Increased bax expression is associated with cell death induced by ganciclovir in a herpes thymidine kinase gene-expressing glioma cell line.

The herpes simplex virus thymidine kinase gene (HSV-tk) was stably transfected into rat C6 glioma cells (C6tk) in order to characterize the mechanisms underlying cell toxicity induced in vitro by the guanosine analog ganciclovir (GCV). The results demonstrate the efficiency of the HSV-tk/GCV system in ablating most of the tumoral cells within 7 to 8 days of treatment with 20 mivroM GCV; however, a few cells still survive. C6tk cells arrest in the S phase of the cell cycle after 2 days of drug treatment before undergoing cell death. Microscopic analysis reveals dying cells with ultrastructural characteristics consistent with apoptosis; we cannot rule out, however, that necrotic cell death may also be occurring. The cytotoxicity induced by GCV is not associated with changes in the expression of p53 protein, suggesting that cell cycle arrest and cell death may occur through a p53-independent pathway. C6tk cells constitutively express Bcl-xL and Bax proteins; when exposed to GCV, Bcl-xL levels do not change but Bax accumulation is rapidly induced. These findings suggest that the balance between Bcl-xL and Bax proteins may be of importance in determining the sensitivity of tumoral cells to GCV.

Identification and characterization of an anti-tyrosine kinase factor in cystic gliomas.

In view of the frequent activation of the epidermal growth factor receptor (EGF-R) in gliomas and autocrine hypothesis, we searched for 'EGF-like' factor(s) in cystic fluids (CFs) associated with gliomas. Membranes of A431 cells, which overexpress EGF-R, were used to explore such activity in 20 CFs. In all cases CFs induced inhibition of EGF-R phosphorylation. Biochemical analysis revealed an anti-tyrosine kinase activity which was identified as a 18 kDa proteic factor. Effectiveness at high dilution and anti-proliferative effect on living cells in culture suggest that this factor may be involved in the negative regulation of glial oncogenesis.

EGF receptor amplification and expression in human brain tumours.

Human epidermal growth factor receptor (EGFr) gene amplification, rearrangements and expression were studied in tumours of the human nervous system. EGFr gene amplification was studied in 46 brain tumours. Gene expression was analysed by northern blot in 37 tumours and binding of its protein to EGF in 27 tumours. The EGFr gene was simultaneously amplified (with arrangements in 12.5% of gliomas) and overexpressed in 53% (9/17) of malignant gliomas, but never in meningiomas. In five high grade gliomas, amplification was always associated with a high level of receptors. However, since high amounts of EGF receptors found in one glioma were not the result of gene amplification, several systems of deregulation in EGFr production may exist and could be located at translational and/or post-translational levels.

A study of the expression of four chemoresistance-related genes in human primary and metastatic brain tumours.

We investigated four mechanisms of intrinsic chemoresistance in a series of 67 human brain tumours including 31 gliomas (one grade I ganglioglioma, nine grade II and 10 grade III astrocytomas, 11 glioblastomas), 13 cerebral metastases, one medulloblastoma, one malignant teratoma, three ependymomas and 18 meningiomas. We studied four genes by northern blotting: multidrug-resistance (MDR 1), glutathione-s transferase (GST pi), dihydrofolate reductase (DHFR), and topoisomerase II (Topo II). The Topo II gene was absent in the normal adult brain (100%) and in 64% of the tumour samples tested. A second gene, GST pi, was found to be overexpressed in 38% of brain tumours. The two other chemoresistance-related genes were occasionally overexpressed in brain tumours (2% for MDR1, 9% for DHFR). Our results provide evidence that chemoresistance is intrinsic to the brain tissue and seems likely to be a multifactorial process.

6-hydroxydopamine-induced nuclear factor-kappa B activation in PC12 cells.

The involvement of nuclear Factor-kappa B (NF-kappa B) transcription factor in PC12 cell death triggered by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was investigated. Results show that oxidative stress generated by 6-OHDA activates NF-kappa B. When the NF-kappa B activation was inhibited by parthenolide, PC12 cell death induced by 6-OHDA was significantly increased, thus suggesting an involvement of this transcription factor in a protective mechanism against 6-OHDA toxicity. To further assess this hypothesis, we studied the involvement of NF-kappa B in the protective effect of two anti-apoptotic genes, bcl-2 and bfl-1. Although Bcl-2 and Bfl-1 expression normally protects PC12 cells from 6-OHDA, parthenolide strongly decreased the beneficial effects afforded by transgene expression. These results suggest: (1) that the transcription factor NF-kappa B is likely associated with the protection of catecholaminergic PC12 cells and (2) that the protective effects afforded by bcl-2 and bfl-1 expression may be dependent on NF-kappa activation.

Met-enkephalin receptors in human gliomas.

Endogenous opioid systems (opioid peptides and receptors) are involved in many functions including the regulation of cell growth. We investigated the presence of Met-enkephalin binding sites in gliomas by displacement assays. Results demonstrated that few gliomas exhibit Met-enkephalin binding sites and that the percentage of tumours which express these binding sites strongly decreases with increasing malignancy. Moreover, we observed a shift from mu Met-enkephalin binding sites in low grade gliomas to delta Met-enkephalin binding sites in high grade gliomas. These results suggest an inactivation of the Met-enkephalinergic system in gliomas which could lead to loss of the inhibitory effect exerted by Met-enkephalin on normal astrocyte growth and thus favour progression of malignancy.

Expression of the preproenkephalin A gene in tumor cells and brain glioma: a northern and in situ hybridization study.

Preproenkephalin A (PPA) mRNA expression was studied by Northern blot and in situ hybridization in cell lines (rat glioma C6, rat hepatoma HTC, human neuroblastoma IMR32, mouse neuroblastoma NS20Y, rat fibroblast FR3T3, human bladder carcinoma EJ, human vulva carcinoma A431, myelocytic leukemia HL60, rat adrenal carcinoma Y1) and in brain tumours (implanted C6 cells). C6 glioma in cell culture, as well as in brain tumours, expressed high levels of PPA mRNA as compared to the caudate nucleus of the rat brain. EJ and FR3T3 cell lines also expressed the PPA mRNA, which was not detectable in A431, Y1, NS20Y, IMR32, HTC, HL60 cell lines as well as in the rat liver. This observation provides an interesting model to study the mechanisms by which the malignant transformation can induce in glial cells the derepression of a gene which is usually expressed in neurons or in neuron-like cells.

p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells.

p53, Bax and Bcl-xL proteins have been implicated in apoptotic neuronal cell death. We have investigated whether those proteins are involved in 6-OHDA-induced PC12 cell death. After a 24-h exposure to the neurotoxin (100 microM), morphological evidence for apoptosis was observed in PC12 cells. Up-regulation of p53 and Bax proteins was demonstrated 4 and 6 h, respectively, after 6-OHDA treatment; in contrast, no change in Bcl-xL levels was found. These findings suggest that p53 and Bax could be relevant markers of neuronal apoptosis as previously described in kainic acid- or ischemia-induced neuronal cell death and may participate to neuronal degeneration in Parkinson's disease.

Unlike MPP+, apoptosis induced by 6-OHDA in PC12 cells is independent of mitochondrial inhibition.

The mechanisms of 6-hydroxydopamine (6-OHDA) cytotoxicity were studied in vitro using the PC12 cell line. Following a 24 h exposure, this neurotoxin induced apoptosis and a dose-dependent decrease in cell survival. The presence of monoamine oxidase inhibitors, tranylcypromine and clorgyline, together with 6-OHDA had neither synergistic nor protective effects. Unlike 1-methyl-4-phenylpyridinium (MPP+), 6-OHDA toxicity to PC12 cells remained unchanged when glycolysis was prevented by either depleting glucose from the culture medium or growing the cells in low-glucose medium containing 2-deoxy-glucose. These results suggest that the inhibition of mitochondrial respiration is not responsible for the cell death induced by 6-OHDA.

Extracellular toxicity of 6-hydroxydopamine on PC12 cells.

6-hydroxydopamine (6-OHDA) is usually thought to cross cell membrane through dopamine uptake transporters, to inhibit mitochondrial respiration and to generate intracellular reactive oxygen species. In this study, we show that the anti-oxidants catalase, glutathione and N-acetyl-cysteine are able to reverse the toxic effects of 6-OHDA. These two latter compounds considerably slow down 6-OHDA oxidation in a cell free system suggesting a direct chemical interaction with the neurotoxin. Moreover, desipramine does not protect PC12 cells and 6-OHDA is also strongly toxic towards non-catecholaminergic C6 and NIH3T3 cells. These results thus suggest that 6-OHDA toxicity on PC12 cells mainly involves an extracellular process.

Antineoplastic activity of two taxol derivatives on an ovarian tumor xenografted into nude mice.

In order to compare the antineoplastic activities of taxol A, taxol B, a mixture of the two (taxol A 72%) and vinblastine, a human ovarian tumor serially transplanted into 104 female athymic mice was used. In the first experiment (11th passage), the antineoplastic activities of taxol A, taxol B and the mixture taxol AB were tested. The same dose was used in each case (12.5 mg/kg i.e. 1/20 of the evaluated LD50 value). It was administered subcutaneously for 5 consecutive days. Three courses of treatment were performed, with 2 rest periods of 1 week in between. All the taxol derivatives produced a statistically significant delay in the tumor growth. However, taxol B had the lowest chemotherapeutic response. In the second experiment (18th passage), different dose levels were administered (mixture 12.5 mg/kg/day x 4 - taxol A 8.8. mg/kg/day x 4 - taxol B 3.5 mg/kg/day x 4 - vinblastine 0.5 mg/kg/day x 2). For all the taxol derivatives 4 treatment courses with 3 rest periods of 4 days were used, and for vinblastine 4 treatment courses with 3 rest periods of 1 week. At the end of the second experiment, vinblastine, taxol A and a mixture of the two showed similar significant activity, whereas no objective antitumor response was observed following the taxol B treatment at the dose level chosen. The experimental results obtained clearly demonstrate that, in the taxane system, the greatest degree of antineoplastic activity can be attributed to taxol A.

[3 experimental models applied to the study of urothelial tumors of the bladder]. / Trois modèles expérimentaux appliqués à l'étude des tumeurs urothéliales de vessie.

Three important models can be used in experimental carcinogenesis: Clonogenic assays which can be used to study tumor cells in vitro. This method is particularly useful in order to investigate biochemical markers and chromosomal spreads. Chemically-induced bladder tumors in various animals are used to analyse the mechanisms of induction and development of these cancers. The athymic mice have provided, over the last twenty years, an immunological system suitable for the heterotransplantation of human tumor tissues. These three experimental models, which are complementary, are reviewed and discussed.

[The EGF receptor pathway in human cerebral tumors]. / Etude de la voie du récepteur a l'E.G.F. dans les tumeurs cérébrales humaines.

The epidermal growth factor receptor gene is the most frequently involved proto-oncogene in human glial brain tumors, in the present series in agreement with previous reports in literature. It is therefore important to study this gene from DNA to the protein product. The vicinity of cystic fluid (C.F.) to tumor cells of the cystic wall has suggested investigation of possible "E.G.F.-like" autocrine activities in C.F. In 40% of gliomas, E.G.F.-R. gene is amplified and overexpressed. This is never observed in low grade astrocytomas. In 12% of the cases, mutations of the E.G.F.-R. gene are observed. In correlation with genomic abnormalities, E.G.F.-R. is immunoprecipitated in 40% gliomas. The basal phosphorylation of the receptor is increased in 50% gliomas. In C.F., unexpectedly, E.G.F.-R. phosphorylation inhibitory effect is observed. Its biochemical analysis suggests an anti-tyrosine kinase activity. The observation of anti-tyrosine kinase activity in C.Fs suggests the presence of negative modulatory factors of the proto-oncogene activation in tumor tissues. This could have therapeutical interest.

A cautionary note on the use of stable transformed cells.

Gene transfection and ectopic expression is a widely used method in experimental biology. In the present report, we would like to point out that this approach may, in certain circumstances, lead to a modification of the transfected cell phenotype. Indeed, we observed that after transfection of bcl-2 gene in the neuronal PC12 cell line some of the selected clones have lost their neuronal and catecholaminergic characteristics, i.e. TH expression and ability to grow neurites in response to NGF. Thus, the resistance of some PC12-Bcl-2 clones against neurotoxic insults may not necessarily reflect the potential benefit afforded by Bcl-2 expression. We therefore encouraged authors to verify cell phenotype after stable transfection to avoid misinterpretation of their results.

Infection with Toxoplasma gondii does not Alter TNFalpha and IL-6 Secretion by A human Astrocytoma Cell Line.

The secretion of tumour necrosis factor-alpha (TNFalpha), interleukin-1alpha (IL-alpha) and interleukin-6 (IL-6) by a human astrocytoma cell fine was studied 1 h, 3 h, 6 h and 24 h after infection with tachyzoites from three Toxoplasma gondii strains (virulent, RH; cystogentc, 76K and Prugniaud strains). The astrocytoma cell fine constitutively secreted TNFalpha and IL-6, but no IL-1alpha. A positive control was obtained by stimulation with phorbol esters inducing a significant increase (p < 0.05) in TNFalpha and IL- 6 secretion but not in IL-1alpha, while lipopolysaccharide (alone and after priming), interferon gamma, ionophore A 23187 and sera positive to T. gondii did not induce any increase in cytokine levels. None of the tachyzoites, whatever their virulence, induced a significant increase in cytokine production at any time in the study. Tachyzoites did not inhibit the secretion induced by phorbol esters.

Therapeutic efficacy of the thymidine kinase/ganciclovir system on large experimental gliomas: a nuclear magnetic resonance imaging study.

Contradictory experimental results and human trials have questioned the clinical relevance of the HSVtk/ganciclovir system. To bypass the problem of transfection efficiency, we used a glioma cell line stably expressing the HSVtk gene, which was also fully characterized from gene to protein. We also designed a more clinically relevant experimental protocol, consisting of late GCV delivery on large tumor formations. In short-term studies, histological examination revealed a significant decrease in tumor volume in GCV-treated animals from day 1 or from day 10 after cell inoculation. We observed that late GCV delivery is as efficient as early delivery, probably because GCV can reach tumor cells more easily when neoangiogenesis occurs. In long-term experiments, the survival of treated rats bearing 15-day tumors was improved by 60% compared with C6 control animals. Surprisingly, a 30% survival rate was observed in C6TK control animals. Nuclear magnetic resonance imaging demonstrated, in all surviving animals, a complete regression of tumors without mass effect. These results clearly demonstrate that the HSVtk/GCV system remains a potent therapeutic strategy, even when tested in large tumors, in contrast with the microscopic tumor formations previously reported.

[Multiple correlative studies of stereotaxic biopsies of brain tumors]. / Etudes correlatives multiples sur biopsies stéreótaxiques de tumeurs cérébrales.

The opportunity of having several samples at the same site which could be spatially localized allows an intensive exploitation of stereotactic biopsies of brain tumors: the pathological data may be correlated to other measures, performed at the same site (electrical impedance X ray absorption coefficient) or on other samples (NMR relaxation times, water content, nucleic acids). These samples are available for oncology experiments in cellular biology (cell cultures, grafts on nude mice) or in molecular biology (DNA and RNA hybridization with specific nucleic acid probes). We were therefore able: 1) to study the diagnostic homologies between pathology and histology examinations; 2) to show that T1 and T2 NMR relaxation times are 2 times longer in tumor tissues than in normal brain; 3) to show that the electrical impedance is decreased by a factor 2 in brain tumors; 4) to show the absence of integrated viral genomic sequences and the existence of oncogenes association patterns in brain tumors by hybridization of specific sequences; 5) to establish permanent cell lines, the tumorigenicity of which is assayed by grafting on nude mice. Therefore, stereotactic biopsies appear to be, provided they are intensively and rationally exploited, a major research tool in an area which remains unsensitive to the various therapeutic approaches.