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Two novel mucin genes Muc11 and MUC are expressed normally in ulcerative colitis
AGAAl129
April 2000
testinal oxidative stress. Methods: Rats were studied 2 or 8 wks after streptozotocin (60mglkg ip) to induce diabetes sensitivity, Rats underwent no operation (CONT), sham operation, or 30 min SMA occlusion and 4 hrs of reperfusion (euglycemics only). I hour prior to sacrifice, Evan's blue was injected to assess capillary leak based on dye concentration in serum, pulmonary lavages. PMN priming state was defined as the product of PMN H Z0 2 production as determined by flow cytometry, and percent PMN (PMNIWBC), Results: The percent of circulating PMN (PMNIWBC) was increased by surgery alone, but there were no differences between euglycemic and diabetic rats (table), Thus, changes in PMN priming state can be attributed to changes in PMN HzOz production. In euglycemic rats, the priming state was increased by sham operation alone and was elevated further by IR240. In these rats, the development of capillary leak afer [R240 was associated with 100% increase in the priming state over sham operation. In contrast, significant capillary leak was observed in response to a sham operation in both 2 and 8 wk diabetic rats, while the priming state was no higher than that in euglycemic shams. Conclusions: Sham operation alone elevates the percent of circulating PMN (PMNIWBC)in both euglycemic and diabetic rats. In euglycemia rats, the subsequent increase in PMN H 20 2 production after IR leads to capillary leak. Diabetics appear to have an increased sensitivity to PMN H2 0 2 production and develop capillary leak at lower priming states. PMNIWSC(%) EU CONT EU SHAM EU 1R240 DIASCONT 2WKSHAM 8WKSHAM
118± 06 62.7 ± 6.5734±3119.6±3.5 40.1±8.643.9±2.3-
SAL
4± 1 20±414±216+2-
PRIMING STATE
20± 2 110 ±17218± 3639±965±20123±13-
5205 AN ARRAY OF PRO-INFLAMMATORY CYTOKINES STlMU· LATE RELEASE OF CCK FROM THE INTESTINAL ENTEROEN· DOCRINE CELL LINE, STC-l. Fiona C. Leslie, Geoff Warhurst, Graham J, Dockray, David G. Thompson, Univ of Manchester, Manchester, United Kingdom; Univ of Liverpool, Liverpool, United Kingdom. Aims: To study the effect of an array of pro-inflammatory cytokines on the cholecystokinin(CCK)-secreting cell line, STC-1. Methods: The CCKsecreting cell line STC-I was incubated with an array of pro-inflammatory cytokines IFN-'}', [L-I J3 and TNF-a at varying concentrations for 2 hours, followed by a standard 15 minute stimulation period with lauric acid (C 12). The supernatant was collected and CCK levels were measured by radioimmunoassay, Results: After incubation with IFN-'}' (1.25U/ml), lipidinduced CCK secretion was increased by 74.6 ::t: 28,4% compared to control (p
·P,;0.05 VS EU CONT; SAL= Sronchoalveolar lavage
5206
5204
POLYAMINE DEPLETION INCREASES THE SUSCEPTIBILITY OF INTESTINAL EPITHELIAL CELLS TO APOPTOSIS VIA ACTIVATION BUT NOT SUPPRESSION OF NF-KB ACTIVITY,
TWO NOVEL MUCIN GENES MUCH AND MUC12 ARE EXPRESSED NORMALLY IN ULCERATIVE COLITIS. Keith Leiper, Jonathan M. Rhodes, Barry J. Campbell, Dept of Medicine, Univ of Liverpool, Liverpool, United Kingdom. Background Two novel mucin core genes have recently been described, MUC II and MUC 12, both of which are downregulated in colon cancer (Williams SJ Cancer Research 1999;59:4083). Both genes are located on chromosome 7q22 which has been identified as a candidate 18D locus in recent genetic studies. It is well recognised that rnucins are abnormal in ulcerative colitis (UC) where the mucus layer is thinner, less glycosylated and less sulphated. MUC II or MUC 12 may therefore represent potential candidate genes for UC, The aim of this study was to assess mRNA abundance of MUC II and MUC 12 in UC using Northern analysis and serni-quantatative RT-PCR. Methods With informed consent, colonic biopsies were taken from patients with ulcerative colitis and patients with normal colon. Total RNA was extracted using a commercially available kit. For Northern analysis, 20JLg total RNA was electrophoresed and transferred onto nylon membranes. Membranes were then sequentially hybridised with 32p labelled PCR generated cDNA probes for MUC II,MUC 12 and 28S ribosomal RNA. mRNA abundance of MUC II and MUC 12 was expressed as a ratio of 28S RNA expression by phosphorimage analysis. For semi-quantatative RT-PCR 5JLg of total RNA was subjected to first strand eDNA synthesis and then PCR was performed using specific oligonucleotide primers for MUC II, MUC 12 and cytokeratin 20 (a marker of epithelial abundance). PCR band intensity was quantified and expressed as a ratio of cytokeratin 20 abundance, All results were expressed as a percentage of control normal colon (100%). Results Northern analysis for MUC II and MUCI2 revealed a polydisperse signal typical of mucins, Quantification showed a wide variation in expression of both mucin with no significant difference between groups: MUC I I (mean::t:SE) normals (n=IO) 100::t:35%, UC (n=18) 144::t: 30,3, MUC 12 normals 100::t:19.7, UC 120::t: 16.8. Semiquantative PCR likewise showed no significant differences between groups: MUC II normals (n=6) lOO::t: 18%, UC (n=6) 90::t:38% and MUC 12 normals (n=6) 100::t: 54%, UC (n=6) 76::t: 23. Preliminary data has confirmed the down regulation of MUC 11 and MUC 12 in colon neoplasia by Northern analysis (n=2) and RT-PCR (n=2). Discussion Although MUC 11 and MUC 12 appear to be downregulated in colonic neoplasia, they are normally expressed in ulcerative colitis. It is unlikely that these are the candidate genes for IBD on the chromosome 7 locus.
Li Li, Rao N, Jaladanki, Barbara L. Bass, Jian-Ying Wang, Univ of Maryland, Baltimore, MD. A balance between cell renewal and cell death including apoptosis determines epithelial integrity in the gastrointestinal mucosa. Apoptosis is regulated both positively and negatively in response to a variety of stimuli in the body. Polyamines are absolutely required for the maintenance of intestinal epithelial integrity, but the precise mechanism responsible for polyamines in the regulation of mucosal growth is still unclear. The current study was designed to determine changes in susceptibility of intestinal epithelial cells (IEC-6Iine) to apoptosis after polyamine depletion and then to examine the role of NF-kB, a inducible transcription factor that has cell type-speci fie, proapoptotic, or antiapoptotic functions, in this process. Methods: IEC-6 cells were grown in the presence or absence of 5 mM DFMO, a specific inhibitor for polyamine synthesis, for 6 days and apoptosis is induced by staurosporine (STS). Results: Administration of DFMO for 6 days depleted cellular polyamines putrescine, spermidine and spermine, Although DFMO did not directly induce apoptosis, susceptibility of the polyamine-deficient cells to apoptosis induced by STS was dramatically increased as measured by changes in morphological features and DNA fragmentation. Increased sensitivity to apoptosis was associated with marked increases in both NF-kB protein level and its sequence-specific DNA binding activity. Inhibition of NF-kB binding activity by using sulfasalazine, a potent and specific inhibitor of NF-kB, prevented increased sensitivity to apoptosis induced by STS in polyamine-deficient cells. Conclusions: 1) Inhibition of polyamine synthesis sensitizes IEC-6 cells to apoptosis induced by STS. 2) Increased susceptibility of polyamine-deficient cells to apoptosis is associated with an increase in NF-kB activity. These findings suggest that NF-kB is a proapoptotic factor in intestinal epithelial cells and that increased susceptibility to apoptosis following polyamine depletion may be due to activation, but not suppression. of NF-kB activity.
April 2000
testinal oxidative stress. Methods: Rats were studied 2 or 8 wks after streptozotocin (60mglkg ip) to induce diabetes sensitivity, Rats underwent no operation (CONT), sham operation, or 30 min SMA occlusion and 4 hrs of reperfusion (euglycemics only). I hour prior to sacrifice, Evan's blue was injected to assess capillary leak based on dye concentration in serum, pulmonary lavages. PMN priming state was defined as the product of PMN H Z0 2 production as determined by flow cytometry, and percent PMN (PMNIWBC), Results: The percent of circulating PMN (PMNIWBC) was increased by surgery alone, but there were no differences between euglycemic and diabetic rats (table), Thus, changes in PMN priming state can be attributed to changes in PMN HzOz production. In euglycemic rats, the priming state was increased by sham operation alone and was elevated further by IR240. In these rats, the development of capillary leak afer [R240 was associated with 100% increase in the priming state over sham operation. In contrast, significant capillary leak was observed in response to a sham operation in both 2 and 8 wk diabetic rats, while the priming state was no higher than that in euglycemic shams. Conclusions: Sham operation alone elevates the percent of circulating PMN (PMNIWBC)in both euglycemic and diabetic rats. In euglycemia rats, the subsequent increase in PMN H 20 2 production after IR leads to capillary leak. Diabetics appear to have an increased sensitivity to PMN H2 0 2 production and develop capillary leak at lower priming states. PMNIWSC(%) EU CONT EU SHAM EU 1R240 DIASCONT 2WKSHAM 8WKSHAM
118± 06 62.7 ± 6.5734±3119.6±3.5 40.1±8.643.9±2.3-
SAL
4± 1 20±414±216+2-
PRIMING STATE
20± 2 110 ±17218± 3639±965±20123±13-
5205 AN ARRAY OF PRO-INFLAMMATORY CYTOKINES STlMU· LATE RELEASE OF CCK FROM THE INTESTINAL ENTEROEN· DOCRINE CELL LINE, STC-l. Fiona C. Leslie, Geoff Warhurst, Graham J, Dockray, David G. Thompson, Univ of Manchester, Manchester, United Kingdom; Univ of Liverpool, Liverpool, United Kingdom. Aims: To study the effect of an array of pro-inflammatory cytokines on the cholecystokinin(CCK)-secreting cell line, STC-1. Methods: The CCKsecreting cell line STC-I was incubated with an array of pro-inflammatory cytokines IFN-'}', [L-I J3 and TNF-a at varying concentrations for 2 hours, followed by a standard 15 minute stimulation period with lauric acid (C 12). The supernatant was collected and CCK levels were measured by radioimmunoassay, Results: After incubation with IFN-'}' (1.25U/ml), lipidinduced CCK secretion was increased by 74.6 ::t: 28,4% compared to control (p
·P,;0.05 VS EU CONT; SAL= Sronchoalveolar lavage
5206
5204
POLYAMINE DEPLETION INCREASES THE SUSCEPTIBILITY OF INTESTINAL EPITHELIAL CELLS TO APOPTOSIS VIA ACTIVATION BUT NOT SUPPRESSION OF NF-KB ACTIVITY,
TWO NOVEL MUCIN GENES MUCH AND MUC12 ARE EXPRESSED NORMALLY IN ULCERATIVE COLITIS. Keith Leiper, Jonathan M. Rhodes, Barry J. Campbell, Dept of Medicine, Univ of Liverpool, Liverpool, United Kingdom. Background Two novel mucin core genes have recently been described, MUC II and MUC 12, both of which are downregulated in colon cancer (Williams SJ Cancer Research 1999;59:4083). Both genes are located on chromosome 7q22 which has been identified as a candidate 18D locus in recent genetic studies. It is well recognised that rnucins are abnormal in ulcerative colitis (UC) where the mucus layer is thinner, less glycosylated and less sulphated. MUC II or MUC 12 may therefore represent potential candidate genes for UC, The aim of this study was to assess mRNA abundance of MUC II and MUC 12 in UC using Northern analysis and serni-quantatative RT-PCR. Methods With informed consent, colonic biopsies were taken from patients with ulcerative colitis and patients with normal colon. Total RNA was extracted using a commercially available kit. For Northern analysis, 20JLg total RNA was electrophoresed and transferred onto nylon membranes. Membranes were then sequentially hybridised with 32p labelled PCR generated cDNA probes for MUC II,MUC 12 and 28S ribosomal RNA. mRNA abundance of MUC II and MUC 12 was expressed as a ratio of 28S RNA expression by phosphorimage analysis. For semi-quantatative RT-PCR 5JLg of total RNA was subjected to first strand eDNA synthesis and then PCR was performed using specific oligonucleotide primers for MUC II, MUC 12 and cytokeratin 20 (a marker of epithelial abundance). PCR band intensity was quantified and expressed as a ratio of cytokeratin 20 abundance, All results were expressed as a percentage of control normal colon (100%). Results Northern analysis for MUC II and MUCI2 revealed a polydisperse signal typical of mucins, Quantification showed a wide variation in expression of both mucin with no significant difference between groups: MUC I I (mean::t:SE) normals (n=IO) 100::t:35%, UC (n=18) 144::t: 30,3, MUC 12 normals 100::t:19.7, UC 120::t: 16.8. Semiquantative PCR likewise showed no significant differences between groups: MUC II normals (n=6) lOO::t: 18%, UC (n=6) 90::t:38% and MUC 12 normals (n=6) 100::t: 54%, UC (n=6) 76::t: 23. Preliminary data has confirmed the down regulation of MUC 11 and MUC 12 in colon neoplasia by Northern analysis (n=2) and RT-PCR (n=2). Discussion Although MUC 11 and MUC 12 appear to be downregulated in colonic neoplasia, they are normally expressed in ulcerative colitis. It is unlikely that these are the candidate genes for IBD on the chromosome 7 locus.
Li Li, Rao N, Jaladanki, Barbara L. Bass, Jian-Ying Wang, Univ of Maryland, Baltimore, MD. A balance between cell renewal and cell death including apoptosis determines epithelial integrity in the gastrointestinal mucosa. Apoptosis is regulated both positively and negatively in response to a variety of stimuli in the body. Polyamines are absolutely required for the maintenance of intestinal epithelial integrity, but the precise mechanism responsible for polyamines in the regulation of mucosal growth is still unclear. The current study was designed to determine changes in susceptibility of intestinal epithelial cells (IEC-6Iine) to apoptosis after polyamine depletion and then to examine the role of NF-kB, a inducible transcription factor that has cell type-speci fie, proapoptotic, or antiapoptotic functions, in this process. Methods: IEC-6 cells were grown in the presence or absence of 5 mM DFMO, a specific inhibitor for polyamine synthesis, for 6 days and apoptosis is induced by staurosporine (STS). Results: Administration of DFMO for 6 days depleted cellular polyamines putrescine, spermidine and spermine, Although DFMO did not directly induce apoptosis, susceptibility of the polyamine-deficient cells to apoptosis induced by STS was dramatically increased as measured by changes in morphological features and DNA fragmentation. Increased sensitivity to apoptosis was associated with marked increases in both NF-kB protein level and its sequence-specific DNA binding activity. Inhibition of NF-kB binding activity by using sulfasalazine, a potent and specific inhibitor of NF-kB, prevented increased sensitivity to apoptosis induced by STS in polyamine-deficient cells. Conclusions: 1) Inhibition of polyamine synthesis sensitizes IEC-6 cells to apoptosis induced by STS. 2) Increased susceptibility of polyamine-deficient cells to apoptosis is associated with an increase in NF-kB activity. These findings suggest that NF-kB is a proapoptotic factor in intestinal epithelial cells and that increased susceptibility to apoptosis following polyamine depletion may be due to activation, but not suppression. of NF-kB activity.