Oligodendroglia from ADSL-deficient patient produce SAICAribotide and SAMP.

Succinylpurines accumulate in the body fluids of patients with adenylosuccinate lyase (ADSL) deficiency but their source in the cerebrospinal fluid remains obscure. Study based on the incorporation of 13C-stable isotope-labeled glycine into cultured oligodendroglia from ADSL-deficient patient and the measurement of labeled products by LC/MS/MS showed total intracellular concentrations of succinylpurines from 45 to 99µmol/l and so these results suggest that these cells can be the source of the compounds in vivo.

Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas.

Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low-oxygen culture conditions and in clinical specimens of both low- and high-grade tumors. The observed global checkpoint signaling, in contrast to only focal areas of overabundant p53 (indicative of p53 mutation) in grade II astrocytomas, are consistent with DDR activation being an early event in gliomagenesis, initially limiting cell proliferation (low Ki-67 index) and selecting for mutations of p53 and likely other genes that allow escape (higher Ki-67 index) from the checkpoint and facilitate tumor progression. Overall, these results support the potential role of the DDR machinery as a barrier to gliomagenesis and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.

BRAFV600E mutation in the pathogenesis of a large series of papillary thyroid carcinoma in Czech Republic.

BACKGROUND: Activating point mutation of the BRAF gene, the most common genetic alteration reported in papillary thyroid carcinomas (PTC), has been associated with poor prognostic characteristics. AIM: Our objective was to determine the frequency of BRAFV600E mutation in PTC tumor tissues from the period 1960-2007 and to correlate it with clinicopathological parameters. SUBJECTS AND METHODS: DNAs were extracted from 242 PTCs, 23 sporadic medullary carcinomas, one anaplastic carcinoma and 6 poorly differentiated carcinomas. The presence of BRAFV600E mutation was determined using single strand conformation polymorphism method and verified by direct sequencing. RESULTS: BRAFV600E mutation was detected in 81 of 242 PTCs (33.5%), in one of 6 poorly differentiated carcinomas (16.7%) and in anaplastic carcinoma. BRAFV600E mutation was much less frequent in the follicular variant compared to classical variant and mixed follicular- classical variant of PTCs (p=0.001). BRAFV600E mutation was significantly associated with presence of nodal metastasis (p=0.029), more advanced TNM stage (p=0.014) and recurrence of disease (p=0.008). The mutation correlated with a higher age at diagnosis (p=0.049) and with a greater tumor size (p=0.041). Multivariate analysis confirmed these findings. The prevalence of BRAFV600E mutation before 1986 was significantly lower than after it (p=0.008). CONCLUSIONS: Our data suggest that BRAFV600E mutation is associated with high-risk clinicopathological characteristics of PTC and worse prognosis of patients. The frequency of the mutation significantly varied during the observed period but rather because of the different age distribution of patients in particular periods than as a consequence of Chernobyl accident.

Analysis of VEGF, Flt-1, Flk-1, nestin and MMP-9 in relation to astrocytoma pathogenesis and progression.

Astrocytomas, particularly high grade astrocytoma, are brain tumors with potent angiogenic activity. Our immnunohistochemical study assessed vascular endothelial growth factor (VEGF), VEGF receptors (Flk-1, and Flt-1), the intermediate filamental protein nestin which plays a role in central nervous system development, and MMP-9, which belongs the family of matrix metalloproteinases implicated in tumor invasion and angiogenesis regulation. We investigated the expression of VEGF, its receptors, nestin and MMP-9 in astrocytomas and their correlation with tumor grade. We used paraffin-embedded samples from 66 patients, 29 with low grade (WHO-grade II) and 37 with high grade (WHO-grade III and IV) astrocytomas. Antibodies against VEGF, Flk-1, Flt1, nestin, CD34 and MMP-9 were used, followed by standard indirect immunohistochemical methods. Expression of Flt-1 and Flk-1 showed no significant differences between low and high grade tumor groups. Expression of VEGF and MMP-9 was increased in the high grade group (p equal to or less than 0.026 and 0.024). Nestin expression in tumor astrocytes and endothelial cells increased in high grade group (p same 0.007 and 0.003). Higher expression of VEGF in high grade astrocytomas may subsequently lead to activation of survival, angiogenesis and migration. Expression of nestin and MMP-9 also suggest their likely role in astrocytoma vascular development and proliferation.

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.

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine.

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21(Waf1/Cip1) and p27(Kip1) in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.

Synergic effects of the cyclin-dependent kinase (CDK) inhibitor olomoucine and androgen-antagonist bicalutamide on prostatic cancer cell lines.

Currently, mechanisms leading to both apoptosis induction and the development of hormone-independence of prostate carcinoma cells are intensively studied. Attention is also given to the possibility of restoring cell sensitivity to hormone-antagonists. The present study focuses on the effect of the combined synthetic cyclin-dependent kinase [CDK] inhibitor, olomoucine and the antiandrogen bicalutamide on hormone-insensitive (DU-145) and hormone-sensitive (LNCaP) prostate cancer cell lines. In both cell lines reduction in cell viability was significantly higher when olomoucine and bicalutamide were applied in combination when compared to separate application of both these drugs. The setting of optimal concentrations for both substances was important for the final effect on both cell lines. The proliferation arrest was accompanied by a decrease in cyclin D1 expression and the activation of p21Waf1/Cip1 and p27Kip1 pathways in both cell lines. Contrary to the previously described effect of 200 microM olomoucine, weak AR induction after treatment with effective concentrations of olomoucine was not seen in the hormone- insensitive cell line DU-145. The related reaction of DU-145 and LNCaP cell lines to treatment with combined olomoucine and bicalutamide likely provides evidence that the inhibitory effect of bicalutamide may not only be associated with its antiandrogenic properties. The tested substances probably influence different regulatory pathways and these have co-operative impact on the cell cycle outcome. Understanding antitumor and antihormone actions of both agents is essential for the development of novel therapeutic schemes integrating substances with different action. Our results show that the combination of synthetic CDK inhibitors and hormone- antagonists may be one of a number of possible alternatives.

Synthetic inhibitors of CDKs induce different responses in androgen sensitive and androgen insensitive prostatic cancer cell lines.

AIMS: Because of the high prevalence of prostatic cancer and the limitations of its treatment, enormous effort has been put into the development of new therapeutic modalities. One potential tool is the use of cyclin dependent kinase (CDK) inhibitors, which are based on the trisubstituted derivatives of purine. The aim of this study was to analyse alterations of the regulatory pathways in both androgen sensitive and androgen insensitive prostatic cancer cell lines (LNCaP and DU-145, respectively) after blockage of the cell cycle by the synthetic CDK inhibitors, olomoucine and bohemine. METHODS: The effects of olomoucine and bohemine were studied on the following parameters: (1) cell proliferation, by measurement of DNA content; (2) viability, by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and/or XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) test; and (3) the expression of p53, pRB, Bcl-2, Bax, p16, p21, p27, cyclins A, B, D1, E, p34(cdc2), and the androgen receptor (AR), by western blot analysis. RESULTS: Both olomoucine and bohemine were potent inhibitors of growth and viability; however, bohemine was two to three times more effective than olomoucine. The sensitivity of LNCaP cells to both agents was significantly higher. After treatment, both cell lines revealed quite different spectra of protein expression. CONCLUSIONS: These results indicate the existence of specific cell cycle regulating pathways in both cell lines, which may be associated with both p53 and AR status. CDK inhibitors exhibited valuable secondary effects on the expression of numerous regulators and thus may modulate the responsiveness of tumour cells to treatment, including treatment with hormone antagonists.

Nuclear receptors in early hormone refractory prostate cancer and their relationship to apoptosis-related proteins.

The expression of several genes involved in apoptosis and cell cycle control can be regulated by steroid hormones and related agents via their nuclear receptors. Members of the bcl-2 gene family participate in the regulation of apoptosis in a diverse range of cell types and are implicated in the development of hormone refractory prostate cancer and resistance to anti-cancer therapy. The aim of this study, therefore, was to examine the expression of several nuclear receptors in relation to the expression of apoptosis and cell cycle related proteins in a series of patients with early hormone refractory prostate cancer. Analysis of protein expression revealed only a weak association between Bcl-2 and AR. Bax positivity and p27Kip1 expression were significantly more frequent in the AR-positive tumors, whereas RXRbeta expression was more frequently observed in the AR-negative group. The expression of AR, Bax and p27Kip1 was inversely related, and the expression of RXRbeta directly related, to Gleason pattern status. These results suggest that the immunophenotype of early hormone refractory prostate cancer may be different to that seen in more advanced stage disease. Androgen withdrawal therapy employing anti-androgens may elicit different signalling pathways that may be dependent on ARstatus and ARsensitivity.

Androgen sensitivity related proteins in hormone-sensitive and hormone-insensitive prostate cancer cell lines treated by androgen antagonist bicalutamide.

Members of the bcl-2 gene family and endogenous inhibitors of cyclin-dependent kinases participate in the regulation of apoptosis and cell cycle in a diverse range of cell types and are implicated in the development of hormone refractory prostate cancer and resistance to anti-cancer therapy. The expression of several of these genes can be regulated by steroid hormones and related agents via their nuclear receptors. However, insufficient information considering the protein expression after the treatment by hormone antagonists is available. The aim of this study was to evaluate the expression of anti- and pro-apoptotic proteins, (Bcl-2, Bax), and to correlate this with the appearance of some nuclear receptors and cell cycle related proteins in androgen sensitive and androgen insensitive prostate cancer cell lines, LNCaP and DU-145, after the treatment by androgen antagonist bicalutamide. Our results revealed that androgen receptor (AR) expression in LNCaP cells decreased, however in DU-145 cells AR slightly increased following anti-androgen treatment. The same agent stimulated expression of p21Waf1/Cip5 and p27Kip1 in LNCaP, as well as in DU-145 cell lines. Bcl-2 level increased slightly in LNCaP cells and, in DU-145 cells was almost undetectable. Bax expression was not changed in LNCaP but significantly decreased in DU-145 cells. Similarly, retinoid X receptor beta (RXRbeta) level was significantly down regulated after 24 hours in DU-145 and also in LNCaP cells after 72 hours. These results confirm that androgen withdrawal therapy employing anti-androgens may elicit different signalling pathways in various types of prostate cancer that may be dependent on AR status and AR sensitivity.

The expression of apoptosis-related proteins and the apoptotic rate in glial tumors of the brain.

Modern molecular biology methods allow a more precise analysis of the biological characteristics of tumors and, consequently, a more precise treatment plan. The determination of apoptotic rate and expression of apoptosis-related proteins belong among the important prognostic/diagnostic markers in many tumors. The validity of these factors had not yet been sufficiently analyzed in astroglial tumors. The aim of this work was therefore to study mutual relationships between apoptotic rate, expression of apoptosis-regulating proteins and some clinical and histopathological data. The TUNEL method was used for the determination of apoptosis in 44 astroglial tumor specimens. The percentage of TUNEL positive cells was expressed by the TUNEL index (TI). The TI data was compared with the immunohistochemically detected expression of proteins involved in apoptosis (BCL-2, FAS, FAS-L, and caspase 1), with grading, age, proliferative activity (assessed by PCNA expression analysis) and overall survival of patients. The statistical evaluation of results was done by two-way sample analysis of variance. We have demonstrated significantly higher values of both TI and expression of FAS-L and caspase 1 in low grade tumors, which were characterized by a longer survival, lower average age and a lower expression of PCNA. FAS-L expression correlated significantly with the expression of the caspase 1. No significant difference was found between the expression of BCL-2 and FAS. These results suggest that the determination of TI in astroglial tumors may be an important prognostic marker. The expression of FAS-L and caspase 1 in low grade astroglial tumors could indicate the increased readiness to apoptosis via the FAS/FAS-L cascade.

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity.

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.

Differences in p53 and Bcl-2 expression in relation to cell proliferation during the development of human embryos.

AIMS: To study the patterns of p53 and Bcl-2 expression in relation to cell proliferation during human embryogenesis in order to help elucidate their potential roles in the regulation of cell proliferation and apoptosis during morphogenesis. METHODS: Immunohistochemistry for p53, Bcl-2, and proliferating cell nuclear cell antigen (PCNA) proteins was performed, using a variety of monoclonal antibodies, on paraffin was embedded sections of tissues from 68 human embryos and fetuses of between 4 and 30 weeks gestation. RESULTS: Positive relations between sites of proliferative activity (as detected by PCNA expression) and p53 expression were found in the kidney, early developmental stages of intestine and lungs, liver, pancreas, heart, and in embryonic osteoblasts. On the other hand, positive relations between proliferative activity and Bcl-2 expression were found in the gonads, adrenal glands, in the cells of the dental lamina, hair follicles, syncytiotrophoblast, chondrocytes, and more advanced stages of intestinal development. In tissues of the central nervous system, p53 and Bcl-2 were co-expressed at the same sites but there was an inverse relation between p53/Bcl-2 expression and proliferative activity. CONCLUSIONS: These data suggest that p53 and Bcl-2 have tissue specific and stage specific functions during embryogenesis.

Induction of apoptosis and regression of spontaneous dog melanoma following in vivo application of synthetic cyclin-dependent kinase inhibitor olomoucine.

This case report describes a dog with spontaneous melanoma of the orofacial region which was treated by a synthetic inhibitor of cyclin-dependent kinases, i.e. olomoucine (OC). The drug was applied i.v. in a single dose of 8 mg/kg/day for 7 days in succession. Repeated bioptic examinations of metastatic cervical lymph nodes showed rapid induction of apoptosis in tumor cells as early as on the third day of treatment. Standard clinical and laboratory examinations did not reveal side effects of the therapy. There were no detectable manifestations of myelosuppression, hepatotoxicity, nephrotoxicity or neurotoxicity. However, transient anemia developed following bleeding from a devitalized tumor mass. For this reason, the dog underwent surgery to minimize tumor load as well as to eliminate the source of bleeding. Two kilograms of primary tumor were extirpated in the course of surgery, including cervical node metastases. Unfortunately, the dog died soon after surgery due to respiratory depression. Histological examinations of the tumor tissue showed marked apoptosis of melanoma cells in both the primary tumor and metastases. The induction of programmed cell death of cancer cells by OC resulted in rapid eradication of at least 68% of the tumor cells. The remaining melanoma cells retained at least equally well in vitro sensitivity to OC as to drugs currently used in clinical practice.