pp60c-Src Kinase Mediates Growth Effects of the Full-Length Precursor Progastrin1–80 Peptide on Rat Intestinal Epithelial Cells, in Vitro
Abstract\, http://www.100md.com
Growth factor effects of precursor forms of gastrins have become evident in recent years. However, intracellular pathways that mediate growth effects of the precursor molecules are not known. In previous studies, we reported an increase in Tyr phosphorylation of pp60c-Src in intestinal epithelial cells (IEC) in response to the fully processed form of gastrin [gastrin1–17 (G17)]. We have now examined whether c-Src kinase is similarly phosphorylated and activated in response to the full-length precursor molecule, progastrin (PG)1–80, (recombinant human PG) in IEC cells. We found a significant increase in pp60c-Src kinase activity in response to both G17 and PG (0.1–1.0 nM), suggesting that growth effects of both the precursor and fully processed gastrin molecules may be mediated via similar pathways. On the other hand, pp62c-Yes was not phosphorylated or activated in response to either G17 or PG. To examine whether c-Src kinase mediates proliferative effects of PG, IEC cells were microinjected with anti-Src-IgG and 3H-thymidine (3H-Tdr) uptake of the cells measured. Control cells received nonimmune IgG. The 3H-Tdr uptake of cells stimulated with 1.0 nM PG was significantly reduced in cells microinjected with anti-c-Src-IgG; control IgG had no effect. In cells stimulated with 1.0% fetal calf serum, microinjection with c-Src-IgG had no effect on 3H-Tdr uptake. The specificity of the effect was further confirmed by blocking the inhibitory effect of anti-c-Src-IgG with antigenic Src peptide. These results suggest that activation of c-Src kinase likely represents a critical step in mediating proliferative effects of both the precursor and fully processed forms of gastrins on IEC.
Introduction8l00)u1, 百拇医药
THE GASTRIN GENE is expressed by G entero endocrine cells in the antrum of the stomach. Gastrin gene products (termed: gastrins) are processed by endopeptidases within G cells, converting the full-length progastrin (PG) precursor peptide (80 amino acids) into glycine-extended forms of gastrins. Subsequent amidation at the C-terminal end generates amidated forms of gastrins [gastrin1–17 (G17), G34)]. Under physiological conditions, G17 and G34 are the major circulating forms of gastrin, and they play an important role in acid secretion from parietal cells (1). Gastrins are also trophic for gastrointestinal mucosal cells and play an important role in the renewal of colonic mucosa (2, 3). However, C-terminal amidation is not crucial for measuring biological effects (4). Mitogenic effects of glycine-extended gastrin have been reported on rat intestinal cells (IEC-6), Swiss 3T3 fibroblasts (5), rat pancreatic cancer cells (AR42J) (6), and human colon cancer cells (DLD-1) (7). Migratory effects of gly-gastrin were reported on mouse gastric epithelial cells (IMGE-5) (8). In recent years, the full-length precursor molecule (PG) was reported to be biologically active. Transgenic mice overexpressing PG demonstrated hyperproliferation of colonic crypts (9). PG-expressing mice were at a higher risk for developing preneoplastic and neoplastic lesions in the colonic mucosa in response to azoxymethane (10, 11). Treatment with gly-gastrin increased the risk for developing aberrant crypt foci in rats (12).
Intracellular mechanisms mediating mitogenic effects of G17 and the precursor PG peptide on intestinal epithelial cells (IEC) remain unknown. In previous studies, G17 was reported to stimulate a significant increase in the levels of protein Tyr kinases in colonic mucosal cells (3) and IEC-6 cells (13, 14) and significantly increase Tyr phosphorylation of pp60c-Src kinase molecules (13, 14, 15). However, it is not known whether these changes result in an increased activity of Src kinases in IEC cells in response to G17. In the current studies, we examined this important question and observed an increase in the activity of c-Src kinases in response to 0.1–10.0 nM G17 in IEC cells. Because both c-Src and c-Yes kinases have been implicated in colon carcinogenesis (16, 17, 18), we also measured changes in the activity of c-Yes kinase protein. We were surprised to learn that only the activity of c-Src kinase, but not that of c-Yes kinase, was specifically increased in response to G17 in IEC cells.
The precursor PG1–80 peptide, compared with the fully processed amidated G17 peptide, is extended by 8 amino acids at the C-terminal end and by 55 amino acids at the N-terminal end. Extension of G17 by only 1 amino acid at the C-terminal end (as in gly-extended G17) results in significantly reducing acid secretory activity of the peptide (reviewed in Ref. 19). The contribution of the additional amino acids at the C- and N-terminal ends of the PG peptide, in acid secretory and growth stimulatory effects of the peptides, remains to be identified. Our preliminary studies suggest that acid stimulatory effects of the precursor PG1–80 peptide are almost completely abolished, which confirms the previous notion that precursor peptides lack acid secretory effects (1). Mice overexpressing the full-length PG molecule demonstrated a significant increase in the proliferation of large IEC (9, 10), suggesting that PG may be functioning as a growth factor for IEC. More recently, growth stimulatory effects of a truncated PG peptide6–80 were demonstrated on a temperature-dependent transformed gastric epithelial cell line (YMAC cells) (20), suggesting that PG-like peptides may exert direct growth effects on the target cells. In the current study, we generated the full-length precursor peptide1–80 and examined growth stimulatory effects of PG on nontransformed IEC. Our results suggest that the precursor PG peptide is more potent than G17 as a growth factor for IEC cells. We next examined whether Src kinases are activated in response to the precursor PG molecule in IEC cells. Based on these studies, we report, for the first time, that the precursor PG peptide is more effective than G17 in specifically stimulating the activation of c-Src, but not c-Yes, kinase in IEC cells.
An activation of c-Src kinases, however, may or may not be required for measuring the observed growth effects of PG and G17 peptides on IEC cells. An obligatory role of Src-like kinases in mediating proliferative effects of PDGF, CSF-1, and EGF was demonstrated by microinjecting anti-Src antibodies (Abs) into fibroblasts (21, 22). On the other hand, c-Src activation plays no role in mediating the mitogenic effects of G protein associated receptor proteins like bombesin receptors and lysophosphatidic acid receptors (21). Activation of c-Src kinases has been reported in pancreatic acinar cancer (AR42J) cells in response to G17 (23, 24), but a mediatory role of c-Src kinases was not established in the stipulated growth effects of G17 on AR42J cells. In the current studies, we used the method of microinjecting c-Src-Abs in a single-cell assay and learned that activation of c-Src kinases is required for measuring growth effects of both the precursor PG peptide and the fully processed G17 peptide on IEC cells, because microinjection of anti-c-Src-Abs almost completely abolished 3H-thymidine (3H-Tdr) uptake in response to both the peptides.
Materials and Methods|m%1p+^, 百拇医药
Generation of the full-length recombinant human PG (rhPG)1–80 in Escherichia coli expression system|m%1p+^, 百拇医药
The full-length rhPG1–80 was generated, using standard procedures, as described briefly below. cDNA-encoding hPG was synthesized by PCR-mediated assembly of oligonucleotides. Codons were optimized for efficient tRNA usage in E. coli (25). The synthesized hPG cDNA was cloned in-frame into pET32 (NspV-HindIII), downstream of thioredoxin gene and His tag sequences, under the control of a phage T7 RNA polymerase promoter. The cDNA for the seven-amino-acid recognition site of rTEV protease was incorporated just before PG cDNA, creating a unique cleavage site within the thioredoxin-PG fusion protein (FP) product. The construct was confirmed by DNA sequencing and was transfected into a BL21 (DE3) E. coli strain containing the engineered isopropyl-ß-D-galactopyranoside-inducible T7 RNA polymerase gene. The FP was mostly in the soluble form and was purified from large-scale cultures of recombinant E. coli by the protein core facility in University of Texas Medical Branch, using column chromatography. The rTEV protease His-tag (Sigma, St. Louis, MO) was added to the eluate and cleaved, at room temperature, for 1 h. After adjusting to 1x washing buffer, the cleavage mixture was once again passed through a Ni-agarose column to remove TEV protease, uncleaved FP, and other contaminants. The flow-through was desalted, lyophilized, and resuspended in 10% acetonitrile and 0.1% trifluoracetric acid, for further separation by FP-HPLC. The peak containing rhPG was lyophilized and analyzed by 15% SDS-PAGE, mass-spectrometry, amino acid sequencing, and Western immunoblotting (data not shown). The final rhPG product was more than 99% pure. The standard yield obtained was approximately 1 mg/liter of culture.
Cell culturege, http://www.100md.com
IEC-6 cells and IEC-18 cells (American Type Culture Collection, Rockville, MD) were grown as monolayer cultures in DMEM (Life Technologies, Inc., Grand Island, NY) growth medium as described previously (13). For cell counting and subculturing, the cells were dispersed with a solution of 0.05% trypsin and 0.02% EDTA.ge, http://www.100md.com
Growth assays to measure an increase in cell numbersge, http://www.100md.com
Cell numbers were measured directly by counting the total number of cells, using a Coulter electronic particle counter (model Z1), as described previously (13). Briefly, an optimal number of IEC cells (4 x 104 cells) were plated in 35-mm dishes with 2 ml of normal growth medium containing 10% fetal calf serum (FCS). After 24 h, the medium was changed to serum-free medium (SFM), and cells were cultured for an additional 48 h. Cells were then treated with increasing concentrations (0.01–1000.0 nM) of either G17 or PG in SFM for 48 h. At the end of the treatment, cells were disbursed with trypsin-EDTA solution as given above, and cells were counted using either a Coulter electronic counter or with the help of a hemacytometer under light microscopy. In preliminary studies, we confirmed that PG was not processed into G17 in the extracellular medium of IEC cells that were incubated with PG for 30 min to 24 h, using specific Abs against the C-terminal ends of either the PG peptide or the G17 peptide, by our published methods (10, 26) (data not shown).
Immunoprecipitation of proteins and Src kinase assays2#?;afi, http://www.100md.com
Relative levels of activated Src kinases in IEC-6 and IEC-18 cells were measured by a slight modification of published methods (21, 22). Briefly, cells in culture at approximately 60% confluency were serum starved for 48 h to obtain quiescence. Quiescent cells were treated with increasing concentrations of either G17 or rhPG (0.0–10 nM) at 37 C for 10–15 min. The stimulation was terminated by aspirating the medium and washing the cells twice with ice-cold washing buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM sodium vanadate, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF)]. The washed cells were then solubilized in lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM dithiothreitol, 1 µg/ml leupeptin, 1% aprotinin, 1 mM PMSF, 10 mM NaF] on ice for 10 min at 4 C, and the lysates were centrifuged at 4 C for 10 min at 13,000 x g in a GS-15R centrifuge (Beckman Coulter, Fullerton, CA). After centrifugation, the supernatants were transferred to fresh tubes and preclarified with normal rabbit serum for 30 min at 4 C. Anti-c-Src- or anti-Yes-Abs were used to immunoprecipitate Src- or Yes-related kinases from the samples, as described in figure legends. Briefly, lysates were treated with primary Abs, at 4 C for 1 h, with gentle mixing at repeated intervals. To immunoprecipitate the kinase-Ab complex, we either used protein A (S. aureus) (Sigma) or Pansorbin in the presence of a second Ab. Immunoprecipitation with either protein A or Pansorbin was carried out, at 4 C for 30 min, with constant agitation. The immunoprecipitate was then washed three times by centrifugation for 5 min at 4 C in an Eppendorf (Brinkmann Instruments, Westbury, NY) microcentrifuge at 10,000 x g using the immunoprecipitate washing buffer (10 mM Tris-Cl, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM/ml Na3Vo4).
The immunoprecipitates were resuspended in 20 µl kinase buffer [20 mM HEPES (pH 7.5), 8 mM MgCl2, 8 mM MnCl2, 0.1 mM Na3Vo4]; and the reaction was started by the addition of kinase substrate, enolase (ENO) (Boehringer Mannheim, Indianapolis, IN) followed by the addition of 10 µCi of {gamma} 32P ATP (ICN Pharmaceuticals, Inc., Costa Mesa, CA), on ice for 15 min. Before adding ENO to the reaction buffer, it was denatured in 20 mM CH3COOH at 37 C for 5 min. The reaction was stopped by adding an equal volume of 4x Laemli sample buffer, and the samples were heated to 95 C for 3 min and separated by SDS-PAGE, using 9.5% polyacrylamide gels under reducing conditions as previously described (27, 28). In some cases, the gels after electrophoresis were additionally treated with 1 mM KOH, at 60 C for 1 h, before autoradiographic development (13). The autoradiograms were densitometrically analyzed with the help of a laser densitometer, using a Documentation Analysis System (Model AlphaImager 2000; Alpha Innotech Corporation, San Leandro, CA).
Determination of percent labeling (3H-Tdr uptake) of control and peptide-stimulated IEC cells in single-cell assaysk, http://www.100md.com
Rat IEC (IEC-6, IEC-18) are known to express autocrine IGFs (29, 30) that are increasingly expressed by the IEC cells, in relation to increasing cell numbers (31). IEC cells, however, do not express the gastrin gene (unpublished data from our laboratory). Because these cells express IGFs, the cells continue to divide in culture, even under serum-free conditions, for more than 48–72 h, until IGF expression in reduced to basal levels. To conduct single-cell growth assays in response to PG and G17, in preliminary studies, we determined the optimal cell culture conditions that would allow us to measure significant growth effects in terms of 3H-Tdr uptake in the nuclei of the cells. Exposure of the cells to SFM for 72 h significantly reduced the percent of cells that were positive for 3H-Tdr uptake, to less than 50%, without effecting viability of the cells. Importantly, after 48–72 h of serum starvation, we measured a significant growth response to both FCS and gastrins, wherein 3H-Tdr labeling of the cells was increased to more than 70–95%, compared with less than 50% in the absence of peptide/FCS stimulation. IEC-18 cells were therefore cultured for 72 h under serum-free conditions, which was considered optimal for measuring maximal growth response to exogenous growth factors. Briefly, IEC cells were plated in 35-mm2 tissue culture dishes (25,000 cells/dish) in 2 ml complete growth medium for 24 h, followed by incubation for 72 h in SFM to induce relative quiescence. Cells were then stimulated with either G17 or PG (0.1–1.0 nM) or 1.0% FCS in DMEM growth medium. To confirm growth effects in terms of 3H-Tdr uptake, 1 µCi/ml 3H-Tdr was included in the culture medium to determine the percent of cells that entered into S phase as a result of peptide stimulation. Cells were incubated for 48 h at 37 C and washed with PBS. The cells were then fixed with methanol, air dried, and subjected to autoradiography. For autoradiography, nuclear track emulsion (NTB2; Eastman Kodak Co., Dallas, TX) was used to coat the dishes, which were then stored in the dark at 4 C for 7 d. After development of the emulsion according to the manufacturers suggested protocol, approximately 200 cells/dish were scored for labeling.
Microinjection of cells with Abs-(h, 百拇医药
Injection needles were pulled from borosilicate capillaries using a Flaming/Brown Micropipette Puller, Model P-97 (Sutter Instrument Co., Novato, CA) with a range of outer tip diameters from 2.5–3 microns, as determined by scanning electron microscopy. Cells were visualized throughout the injection procedure using an Olympus Corp. (Melville, NY) IX70 inverted microscope equipped with a temperature-controlled stage kept at 37 C, and injected with the indicated Abs (as described below) using the electronically interfaced Eppendorf Micromanipulator (Model 5171) and Transjector (Model 5246). Manual cytoplasmic injection of approximately 5–10 femtoliters into each cell was performed (32). All experiments were done in duplicate or triplicate, with 50 cells per dish being injected. Only cells within an etched boundary were injected, to allow for easy localization.-(h, 百拇医药
Single-cell assay of postinjection viability and percent inhibition of DNA synthesis
All Ab samples were dialyzed against PBS buffer before injections. Depending on the experiment, cells were injected with either: 1) nonimmune mouse IgG (ICN Pharmaceuticals, Inc. Biochemical Research Products) at a concentration of 10 mg/ml as a marker protein for identifying injected cells; 2) a mixture of nonimmune mouse IgG (10 mg/ml) and an anti-cSrc-Ab (0.1 mg/ml) (Src-2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); or 3) a mixture of nonimmune mouse IgG (10 mg/ml) and anti-cSrc-Ab (0.1 mg/ml) in the presence of excess competing peptide. The anti-cSrc-Ab used in this study was an affinity-purified rabbit polyclonal Ab raised against an Src peptide (sc-18), mapping at the C terminus of human pp60c-Src, a region identical to the corresponding mouse sequence. The blocking peptide used to inactivate the anti-cSrc-Ab was the sc-18 Src peptide (Santa Cruz Biotechnology, Inc.), that had been used to generate the polyclonal anti-cSrc-Ab by the company. Anti-Src-IgG was prepared from anti-Src-Ab using protein A Sepharose (Sigma) before injecting the cells. After injection, quiescent IEC-18 cells were stimulated with the indicated peptides or FCS as described above. For all experiments, 3H-Tdr was added to the medium at a concentration of 1 µCi/ml. Cells were incubated in a 37 C, 5% CO2 incubator for 72 h, washed with PBS, and fixed with methanol and air-dried. The cells were then stained with FITC-conjugated goat antimouse IgG (100-fold dilution of Ab with PBS), and autoradiography was performed as described above. After autoradiography of each dish was complete, 200 noninjected cells were scored for label (scoring only the cells outside of the etched circles), and the percentage of labeled cells was determined.
The injected cells were visualized on a Olympus Corp. IX70 inverted microscope using fluorescence microscopy, scanning the area inside the etched circles for fluorescent cells. Upon identification of fluorescent (injected) cells, the autoradiograph was observed using either bright field and/or phase contrast microscopy to determine the percentage of injected cells that were labeled.$y+7p/, 百拇医药
The percent viability of cells post injection was determined for each dish (50 cells injected per dish) as: (number of fluorescent cells/50) x 100. Post injection viability of injected cells averaged 51.5 ± 1.9% (SE, n = 12), with no significant differences in percent post injection viability whether cells were stimulated by G17, PG, or FCS (data not shown).$y+7p/, 百拇医药
To assess the effect of Ab injections on entry of the cells into S phase in response to the peptides/FCS, percent inhibition of DNA synthesis was determined using the following formula:$y+7p/, 百拇医药
fig.ommitteed
Image capture7^, 百拇医药
The images of the fluorescently injected cells and their autoradiographs were acquired via video imaging using a Model Mli308 low-light camera (Microimage Video Systems, Houston, TX) linked to a computer containing the image capture and analysis software (Meyers Instruments, Houston, TX).7^, 百拇医药
Statistical analysis7^, 百拇医药
In all cases, data are presented as the mean of the sample populations ± SE. To test for significant differences between means, the nonparametric Mann-Whitney test was employed using Statview 4.1 (Abacus Concepts, Inc., Berkeley, CA); P values were considered to be statistically significant if less than 0.05.7^, 百拇医药
Results7^, 百拇医药
In vitro growth effects of G17 and rhPG on IEC cells7^, 百拇医药
In previous studies, we reported significant dose-dependent growth effects of G17 on IEC-6 cells in culture, wherein G17, at concentrations of 0.1–1.0 nM, increased the growth of IEC-6 cells significantly, with 1 nM G17 being the most effective (13). In the current study, we compared the growth effects of rhPG with those of G17 on IEC-6 and IEC-18 cells, and representative data from one of a total of three to six experiments is presented in Fig. 1. IEC-18 cells were more responsive than IEC-6 cells, especially to PG (Fig. 1). Both peptides were maximally effective at concentrations of 0.1–1.0 nM on the two cell lines. At equimolar concentrations, rhPG was generally more effective than G17 as a growth-promoting agent (Fig. 1). At lower concentrations of 0.01–0.05 nM (10–50 pM), only minimal growth effects were measured; and the results were not statistically significant, compared with control values (data not shown). The minimum dose at which we measured significant growth effects of both G17 and PG was 100 pM (0.1 nM), as shown in Fig. 1. Normally, under basal (nonstimulated) conditions, one measures approximately 30 pM amidated gastrins (G17 and G34) in the circulation of most animals (1). However, on stimulation (such as after meal ingestion), approximately 60–120 pM amidated gastrins are measured in the circulation. Thus, G17 can potentially be expected to exert significant (though suboptimal) growth effects on IEC postprandially. PGs are not normally detected in the circulation before or after meals. In certain pathological conditions (such as Zollinger-Ellison syndrome and in patients with colorectal cancers), however, high concentrations of PG-like peptides (1.0–>10 nM) are measured in the circulation (reviewed in Ref. 19), suggesting that PG can potentially exert significant growth effects on IEC in patients with the above specific pathologies.
fig.ommitteed\]+oyz, 百拇医药
Figure 1. Growth effects of rhPG, in relation to that of G17, on IEC-6 and IEC-18 cells. The growth of IEC cells in response to either 0.1% FCS (positive control) or G17 or rhPG is shown. The increase in cell number measured in each dish, compared with that measured in the control dishes (arbitrarily assigned a zero value), is presented. Each bar graph represents data from three to four dishes/experiment and is representative of three to six experiments. *, P < 0.05 vs. control values.\]+oyz, 百拇医药
Phosphorylation and activation of c-Src kinase in IEC-6 cells, in response to increasing doses of G17\]+oyz, 百拇医药
The c-Src kinase activity was measured in an in vitro kinase assay as described in Materials and Methods, with ENO as a substrate. Autoradiographic data from representative experiments are shown in Fig. 2, A–C. In the majority of the experiments, gels were prewashed with KOH to measure phosphorylation of Tyr moieties and to reduce phosphorylation of all other moieties (13) (Fig. 2A). In a few experiments, gels were not prewashed with KOH; representative results are presented in Fig. 2C. In the absence of KOH washing, one or more nonspecifically phosphorylated protein bands were detected (56–68 kDa). However, with KOH washing, the nonspecifically phosphorylated proteins were not detected (Fig. 2A), which allowed us to more accurately analyze the changes in phosphorylation of c-Src and its substrate, ENO, in response to increasing doses of G17. Immunoprecipitation of an equivalent concentration of c-Src protein in different samples was confirmed by Western immunoblot analysis using anti-c-Src-Abs (Fig. 2B). The data in Fig. 2A were corrected for c-Src levels in Fig. 2B before calculating the percent change in phosphorylation levels as presented in Fig. 2D. The increase in phosphorylation of c-Src protein and ENO was determined densitometrically, as described in Materials and Methods, and data from two to six experiments are presented as bar graphs in Fig. 2D. The c-Src kinase activity, measured in terms of increase in phosphorylation of c-Src and ENO, was significantly increased in response to 0.1–1.0 nM gastrin, with 1.0 nM concentration being the most effective. At higher concentrations (10–100 nM), no significant increase in c-Src kinase activity was observed. In a previous study, we similarly measured a bi-phasic effect of G17 on proliferation and Tyr phosphorylation of c-Src in IEC-6 cells (13).
fig.ommitteede, 百拇医药
Figure 2. Activation of c-Src kinase in response to increasing doses of G17. IEC-6 cells were treated with increasing doses of G17 and processed for measuring c-Src kinase activity in terms of phosphorylation of the substrate (ENO) and autophosphorylation of c-Src, as described in Materials and Methods. Representative data from one of six experiments are presented in A. These data are from a gel that was prewashed with KOH before autoradiography, as described in Materials and Methods. B, Samples from duplicate gels were transblotted onto nitroceullose membranes and processed for Western immunoblotting with anti-src-Ab (SC-18; Santa Cruz Biotechnology, Inc.). Immunoblots were developed with the ECL detection kit (Amersham, Piscataway, NJ), demonstrating equivalent amounts of c-Src protein in samples used in the kinase assays shown in A. Other than lane 5 (stimulated with 100 nM G17), all other samples contained equivalent levels of c-Src protein. C, Gels that were not prewashed with KOH almost always demonstrated the presence of other phosphorylated proteins (~ 56–58 kDa), whose nature is not known. These bands likely represent Ser- or Thr-phosphorylated proteins, because KOH treatment dephosphorylates Ser and Thr moieties. D, Data from two to six experiments, in which dose-dependent effects of G17 were measured on src kinase activity. Even at low concentrations, G17 was effective in significantly increasing c-Src kinase activity, in terms of phosphorylation of ENO (lower panel) and phosphorylation of c-Src (upper panel).
Effects of G17 on activation of c-Src kinase and c-Yes kinaseau, 百拇医药
To examine whether the activation of c-Src kinase in response to G17 was specific, we additionally measured activation of c-Yes kinase, using methods essentially similar to that described above for c-Src kinase. Anti-c-Yes-Abs were used during the immunoprecipitation step to measure c-Yes kinase activity levels. Because 1.0 nM G17 was maximally effective in inducing an increase in c-Src kinase activity (Fig. 2), we used this concentration for comparing the effects of G17 on the activity of the two kinases, as shown in Fig. 3. G17 was ineffective in inducing an increase in Tyr phosphorylation and activation of c-Yes kinase protein (Fig. 3B). However, c-Src kinase activity was increased significantly in the presence of 1.0 nM G17 in the same experiments (Fig. 3A).au, 百拇医药
fig.ommitteedau, 百拇医药
Figure 3. Effect of G17 on activation of c-Src kinase and c-Yes kinase. IEC cells were treated with the optimally effective concentration of G17 (1.0 nM) and immunoprecipitated with either anti-c-Src-Ab (SC-18; Santa Cruz Biotechnology, Inc.) or anti-c-Yes-Ab (3H9; Wako Pure Chemical Industries Ltd., Richmond, VA). The immunoprecipitates containing either c-Src or c-Yes were subjected to an in vitro kinase assay in the presence of ENO, as described in Materials and Methods. At the end of the kinase assay, the samples were separated by SDS-PAGE, subjected to KOH treatment, and autoradiographically developed as described in Materials and Methods. The effect of G17 treatment on c-Src kinase activity (A) and c-Yes kinase activity (B) is shown. Duplicate gels were processed for Western immunoblotting with either anti-c-Src or anti-c-Yes-Ab to confirm equal loading of the lanes with the indicated kinase protein. In all experiments, the relative concentrations of either c-Src (in immunoprecipitates of c-Src-Ab) or c-Yes (in immunoprecipitates of c-Yes-Ab) were similar (data not shown).
Dose-dependent effects of PG on c-Src and c-Yes kinase activity{e5+, 百拇医药
Early passage numbers of IEC-6 cells, used in the studies with G17, were not available by the time we began to examine effects of PG on Src kinases. We therefore used IEC-18 cells that were determined to be equally responsive to growth effects of G17 and PG (Fig. 1, Table 1); PG significantly increased Tyr phosphorylation of both c-Src protein and its substrate ENO at 0.1–1.0 nM concentrations (Fig. 4). At the lower concentration of 0.01 nM, no significant effect of PG was measured on phosphorylation of ENO (data not shown). In the absence of KOH treatment (Fig. 4C), we once again observed phosphorylation of nonspecific bands (56–58 kDa). The activity of c-Yes kinase remained unchanged after treatment of the cells with 1.0 nM PG (Fig. 4F). PG was approximately 1.5–2.0 times more effective than G17 in stimulating c-Src kinase activity at the optimal concentrations of 0.1–1.0 nM (Fig. 5). The relative growth effects of PG and G17 on IEC-18 cells matched the relative potency of the two peptides in activating c-Src kinase activity (Table 1), providing further evidence that c-Src kinases mediate proliferative effects of both the peptides. In a control experiment, excess anti-c-Src-Ab was added to the kinase buffer, before the addition of ENO, which resulted in significantly reducing the phosphorylation of ENO in peptide-stimulated samples (data not shown), strongly suggesting that significant increase in the phosphorylation of the substrate, ENO, in the presence of the peptide-stimulated samples was attributable to the presence of activated c-Src kinases in these samples.
fig.ommitteedpv-&n#7, http://www.100md.com
Table 1. Proliferation of IEC-18 cells and an increase in c-Src kinase activity in response to PG and G17pv-&n#7, http://www.100md.com
fig.ommitteedpv-&n#7, http://www.100md.com
Figure 4. Effect of PG treatment on c-Src and c-Yes kinase activity. IEC-18 cells were treated with increasing concentrations of PG (0.1–10.0 nM), and the c-Src kinase activity in the cells was measured in an in vitro kinase assay as described in Materials and Methods. At the end of the kinase assay, the samples were electrophorically separated by SDS-PAGE, KOH-treated, and examined autoradiographically as described in Materials and Methods. Data from a representative experiment (from a total of three separate experiments) are shown in A. Duplicate samples were processed for immunoblotting with anti-c-Src-Ab, as described under Fig. 3, and the c-Src immunoblot data for samples presented in A are shown in B. As shown in B, c-Src protein levels were equivalent in all samples other than lane 5. In one experiment, the gel was not treated with KOH, and these autoradiographic data are shown in C. D, Data in A (from four separate blots from two experiments) are presented as percent change in densitometric readings of the indicated bands; the readings in the control lanes (not treated with PG) were arbitrarily given a zero value. E, In one experiment, c-Yes kinase activity was also measured in response to 1.0 nM PG, as described for G17 in Fig. 2. Data from one of two blots is shown in E.
fig.ommitteed3![z2n, 百拇医药
Figure 5. Comparison of the effects of G17 and PG on c-Src kinase activity in IEC-18 cells. IEC-18 cells were treated with 0.1 or 1.0 nM G17 or PG, and c-Src kinase activity was measured as described in the legend of Fig. 2. At the end of the reaction, the samples were electrophoretically separated by SDS-PAGE and subjected to KOH treatment and developed autoradiographically. Data from representative experiments (from a total of three similar experiments) are shown in A and B. Data from all three experiments are presented as bar graphs in C. The percent increase in Tyr phosphorylation of c-Src (upper panel) and ENO (lower panel) in response to G17 was arbitrarily assigned a 100% value, and the increase in response to PG is presented as a percent of that measured with G17. *, P < 0.05 vs. G17 values; lane C, control samples from cells not treated with the peptide.3![z2n, 百拇医药
Effects of anti-c-Src Abs on 3H-Tdr uptake by IEC-18 cells in response to gastrins and FCS3![z2n, 百拇医药
Serum-starved IEC-18 cells were microinjected with either control, nonimmune IgG, or specific anti-c-Src-IgG and were stimulated to grow in the presence of optimal concentration of either G17 or PG (1.0 nM), as described in Materials and Methods; as a positive control, cells were also stimulated with 1.0% FCS. Data from representative cells, demonstrating microinjection of the indicated Ab, and the resulting 3H-Tdr uptake in the cells in response to either FCS or gastrins are presented in Figs. 6 and 7. The percent of cells labeled with 3H-Tdr, as a result of the various treatments, was calculated as described in Materials and Methods, and data from several experiments are presented as bar graphs in Fig. 8A. Based on the data presented in Fig. 8A, the percent inhibition of DNA synthesis in the Ab injected vs. noninjected cells was calculated as described in Materials and Methods, and data from several experiments are presented in Fig. 8B. The data presented in Figs. 6 ad 8 demonstrate that microinjection of cells with the specific c-Src-Ab significantly reduced 3H-Tdr labeling of the cells in response to both G17 and PG.
fig.ommitteedt\v}#, 百拇医药
Figure 6. Effect of microinjecting anti-c-Src-Abs on 3H-Tdr up-take by IEC-18 cells in response to PG. IEC-18 cells were microinjected with either anti-c-Src-IgG or control nonimmune IgG and subjected to a single-cell growth assay in response to 1.0 nM PG as described in Materials and Methods. A fluorescently-labeled second Ab was used to identify injected cells as described in Materials and Methods. Autoradiography of the same cells was used to monitor 3H-Tdr uptake as described in Materials and Methods. A1 (phase contrast microscopy) and A2 (fluorescent microscopy) are low magnification (100x) pictures of the same field. At low power, one cannot tell whether a cell is labeled, because one cannot differentiate phase dark chromatin and nucleoli from label. B1 (phase contrast microscopy), B2 (fluorescent microscopy), and B3 (bright field microscopy) are high-magnification (400x) pictures of the field of cells contained within the box shown in A1 and A2. The fluorescent cells shown in A2 and B2 were coinjected with nonspecific mouse IgG and anti-c-Src-IgG. At higher magnification, cells 1, 4, 6, 7, and 9 were injected, and only cell 6 incorporated tritiated thymidine (B1–B3). Cells 2, 3, 5, and 8 were not injected, and all four cells incorporated tritiated thymidine (B1–B3), demonstrating that the anti-c-Src-IgG prevents a significant number of injected cells from cell cycle progression. C1 (phase contrast microscopy) and C2 (fluorescent microscopy) are high-magnification (400x) pictures of the same field. The two fluorescent cells shown in C2 were injected with only nonspecific mouse IgG. In C1, all cells are shown to be labeled, demonstrating that the nonspecific mouse IgG has no effect on cell cycle progression.
fig.ommitteed6q*;.4w, 百拇医药
Figure 7. Specificity of effects of microinjecting anti-c-Src-Abs on 3H-Tdr uptake by IEC 18 cells in response to G17: comparison of effects in response to 1% FCS. IEC-18 cells were microinjected with either anti-c-Src-IgG or a control nonimmune mouse IgG and subjected to a single-cell growth assay in response to G17 or FCS as described in Materials and Methods. Fluorescently-labeled second Ab was used to identify injected cells as described in Materials and Methods. Autoradiography of the same cells was used to monitor 3H-Tdr uptake as described in Materials and Methods. A–C, Cells microinjected with nonimmune mouse IgG and treated with 1.0 nM G17; D–F, cells microinjected with nonimmune mouse IgG and treated with 1% FCS; G–I, cells microinjected with nonimmune mouse IgG plus specific anti-c-Src IgG and treated with 1.0 nM G17. A–C, D–F, and G–I represent cells in the same field of view, respectively. A, D, and G, fluorescent microscopy used to identify injected cells; B, E, and H, bright field microscopy of autoradiographs used to identify dark grains over nuclei of cells that entered into S-phase of the cell cycle; C, F, and I, phase contrast microscopy to delineate the spread of the cells. Thin lines point to the nuclei of injected cells. Lines with arrows point to the nuclei of noninjected cells.
fig.ommitteed%, 百拇医药
Figure 8. Effect of microinjecting anti-c-Src-IgG on 3H-Tdr uptake by IEC-18 cells in response to either 1% FCS or 1.0 nM PG or 1.0 nM G17. A. The cells were treated as described in the legend of Fig. 7. Data from three to five separate experiments are presented as bar graphs, comparing 3H-Tdr labeling of cells with and without injection with indicated Abs, in the presence of the indicated stimulants. B. The data in A were used to determine the percent inhibition of DNA synthesis as described in Materials and Methods. Each bar graph represents the mean ± SEM of data from three to five separate experiments. *, P < 0.05 vs. corresponding values for the cells injected with nonimmune IgG; C-IgG, control nonimmune IgG.%, 百拇医药
Our in vitro Src kinase assay results suggested that anti-c-Src-Ab (used for immunoprecipitation of c-Src from cell lysates) did not block kinase activity of the c-Src protein (Figs. 2–5). The mechanism by which anti-c-Src-Abs inhibit the kinase activity of c-Src in vivo, when microinjected into the IEC cells, is therefore not understood. Because the addition of excess anti-c-Src-Ab to the in vitro kinase assay buffer (containing immunoprecipitated c-Src proteins) significantly reduced the phosphorylation of ENO (Table 1), it suggests that c-Src-Abs can potentially inhibit the activation of c-Src kinases in vitro, as observed in the microinjection experiments in vivo (Figs. 6–8). It is possible that, in the presence of excess substrate (ENO) in the in vitro kinase buffer (Figs. 2–5), the immunoprecipitated c-Src protein gets preferentially bound to the excess substrate rather than c-Src-Ab (that is bound to the second Ab/Pansorbin complex and is perhaps less available for binding or inhibiting the kinase activity of c-Src in the in vitro kinase assay buffers). At least one other group of investigators (21, 22) has used anti-c-Src-Abs (similar to the Ab used by us in the current studies) and reported immunoprecipitation of kinase-active c-Src proteins from various cellular samples; at the same time, the authors observed significant inhibition of the c-Src kinase activity in vivo in cells microinjected with anti-c-Src-Abs (21). The published studies of Roche et al. (21) thus provide strong support for our seemingly divergent observations with the c-Src-Abs in vitro vs. in vivo.
An important finding was that microinjection of the cells with anti-c-Src-IgG had no effect on either basal (nonstimulated) 3H-Tdr labeling, or labeling in response to 1% FCS. The latter results strongly indicate the specificity of the effects of anti-c-Src-Abs in reducing 3H-Tdr uptake in response to gastrins. To further confirm the specificity of the effects of anti-c-Src-Abs, the activity of the Abs was blocked with the antigenic Src peptide (used for generating the Abs), as described in Materials and Methods. The effect of inactivated/blocked Abs was examined in single-cell assays as described above. Results with the blocked Ab, in comparison with results with the untreated Ab, are shown in Figs. 9 and 10. Data from representative cells from one of two separate experiments, demonstrating 3H-Tdr labeling in cells microinjected with either the blocked or the untreated c-Src-IgG, are shown in Fig. 9. The percent inhibition of DNA synthesis in the injected vs. noninjected cells was calculated from both the experiments and is presented as bar graphs in Fig. 10. As can be seen from Figs. 9 and 10, blocking the activity of the anti-c-Src-IgG with the antigenic Src peptide completely abolished the inhibitory effects of anti-c-Src IgG on 3H-Tdr labeling of cells in response to PG (Fig. 9, C–F; and Fig. 10). On the other hand, neither microinjection of the active anti-c-Src-IgG or of the blocked anti-c-Src-IgG had any effect on the 3H-Tdr labeling in response to 1% FCS (Fig. 9, A and B; and Fig. 10), similar to results presented in Figs. 6–8. These results confirm the specificity of the inhibitory effects of anti-c-Src-Abs in reducing 3H-Tdr labeling (and hence proliferation) in response to gastrins but not to other growth-promoting agents contained in FCS.
fig.ommitteedpx^]v, 百拇医药
Figure 9. Effects of blocking the activity of anti-c-Src-IgG on 3H-Tdr uptake by IEC-18 cells in response to either 1% FCS or 1.0 nM PG. Cells were microinjected with the anti-c-Src-IgG with and without peptide blocker and subjected to single-cell growth assays as described in the legend of Fig. 6. Data from representative cells, treated as indicated below, are presented in A–F. A and B, Cells microinjected with anti-c-Src IgG and treated with 1% FCS; C and D, cells injected with anti-c-Src IgG and treated with 1.0 nM PG; E and F, cells injected with inactivated (peptide blocked) anti-c-Src IgG and treated with 1.0 nM PG. A and B, C and D, and E and F represent cells in the same field of view, respectively. Fluorescent microscopy was used to identify injected cells in A–E. Phase contrast microscopy was used to determine whether the injected cells were labeled with 3H-Tdr (indicating entry into the S phase of the cell cycle), in B, D, and F. Thin lines with arrows point to the nuclei of injected cells. Thick lines with arrows point to the nuclei of noninjected cells.px^]v, 百拇医药
fig.ommitteed
Figure 10. Effects of blocking the activity of anti-c-Src IgG on 3H-Tdr uptake by cells in response to either 1.0% FCS or 1.0 nM PG. Cells were microinjected with either the blocked (inactivated) or nontreated anti-c-Src-IgG or with the control nonimmune IgG and stimulated with either 1.0% FCS or 1.0 nM PG, as described in Materials and Methods and in the legend of Fig. 9. Then, 3H-Tdr uptake by the injected vs. noninjected cells was determined, and the percent inhibition of DNA synthesis was calculated from these data as described in Materials and Methods. Each bar graph represents mean ± SEM from duplicate/triplicate measurements from two to three separate experiments. *, P < 0.05 vs. 3H-Tdr uptake in corresponding cells injected with nonimmune IgG.%u]x8, http://www.100md.com
Discussion%u]x8, http://www.100md.com
In the current studies, we demonstrate a critical role of pp60c-Src kinase in mediating mitogenic effects, of both the fully processed form of gastrin (G17) and the full-length precursor form of gastrin (PG), on fetal rat IEC. An important finding was that the mediatory role of c-Src kinase seemed to be specific, because a closely related kinase, pp62c-Yes, was not activated in response to either G17 or PG in IEC cells.
In all previous studies, pharmacological inhibitors of MAP kinases, PI-3 kinase, and Src kinases were used to examine the role of kinases in mediating the stipulated growth effects of G17 and gly-extended G17 on a rat pancreatic cancer cell line (AR42J) (23, 24, 33, 34) and a pituitary cell line (35). Pharmacological inhibitors of Src kinases are not very specific and are known to inhibit the activity of several other members of the Src family of kinases (21, 36). A more precise method for dissecting out the role of various intracellular molecules/kinases currently being used is microinjection of inhibitory Abs (32, 37). Microinjection of anti-Src-Abs was used to confirm a role for Src-kinases in mediating growth effects of various growth factors on fibroblasts (21, 22). Using this method, it was possible, for the first time, to separate the roles of PI-3 kinase and PI-3 kinase ß in DNA synthesis by human colon carcinoma cell lines (38). Similarly the role of various isoforms of protein kinase C in calcium signaling in the IEC could be dissected by microinjecting inhibitory or blocking Abs against the various forms of the enzyme (39). Therefore, in the current studies, we have, for the first time, examined the role of pp60c-Src kinase in mediating growth effects of G17 and PG on IEC-18 cells in single-cell assays by microinjecting c-Src-Abs to block the activity of c-Src kinase. The single-cell assay method is now being used by many investigators to examine the role of various intracellular molecules (40). In the current study, microinjection of anti-c-Src-Abs in IEC cells resulted in the loss of 3H-Tdr up-take in response to both the precursor (PG) and processed (G17) forms of gastrin, to basal (control) levels. The specificity of the effects of the Ab was confirmed by the use of blocking peptides, which completely abolished the inhibitory effects of the Abs. We have thus, for the first time, documented a critical role of pp60c-Src kinase in mediating mitogenic effects of both PG and G17 on IEC cells.
In previous studies, we and others demonstrated a significant increase in protein tyrosine kinase (PTK) activity in response to G17, both in the colonic mucosal cells (3) and IEC-6 cells (13, 14). Intracellular proteins, including pp60c-Src, were Tyr-phosphorylated in response to physiological concentrations of G17 (13). In the current study, we further demonstrated that activity of c-Src kinase was increased in response to increasing doses (0.1–1.0 nM) of G17. In rat colonic epithelial cells, G17 induced IP3 formation by up-regulating phospholipase C {gamma} which similarly resulted in the activation of pp60c-Src kinase (14).rr[7n, 百拇医药
Src-like PTKs are now believed to play a central role in mediating proliferative effects of several growth factors, including PDGF (platelet-derived growth factor), CSF (colon stimulating factor)-1, and EGF (epidermal growth factor) (21, 22, 36). An obligatory role of Src-like PTKs in mediating proliferative effects of PDGF, CSF-1, and EGF was demonstrated by microinjecting Src-Abs into fibroblasts (21); however, Src activation was not required for mediating mitogenic effects of BBS (bombesin) and lysophosphatidic acid that bind G-protein-associated receptor proteins. The current study provides convincing evidence that Tyr phosphorylation and activation of pp60c-Src kinase is significantly up-regulated in response to both G17 and rhPG in IEC cells. The relative potency of the two peptides, in increasing Tyr phosphorylation and kinase activity of c-Src, was equivalent to the relative potency of the two peptides in stimulating growth of IEC-18 cells, suggesting that c-Src may be mediating growth effects of both of the peptides on intestinal cells.
The Src kinase family contains several closely related members (Src, Fyn, Yes, Lck, Hck, Fgr, Lyn, and Yrk) that perform similar, but specific, functions in different cell types (36). In human colon carcinomas, the activity of only two Src-like kinases, pp60c-Src and pp62c-Yes, is significantly increased (16, 17, 18, 41). We now know that a large percentage of human colon cancers express the gastrin gene and release significant concentrations of PG into the circulation (reviewed in Ref. 19). Because the results of the present study suggest that pp60c-Src kinase is specifically and significantly activated in IEC cells in response to both the precursor and the fully processed forms of gastrin, it is possible that the observed increase in the activity of at least one of the kinases (pp60c-Src) in colorectal cancers may be mediated via autocrine gastrins expressed by these cancers. In preliminary studies with a human colon cancer cell line (HCT-116), we observed that an increase in the activity of pp60c-Src kinase, but not pp62c-Yes kinase, may be secondary to the expression of autocrine gastrins (42). The proliferative and tumorigenic potential of antisense (AS) HCT-116 clones, expressing the AS gastrin RNA, was significantly reduced in vivo and in vitro, compared with that of control HCT-116 clones (43); this may be attributable to a loss in the activity of c-Src kinase in AS HCT-116 clones (42). Thus, based on our current studies with the IEC cells and our preliminary studies with the control and AS clones of HCT-116 cells, it seems possible that both endocrine and autocrine gastrins (irrespective of the molecular form of gastrins) may increase proliferation of intestinal and colon cancer cells via up-regulation of c-Src kinase activity.
c-Src kinases are activated in many other cancers, such as breast cancer (44, 45). Because many cancers express autocrine growth factors, such as TGF-{alpha} and PG, it is possible that the increase in c-Src kinase activity observed in these cancers may be secondary to the expression of autocrine growth factors. However, c-Src kinase does not mediate growth effects of all growth factors, considering that 3H-Tdr up-take by IEC cells, in response to autocrine IGFs and/or 1% FCS, was not inhibited by microinjection of anti-c-Src-Ab (current studies). IEC-18 cells, may therefore provide an excellent in vitro model for examining intracellular mechanisms that mediate growth effects of PG and gastrin-like peptides, that may be relevant to the growth of IEC and colon cancer cells in situ.9hdq)-c, http://www.100md.com
Acknowledgments9hdq)-c, http://www.100md.com
We would like to acknowledge Gene-Cell, Inc. for generously providing all the glass injection needles used in the microinjection experiments, and we gratefully acknowledge the secretarial support of Pat Gazzoli. We would also like to thank David Konkel, Ph.D., for critically reading this manuscript.
Received May 10, 2002.), http://www.100md.com
Accepted for publication September 17, 2002.), http://www.100md.com
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Growth factor effects of precursor forms of gastrins have become evident in recent years. However, intracellular pathways that mediate growth effects of the precursor molecules are not known. In previous studies, we reported an increase in Tyr phosphorylation of pp60c-Src in intestinal epithelial cells (IEC) in response to the fully processed form of gastrin [gastrin1–17 (G17)]. We have now examined whether c-Src kinase is similarly phosphorylated and activated in response to the full-length precursor molecule, progastrin (PG)1–80, (recombinant human PG) in IEC cells. We found a significant increase in pp60c-Src kinase activity in response to both G17 and PG (0.1–1.0 nM), suggesting that growth effects of both the precursor and fully processed gastrin molecules may be mediated via similar pathways. On the other hand, pp62c-Yes was not phosphorylated or activated in response to either G17 or PG. To examine whether c-Src kinase mediates proliferative effects of PG, IEC cells were microinjected with anti-Src-IgG and 3H-thymidine (3H-Tdr) uptake of the cells measured. Control cells received nonimmune IgG. The 3H-Tdr uptake of cells stimulated with 1.0 nM PG was significantly reduced in cells microinjected with anti-c-Src-IgG; control IgG had no effect. In cells stimulated with 1.0% fetal calf serum, microinjection with c-Src-IgG had no effect on 3H-Tdr uptake. The specificity of the effect was further confirmed by blocking the inhibitory effect of anti-c-Src-IgG with antigenic Src peptide. These results suggest that activation of c-Src kinase likely represents a critical step in mediating proliferative effects of both the precursor and fully processed forms of gastrins on IEC.
Introduction8l00)u1, 百拇医药
THE GASTRIN GENE is expressed by G entero endocrine cells in the antrum of the stomach. Gastrin gene products (termed: gastrins) are processed by endopeptidases within G cells, converting the full-length progastrin (PG) precursor peptide (80 amino acids) into glycine-extended forms of gastrins. Subsequent amidation at the C-terminal end generates amidated forms of gastrins [gastrin1–17 (G17), G34)]. Under physiological conditions, G17 and G34 are the major circulating forms of gastrin, and they play an important role in acid secretion from parietal cells (1). Gastrins are also trophic for gastrointestinal mucosal cells and play an important role in the renewal of colonic mucosa (2, 3). However, C-terminal amidation is not crucial for measuring biological effects (4). Mitogenic effects of glycine-extended gastrin have been reported on rat intestinal cells (IEC-6), Swiss 3T3 fibroblasts (5), rat pancreatic cancer cells (AR42J) (6), and human colon cancer cells (DLD-1) (7). Migratory effects of gly-gastrin were reported on mouse gastric epithelial cells (IMGE-5) (8). In recent years, the full-length precursor molecule (PG) was reported to be biologically active. Transgenic mice overexpressing PG demonstrated hyperproliferation of colonic crypts (9). PG-expressing mice were at a higher risk for developing preneoplastic and neoplastic lesions in the colonic mucosa in response to azoxymethane (10, 11). Treatment with gly-gastrin increased the risk for developing aberrant crypt foci in rats (12).
Intracellular mechanisms mediating mitogenic effects of G17 and the precursor PG peptide on intestinal epithelial cells (IEC) remain unknown. In previous studies, G17 was reported to stimulate a significant increase in the levels of protein Tyr kinases in colonic mucosal cells (3) and IEC-6 cells (13, 14) and significantly increase Tyr phosphorylation of pp60c-Src kinase molecules (13, 14, 15). However, it is not known whether these changes result in an increased activity of Src kinases in IEC cells in response to G17. In the current studies, we examined this important question and observed an increase in the activity of c-Src kinases in response to 0.1–10.0 nM G17 in IEC cells. Because both c-Src and c-Yes kinases have been implicated in colon carcinogenesis (16, 17, 18), we also measured changes in the activity of c-Yes kinase protein. We were surprised to learn that only the activity of c-Src kinase, but not that of c-Yes kinase, was specifically increased in response to G17 in IEC cells.
The precursor PG1–80 peptide, compared with the fully processed amidated G17 peptide, is extended by 8 amino acids at the C-terminal end and by 55 amino acids at the N-terminal end. Extension of G17 by only 1 amino acid at the C-terminal end (as in gly-extended G17) results in significantly reducing acid secretory activity of the peptide (reviewed in Ref. 19). The contribution of the additional amino acids at the C- and N-terminal ends of the PG peptide, in acid secretory and growth stimulatory effects of the peptides, remains to be identified. Our preliminary studies suggest that acid stimulatory effects of the precursor PG1–80 peptide are almost completely abolished, which confirms the previous notion that precursor peptides lack acid secretory effects (1). Mice overexpressing the full-length PG molecule demonstrated a significant increase in the proliferation of large IEC (9, 10), suggesting that PG may be functioning as a growth factor for IEC. More recently, growth stimulatory effects of a truncated PG peptide6–80 were demonstrated on a temperature-dependent transformed gastric epithelial cell line (YMAC cells) (20), suggesting that PG-like peptides may exert direct growth effects on the target cells. In the current study, we generated the full-length precursor peptide1–80 and examined growth stimulatory effects of PG on nontransformed IEC. Our results suggest that the precursor PG peptide is more potent than G17 as a growth factor for IEC cells. We next examined whether Src kinases are activated in response to the precursor PG molecule in IEC cells. Based on these studies, we report, for the first time, that the precursor PG peptide is more effective than G17 in specifically stimulating the activation of c-Src, but not c-Yes, kinase in IEC cells.
An activation of c-Src kinases, however, may or may not be required for measuring the observed growth effects of PG and G17 peptides on IEC cells. An obligatory role of Src-like kinases in mediating proliferative effects of PDGF, CSF-1, and EGF was demonstrated by microinjecting anti-Src antibodies (Abs) into fibroblasts (21, 22). On the other hand, c-Src activation plays no role in mediating the mitogenic effects of G protein associated receptor proteins like bombesin receptors and lysophosphatidic acid receptors (21). Activation of c-Src kinases has been reported in pancreatic acinar cancer (AR42J) cells in response to G17 (23, 24), but a mediatory role of c-Src kinases was not established in the stipulated growth effects of G17 on AR42J cells. In the current studies, we used the method of microinjecting c-Src-Abs in a single-cell assay and learned that activation of c-Src kinases is required for measuring growth effects of both the precursor PG peptide and the fully processed G17 peptide on IEC cells, because microinjection of anti-c-Src-Abs almost completely abolished 3H-thymidine (3H-Tdr) uptake in response to both the peptides.
Materials and Methods|m%1p+^, 百拇医药
Generation of the full-length recombinant human PG (rhPG)1–80 in Escherichia coli expression system|m%1p+^, 百拇医药
The full-length rhPG1–80 was generated, using standard procedures, as described briefly below. cDNA-encoding hPG was synthesized by PCR-mediated assembly of oligonucleotides. Codons were optimized for efficient tRNA usage in E. coli (25). The synthesized hPG cDNA was cloned in-frame into pET32 (NspV-HindIII), downstream of thioredoxin gene and His tag sequences, under the control of a phage T7 RNA polymerase promoter. The cDNA for the seven-amino-acid recognition site of rTEV protease was incorporated just before PG cDNA, creating a unique cleavage site within the thioredoxin-PG fusion protein (FP) product. The construct was confirmed by DNA sequencing and was transfected into a BL21 (DE3) E. coli strain containing the engineered isopropyl-ß-D-galactopyranoside-inducible T7 RNA polymerase gene. The FP was mostly in the soluble form and was purified from large-scale cultures of recombinant E. coli by the protein core facility in University of Texas Medical Branch, using column chromatography. The rTEV protease His-tag (Sigma, St. Louis, MO) was added to the eluate and cleaved, at room temperature, for 1 h. After adjusting to 1x washing buffer, the cleavage mixture was once again passed through a Ni-agarose column to remove TEV protease, uncleaved FP, and other contaminants. The flow-through was desalted, lyophilized, and resuspended in 10% acetonitrile and 0.1% trifluoracetric acid, for further separation by FP-HPLC. The peak containing rhPG was lyophilized and analyzed by 15% SDS-PAGE, mass-spectrometry, amino acid sequencing, and Western immunoblotting (data not shown). The final rhPG product was more than 99% pure. The standard yield obtained was approximately 1 mg/liter of culture.
Cell culturege, http://www.100md.com
IEC-6 cells and IEC-18 cells (American Type Culture Collection, Rockville, MD) were grown as monolayer cultures in DMEM (Life Technologies, Inc., Grand Island, NY) growth medium as described previously (13). For cell counting and subculturing, the cells were dispersed with a solution of 0.05% trypsin and 0.02% EDTA.ge, http://www.100md.com
Growth assays to measure an increase in cell numbersge, http://www.100md.com
Cell numbers were measured directly by counting the total number of cells, using a Coulter electronic particle counter (model Z1), as described previously (13). Briefly, an optimal number of IEC cells (4 x 104 cells) were plated in 35-mm dishes with 2 ml of normal growth medium containing 10% fetal calf serum (FCS). After 24 h, the medium was changed to serum-free medium (SFM), and cells were cultured for an additional 48 h. Cells were then treated with increasing concentrations (0.01–1000.0 nM) of either G17 or PG in SFM for 48 h. At the end of the treatment, cells were disbursed with trypsin-EDTA solution as given above, and cells were counted using either a Coulter electronic counter or with the help of a hemacytometer under light microscopy. In preliminary studies, we confirmed that PG was not processed into G17 in the extracellular medium of IEC cells that were incubated with PG for 30 min to 24 h, using specific Abs against the C-terminal ends of either the PG peptide or the G17 peptide, by our published methods (10, 26) (data not shown).
Immunoprecipitation of proteins and Src kinase assays2#?;afi, http://www.100md.com
Relative levels of activated Src kinases in IEC-6 and IEC-18 cells were measured by a slight modification of published methods (21, 22). Briefly, cells in culture at approximately 60% confluency were serum starved for 48 h to obtain quiescence. Quiescent cells were treated with increasing concentrations of either G17 or rhPG (0.0–10 nM) at 37 C for 10–15 min. The stimulation was terminated by aspirating the medium and washing the cells twice with ice-cold washing buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM sodium vanadate, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF)]. The washed cells were then solubilized in lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM dithiothreitol, 1 µg/ml leupeptin, 1% aprotinin, 1 mM PMSF, 10 mM NaF] on ice for 10 min at 4 C, and the lysates were centrifuged at 4 C for 10 min at 13,000 x g in a GS-15R centrifuge (Beckman Coulter, Fullerton, CA). After centrifugation, the supernatants were transferred to fresh tubes and preclarified with normal rabbit serum for 30 min at 4 C. Anti-c-Src- or anti-Yes-Abs were used to immunoprecipitate Src- or Yes-related kinases from the samples, as described in figure legends. Briefly, lysates were treated with primary Abs, at 4 C for 1 h, with gentle mixing at repeated intervals. To immunoprecipitate the kinase-Ab complex, we either used protein A (S. aureus) (Sigma) or Pansorbin in the presence of a second Ab. Immunoprecipitation with either protein A or Pansorbin was carried out, at 4 C for 30 min, with constant agitation. The immunoprecipitate was then washed three times by centrifugation for 5 min at 4 C in an Eppendorf (Brinkmann Instruments, Westbury, NY) microcentrifuge at 10,000 x g using the immunoprecipitate washing buffer (10 mM Tris-Cl, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM/ml Na3Vo4).
The immunoprecipitates were resuspended in 20 µl kinase buffer [20 mM HEPES (pH 7.5), 8 mM MgCl2, 8 mM MnCl2, 0.1 mM Na3Vo4]; and the reaction was started by the addition of kinase substrate, enolase (ENO) (Boehringer Mannheim, Indianapolis, IN) followed by the addition of 10 µCi of {gamma} 32P ATP (ICN Pharmaceuticals, Inc., Costa Mesa, CA), on ice for 15 min. Before adding ENO to the reaction buffer, it was denatured in 20 mM CH3COOH at 37 C for 5 min. The reaction was stopped by adding an equal volume of 4x Laemli sample buffer, and the samples were heated to 95 C for 3 min and separated by SDS-PAGE, using 9.5% polyacrylamide gels under reducing conditions as previously described (27, 28). In some cases, the gels after electrophoresis were additionally treated with 1 mM KOH, at 60 C for 1 h, before autoradiographic development (13). The autoradiograms were densitometrically analyzed with the help of a laser densitometer, using a Documentation Analysis System (Model AlphaImager 2000; Alpha Innotech Corporation, San Leandro, CA).
Determination of percent labeling (3H-Tdr uptake) of control and peptide-stimulated IEC cells in single-cell assaysk, http://www.100md.com
Rat IEC (IEC-6, IEC-18) are known to express autocrine IGFs (29, 30) that are increasingly expressed by the IEC cells, in relation to increasing cell numbers (31). IEC cells, however, do not express the gastrin gene (unpublished data from our laboratory). Because these cells express IGFs, the cells continue to divide in culture, even under serum-free conditions, for more than 48–72 h, until IGF expression in reduced to basal levels. To conduct single-cell growth assays in response to PG and G17, in preliminary studies, we determined the optimal cell culture conditions that would allow us to measure significant growth effects in terms of 3H-Tdr uptake in the nuclei of the cells. Exposure of the cells to SFM for 72 h significantly reduced the percent of cells that were positive for 3H-Tdr uptake, to less than 50%, without effecting viability of the cells. Importantly, after 48–72 h of serum starvation, we measured a significant growth response to both FCS and gastrins, wherein 3H-Tdr labeling of the cells was increased to more than 70–95%, compared with less than 50% in the absence of peptide/FCS stimulation. IEC-18 cells were therefore cultured for 72 h under serum-free conditions, which was considered optimal for measuring maximal growth response to exogenous growth factors. Briefly, IEC cells were plated in 35-mm2 tissue culture dishes (25,000 cells/dish) in 2 ml complete growth medium for 24 h, followed by incubation for 72 h in SFM to induce relative quiescence. Cells were then stimulated with either G17 or PG (0.1–1.0 nM) or 1.0% FCS in DMEM growth medium. To confirm growth effects in terms of 3H-Tdr uptake, 1 µCi/ml 3H-Tdr was included in the culture medium to determine the percent of cells that entered into S phase as a result of peptide stimulation. Cells were incubated for 48 h at 37 C and washed with PBS. The cells were then fixed with methanol, air dried, and subjected to autoradiography. For autoradiography, nuclear track emulsion (NTB2; Eastman Kodak Co., Dallas, TX) was used to coat the dishes, which were then stored in the dark at 4 C for 7 d. After development of the emulsion according to the manufacturers suggested protocol, approximately 200 cells/dish were scored for labeling.
Microinjection of cells with Abs-(h, 百拇医药
Injection needles were pulled from borosilicate capillaries using a Flaming/Brown Micropipette Puller, Model P-97 (Sutter Instrument Co., Novato, CA) with a range of outer tip diameters from 2.5–3 microns, as determined by scanning electron microscopy. Cells were visualized throughout the injection procedure using an Olympus Corp. (Melville, NY) IX70 inverted microscope equipped with a temperature-controlled stage kept at 37 C, and injected with the indicated Abs (as described below) using the electronically interfaced Eppendorf Micromanipulator (Model 5171) and Transjector (Model 5246). Manual cytoplasmic injection of approximately 5–10 femtoliters into each cell was performed (32). All experiments were done in duplicate or triplicate, with 50 cells per dish being injected. Only cells within an etched boundary were injected, to allow for easy localization.-(h, 百拇医药
Single-cell assay of postinjection viability and percent inhibition of DNA synthesis
All Ab samples were dialyzed against PBS buffer before injections. Depending on the experiment, cells were injected with either: 1) nonimmune mouse IgG (ICN Pharmaceuticals, Inc. Biochemical Research Products) at a concentration of 10 mg/ml as a marker protein for identifying injected cells; 2) a mixture of nonimmune mouse IgG (10 mg/ml) and an anti-cSrc-Ab (0.1 mg/ml) (Src-2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); or 3) a mixture of nonimmune mouse IgG (10 mg/ml) and anti-cSrc-Ab (0.1 mg/ml) in the presence of excess competing peptide. The anti-cSrc-Ab used in this study was an affinity-purified rabbit polyclonal Ab raised against an Src peptide (sc-18), mapping at the C terminus of human pp60c-Src, a region identical to the corresponding mouse sequence. The blocking peptide used to inactivate the anti-cSrc-Ab was the sc-18 Src peptide (Santa Cruz Biotechnology, Inc.), that had been used to generate the polyclonal anti-cSrc-Ab by the company. Anti-Src-IgG was prepared from anti-Src-Ab using protein A Sepharose (Sigma) before injecting the cells. After injection, quiescent IEC-18 cells were stimulated with the indicated peptides or FCS as described above. For all experiments, 3H-Tdr was added to the medium at a concentration of 1 µCi/ml. Cells were incubated in a 37 C, 5% CO2 incubator for 72 h, washed with PBS, and fixed with methanol and air-dried. The cells were then stained with FITC-conjugated goat antimouse IgG (100-fold dilution of Ab with PBS), and autoradiography was performed as described above. After autoradiography of each dish was complete, 200 noninjected cells were scored for label (scoring only the cells outside of the etched circles), and the percentage of labeled cells was determined.
The injected cells were visualized on a Olympus Corp. IX70 inverted microscope using fluorescence microscopy, scanning the area inside the etched circles for fluorescent cells. Upon identification of fluorescent (injected) cells, the autoradiograph was observed using either bright field and/or phase contrast microscopy to determine the percentage of injected cells that were labeled.$y+7p/, 百拇医药
The percent viability of cells post injection was determined for each dish (50 cells injected per dish) as: (number of fluorescent cells/50) x 100. Post injection viability of injected cells averaged 51.5 ± 1.9% (SE, n = 12), with no significant differences in percent post injection viability whether cells were stimulated by G17, PG, or FCS (data not shown).$y+7p/, 百拇医药
To assess the effect of Ab injections on entry of the cells into S phase in response to the peptides/FCS, percent inhibition of DNA synthesis was determined using the following formula:$y+7p/, 百拇医药
fig.ommitteed
Image capture7^, 百拇医药
The images of the fluorescently injected cells and their autoradiographs were acquired via video imaging using a Model Mli308 low-light camera (Microimage Video Systems, Houston, TX) linked to a computer containing the image capture and analysis software (Meyers Instruments, Houston, TX).7^, 百拇医药
Statistical analysis7^, 百拇医药
In all cases, data are presented as the mean of the sample populations ± SE. To test for significant differences between means, the nonparametric Mann-Whitney test was employed using Statview 4.1 (Abacus Concepts, Inc., Berkeley, CA); P values were considered to be statistically significant if less than 0.05.7^, 百拇医药
Results7^, 百拇医药
In vitro growth effects of G17 and rhPG on IEC cells7^, 百拇医药
In previous studies, we reported significant dose-dependent growth effects of G17 on IEC-6 cells in culture, wherein G17, at concentrations of 0.1–1.0 nM, increased the growth of IEC-6 cells significantly, with 1 nM G17 being the most effective (13). In the current study, we compared the growth effects of rhPG with those of G17 on IEC-6 and IEC-18 cells, and representative data from one of a total of three to six experiments is presented in Fig. 1. IEC-18 cells were more responsive than IEC-6 cells, especially to PG (Fig. 1). Both peptides were maximally effective at concentrations of 0.1–1.0 nM on the two cell lines. At equimolar concentrations, rhPG was generally more effective than G17 as a growth-promoting agent (Fig. 1). At lower concentrations of 0.01–0.05 nM (10–50 pM), only minimal growth effects were measured; and the results were not statistically significant, compared with control values (data not shown). The minimum dose at which we measured significant growth effects of both G17 and PG was 100 pM (0.1 nM), as shown in Fig. 1. Normally, under basal (nonstimulated) conditions, one measures approximately 30 pM amidated gastrins (G17 and G34) in the circulation of most animals (1). However, on stimulation (such as after meal ingestion), approximately 60–120 pM amidated gastrins are measured in the circulation. Thus, G17 can potentially be expected to exert significant (though suboptimal) growth effects on IEC postprandially. PGs are not normally detected in the circulation before or after meals. In certain pathological conditions (such as Zollinger-Ellison syndrome and in patients with colorectal cancers), however, high concentrations of PG-like peptides (1.0–>10 nM) are measured in the circulation (reviewed in Ref. 19), suggesting that PG can potentially exert significant growth effects on IEC in patients with the above specific pathologies.
fig.ommitteed\]+oyz, 百拇医药
Figure 1. Growth effects of rhPG, in relation to that of G17, on IEC-6 and IEC-18 cells. The growth of IEC cells in response to either 0.1% FCS (positive control) or G17 or rhPG is shown. The increase in cell number measured in each dish, compared with that measured in the control dishes (arbitrarily assigned a zero value), is presented. Each bar graph represents data from three to four dishes/experiment and is representative of three to six experiments. *, P < 0.05 vs. control values.\]+oyz, 百拇医药
Phosphorylation and activation of c-Src kinase in IEC-6 cells, in response to increasing doses of G17\]+oyz, 百拇医药
The c-Src kinase activity was measured in an in vitro kinase assay as described in Materials and Methods, with ENO as a substrate. Autoradiographic data from representative experiments are shown in Fig. 2, A–C. In the majority of the experiments, gels were prewashed with KOH to measure phosphorylation of Tyr moieties and to reduce phosphorylation of all other moieties (13) (Fig. 2A). In a few experiments, gels were not prewashed with KOH; representative results are presented in Fig. 2C. In the absence of KOH washing, one or more nonspecifically phosphorylated protein bands were detected (56–68 kDa). However, with KOH washing, the nonspecifically phosphorylated proteins were not detected (Fig. 2A), which allowed us to more accurately analyze the changes in phosphorylation of c-Src and its substrate, ENO, in response to increasing doses of G17. Immunoprecipitation of an equivalent concentration of c-Src protein in different samples was confirmed by Western immunoblot analysis using anti-c-Src-Abs (Fig. 2B). The data in Fig. 2A were corrected for c-Src levels in Fig. 2B before calculating the percent change in phosphorylation levels as presented in Fig. 2D. The increase in phosphorylation of c-Src protein and ENO was determined densitometrically, as described in Materials and Methods, and data from two to six experiments are presented as bar graphs in Fig. 2D. The c-Src kinase activity, measured in terms of increase in phosphorylation of c-Src and ENO, was significantly increased in response to 0.1–1.0 nM gastrin, with 1.0 nM concentration being the most effective. At higher concentrations (10–100 nM), no significant increase in c-Src kinase activity was observed. In a previous study, we similarly measured a bi-phasic effect of G17 on proliferation and Tyr phosphorylation of c-Src in IEC-6 cells (13).
fig.ommitteede, 百拇医药
Figure 2. Activation of c-Src kinase in response to increasing doses of G17. IEC-6 cells were treated with increasing doses of G17 and processed for measuring c-Src kinase activity in terms of phosphorylation of the substrate (ENO) and autophosphorylation of c-Src, as described in Materials and Methods. Representative data from one of six experiments are presented in A. These data are from a gel that was prewashed with KOH before autoradiography, as described in Materials and Methods. B, Samples from duplicate gels were transblotted onto nitroceullose membranes and processed for Western immunoblotting with anti-src-Ab (SC-18; Santa Cruz Biotechnology, Inc.). Immunoblots were developed with the ECL detection kit (Amersham, Piscataway, NJ), demonstrating equivalent amounts of c-Src protein in samples used in the kinase assays shown in A. Other than lane 5 (stimulated with 100 nM G17), all other samples contained equivalent levels of c-Src protein. C, Gels that were not prewashed with KOH almost always demonstrated the presence of other phosphorylated proteins (~ 56–58 kDa), whose nature is not known. These bands likely represent Ser- or Thr-phosphorylated proteins, because KOH treatment dephosphorylates Ser and Thr moieties. D, Data from two to six experiments, in which dose-dependent effects of G17 were measured on src kinase activity. Even at low concentrations, G17 was effective in significantly increasing c-Src kinase activity, in terms of phosphorylation of ENO (lower panel) and phosphorylation of c-Src (upper panel).
Effects of G17 on activation of c-Src kinase and c-Yes kinaseau, 百拇医药
To examine whether the activation of c-Src kinase in response to G17 was specific, we additionally measured activation of c-Yes kinase, using methods essentially similar to that described above for c-Src kinase. Anti-c-Yes-Abs were used during the immunoprecipitation step to measure c-Yes kinase activity levels. Because 1.0 nM G17 was maximally effective in inducing an increase in c-Src kinase activity (Fig. 2), we used this concentration for comparing the effects of G17 on the activity of the two kinases, as shown in Fig. 3. G17 was ineffective in inducing an increase in Tyr phosphorylation and activation of c-Yes kinase protein (Fig. 3B). However, c-Src kinase activity was increased significantly in the presence of 1.0 nM G17 in the same experiments (Fig. 3A).au, 百拇医药
fig.ommitteedau, 百拇医药
Figure 3. Effect of G17 on activation of c-Src kinase and c-Yes kinase. IEC cells were treated with the optimally effective concentration of G17 (1.0 nM) and immunoprecipitated with either anti-c-Src-Ab (SC-18; Santa Cruz Biotechnology, Inc.) or anti-c-Yes-Ab (3H9; Wako Pure Chemical Industries Ltd., Richmond, VA). The immunoprecipitates containing either c-Src or c-Yes were subjected to an in vitro kinase assay in the presence of ENO, as described in Materials and Methods. At the end of the kinase assay, the samples were separated by SDS-PAGE, subjected to KOH treatment, and autoradiographically developed as described in Materials and Methods. The effect of G17 treatment on c-Src kinase activity (A) and c-Yes kinase activity (B) is shown. Duplicate gels were processed for Western immunoblotting with either anti-c-Src or anti-c-Yes-Ab to confirm equal loading of the lanes with the indicated kinase protein. In all experiments, the relative concentrations of either c-Src (in immunoprecipitates of c-Src-Ab) or c-Yes (in immunoprecipitates of c-Yes-Ab) were similar (data not shown).
Dose-dependent effects of PG on c-Src and c-Yes kinase activity{e5+, 百拇医药
Early passage numbers of IEC-6 cells, used in the studies with G17, were not available by the time we began to examine effects of PG on Src kinases. We therefore used IEC-18 cells that were determined to be equally responsive to growth effects of G17 and PG (Fig. 1, Table 1); PG significantly increased Tyr phosphorylation of both c-Src protein and its substrate ENO at 0.1–1.0 nM concentrations (Fig. 4). At the lower concentration of 0.01 nM, no significant effect of PG was measured on phosphorylation of ENO (data not shown). In the absence of KOH treatment (Fig. 4C), we once again observed phosphorylation of nonspecific bands (56–58 kDa). The activity of c-Yes kinase remained unchanged after treatment of the cells with 1.0 nM PG (Fig. 4F). PG was approximately 1.5–2.0 times more effective than G17 in stimulating c-Src kinase activity at the optimal concentrations of 0.1–1.0 nM (Fig. 5). The relative growth effects of PG and G17 on IEC-18 cells matched the relative potency of the two peptides in activating c-Src kinase activity (Table 1), providing further evidence that c-Src kinases mediate proliferative effects of both the peptides. In a control experiment, excess anti-c-Src-Ab was added to the kinase buffer, before the addition of ENO, which resulted in significantly reducing the phosphorylation of ENO in peptide-stimulated samples (data not shown), strongly suggesting that significant increase in the phosphorylation of the substrate, ENO, in the presence of the peptide-stimulated samples was attributable to the presence of activated c-Src kinases in these samples.
fig.ommitteedpv-&n#7, http://www.100md.com
Table 1. Proliferation of IEC-18 cells and an increase in c-Src kinase activity in response to PG and G17pv-&n#7, http://www.100md.com
fig.ommitteedpv-&n#7, http://www.100md.com
Figure 4. Effect of PG treatment on c-Src and c-Yes kinase activity. IEC-18 cells were treated with increasing concentrations of PG (0.1–10.0 nM), and the c-Src kinase activity in the cells was measured in an in vitro kinase assay as described in Materials and Methods. At the end of the kinase assay, the samples were electrophorically separated by SDS-PAGE, KOH-treated, and examined autoradiographically as described in Materials and Methods. Data from a representative experiment (from a total of three separate experiments) are shown in A. Duplicate samples were processed for immunoblotting with anti-c-Src-Ab, as described under Fig. 3, and the c-Src immunoblot data for samples presented in A are shown in B. As shown in B, c-Src protein levels were equivalent in all samples other than lane 5. In one experiment, the gel was not treated with KOH, and these autoradiographic data are shown in C. D, Data in A (from four separate blots from two experiments) are presented as percent change in densitometric readings of the indicated bands; the readings in the control lanes (not treated with PG) were arbitrarily given a zero value. E, In one experiment, c-Yes kinase activity was also measured in response to 1.0 nM PG, as described for G17 in Fig. 2. Data from one of two blots is shown in E.
fig.ommitteed3![z2n, 百拇医药
Figure 5. Comparison of the effects of G17 and PG on c-Src kinase activity in IEC-18 cells. IEC-18 cells were treated with 0.1 or 1.0 nM G17 or PG, and c-Src kinase activity was measured as described in the legend of Fig. 2. At the end of the reaction, the samples were electrophoretically separated by SDS-PAGE and subjected to KOH treatment and developed autoradiographically. Data from representative experiments (from a total of three similar experiments) are shown in A and B. Data from all three experiments are presented as bar graphs in C. The percent increase in Tyr phosphorylation of c-Src (upper panel) and ENO (lower panel) in response to G17 was arbitrarily assigned a 100% value, and the increase in response to PG is presented as a percent of that measured with G17. *, P < 0.05 vs. G17 values; lane C, control samples from cells not treated with the peptide.3![z2n, 百拇医药
Effects of anti-c-Src Abs on 3H-Tdr uptake by IEC-18 cells in response to gastrins and FCS3![z2n, 百拇医药
Serum-starved IEC-18 cells were microinjected with either control, nonimmune IgG, or specific anti-c-Src-IgG and were stimulated to grow in the presence of optimal concentration of either G17 or PG (1.0 nM), as described in Materials and Methods; as a positive control, cells were also stimulated with 1.0% FCS. Data from representative cells, demonstrating microinjection of the indicated Ab, and the resulting 3H-Tdr uptake in the cells in response to either FCS or gastrins are presented in Figs. 6 and 7. The percent of cells labeled with 3H-Tdr, as a result of the various treatments, was calculated as described in Materials and Methods, and data from several experiments are presented as bar graphs in Fig. 8A. Based on the data presented in Fig. 8A, the percent inhibition of DNA synthesis in the Ab injected vs. noninjected cells was calculated as described in Materials and Methods, and data from several experiments are presented in Fig. 8B. The data presented in Figs. 6 ad 8 demonstrate that microinjection of cells with the specific c-Src-Ab significantly reduced 3H-Tdr labeling of the cells in response to both G17 and PG.
fig.ommitteedt\v}#, 百拇医药
Figure 6. Effect of microinjecting anti-c-Src-Abs on 3H-Tdr up-take by IEC-18 cells in response to PG. IEC-18 cells were microinjected with either anti-c-Src-IgG or control nonimmune IgG and subjected to a single-cell growth assay in response to 1.0 nM PG as described in Materials and Methods. A fluorescently-labeled second Ab was used to identify injected cells as described in Materials and Methods. Autoradiography of the same cells was used to monitor 3H-Tdr uptake as described in Materials and Methods. A1 (phase contrast microscopy) and A2 (fluorescent microscopy) are low magnification (100x) pictures of the same field. At low power, one cannot tell whether a cell is labeled, because one cannot differentiate phase dark chromatin and nucleoli from label. B1 (phase contrast microscopy), B2 (fluorescent microscopy), and B3 (bright field microscopy) are high-magnification (400x) pictures of the field of cells contained within the box shown in A1 and A2. The fluorescent cells shown in A2 and B2 were coinjected with nonspecific mouse IgG and anti-c-Src-IgG. At higher magnification, cells 1, 4, 6, 7, and 9 were injected, and only cell 6 incorporated tritiated thymidine (B1–B3). Cells 2, 3, 5, and 8 were not injected, and all four cells incorporated tritiated thymidine (B1–B3), demonstrating that the anti-c-Src-IgG prevents a significant number of injected cells from cell cycle progression. C1 (phase contrast microscopy) and C2 (fluorescent microscopy) are high-magnification (400x) pictures of the same field. The two fluorescent cells shown in C2 were injected with only nonspecific mouse IgG. In C1, all cells are shown to be labeled, demonstrating that the nonspecific mouse IgG has no effect on cell cycle progression.
fig.ommitteed6q*;.4w, 百拇医药
Figure 7. Specificity of effects of microinjecting anti-c-Src-Abs on 3H-Tdr uptake by IEC 18 cells in response to G17: comparison of effects in response to 1% FCS. IEC-18 cells were microinjected with either anti-c-Src-IgG or a control nonimmune mouse IgG and subjected to a single-cell growth assay in response to G17 or FCS as described in Materials and Methods. Fluorescently-labeled second Ab was used to identify injected cells as described in Materials and Methods. Autoradiography of the same cells was used to monitor 3H-Tdr uptake as described in Materials and Methods. A–C, Cells microinjected with nonimmune mouse IgG and treated with 1.0 nM G17; D–F, cells microinjected with nonimmune mouse IgG and treated with 1% FCS; G–I, cells microinjected with nonimmune mouse IgG plus specific anti-c-Src IgG and treated with 1.0 nM G17. A–C, D–F, and G–I represent cells in the same field of view, respectively. A, D, and G, fluorescent microscopy used to identify injected cells; B, E, and H, bright field microscopy of autoradiographs used to identify dark grains over nuclei of cells that entered into S-phase of the cell cycle; C, F, and I, phase contrast microscopy to delineate the spread of the cells. Thin lines point to the nuclei of injected cells. Lines with arrows point to the nuclei of noninjected cells.
fig.ommitteed%, 百拇医药
Figure 8. Effect of microinjecting anti-c-Src-IgG on 3H-Tdr uptake by IEC-18 cells in response to either 1% FCS or 1.0 nM PG or 1.0 nM G17. A. The cells were treated as described in the legend of Fig. 7. Data from three to five separate experiments are presented as bar graphs, comparing 3H-Tdr labeling of cells with and without injection with indicated Abs, in the presence of the indicated stimulants. B. The data in A were used to determine the percent inhibition of DNA synthesis as described in Materials and Methods. Each bar graph represents the mean ± SEM of data from three to five separate experiments. *, P < 0.05 vs. corresponding values for the cells injected with nonimmune IgG; C-IgG, control nonimmune IgG.%, 百拇医药
Our in vitro Src kinase assay results suggested that anti-c-Src-Ab (used for immunoprecipitation of c-Src from cell lysates) did not block kinase activity of the c-Src protein (Figs. 2–5). The mechanism by which anti-c-Src-Abs inhibit the kinase activity of c-Src in vivo, when microinjected into the IEC cells, is therefore not understood. Because the addition of excess anti-c-Src-Ab to the in vitro kinase assay buffer (containing immunoprecipitated c-Src proteins) significantly reduced the phosphorylation of ENO (Table 1), it suggests that c-Src-Abs can potentially inhibit the activation of c-Src kinases in vitro, as observed in the microinjection experiments in vivo (Figs. 6–8). It is possible that, in the presence of excess substrate (ENO) in the in vitro kinase buffer (Figs. 2–5), the immunoprecipitated c-Src protein gets preferentially bound to the excess substrate rather than c-Src-Ab (that is bound to the second Ab/Pansorbin complex and is perhaps less available for binding or inhibiting the kinase activity of c-Src in the in vitro kinase assay buffers). At least one other group of investigators (21, 22) has used anti-c-Src-Abs (similar to the Ab used by us in the current studies) and reported immunoprecipitation of kinase-active c-Src proteins from various cellular samples; at the same time, the authors observed significant inhibition of the c-Src kinase activity in vivo in cells microinjected with anti-c-Src-Abs (21). The published studies of Roche et al. (21) thus provide strong support for our seemingly divergent observations with the c-Src-Abs in vitro vs. in vivo.
An important finding was that microinjection of the cells with anti-c-Src-IgG had no effect on either basal (nonstimulated) 3H-Tdr labeling, or labeling in response to 1% FCS. The latter results strongly indicate the specificity of the effects of anti-c-Src-Abs in reducing 3H-Tdr uptake in response to gastrins. To further confirm the specificity of the effects of anti-c-Src-Abs, the activity of the Abs was blocked with the antigenic Src peptide (used for generating the Abs), as described in Materials and Methods. The effect of inactivated/blocked Abs was examined in single-cell assays as described above. Results with the blocked Ab, in comparison with results with the untreated Ab, are shown in Figs. 9 and 10. Data from representative cells from one of two separate experiments, demonstrating 3H-Tdr labeling in cells microinjected with either the blocked or the untreated c-Src-IgG, are shown in Fig. 9. The percent inhibition of DNA synthesis in the injected vs. noninjected cells was calculated from both the experiments and is presented as bar graphs in Fig. 10. As can be seen from Figs. 9 and 10, blocking the activity of the anti-c-Src-IgG with the antigenic Src peptide completely abolished the inhibitory effects of anti-c-Src IgG on 3H-Tdr labeling of cells in response to PG (Fig. 9, C–F; and Fig. 10). On the other hand, neither microinjection of the active anti-c-Src-IgG or of the blocked anti-c-Src-IgG had any effect on the 3H-Tdr labeling in response to 1% FCS (Fig. 9, A and B; and Fig. 10), similar to results presented in Figs. 6–8. These results confirm the specificity of the inhibitory effects of anti-c-Src-Abs in reducing 3H-Tdr labeling (and hence proliferation) in response to gastrins but not to other growth-promoting agents contained in FCS.
fig.ommitteedpx^]v, 百拇医药
Figure 9. Effects of blocking the activity of anti-c-Src-IgG on 3H-Tdr uptake by IEC-18 cells in response to either 1% FCS or 1.0 nM PG. Cells were microinjected with the anti-c-Src-IgG with and without peptide blocker and subjected to single-cell growth assays as described in the legend of Fig. 6. Data from representative cells, treated as indicated below, are presented in A–F. A and B, Cells microinjected with anti-c-Src IgG and treated with 1% FCS; C and D, cells injected with anti-c-Src IgG and treated with 1.0 nM PG; E and F, cells injected with inactivated (peptide blocked) anti-c-Src IgG and treated with 1.0 nM PG. A and B, C and D, and E and F represent cells in the same field of view, respectively. Fluorescent microscopy was used to identify injected cells in A–E. Phase contrast microscopy was used to determine whether the injected cells were labeled with 3H-Tdr (indicating entry into the S phase of the cell cycle), in B, D, and F. Thin lines with arrows point to the nuclei of injected cells. Thick lines with arrows point to the nuclei of noninjected cells.px^]v, 百拇医药
fig.ommitteed
Figure 10. Effects of blocking the activity of anti-c-Src IgG on 3H-Tdr uptake by cells in response to either 1.0% FCS or 1.0 nM PG. Cells were microinjected with either the blocked (inactivated) or nontreated anti-c-Src-IgG or with the control nonimmune IgG and stimulated with either 1.0% FCS or 1.0 nM PG, as described in Materials and Methods and in the legend of Fig. 9. Then, 3H-Tdr uptake by the injected vs. noninjected cells was determined, and the percent inhibition of DNA synthesis was calculated from these data as described in Materials and Methods. Each bar graph represents mean ± SEM from duplicate/triplicate measurements from two to three separate experiments. *, P < 0.05 vs. 3H-Tdr uptake in corresponding cells injected with nonimmune IgG.%u]x8, http://www.100md.com
Discussion%u]x8, http://www.100md.com
In the current studies, we demonstrate a critical role of pp60c-Src kinase in mediating mitogenic effects, of both the fully processed form of gastrin (G17) and the full-length precursor form of gastrin (PG), on fetal rat IEC. An important finding was that the mediatory role of c-Src kinase seemed to be specific, because a closely related kinase, pp62c-Yes, was not activated in response to either G17 or PG in IEC cells.
In all previous studies, pharmacological inhibitors of MAP kinases, PI-3 kinase, and Src kinases were used to examine the role of kinases in mediating the stipulated growth effects of G17 and gly-extended G17 on a rat pancreatic cancer cell line (AR42J) (23, 24, 33, 34) and a pituitary cell line (35). Pharmacological inhibitors of Src kinases are not very specific and are known to inhibit the activity of several other members of the Src family of kinases (21, 36). A more precise method for dissecting out the role of various intracellular molecules/kinases currently being used is microinjection of inhibitory Abs (32, 37). Microinjection of anti-Src-Abs was used to confirm a role for Src-kinases in mediating growth effects of various growth factors on fibroblasts (21, 22). Using this method, it was possible, for the first time, to separate the roles of PI-3 kinase and PI-3 kinase ß in DNA synthesis by human colon carcinoma cell lines (38). Similarly the role of various isoforms of protein kinase C in calcium signaling in the IEC could be dissected by microinjecting inhibitory or blocking Abs against the various forms of the enzyme (39). Therefore, in the current studies, we have, for the first time, examined the role of pp60c-Src kinase in mediating growth effects of G17 and PG on IEC-18 cells in single-cell assays by microinjecting c-Src-Abs to block the activity of c-Src kinase. The single-cell assay method is now being used by many investigators to examine the role of various intracellular molecules (40). In the current study, microinjection of anti-c-Src-Abs in IEC cells resulted in the loss of 3H-Tdr up-take in response to both the precursor (PG) and processed (G17) forms of gastrin, to basal (control) levels. The specificity of the effects of the Ab was confirmed by the use of blocking peptides, which completely abolished the inhibitory effects of the Abs. We have thus, for the first time, documented a critical role of pp60c-Src kinase in mediating mitogenic effects of both PG and G17 on IEC cells.
In previous studies, we and others demonstrated a significant increase in protein tyrosine kinase (PTK) activity in response to G17, both in the colonic mucosal cells (3) and IEC-6 cells (13, 14). Intracellular proteins, including pp60c-Src, were Tyr-phosphorylated in response to physiological concentrations of G17 (13). In the current study, we further demonstrated that activity of c-Src kinase was increased in response to increasing doses (0.1–1.0 nM) of G17. In rat colonic epithelial cells, G17 induced IP3 formation by up-regulating phospholipase C {gamma} which similarly resulted in the activation of pp60c-Src kinase (14).rr[7n, 百拇医药
Src-like PTKs are now believed to play a central role in mediating proliferative effects of several growth factors, including PDGF (platelet-derived growth factor), CSF (colon stimulating factor)-1, and EGF (epidermal growth factor) (21, 22, 36). An obligatory role of Src-like PTKs in mediating proliferative effects of PDGF, CSF-1, and EGF was demonstrated by microinjecting Src-Abs into fibroblasts (21); however, Src activation was not required for mediating mitogenic effects of BBS (bombesin) and lysophosphatidic acid that bind G-protein-associated receptor proteins. The current study provides convincing evidence that Tyr phosphorylation and activation of pp60c-Src kinase is significantly up-regulated in response to both G17 and rhPG in IEC cells. The relative potency of the two peptides, in increasing Tyr phosphorylation and kinase activity of c-Src, was equivalent to the relative potency of the two peptides in stimulating growth of IEC-18 cells, suggesting that c-Src may be mediating growth effects of both of the peptides on intestinal cells.
The Src kinase family contains several closely related members (Src, Fyn, Yes, Lck, Hck, Fgr, Lyn, and Yrk) that perform similar, but specific, functions in different cell types (36). In human colon carcinomas, the activity of only two Src-like kinases, pp60c-Src and pp62c-Yes, is significantly increased (16, 17, 18, 41). We now know that a large percentage of human colon cancers express the gastrin gene and release significant concentrations of PG into the circulation (reviewed in Ref. 19). Because the results of the present study suggest that pp60c-Src kinase is specifically and significantly activated in IEC cells in response to both the precursor and the fully processed forms of gastrin, it is possible that the observed increase in the activity of at least one of the kinases (pp60c-Src) in colorectal cancers may be mediated via autocrine gastrins expressed by these cancers. In preliminary studies with a human colon cancer cell line (HCT-116), we observed that an increase in the activity of pp60c-Src kinase, but not pp62c-Yes kinase, may be secondary to the expression of autocrine gastrins (42). The proliferative and tumorigenic potential of antisense (AS) HCT-116 clones, expressing the AS gastrin RNA, was significantly reduced in vivo and in vitro, compared with that of control HCT-116 clones (43); this may be attributable to a loss in the activity of c-Src kinase in AS HCT-116 clones (42). Thus, based on our current studies with the IEC cells and our preliminary studies with the control and AS clones of HCT-116 cells, it seems possible that both endocrine and autocrine gastrins (irrespective of the molecular form of gastrins) may increase proliferation of intestinal and colon cancer cells via up-regulation of c-Src kinase activity.
c-Src kinases are activated in many other cancers, such as breast cancer (44, 45). Because many cancers express autocrine growth factors, such as TGF-{alpha} and PG, it is possible that the increase in c-Src kinase activity observed in these cancers may be secondary to the expression of autocrine growth factors. However, c-Src kinase does not mediate growth effects of all growth factors, considering that 3H-Tdr up-take by IEC cells, in response to autocrine IGFs and/or 1% FCS, was not inhibited by microinjection of anti-c-Src-Ab (current studies). IEC-18 cells, may therefore provide an excellent in vitro model for examining intracellular mechanisms that mediate growth effects of PG and gastrin-like peptides, that may be relevant to the growth of IEC and colon cancer cells in situ.9hdq)-c, http://www.100md.com
Acknowledgments9hdq)-c, http://www.100md.com
We would like to acknowledge Gene-Cell, Inc. for generously providing all the glass injection needles used in the microinjection experiments, and we gratefully acknowledge the secretarial support of Pat Gazzoli. We would also like to thank David Konkel, Ph.D., for critically reading this manuscript.
Received May 10, 2002.), http://www.100md.com
Accepted for publication September 17, 2002.), http://www.100md.com
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