- Email: [email protected]
Protein kinase C delta (PKC-δ) regulates post-transcriptional expression of cyclin D1 and Bcl-2 in CaCo-2 cells
trophic action. In the present study, we have further elucidated the mechanism by which PGE~ exerts growth-stimulatory action in LS-174 cells. Methods: LS-174 cells were grown in McCoys 5A medium. Protein kinase A (PKA) activity was assayed using PKA assay kit from Calbiochem Expression of amphiregnlin (AR) was determined by Northern analysis. Results: EP~ receptor signals via generation of IP3 and increase cellular Ca2+. EP2 and EP4 receptors are coupled to stimulatory G (Gs) proteins and signal through increased cyclic AMP (cAMP), whereas the EP3 receptor is coupled to inhibitory G (Gi) proteins which inhibit the generation of cAMP. PGE2 treatment did not alter intracelfular Ca2+ levels. In contrast, PGE2 activated the cAMP/protein kinase A (PKA) signaling pathway determined by PKA kinase assay. Stimulation with PGE~ rapidly increased PKA activity in LS-174 cells. Similar results were produced by the treatment of LS-174 cells with Dibutyryl cAMP, suggesting PGE2 activated PKA through induction of cAMP. utilizing a high density oligonucleotide microarray technique we found that the expression of amphiregulin (AR) is markedly increased following PGE2 treatment, which is a member of epidermal growth factor (EGF) family and plays an important role in colorectal carcinogenesis. Levels of AR mRNA were significantly increased (9-fold) in LS-174 cells following treatment with PGE2 for 2 h. AR treatment stimulated LS-174 cell proliferation and induced PI3K/Akt activation which was required for PGE2 stimulation ofLS-174 cell proliferation. An EGF receptor (EGFR) inhibitor, PD-I53035, blocked PGE2 induced proliferation of LS-174 ceils and attenuated PGE~ activation of PI3K. Conclusion: A. PGE2 signals through activation of cAMP/PKA pathway in LS174 cells. B. Induction of amphiregnlin expression and transactivation of receptor-tyrosin kinase- dependent signaling pathways represents a potential mechanism by which cyclooxygenase-2 promotes colorectal carcinogenesis.
Ik-l-induced NF-KB transcriptional activation. Conclusion: IL-1-induced COX-2 expression in IMF occurs, in part, vua a PKCg-dependent pathway. Contrary to other cell types, neither inhibition of PKC nor treatment with antioxidants affected NF-/cB transcription in IMF. These data suggest that PKC~ and ROS function via a pathway that works in conjunction with NF-KB and that both of these pathways are necessary for IL-l-induced COX-2 expression. Supported by NIDDK and the Gulf Coast DDC
M968
Downregulation of ERK Signaling Mediates the Apoptotie Effect of both NSAIDs and Cyclic GMP Phosphdiesterase (cG-PDE) Inhibitors Fernando Carreira, Pamela Rice, Dennis J. Ahnen NSA1Ds and related compounds induce apoptotic cell death of colon cancer cells in culture. Several biochemical targets for the effect of these drugs have been proposed including cyclooxygenase (COX) inhibition, cG-PDE inhibition leading to protein kinase G (PKG) activation and direct inhibition of ras-signaling but the ultimate biochemical pathway to apoptosis is not known. We have shown that both NSAIDs and cG-PDE inhibitors downregulate MEK and ERK activity. Purpose- To determine if downregulation of MEK/ERK signaling is necessary and sufficient for the apoptotic effect of NSAIDs and cG-PDE inhibitors and to determine which of the proposed biochemical targets is responsible for the effect on MEK/ ERK. Methods- Colon cancer cell lines (HT29, SW480, HCT116, HCT15) and HT29 cells stably transfected with active MEK1 were treated with NSAIDs [sulindac sulfide (SS), indomethacin and resveratrol (Res)] and the non-NSAID, cG-PDE inhibitors exisulind (EX), CP248 and CP461, at doses that induce apoptotic cell death, in the presence or absence of EGF (10 ng/ml), the MEK inhibitor UO126 (10-50 uM), the caspase inhibitors (Boc-AspOMe-FMK, Z-VAD-FMK; 25uM), and/or the PKG activator YC-I (2-5 uM). Apoptosis was measured by caspase-3 cleavage and morphology. MEK and ERK activity were measured by western blots with phosphospecific antibodies. Results- Both the NSAIDs and cG-PDE inhihitors inhibit MEK and ERK activity by up to 90% at times and doses that induce apoptosis; the apoptnsis but not the ERK inhibition was prevented by caspase inhibitors. Both basal and EGF induced MEK and ERK activity are inhibited by SS, Res and EX. The MEK inhibitor, UO126, induces apoptotic cell death in colon cancer cells at doses that inhibit ERK activity. HT29 cells transfected with activated MEK1 are resistant to both ERK inhibition and apoptosis by SS, Res and EX. The apoptotic and ERK inhibitory effects of these drugs are independent of COX inhibition in that they occur in cells that do not express COX-1 or -2 (HCT15 cells) and with drugs that do not inhibit COX activity (EX, CP248, CP461). ERK inhibition by these drugs is also independent of PKG activation in that ERK is not downregnlated by YC-1. Conclusions- ERK inhibition is both necessary and sufficient for the apoptotic effect of both NSAIDs and the cG-PDE inhibitors. The ERK inhibition is not due to either COX inhibition or PKG activation and appears to be an independent effect of these drugs on ras signaling.
M971 Transforming Growth Factor-or Knock-Out Reduces Tumour Initiation but does not Affect Tumour Promotion Omar Bashir, Robert A. Goodlad Background: TGFa may be the main ligand for the EGF receptor in the gut and thus play an important role m maintaining the integrity of the gastrointestinal tract. TGFc~ null mice show some changes in the morphometry of the colon and also have an increased susceptibility to colitis. TGFct expression is increased in humans with adenomas and autocrine stimulation of the EGF-receptor by TGF-ct has been linked to the growth of adenoma cell lines. Aims: To determine if TGFa knock out lead to altered susceptibility to carcinogen induced colonic tnmours. Methods: TGFc~ null mice & appropriate wild-type mice were either injected with saline or dimethylhydrazine (DMH) for 16 weeks. Two weeks after the last injection they were given BrdU and vincristine and then antopsied. Polyp number, cell proliferation and crypt fission were then assayed. Results: Small bowel weight was significantly greater m the TGFa null mice (p<0.001), but colon weight was not changed. DHM had no effect on small bowel weight but significantly increased colon weight and cell proliferation (p<0.001). There was a small but non-significant reduction in polyp number (7.1 +/-0.3 vs 6.1 +/0.6 WT/KO) but the number of aberrant crypt foci was reduced from 97.5 +/-7.4 to 53.2 +/-4.1 (p<0.001) in the null mice. Discussion: TGFct knock out mice had half the number of aberrant crypt foci of the wild type mice, but polyp number was not altered, suggesting that TGFct is involved m the initiation rather than the promotional stages of carcinogenesis.
M969
Protein Kinase C Delta (PKC-8) Regulates Post-transcriptional Expression of Cyclin D1 and Bcl-2 in CaCo-2 cells Soma R. Cerda, Greg Cohen, Christopher M. Moore, Reba Mnstafi, Marc Bissonnette
M972
PKC-8, a serine/threonine kinase, is a member of the "novel" subgroup of the PKC isoenzymes that mediate diverse cellular signal transduction pathways. Previous studies from our laboratory demonstrated that increased expression of PKC-8 inhibited cell growth, while inducing cellular differentiation, and limiting the survival of CaCo-2 cells, derived from a human colonic adenocarcinoma (Gastroenterology 2001). In this study, we futher characterized the signaling pathways involved in the actions of this isoenzyme to induce apoptosis and arrest cell cycling in CaCo-2 cells stably tmnsfected with PKC-B under a Zn2+-inducible metallothionein-regnlated expression vector. Methods: PKC-B expression was induced in CaCo-2 transfectants by treatment with 175 IzM Zn 2+, and cell cycle distributions were determined by flow cytometry, mRNA and protein expression were assessed by real time PCR and Western blotting, respectively. Apoptosis was measured in pre-conflnent CaCo-2 cells by DAP[ staining. Results: Treatment of PKC-8 transfectants with the PKC agonist, phorbo112-mynstate 13-acetate (TPA, 100 nM), significantly enhanced apoptosis as assessed by DAPI staining, compared with empty vector (EV) cells. There was in addition a significant G0/G1 arrest (42-52%), with a decrease in the number of ceils in the S and G2/M phases of the cell cycle by 16 hr in PKC-8 transfectants but not in EV cells. Overexpression of PKC-8 altered G1/S cell cycle regulators, causing rapid downregulation of cychn D1 and E proteins (50% decrease), while mRNA levels remained unchanged. While similar levels of p21 w~ were present in PKC-8 and EV transfectants, TPA treatment induced higher protein and mRNA levels of this cyclin-dependent kinase inhibitor (~2-fold increase) in PKC-8 transfectants. These effects are predicted to inhibit G1/S cychn kinases. Overexpression of PKC-8 resulted in upregulation of pro-apoptotic protein BAX at mRNA and protein levels (~2-fold increase), while anti-apoptotic Bcl-2 protein was downregnlated at a post-transcriptional level (50% decrease). Together, these data indicate a critical role for PKC-B as a key regulator of cell cycle progression and apoptosis in intestinal cancer cells. Furthermore, they underscore the role of this isoenzyme as a tumor suppressor in colonic carcinogenesis.
HGF/Met Induces Downregulation of E-Cadherin and Increases Tef/Beta-Catenin Signaling in Esophageal Adenocardnoma Mark R. Anderson, Moray Campbell, Jannsz Jankowski BACKGROUND: The development of esophageal adenocarcinoma is characterised by progression along the Barrett's metaplasia - dysplasia - carcinoma sequence. The HGF receptor, Met, shows increased expression along this sequence and patients with adenocarcinomas that overexpress Met exhibit poorer short term survival. The perturbation of Cadherin / Catenin complexes has been shown in esophageal adenocarcinoma with downregulation of E-Cadherin a common finding. We sought to investigate a link between Met activation and Cadhenn/Catemn biology. AIMS: To investigate the effect of Met activation on E-Cadberin expression and on Beta-Catemn nuclear signaling in oesophageal cancer cell lines. METHODS: Two cell lines that express Met (OE33 and SEG-1) and one cell line that does not (TE-7) were incubated with HGF at doses ranging from 1 ng/ml to 500 ng/ml. At set time points from 30 minntes to 24 hours, mRNA and protein were harvested. Real time PCR was used to assess levels of E-Cadherin mRNA. Western blotting was used to assess levels of ECadherin protein. Levels of nuclear tcf/beta-catemn signaling were assessed following transient transfection with a tcf/luciferase reporter construct. An ELISA was used to measure levels of HGF in the culture media at different time points to exclude any endogenous synthesis of HGF by the cell lines themselves. RESULTS: OE33 and SEG-1 showed a 37% and 69% reduction m E-Cadherin mRNA following 30 minutes stimulation with HGF at 100 ng/ml (p
M970
Prostaglandin E2 Stimulates Colon Cancer Cell Growth Through Induction of Amphiregulin Jinyi Shao, Hongmiao Sheng, Raymond N. Dubois Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) results in a significant reduction of risk and mortality from colorectal cancer in humans. All of the mechanism(s) by which NSAIDs exert their protective effects are not completely understood, but they are known to inhibit cyclooxygenase activity. We have demonstrated that PGE2 treatment of LS-174 human colorectal carcinoma cells leads to increased proliferation and growth. Prostaglandin EP~ receptor-signaling pathway appears to play a role in transducing signals for this
AGA Abstracts
A-284
Ik-l-induced NF-KB transcriptional activation. Conclusion: IL-1-induced COX-2 expression in IMF occurs, in part, vua a PKCg-dependent pathway. Contrary to other cell types, neither inhibition of PKC nor treatment with antioxidants affected NF-/cB transcription in IMF. These data suggest that PKC~ and ROS function via a pathway that works in conjunction with NF-KB and that both of these pathways are necessary for IL-l-induced COX-2 expression. Supported by NIDDK and the Gulf Coast DDC
M968
Downregulation of ERK Signaling Mediates the Apoptotie Effect of both NSAIDs and Cyclic GMP Phosphdiesterase (cG-PDE) Inhibitors Fernando Carreira, Pamela Rice, Dennis J. Ahnen NSA1Ds and related compounds induce apoptotic cell death of colon cancer cells in culture. Several biochemical targets for the effect of these drugs have been proposed including cyclooxygenase (COX) inhibition, cG-PDE inhibition leading to protein kinase G (PKG) activation and direct inhibition of ras-signaling but the ultimate biochemical pathway to apoptosis is not known. We have shown that both NSAIDs and cG-PDE inhibitors downregulate MEK and ERK activity. Purpose- To determine if downregulation of MEK/ERK signaling is necessary and sufficient for the apoptotic effect of NSAIDs and cG-PDE inhibitors and to determine which of the proposed biochemical targets is responsible for the effect on MEK/ ERK. Methods- Colon cancer cell lines (HT29, SW480, HCT116, HCT15) and HT29 cells stably transfected with active MEK1 were treated with NSAIDs [sulindac sulfide (SS), indomethacin and resveratrol (Res)] and the non-NSAID, cG-PDE inhibitors exisulind (EX), CP248 and CP461, at doses that induce apoptotic cell death, in the presence or absence of EGF (10 ng/ml), the MEK inhibitor UO126 (10-50 uM), the caspase inhibitors (Boc-AspOMe-FMK, Z-VAD-FMK; 25uM), and/or the PKG activator YC-I (2-5 uM). Apoptosis was measured by caspase-3 cleavage and morphology. MEK and ERK activity were measured by western blots with phosphospecific antibodies. Results- Both the NSAIDs and cG-PDE inhihitors inhibit MEK and ERK activity by up to 90% at times and doses that induce apoptosis; the apoptnsis but not the ERK inhibition was prevented by caspase inhibitors. Both basal and EGF induced MEK and ERK activity are inhibited by SS, Res and EX. The MEK inhibitor, UO126, induces apoptotic cell death in colon cancer cells at doses that inhibit ERK activity. HT29 cells transfected with activated MEK1 are resistant to both ERK inhibition and apoptosis by SS, Res and EX. The apoptotic and ERK inhibitory effects of these drugs are independent of COX inhibition in that they occur in cells that do not express COX-1 or -2 (HCT15 cells) and with drugs that do not inhibit COX activity (EX, CP248, CP461). ERK inhibition by these drugs is also independent of PKG activation in that ERK is not downregnlated by YC-1. Conclusions- ERK inhibition is both necessary and sufficient for the apoptotic effect of both NSAIDs and the cG-PDE inhibitors. The ERK inhibition is not due to either COX inhibition or PKG activation and appears to be an independent effect of these drugs on ras signaling.
M971 Transforming Growth Factor-or Knock-Out Reduces Tumour Initiation but does not Affect Tumour Promotion Omar Bashir, Robert A. Goodlad Background: TGFa may be the main ligand for the EGF receptor in the gut and thus play an important role m maintaining the integrity of the gastrointestinal tract. TGFc~ null mice show some changes in the morphometry of the colon and also have an increased susceptibility to colitis. TGFct expression is increased in humans with adenomas and autocrine stimulation of the EGF-receptor by TGF-ct has been linked to the growth of adenoma cell lines. Aims: To determine if TGFa knock out lead to altered susceptibility to carcinogen induced colonic tnmours. Methods: TGFc~ null mice & appropriate wild-type mice were either injected with saline or dimethylhydrazine (DMH) for 16 weeks. Two weeks after the last injection they were given BrdU and vincristine and then antopsied. Polyp number, cell proliferation and crypt fission were then assayed. Results: Small bowel weight was significantly greater m the TGFa null mice (p<0.001), but colon weight was not changed. DHM had no effect on small bowel weight but significantly increased colon weight and cell proliferation (p<0.001). There was a small but non-significant reduction in polyp number (7.1 +/-0.3 vs 6.1 +/0.6 WT/KO) but the number of aberrant crypt foci was reduced from 97.5 +/-7.4 to 53.2 +/-4.1 (p<0.001) in the null mice. Discussion: TGFct knock out mice had half the number of aberrant crypt foci of the wild type mice, but polyp number was not altered, suggesting that TGFct is involved m the initiation rather than the promotional stages of carcinogenesis.
M969
Protein Kinase C Delta (PKC-8) Regulates Post-transcriptional Expression of Cyclin D1 and Bcl-2 in CaCo-2 cells Soma R. Cerda, Greg Cohen, Christopher M. Moore, Reba Mnstafi, Marc Bissonnette
M972
PKC-8, a serine/threonine kinase, is a member of the "novel" subgroup of the PKC isoenzymes that mediate diverse cellular signal transduction pathways. Previous studies from our laboratory demonstrated that increased expression of PKC-8 inhibited cell growth, while inducing cellular differentiation, and limiting the survival of CaCo-2 cells, derived from a human colonic adenocarcinoma (Gastroenterology 2001). In this study, we futher characterized the signaling pathways involved in the actions of this isoenzyme to induce apoptosis and arrest cell cycling in CaCo-2 cells stably tmnsfected with PKC-B under a Zn2+-inducible metallothionein-regnlated expression vector. Methods: PKC-B expression was induced in CaCo-2 transfectants by treatment with 175 IzM Zn 2+, and cell cycle distributions were determined by flow cytometry, mRNA and protein expression were assessed by real time PCR and Western blotting, respectively. Apoptosis was measured in pre-conflnent CaCo-2 cells by DAP[ staining. Results: Treatment of PKC-8 transfectants with the PKC agonist, phorbo112-mynstate 13-acetate (TPA, 100 nM), significantly enhanced apoptosis as assessed by DAPI staining, compared with empty vector (EV) cells. There was in addition a significant G0/G1 arrest (42-52%), with a decrease in the number of ceils in the S and G2/M phases of the cell cycle by 16 hr in PKC-8 transfectants but not in EV cells. Overexpression of PKC-8 altered G1/S cell cycle regulators, causing rapid downregulation of cychn D1 and E proteins (50% decrease), while mRNA levels remained unchanged. While similar levels of p21 w~ were present in PKC-8 and EV transfectants, TPA treatment induced higher protein and mRNA levels of this cyclin-dependent kinase inhibitor (~2-fold increase) in PKC-8 transfectants. These effects are predicted to inhibit G1/S cychn kinases. Overexpression of PKC-8 resulted in upregulation of pro-apoptotic protein BAX at mRNA and protein levels (~2-fold increase), while anti-apoptotic Bcl-2 protein was downregnlated at a post-transcriptional level (50% decrease). Together, these data indicate a critical role for PKC-B as a key regulator of cell cycle progression and apoptosis in intestinal cancer cells. Furthermore, they underscore the role of this isoenzyme as a tumor suppressor in colonic carcinogenesis.
HGF/Met Induces Downregulation of E-Cadherin and Increases Tef/Beta-Catenin Signaling in Esophageal Adenocardnoma Mark R. Anderson, Moray Campbell, Jannsz Jankowski BACKGROUND: The development of esophageal adenocarcinoma is characterised by progression along the Barrett's metaplasia - dysplasia - carcinoma sequence. The HGF receptor, Met, shows increased expression along this sequence and patients with adenocarcinomas that overexpress Met exhibit poorer short term survival. The perturbation of Cadherin / Catenin complexes has been shown in esophageal adenocarcinoma with downregulation of E-Cadherin a common finding. We sought to investigate a link between Met activation and Cadhenn/Catemn biology. AIMS: To investigate the effect of Met activation on E-Cadberin expression and on Beta-Catemn nuclear signaling in oesophageal cancer cell lines. METHODS: Two cell lines that express Met (OE33 and SEG-1) and one cell line that does not (TE-7) were incubated with HGF at doses ranging from 1 ng/ml to 500 ng/ml. At set time points from 30 minntes to 24 hours, mRNA and protein were harvested. Real time PCR was used to assess levels of E-Cadherin mRNA. Western blotting was used to assess levels of ECadherin protein. Levels of nuclear tcf/beta-catemn signaling were assessed following transient transfection with a tcf/luciferase reporter construct. An ELISA was used to measure levels of HGF in the culture media at different time points to exclude any endogenous synthesis of HGF by the cell lines themselves. RESULTS: OE33 and SEG-1 showed a 37% and 69% reduction m E-Cadherin mRNA following 30 minutes stimulation with HGF at 100 ng/ml (p
M970
Prostaglandin E2 Stimulates Colon Cancer Cell Growth Through Induction of Amphiregulin Jinyi Shao, Hongmiao Sheng, Raymond N. Dubois Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) results in a significant reduction of risk and mortality from colorectal cancer in humans. All of the mechanism(s) by which NSAIDs exert their protective effects are not completely understood, but they are known to inhibit cyclooxygenase activity. We have demonstrated that PGE2 treatment of LS-174 human colorectal carcinoma cells leads to increased proliferation and growth. Prostaglandin EP~ receptor-signaling pathway appears to play a role in transducing signals for this
AGA Abstracts
A-284