The "comparative growth assay": examining the interplay of anti-cancer agents with cells carrying single gene alterations - PubMed (original) (raw)
The "comparative growth assay": examining the interplay of anti-cancer agents with cells carrying single gene alterations
P Hausner et al. Neoplasia. 1999 Oct.
Abstract
We have developed a "comparative growth assay" that complements current assays of drug effects based on cytotoxicity. A co-culture of two cell lines, one of which is fluorescently labeled, is exposed to a cytotoxic agent and the proportion of fluorescent cells is compared with that of a baseline unexposed co-culture. For demonstration purposes, two HCT116 cell lines (an hMLH1 homozygous and an hMLH1 heterozygous mutant), altered by insertion of vector alone or the same vector carrying an insert for the expression of enhanced green fluorescent protein (EGFP), were exposed to numerous "anti-cancer" agents. The assay was further validated in a system of two cell lines differing only in the expression of the breast cancer resistance protein (BRCP). The assay allowed the estimation of the duration of action of a particular agent. Assessment of the agent's differential activity over a given time in culture could be expressed as a selection rate, which we chose to describe on an "average selection per day" basis. We conclude that this assay: 1) provides insight into the differential dynamic effects of chemotherapeutic agents or radiation; and 2) allows, through the use of matched cell lines, the investigation of critical physiologic features that govern cell sensitivity.
Figures
Figure 1
A schematic depiction of a typical comparative growth assay. The comparative growth assay was designed to measure the differential growth inhibition, or the selective pressure cytotoxic agents exert on two distinct cell populations. Co-cultures of a nonfluorescent cell line (-) mixed with a cell line tagged with a fluorescent marker (+) are seeded. One day later, one of two identical co-cultures is exposed to a study agent and the other one (baseline) is not. After a period of exposure (e.g., 1 hour), the agent is washed away and the co-cultures are grown for 3 more days. The ratio of fluorescent-to-nonfluorescent cells is determined by flow cytometry (insets). If the studied agent is more growth-inhibitory against one cell type than against the other, then the ratio of fluorescent-to-nonfluorescent cells becomes different in the exposed co-culture compared to the ratio in the undisturbed baseline co-culture. The selection rate expressed as an average selection per day is calculated for the given example. The calculation assumes a 3-day observation period (see Appendix A for derivation of this equation).
Figure 2
Log odds ratio plotted against time depicts the duration of the selective effect (N+/O co-culture). To establish the duration of agents' selective effects on two cell lines differing in their MMR proficiency, the logarithm of the ratio of odds of finding a green cell in a N+/O co-culture exposed to MNNG at different concentrations and compared to an unexposed N+/O co-culture was plotted against the MNNG concentration. All co-cultures were exposed to MNNG for 2 hours and further incubated for 48 to 120 hours. The selective effect, evident as a downward slope of the line (see Appendix) lasts for at least 120 hours for the MNNG concentration of 3 µM (triangles) and at least 72 hours for the MNNG concentration of 1 µM (triangles).
Figure 3
The comparative growth assay allows for estimates of the differential growth inhibitory effect of an agent that can be independent of the interval since initiation of exposure. Co-cultures of two cell lines, “N” (MMR-competent) and “O” (MMR-defective), differing in the status of their hMLH1 gene were exposed for 1 hour to varying concentrations of MNNG or media containing only diluent. They were subsequently grown in the absence of the cytotoxic agent. N was carrying a vector-expressing EGFP rendering the cell line fluorescent green (N+, see Materials and Methods section). The odds of finding a green cell in the exposed culture was compared to the odds of finding a green cell in the control culture and expressed as an odds ratio. The co-cultures were assayed 2, 3, 4, and 5 days after initiating exposure and the odds of fluorescent/nonfluorescent established. The D (days elapsed since exposure) root of the odds ratio is the selection rate per day, S. The data show selection by MNNG against the MMR-competent fluorescent cell line (N+) (the comparative chance of finding a green cell in the exposed co-culture decreases as the concentration of MNNG increases). S depends on the concentration of MNNG and not on the time of evaluation (at least over the course of this 5-day experiment) with the possible exception of 3 µM concentration of MNNG analyzed at 48 hours following exposure. Using selection rate (S) expressed as an average on a per day basis allows comparisons of experiments done with different follow-up times and reflects the time span of the drug's selective activity.
Figure 4
Establishing the optimal ratio of fluorescent/nonfluorescent cells in co-cultures for reproducible flow cytometry analyses. The comparative growth assay, which is based on co-culture of fluorescent and nonfluorescent cell lines, uses their ratio (expressed as odds) for evaluation. The odds at evaluation are obviously dependent not only on the differential growth rate of the observed cell lines but on the initial (seeding) ratio as well. To establish a range of odds at the time of FACS analyses associated with a low variation, data from preliminary experiments with a 1 hour MNNG exposure of an O+/N co-culture done in triplicates were utilized. The variation coefficient (CV=100x standard deviation÷mean) of triplicate odds was plotted against its average odds. The scatter diagram shows that the lowest variation (1% to 6%) is achieved when the odds fluorescent/nonfluorescent at the time of evaluation by flow cytometry are above 0.18. There was more variation within the measurement if the ratio of the fluorescent cells to the nonfluorescent ones at the time of flow cytometry was below 0.18. As the same variation can be expected in the inverse situation of scarce nonfluorescent cells, the same restriction applies on the upper end of the scale. To generate results with low variation, the experiment has to be set up (by adjusting the seeding ratio) to keep the odds ratio at evaluation in the range of 0.18 to (1-0.18).
Figure 5
Dose-effect relationship for MNNG. The differential growth inhibitory effect of MNNG was studied by utilizing the comparative growth assay in which the growth of fluorescently labeled cells (+) is compared with unlabelled cells in the presence or absence (baseline) of the tested agent. Four different co-cultures, two experimental, O+/N, N+/O, and two control, N+/N and O+/O, were exposed for 1 hour to different concentrations of MNNG and compared after 3 days to the same co-cultures which were left untreated. Means of the calculated selection factor averaged per day (S) with 95% confidence limits are plotted against concentrations of MNNG. MNNG favors the growth or survival of MMR-deficient O cells, so that fluorescently tagged O cells become more prevalent in a co-culture (O+/N) (solid line with triangles) as compared to the baseline unexposed co-culture. Inversely fluorescently tagged N cells (N+) decrease in prevalence in a N+/O co-culture (solid line with squares). The prevalence of fluorescent cells in a mix of fluorescent and nonfluorescent MMR-deficient cells (O+/O) (dashed line with triangles) changed orders of magnitude less significantly. The same was true for the prevalence of fluorescent cells in a mix of fluorescent and nonfluorescent cells MMR-competent cells (N+/N) (dashed line with rectangles). MNNG is ineffective at O.1 µM and effective between 1 and 10 µM. At high concentration (10 µM), the differential sensitivity is diminished, as the cytotoxic effect of the agent for each individual cell line is more pronounced. *p<.0001. A possible negative selective interaction of EGFP and MNNG is suggested by the N+/N combination at 1 µM MNNG and the O+/O combination at 10 µM MNNG.
Figure 6
Drugs selecting in vitro for MMR-deficient cells and drugs selecting for MMR-proficient cells. The differential growth inhibitory effects against MMR-deficient and proficient cells of cytotoxic agents that are components of common anticancer regimens were investigated by the comparative growth assay. Co-cultures of fluorescently labeled MMR-deficient O cells and unlabelled MMR-proficient N cells (O+/N, continuous line) and vice versa (N+/O, dotted line) were exposed to 6-thioguanine for 2 hours at the indicated concentrations and to all other drugs for 48 hours. Assay times varied between 48 and 72 hours. The selection factor averaged per day (S) with 95% confidence limits was plotted against the drug concentration. Drugs selecting for MMR-deficient cells: MNNG and 6-thioguanine were evaluated after a 1-hour incubation and cisplatin, doxorubicin, paclitaxel, etoposide, irinotecan, 5-fluorouracil evaluated after a 48-hour exposure. Drugs selecting for MMR-deficient cells: Fludarabine (rectangles) at a concentration of 10 µM and gemcitabine (triangles) at concentrations of 100 µM. *p<.005; #p<.05.
Figure 7
Differential effect of radiation on co-cultures of MMR-deficient (O) and MMR-proficient (N) cell lines. The differential effect of radiation on a MMR-deficient and a MMR-proficient cell line was determined by the comparative growth assay. The cell lines were cultured for 72 hours after the radiation exposure. The selection rate is shown plotted against the radiation dose. Control co-cultures (dashed lines), O+/N co-culture (rectangles), N+/O co-culture (triangles). A selection for MMR-deficient cells is observed with irradiation. A dose-effect relationship is seen. *p<.0001.
Figure 8
Relative resistance to mitoxantrone provided by BCRP overexpression. Using a quadruplet of cell lines either overexpressing the breast cancer resistance protein (B) or transfected with a control vector only (V), made either to express the EGFP (+) or no fluorescent proteins, differential toxicity to mitoxantrone was studied. Whereas control combinations not differing in the expression of BCRP show no selection for green cells by mitoxantrone, green cells overexpressing BCRP (B+) are selected for, and green cells transfected with the vector only are selected against (V+).
References
- Weinstein JN, Myers TG, PM OC, Friend SH, Fornace AJ, Jr, Kohn KW, Fojo T, Bates SE, Rubinstein LV, Anderson NL, Buolamwini JK, van Osdol WW, Monks AP, Scudiero DA, Sausville EA, Zaharevitz DW, Bunow B, Viswanadhan VN, Johnson GS, Wittes RE, Paull KD. An information-intensive approach to the molecular pharmacology of cancer. Science. 1997;275:343–349. - PubMed
- Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994;263:802–805. - PubMed
- Prasher DC. Using GFP to see the light. Trends Genet. 1995;11:320–323. - PubMed
- Fink D, Zheng H, Nebel S, Norris PS, Aebi S, Lin TP, Nehme A, Christen RD, Haas M, MacLeod CL, Howell SB. In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res. 1997;57:1841–1845. - PubMed
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