Gustavo V. Mallo

Inducible Antibacterial Defense System in C. elegans

The term innate immunity refers to a number of evolutionary ancient mechanisms that serve to defend animals and plants against infection. Genetically tractable model organisms, especially Drosophila, have contributed greatly to advances in our understanding of mammalian innate immunity. Essentially, nothing is known about immune responses in the nematode Caenorhabditis elegans. Using high-density cDNA microarrays, we show here that infection of C. elegans by the Gram-negative bacterium Serratia marcescens provokes a marked upregulation of the expression of many genes. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Certain infection-inducible genes are under the control of the DBL-1/TGFbeta pathway. We found that dbl-1 mutants exhibit increased susceptibility to infection. Conversely, overexpression of the lysozyme gene lys-1 augments the resistance of C. elegans to S. marcescens. These results constitute the first demonstration of inducible antibacterial defenses in C. elegans and open new avenues for the investigation of evolutionary conserved mechanisms of innate immunity.

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 under‐expressed 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. Int. J. Cancer 74:35‐44.

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.

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 α induced an increase in PAP I mRNA expression, and interferon 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.

p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis.

p8 is a stress-induced DNA-binding protein, biochemically related to the architectural chromatin binding HMG protein family and whose function is presently unknown. We obtained fibroblast from mice lacking p8 and found that p8 is involved in cell growth regulation and in apoptosis. p8−/− mouse embryonic fibroblasts (MEFs) grow more rapidly than p8+/+ MEFs. This might be explained by the higher intracellular level and activity of the Cdk2 and Cdk4 observed in p8−/− MEFs, which in turn may result, at least in part, from the concomitant decrease observed in the amount of cyclin-dependent kinase inhibitor p27. We also report that p8 mRNA expression is strongly activated in fibroblasts after cell growth arrest induced by serum deprivation or confluence. As expected, MEFs expressing p8 arrest their growth more rapidly after serum deprivation than MEFs lacking p8, which strongly suggests that p8 over-expression is implicated in cell growth arrest. On the other hand, p8+/+ MEFs are more sensitive than p8−/− MEFs to the apoptosis induced by adriamycin treatment. p53 might be involved, as p8 expression increases its intracellular amount and trans-activation capacity. Finally, demonstration that p53 is a negative trans-activator of p8 suggests the presence of a complex autoregulatory loop. In conclusion, p8 is a cell growth inhibitor that facilitates apoptosis induced in fibroblasts by DNA damage.

pap, reg Iα and reg Iβ 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 Iα and reg Iβ, 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 Iα 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 Iα. No significant relationship was found between concentrations of pap, reg Iα or reg Iβ 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 IFNγ 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. Int. J. Cancer 81:688–694, 1999.

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.

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.

Cloning and expression of the rat VMP1 (vacuole membrane protein 1) mRNA, a new gene activated in pancreas with acute pancreatitis, which promotes vacuole formation

To characterize the emergency program set up by pancreatic cells in response to pancreatitis, we established the phenotype of the pancreatitis-affected pancreas by characterizing a large number of its transcripts. In this report, we describe the cloning, sequencing, and expression pattern of a new gene, named VMP1 (vacuole membrane protein 1). The VMP1 mRNA codes for a putative protein of 406 amino acids. In situ hybridization studies revealed that pancreatic expression of VMP1 mRNAs was restricted to the acinar cells. Interestingly, VMP1 mRNA was also overexpressed in kidney after transient ischemic injury. However, many healthy tissues express VMP1 mRNA. Structure analysis suggested that VMP1 is a transmembrane protein with six hydrophobic regions. VMP1/EGFP fusion protein was located to the Golgi apparatus and the endoplasmic reticulum area. Expression of this protein promoted the formation of intracytoplasmatic vacuoles and VMP1/EGFP was located to the membranes of these vacuoles. Cells overexpressing this protein died after 48 h. In conclusion, we have identified a new stress-induced gene which codes for a transmembrane protein that, when overexpressed, promotes formation of intracellular vacuoles followed by cell death.