Cardiac T2* magnetic resonance for prediction of cardiac complications in thalassemia major.

BACKGROUND: The goal of this study was to determine the predictive value of cardiac T2* magnetic resonance for heart failure and arrhythmia in thalassemia major. METHODS AND RESULTS: We analyzed cardiac and liver T2* magnetic resonance and serum ferritin in 652 thalassemia major patients from 21 UK centers with 1442 magnetic resonance scans. The relative risk for heart failure with cardiac T2* values <10 ms (compared with >10 ms) was 160 (95% confidence interval, 39 to 653). Heart failure occurred in 47% of patients within 1 year of a cardiac T2* <6 ms with a relative risk of 270 (95% confidence interval, 64 to 1129). The area under the receiver-operating characteristic curve for predicting heart failure was significantly greater for cardiac T2* (0.948) than for liver T2* (0.589; P<0.001) or serum ferritin (0.629; P<0.001). Cardiac T2* was <10 ms in 98% of scans in patients who developed heart failure. The relative risk for arrhythmia with cardiac T2* values <20 ms (compared with >20 ms) was 4.6 (95% confidence interval, 2.66 to 7.95). Arrhythmia occurred in 14% of patients within 1 year of a cardiac T2* of <6 ms. The area under the receiver-operating characteristic curve for predicting arrhythmia was significantly greater for cardiac T2* (0.747) than for liver T2* (0.514; P<0.001) or serum ferritin (0.518; P<0.001). The cardiac T2* was <20 ms in 83% of scans in patients who developed arrhythmia. CONCLUSIONS: Cardiac T2* magnetic resonance identifies patients at high risk of heart failure and arrhythmia from myocardial siderosis in thalassemia major and is superior to serum ferritin and liver iron. Using cardiac T2* for the early identification and treatment of patients at risk is a logical means of reducing the high burden of cardiac mortality in myocardial siderosis. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00520559.

Joining the two domains of a group I ribozyme to form the catalytic core.

Self-splicing group I introns, like other large catalytic RNAs, contain structural domains. Although the crystal structure of one of these domains has been determined by x-ray analysis, its connection to the other major domain that contains the guanosine-binding site has not been known. Site-directed mutagenesis and kinetic analysis of RNA splicing were used to identify a base triple in the conserved core of both a cyanobacterial (Anabaena) and a eukaryotic (Tetrahymena) group I intron. This long-range interaction connects a sequence adjacent to the guanosine-binding site with the domain implicated in coordinating the 5' splice site helix, and it thereby contributes to formation of the active site. The resulting five-strand junction, in which a short helix forms base triples with three separate strands in the Tetrahymena intron, reveals exceptionally dense packing of RNA.

A randomized, placebo-controlled, double-blind trial of the effect of combined therapy with deferoxamine and deferiprone on myocardial iron in thalassemia major using cardiovascular magnetic resonance.

BACKGROUND: Cardiac complications secondary to iron overload are the leading cause of death in beta-thalassemia major. Approximately two thirds of patients maintained on the parenteral iron chelator deferoxamine have myocardial iron loading. The oral iron chelator deferiprone has been demonstrated to remove myocardial iron, and it has been proposed that in combination with deferoxamine it may have additional effect. METHODS AND RESULTS: Myocardial iron loading was assessed with the use of myocardial T2* cardiovascular magnetic resonance in 167 patients with thalassemia major receiving standard maintenance chelation monotherapy with subcutaneous deferoxamine. Of these patients, 65 with mild to moderate myocardial iron loading (T2* 8 to 20 ms) entered the trial with continuation of subcutaneous deferoxamine and were randomized to receive additional oral placebo (deferoxamine group) or oral deferiprone 75 mg/kg per day (combined group). The primary end point was the change in myocardial T2* over 12 months. Secondary end points of endothelial function (flow-mediated dilatation of the brachial artery) and cardiac function were also measured with cardiovascular magnetic resonance. There were significant improvements in the combined treatment group compared with the deferoxamine group in myocardial T2* (ratio of change in geometric means 1.50 versus 1.24; P=0.02), absolute left ventricular ejection fraction (2.6% versus 0.6%; P=0.05), and absolute endothelial function (8.8% versus 3.3%; P=0.02). There was also a significantly greater improvement in serum ferritin in the combined group (-976 versus -233 microg/L; P<0.001). CONCLUSIONS: In comparison to the standard chelation monotherapy of deferoxamine, combination treatment with additional deferiprone reduced myocardial iron and improved the ejection fraction and endothelial function in thalassemia major patients with mild to moderate cardiac iron loading.

[Hospitalization period and nutritional status in hospitalized patients]. / Tiempo de hospitalización y estado nutricional en pacientes hospitalizados.

With the objective of studying the nutritional status and its relationship with hospitalization period, a cross-sectional study was done with patients from a private hospital representing a population with a better socioeconomic condition. The anthropometric data of 267 patients, 46% males and 54% females ranging from 20 to 80 years of age, were assessed on the second day of hospitalization. Hospitalization period associated with nutritional status. The data were analyzed by the software Excel and Sigma Stat, using Fisher's exact test and the chi-square test. The studied population presented a body mass index of 25.9 +/- 5.3 and most patients lost weight during hospitalization. The longest hospitalization periods were found among patients with lung diseases (13 days), some being pre-obese (40%) with a small prevalence of undernutrition (4%). The percentage distribution of nutritional status among the groups according to diagnosis was different (P < 0.01) when assessed by the Fisher's exact test and the percentage distribution in weight variation between men and women was different (P < 0.02) when assessed by the chi-square test. When the population was segmented according to age, the percentage distribution of the nutritional status between > 60 and < or = 60 did not present a difference when assessed by the chi-square test. The results of this study show that the nutritional status in some diseases deserves special attention given the greater risk found in these situations, contributing to a longer hospitalization period.

Irradiation and prolactin effects on rat mammary carcinogenesis: intrasplenic pituitary and estrone capsule implants.

The implantation of silicone capsules that contained estrone and that were adjacent to grafts of anterior pituitary tissue in the spleens of adrenalectomized glucocorticoid-deficient inbred F344 rats resulted in high circulating prolactin (Prl) levels without the untoward effects of chronic hyperestrogenism or of grafts of Prl-secreting pituitary tumors. All peripheral serum estrone titers were below the titers in sera of proestrous untreated intact rats. Peripheral serum estrone and Prl levels were, however, a function of capsule surface area over the capsule sizes tested (12-74 mm2); the elevated Prl levels persisted for as long as 700 days. In adrenalectomized glucocorticoid-deficient female rats, both 5 Gy gamma-irradiation alone and intrasplenic pituitary-estrone implants alone induced mammary carcinomas; the combination of these treatments induced a greater incidence of first carcinomas and reduced first carcinoma latency. There were, however, no marked differences in tumor incidence or latency due to differences in estrone capsule size. Finally, ovariectomy reduced first carcinoma risk in irradiated, pituitary-estrone-implanted rats but did not change the time of maximum risk. Ovarian secretory activity thus persisted in such rats and ovarian hormones synergized with Prl in mammary carcinoma induction.

Regression of rat primary mammary tumors following dietary d-limonene.

The effects of d-limonene on rat mammary tumors were investigated. Following the appearance of mammary tumors induced by 7,12-dimethylbenz[a]anthracene in female (W/Fu X F344)F2 rats, animals were assigned to pairs in which 1 rat was fed a diet containing 10% d-limonene and the other was pair fed an isocaloric diet containing 10% cellulose. There was a highly significant increase in the regression of the first tumors in the rats fed d-limonene. In addition, d-limonene given at this time inhibited the formation of subsequent multiple tumors in these rats.

Sensitization of nitrosourea-resistant Mer+ human tumor cells to N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea by mild (41 degrees C) hyperthermia.

Experiments were designed to determine whether heat treatment could sensitize nitrosourea-resistant human tumor cell lines expressing a repair system (O6-alkylguanine DNA alkyltransferase; Mer+) capable of removing monoadducts from the DNA of treated cells prior to the formation of lethal interstrand cross-links. Effects of temperatures compatible with systemic hyperthermia were of particular interest, and, consequently, the effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) exposure in vitro for 4 h at 37 degrees C was compared with that for 1 h at 41 degrees C followed by 3 h at 37 degrees C. CCNU toxicity was significantly enhanced by heat treatment in the Mer+ HT-29 human colon carcinoma, and in HeLa-S3 and HeLa-CCL2 cell lines [thermal enhancement factor (ratio of CCNU doses required to reduce cell survival to 0.001 at 37 degrees C and 41 degrees C) = 1.3-1.4]. Pharmacokinetic studies indicated that the effect of heat treatment on CCNU toxicity was not attributable to exposure to increased concentrations of reactive species, nor was the enhancement due to a direct effect of heat and/or drug on alkyltransferase activity. A similar enhancement of CCNU toxicity was also observed in a Mer- line, HeLa-MR (thermal enhancement factor = 1.3). Heat-sequencing experiments clearly demonstrate that heat and CCNU must be administered concurrently. Alkaline elution experiments were designed to examine DNA-DNA cross-link formation in Mer+ and Mer- cells exposed to CCNU at 37 degrees C and 41 degrees C, but quantitation of cross-link formation was not possible owing to the persistence of single strand breaks in the DNA of drug-treated cells. Nevertheless, collectively the data indicate that thermal enhancement of CCNU toxicity is independent of effects on alkytransferase activity and indicate that hyperthermia could provide an effective strategy for improving the nitrosourea response of resistant Mer+ tumors.

Assessment of radiogenic cancer initiation frequency per clonogenic rat mammary cell in vivo.

Radiogenic initiation of mammary cancer is here shown to be a common cellular event. With the aid of a rat mammary clonogen transplant system designed to maximize progression of initiated cells to overt neoplasia, the estimated absolute cancer risk per surviving 7 Gy-irradiated mammary clonogen was [4.1 +/- 1.2 (SE)] X 10(-3), that is (5.8 +/- 1.7) X 10(-4) initiated cells per clonogen-Gy. To maximize neoplastic progression, all clonogen graft recipient rats were glucocorticoid deficient (adrenalectomized with minimal mineralocorticoid replacement) and hyperprolactinemic (implanted intrasplenically with pituitary tissue and an estrone capsule). Each rat was grafted with 4 X 10(6) "morphologically intact" monodispersed mammary cells in the interscapular white fat pad. Group A received cells which had been irradiated 1 day earlier with 7 Gy 137Cs gamma-rays. Groups B and C received unirradiated mammary cells. On day 35 after transplantation, the graft sites of group B were locally exposed to 7 Gy 140 kVp X-rays. Kaplan-Meier estimates (11) of the survivor functions were used to calculate the final tumor incidences corrected for intercurrent animal loss. Estimated mammary carcinoma frequencies so calculated were 65 tumors/131 graft sites in group A, 93/119 in group B, and 13/129 in group C. The relative cancer risks per rat due to the radiation exposure of the grafted cells were 5.0 for group A and 7.8 for group B. These data on neoplasia incidence in grafted mammary clonogens and data on autologous neoplasia in the host rat mammary glands were subjected to statistical analysis. The results indicate that both neoplasm frequency and latency are in large part dependent on initiation target cell number. The high frequency of radiogenic initiation per mammary clonogen observed in this study is in accord with findings with a similar thyroid clonogen transplant system.

Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin.

The effects of dietary supplementation of flavonol quercetin on both 7,12-dimethylbenz(a)anthracene (DMBA)- and N-nitrosomethylurea-induced mammary cancer in female Sprague-Dawley rats were determined. Quercetin diet was started 1 wk before intragastric instillation of DMBA (65 mg/kg of body weight) or i.v. injection of N-nitrosomethylurea (50 mg/kg of body weight) and was continued during the entire period (20 wk) of the experiment. Dietary quercetin inhibited both the incidence and the number of palpable rat mammary tumors; rats fed on 2% quercetin had 25% less incidence of mammary cancer, while the average number of mammary tumors per rat was reduced by 39% at 20 wk post-DMBA administration compared to animals on a control diet. In a separate experiment, a 5% quercetin diet elicited a greater inhibitory effect on the induction of rat mammary tumors by DMBA than was observed with a 2% quercetin diet. The inhibitory effect of quercetin on mammary tumor incidence in rats on 2% and 5% diets and on tumor multiplicity in animals on a 5% diet was statistically significant (P less than 0.05). In addition, the risk of the development of a palpable tumor (as determined by the nonparametric estimate of the hazard function) in the quercetin-fed group was lower than the group on control diet throughout the course of the experiment. Furthermore, 5% dietary quercetin significantly inhibited (P less than 0.05), although to a lesser extent than observed in DMBA-induced tumor formation, both the incidence and the number of palpable mammary tumors per rat induced by N-nitrosomethylurea. Dietary quercetin did not elicit any detectable sign of toxicity. The gain in body weight in rats on the quercetin diet and the quantity of diet consumed per rat per week were similar to those for rats on the control diet.

Interactions of thymidine, hyperthermia, and cis-diammine-1,1-cyclobutane dicarboxylate platinum (II) in human T-cell leukemia.

Using JM and MOLT3, two human T-cell acute lymphoblastic leukemia cell lines, we investigated the ability of 24-h thymidine exposures to enhance the cytotoxicity of cis-diammine-1,1-cyclobutane dicarboxylate platinum (II) (carboplatin). Clinically achievable thymidine concentrations (for 24 h) significantly enhanced carboplatin killing. Unexpectedly, thymidine-carboplatin enhancement was as great at a relatively low 200-micrograms thymidine/ml as at the clinically much more toxic range of 1000 micrograms/ml. For a constant thymidine concentration (500 micrograms/ml), thymidine-carboplatin interaction increased with longer thymidine exposures until about 16 to 24 h. Thymidine and 41.8 degrees C hyperthermia (for 1 h) together enhanced carboplatin killing significantly more than did hyperthermia-carboplatin or thymidine-carboplatin combinations. These results show that relatively brief, presumptively nonmyelosuppressive thymidine exposures can significantly increase carboplatin killing. Carboplatin-thymidine killing can then be further augmented by 41.8 degrees C hyperthermia.

Systematic optimization of the clonal growth of human primary breast carcinoma cells.

Human breast carcinomas have been one of the most difficult tumor types to culture in agar-based clonogenic assays. This fact has limited their clinical applicability. We have used statistically motivated experimental designs to systematically improve the clonal culture of enzymatically monodispersed primary human carcinoma cells in an anchorage-independent agar system. Based upon an initial comparison of two basal media, we selected one which gave the best colony growth and then sought to optimize the individual additives in the medium. Hydrocortisone, fetal bovine serum, and red blood cells all improved both plating efficiency and median size of colonies derived from breast carcinoma cells. Next, the concentrations of these three components were simultaneously idealized using response surface methodology. By these methods, it was found that the optimal concentration of hydrocortisone was 0.35 microgram/ml, fetal bovine serum was 6.5%, and red blood cells was 2.1 X 10(7) cells/ml. Using these culture conditions, we have achieved plating efficiencies of 0.39% and 0.19% for colonies with diameters greater than 50 (50 cells) or 70 (130 cells) micron, respectively.

Factors influencing options in primary breast cancer treatment.

Primary breast cancer treatment is determined by tumor factors and by patient preference. Breast cancer treatments that preserve the cosmetic appearance of the breast are appealing and effective for appropriately selected patients; long-term survival following tumor excision and breast irradiation appears to be comparable to that for mastectomy. Since April 1981, when a protocol was developed and treatment options were offered, factors influencing treatment selection have been analyzed in 206 consecutive primary breast cancer patients. Mastectomy was dictated by tumor-related factors in 96 patients (47%); 110 patients (53%) had the option of mastectomy or conservation--tumor excision plus radiotherapy to the breast. Among these 110 eligible patients, 54 chose conservation (49%) and 56 chose mastectomy (51%). Intraoperative findings for ten patients electing conservation necessitated mastectomy, so conservation was accomplished for 44 (21%) of those treated for breast cancer. Beginning in July 1982, breast cancer patients took a battery of psychosexual assessments before any operation (Profile of Mood States [POMS], Health Locus of Control Scale [HLCS] Locke-Wallace Marital Adjustment Test [MAT], Psychosocial Adjustment to Illness Scale [PAIS], Derogatis Sexual Function Inventory [DSFI], Millon Clinical Multiaxial Inventory [MCMI], and a Breast Cancer Information Test [BCIT]). Comparisons of psychologic and demographic variables were made between patients who chose mastectomy and those who chose conservation. No demographic variable was statistically significantly related to choice, although older women tended to select mastectomy more than younger women. Compared with those who elected conservation, women who elected mastectomy were more tense and anxious (P less than .01), more introverted (P less than .01), felt more depressed and dejected (P less than .05), and reported more sexual problems (P less than .05). Those who elected conservation valued their physical appearance more highly (P less than .01) and were generally more self-interested (P less than .05). Mastectomy was dictated by medical considerations for approximately half of patients with breast cancer. Among candidates for breast conservation, the importance of retaining the breast appeared to be determined to a significant degree by measurable psychological factors.

One hundred autotransplants for relapsed or refractory Hodgkin's disease and lymphoma: value of pretransplant disease status for predicting outcome.

PURPOSE: One hundred autotransplants for Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL) were examined prospectively to identify variables with prognostic significance. PATIENTS AND METHODS: Ninety-six patients with relapsed or refractory HD or NHL underwent 100 autotransplants. Patients received high-dose carmustine (BCNU), etoposide, cytarabine, and cyclophosphamide (BEAC) followed by unpurged autologous stem-cell rescue. RESULTS: The 3-year actuarial event-free survival (EFS) rate for the 47 HD patients is 49%, with a median followup duration of 2 years. For the 53 NHL patients, the 3-year actuarial EFS rate is 40%, with a median follow-up duration of 19 months. By multivariate analysis, minimal disease on admission (all areas < or = 2 cm) is associated with improved EFS (HD, P = .003, NHL, P = .03). The projected EFS rate for HD patients entering with minimal disease is 70% versus 15% for patients with bulky disease (P = .0001). The projected EFS rate for NHL patients with minimal disease is 48% versus 25% for patients with bulky disease (P = .04). Posttransplant involved-field radiotherapy, administered to 26 of the last 61 patients, was associated with an improved EFS rate for NHL patients (P = .015). The BEAC regimen was well tolerated by patients who entered the study with minimal disease (mortality rate, < 5%), but caused significant toxicity in patients with bulky disease (mortality rate, 25%). CONCLUSION: Disease burden before autotransplantation is an important predictor of regimen-related toxicity and EFS. Posttransplant involved-field radiotherapy may improve outcomes in select patients with NHL. The BEAC regimen is safe and effective, particularly for patients with minimal disease.

Effects of oxygen tension on endothelium dependent responses in canine coronary microvessels.

STUDY OBJECTIVE: The aim was to determine the direct effects of oxygen tension on endothelium dependent vasodilator responses in canine coronary microvessels. DESIGN: Coronary microvessels were isolated and studied in vitro in a no flow constant pressure state using a video dimension analysing system. Microvessels were exposed to different partial pressures of oxygen. Endothelium dependent responses to acetylcholine and A23187 calcium ionophore were obtained with and without indomethacin during hyperoxia and normoxia, and compared to responses during hypoxia. Dose-response curves were also obtained to the direct smooth muscle dilator nitroprusside during normoxia and hypoxia. The reversibility of the effects of hypoxia on the acetylcholine response was studied after return to hyperoxic conditions following hypoxia. EXPERIMENTAL MATERIAL: Coronary microvessels (58-150 micron diameter) were obtained from adult mongrel dogs of either sex. MEASUREMENTS AND MAIN RESULTS: Exposure of preconstricted microvessels to hypoxia alone [PO2 5.8(0.4)kPa] resulted in a 25.9(SEM 6.8)% relaxation that was abolished by indomethacin [0.35(2.9)% relaxation]. Acetylcholine elicited dose dependent vasodilatation, with no significant differences in sensitivity between normoxia [PO2 14.6(0.04) kPa] and hypoxia: EC50 = 0.023 v 0.027 mumol.litre-1, respectively. During hyperoxia [PO2 80.2(6.0) kPa] there was a significant increase in the EC50 value to 0.09 mumol.litre-1 (hypoxia and normoxia v hyperoxia). After inhibition of prostaglandin synthesis with indomethacin, the sensitivity to acetylcholine was significantly decreased during hypoxia (EC50 = 0.16 mumol.litre-1) when compared to normoxia and hyperoxia. Indomethacin alone did not alter the acetylcholine response during normoxia and hyperoxia. As with acetylcholine, the sensitivity of indomethacin treated microvessels to A23187 was also decreased during hypoxia when compared to hyperoxia. There was no difference in the nitroprusside response during hypoxia and hyperoxia. The decreased vasodilator response to acetylcholine after hypoxia was persistent up to 2 h after return to hyperoxic conditions. CONCLUSIONS: Hypoxia decreases vasodilatation due to endothelium dependent relaxing factor, and oxygen tension has an important influence on both receptor dependent and receptor independent endothelium dependent vasodilator responses in coronary microvessels. Hypoxia also induces a prostaglandin mediated dilatation of preconstricted coronary microvessels. The effects of hypoxia on endothelium dependent responses are persistent up to 2 h.

Prostaglandin-mediated inhibition of nitric oxide production by bovine aortic endothelium during hypoxia.

OBJECTIVE: The present study utilized a monoculture of vascular endothelium to: (1) determine if nitric oxide (NO) production was decreased during hypoxia, (2) ascertain if specific prostaglandins were released in response to hypoxia, and (3) determine if cyclo-oxygenase inhibition would modulate hypoxia-induced decreases in NO production. METHODS: Bovine aortic endothelial cells (BAE cells) were grown to confluence on microcarrier beads. NO released in response to the receptor-independent agonist, A23187 calcium ionophore, was directly and continuously measured using a sensitive and specific chemiluminescence method. Cells were exposed to either "hypoxia" (pO2 = 10 mmHg) or "normoxia" (pO2 = 160 mmHg) for 30 min. NO was quantitatively measured with and without indomethacin (1.7 microM) in the incubation medium, and also following incubation with the prostacyclin analog, iloprost. The prostaglandins PGI2 and PGE2 released in response to hypoxia were quantitated using an enzyme immunoassay. RESULTS: Hypoxia significantly decreased NO production, resulting in a 22.8(2.1)% reduction in NO from 94.3(5.3) nmol/micrograms protein (during normoxia) to 73.5(2) nmol/micrograms protein (during hypoxia). Hypoxia significantly stimulated the production of PGI2 and PGE2, in excess of that released in response to A23187 when compared with normoxia. Following cyclo-oxygenase inhibition, the hypoxia-induced decrease in NO production was abolished (0.13 [2.7] % change relative to controls). Furthermore, iloprost (10 nM) directly inhibited NO production. CONCLUSIONS: The results demonstrate that ionophore-stimulated NO production is sensitive to oxygen tension, decreasing in response to hypoxia. Inhibition of prostaglandin synthesis restores NO production during hypoxia, while iloprost directly suppresses NO production. Thus, endothelium-derived prostanoids produced in response to hypoxia may modulate NO production via an autocrine negative feedback mechanism.

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells.

OBJECTIVE: Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. METHODS: Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2 = 10 mmHg) or normoxia (pO2 = 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. RESULTS: Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol . micrograms-1 protein, hypoxia vs. normoxia respectively, n = 8, P < 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol . microgram-1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol . micrograms-1 protein (n = 6, p < 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. CONCLUSIONS: These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide.

Specific activity of skeletal alkaline phosphatase in human osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase activity inversely regulated by calcium.

We assessed the significance of Ca and phosphate (P(i)) as determinants of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJb medium containing 0.25-2 mM P(i), the specific activity of ALP in SaOS-2 cells was proportional to P(i) concentration (r = 0.99, p < 0.001). The P(i)-dependent increase in ALP activity was time dependent (evident within 6 h) and could not be attributed to decreased ALP release, since P(i) also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25-2.0 mM) showed that the amount of ALP activity released from SaOS-2 cells was inversely proportional to the concentration of Ca (r = -0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) and could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinositol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol-glycan-anchored ALP, released by membrane vesicle formation). The range of Ca-dependent changes in ALP-specific activity was much smaller than the range of P(i)-dependent changes. The observed correlation between skeletal ALP-specific activity and P(i) was not unique to osteosarcoma cells or to P(i). Similar effects were seen in normal human bone cells in response to P(i) (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of P(i) esters and analogs (e.g., beta-glycero-P(i) and molybdate). Further studies indicated that the effects of phosphoryl compounds on ALP-specific activity could not be correlated with effects on ALP reaction kinetics, cell proliferation, or acid phosphatase activity and that the beta-glycero-P(i)-dependent increase in ALP activity was blocked by cycloheximide but not actinomycin D. Together these data suggest that the function of skeletal ALP may be regulated by P(i) and that Ca may be involved in ALP release.

A comparison of tocopherol and tocotrienol for the chemoprevention of chemically induced rat mammary tumors.

Two forms of vitamin E, tocopherol and tocotrienol, were tested for chemopreventive activity in two chemically induced rat mammary-tumor models. When mammary tumors were induced by 7,12-dimethylbenz(a)anthracene (DMBA, 50 mg/kg), only the tocotrienol group had a statistically significant increase in tumor latency. There was no effect of either compound on tumor multiplicity. When tumors were induced by N-nitrosomethylurea (NMU, 30 mg/kg), neither analogue of vitamin E modified latency, whereas tocotrienol increased tumor multiplicity. In summary, neither vitamin analog had a major impact on mammary-tumor development after tumor induction with either DMBA or NMU.

Radiation survival of human mammary carcinoma cells: criteria for an agar-based clonogenic assay.

A human in vivo-in vitro model for breast cancer has been developed based on culture methods that were systematically optimized for solid breast carcinomas. A V-79 cell clonal agar-based model was used to define the analysis problem encountered with agar based clonogenic survival assays. Based on this, we established methods for the generation and analysis of the survival of primary and recurrent breast carcinomas following irradiation. Four out of five primary breast carcinomas studied had similar radiation-cell survival curves. The fifth tumor was more radioresistant. Interestingly, a recurrent breast carcinoma arising in a heavily irradiated chest wall was no more radioresistant than our series of unirradiated primary carcinomas. These methods may be useful both for the study of the radiobiology of human neoplasms and for customizing the treatments and prognosis of individual patients.

Ethanol increases endothelial nitric oxide production through modulation of nitric oxide synthase expression.

Moderate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO.