Adenoviral-mediated delivery of the herpes simplex virus thymidine kinase gene selectively sensitizes human ovarian carcinoma cells to ganciclovir

One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.     

Nitric oxide signaling in ischemic heart

OBJECTIVE: Several recent studies have implicated a role of endogenous nitric oxide (NO) in the pathophysiology of myocardial ischemic/reperfusion injury. However, the mechanism by which NO exerts its beneficial/detrimental effects remains unknown. This study examined the intracellular signaling of NO by studying the role of the NO-cGMP signaling pathway on the phospho-diesteratic breakdown and turnover of phosphoinositides during myocardial ischemia and reperfusion. METHODS: Isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H]myoinositol and [14C]arachidonic acid. Hearts were then perfused for 10 min in the presence or absence of L-arginine via the Langendorff mode. Ischemia was induced for 30 min followed by 30 min of reperfusion. Experiments were terminated before L-arginine and after L-arginine treatment, after ischemia, and during reperfusion. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. RESULTS: The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-Arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. cGMP, which remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The cGMP level persisted up to 10 min of reperfusion and then dropped slightly. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [14C]-arachidonic acid resulted in modest increases in [14C]diacylglycerol and [14C]phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfusion the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine-treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. CONCLUSIONS: The results suggest for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. The signaling seems to be transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. This signaling effect appears to be reduced as reperfusion progresses. These results, when viewed in the light of free radical chemistry of NO, suggest that such on- and off-signaling of NO may be linked to its interaction with the superoxide radical generated during the reperfusion of ischemic myocardium.     

The association of peripheral monocyte derived interleukin 1beta (IL-1beta), IL-1 receptor antagonist, and tumor necrosis factor-alpha with osteoarthritis in the elderly

OBJECTIVE: To examine the association of peripheral blood mononuclear cell (PBMC) derived interleukin 1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor alpha (TNF-alpha), and radiographic osteoarthritis (OA) in the elderly. METHODS: A total of 703 subjects (436 women, 267 men, mean age 78.5+/-4.5 yrs) had both knee and hand radiographs, and cytokines were measured during the 22nd biennial examination of the Framingham Cohort. PBMC derived IL-1beta , IL-1Ra, and TNF-alpha production was assessed using a non-cross reacting polyclonal radioimmunoassay. Knee OA was defined as a score of > 2 using a modified Kellgren and Lawrence scale. The presence of osteophytes and joint space narrowing were scored separately on a 0-3 scale, in which disease was defined a priori as a score > 0 for each feature. Sex-specific odds ratios were calculated for knee OA after adjusting for weight, history of knee injury, and use of estrogen and nonsteroidal antiinflammatory drugs. RESULT: No uniform associations were found for IL-1beta or IL-1Ra in men, or for TNF-alpha production and radiographic OA in either sex. We found possible associations for the highest levels of IL-1beta production and the presence of knee osteophytes [OR=2.0 (1.2-3.5)] and joint space narrowing [OR=1.7 (1.1-2.8)] in women. Our data suggested a possible protective effect for IL-1Ra production and hand OA in women [OR=0.6 (0.4-1.0)]. CONCLUSION: We found no consistent association of PBMC cytokine production and radiographic OA. However, women with the highest production of IL-1beta and IL-1Ra had respectively higher rates of knee OA and lower rates of hand OA than expected.     

Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression

Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.     

Specific interaction of wild-type and truncated mouse N-methylpurine-DNA glycosylase with ethenoadenine-containing DNA

BACKGROUND: The present study was designed to test the biocompatibility of a new vitamin E-modified multi-layer membrane (CL-E filter), as well as its ability to protect against oxygen free radicals during hemodialysis (HD). METHODS: We investigated, both in vitro and in vivo, the bioreactivity of the filter with respect to the blood antioxidants and its ability to prevent lipoperoxidation. The effects on the leukocyte respiratory burst were also studied. Cuprammonium rayon was used as a comparison material (CL-S filter). RESULTS: The in vitro results demonstrated that, under controlled conditions, CL-E is able to preserve blood antioxidants, and particularly vitamin E, from the spontaneous consumption observed in the incubation with CL-S filters and in control incubations. In accordance with this observation, the rate of the oxidative demolition of lipids either in plasma and red blood cells (RBC) or from rat brain homogenate decreased after the exposure to CL-E filters in comparison with the CL-S filter. Moreover, in the absence of any significant cytotoxic effects due to both the types of material studied, the production of oxygen free radicals and nitric oxide (NO) by leukocytes was higher after their in vitro exposure to CL-S, but was quite similar to that of the control leukocytes after exposure to CL-E. In vivo, a one-month treatment with the CL-E filter increased plasma vitamin E by 84.3% with respect to treatment with CL-S; this gain slightly decreased to 68.9% when CL-E treatment was prolonged to three months. In the RBC, vitamin E was found to have increased by 76.7% and 113.4% at one and three months, respectively. Plasma glutathione (GSH) levels determined at three months were significantly increased from 0.10 +/- 0.02 to 0.33 +/- 0.12 mumol/ml, while the erythrocyte GSH was only slightly increased. The leukocyte function estimated as responsiveness to soluble chemical stimuli in CL-S-treated patients was significantly improved both qualitatively and quantitatively after CL-E treatment. The presence of an increased number of mononuclear cells undergoing programmed cell death (apoptosis) in CL-S-treated patients (18.8 +/- 1.7% vs. a control value of 6.5 +/- 2.3%) as well as the apoptogenic effect of their plasma in vitro on U937 cells was significantly corrected after CL-E treatment (mean decrease in apoptotic mononuclear cells at 24 hours of culture, 25.5% and 27.1% at 1 and 3 months, respectively). The anti-apoptogenic effect of CL-E treatment showed a close dependence on the increase in vitamin E in the blood cell compartment. CONCLUSIONS: This study suggests that this vitamin E-modified membrane can be considered a highly biocompatible material, the antioxidant properties of which can exert a site-specific and timely scavenging function against oxygen free radicals in synergy with a hypostimulatory action on the PMN respiratory burst.     

Nuclear estradiol receptor in the adult rat uterus: a new exchange assay

A protamine exchange assay has been developed to measure uterine nuclear estrogen receptor in mature rats exposed to estradiol (E). After ovariectomized-adrenalectomized mature rats are injected with E, estrogen receptor (RnE) is extracted from uterine nuclei with 0.6 M potassium chloride, diluted, and quantitatively precipitated with protamine sulfate. The precipitate is subjected to a ligand exchange with radiolabeled estradiol (E), with or without unlabeled diethylstilbesterol, to determine nonspecific binding. At 37 degrees C complete exchange of E for E in RnE is observed at 2.5 h; virtually no receptor degradation occurs up to at least 5 h. Exchange does not occur at 4 degrees C. Using the protamine assay, the depletion of cytoplasmic estrogen receptor (Rc) and the accumulation of RnE were studied at various doses of E at specific time points. Increasing doses of E result in a decrease of Rc with an equal increase of RnE. At the highest dose of E (10 mug) Rc is completely depleted within 10 min, by 6 h it is 25% replenished, and by 24 h returns to slightly above control levels. Within 10 min after the injection, RnE increases to 80-90% of the original cytoplasmic level of receptor (approximately 2-3 pmol/mg of DNA or approximately 1.5 pmol/100 mg of uterus). At 6 h RnE is 75% depleted and it is completely absent at 24 h. The protamine assay permits precise quantitative studies of nuclear estrogen receptor and avoids the problems of receptor degradation and excessive nonspecific binding often found in exchange reactions at elevated temperatures.