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.

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.

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.

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.

Diary of hot flashes reported upon occurrence: results of a randomized double-blind study of raloxifene, placebo, and paroxetine.

OBJECTIVE: This trial examined diaries of hot flash events reported upon occurrence to assess the test/retest reliability of the diaries and their ability to measure treatment effects on hot flash frequency and severity. METHODS: Forty-two postmenopausal women (aged ≥40 y; 5-50 hot flashes/wk) were randomized (3:3:1) to placebo, raloxifene 60 mg, or paroxetine 20 mg daily for 12 weeks. Diaries of hot flash frequency and severity were evaluated at 1-week intervals (twice before study treatment and thrice during study treatment). RESULTS: Forty-one women were evaluated. Baseline characteristics were similar between groups (eg, mean, 29.8 hot flashes/wk). Concordance correlation coefficients between screening (week -2) and baseline (week -1) measures of hot flash frequency and severity were 0.73 and 0.71, respectively. After 12 weeks, the mean (95% CI) percent changes from baseline in weekly hot flash frequency were as follows: placebo, -37.4% (-60.9 to -14.0); raloxifene, -14.2% (-37.7 to 9.3); paroxetine, -49.8% (-88.6 to -11.0); the mean (95% CI) percent changes in hot flash severity were as follows: placebo, -39.9% (-69.1 to -10.8); raloxifene, -9.6% (-38.8 to 19.6); paroxetine, -36.6% (-84.7 to 11.5). There were no significant differences in hot flash diary results between treatment groups. CONCLUSIONS: Measures of hot flash frequency and severity show acceptable test/retest reliability between screening and baseline. Reductions in vasomotor symptoms by raloxifene are numerically less than those seen with placebo, but no statistically significant treatment differences have been documented in this small study. The large effect of placebo and the significant reduction in vasomotor symptoms by paroxetine are consistent with other studies. The diary seems to be suitable for use in hot flash clinical trials.

The Influence of Hypoxia and pH on Bioluminescence Imaging of Luciferase-Transfected Tumor Cells and Xenografts.

Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.

ATM kinase inhibition preferentially sensitizes p53-mutant glioma to ionizing radiation.

PURPOSE: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12 to 15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of patients with GBM is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. Ataxia-telangiectasia mutated (ATM) is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, and growth, and potently radiosensitized human glioma cells in vitro. EXPERIMENTAL DESIGN: Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intracranially, and KU-60019 was administered intratumorally by convection-enhanced delivery or osmotic pump. RESULTS: Our results show that the combined effect of KU-60019 and radiation significantly increased survival of mice 2- to 3-fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. CONCLUSIONS: Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.

Parametric estimation of 3D tubular structures for diffuse optical tomography.

We explore the use of diffuse optical tomography (DOT) for the recovery of 3D tubular shapes representing vascular structures in breast tissue. Using a parametric level set method (PaLS) our method incorporates the connectedness of vascular structures in breast tissue to reconstruct shape and absorption values from severely limited data sets. The approach is based on a decomposition of the unknown structure into a series of two dimensional slices. Using a simplified physical model that ignores 3D effects of the complete structure, we develop a novel inter-slice regularization strategy to obtain global regularity. We report on simulated and experimental reconstructions using realistic optical contrasts where our method provides a more accurate estimate compared to an unregularized approach and a pixel based reconstruction.

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.

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control.

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.

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.

Mutations in the BRCT binding site of BRCA1 result in hyper-recombination.

We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. Thesein vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer.

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.

ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks.

Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU) - substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose- dependent generation of DSBs equivalent to radiation doses between 0.2 - 20 Gy, as determined by pulsed-field gel electrophoresis, and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68), and p53 (ser-15) phosphorylation. Interestingly, low levels (≤ 2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (> 2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.

Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion.

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with K(i) and IC(50) values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells.

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.

Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells.

To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM(+), p53(+)) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G(0)/G(1), suggesting that ATM is also important for HRR outside of the S and G(2) cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.

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.