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Asparagine deamidation as a novel posttranslational modification of Bcl-xL
AGAA443
April 2000
expression. This is clinically significant since human colorectal tumors frequently display increased expression of these proteins. 4. The AS-bak 1EC18 cell model system may be useful for understanding the role of bak in colorectal tumorginesis
action of 53BP2 in mitochondria. Thus, 53BP2 gene transfer may be a useful tool to induce cell death in cancer cells with mutated p53 gene.
2431
ASPARAGINE DEAMIDATION AS A NOVEL POSTTRANSLATIONAL MODIFICATION OF BCL·XL. Tetsuo Takehara, Hiroshi Takahashi, MA Gen Hosp, Harvard Med Sch, Boston, MA.
APOPTOSIS AS AN ANTI-NEOPLASTIC MECHANISM IN MICE EXPRESSING A GOBLET CELL-SPECIFIC HYBRID ONCOGENE. James R. Gum, James W. Hicks, Anne-Marie Gillespie, Elaine J. Carlson, Jose L. Rius, Charles 1. Epstein, Young S. Kim, Vamc, San Francisco, CA; UCSF, San Francisco, CA. Background: We have demonstrated that bases -2864 to + 17 of the MUC2 gene promoter drives reporter (hGH) gene expression at high levels and with high specificity in still dividing, deep crypt goblet cells of the distal small intestine (DS1) of transgenic mice (Gum et al., Am. 1. Physiol. 276: G666-G676, 1999). To develop an in vivo model for possible phenotypes including intestinal goblet cell ablation, altered cell lineage allocation, apoptosis, and neoplasia we employed the same MUC2 promoter to drive SV40 T antigen (Tag) expression in mice. Methods: Standard techniques were used to produce mice containing the 2864 base MUC2 promoter-SV 40 Tag hybrid oncogene. Intestinal goblet cell number and size was estimated following Mayers-PAS staining using a Zeiss research microscope fitted with a Hitachi video camera using Scion Image software. Apoptosis in situ was evaluated using the TUNEL assay with results confirmed using standard histological criteria. Results: Five independent transgenic mice were produced that incorporated I to ~ 8 copies of the hybrid oncogene. All of these mice died within 6 months with tumors growing in various locations including the salivary gland, DSI, cervix, spleen, and colon. A line was established from one mouse (MUCTag6) prior to death allowing detailed analysis of intestinal epithelial alterations in pre-pathological offspring. Similar to the previous MUC2lhGH transgenies, MUCTag6 mice expressed transgene product (Tag) at high levels in the middle and DSI. Message levels of absorptive cell lineage markers dppiv and fabi were unchanged between MUCTag6 and non-transgenic littermates while muc2, a goblet cell marker, was decreased to about 113 the normal level. Marked goblet cell involution was apparent in MUCTag6 DSI, the average length of the mucus (PAS)-staining supranuclear region was 4.l:t1.4 JAM in MUCTag6 vs 7.4:t1.8 JAM in normal (p
2433
Posttranslational modification including phosphorylation, caspase-mediated cleavage, and conformational changes has been proposed to regulate the function of BcI-2 family molecules. Here we show evidence that BcI-xL, an anti-apoptotic member of the BcI-2 family, is deamidated in vivo, and that its alteration may be involved in hepatocarcinogenesis. We employed 20 surgically resected hepatocellular carcinomas (HCCs) and adjacent non-tumorous livers as well as 10 normal livers to investigate expression of BcI-xL. BcI-xL was detected in all liver tissues by Western blot analysis and its expression was higher in HCC than in non-tumorous or normal livers. BcI-xL migrated as 30 and 32 kDa doublet bands; the 32 kDa band was the major form of BcI-xL in normal and non-tumor livers, while the 30 kDa band was predominantly found in a majority of HCCs. Similarly, human hepatoma cell lines expressed BcI-xL as doublet bands, and 30 kDa band was more intense than 32 kDa band. Since vincristine and paclitaxel have been shown to phosphorylate BcI-xL, we treated human hepatoma cells with these rnicrotuble-disrupting drugs. Phosphorylated BcI-xL migrated more slowly and was detected as a third band on Western blot. This third band was completely eliminated, when cell Iysates were treated with phosphatase. To the contrary, the same treatment did not affect the density or migration pattern of the 30 and 32 kDa bands, indicating that these BcI-xL bands are not phosphorylated. Human BcI-xL protein contains three -Asn-Gly- sequences, which are susceptible to asparagine deamidation. To examine the possibility that the difference in electrophoretic mobility may result from deamidation of BcI-xL, the Iysates from liver tissues and hepatoma cell lines were treated with an alkaline buffer that promotes asparagine deamidation, and then subjected to Western analysis. The alkaline treatment resulted in an electrophoretic mobility shift of the 30 kDa band to 32 kDa. More importantly, deamidatin was confirmed by the detection of isoaspartate, a product of asparagine deamidation, in recombinant BcI-xL that was treated with the same alkaline buffer. These results indicate that the 32 kDa molecule is mainly generated from the 30 kDa BcI-xL as a result of asparagine deamidation. This is the first demonstration of BcI-xL deamidation in vivo and its modified expression in human HCCs. Since deamidation plays a critical role in protein conformation, function and degradation, its alteration may be tied to anti-apoptotic function of BcI-xL during hepatocarcinogenesis. 2434 FAS LIGAND MEDIATES IMMUNE PRIVILEGE AND NOT NEUTROPHIL RECRUITMENT IN COLON CANCER IN VIVO. Aileen Houston, Frank Waldron-Lynch, Michael W. Bennett, Desmond Roche, Gerald C. O'Sullivan, John K. Collins, Fergus Lj Shanahan, Joe O'Connell, Nui, Cork, Ireland. Background: Fas ligand (FasL) triggers apoptosis - via its cognate receptor, Fas (APO-IICD95) - of activated lymphocytes entering sites of immune privilege. In contrast to evidence that FasL helps to maintain colon cancers in a state of immune privilege, overexpression of recombinant FasL in murine tumor allografts revealed a potential antitumor effect of Fasl., via recruitment of a neutrophilic infiltrate. Aim: To determine if FasL expressed in colon cancer mediates immune privilege or neutrophil recruitment. Methods: A reference section was immunohistochemically stained to identify FasL-positive and FasL-negative tumor nests within colonic adenocarcinomas (Dukes' A-D). Within both FasL-positive and FasL-negative tumor nests, we counted: (I) tumor-infiltrating lymphocytes (TILs) per 2000 total nuclei; immunohistochemical staining for CD45 facilitated the identification of TILs; (2) TILs that were undergoing apoptosis, per 500 total TILs; apoptotic TILs were identified by dual staining combining TUNEL with immunohistochemistry for CD45; (3) neutrophils per 2000 total nuclei; immunohistochemical staining for the neutrophil marker, lactoferrin, plus their characteristic multi-lobed nuclei, facilitated the identification of neutrophils. Statistics: Wilcoxon Signed Rank. Results: There were consistently fewer TILs within FasL-expressing relative to FasLnegative tumor nests within each tumor examined (mean=4.0-fold, range 1.8- to 33-fold, P < 0.001, n= 16). There was consistently two-fold more apoptosis occurring in TILs within tumor nests that expressed FasL relative to those nests that did not express FasL (mean = 1.9-fold, range 1.6- to 2.5-fold, P < 0.001, n= 14). The overall level of neutrophils within the tumors was low (mean = 0.3%, n = 16). Overall, there were twofold fewer neutrophils in tumor nests that expressed FasL compared to nests that did not express FasL (n = 16, p<0.062). Conclusions: FasL expression was consistently and significantly associated with increased apoptosis of TILs, leading to dramatically diminished TILs in FasL-positive versus -negative areas in all tumors. FasL mediates immune privilege and not neutrophil recruitment in human colon cancer.
April 2000
expression. This is clinically significant since human colorectal tumors frequently display increased expression of these proteins. 4. The AS-bak 1EC18 cell model system may be useful for understanding the role of bak in colorectal tumorginesis
action of 53BP2 in mitochondria. Thus, 53BP2 gene transfer may be a useful tool to induce cell death in cancer cells with mutated p53 gene.
2431
ASPARAGINE DEAMIDATION AS A NOVEL POSTTRANSLATIONAL MODIFICATION OF BCL·XL. Tetsuo Takehara, Hiroshi Takahashi, MA Gen Hosp, Harvard Med Sch, Boston, MA.
APOPTOSIS AS AN ANTI-NEOPLASTIC MECHANISM IN MICE EXPRESSING A GOBLET CELL-SPECIFIC HYBRID ONCOGENE. James R. Gum, James W. Hicks, Anne-Marie Gillespie, Elaine J. Carlson, Jose L. Rius, Charles 1. Epstein, Young S. Kim, Vamc, San Francisco, CA; UCSF, San Francisco, CA. Background: We have demonstrated that bases -2864 to + 17 of the MUC2 gene promoter drives reporter (hGH) gene expression at high levels and with high specificity in still dividing, deep crypt goblet cells of the distal small intestine (DS1) of transgenic mice (Gum et al., Am. 1. Physiol. 276: G666-G676, 1999). To develop an in vivo model for possible phenotypes including intestinal goblet cell ablation, altered cell lineage allocation, apoptosis, and neoplasia we employed the same MUC2 promoter to drive SV40 T antigen (Tag) expression in mice. Methods: Standard techniques were used to produce mice containing the 2864 base MUC2 promoter-SV 40 Tag hybrid oncogene. Intestinal goblet cell number and size was estimated following Mayers-PAS staining using a Zeiss research microscope fitted with a Hitachi video camera using Scion Image software. Apoptosis in situ was evaluated using the TUNEL assay with results confirmed using standard histological criteria. Results: Five independent transgenic mice were produced that incorporated I to ~ 8 copies of the hybrid oncogene. All of these mice died within 6 months with tumors growing in various locations including the salivary gland, DSI, cervix, spleen, and colon. A line was established from one mouse (MUCTag6) prior to death allowing detailed analysis of intestinal epithelial alterations in pre-pathological offspring. Similar to the previous MUC2lhGH transgenies, MUCTag6 mice expressed transgene product (Tag) at high levels in the middle and DSI. Message levels of absorptive cell lineage markers dppiv and fabi were unchanged between MUCTag6 and non-transgenic littermates while muc2, a goblet cell marker, was decreased to about 113 the normal level. Marked goblet cell involution was apparent in MUCTag6 DSI, the average length of the mucus (PAS)-staining supranuclear region was 4.l:t1.4 JAM in MUCTag6 vs 7.4:t1.8 JAM in normal (p
2433
Posttranslational modification including phosphorylation, caspase-mediated cleavage, and conformational changes has been proposed to regulate the function of BcI-2 family molecules. Here we show evidence that BcI-xL, an anti-apoptotic member of the BcI-2 family, is deamidated in vivo, and that its alteration may be involved in hepatocarcinogenesis. We employed 20 surgically resected hepatocellular carcinomas (HCCs) and adjacent non-tumorous livers as well as 10 normal livers to investigate expression of BcI-xL. BcI-xL was detected in all liver tissues by Western blot analysis and its expression was higher in HCC than in non-tumorous or normal livers. BcI-xL migrated as 30 and 32 kDa doublet bands; the 32 kDa band was the major form of BcI-xL in normal and non-tumor livers, while the 30 kDa band was predominantly found in a majority of HCCs. Similarly, human hepatoma cell lines expressed BcI-xL as doublet bands, and 30 kDa band was more intense than 32 kDa band. Since vincristine and paclitaxel have been shown to phosphorylate BcI-xL, we treated human hepatoma cells with these rnicrotuble-disrupting drugs. Phosphorylated BcI-xL migrated more slowly and was detected as a third band on Western blot. This third band was completely eliminated, when cell Iysates were treated with phosphatase. To the contrary, the same treatment did not affect the density or migration pattern of the 30 and 32 kDa bands, indicating that these BcI-xL bands are not phosphorylated. Human BcI-xL protein contains three -Asn-Gly- sequences, which are susceptible to asparagine deamidation. To examine the possibility that the difference in electrophoretic mobility may result from deamidation of BcI-xL, the Iysates from liver tissues and hepatoma cell lines were treated with an alkaline buffer that promotes asparagine deamidation, and then subjected to Western analysis. The alkaline treatment resulted in an electrophoretic mobility shift of the 30 kDa band to 32 kDa. More importantly, deamidatin was confirmed by the detection of isoaspartate, a product of asparagine deamidation, in recombinant BcI-xL that was treated with the same alkaline buffer. These results indicate that the 32 kDa molecule is mainly generated from the 30 kDa BcI-xL as a result of asparagine deamidation. This is the first demonstration of BcI-xL deamidation in vivo and its modified expression in human HCCs. Since deamidation plays a critical role in protein conformation, function and degradation, its alteration may be tied to anti-apoptotic function of BcI-xL during hepatocarcinogenesis. 2434 FAS LIGAND MEDIATES IMMUNE PRIVILEGE AND NOT NEUTROPHIL RECRUITMENT IN COLON CANCER IN VIVO. Aileen Houston, Frank Waldron-Lynch, Michael W. Bennett, Desmond Roche, Gerald C. O'Sullivan, John K. Collins, Fergus Lj Shanahan, Joe O'Connell, Nui, Cork, Ireland. Background: Fas ligand (FasL) triggers apoptosis - via its cognate receptor, Fas (APO-IICD95) - of activated lymphocytes entering sites of immune privilege. In contrast to evidence that FasL helps to maintain colon cancers in a state of immune privilege, overexpression of recombinant FasL in murine tumor allografts revealed a potential antitumor effect of Fasl., via recruitment of a neutrophilic infiltrate. Aim: To determine if FasL expressed in colon cancer mediates immune privilege or neutrophil recruitment. Methods: A reference section was immunohistochemically stained to identify FasL-positive and FasL-negative tumor nests within colonic adenocarcinomas (Dukes' A-D). Within both FasL-positive and FasL-negative tumor nests, we counted: (I) tumor-infiltrating lymphocytes (TILs) per 2000 total nuclei; immunohistochemical staining for CD45 facilitated the identification of TILs; (2) TILs that were undergoing apoptosis, per 500 total TILs; apoptotic TILs were identified by dual staining combining TUNEL with immunohistochemistry for CD45; (3) neutrophils per 2000 total nuclei; immunohistochemical staining for the neutrophil marker, lactoferrin, plus their characteristic multi-lobed nuclei, facilitated the identification of neutrophils. Statistics: Wilcoxon Signed Rank. Results: There were consistently fewer TILs within FasL-expressing relative to FasLnegative tumor nests within each tumor examined (mean=4.0-fold, range 1.8- to 33-fold, P < 0.001, n= 16). There was consistently two-fold more apoptosis occurring in TILs within tumor nests that expressed FasL relative to those nests that did not express FasL (mean = 1.9-fold, range 1.6- to 2.5-fold, P < 0.001, n= 14). The overall level of neutrophils within the tumors was low (mean = 0.3%, n = 16). Overall, there were twofold fewer neutrophils in tumor nests that expressed FasL compared to nests that did not express FasL (n = 16, p<0.062). Conclusions: FasL expression was consistently and significantly associated with increased apoptosis of TILs, leading to dramatically diminished TILs in FasL-positive versus -negative areas in all tumors. FasL mediates immune privilege and not neutrophil recruitment in human colon cancer.