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Background Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells. Methods and Results Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [ 3 H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2×10 4 to 8×10 4 receptors per cell and a K d of ≈5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex. Conclusions These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.

DNA double strand breaks (DSBs) are the most cytotoxic lesions induced by topoisomerase II poisons. Enzyme-mediated repair of DNA DSBs is a major mechanism of resistance to DNA DSB-inducing agents. Non-homologous end joining (NHEJ) is the major pathway for the repair of DSBs and requires DNA-PK activity. DNA-PK has close structural similarities to PI-3K, which promotes cell survival and proliferation and is up-regulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. We investigated the therapeutic utility of KU-0060648, in models of human cancer. KU-0060648 was investigated in a panel of human breast and colon cancer cells, it inhibited cellular DNA-PK auto-phosphorylation with IC50 values of 0.019 µM (MCF7 cells) and 0.17 µM (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC50 values of 0.039 µM (MCF7 cells) and >10 µM (SW620 cells). 5-day exposure to 1 µM KU-0060648 inhibited cell proliferation by > 95% in MCF7 cells, but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitisation were maintained within the tumour for at least 4 hours at non-toxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumour growth delay in both SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof of principle in vitro and in vivo chemosensitisation with KU-0060648 justifies further evaluation and characterisation of dual DNA-PK and PI-3K inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2011-2548

beta thalassemia major, a severe genetic disorder of the red blood cell, affects about 1,400 people in the United States, including children. Clinical management of this disorder has improved significantly in recent years, and ongoing research will lead to an even brighter outlook in the future.

One of the most prevalent of cancers, breast cancer, is characterized by hormonal control of its growth. Expression of the estrogen receptor (ER) in MCF-7 breast cancer cells appears to be a complex process involving multiple steps subject to hormonal regulation by estrogen. Treatment of MCF-7 cells with estradiol results in the suppression of estrogen receptor protein. By 6 hours, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA and remained suppressed for 24-48 hours. Similar results were obtained with an estrogen receptor binding assay. Estrogen treatment also resulted in a decrease of receptor mRNA to approximately 10% of control values by 6 hours. Estrogen receptor remained at the suppressed level for up to 48 hours. Transcription run-on experiments demonstrated a transient decrease of about 90% in receptor gene transcription after 1 hour. By 3-6 hours transcription increased approximately 2-fold and remained elevated for at least 48 hours. These data suggest that estrogen suppresses ER mRNA by inhibition of ER gene transcription at early times and by a post-transcriptional effect on receptor mRNA at later times. To determine whether post-transcriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study the regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the post-transcriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis.

Ataxia telangiectasia mutated (ATM) is an important serine/threonine kinase involved in the DNA damage response and cell cycle checkpoint control. The 350kDa protein is a member of the PI3-kinase like kinase (PIKK) family and is mutated in the neurodegenerative disorder, Ataxia telangiectasia. ATM exists in the inactive form as a dimer but following DNA damage is phosphorylated at a serine residue at position 1981 and dissociates to become an active monomer. It detects DNA double strand breaks (DSBs) through the Mre11/Rad50/NBS1 (MRN) complex and signals this information downstream to the cell cycle machinery, for example by the phosphorylation of p53 (Ser15) and Chk2 (Thr68). ATM is a key signalling molecule for DNA double strand breaks that can regulate the homologous recombination repair pathway. Inhibition of ATM activity sensitises cells to the cytotoxic effects of DNA damaging agents such as some chemotherapies or ionising radiation that either directly or indirectly induce double stand breaks. Here we present the biological profile of KU-0060019, a potent and specific novel small molecule inhibitor of ATM kinase activity. The compound has an IC50 value against the enzyme of 5 nM with above 1 μM IC50 value against DNA-PK and inhibits cellular phosphorylation of p53 at serine 15 with an EC50 value of 150 nM. It sensitises cells to chemotherapeutics, in particular topoisomerase II inhibitors, as well as radiotherapeutics in vitro in a variety of cancer cell lines. We also demonstrate potentiation of chemotherapeutics in in vivo mouse xenograft tumour models. Using the novel ATM inhibitor KU-0060019 these studies demonstrate the potential of targeting ATM as a sensitising mechanism by a small molecule in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3928.

Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) has shown anti-tumor activity in patients with hereditary BRCA mutations and provides positive evidence for synthetic lethal approaches for the treatment of cancers with DNA repair defects. The BRCA genes are regulators of homologous recombination (HR) DNA repair pathway but deficiency in other mediators of this pathway, such as the MRE11-NBS1-Rad50 (MRN) complex have also been demonstrated to be synthetically lethal with PARP inhibition in pre-clinical models. Cells deficient in HR DNA repair or signalling pathways are potentially sensitive to PARP inhibition and have been termed homologous recombination deficient (HRD). Examples of HRD include inactivating MRE11 mutations which have been linked to mismatch repair (MMR) deficient, microsatellite instable (MSI) colorectal and endometrial cancers. Previous studies have indicated a high frequency (up to 87% of MSI colorectal cancers) of −1/−2 frameshifts within the poly(T)11 microsatellite repeat sequence and a corresponding reduction in expression. We have therefore undertaken a pre-clinical study to determine whether MRE11-deficient colorectal cancer (CRC) respond to olaparib treatment. Methods: CRC cell line panels with known MSI status as well as isogenic matched cell lines models of MRE11 and MMR-deficiency were assessed for their response to olaparib by both MTS viability and clonogenic survival assays. Quantitative RT-PCR mRNA and protein expression analysis of MRE11 and DNA damage response genes were obtained for each cell line and HRD status determined. Expression of Pgp (ABCB1) multi-drug transporter, a pre-clinical resistance factor for olaparib and whose over-expression is associated with CRC, was also determined. Results: MTS viability assays showed a limited range of sensitivities between CRC cell lines and only modest correlation with molecular profiles. Analysis using sensitive long term clonogenic survival assays showed a wider range of sensitivities and revealed that HRD (MRE11 mutant) and MSI CRC cells were sensitive to olaparib treatment while microsatellite stable (MSS) and non-HRD (MRE11 wild-type) cells remained insensitive. Using isogenic cell line models we confirmed that loss of MRE11 expression can confer sensitivity to olaparib whereas MMR-deficiency did not. CRC cells which were HRD but overexpressed Pgp were more resistant to olaparib but could be re-sensitized to treatment by co-administration of the Pgp inhibitors or through the use of alternative non-Pgp substrate PARP inhibitors. Conclusions: Here we demonstrate that CRC cell lines with HRD due to MRE11 mutation and loss of protein expression are highly sensitive to the PARP inhibitor olaparib. These data provide evidence that patients with MSI and MRE11-deficient colorectal tumors could be suitable for treatment with olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A114.

DNA double strand breaks (DSBs) are profoundly cytotoxic unless rapidly repaired. Non-homologous end joining (NHEJ) is the predominant pathway of DSB repair in mammalian cells. DNA-dependent protein kinase (DNA-PK), a member of the PI3-kinase like kinase (PIKK) family, is an essential component of NHEJ, and DNA-PK inhibition in combination with radio- or chemotherapy therefore represents an attractive therapeutic approach in cancer therapy. Through medicinal chemistry, a series of DNA-PK inhibitors of increasing potency was identified, resulting in KU-0060648, a potent DNA-PK inhibitor with an IC50 value of 10nM against the enzyme and high selectivity for DNA-PK over other PIKKs and a panel of 60 kinases. In cellular assays, KU-0060648 significantly increases cell death induced by ionising radiation (IR) or chemotherapeutic treatments, such as doxorubicin or etoposide. KU-0060648 does not sensitise DNA-PK-deficient cells to IR or chemotherapeutic treatments, further highlighting its selective nature. The compound was also found to preferentially kill DNA repair-defective cells in combination with IR or chemotherapy, indicating a therapeutic advantage for this class of agent in DNA repair-compromised tumor cells. Auto-phosphorylation of DNA-PK at serine 2056 is inhibited by KU-0060648, and significant changes in IR- or chemotherapy-induced H2AX- and KAP-1 phosphorylation in the presence KU-0060648 over time clearly indicates inhibition of DNA damage repair by this compound. Pharmacokinetic and pharmacodynamic profiling of KU-0060648 revealed that the compound has very good oral bioavailability and a favourable half life. In a fractionated IR xenograft model, dosing with KU-0060648 caused significant tumor growth delay compared to the control group. PK/PD relationships were established to define the exposures and level of DNA-PK inhibition required for effective radiosensitisation. Our results to date support further in vitro and in vivo evaluation of this novel class of therapeutic agents as a potential clinical radio- and chemosensitiser. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A116.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the regulation of the estrogen receptor (ER) was investigated in this study. After treatment with 100 nM TPA the concentration of receptor protein was measured using an enzyme immunoassay. By 24 h the receptor protein declined by about 80% from a level of approximately 236 fmol of ER/mg of protein in control cells to 50 fmol of ER/mg of protein in cells treated with TPA. Similar results were obtained with an estrogen receptor ligand binding assay. After removal of TPA, the level of ER returned to control values. 4-alpha-Phorbol, a compound related to TPA, had no effect on ER. The effects of TPA on ER expression appear to be mediated by activation of protein kinase C as H-7, an inhibitor of protein kinase C, blocks these effects. In addition to the effect on ER protein, TPA treatment also resulted in a decrease in the steady-state level of ER mRNA as determined by a RNase protection assay. The metabolic inhibitor cycloheximide was unable to prevent the TPA-induced decrease in ER mRNA. Transcription run-off experiments demonstrated that TPA had no effect on ER gene transcription. A half-life study demonstrated that TPA decreased ER mRNA half-life by a factor of 6 from approximately 4 h in control cells to 40 min in TPA-treated cells. These data suggest that the decline in ER expression is mediated by post-transcriptional destabilization of ER mRNA.

Beta-thalassemia major and sickle cell disease are genetic disorders of red blood cells, caused by abnormal hemoglobin. These hemoglobinopathies affect males and females equally. Both are chronic disorders requiring lifelong treatment for affected individuals and education and support for them and their families. Special nursing considerations for the care of women facing the unique challenges of these disorders will be discussed.

In the article on how Brca2 deficiency sensitizes cells to PARP inhibition in the November 15, 2005 issue of Cancer Research ( 1), the grant support in the Acknowledgment should have read as follows: Association for International Cancer Research grant no. 03–336, KuDOS Pharmaceuticals Ltd., and Wales Gene Park.

The nature of the carbonyl and nitrogen substituents of hydroxamic acids has a major influence on the biological profile of these compounds. Hydroxamates with small groups such as methyl appended to the carbonyl and relatively large nitrogen substituents generally have longer duration in vivo, produce greater plasma concentrations, and often are more potent inhibitors of in vivo leukotriene biosynthesis than hydroxamic acids with the opposite arrangement. The structure-activity relationships that describe in vitro 5-lipoxygenase inhibitory activity and in vivo leukotriene biosynthesis inhibitory potency for a group of these hydroxamic acids were investigated. While most of the compounds examined were potent in vitro inhibitors of 5-lipoxygenase, their in vivo potencies varied widely. This discrepancy was usually attributable to differences in bioavailability. Substitution patterns are described that produce potent, orally active inhibitors of leukotriene biosynthesis.

Directional correlation and polarization-direction correlation measurements have been done on gamma rays following the decay of Ge77. The results are found to be consistent with previous spin assignments and a spin of 7 2 can be assigned to the 1.19 MeV level. The quadrupole content of several gamma transitions is given.

The pathophysiology of pulmonary inhalation injury, a major cause of morbidity and mortality from fires, is poorly understood. To examine the effects of colloid and crystalloid resuscitation on extravascular lung water (EVLW) during a standard smoke inhalation injury, we subjected 12 sheep to 8 minutes of cool pine smoke inhalation. The animals were then resuscitated to a pulmonary capillary wedge pressure (PCWP) of 10 +/- 1.5 mm Hg with either lactated Ringer's solution or plasma protein derivative. EVLW, cardiac output, vascular resistance, colloid oncotic pressure (COP), arterial and pulmonary artery pressures, PCWP, and blood gases were monitored during 4 hours of resuscitation. In colloid-treated animals, EVLW increased from 8.3 +/- 1.2 to 11.1 +/- 0.9 ml/kg with injury; it increased only to 12.5 +/- 1.3 ml/kg during resuscitation. In crystalloid-treated animals, EVLW increased from 8.0 +/- 1.0 to 10.3 +/- 0.8 ml/kg with injury and further increased to 17.4 +/- 1.6 ml/kg during resuscitation, a level significantly higher than that in the colloid group (P less than 0.05). The increases in EVLW were associated with progressive hypoxia, which was worse in the crystalloid group. In the crystalloid group, COP decreased from 27.3 +/- 0.9 to 14.2 +/- 0.4 mm Hg and intravascular driving force (COP-PCWP) dropped from 17.6 to 3.26 +/- 1.5 mm Hg; COP and COP-PCWP were maintained in the colloid group. These data demonstrate that supporting serum COP minimizes the increase in EVLW with smoke inhalation injury and suggests that smoke inhalation does not lead to a dramatic increase in alveolar capillary membrane permeability to protein.

Despite advances in wound healing, fascial dehiscence continues to be a problem in celiotomy wounds. Experimental and clinical studies on suture material and on patient-related factors in wound disruption are abundant, but little attention has been given to mechanical factors in wound closure, although they may be of greater importance. A midline abdominal wound was made in 120 Harlan Sprague-Dawley rats, and the fascia was closed with Dacron in one of six ways: simple interrupted, interrupted figure-of-eight, and running, each tied loosely in one half of each group and tightly in the other half. One week later, the rats were reanesthetized and wound-bursting strength was measured. In general, the running suture technique resulted in the greatest wound-bursting pressures. A loosely tied figure-of-eight technique was nearly as good as a loosely tied running stitch, but tying figure-of-eight sutures tightly caused a significant decrease in wound-bursting pressure. The simple interrupted technique was unaffected by suture tension but was generally inferior to the running stitch in terms of wound-bursting strength. Histologic studies were performed, but most of the disparity in wound strength among the suture techniques apparently was due solely to mechanical factors. Closing midline abdominal fascial wounds with a running suture may be a superior method of closure in clean, incised wounds.

A midline abdominal wound was made in 135 Harlan Sprague-Dawley rats, and the fascia was then closed by the continuous suture technique with 4-0 absorbable monofilament polydioxanone sutures (n = 45 rats), 4-0 permanent monofilament polypropylene sutures (n = 45 rats), or 4-0 Teflon-coated braided Dacron sutures (n = 45 rats). At 1-, 2-, and 6-month intervals, wound-bursting pressures were determined in 15 animals from each group. At 1 month and at 6 months, there was no significant difference in wound-bursting strength among the three types of sutures. At 2 months, wounds closed with Teflon-coated braided Dacron were significantly stronger than wounds closed with the other types of sutures. In most of the braided Dacron suture animals, the fascia burst in areas other than the abdominal wound, indicating that the wound was stronger at that time than was the surrounding fascia. There is no advantage to that added strength, and braided Dacron sutures have been shown to cause greater tissue reaction than monofilament sutures. Therefore, except in patients with poor wound healing who are highly susceptible to dehiscence and in whom permanent low-tissue-reaction sutures may be beneficial, the authors recommend a running suture technique using absorbable monofilament suture, such as polydioxanone, for the routine closure of clean abdominal wounds.

The effects of estrogen on liver gene expression in Xenopus laevis were examined using a nuclear transcription "run-on" assay. Vitellogenin transcription was detected 2 h after a single dose of estradiol and reached a maximum on day 4. By 12 days after hormone treatment vitellogenin transcription declined to low levels. Within 3 h after estrogen, total transcriptional activity increased 9-fold relative to control values, reaching a maximum level of 40-fold by 12 h. Total transcription remained elevated throughout the following 12-day time course. These data indicate that changes in vitellogenin transcription and total nuclear transcription are uncoupled. Steady-state levels of vitellogenin mRNA demonstrated a close correlation with the level of vitellogenin gene transcription at all time points. In another series of experiments, animals treated repeatedly with estrogen demonstrated an elevated steady-state level of vitellogenin transcription, an elevated steady-state level of vitellogenin mRNA, and a constant elevated level of total nuclear transcriptional activity. Neither hormonal treatment regimen had an effect on the transcription of actin or induced the embryonic gene DG42. Finally, the increase in total transcriptional activity is associated with increased activities of RNA polymerase I and/or III in addition to stimulation of RNA polymerase II activity.

One thousand four hundred cases of colorectal carcinoma were treated primarily at the Wake Forest University Medical Center between 1945 and 1985. The surgical approach was constant in all patients without obvious stage IV disease: wide resection, including at least the primary-level and intermediate-level lymph nodes. There were 812 women and 588 men in the series. Sixty-eight per cent of the 1400 cancers occurred in the rectosigmoid, but only 53 per cent of the last 300 cases were in this region. Initial staging showed 560 cases (40%) of local disease, 504 cases (36%) of regional disease, and 336 cases (24%) of distant disease. Cecal, ascending, hepatic, and transverse lesions were most often associated with stage IV disease. Among the 1115 patients with long-term follow-up, 44 per cent with stage I disease, 37 per cent with stage II disease, 24 per cent with stage III disease, and 6 per cent with stage IV disease had survived for 5 years or longer. There were no differences when 5-year survival was correlated with site. This review provided no evidence that wide resection leads to increased long-term survival.

Polytetrafluoroethylene (PTFE) grafts, 1 mm in internal diameter, were interpositioned in the infrarenal aortas of 20 adult Sprague-Dawley rats with either continuous or interrupted sutures. The interrupted-suture (I-S) technique was used in ten rats, the continuous-suture (C-S) technique in the other ten. After followup of 90 days, the overall patency rate for the 20 rats was 95% (19/20); all grafts in the C-S group were patent; one graft in the I-S group had occluded on the day of operation. Since excellent patency rates were obtainable with these minute PTFE grafts placed in the rat aorta by either technique, the speed with which the C-S technique can be accomplished appears to make it preferable.

This retrospective study analyzes the results of 198 consecutive greater curvature gastroplasties performed in 23 male and 175 female patients with morbid obesity. The gastroplasty consisted of the creation of a 30-50 cm3 proximal gastric pouch by a double application of the TA-90 stapler, modified by removing three staples near the pin and trimming the cartridge to prevent crushing of the stomach at the outlet. The outlet was calibrated to a diameter of 1 cm and reinforced with a polypropylene seromuscular suture. No deaths occurred; postoperative complications included 32 superficial wound infections (16.2%), 24 incisional hernias (12.0%), 15 staple-line disruptions (7.6%), seven gastric outlet obstructions (3.5%), five subphrenic abscesses (2.5%), three perforations (1.5%), one splenic infarction, and one enterocutaneous fistula. One hundred sixty-one (81%) patients have been followed for a mean of 34 months (range 24 to 56 months); their mean weight loss is 36.4 per cent +/- 32.9 per cent of excess body weight. Only 45.5 per cent of these patients can be considered to have had satisfactory weight loss. Because this is not as effective as other forms of surgical therapy, the authors do not recommend greater curvature gastroplasty for the treatment of morbid obesity.

The pseudocalculus formation was first noted by Caroli in 1960, and was first called a sign by Beneventano and Schein in 1968. It is a cholangiographic illusion that appears as a filling defect in the distal common bile duct when static or "spot" films are exposed during the contractile phase of choledochal sphincter activity. Although first noted and most frequently seen in T-tube cholangiography, it is seen also during intravenous and intraoperative cholangiography. Because it mimics radiographically an impacted stone in the distal common bile duct, its delineation is critical to avoid unnecessary instrumentation of the common bile duct or even reoperation. In this review the authors discuss briefly the anatomy and physiology of the distal common bile duct and the etiology, differential diagnosis, and recognition of the pseudocalculus sign.

The effects of ibuprofen (Motrin), dipyridamole (Persantine), and prostacyclin on the deposition of platelets on polytetrafluoroethylene grafts (5.5 cm long, 4 mm internal diameter implanted into both femoral arteries of 21 dogs) were studied by harvesting autologous platelets, labeling them with 51Cr, and reinjecting them 15 minutes before cross-clamping. Arterial flow was adjusted to 65 ml/min and monitored continuously. Ibuprofen (12.5 mg/kg) and dipyridamole (2.5 mg/kg) were each administered as a single intravenous injection in four dogs (eight grafts) and five dogs (10 grafts), respectively. Prostacyclin (50 ng/kg/min) was administered by continuous intravenous infusion in five dogs (10 grafts). Seven dogs (14 grafts) served as controls. Grafts were removed 2 hours after implantation, and radioactivity (counts per 10 minutes) was determined in four segments of each graft (including both anastomoses) with a gamma-counter. Counts were averaged for each group of dogs. Significance was calculated by the Student t test. Platelet deposition in control dogs averaged 10,033.9 +/- 1134.2 SE; that in prostacyclin-treated dogs averaged 2513.7 +/- 276 SE (p less than 0.001); that in ibuprofen-treated dogs averaged 5453.4 +/- 1336.3 SE (p = 0.02); that in dipyridamole-treated dogs averaged 11,213.7 +/- 1632.5 SE (p = 0.55). These data demonstrate the effectiveness of prostacyclin and ibuprofen in reducing platelet deposition on polytetrafluorethylene grafts in dogs.

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