Jacques Abello

Characterization of calcitonin gene-related peptide receptors and adenylate cyclase response in the murine macrophage cell line P388 D1

Specific receptors for calcitonin gene-related peptide (CGRP) were identified and characterized on plasma membranes from the interleukin-1 secreting murine macrophage-like cells line P388 D1. The binding of [125I]-rat CGRP I was time-dependent, reversible and the rate of dissociation of [125I]-rat CGRP I increased in the presence of GTP. Scatchard analysis was consistent with a single class of binding sites, with an apparent dissociation constant of 1.76 nM and a maximal binding capacity of 85.48 fmol/mg protein. In competitive displacement studies, rat CGRP I, human CGRP I and human CGRP II were equipotent to inhibit the binding of [125I]-rat CGRP I (IC50 = 4 nM) while rat CGRP II and the synthetic analogue [tyr(o)]-human CGRP I were ten-fold less potent. Porcine calcitonin and VIP did not inhibit tracer binding. In the presence of GTP, CGRP stimulation of adenylate cyclase was dose-dependent and strongly correlated with receptor occupation. These results indicate that the P388 D1 macrophage-like cell line expresses CGRP specific receptors functionally coupled to adenylate cyclase, which may be involved in CGRP-mediated macrophage immune response.

Differential involvement of destrin and cofilin-1 in the control of invasive properties of Isreco1 human colon cancer cells.

Actin depolymerizing factor (ADF)/cofilin family proteins are key regulators of actin filament turnover and cytoskeleton reorganization. The role of cofilin‐1 in cell motility has been demonstrated in several cell types but remained poorly documented in the case of colon cancer. In addition, the putative function of destrin (also known as ADF) had not been explored in this context despite the fact that it is expressed in all colon cancer cell lines examined. We were therefore prompted to evaluate the respective contributions of these proteins to the invasive properties of the human colon cancer Isreco1 cell line, which expresses a comparatively high destrin/cofilin ratio. Reduction of cofilin‐1 or destrin expression in Isreco1 cells using RNA interference led to an increase of the number of multinucleated cells and altered polarized lamellipodium protrusion and distribution of paxillin‐containing adhesions. Both cofilin‐1 and destrin silencing enhanced cell adhesion to extracellular matrix components. However, only destrin appeared to be required for cell migration on collagen I and for cell invasion through Matrigel in response to the proinvasive neuroendocrine peptide bombesin. This differential functional involvement was supported by a destrin‐dependent, cofilin‐independent phosphorylation of p130Crk‐associated substrate (p130Cas) upon cell adhesion to collagen I or Matrigel. Taken together, our results suggest that destrin is a significant regulator of various processes important for invasive phenotype of human colon cancer Isreco1 cells whereas cofilin‐1 may be involved in only a subset of them.

Binding and in vitro modulation of human epidermal Langerhans cell functions by substance P.

Substance P (SP) is distributed in both the central and peripheral nervous system. It has various effects on immunocompetent cells, such as macrophages and lymphocytes. The aim of our study was to search for the presence of SP receptors (SP-R) on human cutaneous Langerhans cells (LC), and to determine the effects of SP on LC immunological functions in a model of mixed epidermal cell-lymphocyte reaction (MELR). Radioligand binding studies showed that LC-enriched epidermal cell suspensions reversibly bound SP, and that the specific binding increased with the percentage of LC. Functional assays showed that SP had no effect when added at concentrations from 10–6M to 10–12M to the MELR. The addition of SP at concentrations of 10–4M and 10–5M was able to inhibit the allogeneic T-cell response (98.3 ± 1.8% and 92.8 ± 8.9% inhibition, respectively) without modifying the cell viability. This inhibition was through an effect of SP on both T-cell and LC function. We conclude that SP has receptors on LC and may inhibit antigen presentation.

p53-Mediated upregulation of DcR1 impairs oxaliplatin/TRAIL-induced synergistic anti-tumour potential in colon cancer cells

Oxaliplatin has emerged as a major chemotherapeutic drug in the treatment of advanced colorectal cancer, yet like most conventional cancer therapeutics, its efficacy is often compromised due to p53 mutations. Unlike oxaliplatin, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a p53-independent manner, and chemotherapy is known to overcome tumour resistance to TRAIL-induced cell death in most cancer cells. Using a panel of colon cancer cell lines, we assessed the ability of oxaliplatin to sensitize to TRAIL-induced apoptosis. We demonstrate that while both drugs additively or synergistically induced apoptosis in almost all cell lines tested, p53 wild-type colon cancer cells such as HCT116, LS513 or LS174T remained resistant. Impaired TRAIL-induced cell death resulted from a strong p53 dependent, oxaliplatin-mediated, DcR1 receptor expression increase. According to our finding, downregulation of DcR1 using siRNA, in p53 wild-type colon cancer cells, restored oxaliplatin/TRAIL synergistic apoptotic activity. On the contrary, exogenous DcR1 overexpression in SW480, a p53-mutated cell line, abolished the synergy between the two drugs. Altogether we demonstrate for the first time that p53 negatively regulates oxaliplatin-mediated TRAIL-induced apoptotic activity through DcR1 upregulation. Our findings could have important implications for future therapeutic strategies, and suggest that the association oxaliplatin/TRAIL should be restricted to patients harbouring a non-functional p53 protein.

In colon carcinogenesis, the cytoskeletal protein gelsolin is down-regulated during the transition from adenoma to carcinoma

The actin-binding protein gelsolin is involved in cell motility via the regulation of actin cytoskeleton, and its expression is modified in several human cancers. However, the potential implication of this protein in colorectal carcinogenesis is debated. By using immunohistochemistry, we studied gelsolin expression in 69 cases of colon adenocarcinomas and in 72 lesions representative of the different stages of colonic tumorigenesis. In addition, we performed Northern blot analysis of gelsolin messenger RNA in 12 paired samples of human colon cancer and normal corresponding mucosa. Gelsolin protein and messenger RNA expressions were severely down-regulated in all adenocarcinomas tested. Moreover, gelsolin protein was down-regulated in a large proportion of high-grade adenomas (14/16) before the acquisition of invasive properties but in only a small proportion of low grade adenomas and serrated adenomas (2/30) and in none of the 9 cases of nonneoplastic hyperplastic polyps tested. Our results therefore demonstrate that gelsolin down-regulation is an early and almost constant event in colon carcinogenesis and is associated with the transition from adenoma to carcinoma.

Expression of SNARE proteins in enteroendocrine cell lines and functional role of tetanus toxin-sensitive proteins in cholecystokinin release

In neurons, synaptic vesicle exocytosis involves the formation of a core complex particle including syntaxin‐1, synaptosomal‐associated protein of 25 kDa (SNAP‐25) and vesicle‐associated membrane protein (VAMP)‐2/synaptobrevin. The expression of these proteins was investigated in a panel of cell lines, including lines of endocrine and intestinal origin, by Western blotting and/or immunocytochemistry. The three core complex proteins were detected in the enteroendocrine, cholecystokinin (CCK)‐secreting, cell lines STC‐1 and GLUTag, and in the endocrine non‐intestinal cell lines CA‐77 and HIT‐T15. In contrast, SNAP‐25 and syntaxin‐1 were undetected in the intestinal non‐endocrine cell lines IEC‐6, HT‐29 and Caco‐2, whereas a slight expression of VAMP‐2 was documented in IEC‐6 and HT‐29 cells. Co‐immunoprecipitation experiments indicated that syntaxin‐1, SNAP‐25 and VAMP‐2 were present in a complex similar to that identified in brain. In the STC‐1 cell line, treatment of streptolysin‐O‐permeabilized cells with tetanus toxin (Tetx) selectively cleaved VAMP‐2 and VAMP‐3/cellubrevin, and simultaneously abolished Ca2+‐induced CCK secretion (IC50∼12 nM). These results show that endocrine cell lines of intestinal origin express syntaxin‐1, SNAP‐25 and VAMP‐2, and suggest a key role for a Tetx‐sensitive protein (for example VAMP‐2 and/or VAMP‐3) in the CCK secretion by STC‐1 cells.

Rab3a controls exocytosis in cholecystokinin-secreting cells.

The expression of rab3A and rab3D isoforms in the enteroendocrine, cholecystokinin‐secreting, cell lines STC‐1 and GLUTag is here demonstrated. In contrast, rab3B is undetectable in these two cell lines, and rab3C is only slightly expressed in GLUTag cells. Using a transient co‐transfection system with human growth hormone as reporter protein, we show that overexpression of the GTPase‐deficient mutant rab3AQ81L, but not rab3DQ81L, significantly decreases human growth hormone secretory responses to various agonists in STC‐1 cells. These results indicate that endocrine cell lines of intestinal origin express rab3A and rab3D proteins, but the GTP‐bound form of rab3A only acts as a negative modulator in the control of cholecystokinin secretion from STC‐1 cells.

Release of ileal neurotensin in the rat by neurotransmitters and neuropeptides

To investigate the functional relationship between the enteric nervous system and the intestinal neurotensin (N) cells, the release of neurotensin (NT) was measured upon vascular 8-min infusion periods of various neurotransmitters and neuropeptides in an isolated vascularly perfused rat jejunoileum. NT-like immunoreactivity (NT-LI) was measured with an antiserum that specifically recognizes intact NT. The cholinergic agonists methacholine and carbachol produced a strong release of NT-LI (250% and 700% of basal, respectively at 10(-5) M). The infusion of a lower dose (10(-7) M) was less effective in both cases. The nicotinic receptor agonist DMPP (10(-4) M) had no significant effect on NT-LI release. Norepinephrine (10(-6) M) produced a moderate and well-sustained secretion of NT (200% of basal). Infusion of higher doses of these neurotransmitters dramatically increased the arterial pressure. G-amino-n-butyric acid (GABA), histamine, serotonin and dopamine administered at final concentrations up to 10(-5) M had no effect on NT-LI release. In contrast, gastrin-releasing peptide and bombesin induced a dose-dependent transient increase of portal NT-LI (maximal value at 10(-7) M: 1000% of basal) followed by a rapid return to near basal values. Substance P (10(-7) M) evoked a prompt release of NT-LI with a peak at 600% of basal followed by a decline to 200% of basal at the end of the session. Leu-enkephalin and calcitonin-gene-related-peptide (CGRP, 10(-7) M) produced a small rise in portal NT-LI, while Met-enkephalin, dynorphin, vasoactive intestinal peptide (VIP), galanin, neuropeptide Y (NPY), peptide histidine isoleucine (PHI), neuromedin U and thyrotropin releasing hormone (TRH) had no stimulatory effect. Our results indicate that additionally to the secretion of NT induced by cholinergic agents and bombesin, substance P and to a lesser extent Leu-enkephalin are capable of stimulating NT release in the rat.

Huan normal dermal fibroblasts express somatostain receptors

Abstract: The hormone/neuropeptide somatostatin (SOM) exerts multiple functions in the central nervous system, the immune system, the hypothalamo‐pituitary axis, the gastrointestinal tract, and the pancreas. Endogenous SOM occurs in 2 biologically active forms, with 14 or 28 amino acids. Five subtypes of SOM reporters have been cloned. SOM is present in human skin. We have investigated the expression of SOM receptors on human dermal normal fibroblasts. Biotinyl‐SOM allowed the visualization of SOM receptors on human dermal fibroblasts. Radioligand binding with (3‐[125I]iodotyrosy]11)‐SOM‐14 were performed on these cells and the effect of SOM‐14 on the DNA synthesis by fibroblasts was evaluated by measuring [3H]‐methyl thymidine incorporation. Saturation curve, and Scatchard plot showed a homogeneous class of receptors with a Bmax of 0.055±0.023 nM and KD of 2.0±0.4 nM (values: mean±SEM). Fibroblasts expressed 3,317±1,385 binding sites per cell. Commpetitive displacement experiments showed that SOM‐14 IC50 was 69.3±4.5 nM (mean±SEM), for SOM‐28 33.2±6.0 nM and for octreotide 36.5±3.3 nM. The KI values calculated from these IC50 were, respectively: 62.4±4.1 nM; 29.9±5.4 nm; 32.9±2.9 nM. We conclude that subtype 2 or 3 SOM receptros is present on human normal dermal fibroblasts. A weak effect of SOM‐14 on DNA synthesis was observed with SOM concentrations of 10‐7 and 10‐6 M.

CCK expression in enteroendocrine cell is regulated by soluble factor(s) from underlying fibroblasts.

Studies on the cross-talk between the intestinal epithelium and the underlying connective tissue have concentrated on enterocytes. In contrast, little is known about the interactions between the mesenchymal compartment and the enteroendocrine cells, scattered among the other cell types of the epithelium. To address this question, a panel of coculture systems between the enteroendocrine STC-1 cell line and three intestinal myofibroblastic cell lines (MIC) was used in order to assess different levels of regulation, namely cell-cell and cell-matrix interactions, and the role of diffusible factors. We demonstrate that the expression of cholecystokinin, a typical intestinal hormone produced by STC-1 cells, is up-regulated in the presence of a fibroblastic environment through a paracrine pathway involving FGF2. Concomitantly, STC-1 cell morphology and proliferation were also modulated, but through distinct mechanisms according to the origin of fibroblasts. The results reveal definite epithelio-mesenchymal interactions that may be critical for the maintenance of phenotype and function of enteroendocrine cells.