To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2 0 -deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription-PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1 domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains towards the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control, and are broadly applicable tools to study DAG signaling.

The pathogenesis of breast cancer involves multiple genetic and epigenetic events. In this study, we report an epigenetic alteration of DFNA5 in human breast cancer. DFNA5 gene was silenced in breast cancer cell lines that were methylated in the DFNA5 promoter, and restored by treatment with the demethylating agent, 5-aza-dC, and gene knock-down of DFNA5 increased cellular invasiveness in vitro. The mRNA expression of DFNA5 in breast cancer tissues was downregulated as compared to normal tissues. Moreover, the DFNA5 promoter was found to be methylated in primary tumor tissues with high frequency (53%, 18/34). Quantitative methylationspecific PCR of DFNA5 clearly discriminated primary breast cancer tissues from normal breast tissues (15.3%, 2/13). Moreover, methylation status of DFNA5 was correlated with lymph node metastasis in breast cancer patients. Our data implicate DFNA5 promoter methylation as a novel molecular biomarker in human breast cancer. Keywords DFNA5; The human deafness; Autosomal dominant 5 gene; Promoter methylation; Breast cancerAlthough breast cancer mortality declined significantly in recent years, in part due to early diagnosis by screening mammography, the limitations of mammography are wellrecognized [1,2]. Therefore, novel approaches for the early detection of breast cancer are necessary. The pathogenesis of breast cancer involves multiple genetic and epigenetic events. In spite of recent advances in the assessment of breast cancer risk, the identification of crucial susceptibility genes such as BRCA1/2, PTEN, and p53 account for only about 5% of all breast cancer cases [3]. The marked cytogenetic complexity seen in advanced breast cancer precludes investigators from readily identifying primary causative genetic and epigenetic events in breast cell carcinogenesis [4].

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Graphical Abstract Highlights d Presence or absence of food promotes the dwelling or dispersal behavior of C. elegans d Dopamine signals to peptidergic interneurons in response to food d Peptidergic interneurons antagonize each other to inhibit or excite motoneurons d Cholecystokinin and RFamide modulate motoneurons to generate food response behavior SUMMARY Finding food and remaining at a food source are crucial survival strategies. We show how neural circuits and signaling molecules regulate these foodrelated behaviors in Caenorhabditis elegans. In the absence of food, AVK interneurons release FLP-1 neuropeptides that inhibit motorneurons to regulate body posture and velocity, thereby promoting dispersal. Conversely, AVK photoinhibition promoted dwelling behavior. We identified FLP-1 receptors required for these effects in distinct motoneurons. The DVA interneuron antagonizes signaling from AVK by releasing cholecystokinin-like neuropeptides that potentiate cholinergic neurons, in response to dopaminergic neurons that sense food. Dopamine also acts directly on AVK via an inhibitory dopamine receptor. Both AVK and DVA couple to head motoneurons by electrical and chemical synapses to orchestrate either dispersal or dwelling behavior, thus integrating environmental and proprioceptive signals. Dopaminergic regulation of foodrelated behavior, via similar neuropeptides, may be conserved in mammals.

Key words: p53 polymorphism; HPV; oral cancerOral squamous cell carcinoma (OSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality worldwide. The overall survival percentage has not changed in recent years, despite extensive research on the biological and molecular aspects of oral SCC. Among the more pressing problems in clinical management are the lack of early detection and the high incidence of local-regional recurrence, even with aggressive surgical therapy. OSCC incidence accounts for up to 40% of all malignancies in India and South East Asia. 1 Moreover differences have been observed in the clinico-pathological and molecular pathological profile in the tobacco smoking and alcoholassociated oral cancers in the USA, UK, France, Japan, etc. and the chewing tobacco-associated oral cancers, particularly in the Indian sub-continent. 2 In India, oral leukoplakias have been reported to show a significant tendency to malignant transformation from 0.13 to 6% and rising to 14% or higher in cases of dysplasia. 3,4 Epidemiological studies reveal the betel quid and tobacco chewing habit as an important risk factor in the etiology of oral cancer in India. Since carcinogenesis is a multi-step process, therefore, in addition to insult by tobacco-associated intra oral carcinogens, several additional factors, such as genetic susceptibility of individuals and external agents, such as dietary factors and viruses (human papillomavirus, HPV, and Epstein-Baar virus, EBV), may play a synergetic role in oral tumorigenesis. [5][6][7][8] HPVs are a very heterogenous group of DNA viruses. Almost 90 HPV types have been described; 30 of these types are associated with anogenital cancers, forming either the high-risk types (HPV16 and HPV18) that are associated with anogenital invasive tumour and their precursor lesions or the low-risk types (HPV6 and HPV11) that rarely progress to malignancy. Analysis of viral genome has revealed that 2 early genes, E6 and E7, of high risk HPVs (16/18) are transforming genes that are responsible for maintenance of tumorigenic phenotype. 6,9,10 Genetic polymorphism at the genes involved in tumorigenesis may determine individual susceptibility of cancer. The genotype distribution of p53 codon 72 polymorphism is significantly different among ethnic groups. 11 Storey et al. 12 recently reported the probable correlation between p53 polymorphism and HPV associated cervical tumorigenesis. They showed that population homozygous for Arg-72 (arginine) is about 7 times more susceptible to development of cervical cancer than heterozygotes. However, this has been subject of much debate. Makni et al. 13 recently showed the substantial inter-laboratory variation in the proportion of Arg/Arg, Arg/Pro (proline) and Pro/Pro.In spite of its great relevance, so far only 2 contradictory reports have been proposed on the status of HPV in oral cancer on Indian population. The work on South Indian population has indicated 42% HPV-16 infection and 47% of HPV-18 infection, 7 whereas the r...

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DNp63a associated with its increased phosphorylated status. Since DNp63a usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DNp63a in HNSCC cells upon cisplatin exposure. We found that DNp63a is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DNp63a in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DNp63a residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DNp63a degradation. Finally, we demonstrated that the DNp63a protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DNp63a following DNA damage.