Valproic acid in combination with all-trans retinoic acid and intensive therapy for acute myeloid leukemia in older patients.

The outcome of patients with acute myeloid leukemia who are older than 60 years has remained poor because of unfavorable disease characteristics and patient-related factors. The randomized German-Austrian AML Study Group 06-04 protocol was designed on the basis of in vitro synergistic effects of valproic acid (VPA) and all-trans retinoic acid with chemotherapy. Between 2004 and 2006, 186 patients were randomly assigned to receive 2 induction cycles with idarubicin, cytarabine, and all-trans retinoic acid either with VPA or without (STANDARD). In all patients, consolidation therapy was intended. Complete remission rates after induction tended to be lower in VPA compared with STANDARD (40% vs 52%; P = .14) as a result of a higher early death rate (26% vs 14%; P = .06). The main toxicities attributed to VPA were delayed hematologic recovery and grade 3/4 infections, observed predominantly during the second induction cycle. After restricting VPA to the first induction cycle and reducing the dose of idarubicin, these toxicities dropped to rates observed in STANDARD. After a median follow-up time of 84 months, event-free and overall survival were not different between the 2 groups (P = .95 and P = .57, respectively). However, relapse-free-survival was significantly superior in VPA compared with STANDARD (24.4% vs 6.4% at 5 years; P = .02). Explorative subset analyses revealed that AML with mutated Nucleophosmin 1 (NPM1) may particularly benefit from VPA. This trial was registered at www.clinicaltrials.gov as #NCT00151255.

Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis.

Inappropriate transcriptional repression involving histone deacetylases (HDACs) is a prominent cause for the development of leukemia. We now identify faulty expression of a specific mediator of transcriptional repression in a solid tumor. Loss of the adenomatosis polyposis coli (APC) tumor suppressor induces HDAC2 expression depending on the Wnt pathway and c-Myc. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of APC-deficient mice. HDAC2 is required for, and sufficient on its own to prevent, apoptosis of colonic cancer cells. Interference with HDAC2 by valproic acid largely diminishes adenoma formation in APC(min) mice. These findings point toward HDAC2 as a particularly relevant potential target in cancer therapy.

TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor­associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Histone deacetylase (HDAC) 1 and 2 expression and chemotherapy in gastric cancer.

BACKGROUND: Histone deacetylases (HDACs) modulate chromatin and may influence the effect of DNA-damaging drugs. We investigated HDAC1 and -2 expression in gastric carcinomas (GCs) for an association of patient outcome with conventional neoadjuvant chemotherapy. In vitro, HDAC inhibitors were evaluated as alternative treatment options. METHODS: HDAC1/2 expression was analyzed immunohistochemically in 127 pretherapeutic biopsy samples of neoadjuvant (platinum/5-fluorouracil) chemotherapy-treated GC patients and correlated with response and overall survival (OS). Chemosensitivity of four GC cell lines to cisplatin and the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and valproic acid was determined by XTT assays. Efficiencies of combined drug schedules were analyzed. RESULTS: High expression of HDAC1/2 was found in 69 (54%) of 127 and 108 (85%) of 127 carcinomas, respectively, and was not associated with response or OS. In patients whose disease responded to therapy, high HDAC1 expression was associated with worse OS (P = 0.005). In cell lines, sequential treatment with SAHA and cisplatin showed synergistic effects irrespective of the initial cisplatin sensitivity. CONCLUSIONS: HDAC1 and -2 expression is not suitable to predict response or survival for neoadjuvant-treated GC patients, but HDAC1 expression may be used for risk stratification in patients whose disease responds to therapy. Sequential treatment with SAHA and cisplatin may represent an alternative treatment option for GC patients.

New Frontiers in Drug Discovery: Academia Meets Industry at the International Helmholtz Drug Discovery Conference (HDDC).

The Helmholtz Drug Discovery conference HDDC 2018 was held on April 26-27, 2018 at the Helmholtz Zentrum in Munich, Germany. The meeting covered a wide range of topics. Keynote lectures were presented by 17 internationally renowned speakers from Europe and the US, and an additional 11 talks were selected from 70 submitted abstracts. With over 220 participants, there was overwhelming response and interest by researchers from both academia and industry.

IGFBP-2 overexpression reduces the appearance of dysplastic aberrant crypt foci and inhibits growth of adenomas in chemically induced colorectal carcinogenesis.

Colon cancer patients frequently show increased levels of serum insulin-like growth factor-binding protein-2 (IGFBP-2), however, the pathogenetic relevance of this phenomenon for colorectal cancer is unclear. Therefore, we have used IGFBP-2 transgenic animals which overexpress IGFBP-2 systemically and locally in the intestine to study its role in chemically induced colorectal carcinogenesis. Mice received intraperitoneal injections of 1,2-dimethylhydrazine (DMH) (40 mg/kg body weight) once a week for 6 weeks to selectively induce aberrant crypt foci (ACF) and tumors in the colon. While tumor incidence was comparable in transgenic and control mice, the volume of adenomas in IGFBP-2 transgenic mice was reduced more than 2-fold. Furthermore, serum IGFBP-2 levels negatively correlated with tumor volume in the IGFBP-2 transgenic group. Histological examination showed that IGFBP-2 transgenic mice developed significantly less dysplastic ACF with a high potential to progress to advanced stages. The reduced tumor volume in IGFBP-2 transgenic animals was due to significantly reduced proliferative capacity, evidenced by a lower proportion of cells positive for Ki67. Our results demonstrate for the first time in an experimental model that IGFBP-2 overabundance prior to the onset and during colorectal carcinogenesis reduces tumor growth by inhibition of cell proliferation.

Reduced body size and decreased intestinal tumor rates in HDAC2-mutant mice.

Histone deacetylases (HDAC) reverse the acetylation of histone and nonhistone proteins and thereby modulate chromatin structure and function of nonhistone proteins. Many tumor cell lines and experimental tumors respond to HDAC inhibition. To assess the role of an individual HDAC isoenzyme in physiology and tumor development, HDAC2-mutant mice were generated from a gene trap embryonic stem cell clone. These mice express a catalytically inactive fusion protein of the NH(2)-terminal part of HDAC2 and beta-galactosidase, which fails to integrate into corepressor complexes with mSin3B. They are the first class 1 HDAC mutant mice that are viable although they are approximately 25% smaller than their littermates. Cell number and thickness of intestinal mucosa are reduced. Mutant embryonic fibroblasts fail to respond to insulin-like growth factor I (IGF) by the IGF-I-induced increase in cell number observed in wild-type cells. These data suggest a novel link between HDACs and IGF-I-dependent responses. Crossing of HDAC2-mutant with tumor-prone APC(min) mice revealed tumor rates that are lower in HDAC2-deficient mice by 10% to 100% depending on segment of the gut and sex of the mice. These mice provide evidence that the key functions of HDAC2, although not essential for survival of the organism, play a rate-limiting role for tumor development in vivo.

Electrophoretic analysis of the mitochondrial outer membrane rupture induced by permeability transition.

A pathological increase of the permeability of the mitochondrial membranes may culminate in the irreversible rupture of the mitochondrial outer membrane. Such a permeability transition is lethal because it results in the release of death-inducing molecules from mitochondria and/or metabolic failure. Current methods to assess this outer membrane damage are mostly indirect or scarcely representative of the overall mitochondrial population. Here we present an analytical and preparative approach using free flow electrophoresis to directly distinguish rat liver mitochondria that have undergone the permeability transition from unaffected organelles or from organelles that are damaged to a minor degree. Mitochondrial populations, which considerably differ in outer membrane integrity or cytochrome c content, were separated by this means. We further show that the relative abundance of each population depends on the dose of the permeability transition inducer and the duration of the treatment time. Finally, we have employed this approach to investigate the impairment of mitochondria that were isolated from livers subjected to ischemia/reperfusion damage.

Isolation of highly pure rat liver mitochondria with the aid of zone-electrophoresis in a free flow device (ZE-FFE).

This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium speed centrifugation and is further purified by washing followed by a Nycodenz gradient centrifugation. Lysosomes and microsomes are located at the upper parts of the gradient, whereas mitochondria are found in the medium part of the gradient. A subsequent purification step with ZE-FFE efficiently removes remaining lysosomes and microsomes and, importantly, damaged mitochondrial structures. The resulting purified mitochondria can be concentrated by centrifugation and used for further experiments. Finally, possible modifications of this protocol with respect to the isolation of pure lysosomes are discussed.

Flavonoids alter P-gp expression in intestinal epithelial cells in vitro and in vivo.

Flavonoids are secondary plant metabolites included in our diet but are also provided in a growing number of supplements. They are suggested to interact with intestinal transport systems including phospho-glycoprotein (P-gp) which mediates the efflux of a variety of xenobiotics back into the gut lumen. In human intestinal Caco-2 cells, we tested the effects of 14 different flavonoids on P-gp expression in vitro. Protein expression levels were quantified by Western blotting, flow cytometry, and real-time PCR. Except apigenin, all flavonoids at concentrations of 10 microM increased P-gp expression in Western blotting experiments when cells were exposed to the compounds over 4 wk. Flavone was one of the most effective P-gp inducers in Caco-2 cells and its effects were, therefore, also assessed for changes in P-gp in vivo in the gastrointestinal tract of C57BL/6 mice. P-gp expression was significantly increased by flavone (400 mg/kg body weight x day over 4 wk) in the small intestine but not in the colon which displayed intrinsically the highest expression level. In conclusion, the increase in P-gp expression caused by flavonoids in intestinal epithelial cells in vitro and also in vivo may serve as an adaptation and defense mechanism limiting the entry of lipophilic xenobiotics into the organism.

TCDD induces c-jun expression via a novel Ah (dioxin) receptor-mediated p38-MAPK-dependent pathway.

The aryl hydrocarbon receptor (AhR) has a fundamental role during postnatal liver development and is essential for mediating dioxin toxicity. However, the genetic programs mediating, both, the toxic and physiological effects downstream of the transcription factor AhR are in major parts unknown. We have identified the proto-oncogene c-jun as a novel target gene of AhR. Induction of c-jun depends on activation of p38-mitogen-activated protein kinase (MAPK) by an AhR-dependent mechanism. None of the kinases that are known to phosphorylate p38-MAPK is activated by AhR. Neither the dephosphorylation rate of p38-MAPK is reduced. Furthermore, increased p38-MAPK phosphorylation in response to dioxins does not require ongoing transcription. These findings establish activating 'cross-talk' with MAPK signaling as a novel principle of AhR action, which is apparently independent of the AhR's function as a DNA-binding transcriptional activator.

Langendorff heart: a model system to study cardiovascular effects of engineered nanoparticles.

Engineered nanoparticles (ENPs) are produced and used in increasing quantities for industrial products, food, and drugs. The fate of ENPs after usage and impact on health is less known. Especially as air pollution, suspended nanoparticles have raised some attention, causing diseases of the lung and cardiovascular system. Human health risks may arise from inhalation of ENPs with associated inflammation, dispersion in the body, and exposure of vulnerable organs (e.g., heart, brain) and tissues with associated toxicity. However, underlying mechanisms are largely unknown. Furthermore future use of ENPs in therapeutic applications is being researched. Therefore knowledge about potential cardiovascular risks due to exposure to ENPs is highly demanded, but there are no established biological testing models yet. Therefore, we established the isolated beating heart (Langendorff heart) as a model system to study cardiovascular effects of ENPs. This model enables observation and analysis of electrophysiological parameters over a minimal time period of 4 h without influence by systemic effects and allows the determination of stimulated release of substances under influence of ENPs. We found a significant dose and material dependent increase in heart rate accompanied by arrhythmia evoked by ENPs made of flame soot (Printex 90), spark discharge generated soot, anatas (TiO(2)), and silicon dioxide (SiO(2)). However, flame derived SiO(2) (Aerosil) and monodisperse polystyrene lattices exhibited no effects. The increase in heart rate is assigned to catecholamine release from adrenergic nerve endings within the heart. We propose the isolated Langendorff heart and its electrophysiological characterization as a suitable test model for studying cardiovascular ENP toxicity.

Differential impact of diesel particle composition on pro-allergic dendritic cell function.

Diesel exhaust particles (DEP) were described as potent adjuvant in the induction and maintenance of allergic diseases, suggesting that they might play a role in the increase of allergic diseases in the industrialized countries. However, the cellular basis by which these particles enhance allergic immune responses is still a matter of debate. Thus, we exposed immature murine bone marrow-derived dendritic cells (BMDC) to different particles or particle-associated organic compounds in the absence or presence of the maturation stimuli lipopolysaccharide (LPS) and analyzed the cellular maturation, viability, and cytokine production. Furthermore, we monitored the functionality of particle-exposed BMDC to suppress B cell isotype switching to immunoglobulin (Ig) E. Only highly polluted DEP (standard reference material 1650a [SRM1650a]) but not particle-associated organic compounds or less polluted DEP from modern diesel engines were able to modulate the dendritic cell phenotype. SRM1650a particles significantly suppressed LPS-induced IL-12p70 production in murine BMDC, whereas cell-surface marker expression was not altered. Furthermore, SRM1650a-exposed immature BMDC lost the ability to suppress IgE isotype switch in B cells. This study revealed that highly polluted DEP not only interfere with dendritic cell maturation but also additionally with dendritic cell function, thus suggesting a role in T(h)2 immune deviation.

The specific role of histone deacetylase 2 in adult neurogenesis.

Gene expression changes during cell differentiation are thought to be coordinated by histone modifications, but still little is known about the role of specific histone deacetylases (HDACs) in cell fate decisions in vivo. Here we demonstrate that the catalytic function of HDAC2 is required in adult, but not embryonic neurogenesis. While brain development and adult stem cell fate were normal upon conditional deletion of HDAC2 or in mice lacking the catalytic activity of HDAC2, neurons derived from both zones of adult neurogenesis die at a specific maturation stage. This phenotype is correlated with an increase in proliferation and the aberrant maintenance of proteins normally expressed only in progenitors, such as Sox2, also into some differentiating neurons, suggesting that HDAC2 is critically required to silence progenitor transcripts during neuronal differentiation of adult generated neurons. This cell-autonomous function of HDAC2 exclusively in adult neurogenesis reveals clear differences in the molecular mechanisms regulating neurogenesis during development and in adulthood.

Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357.

BACKGROUND/AIMS: Due to a particular resistance against conventional chemotherapeutics, palliative treatment of hepatocellular carcinomas (HCC) is highly ineffective. Recent demonstration of both proliferation-inhibition and apoptosis of hepatoma cells by a histone deacetylase inhibitor (HDAC-I) treatment opens up a promising new approach. However, little is known about tumor cell death mechanisms and HDAC-I influences on healthy hepatocytes. METHODS: HDAC-I substances with favourable in vivo profiles, valproate (VPA) and ITF2357, were investigated on HCC cell lines and primary human hepatocytes (PHH). Histone acetylation and apoptosis-modulating proteins were investigated by western-blotting, proliferation by sulforhodamin B binding, toxicity by enzyme release, apoptosis by FACS analysis. RESULTS: VPA and ITF2357 inhibited proliferation in HCC cell lines. Both substances induced considerable cellular damage in HCC-derived cells, but PHH tolerated these substances well. A downregulation of anti- and upregulation of proapoptotic factors was found. Moreover, Bcl-X(L) transfection into HCC cells abrogated apoptosis induced by both substances, indicating that modulation of intracellular pro- and anti-apoptotic proteins is a key event in VPA or ITF2357 induced tumor-cell death. CONCLUSIONS: Preferential induction of cell death in HCC-derived cell lines, without toxicity in PHH, demonstrates the potential of VPA and ITF2357 to become promising new tools in the fight against HCC.

Specific and redundant functions of histone deacetylases in regulation of cell cycle and apoptosis.

Inappropriate control of expression of genetic information is the cause of many forms of cancer. Aberrant transcriptional repression by recruitment of histone deacetylases (HDACs) is a key step in pathogenesis of myeloid leukemia. We recently reported that development of colonic cancer involves alterations in the transcriptional repression machinery by increased expression of HDAC2 upon loss of the APC tumor suppressor. Increased expression of HDAC2 is essential for prevention of apoptosis of HT-29 colonic cancer cells. We now discuss whether HDAC2 also plays a role for aberrant cell cycle regulation and expression of the p21(Cip/Waf) cell cycle inhibitor. Whereas inhibition of HDACs by valproic acid or trichostatin A increases p21 expression, selective interference with HDAC2 by siRNA transfection or reconstitution of wildtype APC does not affect p21 expression. Likewise, treatment of HT-29 cells with the HDAC inhibitor valproic acid leads to a moderate inhibition of cell cycle progression in the G1 phase whereas interference with HDAC2 expression does not. Thus, HDAC2 appears to serve a preferential role in the prevention of apoptosis and not in cell cycle control similar to the specific importance of HDAC1 for cell cycle regulation or HDAC 9 for the stress response of the heart.

A nuclear isoform of the focal adhesion LIM-domain protein Trip6 integrates activating and repressing signals at AP-1- and NF-kappaB-regulated promoters.

Glucocorticoid receptor (GR)-mediated transrepression of the transcription factors AP-1 and NF-kappaB, responsible for most of the anti-inflammatory effects of glucocorticoids, is initiated by the tethering of GR to the promoters of target genes. We report that this tethering is mediated by a nuclear isoform of the focal adhesion LIM domain protein Trip6. Trip6 functions as a coactivator for both AP-1 and NF-kappaB. As shown by chromatin immunoprecipitation, Trip6 is recruited to the promoters of target genes together with AP-1 or NF-kappaB. In the presence of glucocorticoids, GR joins the Trip6 complex. Reducing the level of Trip6 by RNA interference or abolishing its interaction with GR by dominant-negative mutation eliminates transrepression. We propose that GR tethering to the target promoter through Trip6 forms the basis of transrepression, and that Trip6 exerts its nuclear functions by acting as a molecular platform, enabling target promoters to integrate activating or repressing signals.

UVA inactivates protein tyrosine phosphatases by calpain-mediated degradation.

UV irradiation causes inflammatory and proliferative cellular responses. We have proposed previously that these effects are, to a large extent, caused by the ligand-independent activation of several receptor tyrosine kinases due to the inactivation of their negative control elements, the protein tyrosine phosphatases (PTPs). We examined the mechanism of this inactivation and found that, in addition to reversible oxidation of PTPs, UV triggers a novel mechanism: induced degradation of PTPs by calpain, which requires both calpain activation and substrate PTP oxidative modification. This as yet unrecognized effect of UV is irreversible, occurs predominantly with UVA and UVB, the range of wavelengths in sunlight that reach the skin surface, and at physiologically relevant doses.

The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2.

Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as efficient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin-proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2.