Expression ratio of CCND1 to CDKN2A mRNA predicts RB1 status of cultured cancer cell lines and clinical tumor samples.

BACKGROUND: The retinoblastoma product (RB1) is frequently deregulated in various types of tumors by mutation, deletion, or inactivation through association with viral oncoproteins. The functional loss of RB1 is recognized to be one of the hallmarks that differentiate cancer cells from normal cells. Many researchers are attempting to develop anti-tumor agents that are preferentially effective against RB1-negative tumors. However, to identify patients with RB1-negative cancers, it is imperative to develop predictive biomarkers to classify RB1-positive and -negative tumors. RESULTS: Expression profiling of 30 cancer cell lines composed of 16 RB1-positive and 14 RB1-negative cancers was performed to find genes that are differentially expressed between the two groups, resulting in the identification of an RB1 signature with 194 genes. Among them, critical RB1 pathway components CDKN2A and CCND1 were included. We found that microarray data of the expression ratio of CCND1 and CDKN2A clearly distinguished the RB1 status of 30 cells lines. Measurement of the CCND1/CDKN2A mRNA expression ratio in additional cell lines by RT-PCR accurately predicted RB1 status (12/12 cells lines). The expression of CCND1/CDKN2A also correlated with RB1 status in xenograft tumors in vivo. Lastly, a CCND1/CDKN2A assay with clinical samples showed that uterine cervical and small cell lung cancers known to have a high prevalence of RB1-decifiency were predicted to be 100% RB1-negative, while uterine endometrial or gastric cancers were predicted to be 5-22% negative. All clinically normal tissues were 100% RB1-positive. CONCLUSIONS: We report here that the CCND1/CDKN2A mRNA expression ratio predicts the RB1 status of cell lines in vitro and xenograft tumors and clinical tumor samples in vivo. Given the high predictive accuracy and quantitative nature of the CCND1/CDKN2A expression assay, the assay could be utilized to stratify patients for anti-tumor agents with preferential effects on either RB1-positive or -negative tumors.

Potent anti-tumor activity of a macrocycle-quinoxalinone class pan-Cdk inhibitor in vitro and in vivo.

Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.

Structures of the PKC-iota kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533-551 in the C-terminal tail and their roles in ATP binding.

Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-theta, PKC-iota and PKC-betaII kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC-substrate complex has been determined. In the previously determined PKC-iota complex, residues 533-551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-iota kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0 A resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-iota are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-iota, which is different from the case of the prototypical AGC-branch kinase PKA.

Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements.

Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets. Due to the selective lethal effect on cancer cells harboring specific genetic alterations, it is expected that targeting synthetic lethal genes would provide wider therapeutic windows compared with cytotoxic chemotherapeutics. Here, we review the current status of the application of synthetic lethal screening in cancer research fields from biological and methodological viewpoints. Very recent studies seeking to identify synthetic lethal genes with K-RAS and p53, which are known to be the most frequently occurring oncogenes and TSGs, respectively, are introduced. Among the accumulating amount of research on synthetic lethal interactions, the synthetic lethality between BRCA1/2 and PARP1 inhibition has been clinically proven. Thus, both preclinical and clinical data showing a preferential anti-tumor effect on BRCA1/2 deficient tumors by a PARP1 inhibitor are the best examples of the synthetic lethal approach of cancer therapeutics. Finally, methodological progress regarding synthetic lethal screening, including barcode shRNA screening and in vivo synthetic lethal screening, is described. Given the fact that an increasing number of synthetic lethal genes for major cancerous genes have been validated in preclinical studies, this intriguing approach awaits clinical verification of preferential benefits for cancer patients with specific genetic alterations as a clear predictive factor for tumor response.

Induction and down-regulation of Sox17 and its possible roles during the course of gastrointestinal tumorigenesis.

BACKGROUND & AIMS: The activation of Wnt/beta-catenin signaling causes the development of gastric and colon cancers. Sox17 represses Wnt/beta-catenin signaling and is down-regulated in colon cancer. This study was designed to elucidate the role of Sox17 during the course of gastrointestinal tumorigenesis. METHODS: Sox17 expression was examined in gastrointestinal tumors of mouse models and humans. The roles of Sox17 in gastric tumorigenesis were examined by cell culture experiments and by construction of Sox17 transgenic mice. RESULTS: Sox17 was induced in K19-Wnt1/C2mE mouse gastric tumors and K19-Wnt1 preneoplastic lesions, where Wnt/beta-catenin signaling was activated. Consistently, Wnt activation induced Sox17 expression in gastric cancer cells. In contrast, Sox17 was rarely detected by immunohistochemistry in gastric and colon cancers, whereas strong nuclear staining of Sox17 was found in >70% of benign gastric and intestinal tumors. Treatment with a demethylating agent induced Sox17 expression in gastric cancer cells, thus indicating the down-regulation of Sox17 by methylation. Moreover, transfection of Sox17 in gastric cancer cells suppressed both the Wnt activity and colony formation efficiency. Finally, transgenic expression of Sox17 suppressed dysplastic tumor development in K19-Wnt1/C2mE mouse stomach. CONCLUSIONS: Sox17 plays a tumor suppressor role through suppression of Wnt signaling. However, Sox17 is induced by Wnt activation in the early stage of gastrointestinal tumorigenesis, and Sox17 is down-regulated by methylation during malignant progression. It is therefore conceivable that Sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis, and down-regulation of Sox17 contributes to malignant progression through promotion of Wnt activity.

Expression levels of NF-Y target genes changed by CDKN1B correlate with clinical prognosis in multiple cancers.

CDK inhibitors CDKN1B (p27) and CDKN2A (p16) inhibit cell cycle progression. A lower expression level of only p27 has been correlated with poorer prognosis in various types of clinical cancers. The difference may be the result of distinct genes downstream of these CDK inhibitors. Here, we report that NF-Y transcription factor-targeted genes specifically down-regulated by p27 correlate with poor prognosis in multiple tumor types. We performed mRNA expression profiling in HCT116 cells over-expressing either p16 or p27 and identified their regulatory genes. In silico transcription factor prediction indicated that most of the genes specifically down-regulated by p27 are controlled by NF-Y. Under the hypothesis that NF-Y-targeted genes are responsible for poor prognosis, we predicted prognosis in four types of cancer based on genes with the NF-Y motif, and found a significant association between the expression of NF-Y-targeted genes and poor prognosis.

Mouse gastric tumor models with prostaglandin E2 pathway activation show similar gene expression profiles to intestinal-type human gastric cancer.

BACKGROUND: Gastric cancers are generally classified into better differentiated intestinal-type tumor and poorly differentiated diffuse-type one according to Lauren's histological categorization. Although induction of prostaglandin E2 pathway promotes gastric tumors in mice in cooperation with deregulated Wnt or BMP signalings, it has remained unresolved whether the gastric tumor mouse models recapitulate either of human gastric cancer type. This study assessed the similarity in expression profiling between gastric tumors of transgenic mice and various tissues of human cancers to find best-fit human tumors for the transgenic mice models. RESULTS: Global expression profiling initially found gastric tumors from COX-2/mPGES-1 (C2mE)-related transgenic mice (K19-C2mE, K19-Wnt1/C2mE, and K19-Nog/C2mE) resembled gastric cancers among the several tissues of human cancers including colon, breast, lung and gastric tumors. Next, classification of the C2mE-related transgenic mice by a gene signature to distinguish human intestinal- and diffuse-type tumors showed C2mE-related transgenic mice were more similar to intestinal-type compared with diffuse one. We finally revealed that induction of Wnt pathway cooperating with the prostaglandin E2 pathway in mice (K19-Wnt1/C2mE mice) further reproduce features of human gastric intestinal-type tumors. CONCLUSION: We demonstrated that C2mE-related transgenic mice show significant similarity to intestinal-type gastric cancer when analyzed by global expression profiling. These results suggest that the C2mE-related transgenic mice, especially K19-Wnt1/C2mE mice, serve as a best-fit model to study molecular mechanism underlying the tumorigenesis of human gastric intestinal-type cancers.

Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines.

BACKGROUND: The Hedgehog (HH) pathway promotes tumorigenesis in a diversity of cancers. Activation of the HH signaling pathway is caused by overexpression of HH ligands or mutations in the components of the HH/GLI1 cascade, which lead to increased transactivation of GLI transcription factors. Although negative kinase regulators that antagonize the activity of GLI transcription factors have been reported, including GSK3beta, PKA and CK1s, little is known regarding positive kinase regulators that are suitable for use on cancer therapeutic targets. The present study attempted to identify kinases whose silencing inhibits HH/GLI signalling in non-small cell lung cancer (NSCLC). RESULTS: To find positive kinase regulators in the HH pathway, kinome-wide siRNA screening was performed in a NSCLC cell line, A549, harboring the GLI regulatory reporter gene. This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity. The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability. We next investigated the mechanism for p70S6K2-mediated inhibition of GLI1 transcription by hypothesizing that GSK3beta, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3beta (Ser9) was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein. Finally, to further confirm the involvement of p70S6K2 in GLI1 signaling, down-regulation in GLI-mediated transcription by PI3KCA-inhibition was confirmed, establishing the pivotal role of the PI3K/p70S6K2 pathway in GLI1 cascade regulation. CONCLUSION: We report herein that inhibition of p70S6K2, known as a downstream effector of the PI3K pathway, remarkably decreases GLI-mediated transactivation in NSCLC by reducing phosphorylated-GSK3beta followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.

Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor.

BACKGROUND: Wee1 is a tyrosine kinase regulating S-G2 cell cycle transition through the inactivating phosphorylation of CDC2. The inhibition of Wee1 kinase by a selective small molecule inhibitor significantly enhances the anti-tumor efficacy of DNA damaging agents, specifically in p53 negative tumors by abrogating S-G2 checkpoints, while normal cells with wild-type p53 are not severely damaged due to the intact function of the G1 checkpoint mediated by p53. Since the measurement of mRNA expression requires a very small amount of biopsy tissue and is highly quantitative, the development of a pharmacodynamic (PD) biomarker leveraging mRNA expression is eagerly anticipated in order to estimate target engagement of anti-cancer agents. RESULTS: In order to find the Wee1 inhibition signature, mRNA expression profiling was first performed in both p53 positive and negative cancer cell lines treated with gemcitabine and a Wee1 inhibitor, MK-1775. We next carried out mRNA expression profiling of skin samples derived from xenograft models treated with the Wee1 inhibitor to identify a Wee1 inhibitor-regulatory gene set. Then, the genes that were commonly modulated in both cancer cell lines and rat skin samples were extracted as a Wee1 inhibition signature that could potentially be used as a PD biomarker independent of p53 status. The expression of the Wee1 inhibition signature was found to be regulated in a dose-dependent manner by the Wee1 inhibitor, and was significantly correlated with the inhibition level of a direct substrate, phosphorylated-CDC2. Individual genes in this Wee1 inhibition signature are known to regulate S-G2 cell cycle progression or checkpoints, which is consistent with the mode-of-action of the Wee1 inhibitor. CONCLUSION: We report here the identification of an mRNA gene signature that was specifically changed by gemcitabine and Wee1 inhibitor combination treatment by molecular profiling. Given the common regulation of expression in both xenograft tumors and animal skin samples, the data suggest that the Wee1 inhibition gene signature might be utilized as a quantitative PD biomarker in both tumors and surrogate tissues, such as skin and hair follicles, in human clinical trials.

High-throughput assay for long chain fatty acyl-CoA elongase using homogeneous scintillation proximity format.

Elongase of very-long-chain fatty acid (Elovl) 6 is a rate-limiting enzyme that is responsible for the elongation of long-chain fatty acids such as palmitoic acid (C16). Elovl6 is abundantly expressed in liver and adipose tissue, and the expression levels in these tissues are up-regulated in obese animals. Furthermore, Elovl6-deficient mice display improved glucose homeostasis and insulin sensitivity, suggesting that Elovl6 might be a potential therapeutic target for metabolic disorders. From the drug discovery point of view, it is critical to establish a high-throughput screening (HTS) assay for the identification of therapeutic agents. Conventional assay methods for fatty acid elongases include an extraction step for respective radioactive products from the reaction mixtures, which is labor-intensive and not feasible for HTS. In this study, we utilized the acyl-coenzyme A (CoA) binding protein (ACBP) as a molecular probe to detect radioactive long-chain acyl-CoA, a direct product of Elovl6. Recombinant ACBP binds stearoyl-CoA but not malonyl-CoA, enabling specific detection of the radioactive product in the homogenous reaction mixture without the liquid extraction step. Finally, combination of ACBP and scintillation proximity assay beads led to specific detection of Elovl6 activity with appropriate window and reproducibility amenable to HTS (signal-to-background noise ratio of approximately 13.0-fold, Z' = 0.85). The assay system described here has the potential to enable identification of small compounds that modify fatty acid elongase activity and assessment of the therapeutic potential of acyl-CoA elongases.

Imidazopyridine derivatives as potent and selective Polo-like kinase (PLK) inhibitors.

A novel class of imidazopyridine derivatives was designed as PLK1 inhibitors. Extensive SAR studies supported by molecular modeling afforded a highly potent and selective compound 36. Compound 36 demonstrated good antitumor efficacy in xenograft nude rat model.

Integrated genomic and pharmacological approaches to identify synthetic lethal genes as cancer therapeutic targets.

Various types of cancers are generated through mutations or dysregulations of oncogenes/tumor suppressor genes involved in cell cycles and signaling transduction pathways. To identify cancer therapeutic targets whose inhibition selectively kills cancer cells, synthetic lethal screening is being developed to identify genes whose intervention suppresses tumor progression only when combined with the dysregulation of the genes. The recent emergence of genomic technologies, including microarray, RNA interference and chemogenomics, provides platforms to realize this concept. This review introduces the research that could successfully identify synthetic lethal genes in cancer cells harboring major gene alterations such as p53, RB, K-Ras, or Myc. We also illustrate remarkable candidate targets that were identified by synthetic lethal screening to find chemosensitizers for paclitaxel and cisplatin. Next, we introduce the chemogenomics approaches that explore chemical compounds that exhibit synthetic lethality to cancer gene alterations. Although the synthetic lethal compounds are of great interest in terms of cancer drug development, a method of identifying target proteins for the phenotypic compounds has been elusive. Finally, we demonstrate several noteworthy techniques to identify target proteins for the compounds: a Connectivity Map that compares expression profiles of compound-treated cells by pattern-matching algorithms; an siRNA/compound co-treatment assay to find enhancer genes for the phenotypes of compounds; and a state-of-the-art proteomics approach that modifies classical compound-immobilized affinity chromatography. The integration of genomic and pharmacological analyses would significantly accelerate the identification of cancer-specific synthetic lethal targets.

Role of the Toll-like receptor 4/NF-kappaB pathway in saturated fatty acid-induced inflammatory changes in the interaction between adipocytes and macrophages.

OBJECTIVE: Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-alpha derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. METHODS AND RESULTS: Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-kappaB (NF-kappaB), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-kappaB markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFalpha production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the beta3-adrenergic stimulation, resulted in the activation of the TLR4/NF-kappaB pathway. CONCLUSIONS: These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-kappaB activation.

Can systems biology understand pathway activation? Gene expression signatures as surrogate markers for understanding the complexity of pathway activation.

Cancer is thought to be caused by a sequence of multiple genetic and epigenetic alterations which occur in one or more of the genes controlling cell cycle progression and signaling transduction. The complexity of carcinogenic mechanisms leads to heterogeneity in molecular phenotype, pathology, and prognosis of cancers.Genome-wide mutational analysis of cancer genes in individual tumors is the most direct way to elucidate the complex process of disease progression, although such high-throughput sequencing technologies are not yet fully developed. As a surrogate marker for pathway activation analysis, expression profiling using microarrays has been successfully applied for the classification of tumor types, stages of tumor progression, or in some cases, prediction of clinical outcomes. However, the biological implication of those gene expression signatures is often unclear. Systems biological approaches leverage the signature genes as a representation of changes in signaling pathways, instead of interpreting the relevance between each gene and phenotype. This approach, which can be achieved by comparing the gene set or the expression profile with those of reference experiments in which a defined pathway is modulated, will improve our understanding of cancer classification, clinical outcome, and carcinogenesis. In this review, we will discuss recent studies on the development of expression signatures to monitor signaling pathway activities and how these signatures can be used to improve the identification of responders to anticancer drugs.

Mutant p53 induces the GEF-H1 oncogene, a guanine nucleotide exchange factor-H1 for RhoA, resulting in accelerated cell proliferation in tumor cells.

The tumor suppressor gene p53 is known to induce G1-S and G2-M cell cycle arrest and apoptosis by transactivating various wild-type (WT) p53 regulatory genes. Mutational inactivation of p53 is detected in more than half of human cancers, depriving the p53 protein of its tumor-suppressive functions. Recent studies have shown that mutant p53 provides tumor cells with gain-of-function properties, such as accelerated cell proliferation, increased metastasis, and apoptosis resistance. However, the mechanism underlying the elevated tumorigenicity by p53 mutation remains to be elucidated. In the present study, we showed that GEF-H1, a guanine exchange factor-H1 for RhoA, is transcriptionally activated by the induction of mutant p53 proteins, thereby accelerating tumor cell proliferation. Osteosarcoma U2OS cell lines, which express inducible p53 mutants (V157F, R175H, and R248Q), were established, and the expression profiles of each cell line were then analyzed to detect genes specifically induced by mutant p53. We identified GEF-H1 as one of the consensus genes whose expression was significantly induced by the three mutants. The GEF-H1 expression level strongly correlated with p53 status in a panel of 32 cancer cell lines, and GEF-H1 induction caused activation of RhoA. Furthermore, growth of mutant p53 cells was dependent on GEF-H1 expression, whereas that of WT p53 cells was not. These results suggest that increased GEF-H1 expression contributes to the tumor progression phenotype associated with the p53 mutation.

Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors.

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase (MAPK). RSK1, a member of the RSK family, contains two distinct kinase domains in a single polypeptide chain, the regulatory C-terminal kinase domain (CTKD) and the catalytic N-terminal kinase domain (NTKD). Autophosphorylation of the CTKD leads to activation of the NTKD that subsequently phosphorylates downstream substrates. Here we report the crystal structures of the unactivated RSK1 NTKD bound to different ligands at 2.0 A resolution. The activation loop and helix alphaC, key regulatory elements of kinase function, are disordered. The DFG motif of the inactive RSK1 adopts an "active-like" conformation. The beta-PO(4) group in the AMP-PCP complex adopts a unique conformation that may contribute to inactivity of the enzyme. Structures of RSK1 ligand complexes offer insights into the design of novel anticancer agents and into the regulation of the catalytic activity of RSKs.

Targeted disruption of H3 receptors results in changes in brain histamine tone leading to an obese phenotype.

Histamine is an aminergic neurotransmitter that is localized in the CNS and in peripheral tissues. To date, four histamine receptors have been identified, and the H3 receptor, which was recently cloned, is predominantly expressed in the CNS. The peripheral functions of histamine have been investigated intensively using available molecular and pharmacological tools, and the molecular identification of the H3 receptor opens up new possibilities for investigating the role of histamine in central tissues. To understand the biological function of the histamine presynaptic autoreceptor H3, we inactivated the receptor through homologous recombination. H3(-/-) mice manifest mild obese phenotypes that are characterized by increases in body weight, food intake, and adiposity and by reductions in energy expenditure. Consistent with these observations, homozygous null mice have insulin and leptin resistance, increased levels of plasma leptin and insulin, and decreased levels of histamine in the hypothalamic/thalamic region of their brains coupled with increased histamine turnover. The expression of UCP1 in brown adipose tissue and of UCP3 in brown adipose tissue, white adipose tissue, and skeletal muscle is decreased in H3(-/-) mutants, and the anorexigenic activity of thioperamide is not observed. These results suggest that neuronal histamine is a mediator of body-weight homeostasis and that neuronal histamine functions through H3 receptors in mice.

Impaired drinking response in histamine H3 receptor knockout mice following dehydration or angiotensin-II challenge.

Histamine H3 receptors (H3Rs) are presynaptic receptors that negatively regulate the release of histamine. The present study examined the physiological role of H3Rs in drinking behavior. In water-replete rats, intracerebroventricular (i.c.v.) administration of R-alpha-methylhistamine (RalphaMeHA), an H3R agonist, elicited drinking behavior. In contrast, i.c.v. administration of thioperamide, an H3R inverse agonist, significantly attenuated the drinking behavior elicited by either overnight dehydration or i.c.v. administration of angiotensin-II (AT-II). Inhibition of histamine release with alpha-fluoromethylhistidine, an inhibitor of histidine decarboxylase, did not elicit drinking behavior. Moreover, the inhibitory effects of thioperamide on drinking behavior in water-depleted rats were not mimicked by i.c.v. administration of histamine. These results suggest that the predominant effects of H3Rs on drinking behavior are not mediated by the modulation of histamine release. In H3R-deficient (H3RKO) mice, drinking behavior induced by overnight dehydration or i.c.v. administration of AT-II was significantly impaired compared to wild type mice. Collectively, these observations suggest that brain H3Rs play a pivotal role in drinking behavior in response to dehydration and AT-II, and these effects may be largely independent of the modulation of histaminergic tone.

Enhanced histaminergic neurotransmission and sleep-wake alterations, a study in histamine H3-receptor knock-out mice.

Long-term abolition of a brain arousal system impairs wakefulness (W), but little is known about the consequences of long-term enhancement. The brain histaminergic arousal system is under the negative control of H3-autoreceptors whose deletion results in permanent enhancement of histamine (HA) turnover. In order to determine the consequences of enhancement of the histaminergic system, we compared the cortical EEG and sleep-wake states of H3-receptor knockout (H3R-/-) and wild-type mouse littermates. We found that H3R-/-mice had rich phenotypes. On the one hand, they showed clear signs of enhanced HA neurotransmission and vigilance, i.e., a higher EEG θ power during spontaneous W and a greater extent of W or sleep restriction during behavioral tasks, including environmental change, locomotion, and motivation tests. On the other hand, during the baseline dark period, they displayed deficient W and signs of sleep deterioration, such as pronounced sleep fragmentation and reduced cortical slow activity during slow wave sleep (SWS), most likely due to a desensitization of postsynaptic histaminergic receptors as a result of constant HA release. Ciproxifan (H3-receptor inverse agonist) enhanced W in wild-type mice, but not in H3R-/-mice, indicating a functional deletion of H3-receptors, whereas triprolidine (postsynaptic H1-receptor antagonist) or α-fluoromethylhistidine (HA-synthesis inhibitor) caused a greater SWS increase in H3R-/- than in wild-type mice, consistent with enhanced HA neurotransmission. These sleep-wake characteristics and the obesity phenotypes previously reported in this animal model suggest that chronic enhancement of histaminergic neurotransmission eventually compromises the arousal system, leading to sleep-wake, behavioral, and metabolic disorders similar to those caused by voluntary sleep restriction in humans.

MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil.

MK-1775 is a potent and selective small molecule Wee1 inhibitor. Previously we have shown that it abrogated DNA damaged checkpoints induced by gemcitabine, carboplatin, and cisplatin and enhanced the anti-tumor efficacy of these agents selectively in p53-deficient tumor cells. MK-1775 is currently in Phase I clinical trial in combination with these anti-cancer drugs. In this study, the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging agents with different modes of action were determined. MK-1775 enhanced the cytotoxic effects of 5-FU in p53-deficient human colon cancer cells. MK-1775 inhibited CDC2 Y15 phosphorylation in cells, abrogated DNA damaged checkpoints induced by 5-FU treatment, and caused premature entry of mitosis determined by induction of Histone H3 phosphorylation. Enhancement by MK-1775 was specific for p53-deficient cells since this compound did not sensitize p53-wild type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 potentiated the anti-tumor efficacy of 5-FU or its prodrug, capecitabine, at tolerable doses. These enhancements were well correlated with inhibition of CDC2 phosphorylation and induction of Histone H3 phosphorylation in tumors. In addition, MK-1775 also potentiated the cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA-damaging agents in cancer patients.