Human Epithelial Colorectal Cells Research Articles

Cholera toxin inhibits HIV-1 replication in human colorectal epithelial HT-29 cells through adenylate cyclase activation

Mixed feeding, combining breast milk and nonhuman milk and/or solid food, is a common practice in developing countries that increases the risk of vertical HIV-1 transmission. It also enhances the risk of infection by waterborne microorganisms such as Vibrio cholerae, a diarrhoea-causing pathogen that frequently infects children below 18 months of age. Although both HIV-1 and V. cholerae affect young children and target intestinal epithelial cells, no information is currently available on possible interactions between these two pathogens. In this study, we show for the first time that cholera toxin (CTx), at a concentration as low as 100 pg/ml, inhibits HIV-1 infection of HT-29, a human colorectal epithelial cell line. The CTx-mediated inhibitory effect does not result from a down-regulation of receptor/co-receptor expression or a modulation of viral transcription. Nevertheless, additional experiments indicate that a yet to be identified early step in the virus life cycle is targeted by CTx since the enterotoxin similarly reduces infection of HT-29 cells with AMLV-I, HTLV-I and HIV-1 pseudotyped viruses while exerting no effect on infection with VSV-G pseudotypes. Furthermore, our results indicate that the CTx-dependent suppression is not due to the cholera toxin subunit B but linked instead to the action of cholera toxin subunit A (CTA). Altogether our data indicate that the CTA subunit of CTx is negatively affecting an early event in HIV-1 replication in human colon cancer HT-29 cells.

Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model.

Contributions of the alternative sigma factor sigmaB to Listeria monocytogenes infection were investigated using strains bearing null mutations in sigB, prfA, or inlA or in selected inlA or prfA promoter regions. The DeltaP4inlA strain, which has a deletion in the sigmaB-dependent P4inlA promoter, and the DeltasigB strain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially sigmaB dependent P2prfA promoter region did not differ from that of the wild type. The virulence of the DeltasigB and DeltaP4inlA strains was attenuated in intragastrically inoculated guinea pigs, with the DeltasigB strain showing greater attenuation, while the virulence capacity of the DeltaP2prfA strain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the DeltasigB mutation does not result from loss of sigmaB-dependent prfA transcription. Our results show that sigmaB-dependent activation of inlA is important for cell invasion and gastrointestinal infection and suggest that sigmaB-regulated genes in addition to inlA appear to contribute to gastrointestinal infection. Interestingly, the virulence of the DeltasigB strain was not attenuated in intravenously infected guinea pigs. We conclude that (i) L. monocytogenes sigmaB plays a critical role in invasion of human host cells, (ii) sigmaB-mediated contributions to invasion are, in part, due to direct effects on inlA transcription but not on prfA transcription, and (iii) sigmaB plays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.

Alterations in Connexin26 Expression during Colorectal Carcinogenesis

Background: Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions, which consist of transmembrane proteins called connexins (Cxs). Human colorectal epithelial cells express Cx32 and Cx43. Methods: Tissue samples (152 from colorectal cancer and 75 from adenoma) were investigated by immunohistochemistry, using the antibody for Cx26. Moreover, Cx26 was assessed in normal epithelium of the colon and rectum, adjacent to colorectal cancer. Results: In normal epithelium and adenomas, intercellular, punctate staining for Cx26 was observed. In adenomas with severe dysplasia, focally decreased expression of Cx26 was observed. Among 152 colorectal cancers, 55.9% classified only as adenocarcinoma stained positive for Cx26, but mainly cytoplasmic staining was found. We observed a positive correlation between Cx26 expression and tumor G2 grade (p < 0.005). The expression of Cx26 did not correlate with age, sex of patients, tumor localization, lymph node status or tumor size. Conclusions: Our results show that during colorectal carcinogenesis, loss of normal intercellular Cx expression occurs. Furthermore, the cytoplasmic presence of Cx26 could indicate a different role of Cx26 in neoplastic cells than participation in GJIC.

Expression of prostaglandin D 2 receptors DP1 and DP2 by human colorectal cancer cells

The expression and function of prostaglandin (PG) D 2 DP receptors during colorectal carcinogenesis has not been elucidated. Therefore, we studied expression of DP1 and DP2 receptors by reverse transcription-polymerase chain reaction analysis of receptor mRNA levels in five human colorectal cancer cell lines (HT-29, HCA-7, HCT116, SW480 and SW48) and VACO-235 human colorectal adenoma cells. DP1 receptor transcripts were present only in HT-29 cells. In addition, none of the human colorectal epithelial cell lines tested expressed DP2 receptor mRNA. Therefore, PGD 2 is unlikely to have direct activity on neoplastic colorectal epithelial cells via cell surface DP receptors.

Effect of PPAR Activators on Cytokine-Stimulated Cyclooxygenase-2 Expression in Human Colorectal Carcinoma Cells

Cyclooxygenase-2 (COX-2) expression is up-regulated in colorectal cancer tissue. Peroxisome proliferator-activated receptors (PPARs) are expressed in human colorectal tissue and activation of PPARs can alter COX-2 expression. In macrophages, activation of PPARs down-regulates COX-2 expression. We examined the effect of PPARα and PPARγ ligands on untreated and TNF-α-induced COX-2 expression in the human colorectal epithelial cell line HT-29. The expression of PPARα and PPARγ was confirmed in these cells. TNF-α, an inflammatory cytokine, increased COX-2 expression via the NFκB pathway. In the absence of TNF-α, WY14643 (PPARα activator) caused an increase, while BRL49653 (PPARγ activator) did not alter COX-2 expression. When HT-29 cells were incubated with TNF-α and WY14643, a further increase in COX-2 expression was detected. Incubation with TNF-α and BRL49653 caused an additional twofold increase in COX-2 expression. Our results suggest that both PPARα signaling and TNF-α signaling increase COX-2 expression by independent pathways, while PPARγ stimulates COX-2 expression by up-regulation of the TNF-α pathway.

Transcriptional down-regulation of the retinoblastoma protein is associated with differentiation and apoptosis in human colorectal epithelial cells.

The aim of this study was to investigate the regulation of Rb protein expression in relation to increased differentiation and induction of apoptosis in colonic epithelial cells. In vivo, Rb protein expression was found to be down-regulated towards the top of the normal colonic crypt, coincident with the region of differentiation and apoptosis, but highly expressed in colonic carcinoma tissue. Using in vitro models to study the regulation of Rb expression in pre-malignant colonic epithelial cells, we have been able to show for the first time that Rb protein expression is transcriptionally down-regulated in differentiated pre-malignant cells (in post-confluent cultures) but not in malignant colorectal epithelial cells. Furthermore, suppression of rb protein function by the HPV-E7 viral oncoprotein increased both spontaneous and DNA damage-induced apoptosis. These results suggest that Rb is able to act as a survival factor in colonic epithelial cells by suppressing apoptosis, and that over-expression of pRb in colorectal tumour cells can cause a loss of sensitivity to apoptotic signalling, resulting in aberrant cell survival and resistance to therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com

Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer.

Uridine diphosphate (UDP)-GalNAc: polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) catalyzes the initial step in mucin type O-glycosylation, and its expression has been assumed to be altered between normal epithelial cells and cancer cells. We studied the alteration of GalNAc transferase expression during the carcinogenesis of human colorectal epithelial cells. We produced polyclonal antibodies against synthetic polypeptides with specific sequence to two GalNAc transferase isozymes, T1 and T2. Surgically resected specimens from 50 patients with colorectal cancer were immunohistochemically stained, and the staining grade (percentage of positively stained cells) was compared between cancer and its normal counterpart in the same specimen. Significant signals for both T1 and T2 expression were seen in the supranuclear region of normal and cancer cells, indicating the subcellular localization of the enzymes in the Golgi apparatus. The prevalence of positive staining for T1 and T2 expression in colorectal cancer was significantly higher than that in normal epithelium (P < 0.05). However, the difference in staining grades between cancer and normal tissues varied in each patient. These results indicate that there is variability in the expression patterns of GalNAc transferase isozymes in normal and cancerous cells colorectal among individuals.

Analysis by High Density cDNA Arrays of Altered Gene Expression in Human Intestinal Epithelial Cells in Response to Infection with the Invasive Enteric BacteriaSalmonella

Many clinically important enteric pathogens initiate disease by invading and passing through the intestinal epithelium, a process accompanied by increased epithelial expression of proinflammatory cytokines. To further define the role intestinal epithelial cells play in initiating and modulating the host response to infection with invasive bacteria, hybrid selection on high density cDNA arrays was used to characterize the mRNA expression profile of approximately 4,300 genes in human intestinal epithelial cells after infection with the prototypic invasive bacteria, Salmonella. Selected findings were further evaluated by reverse transcription-polymerase chain reaction, Northern blot analysis, and protein assays. Epithelial infection with Salmonella significantly up-regulated mRNA expression of a relatively small fraction of all genes tested. Of these, several cytokines (granulocyte colony-stimulating factor, inhibin A, Epstein-Barr virus-induced gene 3, interleukin-8, macrophage inflammatory protein-2alpha), kinases (TKT, Eck, HEK), transcription factors (interferon regulatory factor-1), and HLA class I were the most prominent. Furthermore, the transcription factor NF-kappaB is shown to be important for inducible mRNA expression for a broad group of genes tested. These findings expand the repertoire of known epithelial cell responses to infection with an invasive enteric pathogen. The results also show that evaluation of mRNA expression profiles by cDNA array analysis is a powerful approach to characterizing and understanding host-pathogen interactions.

CD44 is associated with proliferation in normal and neoplastic human colorectal epithelial cells

Flash-frozen biopsies obtained from surgical specimens of three adenomatous polyps and 22 colorectal adenocarcinomas (19 primary and three metastatic) were tested by immunohistochemistry for CD44 expression using F10-44-2 monoclonal antibody. CD44 positivity was correlated with proliferative status defined by Ki-67 monoclonal antibody reactivity. In normal colonic mucosa, CD44 was expressed in the proliferative zone of crypts. In tumours, CD44 expression was associated with proliferative areas irrespective of tumour stage or differentiation. Non-proliferating areas of the carcinomatous epithelium did not express CD44 although non-proliferating stromal lymphoid tissue did. There was no apparent association with tumour progression. F10-44-2-defined CD44 is consistently expressed during proliferation by normal colorectal epithelial cells and by both benign and malignant colorectal tumour cells.