In-vitro schedule-dependency of eo9 and miltefosine in comparison to standard drugs in colon-cancer cells.

In vitro screening of new antitumor drugs is often limited to fast chemosensitivity assays. The potency of drugs is studied by measuring growth inhibition at the end of the assay after a fixed drug exposure time. In this study, the human colon cancer cell lines HT29 and SW620, were exposed to drugs for 1 h, 24 h (followed by culture in drug-free medium), 72 h and for 72 h with drug renewal every 24 h. Growth inhibition was evaluated at 48 and 72 h after initial drug addition, using the sulforhodamine B (SRB) assay. Chemosensitivity profiles of the investigational drugs EO9, a bioreductive alkylator, and the ether lipid miltefosine (HPC), were compared to those of drugs with different mechanisms of action: doxorubicin, cisplatin (DDP) and 5-fluorouracil. HPC displayed recovery from growth inhibition, in both cell lines, after drug exposures of 1 and 24 hours. DDP showed an increase for both cell lines (p < 0.05) of growth inhibition when drug was being refreshed every 24 h, compared to 72 h continuous drug exposure. Doxorubicin, 5-fluorouracil and EO9 were more potent in SW620 cells. These data suggest that more initial information can be obtained from drug screening assays, when both continuous and short-term drug exposures are studied, instead of one fixed drug exposure time, and indicates that daily renewal of drugs, may reveal possible drug stability and availability problems.

Transformation of mouse fibroblasts with the oncogenes H-ras OR trk is associated with pronounced changes in drug sensitivity and metabolism.

Malignant activation of oncogenes ras or trk is implicated in a number of solid tumors and leukemias. We determined the chemosensitivity profile of wild-type mouse NIH-3T3 fibroblasts, and that of NIH-3T3 lines transformed by the H-ras (S2-721) and trk (106-632) oncogenes, against 11 different drugs from various classes. Differences in sensitivity were related to drug accumulation and metabolism. Both ras- and trk-transformed cell lines were less sensitive to cisplatin (CDDP) and doxorubicin (DXR) than the wild type. NIH-3T3 transformants expressing H-ras were less sensitive than those expressing trk or the wild type to the indoloquinone EO9, methotrexate and arabino-furanosylcytosine. No clear difference in sensitivity was observed for vincristine, VP-16, or the new cytidine analog 2',2'-difluoro-deoxycytidine. In both ras- and trk-transformed cell lines sensitivity to 5FU was increased moderately, but sensitivity to 5'deoxy-5-fluorouridine (5'dFUR) was increased markedly. Only the trk-transformed line NIH-3T3 was more sensitive to 2'deoxy-5-fluorouridine. Expression of P-glycoprotein was not different between the 3 cell lines but DXR accumulation in both mutants was decreased, indicating a non-P-glycoprotein-associated difference in sensitivity. Conversion of 5'dFUR to 5FU (catalyzed by pyrimidine nucleoside phosphorylases) was 5-10 times higher in both mutants than in the wild type. The activity of the phosphoribosyl-transferase (direct conversion of 5FU to FUMP) was comparable, but the rate of conversion of 5FU to fluorouridine (FUR) was lower in the wild type, as well as that of 5FU to FUMP via FUR. In contrast, the activity of thymidylate synthase, the target enzyme for fluoropyrimidines, was higher in the wild-type cells. The concentrations of both purine and pyrimidine nucleotides were lower in cells expressing trk. In conclusion, transformation of cells with the H-ras or trk oncogenes can markedly influence sensitivity to several drugs and affect normal metabolism and that of several anti-cancer agents.

Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing.

The sulforhodamine B (SRB) protein stain assay was compared with the tetrazolium (MTT) colorimetric assay for in vitro chemosensitivity testing of various human tumour cell lines. The SRB assay provided a better linearity with cell number and a higher sensitivity, and its staining was not cell-line dependent. In contrast to the MTT assay, the SRB assay stained recently lysed cells. Cell debris, however, was not stained by SRB and therefore the drug sensitivity data were not affected.

Complete and partial deficiencies of complement factor D in a Dutch family.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.