Neuropathic pain is a chronic disease resulting from dysfunction of the nervous system often due to peripheral nerve injury. Hypersensitivity to sensory stimuli (mechanical, thermal or chemical) is a common source of pain in patients and ion channels involved in detecting these stimuli are possible candidates for inducing and/or maintaining the pain. Transient receptor potential (TRP) channels expressed on nociceptors respond to different sensory stimuli and a few of them have been studied previously in the models of neuropathic pain. Using real-time PCR for quantification of all known TRP channels we identified several TRP channels, which have not been associated with nociception or neuropathic pain before, to be expressed in the DRG and to be differentially regulated after spared nerve injury (SNI). Of all TRP channel members, TRPML3 showed the most dramatic change in animals exhibiting neuropathic pain behaviour compared to control animals. In situ hybridisation showed a widespread increase of expression in neurons of small, medium and large cell sizes, indicating expression in multiple subtypes. Co-localisation of TRPML3 with CGRP, NF200 and IB4 staining confirmed a broad subtype distribution. Expression studies during development showed that TRPML3 is an embryonic channel that is induced upon nerve injury in three different nerve injury models investigated. Thus, the current results link for the first time a re-expression of TRPML3 with the development of neuropathic pain conditions. In addition, decreased mRNA levels after SNI were seen for TRPM6, TRPM8, TRPV1, TRPA1, TRPC3, TRPC4 and TRPC5.

MIM/MTSS1 was initially described as a gene missing in invasive bladder cancer cell lines. Functional analysis revealed that MIM is an actin binding protein involved in the regulation of actin cytoskeleton dynamics. MIM was shown to be sonic hedgehog (Shh) signaling dependent and synergizes with the effects of Gli transcription factors. Overexpression of MIM in cell lines leads to the inhibition of cell proliferation. In this study, we showed that the inhibition of cell growth by MIM is anchorage independent. We identified and cloned the promoter region of MIM and located the main promoter activity to 276 bp of 5 0 flanking sequence sited within a CpG island. Analysis of DNA methylation using bisulphite sequencing revealed that MIM promoter is methylated in its 5 0 region in cells and tissue samples with reduced endogenous MIM expression. Using luciferase reporter assay, we demonstrated that nonmethylated MIM promoter has a similar activity in cell lines with different endogenous MIM expression. Inhibition of DNA methylation by 5-Aza-2 0 -deoxycytidine led to upregulation of MIM expression in a low expressing cell line. In conclusion, we clearly demonstrate here that the expression of metastasis suppressor MIM is regulated by DNA methylation of a CpG island within its promoter region. ' 2006 Wiley-Liss, Inc.Key words: actin; tumor suppressor; carcinoma; 5-Aza-2 0 -deoxycytidine; BEG4Since decades DNA methylation is known to play an essential role during tumor initiation and progression. Two main directions of DNA methylation changes are associated with carcinogenesis: general DNA hypomethylation and regional DNA hypermethylation. 1 Hypomethylation leads to a general destabilization of the genome and increases the frequency of chromosomal aberrations, 2 while the regional DNA hypermethylation is described as an alternative for mutation or deletion of the tumor suppressor gene leading to the loss of function. 3-5 Structurally, the promoters of genes regulated by DNA methylation may be divided into 2 groups: 1--containing CpG island (a region with increased density of CpG dinucleotides) and 2--lacking CpG island. Although the nonmethylated state of a CpG island does not necessarily indicate the transcriptionally active promoter, methylated state of a CpG island usually leads to inactivation of the promoter. 3 During carcinogenesis de novo methylation of normally nonmethylated CpG island causes deacetlytion of the histones, and formation of transcriptionally inactive chromatin. 3 Following tumor suppressors are examples of the genes that can be inactivated by de novo methylation within the promoter region CpG island: pRb in retinoblastoma, 6 p16INK4a in melanoma and others, 7 p15INK4b in haematological malignancies, 8 hMLH1 and APC in colorectal carcinoma 9,10 and BRCA1 in breast carcinoma. 11 Although usually the aberrant methylation of CpG island containing promoters is associated with carcinogenesis, changes in methylation pattern of promoters lacking CpG island were also reported. 12,13 MIM (Missing in metastas...