- Email: [email protected]
Genetic alterations in colorecial carcinoma detected by comparative genomic hybridization (CGH)
1533
repair defect (MMR), which reduces their sensitivity to chemotherapeuticdrugs. We investigated if and how MMR affects the sensitivity to UCN-01. Methods: The sensitivity to UCN01 was determined in a clonogenic assay after 48 h of treatment of the cell line HCT116 (MMR-) and of the isogonic cell line HCT116+ ch3 (MMR+). Protein expressionwas detected in Western blot, cell cycle was analysedwith FACScan.Results: The MMR cell line was more resistant to UCN-Ol in clonogenic assay than the MMR+ cell line. In the resistant cell line apoptosis was detectable by PARPfragmentation and cell death detection ELISA assay but little GO/G1arrest. The presenceof both p53wt and a defect in MMR appearedto be necessary for apoptosis induction. The susceptible, MMR+ cells reacted to treatment with a massive GO/G1 arrest, without apoptosis. The arrest was concomitant with a decreaseof the relative amount of phosphorylated Rb protein. Cells lacking both MMR and p53 (HCT116, p53-~-) showed neither apoptosis nor GO/G1 arrest after treatment. Conclusion: Mismatch repair defect causes an increase of resistance of colon carcinoma cells to treatment with UCN-Ol. The maintenanceof Rb protein phosphorylation in the MMR- cells prevents an effective GO/ G1 arrest and allows proliferation of these cells. The defects of mismatch repair in sporadic colon carcinoma as well as in patients with HNPCCmay therefore reduce the effectiveness of UCN-01 treatment.
Genetic Alterations in Colorectal Carcinoma Detected by Comparative Genomic Hybridization(CGR) Seunja Park, Kosin Univ, Gospel Hosp, Pusan South Korea; Sunhoe Koo, Chungnam National Univ, DaejeonSouth Korea; Mooin Park, Jayoung Koo, Kosin Univ, Gospel Hosp, Pusan South Korea Backgrounds: Comparativegenomic hybridization(CGH)is a powerful new technique for the molecular cytogeneticanalysisof cancer, and used to detect amplified and/or deletedchromosomal regions in tumors by mapping their locations on normal metaphasechromosomes. Methods : Twenty-five colorectal carcinomas were screened for chromosomal aberrations using CGH. Results : 1) All carcinomas had chromosomal aberrations,and the mean number of chromosomal aberrations per tumor was 5.3. Gain of chromosomal material was more common than loss (gain/loss ratio 1.3:1). 2) Therewas no differences in gonenumber changes according to the status of CEA value, histological differentiation, but the cases with changes in total gene number and amplification were significantly higher in lymph node-positivegroup and advancedstage group(Ill-IV), compared to lymph node-negativegroup and early stage group(stage I-II), respectively(p
1535 Frequent Frsmeshifl Mutations of the RAD50 Recombinational DNA Repair Gene in Colorental Cancers with Microsatellite Instability Tsuneo Ikenoue,the Institute for Adult Diseases, Asahi Life Fdn, Tokyo Japan; Goichi Togo, Hideaki ijichi, Jun Kato, Yutaka Yamaji, Makoto Okamoto, Naoya Kato, Univ of Tokyo, Tokyo Japan; Atsushi Tanaka, Masayuki Matsumura, the Institute for Adult Diseases, Asahi Life Fdn, Tokyo Japan; Takao Kawabe,Yasushi Shiratori, Masao Omata, Univ of Tokyo, Tokyo Japan (BACKGROUND)Microsatelliteinstability (MSI) is found in hereditarynon-polyposis colorectal cancers (HNPCC),as well as in some sporadic colorectal cancers. A protein encoded by the RAD50 gone plays an important role in the recombinational DNA repair which is related to chromosomal stability. In its coding region this protein presents an (A)9 repeat and an (A)8 repeatthat are potential targets of mutations in tumors with MSI. (METHODS)Nine coloractal cancer cell lines (5 USI-positive and 4 MSl-negative) and 49 primary colorectal tumors (13 MSI-H, 8 MSI-L, and 28 MSI-negative)were examined.PCR primers for (A)9 and (A)8 tracts were preparedon the basis of the sequenceobtainedfrom Genbankdatabase.Forwardprimers were fluuorascently labeled. Length of the PCR products were analysed on a denaturing polyacrylamide gel electrophorasis. Expression of RAD50 proteins in the cell lines were examined by immunoblot. (RESULTS)Sixty percent (3 out of 5) of MSI-H coloreetal cancer cell lines and 46% (6 out of 13) of MSI-H primary colorectal tumors were found to have a heterozygous 1-bp deletion or insertion in the (A)9 repeat but not in the (A)8 repeat of this gene. In contrast, frameshift muations in either the (A)9 or (A)8 repeat were not found in any of the 5 MSI-negativecolorectal cancer cell lines, 8 MSI-L or 28 MSl-negativecolorectal tumors. The expression of mutant RADSOproducts was suppressed in RAD60 mutated cell lines, while the expressionof normal RAD50 products in these cell lines was lower than that in RAD50 wild-type cell lines. (CONCLUSION)These results suggest that RAD50 frameshift mutations may play a role in the tumorigenesis of MSI-H colorectal cancers.
1534 Downregulation of Hnmeobox Genes HOXl, HOX2 and HOX7 in Metastatic Colon Cancer Sergio Huerta, Jeff Sebastian, David Heber, UCLA Ctr for Human Nutrition, Los Angeles, CA; Julio Licinio, Ma-Li Wong, Jonas Hannestad,UCLA, Los Angeles, CA; Edward H. Livingston, VA Greater Los Angeles and UCLA, Los Angeles, CA Background/Objectives:Homeoboxproteins are transcription factors that simultaneouslyregulate entire systems of gene expression.During embryogenesisthey mediatetissue differentiation. In adults they establish cell and organ identity. The dedifferantiation occurring with malignancy resembles reversed embryogenesis and disordered homeobox expression has recently been described for colon cancer (Vider et al. Bioch. Biophys. Res. Com. 272:513518, 2000). We applied large scale expression array analysis to a model of colon cancer progression to determine what changes occur in homeohox genes as these cancers develop. Methods: Cell lines SW480 (primary colon cancer) and SW620 (metastatic lesion from the same patient) were obtained from the ATCC and grown under standard conditions, mRNA was extracted as previously described (Roesler et al. Am. J. Surg 174:251-257, 1997.), converted to cDNA then hybridized to a GeneChipas per standard protocol for the device. CDNA was also hybridized to an Atlas-both that contains 590 genes to verify the GeneChip results. Results: See Table. No expression for HOX genes 3, 5, 11, and 13 were detected with the microarray. The Atlas blot detected 8.0 % of GAPDH control expression for HOX11 in SW480 that was reduced to 5.1% in SW620 Conclusions: Both techniques confirmed strong expression of HOX 7 in SW480 cells that was markedly reduced in cells from the metastatic cell line SW620. The Atlas technique revealed HOXl expression in SW480 that was reduced in SW620. These findings demonstrate that HOX1, 2, and 7 expression is lost during the progression from primary to metastatic colon cancer. Becauseof the importance in regulating entire genetic systems these changes may account for many of the genetic alterations occurring when cells acquire the metastatic phenotype.
1537 Novel Pathways to Regulate Wild Type APC Expression in a Native Hyperprolireraflng Colonic Mucosa. Shahid Umar, Andrew P. Morris, Joseph H. Sellin, Univ of Texas, Houston, TX Background: Mutated adenomatous polyposis coil (APC) protein is integral to the genesis of colon cancer. However,the role of wild type APC (wtAPC)in native epithelia in the regulation of cytosolic levels of ~catenin is poorly understood. Previously, in an in vivo model of transmissible murine colonic hyperplasia (TMCH; Gastro 118:A878,2000), we observed/~ceteninabundanceto increaseat day 1 following Citrobacterrodentium infection, with progressive rise over a period of 12 days.This was coupledwith increasesin expressionof downstream targets including cyclin D1 and c-myc, underlyingprollteration.Aim: To determinerelationships between wtAPC and ~catenin expression during pro-neoplastic hyperproliferation of the colonic mucosa. Methods: Distal colonic crypts isolated from normal or TMCH mice were fractionated into cytosolic and nuclear componentsfor Western blotting and immunoprecipitation (IP). Results: TMCH doubled crypt length within 12 days. Analysis of protein extracts at 0,1,3,6,9, and 12 days postinfection with an N-terminal APC antibody (e.APC,) revealedthe following:/) a ~312 kDa protein (p312) correspondingto full lengthAPC increasedsignificantly by day 6 (4.0-+0.85 fold), peakedby day 9 and declinedby day 12, ii) an orAPC,immunoreactive band at ~110 kDa (p110) appeared at day 9 and increased at day 12 (n=5). Re-probing with a C-terminal antibody detected only p312, suggesting that p110 is part of p312 Nterminal domain. Indeed, peptide mapping of both p312 and p110 revealedseveral common pepUdes. Following co-IP with oAPC,, ~catenin/APC association decreasedat day 12 compared to normal. Further, IP with anti/~--catenin followed by ~yAPC,blotting detected p312 but not p110, indicating lack of ~catenin binding to the lighter fragment. Nucleartranslocation of APC was undetectablein the normal crypts. TMCH crypts exhibited nuclear accumulation of p110 but not p312. Nuctearlevelswere however,much lower than cytoplasmic.Conclusions: (i) wtAPC increasesin responseto hyperproliferationduring TMCH, (ii) increasedwtAPC lags behind/}-catenin and may represent an adaptive response, (iii) cleavageof wtAPC to p110 may be a novel mechanismto maintain elevated~catenin levels during sustained colonocyte hyperproliferation. Significance: Novel alterations in APC abundanceand proteolysis during TMCH may represent a previously unrecognized mechanism by which the normal mucosa may regulate prollteration.
Expressionof HOXgenes(% ExpressionRelativeto GADPH) Cell Line
HOXI
HOX2
HOX4
HOX7
Cdx2
SW480(Array) SW62O(Array) SW480(Atlas) SW620(Atlas)
0 0 23.0 12.0
9.6 0.3 14.1 5.7
9.7 6.9 0 0
44.3 2.29 25.0 6.0
1.6 0 N/A N/A
1535 Defects in Mismatch Repair System Increase the Resistance of Colon Carcinoma Cells to the New Chemotherapeutic Agent UCN-01 (7-Hydrexystauresporine) Roberta Magrini, Mandar Bhonde, Miriam Lenz, Marie Luise Hanski, Ursula Kobalz, Michael Notter, Ernst Otto Riecken, Christoph Hanski, Universit~"tsklinikum Benjamin Franklin, Berlin Germany Introduction: UCN-01 (7-hydoxystaurosporine)is presently in Phase I clinical trials as a new antineoplastic drug as well as an enhancer of the conventional drugs like 5-FU or CPT-11. Its mechanismof action is not known. 10-15% of sporadiccoloractal cancershavea mismatch
A-297
repair defect (MMR), which reduces their sensitivity to chemotherapeuticdrugs. We investigated if and how MMR affects the sensitivity to UCN-01. Methods: The sensitivity to UCN01 was determined in a clonogenic assay after 48 h of treatment of the cell line HCT116 (MMR-) and of the isogonic cell line HCT116+ ch3 (MMR+). Protein expressionwas detected in Western blot, cell cycle was analysedwith FACScan.Results: The MMR cell line was more resistant to UCN-Ol in clonogenic assay than the MMR+ cell line. In the resistant cell line apoptosis was detectable by PARPfragmentation and cell death detection ELISA assay but little GO/G1arrest. The presenceof both p53wt and a defect in MMR appearedto be necessary for apoptosis induction. The susceptible, MMR+ cells reacted to treatment with a massive GO/G1 arrest, without apoptosis. The arrest was concomitant with a decreaseof the relative amount of phosphorylated Rb protein. Cells lacking both MMR and p53 (HCT116, p53-~-) showed neither apoptosis nor GO/G1 arrest after treatment. Conclusion: Mismatch repair defect causes an increase of resistance of colon carcinoma cells to treatment with UCN-Ol. The maintenanceof Rb protein phosphorylation in the MMR- cells prevents an effective GO/ G1 arrest and allows proliferation of these cells. The defects of mismatch repair in sporadic colon carcinoma as well as in patients with HNPCCmay therefore reduce the effectiveness of UCN-01 treatment.
Genetic Alterations in Colorectal Carcinoma Detected by Comparative Genomic Hybridization(CGR) Seunja Park, Kosin Univ, Gospel Hosp, Pusan South Korea; Sunhoe Koo, Chungnam National Univ, DaejeonSouth Korea; Mooin Park, Jayoung Koo, Kosin Univ, Gospel Hosp, Pusan South Korea Backgrounds: Comparativegenomic hybridization(CGH)is a powerful new technique for the molecular cytogeneticanalysisof cancer, and used to detect amplified and/or deletedchromosomal regions in tumors by mapping their locations on normal metaphasechromosomes. Methods : Twenty-five colorectal carcinomas were screened for chromosomal aberrations using CGH. Results : 1) All carcinomas had chromosomal aberrations,and the mean number of chromosomal aberrations per tumor was 5.3. Gain of chromosomal material was more common than loss (gain/loss ratio 1.3:1). 2) Therewas no differences in gonenumber changes according to the status of CEA value, histological differentiation, but the cases with changes in total gene number and amplification were significantly higher in lymph node-positivegroup and advancedstage group(Ill-IV), compared to lymph node-negativegroup and early stage group(stage I-II), respectively(p
1535 Frequent Frsmeshifl Mutations of the RAD50 Recombinational DNA Repair Gene in Colorental Cancers with Microsatellite Instability Tsuneo Ikenoue,the Institute for Adult Diseases, Asahi Life Fdn, Tokyo Japan; Goichi Togo, Hideaki ijichi, Jun Kato, Yutaka Yamaji, Makoto Okamoto, Naoya Kato, Univ of Tokyo, Tokyo Japan; Atsushi Tanaka, Masayuki Matsumura, the Institute for Adult Diseases, Asahi Life Fdn, Tokyo Japan; Takao Kawabe,Yasushi Shiratori, Masao Omata, Univ of Tokyo, Tokyo Japan (BACKGROUND)Microsatelliteinstability (MSI) is found in hereditarynon-polyposis colorectal cancers (HNPCC),as well as in some sporadic colorectal cancers. A protein encoded by the RAD50 gone plays an important role in the recombinational DNA repair which is related to chromosomal stability. In its coding region this protein presents an (A)9 repeat and an (A)8 repeatthat are potential targets of mutations in tumors with MSI. (METHODS)Nine coloractal cancer cell lines (5 USI-positive and 4 MSl-negative) and 49 primary colorectal tumors (13 MSI-H, 8 MSI-L, and 28 MSI-negative)were examined.PCR primers for (A)9 and (A)8 tracts were preparedon the basis of the sequenceobtainedfrom Genbankdatabase.Forwardprimers were fluuorascently labeled. Length of the PCR products were analysed on a denaturing polyacrylamide gel electrophorasis. Expression of RAD50 proteins in the cell lines were examined by immunoblot. (RESULTS)Sixty percent (3 out of 5) of MSI-H coloreetal cancer cell lines and 46% (6 out of 13) of MSI-H primary colorectal tumors were found to have a heterozygous 1-bp deletion or insertion in the (A)9 repeat but not in the (A)8 repeat of this gene. In contrast, frameshift muations in either the (A)9 or (A)8 repeat were not found in any of the 5 MSI-negativecolorectal cancer cell lines, 8 MSI-L or 28 MSl-negativecolorectal tumors. The expression of mutant RADSOproducts was suppressed in RAD60 mutated cell lines, while the expressionof normal RAD50 products in these cell lines was lower than that in RAD50 wild-type cell lines. (CONCLUSION)These results suggest that RAD50 frameshift mutations may play a role in the tumorigenesis of MSI-H colorectal cancers.
1534 Downregulation of Hnmeobox Genes HOXl, HOX2 and HOX7 in Metastatic Colon Cancer Sergio Huerta, Jeff Sebastian, David Heber, UCLA Ctr for Human Nutrition, Los Angeles, CA; Julio Licinio, Ma-Li Wong, Jonas Hannestad,UCLA, Los Angeles, CA; Edward H. Livingston, VA Greater Los Angeles and UCLA, Los Angeles, CA Background/Objectives:Homeoboxproteins are transcription factors that simultaneouslyregulate entire systems of gene expression.During embryogenesisthey mediatetissue differentiation. In adults they establish cell and organ identity. The dedifferantiation occurring with malignancy resembles reversed embryogenesis and disordered homeobox expression has recently been described for colon cancer (Vider et al. Bioch. Biophys. Res. Com. 272:513518, 2000). We applied large scale expression array analysis to a model of colon cancer progression to determine what changes occur in homeohox genes as these cancers develop. Methods: Cell lines SW480 (primary colon cancer) and SW620 (metastatic lesion from the same patient) were obtained from the ATCC and grown under standard conditions, mRNA was extracted as previously described (Roesler et al. Am. J. Surg 174:251-257, 1997.), converted to cDNA then hybridized to a GeneChipas per standard protocol for the device. CDNA was also hybridized to an Atlas-both that contains 590 genes to verify the GeneChip results. Results: See Table. No expression for HOX genes 3, 5, 11, and 13 were detected with the microarray. The Atlas blot detected 8.0 % of GAPDH control expression for HOX11 in SW480 that was reduced to 5.1% in SW620 Conclusions: Both techniques confirmed strong expression of HOX 7 in SW480 cells that was markedly reduced in cells from the metastatic cell line SW620. The Atlas technique revealed HOXl expression in SW480 that was reduced in SW620. These findings demonstrate that HOX1, 2, and 7 expression is lost during the progression from primary to metastatic colon cancer. Becauseof the importance in regulating entire genetic systems these changes may account for many of the genetic alterations occurring when cells acquire the metastatic phenotype.
1537 Novel Pathways to Regulate Wild Type APC Expression in a Native Hyperprolireraflng Colonic Mucosa. Shahid Umar, Andrew P. Morris, Joseph H. Sellin, Univ of Texas, Houston, TX Background: Mutated adenomatous polyposis coil (APC) protein is integral to the genesis of colon cancer. However,the role of wild type APC (wtAPC)in native epithelia in the regulation of cytosolic levels of ~catenin is poorly understood. Previously, in an in vivo model of transmissible murine colonic hyperplasia (TMCH; Gastro 118:A878,2000), we observed/~ceteninabundanceto increaseat day 1 following Citrobacterrodentium infection, with progressive rise over a period of 12 days.This was coupledwith increasesin expressionof downstream targets including cyclin D1 and c-myc, underlyingprollteration.Aim: To determinerelationships between wtAPC and ~catenin expression during pro-neoplastic hyperproliferation of the colonic mucosa. Methods: Distal colonic crypts isolated from normal or TMCH mice were fractionated into cytosolic and nuclear componentsfor Western blotting and immunoprecipitation (IP). Results: TMCH doubled crypt length within 12 days. Analysis of protein extracts at 0,1,3,6,9, and 12 days postinfection with an N-terminal APC antibody (e.APC,) revealedthe following:/) a ~312 kDa protein (p312) correspondingto full lengthAPC increasedsignificantly by day 6 (4.0-+0.85 fold), peakedby day 9 and declinedby day 12, ii) an orAPC,immunoreactive band at ~110 kDa (p110) appeared at day 9 and increased at day 12 (n=5). Re-probing with a C-terminal antibody detected only p312, suggesting that p110 is part of p312 Nterminal domain. Indeed, peptide mapping of both p312 and p110 revealedseveral common pepUdes. Following co-IP with oAPC,, ~catenin/APC association decreasedat day 12 compared to normal. Further, IP with anti/~--catenin followed by ~yAPC,blotting detected p312 but not p110, indicating lack of ~catenin binding to the lighter fragment. Nucleartranslocation of APC was undetectablein the normal crypts. TMCH crypts exhibited nuclear accumulation of p110 but not p312. Nuctearlevelswere however,much lower than cytoplasmic.Conclusions: (i) wtAPC increasesin responseto hyperproliferationduring TMCH, (ii) increasedwtAPC lags behind/}-catenin and may represent an adaptive response, (iii) cleavageof wtAPC to p110 may be a novel mechanismto maintain elevated~catenin levels during sustained colonocyte hyperproliferation. Significance: Novel alterations in APC abundanceand proteolysis during TMCH may represent a previously unrecognized mechanism by which the normal mucosa may regulate prollteration.
Expressionof HOXgenes(% ExpressionRelativeto GADPH) Cell Line
HOXI
HOX2
HOX4
HOX7
Cdx2
SW480(Array) SW62O(Array) SW480(Atlas) SW620(Atlas)
0 0 23.0 12.0
9.6 0.3 14.1 5.7
9.7 6.9 0 0
44.3 2.29 25.0 6.0
1.6 0 N/A N/A
1535 Defects in Mismatch Repair System Increase the Resistance of Colon Carcinoma Cells to the New Chemotherapeutic Agent UCN-01 (7-Hydrexystauresporine) Roberta Magrini, Mandar Bhonde, Miriam Lenz, Marie Luise Hanski, Ursula Kobalz, Michael Notter, Ernst Otto Riecken, Christoph Hanski, Universit~"tsklinikum Benjamin Franklin, Berlin Germany Introduction: UCN-01 (7-hydoxystaurosporine)is presently in Phase I clinical trials as a new antineoplastic drug as well as an enhancer of the conventional drugs like 5-FU or CPT-11. Its mechanismof action is not known. 10-15% of sporadiccoloractal cancershavea mismatch
A-297