An Approach to Adapting a Community-Based Cancer Control Intervention to Organizational Context.

There has been increasing attention in implementation science to optimizing the fit of evidence-based interventions to the organizational settings where they are delivered. However, less is known about how to maximize intervention-context fit, particularly in community-based settings. We describe a new strategy to customize evidence-based health promotion interventions to community sites. Specifically, leaders in African American churches completed a memorandum of understanding where they were asked to identify two or more health promotion implementation strategies from a menu of 20 and select a planned implementation time frame for each. In a pilot phase with three churches, the menu-based strategy and protocols were successfully implemented and finalized in preparation for a subsequent randomized trial. The three pilot churches identified between two and nine strategies (e.g., form a health ministry, allocate space or budget for health activities, include health in church communications/sermons). The selected strategies varied widely, reinforcing the need for interventions that can be customized to fit the organizational context. Despite the challenges of integrating health promotion activities into non-health focused organizations, this approach has promise for fostering sustainable health activities in community settings.

CXCR2 Antagonist MK-7123. A Phase 2 Proof-of-Concept Trial for Chronic Obstructive Pulmonary Disease.

RATIONALE: An antagonist (MK-7123) of the cytokine receptor CXCR2 reduces neutrophil chemotaxis and thus may alleviate airway inflammation in chronic obstructive pulmonary disease (COPD). OBJECTIVES: To assess the efficacy, safety, and tolerability of three dose levels of MK-7123, compared with placebo, in patients with moderate to severe COPD. METHODS: This 6-month, double-blind study randomized patients with moderate to severe COPD (already on standard therapy) to daily MK-7123 at 10, 30, or 50 mg or placebo. The primary endpoint was change from baseline in post-bronchodilator FEV1. MEASUREMENTS AND MAIN RESULTS: A total of 616 patients (71% male; mean age, 63 yr; 45% current smokers; baseline FEV1 [SD], 1.43 L [0.45]; mean FEV1 percent predicted, 43.9%) were randomized. Only MK-7123 50 mg led to significant improvement in FEV1 over placebo (mean difference [SE], 67 ml [32]). Reduced sputum neutrophil count was observed among the 122 patients examined; P = 0.003 (3 mo) and P = 0.092 (6 mo) (MK-7123 50 mg vs. placebo). The stratum of current smokers, but not that of nonsmokers, showed significant improvement versus placebo in FEV1 (168 ml) and time-to-first exacerbation, and showed numerical improvement in St. George's Respiratory Questionnaire for COPD score. MK-7123 caused a dose-dependent decrease in absolute neutrophil count (ANC) and reduced inflammatory biomarkers matrix metallopeptidase-9 and myeloperoxidase in plasma and sputum; ANC lower than 1.5 × 10(9)/L led to discontinuations with higher doses of MK-7123 (18% in the MK-7123 50-mg group vs. 1% in placebo). Plasma C-reactive protein and fibrinogen increased with MK-7123 treatment. Rates of infections at 6 months were similar in all groups. CONCLUSIONS: Treatment with MK-7123 50 mg versus placebo led to significant improvement in FEV1 in patients with COPD, suggesting clinically important antiinflammatory effects with CXCR2 antagonism, although dose-related discontinuations were observed because of ANC decreases with MK-7123. Greater response was observed in smokers versus ex-smokers. Clinical trial registered with www.clinicaltrials.gov (NCT 01006616).

The effects of the CXCR2 antagonist, MK-7123, on bone marrow functions in healthy subjects.

The CXCR2 antagonist MK-7123 causes dose-dependent reductions in absolute neutrophil counts (ANC) and decreases neutrophil tissue responses, but its effects on bone marrow functions are not yet known. We conducted a double-blind, randomized study in 18 healthy subjects comparing the effects of either MK-7123 (30mg, po, daily for 28days) or placebo on peripheral blood counts and bone marrow myeloid cell populations. MK-7123 caused a reversible decrease (approximately 50%) in the ANC as demonstrated on days 1 and 28, the first and last days of the treatment period. Bone marrow aspirate smears and biopsy imprints did not differ in the proportion of mature neutrophils in pretreatment, day 28, day 56 or placebo samples. There were no treatment effects on biopsy or aspirate clot cellularity, myeloid to erythroid or myeloid post-mitotic to mitotic ratios; flow-cytometric analyses of aspirate cells; or bone marrow fat to cell balance as assessed by MRI. MK-7123 was generally well tolerated with neutropenia being the most common adverse event; however, there were no clinical symptoms associated with decreased ANCs. These findings indicate that the CXCR2 antagonist MK-7123 causes rapidly reversible decrease in the ANC without measurable myelosuppressive effects. The results support the development of CXCR2 antagonists as potentially useful anti-inflammatory agents, primarily interrupting neutrophil trafficking.

Vitamin D status and bone mineral density changes during alendronate treatment in postmenopausal osteoporosis.

Vitamin D supplementation is recommended for women with osteoporosis. In the FOCUS-D trial comparing the combination tablet alendronate plus vitamin D3 5,600 IU (ALN/D) with standard care (SC) prescribed by patients' personal physicians, ALN/D was more effective in improving serum 25(OH)D and bone turnover markers by 6 months and increasing spine and hip bone mineral density (BMD) after 1 year than SC. This post hoc analysis examined the relationship between BMD gain and 25(OH)D in women in SC receiving alendronate (SC/ALN, n = 134, 52% of the SC group) and in the ALN/D group (n = 257). At baseline, participants were of mean age 73 years and 72% were Caucasian, with a mean 25(OH)D of 14.9 ng/mL. In the SC/ALN group, most received vitamin D, although intake of vitamin D varied extensively (51% received <400 µg/day). In this group, end-of-study 25(OH)D correlated positively with mean percent increases from baseline in lumbar spine and femoral neck BMD [Pearson correlation coefficients (95% CI) = 0.23 (0.02-0.41) and 0.24 (0.03-0.41), respectively]. Baseline 25(OH)D correlated with increases in only lumbar spine BMD [Pearson correlation coefficient (95% CI) = 0.22 (0.01-0.40)]. No correlations between mean BMD change and 25(OH)D were seen with ALN/D. In conclusion, in postmenopausal women with osteoporosis and low 25(OH)D receiving alendronate and a wide range of vitamin D doses, the increase in lumbar spine and femoral neck BMD was positively correlated with serum 25(OH)D achieved by the end of the study and, to some extent, with 25(OH)D concentrations at baseline. The degree of success of alendronate therapy for osteoporosis may depend on the vitamin D status of patients.

In vitro and in vivo responses to high and low doses of nitrogen-containing bisphosphonates suggest engagement of different mechanisms for inhibition of osteoclastic bone resorption.

The effects of nitrogen-containing bisphosphonates (N-BPs) on osteoclasts (Ocs) may differ with dose and regimen. N-BPs reduce Oc bone resorption by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), an effect counteracted by geranylgeraniol (GGOH), which restores geranylgeranylation downstream of FPPS. We assessed GGOH effects on inhibition of bone resorption by the N-BPs alendronate (ALN), ibandronate (IBN), and zoledronate (ZOL) in an assay of rabbit Oc resorption of bovine cortical bone. GGOH blocked inhibition of resorption at low, but not high, N-BP concentrations, with a 14- to 20-fold increase in IC50 values for each N-BP. In vivo, growing male rats were administered doses calculated to mimic bioavailable exposures in daily (ALN, IBN), weekly (ALN), monthly (IBN), and yearly (ZOL) clinical regimens. Tibiae were harvested at 48 h, and metaphyses were analyzed. With lower ALN and IBN doses, Oc numbers rose by 26-48 %, morphology was normal, and there was no increase in apoptotic Ocs. In contrast, with higher IBN and ZOL doses, bone-associated Ocs were generally rounded in appearance and numbers of nuclei/Oc versus vehicle increased 42 and 31 %, respectively (P < 0.05). With ZOL, there was no rise in Oc number, but there was a 6.5-fold increase in apoptotic Ocs versus vehicle and a ≥13.5-fold increase versus lower-dose ALN or IBN (P < 0.05). With higher-dose IBN there was no rise in Oc number but 7- and 14-fold increases in Oc apoptosis versus low-dose ALN and IBN (P < 0.02). These results suggest that different mechanisms may come into play across the dosing spectrum of N-BPs.

A phase I trial of MK-0731, a kinesin spindle protein (KSP) inhibitor, in patients with solid tumors.

PURPOSE: The kinesin spindle protein (KSP) is essential for separation of spindle poles during mitosis. Its inhibition results in mitotic arrest. This phase I trial examined safety, tolerability, dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetic parameters, and anti-tumor activity of MK-0731, a potent inhibitor of KSP. EXPERIMENTAL DESIGN: In part 1, patients with advanced solid tumors received MK-0731 intravenously over 24 h every 21 days starting at 6 mg/m(2), escalating until MTD was reached. In part 2, patients with taxane-resistant tumors received the MTD. Plasma samples were collected to analyze the pharmacokinetics of MK-0731. Tumor response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) v1.0. RESULTS: In part 1, 21 patients (median age 63 years) were treated with MK-0731 at doses ranging from 6 to 48 mg/m(2)/24 h for median four cycles. The dose-limiting toxicity was neutropenia and the MTD was 17 mg/m(2)/24 h. At the MTD, AUC (±SD) was 10.5 (±7.3) µM × hour, clearance (±SD) was 153 mL/min (±84), and t(1/2) was 5.9 h. In part 2, 22 patients received the MTD and there were no DLTs. Although there were no objective tumor responses, four patients (with cervical, non-small cell lung, and ovarian cancers) had prolonged stable disease. CONCLUSIONS: MK-0731 at the MTD of 17 mg/m(2)/day every 21 days in patients with solid tumors had few grade 3 and 4 toxicities with the major DLTs at higher doses being myelosuppression. Anti-tumor efficacy was suggested by the length of stable disease in selected patients with taxane-resistant tumors.

The osteoclast, bone remodelling and treatment of metabolic bone disease.

BACKGROUND: Bone remodelling maintains skeletal integrity by osteoclasts removing foci of damaged bone and osteoblasts replacing them with new bone. Diseases associated with increased bone resorption have increased remodelling often with inadequate bone formation and increased risk of fracture. New therapies are needed for these diseases to reduce resorption and increase formation. DESIGN: The molecular mechanisms regulating osteoclast and osteoblast functions have become better understood in the past 20 years and have led to questioning of the long-held notion that osteoblastic cells have the dominant regulatory role over osteoclastic cells in bone remodelling. Here, we review current knowledge of how osteoclast formation and functions are regulated and describe how enhanced understanding of these has led to development of new drugs for the management of common bone diseases characterized by increased bone resorption. RESULTS: Osteoclast formation and functions are regulated by cytokines, especially receptor activator of NF-κB ligand (RANKL) and macrophage-colony-stimulating factor (M-CSF). The differentiation, activity and lifecycle of osteoclasts are regulated in part by other cells that reside within the bone. These include osteoblasts, osteocytes and immune cells, which express these cytokines in response to most factors that promote bone resorption. RANKL and M-CSF activate numerous signalling pathways, which are potential targets for therapeutic intervention. Importantly, osteoclastic cells also function as positive and negative regulators of osteoblastic bone formation. CONCLUSIONS: There are multiple targets within osteoclasts for pharmacologic intervention to prevent bone loss in osteoporosis and other resorptive bone diseases. However, novel therapies could also affect osteoblastic cell functions.

Inhibition of cathepsin K for treatment of osteoporosis.

Cathepsin K is the protease that is primarily responsible for the degradation of bone matrix by osteoclasts. Inhibitors of cathepsin K are in development for treatment of osteoporosis. Currently available antiresorptive drugs interfere with osteoclast function. They inhibit both bone resorption and formation, due to the coupling between these processes. Cathepsin K inhibitors, conversely, target the resorption process itself and may not interfere with osteoclast stimulation of bone formation. In fact, when cathepsin K is absent or inhibited in mice, rabbits, or monkeys, bone formation is maintained or increased. In humans, inhibition of cathepsin K is associated with sustained reductions in bone resorption markers but with smaller and transient reductions in bone formation markers. The usefulness of cathepsin K inhibitors in osteoporosis is now being examined in phase 2 and phase 3 clinical trials of postmenopausal osteoporotic women.

Pharmacokinetics and metabolism in rats, dogs, and monkeys of the cathepsin k inhibitor odanacatib: demethylation of a methylsulfonyl moiety as a major metabolic pathway.

Odanacatib is a potent cathespin K inhibitor that is being developed as a novel therapy for osteoporosis. The disposition and metabolism of odanacatib were evaluated in rats, dogs, and rhesus monkeys after intravenous and oral administration of [¹4C]odanacatib. Odanacatib was characterized by low systemic clearance in all species and by a long plasma half-life in monkeys (18 h) and dogs (64 h). The oral bioavailability was dependent on the vehicle used and ranged from 18% (monkey) to ~100% (dog) at doses of 1 to 5 mg/kg, using nonaqueous vehicles. After intravenous and oral administration to intact rats and monkeys > 90% of the dose was recovered, mainly in the feces. Studies in bile duct-cannulated animals indicated that biliary secretion was the major mode of elimination of radioactivity; odanacatib also underwent some intestinal secretion. In monkeys, odanacatib was almost completely eliminated by metabolism; metabolism also played a major role in the clearance of odanacatib in rats and dogs. The major metabolic pathways were methyl hydroxylation (formation of M8 and its derivatives), methyl sulfone demethylation (formation of M4 and its derivative M5), and glutathione conjugation (formation of the cyclized cysteinylglycine adduct M6 after addition of glutathione to the nitrile group of odanacatib). The major metabolites in rats [M4 (parent-14 Da) and M5 (oxygenated derivative of M4)] were determined to arise from a novel pathway that involved oxidative demethylation of the methylsulfonyl moiety of odanacatib. Overall, odanacatib displayed species-dependent metabolism, which explains, at least in part, the divergent plasma half-life observed.

Randomized trial of alendronate plus vitamin D3 versus standard care in osteoporotic postmenopausal women with vitamin D insufficiency.

Vitamin D insufficiency is common in patients with osteoporosis. We conducted a randomized trial comparing alendronate 70 mg combined with vitamin D(3) 5,600 IU in a single tablet (ALN/D5600, n = 257) with standard care chosen by the patients' personal physicians (n = 258) in patients with postmenopausal osteoporosis (BMD T score ≤2.5 or ≤1.5 and a prior fragility fracture) who had vitamin D insufficiency (serum 25[OH]D values 8-20 ng/ml) and who were at risk of falls. Virtually all patients randomized to standard care received bisphosphonate therapy, and in approximately 70% of cases this was combined with vitamin D supplements. However, only 24% took ≥800 IU/day of supplemental vitamin D. At 6 months the proportion of patients with vitamin D insufficiency was 8.6% in the ALN/D5600 group compared with 31.0% in the standard care group (P < 0.001). Those in the ALN/D5600 group also had a greater reduction in urinary NTX/creatinine ratio (-57% vs. -46%, P < 0.001) and bone-specific alkaline phosphatase (-47% vs. -40%, P < 0.001). In the ALN/5600 group, by 12 months the increase in BMD was greater at the lumbar spine (4.9% vs. 3.9%, P = 0.047) and the total hip (2.2% vs. 1.4%, P = 0.035), significantly fewer patients were vitamin D-insufficient (11.3% vs. 36.9%, P < 0.001), and bone turnover marker (BTM) results were similar to those at 6 months. There was no difference between groups in those who experienced falls or fractures, and adverse events were similar. Based on the finding that ALN/D5600 was more effective than standard care at correcting vitamin D insufficiency, increasing BMD, and reducing BTMs in this patient group, greater attention needs to be directed toward optimizing the treatment of osteoporosis and correcting vitamin D deficiency in postmenopausal women.

Evaluation of alternative diluents for clinical use of collagenase clostridium histolyticum (CCH-aaes).

BACKGROUND: Collagenase clostridium histolyticum (CCH-aesthetic formulation [CCH-aaes]; QWO™ [Endo Aesthetics, Malvern PA, USA] is approved as a subcutaneous injection for treatment of cellulite. In the aesthetic practice, dilution of marketed products is commonly employed to tailor treatments to individual patients or off-label locations. Dilution beyond the 0.23 mg/ml achievable with the proprietary diluent supplied with the CCH-aaes lyophilized powder requires diluents readily available in clinic. AIM: To characterize the functionality and stability of CCH-aaes when reconstituted and/or diluted with alternative diluents, including normal saline, bacteriostatic saline, and/or proprietary diluent. PATIENTS/METHODS: Each dilution was assessed for purity using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), activity using collagenase (AUX-I) and gelatinase (AUX-II) assays, and aggregation using size-exclusion chromatography. RESULTS: When reconstituted with either saline or proprietary diluent, and diluted with proprietary diluent or saline, purity, activity, and stability of CCH-aaes is maintained for up to 24 h at 5°C or 25°C. In contrast, use of bacteriostatic saline to reconstitute and/or dilute CCH-aaes results in up to a 40% decrease in activity and aggregation of 5.3% of CCH-aaes protein. Importantly, inclusion of 2% lidocaine and 1:200 000 epinephrine does not negatively impact CCH-aaes purity, concentration, or activity for up to 24 h at 5°C or 25°C. CONCLUSIONS: From an efficacy and safety perspective, CCH-aaes must not be/should not be reconstituted and/or diluted with bacteriostatic saline to avoid injection of protein aggregates. Ideally, CCH-aaes should be reconstituted in proprietary diluent: further dilution with normal saline and addition of lidocaine and epinephrine is acceptable.

A multicenter, double-blind, randomized, placebo controlled trial of a neurokinin-1 receptor antagonist for overactive bladder.

PURPOSE: Neurokinin-1 receptor dependent mechanisms may regulate urinary frequency and urgency. We conducted this study to assess the efficacy and tolerability of the neurokinin-1 receptor antagonist serlopitant vs placebo or tolterodine in patients with overactive bladder. MATERIALS AND METHODS: This randomized, double-blind, 69-center trial enrolled adults with overactive bladder (8 or more average daily micturitions and 1 or more daily urge incontinence episodes). After a 1-week placebo run-in the patients were randomized to 8 weeks of daily 0.25, 1 or 4 mg serlopitant, 4 mg tolterodine extended release or placebo. Patients kept 7-day voiding diaries. The primary end point was change from baseline in micturitions per day. Secondary end points included urgency, total incontinence, urge incontinence episodes and incidence of dry mouth. RESULTS: Of 557 patients randomized 476 completed the trial and had valid efficacy data for analysis. Mean change from baseline in daily micturitions was significantly greater for 0.25 (-1.1) and 4 mg (-1.1) serlopitant, and for tolterodine (-1.5) than for placebo (-0.5), but not for 1 mg serlopitant (-0.8). No serlopitant dose response was demonstrated. Tolterodine was numerically superior to all doses of serlopitant in mean micturitions per day and secondary end points. The incidence of dry mouth on serlopitant (3.3%) was comparable to placebo (4.6%) and lower than tolterodine (8.8%). Serlopitant was generally well tolerated. CONCLUSIONS: Serlopitant (0.25 and 4 mg) significantly decreased the primary end point of daily micturitions but not the secondary end points compared with placebo. Serlopitant was generally well tolerated. Thus, NK-1 receptor antagonists may have a role in the treatment of overactive bladder but this compound does not offer advantages in efficacy compared to tolterodine.

Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated, homologous recombination repair, and the DNA damage response.

The accurate joining of DNA double-strand breaks by homologous recombination repair (HRR) is critical to the long-term survival of the cell. The three major mitogen-activated protein (MAP) kinase (MAPK) signaling pathways, extracellular signal-regulated kinase (ERK), p38, and c-Jun-NH(2)-kinase (JNK), regulate cell growth, survival, and apoptosis. To determine the role of MAPK signaling in HRR, we used a human in vivo I-SceI-based repair system. First, we verified that this repair platform is amenable to pharmacologic manipulation and show that the ataxia telangiectasia mutated (ATM) kinase is critical for HRR. The ATM-specific inhibitor KU-55933 compromised HRR up to 90% in growth-arrested cells, whereas this effect was less pronounced in cycling cells. Then, using well-characterized MAPK small-molecule inhibitors, we show that ERK1/2 and JNK signaling are important positive regulators of HRR in growth-arrested cells. On the other hand, inhibition of the p38 MAPK pathway generated an almost 2-fold stimulation of HRR. When ERK1/2 signaling was stimulated by oncogenic RAF-1, an approximately 2-fold increase in HRR was observed. KU-55933 partly blocked radiation-induced ERK1/2 phosphorylation, suggesting that ATM regulates ERK1/2 signaling. Furthermore, inhibition of MAP/ERK kinase (MEK)/ERK signaling resulted in severely reduced levels of phosphorylated (S1981) ATM foci but not gamma-H2AX foci, and suppressed ATM phosphorylation levels >85% throughout the cell cycle. Collectively, these results show that MAPK signaling positively and negatively regulates HRR in human cells. More specifically, ATM-dependent signaling through the RAF/MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation, suggestive of a regulatory feedback loop between ERK and ATM.

A cross-sectional study of bone turnover markers in healthy premenopausal women.

BACKGROUND: Biochemical markers of bone turnover (BTMs) provide useful information in the diagnosis and management of metabolic bone diseases. Currently, there exist few published reference ranges for bone markers in healthy premenopausal women using the newer, automated assays of bone turnover. This cross-sectional study of healthy premenopausal women was performed to determine reference ranges for four different markers of bone turnover and to compare reference ranges in users and non-users of oral contraceptives (OCs). METHODS: Urinary N-telopeptide of type 1 collagen (NTX) was determined from fasting second morning-void urine of healthy premenopausal women. In addition, fasting serum was collected for determination of C-telopeptide of type I collagen (CTX), bone-specific alkaline phosphatase (bone ALP), and N-terminal propeptide of type 1 procollagen (PINP). Subjects underwent central dual energy X-ray absorptiometry and completed a questionnaire regarding medical history and activities known to affect bone health. RESULTS: Serum and urine samples were collected from 237 healthy premenopausal women (119 OC users and 118 non-users) between the ages of 28 and 45 years. The mean age of subjects was 37 years, with a mean bone mineral density T-score of -0.1 at the lumbar spine and 0.0 at the total hip. Logarithmic transformation produced normal distributions for all markers but NTX. Mid-95% ranges for each marker were generally consistent with those reported by manufacturers. For each BTM examined, values were skewed toward the lower end of the range. Median NTX levels for OC users and non-users were 16.0 and 29.0 nmol/mmol creatinine, respectively. The mid-95% ranges for NTX in OC users and non-users were 3-60 and 4-64 nmol/mmol creatinine, respectively. Median levels of CTX, bone ALP, and PINP were also lower in OC users than non-users. The mean level of each BTM was significantly lower in OC users than non-users (P<0.01), whereas reference ranges (geometric mean+/-2 SD) were somewhat similar for the two groups. CONCLUSION: Values obtained from this well-characterized population provide reference ranges for BTMs in healthy premenopausal women. Median and mean BTM levels for OC users were consistently lower compared with non-users; thus, separate reference ranges are required for these two groups of premenopausal women. The relevance of premenopausal reference ranges for postmenopausal women remains uncertain.

A role for C/EBP delta in human surfactant protein A (SP-A) gene expression.

C/EBP delta, a member of the leucine zipper transcription factor family, is expressed at higher levels in the lung than in any other tissue. We detected C/EBP delta mRNA and protein in NCI-H441 cells, a cell line derived from a human adenocarcinoma that produces surfactant protein A (SP-A). NCI-H441 cells were exposed to phosphorothioate-substituted antisense oligonucleotides directed against C/EBP delta. After exposure to the oligonucleotides, cells were harvested, total RNA prepared, and levels of mRNA for C/EBP delta, SP-A and a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were quantified from Northern blots. An oligonucleotide that overlapped the translational start was effective in reducing C/EBP delta mRNA. Oligonucleotides that corresponded to regions upstream and downstream from the translational start were not as effective. The loss of C/EBP delta was accompanied by a decrease in the level of SP-A mRNA. The overlapping oligonucleotide was tested more extensively. After 72 h, antisense oligonucleotide at 3 and 5 microM reduced the level of C/EBP delta mRNA and protein by 50% or more as compared with sense and scrambled controls. The SP-A mRNA level was reduced even more, by about 75%. GAPDH mRNA was not affected. We conclude that C/EBP delta plays a role in the regulation of SP-A gene expression.

Members of the C/EBP transcription factor family stimulate expression of the human and rat surfactant protein A (SP-A) genes.

Members of the CCAAT enhancer binding protein (C/EBP) transcription factor family were detected in fetal lung of both human and rat. In rat lung, the level of C/EBPs increased with time of gestation, peaking around birth. In adult rat lung, C/EBPs were localized to the alveolar type II cells. The effect of C/EBPs on pulmonary surfactant protein A (SP-A), which is also expressed late in gestation, was investigated. In contrast to control plasmids, C/EBP delta expressing plasmids reversed the action of a transcriptional silencer just upstream of the rat SP-A promoter. In order to test the effect of C/EBPs on endogenous SP-A gene expression, cells that express SP-A were exposed to a phosphorothioate-substituted, double-stranded oligonucleotide matching the consensus C/EBP binding site (decoy oligonucleotide) at concentrations from 0.5 to 10 microM for 72 h. A mutant oligonucleotide with an 8-base pair (bp) substitution served as a control. The decoy oligonucleotide reduced SP-A mRNA as much as 75% compared to a mutant oligonucleotide both in the human lung cell line, NCI-H441, and in primary human fetal alveolar type II cells. The data indicate that C/EBPs facilitate SP-A gene expression, possibly by overcoming transcriptional silencing.

MDA-7 (interleukin-24) inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity.

The median survival of metastatic renal cell carcinoma (RCC) is 12 months, and the majority of treatment options are palliative. MDA-7 (interleukin-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound antiproliferative and cytotoxic effects in a wide variety of tumor cells but not in nontransformed cells. The studies in this study examined the impact of MDA-7 on RCC proliferation and survival. RCC lines (A498 and UOK121N), but not primary renal epithelial cells, were resistant to adenoviral infection that correlated with a lack of coxsackievirus and adenovirus receptor expression. Additional studies were performed using purified preparations of bacterially synthesized glutathione S-transferase (GST)-MDA-7 protein. GST-MDA-7, but not GST, caused a dose-dependent inhibition of RCC proliferation but not of primary renal epithelial cells. Clinically achievable concentrations of the novel therapeutic agent arsenic trioxide (0.5-1 micro M) were found to have little effect on RCC growth. However, the combination of GST-MDA-7 and arsenic trioxide resulted in a greater than additive reduction in cell growth that correlated with a large increase in tumor cell death. The free radical scavenger N-acetyl cysteine abolished the potentiating effect of arsenic trioxide. Although pro-caspase 3, poly(ADP-ribose) polymerase, and Bcl-(XL) levels, as well as nucleosomal DNA integrity, were reduced by combined treatment, cell killing was predominantly nonapoptotic. Combined treatment of RCC lines with GST-MDA-7 and arsenic trioxide also resulted in a substantial reduction in clonogenic survival compared with either treatment individually. Collectively, these findings demonstrate that MDA-7 protein, in combination with agents that generate free radicals, may have potential in the treatment of RCC.

Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

Radiosensitization of human glioma cells in vitro and in vivo with acyclovir and mutant HSV-TK75 expressed from adenovirus.

PURPOSE: We recently reported that an adenovirus-expressing mutant HSV-TK75 (AdCMV-TK75) radiosensitized rat syngeneic gliomas in combination with low concentrations of acyclovir (ACV) much more effectively than a virus expressing wild-type herpes simplex virus thymidine kinase (HSV-TK). In this report we have examined whether similar radiosensitizing effects are also seen with human glioma cells in vitro and in vivo. METHODS AND MATERIALS: Human U87 MG glioma cells were transduced with AdCMV-TK75 and exposed to ACV followed by single-dose irradiation and colony-forming survival assays. Similarly, U87 MG xenografts were infused with AdCMV-TK75 or Adbetagal control virus, followed by ACV administration and fractionated irradiation. Therapeutic efficacy was monitored by tumor growth. RESULTS: U87 MG cells transduced with AdCMV-TK75 were significantly more sensitive to ACV (3 microM) than cells transduced with either wild-type HSV-TK or control virus. To determine whether human cells also demonstrate improved radiosensitization similar to that seen with rat glioma cells and tumors, we transduced U87 MG cells with either AdCMV-TK75, AdCMV-TK, expressing wild-type HSV-TK, or Adbetagal and then treated the cells with 3 microM of ACV. Cells transduced with AdCMV-TK75 were significantly more radiosensitive (dose enhancement ratio [D(37)]: 2.6) by colony-forming survival assay than cells transduced with either AdCMV-TK or Adbetagal. Furthermore, we found that U87 MG xenografts infused with AdCMV-TK75 by slow positive pressure infusion were more radiosensitive after administration of ACV than tumors infused with Adbetagal. A more dramatic result was achieved when fractionated irradiation was carried out concurrently with ACV administration, in which case AdCMV-TK75-treated tumors did not grow at all. CONCLUSIONS: These results demonstrate that transduction of human glioma cells in vitro and infusion of xenografts in vivo with AdCMV-TK75 and treatment with concentrations of ACV that can be achieved in vivo produce similar radiosensitization, as previously reported with rat glioma cells and intracerebral syngeneic tumors. In addition, concurrent treatment with ACV and radiation therapy is more efficient than when ACV is administered before radiation therapy.

Sensitivity of nucleotide excision repair-deficient human cells to ionizing radiation and cyclophosphamide.

Nucleotide excision repair (NER)-deficient rodent and human cells (such as those derived from patients with xeroderma pigmentosum, XP) are hypersensitive to UV light. Some of these cell lines, specifically certain rodent mutants with severe defects in the ERCC1 and XPF genes, are dramatically sensitive to crosslinking agents such as phosphoramide mustard (PM). These crosslink-sensitive rodent mutants also exhibit sensitization to gamma-rays under hypoxic (but not under aerated) conditions. Like their rodent counterparts, human XP cells are highly sensitive to UV light; however, none of the human XP lines, even XPF, displays extreme hypersensitivity to crosslinking agents. Studying XP cells, therefore, allows us to further assess the extent to which the phenotypic characteristic of hypoxia-specific radiosensitization of mammalian cells tracks with defects in crosslink repair (as opposed to NER). The sensitivity to PM and gamma-rays of normal human fibroblasts and human XP fibroblasts from two complementation groups, XPA and XPF, was assessed using a clonogenic survival assay. Compared with normal cells, XPA cells were not appreciably hypersensitive to PM or to gamma-rays under either aerated or hypoxic conditions. XPF cells were modestly (approximately 1.75-fold) sensitive to PM but showed no significant radiosensitization under either aerated or hypoxic conditions. Thus, although the phenotype of human XPF cells is quite different from that of "severe" rodent XPF mutants such as UV41, the characteristic of hypoxia-specific radiosensitization consistently tracks with extreme hypersensitivity to crosslinking agents and is separable from UV sensitivity (and thus from defects in NER).