Detection of subpopulations resistant to DNA-damaging agents in spheroids and murine tumours

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Abstract

Chinese hamster V79 monolayers, V79 spheroids, and SCCVII murine tumours were examined for DNA damage using the alkaline comet assay and for cell killing by measuring clonogenicity following a 1-h exposure to doxorubicin, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-N-oxide (4-NQO), etoposide, or 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine). Greater heterogeneity in DNA damage was evident in spheroids compared to monolayers exposed to these drugs, and cell survival was correlated with the fraction of cells which lacked sufficient DNA damage following treatment with tirapazamine or doxorubicin. Cell sorting experiments verified that subpopulations of cells resistant to DNA damage were also more resistant to cell killing. Significant heterogeneity was observed in cells from SCCVII tumours exposed to tirapazamine and etoposide, and comet DNA content was used to independently assess DNA damage to aneuploid tumour cells and diploid host cells. These results suggest that, for some drugs, the comet assay may be an effective method of identifying drug-resistant cells in solid tumours.

Introduction

Many methods have been developed and applied to measure numbers of DNA single- and double-strand breaks produced by a wide variety of genotoxic agents (Ahnstrom, 1988; Iliakis, 1991). With few exceptions, these are population-based assays and it must be assumed that all cells within a population will respond identically to a DNA-damaging agent. While, generally, a reasonable assumption for cells in tissue culture incubated with an agent in a well-controlled environment, a global measure of DNA strand breakage is unlikely to be adequate to describe damage produced in cells exposed in solid tumours. The tumour microenvironment can be heterogeneous in terms of the cell types present, degree of cell packing, pH, oxygenation, and nutrient status, and these, in turn, may affect proliferation rate, drug metabolism, drug accessibility, and DNA repair (Sutherland, 1988; Durand, 1994). While several methods can be used to measure DNA damage to individual cells, only the comet assay provides adequate resolution to detect resistant subpopulations which differ in damage by as little as a factor of 2 (Olive et al., 1993b). This is an important strength of this method, and one which separates the comet assay from other methods for measuring DNA damage. This paper examines heterogeneity in DNA damage measured in two tumour models: multicell spheroids composed of Chinese hamster V79 cells; and a squamous cell carcinoma, SCCVII, grown subcutaneously in C3H mice. Spheroids and tumours were exposed to drugs which have different mechanisms of action: (1) MNNG, a direct-acting alkylating agent; (2) etoposide, a topoisomerase II inhibitor which selectively damages proliferating cells of spheroids (Olive et al., 1993a); (3) 4-NQO, a carcinogen which is rapidly metabolized and penetrates poorly into spheroids (Olive and Durand, 1983); (4) tirapazamine, a bioreductive drug which is preferentially toxic to hypoxic cells (Zeman et al., 1986; Brown, 1993); and (5) doxorubicin, a chemotherapeutic agent which is absorbed predominantly by the external cells of spheroids (Durand, 1981). Experiments were designed to examine the degree of heterogeneity in DNA damage following exposure to these agents, and to determine whether DNA damage measured using the comet assay could be predictive for cell killing.

Section snippets

Chemicals

Tirapazamine (SR-4233), kindly provided by Dr. Martin Brown at Stanford, was dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg/ml (5.56 mM). Doxorubicin and MNNG were purchased from Sigma, St. Louis, MO, and 4-NQO was obtained from Aldrich Chemical Co, Milwaukee, WI. Doxorubicin was dissolved in distilled water and kept as a frozen stock (2 mg/ml). Both 4-NQO and MNNG were maintained as frozen stock solutions (2 mg/ml) in dimethylsulphoxide.

Spheroid culture

Chinese hamster V79-171b lung

Results

The response of Chinese hamster V79 monolayer cells was examined after exposure to the alkylating agent, MNNG. Cell survival, measured using clonogenicity as the endpoint, was exponential with dose (Fig. 1a), and the relationship between dose and average DNA damage measured using the alkaline comet assay was linear over this range (Fig. 1c). Heterogeneity in response between individual cells of the exposed population was relatively small (Fig. 1b). However, as the dose of MNNG increased, there

Discussion

The good correlation between cell survival measured using clonogenicity and DNA damage for tirapazamine and doxorubicin using the comet assay (Fig. 6) demonstrates the potential of a method which can detect DNA damage in individual cells. When the goal is to sterilize the tumour, the critical factor is not whether all cells sustain an appropriate level of DNA damage, but whether some cells escape sufficient damage. These will be the cells which repopulate the tumour. Similarly, when damage to

Acknowledgements

This work was supported by grants from the National Cancer Institute of Canada and by USPHS Grant CA-37879.

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