Variants of the CNTNAP2 5' promoter as risk factors for autism spectrum disorders: a genetic and functional approach.

Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5' promoter. Here, we directly sequenced the CNTNAP2 5' promoter region of 236 German families with one child with ASD and detected four novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG[7] with language development. The four novel and the three known minor alleles of the identified variants were predicted to alter transcription factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specific and differentiation stage-dependent effects at the level of promoter activation. The novel potential rare risk-variant M2, a G>A mutation -215 base pairs 5' of the transcriptional start site, significantly reduced promoter efficiency in HEK293T and in undifferentiated and differentiated neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus confirm the hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD.

Molecular targeted therapies for rhabdomyosarcoma: focus on hedgehog and apoptosis signaling.

Dysfunction of cell death and proliferation pathways can contribute to rhabdomyosarcomagenesis, tumor progression and treatment resistance. Therefore, the identification of key signaling hubs and molecules that govern the decision between life and death of a cancer cell is expected to open new perspectives for drug discovery. For example, programmed cell death pathways can be engaged in rhabdomyosarcoma (RMS) cells by recombinant soluble proteins, monoclonal antibodies or small-molecule inhibitors. In addition, the hedgehog (Hh) cascade is often aberrantly activated in RMS and represents a promising target for therapeutic intervention. The development of molecular targeted cancer therapeutics will likely lead to more effective treatment options for patients with RMS.

Impact of daytime sleepiness on rehabilitation outcome in the elderly.

Daytime sleepiness (DS) is associated with poor health, impaired physical functioning, as well as somatic and psychiatric morbidity. The impact of DS on functional outcome in the elderly is unknown. We investigated whether observed daytime sleepiness in geriatric patients with moderate to severe functional impairment was associated with functional clinical outcomes. We addressed the issue by determining the impact of observed daytime sleepiness, by means of the Essener Questionnaire of Age and Sleepiness (EQAS), on improvement in functional status - measured by the Barthel ADL Index - among disabled geriatric in-patients. We included 129 patients, 28 (22%) were male and 101 (78%) were female. Sleepiness according to EQAS scale was absent in 27 (21%) patients, mild in 71 (55%) patients and moderate to severe in 31 (24%) patients. The three patient groups did not differ in the Barthel ADL Index (BI) on admission or co-morbid conditions. Geriatric treatment was comparable across groups. Improvement in the BI of at least 1 standard deviation (SD) occurred in 23/27 (85%) of subjects without sleepiness, in 53/71 (75%) of subjects with mild to moderate sleepiness and in 15/31 (44%) of subject with severe sleepiness (p < 0.01). BI increased at least 2 SD in 20/27 (74%), 38/71 (54%) and 11/31 (35%) individuals, respectively (p < 0.02). We conclude that the daytime sleepiness predicts a poorer functional recovery rate in older patients during geriatric in-hospital rehabilitation. Furthermore, we found a significant association and a dose response relationship between severity of daytime sleepiness and improvement in Barthel ADL Index.

Validation of the Essener Questionnaire of Age and Sleepiness in the elderly using pupillometry.

In the elderly population, daytime sleepiness (DS) is a burden that affects quality of life, cognitive and physical functioning as well as health status and morbidity. The measurement of DS in older subjects continues to be a challenge, as there are only few elderly-specific assessment tools available. Therefore, we compared the newly developed Essener Questionnaire of Age and Sleepiness (EQAS) with pupillography, a physiological measure of sleepiness. The aim was to identify EQAS cut-off values for increased daytime sleepiness. For the validation study, we determined EQAS scores and the pupillary unrest index (PUI) of the pupillographic sleepiness test (PST) in 88 geriatric in-patients. We also collected data on age, gender, co-morbidities, and geriatric assessment in these subjects. Of all included patients 37 (42%) completed the PST. Fourteen (16%) subjects refused to participate and 37 (42%) subjects could not complete 11 min required for a valid PUI. Subjects with complete and incomplete pupillometry did not differ in basic assessment parameters of health status or cognitive functioning. EQAS scores correlated significantly with PUI values (r = 0.70; p < 0.001) demonstrating a dose-response relationship. Based on ROC analysis, an EQAS score above 3 was optimal to distinguished sleepy from non-sleepy participants with sensitivity of 67%, specificity of 93% and positive and negative predictive values of 75% and 90%, respectively. In conclusion, the high negative and positive predictive values of the EQAS indicate that this instrument is a useful and valid assessment tool for daytime sleepiness in the elderly. The easy administration of this observational instrument favors its adoption in geriatric medicine.

CUX1: target of Akt signalling and mediator of resistance to apoptosis in pancreatic cancer.

BACKGROUND AND AIMS: The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer. METHODS: The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues. RESULTS: Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts. CONCLUSION: CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.

Cell death and survival signaling in oncogenesis.

One of the hallmarks of human cancers is the deregulation of signal transduction pathways that regulate fundamental processes such as cell proliferation and cell death. This leads to the disturbance in the physiological control of tissue homeostasis and a concomitant net increase in cell number. Thus, deregulation of intracellular signaling events can contribute to tumorigenesis and tumor progression. In addition, this may also confer treatment resistance, since the response of cancer cells towards most cytotoxic therapies including chemo-, radio- or immunotherapy critically depends on intact signaling cascades that eventually lead to cell death. Hence, a better understanding of the molecular mechanisms that regulate cell death and survival programs in cancer cells is expected to provide the basis for the development of new diagnostic and therapeutic strategies.

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.

Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance.

Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. Although it usually promotes tumor cell survival under hypoxia, it has also been implied to trigger apoptosis. Although the impact of hypoxia has been extensively studied in many adult solid tumors, its role in most childhood tumors, for example, in rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), has not yet been addressed. Here, we report that hypoxia protects A204 RMS and A673 ES cells against anticancer drug- or tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and that Hif-1alpha plays a key role in conferring apoptosis resistance under hypoxia. Although a functional HIF-1 pathway and proapoptotic proteins such as p53 and Bcl-2/E1B 19 kDa interacting protein 3 were activated under hypoxia in both A204 RMS and A673 ES cells, these cells remained refractory to apoptosis. Concomitant analysis of antiapoptotic proteins revealed that hypoxia induced expression of Bcl-2 and inhibitor of apoptosis proteins (IAP)-2 as well as proteins associated with anaerobic metabolism such as the glucose transporter protein GLUT-1 and the glycolytic enzyme Aldolase A. Specific downregulation of Hif-1alpha by RNA interference significantly enhanced apoptosis under hypoxia by preventing the hypoxia-mediated increase in GLUT-1 expression without altering expression levels of the antiapoptotic proteins Bcl-2 or cIAP-2. Moreover, glucose deprivation-induced apoptosis of A204 RMS and A673 ES cells was inhibited under hypoxic conditions in a Hif-1alpha-dependent manner. As GLUT-1 was induced via Hif-1alpha under hypoxia in A204 RMS and A673 ES, these findings suggest that the Hif-1alpha-mediated increase in glucose uptake plays an important role in conferring apoptosis resistance. Thus, hypoxia-inducible genes may represent novel targets for therapeutic intervention in some pediatric tumors, which warrants further investigation.

Sensitization of pancreatic carcinoma cells for gamma-irradiation-induced apoptosis by XIAP inhibition.

Resistance of pancreatic cancer to current treatments including radiotherapy remains a major challenge in oncology and may be caused by defects in apoptosis programs. Since 'inhibitor of apoptosis proteins' (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting caspases, therapeutic modulation of IAPs could tackle a key resistance mechanism. Here, we report that targeting X-linked inhibitor of apoptosis (XIAP) by RNA-interference-mediated knockdown or overexpression of second mitochondria-derived activator of caspase significantly enhanced apoptosis and markedly reduced clonogenic growth of pancreatic carcinoma cells upon gamma-irradiation. Analysis of signaling pathways revealed that antagonizing XIAP increased activation of caspase-2, -3, -8 and -9 and loss of mitochondrial membrane potential upon gamma-irradiation. Interestingly, inhibition of caspases also reduced the cooperative effect of XIAP targeting and gamma-irradiation to trigger mitochondrial perturbations, suggesting that XIAP controls a feedback mitochondrial amplification loop by regulating caspase activity. Importantly, our data demonstrate for the first time that small molecule XIAP inhibitors sensitized pancreatic carcinoma cells for gamma-irradiation-induced apoptosis, whereas they had no effect on gamma-irradiation-mediated apoptosis of non-malignant fibroblasts indicating some tumor specificity. In conclusion, targeting XIAP, for example by small molecules, is a promising novel approach to enhance radiosensitivity of pancreatic cancer that warrants further investigation.

Regulation of TRAIL-induced apoptosis by XIAP in pancreatic carcinoma cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapy because of its relative tumor selectivity. However, many cancers including pancreatic cancer remain resistant towards TRAIL. To develop TRAIL for cancer therapy of pancreatic carcinoma, it will therefore be pivotal to elucidate the molecular mechanisms of TRAIL resistance. Here, we identify X-linked inhibitor of apoptosis (XIAP) as a regulator of TRAIL sensitivity in pancreatic carcinoma cells. Full activation of effector caspases, loss of mitochondrial membrane potential and cytochrome c release following TRAIL treatment were markedly impaired in pancreatic carcinoma cell lines, which poorly responded to TRAIL (PaTuII, PancTu1, ASPC1, DanG), compared to TRAIL-sensitive Colo357 pancreatic carcinoma cells. Stable downregulation of XIAP by RNA interference significantly reduced survival and enhanced TRAIL-induced apoptosis in pancreatic carcinoma cells. Also, downregulation of XIAP significantly increased CD95-induced cell death. Importantly, knockdown of XIAP strongly inhibited clonogenicity of pancreatic cancer cells treated with TRAIL indicating that XIAP promotes clonogenic survival of pancreatic carcinoma cells. Thus, our findings for the first time indicate that targeting XIAP represents a promising strategy to enhance the antitumor activity of TRAIL in pancreatic cancer, which has important clinical implications.

Thirty-eight-negative kinase 1 (TNK1) facilitates TNFalpha-induced apoptosis by blocking NF-kappaB activation.

Thirty-eight-negative kinase 1 (TNK1) is a member of the ACK-family of nonreceptor tyrosine kinases and was originally cloned from CD34+/Lin-/CD38-hematopoietic stem/progenitor cells. The signaling pathways induced by TNK1 are largely unknown. Here, we report that expression and consequent activation of TNK1 enables tumor necrosis factor alpha (TNFalpha)-induced apoptosis by selectively inhibiting TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB). TNK1 has no effect on NF-kappaB DNA binding or the composition of the NF-kappaB complex; however, the kinase markedly prevents TNFalpha-induced NF-kappaB transactivation. TNK1 therefore acts as a novel molecular switch that can determine the properties of TNFalpha signaling and therefore cell death.

NF-kappaB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

Depression, comorbidities and the TNF-alpha system.

Depression has frequently been reported to be associated with other physical diseases and changes in the cytokine system. We aimed to investigate associations between a medical history of depression, its comorbidities and cytokine plasma levels in the Bavarian Nutrition Survey II (BVS II) study sample and in patients suffering from an acute depressive episode. The BVS II is a representative study of the Bavarian population aged 13-80years. The disease history of its 1050 participants was assessed through face-to-face interviews. A sub-sample of 568 subjects and 62 additional acutely depressed inpatients of the Max Planck Institute of Psychiatry participated in anthropometric measurements and blood sampling. Tumor necrosis factor-alpha (TNF-alpha) and soluble TNF receptor (sTNF-R) p55 and sTNF-R p75 plasma levels were measured using enzyme-linked immunosorbent assays. A history of depression was associated with a higher incidence of high blood pressure, peptic ulcer, dyslipoproteinemia, osteoporosis, allergic skin rash, atopic eczema and thyroid disease. Within the BVS II sample, participants with a history of depression differed from subjects who had never had depression with regard to sTNF-R p55 and sTNF-R p75 levels even when controlling for age, BMI and smoking status. Acutely depressed inpatients showed even higher levels of sTNF-R p55 and sTNF-R p75 than subjects in the normal population. TNF-alpha levels were also significantly elevated in acutely depressed patients. These results confirm earlier studies regarding the comorbidities of depression and support the hypothesis that activation of the TNF-alpha system may contribute to the development of a depressive disorder.

Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy.

Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. One of the most important advances in cancer research in recent years is the recognition that cell death mostly by apoptosis is crucially involved in the regulation of tumor formation and also critically determines treatment response. Killing of tumor cells by most anticancer strategies currently used in clinical oncology, for example, chemotherapy, gamma-irradiation, suicide gene therapy or immunotherapy, has been linked to activation of apoptosis signal transduction pathways in cancer cells such as the intrinsic and/or extrinsic pathway. Thus, failure to undergo apoptosis may result in treatment resistance. Understanding the molecular events that regulate apoptosis in response to anticancer chemotherapy, and how cancer cells evade apoptotic death, provides novel opportunities for a more rational approach to develop molecular-targeted therapies for combating cancer.

5-Aza-2'-deoxycytidine and IFN-gamma cooperate to sensitize for TRAIL-induced apoptosis by upregulating caspase-8.

Resistance of tumors to cytotoxic therapy remains a major obstacle in cancer treatment and is often caused by defects in apoptosis programs. Caspase-8, a key mediator of death receptor-induced apoptosis, has previously been reported to be frequently inactivated by epigenetic silencing in many tumors, for example in neuroblastoma or medulloblastoma. Here, we provide for the first time evidence that combined treatment with suboptimal concentrations of the demethylating agent 5-Aza-2'-deoxycytidine (5-dAzaC) and interferon-gamma (IFN-gamma) cooperated to upregulate caspase-8 expression in neuroblastoma and medulloblastoma cells lacking caspase-8. Consequently, activation of caspase-8 and downstream caspases upon addition of TNF-related apoptosis-inducing ligand (TRAIL) was restored by pretreatment with 5-dAzaC and IFN-gamma. Importantly, pretreatment with 5-dAzaC and IFN-gamma acted in concert to significantly enhance TRAIL-induced apoptosis in neuroblastoma and medulloblastoma cells. Inhibition of caspase-8 by dominant-negative caspase-8 or by the relatively specific caspase-8 inhibitior zIETD.fmk inhibited the increase in apoptosis provided by 5-dAzaC and IFN-gamma, indicating that caspase-8 is a key mediator of this sensitization effect. Thus, by demonstrating that 5-dAzaC and IFN-gamma at relatively low individual concentrations cooperate to restore caspase-8 expression and sensitize resistant neuroblastoma and medulloblastoma cells to TRAIL-induced apoptosis, our findings have important implications for novel strategies targeting defective apoptosis pathways in neuroectodermal tumors.

Smac Mimetics to Therapeutically Target IAP Proteins in Cancer.

Inhibitor of Apoptosis (IAP) proteins are overexpressed in a variety of human cancers. Therefore, they are considered as promising targets for the design of therapeutic strategies. Smac mimetics mimic the endogenous mitochondrial protein Smac that antagonizes IAP proteins upon its release into the cytosol. Multiple preclinical studies have documented the ability of Smac mimetics to either directly induce cell death of cancer cells or to prime them to agents that trigger cell death. At present, several Smac mimetics are being evaluated in early clinical trials. The current review provides an overview on the potential of Smac mimetics as cancer therapeutics to target IAP proteins for cancer therapy.