relationship between apc and cox-2 gene expression in colorectal cancer

1
AM AGA ABSTRACTS GASTROENTEROLOGY Vol. 118, No.4 Mean Basal and Peak PGE" PGHS-2 Protein and mRNA Levels RLU-Relative Light Units pvalues are forcomparison with corresponding Group I basal orpeak level. Group I (n=2) II (n=13) III(n=4) PGE2 Basal 7±2 23±7 (p=0,12) 59±27 (p=0.02) (ng/mg protein) Peak 33±6 190±29 (p=0.001) 269±47 (p<0.0001) PGHS·2 protein (RLU) Basal 6±3 34±11 (p=0.07) 124±97 (p=0.01) Peak 10±0 357±163 (p<0.OOO1) 221±112 (p=0.0002) PGHS·2 mRNA (RLU) Basal 4±0 121±61 (p=0.002) 28±19 (p=0.009) Peak 32±0 878±287 (p=0.0004) 1205±953 (p=0.0002) 483 CHARACTERIZATION OF THE PPAR-,1 ACTIVATION FUNC· TION-2 DOMAIN. Wade F. Krause, Rajnish A. Gupta, Raymond N. DuBois, Vanderbilt Univ Med Ctr, Nashville, TN; Vanderbilt Univ Sch of Medicine, Nashville, TN. Introduction: PPARIl is a member of the nuclear receptor superfamily. Recent studies have shown that PPARIl is downstream of the APC signal- ing pathway and upregulated in colorectal tumors. This points to the possibility that PPARIl may be involved in colorectal carcinogenesis. Here we utilized site-directed mutagenesis to characterize the role of the C- terminal Activator Function-2 (AF-2) domain of PPARIl in ligand depen- dent transactivation, as well as to identify dominant negative acting PPARIl mutants that can be used to explore the role of PPARIl in colorectal cancer. Methods: High fidelity PCR was employed to amplify both wild type(WT) and mutant forms of the human PPARIl coding region. Each amplicon was 500 04 HT·29APC 200 .01 500 .139 HT.29f3gal 200 .045 500 .52 HT·29 200 .57 eeliline Cone. of Zine(llm) MeanMCt MCtvalues 482 VARYING LEVELS OF PROSTANOID SYNTHESIS IN FmRO- BLASTS FROM NORMAL AND NEOPLASTIC COLON. Edward C. Kim, Shaopeng Zheng, Yingting Zhu, Peter Lance, VA Med Ctr, Buffalo, NY; SUNY and VA Med Ctr, Buffalo, NY. Colonic fibroblasts are a potent source of prostanoids and it is increasingly clear that they are crucial determinants of epithelial cell behavior. In this study we have further characterized prostanoid biogenesis in an expanded panel of human fibroblast strains established from colonoscopic biopsies taken from normal and diseased mucosa. Prostaglandin (PG) E z production in fibroblast cultures was measured, by radioimmunoassay, before and after stimulation with interleukin (IL)-113 (10 ng/ml). PGH synthase-Z (PGHS-2 or COX-2) mRNA and protein levels, respectively, were assessed by Northern and Western analysis. Fibroblast strains were classified according to the clinical characteristics of the patient and the site of colonoscopic biopsy: Group I strains were initiated from normal mucosa obtained at a normal total colonoscopy, Group II strains were from normal colonic mucosa remote from an advanced colorectal adenoma(s) or carcinoma, and Group III strains were directly from neoplastic mucosa. As depicted in the table below, mean peak levels of PGE z, PGHS-2 protein and mRNA in Groups II and III were significantly higher than in Group I. Mean basal levels of PGE z, PGHS-2 protein and mRNA in Group III were also significantly higher than in Group I. Mean basal levels of PGE z, PGHS-2 protein and mRNA in Group II were higher than in Group I but reached significance only with respect to mRNA levels. These data reinforce the hypothesis that colonic epithelial neoplastic change is associated with increased fibroblast (submucosal) prostanoid synthetic capacity. "Field effect" alterations in fibroblast biology may contribute to development of colorectal cancer. APC delta 716 knock out mice study.( Oshima et al;Cell nov 96;87(5); 803-809)We used the HT-Z9 cell line with a zinc inducible wild type APC gene to study the relationship between APC and COX-Z. Methods: HT- Z9,HT-Z9 APC and HT-Z9 I3gal cell lines were kindly provided by Prof. Bert Vogelstein and maintained in RPMI1640 + 10% FCS medium. ZOO and 500 ILM Zn concentrations were used to induce APC and the same concentrations of Mg was used as a negative control. Total RNA was extracted;cDNA was synthesised according to standard protocols and real time PCR performed using a reaction mix containing standardised GAPDH and COX-Z primers. L1L1Ct values were obtained after normalising for GAPDH expression. Results: COX-Z gene expression was reduced after induction of APC in HT-Z9 APC cell line. Zinc up-regulated COX-Z gene levels in negative control cell lines and a similar concentration of Mg. had the same effect. Mg up-regulated COX-Z gene expression in the HT-Z9 APC cell line,indicating the down-regulation of COX-Z was specifically due to the induction of the APC gene by Zinc. Conclusion: APC induction seems to reduce the COX-Z gene levels in a colorectal tumour cell line. Identification of APC mutations and other genetic events will pave the way for designing new anti-cancer drugs 480 NICOTINE UPREGULATES COX·2 AND BFGF IN RAS-TRANS- FORMED INTESTINAL CELLS, BUT NOT IN NON-TRANS- FORMED CELLS - POSSmLE ROLE OF SMOKING ON TUMOR ANGIOGENESIS -. Yoshimi Kakiuchi, Shingo Tsuji, Masahiko Tsujii, Naoki Kawai, Masakazu Yasumaru, Arata Kimura, Yutaka Sasaki, Masatsugu Hori, Sunao Kawano, Osaka Univ Grad Sch of Medicine, Suita, Japan. Background and Aim: Recent studies suggest that cyclooxygenase (COX) and ras-activation are involved in colon carcinogenesis. Basic fibroblast growth factor (bFGF) stimulates tumor angiogenesis and also may play a role in progression of cancer. Although smoking has been thought to be one of the risk factors of gastro-intestinal cancer, the influences of smoking on COX-Z and tumor angiogenesis have not been clarified. We investigated the effects of nicotine on COX-Z expression and on bFGF release in non-transformed and ras-transformed intestinal epithelial cells. Materials and Methods: Non-transformed rat intestinal epithelial (RIE) cells were transfected with pCB-7/neo vector containing Il-ras, and designated as RIE-ras. RIE cells transfected with the vector (RIE-neo) were used as the control. After serum starvation for 3 days, both cell lines were stimulated with 10% fetal calf serum in the presence or the absence of nicotine. The COX-Z expression was examined using Western blotting analysis. bFGF levels were measured using ELISA. Results: Incubation with fetal calf serum for 6 hours increased COX-Z expression in RIE-neo and in RIE-ras. Nicotine enhanced COX-Z expression in RIE-ras, while it inhibited COX-Z expression in non-transformed RIE-neo. Incubaton with fetal calf serum for 6 hours increased bFGF release. Nicotine stimulated bFGF release in RIE-ras, while it inhibited bFGF release in RIE-neo. Conclusion: Nicotine induced COX-Z expression in ras-transformed cells leading to increased bFGF production, while it inhibited COX-Z expression and decreased bFGF production in non-transformed epithelial cells. Therefore, smoking have an important role in promoting tumor angiogenesis possibly by upregulating COX-Z' 481 RELATIONSHIP BETWEEN APC AND COX-2 GENE EXPRES- SION IN COLORECTAL CANCER. Giridhar Katti, Daniel F. McWilliams, Sue A. Watson, Univ Hosp, Not- tingham, United Kingdom; Cancer Study Unit, Nottingham, United King- dom. Background: APC mutations occur very early in most colorectal cancers (CRC). COX-Z is over expressed in more than 80% of CRC. Identification of APC mutations and their down stream genetic events is essential. The role of COX-Z in colorectal carcinogenesis was well demonstrated by the 479 EXPRESSION OF CYCLOOXYGENASE·2 IN HUMAN COLO· RECTAL ADENOMAS. Kumi Hasegawa, Takeshi Fujita, Keigo Yoshinaga, Hidefumi Tsunozaki, Ryo Ohno, Jun Kaneko, Hideaki Iseki, Yohko Takagi, Wataru Ichikawa, Kenichi Sugihara, Tokyo Med and Dental Univ, Tokyo, Japan. Cyclooxygenase-2(COX-2) is involved in the pathway of colorectal tumor- igenesis. The molecular mechanisms underlying COX-Z's contribution to colorectal tumorigenesis are still not clear. The purpose of this study was to evaluate the role of COX-2 in human sporadic colorectal adenoma by using reverse transcription-polymerase chain reaction (RT-PCR) and im- munohistochemical study. Materials and methods: Colorectal polyps com- pletely excised by endoscopic polypectomy or surgical resection were cut vertically into halves. One half of the polyp was stored at -80 0 for extraction ofmRNA. The other half was fixed in 10% buffered formalin for histological and immunohistochemical examination. Histologically veri- fied 63 sporadic adenoma specimens and 46 normal colorectal mucosae were examined for the COX-Z mRNA expression. To estimate the COX-Z mRNA levels, RT-PCRs were performed for both COX-l and COX-Z concomitantly in the same tube on each frozen sample, and COX-Z index was designated by the band density ratio of COX-Z/COX-l, because COX-l mRNA is expressed constitutively. To investigate the correlation between the COX-2 mRNA and protein, 50 specimens of adenomas and 40 colorectal carcinomas were examined by immunohistochemical staining for COX-2 protein. All results are shown as means ± SE. Results: The COX-Z mRNA expression was detected in all adenomas. The COX-2 indices of adenomas were significantly higher than that of normal mucosae (adenoma; 1.7 ± O.Z, normal mucosa 0.3 ± 0.1; p < 0.001). No correlation was observed between the COX-2 indices and macroscopic shape, location or dysplasia. The COX-Z indices of small adenomas(O -s 6 mm in maximum diameter) , medium(6 mm<O:::;IZ mm), and large(O<IZ mm) were 1.1 ± 0.1, 1.7 ± 0.3 and Z.5 ± 0.5 , respectively; The COX-Z mRNA in adenomas increased in a size-dependent manner (p = 0.019). In immu- nohistochemistry, the staining intensities of COX-2 protein correlated with the COX-2 mRNA levels and the size of adenoma. The location of COX-Z protein differed by the size of adenoma; In large adenomas, as well as carcinomas, COX-Z protein mainly located in cytoplasmic regions of tumor cells. While in small adenomas, the COX-Z protein was found in the interstitial cells, rather than in adenoma epithelium, itself. Conclusion: The COX-Z expression in terms of mRNA and protein in colorectal adenoma increased in a size dependent manner. These results suggest that COX-Z plays an important role in growth of human sporadic colorectal adenoma.

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AM AGA ABSTRACTS GASTROENTEROLOGY Vol. 118, No.4

Mean Basal and Peak PGE" PGHS-2 Protein and mRNA Levels

RLU-Relative Light Unitspvalues are forcomparison with corresponding Group I basal orpeak level.

Group I (n=2) II(n=13) III(n=4)

PGE2 Basal 7±2 23±7(p=0,12) 59±27 (p=0.02)(ng/mg protein) Peak 33±6 190±29 (p=0.001) 269±47 (p<0.0001)PGHS·2 protein (RLU) Basal 6±3 34±11 (p=0.07) 124±97 (p=0.01)

Peak 10±0 357±163 (p<0.OOO1) 221±112 (p=0.0002)PGHS·2 mRNA (RLU) Basal 4±0 121±61 (p=0.002) 28±19 (p=0.009)

Peak 32±0 878±287 (p=0.0004) 1205±953 (p=0.0002)

483

CHARACTERIZATION OF THE PPAR-,1 ACTIVATION FUNC·TION-2 DOMAIN.Wade F. Krause, Rajnish A. Gupta, Raymond N. DuBois, Vanderbilt UnivMed Ctr, Nashville, TN; Vanderbilt Univ Sch of Medicine, Nashville, TN.

Introduction: PPARIl is a member of the nuclear receptor superfamily.Recent studies have shown that PPARIl is downstream of the APC signal­ing pathway and upregulated in colorectal tumors. This points to thepossibility that PPARIl may be involved in colorectal carcinogenesis. Herewe utilized site-directed mutagenesis to characterize the role of the C­terminal Activator Function-2 (AF-2) domain of PPARIl in ligand depen­dent transactivation, as well as to identify dominant negative acting PPARIlmutants that can be used to explore the role of PPARIl in colorectal cancer.Methods: High fidelity PCR was employed to amplify both wild type(WT)and mutant forms of the human PPARIl coding region. Each amplicon was

50004

HT·29APC

200.01

500.139

HT.29f3gal

200.045

500.52

HT·29

200.57

eeliline

Cone. ofZine(llm)MeanMCt

MCtvalues

482

VARYING LEVELS OF PROSTANOID SYNTHESIS IN FmRO­BLASTS FROM NORMAL AND NEOPLASTIC COLON.Edward C. Kim, Shaopeng Zheng, Yingting Zhu, Peter Lance, VA MedCtr, Buffalo, NY; SUNY and VA Med Ctr, Buffalo, NY.

Colonic fibroblasts are a potent source of prostanoids and it is increasinglyclear that they are crucial determinants of epithelial cell behavior. In thisstudy we have further characterized prostanoid biogenesis in an expandedpanel of human fibroblast strains established from colonoscopic biopsiestaken from normal and diseased mucosa. Prostaglandin (PG) Ez productionin fibroblast cultures was measured, by radioimmunoassay, before and afterstimulation with interleukin (IL)-113 (10 ng/ml). PGH synthase-Z (PGHS-2or COX-2) mRNA and protein levels, respectively, were assessed byNorthern and Western analysis. Fibroblast strains were classified accordingto the clinical characteristics of the patient and the site of colonoscopicbiopsy: Group I strains were initiated from normal mucosa obtained at anormal total colonoscopy, Group II strains were from normal colonicmucosa remote from an advanced colorectal adenoma(s) or carcinoma, andGroup III strains were directly from neoplastic mucosa. As depicted in thetable below, mean peak levels of PGEz, PGHS-2 protein and mRNA inGroups II and III were significantly higher than in Group I. Mean basallevels of PGEz, PGHS-2 protein and mRNA in Group III were alsosignificantly higher than in Group I. Mean basal levels of PGEz, PGHS-2protein and mRNA in Group II were higher than in Group I but reachedsignificance only with respect to mRNA levels. These data reinforce thehypothesis that colonic epithelial neoplastic change is associated withincreased fibroblast (submucosal) prostanoid synthetic capacity. "Fieldeffect" alterations in fibroblast biology may contribute to development ofcolorectal cancer.

APC delta 716 knock out mice study.( Oshima et al;Cell nov 96;87(5);803-809)We used the HT-Z9 cell line with a zinc inducible wild type APCgene to study the relationship between APC and COX-Z. Methods: HT­Z9,HT-Z9 APC and HT-Z9 I3gal cell lines were kindly provided by Prof.Bert Vogelstein and maintained in RPMI1640 + 10% FCS medium. ZOOand 500 ILM Zn concentrations were used to induce APC and the sameconcentrations of Mg was used as a negative control. Total RNA wasextracted;cDNA was synthesised according to standard protocols and realtime PCR performed using a reaction mix containing standardised GAPDHand COX-Z primers. L1L1Ct values were obtained after normalising forGAPDH expression. Results: COX-Z gene expression was reduced afterinduction of APC in HT-Z9 APC cell line. Zinc up-regulated COX-Z genelevels in negative control cell lines and a similar concentration of Mg. hadthe same effect. Mg up-regulated COX-Z gene expression in the HT-Z9APC cell line,indicating the down-regulation of COX-Z was specificallydue to the induction of the APC gene by Zinc. Conclusion: APC inductionseems to reduce the COX-Z gene levels in a colorectal tumour cell line.Identification of APC mutations and other genetic events will pave the wayfor designing new anti-cancer drugs

480

NICOTINE UPREGULATES COX·2 AND BFGF IN RAS-TRANS­FORMED INTESTINAL CELLS, BUT NOT IN NON-TRANS­FORMED CELLS - POSSmLE ROLE OF SMOKING ON TUMORANGIOGENESIS -.Yoshimi Kakiuchi, Shingo Tsuji, Masahiko Tsujii, Naoki Kawai,Masakazu Yasumaru, Arata Kimura, Yutaka Sasaki, Masatsugu Hori,Sunao Kawano, Osaka Univ Grad Sch of Medicine, Suita, Japan.

Background and Aim: Recent studies suggest that cyclooxygenase (COX)and ras-activation are involved in colon carcinogenesis. Basic fibroblastgrowth factor (bFGF) stimulates tumor angiogenesis and also may play arole in progression of cancer. Although smoking has been thought to be oneof the risk factors of gastro-intestinal cancer, the influences of smoking onCOX-Z and tumor angiogenesis have not been clarified. We investigatedthe effects of nicotine on COX-Z expression and on bFGF release innon-transformed and ras-transformed intestinal epithelial cells. Materialsand Methods: Non-transformed rat intestinal epithelial (RIE) cells weretransfected with pCB-7/neo vector containing Il-ras, and designated asRIE-ras. RIE cells transfected with the vector (RIE-neo) were used as thecontrol. After serum starvation for 3 days, both cell lines were stimulatedwith 10% fetal calf serum in the presence or the absence of nicotine. TheCOX-Z expression was examined using Western blotting analysis. bFGFlevels were measured using ELISA. Results: Incubation with fetal calfserum for 6 hours increased COX-Z expression in RIE-neo and in RIE-ras.Nicotine enhanced COX-Z expression in RIE-ras, while it inhibited COX-Zexpression in non-transformed RIE-neo. Incubaton with fetal calf serum for6 hours increased bFGF release. Nicotine stimulated bFGF release inRIE-ras, while it inhibited bFGF release in RIE-neo. Conclusion: Nicotineinduced COX-Z expression in ras-transformed cells leading to increasedbFGF production, while it inhibited COX-Z expression and decreasedbFGF production in non-transformed epithelial cells. Therefore, smokinghave an important role in promoting tumor angiogenesis possibly byupregulating COX-Z'

481

RELATIONSHIP BETWEEN APC AND COX-2 GENE EXPRES­SION IN COLORECTAL CANCER.Giridhar Katti, Daniel F. McWilliams, Sue A. Watson, Univ Hosp, Not­tingham, United Kingdom; Cancer Study Unit, Nottingham, United King­dom.

Background: APC mutations occur very early in most colorectal cancers(CRC). COX-Z is over expressed in more than 80% of CRC. Identificationof APC mutations and their down stream genetic events is essential. Therole of COX-Z in colorectal carcinogenesis was well demonstrated by the

479

EXPRESSION OF CYCLOOXYGENASE·2 IN HUMAN COLO·RECTAL ADENOMAS.Kumi Hasegawa, Takeshi Fujita, Keigo Yoshinaga, Hidefumi Tsunozaki,Ryo Ohno, Jun Kaneko, Hideaki Iseki, Yohko Takagi, Wataru Ichikawa,Kenichi Sugihara, Tokyo Med and Dental Univ, Tokyo, Japan.

Cyclooxygenase-2(COX-2) is involved in the pathway of colorectal tumor­igenesis. The molecular mechanisms underlying COX-Z's contribution tocolorectal tumorigenesis are still not clear. The purpose of this study wasto evaluate the role of COX-2 in human sporadic colorectal adenoma byusing reverse transcription-polymerase chain reaction (RT-PCR) and im­munohistochemical study. Materials and methods: Colorectal polyps com­pletely excised by endoscopic polypectomy or surgical resection were cutvertically into halves. One half of the polyp was stored at -800 forextraction ofmRNA. The other half was fixed in 10% buffered formalin forhistological and immunohistochemical examination. Histologically veri­fied 63 sporadic adenoma specimens and 46 normal colorectal mucosaewere examined for the COX-Z mRNA expression. To estimate the COX-ZmRNA levels, RT-PCRs were performed for both COX-l and COX-Zconcomitantly in the same tube on each frozen sample, and COX-Z indexwas designated by the band density ratio of COX-Z/COX-l, becauseCOX-l mRNA is expressed constitutively. To investigate the correlationbetween the COX-2 mRNA and protein, 50 specimens of adenomas and 40colorectal carcinomas were examined by immunohistochemical stainingfor COX-2 protein. All results are shown as means ± SE. Results: TheCOX-Z mRNA expression was detected in all adenomas. The COX-2indices of adenomas were significantly higher than that of normal mucosae(adenoma; 1.7 ± O.Z, normal mucosa 0.3 ± 0.1; p < 0.001). No correlationwas observed between the COX-2 indices and macroscopic shape, locationor dysplasia. The COX-Z indices of small adenomas(O -s 6 mm inmaximum diameter) , medium(6 mm<O:::;IZ mm), and large(O<IZ mm)were 1.1 ± 0.1, 1.7 ± 0.3 and Z.5 ± 0.5 , respectively; The COX-Z mRNAin adenomas increased in a size-dependent manner (p = 0.019). In immu­nohistochemistry, the staining intensities of COX-2 protein correlated withthe COX-2 mRNA levels and the size of adenoma. The location of COX-Zprotein differed by the size of adenoma; In large adenomas, as well ascarcinomas, COX-Z protein mainly located in cytoplasmic regions of tumorcells. While in small adenomas, the COX-Z protein was found in theinterstitial cells, rather than in adenoma epithelium, itself. Conclusion: TheCOX-Z expression in terms of mRNA and protein in colorectal adenomaincreased in a size dependent manner. These results suggest that COX-Zplays an important role in growth of human sporadic colorectal adenoma.