Biomedical subjects

M Bache

Publications and source records attributed to M Bache.

32 records · Page 2Linked to original sources

PubMed1998

NT-3 attenuates functional and structural disorders in sensory nerves of galactose-fed rats.

The present study investigated the effect of NT-3, a neurotrophin expressed in nerve and skeletal muscle, on myelinated fiber disorders of galactose-fed rats. Adult, female Sprague-Dawley rats were fed diets containing complete micronutrient supplements and either 0% D-galactose (control) or 40% D-galactose. Treated controls received 20 mg/kg NT-3 and treated galactose-fed rats received 1, 5, or 20 mg/kg NT-3 three times per week by subcutaneous injections. After 2 months, sciatic and saphenous sensory nerve conduction velocity (SNCV) and sciatic motor nerve conduction velocity (MNCV) were measured and the sciatic, sural, peroneal and saphenous nerves and dorsal and ventral roots processed for light microscopy. Treatment of control animals with NT-3 had no effect on any functional or structural parameter. Compared to control values, galactose feeding induced a sensory and motor nerve conduction deficit and a reduction in axonal caliber. Treatment with 5 and 20 mg/kg NT-3 ameliorated deficits in sciatic and saphenous SNCV in galactose-fed rats but had no effect on the MNCV deficit. NT-3 treatment also attenuated the decrease in mean axonal caliber in the dorsal root and sural nerve but not in the saphenous nerve, ventral root and peroneal nerve. These observations show that NT-3 can selectively attenuate the sensory conduction deficit of galactose neuropathy in a dose-dependent manner that depends only in part on restoration of axonal caliber of large-fiber sensory neurons.

Animals↗

PubMed1998

Detection of numerical chromosomal changes in 20 malignant fibrous histiocytomas by FISH.

We investigated 20 malignant fibrous histiocytomas (MFHs) with the help of specific centromeric probes for chromosomes 1, 3, 4, 6, 8, 9, 12, 16, 17 and 18. The results show a broad variation in the number of signals per nucleus. However, tumors can be assigned into four groups: i) with mostly disomic clones, ii) with a high percentage of polysomic clones, iii) with a considerable amount of monosomic and nullisomic clones and iv) with a tendency in both directions. A gain of spots per nucleus takes place in 75-100% of the investigated tumors - the highest incidence occurring with respect to chromosome 3. A loss of spots per nucleus occurred in 20-60% of the tumors - predominantly with respect to chromosome 1.

Aged↗

PubMed1998

Molecular characterization and liposomal transfection of a p53-mutated cell line established from a poorly differentiated leiomyosarcoma.

A human cell line LMS6-93 has been established from a leiomyosarcoma (LMS). Characteristics for ultrastructure, growth characteristics, cell cycle distribution, karyotype, protein expression detected by immunohistochemistry (IHC), p53 mutational status and liposomal transfection behaviour were studied and determined. The primary tumor was clearly positive for á-smooth muscle type actin and desmin in moderately differentiated areas and indicated a loss of myogenic differentiation in other regions and therefore was classified as a poorly differentiated LMS. The cell line LMS6-93 contains mainly polymorphic spindle shaped or polygonal tumor cells which possess the characteristics of primitive mesenchymal cells, based on their morphology and positive reaction with an antibody to vimentin. IHC staining for S100, synaptophysin A, NSE, neurofilament proteins and cytokeratins were negative. Cytogenetic analysis revealed in the cell line diploid karyotypes comparatively close to several structural and numerical aberrations for chromosomes 2, 5, 6, 9, 10, 12, 14, 17, 18, 20, 22, and Y. IHC positivity was found for the tumor suppressor protein Rb and the oncogene product MDM2. In a p53 mutational analysis a 1 bp insertional mutation in exon 6 (G insertion in codon 215) was detected and confirmed in the original primary tumor. The other p53 allele appears to be wild-type as indicated in Western hybridization. Using different cationic lipid formulations complexed with a reporter expression vector (GFP) successful transfection into LMS6-93 cells was observed. The highest transfection rates (20-30% GFP expression in the viable cell population) were obtained with lipofectin. These results suggest that LMS6-93 functions as a good in vitro model for transfection studies on an LMS cell line carrying a heterozygous p53-frameshift mutation.

Blotting, Western↗

PubMed1998

[Liposomal DNA transfection of human sarcoma cells with p53 alterations].

An vital assay allows to optimize liposomal transfection for human tumor cells via FACS. Various cationic lipids were tested to analyse the reporter gene expression (green fluorescent protein, GFP) in different soft tissue sarcoma (STS) cells with known genetic alterations. Furthermore, the cellular uptake of fluorescence-labeled oligodeoxynucleotides (ODN's) was determined. The results obtained with two self-established sarcoma cell lines (LMS6-93, US8-93) were compared with ATCC sarcoma cell lines (Saos-2, A-204, RD) and fibroblast cells. We found maximal 37% cells expressing GFP 24 h post-transfection. All mesenchymal (tumor) cells but not fibroblast cells could be transfected in a cell-specific and lipid-dependent manner. In kinetic studies highest transfection rates were determined between 24 and 48 h, whereas the GFP expression is downregulated after 72 h. Furthermore, we found transfectability is p53 mutation-independent and a relative low toxicity of the new lipids (Lipotaxi and Clonfectin) in comparison to other lipids (Lipofectin, Lipofectamine). By a cell sorting system sarcoma cell lines expressing the reporter gene could be enriched up to 84% of the living cell population. Labeled ODN's were taken up more efficiently (> 90%) when they were mixed with lipids before, but ODN's alone were incorporated into sarcoma cells only in a low percentage (< 10%) and concentration. STS cell cultures showed also a relative high ODN uptake compared with cell lines. We propose the liposomal transfection strategy as an efficient method which can be applied to adherent-growing tumor cells. The method allows simultaneously to study transfection rates, apoptosis and cell cycle alterations in vitro. Furthermore, in future, extension on ex vivo and in vivo transgene expression (xenotransplanted sarcomas) will be evaluated.

Gene Expression Regulation, Neoplastic↗

PubMed1998

[Frequency, distribution and prognostic relevance of p53 mutations in soft tissue sarcomas].

Soft tissue sarcomas although rarely occurring (about 1% of malignant tumors), are because of their histo-morphological diversity and often similar appearance to tumor-like lesions difficult to characterize and estimate in their tumor biological behaviour. Analysis of molecular characteristics as alterations in tumor-suppressor and oncogenes may allow insight in STS genesis. We have chosen the in carcinomas well, but in STS not comprehensively investigated tumor-suppressor gene p53 for mutational analysis. In 16 out of 146 STS patients we could identify p53-mutations. In a multivariate Cox-regression analysis prognosis was correlated with the p53-mutation type. However, only patients with non-frameshift mutations possessed a poorer prognosis (RR = 2.42; p = 0.014) in comparison to patients without mutations, but frameshift-mutations didn't seem to affect prognosis negatively. Compiling our results and those of the literature an overall frequency of 16.3% of p53-mutations in STS, with various frequencies in different entities is detectable. STS specific hotspots are not recognizable. Rather mutational hotspots in codons 175, 245, 248 and 273 well known from studies in carcinomas are also apparent in STS. Summarizing, we want to state that the occurrence of p53-mutations (non-frameshift mutations) is of prognostic importance in STS. Combination of histo-pathological, clinical and molecular characteristics may allow to distinguish in future different groups of patients for an individual treatment.

Codon↗

PubMed1997

BDNF attenuates functional and structural disorders in nerves of galactose-fed rats.

Galactose intoxication of rats was used to disrupt metabolism of Schwann cells and skeletal muscle, two sites that contain the polyol-forming enzyme aldose reductase (AR). Galactose-fed rats develop a neuropathy characterized by nerve conduction deficits and axonal atrophy. To investigate the possibility that galactose metabolism by AR influences axonal function and structure by altering production of neurotrophic factors, the impact of galactose intoxication on nerve and muscle BDNF levels and the effects of exogenous BDNF treatment on galactose neuropathy were examined using biochemical, electrophysiologic and morphometric techniques. Galactose feeding increased BDNF protein in peripheral nerve and muscle. Exogenous BDNF treatment attenuated motor nerve conduction velocity deficits in the sciatic nerve of galactose-fed animals and myelin splitting of motor axons in the ventral root. In contrast, sensory nerve conduction velocity (SNCV) deficits in the sciatic nerve and myelin splitting in the central projections of sensory neurons were not prevented by BDNF treatment. BDNF treatment did not attenuate reduced axonal caliber in the sciatic nerve, but did ameliorate the diminution of the caliber of central sensory projections in the dorsal root. These findings point to the potential use of BDNF in the treatment of peripheral neuropathies.

Aldehyde Reductase↗

PubMed1997

Prognostic relevance of C-terminal Mdm2 detection is enhanced by p53 positivity in soft tissue sarcomas.

We examined the clinical value of immunohistochemical (IHC) Mdm2 detection by an N-terminal (IF2) and a C-terminal (19E3) binding monoclonal antibody (Ab) in soft tissue sarcomas (STSs) with regard to the p53 status. Therefore, we investigated a cohort of 198 patients with STSs of six entities with known p53 IHC by using a multivariate Cox regression model to determine the prognostic value of Mdm2 staining. Only positivity with the 19E3 Ab correlated multivariately significantly with survival (RR = 2.32, p = 0.0035). We stratified the C-terminal Mdm2 staining (19E3) according to p53 IHC (DO-1) and found patients could be divided into three groups with an increasing risk: (a) patients with Mdm2 (19E3)-negative as well as p53 (DO-1)-negative tumors, (b) patients with tumors that were either Mdm2 (19E3) or p53 (DO-1) positive, and (c) patients with tumors that were Mdm2 (19E3) as well as p53 (DO-1) positive. Positive staining for both Mdm2 and p53 meant a very poor prognosis with a relative risk of 4.63 (p = 0.00001). This points to the possibility that--in addition to the p53-dependent pathway--Mdm2 could have an effect through a p53-independent pathway. Thus, our results indicate that C-terminal Mdm2 staining (19E3) constitutes an independent prognostic marker in STS.

Adolescent↗

PubMed1996

Frequent occurrence of p53 mutations in rhabdomyosarcoma and leiomyosarcoma, but not in fibrosarcoma and malignant neural tumors.

We have analyzed soft-tissue sarcomas (STS) molecularly for mutations in the tumor-suppressor gene p53 and immunohisto-chemically for expression of p53 and mdm2 proteins. In this study, tumor samples from 3 groups of soft-tissue sarcomas, i.e., fibrosarcomas, myogenic sarcomas and malignant neural tumors (MNT), were investigated. The methods applied encompass immunohistochemistry on 198 tumor samples using p53 antibodies (DO-1 and DO-7) and an mdm2 antibody (IF-2). Out of these, 100 samples were subjected to non-radioactive PCR-SSCP-sequencing analysis. Immunohistochemical detection rate for p53 (range of 57% to 67%) and for mdm2 proteins (range of 19 to 44%) was similar in all 3 groups. In higher tumor grades, an increased rate of immunopositivity was found for p53 but not for mdm2. Investigation of p53 mutational status revealed 6 mutations in myogenic sarcomas but none in malignant neural tumors or fibrosarcomas, suggesting different roles of p53 in the 3 STS groups. Interestingly, a G-->A transition in codon 245 (a CpG site) was found in 3 myogenic sarcomas. Our results and those of others suggest p53 codon 245 as a mutational hotspot in sarcomas, as recognized in carcinomas.

Base Sequence↗

PubMed1994

Inhibition of macrophage chemotaxis and peripheral nerve regeneration in normal and hyperglycemic rats by the aldose reductase inhibitor Tolrestat.

This study examined the effect of Tolrestat, an inhibitor of aldose reductase, on the regenerative capacity and macrophage chemotactic property of crush-injured sciatic nerve in normal and galactose-fed rats. Galactose intoxication reduced the incidence of regeneration but did not alter the regeneration distance or the injury-induced increase in vasoactive intestinal polypeptide content of dorsal root ganglia. Tolrestat improved the incidence of regeneration in galactose-fed rats but significantly (P < 0.05) reduced the distance of nerve regeneration in both control and galactose-fed rats. Galactose intoxication enhanced the ability of homogenates of nerve undergoing Wallerian degeneration to attract macrophages, whereas chemotaxis toward nerve homogenates from Tolrestat-treated rats was absent. Tolrestat, but not the structurally dissimilar aldose reductase inhibitors Ponalrestat and Sorbinil, exhibited a reversible, dose-dependent inhibition of macrophage chemotaxis induced by polyinosinic acid. These data suggest that exaggerated sugar metabolism by aldose reductase may restrict the ability of nerve to initiate regeneration but is not responsible for the reduced distance of nerve regeneration or attenuated increase in vasoactive intestinal polypeptide production that occur after crush injury of diabetic rats. Inhibition of macrophage responses to chemotactic signals by Tolrestat may impede regeneration and other reparative mechanisms.

Aldehyde Reductase↗

PubMedDate not supplied

Radiosensitization in sarcoma cell lines with a p53 missense mutation correlates with prevention of irradiation G2/M arrest but not with induction of apoptosis.

We analysed the effects of caffeine and taxol on the radiobiological behaviour of two human sarcoma cell lines (RD, SK-LMS-1) each with a p53 missense mutation. Treatment with 2 mM caffeine resulted in an inhibition of the irradiation induced G2/M arrest in both cell lines. This effect was coupled with a radiosensitization in cell line SK-LMS-1 after an irradiation with 6 Gy (enhancement factor of 5.0). However, the effect of radiosensitization was not correlated with an induction of apoptosis. Incubation with 20 nM taxol increased the irradiation induced apoptosis almost 3-fold in cell line SK-LMS-1, but not in cell line RD. However, taxol had no effect on the irradiation induced G2/M arrest or radiosensitivity in either cell line. The results support the hypothesis that the prevention of irradiation induced G2/M arrest but not the induction of apoptosis plays a critical role in determining radiosensitivity in sarcoma cell lines with p53 mutations.

Apoptosis↗

PubMedDate not supplied

G2/M checkpoint is p53-dependent and independent after irradiation in five human sarcoma cell lines.

To determine the role of p53 in G2/M arrest, G2/M transition and apoptosis, we investigated five human sarcoma cell lines with different p53 gene status in their response to X-rays. The p53 status of the cell lines was mutant (US 8-93, LMS 6-93 and RD), null (SAOS-2) and wildtype (A-204). Clonogenic survival of the cell lines varied as the survival fraction at 2 Gy (SF2) ranged from 0.28 to 0.79. Compared with the mutated p53 cell lines (SF2 with a range from 0.46 to 0.79) the clonogenic survival of the wildtype p53 (wt-p53) cell line A-204 (SF2 = 0.34) was lower. The p53 null cell line (SAOS-2) was also sensitive to X-rays (SF2 = 0.28). We detected, in all cell lines a similar behavior in their response to irradiation with G2/M arrest and apoptosis. However, the maximal rate of apoptosis with a range from 7.0 to 18.0% was rather small. The decrease of G2/M cells was coupled with an increased percentage of apoptotic cells. However, a different delay in G2/M did not result in a change of radiation sensitivity. Western analyses showed an increased P53 level only for the cell line A-204 (wt-p53) after irradiation. Our results point out that there is not always a simple relationship between p53 gene status and radiation sensitivity. We suggest, that wt-p53 plays an active role in G2/M arrest and in decreasing the number of G2/M cells as a response to apoptosis. Therefore, p53-dependent regulation in G2/M may be as important as p53-independent mechanisms.

Apoptosis↗

PubMedDate not supplied

A multifactorial prognostic model for adult soft tissue sarcoma considering clinical, histopathological and molecular data.

Soft tissue sarcomas (STS) are malignant mesenchymal lesions with a high degree of prognostic variability. Different prognostic markers such as grading, staging, tumour type and localisation are known. The establishment of these markers was based on the evaluation at results of extensive cohorts of patients. Therefore, only the established markers provide us with information about probabilities in relation to other qualities. Considering as many different markers as possible in one prognostic statement should increase the value of the resultant information. Therefore, we developed a model involving known prognostic markers to formulate an individual prognostic index. In a retrospective analysis, different prognostic factors of 198 adult STS patients with histological tumour free resection margins were evaluated using a multifactorial analysis. On the basis of a Cox-Regression-Model with proportional hazards, the prognostic factors (tumour type, staging, localisation and type of surgical resection) were selected using previous knowledge and a statistical step backward selection procedure adjusting the immunohistochemical status of p53/Mdm2 expression. On the basis of the baseline survival function of our cohort (S0 (t)), the cumulative probability of survival for two S (2) and five S (5) years was estimated. As a result of our analysis the equations S (2) = (e-00393)P and S (5) = (e-00869)P can be used to estimate the individual two and five-year probability of survival in our cohort. Here p is the result of the amount of the estimated regression-coefficients of the exact variables of the respective individual patient. This model makes it possible to include all the evaluated prognostic factors which, in turn, increases the accuracy of the prognostic information for individual patients underlining the proportional hazards assumption.

Abdominal Neoplasms↗

PubMedDate not supplied

Transfection with mdm2-antisense or wtp53 results in radiosensitization and an increased apoptosis of a soft tissue sarcoma cell line.

Soft tissue sarcomas (STS) are mostly resistant after radiation treatment and are characterized by a rather low rate of apoptosis. The aim of this study was to test, in the p53 mutant STS cell line US8-93, the effect of a combined treatment with DNA transfection--either with mdm2 antisense oligodesoxynucleotides (mdm2-AS) or with a wild-type p53-plasmid (wtp53)--and the effects of irradiation on radiosensitivity. Mdm2-sense oligodesoxynucleotides (mdm2-SE) and a GFP-plasmid (GFP) were applied as controls. In order to evaluate the treatment radiation sensitization (clonogenic survival), apoptotic cell death and P53/MDM2-protein expression were determined. A moderately increased radiation sensitization was observed when comparing clonogenic survival after 2 Gy irradiation between cells transfected either with the control mdm2-SE (48%) or with mdm-2 AS (30%). At the same irradiation dose, clonogenic survival of wtp53-plasmid transfected cells (32%) was about 2-fold less than in the cells transfected with the control GFP-plasmid (61%). This enhancement factor of radiation sensitization was increased by about 3-fold at 4 Gy irradiation. Furthermore, an increase in apoptotic cells was already detectable by up to 7.7% (mdm2-AS) in comparison to 3.1% (mdm2-SE control) 72 hours after transfection. In parallel, the percentage of apoptotic cells could be further elevated after subsequent irradiation with 12 Gy by up to 15% (mdm2-AS) compared to 5.7% (mdm2-SE control). A striking result was obtained with the combined treatment of a wtp53 and 12 Gy irradiation which produced in 25% and 38.9% of apoptotic cells 48 hours and 72 hours after transfection, respectively. We can therefore conclude that the sensitivity of radiation therapy is enhanced by DNA transfection with wtp53 or mdm-2 AS ODNs for the correction of the p53-mdm2 balance in STS in vitro.

Apoptosis↗

PubMedDate not supplied

The p53 gene in soft tissue sarcomas: prognostic value of DNA sequencing versus immunohistochemistry.

BACKGROUND: The aim of this study was to compare sequencing and immunohistochemistry (IHC) for their ability to detect p53 mutations and to assess their prognostic value in soft tissue sarcomas (STS). MATERIAL AND METHODS: 146 STS samples were investigated by single strand DNA conformation polymorphism (SSCP)-sequencing for p53 mutations. Additionally, IHC with five p53 antibodies (CM-1, Pab1801, Pab240, DO1, DO-7) was performed. RESULTS: In 65 to 131 tumor samples (44.5% to 90.4%) elevated levels of p53 protein were recorded by IHC, depending on the antibody applied. Sixteen out of 146 STS tumor samples (11%) had p53 gene mutations. 13 cases (81.2%) of the 16 tumors with p53 mutations could be detected by DO-1. However, of the 93 DO-1 positive tumors 79 were negative by SSCP-sequencing analysis. Using multivariate regression analysis (Cox proportional hazards model) both p53 mutations (non-frameshift mutations) and IHC detection of p53 protein overexpression were prognostic predictors for poor survival. CONCLUSIONS: This study suggests that IHC is valuable for assessing p53 mutations in STS, but sequencing provides additional important information on the molecular characteristics of the alterations.

DNA, Neoplasm↗