Transcriptional up-regulation of transforming growth factor-β (TGF-β) receptors by TGF-β1...
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A626 AGA ABSTRACTS • G2574 EXPRESSION OF Fas LIGAND AND PROGRESSION OF THE HUMAN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS. Y. Kitanawa. M. Ueda, N. Ando, S. Ozawa, M. Kitajima, Department of Surgery~ Keio University, Tokyo, Japan FasL and its receptor, Fas regulate immune system through the induction of apoptosis in the target cells. Recently, expression of FasL in several human cancer cell lines and resected tumors has been demonstrated. However, the expression of FasL in human esophageal squamous cell carcinomas (ESCC) and its clinical significance are still unknown. In the present study, the relationship between the expression of FasL and clinicopathological parameters of human ESCC has been investigated. Methods: Expression of FasL in the primary tumors of 54 cases with ESCC has been examined by immunohistochemistry using monoclonal antibody raised to residues 116-277 of human FasL (Transduction Lab). The killing effect of ESCC cell lines (TE 2, 4, 5, 8, 9) in the coculture with Fas-sensitive human T cell line (Jurkat) was tested according to the previously described methods 1). Results: Twenty eight of 54 cases (52%) were scored FasL positive (cytoplasmic staining was seen in greater than 10% of cancer cells). The incidence of FasL expression was significantly (p<0.01) higher in the cases with lymph node metastasis (69%) than that of the case without lymph node metastasis(27%). Also the expression of FasL was significantly correlated to the depth of invasion (p<0.01). In the coculture experiments, the killing effect of TE cell lines to Jurkat was compatible to the expression level of FasL(TE 4 with relatively high level of FasL expression killed 62% of Jurkat ceils by 24 h coculture). Conclusions: These results suggest the possibility that FasL expression is closely correlated to the progression of human ESCC through the protection of tumor ceils from immune response by T cells which are sensitive to the FasL-Fas mediated apnptosis. [1] Niehans GA et al; Cancer Res. 57, 1007-1012, 1997 • G2575 TRANSCRIPTIONAL UP-REGULATION OF TRANSFORMING GROWTH FACTORq3 (TGFq3) RECEPTORS BY TGF-~I CORRELATES WITH EXPRESSION OF CYCLIN D-DEPENDENT KINASE INFIIBITORS. Jt~rg Kleeff and Murray Korc, Departments of Medicine, Biological Chemistry and Pharmacology, University of California, Irvine, California 92697, USA AIM: TGF-[31 is a multifunctional polypeptide that binds to specific cell surface receptors and suppresses epithelial cell growth. In the present study we investigated the actions of TGF-131 on cell proliferation in relation to cyclin D-dependent kinase inhibitor expression and ,TGF-13 receptor modulation in COLO-357 pancreatic cancer cells. RESULTS: TGF-131 inhibited the growth of COLO-357 cells in a time and dose dependent manner. TGF-131 also caused a delayed but sustained increase in protein levels of the cyclin D-dependent kinase inhibitors p15 Inlc4B, p21C|pl, and p27 l°pl as well as a delayed but sustained increase in TI3RI and TI3RII mRNA and protein levels. In contrast, TGF-131 caused a rapid but transient increase in plasminogen activator inhibitor-I (PAl-I) and insulin like growth factor binding protein-3 (IGFBP-3) mRNA levels. The protein synthesis inhibitor cycloheximide completely blocked the TGF-131 mediated increase in TI3RI and TI3RII expression. Furthermore, a nuclear runoff transcription assay revealed that the increase in receptor mRNA levels was due to newly transcribed RNA. There was a significant increase in TI3RI and TI3RII mRNA levels in confluent cells in comparison to subconfluent (< 80% confluent) controls, as well as in serum starved ceils when compared with cells incubated in medium containing 10% FBS. COLO-357 cells expressed a normal SMAD-4 gene as determined by Northern blot analysis and sequencing. CONCLUSION: These results indicate that TGF-131 is capable of modulating a variety of functions in COLO-357 pancreatic cancer cells and that cell density and serum can modulate TGF-13 receptor expression. In addition, the kinetics of the TGF-131-mediated transcriptional up-regulation of TGF-13 receptors suggests that this up-regulation may have the potential to maximize TGF-131-dependent antiproliferative responses. • G2576 PKC ISOFORMS IN INTESTINAL MUCOSA AND ADENOMAS OF APC MIN MICE. Irene K. Klein. Steven R. Ritland, Steven C. Ziesmer, Lawrence J. Burgart, Patrick C. Roche, Sandra J. Gendler, William E. Karnes, Jr., M.D. Colorectal Neoplasia Group, Mayo Clinic, Rochester, MN; Mayo Clinic, Scottsdale, AZ; Brown University, Providence, RI. BACKGROUND: Protein kinase C (PKC) PKC is reported to regulate beta-catenin levels in conjunction with the adenomatous pnlyposis coli (APC) gene product. To gain insight into the potential roles of PKC isoforms in adenomatous transformation related to APC loss, we compared the levels of PKC isoforms in normal and adenomatous mucosa of APC MIN mice and wild- type C57B1/6 mice. METHODS: Eight wild-type and 8 APC MtN mice were sacrificed at 70-100 days of age. Samples of normal colon, ileum and adenomas (when present) were snap frozen and analyzed for PKC isoform expression by immunohistochemistry with and without competing peptides to assess binding specificity. Scoring (0-4) was performed blindly. RESULTS: APCMIN and wild-type mice were indistinguishable with respect to the distributions and intensities of specific cytoplasmic staining for PKC isoforms in normal mucosa. In the colon, most isoforms showed strongest staining in the lower 3rd of the crypts and weakest staining in the upper 3rd of the crypts. GASTROENTEROLOGY Vol. 114, No. 4 Compared to the normal colonic mucosa, staining intensities in colonic adenomas were lower for PKCs ct and ~ and higher for PKCs "/and In. Staining for PKCs "t and la was predominantly cytoplasmic in normal mucosa and predominately nuclear in colonic adenomas. In the ileum, the gradient of PKC isoform expression was reversed for most isoforms, being lowest in the crypts. As in the case for colon adenomas, ileal adenomas exhibited significantly weaker staining for PKCs a and ~ and significantly stronger staining for PKCs la and ~/compares to normal ileal mucosa. A shift toward nuclear staining was again evident for PKCs ~ and T in ileal adenomas. ~-~ muoo~ ~ IIQum muc~a 0 ( ~ n ade~oma 0 Ileum aderoma 4,, a Ill 112 6 ~ n y ~ # CONCLUSIONS: 1) PKC gradients along the crypt-villous axis are consistent with their known roles in cell growth and differentiation; 2) heterozygous loss of APC does not influence these gradients (no differences between wild-type and APC MIN mice); and 3) the decrease of PKCs ct and ~ and increase (with nuclear translocation) of PKCs "1' and g expression in adenomas of APCMIN mice suggests a role of these isoforms in adenomatous transformation resulting from loss of both APC alleles. This research was funded by NCI R29 CA71974, the Mayo Comprehensive Cancer Center and the Mayo Foundation. Irene Kline was funded by S.U.R.F. • G2577 THE ROLE OF p161NK4A PROMOTER HYPERMETHYLATION DURING NEOPLASTIC PROGRESSION IN BARRETr'S ESOPHAGUS. B. Klumv. CJ. Hsieh, K. Holzmann, M. Gregor, R. Porschen. Department of Internal Medicine I, University Hospital Tuebingen, Germany Barrett's esophagus is considered as a premalignant condition predisposing to the development of esophageal adenocarcinoma. Transcriptional silencing of the tumor suppressor gene pl6INK4A by hypermethylation of its promoter region has been reported to be a predominant mechanism of gene inactivation in esophageal adenocarcinoma. This study sought to identify a possible significance of pl6INK4A hypermethylation during the process of neoplastic progression in Barrett's esophagus. 95 paraffin-embedded specimens were retrieved from 14 patients with Barrett's esophagus, who were followed over a mean time of 49.5 months. A methylation-specific PCR protocol was applied. Methylation status of the pI61NK4A promoter region was compared to histomorphological findings classified as indefinite for dysplasia, low grade or high grade dysplasia and carcinoma. 16 of 28 (57.1%) specimens with dysplasia but merely 2 of 67 specimens (3.0%) without dysplastic lesions showed hypermethylation of the p16 promoter region. Hypermethylation was detected in the majority of specimens indefinite for dysplasia (60%) and in a non-dysplastic specimen of a patient who developed dysplasia 24 months later. These data suggest that promoter hypermethylation is a common mechanism of pl61NK4a gene inactivation during neoplastic progression in Barrett's esophagus, p16 promoter hypermethylation seems to be a frequent and early occurring epigenetic abnormality during this process. Its application in the surveillance of patients with Barrett's esophagus has to be scrutinized in prospective studies. Supported by BMFT (01 KS 9602) • (;2578 UPTAKE MECHANISM OF IRINOTECAN (CPT-11) AND ITS METABOLITE (SN-38) BY HAMSTER INTESTINAL CELLS. K. KobayashP,~, B. Bouscarel l, Y. Matsuzaki3, S. Ceryak I H. FrommL I. Div. Gastroenterology, Dept. Medicine, The George Washington Univ., Washington, D.C. 2. The 4th Dept. of Internal Med., Nippon Med. School, Japan, 3. Div. Gastroenterology, Inst. Clin. Med., Univ. Tsukuba, Japan. BACKGROUND: CPT-11 is characterized by the novel mechanism of inhibiting DNA topoisomerase I and has shown promising antitumor activity clinically. CPT-11 and its active metabolite, SN-38, are eliminated by the liver, reabsorbed by the intestine, and enter enterohepatic circulation. Thus, many pharmacokinetic analyses in humans have shown its large interpatient variability. Furthermore, diarrhea (about 60 % incidence) is now recognized as the major dose-limiting toxicity of this drug. AIM: The objective was to investigate the comparative intestinal transport mechanism of CPT-11 and SN-38, in order to design new approaches to prevent the variability in pharmacokinetics and diarrhea. METHODS: The uptake rate of 14C-CPT-11 and 14C-SN-38, both as respective lactone (lact.) forms in acidic pH and anion-charged carboxylate (carbo.) forms in alkaline pH, were investigated. The rapid vacuum filtration technique was used with isolated intestinal epithelial cells of jejunum, ileum, cecum and colon from Golden Syrian hamsters. RESULTS: Comparative uptake rates (n mol / 106 cells /sec) were: jejunum ileum cecum colon lact. carbo, lact. carbo, lact. carbo, lact. carbo. CPT-11 (20 pM) 279 41.1 268 56.5 314 60.7 258 5.1 Lp = o.o04J Lp= o.o04J LP= o.o17J /v = o.o08J SN-38 (2 pM) 18.1 1.49 21.9 3.46 37.4 4.04 16.5 1.57 [1'=0.008-] Lp=o.016-] Lpfo.oosJ Lp=o.oo8J
Transcript of Transcriptional up-regulation of transforming growth factor-β (TGF-β) receptors by TGF-β1...
A626 AGA ABSTRACTS
• G2574
EXPRESSION OF Fas L I G A N D AND PROGRESSION OF THE HUMAN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS. Y. Kitanawa. M. Ueda, N. Ando, S. Ozawa, M. Kitajima, Department of Surgery~ Keio University, Tokyo, Japan
FasL and its receptor, Fas regulate immune system through the induction of apoptosis in the target cells. Recently, expression of FasL in several human cancer cell lines and resected tumors has been demonstrated. However, the expression of FasL in human esophageal squamous cell carcinomas (ESCC) and its clinical significance are still unknown. In the present study, the relationship between the expression of FasL and clinicopathological parameters of human ESCC has been investigated. Methods: Expression of FasL in the primary tumors of 54 cases with ESCC has been examined by immunohistochemistry using monoclonal antibody raised to residues 116-277 of human FasL (Transduction Lab). The killing effect of ESCC cell lines (TE 2, 4, 5, 8, 9) in the coculture with Fas-sensitive human T cell line (Jurkat) was tested according to the previously described methods 1). Results: Twenty eight of 54 cases (52%) were scored FasL positive (cytoplasmic staining was seen in greater than 10% of cancer cells). The incidence of FasL expression was significantly (p<0.01) higher in the cases with lymph node metastasis (69%) than that of the case without lymph node metastasis(27%). Also the expression of FasL was significantly correlated to the depth of invasion (p<0.01). In the coculture experiments, the killing effect of TE cell lines to Jurkat was compatible to the expression level of FasL(TE 4 with relatively high level of FasL expression killed 62% of Jurkat ceils by 24 h coculture). Conclusions: These results suggest the possibility that FasL expression is closely correlated to the progression of human ESCC through the protection of tumor ceils from immune response by T cells which are sensitive to the FasL-Fas mediated apnptosis. [1] Niehans GA et al; Cancer Res. 57, 1007-1012, 1997
• G2575 TRANSCRIPTIONAL UP-REGULATION OF TRANSFORMING GROWTH FACTORq3 (TGFq3) RECEPTORS BY TGF-~I CORRELATES W I T H EXPRESSION OF CYCLIN D-DEPENDENT KINASE INFIIBITORS. Jt~rg Kleeff and Murray Korc, Departments of Medicine, Biological Chemistry and Pharmacology, University of California, Irvine, California 92697, USA
AIM: TGF-[31 is a multifunctional polypeptide that binds to specific cell surface receptors and suppresses epithelial cell growth. In the present study we investigated the actions of TGF-131 on cell proliferation in relation to cyclin D-dependent kinase inhibitor expression and ,TGF-13 receptor modulation in COLO-357 pancreatic cancer cells. RESULTS: TGF-131 inhibited the growth of COLO-357 cells in a time and dose dependent manner. TGF-131 also caused a delayed but sustained increase in protein levels of the cyclin D-dependent kinase inhibitors p15 Inlc4B, p21C|pl, and p27 l°pl as well as a delayed but sustained increase in TI3RI and TI3RII mRNA and protein levels. In contrast, TGF-131 caused a rapid but transient increase in plasminogen activator inhibitor-I (PAl-I) and insulin like growth factor binding protein-3 (IGFBP-3) mRNA levels. The protein synthesis inhibitor cycloheximide completely blocked the TGF-131 mediated increase in TI3RI and TI3RII expression. Furthermore, a nuclear runoff transcription assay revealed that the increase in receptor mRNA levels was due to newly transcribed RNA. There was a significant increase in TI3RI and TI3RII mRNA levels in confluent cells in comparison to subconfluent (< 80% confluent) controls, as well as in serum starved ceils when compared with cells incubated in medium containing 10% FBS. COLO-357 cells expressed a normal SMAD-4 gene as determined by Northern blot analysis and sequencing. CONCLUSION: These results indicate that TGF-131 is capable of modulating a variety of functions in COLO-357 pancreatic cancer cells and that cell density and serum can modulate TGF-13 receptor expression. In addition, the kinetics of the TGF-131-mediated transcriptional up-regulation of TGF-13 receptors suggests that this up-regulation may have the potential to maximize TGF-131-dependent antiproliferative responses.
• G2576 PKC ISOFORMS IN INTESTINAL MUCOSA AND ADENOMAS OF APC MIN MICE. Irene K. Klein. Steven R. Ritland, Steven C. Ziesmer, Lawrence J. Burgart, Patrick C. Roche, Sandra J. Gendler, William E. Karnes, Jr., M.D. Colorectal Neoplasia Group, Mayo Clinic, Rochester, MN; Mayo Clinic, Scottsdale, AZ; Brown University, Providence, RI.
BACKGROUND: Protein kinase C (PKC) PKC is reported to regulate beta-catenin levels in conjunction with the adenomatous pnlyposis coli (APC) gene product. To gain insight into the potential roles of PKC isoforms in adenomatous transformation related to APC loss, we compared the levels of PKC isoforms in normal and adenomatous mucosa of APC MIN mice and wild- type C57B1/6 mice. METHODS: Eight wild-type and 8 APC MtN mice were sacrificed at 70-100 days of age. Samples of normal colon, ileum and adenomas (when present) were snap frozen and analyzed for PKC isoform expression by immunohistochemistry with and without competing peptides to assess binding specificity. Scoring (0-4) was performed blindly. RESULTS: APC MIN and wild-type mice were indistinguishable with respect to the distributions and intensities of specific cytoplasmic staining for PKC isoforms in normal mucosa. In the colon, most isoforms showed strongest staining in the lower 3rd of the crypts and weakest staining in the upper 3rd of the crypts.
GASTROENTEROLOGY Vol. 114, No. 4
Compared to the normal colonic mucosa, staining intensities in colonic adenomas were lower for PKCs ct and ~ and higher for PKCs "/and In. Staining for PKCs "t and la was predominantly cytoplasmic in normal mucosa and predominately nuclear in colonic adenomas. In the ileum, the gradient of PKC isoform expression was reversed for most isoforms, being lowest in the crypts. As in the case for colon adenomas, ileal adenomas exhibited significantly weaker staining for PKCs a and ~ and significantly stronger staining for PKCs la and ~/compares to normal ileal mucosa. A shift toward nuclear staining was again evident for PKCs ~ and T in ileal adenomas.
~ - ~ muoo~ ~ IIQum muc~a 0 ( ~ n ade~oma 0 Ileum aderoma 4,,
a Ill 112 6 ~ n y ~ #
CONCLUSIONS: 1) PKC gradients along the crypt-villous axis are consistent with their known roles in cell growth and differentiation; 2) heterozygous loss of APC does not influence these gradients (no differences between wild-type and APC MIN mice); and 3) the decrease of PKCs ct and ~ and increase (with nuclear translocation) of PKCs "1' and g expression in adenomas of APC MIN mice suggests a role of these isoforms in adenomatous transformation resulting from loss of both APC alleles. This research was funded by NCI R29 CA71974, the Mayo Comprehensive Cancer Center and the Mayo Foundation. Irene Kline was funded by S.U.R.F.
• G2577
THE ROLE OF p161NK4A PROMOTER HYPERMETHYLATION DURING NEOPLASTIC PROGRESSION IN BARRETr'S ESOPHAGUS. B. Klumv. CJ. Hsieh, K. Holzmann, M. Gregor, R. Porschen. Department of
Internal Medicine I, University Hospital Tuebingen, Germany
Barrett's esophagus is considered as a premalignant condition predisposing to the development of esophageal adenocarcinoma. Transcriptional silencing of the tumor suppressor gene pl6INK4A by hypermethylation of its promoter region has been reported to be a predominant mechanism of gene inactivation in esophageal adenocarcinoma. This study sought to identify a possible significance of pl6INK4A hypermethylation during the process of neoplastic progression in Barrett's esophagus. 95 paraffin-embedded specimens were retrieved from 14 patients with Barrett's esophagus, who were followed over a mean time of 49.5 months. A methylation-specific PCR protocol was applied. Methylation status of the pI61NK4A promoter region was compared to histomorphological findings classified as indefinite for dysplasia, low grade or high grade dysplasia and carcinoma. 16 of 28 (57.1%) specimens with dysplasia but merely 2 of 67 specimens (3.0%) without dysplastic lesions showed hypermethylation of the p16 promoter region. Hypermethylation was detected in the majority of specimens indefinite for dysplasia (60%) and in a non-dysplastic specimen of a patient who developed dysplasia 24 months later. These data suggest that promoter hypermethylation is a common mechanism of pl61NK4a gene inactivation during neoplastic progression in Barrett's esophagus, p16 promoter hypermethylation seems to be a frequent and early occurring epigenetic abnormality during this process. Its application in the surveillance of patients with Barrett's esophagus has to be scrutinized in prospective studies. Supported by BMFT (01 KS 9602)
• (;2578
UPTAKE MECHANISM OF IRINOTECAN (CPT-11) AND ITS METABOLITE (SN-38) BY HAMSTER INTESTINAL CELLS. K. KobayashP ,~, B. Bouscarel l, Y. Matsuzaki 3, S. Ceryak I H. FrommL I. Div. Gastroenterology, Dept. Medicine, The George Washington Univ., Washington, D.C. 2. The 4th Dept. of Internal Med., Nippon Med. School, Japan, 3. Div. Gastroenterology, Inst. Clin. Med., Univ. Tsukuba, Japan.
BACKGROUND: CPT-11 is characterized by the novel mechanism of inhibiting DNA topoisomerase I and has shown promising antitumor activity clinically. CPT-11 and its active metabolite, SN-38, are eliminated by the liver, reabsorbed by the intestine, and enter enterohepatic circulation. Thus, many pharmacokinetic analyses in humans have shown its large interpatient variability. Furthermore, diarrhea (about 60 % incidence) is now recognized as the major dose-limiting toxicity of this drug. AIM: The objective was to investigate the comparative intestinal transport mechanism of CPT-11 and SN-38, in order to design new approaches to prevent the variability in pharmacokinetics and diarrhea. METHODS: The uptake rate of 14C-CPT-11 and 14C-SN-38, both as respective lactone (lact.) forms in acidic pH and anion-charged carboxylate (carbo.) forms in alkaline pH, were investigated. The rapid vacuum filtration technique was used with isolated intestinal epithelial cells of jejunum, ileum, cecum and colon from Golden Syrian hamsters. RESULTS: Comparative uptake rates (n mol / 106 cells /sec) were:
jejunum ileum cecum colon lact. carbo, lact. carbo, lact. carbo, lact. carbo.
CPT-11 (20 pM) 279 41.1 268 56.5 314 60.7 258 5.1 Lp = o.o04J Lp = o.o04J LP= o.o17J /v = o.o08J
SN-38 (2 pM) 18.1 1.49 21.9 3.46 37.4 4.04 16.5 1.57 [1'=0.008-] Lp=o.016-] Lpfo.oosJ Lp=o.oo8J
