A method for sequential extraction of RNA and DNA from the same sample, specially designed for a limited supply of biological material.
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Animals↗
Biomedical subjects
S Chevillard
Publications and source records attributed to S Chevillard.
Doxorubicin-loaded nanospheres bypass tumor cell multidrug resistance.
We have demonstrated that in vitro resistance of tumor cells to doxorubicin (Dox) can be fully circumvented by using doxorubicin-loaded nanospheres (Dox-NS), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of Dox per 31.5 mg of polymer. Five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: Dox-R-MCF7, a human breast adenocarcinoma; SKVBL1, a human ovarian adenocarcinoma; K562-R, a human erythroleukemia; and two murine lines: P388-Adr-R, a monocytic leukemia of DBA2 mouse, and LR73MDR, a Chinese hamster ovarian cell line. These lines were 38.7, 210, 232, 143 and 20 times more resistant than their corresponding sensitive counterparts, respectively. Using Dox-NS, we obtained complete reversion of drug resistance in vitro, i.e. cell growth inhibition comparable with that obtained with sensitive cells exposed to free Dox. In vivo, we significantly prolonged the survival of DBA2 mice which had previously received P388-Adr-R cells by i.p. injections of Dox-NS, while free Dox injection was ineffective toward this rapidly growing tumor. (Prolongation of survival time: 115% vs 167% after Dox vs Dox-NS treatment, respectively.) Using the MCF7 cell line and its resistant variant, we studied the intracellular concentration and the cytoplasmic and nuclear distribution of Dox by laser microspectrofluorometry (LMSF). In sensitive cells, we observed a similar accumulation and distribution of Dox whatever the form of Dox delivery, i.e. whether free or carried by nanospheres. Analysis by LMSF showed that 99% of intranuclear Dox was bound to DNA after treatment with both forms of Dox. Of Dox, 81 and 83% were found in the intranuclear compartment of sensitive cells incubated with free Dox and Dox-NS, respectively. Resistant cells incubated with Dox-NS accumulated the same amount of Dox as sensitive cells incubated with free Dox or with Dox-NS. Dox, when loaded in nanospheres, bypasses the efflux mechanism responsible for multidrug resistance. LMSF analysis showed that Dox, transported and released by nanospheres, interacts with DNA identically in sensitive and resistant cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Cytotoxic effects and pharmacokinetic analysis of combined adriamycin and X-ray treatments in human organotypic cell cultures.
Organotypic cultures of human A549 cells were used as a tumor model to investigate sequence effects for combination treatments with adriamycin (ADR) and X-irradiation. Initial drug exposure led to the greatest cytotoxic effect especially when X-rays were delivered 24 h later and this subsequent irradiation did not significantly modify the intracellular ADR concentration. In contrast, post-irradiation drug exposure gave rise to a lower cytotoxic effect, and induced a marked reduction of intracellular and more specifically intranuclear ADR uptake and retention, especially when the drug was given 24 h later.
Adenocarcinoma↗
Adriamycin uptake and metabolism in organotypic culture of A549 human adenocarcinoma cells according to the exposure time.
In organotypic cultures (nodules) of A 549 human lung adenocarcinoma cells, the long-term cytotoxicity of Adriamycin is strongly improved by shortening the exposure time to the drug. In order to gain insight into the mechanisms of Adriamycin toxicity in this system, we have examined the drug uptake, retention and metabolism by fluorescence microscopy and HPLC analysis. A 549 nodules efficiently metabolize Adriamycin, two major metabolites, adriamycinol and an aglycone derivative, as yet chemically unidentified, are formed and efficiently excreted. Kinetic data show that a long exposure to Adriamycin triggers its efflux from both the nucleus and the cytoplasm while stimulating its metabolism. Therefore, a long exposure time to the drug appears to trigger a process of cellular detoxification by favouring its excretion from the cells via increased metabolism.
Adenocarcinoma↗
Ki67 index and S-phase fraction in human breast carcinomas. Comparison and correlations with prognostic factors.
In a prospective study of 148 consecutive breast adenocarcinomas, proliferative indices of the same surgical tumor sample were performed by immunohistologic staining (Ki67 index) with the use of the Ki67 monoclonal antibody, which binds to a nuclear antigen only expressed in cycling cells, and by flow cytometry-derived S-phase fraction (SPF). Measurable Ki67 and SPF indices were obtained in 142 cases and 99 cases, respectively, and in 96 cases by both methods. In aneuploid tumors, a significant but low (P less than 0.05, r = 0.3) relationship was observed between Ki67 index and SPF. When compared with clinical, pathologic, and biochemical parameters these two proliferative indices were shown to be associated with nuclear grading and mitotic index. Additionally, correlations were observed between Ki67 index and node involvement (P less than 0.02) and between SPF and estrogen receptors (P = 0.002). These results show that (1) proliferative indices are obtained in 96% of surgical samples with Ki67 versus 67% with SPF and that (2) Ki67 index and SPF may provide complementary data with respect to prognosis.
Adenocarcinoma↗
Effects of MDR reversing agent combinations on the 3H-daunomycin accumulation in drug-sensitive and drug-resistant human cancer cells.
BACKGROUND: As multidrug resistant (MDR) tumour cells generally exhibit a drug accumulation deficit, the effects of three prototype modulators and their combinations were investigated by studying the modulation of 3H-dounomycin cellular accumulation. MATERIALS AND METHODS: Two cell lines derived from a rhino-pharingeal human carcinoma, either sensitive (KB-3-1) or selected as MDR (KB-A1) were used. Verapamil (10mumol.L-1), PSC 833 (lmumol.L-1) and S9788 (5mumol.L-1) were tested alone or in association two by two. The cells were characterized by reverse transcriptase polymerase chain reaction (RT-PCR) in terms of pleiotropic resistance gene expression. RESULTS: A strong mdr1 and a light LRP gene expression were found in KB-A1 resistant cells compared to KB-3-1, whereas MRP expression was found to a similar extent. Relative to the KB-3-1, cells, accumulation of 3H-daunomycin was reduced to 31 +/- 5% in the KB-A1 cells. In these KB-A1 cells, the three agents tested significantly increased the 3H-daunomycin intracellular concentration, S9788 being the most active (311 +/- 37%) and inducing a near complete reversion to the basal level of the sensitive cells. Verapamil and PSC 833 demonstrated an additive effect (252 +/- 69% compared to 188 +/- 33% and 126 +/- 27%, respectively). On KB-3-1 sensitive cells, S9788 had no effect, while verapamil or PSC 833 moderately increased the 3H-daunomycin accumulation, without additive effect. CONCLUSION: These results show a strong MDR reversing effect of S9788, which appears specific to P-glycoprotein (Pgp) and an additive effect between verapamil and PSC 833, suggesting a better therapeutic efficiency if used in well defined combinations.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
A single 24h contact time with adriamycin provokes the emergence of resistant cells expressing the Gp 170 protein.
We investigate here, in A549 cells, the influence of a single short (1h) or long (24h) adriamycin (ADR) contact time on the long-term cytotoxicity of the drug and on the emergence of resistant cells. In contrast to a 1 h ADR contact, a 24 h treatment provokes the emergence of resistant cells overexpressing the Gp170 protein and this overexpression is maintained for at least three months without any drug selection pressure.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Growth stimulation by misonidazole of lung carcinoma cells maintained in continuous organotypic and monolayer cultures.
Inasmuch as misonidazole is a drug used in clinical trials for sensitizing radioresistant hypoxic cells in solid tumors, it seemed of interest to study its effects in human tumor cells maintained in tridimensional organotypic cultures. This type of culture involves: spatial organisation of the cells with fairly undisturbed differentiation patterns, minimal traumatizing culture conditions, and offers the possibility to follow post-treatment growth patterns over several months without disturbing the cultures. Misonidazole exhibited a radiosensitizing effect on irradiated nodules derived from a lung adenocarcinoma, and on cells of this tumor growing in monolayers. However, after a 4 hour contact with misonidazole at concentrations corresponding to the range of those found in the serum of treated patients, a significant stimulation of nodule growth was repeatedly observed, together with a strong increase in the frequency of sister chromatid exchanges. Similarly, after treatment of the same tumor cells in confluent monolayers, their colony forming ability was increased. These observations may account for some of the non- convincing therapeutic results obtained in clinical trials.
Adenocarcinoma↗