CpG PatternFinder: a Windows-based utility program for easy and rapid identification of the CpG methylation status of DNA.

The bisulfite genomic sequencing technique is one of the most widely used techniques to study sequence-specific DNA methylation because of its unambiguous ability to reveal DNA methylation status to the order of a single nucleotide. One characteristic feature of the bisulfite genomic sequencing technique is that a number of sample sequence files will be produced from a single DNA sample. The PCR products of bisulfite-treated DNA samples cannot be sequenced directly because they are heterogeneous in nature; therefore they should be cloned into suitable plasmids and then sequenced. This procedure generates an enormous number of sample DNA sequence files as well as adding extra bases belonging to the plasmids to the sequence, which will cause problems in the final sequence comparison. Finding the methylation status for each CpG in each sample sequence is not an easy job. As a result CpG PatternFinder was developed for this purpose. The main functions of the CpG PatternFinder are: (i) to analyze the reference sequence to obtain CpG and non-CpG-C residue position information. (ii) To tailor sample sequence files (delete insertions and mark deletions from the sample sequence files) based on a configuration of ClustalW multiple alignment. (iii) To align sample sequence files with a reference file to obtain bisulfite conversion efficiency and CpG methylation status. And, (iv) to produce graphics, highlighted aligned sequence text and a summary report which can be easily exported to Microsoft Office suite. CpG PatternFinder is designed to operate cooperatively with BioEdit, a freeware on the internet. It can handle up to 100 files of sample DNA sequences simultaneously, and the total CpG pattern analysis process can be finished in minutes. CpG PatternFinder is an ideal software tool for DNA methylation studies to determine the differential methylation pattern in a large number of individuals in a population. Previously we developed the CpG Analyzer program; CpG PatternFinder is our further effort to create software tools for DNA methylation studies.

Protein kinase Cepsilon interacts with signal transducers and activators of transcription 3 (Stat3), phosphorylates Stat3Ser727, and regulates its constitutive activation in prostate cancer.

Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cepsilon (PKCepsilon), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCepsilon expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCepsilon mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCepsilon. The interaction of PKCepsilon with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCepsilon. Localization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCepsilon with Stat3 was PKCepsilon isoform specific. Inhibition of PKCepsilon protein expression in DU145 cells using specific PKCepsilon small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCepsilon activation is essential for constitutive activation of Stat3 and prostate cancer progression.

Protein kinase Cepsilon interacts with Stat3 and regulates its activation that is essential for the development of skin cancer.

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)-dependent serine/threonine protein kinases. At least six PKC isoforms (alpha, delta, epsilon, eta, micro, and zeta) are expressed in epidermis. PKC is a major intracellular receptor for 12-O-tetradecanoylphorbol-13-acetate (TPA) and is also activated by a variety of stress factors including ultraviolet radiation (UVR). PKC isozymes (alpha, delta, epsilon, and eta), exhibit specificities to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform, is linked to the development of squamous cell carcinoma (SCC) elicited either by the 7,12-Dimethylbenzanthracene (DMBA)-TPA protocol or by repeated exposures to UVR. PKCepsilon overexpressing transgenic mice, when treated either with TPA or exposed to UVR, elicit similar responses such as inhibition of apoptosis, promotion of cell survival, and development of SCC. PKCepsilon overexpression increases Stat3 activation after either TPA treatment or UVR exposure. Both PKCepsilon and signal transducers and activators of transcription-3 (Stat3) are implicated in the development of SCC. However, the link between PKCepsilon and Stat3 remains elusive. We found that PKCepsilon interacts with Stat3. PKCepsilon interaction with Stat3 was dependent upon UVR treatment. In reciprocal immunoprecipitation/blotting experiments, Stat3 coimmunoprecipitated with PKCepsilon. Colocalization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. PKCepsilon interaction with Stat3 was PKCepsilon isoform specific and was not observed with other protein kinases. As observed in vitro with immunocomplex kinase assay with immunopurified PKCepsilon and Stat3, PKCepsilon phosphorylated Stat3 at the serine 727 residue. PKCepsilon depletion prevented Stat3Ser727 phosphorylation, Stat3 DNA binding, and transcriptional activity. The results presented indicate that PKCepsilon mediates Stat3 activation.

Protein kinase C epsilon, which sensitizes skin to sun's UV radiation-induced cutaneous damage and development of squamous cell carcinomas, associates with Stat3.

Chronic exposure to UV radiation (UVR) is the major etiologic factor in the development of human skin cancers including squamous cell carcinoma (SCC). We have shown that protein kinase C(epsilon) (PKC(epsilon)), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is an endogenous photosensitizer. PKC(epsilon) is among the six isoforms (alpha, delta, epsilon, eta, mu, and zeta) expressed in both mouse and human skin. PKC(epsilon) transgenic mice, which overexpress PKC(epsilon) in the basal epidermal cells and cells of the hair follicle, are highly sensitive to UVR-induced cutaneous damage and development of SCC. We now present that PKC(epsilon)-overexpressing, but not PKC(delta)-overexpressing, transgenic mice, when exposed to a single (4 kJ/m(2)) or repeated (four doses, 2 kJ/m(2)/dose, thrice weekly) UVR, emitted by Kodacel-filtered FS-40 sun lamps, elicit constitutive phosphorylation of signal transducers and activators of transcription 3 (Stat3) at both Tyr705 and Ser727 residues. UVR-induced phosphorylation of Stat3 accompanied increased expression of Stat3-regulated genes (c-myc, cyclin D1, cdc25A, and COX-2). In reciprocal immunoprecipitation/blotting experiments, phosphorylated Stat3 co-immunoprecipitated with PKC(epsilon). As observed in vivo using PKC(epsilon) knockout mice and in vitro in an immunocomplex kinase assay, PKC(epsilon) phosphorylated Stat3 at Ser727 residue. These results indicate for the first time that (a) PKC(epsilon) is a Stat3Ser727 kinase; (b) PKC(epsilon)-mediated phosphorylation of StatSer727 may be essential for transcriptional activity of Stat3; and (c) UVR-induced phosphorylation of Ser727 may be a key component of the mechanism by which PKC(epsilon) imparts sensitivity to UVR-induced development of SCC.