P8 expression is induced in acinar cells during chronic pancreatitis.

The p8 gene is barely expressed in the normal pancreas, but is overexpressed in acute pancreatitis. To elucidate the dynamic expression of p8 mRNA and its significance in the course of chronic pancreatitis, we investigated the p8 expression in spontaneous chronic pancreatitis in the WBN/Kob rat as well as in humans and arginine-treated rat pancreatic acinar AR4-2J cells. p8 mRNA was significantly increased at 12 weeks when chronic pancreatitis first appeared in the WBN/Kob rats. p8 was immunolocalized in the acinar cell nuclei. Acinar cell apoptosis was significantly increased at 12 and 20 weeks in the WBN/Kob rats. In AR4-2J cells, p8 mRNA was significantly induced at 4 hr after arginine addition. Apoptosis of AR4-2J cells was not increased during the strong expression of p8 mRNA. These results suggest that p8 is induced in the acinar cells during chronic pancreatitis as the self-defence mechanism against proapoptotic insults.

Human p8 is a HMG-I/Y-like protein with DNA binding activity enhanced by phosphorylation.

We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mm concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

Expression of the stress-induced p8 mRNA is transiently activated after culture medium change.

We report here that the mere fact of changing culture medium for fresh medium induced in several cell lines the expression of stress-activated genes including protein kinases p38, JNK and ERK1/2 and the transcription factor C/EBPbeta. As a consequence, p8, a gene induced by stress in several tissues, was strongly up-regulated. Induction did not occur after change for cell-conditioned medium. Induction was however transient, with a peak at 60 min for p38, at 15-30 min for JNK and at 15 min for ERK1/2, at 2-3 hours for C/EBPbeta and at 4-6 hours for p8. Repression of the induction was due to the secretion of thermolabile molecule(s) that progressively conditioned the medium. As low as 25% of conditioned medium added to fresh culture medium was sufficient to abolish the stress response. Taken together, our data indicate that the renewal of culture medium induces a transient cellular stress that may be a source of artifacts in experiments performed shortly after a change of culture medium.

Overexpression of Cdx1 and Cdx2 homeogenes enhances expression of the HLA-I in HT-29 cells.

We have previously reported that down-regulation of Cdx1 and Cdx2 mRNA expression is associated with colon carcinogenesis, and that coordinated reexpression of these genes in the HT29 colon cancer-derived cell line leads to a reduced malignant phenotype. Here we show that restoring Cdx1 and Cdx2 expression in HT29 cells enhanced the antigen presentation system, as reflected by a strong induction of the concentration of HLA-I molecules at the cell surface, resulting from increased expression of the HLA-I mRNA. Expression of the LMP2 proteasomal protein was also strongly induced by Cdx1 and Cdx2 at the transcriptional level, whereas TAP1 expression which is under the control of the same bidirectional promoter as LMP2 remained unchanged. Furthermore, expression of the adhesion molecule ICAM-1, which works in concert with HLA-I, and of the cell death promoter Fas was also increased upon Cdx1 and Cdx2 expression. Taken together, these results suggest that loss of Cdx1 and Cdx2 expression during colorectal carcinogenesis could favor the escape of tumor cells from the immune system. In conclusion, restoration of Cdx1 and Cdx2 expression should be considered in immunotherapeutic strategies for colorectal cancer.

Cdx1 promotes differentiation in a rat intestinal epithelial cell line.

BACKGROUND & AIMS: Homeobox genes are involved in establishing and maintaining differentiated patterns in adult tissues. Cdx1 might carry out that function in the intestinal epithelium because its expression is specific to that tissue and increases during development. METHODS: Cdx1 expression was induced in IEC-6 intestinal epithelial cells by stable transfection, and subsequent changes in cell growth, resistance to apoptosis, migration, and differentiation were monitored. RESULTS: Compared with control, IEC-6/Cdx1 cells proliferated more rapidly, were more resistant to apoptosis, and migrated 3-4 times faster, as shown by an in vitro wound assay. IEC-6/Cdx1 cells in culture formed multilayers. Morphology of the top layer was similar to that of columnar epithelium, with cells showing typical features of differentiated enterocytes, including complex junctions and well-developed microvilli with glycocalix. Expression of 2 markers of enterocyte differentiation, aminopeptidase N and villin, was induced in IEC-6/Cdx1 cells. Aminopeptidase N was targeted to the basolateral membrane, and villin was localized to the cytoplasm. Actin filaments, which were mostly present in transcytoplasmic stress fibers in control cells, were redistributed to the cortex in Cdx1-transfected cells. CONCLUSIONS: Cdx1 expression in IEC-6 cells induces phenotypic changes characteristic of differentiating enterocytes, suggesting an important role for Cdx1 in the transition from stem cells to proliferating/transit cells.

Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter.

Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5' flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPalpha or C/EBPbeta expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPalpha and C/EBPbeta. Co-transfection with C/EBPalpha or C/EBPbeta expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPalpha or C/EBPbeta still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPalpha and C/EBPbeta trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.

pap, reg Ialpha and reg Ibeta mRNAs are concomitantly up-regulated during human colorectal carcinogenesis.

We have established the phenotype of a colorectal tumor by partial sequencing of 2166 transcripts that were eventually arrayed on high-density filters. These filters were used for differential screening with mRNAs of colorectal cancer and normal adjacent mucosa to characterize genes whose expression is altered in colorectal carcinoma. Three genes encoding related proteins, PAP, reg Ialpha and reg Ibeta, were over-expressed in cancer. Northern-blot analysis confirmed that their expression was very low in normal colonic epithelial cells, but elevated in 75% of tumors. Western blotting with specific antibodies to pap and reg Ialpha revealed in tumors a single band of the expected size ( 15-16 kDa), demonstrating synthesis of the proteins. Pap was localized by immunohistochemistry to the cytoplasm of epithelial cells. In cancerous tissue, many cells showed a strong staining signal, but the proportion of stained cells was variable among patients. In normal mucosa, staining was light and restricted to a few cells scattered in the epithelium. Similar results were obtained with antibodies against reg Ialpha. No significant relationship was found between concentrations of pap, reg Ialpha or reg Ibeta and clinical outcome. We looked at potential effectors of pap/reg gene over-expression by testing, in 2 adenocarcinoma cell lines, the efficacy of the pap promoter at driving a reporter gene; strong induction was observed upon exposure to IFNgamma and IL-6. By analogy with observations in hepatocellular carcinoma, our results suggest that prevention of PAP/reg expression in normal colon cells by silencing their gene promoters is relieved during colon carcinogenesis, allowing their up-regulation by mediators such as cytokines.

Overexpression of the PC3/TIS21/BTG2 mRNA is part of the stress response induced by acute pancreatitis in rats.

We have previously shown that the acute phase reaction of the pancreas is a powerful emergency mechanism which protects the organism against further pancreatic aggression. In an attempt to understand the mechanisms involved in this protective effect we tried to characterize at the molecular level the phenotypic changes of the pancreatic cell during acute stress. Using a systematic approach, we identified the PC3/TIS21/BTG2 mRNA as strongly overexpressed in pancreas during the acute phase of pancreatitis. PC3/TIS21/BTG2 mRNA is also overexpressed in liver and kidney during acute pancreatitis but not in the other tissues analyzed. In addition, PC3/TIS21/BTG2 mRNA is overexpressed in kidney after a 30-min ischemia. Since acute pancreatitis and kidney ischemia-reperfusion-induced injury were associated with apoptosis, and PC3/TIS21/BTG2 has an antiapoptotic activity, we speculate that this protein may play a role in the control of apoptosis progression in these tissues.

Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development.

Molecular mechanisms associated with apoptosis in pancreas remain largely unknown. Clusterin mRNA is induced in several tissues in response to most apoptotic stimuli. In these tissues, clusterin has an antiapoptotic activity. The aim of this work was to test whether clusterin, which is not expressed in normal pancreas, was induced in pancreas during pancreatitis and pancreatic development. Clusterin mRNA levels were strongly increased 6 h after pancreatitis induction. Maximal expression happened between 24-48 h and decreased progressively to undetectable levels at day 5. Clusterin mRNA was expressed with similar intensity in oedematous caerulein-induced pancreatitis and in response to various degrees of necrohaemorrhagic taurocholate-induced pancreatitis, indicating a maximal gene activity in all types of pancreatitis; in situ hybridization showed that the acinar cells and some ducts expressed clusterin mRNA. A single band of about 35-38 kDa was detected by western blot in pancreatic homogenates and in pancreatic juice from rats with acute pancreatitis, but not from control rats. Clusterin mRNA expression was strong in late fetal life and remains high until day 11 post-partum, then decreased progressively with a minimum from 35 to 90 days post-partum. Clusterin mRNA levels were strongly induced in pancreatic acinar AR4-2J cells in response to various apoptotic stimuli (i.e., cycloheximide, staurosporine, ceramide and H2O2) but not with interleukin (IL)-1, IL-4 or IL-6 or heat shock, which do not induce apoptosis in AR4-2J cells. In conclusion, we demonstrated that clusterin is synthesized and released by the pancreas. Its strong expression during acute pancreatitis suggests its involvement in the pancreatic response to injury. Clusterin is also induced during pancreatic development. Because these situations are associated with apoptosis and clusterin was shown to protect against apoptosis, we speculate that clusterin could be involved in the control of acinar cell apoptosis.

Expression of the Cdx1 and Cdx2 homeotic genes leads to reduced malignancy in colon cancer-derived cells.

We have previously described an inverse relationship between Cdx1 and Cdx2 mRNA levels and the extent of dysplasia and severity of clinical outcome in colorectal carcinoma, suggesting that altered expression of these genes was associated with colorectal carcinogenesis or tumor progression. To investigate further their involvement in the physiopathology of colorectal cancer, HT29 colon carcinoma cells that show very low Cdx expression were transfected with Cdx1 and/or Cdx2 cDNA to elicit their overexpression. Growth rate, tumorigenicity, resistance to apoptosis, and migration potential of the corresponding cells were analyzed. Growth rate of cells overexpressing Cdx2 decreased by half, whereas overexpression of Cdx1 had no effect. However, cells overexpressing both Cdxs had a growth rate reduced to 20% of control. In cells overexpressing Cdx1 or Cdx2, tumorigenicity and resistance to apoptosis induced by serum starvation, ceramide, or staurosporine were not changed compared with control cells; yet phorbol ester-stimulated cell migration was decreased by 50%. In cells overexpressing both Cdx1 and Cdx2, tumorigenicity was decreased by 50%, resistance to apoptosis was significantly lowered, and stimulated cell migration was further decreased to 15% of control compared with cells expressing Cdx1 or Cdx2. Finally, cells overexpressing both Cdxs showed strongly decreased Bcl-2 expression, which could account for their increased sensitivity to apoptosis. These findings show that, in HT29 cells, both Cdx1 and Cdx2 genes must be expressed to reduce tumorigenic potential, to increase sensitivity to apoptosis, and to reduce cell migration, suggesting that the two genes control the normal phenotype by independent pathways. This may explain why loss of Cdx1 or Cdx2 expression is associated with tumor development and invasiveness in colorectal tumors.

Cloning and expression of the mRNA of human galectin-4, an S-type lectin down-regulated in colorectal cancer.

We are interested in the characterization of genes whose expressions in the colon are modified during colorectal carcinogenesis. Our approach was to establish the phenotype of a colon tumor by partial sequencing of a large number of transcripts, then to select mRNAs of potential interest by differential screening with complex probes from normal or cancerous colon. In this paper, we report the cloning and sequencing of a mRNA strongly underexpressed in colorectal cancer. It corresponded to a protein comprising 323 amino acids, that appeared to be human galectin-4 on the basis of 76% and 79% amino acid identity to the rat and pig counterparts, respectively. Tissue distribution analysis showed that its expression was restricted to the small intestine, colon and rectum. Galectin-4 expression was compared in tumor and normal adjacent colon of 19 patients. In 18 patients, the mRNA concentration was 1.5-50-times lower in the tumor. No significant correlation was observed between decreased expression of galectin-4 and the degree of differentiation of the tumor or Duke's state. These results suggest that decreased galectin-4 mRNA expression may be an early event in colon carcinogenesis. Among five cell lines derived from colon carcinoma, only two (HT29 and LS174T) expressed galectin-4 mRNA.

Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis.

Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal-cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high-density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty-one clones were found over-expressed and 23 were underexpressed in the colorectal-cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain-containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT-PCR approach, complete sequence of both types of homeobox-containing cDNA was obtained. The amino-acid sequence of the human Cdx1 is 85% identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue-distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Twelve tissue samples from colorectal adenocarcinomas and the corresponding normal mucosa were analyzed by Northern blot. Expression of the 2 types of mRNA was either reduced or absent in 10 of them. Several colon-cancer cell lines were also analyzed. Cdx2 mRNA was absent from LS174T cells and Cdx1 mRNA was absent in PF11, TC7 and SW480 cells; none was detected in HT29 cells. It was concluded that decrease in human Cdx1 and/or Cdx2 expression is associated with colorectal tumorigenesis.

Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth.

To characterize at the molecular level the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis, we have developed a strategy in which the phenotype of the pancreatitis affected pancreas is established by characterization of a large number of its transcripts. Herein, we describe the cloning, sequence, and expression of a new gene, named p8, which is strongly activated in pancreatic acinar cells during the acute phase of pancreatitis, in developing pancreas and during pancreatic regeneration. In acinar cells, p8 mRNA is expressed rapidly and specifically in response to cellular pancreatitis-induced injury; its induction occurred almost similarly in edematous and necrohemorrhagic pancreatitis, indicating that p8 mRNA is maximally activated even in response to a mild pancreatic injury. Furthermore, in vitro studies suggest that p8 mRNA is induced in pancreatic and non-pancreatic cells in response to some apoptotic stimuli. p8 acts as a promoter of cellular growth factor when its cDNA is transfected into COS-7 and AR4-2J cells. Although we failed to identify p8-related sequences, analysis of its primary and secondary structure suggests that p8 is a basic helix-turn-helix-containing gene with slight homology to several homeotic genes and sufficient signal to be targeted to the nucleus. We therefore propose p8 as a putative transcriptional factor which can regulate pancreatic growth.

Induction of lithostathine/reg mRNA expression by serum from rats with acute pancreatitis and cytokines in pancreatic acinar AR-42J cells.

During the acute phase of pancreatitis, expression of most pancreatic enzymes decreases, whereas mRNAs of pancreatitis associated protein and lithostathine/reg increase dramatically. In the present study we have investigated the effect of serum from rats with acute pancreatitis (SAP) and cytokines on the lithostathine/reg mRNA expression in AR-42J cells. Lithostathine/reg mRNA was strongly induced by SAP in a dose-dependent manner. Induction was abolished by preheating the SAP or by treating the cells with cycloheximide. Treatment with interleukins (IL) IL-1 or IL-6 or dexamethasone alone was ineffective. Combination of IL-1 with IL-6 was also ineffective. Combination of IL-6 with dexamethasone resulted in strong induction of the lithostathine/reg gene, but the further addition of IL-1 to the mixture reduced induction. Treatment with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma) induced lithostathine/reg mRNA expression. Combination of dexamethasone with TNFalpha or IFNgamma showed an inhibitory effect on lithostathine/reg mRNA expression. These findings suggest that expression of the lithostathine/reg mRNA during acute pancreatitis could be mediated by specific combinations of cytokines and/or glucocorticoids.

Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region.

Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238-243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor alpha induced an increase in PAP I mRNA expression, and interferon gamma caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids.