Could changes in the regulation of the PI3K/PKB/Akt signaling pathway and cell cycle be involved in astrocytic tumor pathogenesis and progression?

The most frequent alterations found in astrocytomas are two major groups of signaling proteins: the cell cycle and the growth factor-regulated signaling pathways. The aim of our study was to detect changes in expression of the following proteins: the tumor suppressors PTEN, p53, and p21Waf1/Cip1, glial fibrillary acidic protein (GFAP, as a marker of astroglial differentiation), the phosphorylated form of protein kinase B/Akt (PKB/Akt), which is downstream to the epidermal growth factor receptor (EGFR), and MDM2, which degrades p53. Paraffin-embedded astrocytoma tissue samples from 89 patients were divided into low grade (grade I-II; 42 samples) and high grade astrocytomas (grade III-IV; 47 samples). Mouse monoclonal antibodies against GFAP, PTEN, PKB/Akt phosphorylated on serine 473, EGFR, p53, p21Waf1/Cip1 and MDM2 were used, followed by standard indirect immunohistochemical method. EGFR protein was detected in 29 % of low grade and in 60 % of high grade astrocytomas. The expression of phosphorylated PKB/Akt was found in roughly the same proportions: in 86% of low grade and in 79% of high grade astrocytomas. PTEN was not found in most of astrocytomas, 64% of low grade and 74% of high grade tumors showed no PTEN staining. Overexpression of the mutated form of p53 or loss of p53 expression, however, was found in about 63% in both groups of astrocytomas with no differences between them. GFAP expression was decreased in tumor astrocytes compared to normal astrocytes and this decreased with grading. GFAP positive tumor cells were detected in only 50% of low grade, and 32% of high grade astrocytomas. The level of MDM2 expression was similar in both grades. Loss of p21Waf1/Cip1 expression was shown in 20% of low and in 45% of high grade tumors. In the subgroup of high grade tumors with wild type p53, 86% showed p21Waf1/Cip1 expression, whereas in the subgroup of high grade tumors with altered p53, only 35% displayed p21Waf1/Cip1. We conclude that EGFR expression increases with astrocytoma grading. EGFR activation may subsequently lead to stimulation of the PKB/Akt survival pathway. PTEN defects may also participate in aggressive tumor behaviour through activation of the PKB/Akt pathway. The alteration of p53 supports the finding that the cell cycle regulation is also disrupted during development of astrocytomas. The changes in PTEN and p53 expression, and activation of PKB/Akt are events in the early stages of astrocytomagenesis. EGFR is one of the factors, which drives the progression of astrocytomas from low to high grade stage.

[Immunohistochemical study of the apoptotic and proliferative mechanisms in the intestinal mucosa during coeliac disease]. / Imunohistochemická studie mechanismu apoptózy a proliferace ve sliznici tenkého streva u celiakální sprue.

Mechanisms leading to morphological changes of the small intestine during coeliac disease are not yet completely recognized, however, two main processes have been suggested recently: remodelling of mucosa by matrix metalloproteinases, and mucosal atrophy by apoptosis. The aim of this study was to analyze the expression of proteins regulating apoptosis and some markers of proliferation in the mucosa of the small intestine of children with active (ACD) and latent form (LCD) of coeliac disease (CD). Intestinal biopsies of 43 children with ACD and LCD were analyzed by standard indirect immunohistochemical technique for Fas, Fas ligand (Fas-L), tissue transglutaminase (tTG), Bcl-2, Bid, glutathione S-transferase (GST), CAS 3, CAS 8, PARP, Ki-67, Topoisomerase IIa, PCNA expression. We found significantly lower numbers of Fas-expressing enterocytes in ACD patients than in LCD patients and controls. The number of Fas-positive mucosal lymphocytes was decreased in ACD when compared with LCD. Fas-L expression in enterocytes and mucosal lymphocytes was higher in ACD and LCD compared to controls. We found significantly more Bcl-2 negative lymphocytes in ACD than in LCD and controls. Bid expression in enterocytes was higher in LCD compared to ACD and controls. In intraepithelial lymphocytes, there was higher Bid expression in LCD than in ACD and controls compared to expression in mucosal lymphocytes, where was found higher number of positive cells in controls than in ACD and LCD. Expression of CAS 8 in mucosal lymphocytes was significantly higher in ACD compared to LCD. The expression of tTG in extracellular matrix and basal lamina was significantly higher in LCD and ACD when compared to controls. Expression of tTG was higher in the group of ACD and LCD in the enterocytes and in the lymphocytes. Our findings showed that Fas/Fas-L, Bcl-2, and CAS 8 may be involved in modulation of apoptosis during CD. Increased apoptotic elimination of IEL in LCD can partially explain preservation of the normal villous architecture. Increased tTG expression may be an early sign of increased apoptosis or may be related to its role in CD pathogenesis.

Immunohistochemical detection of the hMLH1 and hMSH2 proteins in hereditary non-polyposis colon cancer and sporadic colon cancer.

Defects in DNA mismatch repair system are involved in carcinogenesis of sporadic and inherited human cancers. We assessed the feasibility of using immunohistochemistry to detect tumors with DNA mismatch repair deficiency. We analyzed 81 samples (74 colon cancers (CC), 1 colon dysplasia and 6 extracolonic cancers) for hMLH1 and hMSH2 protein expression, microsatellite instability (MSI) and/or mutational analysis. A meta-analysis of the published data on immunohistochemistry of hMLH1/hMSH2 proteins was performed. Sensitivity and specificity of the method was calculated. Twenty four of 29 tumors from hMLH1/hMSH2 mutation carriers and 10 of 13 sporadic high frequency MSI tumors lost one of the proteins. None of the 42 tumors with stable microsatellites or low frequency MSI lost the proteins. Based on literature review of 49 publications on colorectal cancer, hMLH1 immunohistochemistry was able to detect 136 of 154 tumors from hMLH1 germline mutation carriers (the sensitivity of 88.3% [95%CI, 85.8-90.8%]), hMSH2 immunohistochemistry detected 99 of 109 tumors from hMSH2 mutation carriers (the sensitivity of 90.8% [95%CI, 88.5-93.1%]), and hMLH1/hMSH2 immunohistochemistry identified 1262 of 1382 tumors with high-frequency microsatellite instability not correlated with mutational analysis (the sensitivity of 91.3% [95%CI, 90.4-92.2%]). The specificity of the method was 99.4% (95%CI, 99.2-99.6%). In conclusion, immunohistochemistry of hMLH1 and hMSH2 proteins is a useful method to predict the presence of mismatch repair deficiency, although its sensitivity is lower than that of MSI analysis.

Proto-oncogene c-myc in uterine cervix carcinogenesis.

The biological behaviour of precancerous and early stages of uterine cervix carcinoma is not always easily predictable. It is important therefore to identify new biological markers which could more reliably predict the evolution of the disease or provide important therapeutic targets. To establish the role of the proto-oncogene c-myc in uterine cervix tumorigenesis, we examined 96 tissue samples of different degrees of cervical intraepithelial neoplasia (CIN1-CIN 3), in situ (CIS) or invasive squamous cell carcinoma (ISCC) and control cases. Indirect immunohistochemical techniques were used to detect the c-myc expression. Significantly higher levels of Myc protein were found in keratinocytes of high-grade dysplasias in comparison to low-grade dysplasias and control cases. There was no difference between low-grade CIN and a control group of patients. The same significant changes between above mentioned groups were seen in surrounding stromal cells (fibrocytes, fibroblasts, some endothelial cells and lymphocytes). We confirm that expression of c-Myc protein is increased not only in uterine cervix cancer but also in the premalignant lesions. Problem for discussion seems there for whether increased Myc expression in stromal cells might create a more tumor promoting microenvironment which may support the growth and proliferation of transformed cells.

Effect of boar seminal plasma and seminal vesicle fluid on porcine lymphocytes.

Boar seminal plasma and seminal vesicle fluid inhibited mitogen-induced blastic transformation of porcine lymphocytes. Chromatographic separation of seminal vesicle fluid on Sephadex G-100 yielded four fractions. Only the SV-1 fraction displayed significant inhibition of blastic transformation. The results demonstrated that the immunosuppressive factor(s) in seminal plasma is secreted in seminal vesicles. Increased inhibition of blastic transformation of lymphocytes stimulated with pokeweed mitogen indicates that seminal plasma inhibits mainly B lymphocytes. Both seminal plasma and seminal vesicle fluid inhibited unstimulated lymphocytes.

The effect of haemagglutinating factor from boar seminal vesicle fluid on leucocytes.

Complete agglutination of porcine, bovine, ovine, and rabbit leucocytes and of porcine and bovine thrombocytes was observed after their exposure to a 1% solution of the haemagglutinating protein isolated from boar seminal fluid. Bull seminal vesicle fluid had the same agglutinating effect on leucocytes, but did not agglutinate thrombocytes. Ram seminal plasma and other fluids from the reproductive tract of boar and bull did not agglutinate either leucocytes or thrombocytes. A viability test showed that the agglutinin of boar seminal vesicle fluid, bull seminal vesicle fluid, and boar prostate fluid were all lethal for leucocytes.

First attempt to characterize 3H-fucose acrosomal label in spermatoza.

Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-N-alpha-benzoyl-L-agrinine ethyl ester splitting), akin to that exhibited by acrosin.

Isolation of basic acrosin inhibitor from bull seminal plasma (BUSI II).

An acrosin inhibitor was isolated from bull seminal plasma by gel filtration on Sephadex G-50 fine and ion-exchange chromatography on CM-Sephadex. The inhibitor is a basic polypeptide (pl greater than or equal to 10.5) of molecular weight 6 200 (calculated from amino acid composition). Its N-terminal amino group is blocked. The inhibitor is not strictly specific in its effect since it also inhibits trypsin and to a lesser degree chymotrypsin, in addition to bull and boar acrosin.

Isolation of acidic acrosin isoinhibitors (BUSI I A, BUSI IB1 and BUSI I B2) from bull seminal plasma.

Three natural proteinase isoinhibitors with low isoelectric points BUSI I A (pI = 3.9), BUSI I B1 (pI = 3.4 and BUSI I B2 (pI = 3.7) were isolated from bull seminal plasma by gel filtration on Sephadex G-50 and ion exchange chromatography on DEAE-Sephadex and SE-Sephadex. Isoinhibitors Bl and B2 have identical amino acid composition. Isoinhibitor A contains six amino acid residues less than isoinhibitors B1 and B2. Since sugars have been detected in the isoinhibitors, heterogeneity may also be due to the sugar component. The isoinhibitors show the same inhibitory properties; all of them inhibit acrosin, trypsin and chymotrypsin. Glandular kallikrein is also inhibited, but to a very low extent only. The molecular weight (Mr approximately 8 900) was determined by gel filtration.

Plasma saluretic activity: its nature and relation to oxytocin analogs.

Plasma natriuretic activity was evoked in cows and dogs by infusion of saline with or without dextran. Deproteinized samples were fractionated on both Sephadex and Bio-Gel columns; the activity was separated, the approximate molecular weight being in the region of 1000. Incubation with chymotrypsin destroyed the activity, suggesting that it might be a polypeptide. A similar activity in blood resulted from intracarotid injection of either oxytocin or either of two synthetic analogs. Possibly the latter are saluretic by virtue of a releasing action on some intracranial structure for another natriuretic peptide.