Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY USA 14263
Tel: 716 - 845 - 5766
Fax: 716 - 845 - 8857
E-mail: jennifer.black@roswellpark.org

Program: Intestinal Epithelial Cell Growth; Differentiation and Transformation; Signal Transduction/Protein Kinase C; Cell Cycle Control; Cell Morphology
Dysregulation of the cellular signaling systems involved in control of normal cell proliferation, differentiation, and death is a key mechanism underlying the development of a transformed phenotype. An approach used in the therapy of cancer is to target the signals that are subverted during malignancy and thus restore normal control mechanisms. Current studies in this laboratory focus on the regulation and function of members of the protein kinase C (PKC) family of signal transduction molecules, key players in the cellular responses mediated by the second messenger diacylglyerol and the phorbol ester tumor promoters. The PKC family consists of at least 10 structurally-related serine/threonine kinases which are expressed in tissue- and cell-type specific patterns and are believed to play distinct role(s) in cell signaling. Individual PKC isozymes have been implicated in control of at least three key cellular processes that are altered in malignant cells including cell cycle progression, differentiation, and apoptosis. This laboratory is investigating the involvement of PKC isozymes in these processes using the intestinal epithelium as a model system. The lining of the mammalian intestine represents a unique system for these studies since it undergoes continuous and rapid renewal, with an actively mitotic cell population in the crypts of Lieberkuhn giving rise to the differentiated cells on the villi. The non-dividing, differentiating cells migrate upward from the crypts, and eventually undergo apoptosis and are shed into the intestinal lumen from the villus tips. The entire sequence of developmental events is, therefore, displayed along the crypt-to-villus axis at any moment in time. Cells at different developmental stages can be isolated for biochemical and molecular analysis and complementary in vitro models, which can be manipulated more readily than in vivo systems, are available for comparative studies. In addition, since each intestinal crypt is derived from a single cell, the intestinal epithelium represents a unique system for the analysis of the effects of targeted genetic alterations in transgenic-chimeric mice in vivo. Finally, the large intestine is an important site for tumor development in humans.

The specific aims of this program are: a) To define the role(s) of the PKC family of signal transduction molecules in control of intestinal self-renewal, b) To examine the interplay between specific PKC isozyme signaling and control of the cell cycle machinery in intestinal epithelial cells at the molecular level, c) To understand the mechanisms regulating PKC isozyme activity and localization in cells, and d) To determine the contribution of alterations in PKC signaling to the development of intestinal tumors and to evaluate PKC as a target for cancer therapy. During the past three years, our laboratory has also been involved in studies on the mechanism(s) underlying cytokine [Interleukin-15 (IL-15)]-mediated protection of the intestinal mucosa against chemotherapy-induced toxicity in a rat model system.

Progress
A major focus of this program is to address the role of individual PKC isozymes in development and maintenance of the intestinal epithelium. In previous studies, our laboratory demonstrated that activation of several members of the PKC family coincides precisely with cell growth arrest/onset of differentiation in both the small intestine and colon in situ. The data obtained from these studies represented the first demonstration of naturally occurring changes in PKC isozyme activation status at the individual cell level within the context of a developing tissue, and laid the foundation for understanding the role(s) of individual isozymes in maintenance of a complex tissue system. A direct role for PKC isozymes in enterocyte post-mitotic events was demonstrated using an immature intestinal crypt cell line (IEC-18 cells) as a model system. Activation of PKC in IEC-18 cells was found to result in cell growth cessation and inhibition of cell cycle progression in G0/G1 and G2/M phases. In-depth studies of the molecular basis for the cell cycle-specific effects of PKC activation in IEC-18 cells revealed for the first time that PKCα signaling, in particular, is capable of inducing hallmark molecular events of cell cycle withdrawal into G0, including rapid downregulation of cyclin D1, differential induction of Cip/Kip cyclin-dependent kinase (cdk) inhibitors, inhibition of cdk activity, and coordinated alterations in the expression and phosphorylation of members of the pocket protein family. A comprehensive morphological/biochemical analysis of the developmental control of G1 regulatory molecules along the crypt/villus axis revealed that PKCa ?activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Studies in the IEC-18 cell line using the PKC inhibitor Gö6976, which specifically blocks the activity of the classical PKC isozymes (i.e., PKCα in IEC-18 cells), suggest that only PKCα, and not PKCδ or PKCε, is capable of signaling cell cycle exit in intestinal epithelial cells. Together, the data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

During the past year, biochemical and molecular approaches were used to examine the regulation of cyclin D1 expression levels by PKCα signaling in IEC-18 cells. Downregulation of cyclin D1 protein was evident by 60-90 min following PKC activation. Northern analysis revealed no concomitant alterations in cyclin D1 mRNA levels, indicating that the PKC-induced reduction in cyclin D1 protein does not involve transcriptional regulation of the cyclin D1 gene or changes in mRNA stability. Downregulation of cyclin D1 protein was found to be proteasome-dependent; however, use of Li2+ treatment, a T286A cyclin D1 mutant, or 'destruction box' cyclin D1 mutants excluded the involvement of GSK3-β and the amino terminus RxxL destruction box motif in PKC-induced loss of cyclin D1. Taken together with evidence that cyclin D1 downregulation is also calpain-independent, the data point to a novel mechanism of cyclin D1 control by PKC signaling; current efforts are being directed towards identification of the mechanism involved. Interestingly, depletion of PKC protein or inhibition of PKC activity in IEC-18 cells resulted in a marked accumulation of cyclin D1 and significant acceleration of cell growth, further pointing to a role of PKC signaling pathways in maintaining intestinal epithelial growth control via regulation of cyclin D1 protein levels. The accumulation of cyclin D1 protein associated with absence of PKC signaling was found to involve alterations in cyclin D1 mRNA levels, although translational and/or post-translational mechanisms may also contribute to the observed effect. These data provide a perspective on the finding that adenomas of the APCMin mouse show complete absence of PKCα protein, accompanied by markedly elevated levels of cyclin D1. Crosstalk between PKC signaling pathways and cyclin D1 regulation may, thus, have important implications for the development and progression of tumors of the intestinal tract. We hypothesize that strategies to modulate the expression of PKCα may prove useful in the prevention and/or therapy of colon carcinoma.

The role of the MAPK/ERK signaling cascade in PKC-mediated intestinal epithelial cell cycle withdrawal was also investigated. PKC agonist treatment of IEC-18 cells was found to result in strong and sustained ERK activation relative to that produced by proliferative signals (e.g., serum boost) in these cells (e.g., 6h vs. ~30 min). Use of the MEK inhibitors PD098059 and UO126 revealed that PKC-triggered cyclin D1 downregulation and p21waf1/cip1 induction, as well as cell cycle exit, are dependent on the MAPK pathway in these cells. The duration of MAPK activation was also found to be critical in sustaining PKC-mediated cell cycle withdrawal in intestinal epithelial cells; prolonged cell cycle exit correlated with sustained MAPK kinase activation which, in turn, correlated with sustained activation of PKC. Taken together with immunohistochemical evidence that post-mitotic intestinal epithelial cells express increased levels of phospho-ERK relative to proliferating crypt cells, these findings support a role for the MAPK/ERK signal transduction pathway in the induction and maintenance of PKC-mediated negative growth regulation in the intestinal epithelium.

Resiniferatoxin (RTX), an ultrapotent analog of capsaicin that has been reported to function as a PKC agonist, was also used to determine the effects of PKC activation on the MAPK/ERK pathway and on cell growth in intestinal epithelial cells. In initial studies, RTX appeared to strongly activate PKC isozymes in IEC-18 cells, and to produce a prolonged growth arrest (>24 hours) that was associated with prolonged activation of ERK1/2 as well as sustained p21waf1/cip1 induction and cyclin D1 downregulation. However, more recent studies using different preparations of RTX demonstrated that this agent produces prolonged (>16 hours) growth arrest in IEC-18 cells through a PKC-independent mechanism. Efforts to resolve this discrepancy revealed that resiniferonol 9, 13, 14-orthophenyl acetate (ROPA), a derivative of RTX and a weak tumor promoter, is a potent activator of PKC isozymes in IEC-18 cells which produces PKC-dependent growth arrest for approximately 12 h. Combinations of RTX and ROPA activated PKC isozymes in IEC-18 cells and produced sustained erk1/2 activation, p21waf1/cip1 induction, and cyclin D1 downregulation, as well as prolonged growth arrest (>24 hours), thus recapitulating the effects of early RTX preparations. Based on these findings, we hypothesize that the RTX preparations used in our initial studies also contained significant amounts of ROPA. These findings indicate that caution should be exercised in the use of this compound when acquired from commercial sources.

Capsaicin has been shown to have chemopreventive effects in a colon cancer model. Thus, in addition to its current use in pain management and treatment of overactive bladder, this agent and its analogs (e.g., resiniferatoxin) may be useful in the chemoprevention and/or chemotherapy of colon tumors. Studies performed by Dr. Adrian R. Black (Pharmacology and Therapeutics) have demonstrated that both capsaicin and resiniferatoxin potently downregulate cyclin D1 mRNA and protein in IEC-18 cells in a PKC-independent manner. The ability of these agents to regulate cyclin D1 expression may prove useful in the management of intestinal adenomas (e.g., patients with Familial Adenomatous Polyposis), since these lesions have been demonstrated to express markedly increased levels of cyclin D1, while lacking PKCα expression.
Progress has also been made in the generation of mice with targeted expression of PKCa ?in the intestine. In collaboration with Dr. Paul Soloway, constructs in which PKCa or PKCα/GFP cDNA was placed under the control of an intestine-specific promoter [nucleotides -596 to +21 of rat liver fatty acid binding protein (L-FABP)] were electroporated into ES cells for generation of chimeric-transgenic mice. In-depth analysis of the resulting animals is in progress. These mice were also bred for germ-line transmission of the transgene, and positive offspring have been identified. These transgenic animals will be evaluated for alterations in intestinal homeostasis and susceptibility to carcinogen-induced tumorigenesis.

Studies are also in progress to determine the role of phosphorylation in regulation of PKC isozyme downregulation. In contrast to published studies, we have found that phosphorylation accelerates PKC isozyme ubiquitination and degradation by the proteasome. Our findings suggest that phosphorylation of PKC at one or more specific site(s) serves to target the activated enzyme for efficient proteasome-mediated desctruction.

Students

Dysregulation of the cellular signaling systems involved in control of normal cell proliferation, differentiation, and death is a key mechanism underlying the development of a transformed phenotype. An approach used in the therapy of cancer is to target the signals that are subverted during malignancy and thus restore normal control mechanisms. Current studies in this laboratory focus on the regulation and function of members of the protein kinase C (PKC) family of signal transduction molecules, key players in the cellular responses mediated by the second messenger diacylglyerol and the phorbol ester tumor promoters. The PKC family consists of at least 11 structurally-related serine/threonine kinases which are expressed in tissue- and cell-type specific patterns and are believed to play distinct role(s) in cell signaling. Individual PKC isozymes have been implicated in control of at least three key cellular processes that are altered in malignant cells including cell cycle progression, differentiation, and apoptosis. This laboratory is investigating the involvement of PKC isozymes in these processes using the intestinal epithelium as a model system. The specific aims of this program are: a) To define the role(s) of the PKC family of signal transduction molecules in control of intestinal self-renewal, b) To examine the interplay between specific PKC isozyme signaling and control of the cell cycle machinery in intestinal epithelial cells at the molecular level, c) To understand the mechanisms regulating PKC isozyme activity and localization in cells, and d) To determine the contribution of alterations in PKC signaling to the development of intestinal tumors and to evaluate PKC as a target for cancer therapy.

Key Publications

 

• Serin G, Gersappe A, Black JD, Aronoff R, Maquat LE. Identification and characterization of human orthologues to S. cerevisiae Upf2 protein and S. cerevisiae Upf3 protein/C. elegans SMG-4. Mol. Cell. Biol. 21:209-223, 2001.
• Frey MR, Leontieva O, Watters D, Black JD. Bistratene A stimulates protein kinase C-dependent and -independent signaling pathways in intestinal epithelial cells. Biochem. Pharmacol. 61: 1093-1100, 2001.
• Black AR, Black JD, Azizkhan-Clifford J. The Sp1 and Kruppel-like family of transcription factors in cell growth regulation and cancer. J. Cellular Physiology 188: 143-160, 2001. Black JD. Protein kinase C isozymes in colon carcinogenesis: Guilt by Omission. Gastroenterology 120: 1868-1872, 2001 - Invited Editorial.