Pyroglutamyl-histidyl-glycine, the endogenous colon mitosis inhibitor, regulates cyclic AMP level in non-tumorigenic colonic epithelial cells.

BACKGROUND: We have proposed that the mitosis inhibiting peptide, pyroGlu-His-Gly (pEHG), a colon-specific negative feedback regulator of cell proliferation, works through a G protein-coupled receptor (GPCR), as do many other pyroglutamyl-peptides. MATERIALS AND METHODS: Non-tumorigenic YAMC (colon mucosa of Immorto mice), IMCE (Immorto-Min mouse hybrid) and human hepatoma (HepG2) cell lines were exposed to pEHG. cAMP concentrations were measured with a protein binding assay, mRNA levels with real-time PCR and Ca2+ concentration with an inverted fluorescence microscope on Fura-2/AM-loaded cells. RESULTS: pEHG (1 nM) increased the intracellular concentration of cAMP after 5-10 min in YAMC cells, but not in HepG2 cells. No effect was seen on cytosolic Ca2+, or in the expression of the proliferation and differentiation regulatory genes c-fos, egr-1 or fosB in YAMC or IMCE cells. CONCLUSION: pEHG stimulates the second messenger cAMP, but has no effect on intracellular Ca2+ or the gene expression of c-fos, egr-1 or fosB.

The colon mitosis inhibitor pyroglutamyl-histidyl-glycine inhibits growth of non-tumorigenic colonic epithelial cells.

BACKGROUND: The colon mitosis inhibiting peptide pyroglutamyl-histidyl-glycine (pEHG) increases the expression of c-fos, fosB and egr-1 genes in the colon carcinoma cell line HT-29. However, the effect on non-tumorigenic colonic cells has not been investigated. MATERIALS AND METHODS: After exposure of the cell lines YAMC (from colon mucosa of Immorto mice) and IMCE (fromn Immorto-Min mouse hybrid) to pEHG, DNA-synthesis was analysed by H3-thymidine incorporation, apoptosis and necrosis by fluorescence microscopy, and cell cycle distribution by flow cytometry. RESULTS: pEHG inhibited DNA-synthesis with a maximal effect at 10(-8)-10(-9) M, but stimulated at 10(-4) M. It blocked cell flow through the cell cycle at GC/M after 8 h of treatment, but had no effect on apoptosis or necrosis at any concentration. A low concentration of ascorbic acid stabilised the cells, maybe as a free radical scavanger. CONCLUSION: pEHG inhibits flux at the G2/M transition, but has no effect on cell death.

Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols.

BACKGROUND: Established colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth- and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance. RESULTS: HCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment. CONCLUSION: All HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols.

The effects of a growth-inhibiting tripeptide, acetylGlu-Ser-GlyNH2 (Ac-ESG), on gene expression and cell cycle progression of two lymphoma cell lines.

BACKGROUND: We recently isolated and characterized a tripeptide, acetylGlu-Ser-GlyNH2 (Ac-ESG), which inhibits proliferation of lymphoid cells. In this paper we describe the effects of Ac-ESG on growth-related gene expression and cell cycle progression in two lymphoma cell lines, Ramos and Molt, representing B and T lymphocytes, respectively. MATERIALS AND METHODS: RNA was extracted from Molt and Ramos cells with or without the tripeptide treatment. Gene expression was examined by semi-quantitative RT-PCR and Northern blot hybridization, and cell cycle progression was detected by flow cytometry. RESULTS: In the Molt cells, p53 gene expression was increased following treatment while c-myc was decreased after treatment shorter than 24 hours; N-ras expression was significantly reduced at picomolar concentration for 24-hour treatment; cyclinD1 and cdk4 expression did not show any change; DNA flow cytometry demonstrated that Molt cells were arrested or delayed predominantly in G2-M. Untreated Ramos cells had higher gene expression levels of c-myc, N-Ras and cdk4 than Molt cells, but lower p53 expression. These cells were not sensitive to Ac-ESG. CONCLUSION: The tripeptide Ac-ESG alters the expression of several growth-related genes in the Molt cell line and brings about an arrest or delay of cells in G2-M.

The colon mitosis-inhibitor pyroglutamyl-histidyl-glycine alters expression of immediate-early cancer-related genes in HT-29 cells.

BACKGROUND: The intestinal mitosis-inhibiting peptide pyroglu-His-GlyOH (pEHG), inhibits normal intestinal epithelial cells and the human colon adenocarcinoma cell line HT-29 and increases the expression of c-fos (1). In this study, we investigated the mechanisms of the growth-inhibiting effects of pEHG. MATERIALS AND METHODS: cDNA expression array was hybridized with cDNA from HT-29 cells exposed to pEHG or control. The results were confirmed with Northern blot or real-time PCR. RESULTS: pEHG(1 nM) provoked a significant increase in the expression of the early growth response protein 1 (egr-1) after an incubation of 20 minutes, while c-fos was confirmed up-regulated by the same treatment. We further studied the expression of fosB, c-jun and junB, in the AP-1 complex. fosB was up-regulated 20-fold, but only minor effects on jun variants were observed. CONCLUSION: pEHG stimulates the gene expression of some immediate-early transcription factors involved in cell proliferation.

Early oncogene mRNA expression in HT-29 cells treated with the endogenous colon mitosis inhibitor pyroglutamyl-histidyl-glycine.

BACKGROUND: The tripeptide pyroGlu-His-GlyOH (pEHG), isolated from intestinal extracts (1), suppresses growth of human colonic epithelial cells and human colorectal adenocarcinoma cells (HT-29) both in vitro and in vivo. The present study represents the first attempt to relate alterations in relevant gene expression to the effect on cell growth. MATERIALS AND METHODS: Northern blot with RNA, extracted from HT-29 cells exposed to pEHG, was hybridised with c-fos and c-myc probes. RESULTS: c-fos gene expression was doubled in HT-29 cells after 20 minutes of incubation with pEHG and decreased to half the initial value after 2 hours. The increase at 20 minutes was concentration-dependent at a peptide concentration range from 10(-6)-10(-12) M. The expression of the oncogene c-myc showed only marginal alternations at the concentrations and times tested. CONCLUSION: The colon mitosis-inhibiting peptide pEHG increases gene expression of c-fos, but not that of c-myc.