, causes selective blood flow reduction in tumor tissue.

We previously reported that a synthetic low-toxicity lipid A analog, DT-5461a, exhibited a significant antitumor effect was characteristically accompanied by extensive tumor necrosis, suggesting that DT-5461a causes a local circulatory disturbance in tumor tissues. In this study, we investigated the effect of DT-5461a on regional blood flow in various organs including tumor tissue with a radiolabeled tracer-distribution technique using 14C-iodoantipyrine. Intravenous administration of DT-5461a induced blood flow reduction in Meth A tumor subcutaneously implanted into BALB/c mice, but not in liver, spleen or lung of these mice. This tumor tissue-specific reduction in blood flow was significantly inhibited by pretreatment with antisera against tumor necrosis factor (TNF) alpha, interferon (IFN) alpha/beta, and IFN gamma. These results indicate that endogenously induced cytokines, namely TNF alpha and IFNs, are involved in the intratumor blood flow reduction caused by DT-5461a.

Effects of DN-9693, a new metastasis inhibitor, on murine hematogenous metastasis.

1,5-Dihydro-7-(1-piperidinyl)-imidazo[2,1-b]quinazolin-2(3H)-one dihydrochloride hydrate (DN-9693), a new c-AMP: phosphodiesterase inhibitor was examined for its inhibitory effects on platelet aggregation induced by metastasizing tumor cells and on blood-borne metastases of these tumors. 1-3 microM of DN-9693 completely inhibited platelet aggregation induced by B16 melanoma subline BL6 and Lewis lung carcinoma (3LL) cells. Platelets prepared from mice intravenously or orally administered with DN-9693 failed to aggregate after the addition of BL6 cells. Intravenous injection of DN-9693 was effective in protecting the mice inoculated with 1 X 10(6) BL6 cells against acute pulmonary embolic death. Either intravenous or oral administration of DN-9693 (1-10 mg/kg) sufficiently suppressed thrombus formation and subsequent pulmonary metastasis caused by intravenously inoculated BL6 or 3LL cells. Spontaneous pulmonary metastasis of 3LL was also inhibited by DN-9693. Continuous administration of DN-9693 during and after surgical excision of the primary tumors was the most effective treatment against the development of pulmonary metastases of 3LL.

Quantification of experimental pulmonary metastases by image analyzer.

Image analysis techniques for quantifying experimental pulmonary metastases were developed using Lewis lung carcinoma (3LL) and B16 melanoma. Binary images of the metastatic colonies were differentiated by staining the lung parenchyma. Thus, it became possible for the image analyzer to determine automatically the size and number of the metastatic foci. Image analysis was used successfully to evaluate the inhibitory effect of adriamycin on spontaneous metastases of 3LL. Although the inhibitory effect was not detected by the usual counting method, a significant reduction of the area of metastatic foci was detected by image analysis.

Antitumour activity of DX-8951f: a new camptothecin derivative.

DX-8951f is a water-soluble camptothecin analogue with a unique hexacyclic structure. Compared to other current camptothecin derivatives, DX-8951f is the most effective topoisomerase I (topo I) inhibitor and has the most potent cytotoxic activity against various tumour cell lines in vitro. Of particular interest is DX-8951f's significant effect on certain tumour cell lines resistant to other camptothecin derivatives, as well as on multi-drug resistant variants that overexpress P-glycoprotein. In addition, in in vivo xenograft systems using nude mice, DX-8951f strongly inhibits the growth of human solid tumours, including resistant tumours. Its antitumour effects and resulting life prolongation in tumour-bearing mice have also been confirmed in several metastasis models. DX-8951f provides greater therapeutic efficacy and broader effective dose ranges using multiple injections than with a bolus injection and simple intermittent applications. The in vivo effects of the compound are superior to those of CPT-11 and SK&F104864, suggesting that DX-8951f is a promising therapeutic agent for the treatment of cancer patients. Phase I clinical trials are ongoing in Europe, the USA and Japan.

Effect of combined administration of a synthetic low-toxicity lipid A derivative, DT-5461a, and indomethacin in various experimental tumor models of colon 26 carcinoma in mice.

We investigated the antitumor effects of a synthetic lipid A derivative, DT-5461a, in combination with indomethacin in three experimental tumor models (peritoneal carcinomatosis, liver tumor, and lung tumor models) of transplanted colon 26 carcinoma in mice. This carcinoma produces the immunosuppressive prostaglandin E2 (PGE2). Intravenous administration of DT-5461a alone resulted in little or no prolongation of survival time [increase in life span (ILS): -2%-22%]. When indomethacin was given in drinking water a slight or moderate increase in survival time was seen (ILS: 4%-45%). In contrast, the combination of DT-5461a and indomethacin induced an additive increase in life span (ILS: 16% to more than 193%). The strongest antitumor effect of this combined therapy was seen in the peritoneal carcinomatosis model; in this model, plasma PGE2 concentrations were considerably higher than in normal mice, and concentrations were further but transiently increased by DT-5461a administration. Following oral indomethacin administration, these elevated PGE2 concentrations were reduced to the level in untreated normal mice. Furthermore, intratumoral tumor necrosis factor (TNF) activity in the group receiving the combined therapy was significantly higher than that in the DT-5461a-treated group. No TNF production was induced by the administration of indomethacin alone. These results suggest that the antitumor effect of DT-5461a can be enhanced by combination with indomethacin, and that the inhibition of PGE2 production may have a role in this antitumor effect.

Cytostatic action of human fibroblast interferon in direct growth inhibition: effect on human malignant melanoma.

The mode of action of the direct antiproliferative effect of human fibroblast interferon (HuIFN-beta) on tumor cells was analyzed in vitro with a human malignant melanoma cell line, HMV-1. HuIFN-beta inhibited the growth of HMV-1 cells in a time-dependent fashion (50%-inhibition concentration: less than 50 IU/ml). Its action was cytostatic. DNA synthesis was inhibited before the antitumor effect appeared, and apparent dose-dependent inhibitions of RNA and protein synthesis were induced. An increase of cell protein content was seen with the occurrence of growth inhibition. Increase of cell volume and prolongation of doubling time were seen from the second day after the addition, which corresponded well with the effects of HuIFN-beta on the cell cycle (the accumulation of cells in the S phase). These results indicate that the direct antitumor effect of HuIFN-beta on HMV-1 cells may result from cell cycle retardation mediated through obstruction of DNA synthesis.