Genomic analyses of germline and somatic variation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 557 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations (SCNA). RESULTS: Approximately one-third of tumors had loss-of-function (LOF) germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM, and PALB2. LOF germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP, and NF1. In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of LOF variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536, and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. CONCLUSIONS: From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 557 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Genomic Analyses of Germline and Somatic Variation in High-Grade Serous Ovarian Cancer.

Background High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 577 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations. Results Approximately one-third of tumors had loss-of-function germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM , and PALB2 . Loss-of-function germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP , and NF1 . In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of loss-of-function variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536 , and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. Conclusions From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 577 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Relations of osteoporosis and follow-up duration to recurrent falls in older men and women.

SUMMARY: There were differences in risk factors between men and women and between two follow-up time lengths. Osteoporosis was significantly associated with recurrent falls for women but not for men. The relationship of osteoporosis with falls in the past year decreased during follow-up, while those of sedatives and hypnotics remained. INTRODUCTION: A prospective study to investigate relationships between osteoporosis and recurrent falls at two follow-up lengths of 6 and 12 months in older men and women. METHODS: In total, 204 men and 447 women who visited an emergency department due to a fall were recruited. RESULTS: For men, the risk of falling was not significantly associated with osteoporosis at 6 or 12 months. Men with a fall history were 127 and 100 %, respectively, more likely to have a fall at 6 and 12 months than those without. Men who did not use walking aids were 97 % more likely to have a fall at 12 months than those who did. Women with osteoporosis were 246 and 104 %, respectively, more likely to have a fall at 6 and 12 months than those without. Women with a fall history were 129 and 66 %, respectively, more likely to have a fall at 6 and 12 months than those without. Women taking sedatives and hypnotics were 75 and 102 %, respectively, more likely to have a fall at 6 and 12 months than their counterparts. Women with depression were 138 % more likely to have a fall at 6 months and those using walking aids were 59 % more likely to have a fall at 12 months, compared to their counterparts. CONCLUSIONS: Osteoporosis is association with falls for older women but not for older men. Identifying risk factors for recurrent falls in older people may be affected by the follow-up length, as their associations are reduced over time.

Effect of triethylene glycol dimethacrylate on the cytotoxicity, cyclooxygenase-2 expression and prostanoids production in human dental pulp cells.

AIM: To evaluate the effect of TEGDMA on cell cycle progression as well as alterations of cell cycle-related gene and protein expression. METHODOLOGY: Human dental pulp cells were exposed to 0-5 mmol L(-1) TEGDMA for 24 h. Cytotoxicity was evaluated by 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell cycle progression was analysed by propidium iodide (PI) flow cytometry. Cell death pathway was surveyed by annexin V/PI dual-staining flow cytometry. The mRNA expression of cell cycle-related genes (cdc2, cyclinB1 and p21) and COX-2 was evaluated by reverse transcriptase-polymerase chain reaction, and their protein expression was evaluated by Western blotting. The production of PGE(2) and PGF(2α) in the culture medium was determined by enzyme-linked immunosorbent assay. RESULTS: Triethylene glycol dimethacrylate inhibited cellular growth and induced cell cycle deregulation in dental pulp cells. High-dose exposure provoked both necrotic and apoptotic cell death. The gene and protein expression of cdc2, cyclin B1 and cdc25C declined obviously whilst cells treated with 2.5 mmol L(-1) TEGDMA concurrent with the elevated expression of p21. The mRNA and protein expression of COX-2, along with production of PGE(2) and PGF(2α), are drastically raised by 2.5-5 mmol L(-1) TEGDMA. CONCLUSIONS: Triethylene glycol dimethacrylate induced cytotoxicity, cell cycle arrest and apoptosis in dental pulp cells, which was associated with the decline of cdc2, cyclin B1, cdc25C expression and elevation of p21 expression. Concomitantly, COX-2 expression, PGE(2) and PGF(2α) production increased. These effects may contribute to explain the pulpal damage and inflammation induced by TEGDMA after operative procedures.

Identification of risk factors of Coxiella burnetii (Q fever) infection in veterinary-associated populations in southern Taiwan.

The first case of Q fever in Taiwan was reported in 1993. The disease is considered to be emerging in Taiwan, but the route of transmission has remained unclear. The annual number of confirmed Q fever cases has been increasing up to more than 100 cases since 2005, comparing with less than 30 before 2003. The purpose of this study was to determine the seroprevalence and risk factors of Coxiella burnetii infection in veterinary-associated populations in southern Taiwan. A total of 228 serum samples of high risk individuals engaging in veterinary-related work or animal-farm work, were collected between March and June in 2007. The study individuals were interviewed by a structured questionnaire designed for Q fever investigation. Serum samples from different animal species were also obtained for Q fever analysis in the same study areas. Serological test was conducted by indirect immunofluorescence antibody assay (IFA). The result demonstrated the overall seroprevalence of Q fever was 26.3% in individuals engaging in veterinary and animal-related work in southern Taiwan. After multiple logistic regression analysis, goat exposure was significantly associated with seropositivity of Q fever in the study population in southern Taiwan (adjusted odds ratio: 2.62; 95% CI: 1.06-6.46). In addition, the highest seroprevalence (43.8%) of Q fever was identified in goats (P < 0.05). Finally, this study documented that people with prior knowledge of Q fever were less likely to be seropositive for C. burnetii. It was concluded that goat exposure was the most important risk factor associated with C. burnetii infection and appropriate health education could be useful to prevent high risk individuals from the infection in southern Taiwan.

Anti-erbB receptor strategy as a gene therapeutic intervention to improve radiotherapy in malignant human tumours.

PURPOSE: We explored and quantified the therapeutic potential of using dominant-negative EGFR transduction with replication-incompetent adenovirus (Ad-EGFR-CD533 or Ad-CD533) as a genetic approach for radiosensitization in different carcinoma and malignant glioma cell lines in vitro and in established tumour xenografts in vivo. MATERIAL AND METHODS: The cell lines MDA-MB-231, A-431, U-373 MG, U-87 MG and T47D were used. The ErbB expression profiles were quantified by Western blotting. MAPK immune complex assay measured MAPK activity with or without EGFR-CD533 expression after ionizing radiation. Radiosensitization was determined and quantified in vitro by colony-formation assays, in vivo by use of an ex vivo-in vitro colony-formation assay after intratumoral infusion of the adenoviral vectors expressing EGFR-CD533 or the control LacZ. RESULTS: Western blotting demonstrated widely varied expression levels of the ErbB receptors in the tested cell lines. Expression of EGFR-CD533 effectively blocked the radiation-induced activation of MAPK, leading to significant radiosensitization in vitro and in vivo. CONCLUSIONS: The radiation-induced ErbB activation can be effectively modulated by a gene therapeutic approach of over-expressing EGFR-CD533 leading to tumour cell radiosensitization after single and repeated radiation exposures both in vitro and in vivo.

Adenovirus-mediated overexpression of dominant negative epidermal growth factor receptor-CD533 as a gene therapeutic approach radiosensitizes human carcinoma and malignant glioma cells.

PURPOSE: Epidermal growth factor receptor (EGFR) and other members of the ErbB family of receptor tyrosine kinases (RTK) mediate autocrine growth regulation in a wide spectrum of human tumor cells. We have previously demonstrated that in stably transfected mammary carcinoma cells a dominant negative (DN) mutant of EGFR, EGFR-CD533 is a potent inhibitor of EGFR and its cytoprotective signaling after exposure to ionizing radiation. In the present study, we further investigate the capacity of a genetic approach, using replication-incompetent adenovirus (Ad)-mediated transfer of EGFR-CD533 (Ad-EGFR-CD533), to enhance the radiosensitivity in vitro of four cell lines representative of three major cancer phenotypes. METHODS AND MATERIALS: The cell lines MDA-MB-231 and T-47D mammary carcinoma, A-431 squamous carcinoma, and U-373 MG malignant glioma cells were used. The ErbB expression profiles and the EGFR tyrosine phosphorylation (Tyr-P) levels following irradiation were quantified by Western blotting. The relative radiosensitivities of tumor cells were assessed by standard colony formation assays after infection with control vector (Ad-LacZ) or Ad-EGFR-CD533. RESULTS: The expression profiles demonstrated varying levels of EGFR, ErbB2, ErbB3, and ErbB4 expression. The overexpression of EGFR-CD533 after infection with Ad-EGFR-CD533 completely inhibited the radiation-induced stimulation of EGFR Tyr-P relative to the immediate 2.4- to 3.1-fold increases in EGFR Tyr-P in control infected cells (Ad-LacZ). Ad-EGFR-CD533-infected cells demonstrated significant (p < 0.001) radiosensitization over a range of radiation doses (1-8 Gy), yielding dose-enhancement ratios (DER) between 1.4 and 1.7. This radiosensitization was maintained under conditions of repeated radiation exposures, using 3 x 2 Gy, yielding DERs of 1.6 and 1.7 for MDA-MB-231 and U-373 cells, respectively. CONCLUSIONS: Overexpression of EGFR-CD533 significantly sensitizes human carcinoma and glioma cells to single and repeated radiation exposures irrespective of their ErbB expression levels. Therefore, transduction of human tumor cells with EGFR-CD533 holds promise as a gene therapeutic approach for the radiosensitization of neoplastic cells that are growth-regulated by EGFR or other ErbB receptors.

Epidermal growth factor receptor as a genetic therapy target for carcinoma cell radiosensitization.

BACKGROUND: Exposure of human cancer cells to ionizing radiation activates the epidermal growth factor receptor (EGFR), which, in turn, mediates a cytoprotective response that reduces the cells' sensitivity to ionizing radiation. Overexpression of a dominant-negative EGFR mutant, EGFR-CD533, disrupts the cytoprotective response by preventing radiation-induced activation of the receptor and its downstream effectors. To investigate whether gene therapy with EGFR-CD533 has the potential to increase tumor cell radiosensitivity, we introduced an adenoviral vector containing EGFR-CD533 into xenograft tumors in nude mice and evaluated the tumor response to ionizing radiation. METHODS: Xenograft tumors established from the human mammary carcinoma cell line MDA-MB-231 were transduced via infusion with the adenoviral vector Ad-EGFR-CD533 or a control vector containing the beta-galactosidase gene, Ad-LacZ. The transduced tumors were then exposed to radiation in the therapeutic dose range, and radiation-induced EGFR activation was assessed by examining the tyrosine phosphorylation of immunoprecipitated EGFR. Radiosensitization was determined in vitro by colony-formation assays. All statistical tests were two-sided. RESULTS: The transduction efficiency of MDA-MB-231 tumors by Ad-LacZ was 44%. Expression of EGFR-CD533 in tumors reduced radiation-induced EGFR activation by 2.94-fold (95% confidence interval [CI] = 2.23 to 4.14). The radiosensitivity of Ad-EGFR-CD533-transduced tumors was statistically significantly higher (46%; P<.001) than that of Ad-LacZ-transduced tumors, yielding a dose-enhancement ratio of 1.85 (95% CI = 1.54 to 2.51). CONCLUSIONS: Transduction of MDA-MB-231 xenograft tumors with Ad-EGFR-CD533 conferred a dominant-negative EGFR phenotype and induced tumor radiosensitization. Therefore, disruption of EGFR function through overexpression of EGFR-CD533 may hold promise as a gene therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation.

Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen.

Transient generation of reactive oxygen or nitrogen (ROS/RNS), detected with dihydrodichlorofluoroscein by fluorescence microscopy, occurs within minutes of exposing cells to ionizing radiation. In the 1-10 Gy dose range, the amount of ROS/RNS produced/cell is constant, but the percentage of producing cells increases with dose (20 to 80%). Reversible depolarization of the mitochondrial membrane potential () and decrease in fluorescence of a mitochondria-entrapped dye, calcein, are observed coincidentally. Radiation-induced ROS/RNS, depolarization, and calcein fluorescence decrease are inhibited by the mitochondrial permeability transition inhibitor, cyclosporin A, but not the structural analogue, cyclosporin H. Radiation-stimulated ROS/RNS is also inhibited by overexpressing the Ca(2+)-binding protein, calbindin 28K, or treating cells with an intracellular Ca(2+) chelator. Radiation-induced ROS/RNS is observed in several cell types with the exception of rho(o) cells deficient in mitochondrial electron transport. rho(o) cells show neither radiation-induced ROS/RNS production nor depolarization. We propose that radiation damage in a few mitochondria is transmitted via a reversible, Ca(2+)-dependent mitochondrial permeability transition to adjacent mitochondria with resulting enhanced ROS/RNS generation. Measurements of radiation-induced mitogen-activated protein kinase activity indicate that this sensing/amplification mechanism is necessary for activation of some cytoplasmic signaling pathways by low doses of radiation.

Radiosensitization of malignant glioma cells through overexpression of dominant-negative epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) plays an important role in neoplastic growth control of malignant gliomas. We have demonstrated that radiation activates EGFR Tyr-phosphorylation (EGFR Tyr-P) and the proliferation of surviving human carcinoma cells, a likely mechanism of accelerated cellular repopulation, a major cytoprotective response after radiation. We now investigate the importance of radiation-induced activation of EGFR on the radiosensitivity of the human malignant glioma cells U-87 MG and U-373 MG. The function of EGFR was inhibited through a genetic approach of transducing cells with an Adenovirus (Ad) vector containing dominant-negative (DN) EGFR-CD533 (Ad-EGFR-CD533) at efficiencies of 85-90%. The resulting cells are referred to as U-87-EGFR-CD533 and U-373-EGFR-CD533. After irradiation at 2 Gy, both of the cell lines exhibited a mean 3-fold increase in EGFR Tyr-P. The expression of EGFR-CD533 completely inhibited the radiation-induced activation of EGFR. In clonogenic survival assays after a single radiation exposure, the radiation dose for a survival of 37% (D37) for U-87-EGFR-CD533 cells was 1.4- to 1.5-fold lower, relative to cells transduced with AdLacZ or untransduced U-87 MG cells. This effect was amplified with repeated radiation exposures (3 x 2 Gy) yielding a D37 ratio of 1.8-2.0. In clonogenic survival studies with U-373 MG cells, the radiosensitizing effect of EGFR-CD533 was similar. Furthermore, in vivo studies with U-87 MG xenografts confirmed the effect of EGFR-CD533 on tumor radiosensitization (dose enhancement ratio, 1.8). We conclude that inhibition of EGFR function via Ad-mediated gene transfer of EGFR-CD533 results in significant radiosensitization. As underlying mechanism, we suggest the disruption of a major cytoprotective response involving EGFR and its downstream effectors, such as mitogen-activated protein kinase. The experiments demonstrate for the first time that radiosensitization of malignant glioma cells through disruption of EGFR function may be achieved by genetic therapy approaches.

L-arginine enhances aerobic exercise capacity in association with augmented nitric oxide production.

We tested whether supplementation with L-arginine can augment aerobic capacity, particularly in conditions where endothelium-derived nitric oxide (EDNO) activity is reduced. Eight-week-old wild-type (E(+)) and apolipoprotein E-deficient mice (E(-)) were divided into six groups; two groups (LE(+) and LE(-)) were given L-arginine (6% in drinking water), two were given D-arginine (DE(+) and DE(-)), and two control groups (NE(+) and NE(-)) received no arginine supplementation. At 12-16 wk of age, the mice were treadmill tested, and urine was collected after exercise for determination of EDNO production. NE(-) mice demonstrated a reduced aerobic capacity compared with NE(+) controls [maximal oxygen uptake (VO(2 max)) of NE(-) = 110 +/- 2 (SE) vs. NE(+) = 122 +/- 3 ml O(2). min(-1). kg(-1), P < 0.001]. This decline in aerobic capacity was associated with a diminished postexercise urinary nitrate excretion. Mice given L-arginine demonstrated an increase in postexercise urinary nitrate excretion and aerobic capacity in both groups (VO(2 max) of LE(-) = 120 +/- 1 ml O(2). min(-1). kg(-1), P < 0.05 vs. NE(-); VO(2 max) of LE(+) = 133 +/- 4 ml O(2). min(-1). kg(-1), P < 0.01 vs. NE(+)). Mice administered D-arginine demonstrated an intermediate increase in aerobic capacity in both groups. We conclude that administration of L-arginine restores exercise-induced EDNO synthesis and normalizes aerobic capacity in hypercholesterolemic mice. In normal mice, L-arginine enhances exercise-induced EDNO synthesis and aerobic capacity.

Substantially improved in vivo radiosensitization of rat glioma with mutant HSV-TK and acyclovir.

We recently demonstrated in vitro that a mutant HSV-TK (mutant 75) expressed from an adenovirus (AdCMV-TK75) radiosensitized rat RT2 glioma cells significantly better than wild type HSV-TK (AdCMV-TK) in combination with acyclovir (ACV). To examine whether a similar improvement could also be observed in vivo, we tested these viruses in a syngeneic rat glioma tumor model (RT2/Fischer 344). First, we demonstrate that treatment with AdCMV-TK and ACV significantly radiosensitizes implanted gliomas and roughly doubles the mean survival time to 37 days, compared to 20 days for control animals implanted with Adbetagal-transduced cells (P<.02). Second, it was important to first examine the effect of AdCMV-TK75 and ACV on survival without any irradiation. We found that AdCMV-TK75 appeared to sensitize gliomas more efficiently than AdCMV-TK, although this difference was not significant ( P= .19 ). Third, and most importantly, in combined HSV-TK, ACV and irradiation experiments, we demonstrate that AdCMV-TK75 is superior over AdCMV-TK and significantly (P<.005) prolonged the survival of treated animals. Our results suggest that AdCMV-TK75 is far more efficient than AdCMV-TK in radiosensitizing rat glioma when administered in combination with ACV.

Cholesterol-induced upregulation of angiotensin II and its effects on monocyte-endothelial interaction and superoxide production.

Atherogenesis involves an early endothelial dysfunction hallmarked by elevated free radical production and increased adhesiveness for monocytes. It was hypothesized that activation of the tissue renin angiotensin system may contribute to the endothelial alteration. To test this hypothesis, thoracic aortae were isolated from normocholesterolemic (NC; n = 6) and hypercholesterolemic (HC; n = 6; diet: 0.5% cholesterol; 6 weeks) New Zealand white rabbits, and incubated for 2 h with the angiotensin II (Ang II) receptor antagonist Sar-1,Ile-8-Ang II, the antioxidant pyrolidine dithiocarbamate (PDTC) and the protein kinase C (PKC) antagonist staurosporin. Superoxide production from aortic segments was measured by lucigenin-enhanced chemiluminescence. In comparison to the normocholesterolemic state, hypercholesterolemia led to a significant increase in superoxide production (221 +/- 44%, p < 0.02); this was reduced by ex vivo treatment of the vessel segment with Ang II-antagonist (to 130 +/- 29%; p < 0.04 vs HC), or PKC-antagonist (to 86 +/- 26%; p < 0.001 vs HC), or PDTC (to 103 +/- 27%; p < 0.02 vs HC). Monocyte-endothelial interaction was assessed by functional binding assay. When compared to normocholesterolemic rabbits, hypercholesterolemia led to a twofold increase in monocyte binding (74 +/- 13 vs 37 +/- 4 monocytoid cells per high power field (m/hpf); p < 0.03). The Ang II-antagonist and the PKC-antagonist led to a normalization of monocyte-endothelial binding (Ang II-antagonist: 37 +/- 9 m/hpf; PKC-antagonist: 41 +/- 17 m/hpf; p < 0.05). In conclusion, these results indicate that hypercholesterolemia activates the tissue renin angiotensin system, which results in an increased endothelial production of superoxide and monocyte adhesiveness. Ang II-antagonist inhibits free radical production and monocyte adhesion through a mechanism which may include PKC.

An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes.

OBJECTIVES: We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes. BACKGROUND: Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase. METHODS: Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2-) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-KB by electromobility gel shift assay. RESULTS: Incubation of endothelial cells with ADMA (0.1 microM to 100 microM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 microM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2- to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-KB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody. CONCLUSIONS: We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

Chronic exposure to bryostatin-1 increases the radiosensitivity of U937 leukaemia cells ectopically expressing Bcl-2 through a non-apoptotic mechanism.

PURPOSE: Ionizing radiation (IR) produced a dose-dependent increase in apoptosis in U937/pCEP4 cells which was attenuated by the stable over expression of Bcl-2 (U937/Bcl-2). A dose of 2 Gy IR was selected for further analyses to determine if subsequent exposure to 10nM bryostatin- would overcome the resistance to IR-induced apoptosis conferred by Bcl-2 over expression. METHODS AND RESULTS: Although bryostatin- did not increase IR-induced apoptosis in U937/pCEP4 or U937/Bcl-2 cells, it impaired mitochondrial function and increased the antiproliferative effects of IR in both cell lines. The effects were more pronounced in U937/Bcl-2 cells. Bryostatin-1 also exerted differential effects on cell-cycle distributions of U937 transfectant cells, producing a significant G0/G1 arrest in U937/Bcl-2 cells, while decreasing IR-induced G2/M arrest in U937/pCEP4 cells. Although Bcl-2 over expression attenuated IR-induced apoptosis, clonogenic survival was similar in U937/pCEP4 and U937/Bcl-2 cells following 2 Gy IR treatment. Treatment with 10nM bryostatin-1 after 2 Gy IR further reduced clonogenic survival in both cell lines. Moreover, U937/Bcl-2 cells were more susceptible to the growth-inhibitory effects of IR/bryostatin-1 than U937/pCEP4 cells. CONCLUSIONS: Bryostatin-1 increased the radiosensitivity of U937 transfectant cell lines without enhancing apoptosis; furthermore, U937/Bcl-2 cells were more susceptible to IR/bryostatin-1-mediated antiproliferative effects than their empty-vector counterparts.

Gene expression after intrarenal injection of plasmid DNA in the rat.

Effective gene therapy requires efficient delivery and expression of the necessary genetic information to the target tissue. We demonstrate here that plasmid DNA, injected as naked, uncomplexed DNA into the cortical region of rat kidney, or intravenously, is localized and expressed in the kidney. The plasmid pRSVZ contained the Rous sarcoma virus promoter and a reporter gene, the beta-galactosidase gene, derived from bacteria. The beta-galactosidase gene hydrolyzes the artificial substrate X-gal to produce an intense blue color in cells that have taken up and expressed the plasmid genes. We have used X-gal staining and Western blotting to study plasmid gene expression 1, 4, and 8 days and 6 months after intrarenal injection of 50 microg of plasmid DNA and at 1 and 4 days after intravenous injection. Expression was apparent in the kidneys and several other tissues 24 h after injection and persisted for at least 8 days; expressed proteins could still be detected in the injected kidney 6 months later. These observations were corroborated by use of a plasmid, pEGFP-Puro, harboring the cytomegalovirus promoter in conjunction with a different reporter gene, the green fluorescent protein (GFP). Histological localization and Western blotting analysis of GFP expression after intrarenal injection of pEGFP-Puro paralleled results obtained with the plasmid pRSVZ. Our findings support the suggestion that intrarenal or intravenous injection of naked plasmid DNA may be an effective means of delivering therapeutic genes to the kidney and several other tissues.

[Clinical analysis of 35 patients with orbital maxillary zygoma complex fractures].

OBJECTIVE: To assess the treatment result of reposition and fixation to orbital maxillary zygoma complex fractures. METHOD: The records of 35 patients, who were treated in 1996-1998 at our hospital, were analyzed retrospectively. The patients of open fracture operated debridement, at the same time, operated reposition and fixation. The patients of closed fracture operated hairline approach and sublabial approach or use two approach for reposition and fixation. RESULT: We had 35 patients of operation. After operation, two of them had malocclusion, one of them had facial deformity, two of them had restriction of mouth opening. The treatment results of the rest appeared to be reasonably satisfactory. CONCLUSION: To know topographic anatomy, earlier reposition and sturdy fixation is the key for curing orbital maxillary zygoma complex fractures.

Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction.

OBJECT: The goal of this study was to determine whether adenoviral vector-mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53. The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis. METHODS: Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase-polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation. The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p<0.01). CONCLUSIONS: The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

Impaired aerobic capacity in hypercholesterolemic mice: partial reversal by exercise training.

The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised (E+ex, n = 13; E-ex, n = 7) and high-fat chow, sedentary (E+chol, n = 6; E-chol, n = 8) or exercised (E+chol-ex, n = 6; E-chol-ex, n = 7). Mice were trained on a treadmill 2 x 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake (r = -0.35; P < 0. 02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match E+ex (all P < 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold (r = 0.36; P < 0.025) and maximal distance run (r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.

The current status of surgical staging of ovarian serous borderline tumors.

BACKGROUND: The purpose of the current study was to evaluate the current practice of surgical staging of ovarian serous borderline tumors. METHODS: Women with a diagnosis of ovarian serous borderline tumors whose pathology slides were sent to the M. D. Anderson Cancer Center for second-opinion diagnostic consultation between 1990-1996 were identified. The original pathology reports and M. D. Anderson Cancer Center consultation reports of 255 cases were reviewed for the frequencies of frozen-section analyses and staging biopsies, biopsy results, the specialty of the surgeon, and hospital type. RESULTS: The majority (78%) of ovarian borderline tumors primarily were encountered and staged by general obstetrician-gynecologists. Overall, 66% of patients had at least 1 staging biopsy performed. Approximately 12% of subjects underwent complete surgical staging, defined as having biopsy samples taken from pelvic and abdominal peritoneum, omentum, and retroperitoneal lymph nodes. Gynecologic oncologists performed complete staging in 50% of cases, obstetrician-gynecologists performed complete staging in 9% of cases, and general surgeons performed complete staging in 0% cases. The overall frequency of a positive staging biopsy was 37%. Approximately 47% (80 of 169) of patients who underwent biopsies were upstaged as a result of positive biopsies, - with 41% (70 of 169) having extrapelvic spread. CONCLUSIONS: Currently, surgical staging for women with ovarian serous borderline tumors remains inadequate, although a significant proportion of patients who undergo staging are noted to have extrapelvic spread.