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 CE (PKCE), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCE expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCE 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 PKCE. The interaction of PKCE 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 PKCE. Localization of PKCE with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCE with Stat3 was PKCE isoform specific. Inhibition of PKCE protein expression in DU145 cells using specific PKCE small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNAbinding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCE activation is essential for constitutive activation of Stat3 and prostate cancer progression. [Cancer Res 2007;67(18):8828-38]

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 CE (PKCE), a Ca 2+ -independent, phospholipiddependent serine/threonine kinase, is an endogenous photosensitizer. PKCE is among the six isoforms (A, D, E, H, M, and Z) expressed in both mouse and human skin. PKCE transgenic mice, which overexpress PKCE 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 PKCE-overexpressing, but not PKCD-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 coimmunoprecipitated with PKCE. As observed in vivo using PKCE knockout mice and in vitro in an immunocomplex kinase assay, PKCE phosphorylated Stat3 at Ser727 residue. These results indicate for the first time that (a) PKCE is a Stat3Ser727 kinase; (b) PKCE-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 PKCE imparts sensitivity to UVR-induced development of SCC. [Cancer Res 2007;67(3):1385-94]

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 (PKC) represents a large family of phosphatidylserine (PS)-dependent serine/threonine protein kinases. At least five PKC isoforms (alpha, delta, epsilon, eta, and zeta) are expressed in epidermal keratinocytes. PKC isoforms are differentially expressed in proliferative (basal layer) and nonproliferative compartments (spinous, granular, cornified layers), which exhibit divergence in their roles in the regulation of epidermal cell proliferation, differentiation, and apoptosis. Immunocytochemical localization of PKC isoforms indicate that PKCalpha is found in the membranes of suprabasal cells in the spinous and granular layers. PKCepsilon is mostly localized in the proliferative basal layers. PKCeta is localized exclusively in the granular layer. PKCdelta is detected throughout the epidermis. PKC isozymes exhibit specificities in their signals to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform mediates the induction of squamous cell carcinoma (SCC) elicited either by the DMBA-TPA protocol or by repeated exposures to ultraviolet radiation (UVR). PKCepsilon overexpression, which sensitizes skin to UVR-induced carcinogenesis, suppresses UVR-induced sunburn (apoptotic) cell formation, and enhances both UVR-induced levels of TNFalpha and hyperplasia. UVR-induced sunburn cell formation is mediated by Fas/Fas-L and TNFalpha NFR1 extrinsic apoptotic pathways. The death adaptor protein termed Fas-associated death domain (FADD) is a common adaptor protein for both of these apoptotic pathways. PKCepsilon inhibits UVR-induced expression of FADD leading to the inhibition of both apoptotic pathways. It appears that PKCepsilon sensitizes skin to the development of SCC by UVR by transducting signals, which inhibit apoptosis on one hand, and enhances proliferation of preneoplastic cells on the other hand.

Previous studies in this laboratory have demonstrated that the earliest cytogenetic alteration in the development of hepatic neoplasms in a transgenic strain of rats bearing the albumin Simian virus 40 T antigen (Alb SV40 T Ag) construct was a duplication of the chromosome 1q4.1-1q4.2 band. In this region, in the rat genome a cluster of linked imprinted genes occurs. One of these imprinted genes, H19, which is expressed in fetal liver but not in adult liver, was found to be expressed in virtually all neoplasms investigated. A single-nucleotide polymorphic marker in the H19 coding sequence was identified in two rat strains and utilized for the investigation of H19 imprinting. Our results reveal monoallelic expression of the maternal gene in fetal liver, but biallelic expression of the H19 gene in liver neoplasms, thus demonstrating the basis for the deregulation of the imprinted gene expression during hepatocarcinogenesis. These results suggest that the loss of genomic imprinting of the H19 gene found in the liver neoplasms of the Alb SV40 T Ag rat may result not from allelic loss, but from adverse changes in the epigenetic imprints present in the 5'-upstream region of the H19 promoter of the parental alleles.

Monoallelic expression of the imprinted H19 and insulin-like growth factor-2 (Igf2) genes depends on the hypomethylation of the maternal allele and hypermethylation of the paternal allele of the H19 upstream region. Previous studies from our laboratory on liver carcinogenesis in the F1 hybrid of Fischer 344 (F344) and Sprague-Dawley Alb SV40 T Ag transgenic rat (SD) strains revealed the biallelic expression of H19 in hepatomas. We undertook a comparative study of the DNA methylation status of the upstream region of H19 in fetal, adult, and neoplastic liver. Bisulfite DNA sequencing analysis of a 3.745-kb DNA segment extending from 2950 to 6695 bp of the H19 upstream region revealed marked variations in the methylation patterns in fetal, adult, and neoplastic liver. In the fetal liver, equal proportions of hyper- and hypomethylated strands revealed the differentially methylated status of the parental alleles, but in neoplastic liver a pronounced change in the pattern of methylation was observed with a distinct change to hypomethylation in the short segments between 2984 and 3301 bp, 6033-6123 bp, and 6518-6548 bp. These results indicated that methylation of all cytosines in this region may contribute to the imprinting status of the rat H19 gene. This phenomenon of differential methylation-related epigenetic alteration in the key cis-regulatory domains of the H19 promoter influences switching to biallelic expression in hepatocellular carcinogenesis. Similar to mouse and human, we showed that the zinc-finger CCTCC binding factor (CTCF) binds to the unmethylated CTCF binding site in the upstream region to influence monoallelic imprinted expression in fetal liver. CTCF does not appear to be rate limiting in fetal, normal, and neoplastic liver. 3' to the CTCF binding sites, another DNA region exhibits methylation of CpG's in both DNA strands in adult liver, retention of the imprint in fetal liver, and complete demethylation in neoplastic liver. In this region is also a putative binding site for a basic helix-loop-helix leucine-zipper transcription factor, TFEB. The differential CpG methylation seen in the adult that involves the TFEB binding site may explain the lack of expression of the H19 gene in adult normal liver. Furthermore, these findings demonstrate that the loss of imprinting of the H19 gene in hepatic neoplasms of the SD Alb SV40 T Ag transgenic rat is directly correlated with and probably the result of differential methylation of CpG dinucleotides in two distinct regions of the gene that are within 4 kb 5' of the transcription start site. Cytogenetic analysis of hepatocytes in the transgenic animal prior to the appearance of nodules or neoplasms indicates a role of such loss of imprinting in the very early period of neoplastic development, possibly the transition from the stage of promotion to that of progression.

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.

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