Influence of Cytochrome P450, ABC and SLC Gene Polymorphisms on Imatinib Therapy Outcome of Patients with Gastrointestinal Stromal Tumours (GIST).

The efficacy of imatinib-based therapy depends on the proteins involved in its metabolism and transportation. Therefore, the aim of our study was to investigate the possible correlation of selected P450, ABC and SLC polymorphic variants and the outcome of imatinib therapy. A total of 101 patients with advanced, KIT/PDGFRA(+) GIST treated with imatinib were enrolled to the study. DNA was extracted from peripheral blood samples and genotypes were determined by PCR-RFLP and direct sequencing. Deviation from the Hardy-Weinberg equilibrium was only observed for rs2740574. None of the studied SNPs was associated with GIST time to progression. No significant correlation between any specific variant and time to progression was found in the group with KIT exon 11 mutation. However, individuals of at least three potentially unfavourable genotypes presented significantly shorter time to progression in comparison to patients with two or less unfavourable genotypes.

Aberrant DNA methylation of alternative promoter of DLC1 isoform 1 in meningiomas.

DLC1 encodes GTPase-activating protein with a well-documented tumor suppressor activity. This gene is downregulated in various tumors through aberrant promoter hypermethylation. Five different DLC1 isoforms can be transcribed from alternative promoters. Tumor-related DNA methylation of the DLC1 isoform 1 alternative promoter was identified as being hypermethylated in meningiomas in genome-wide DNA methylation profiling. We determined the methylation pattern of this region in 50 meningioma FFPE samples and sections of 6 normal meninges, with targeted bisulfite sequencing. All histopathological subtypes of meningiomas showed similar and significant increase of DNA methylation levels. High DNA methylation was associated with lack of DLC1 protein expression in meningiomas as determined by immunohistochemistry. mRNA expression levels of 5 isoforms of DLC1 transcript were measured in an additional series of meningiomas and normal meninges. The DLC1 isoform 1 was found as the most expressed in normal control tissue and was significantly downregulated in meningiomas. Transfection of KT21 meningioma cell line with shRNA targeting DLC1 isoform 1 resulted in increased activation of RHO-GTPases assessed with pull-down assay, enhanced cell migration observed in scratch assay as well as slight increase of cell metabolism determind by MTT test. Results indicate that isoform 1 represents the main pool of DLC1 protein in meninges and its downregulation in meningiomas is associated with hypermethylation of CpG dinucleotides within the corresponding promoter region. This isoform is functional GAP protein and tumor suppressor and targeting of its expression results in the increase of DLC1 related cell processes: RHO activation and cell migration.

[Multikinase inhibitors as a new approach in neovascular age-related macular degeneration (AMD) treatment: in vitro safety evaluations of axitinib, pazopanib and sorafenib for intraocular use]. / Multikinase-inhibitoren als therapeutischer ansatz bei neovaskulärer AMD: in-vitro-evaluation der sicherheit von axitinib, pazopanib und sorafenib zur intraokularen anwendung.

BACKGROUND: Multikinase inhibitors (MKI) interfere effectively at different levels of the neovascularisation cascade. Early clinical and experimental data suggest that MKIs represent a promising novel approach for the treatment of neovascular age-related macular degeneration (AMD). However, so far little is known about the biocompatibility of MKIs regarding human ocular cells. This in vitro study investigates and compares the biocompatibility of three MKIs, axitinib, pazopanib, and sorafenib regarding ocular cells of the anterior and posterior segments, as well as organ-cultured donor corneas. METHODS: Primary human optic nerve head astrocytes (ONHA), trabecular meshwork cells (TMC), and retinal pigment epithelium (RPE), human corneal endothelial and lens epithelial cells (CEC and LEC) were treated with different concentrations of axitinib, pazopanib, or sorafenib (0.1 to 100 µg/mL). To simulate oxidative stress, the cells were additionally co-incubated with 400 µM hydrogen peroxide. Induction of cell death and cellular viability were examined by live-dead assay and tetrazolium dye reduction assay (MTT). In addition, the influence of the three substances on human corneal endothelium was evaluated in seropositive donor corneas in organ culture by phase contrast microscopy. RESULTS: Up to a concentration of 7.5 mg/mL of the substances tested in any cell type examined, no toxic effects were found. Even after 10 days of incubation of organ-cultured donor corneas with 7.5 µg/mL, axitinib, pazopanib, or sorafenib, no evidence for endothelial toxicity was found. CONCLUSION: All three MKIs tested, axitinib, pazopanib, and sorafenib showed a good biocompatibility on the investigated ocular cells. Even under conditions of oxidative stress, there were no toxic effects up to a concentration of 7.5 µg/mL. Only at higher concentrations, there was a dose-dependent decrease in cellular viability and pronounced induction of cell death. These effects on cellular viability and induction of cell death appeared to be stronger with pazopanib, followed by sorafenib, than with axitinib.

Prognostic value of KIT/PDGFRA mutations in gastrointestinal stromal tumours (GIST): Polish Clinical GIST Registry experience.

BACKGROUND: Majority of gastrointestinal stromal tumours (GISTs) are characterised by KIT-immunopositivity and the presence of KIT/platelet-derived growth factor receptor alpha (PDGFRA) activating mutations. PATIENTS AND METHODS: Spectrum and frequency of KIT and PDGFRA mutations were investigated in 427 GISTs. Univariate and multivariate analysis of relapse-free survival (RFS) was conducted in relation to tumours' clinicopathologic features and genotype. RESULTS: Mutations were found in 351 (82.2%) cases, including 296 (69.3%) KIT and 55 (12.9%) PDGFRA isoforms. Univariate analysis revealed higher 5-year RFS rate in women (37.9%; P = 0.028) and in patients with gastric tumours (46.3%; P < 0.001). In addition a better 5-year RFS correlated with smaller tumour size ≤ 5 cm (62.7%; P < 0.001), tumours with mitotic index ≤ 5/50 high-power fields (60%; P < 0.001), and characterised by (very) low/moderate risk (70.2%; P = 0.006). Patients with GISTs bearing deletions encompassing KIT codons 557/558 had worse 5-year RFS rate (23.8%) than those with any other KIT exon 11 mutations (41.8%; P < 0.001) or deletions not involving codons 557/558 (33.3%; P = 0.007). Better 5-year RFS characterised patients with KIT exon 11 point mutations (50.7%) or duplications (40%). By multivariate analysis, tumours with PDGFRA mutations and KIT exon 11 point mutations/other than 557/558 deletions had lower risk of progression than with KIT exon 11 557/558 deletions (both Ps = 0.001). CONCLUSIONS: KIT/PDGFRA mutational status has prognostic significance for patients' outcome and may help in management of patients with GISTs.

[Proliferative vitreoretinopathy (PVR) surgery: Scar Wars : Five steps to successful treatment of PVR detachment]. / Proliferative Vitreoretinopathie(PVR)-Chirurgie: "Scar Wars" : Fünf Schritte zur erfolgreichen Therapie der PVR-Amotio.

BACKGROUND: After initially successful surgery of retinal detachment, proliferative vitreoretinopathy (PVR) is the most common cause of renewed retinal detachment. With an incidence of 5-20% it represents a frequent surgical challenge based on a pronounced epiretinal, subretinal and intraretinal scar formation. MATERIAL AND METHODS: The five most important steps leading to a successful repair of a PVR retinal detachment are described. RESULTS: 1. The basic prerequisite is the complete removal of the vitreous body in order to remove the substrate for proliferation of pathological cells. 2. Furthermore, the complete removal of all tractional PVR membranes is necessary. Subretinal PVR membranes that show no traction can be left in place. 3. The professional care of the macular is still important. As approximately 12% of all patients who undergo surgery for retinal detachment develop an epiretinal gliosis/macular pucker, peeling of the internal limiting membrane (ILM) is obligatory in cases of PVR. 4. Particularly in PVR detachment the mentioned surgical procedure is facilitated by the selection of suitable modern instruments, including wide-angle optics, such as the binocular indirect ophthalmomicroscope (BIOM), chandelier lights, perfluorocarbons (PFCL) and silicone oil. 5. Last but not least, the credo as much as necessary, as little as possible is of essential importance, as PVR eyes have usually been previously operated on and any further surgical intervention leads to subsequent inflammation and a persisting stimulation of the PVR reaction and further damage. CONCLUSION: Following a few decisive rules and tips is a prerequisite for a successful reattachment in cases of PVR retinal detachment.

Presence of t(14;18) positive cells in blood and bone marrow does not predict outcome in follicular lymphoma.

Follicular Lymphoma International Prognostic Index-FLIPI is an established clinical predictor for outcome in follicular lymphoma. The role of molecular abnormalities in blood and bone marrow of follicular lymphoma patients including t(14;18) is less clear. Seventy-five patients from a single institution diagnosed with follicular lymphoma between1999 and 2005 were included into the study. Diagnosis was based on lymph node biopsy in 62 cases (83%). Thirty-nine patients (52%) had G1 histological grade and 47 (63%) had entirely follicular growth pattern, as well as 9 patients (12%) had systemic symptoms and 33 (44%) were assigned to a good risk according to FLIPI. Median age of patients was 53 years. During a median observation time of 3 years 63 patients (84%) required initiating anti-lymphoma treatment. Seventy-five samples of peripheral blood and 65 samples of bone marrow were collected at the diagnosis. Bcl2 rearrangements including major breakpoint region and minor breakpoint cluster region were investigated using nested polymerase chain reaction technique. The primary end points of the study were time to first line lymphoma treatment and progression-free survival. Cells carrying t(14;18) were found in 31 cases (41%) including 29 samples of peripheral blood and 26 samples of bone marrow. Detection of t(14;18) in blood and bone marrow at diagnosis had no influence on clinical outcome. Age, follicular growth pattern systemic symptoms, and FLIPI score above 1 were predictive for initiation of the first lymphoma therapy. Follicular growth pattern, initial nodal involvement, serum LDH level, and FLIPI score above 1 were predictive for longer progression-free survival.

Molecular and biochemical testing in stage III melanoma: multimarker reverse transcriptase-polymerase chain reaction assay of lymph fluid after lymph node dissection and preoperative serum lactate dehydrogenase level.

BACKGROUND: There is a need for biomarkers to identify patients at risk for disease progression after resection of melanoma regional lymph node metastasis. OBJECTIVES: This study assessed the prognostic value of multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) assay in lymphatic drainage (LY) after lymph node dissection (LND) and of preoperative serum lactate dehydrogenase (LDH) levels in American Joint Committee on Cancer (AJCC) stage III melanoma patients. METHODS: We collected 24-h LY from 255 stage III melanoma patients after radical LND [114, completion LND after positive sentinel node biopsy (CLND); 141, therapeutic LND for clinically/cytologically detected regional nodal metastases (TLND)]. For detection of melanoma cells, RT-PCR assays with primers specific for tyrosinase, MART1 (MelanA) and uMAGE mRNA were conducted. The LY sample was deemed positive if at least one marker was detected. In 244 patients, the preoperative serum LDH level was available. Median follow-up time was 25 months (range 5-60). RESULTS: The LY multimarker RT-PCR assay results were positive in 82 of 255 patients (32%). A significantly higher rate of melanoma recurrence was observed in patients with positive LY multimarker RT-PCR results (P = 0.01). Significant relationships were observed between positive LY multimarker RT-PCR results and shorter 3-year overall survival (OS) and disease-free survival (DFS), both in univariate and multivariate analyses (P = 0.007). Preoperative serum LDH level was increased in 79 of 244 patients (32%) [40.5% in TLND group and 23.0% in CLND group (P = 0.003)]. There were significant differences in OS between patients with normal and high preoperative LDH levels (P = 0.007), and these differences were seen mainly in patients in the TLND group. CONCLUSIONS: The multimarker RT-PCR assay detected melanoma cells in approximately 32% of LY after LND, which correlated significantly with early melanoma recurrence and shorter survival. Increased pre-LND serum LDH level had an additional negative impact on OS of melanoma patients with palpable nodal metastases after TLND.

[Histopathological correlation of epiretinal tissue in lamellar macular holes and macular pseudoholes]. / Histopathologische Korrelation epiretinalen Gewebes bei Schichtforamen und Pseudoforamen der Makula.

BACKGROUND: Using high-resolution spectral domain optical coherence tomography (SD-OCT), morphologically different types of epiretinal tissue can be distinguished in lamellar macular holes (LMH) and macular pseudoholes (MPH). OBJECTIVE: This article presents the results of histopathological characterization and differentiation of epiretinal tissue in eyes with LMH and MPH, which are classified based on a morphological differentiation in SD-OCT. MATERIAL AND METHODS: This review is based on the currently available literature and own data analyses. Using SD-OCT, a differentiation into hyporeflective epiretinal tissue and contractile epiretinal membranes (ERM) was performed. For fluorescence and transmission electron microscopic analyses, epiretinal tissue harvested by pars plana vitrectomy and peeling of epiretinal tissue was processed. RESULTS: By SD-OCT hyporeflective tissue appears as a thick homogeneous layer of hypodense material located directly on the surface of the inner retina and has no visible signs of traction. Using immunocytochemistry, hyalocytes and glial cells showing no contractile activity are dominant; however, in contractile ERM in MPH, anti-alpha SMA-positive myofibroblasts are predominantly found representing the contractile element. CONCLUSION: The results of ultrastructual investigations demonstrate that morphological cell components of hyporeflective epiretinal tissue from LMH have less contractile properties than cells of contractile ERM. It can therefore be assumed that there are differences in the pathogenesis of epiretinal cell proliferation in LMH. Histopathological investigations support the hypothesis that hyporeflective epiretinal tissue represents modified material from the outer layer of the vitreous body.

Inhibition of calf thymus DNA polymerase alpha and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine.

6-(p-n-Butylanilino)uracil and N2-(p-butylphenyl)guanine inhibited the activity of DNA polymerase alpha from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases beta and gamma) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5'-triphosphate and did not occur in the reaction of DNA polymerase alpha with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases alpha.

Levels and size complexity of DNA polymerase beta mRNA in rat regenerating liver and other organs.

A cDNA probe encoding DNA polymerase beta (beta-pol) was used to study the level and size complexity of beta-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in beta-pol mRNA was observed 18-24 h after partial hepatectomy. In most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of beta-pol mRNA was low. In contrast, young brain and testes exhibited beta-pol mRNA levels 5- and 15-times higher, respectively. The observed changes in the level of beta-pol mRNA in regenerating rat liver and in developing brain are correlated with reported changes in DNA polymerase beta activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to beta-pol probe were found in all tissues examined. The 4.0 kb transcript was dominant for young and adult brain, whereas the 1.4 kb transcript was dominant for testes. The significance of these transcripts is discussed.

Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta.

Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.

Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases.

Hypoxanthine¿xanthine oxidase¿Fe3+¿ethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA. The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol. Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands. When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced. This suggests that FapyAde, when present in DNA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase. FapyGua might possess similar properties.

Proto-oncogene c-fos induction in rat hippocampus.

The apparently non-specific accumulation of c-fos proto-oncogene mRNA was found in rat hippocampus as a result of injection of either glutamate, noradrenaline, or physiological saline. There was no c-fos gene induction following an electroconvulsive shock.

Purification and properties of DNA polymerase gamma from rabbit intestinal epithelial cells.

1. DNA polymerase gamma from the cytoplasmic fraction of rabbit intestinal epithelial cells has been purified 120 000-fold and was free of phosphatase and nuclease activities towards deoxyribonucleoside-5'-triphosphates and polynucleotides. 2. The enzyme exhibited maximal activity for activated DNA and poly(A) . oligo(dT)12--18 at pH 8.5 IN 0.25 AND 0.15 M-KCl, respectively. Km values for dTTP with these two templates were 0.5 and 3.8 microM, respectively. 3. In contrast to DNA polymerases alpha and beta, the enzyme replicated poly(A) . oligo(dT)12--18 10 times faster and poly(dA) . oligo(dT)12--18 5 times slower than activated DNA. 4. DNA polymerase gamma did not replicate poly(C) . oligo(dG)12--18 or poly(Cm) . oligo(dT)12--18. The reaction with poly(I) and poly(U) did not exceed 1% of that observed with poly(A). 5. The enzyme was inhibited in 60% by antiserum against DNA polymerase gamma from human lymphoblasts. 6. The nuclear fraction of rabbit intestinal epithelial cells contained DNA polymerase gamma with the same characteristics.

Role of Mn2+ in the reaction of polynucleotide phosphorylase with 2'-O-methylated substrates.

Polymerization of 2'-O-methylcytidine-5'-diphosphate (CmDP) with polynucleotide phosphorylase in the presence of Mn2+ proceeds with 65% yield after 72 h, and in the presence of Mg2+ the yield does not exceed 10%. Phosphorolysis of poly 2'-O-methylcytidylic acid and poly 2'-O-methyluridylic acid, as well as exchange of the beta-phosphate group of CmDP in the presence of Mn2+ and Mg2+, proceed with a yield of only a few percent. A possible mechanism of Mn2+ action on CmDP polymerization is discussed.

The ability of DNA polymerase beta to synthesize DNA beyond the gap with displacement of the non-replicated strand.

Study was made of the ability of calf thymus DNA polymerases alpha and beta to replicate templates containing a small gap. It was found that during extensive replication of activated DNA or synthetic template.primers or specially prepared circular DNA containing a small gap, catalyzed by DNA polymerase alpha, the levels of incorporated nucleotides corresponded to the amounts of the single-stranded fraction of these templates. In contrast, in the reaction catalyzed by DNA polymerase beta the amounts of products were several times greater. The ability to synthesize the product in a great excess was a specific feature of the latter enzyme. An analysis of the gap-filling products by sucrose gradient centrifugation, gel electrophoresis and Southern hybridization showed that, contrary to DNA polymerase alpha, DNA polymerase beta exhibited the ability to synthesize DNA not only within but also beyond the gap. The "net" DNA product is complementary to the template strand. It is suggested that DNA was synthesized beyond the gap by displacement of the non-replicated strand.

Cloning and characterization of the 5' part of the high molecular weight transcript of rat DNA polymerase beta.

A rat brain cDNA library has been constructed. Three identical clones of about 2.2 kb representing high molecular weight DNA polymerase beta transcript were found. Sequencing proved our earlier suggestion that both high and low molecular weight DNA polymerase beta transcripts have the same open reading frame and differ mainly at the 3' end. Because of that, alternative polyadenylation is discussed as a possible mechanism for tissue- and development-specific regulation of the DNA polymerase beta gene expression.

DNA polymerase gamma of rabbit intestinal epithelial cells.

1. DNA-directed DNA polymerase gamma was isolated from epithelial cells of the rabbit small intestine, and characterized. 2. The molecular weight of the enzyme, determined by Sephadex G-200 filtration was 105 000 +/- 15%. 3. The enzyme showed preference for poly(A) replication on poly(A)-poly(dT) or poly(A)-oligo(dT) 12-18 templates, as compared with activated DNA. Poly(C)-oligo(dG)12-18 replication was not observed. 4. The Michaelis constants for dTTP in replication of activated DNA and poly(A)-oligo(dT)12-18 were 3.3 and 2.0 micron, respectively.

DNA polymerases of murine LBN/b leukemic cells.

In the post-mitochondrial fraction of murine LBN/b leukemic cells, four fractions with DNA polymerase activity (I, II, III, IV) were found. On the basis of ion exchanger affinity and poly(A), poly(C) and poly(Cm) replication ability, fraction I was classified as RNA-directed DNA polymerase of viral origin. On the basis of the differences in the ion exchanger affinity, molecular weight, template requirement, pH-dependence of enzymatic activity and NaCl concentration, divalent ion requirements and susceptibility to N-ethylmaleimide inhibition, fractions II, III and IV were classified as DNA-directed DNA polymerases beta, alpha and gamma, respectively. Three fractions, i.e. reverse transcriptase, and DNA-directed DNA polymerases beta and gamma, were found to incorporate dTMP on a poly(A)-oligo(dT) template-primer. Despite the similarity of the reaction of DNA polymerases beta and gamma with poly(A)-oligo(dT), some other properties of these enzymes suggest that they represent distinct enzymatic entities.

Net DNA synthesis catalysed by calf thymus DNA polymerase beta.

1. The amounts of deoxynucleotides incorporated during an extensive replication by DNA polymerase alpha into poly(dA)-oligo(dT)12-18 and damaged DNA containing 2.5 incisions per molecule and 3.7% single-stranded DNA, corresponded to the amounts of non-complexed poly(dA) and single-stranded fraction of DNA, respectively. The amounts of the corresponding DNA polymerase beta products were several times higher. In the case of activated DNA they exceeded input DNA. The DNA polymerase beta reaction on this template was continued till substrate exhaustion. 2. The reaction of DNA polymerase beta with activated DNA, leading to net DNA synthesis, was template-directed, required Mg2+ and four deoxynucleoside triphosphates; was not inhibited by DNA polymerase alpha inhibitors, but was sensitive to 2',3'-dideoxythymidine triphosphate. The DNA product was completely digestable by DNAse I and showed a base ratio typical of calf thymus DNA. 3. The essential difference in the reaction mechanism between DNA polymerase alpha and beta suggests the ability of the latter enzyme to synthesize DNA with displacement of the non-replicated strand.