Oncogramme responses of breast tumour cells treated with herceptin correlate with HER2/C-ERB B2 pathological status.

BACKGROUND: Among targeted therapies, Herceptin is a monoclonal antibody successfully used on patients with breast cancer expressing Human Epidermal Growth Factor Receptor-2 (HER2 receptors). Oncogramme is a method developed to predict anticancer activity of molecules and thus individualize chemotherapeutic strategies. Before this ex vivo test enters clinical validation, it was desirable to correlate breast cancer cell responses to Herceptin observed through Oncogramme with HER2 expression by these cells. MATERIALS AND METHODS: Breast tumour fragments were dissociated and obtained cells were cultured in defined medium. After Herceptin treatment, cytotoxicity was detected by cell death analysis, and responses compared to tumour HER2 status were determined by pathologists. RESULTS: Cell responses to increasing doses of Herceptin obtained with Oncogramme were in correlation with HER2 expression. CONCLUSION: Comparison between Herceptin responses obtained with Oncogramme and HER2 status of breast tumour cells confirmed that Oncogramme is a reliable method for prediction of patient cell sensitivity to anticancer drugs.

Oncogramme, a new promising method for individualized breast tumour response testing for cancer treatment.

BACKGROUND: Breast cancer is the most widely spread cancer in the world, attracting much research and individualized tumour response testing (ITRT) methods are now used to individualize patient chemotherapeutic administrations. A new ITRT method was developed with optimized processing. MATERIALS AND METHODS: Breast tumour fragments were separated and the cells seeded in a foetal calf serum-free defined medium. After various chemotherapeutic treatments, cytotoxicity was determined by cell death detection with calcein acetoxymethyl and ethidium homodimer labelling. RESULTS: The culture medium allowed breast tumour cell proliferation in culture, while preventing fibroblastic cell survival. Moreover, the cell death analysis gave rise to a chemoresistance profile called an Oncogramme, with statistically significant values. CONCLUSION: The Oncogramme is a new ITRT method which can predict patient cell sensitivities to chemotherapeutics and should be validated by a new phase I clinical trial.

Increase of Fas-induced apoptosis by inhibition of extracellular phosphorylation of Fas receptor in Jurkat cell line.

Apoptosis signalling through the Fas pathway requires several steps of aggregation of the Fas receptor in the membrane, including aggregation that may occur in the absence of Fas ligand. Association of Fas domains is determinant to signal transmission following Fas ligand binding to a specific domain. The domains involved in Fas aggregation are located in its extracellular region and contain three potential protein kinase C-binding motifs. We therefore studied the possibility that phosphorylation of the extracellular region of Fas might be implicated in the regulation of Fas-mediated apoptosis. Inhibition experiments of extracellular phosphorylation were performed in human Jurkat T leukemia cells with K252b, an impermeant protein-kinase inhibitor. Extracellular phosphorylation of Fas receptor was related to ecto-kinase, as assessed by the [gamma-(32)P] ATP labelling of Fas-116 kDa aggregates, suppressed by K252b inhibitor which significantly increased the sensitivity to Fas-mediated apoptosis. Ecto-PKC involvement was demonstrated by bisindolylmaleimide VIII, a selective inhibitor of protein kinase C which significantly increased both Fas aggregation in the membrane and Fas-mediated apoptosis and by the addition of the PKC pseudo-substrate 19-36 which inhibited the phosphorylation of 116 kDa Fas aggregates. These data support a role for Fas phosphorylation in the decreased sensitivity to apoptosis in the Jurkat T leukemia cell line.

Modulation of Fas-induced apoptosis by p75 neurotrophin receptor in a human neuroblastoma cell line.

Fas and p75 neurotrophin receptors (p75(NTR)) are death receptors that alone induce apoptosis of SH-SY5Y neuroblastoma cell line respectively by Fas ligand or brain-derived neurotrophic factor (BDNF, a p75(NTR) ligand). We report on the modulation of Fas-mediated apoptosis by concomitant p75(NTR) activation. The exposure to both ligands suppressed the apoptotic effect. A co-localisation of Fas and p75(NTR) receptors was evidenced by co-capping and immunoprecipitation assays. Moreover, a caspase-8 inhibitor suppressed the protective effect of the concomitant BDNF and Fas ligand stimulation, suggesting that p75(NTR) and Fas receptors could share common signalling pathways.

Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line.

The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.

Expression of a functional Fas death receptor by human foetal motoneurons.

The expression of the apoptosis inducer Fas (CD95/APO-1) surface receptor by human foetal neurons was investigated in vitro and ex vivo. Immunofluorescence studies of brain and spinal cord cells in primary cultures and of cryosections obtained from 9- and 10-week-old human foetuses, respectively, showed that all Fas-expressing cells were motoneurons (5.3 and 4.2% of the neurons in brain or spinal cord cultures, respectively) on the basis of morphology, reactivity with the monoclonal antibody SMI-32, a mostly motoneuronal marker and acetylcholine esterase expression. Fas was undetectable on the other cell types in culture. The ability of Fas to induce apoptosis of cultured cells from both tissues was determined by using the terminal transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) method combined with the same double-staining procedure. Under basal culture conditions, about 9% of cells, all glial fibrillary acidic protein-expressing astrocytes, were apoptotic. After a 48-h incubation with Fas ligand, mean 28.5% of brain motoneurons and 29.4% of spinal motoneurons underwent apoptosis, with an inhibition by Z-IETD-FMK, a caspase-8 inhibitor. Hence, Fas appears to be functional through a caspase-8-dependent pathway in a subpopulation of human foetal motoneurons.

In vitro induction of neuronal apoptosis by anti-Fas antibody-containing sera from amyotrophic lateral sclerosis patients.

Sera from 26% of patients with sporadic amyotrophic lateral sclerosis (ALS) induced in vitro apoptosis of a human neuroblastoma cell line, as detected by two methods, and most contained anti-Fas autoantibodies. In contrast, Alzheimer sera (studied as controls) very rarely induced apoptosis and did not contain detectable anti-Fas antibodies. Soluble Fas-ligand levels in ALS sera were not different from those in normal sera, except for slightly higher levels in a single case. In mixed cultures of rat embryonic brain and spinal cord cells, ALS sera (and agonistic anti-Fas monoclonal antibodies and soluble Fas-ligand) induced the apoptosis of a subpopulation of neurons. These neurons were motoneurons on the basis of staining with the monoclonal antibody SMI 32 and Fas expression was restricted to these SMI 32-positive neurons. These data are compatible with the hypothesis of the participation of an autoimmune mechanism possibly related to anti-Fas autoantibodies in certain ALS patients.