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Polyamines negatively regulate posttranscription of the P53 gene in small intestinal crypt cells
April 1998
MV. rubella, and CMV was measured by ELISA in sera from 22 affected children and 32 normal controls of similar age and sex, and cerebrospinal fluid in 6 affected children. Immunocytological profiles of PBMC were examined by flow cytometry. Results: MV N-protein antigen but no other viral antigen, was detected in tissues from 5 of 7 affected children: staining was present exclusively in follicular dendritic cells in foci of ileal follicular hyperplasia, confirmed by double immunolabelling with CD21. MV staining was not seen in either 9 of 10 age-matched control ileocolonic biopsy series, ileal lymphoid follicles in 10 children with Crohn's disease, or 6 of 6 lymph nodes from adults with AIDS. One control - ulcerative colitis with ileal LNH - showed identical staining in a focus of reactive follicular hyperplasia. MV haemagglutinin - but not N-gene. consistent with Schwarz vaccine-strain, was detected in coded. duplicate PBMC samples from 1 of 2 affected children but from neither 2 cases of subacute selerosing panencephalitis (SSPE) nor control RNA samples. Serum MV IgG immunoreactivity, but neither MV IgM nor rubella or CMV antibody immunoreactivity, was significantly elevated in affected children compared with normal controls (3688 MIU/ml vs. 2042 MIU/mI: p < 0.02). Total IgG was not elevated in affected children, and there was no significant relationship between MV IgG and either total IgG or rubella IgG (p > 0.4). Virus-specific immunoglobulin was not detected in CSF. Eleven of 12 affected children had low absolute numbers of either CD4- and CD8- T cells. B cells, or natural killer cells. CD4:CD8 ratios were within normal limits. Conclusion: The data indicate the possibility of an acquired immunodeficiency and persistent MV infection of ileal lymphoid tissue, in children with non-specific colitis and autism. • G1755 POLYAMINES NEGATIVELY REGULATE POSTTRANSCRIPTION OF THE pS3 GENE IN SMALL INTESTINAL CRYPT CELLS. L-Y. Wang. J. Li, AR. Patel, L. Li, and JN. Rao. Dept. of Surgery, Univ. of MD and Baltimore VA Med. Center, MD 21201 The p53 gene encodes the nuclear phosphoprotein that prevents the transition from G1 to S phase of the cell cycle. Our previous studies have shown that polyamines play a major role in the regulation of intestinal mucosal growth and that decreasing cellular polyamine levels inhibits cell proliferation. We have recently demonstrated that growth inhibition following polyamine depletion is associated with an increased expression of the p53 gene in intestinal epithelial cells (IEC-6 cell line). In the current study, we have examined whether polyamines are involved in the regulation of transcription and posttranseription of the p53 gene in IEC-6 cells. Methods: Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence or absence of DFMO, a specific inhibitor of polyamine biosynthesis. The levels of p53 mRNA and protein, rate of the p53 gene transcription and the stability of the mRNA were measured at different times after initial plating. Results: Exposure to DFMO for 4, 6 and 12 days almost completely depleted cellular polyamine putrescine, spermidine, and spermine. Polyamine depletion also significantly increased the mRNA levels of the p53 gene. Increased p53 mRNA in DFMOtreated cells was paralleled by an increase in p53 protein. The activation of p53 gene expression following polyamine depletion was associated with a significant decrease in cell division. Using nuclear run on transcription technique, we demonstrated that there were no significant differences in rates of the p53 gene transcription between control cells and the ceils exposed to DFMO for 4, 6, and 12 days. However, polyamine depletion dramatically increased the stability of p53 mRNA. The half-life of p53 mRNA in control cells was ~45 min and increased to more than 16 h in cells exposed to DFMO. Spermidine, when given together with DFMO, returned the increased half-life of p53 mRNA to normal. Conclusions: 1) polyamine depletion has no effects on the transcriptional regulation of the p53 gene and 2) increased levels of p53 mRNA in polyamine-deficient cells primarily result from a delay in the rate of mRNA degradation. These findings suggest that polyamines are required for gut mucosal growth by virtue of their ability to negatively regulate posttranscription of the p53 gene. • G1756 ZONULA OCCLUDENS TOXIN (ZOT) REPRESENTS A NEW MEMBER OF THE MICROTUBULE-ASSOCIATED P R O T E I N S (MAPS) FAMILY. W. Wang, R. Lu, Y. Pang, A. Fasano. University of Maryland, Baltimore, MD 21201. Zonula Occludens Toxin (Znt) is a novel toxin elaborated by Vibrio cholerae that increases the intestinal permeability. Zot effect is mediated by a complex intracellular signaling that involves the activation of PKC-~ with the subsequent rearrangement of the cellular cytoskeleton strategically located to control the tight junction permeability. However, the initial steps of Zot signaling after the protein internalization remain to be established. Microtubules (Mt) are intracellular structures functionally and anatomically related to the cell cytoskeleton. Numerous proteins named MicrotubuleAssociated Proteins (MAPs) have been described to bind tO Mt and affect cell functions. MAPs can either Stabilize Mt and/or modify the transport of molecules along Mt. Methods: A Zot affinity column was prepared by coupling 1.0 mg of purified Zot to a resin snpport at 4°C overnight. The
Intestinal Disorders A431
column was then loaded with crude intestinal cell lysates obtained from human cadavers. After 90 rain of incubation at room temperature, the column was washed five times with PBS and the proteins which bound to the Zot affinity column were eluted by step gradient with NaCI (0.3-0.5 M) containing 0.1% (v/v) Triton X-100 in PBS, pH 7.4. The fractions were subjected to 6.0-15.0% (w/v) gradient SDS-PAGE under reducing conditions and then transferred to a PVDF membrane for N-terminal sequencing. Purified Zot was also transferred on nitrocellulose filters and Western immunoblotting were performed by using both polyclonal anti-Zot and antiTan (a well described MAP) antibodies. Finally, purified Zot was tested in an in vitro tubulin binding assay. Results: Fractions from human intestine loaded on the Zot affinity column yielded two protein bands with a M, of 60 kDa and 45 kDa ca, respectively. The N-terminal sequence of the 60 kDa band resulted identical to I]-tubulin. Purified Zot also cross-react with antibodies against Tan, a well described MAP isolated from mammalian brain, suggesting that the two proteins are structurally similar. Finally, when tested in the tubulin binding assay, Zot co-precipitated with Mt, further indicating that Zot possesses tubulin-binding properties. Conclusions: Taken together, these results suggest that Zot regulates the permeability of intestinal tight junctions by binding to intracellular Mt, with the subsequent activation of the intracellular signaling leading to the permeabilization of intercellular tight junctions. • G1757 IDENTIFICATION OF THE NUCLEOSIDE TRANSPORTERS IN HUMAN SMALL INTESTINE AND COLONIC EPITHELIAL CELL LINES. J.L. Ward and C.M. Tse, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Physiological studies have shown that intestinal epithelial cells have Na÷dependent nucleoside transporters on the brush border membranes (BB) and Na÷-independent nucleoside transporters on their basolateral (BL) membranes. Three Na÷-dependent nucleoside transport systems (N1, pyrimidine specific; N2, purine specific; and N3, broadly specific) and two Na÷-independent nucleoside transporters [nitrobenzylthioinosine-sensitive (ES) and insensitive (El)] have been physiologically characterized in the intestine. Recently, two Na÷-dependent nucleoside transporters (cNT1, encoding for the N2 system, and SPNT, encoding for the N1 system) and two Na÷-independent nucleoside transporters (ENT1, encoding ES, and ENT2, encoding El) were cloned. Previous nucleoside transport studies in Caco-2 cells have reached conflicting conclusions suggesting either an N2 or an N3 system on the BB. Physiological studies in T84 cells have suggested that these secretory epithelial cells lack apical Na÷-dependent nucleoside transporters, but have BL Na÷-independent transporters that are involved in regulating the local concentration of adenosine, which acts through the A2b receptor to elicit CIsecretion. The purpose of this study was 1) to identify, by RT-PCR, the nucleoside transporter isoforms expressed in human small intestine, Caco-2 ceils, and T84 cells, and 2) to correlate the identified nucleoside transporter isoforms with the previously characterized physiologic responses in these cell models and tissues. RT-PCR was performed by oligo(dT) and random priming Of total RNA isolated from human small intestine, Caco-2, and T84 ceils, followed by amplification of first-strand eDNA with specific forward and reverse oligonucleotide primers based on the available sequences of cloned human nucleoside transporters. Amplification products were analyzed by agarose gel electrophoresis, and, in positive reactions, all primer pairs yielded single PCR products of the predicted size using an annealing temperature of 55 ° C and 30 amplification cycles. The identity of all products was confirmed by subcloning of amplified fragments and DNA sequencing. The results showed no PCR amplification of cDNA from Caco-2 and T84 cells when using either of the two primer pairs specific for the cloned Na÷-dependent transporters, cNT1 and SPNT. Both cell lines, however, demonstrated specific amplification of fragments of the Na÷-independent ENT1 and ENT2. In contrast, the human small intestine yielded PCR fragments using each of the four primer pairs specific for the cloned transporters. In conclusion: 1) in human small intestine, the Na+-dependent transporters, cNT1 and SPNT, and the Na÷-independent transporters, ENT1 and ENT2, are among the transport mechanisms responsible for previously characterized functions including nucleoside salvaging and vectorial transport of nucleosides and nucleoside analogs across intestinal epithelium; 2) the colonic epithelial cell lines T84 and Caco-2 do not express the Na÷-dependent nucleoside transporters cNT1 and SPNT; 3) the physiologically characterized N3 system in Caco-2 cells represents an as yet uncloned novel isoform of the Na÷-dependent nucleoside transporter; and 4) in T84 cells, ENT1 (ES) and ENT2 (EI) are likely to be the BL Na÷-independent nucleoside transporters that were previously shown to help regulate local adenosine concentrations and adenosine-mediated C1- secretion.
MV. rubella, and CMV was measured by ELISA in sera from 22 affected children and 32 normal controls of similar age and sex, and cerebrospinal fluid in 6 affected children. Immunocytological profiles of PBMC were examined by flow cytometry. Results: MV N-protein antigen but no other viral antigen, was detected in tissues from 5 of 7 affected children: staining was present exclusively in follicular dendritic cells in foci of ileal follicular hyperplasia, confirmed by double immunolabelling with CD21. MV staining was not seen in either 9 of 10 age-matched control ileocolonic biopsy series, ileal lymphoid follicles in 10 children with Crohn's disease, or 6 of 6 lymph nodes from adults with AIDS. One control - ulcerative colitis with ileal LNH - showed identical staining in a focus of reactive follicular hyperplasia. MV haemagglutinin - but not N-gene. consistent with Schwarz vaccine-strain, was detected in coded. duplicate PBMC samples from 1 of 2 affected children but from neither 2 cases of subacute selerosing panencephalitis (SSPE) nor control RNA samples. Serum MV IgG immunoreactivity, but neither MV IgM nor rubella or CMV antibody immunoreactivity, was significantly elevated in affected children compared with normal controls (3688 MIU/ml vs. 2042 MIU/mI: p < 0.02). Total IgG was not elevated in affected children, and there was no significant relationship between MV IgG and either total IgG or rubella IgG (p > 0.4). Virus-specific immunoglobulin was not detected in CSF. Eleven of 12 affected children had low absolute numbers of either CD4- and CD8- T cells. B cells, or natural killer cells. CD4:CD8 ratios were within normal limits. Conclusion: The data indicate the possibility of an acquired immunodeficiency and persistent MV infection of ileal lymphoid tissue, in children with non-specific colitis and autism. • G1755 POLYAMINES NEGATIVELY REGULATE POSTTRANSCRIPTION OF THE pS3 GENE IN SMALL INTESTINAL CRYPT CELLS. L-Y. Wang. J. Li, AR. Patel, L. Li, and JN. Rao. Dept. of Surgery, Univ. of MD and Baltimore VA Med. Center, MD 21201 The p53 gene encodes the nuclear phosphoprotein that prevents the transition from G1 to S phase of the cell cycle. Our previous studies have shown that polyamines play a major role in the regulation of intestinal mucosal growth and that decreasing cellular polyamine levels inhibits cell proliferation. We have recently demonstrated that growth inhibition following polyamine depletion is associated with an increased expression of the p53 gene in intestinal epithelial cells (IEC-6 cell line). In the current study, we have examined whether polyamines are involved in the regulation of transcription and posttranseription of the p53 gene in IEC-6 cells. Methods: Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence or absence of DFMO, a specific inhibitor of polyamine biosynthesis. The levels of p53 mRNA and protein, rate of the p53 gene transcription and the stability of the mRNA were measured at different times after initial plating. Results: Exposure to DFMO for 4, 6 and 12 days almost completely depleted cellular polyamine putrescine, spermidine, and spermine. Polyamine depletion also significantly increased the mRNA levels of the p53 gene. Increased p53 mRNA in DFMOtreated cells was paralleled by an increase in p53 protein. The activation of p53 gene expression following polyamine depletion was associated with a significant decrease in cell division. Using nuclear run on transcription technique, we demonstrated that there were no significant differences in rates of the p53 gene transcription between control cells and the ceils exposed to DFMO for 4, 6, and 12 days. However, polyamine depletion dramatically increased the stability of p53 mRNA. The half-life of p53 mRNA in control cells was ~45 min and increased to more than 16 h in cells exposed to DFMO. Spermidine, when given together with DFMO, returned the increased half-life of p53 mRNA to normal. Conclusions: 1) polyamine depletion has no effects on the transcriptional regulation of the p53 gene and 2) increased levels of p53 mRNA in polyamine-deficient cells primarily result from a delay in the rate of mRNA degradation. These findings suggest that polyamines are required for gut mucosal growth by virtue of their ability to negatively regulate posttranscription of the p53 gene. • G1756 ZONULA OCCLUDENS TOXIN (ZOT) REPRESENTS A NEW MEMBER OF THE MICROTUBULE-ASSOCIATED P R O T E I N S (MAPS) FAMILY. W. Wang, R. Lu, Y. Pang, A. Fasano. University of Maryland, Baltimore, MD 21201. Zonula Occludens Toxin (Znt) is a novel toxin elaborated by Vibrio cholerae that increases the intestinal permeability. Zot effect is mediated by a complex intracellular signaling that involves the activation of PKC-~ with the subsequent rearrangement of the cellular cytoskeleton strategically located to control the tight junction permeability. However, the initial steps of Zot signaling after the protein internalization remain to be established. Microtubules (Mt) are intracellular structures functionally and anatomically related to the cell cytoskeleton. Numerous proteins named MicrotubuleAssociated Proteins (MAPs) have been described to bind tO Mt and affect cell functions. MAPs can either Stabilize Mt and/or modify the transport of molecules along Mt. Methods: A Zot affinity column was prepared by coupling 1.0 mg of purified Zot to a resin snpport at 4°C overnight. The
Intestinal Disorders A431
column was then loaded with crude intestinal cell lysates obtained from human cadavers. After 90 rain of incubation at room temperature, the column was washed five times with PBS and the proteins which bound to the Zot affinity column were eluted by step gradient with NaCI (0.3-0.5 M) containing 0.1% (v/v) Triton X-100 in PBS, pH 7.4. The fractions were subjected to 6.0-15.0% (w/v) gradient SDS-PAGE under reducing conditions and then transferred to a PVDF membrane for N-terminal sequencing. Purified Zot was also transferred on nitrocellulose filters and Western immunoblotting were performed by using both polyclonal anti-Zot and antiTan (a well described MAP) antibodies. Finally, purified Zot was tested in an in vitro tubulin binding assay. Results: Fractions from human intestine loaded on the Zot affinity column yielded two protein bands with a M, of 60 kDa and 45 kDa ca, respectively. The N-terminal sequence of the 60 kDa band resulted identical to I]-tubulin. Purified Zot also cross-react with antibodies against Tan, a well described MAP isolated from mammalian brain, suggesting that the two proteins are structurally similar. Finally, when tested in the tubulin binding assay, Zot co-precipitated with Mt, further indicating that Zot possesses tubulin-binding properties. Conclusions: Taken together, these results suggest that Zot regulates the permeability of intestinal tight junctions by binding to intracellular Mt, with the subsequent activation of the intracellular signaling leading to the permeabilization of intercellular tight junctions. • G1757 IDENTIFICATION OF THE NUCLEOSIDE TRANSPORTERS IN HUMAN SMALL INTESTINE AND COLONIC EPITHELIAL CELL LINES. J.L. Ward and C.M. Tse, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Physiological studies have shown that intestinal epithelial cells have Na÷dependent nucleoside transporters on the brush border membranes (BB) and Na÷-independent nucleoside transporters on their basolateral (BL) membranes. Three Na÷-dependent nucleoside transport systems (N1, pyrimidine specific; N2, purine specific; and N3, broadly specific) and two Na÷-independent nucleoside transporters [nitrobenzylthioinosine-sensitive (ES) and insensitive (El)] have been physiologically characterized in the intestine. Recently, two Na÷-dependent nucleoside transporters (cNT1, encoding for the N2 system, and SPNT, encoding for the N1 system) and two Na÷-independent nucleoside transporters (ENT1, encoding ES, and ENT2, encoding El) were cloned. Previous nucleoside transport studies in Caco-2 cells have reached conflicting conclusions suggesting either an N2 or an N3 system on the BB. Physiological studies in T84 cells have suggested that these secretory epithelial cells lack apical Na÷-dependent nucleoside transporters, but have BL Na÷-independent transporters that are involved in regulating the local concentration of adenosine, which acts through the A2b receptor to elicit CIsecretion. The purpose of this study was 1) to identify, by RT-PCR, the nucleoside transporter isoforms expressed in human small intestine, Caco-2 ceils, and T84 cells, and 2) to correlate the identified nucleoside transporter isoforms with the previously characterized physiologic responses in these cell models and tissues. RT-PCR was performed by oligo(dT) and random priming Of total RNA isolated from human small intestine, Caco-2, and T84 ceils, followed by amplification of first-strand eDNA with specific forward and reverse oligonucleotide primers based on the available sequences of cloned human nucleoside transporters. Amplification products were analyzed by agarose gel electrophoresis, and, in positive reactions, all primer pairs yielded single PCR products of the predicted size using an annealing temperature of 55 ° C and 30 amplification cycles. The identity of all products was confirmed by subcloning of amplified fragments and DNA sequencing. The results showed no PCR amplification of cDNA from Caco-2 and T84 cells when using either of the two primer pairs specific for the cloned Na÷-dependent transporters, cNT1 and SPNT. Both cell lines, however, demonstrated specific amplification of fragments of the Na÷-independent ENT1 and ENT2. In contrast, the human small intestine yielded PCR fragments using each of the four primer pairs specific for the cloned transporters. In conclusion: 1) in human small intestine, the Na+-dependent transporters, cNT1 and SPNT, and the Na÷-independent transporters, ENT1 and ENT2, are among the transport mechanisms responsible for previously characterized functions including nucleoside salvaging and vectorial transport of nucleosides and nucleoside analogs across intestinal epithelium; 2) the colonic epithelial cell lines T84 and Caco-2 do not express the Na÷-dependent nucleoside transporters cNT1 and SPNT; 3) the physiologically characterized N3 system in Caco-2 cells represents an as yet uncloned novel isoform of the Na÷-dependent nucleoside transporter; and 4) in T84 cells, ENT1 (ES) and ENT2 (EI) are likely to be the BL Na÷-independent nucleoside transporters that were previously shown to help regulate local adenosine concentrations and adenosine-mediated C1- secretion.