Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms.

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC(50)) of <1 n, and had 30-50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL-JAK2 fusion gene; IC(50)=11-120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.

C1s and O1s photoelectron satellite spectra of CO with symmetry-dependent vibrational excitations.

The photoelectron shake-up satellite spectra that accompany the C1s and O1s main lines of carbon monoxide have been studied by a combination of high-resolution x-ray photoelectron spectroscopy and accurate ab initio calculations. The symmetry-adapted cluster-expansion configuration-interaction general-R method satisfactorily reproduces the satellite spectra over a wide energy region, and the quantitative assignments are proposed for the 16 and 12 satellite bands for C1s and O1s spectra, respectively. Satellite peaks above the pi(-1)pi(*) transitions are mainly assigned to the Rydberg excitations accompanying the inner-shell ionization. Many shake-up states, which interact strongly with three-electron processes such as pi(-2)pi(*2) and n(-2)pi(*2), are calculated in the low-energy region, while the continuous Rydberg excitations are obtained with small intensities in the higher-energy region. The vibrational structures of low-lying shake-up states have been examined for both C1s and O1s ionizations. The vibrational structures appear in the low-lying C1s satellite states, and the symmetry-dependent angular distributions for the satellite emission have enabled the Sigma and Pi symmetries to be resolved. On the other hand, the potential curves of the low-lying O1s shake-up states are predicted to be weakly bound or repulsive.

Synthesis of bioactive PMMA bone cement via modification with methacryloxypropyltri-methoxysilane and calcium acetate.

Bone cement consisting of polymethylmethacrylate (PMMA) powder and methylmethacrylate (MMA) liquid is clinically used for fixation of implants such as artificial hip joints. However, it does not show bone-bonding ability, i.e., bioactivity. The lack of bioactivity would be one of factors which cause loosening between the cement and the implant. The present authors recently showed the potential of bioactive PMMA-based bone cement through modification with gamma-methacryloxypropyltrimethoxysilane (MPS) and calcium acetate. In this study, the effects of the kinds of PMMA powder on setting time, apatite formation and compressive strength were investigated in a simulated body fluid (Kokubo solution). The cement modified with calcium acetate calcined at 220 degrees C could set within 15 min when the PMMA powder had an average molecular weight of 100,000 or less. The addition of calcium acetate calcined at 120 degrees C in the PMMA powder required a much longer period for setting. The modified cements formed an apatite layer after soaking in the Kokubo solution within 1 day for cement starting from PMMA powder with a molecular weight of 100,000 or less. Compressive strengths of the modified cements were more than 70 MPa for cements starting from 100,000 and 56,000 in molecular weight. After soaking in Kokubo solution for 7 days, the modified cement consisting of PMMA powder of 100,000 in molecular weight showed a smaller decrease in compressive strength than that consisting of 56,000 in molecular weight. These results indicate that bioactive PMMA cement can be produced with appropriate setting time and mechanical strength when PMMA powders with a suitable molecular weight are used. Such a type of design of bioactive PMMA bone cement leads to a novel development of bioactive material for bone substitutes.

Chromosomal instability and radiosensitivity in myelodysplastic syndrome cells.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells. To investigate whether chromosomal instability and/or DNA repair defects are involved in the development of MDS, we measured the micronucleus (MN) frequency in peripheral blood lymphocytes exposed to various doses of X-rays, using a cytokinesis-block micronucleus assay. The spontaneous MN frequencies in RAEB and RAEB-T patients were significantly higher than those in normal individuals (P = 0.0224, P = 0.008, respectively). Also, the X-ray-induced MN frequencies in RA/RARS, RAEB, and RAEB-T patients were significantly higher than those in normal individuals (P = 0.007, P = 0.003, P = 0.003, respectively, at 2 Gy). In order to elucidate the cause of unusual radiosensitivity, we measured the expression levels of nucleotide excision repair (NER) genes in peripheral blood mononuclear cells using a RT-PCR method. Reduction of NER gene expression was found in only one of 10 patients with low risk MDS, but in four of 11 patients with high risk MDS. Our data suggest that chromosomal instability and DNA repair defects may be involved in the pathophysiology of disease progression of MDS.

Tolerability and safety of a calcium channel blocker in comparison with a diuretic in the treatment of elderly patients with hypertension: secondary analysis of the NICS-EH.

A randomized prospective controlled study, the National Interventional Cooperative Study in Elderly Hypertensives (NICS-EH), previously demonstrated that the preventive effect of the long-acting calcium channel blocker nicardipine on the cardiovascular endpoint was similar to that of the diuretic, trichlormethiazide. The present report is a sub-analysis in which we compare the tolerability and safety of the calcium channel blocker with that of a diuretic in the long-term treatment of elderly hypertensives. A total of 429 elderly patients with hypertension were assigned to the nicardipine group or the diuretic group by the double-dummy method and were followed up for 5 years. Two hundred four patients in the nicardipine group and 210 patients in the diuretic group were analyzed. The incidences of fatal and nonfatal cardiovascular (CV) events in the two groups were comparable, and there was no significant difference in the cumulative event-free rate. However, the total incidence of adverse reactions, including non-CV events and unfavorable BP changes, was 31 cases (15.2%) in the nicardipine group, which was significantly lower than the 47 cases (22.4%) in the diuretic group (log-rank: p=0.026, G. Wilcoxon: p=0.01). The total number of medical endpoints, including CV events, the withdrawal of the patient from the study, was 52 (25.5%) in the nicardipine group, which was significantly lower than the 65 (31.0%) in the diuretic group (log-rank: p=0.078, G. Wilcoxon: p=0.044). It was concluded that sustained-release nicardipine is better tolerated, as it exhibits a lower incidence of medical-related withdrawals such as adverse drug reactions, non-cardiovascular events and unfavorable BP responses during the treatment.

Elevated levels of inhibin-A and immunoreactive inhibin in aged male Wistar rats with testicular Leydig cell tumor.

This study was undertaken to investigate the endocrine changes that occur in male Tig:Wistar rats with Leydig cell tumors, with special reference to immunoreactive inhibin (ir-inhibin) and its dimeric forms. Adult male rats from 2 to 28 months of age were used. Blood samples were taken to measure plasma concentrations of ir-inhibin, inhibin-A, inhibin-B, 17beta-estradiol (E2), testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Inhibin bioactivity in both peripheral plasma and testicular extracts was also measured. Rats aged 18 months and older had testicular Leydig cell tumor. Testicular tissue sections from 27-month-old rats examined immunohistochemically showed strong positive staining for all 3 inhibin subunits, in particular inhibin alpha and betaA subunits, in the tumor cells. Plasma concentrations of ir-inhibin began to rise significantly (P < .05) at 18 months of age. High bioactivity of inhibin was detected not only in testicular extracts but also in peripheral plasma of aged rats. Thus, plasma concentrations of bioactive inhibin-A, but not inhibin-B, were significantly elevated with increasing age. The concentrations were significantly higher than those in normal male (P < .01) or normal female (P < .05) rats. Plasma concentrations of E2 were significantly (P < .05) elevated only at 23-24 months of age. A marked reduction (P < .05 to .001) in plasma LH and FSH concentrations was observed at 18 months of age and older. Plasma concentrations of testosterone were highest at 2 months of age and then decreased gradually and significantly (P < .05 to .001) afterward. Significant (P < .05 to .001) positive (testosterone vs LH) and negative (ir-inhibin vs FSH, ir-inhibin vs LH, and E2 vs FSH) correlations were observed. It is suggested that plasma inhibin-A levels are elevated in male Tig:Wistar rats with Leydig cell tumor, and thus inhibin-A may be used as a specific marker of testicular Leydig cell tumors. The present results also suggest that the age-related decline in plasma gonadotropins and thus testosterone levels in Tig:Wistar rats may be due to the development of tumors of the Leydig cells rather than to aging per se.

Establishment and characteristics of four sub-strains of F344 rats reared on various low protein and low energy diets.

Four sub-strains, reared by sib-mating and having for their origin the F344/DuCrj strain of rats, were established by feeding with different levels of low protein and low energy diets, and their characteristics investigated. The amounts of crude protein (CP) and digestible energy (DE) in the four diets were 17.6%-3.0 kcal, 10.5%-2.5 kcal, 8.4%-2.0 kcal, and 10.5%-2.5 kcal, respectively, and the four sub-strains established here were provisionally designated as F344/Tig1, F344/Tig2, F344/Tig3 and F344/Tig4, respectively. Intakes of nitrogen-corrected metabolizable energy (MEn) did not differ, and a large intake of digestible crude protein (DCP) was observed in F344/Tig1 rats. The body weight of rats provided with lower-nutrient diets showed a tendency to decrease until the F2 generation, but no change among the generations was seen subsequently, and the same compiled differences in protein content were maintained. Similar transitions were observed in the lifetime rearing test. Though F344/Tig3 rats, which were reared on minimum nutrients, showed a tendency to delayed puberty, we were easily able to breed four pairs in every generation using procedures similar to those used for other strains of rats. There were no differences among the F344/Tig1 to -3 strains of rats in body length, digestive tract length, or organ weight per body weight, and all the rats had a normal range of biochemical values. But the F344/Tig4 showed a high glutamic-oxaloacetic transaminase (GOT), and a tendency to decreased liver function and a shorter lifespan. These sub-strains of F344 rats clarified differences in fatty acid compositions, such as docosahexaenoic acid (DHA) in serum, liver and the brain. The rats were intended to be useful animal models for the study of nutritional environments and their influence on the memory and learning.

[MALT lymphoma producing IgG-kappa type M-protein].

A 72-year-old woman, who has been administered prednisolone and azathioprine with diagnoses of idiopathic thrombocytopenic purpura (ITP) and autoimmune hepatitis (AIH), underwent a complete medical examination because of monoclonal gammopathy (IgG-kappa). Tumors were found in the ileum and descending colon. Pathological examination of biopsy specimens suggested a diagnosis of marginal zone B-cell lymphoma of the MALT type with a high-grade component. Flow cytometric analysis by two-color staining revealed that the neoplastic B cells expressed CD38, CD19, IgG and kappa, but not CD5 or CD10. There were no abnormal plasma cells in bone marrow smears. The patient achieved complete remission after receiving three cycles of THP-COP chemotherapy, which resulted in a decrease of the IgG level to within the normal range. These findings indicated that monoclonal IgG-kappa might be produced by lymphoma cells. However, the relationship of the immunosuppressive agents to the pathogenesis of the MALT lymphoma remains to be clarified.

Transcatheter embolization of mycotic aneurysm of the subclavian artery with metallic coils.

Mycotic aneurysms of the subclavian artery are rare. This report describes an experience of 2 rare cases in which transcatheter embolization with metallic coils was performed for the management of these lesions alternative to surgery. Two patients who had been treated with chemotherapy for malignant neoplasms were diagnosed as having mycotic aneurysms of the left subclavian artery. The causes of these lesions were presumed to be the invasion of the arterial wall by the pulmonary abscess in case 1, and wound infection after placement of the reservoir for intraarterial chemotherapy in case 2. In both cases, proximal and distal sites of the aneurysm were embolized with metallic coils. In case 1, the vertebral artery was also embolized with Guglielmi detachable coils to avoid retrograde blood flow. Both aneurysms were completely occluded by a single embolization. In case 1, although weakness and paresthesia of the left hand remained, lethal hemoptysis due to aneurysmal fistulization to the lung parenchyma ceased. In case 2, no neurological deficit except for mild paresthesia in the left thumb had been observed. Both patients died of primary disease 10 and 5 months after the procedure. Transcatheter embolization is technically feasible and effective enough to treat the mycotic aneurysm of the subclavian artery even in the situation in which the surgical option seems to be difficult or risky.

Effect of long-term treatment with antihypertensive drugs on quality of life of elderly patients with hypertension: a double-blind comparative study between a calcium antagonist and a diuretic. NICS-EH Study Group. National Intervention Cooperative Study in Elderly Hypertensives.

We investigated the effect of long-term treatment with a calcium antagonist (nicardipine hydrochloride retard tablet) and a diuretic (trichlormethiazide) on quality of life (QOL) in elderly hypertensives in a multicenter, randomized, double-blind, comparative study (National Intervention Cooperative Study in Elderly Hypertensives Study Group). The percentage of patients who experienced side effects was 17.2% in the nicardipine group and 18.1% in the trichlormethiazide group and 2.9% and 4.3% of participants, respectively, withdrew due to those side effects. These results suggested that nicardipine was tolerated slightly better than trichlormethiazide. There were no significant differences between the two groups in terms of total QOL score or in degree of change (delta score) before and after calcium antagonist or diuretic administration. Lower score was seen in 3 categories (general symptoms, sleep scale, and sexual function) in the trichlormethiazide group (p< 0.05) and in one category (cognitive function) in the nicardipine group, but there was no significant difference in delta score in any of the individual items. In conclusion, the two anti-hypertensive agents had nearly equivalent effects on QOL in the long-term treatment of hypertension in the elderly and that neither resulted in a deterioration in QOL.

ZK7, a novel zinc finger gene, is induced by vascular endothelial growth factor and inhibits apoptotic death in hematopoietic cells.

Vascular endothelial growth factor (VEGF) inhibits radiation-induced apoptosis in the leukemia cell line, CMK86 (O. Katoh et al., Cancer Res., 55: 5687-5692, 1995). To elucidate the molecular mechanisms underlying this inhibitory effect of VEGF, we attempted to identify a transcription factor involved in the cellular response to VEGF stimulation. We cloned the cDNA of a novel Kruppel-type zinc finger (ZNF) gene, ZK7, from a cDNA library constructed from the human leukemia cell line CMK86 after incubation with VEGF. This cDNA encoded a protein of 289 amino acids, which contained a Kruppel-associated box A-box domain at the NH2 terminus and seven ZNF motifs at the COOH terminus. These ZNF motifs were identical to those of HZF16 (M. Saleh et al., Am. J. Hum. Genet., 52: 192-203, 1993). ZK7 and HZF16 genes appear to be the splice variants transcribed from the same gene. Northern blotting and quantitative reverse transcription-PCR analysis revealed that expression of ZK7 mRNA in CMK86 cells and human hematopoietic progenitor cells was increased after VEGF stimulation, whereas that of HZF16 mRNA remained unchanged. To examine the function of ZK7 protein, we generated clones of a human monocytoid leukemia cell line, U937, which were stably transfected with ZK7 or HZF16 cDNA. Importantly, ZK7-overexpressing cells had lower sensitivity to ionizing radiation and the chemotherapeutic agent etoposide than U937 parent cells or HZF16-overexpressing cells. Therefore, ZK7 protein may be involved in the inhibitory effect of VEGF on apoptotic cell death in human hematopoietic cells.

Influence of concomitant miso or NaCl treatment on induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

Six-week old male Sprague-Dawley (CD) rats were treated with 100 ppm N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 16 weeks in their drinking water with control, miso or sodium chloride (NaCl) supplemented diets. All animals were autopsied 12 months after the beginning of the MNNG treatment. Despite higher intake of MNNG in the high dose miso and NaCl groups, the total tumor incidences were decreased compared to middle and lowest values. The glandular stomach adenocarcinoma incidences in the 10% and 5% miso groups were significantly decreased as compared to those in the 2.2% or 1.1% NaCl groups, with the same concentration of NaCl.

Gastric tumor induction by 1,2-dimethylhydrazine in Wistar rats with intestinal metaplasia caused by X-irradiation.

Five-week-old male Wistar rats were X-irradiated with a total of 20 Gy in 2 equal fractions at a 3-day interval. 1,2-Dimethylhydrazine (DMH) solution was injected i.m. into the back musculature at a dose of 20 mg/kg body weight weekly for 10 weeks, beginning 20 weeks after the final irradiation. Twelve months after the initial carcinogen treatment, tumors in the fundus of the glandular stomach were observed in 5 of 23 animals receiving both X-irradiation and DMH treatment. No tumors of the glandular stomach were observed in the DMH and X-ray alone or nontreatment groups. It is concluded that the presence of intestinal metaplasia may increase sensitivity to the induction of gastric tumors by carcinogens like DMH.

Vascular endothelial growth factor inhibits apoptotic death in hematopoietic cells after exposure to chemotherapeutic drugs by inducing MCL1 acting as an antiapoptotic factor.

We reported previously that vascular endothelial growth factor (VEGF) inhibits the apoptotic death of hematopoietic cells that is induced by exposure to ionizing radiation (O. Katoh et al., Cancer Res., 55: 5687-5692, 1995). In this study, we show that VEGF also inhibits apoptotic cell death that is induced by exposure to the chemotherapeutic drugs etoposide and doxorubicin. To elucidate the molecular mechanisms underlying this inhibitory effect of VEGF, we examined expression levels of BCL2 family proteins in CMK86, a human leukemia cell line, after treatment with VEGF. Northern blotting and immunoblotting analyses revealed that the expression level of MCL1, a member of the BCL2 family, was increased by VEGF. Moreover, to examine the effects of MCL1 on apoptotic cell death induced by exposure to etoposide, we generated a clonal U937 myeloid leukemia cell line transfected with vectors that promoted the constitutive expression of MCL1. MCL1 decreased the caspase 3 activity induced by exposure to etoposide and increased the viability of the transfected cells after etoposide exposure. Therefore, MCL1 may be involved in the inhibitory effect of VEGF on apoptotic cell death.

The Krüppel-type zinc finger family gene, HKR1, is induced in lung cancer by exposure to platinum drugs.

To investigate the molecular mechanism associated with the signaling pathway of platinum drug administration, we focused on the C2H2-type zinc finger (ZNF) transcription factor gene family. Here we show cloning of a Krüppel-type ZNF gene, HKR1, which contains Krüppel-associated box (KRAB) domain and ZNF motifs. We found that mRNA expression of the HKR1 gene was induced in lung-cancer cell lines by exposure to cisplatin using Northern blot analysis. Moreover, we also found that HKR1 mRNA expression levels in lung cancers were higher than those in normal lung tissues, and that high expression levels in lung cancers were associated with antemortem platinum drug administration. These results suggest that HKR1 may be associated with the regulation of a signaling pathway involved in the progression of lung cancer or the acquisition of resistance to platinum drugs.

No susceptibility of colon tissue implanted into the glandular stomach of rats to N-nitroso-N-methylurea carcinogenesis.

The present study was undertaken to determine the response of colonic mucosa implanted into the stomach fundus in 6-week-old male F344 rats to oral administration of N-nitroso-N-methylurea (MNU). In graft + MNU, 15% animals had tumors in the implanted colon tissue whereas a 26% incidence of gastric tumors was observed for the pyloric mucosa. In MNU alone, 30% of rats demonstrated gastric tumors. In graft alone 10% of tumors were observed in the grafted site but none in the pylorus. Thus while graft tissue may intrinsically have an elevated risk of tumor development, no susceptibility to MNU was observed in the present study.

Induction of ovarian tumors by heavy ion irradiation in B6C3F1 mice.

Six-week-old B6C3F1 mice were exposed to 0.439 Gy heavy ion irradiation as a 290 MeV/u carbon-ion beam (LET 10 keV/micron) at 2 cm from the upper proximal point of a spread Bragg beam and autopsied 13.5 months after the irradiation. In males total tumor incidences, mainly liver tumors, were 37.0% in irradiated group and 25.0% in control (P>0.05). In females the total tumor incidences were 32.3%, mainly ovarian tumors, in the irradiated group and 0% in the controls. These results indicate that heavy ion irradiation induces ovarian tumors in females but does not target any organ in males.