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Background: Because cervical intraepithelial neoplasia (CIN, or dysplasia) is associated with behavioral factors, health education is an important part of the care of patients at risk for this disease. Smoking, which is strongly associated with CIN and cancers, is particularly important because smoking cessation, even after the onset of CIN, improves outcomes. This study is part of an effort to identify effective ways to convey information about the association between smoking and CIN to at-risk women.Methods: We created 8 images to systematically incorporate a range of artistic approaches to inform viewers about the relationship between smoking and CIN. College women smokers were recruited to participate. Participants rated the images on the extent to which they attracted attention and motivated smoking cessation.Results: Significant differences were identified in the response of this population to visual images differing in content and style. Informative images were more compelling than those that were emotive.Conclusions: Further exploration is warranted to improve our understanding of how visual forms of health education can impact smoking cessation by those at risk for cervical cancer. [ABSTRACT FROM AUTHOR]

Cervical cancer is a well-established smoking-related illness, but many at-risk women are unaware of this link. Objective: The authors designed this study to (1) investigate the relationship of smoking behavior with the history of abnormal Pap test results, sexual history, and perceived risk of cervical cancer and (2) determine whether self-classified smoking status (and hence perceived risk) corresponds with actual smoking behavior in a college student population. Participants and Method Summary: College women students (N = 135) completed a survey assessing smoking history, health history, sexual risk behavior, and risk awareness. Results: Relative to those who had not smoked in the past month, current smokers (n = 36, or 27% of the total sample) perceived themselves to be at higher risk for developing cervical cancer, but did not demonstrate increased awareness of specific cervical cancer risk factors, including smoking. Twenty-eight per-cent (10 of 36) of past-month smokers did not define themselves as current smokers. Conclusion: The authors conclude that anti-smoking and health-related messages targeting smokers may misfire for individuals who do not define themselves as smokers but are nonetheless at risk for smoking-related consequences and escalating use. [ABSTRACT FROM AUTHOR]

Background and Aims: Inflammatory bowel diseases (IBD) are characterized by mucosal inflammation and sequential fibrosis formation, but the exact role of the hyperactive NLRP3 inflammasome in these processes is unclear. Thus, we studied the expression and function of the NLRP3 inflammasome in the context of inflammation and fibrosis in IBD., Methods: We analysed intestinal NLRP3 expression in mucosal immune cells and fibroblasts from IBD patients and NLRP3-associated gene expression via single-cell RNA sequencing and microarray analyses. Furthermore, cytokine secretion of NLRP3 inhibitor treated blood and mucosal cells, as well as proliferation, collagen production, and cell death of NLRP3 inhibitor treated intestinal fibroblasts from IBD patients were studied., Results: We found increased NLRP3 expression in the inflamed mucosa of IBD patients and NLRP3 inhibition led to reduced IL-1β and IL-18 production in blood cells and diminished the bioactive form of mucosal IL-1β. Single cell analysis identified overlapping expression patterns of NLRP3 and IL-1β in classically activated intestinal macrophages and we also detected NLRP3 expression in CD163+ macrophages. In addition, NLRP3 expression was also found in intestinal fibroblasts from IBD patients. Inhibition of NLRP3 led to reduced proliferation of intestinal fibroblasts, which was associated with a marked decrease in production of collagen type I and type VI in IBD patients. Moreover, NLRP3 inhibition in intestinal fibroblasts induced autophagy, a cellular process involved in collagen degradation., Conclusions: In the presented study, we demonstrate that inhibiting NLRP3 might pave the way for novel therapeutic approaches in IBD, especially to prevent the severe complication of intestinal fibrosis formation., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (T H 17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. T H 17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that T H 17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates T H 17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in T H 17s. We found that T H 17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS T H 17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS T H 17s. By contrast, glycolytic T H 17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected T H 17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates T H 17 cell fate and highlights the potential for therapies that target OXPHOS in T H 17-driven diseases.

Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4 + T cells in umbilical cord and peripheral blood. We found that naive CD4 + T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8 + naive T cells were preferentially enriched CD31 + T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.

Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objectives: To identify whether mast cell densities in vulvar biopsies from the vestibule are associated with vulvodynia., Methods: We enrolled 100 women aged 19 to 59 years with confirmed vulvodynia cases, 100 racially matched controls, and 100 black control women. All had vulvar biopsies performed at the 7 o'clock position of the vestibule, which were then immunostained to detect c-KIT protein. The numbers of c-KIT positive mast cells per ×400 magnification field were manually counted, and t tests and logistic regression were used to assess the association with case-control status., Results: Of the biopsies, 235 were adequate samples for c-KIT testing for mast cells. The mast cell density was substantially lower in black control women (13.9 ± 10.9) in comparison to white control women (22.5 ± 13.2 p < 0.001): hence the analysis was confined to white cases and racially matched control women. Compared with racially matched controls, cases were younger, more likely to be married, and reported a higher household income. The average number of mast cells per ×400 magnification field overall was 19.1 ± 13.2 (range, 0-62). There was no difference in the mast cell count between racially matched cases (22.4 ± 13.9 per ×400 field) and controls (22.5 ± 13.2) in either the univariate or multivariable analyses. Within the group of cases, there was no difference in mast cell density based on the presence or absence of a variety of urogenital symptoms., Conclusions: No difference in mast cell density in biopsies of the vestibule was found between white cases and racially matched controls. Black control women have a lower mast cell density compared with white control women.

Background: Resveratrol inhibits the growth of ovarian carcinoma cells in vitro through the inhibition of glucose metabolism and the induction of both autophagy and apoptosis. In the current study, we investigated the metabolic and therapeutic effects of resveratrol in vivo., Methods: A fluorescent xenograft mouse model of ovarian cancer was used. Mice were treated with cisplatin, resveratrol, or vehicle alone. Tumor burden was assessed using whole-body imaging. The effect of resveratrol on glucose uptake in vivo was determined using micro-positron emission tomography scanning. To determine whether resveratrol could inhibit tumor regrowth, tumor-bearing mice were treated with cisplatin followed by either daily resveratrol or vehicle. Autophagic response in resected tumors taken from mice treated with resveratrol was examined by transmission electron microscopy. Glycolysis and mitochondrial respiration in ovarian tumor cells after treatment with resveratrol was assessed., Results: Mice treated with resveratrol and cisplatin were found to have a significantly reduced tumor burden compared with control animals (P<.001). Resveratrol-treated mice demonstrated a marked decrease in tumor uptake of glucose compared with controls. After treatment with cisplatin, "maintenance" resveratrol resulted in the suppression of tumor regrowth compared with mice receiving vehicle alone (P<.01). Tumors resected from mice treated with resveratrol exhibited autophagosomes consistent with the induction of autophagy. Treatment with resveratrol inhibited glycolytic response in ovarian tumor cells with high baseline glycolytic rates., Conclusions: Treatment with resveratrol inhibits glucose uptake and has a significant antineoplastic effect in a preclinical mouse model of ovarian cancer. Resveratrol treatment suppresses tumor regrowth after therapy with cisplatin, suggesting that this agent has the potential to prolong disease-free survival. Cancer 2016;122:722-729. © 2015 American Cancer Society., (© 2015 American Cancer Society.)

Mitochondria are multifunctional organelles that play a central role in cellular homeostasis. Severe mitochondrial dysfunction leads to life-threatening diseases in humans and accelerates the aging process. Surprisingly, moderate reduction of mitochondrial function in different species has anti-aging effects. High-throughput screenings in the nematode Caenorhabditis elegans lead to the identification of several pro-longevity genetic and pharmacological interventions. Large-scale screens, however, are manual, subjective, time consuming and costly. These limitations could be reduced by the identification of automatically quantifiable biomarkers of healthy aging. In this study we exploit the distinct and reproducible phenotypes described in C. elegans upon different levels of mitochondrial alteration to develop an automated high-content strategy to identify new potential pro-longevity interventions. Utilizing the microscopy platform Cellomics ArrayScan Reader, we optimize a workflow to automatically and reliably quantify the discrete phenotypic readouts associated with different degrees of silencing of mitochondrial respiratory chain regulatory proteins, and validate the approach with mitochondrial-targeting drugs known to extend lifespan in C. elegans. Finally, we report that a new mitochondrial ATPase modulator matches our screening phenotypic criteria and extends nematode's lifespan thus providing the proof of principle that our strategy could be exploited to identify novel mitochondrial-targeted drugs with pro-longevity activity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The 2006 National Institutes of Health (NIH) Consensus paper presented recommendations by the Ancillary Therapy and Supportive Care Working Group to support clinical research trials in chronic graft-versus-host disease (GVHD). Topics covered in that inaugural effort included the prevention and management of infections and common complications of chronic GVHD, as well as recommendations for patient education and appropriate follow-up. Given the new literature that has emerged during the past 8 years, we made further organ-specific refinements to these guidelines. Minimum frequencies are suggested for monitoring key parameters relevant to chronic GVHD during systemic immunosuppressive therapy and, thereafter, referral to existing late effects consensus guidelines is advised. Using the framework of the prior consensus, the 2014 NIH recommendations are organized by organ or other relevant systems and graded according to the strength and quality of supporting evidence., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The coinhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly upregulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1(Hi)ROS(Hi) phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels, and PD-1-driven increases in ROS were dependent upon the oxidation of fatty acids, because treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by antioxidants. Furthermore, PD-1-driven changes in ROS were fundamental to establishing a cell's susceptibility to subsequent metabolic inhibition, because blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing ROS in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti-PD-1 therapies in the clinic., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Unlabelled: In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival., Implications: These findings provide an insight into DNA repair fidelity in neuroblastoma and identify components of the alt-NHEJ pathway as promising therapeutic targets., (©2015 American Association for Cancer Research.)

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic (13)C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Activated T cells require increased energy to proliferate and mediate effector functions, but the metabolic changes that occur in T cells following stimulation in vivo are poorly understood, particularly in the context of inflammation. We have previously shown that T cells activated during graft-versus-host disease (GVHD) primarily rely on oxidative phosphorylation to synthesize adenosine 5'-triphosphate. Here, we demonstrate that alloreactive effector T cells (Teff) use fatty acids (FAs) as a fuel source to support their in vivo activation. Alloreactive T cells increased FA transport, elevated levels of FA oxidation enzymes, up-regulated transcriptional coactivators to drive oxidative metabolism, and increased their rates of FA oxidation. Importantly, increases in FA transport and up-regulation of FA oxidation machinery occurred specifically in T cells during GVHD and were not seen in Teff following acute activation. Pharmacological blockade of FA oxidation decreased the survival of alloreactive T cells but did not influence the survival of T cells during normal immune reconstitution. These studies suggest that pathways controlling FA metabolism might serve as therapeutic targets to treat GVHD and other T-cell-mediated immune diseases.

Ku70 was originally described as an autoantigen, but it also functions as a DNA repair protein in the nucleus and as an antiapoptotic protein by binding to Bax in the cytoplasm, blocking Bax-mediated cell death. In neuroblastoma (NB) cells, Ku70's binding with Bax is regulated by Ku70 acetylation such that increasing Ku70 acetylation results in Bax release, triggering cell death. Although regulating cytoplasmic Ku70 acetylation is important for cell survival, the role of nuclear Ku70 acetylation in DNA repair is unclear. Here, we showed that Ku70 acetylation in the nucleus is regulated by the CREB-binding protein (CBP), and that Ku70 acetylation plays an important role in DNA repair in NB cells. We treated NB cells with ionization radiation and measured DNA repair activity as well as Ku70 acetylation status. Cytoplasmic and nuclear Ku70 were acetylated after ionization radiation in NB cells. Interestingly, cytoplasmic Ku70 was redistributed to the nucleus following irradiation. Depleting CBP in NB cells results in reducing Ku70 acetylation and enhancing DNA repair activity in NB cells, suggesting nuclear Ku70 acetylation may have an inhibitory role in DNA repair. These results provide support for the hypothesis that enhancing Ku70 acetylation, through deacetylase inhibition, may potentiate the effect of ionization radiation in NB cells.

For several decades, it has been known that T-cell activation in vitro leads to increased glycolytic metabolism that fuels proliferation and effector function. Recently, this simple model has been complicated by the observation that different T-cell subsets differentially regulate fundamental metabolic pathways under the control of distinct molecular regulators. Although the majority of these data have been generated in vitro, several recent studies have documented the metabolism of T cells activated in vivo. Here, we review the recent data surrounding the differential regulation of metabolism by distinct T-cell subsets in vitro and in vivo and discuss how differential metabolic regulation might facilitate T-cell function vis-à-vis proliferation, survival, and energy production. We further discuss the important therapeutic implications of differential metabolism across T-cell subsets and review recent successes in exploiting lymphocyte metabolism to treat immune-mediated diseases., (© 2012 John Wiley & Sons A/S.)

Ku70 was first characterized as a nuclear factor that binds DNA double-strand breaks in nonhomolog end-joining DNA repair. However, recent studies have shown that Ku70 is also found in the cytoplasm and binds Bax, preventing Bax-induced cell death. We have shown that, in neuroblastoma cells, the binding between Ku70 and Bax depends on the acetylation status of Ku70, such that, when Ku70 is acetylated, Bax is released from Ku70, triggering cell death. Thus, to survive, in neuroblastoma cells, cytoplasmic Ku70 acetylation status is carefully regulated such that Ku70 is maintained in a deacetylated state, keeping Bax complexed with Ku70. We have shown that overexpression of CREB-binding protein (CBP), a known acetyltransferase that acetylates Ku70, releases Bax from Ku70, triggering apoptosis. Although we have shown that blocking deacetylase activity using non-type-specific inhibitors also triggers Ku70 acetylation and Bax-dependent cell death, the targets of these deacetylase inhibitors in neuroblastoma cells remain unknown. Here, we demonstrate that, in neuroblastoma cells, histone deacetylase 6 (HDAC6) binds Ku70 and Bax in the cytoplasm and that knocking down HDAC6 or using an HDAC6-specific inhibitor triggers Bax-dependent cell death. Our results show that HDAC6 regulates the interaction between Ku70 and Bax in neuroblastoma cells and may be a therapeutic target in this pediatric solid tumor.

Background: Persistent infection with high-risk human papillomavirus (HPV) types is necessary for the development of high-grade cervical dysplasia and cervical carcinoma. The presence of HPV DNA in the blood of cervical cancer patients has been reported; however, whether HPV DNA is detectable in the blood of patients with pre-invasive cervical disease is unclear., Objectives: The objectives of this study were to determine if HPV 16 and HPV 18 DNA could be detected in the serum of colposcopy clinic patients, and if serum HPV detection was associated with grade of cervical disease and HPV cofactors., Study Design: Samples were selected from a biorepository collected from non-pregnant, HIV-negative women ages 18-69 attending colposcopy clinics at two urban public hospitals. Cervical disease status was based on review of colposcopy, biopsy and cytology findings. Serum HPV DNA detection was conducted using a novel PCR and mass spectroscopy-based assay., Results: Of the 116 adequate serum samples, all (100%) were negative for HPV 16 and HPV 18. Over half (51.7%) of participants had cervical HPV 16 and/or HPV 18 infection. Nearly one-third (31.1%) had high grade, 10.3% had low grade, and 50.9% had no cervical disease. Nearly one-third (28.5%) had ever regularly smoked cigarettes, 70.7% had early onset of sexual intercourse, and 75% had ever used oral contraceptives., Conclusions: In this colposcopy clinic population with a range of clinical characteristics and established HPV cofactors, HPV DNA was undetectable in their serum. Our findings suggest that serum HPV DNA detection is not a cervical cancer screening tool., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.

Background: Chemoresistance is the major factor limiting long-term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug-resistant phenotype in many tumor types., Methods: A panel of ovarian cancer cell lines was screened for expression and function of the apoptosome components Apaf-1 and caspase-9. Expression levels were analyzed by immunohistochemistry and immunoblotting; Apaf-1 function was determined by assessing the ability of endogenous Apaf-1 to cleave caspase-9 in the presence or absence of cytochrome c. The effect of the histone deacetylase inhibitor trichostatin A on Apaf-1 expression and function was evaluated., Results: The authors report here that the resistance of ovarian cancer cells to the proapoptotic effects of chemotherapy is due in part to deficient Apaf-1 activity. Although Apaf-1 is expressed in most ovarian cancers, the functional activity is impaired, as Apaf-1 has a diminished ability to recruit and activate caspase-9. Treatment of ovarian cancer cells with trichostatin A results in restoration of Apaf-1 function independent of alterations in Apaf-1 expression. Furthermore, treating chemoresistant cells with sublethal doses of trichostatin A restores Apaf-1 function and sensitizes cells to cisplatin-induced apoptosis., Conclusions: Targeting intrinsic pathway defects for therapeutic intervention may result in sensitizing tumors to standard chemotherapy or triggering apoptosis in the absence of other apoptotic signals. The identification of drugs that can use Apaf-1 when it is present, yet can overcome its functional inactivation, may be an important clinical advance., (Copyright © 2010 American Cancer Society.)

Cells generate adenosine triphosphate (ATP) by glycolysis and by oxidative phosphorylation (OXPHOS). Despite the importance of having sufficient ATP available for the energy-dependent processes involved in immune activation, little is known about the metabolic adaptations that occur in vivo to meet the increased demand for ATP in activated and proliferating lymphocytes. We found that bone marrow (BM) cells proliferating after BM transplantation (BMT) increased aerobic glycolysis but not OXPHOS, whereas T cells proliferating in response to alloantigens during graft-versus-host disease (GVHD) increased both aerobic glycolysis and OXPHOS. Metabolomic analysis of alloreactive T cells showed an accumulation of acylcarnitines consistent with changes in fatty acid oxidation. Alloreactive T cells also exhibited a hyperpolarized mitochondrial membrane potential (ΔΨm), increased superoxide production, and decreased amounts of antioxidants, whereas proliferating BM cells did not. Bz-423, a small-molecule inhibitor of the mitochondrial F(1)F(0) adenosine triphosphate synthase (F(1)F(0)-ATPase), selectively increased superoxide and induced the apoptosis of alloreactive T cells, which arrested established GVHD in several BMT models without affecting hematopoietic engraftment or lymphocyte reconstitution. These findings challenge the current paradigm that activated T cells meet their increased demands for ATP through aerobic glycolysis, and identify the possibility that bioenergetic and redox characteristics can be selectively exploited as a therapeutic strategy for immune disorders.

Bz-423 is an inhibitor of the mitochondrial F(1)F(0)-ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water-soluble Bz-423 analog (5-(3-(aminomethyl)phenyl)-7-chloro- 1-methyl-3-(naphthalen-2-ylmethyl)-1H-benzo][e][1,4]diazepin-2(3H)-one); (1) to its target subunit on the enzyme, the oligomycin sensitivity conferring protein (OSCP), by NMR spectroscopy using chemical shift perturbation and cross-relaxation experiments. Titration experiments with constructs representing residues 1-120 or 1-145 of the OSCP reveals that (a) 1 binds to a region of the protein, at the minimum, comprising residues M51, L56, K65, V66, K75, K77, and N92, and (b) binding of 1 induces conformational changes in the OSCP. Control experiments employing a variant of 1 in which a key binding element on the small molecule was deleted; it had no perturbational effect on the spectra of the OSCP, which indicates that the observed changes with 1 represent specific binding interactions. Collectively, these data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP-F(1) interface resulting in disrupted communication between the peripheral stalk and the F(1)-domain of the enzyme.

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a proapoptotic 1,4-benzodiazepine that potently suppresses disease in the murine model of lupus by selectively killing pathogenic lymphocytes. In MRL/MpJ-Fas(lpr) (MRL-lpr) mice, Bz-423 overcomes deficient expression of the Fas death receptor and hyperactivation of antiapoptotic phosphatidylinositol 3-kinase (PI3K)-Akt signaling to specifically kill pathogenic CD4(+) T cells. Bz-423 binds to the oligomycin-sensitivity-conferring protein component of the mitochondrial F(0)F(1)-ATPase, which modulates the enzyme leading to formation of superoxide by the mitochondrial respiratory chain. Scavenging this reactive oxygen species blocks all subsequent components of the apoptotic cascade. To gain insight into how apoptotic signaling activated by Bz-423-induced superoxide contributes to the selective depletion of MRL-lpr CD4(+) T cells, we characterized the death mechanism in a CD4(+) T cell leukemia line (Jurkat). Although Bz-423-induced superoxide indirectly inactivates Akt, this response is not required for T cell death. Apoptosis instead results from parallel increases in levels of the proapoptotic Bcl-2 proteins Noxa and Bak leading to specific activation of Bak, mitochondrial outer membrane permeabilization, and a commitment to apoptosis. By directly up-regulating proteins that trigger loss of mitochondrial outer membrane integrity, Bz-423 bypasses defective Fas function and antiapoptotic PI3K-Akt signaling in MRL-lpr CD4(+) T cells. Moreover, because disease-associated abnormalities should sensitize autoreactive CD4(+) T cells to transcriptional up-regulation of Noxa by redox signals and to Bak-dependent apoptosis, the apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.

Bz-423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. In order to understand some of the features that contribute to the increased sensitivity of lymphocytes, we report the signaling pathway engaged by Bz-423 in a Burkitt lymphoma cell line (Ramos). Following the generation of superoxide, Bz-423-induced apoptosis requires the activation of Bax and Bak to induce mitochondrial outer membrane permeabilization and cytochrome c release. Knockdown of the BH3-only proteins Bad, Bim, Bik, and Puma inhibits Bz-423 apoptosis, suggesting that these proteins serve as upstream sensors of the oxidant stress induced by Bz-423. Treatment with Bz-423 results in superoxide-dependent Mcl-1 degradation, implicating this protein as the link between Bz-423-induced superoxide and Bax and Bak activation. In contrast to fibroblasts, B cell death induced by Bz-423 is independent of c-Jun N-terminal kinase. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a specific apoptotic response that differs across cell types.

The mitochondrial F(1)F(o)-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F(1)F(o)-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the beta subunits in the F(1) domain and inhibits F(1)F(o)-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F(1)F(o)-ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K(i) values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F(1)F(o)-ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F(1)F(o)-ATPase catalyzes ATP synthesis and hydrolysis., ((c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 830-840, 2009.)

Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High-Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.

Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and childhood. Whereas most low-risk patients do well, children with high-risk tumors often fail intensive treatment. Identification of novel biomarkers is critical to improve prognostication, tailor therapy, and develop new therapeutic targets. Differential RNA-level expression between tumor cells with neuroblastic (N-type) and Schwannian stromal (S-type) phenotypes was used to identify genes of potential interest based on tumor cell type-specific regulation. Gene expression microarray analysis revealed marked differences between N-type and S-type cells in their levels of BCL6 messenger RNA, a transcriptional regulator overexpressed in a variety of hematopoietic malignancies. S-type cells express higher levels of Bcl6 RNA and protein than N-type, and protein levels are significantly limited by proteasome function. An NB tumor tissue microarray linked to clinicopathologic data was immunohistochemically stained to measure Bcl6 protein levels. Bcl6 was detected in both the neuroblastic and Schwannian stromal regions, as distinguished histologically, and correlated with outcome. We found that expression in neuroblastic regions differentiates outcomes, in that Bcl6 expression in neuroblastic regions is associated with increased time to relapse and increased overall survival compared with absent expression in neuroblastic regions, regardless of Schwannian stromal expression. Thus, our findings suggest that Bcl6 may be useful as a prognostic marker and might represent a potential therapeutic target for high-risk NB.

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a benzodiazepine that has cytotoxic and cytostatic activity against a variety of cells in vivo and in vitro. In the present study, we demonstrate that Bz-423 (formulated for topical delivery) reduces epidermal hyperplasia in human psoriatic skin after transplantation to severe, combined immunodeficient (scid) mice. Bz-423 also suppresses the hyperplasia that develops in nonpsoriatic human skin as a consequence of transplantation to scid mice. Proliferation of human epidermal keratinocytes in monolayer culture was suppressed by Bz-423 at concentrations of 0.5 to 2.0 muM (noncytotoxic concentrations). Keratinocyte growth inhibition was accompanied by increased oxidant generation in Bz-423-treated cells, and treatment with vitamin E along with Bz-423 reversed the growth inhibition. Growth inhibition was accompanied by a redistribution of beta-catenin from a cytoplasmic pool to the cell membrane and by reduced levels of c-myc and cyclin D1 (two molecules associated with Wnt pathway signaling). Several analogs of Bz-423 were examined for antiproliferative activity against human epidermal keratinocytes and human dermal fibroblasts in monolayer culture. Each of the analogs tested suppressed growth of both cell types, but in all cases, keratinocytes were more sensitive than fibroblasts. Two of the compounds were found to suppress epidermal hyperplasia induced with all-trans retinoic acid in organ cultures of human skin. Taken together, these data show that Bz-423 and certain analogs produce biological responses in skin cells in vitro and in vivo that are consistent with therapeutic goals for treating psoriasis or epidermal hyperplasia resulting from other causes.

Outcomes remain poor in neuroblastoma despite intensive treatment. Agents with potential efficacy can be drawn from anti-neoplastic drugs introduced for other malignancies. Bortezomib, a proteasome inhibitor, modulates cell-signaling molecules leading to apoptosis. Bortezomib, alone and in combination with other agents, was tested across an in vitro panel of neuroblastic, stromal, and chemo-resistant neuroblastoma cell types to determine its effect on cell viability and to assess for interactions between bortezomib and other chemotherapeutic agents that either limit or increase overall response. Each subtype of neuroblastoma was sensitive to bortezomib and killing occurred with EC50 values of approximately 50 nM. When bortezomib was combined with other agents (doxorubicin, etoposide, SN-38, carboplatin, or cisplatin), no antagonism was observed. The bortezomib-doxorubicin combination was especially effective, demonstrating synergy on isobolographic analysis and resulting in a decrease in EC50 from 50 ng/mL with doxorubicin alone to 5 ng/mL with 25 nM bortezomib. Interestingly, the different cell types exhibited varying response patterns to treatment with bortezomib alone and in combination with other drugs suggesting different mechanisms may be engaged. A decision analysis, incorporating these results showing efficacy in all cell types, the synergy obtained in combination, and the available toxicity data, supports a phase II clinical trial of bortezomib in neuroblastoma.

Objectives: Resveratrol is a phytoalexin found in grapes that inhibits the in vitro growth of multiple tumor cell types. We showed previously that resveratrol induces autophagic cell death in ovarian cancer cells. Because autophagy is typically an adaptive response to nutrient starvation, we hypothesized that autophagy would also be triggered when ovarian cancer cells are nutrient deprived and that resveratrol could in fact be acting by inducing a starvation-like signaling response., Methods: Ovarian cancer cells were incubated with normal media, media containing resveratrol, glucose free media, or media lacking amino acids. Growth inhibition was determined using the sulforhodamine assay. Cells were evaluated for autophagocytosis by analyzing cleavage of LC3. Glucose uptake, lactate production, and activation of glycolytic regulators pAkt and pmTOR were analyzed following resveratrol treatment., Results: We show here that epithelial ovarian cancer cells are highly sensitive to glucose-deprivation-induced cell death and like resveratrol, glucose deprivation induces caspase-independent cell death with hallmarks of autophagy. Consistent with the hypothesis that resveratrol treatment results in biochemical conditions that mirror a nutrient deprived state, we found that resveratrol dramatically reduces glucose uptake and lactate production. Moreover, resveratrol reduces the levels of phosphorylated Akt and mTOR, two signals that increase glucose uptake and the rate limiting steps in glycolysis., Conclusions: Our findings are consistent with the hypothesis that resveratrol-induced changes in glucose utilization comprise the mechanism that underlies resveratrol-induced autophagocytosis in ovarian cancer. Inhibition of glycolysis in ovarian cancer with resveratrol or other compounds may be effective therapy for ovarian cancer.

The cytotoxic mechanism of the histone deacetylase inhibitor (HDACI) Trichostatin A (TSA) was explored in a neuroblastoma (NB) model. TSA induces cell death in neuroblastic-type NB cells by increasing the acetylation of Ku70, a Bax-binding protein. Ku70 acetylation causes Bax release and activation, triggering cell death. This response to TSA depends on the CREB-binding protein (CBP) acetylating Ku70. TSA-induced cell death response correlates with CBP expression. In stromaltype NB cell lines with low levels of CBP and relative resistance to TSA, increasing CBP expression disrupts Bax-Ku70 binding and sensitizes them to TSA. Reducing CBP expression in neuroblastic cell types causes resistance. Cytotoxic response to TSA is Bax-dependent. Interestingly, depleting NB cells of Ku70 also triggers Bax-dependent cell death, suggesting that conditions that leave Bax unbound to Ku70 result in cell death. We also show that CBP, Ku70, and Bax are expressed in human NB tumors and that CBP expression varies across cell types comprising these tumors, with the highest expression observed in neuroblastic elements. Together, these results demonstrate that CBP, Bax, and Ku70 contribute to a therapeutic response to TSA against NB and identify the possibility of using these proteins to predict clinical responsiveness to HDACI treatment.

Although PK11195 binds to the peripheral benzodiazepine receptor with nanomolar affinity, significant data exist which suggest that it has another cellular target distinct from the PBR. Here we demonstrate that PK11195 inhibits F(1)F(0)-ATPase activity in an OSCP-dependent manner, similar to the pro-apoptotic benzodiazepine Bz-423. Importantly, our data indicate that cellular responses observed with micromolar concentrations of PK11195, which are commonly attributed to modulation of the PBR, are likely a direct result of mitochondrial F(1)F(0)-ATPase inhibition.

Neuroblastic (N) type neuroblastoma (NB) is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor kappaB (NF-kappaB) is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-kappaB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-kappaB (I-kappaB) alpha and the NF-kappaB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-kappaB activation because cells with inactive p53 were resistant to NF-kappaB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK) 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-kappaB kinase activation. Together, these results demonstrate for the first time how NF-kappaB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents.

Neuroblastic (N) type neuroblastoma (NB) is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor kappaB (NF-kappaB) is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-kappaB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-kappaB (I-KB) alpha and the NF-kappaB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-kappaB activation because cells with inactive p53 were resistant to NF-kappaB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK) 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-kappaB kinase activation. Together, these results demonstrate for the first time how NF-kappaB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents.

A family of 1,4-benzodiazepine-2,5-diones (BZDs) has been synthesized and evaluated against transformed B- and T-cells for lymphotoxic members. A large aromatic group on the C3 position is critical for cytotoxicity. When the C3 moiety contains an electron-rich heterocycle, the resulting BZDs have sub-micromolar potency and are selective for T-cells. Cell death is consistent with apoptosis and does not result from inhibition of the mitochondrial F(o)F1-ATPase, which is the molecular target of recently reported cytotoxic 1,4-benzodiazepines. Collectively, these studies begin to characterize some of the structural elements required for the activity of a novel family of T-cell-selective lymphotoxic agents.

Myc proteins regulate cell growth and are oncogenic in many cancers. Although these proteins are validated molecular anticancer targets, new therapies aimed at modulating myc have yet to emerge. A benzodiazepine (Bz-423) that was discovered in efforts to find new drugs for lupus was found recently to have antiproliferative effects on Burkitt's lymphoma cells. We now show that the basis for the antiproliferative effects of Bz-423 is the rapid and specific depletion of c-myc protein, which is coupled to growth-suppressing effects on key regulators of proliferation and cell cycle progression. c-Myc is depleted as a result of signals coupled to Bz-423 binding its molecular target, the oligomycin sensitivity-conferring protein subunit of the mitochondrial F(1)F(o)-ATPase. Bz-423 inhibits F(1)F(o)-ATPase activity, blocking respiratory chain function and generating superoxide, which at growth-inhibiting concentrations triggers proteasomal degradation of c-myc. Bz-423-induced c-myc degradation is independent of glycogen synthase kinase but is substantially blocked by mutation of the phosphosensitive residue threonine 58, which when phosphorylated targets c-myc for ubiquitination and subsequent proteasomal degradation. Collectively, this work describes a new lead compound, with drug-like properties, which regulates c-myc by a novel molecular mechanism that may be therapeutically useful.

Histone deacetylase inhibitors constitute a promising new treatment for cancer due to their novel site of action and low toxicity. Almost all histone deacetylase inhibitors currently in clinical development have anti-proliferate activities against cells in cultures, and specifically cause cell cycle arrest, differentiation and apoptosis. Interestingly, despite their rapid advance into clinical use, the cellular responses leading to these effects remain unclear. We recently reported that histone deacetylase inhibitor treatment induces apoptosis of neuroblastoma cells by increasing the acetylation of Ku70 in the cytoplasm, resulting in the release of Bax from Ku70. Subsequently, Bax releases cytochrome c from mitochondria causing apoptosis. Here we will discuss these findings and the implications of our model for the further clinical development of histone deacetylase inhibitors in the treatment of cancer.

Melanoma is the most aggressive form of skin cancer and advanced stages are invariably resistant to conventional therapeutic agents. Using bortezomib as a prototypic proteasome inhibitor, we have identified a novel and critical role of the proteasome in the maintenance of the malignant phenotype of melanoma cells that could have direct translational implications. Thus, melanoma cells from early, intermediate, and late stages of the disease could not sustain proteasome inhibition and underwent an effective activation of caspase-dependent and -independent death programs. This effect was tumor cell selective, because under similar conditions, normal melanocytes remained viable. Intriguingly, and despite of interfering with a cellular machinery in charge of controlling the half-life of the vast majority of cellular proteins, bortezomib did not promote a generalized disruption of melanoma-associated survival factors (including NF-kappaB, Bcl-2, Bcl-x(L), XIAP, TRAF-2, or FLIP). Instead, we identified a dramatic induction in vitro and in vivo of the BH3-only protein Noxa in melanoma cells (but not in normal melanocytes) in response to proteasome inhibition. RNA interference validated a critical role of Noxa for the cytotoxic effect of bortezomib. Notably, the proteasome-dependent regulation of Noxa was found to extend to other tumor types, and it could not be recapitulated by standard chemotherapeutic drugs. In summary, our results revealed Noxa as a new biomarker to gauge the efficacy of bortezomib specifically in tumor cells, and provide a new strategy to overcome tumor chemoresistance.

We assayed for the presence of human papilloma virus (HPV) DNA in serum and/or peripheral blood fraction (PBF) of individuals with cervical, head/neck, or bladder cancer due to schistosomiasis. Using mass spectroscopy coupled with competitive PCR, HPV DNA was detected at the individual molecule level by using "MassARRAY" assays. The resultant sensitivity was superior to real-time fluorescent PCR-based assays, while specificity was maintained. Our principal findings were: (i) Virtually all tested cervical cancers and schistosomiasis-associated bladder cancers, and a plurality of head/neck cancers, are associated with HPV DNA in the tumor. (ii) All 27 bladder cancers due to schistosomiasis were associated with the presence of HPV-16 DNA, which can be detected in tumor and serum but not in PBF. In contrast, no serum HPV-16 DNA signal was detected in seven individuals with schistosomiasis-associated bladder cancers after surgical removal of the tumor. (iii) Among the head/neck cancers we studied, anterior tumors were more often associated with HPV DNA in tumor, serum, and/or PBF than posterior tumors. (iv) In cervical cancer, where all tumors contain HPV DNA, viral DNA could be detected often in serum and/or PBF. Further, HPV-16 DNA was detected in serum and/or PBF of most patients with untreated high-grade cervical dysplasia but disappeared if the dysplasia was eliminated. The sensitive, specific, and quantitative MassARRAY technique should make it feasible to monitor cancer occurrence and treatment and recurrence of malignancies and dysplasias associated with HPV DNA.

Bz-423 is a new benzodiazepine that has cytotoxic and cytostatic effects against a number of cell types in culture, and recent studies have shown efficacy in experimental lupus models in rodents. The present study demonstrates that treating human skin in organ culture with Bz-423 suppresses retinoid-induced epidermal hyperplasia. Bz-423 suppresses hyperplasia in organ culture at concentrations that also inhibit keratinocyte proliferation in monolayer culture but that are not cytotoxic for keratinocytes and do not inhibit fibroblast growth. Under conditions in which keratinocyte proliferation is inhibited, there is no measurable effect on epidermal growth factor receptor activation, but there is reduced signaling at the level of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase. Suppression of keratinocyte growth by Bz-423 is associated with generation of intracellular oxidants. However, antioxidant treatment reduces keratinocyte cytotoxicity that occurs at high concentrations of Bz-423, but it does not inhibit growth suppression. Together, these data suggest that Bz-423 has the potential to limit the untoward effects associated with topical retinoid treatment, and in addition, may have therapeutic effects against other forms of epidermal hyperplasia.

Bz-423 is a 1,4-benzodiazepine that suppresses disease in lupus-prone mice by selectively killing pathogenic lymphocytes, and it is less toxic compared to current lupus drugs. Cells exposed to Bz-423 rapidly generate O(2)(-) within mitochondria, and this reactive oxygen species is the signal initiating apoptosis. Phage display screening revealed that Bz-423 binds to the oligomycin sensitivity conferring protein (OSCP) component of the mitochondrial F(1)F(0)-ATPase. Bz-423 inhibited the F(1)F(0)-ATPase in vitro, and reconstitution experiments demonstrated that inhibition was mediated by the OSCP. This target was further validated by generating cells with reduced OSCP expression using RNA interference and studying the sensitivity of these cells to Bz-423. Our findings help explain the efficacy and selectivity of Bz-423 for autoimmune lymphocytes and highlight the OSCP as a target to guide the development of novel lupus therapeutics.

Histone deacetylase inhibitors (HDACIs) are therapeutic drugs that inhibit deacetylase activity, thereby increasing acetylation of many proteins, including histones. HDACIs have antineoplastic effects in preclinical and clinical trials and are being considered for cancers with unmet therapeutic need, including neuroblastoma (NB). Uncertainty of how HDACI-induced protein acetylation leads to cell death, however, makes it difficult to determine which tumors are likely to be responsive to these agents. Here, we show that NB cells are sensitive to HDACIs, and that the mechanism by which HDACIs induce apoptosis involves Bax. In these cells, Bax associates with cytoplasmic Ku70, a protein that typically increases chemotherapy resistance. Our data show that in NB cells Ku70 binds to Bax in an acetylation-sensitive manner. Upon HDACI treatment, acetylated Ku70 releases Bax, allowing it to translocate to mitochondria and trigger cytochrome c release, leading to caspase-dependent death. This study shows that Ku70 is an important Bax-binding protein, and that this interaction can be therapeutically regulated in NB cells. Whereas the Bax-binding ability of Ku70 allows it to block apoptosis in response to certain agents, it is also a molecular target for the action of HDACIs, and in this context, a mediator of NB cell death.

Bz-423 is a 1,4-benzodiazepine with selective lymphotoxic properties and potent therapeutic activity against lupus-like disease in autoimmune mice. In NZB/W lupus-prone mice, Bz-423 specifically kills germinal center B cells, which are the cells that drive disease both in this model and in human systemic lupus erythematosus. In this report, the mechanistic basis for the selective action of Bz-423 is investigated. We show that Bz-423-induces superoxide as an immediate early response and that this reactive oxygen species is more effective as a second messenger death signal in B cells activated by B cell receptor stimulation compared with resting cells. As a result, low [Bz-423] that are not cytotoxic to non-stimulated cells kill stimulated cells in synergy with anti-immunoglobulin M antibodies. Subsequent experiments demonstrated that Bz-423 extends the rise in intracellular calcium that accompanies anti-immunoglobulin M stimulation, and this effect mediates the synergistic death response. Because B cell hyperactivation and altered calcium signaling is a distinguishing feature of autoreactive lymphocytes in lupus, the mechanism by which Bz-423 induces apoptosis preferentially targets disease-causing cells on the basis of their activation state. Thus, molecules like Bz-423 could form the basis for new and selective anti-lupus agents.

Stromal or S-type tumor cells are a distinct lineage found in neuroblastoma tumors and have an important role in the biology of this disease. Anticancer agents induce apoptosis through death receptor- and mitochondria-initiated pathways. The object of this work was to determine the involvement of these pathways in the response to doxorubicin (Dox) and cisplatin (CDDP) in S-type neuroblastoma cells. Both drugs activated caspase-9 and caspase-3 but not caspase-8. Caspase-9-specific inhibition blocked S-type cell death induced by Dox. SH-EP1 cells transfected to express dominant negative mutant caspase-9, but not those expressing DN caspase-8, were resistant to Dox- and CDDP-induced apoptosis. The lack of caspase-8 involvement in chemotherapy-induced death was not the result of an intrinsic inability of these cells to activate this enzyme because when they were treated with tumor necrosis factor-related apoptosis-inducing ligand, caspase-8 was activated. We also found that both drugs up-regulated CD95/Fas expression but that CD95/Fas signaling was not necessary for cell killing. Experiments testing the response of chemotherapy-treated cells to agonists of the CD95/Fas receptor established that Dox and CDDP treatment sensitizes cells to CD95/Fas killing. Together, these results are consistent with a model in which caspase-9 is of central importance in the death mechanism utilized by these drugs in S-type cells. Although the death response is not dependent on CD95/Fas, concomitant stimulation of this receptor amplifies the death response in drug-treated cells.

Resveratrol (3,5,4-trihydroxystilbene), a natural phytoalexin present in grapes, nuts, and red wine, has antineoplastic activities. Several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo. In the present study, the response of ovarian cancer cells to resveratrol is explored. Resveratrol inhibited growth and induced death in a panel of five human ovarian carcinoma cell lines. The response was associated with mitochondrial release of cytochrome c, formation of the apoptosome complex, and caspase activation. Surprisingly, even with these molecular features of apoptosis, analysis of resveratrol-treated cells by light and electron microscopy revealed morphology and ultrastructural changes indicative of autophagocytic, rather than apoptotic, death. This suggests that resveratrol can induce cell death through two distinct pathways. Consistent with resveratrol's ability to kill cells via nonapoptotic processes, cells transfected to express high levels of the antiapoptotic proteins Bcl-x(L) and Bcl-2 are equally sensitive as control cells to resveratrol. Together, these findings show that resveratrol induces cell death in ovarian cancer cells through a mechanism distinct from apoptosis, therefore suggesting that it may provide leverage to treat ovarian cancer that is chemoresistant on the basis of ineffective apoptosis.

Bz-423 is a novel proapoptotic 1,4-benzodiazepine that induces cell death via a superoxide signal. Previous work has shown that Bz-423 ameliorates disease in animal models of systemic lupus erythematosus that also have features of lymphoproliferative disease. Here we describe the effects of Bz-423 against a group of malignant B-cell lines derived from Burkitt's lymphoma. These experiments demonstrate that Bz-423 has cytotoxic activity against all B-cell lines tested, regardless of EBV status or Bcl-2 and Bcl-x(L) expression levels. In addition to its cytotoxic properties, we found that Bz-423 is also a potent antiproliferative agent that induces a G(1)-phase arrest independent of p53. Mechanistically, both the cytotoxicity and growth arrest are mediated by increased reactive oxygen species levels and appear independent of binding to the peripheral benzodiazepine receptor. This work further defines the biological activities of Bz-423 that are consistent with those of other compounds in clinical development for antineoplastic therapies.