Susan P. C. Cole

Rapid chemosensitivity testing of human lung tumor cells using the MTT assay

SummaryNumerous procedures have been described which test the chemosensitivity of tumor cell lines. A major disadvantage of most of these assays is that practical limitations prevent the testing of more than a few variables. We have adapted a rapid and efficient colorimetric assay for testing the chemosensitivity of human lung tumor cells. In this assay, a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, MTT) is converted to a colored formazan product by enzymes active only in living cells. The MTT assay may be carried out entirely in 96-well microtiter plates, so that large experiments examining a number of variables can be readily performed. Thus, drug concentration, time of exposure to drug, length of assay, and cell density can be varied and tested. Moreover, the simplicity of this assay allows simultaneous testing of multiple drugs on multiple cell lines. Finally, the MTT assay is useful for monitoring the development of multidrug-resistant cells in culture.

Use of the mtt assay for rapid determination of chemosensitivity of human leukemic blast cells

A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtitre plate spectrophotometer. In the present study, optimal conditions for chemosensitivity testing of human leukemia samples were determined, and the relative chemosensitivity of five patient samples was tested.

Human monoclonal antibodies.

SummaryThe technology for the production of murine monoclonal antibodies has been refined enormously since its introduction in 1975. However, the technology for generating human monoclonal antibodies has only recently come into its own. In this review, three currently available approaches to the production of human monoclonal antibodies are described. These include the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to ‘immortalize’ antigen-specific human B lymphocytes; and the EBV-hybridoma technique, based on a combination of the first two methods.The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity.

Effect of calcium antagonists on the chemosensitivity of two multidrug-resistant human tumour cell lines which do not overexpress P-glycoprotein

We have examined the ability of eight compounds to enhance adriamycin (ADM) sensitivity of two human tumour cell lines (a small cell lung cancer cell line, NCI-H69, and a fibrosarcoma cell line, HT1080) and their multidrug-resistant variants. The resistant cell lines (H69AR and HT1080/DR4) do not overexpress P-glycoprotein. Verapamil, nicardipine, perhexiline maleate, chloroquine, tamoxifen, clomiphene, prenylamine and trifluoperazine were tested alone and in combination with ADM for their cytotoxic effects. No major differences in sensitivity between the parent and resistant cell lines were noted when these agents were tested alone, except for HT1080/DR4 cells which exhibited a slight collateral sensitivity to nicardipine and H69AR cells which showed cross-resistance to chloroquine and clomiphene. When the chemosensitisers were combined with ADM no enhanced cytotoxicity of either parent cell line was observed. In HT1080/DR4 cells, verapamil showed only a modest dose-dependent chemosensitising effect while the other compounds had no effect. Verapamil and nicardipine enhanced ADM cytotoxicity in H69AR cells slightly but these effects were not dose-dependent. These results demonstrate that the reversal of drug resistance by verapamil and other calcium antagonists in a dose-dependent fashion is not an invariable property of multidrug-resistant tumour cells.

Specific immunoglobulin production and enhanced tumorigenicity following ascites growth of human hybridomas

Human X human hybridomas constructed with the B6 lymphoblastoid clone, which produces antitetanus toxoid (TT) antibody, and the lymphoblastoid cell line KR-4 or human hybrid myeloma KR-12, were adapted to growth as ascites in pristane-treated BALB/c nude mice by a single prior passage as a solid subcutaneous (s.c.) tumor in irradiated nude mice followed by in vitro culture. Both B6 X KR-4 and B6 X KR-12 hybrids produced anti-TT antibody and phenotypically resembled the lymphoblastoid KR-4, or the hybrid myeloma KR-12 parent, respectively. Growth as ascites increased the tumorigenicity of both hybrids in nude mice as measured by tumor incidence and rate of tumor growth. The observed increase in tumorigenicity of these hybrid cells after ascites growth was associated with a substantial loss of chromosomes. Passage of the B6 X KR-4 lymphoblastoid hybrid resulted in several reversible morphological changes characteristic of myeloma cells. These changes correlated with increased human Ig production. These observations provide a system for greatly amplifying human monoclonal antibody production.

Inhibitors of prostaglandin catabolism. I. Differential sensitivity of 9-PGDH, 13-PGR and 15-PGDH to low concentrations of indomethacin

Es zeigt sich, dass jedes der 3 Enzyme, die den Abbau von Prostaglandin in den Nieren erwachsener Ratten verursachen, durch schwache Konzentrationen von Indomethacin in steigendem Ausmass inhibiert werden könne: 9-PGDH>13-PGR>15-PGDH.

Differential inhibition of hepatic ferrochelatase by the isomers of N-ethylprotoporphyrin IX

Abstract The four isomers of N-ethylprotoporphyrin IX have been synthesized. The two isomers with the N-ethyl group on pyrrole rings A or B inhibit rat liver ferrochelatase as effectively as the corresponding N-methyl analogues, whereas those with the N-ethyl moiety on rings C or D are 30–100 times less effective. The ability of N-alkyl porphyrins to inhibit ferrochelatase thus depends not only on the size of the N-alkyl group but also on its precise location on the porphyrin face.

Establishment of a human large cell lung tumor line (QU‐DB) with metastatic properties in athymic mice

A continuous human cell line was established from a patient with large cell anaplastic lung carcinoma. This cell line, designated QU‐DB, has been in culture for over 36 months and grows as an adherent monolayer with a doubling time of 10–12 hours. Its morphology, ultrastructure, karyotype, ability to grow in soft agar and heterotransplantability, indicate it is a large‐cell lung tumor cell line of human origin. Three cell lines were established from metastatic tumors in nude mice receiving subcutaneous injections of QU‐DB cells. The morphology and growth characteristics exhibited by these cell lines were similar to the primary cell line. Karyotypic analysis of cell lines derived from the primary tumor and a metastasis to the diaphragm were similar, but cells from a liver metastasis culture showed additional karyotypic changes. This large cell lung tumor cell line may prove useful as a model system for studies of human tumor progression and metastasis.

Ferrochelatase and N-alkylated porphyrins

SummaryThe final step in heme synthesis is catalyzed by the mitochondrial enzyme, ferrochelatase. Characterization of this enzyme has been complicated by a number of factors including the dependence of enzyme activity on lipids. Purification of ferrochelatase from rat and bovine sources has been achieved only relatively recently using blue Sepharose CL-6B chromatography. When 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is given to animals, it produces a hepatic porphyria resembling human variegate porphyria thus providing an experimental system in which to study this disease. DDC has been found to cause the accumulation of a green pigment, identified as N-methyl protoporphyrin IX (N-MePP), which is a potent inhibitor of ferrochelatase. The source of the N-methyl substituent of N-MePP was found to be the 4-methyl group of DDC. Considerable evidence indicates that the protoporphyrin IX moiety of N-MePP originates from the heme moiety of cytochrome P-450 and that DDC is a suicide substrate for this hemoprotein. Some studies suggest that cytochrome P-450 isozymes differ in their susceptibility to destruction by DDC and its 4-alkyl analogues. Griseofulvin has also been reported to inhibit hepatic ferrochelatase in rodents but not in the 17-day old chick embryo nor in hepatocyte culture systems. Thus, the mechanism by which griseofulvin produces an experimental porphyria in chick embryo liver cell culture is different from that for rodents.

Drug-induced porphyrin biosynthesis—XIX: Potentiation of the porphyrin-inducing effects of SKF 525-A in the chick embryo liver by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine, an inhibitor of ferrochelatase

Abstract Pretreatment of 17-day-old chick embryos with 2-diethylaminocthyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) resulted in enhancement of hepatic δ-aminolevulinic acid (ALA)-synthetase activity and porphyrin accumulation induced by 3.5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC). The levels of [ 14 C]DDC and its metabolites in chick embryo livers were measured at different times after administration of [ 14 C]DDC in the presence and absence of SKF 525-A. It is concluded that the magnitude of inhibition of DDC metabolism following SKF 525-A pretreatment is too small to account for the enhanced inducing effects of DDC. DDC was found to inhibit ferrochelatase in chick embryo liver at doses considerably less than those required to induce ALA-synthetase activity. A dose of DDC was selected for administration to the chick embryo large enough to produce 95 per cent inhibition of ferrochelatase without affecting ALA-synthetase activity. When SKF 525-A was then administered, a marked synergistic effect was observed on ALA-synthetase activity and porphyrin accumulation. It is concluded that DDC, by inhibiting ferrochelatase, enhances the ability of SKF 525-A to induce ALA-synthetase activity and porphyrin accumulation.