Butyrate-induced alterations of phosphoinositide metabolism, protein kinase C activity and reduced CD44 variant expression in HT-29 colon cancer cells.

Initiation of cell growth and neoplastic transformation frequently involves activation of growth factor receptor-coupled tyrosine kinases and stimulation of the phosphoinositide second messenger system. Altered expression of CD44 variants was reported in several malignant tumor types with possible implications for tumor progression and prognosis. CD44 variant expression was reported to be associated with second messenger activation and differentiation. We therefore investigated the effects of butyrate-induced short-term differentiation on phosphoinositide signaling, phospholipase C and protein kinase C activity and alteration of CD44 variant expression in human HT-29 colon carcinoma cells. HT-29 cells were cultured with sodium butyrate for 6 days. Phosphoinositide turnover was measured by [32P]orthophosphate incorporation and phospholipase C activity by determination of the release of [3H]inositolphosphates from [3H]myoinositol prelabeled cells. Protein kinase C activity was determined by histone III-S phosphorylation, PKC subtype expression by RNase protection analysis, and CD44 variant expression was determined by RT-PCR using variant-specific primers. Treatment of HT-29 human colon carcinoma cells with sodium butyrate caused a dose-dependent inhibition of cell proliferation (IC50, 2.5 mM) with morphologic signs of an enterocytic differentiation following 6 days of treatment. The phosphoinositide turnover as determined by 32P-incorporation under non-equilibrium conditions showed a 30-40% inhibition of labeled phosphoinositides and phosphatidic acid and a dose-dependent inhibition of cholinergically stimulated phospholipase C activity as a secondary event following butyrate-induced enterocytic differentiation. However, long-term incubation of HT-29 cells with phorbol ester or an inhibitor of classical and novel PKC subtypes did not affect cell proliferation. In butyrate-treated HT-29 cells activation of calcium-dependent protein kinase C by cholinergic stimulation or phorbolester treatment induced an increase in membrane-bound cPKC activity, while expression of distinct high- molecular CD44 variant transcripts v3 (670 bp), v5 (940 bp) and v8 (535 bp) were drastically reduced after butyrate pretreatment. Enterocytic differentiation of HT-29 colon carcinoma cells seems to be associated with alterations in phosphoinositide resynthesis, phospholipase C activity and ligand/receptor-induced PKC translocation. The observed reduction of distinct high-molecular CD44v3, v5 and v8 variants following butyrate-induced differentiation indicates an association of specific CD44 variant expression with the malignant phenotype of HT-29 colon cancer cells, thus being possible targets for new diagnostic and therapeutic strategies.

Different behaviour of soluble CD40L concentrations can be reflected by variations of preanalytical conditions.

INTRODUCTION: Biomarkers reflecting an inflammatory or immunological response are increasingly offered to improve the risk stratification of patients. For example, current evidence suggests that soluble CD40 ligand (sCD40L) is elevated in patients with acute coronary syndrome. But only a few data are available to evaluate the influence of preanalytic conditions on sCD40L values. METHODS: Blood samples of seven healthy blood donors were collected in tubes without additives and in EDTA- or citrate-filled tubes at various storage conditions. Platelet count was modified by serum dilution, and sCD40L was measured in platelet-rich-plasma and in whole blood. sCD40L levels were determined by an commercially available ELISA-Kit. RESULTS: Immediately after blood sample assessment, sCD40L levels in serum samples were elevated (1258+/-820 pg/ml) compared to EDTA (64+/-32 pg/ml) and citrate (60+/-8.5 pg/ml) values. Additionally, sCD40L levels were dependent on storage duration. After platelet activation, sCD40L levels were significantly increased to 8278+/-2453 pg/ml and were significantly correlated to platelet count (r=0.96). CONCLUSIONS: Soluble CD40L levels were clearly influenced by preanalytical conditions and were dependent on storage duration, sample technique and platelet count. These influences should be considered by the determination and evaluation of sCD40L concentrations.

Effects of the putatively oncogenic protein kinase Calpha D294G mutation on enzymatic activity and cell growth and its occurrence in human thyroid neoplasias.

A point mutation of protein kinase Calpha (PKCalpha) has been described in pituitary adenomas and in follicular adenomas and thyroid carcinomas. The mutation results in an exchange of aspartic acid into a glycine of the amino acid 294 of PKCalpha, which is located adjacent to the Ca (2+)-binding hinge region and has been proposed as an activation inhibitor. To investigate its biochemical sequelae, we constructed the mutated enzyme and expressed it in human embryonic kidney cells (HEK). The K M of the purified enzyme for Ca (2+) and its K M for the substrate MBP 4 - 14 was not altered by the mutation. Translocation of PKCalpha to HEK cell membranes upon activation was not changed and the mutant potently inhibited cell proliferation upon 5-fold stable overexpression in HEK cells. Thus, loss of function in mutated PKCalpha was excluded. A screen for the mutation using a restriction assay with a sensitivity of at least 8 % for the mutated DNA did not show any mutation in 11 carcinoma and 13 adenomatous thyroid samples. We conclude that the A294G mutation of PKCalpha does not detectably affect its biochemical properties in vitro or in vivo, and is at least rare in thyroid neoplasias, in Germany.

Regulation of protein kinase C by short term hyperglycaemia in human platelets in vivo and in vitro.

AIMS/HYPOTHESIS: Postprandial hyperglycaemia carries an increased risk of macrovascular disease even without Type II (non-insulin-dependent) diabetes mellitus. Chronic hyperglycaemia activates protein kinase C (PKC) in vitro and in vivo but it is not known whether PKC is regulated by short-term post-prandial hyperglycaemia in vivo in humans. We investigated whether PKC is regulated in vivo in hyperglycaemic and hyperinsulinaemic infusion tests and correlated the results to stimulations in vitro. METHODS: Protein kinase C regulation was measured in platelets obtained from 8 healthy subjects who were infused with glucose and insulin for 2 h attaining peak concentrations of 16 mmol/l glucose and in platelets from 8 healthy young subjects, 8 older subjects without diabetes, and 10 older subjects with Type II diabetes after incubation in vitro with 16 mmol/l glucose or glucose and insulin. For precise quantification, a shortened PKC beta1 standard protein was generated by bacterial expression and PKC alpha, beta1, beta2 and delta isoenzyme values were measured by immunoblot analyses. RESULTS: Hyperglycaemic and hyperinsulinaemic in vivo tests increased the amounts of PKC alpha, beta1 and beta2 in the membrane fraction of platelets to 225 +/- 87 %, 164 +/- 22 % and 302 +/- 135 %, respectively, when compared with the baseline values in young healthy volunteers (n = 8, p < 0.05). The expression of PKC delta did not change. In comparison to the recombinant PKC beta1 standard protein, 5 ng PKC beta1/ microg protein was measured before the test and 2 ng/microg were translocated to the membrane fraction after the infusion. No change in the absolute amount of PKC beta1 was detected. In contrast, after incubation in vitro PKC was not regulated by glucose or glucose and insulin in 8 young healthy subjects (age 26 +/- 0.7 years) and in 8 older, healthy subjects (age 64,8 +/- 4 years) although 100 nmol/l 12-O-tetradecanoylphorbol 13-acetate caused maximal activation. In marked contrast, PKC beta1 and PKC beta2, but not PKC alpha or PKC delta, were increased in vitro in the membrane fraction by 292 +/- 61% and 432 +/- 88% (p < 0.05) in 10 subjects with Type II diabetes mellitus matched for age, sex and BMI. CONCLUSION/INTERPRETATION: We found that short-term hyperglycaemia activates PKC alpha, beta1 and beta2 in platelets of healthy persons making them potential candidates for mediating the increased cardiovascular risk of postprandial hyperglycaemia. Hyperglycaemia and hyperinsulinaemia did not cause short-term activation of PKC in platelets in vitro suggesting the existence of additional stimuli. Subjects with Type II diabetes showed a markedly altered reactivity of platelet PKC beta in vitro indicating some diabetes-related regulation.

Decreased expression of epidermal growth factor receptor and mRNA of its ligands in Helicobacter pylori-infected gastric mucosa.

BACKGROUND: Epidermal growth factor (EGF) and TGF-alpha play a central role in maintaining gastric mucosal integrity. Little is known about the regulative role of the four other widely expressed epidermal growth factor receptor ligands, heparin-binding EGF, amphiregulin, betacellulin and cripto in the gastric mucosa. METHODS: Nineteen patients with Helicobacter pylori-positive gastritis and 32 healthy controls were investigated. Mucosal mRNA expression of EGF receptor ligands was determined by quantitative PCR before and after H. pylori eradication. PCR products were analyzed by soft laser scanning densitometry. Moreover, the effect of chronic active gastritis on EGF receptor expression was assessed by [125I] EGF receptor autoradiography. Immunohistochemistry was performed for TGF-alpha to localize growth factor expression. RESULTS: Antral and oxyntic biopsies showed strong mRNA expressions for TGF-alpha, amphiregulin and heparin binding EGF, but not for EGF, cripto and betacellulin. mRNA expression was significantly reduced down to 50% in H. pylori infection, significantly lower compared to normal gastric mucosa, and increased after eradication therapy. Moreover, chronic gastritis was associated with decreased antral EGF receptor binding compared to healthy controls, possibly reflecting reduced autoinduction. Immunohistochemical analyses localized TGF-alpha in the cytoplasma of gastric epithelial cells and revealed its increased expression after H. pylori eradication. CONCLUSIONS: The data presented suggest that amphiregulin, heparin binding EGF and TGF-alpha are important EGF receptor ligands in the gastric mucosa. H. pylori infection apparently suppresses their mRNA as well as receptor expression that is reversed by H. pylori eradication. This deficiency of the gastroprotective EGF system may contribute to the gastric pathogenicity of H. pylori infection.

Anti-proliferative activity of protein kinase C in apical compartments of human colonic crypts: evidence for a less activated protein kinase C in small adenomas.

The protein-kinase-C (PKC) family of iso-enzymes regulates mitogenic signal transduction in colorectal-cell lines. Its function in human colonic mucosal proliferation is controversial. Our study investigated the role of PKC with regard to proliferation and changes of PKC iso-enzyme expression in colonic biopsies compared with small adenomas. In short-term tissue-culture experiments of colonic mucosal biopsies, we found reduced S-phase labeling in the 2 apical compartments of longitudinally sectioned crypts when PKC was activated by 200 nM of the phorbol ester TPA (n = 8). Thus, PKC inhibited growth of differentiated colonocytes which may influence cell homeostasis in colonic crypts. Furthermore, we have determined the expression of PKC alpha, -beta1, -beta2, -delta and -epsilon in colonic adenomas smaller than 1 cm in diameter of 18 patients and found a significant increase of PKC alpha in the cytosolic fraction and decreased membrane levels of PKC beta2 in adenomas compared to normal, neighboring mucosa while protein levels of PKC beta1, -delta and -epsilon were not altered. Moreover PKC delta but not PKC alpha mRNA expression was significantly lowered in adenoma tissue in 7 patients, as determined by ribonuclease-protection analysis. Changes in the regulation patterns of PKC isoforms suggest a decreased activation state of PKC even in small adenomas. This is compatible with an anti-proliferative function of PKC serving to protect mucosa from expanding mutated cells.

Analysis of a protein kinase C-alpha mutation in human pituitary tumours.

It is generally accepted that protein kinase C-alpha (PKC-alpha) is an important enzyme in the cellular regulation of growth and differentiation by phosphorylating proteins. Recent studies have described a point mutation of PKC-alpha (position 908 of the genetic sequence, codon GAC becoming GGC) in invasive human pituitary tumours which leads to an exchange of amino acids in the protein. We investigated 11 human pituitary tumours to evaluate the data obtained previously. cDNA was subcloned and up to ten individual clones were sequenced from each tumour, resulting in 85 clones analyzed in total. All of the pituitary adenomas showed a normal wild-type sequence of PKC-alpha DNA. Even if the tumour was 'invasive' (infiltration of the dura mater) no mutation at position 908 of the sequence was found. Moreover, using Western blot analyses we did not observe any differences in PKC-alpha protein expression in invasive as compared with noninvasive pituitary adenomas. Until now we have been unable to confirm the data of other investigators, suggesting that mutated PKC-alpha is an inconsistent feature of invasive pituitary tumours.

Upregulation of PKC delta- and downregulation of PKC alpha-mRNA and protein by phorbol ester in human T84 cells.

Protein kinase C (PKC) family members are downregulated by chronic activation at the protein level in most cell types. In T84 human epithelia] cells 12-O-tetradecanoyl phorbol 13-acetate (TPA) caused persistent translocation of PKC delta to the membrane compartment and a 400% increase of PKC delta-mRNA after 24 h. In contrast, PKC alpha protein was completely downregulated and its mRNA was decreased to 60% of control levels after 24 h. This is the first report of PKC delta-mRNA upregulation by TPA which was previously only shown for PKCbeta. In view of the antimitogenic actions of PKC delta this pattern of regulation may serve to preserve growth control even in the presence of chronic cell activation.

Activation of human platelet protein kinase C-beta 2 in vivo in response to acute hyperglycemia.

Protein kinase C (PKC) is known to be activated in experimental model systems by elevated glucose and may play an important role in the pathogenesis of diabetic complications. Since there is no information about its role in humans in vivo we investigated the activation of PKC in human thrombocytes during infusion of glucose and insulin in normal controls and in 19 NIDDM patients by determining membrane and cytosol levels of PKC beta 2 using immune blots. In the 27 subjects investigated (8 controls, 19 NIDDM) membrane-associated levels of PKC beta 2 increased significantly after 60 and 150 min (p < 0.005). In controls an increase of membrane and of cytosolic PKC beta 2 occurred upon elevation of glucose by 5.5 mmol/L or more and the membrane association persisted for at least 60 min. In NIDDM glucose was elevated by 7.5-10 mmol/L during infusions. Increases of both membrane and cytosolic PKC beta 2 (< 20%-300%) occurred in 10 NIDDM patients suggesting that both, translocation and increased synthesis of PKC beta 2 were stimulated by glucose. Nine other patients showed no alteration (i.e. < 20%) of PKC beta 2. The 2 groups were similar regarding parameters of diabetes control, baseline glucose and glucose elevation during the test. However, the PKC beta 2 responsive group had lower levels of serum triglycerides (1.39 +/- 0.19 vs. 2.32 +/- 0.34 g/L; p = 0.038). To assess whether absolute levels of PKC were altered in human diabetes, platelet levels of PKC alpha, beta 1 and beta 2 were determined in 22 controls and 25 NIDDM subjects with poorly controlled diabetes (HbA1c = 9.8 +/- 0.36%). Cytosolic levels of PKC alpha were significantly decreased by 27% compared to controls in NIDDM but there was no change of PKC beta 1 or PKC beta 2. We conclude that 1. acute elevation of glucose by 5.5 mmol/L or more can activate PKC beta 2 translocation in controls and NIDDM patients in vivo irrespective of parameters of metabolic control. 2. NIDDM patients differ in their PKC beta 2-responses to glucose and 3. poor metabolic control leads to moderate downregulation of PKC alpha suggesting continued activation.

A single base pair deletion from the inactive octamer-like motif of the 7S K distal sequence element brings full functionality in vivo.

Octamer sequence elements were analyzed for their capacity to induce the 7S K "core" promoter in vivo. The U6 distal sequence element (DSE) which contains a consensus sequence octamer, was able to support efficient 7S K expression in vivo. In contrast, no such function could be attributed to the octamer-like element alone, which is present within the 7S K DSE. However, conversion of this octamer-like element (ATTTaGCAT) to the octamer consensus sequence ATTTGCAT generated a potent DSE, even in the absence of the CACCC box, which constitutes the major functional element of the 7S K DSE. Both the consensus and the octamer-like sequences revealed no cooperativity with the CACCC box. Together, these results demonstrate that the octamer-like element of the wild-type 7S K DSE is definitely not functional in vivo. Furthermore, our experiments indicate that in contrast to the RNA polymerase II-transcribed small nuclear RNA genes, in intact cells a single functional DSE motif is necessary and sufficient for maximal transcription by RNA polymerase III of the 7S K RNA gene.