Herbert Manoharan

Protein Kinase Cε 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 Cε and development of squamous cell carcinoma, the nonmelanoma human skin cancer

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)‐dependent serine/threonine protein kinases. At least five PKC isoforms (α, δ, ε, η, and ζ) 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 PKCα is found in the membranes of suprabasal cells in the spinous and granular layers. PKCε is mostly localized in the proliferative basal layers. PKCη is localized exclusively in the granular layer. PKCδ is detected throughout the epidermis. PKC isozymes exhibit specificities in their signals to the development of skin cancer. PKCε, 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). PKCε overexpression, which sensitizes skin to UVR‐induced carcinogenesis, suppresses UVR‐induced sunburn (apoptotic) cell formation, and enhances both UVR‐induced levels of TNFα and hyperplasia. UVR‐induced sunburn cell formation is mediated by Fas/Fas‐L and TNFα 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. PKCε inhibits UVR‐induced expression of FADD leading to the inhibition of both apoptotic pathways. It appears that PKCε 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.

Protein kinase Cε 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 (α, δ, ε, η, µ, and ζ) 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 (α, δ, ε, and η), exhibit specificities to the development of skin cancer. PKCε, 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. PKCε 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. PKCε overexpression increases Stat3 activation after either TPA treatment or UVR exposure. Both PKCε and signal transducers and activators of transcription‐3 (Stat3) are implicated in the development of SCC. However, the link between PKCε and Stat3 remains elusive. We found that PKCε interacts with Stat3. PKCε interaction with Stat3 was dependent upon UVR treatment. In reciprocal immunoprecipitation/blotting experiments, Stat3 coimmunoprecipitated with PKCε. Colocalization of PKCε with Stat3 was confirmed by double immunofluorescence staining. PKCε interaction with Stat3 was PKCε isoform specific and was not observed with other protein kinases. As observed in vitro with immunocomplex kinase assay with immunopurified PKCε and Stat3, PKCε phosphorylated Stat3 at the serine 727 residue. PKCε depletion prevented Stat3Ser727 phosphorylation, Stat3 DNA binding, and transcriptional activity. The results presented indicate that PKCε mediates Stat3 activation.

Changes in the DNA methylation profile of the rat H19 gene upstream region during development and transgenic hepatocarcinogenesis and its role in the imprinted transcriptional regulation of the H19 gene

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 CpGs 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.

Biallelic expression of the H19 gene during spontaneous hepatocarcinogenesis in the albumin SV40 T antigen transgenic rat

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.

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.

CpG Analyzer, a Windows-based utility program for investigation of DNA methylation

656 BioTechniques Vol. 39, No. 5 (2005) There is substantial evidence that changes in DNA methylation occur during preneoplasia, including both global changes in DNA methylation and changes in CpG dinucleotide methylation sites in specific genes (1). Aberrant DNA methylation within CpG islands is one of the earliest and more common alterations in human malignancies (2). Cytosine methylation in CpG dinucleotides has been observed to be an important control mechanism in development and differentiation (3). The bisulfite genomic sequencing technique (4) has found wide acceptance for the generation of DNA methylation status maps with singlebase resolution. This method is based on the selective deamination (induced by bisulfite treatment) of cytosine to uracil, while 5-methylcytosine residues remain unchanged. This bisulfitemodified DNA sequence is amplified by PCR and then sequenced. The uracils in the sequence are detected as thymines on PCR amplification and complement with adenines on formation of the double strand. Methylation status is obtained by the comparison of bisulfite sequence PCR products with the computer-generated bisulfite-modified sequences. Knowledge of the CpG distribution within the sequence, identifying each CpG location, and generating bisulfite-modified sequences are essential in the use of this method, while the ability to highlight CpGs in the sequence text will greatly speed up the process of sequence comparison. Some programs are available to simplify the process (see the MethDB links web page at 195.83.84.240/links. html). Methtools is a program available only on the UNIX® operating system, thus excluding Microsoft® Windows users from employing it (5). CpG Island Searcher is a web site that has a simple user interface to identify CpG islands from submitted sequences (6). However, this program does not supply the detailed CpG location information and CpGhighlighted text that are important for a DNA methylation map with single base resolution. On the Microsoft Windows platform, Anbazhagan et al. used Microsoft Excel® to identify and mark CpG islands (7). CpGs can also be analyzed and highlighted by searching for “cg” in a sequence using most word processing software. However, since the sequence text commonly used is in GenBank® flat file format and consists of line numbers, spaces, and line breaks, the searching process must be performed after these extraneous characters have been eliminated. This involves repeated use of the find and delete commands. Manual CpG analysis and sequence conversion should be avoided, since not only are these timeconsuming, but mistakes are readily introduced. Singal et al. (8,9) used Microsoft Word® macros to simplify the repetative tasks such as removing numbers, spaces, and line breaks, highlighting CpGs, and generating the bisulfite-modified sequences. However, this method does not record or generate CpG location data. Since the valuable numbers and spaces that indicate the location of CpGs are eliminated from the highlighted text, this makes it difficult to find the exact locations of the highlighted CpGs, which is important for manual inspection later, CpG Analyzer, a Windows-based utility program for investigation of DNA methylation

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST pi protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.

Hepatocellular carcinomas of the albumin SV40 T‐antigen transgenic rat display fetal‐like re‐expression of Igf2 and deregulation of H19

Previous studies in our laboratory have shown that one of the earliest events during hepatocarcinogenesis in the albumin SV40 T antigen (Alb SV40 T Ag) transgenic rat is the duplication of chromosome 1q3.7‐4.3, a region which contains the imprinted and coordinately regulated genes Igf2 and H19. We have also shown that this duplication is associated with the biallelic expression of the normally monoallelically‐expressed H19. These results, however, are seemingly at odds with studies in the mouse that have shown a conservation of fetal regulatory patterns of these two genes in hepatic neoplasms. We therefore aimed in this study to determine the allelic origin of Igf2 expression in hepatocellular carcinomas of the Alb SV40 T Ag transgenic rat. Sprague–Dawley Alb SV40 T Ag transgenic rats and Brown Norway rats were reciprocally mated and the expression of Igf2 in hepatocellular carcinomas of the resulting F1 transgene‐positive female rats was analyzed by Northern blotting and RT‐PCR. We determined that Igf2 was expressed exclusively from the paternal allele, which prompted the study (by the same methods) of the allelic origin of H19 in the same hepatocellular carcinomas in order to determine if the two genes remained coordinately regulated. Our results demonstrate fetal‐like re‐expression of Igf2 and deregulation of H19 in singular hepatocellular carcinomas of the rat. These results imply that another regulatory mechanism other than the generally accepted ICR/CTCF mechanism may play a role in the control of Igf2 and H19 expression.