Michel Pagé

Optimization of the tetrazolium-based colorimetric assay for the measurement of cell number and cytotoxicity

We report some modifications of the semiautomated tetrazolium-based assay for the measurement of anchorage-dependent and -independent mammalian cells. The various factors affecting color production, such as the concentration of tetrazolium, incubation period, the type and volume of solvent, were optimized. Using KCN and daunorubicin as cytotoxic agents, the influence of dead cells was studied on the measurement. The assay was tested with mouse leukemia P388 cells, H69 small cell carcinoma cells growing in suspension and anchorage dependent colon adenocarcinoma cells (LoVo). Centrifugation of the microtitration plate was eliminated by the use of a Skatron supernatant collection system. Although the use of the MTT assay is rapid and precise, we found that care should be taken when using this assay for short-term cytotoxicity assays since non-viable cells also reduce the tetrazolium.

Genetic polymorphism in low-molecular-weight glutenin genes from Triticum aestivum, variety Chinese Spring.

Abstract Low-molecular-weight (LMW) glutenin subunits consist mainly of two domains, one at the N- terminus which contains repeats of short amino-acid motifs, and a non-repetitive one rich in cysteine, at the C- terminal region. In previous reports, polyacrylamide-gel electrophoresis has been used to show that large size variation exists among LMW and HMW glutenin subunits, and it has been suggested that deletions and insertions within the repetitive region are responsible for these variations in length. In this study, PCR-amplification of genomic DNA (Triticum aestivum variety Chinese Spring) was used to isolate three full-length LMW glutenin genes: LMWG-MB1, LMWG-MB2 and LMWG-MB3. The deduced amino-acid sequences show a high similarity between these ORFs, and with those of other LMW glutenin genes. Comparisons indicate that LMWG-MB1 has probably lost a 12-bp fragment through deletion and that LMWG-MB1 and LMWG-MB2 have an insertion of 81 bp within the repetitive domain. The current study has shown direct evidence that insertions and/or deletions provide a mechanistic explanation for the allelic variation, and the resultant evolution, of prolamin genes. Single-base substitutions at identical sites generate stop codons in both LMWG-MB2 and LMWG-MB3 indicating that these clones are pseudogenes.

Improved fluorescent bioassay for the detection of tumor necrosis factor activity

Tumor necrosis factor alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity on mouse L929 fibroblast cells. After an incubation with TNF, the cells were stained with a solution of ethidium homodimer-1, a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. The assay is sensitive, inexpensive and correlates with the already reported TNF assays while measuring the membrane alteration by TNF and not the cell detachment. It requires no rinsing before dye addition which may cause cell loss; there is no interference with culture medium components since the assay is performed in PBS. This method is more rapid and precise for routine measurement of TNF activity.

An approach to ampholyte-displacement chromatography

Abstract An experimental approach to ampholyte-displacement chromatography is described in which a linear pH gradient is obtained by ampholyte displacement on DEAE-Sepharose CL-6B. The results show that a pH gradient is obtained whatever the initial pH of the ion exchanger. Ampholytes are bound selectively to the ion exchanger according to their respective pI values and to their charge in solution. This experimental approach to ampholyte-displacement chromatography demonstrates the applicability of this technique for the separation of closely related proteins.

Measurement of the enzymatic specificity of carboxypeptidase A by capillary zone electrophoresis

Abstract The development of a novel method using capillary zone electrophoresis for monitoring enzymatic assays involving carboxypeptidase A and methotrexate-α-peptides is reported. Since the hydrolysis product, methotrexate, and the substrate have the same absorption spectrum, spectrophotometry could not be used to monitor the reaction. Eleven methotrexate amino acid prodrugs were synthesized and tested as substrates for carboxypeptidase A. Since the product has a charge difference with the substrate, the reaction could be monitored easily by capillary zone electrophoresis. This method may be adapted to various enzyme kinetics.

Development of a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel

We have developed a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel in serum, crude Taxus extracts and Erwinia taxi culture medium. To achieve this, we used an antipaclitaxel monoclonal antibody and an FITC-labeled paclitaxel. Paclitaxel was chemically modified by introducing an amino function to enable coupling with fluorescein isothiocyanate. Paclitaxel competitively inhibited the binding of the monoclonal antibody with FITC-paclitaxel causing a decrease in polarization. We were able to detect paclitaxel in a concentration as low as 2 nM. Fluorescence polarization immunoassay requires only the addition of the fluorescent probe to the antibody, followed by an incubation and measurement of polarization. Both the simplicity and the sensitivity of this method make it useful for estimating the paclitaxel content in yew tree crude extracts and culture medium of paclitaxel producing micro-organisms and a possible assay for monitoring paclitaxel level in patients under treatment.

An Mr 7-kDa membrane protein overexpressed in human multidrug-resistant ovarian cancer cells

We have developed a monoclonal antibody (designated 1D7) which recognizes an M(r) 7-kDa plasma membrane protein overexpressed in ovarian MDR cancer cells. The expression of the M(r) 7-kDa protein in various human multidrug-resistant and drug-sensitive cell lines was analysed by Western blot and flow cytometry methods. The small molecular weight protein was overexpressed in the human ovarian carcinoma cell line, SKVLB which was selected for vinblastine resistance from SKOV3 cells and in OVCAR 4/ADR100 and OVCAR 4/VBL200 which were generated from NIH:OVCAR4 by stepwise selection against adriamycin and vinblastine, respectively. Only a minor amount of the M(r) 7-kDa protein was found in the parent cell line, SKOV3. It was not found in other drug-resistant human cell lines such as the vinblastine-resistant CEM cells (CEM/VLB300), the intrinsic MDR colon cell line HCT15 and the human MDR breast cancer cell line, MCF7/AdrVp. 1D7 specifically inhibited the proliferation of the resistant cells. Our results suggest that the expression of the M(r) 7-kDa protein on the plasma membrane of ovarian MDR cancer cells may be involved in a mechanism related to the proliferation of the drug resistant cancer cells.

Rapid purification method for human recombinant tumor necrosis factor alpha.

Human recombinant tumor necrosis factor alpha was purified in a single step to about 95% purity from Escherichia coli lysate by chromatography on hydroxyapatite. The last traces of contaminants were removed by fast protein liquid chromatography on a Mono Q column. The final product was found to be pure by gel electrophoresis with silver staining. A molecular mass of approximately 17,000 and a specific activity of 4.3 x 10(6) U/mg after a single purification step were found.

Coupling daunorubicin to monoclonal antialphafoetoprotein with a new activated derivative

We report the synthesis of daunorubicin antialphafoetoprotein conjugates, using a new coupling method. At equimolar concentrations on AFP producing cells in vitro, these conjugates were found three times as cytotoxic as the free drug.

New strategy for the production of monoclonal antibodies to external portion of the P-glycoprotein

As early as 1970, Biedler and Riehm have described the MDR phenotype [1]. They characterized Chinese hamster cell lines that showed a high level of resistance to Actinomycin D or Daunorubicin. These cells showed crossresistance to various structurally unrelated natural products such as Vinblastin, Vincristin, Puromycin and Mitomycin C. Some years later, Ling and co-workers reported a similar phenotype in Chinese hamster ovary cell lines [2]. Although the relative resistance may vary quantitatively between different MDR cell lines, the pattern of crossresistance is qualitatively uniform regardless of the selecting drug. The net cellular accumulation of drugs involved in resistance is usually reduced and this presumably accounts, in part, for the decreased cytotoxicity [3]. Although the entry of these drugs appears to occur by simple diffusion, there seems to be an energy dependent drug efflux system. This enhanced efflux appears to be the major determinant of reduced drug accumulation in MDR cell lines.