melphalan and thiazolyl-blue

Increased nuclear factor kappaB (NF-kappaB) activity is associated with increased tumor cell survival in multiple myeloma. The function of NF-kappaB is inhibited through binding to its inhibitor, IkappaB. Release of activated NF-kappaB follows proteasome-mediated degradation of IkappaB resulting from phosphorylation of the inhibitor and, finally, conjugation with ubiquitin. We report that myeloma cells have enhanced IkappaBalpha phosphorylation and increased NF-kappaB activity compared with normal hematopoietic cells. The proteasome inhibitor PS-341 blocked nuclear translocation of NF-kappaB, blocked NF-kappaB DNA binding, and demonstrated consistent antitumor activity against chemoresistant and chemosensitive myeloma cells. The sensitivity of chemoresistant myeloma cells to chemotherapeutic agents was markedly increased (100,000-1,000,000-fold) when combined with a noncytotoxic dose of PS-341 without affecting normal hematopoietic cells. Similar effects were observed using a dominant negative super-repressor for IkappaBalpha. Thus, these results suggest that inhibition of NF-kappaB with PS-341 may overcome chemoresistance and allow doses of chemotherapeutic agents to be markedly reduced with antitumor effects without significant toxicity.

Topics: Active Transport, Cell Nucleus; Adenoviridae; Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cell Division; Cell Nucleus; Cell Survival; Cysteine Endopeptidases; Cytosol; Dose-Response Relationship, Drug; Genes, Dominant; Humans; I-kappa B Proteins; Melphalan; Microscopy, Fluorescence; Multienzyme Complexes; Multiple Myeloma; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Proteasome Endopeptidase Complex; Pyrazines; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Tumor Cells, Cultured; Ubiquitin

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.

Topics: 3T3 Cells; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents, Alkylating; Blotting, Southern; Cell Division; Cytarabine; Cytidine Deaminase; DNA, Complementary; Drug Resistance, Neoplasm; Fibroblasts; Gene Transfer Techniques; Genetic Vectors; Glutathione Transferase; Humans; Mechlorethamine; Melphalan; Mice; Protein Biosynthesis; Rats; Retroviridae; Tetrazolium Salts; Thiazoles; Transduction, Genetic

Acquired resistance is a limiting factor in chemotherapy. We have employed nitrogen mustard resistant B-cell chronic lymphocytic leukemia (B-CLL) as a clinically relevant model to study this phenomenon. Resistance in B-CLL is associated with enhanced repair of nitrogen mustard crosslinks. In order to identify the repair pathway responsible for nitrogen mustard resistance, lymphocytes were screened for cross-resistance to a variety of DNA damaging agents. The MTT assay was used to measure the resistance of B-CLL lymphocytes to various DNA damaging agents, including nitrogen mustards, UV light, methyl methanesulfonate, and mitomycin C. We have shown that B lymphocytes from patients with nitrogen mustard resistant chronic lymphocytic leukemia reflect their clinical status. This assay allows us to classify lymphocytes as nitrogen mustard sensitive or resistant, based on in vitro observations. The resistant population was 5.6 and 4.1 fold more resistant to the nitrogen mustard analogs, chlorambucil and melphalan, respectively. Resistant lymphocytes displayed no increased resistance to either methyl methanesulfonate or UV light, indicating that neither classical base nor nucleotide excision repair is rate-limiting in resistance. Resistant lymphocytes were 6.0 and 2.2 fold more resistant to mitomycin C and cis-diamminedichloroplatinum (II), respectively, suggesting enhanced crosslink repair. Neither glutathione nor glutathione S-transferase levels correlated with resistance. The development of nitrogen mustard drug resistance in B-CLL appears to be associated with cross-resistance to other bifunctional alkylating agents which produce interstrand crosslinks. Our results indicate that resistance to nitrogen mustards in chronic lymphocytic leukemia is associated with enhanced repair of DNA crosslinks which may involve a recombination dependent system. This model should prove very useful in the elucidation of the molecular mechanisms of crosslink repair.

Topics: B-Lymphocytes; Chlorambucil; Cisplatin; Cross-Linking Reagents; DNA Adducts; DNA Damage; DNA Repair; DNA, Neoplasm; Drug Resistance; Humans; Inactivation, Metabolic; Leukemia, Lymphocytic, Chronic, B-Cell; Melphalan; Methyl Methanesulfonate; Mitomycin; Nitrogen Mustard Compounds; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Mosmann's method for measuring the number of viable cells, examination of their growth and function by tetrazolium test, "MTT assay", is widely thought to be reliable. For the purpose to establish a rapid, accurate, in vitro drug sensitivity test, MTT assay was applied and evaluated for clinical application. Based on Mosmann's original MTT assay, optimal and adequate conditions for (1) the number of the cells examined at the starting of cultivation, (2) concentration of anti-tumor agents, doxorubicin, cisplatin, mitomycin C, L-phenylalanine mustard, (3) incubation time with anti-tumor agents, were determined using established cell lines, T-24, RMUG, HeLa, Vero, P 388, and Colon 26 in 96 well microplates. Conclusions are as below: (1) Number of the cells in each well of microplate is 1 x 10(3)-1 x 10(6) cells/ml, that seemed to be theoretically and technically adequate. (2) Anti-tumor agents should be added at the peak plasma concentration. (3) Incubation for 4 to 5 days is preferable. (4) HCl-isopropanol seemed to be advantageous compared to 10% sodium dodecyl sulfate for solubilization of MTT formazan crystal. (5) Results of MTT assay and colony assay were well correlated.

Topics: Antineoplastic Agents; Cisplatin; Doxorubicin; Drug Screening Assays, Antitumor; Melphalan; Mitomycin; Mitomycins; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.

Topics: Animals; Antineoplastic Agents; Autoanalysis; Cell Line; Cisplatin; Clone Cells; Colorimetry; Coloring Agents; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance; Humans; In Vitro Techniques; Lung Neoplasms; Melphalan; Tetrazolium Salts; Thiazoles; Vinblastine