Effect of polysialic acid on the tumor xenografts implanted into nude mice.

Polysialic acid (PSA), which is abundantly expressed in the embryonic brain, plays important roles in neural development and plasticity. PSA is also expressed in tumors of neural crest origin such as neuroblastoma. However, the biologic significance of PSA in these tumors has not been elucidated. In this study, we examined the expression of PSA as well as 2 polysialyltransferases, PST and STX, in various tumor cell lines. PST and STX were simultaneously expressed in all the tumor cells positive for PSA. However, even in the tumor cells negative for PSA, they expressed PSA after transfection of neural cell adhesion molecule (NCAM) cDNA when these cells expressed PST, suggesting that the presence of NCAM was critical for PSA expression. To determine the role of PSA in tumor growth and development, we established tumor sublines expressing or lacking PSA from PC-14 or NCI-H146 cells. Although significant differences of growth rates between the PSA-positive and -negative tumor cells were not detected in vitro, the PSA-positive tumor cells hardly produced detectable tumors when injected into nude mice subcutaneously or intravenously. In addition, the PSA-positive tumor cells adhered less to a basement membrane matrix Matrigel than did the PSA-negative tumor cells. These results altogether suggested that PSA significantly reduced tumor formation in the transplanted xenografts through attenuation of cell-cell or cell-matrix interactions by its large, negatively charged glycans in this particular animal model system.

Orally active docetaxel analogue: synthesis of 10-deoxy-10-C-morpholinoethyl docetaxel analogues.

To improve cytotoxicity of 10-deoxy-10-C-morpholinoethyl docetaxel analogues against various tumor cell lines including resistant cells expressing P-glycoprotein (P-gp), we modified the 7-hydroxyl group to hydrophobic groups (methoxy, deoxy, 6,7-olefin, alpha-F, 7-beta-8-beta-methano, fluoromethoxy). Among these analogues, the 7-methoxy analogue showed the strongest cytotoxicity. This analogue showed potent activity against B16 melanoma BL6 in vivo by oral administration.

Molecular cloning and identification of bottle-nosed dolphin p40(phox), p47(phox) and p67(phox).

The bottle-nosed dolphin NADPH oxidase cytosolic components, p40(phox), p47(phox) and p67(phox) cDNA's were cloned from mitogen stimulated peripheral white blood cell mRNA utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that dolphin p40(phox), p47(phox) and p67(phox) clones contained open reading frames encoding predicted polypeptides of 339, 391 and 526 amino acids, respectively. Analysis of the p47(phox) and p67(phox) amino acid sequences showed two potential Src homology three domains and p40(phox) one. Comparison of the deduced amino acids showed that dolphin p40(phox) sequence shared 88.8% similarity with the human p40(phox), that dolphin p47(phox) sequence shared 87.7% similarity with the bovine p47(phox), and that dolphin p67(phox) shared 88.1% similarity with the bovine p67(phox). Western blot analysis using anti-human p40(phox), p47(phox) and p67(phox) antibodies demonstrated that dolphin neutrophil possesses p40(phox), p47(phox) and p67(phox) with similar molecular masses and structures, to each counterpart in human neutrophils, except for the p67(phox) COOH-terminus. These results suggest that dolphin NADPH oxidase cytosolic components have functional activities equivalent to those of human.

Molecular cloning and functional expression of bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist.

The bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist IL-1ra cDNA was cloned from mitogen-stimulated peripheral blood mononuclear cells (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequence of this cDNA showed that dolphin IL-1ra clones contained open reading frames encoding 177 amino acids. Comparison of the deduced amino acids showed that dolphin IL-1ra sequence shared 87.6, 77.9, 77.4, 77.4, 76.4, and 75.8% similarity with the bovine, rabbit, equine, human, mouse, and rat IL-1ra sequences, respectively. Recombinant glutathione S-transferase (GST) dolphin IL-1ra produced in Escherichia coli (E. coli) was purified. This protein suppressed the cytostatic activity of dolphin IL-1beta on A375S2 cells, indicating that the dolphin IL-1ra cDNA obtained in the present study encodes biologically active dolphin IL-1ra.

Molecular cloning and identification of bottle-nosed dolphin flavocytochrome b gp91(phox) and p22(phox) subunits.

The bottle-nosed dolphin (Tursiops truncatus) gp91(phox) and p22(phox) cDNA were cloned from mitogen stimulated leukocytes RNA utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that dolphin gp91(phox) and p22(phox) clones contained open reading frames encoding 569 and 192 amino acids, respectively. Analysis of the gp91(phox) amino acids sequence showed three potential N-linked glycosylation sites. Comparison of the deduced amino acid showed that dolphin gp91(phox) sequence shared 95.4, 93.8, 91.4 and 89.5% similarity with the bovine, porcine, human and mouse gp91(phox) sequences, respectively. Similarly, the amino acid sequence showed that dolphin p22(phox) shared 89.7, 84.6, 84.1, 83.6 and 83.6% similarity with the bovine, mouse, porcine, human and rattus p22(phox) sequences, respectively. Western blotting analysis with anti-peptide antibodies supported the molecular weights of the dolphin gp91(phox) and p22(phox) homologous proteins predicted from the cDNAs and amino acids sequence data.

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.

Antitumor effect of DT-5461, a lipid A derivative, against human tumor xenografts is mediated by intratumoral production of tumor necrosis factor and affected by host immunosuppressive factors in nude mice.

We previously reported that DT-5461, a synthetic low-toxic lipid A analog, inhibits growth of various murine tumors through activation of host immune systems. In the present study, DT-5461 also exhibited significant antitumor effects against 5 out of 6 human tumor xenografts in nude mice. The antitumor activity was similar to or greater than those of chemotherapeutics. Antitumor effects of DT-5461 significantly correlated with intratumoral levels of tumor necrosis factor (TNF) induced by the compound (r = 0.701, p < 0.05). In vitro TNF production by DT-5461-stimulated macrophages was augmented by tumor cells, and the augmentative effect correlated with TNF activity detected in these tumor tissues. Meanwhile, a weaker therapeutic efficacy of DT-5461 was observed against certain tumors that caused a significant increase in the level of immunosuppressive factors in host blood. These findings support the idea that intratumoral TNF plays a crucial role in the antitumor mechanisms of DT-5461 and suggest that its antitumor action is influenced by an augmentative effect of tumor cells on TNF production and by blood levels of immunosuppressive factors.

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.

Intratumoral production of tumor necrosis factor augmented by endogenous interferons results in potent antitumor effects of DT-5461, a synthetic lipid A analog.

We previously reported that DT-5461 exhibits potent antitumor effects on various murine syngeneic tumors, probably via activation of host immune systems. Of the various systemic administration routes, intravenous (i.v.) administration gave the best antitumor effects. When the total dose was fixed, multiple and intermittent applications resulted in greater therapeutic efficacy than single and daily applications, respectively. The therapeutically effective applications of DT-5461 induced endogenous tumor necrosis factor (TNF) activity in serum and tumor tissue. The TNF activity peaked at 1-2 h after the administration. Although TNF activity in the serum declined to an undetectable level by 4 h, intratumoral TNF activity persisted even at 16 h. TNF-alpha messenger RNA (mRNA) was clearly expressed in the tumor tissues as early as 0.5 h after the DT-5461 administration. DT-5461 also caused increases in interferon activity in tumor-bearing mice. In vivo treatment with anti-interferon-alpha/beta serum or anti-interferon-gamma serum, as well as with anti-TNF-alpha serum, significantly reduced the antitumor effect of DT-5461. DT-5461-induced endogenous TNF production was also inhibited by treatment with either of these anti-interferon antisera alone. These results suggest that intermittent i.v. administration is optimal for cancer treatment with DT-5461, and that the optimal application of DT-5461 causes a long-lasting production of intratumoral TNF-alpha that may play a crucial role in the antitumor mechanisms of this compound. Furthermore, endogenous interferons induced by DT-5461 are involved in the antitumor mechanisms of this compound, probably by regulating the intratumoral TNF induction.

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.

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.

Antitumor effect of DT-5461a, a synthetic low-toxicity lipid A analog, involves endogenous tumor necrosis factor induction subsequent to macrophage activation.

We previously showed that the synthetic lipid A derivative DT-5461a exhibited significant antitumor effects against various murine solid tumors, probably via activation of host immune systems. To clarify the participation of the macrophage-stimulating effect of DT-5461a in the antitumor mechanisms, we studied the ability of this compound to induce cytostatic macrophages and TNF production in murine systems. Cytostatic macrophages were induced by treatment with DT-5461a either in vitro or in vivo. DT-5461a also induced TNF production by resident peritoneal macrophages or spleen cells obtained from untreated mice. When spleen cells prepared from DT-5461a-treated mice were re-stimulated in vitro with DT-5461a, no TNF was produced by cells obtained at 1 day after the treatment. This may be due to transient refractoriness of macrophages to the compound, since the response to re-stimulation with DT-5461a recovered in cells obtained at 3 or 5 days after treatment. Moreover, while the serum TNF production and antitumor effects by DT-5461a decreased on daily administration, they were elicited by intermittent administration at intervals of 3 days or more. This suggests that the antitumor effects of DT-5461a depend on the TNF-producing activity of macrophages. These results indicate that DT-5461a possesses significant macrophage-stimulating activity, and that macrophages so activated mediate the DT-5461a-induced augmentation of host response against solid tumors.

[Effect of phorbol ester on tissue-type plasminogen activator (t-PA) secretion in endometrial carcinoma cell line in vitro].

A plasminogen activator (PA) system is involved in ovulation, implantation, tumor invasion and metastasis. In order to clarify the regulation of this PA system in endometrial cells, we examined which agent affecting cellular function altered tissue-type plasminogen activator (t-PA) secretion by endometrial carcinoma cell line (KLE cells) in vitro. Triiodothyronine, retinoic acid, insulin, 8-bromo-cAMP, PDGF, IGF-I, basic FGF or TNF-alpha did not alter t-PA secretion while the activator of protein kinase C, phorbol myristate acetate (PMA) stimulated t-PA secretion in a dose-dependent fashion (10(-10)-10(-8) M). The time required to give a statistically significant increase in t-PA over control was 3 hours, and the maximal increase was seen after 24 hours of exposure. Another active phorbol ester, PDD also stimulated t-PA secretion while inactive forms of phorbol ester, 4 alpha-PDD and phorbol did not alter it. Cholera toxin or 8-bromo-cAMP did not affect t-PA secretion, but enhanced PMA-stimulated t-PA secretion. Cycloheximide and actinomycin D completely abolished PMA-stimulated t-PA secretion. These results suggest that (1) t-PA secretion in the endometrial carcinoma cell is modulated by a protein kinase C system, (2) This effect is through new RNA production and protein synthesis. (3) There is a complicated relationship between protein the kinase C and protein kinase A system as to the regulation of t-PA secretion. This would be a suitable model to clarify the PA system in endometrial cells.

Uterine leiomyoma with a focus of fatty and cartilaginous differentiation.

A uterine leiomyoma with a focus of fatty and cartilaginous differentiation in a 58-year-old female is reported. The leiomyoma was located in the posterior uterine wall and had a maximum diameter of about 15 cm. A yellow hard nodule, about 5 cm in diameter, was found in the periphery of the leiomyoma and histologically was composed of cartilaginous tissue in islands with lipofibromyomatous tissue surrounding them. Generally so-called lipomatous lesions of the uterus include circumscribed or diffuse lipomatosis of a leiomyoma and pure lipoma. Although appearance of cartilaginous tissue in lipomatous lesions of the uterus has never been reported, this case should be a very special form of circumscribed lipomatosis of a uterine leiomyoma.

[Combination therapy with granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) induced prominent granulocyte increase in an elderly case of myelodysplastic syndrome (MDS)].

A 70-year-old man was admitted to our hospital with pancytopenia. He was diagnosed as having MDS (RA), and therapy with subcutaneous S-CSF (100 micrograms/day) was started. His leukocyte count increased from 800/microliters to 1,400/microliters in two weeks. The dose of G-CSF was raised to 200 micrograms/day in the third week, and leukocytes increased to 2,00/microliters. At the fifth week, intravenous EPO (6,000 U x 3 times/week) was added. His leukocyte count increased to 4,000/microliters. EPO therapy was raised to 12,000 U x 3 times/week at the eighth week, his leukocyte count remained at the same level. G-CSF and EPO was stopped at the eleventh week, and leukocytes decreased to the same level as before administration. Throughout the course, his platelet count and reticulocyte count did not change. G-CSF and EPO are known as the stimulators of granuriod and erythroid progenitor, respectively. However, in this case, combination therapy with G-CSF and EPO induced marked increase of granulocytes only. This was an interesting case in relation to the roles of these cytokines in the hematopoietic system.

Antenatal diagnosis of fetal abnormality by ultrasonic real time scanner.

Recent progress in antenatal diagnosis has made it possible to detect most fetal malformations which can be treated and cured by neonatal surgery. In this study an analysis of antenatal diagnosis by ultrasonography in the author's clinic is reported.

Role of excision repair in UVB-induced depletion and recovery of human epidermal Langerhans cells.

Skin exposure to UVB radiation deprives the antigen-presenting function and OKT6 monoclonal antibody-binding characteristics of Langerhans cells. Decrease of Langerhans cell population could be relevant to immune surveillance disturbance in the UV-exposed skin. Patients with xeroderma pigmentosum exhibit a thousandfold higher risk for sunlight-induced skin cancers than patients with normal skin, and also have various defects in cellular immunity. Therefore, studies of numerical and structural changes in epidermal Langerhans cells of patients with xeroderma pigmentosum after UVB irradiation compared with normal subjects may contribute to understanding the role of antigen-presenting cells in photocarcinogenesis. The effect of UVB radiation on OKT6+ Langerhans cells was studied in epidermal sheets obtained from irradiated normal subjects (36 and 49 years old) and subjects with xeroderma pigmentosum complementation group A (21 years old), complementation group D (32 years old), and variant (35 and 60 years old). Langerhans cell densities in chronically sunlight-exposed skin were remarkably reduced in patients with xeroderma pigmentosum group A but only slightly in those with xeroderma pigmentosum variant and in normal subjects compared with covered skin. Structural changes were substantial in Langerhans cells of chronically exposed patients with xeroderma pigmentosum group A but fewer in subjects with other xeroderma pigmentosum groups and in normal subjects. A single irradiation of three times the minimal erythema dose induced a large reduction of Langerhans cells from 3 to 7 days in all subjects. However, subsequent reappearance and return to preirradiated levels was delayed more in patients with xeroderma pigmentosum group A than in those with xeroderma pigmentosum variant and normal subjects. These results indicate (1) an essential role for excisional repair in the UVB-induced depletion, recovery, and maintenance of the epidermal Langerhans cell population, and (2) a possible, but not confirmed, relationship between depletion of Langerhans cells and earlier photocarcinogenesis in xeroderma pigmentosum.

[Amplification of L-myc oncogene in malignant meningioma. Case report].

Amplification of L-myc oncogene was noticed in a malignant meningioma originating from the right sphenoidal wing of a 54-year-old female. The patient underwent three surgical resections plus radiotherapy over a period of 11 years and then the growth rate of the tumor became much greater with a severely invasive appearance. Using Southern blot hybridization, L-myc amplification was examined on the specimen resected at the fourth operation. As a result, approximately five-fold amplification was confirmed, which has not been previously reported except for that in a small cell carcinoma of the lung. This result may suggest that L-myc amplification is responsible to some extent for the malignant transformation in this meningioma.

[Do OKT6 positive Langerhans cells play a role in the suppression of ultraviolet-induced carcinogenesis?].

Langerhans cells (LC) in epidermis are antigen presenting cells. LC may play a role in immune surveillance system and are considered to suppress development of ultraviolet (UV) induced skin cancers. We studied effect of UVB irradiation to LC of xeroderma pigmentosum (XP) and normal subjects by using OKT6 monoclonal antibody. When 3 minimal erythema dose (MED) of UVB were irradiated, density of OKT6 positive LC of XP began to decrease 6 hours after irradiation, and showed the least numbers on day 2 and returned completely to the pre-irradiation level on day 14. Further, after 3 MED irradiation, LCs of both normal subjects became the least on day 3 and returned to the pre-irradiation level on day 14. In XP variant and normal subjects, the number of LC in chronic sun-exposed skin decreased significantly in a similar way comparing to that of non-exposed skin. These results suggest that epidermal LC may not play an essential role in prevention of UV-induced tumor development.

[Dynamic changes in serum osteocalcin levels in the perinatal periods].

Osteocalcin is a bone-specific protein released into the blood proportional to the rate of new born formation. It is widely accepted that the level of serum osteocalcin is a clinical marker of bone turnover. Nephrogenic cAMP is a specific indirect parameter of the biologically active parathyroid hormone. For analysis of bone metabolism during pregnancy, we measured the concentrations of osteocalcin and nephrogenic cAMP in the maternal serum during pregnancy and in the cord serum at delivery. Nephrogenic cAMP values (n mol/dl GF: mean +/- SEM) increased from the first trimester (1.5 +/- 0.21) to the term (2.11 +/- 0.11). Osteocalcin values (ng/ml: mean +/- S.D.) conversely declined from the first trimester (3.17 +/- 1.66) until the term (1.48 +/- 0.71) and acutely increased in the puerperium (5.91 +/- 2.58). These results might indicate that pregnancy induces a state of secondary hyperparathyroidism, but bone turnover is suppressed. In the cases of uncomplicated deliveries, the concentration of osteocalcin in the umbilical vein was significantly higher than that in the cord artery. This result suggests that a protein immunologically reactive to the osteocalcin antibody might be produced in the human placenta.