Inhibition of the spindle assembly checkpoint kinase Mps-1 as a novel therapeutic strategy in malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients' outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients.

Minimal asbestos exposure in germline BAP1 heterozygous mice is associated with deregulated inflammatory response and increased risk of mesothelioma.

Germline BAP1 mutations predispose to several cancers, in particular malignant mesothelioma. Mesothelioma is an aggressive malignancy generally associated with professional exposure to asbestos. However, to date, we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. We hypothesized that germline BAP1 mutations might influence the asbestos-induced inflammatory response that is linked to asbestos carcinogenesis, thereby increasing the risk of developing mesothelioma after minimal exposure. Using a BAP1(+/-) mouse model, we found that, compared with their wild-type littermates, BAP1(+/-) mice exposed to low-dose asbestos fibers showed significant alterations of the peritoneal inflammatory response, including significantly higher levels of pro-tumorigenic alternatively polarized M2 macrophages, and lower levels of several chemokines and cytokines. Consistent with these data, BAP1(+/-) mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos, doses that rarely induced mesothelioma in wild-type mice. Our findings suggest that minimal exposure to carcinogenic fibers may significantly increase the risk of malignant mesothelioma in genetically predisposed individuals carrying germline BAP1 mutations, possibly via alterations of the inflammatory response.

Aspirin delays mesothelioma growth by inhibiting HMGB1-mediated tumor progression.

High-mobility group box 1 (HMGB1) is an inflammatory molecule that has a critical role in the initiation and progression of malignant mesothelioma (MM). Aspirin (acetylsalicylic acid, ASA) is the most widely used nonsteroidal anti-inflammatory drug that reduces the incidence, metastatic potential and mortality of many inflammation-induced cancers. We hypothesized that ASA may exert anticancer properties in MM by abrogating the carcinogenic effects of HMGB1. Using HMGB1-secreting and -non-secreting human MM cell lines, we determined whether aspirin inhibited the hallmarks of HMGB1-induced MM cell growth in vitro and in vivo. Our data demonstrated that ASA and its metabolite, salicylic acid (SA), inhibit motility, migration, invasion and anchorage-independent colony formation of MM cells via a novel HMGB1-mediated mechanism. ASA/SA, at serum concentrations comparable to those achieved in humans taking therapeutic doses of aspirin, and BoxA, a specific inhibitor of HMGB1, markedly reduced MM growth in xenograft mice and significantly improved survival of treated animals. The effects of ASA and BoxA were cyclooxygenase-2 independent and were not additive, consistent with both acting via inhibition of HMGB1 activity. Our findings provide a rationale for the well documented, yet poorly understood antitumorigenic activity of aspirin, which we show proceeds via HMGB1 inhibition. Moreover, the use of BoxA appears to allow a more efficient HMGB1 targeting while eluding the known gastrointestinal side effects of ASA. Our findings are directly relevant to MM. Given the emerging importance of HMGB1 and its tumor-promoting functions in many cancer types, and of aspirin in cancer prevention and therapy, our investigation is poised to provide broadly applicable information.

[Integrated coronary artery bypass strategy prevents urgent pump conversion during off-pump coronary artery bypass grafting].

Urgent pump conversion during off-pump coronary artery bypass (OPCAB) results in high morbidity and mortality. We retrospectively evaluated if the peri-operative integrated strategy prevents this lethal event in our 400 consecutive OPCAB operations. The patients with preoperative cardiogenic shock and/or ventricular arrhythmias underwent on-pump coronary artery bypass grafting (CABG). All other patients (99% of total CABG) were scheduled to undergo OPCAB (n=400). Prophylactic intraaortic balloon pumping (IABP) was applied to the patients with critical (>95%) left main trunk stenosis or low (<0.35) left ventricular ejection fraction. All the patients received the deep pericardial suture, apex-traction device, suction-type stabilizer, test-clamp of target coronary arteries by micro bulldog clamp, and intra-coronary shunts. Intra-operative IABP was applied in the case of sustained ST-segment change and/or elevated pulmonary artery pressure. Pump conversion was indicated for the patients with ventricular fibrillation and/or cardiogenic shock. Two patients (0.5%) had pump conversion due to ventricular arrhythmia and sustained hypotension, respectively. These pump conversion did not result in hospital mortality. Three hospital deaths (0.7%) occurred due to non-cardiac causes. The integrated strategy using prophylactic or intra-operative IABP in OPCAB produce a low pump conversion rate even during an early period of surgeon's learning curve.

Growth inhibition of MCF-7 cells by estrogen is dependent upon a serum factor.

MCF-7 cell growth is normally dependent upon estrogen, but if cells are maintained in serum-free medium estrogen inhibits cell growth with an IC50 of about 1 nM. Cells adapted to serum-free medium have approximately the same levels of estrogen receptor mRNA as control cells that are stimulated by estrogen. We have identified two serum components, one estrogen dependent and one not, that appear to be responsible for the inhibition of growth by estrogen. Our observations are consistent with the presence of a plasma membrane receptor which negatively regulates MCF-7 cell growth, and that can be inhibited by a serum protein that binds estrogen.

A steroid-binding protein mediates estrogen-dependent inhibition of growth of MCF-7 breast cancer cells.

We have described two different proteins that mediate MCF-7 cell growth inhibition (1). One is estrogen-dependent and is a heterodimer consisting of a heavy (54 kDa) and light (29 kDa) chain. This protein is distinct from cortisol-binding globulin (CBG), sex hormone-binding protein (SBP or SHBG) and albumin (HSA). It may be fetal steroid binding protein (FSBP). The other is not estrogen dependent, but may be related to the estrogen-dependent protein. Since these are large proteins they must be acting at the level of the plasma membrane, not traditional intracellular estrogen receptors, and inhibit MCF-7 cell growth.

Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells.

Solid tumor cells are often exposed to hypoxia in vivo, which has been suggested to promote genetic instability in those cells. Telomere elongation by telomerase is implicated in chromosome stabilization in immortal cells. Here we found that hypoxia enhanced telomerase activity in the solid tumor A2780 and HT-29 cells but not in the leukemia U937 cells. The telomerase activation correlated with activation of mitogen-activated protein kinase (MAPK) and c-fos expression. The MEK1 inhibitor PD98059 repressed telomerase activation in the hypoxic cells. Consistently, a dominant negative MEK1 inhibited telomerase activation by hypoxia. Finally, we found a good correlation between telomerase activation and resistance to apoptotic cell death under hypoxic conditions. These findings indicate that hypoxia up-regulates telomerase activity via MAPK cascade signaling especially in solid tumor cells and suggest that solid tumor cells might enhance the telomerase activity as a stress response against genotoxicity induced by hypoxia.

The leukemic oncogene tal-2 is expressed in the developing mouse brain.

tal-1 (T-cell acute leukemia-1; also known as SCL) and tal-2 genes belong to a family of basic helix-loop-helix transcription factors and were originally isolated from the breakpoints of chromosomal translocations in human T-cell leukemia cell lines. tal-1 is expressed not only in hematopoietic cells but also in several endothelial structures and the central nervous system during development. On the other hand, the detailed function and the sites of expression of tal-2 have remained obscure. We cloned the tal-2 cDNA from a mouse embryonic cDNA library and examined its expression pattern in the mouse, comparing with that of tal-1. In situ analyses revealed that tal-2 transcripts are detected at embryonic day 12.5 in the following regions; 1) the diencephalon-the zona limitans intrathalamica and the pretectum, 2) the mesencephalon-the tectum, and the anterior and posterior tegmentum, 3) the metencephalon-the isthmus and the anterior pons. In the diencephalon and the mesencephalon, the expression sites of tal-2 gene were similar to those of tal-1, and its expression was stronger than that of tal-1. In the metencephalon, tal-2 expression was observed in the anterior pons, whereas tal-1 transcripts were detected in the entire pons, and showed stronger expression than tal-2. The tal-2 messages were barely detectable in the brain at birth. These results suggest that tal-1 and tal-2 are involved in the development of specific areas of the central nervous system.

[A case of congenital tricuspid regurgitation associated with atrial septal defect and peripheral pulmonary stenosis].

This report describes a 5-year-old girl with congenital tricuspid regurgitation associated with an atrial septal defect and peripheral pulmonary stenosis. The girl was diagnosed with the heart murmur at birth and recently developed the cardiomegaly. Cardiac echocardiography and catheterization showed severe tricuspid regurgitation, an atrial septal defect of the secundum type and peripheral pulmonary stenosis. In the operative findings, the tricuspid annulus was dilated to 33 mm in diameter, and leaflets were attached normally to the antomic annulus. There was a large cleft of the anterior leaflet of the tricuspid valve. Suture of the cleft and annuloplasty of the tricuspid valve, suture closure of the atrial septal defect and patch dilatation of peripheral pulmonary stenosis were successfully performed. Including this case, 19 other cases with congenital tricuspid regurgitation undergoing surgery were reported to date.

Cerebral metabolic recovery from deep hypothermic circulatory arrest after treatment with arginine and nitro-arginine methyl ester.

BACKGROUND: Recent studies suggest that nitric oxide is important in the pathogenesis of ischemic brain injury and also has a role in controlling cerebrovascular tone. This study examines the net effects of nitric oxide on cerebral metabolic recovery after deep hypothermic circulatory arrest. METHODS: Two-week-old piglets were supported by cardiopulmonary bypass and cooled to 15 degrees C followed by 1 hour of deep hypothermic circulatory arrest, 45 minutes of reperfusion and rewarming, and then 3 hours of normothermic perfusion. Groups of 10 piglets received one of four treatments before bypass; L-nitro-arginine methyl ester, inhibitor of nitric oxide synthesis, 10 mg/kg intravenously; L-arginine, to enhance nitric oxide synthesis, 30 mg/kg intravenously before bypass and then 10 mg/kg per minute during the first hour of reperfusion; a combination of L-nitro-arginine methyl ester plus L-arginine at these same doses; and no pretreatment (controls). Cerebral high-energy phosphates and pH were measured by magnetic resonance spectroscopy in half the animals. Cerebral blood flow, metabolic rates for oxygen and glucose, and the oxidation/reduction state of cytochrome aa3 and oxygenated and deoxygenated hemoglobin measured by near-infrared spectroscopy were assessed in the other half of the piglets. RESULTS: L-nitro-arginine methyl ester significantly increased cerebral vascular resistance and markedly reduced recovery of high-energy phosphates, pH, and oxidation state of cytochrome aa3, L-arginine increased cerebral blood flow, cerebral glucose and oxygen consumption, and recovery of cytochrome aa3 oxidation and high-energy phosphates. L-Arginine did not reverse completely the effects of L-nitro-arginine methyl ester on cerebral metabolic recovery. CONCLUSION: In a piglet model of deep hypothermic circulatory arrest, L-nitro-arginine methyl ester has a deleterious effect and L-arginine has a beneficial effect on cerebral metabolic recovery. The deleterious metabolic effects of L-nitro-arginine methyl ester are only partially reversed by L-arginine. This fact suggests that there may be mechanisms in addition to inhibition of nitric oxide synthesis contributing to the neurotoxicity of L-nitro-arginine methyl ester in this model.

Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.

The role of experimental aluminum intoxication in allogeneic immunoresponse.

To evaluate the immunological properties of aluminum (Al) in experimental Al intoxication in rats, we performed heart transplantation and in vitro experiments. Lewis (Lew) rats were intoxicated with intraperitoneal injections of AlCl3. heart transplants were performed using Brown-Norway (BN) rats as donors. Isotransplants and normal Lew were used as controls. No differences in survival were observed. Unidirectional mixed lymphocyte cultures (MLC) and Concanavalin A (Con A)-stimulated cultures were prepared using spleen cells from normal and Al-intoxicated Lew rats. No differences were found in unidirectional MLC. Intoxicated cells showed a less intense response to con A than did normal cells. In conclusion, we could not detect an immunosuppressive role of Al intoxication in experimental cardiac transplantation or in MLC. However, the depressed Con A blastogenic response of Al-intoxicated cells may reflect an immunological role yet to be defined.

[Left main coronary artery stenosis following aortic valve replacement using a solid coronary perfusion catheter: report of two cases].

We experienced two cases of iatrogenic left main coronary artery stenosis (IOCS) following double (aortic and mitral) valve replacement (DVR). The solid coronary perfusion catheter may attribute IOCS, with grave consequence. There have been no IOCS since the time we exchanged a solid catheter for a soft one. One case, she was successfully treated percutaneous transluminal coronary angioplasty (PTCA), because she developed angina pectoris about 5 years after PTCA. But she developed angina pectoris again and angiographically left main coronary was severe stenotic. So she was undergone aorto coronary bypass grafting (CABG) to the left anterior descending. The other case, he developed angina pectoris about 3 months after DVR. He was treated with PTCA. Angiographically left mine coronary artery stenosis reduced 50% from 90%. Generally the treatment of IOCS is CABG, but we performed PTCA for 2 patients. Because we thought it was very hazardous for us to perform them open heart surgery. When it is very hazardous to perform patients open heart surgery, they need to be performed PTCA.

[Two cases of atrial septal defect with absence of right superior vena cava and persistent left superior vena cava].

Two women (16-year-old, 54-year-old) of atrial septal defect with absence of right superior vena cava and persistent left superior vena cava were successfully operated on. Electrocardiographic findings show coronary sinus rhythm and atrial fibrillation. When we closed atrial septal defect cannulated to persistent left superior vena cava via the coronary sinus directly, sick sinus syndrome was not appeared postoperatively.

[Two operated cases of mitral stenosis (MS) associated with left atrial ball thrombus].

Mitral stenosis associated with left atrial ball thrombus is rare. Removal of left atrial ball thrombus and mitral valve replacement was performed in two patients successfully. The rate of thromboembolism was high in patient who has left atrial ball thrombus. Also, sudden death was reported in these cases due to incarceration of ball thrombus in the mitral orifice (hole-in-one thrombus). We concluded that we should operate the MS associated with left atrial ball thrombus as soon as possible.

[A case of ruptured aneurysm of Valsalva sinus into right atrium with peculiar findings on aortography].

A 24-year-old man with ruptured aneurysm of sinus of Valsalva into the right atrium originating from the noncoronary sinus is presented. On aortography through the ascending aorta the right atrium in systolic phase and the right ventricle in diastolic phase were opacified. We considered ruptured aneurysm like a streamer (wind sock) entered into the right ventricle in diastolic phase and into the right atrium in systolic phase. Post-aneurysmectomy course was uneventful, and radiographic examination revealed complete repair of the aneurysm.

[A case report of perforated aneurysm of mitral valve with aortic regurgitation].

The patient was a 71-year-old male who complained of palpitation and tachycardia. The echocardiogram showed a bulging of the anterior mitral valve leaflet toward the left atrium that persisted throughout cardiac cycle. The cine angiogram showed deformity of the anterior mitral valve leaflet with severe mitral regurgitation and mild aortic regurgitation. At operation, a perforated aneurysm was recognized at the anterior mitral valve leaflet without thrombus and vegetation. The size of aneurysm was 40 x 25 x 25 mm. The patient underwent MVR + AVR, and the postoperative course was uneventful. Pathological examination of the anterior mitral valve leaflet revealed scar-like fibrosis and old inflammatory change. It was judged a true aneurysm of mitral valve, because the structure of endocardium was kept.

The primary structure of Aspergillus niger acid proteinase A.

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.

Inhibitory effects of cimetidine on graft survival of allotransplanted rats submitted to an active-passive enhancement protocol

The objetive of the presented study was to determine wheter cimetidine, a type-2 histamine receptor antagonist, inhibits the immunological enhancement of allografted rats achieved by treatment with donor antigen plus anti-donor antibody. Groups of rats submitted to this active-passive enhancement protocol and treated ip with 30 (APEC30; Group I; N = 4) or 60 (APEC 60; Group II; N = 8) mg/day cimetidine for 14 days had a significantly shorter graft survival (20.2 ñ 5.1 and 11.1 ñ 2.6 days, respectively) than the control group (animals submitted to the enhancement protocol and killed on day 72 after transplant when the graft was beating normally; APE; Group III; N = 6; P<0.05). On the other hand, these animals had a significantly longer graft survival than rats allotransplanted but not treated for enhancement (ALLO; Group V; N = 5; 8.2 ñ 0.8 days). The surgical control, consisting of isotransplanted animals, had a long-term survival (ISO; Group V; N = 6; rats killed 120 days after transplant with the graft beating normally). Animals treated with cimetidine, but not submitted to the enhancement protocol (AC 60; Group IV, N = 4) had a significantly shorter graft survival (6.25 ñ 0.5) than the allotransplanted animals (Group V). These results indicate inhibition of the suppressor mechanisms which participate in this type of immunological enhancement