Claude Asselin

MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells

The present report delineates the critical pathway in the G1 phase involved in downregulation of p27Kip1, a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27Kip1 were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27Kip1 protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27Kip1 reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27Kip1 downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27Kip1 was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27Kip1 may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27Kip1 downregulation in fibroblasts and epithelial cells.

Regulation of c-myc expression by sodium butyrate in the colon carcinoma cell line Caco-2

The human colon carcinoma cell line Caco‐2 spontaneously undergoes enterocytic differentiation in culture. We used sodium butyrate to modify differentiation and growth properties of this cell line and considered c‐myc expression as a potential target. Degradation of normal c‐myc mRNAs with a half‐life of 20 min is not coupled to translation in this cell line, as determined by cycloheximide treatment. We show that butyrate reduces c‐myc. mRNA levels after a 30 min delay. Butyrate does not affect c‐myc, expression at the level of transcriptional initiation or elongation, as determined by run‐on analysis, but at a post‐transcriptional level. Cycloheximide blocks butyrate‐dependent reduction of c‐myc mRNA levels. Cross‐linking experiments show that a 34 kDa protein binds specifically to the c‐myc AU‐rich instability determinant found in the 3′‐untranslated region (ARE). Binding of this protein to the ARE is not modulated by butyrate or cycloheximide. These experiments suggest that butyrate induces a factor involved in c‐myc. mRNA degradation that differs from the known ARE‐associated proteins. Post‐transcriptional modification of gene expression could be one of the major targets for this anti‐proliferative agent.

Activation of haptoglobin gene expression by cAMP involves CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells

CCAAT/enhancer‐binding protein (C/EBP) isoforms are expressed in rodent intestine and in the rat intestinal epithelial cell line IEC‐6 but their role remains to be determined. Treatment of IEC‐6 cells with the adenylate cyclase activator forskolin led to coordinate induction of C/EBP isoforms α, β and δ at the mRNA and protein levels. Transient transfection assays showed that their expression is controlled at the transcriptional level. Forskolin treatment induced haptoglobin mRNA levels. Electrophoretic mobility shift and supershift assays demonstrated an increase in DNA‐binding activities of the three C/EBP isoforms to the haptoA and haptoC C/EBP DNA‐binding sites of the proximal haptoglobin promoter. Site‐specific mutations of both sites led to a decrease in transcriptional induction by forskolin, suggesting that C/EBP isoforms are involved in the cAMP‐dependent regulation of the acute‐phase protein gene haptoglobin in intestinal epithelial cells.

Expression of the α-5(IV) collagen chain in the fetal human small intestine

Abstract Background/Aims: The basement membrane type IV collagen is a family composed of at least five genetically distinct but structurally similar polypeptide chains, α1–α5. The α1(IV) and α2(IV) chains are ubiq-uitous components of basement membranes, whereas the α3(IV), α4(IV), and α5(IV) chains have a restricted tissue distribution. The aim of this study was to analyze the presence of these minor type IV collagen chains in the small intestinal mucosa. Methods: The expression of type IV collagen chains in the developing and adult human small intestine was determined by indirect immunofluorescence with monoclonal and polyclonal antibodies. Western blotting and Northern hybridization analysis were also used to additionally investigate the expression of theα1(IV) and α5(IV) chains. Results: The α3–α5(IV) chains were absent from the adult epithelium, but, surprisingly, the α5(IV) chain was consistently detected in the fetal mucosa. Its expression was confirmed by Western blotting, complementary DNA polymerase chain-reaction amplification, and Northern hybridization analysis. Conclusions: The α5(IV) chain of collagen is expressed in the fetal but not adult human intestinal epithelium. Its position at the basolateral domain of epithelial cells suggests a potential role for this molecule during development.

Differential Expression of fos and jun Family Members during Murine Postnatal Intestinal Development

We have examined the patterns of expression of members of the fos and jun families of transcription factors during murine postnatal intestinal development. Northern analysis showed increases in fosB, c-fos, and junB mRNA levels in both proximal jejunum and colon. These increases coincide with weaning a period of major changes in intestinal cell proliferation and migration. Administration of dexamethasone to suckling mice resulted in transient induction of c-fos mRNA levels in all gut segments. Thus, modifications in expression of certain members of the fos and jun families in vivo correlate with major maturational changes during murine intestinal development and support a role for these transcription factors in the regulation of intestinal functions.

Phenotypic analysis of human fetal renal cells transformed by the SV40 large T antigen

Dear Editor: The kidney is composed of a variety of highly differentiated cell types with specific transport properties and biochemical functions (15). Development of serum-free hormonally defined medium has allowed growth of primary cultures of kidney epithelial ceils exhibiting several specific functions of the normal parental cells (21). Studies on primm3~ cultures are hampered because of their limited life span and loss of differentiated characteristics after several passages. To circumvent these problems, transformed human renal cell lines have been established. Homogeneous populations of ceils from defined nephron locations have been obtained following transformation with the early region genes of simian virus 40 (SV40), a wellknown oncogenic virus (9). Murine glomerular and cortical duct epithelial cell lines (8,20) or proximal tubule cell lines (3) have been obtained from mice transgenic for the early region of SV40. Rabbit kidney cell lines arising from the thick ascending limb of Henles loop (17) or from the proximal and distal tubule (14,22) and murine (5,23) and human (1,4,12) kidney cell lines have been obtained after transfection in vitro with SV40 early region DNA. These previous studies have shown that long-term culture of established cell lines results in partial loss of some specific functions and that the differentiation program is partially altered by the transforming agent• Little is known about the phenotypic modifications arising during the first passages after SV40 transformation of human kidney derived epithelial cells. In this study, we report the transformation of fetal kidney cells by the SV40 large T gene and demonstrate that these cells retain characteristics of tubular epithelial cells• This work represents a detailed analysis of in vitro transformed human fetal kidney cells before crisis. Eighteen-wk-old human fetal kidneys were obtained after legal abortion. The project complied with requirements from the institutional review committee for the use of human material. Kidneys were washed in phosphate-buffered saline (PBS), minced, and treated for 25 rain at 37 ° C with a dispase-collagenase solution (25 U/ml, 35 U/ ml). Isolated cells were centrifuged, resuspended in Dulbeccos nlodified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 raM glutamine, and plated at 37 ° C. Cells were grown to 90% confluence in an atmosphere of 95% air, 5% CO2. Cells were then trypsinized, washed, and resuspended in 2 ml serumfree Opti-MEM (GIBCO-BRL, Grand Island, NY). Transfection was performed with 20 Hg of plasmid neoA2005 complexed to 50 ~tg of lipofectin (GIBCO-BRL) in a total volume of 100 HI. This plasmid contains the G418 resistance gene linked to the A2005 mutant of the SV40 early region encoding only the SV40 large T gene (18). After a 15-rain incubation at room temperature, cells were plated in serum-free DMEM; 24 h later, the medium was replaced with DMEM containing 10% FBS. Cells were then selected after 24 h in complete medium containing 200 ~tg/ml geneticin (G418, GIBCO-BRL). Twenty days later, G418 resistant clones were isolated and allowed to grow. These cells were characterized by a fibroblastlike morphology. Karyotypic analysis of two randomly selected cell lines showed a normal diploid karyotype after 10 passages (40 doublings) (data not shown). The large T antigen was detected by immunofluorescence in the nuclei of more than 90% of the individual cells. Although SV40 large T expression definitely increased the life span of human fetal kidney ceils, transformed cells showed signs of apoptosis after 20 passages (80 doublings) and eventually died. The phenotype of one clone (HFK-1, human fetal kidney) was further investigated between Passages 8 and 15. We undertook a comparative morphological and ultrastructural analysis of HFK-1 cells at various culture stages. The HFK-1 cells exhibited an absence of contact inhibition, resulting in the formation of multiple layers after 5 and 17 d of culture. The upper cell layer was compact and cells exhibited a more epithelioid shape 17 d after confluence. Lower cell layers displayed large intercellular spaces (data not shown). DNA synthesis occurred in every cell layer, as determined by in situ 3H-thymidine incorporation (data not shown). Uhrastructural analysis by transmission electron microscopy (TEM) showed no evidence of epithelial cell polarization or structural differentiation 5 d after confluence (Fig. 1 A). However, collagen deposits appeared 10 d after confluence in lower cell layers (Fig. 1 B). Seventeen days after confluence, the upper cell layer developed