Gene expression profiles induced by growth factors in in vitro cultured osteoblasts.

OBJECTIVES: Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought. METHODS: Primary human osteoblasts in in vitro culture were treated with IGF1, BMP2 or FGF2 (10 ng/ml) for 24 hours (IGF1) or 48 hours (BMP2 and FGF2). Experiments were performed during the exponential growth phase with approximately 1e7 cells per 75 cm(2) flask. mRNA was reverse transcribed directly and analysed using RT-PCR Taqman assays. Expression levels of key genes involved in cell growth and differentiation (CDH11, TNFRSF11B, RUNX2, POSTN, ALP, WNT5A, LEF1, HSPA5, FOS, p21) were monitored using RT-PCR with gene-specific Taqman probes. RESULTS: Autocrine expression of BMP2 is stimulated by FGF2 and BMP2 itself. BMP2 and FGF2 act as proliferative factors as indicated by reduced expression of ALP and POSTN, whereas IGF1 exhibits a more subtle picture: the Wingless und Int-1 (Wnt) signalling pathway and the Smad pathway, but not p38 mitogen-activated protein (MAP) kinase signalling, were shown to be activated by IGF1, leading to proliferation and differentiation of the cells. CONCLUSIONS: For future use of autologous bone cells in the management of bony defects, new treatment options take advantage of growth factors and differentiation factors. Thus, our results might help to guide the timely application of these factors for the expansion and subsequent differentiation of osteoblastic cells in culture. Cite this article: Bone Joint Res 2014;3:236-40.

The role of memory-related gene polymorphisms, KIBRA and CLSTN2, on replicate memory assessment in the elderly.

The role of the CLSTN2 (rs6439886) and KIBRA (rs17070145) SNPs in cognitive impairment was analysed in a 75-76 years old group. Various memory assessment tests were carried out on individuals at baseline and during follow-up investigations, and biallelic genotyping was performed. No influence of the allele status of either SNPs was observed on any memory test. No increased risk of any type of late development, and cognitive impairment was associated with rs6439886 or rs17070145.

Risk factors for cerebrovascular and cardiovascular diseases beyond age 75 years.

Globally, cardiovascular diseases (CVDs) are the number one cause of all mortalities. Of these deaths, 7.6 million are due to heart attacks, and 5.7 millions are due to stroke. The Vienna Transdanube Aging Study (VITA), a population-based cohort study, enabled us to evaluate associations between the known major risk factors for cerebrovascular and CVDs and their appearance beyond age 75 years. Using a single birth cohort, age was excluded as confounding factor. In the baseline investigations in the Danube Hospital, 606 individuals took part and were examined completely at baseline. After 60 months, 508 patients were re-examined. Each participant underwent an indepth investigation with the duration of 7 h, including neuropsychological testing, as well as analyses of biochemical, clinical chemical and genetic parameters, and magnetic resonance imaging (MRI) of the brain. In the present study, only a history of cerebral and cardiovascular events at the baseline or smoking was associated significantly with the appearance of CVDs. In a multiple model both risk factors-history of cerebral and cardiovascular events at the baseline (p = 0.0003, OR 2.36, 95% CI 1.49-3.76) and smoking (p = 0.0005, OR 1.57, 95% CI 1.22-2.03)-remained significant. However, the predictive value of this assessment model was low. The rescaled r² of the model was 0.088. A significant correlation was found only between exposure to cigarette smoke or a history of previous CVDs, such as stroke or myocardial infarction. Smoking or earlier CVDs greatly increase the risk for further cerebral and cardiovascular events in persons after 75 years.

Serum protein 90K/Mac-2BP is an independent predictor of disease severity during hepatitis C virus infection.

The serum protein designated 90K/Mac-2BP has been found at elevated concentrations in the sera of patients with various types of cancer and viral infections. The importance of the 90K/Mac-2BP serum concentrations in predicting the response towards interferon-alpha treatment for hepatitis C virus (HCV) infection prompted us to utilize a new ELISA for soluble human 90K/Mac-2BP to monitor the serum concentrations of this protein in our HCV-positive patients. Seventy HCV-PCR and anti-HCV antibody positive patients were analyzed for their serum levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, cholinesterase, HCV-viral load, viral subtypes, and 90K/Mac-2BP. On correlation of age and 90K/Mac-2BP levels, we found an apparent correlation that was proved rather to be a strong dependence of 90K/Mac-2BP concentrations on disease severity/duration, which increases with age. Multiple correlation analysis demonstrated the independent nature of 90K/Mac-2BP concentrations, underscoring the potential high utility of this new marker. Our data corroborate the potential of the scavenger receptor family protein 90K/Mac-2BP as an independent predictor of disease severity during HCV infection.

[Laboratory diagnosis of hepatitis C virus infection]. / Labordiagnostik bei Hepatitis-C-Virus-Infektion.

In Austria, the prevalence of hepatitis C virus infections is 0.7% (17). Exclusion of a putative infection as well as diagnosis and continuous monitoring of HCV-disease produce considerable costs for the health system. How many and which patients with HCV infection will acquire life-threatening complications is by far not clear. Also, the causes for viral persistence and liver-complications remain obscure. For certain, complex interactions of viral and immunological mechanisms will determine the individual outcome of the disease (1). These considerations pose decisive demands on clinical diagnostics for HCV infections to be dealt with in detail: methods for qualitative detection of an infection as well as for analysis of subtypes and for quantitative determination of viral copies; monitoring of therapy; estimation of the progress of the disease and/or efficacy of therapy.

Restriction digest PCR (RD-PCR) for the analysis of gene mutations. Application to Ki-ras.

The Kirsten-ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras-mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c-erbB-2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, "enrichment PCR" must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5'primer (...GGA CCT GGT...) for an enzyme (BstNI) (5'CC!WGG 3') to differentiate between wild-type sequence (...GGA GCT GGT...) (during amplification, the G is replaced by a C) and mutated sequences (_...GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the second round altogether. The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.

The N-methyl-D-aspartate (NMDA) receptor glycine site antagonist ACEA 1021 does not produce pathological changes in rat brain.

ACEA 1021 is a potent, selective N-methyl-D-aspartate (NMDA) receptor glycine site antagonist under clinical evaluation as a neuroprotectant for stroke and head trauma. The potential of ACEA 1021 to produce morphologic changes in cerebrocortical neurons of the rat was assessed since it is known that noncompetitive (e.g., MK-801) and competitive (e.g., CGS 19755)NMDA receptor antagonists produce neuronal vacuolization and necrosis in the rat posterior cingulate/retrosplenial cortex. Male and female adult rats were treated intravenously with either vehicle (Tris) or 10 mg/kg or 50 mg/kg ACEA 1021. MK-801 (5 mg/kg, s.c.) served as positive control. Whereas MK-801 produced characteristic neuronal vacuolization and necrosis in the posterior cingulate/retrosplenial cortex, neither dose of ACEA 1021 had any effect on neuronal morphology. The absence of neuropathological changes in rats supports the further clinical evaluation of ACEA 1021 for stroke and head trauma, and suggests that glycine site antagonists may be devoid of neurotoxic potential.

Detection of common hepatitis C virus subtypes with a third-generation enzyme immunoassay.

The causal agent of most posttransfusion non-A and non-B hepatitis infections was characterized in 1989 by molecular biological techniques as a positive-stranded, enveloped RNA virus, designated hepatitis C virus (HCV). Only since 1990 has it been possible to screen for an infection with antibody tests or direct amplification assays for the nucleic acid (i.e., reverse-transcription polymerase chain reaction [PCR]). However, these nucleic acid based tests are time consuming and rather expensive. Recently, third-generation enzyme immunoassays (EIAs) for HCV infection were introduced (i.e., Abbott Laboratories, North Chicago, IL; Ortho Diagnostics, Inc., Raritan, NJ; and Roche Diagnostics, Basel, Switzerland). The Roche Diagnostics EIA has been evaluated with our patient population. To this end, 1,090 samples were assayed by both EIA and PCR; 946 of all samples (87%) were negative, and 107 samples (9.8%) were positive by both tests. Thirty of the patients (2.7%) showed antibodies but no detectable virus, whereas 7 of all patients tested (0.6%) were PCR-positive but had not yet developed antibodies to the virus. Of these 7 patients, only one showed normal serum transaminases. Virus strain subtyping and quantification of viral load on the positive samples in parallel have been performed, and the fact that the EIA detects all the virus subtypes found in our hospital can be inferred. No correlation between subtypes, viral load, and immune response could be measured with this antibody test. Our results indicate that, in most circumstances (except in settings where immunocompromised patients are abundant), this EIA can replace the much more expensive PCR tests for the routine screening for HCV infection.

Human tumor cells resistant to verapamil.

The efficacy of the calcium channel blocker verapamil for enhancing at low concentrations the cytotoxicity of unrelated antineoplastic drugs and for inhibiting at high concentrations cell proliferation has stimulated interest in the underlying mechanisms of these two diverse effects. We have selected two human brain tumor cell lines (a TE671 medulloblastoma and a A172 glioma line) for resistance against 100 uM verapamil to aid in the elucidation of the mechanism of verapamil's antiproliferative effect. Our first experiments on the selected TE671 medulloblastoma cells show that, in the presence of 100 uM verapamil, these cells grow at a rate similar to that observed for the sensitive cells in the absence of verapamil. This resistant clone continues to exhibit resistance toward verapamil for at least three days after the verapamil has been removed from the growth medium. In contrast to the sensitive cells, the resistant cells show only slight cell cycle phase alterations after removal of verapamil from the growth medium. This, together with an unchanged c-myc gene expression after removal of verapamil, indicates a stable phenotypic alteration that is responsible for the exhibited resistance toward the antiproliferative effects of the drug. Experiments designed to elucidate the mechanism of resistance showed that these cells are not cross-resistant to the antineoplastic drugs vincristine and adriamycin. Also, the resistance is not accompanied by increased amounts of the 170-180 kDa P-glycoprotein that has been implicated in resistance phenomena of cancer cells towards antineoplastic drugs.

Effect of verapamil on cell cycle transit and c-myc gene expression in normal and malignant murine cells.

Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines (Schmidt et al., Cancer Res., 48, 3617, 1988). We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action.

Antiproliferative effect of verapamil alone on brain tumor cells in vitro.

Recent studies have shown that the calcium channel blockers, when combined with standard anticancer drugs, help overcome resistance that often develops to those drugs. Little is known about the effects of the calcium channel blockers themselves on tumor cells. We have studied the effects of one calcium channel blocker, verapamil, on human tumor cell lines in vitro. Our results show a reversible, antiproliferative action of verapamil on human medulloblastoma, pinealoblastoma, glioma, and neuroblastoma tumor lines established from pediatric patients. Growth rates are inhibited 10 to 100% by 10 to 100 microM verapamil with 50% inhibition occurring between 25 and 50 microM verapamil. No cell line proliferates in 100 microM verapamil, yet washing the cells after 72 h of incubation with 100 microM verapamil results in resumed cell growth. Growth inhibition is accompanied by dose-dependent decreases in DNA, RNA, and protein synthesis which occur within minutes after addition of verapamil. DNA flow cytometry on propidium iodide-stained nuclei shows that, after incubation for 48 h with 100 microM verapamil, the medulloblastoma and neuroblastoma tumor lines as well as normal, human foreskin and lung fibroblast cell lines are reversibly blocked throughout the cell cycle with slight increases in G1. Verapamil appears to have no effect on nucleic acid precursors or on calcium influx or efflux in human medulloblastoma cells.

Uptake of glutamine antimetabolites 6-diazo-5-oxo-L-norleucine (DON) and acivicin in sensitive and resistant tumor cell lines.

The uptake system for 6-diazo-5-oxo-L-norleucine (DON) was studied in mouse P388 leukemia cells. The DON transport system was found to resemble that of another glutamine antimetabolite, Acivicin, in its strong temperature dependence, utilization of the "L" transport system, inhibition by glutamine but not by glutamate, potent inhibition by p-chloromercuribenzene sulfonate, Na+, and only minimal inhibition by various energy poisons. A Km of approximately 70 microM and a Vmax of 3.4 nmoles/10(6) cells/min was calculated for this cell line. The accumulated DON was not metabolized by P388 cells and moderate efflux occurred at 37 degrees C. The DON transport characteristics of a DON-resistant P388 cell line (100 times ID50 of parent line) were similar to those of the DON-sensitive parent line, indicating that altered drug transport may not be involved in development of resistance to this antimetabolite. The finding that an Acivicin-resistant subline of P388 cells which exhibited good transport of DON showed negligible transport of Acivicin suggests different modes of resistance towards the two glutamine antimetabolites.

Superoxide dismutase-like activities of copper(II) complexes tested in serum.

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.