Lipid hydroperoxides and oxylipins are mediators of denervation induced muscle atrophy.

Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA2 and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy in vivo, we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% in vitro and by over 65% in vivo by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.

Tumor burden negatively impacts protein turnover as a proteostatic process in noncancerous liver, heart, and muscle, but not brain.

Cancer survivors are more susceptible to pathologies such as hypertension, liver disease, depression, and coronary artery disease when compared with individuals who have never been diagnosed with cancer. Therefore, it is important to understand how tumor burden negatively impacts nontumor-bearing tissues that may impact future disease susceptibility. We hypothesized that the energetic costs of a tumor would compromise proteostatic maintenance in other tissues. Therefore, the purpose of this study was to determine if tumor burden changes protein synthesis and proliferation rates in heart, brain, and liver. One million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (PBS, sham) were injected into the hind flank of female mice at ∼4.5 mo of age, and the tumor developed for 3 wk. Rates of proliferation and protein synthesis were measured in heart, brain, liver, and tumor tissue. Compared with sham, rates of protein synthesis (structural/nuclear, cytosolic, mitochondrial, and collagen) relative to proliferation were lower in the heart and liver of LLC mice, but higher in the brain of LLC mice. In the tumor tissue, the ratio of protein synthesis to DNA synthesis was approximately 1.0 showing that protein synthesis in the tumor was used for proliferation with little proteostatic maintenance. We further provide evidence that the differences in tissue responses may be due to energetic stress. We concluded that the decrease in proteostatic maintenance in liver, heart, and muscle might contribute to the increased risk of disease in cancer survivors.NEW & NOTEWORTHY We present data showing that simultaneously measuring protein synthesis and cell proliferation can help in the understanding of protein turnover as a proteostatic process in response to tumor burden. In some tissues, like hepatic, cardiac, and skeletal muscle, there was a decrease in the protein to DNA synthesis ratio indicating less proteostatic maintenance. In contrast, the brain maintained or even increased this protein to DNA synthesis ratio indicating more proteostatic maintenance.

Cancer cachexia in a mouse model of oxidative stress.

BACKGROUND: Cancer is associated with muscle atrophy (cancer cachexia) that is linked to up to 40% of cancer-related deaths. Oxidative stress is a critical player in the induction and progression of age-related loss of muscle mass and weakness (sarcopenia); however, the role of oxidative stress in cancer cachexia has not been defined. The purpose of this study was to examine if elevated oxidative stress exacerbates cancer cachexia. METHODS: Cu/Zn superoxide dismutase knockout (Sod1KO) mice were used as an established mouse model of elevated oxidative stress. Cancer cachexia was induced by injection of one million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (saline) into the hind flank of female wild-type mice or Sod1KO mice at approximately 4 months of age. The tumour developed for 3 weeks. Muscle mass, contractile function, neuromuscular junction (NMJ) fragmentation, metabolic proteins, mitochondrial function, and motor neuron function were measured in wild-type and Sod1KO saline and tumour-bearing mice. Data were analysed by two-way ANOVA with Tukey-Kramer post hoc test when significant F ratios were determined and α was set at 0.05. Unless otherwise noted, results in abstract are mean ±SEM. RESULTS: Muscle mass and cross-sectional area were significantly reduced, in tumour-bearing mice. Metabolic enzymes were dysregulated in Sod1KO mice and cancer exacerbated this phenotype. NMJ fragmentation was exacerbated in tumour-bearing Sod1KO mice. Myofibrillar protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.00847 ± 0.00205; wildtype LLC, 0.0211 ± 0.00184) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0180 ± 0.00118; Sod1KO LLC, 0.0490 ± 0.00132). Muscle mitochondrial oxygen consumption was reduced in tumour-bearing mice compared with saline-injected wild-type mice. Mitochondrial protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.0204 ± 0.00159; wild-type LLC, 0.167 ± 0.00157) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0231 ± 0.00108; Sod1 KO LLC, 0.0645 ± 0.000631). Sciatic nerve conduction velocity was decreased in tumour-bearing wild-type mice (wild-type saline, 38.2 ± 0.861; wild-type LLC, 28.8 ± 0.772). Three out of eleven of the tumour-bearing Sod1KO mice did not survive the 3-week period following tumour implantation. CONCLUSIONS: Oxidative stress does not exacerbate cancer-induced muscle loss; however, cancer cachexia may accelerate NMJ disruption.

Short-term Calorie Restriction and 17α-Estradiol Administration Elicit Divergent Effects on Proteostatic Processes and Protein Content in Metabolically Active Tissues.

17α-Estradiol (17α-E2) is a "non-feminizing" estrogen that extends life span in male, but not female, mice. We recently reported that 17α-E2 had robust beneficial effects on metabolic and inflammatory parameters in aged male mice. However, it remains unclear if 17α-E2 also delays other "hallmarks" of aging, particularly maintaining proteostasis. Here, we used isotope labeling methods in older mice to examine proteostatic mechanisms. We compared weight-matched mild calorie restricted (CR) and 17α-E2 treated male mice with the hypothesis that 17α-E2 would increase protein synthesis for somatic maintenance. 17α-E2 had no effect on protein synthesis or DNA synthesis in multiple tissues, including white adipose tissue. Conversely, mild short-term CR decreased DNA synthesis and increased the protein to DNA synthesis ratio in multiple tissues. Examination of individual protein synthesis and content did not differentiate treatments, although it provided insight into the regulation of protein content between tissues. Contrary to our hypothesis, we did not see the predicted differences in protein to DNA synthesis following 17α-E2 treatment. However, mild short-term CR elicited differences consistent with both lifelong CR and other treatments that curtail aging processes. These data indicated that despite similar maintenance of body mass, 17α-E2 and CR treatments elicit distinctly different proteostatic outcomes.

The surgical treatment of tumors of the fourth ventricle: a single-institution experience.

OBJECTIVE Fourth ventricle tumors are rare, and surgical series are typically small, comprising a single pathology, or focused exclusively on pediatric populations. This study investigated surgical outcome and complications following fourth ventricle tumor resection in a diverse patient population. This is the largest cohort of fourth ventricle tumors described in the literature to date. METHODS This is an 18-year (1993-2010) retrospective review of 55 cases involving patients undergoing surgery for tumors of the fourth ventricle. Data included patient demographic characteristics, pathological and radiographic tumor characteristics, and surgical factors (approach, surgical adjuncts, extent of resection, etc.). The neurological and medical complications following resection were collected and outcomes at 30 days, 90 days, 6 months, and 1 year were reviewed to determine patient recovery. Patient, tumor, and surgical factors were analyzed to determine factors associated with the frequently encountered postoperative neurological complications. RESULTS There were no postoperative deaths. Gross-total resection was achieved in 75% of cases. Forty-five percent of patients experienced at least 1 major neurological complication, while 31% had minor complications only. New or worsening gait/focal motor disturbance (56%), speech/swallowing deficits (38%), and cranial nerve deficits (31%) were the most common neurological deficits in the immediate postoperative period. Of these, cranial nerve deficits were the least likely to resolve at follow-up. Multivariate analysis showed that patients undergoing a transvermian approach had a higher incidence of postoperative cranial nerve deficits, gait disturbance, and speech/swallowing deficits than those treated with a telovelar approach. The use of surgical adjuncts (intraoperative navigation, neurophysiological monitoring) did not significantly affect neurological outcome. Twenty-two percent of patients required postoperative CSF diversion following tumor resection. Patients who required intraoperative ventriculostomy, those undergoing a transvermian approach, and pediatric patients (< 18 years old) were all more likely to require postoperative CSF diversion. Twenty percent of patients suffered at least 1 medical complication following tumor resection. Most complications were respiratory, with the most common being postoperative respiratory failure (14%), followed by pneumonia (13%). CONCLUSIONS The occurrence of complications after fourth ventricle tumor surgery is not rare. Postoperative neurological sequelae were frequent, but a substantial number of patients had neurological improvement at long-term followup. Of the neurological complications analyzed, postoperative cranial nerve deficits were the least likely to completely resolve at follow-up. Of all the patient, tumor, and surgical variables included in the analysis, surgical approach had the most significant impact on neurological morbidity, with the telovelar approach being associated with less morbidity.

Impact of dairy protein during limb immobilization and recovery on muscle size and protein synthesis; a randomized controlled trial.

Muscle disuse results in the loss of muscular strength and size, due to an imbalance between protein synthesis (MPS) and breakdown (MPB). Protein ingestion stimulates MPS, although it is not established if protein is able to attenuate muscle loss with immobilization (IM) or influence the recovery consisting of ambulatory movement followed by resistance training (RT). Thirty men (49.9 ± 0.6 yr) underwent 14 days of unilateral leg IM, 14 days of ambulatory recovery (AR), and a further six RT sessions over 14 days. Participants were randomized to consume an additional 20 g of dairy protein or placebo with a meal during the intervention. Isometric knee extension strength was reduced following IM (-24.7 ± 2.7%), partially recovered with AR (-8.6 ± 2.6%), and fully recovered after RT (-0.6 ± 3.4%), with no effect of supplementation. Thigh muscle cross-sectional area decreased with IM (-4.1 ± 0.5%), partially recovered with AR (-2.1 ± 0.5%), and increased above baseline with RT (+2.2 ± 0.5%), with no treatment effect. Myofibrillar MPS, measured using deuterated water, was unaltered by IM, with no effect of protein. During AR, MPS was increased only with protein supplementation. Protein supplementation did not attenuate the loss of muscle size and function with disuse or potentiate recovery but enhanced myofibrillar MPS during AR. NEW & NOTEWORTHY Twenty grams of daily protein supplementation does not attenuate the loss of muscle size and function induced by 2 wk of muscle disuse or potentiate recovery in middle-age men. Average mitochondrial but not myofibrillar muscle protein synthesis was attenuated during immobilization with no effect of supplementation. Protein supplementation increased myofibrillar protein synthesis during a 2-wk period of ambulatory recovery following disuse but without group differences in phenotype recovery.

10-Year Follow-Up Wear Analysis of Marathon Highly Cross-Linked Polyethylene in Primary Total Hip Arthroplasty.

BACKGROUND: Short-term and intermediate-term wear rates for highly cross-linked polyethylene (HCLPE) liners in total hip arthroplasty (THA) are significantly lower than published rates for traditional polyethylene liners. The aim of this study was to report the longest-to-date follow-up of a specific HCLPE liner. METHODS: A series of 35 THAs using a specific HCLPE liner were reviewed. Anteroposterior radiographs were reviewed for femoral head penetration, the presence of femoral and/or acetabular osteolysis, long-term survival, total wear, and wear rates in all patients. RESULTS: The average patient age at time of surgery was 70 years with an average follow-up of 10 years (118 months; range, 7.2-13.4 years). The mean wear rate in our cohort was 0.07 mm/y. Total wear was 0.71 mm over the study period. No hips showed evidence of osteolysis in any zones. Survivorship at latest follow-up was 100% with all-cause revision as an end point. CONCLUSION: The wear rate of HCLPE liners continues to be lower than published wear rates for traditional polyethylene and continues to reaffirm the acceptably low wear rates using HCLPE acetabular liner in primary THA.

Influence of Nrf2 activators on subcellular skeletal muscle protein and DNA synthesis rates after 6 weeks of milk protein feeding in older adults.

In older adults, chronic oxidative and inflammatory stresses are associated with an impaired increase in skeletal muscle protein synthesis after acute anabolic stimuli. Conjugated linoleic acid (CLA) and Protandim have been shown to activate nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor for the antioxidant response element and anti-inflammatory pathways. This study tested the hypothesis that compared to a placebo control (CON), CLA and Protandim would increase skeletal muscle subcellular protein (myofibrillar, mitochondrial, cytoplasmic) and DNA synthesis in older adults after 6 weeks of milk protein feeding. CLA decreased oxidative stress and skeletal muscle oxidative damage with a trend to increase messenger RNA (mRNA) expression of a Nrf2 target, NAD(P)H dehydrogenase quinone 1 (NQO1). However, CLA did not influence other Nrf2 targets (heme oxygenase-1 (HO-1), glutathione peroxidase 1 (Gpx1)) or protein or DNA synthesis. Conversely, Protandim increased HO-1 protein content but not the mRNA expression of downstream Nrf2 targets, oxidative stress, or skeletal muscle oxidative damage. Rates of myofibrillar protein synthesis were maintained despite lower mitochondrial and cytoplasmic protein syntheses after Protandim versus CON. Similarly, DNA synthesis was non-significantly lower after Protandim compared to CON. After Protandim, the ratio of protein to DNA synthesis tended to be greater in the myofibrillar fraction and maintained in the mitochondrial and cytoplasmic fractions, emphasizing the importance of measuring both protein and DNA synthesis to gain insight into proteostasis. Overall, these data suggest that Protandim may enhance proteostatic mechanisms of skeletal muscle contractile proteins after 6 weeks of milk protein feeding in older adults.

Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals.

Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (1-4), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (5-7). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (5, 6, 8, 9).

Modeling the contribution of individual proteins to mixed skeletal muscle protein synthetic rates over increasing periods of label incorporation.

Advances in stable isotope approaches, primarily the use of deuterium oxide ((2)H2O), allow for long-term measurements of protein synthesis, as well as the contribution of individual proteins to tissue measured protein synthesis rates. Here, we determined the influence of individual protein synthetic rates, individual protein content, and time of isotopic labeling on the measured synthesis rate of skeletal muscle proteins. To this end, we developed a mathematical model, applied the model to an established data set collected in vivo, and, to experimentally test the impact of different isotopic labeling periods, used (2)H2O to measure protein synthesis in cultured myotubes over periods of 2, 4, and 7 days. We first demonstrated the influence of both relative protein content and individual protein synthesis rates on measured synthesis rates over time. When expanded to include 286 individual proteins, the model closely approximated protein synthetic rates measured in vivo. The model revealed a 29% difference in measured synthesis rates from the slowest period of measurement (20 min) to the longest period of measurement (6 wk). In support of these findings, culturing of C2C12 myotubes with isotopic labeling periods of 2, 4, or 7 days revealed up to a doubling of the measured synthesis rate in the shorter labeling period compared with the longer period of labeling. From our model, we conclude that a 4-wk period of labeling is ideal for considering all proteins in a mixed-tissue fraction, while minimizing the slowing effect of fully turned-over proteins. In addition, we advocate that careful consideration must be paid to the period of isotopic labeling when comparing mixed protein synthetic rates between studies.

Effect of a selective COX-2 inhibitor, celecoxib, on heterotopic ossification after total hip arthroplasty: a case-controlled study.

Heterotopic ossification (HO) is a well-known complication of total hip arthroplasty (THA), especially when the direct lateral approach is used. In this study, we examined the effect of the selective COX-2 inhibitor, celecoxib, on the rates of HO after THA. A control group consisting of 108 patients that did not receive celecoxib was compared with a study group consisting of 106 patients that did receive celecoxib. We assessed the presence and grade of HO using the Brooker classification and Harris hip scores were determined pre- and postoperatively to better quantify clinical outcomes. In this retrospective study of prospectively collected data, celecoxib is associated with a significant reduction in the incidence of HO in patients undergoing THA.

10-year follow-up wear analysis of first-generation highly crosslinked polyethylene in primary total hip arthroplasty.

Our goal was to report a 10-year follow up of linear penetration rates for HCLPE, and to determine whether a difference exists between penetrations measured on pelvis or hip anterior-posterior radiographs. We reviewed 48 total hip arthroplasties where a first-generation HCLPE liner was used. Femoral head penetration was measured on both AP pelvis and hip radiographs. Total wear and wear rate at 10 years were 1.26 mm and 0.122 mm/y, respectively. The rate decreased significantly after the first 2-3 years, plateauing at a wear rate of 0.05 mm/y for the last 5 years. The AP hip total wear and wear rate were 1.38 mm and 0.133 mm/y respectively, while rates were 1.13 mm and 0.109 mm/y respectively for the pelvis radiographs (P<.05). We found a significant difference in measurements of linear penetration when comparing AP pelvis vs. hip radiographs with lower rates recorded using an AP pelvis.

Non-O1, non-O139 Vibrio cholerae sepsis in a patient with nephrotic syndrome.

Non-O1, non-O139 Vibrio cholerae is an encapsulated bacterium, ubiquitous in the marine environment and generally considered to be non-pathogenic. However, it is known to cause diarrheal illness, wound infection, and bacteremia in immunocompromised hosts. Here we have describe non-O1, non-O139 V. cholerae sepsis in a patient with nephrotic syndrome following exposure to sea-water. Interestingly, the exposure occurred remotely 4 months prior to the onset of nephrotic syndrome. The occurrence of florid sepsis after a prolonged interval from the time of exposure is peculiar and raises the possibility of an association between occult Vibrio sepsis and nephrotic syndrome.

Treating a physician patient with psychosis.

The authors present a case of a psychotic female patient who is a former graduate of a locally prestigious medical school and has subsequently been diagnosed with schizophrenia. The patient entered treatment in an outpatient clinic following discharge from her 11th hospitalization. This hospitalization was initiated after the patient's physician friend had called the police and notified them that the patient was significantly disorganized to warrant further evaluation. Treatment was characterized by significant transference and counter-transference reactions amongst her clinicians - both treatment-promoting and treatment-interfering - based on her status as a physician. The problem of insight was a significant hurdle in the treatment of the patient as her medical knowledge of mental illness was substantially greater than her insight into her own mental illness. Throughout treatment, a number of medical-legal and ethical issues arose. Initially, the question was raised as to the legality of the actions by the patient's friend-having made a clinical assessment without having a clinical role in the patient's care. As the patient's clinical status improved and she sought to re-enter the medical field as a resident, new medical legal issues surfaced. What were the roles of the patient's treaters in maintaining confidentiality and simultaneously ensuring the safety of patients that the psychotic physician might care for? This case highlights the universality of psychiatric vulnerability. Insight in psychosis as well as the transference and counter-transference issues involved in caring for a psychotic physician are discussed. Additionally, a thorough medical-legal discussion addresses the various complexities of caring for a psychotic physician.

Revision total hip arthroplasty using a modular femoral implant in Paprosky type III and IV femoral bone loss.

The purpose of this study was to compare results of patients with Paprosky type I and II femoral defects vs type IIIA, IIIB, and IV defects in patients undergoing revision hip arthroplasty. There were 64 patients in the group with type I and II defects with an average age of 68 years. There were 52 patients with Paprosky type IIIA, IIIB, and IV defects with an average age of 67 years. There were 8 intraoperative fractures in the type III and IV group, whereas there were 9 in the type I and II group. There were no differences between the 2 groups with respect to subsidence, loosening, dislocation, infection, and medical complications. Survivorship for the whole group was 96.9% at 5 years. Modular femoral implants provide several intraoperative options to restore leg length, offset, and stability despite femoral defects. We did not realize a higher failure rate in patients with type III or IV defects.

Novel disubstituted chrysene as a potent agent against colon cancer.

Research on polycyclic aromatic hydrocarbons and their derivatives has received significant attention from the scientific community. The present study involved the synthesis of several novel 6,12-disubstituted chrysene derivatives. Nitration of chrysene with nitric acid produced 6,12-dinitrochrysene which when reduced yielded 6,12-diaminochrysene. A coupling reaction of 6,12-diaminochrysene with an acid in the presence of isobutylchloroformate produced amide. The reduction of amide produced an amine. The amino was converted to a hydrochloride salt. The new compounds were characterized through different types of analytical data. One of these compounds demonstrated marked activity in vivo against a colon cancer cell line. Inhibition of the growth of this tumor was best noted at day 20 when each treatment regimen inhibited the average tumor volume by 50%. In a number of in vivo tests in various regimens, the hydrochloride salt demonstrated consistent inhibition of the growth of the cancer HT-29 cell line. Despite the research progress in polycyclic aromatic compounds, the use of these types of molecules as anticancer agents has not been reported systematically.

Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

Inhibition of radiation-induced nuclear factor-kappaB activation by an anti-Ras single-chain antibody fragment: lack of involvement in radiosensitization.

We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-kappaB (NF-kappaB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-kappaB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IkappaB kinase activity and IkappaBalpha phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-kappaB. Whereas AV1Y28 inhibited NF-kappaB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-alpha-induced NF-kappaB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-kappaB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-kappaB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-kappaB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-kappaB activation. However, inhibition of NF-kappaB activation via the expression of a dominant-negative form of IkappaBalpha in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-kappaB activation, this process does not appear to play a direct role in its radiosensitizing actions.