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The tetracycline-controlled gene expression system utilizes the control elements of the tetracycline resistance operon encoded in TnlO of Escherichia coli to control gene expression in eukaryotic cells. Here we demonstrate the quantitative control of the expression of the luciferase gene, dihydrofolate reductase gene, and bcl-2 gene in HeLa S3 or Chinese hamster ovary AA8 cells using the tetracycline-controlled gene expression system. Regardless of the host cell lines or the genes being expressed, there is a common range of tetracycline concentration within which the expression of genes is most sensitively regulated. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system is higher than that of the wild-type CMV promoter/enhancer-driven system. Nonetheless, careful selection of stably transfected clones is necessary to achieve the optimally regulated gene expression using this system.

We have studied cell lines of rodent and human origin for their propensity to become resistant to antifolates (methotrexate, trimetrexate), phosphonacetyl-L-aspartate (PALA), and colcemid, resistances associated with amplification of the DHFR, CAD, and MDR1 genes, respectively. We have employed two different methods: (1) a shallow step-wise selection protocol, where time to attain specified resistance is the quantitative measure, (2) the frequency of resistant colonies at specified drug concentrations. Although there are advantages and disadvantages to both methods, the two methods gave the same relative ranking of cell lines. Striking differences in the propensity for gene amplification (resistance) were found: human cell lines were less prone to amplify genes than Chinese hamster ovary (CHO) cells. This ranking was similar with all of the agents employed. Additionally, we observed that whereas PALA resistance in CHO cells is associated with amplification of the CAD gene, PALA resistance in the two human cell lines studied (HeLaS3 and VA13) was not associated with amplification and/or overexpression of the CAD gene, and thus this resistance to PALA occurs by an unknown mechanism.

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured as a function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction (approximately 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure to UV light, measured by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.

We describe a bioassay for the quantitation of plasma methotrexate and trimetrexate levels employing intact cells. This assay is based on the intracellular saturation of dihydrofolate reductase with fluorescein-methotrexate (F-MTX) and its dose-dependent displacement by methotrexate or trimetrexate as monitored by flow cytometry. Serially diluted methotrexate-containing plasma, representing a wide chemotherapeutic range, produces F-MTX displacement curves similar to those of standard methotrexate solutions. There is no interference by normal plasma components such as folate and its reduced forms. Plasma methotrexate or trimetrexate concentration is the product of the 50% displacing concentration of standard antifolate (IC50) and the reciprocal of the plasma dilution which yields the same displacement. F-MTX competition with standard methotrexate displayed linear displacement from 18.0 +/- 3.1 to 33.7 +/- 1.5 nM (n = 10). The standard trimetrexate calibration curve was linear from 0.28 +/- 0.03 to 1.5 +/- 0.33 nM (n = 8). Thus, the bioassay sensitivities for methotrexate and trimetrexate are at least 18 and 0.3 nM, respectively. Comparison of methotrexate levels in 10 plasma specimens from cancer patients determined by the clinical enzyme inhibition assay and by our bioassay showed a high degree of correlation (r = 0.987).