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Carcinogenesis, Vol. 21, No. 8, 1611-1618, August 2000
© 2000 Oxford University Press

Carcinogenesis

Diverse chemical carcinogens fail to induce G1 arrest in MCF-7 cells

Qasim A. Khan2 and Anthony Dipple1

Laboratory of Comparative Carcinogenesis and
1 ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of three reactive potent chemical carcinogens on the passage of MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G1 after treatment with actinomycin D (a positive control), treatment with anti-benzo[a]pyrene dihydrodiol epoxide, N-acetoxy-N-2-fluorenylacetamide or N-methyl-N'-nitro-N-nitrosoguanidine, at doses consistent with survival of significant numbers of cells, caused the cells to accumulate in S phase, with little increase in those in G1. This property of these three reactive potent carcinogens, of diverse chemical types, to induce evasion of G1 arrest (the stealth property) presumably increases the likelihood of malignant change, because DNA replication continues on a damaged template. This stealth characteristic may be a major contributor to the tumorigenicity of DNA-adducting chemical carcinogens in general.

Abbreviations: AAF, 2-acetylaminofluorene; AcOAAF, N-acetoxy-N-2-fluorenylacetamide; BaPDE, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BP, benzo[a]pyrene; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; PBS, phosphate-buffered saline.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The chemical induction of cancer in experimental animals, first reported in 1915 (1), has allowed the identification of a large number of compounds from various chemical classes as potential carcinogenic hazards for man (2). Most potent chemical carcinogens are either intrinsically reactive towards cellular DNA or are metabolized to such reactive species in cellular systems (3,4). Thus, they are capable of mutating oncogenes (5) and/or tumor suppressor genes (6,7) to effect carcinogenic transformation of mammalian cells. However, species and tissue specificity in chemical carcinogenesis is only partially explained by levels of DNA damage and capacity for DNA repair; DNA alterations may be necessary, but usually are not sufficient, for causation of neoplasia (8). Other factors are clearly important, but are poorly understood.

One such factor may relate to the cellular defense mechanism provided by checkpoints, which can arrest the progress of cells with damaged DNA through the cell cycle, prior to entry into critical phases such as DNA replication (G1 arrest) and cell division (G2 arrest) (9–14). Tumor suppressor proteins such as p53, p21waf1/cip1 and pRb play crucial roles in the protective cellular response pathway of G1 arrest (15–25). For example, in the absence of functional p53 the G1 arrest function is lost, leading to increases in genomic instability and cell survival (26–29) that can accelerate the rate of oncogenesis (30).

Earlier, we observed that protective G1 arrest did not occur in human mammary carcinoma MCF-7 cells exposed to the polycyclic aromatic hydrocarbon carcinogen reactive metabolites benzo[g]chrysene dihydrodiol epoxide and 5-methylchrysene dihydrodiol epoxide (31–35). This ability of carcinogens to evade the cellular defense mechanism of G1 arrest (termed the stealth property) could make an important contribution to their carcinogenic potency. To determine if the stealth property is a general characteristic of potent chemical carcinogens, we have now studied three additional chemicals of diverse types of relevance to human cancer causation.

The polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) was among the first pure compounds recognized to exhibit carcinogenic activity in mice (36). This carcinogen is now known to be present in cigarette smoke (20–40 ng/cigarette) and has been implicated in the development of smoking-related lung cancer (7,37). BP undergoes metabolic activation to the carcinogenic metabolite anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE), which reacts predominantly with the N2 position of guanine residues and to a small extent with the N6 position of adenine residues in DNA (38–41). We have also studied N-acetoxy-N-2-fluorenylacetamide (AcOAAF), a reactive derivative of the liver carcinogen N-2-fluorenylacetamide (42) and representative of the aryl amine class of carcinogens. AcOAAF is carcinogenic (43) and it primarily modifies the 8 position (44) and, to a lesser extent, the N2 position (45) of guanine residues in nucleic acids. The third agent examined is the directly reactive alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (46), which is known to react to form mostly 7-substituted guanine, lesser amounts of 3-substituted adenine and some O6-substituted guanine residues in DNA (47). It represents the alkylating agents present in tobacco smoke and other environmental sources.

Using MCF-7 cells, which undergo p53-mediated G1 arrest following {gamma}-irradiation, UV or actinomycin D treatment (16,17), we show that BaPDE, AcOAAF and MNNG, at doses consistent with survival of a significant percentage of cells, fail to induce cell cycle arrest in the G1 phase. Thus, the stealth property appears to be associated with a wide range of types of potent chemical carcinogens.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
Racemic anti-BaPDE, MNNG and AcOAAF were obtained from the NCI Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO) and were stored at –80°C until use. Actinomycin D, nocodazole and propidium iodide were purchased from Sigma Chemical Co. (St Louis, MO).

Cells and media
Human mammary carcinoma MCF-7 cells, kindly provided by Dr D.A. Scudiero, were grown in RPMI 1640 medium containing 1% L-glutamine and 5% fetal bovine serum. The various chemicals used in these studies were added to cell cultures (5 ml) in 1.6 µl of acetone (0.1–1.5 µM BaPDE), 1.6 µl of DMSO (0.1–10 µM MNNG or 1–10 µM AcOAAF) or in 5 µl of water (actinomycin D).

Flow cytometry analyses
Cells were grown to exponential phase, treated with the indicated concentrations of BaPDE, MNNG, AcOAAF or actinomycin D or vehicle for 3 h and then grown in the presence (or absence) of nocodazole (0.4 µg/ml) for an additional 18 h. Cells were harvested, fixed in ice-cold 70% ethanol, stored at 4°C, washed with phosphate-buffered saline (pH 7.2) (PBS), treated with RNase A (3 U/ml) at 37°C for 15 min and stained with propidium iodide (50 µg/ml) for 20 min. DNA content of 10 000 cells/analysis was monitored with Becton-Dickinson fluorescence-activated cell analyzers, Model CellFit Cell-Cycle Analysis v.2.01.2, which presented the data as open graphs (Figures 2, 4 and 5GoGoGo), or Model Sync Wizard ModFit LT v.2.0, with data as closed graphs (Figures 7 and 8GoGo). Quantification was based on software programs provided by the manufacturer, as described (31).



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  		Fig. 2. Representative flow cytometry analyses of cultured MCF-7 cells grown in the absence of nocodazole (a), vehicle in the presence of nocodazole (b), 5 nM actinomycin D followed by nocodazole(c) or racemic BaPDE at the concentrations indicated followed by nocodazole (d–i). Exposure to actinomycin D or racemic BaPDE was for 3 h, followed by an 18 h nocodazole treatment. Data collected from three or four repetitions of this experiment are shown in Figure 3Go.

 


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  		Fig. 4. Representative flow cytometry analyses of MCF-7 cells grown in the presence of nocodazole for 18 h after a 3 h treatment with: (a) vehicle; (b) 5 nM actinomycin D; (c–f) (+)-anti-BaPDE at the concentrations indicated. A second experiment gave similar results, with corresponding G0/G1, S and G2/M values of 6, 5 and 89% for controls, 55, 5 and 40% for actinomycin D, 4, 5 and 91% for 0.01 µM (+)-anti-BaPDE, 5, 8 and 87% for 0.05 µM (+)-anti-BaPDE, 8, 37 and 55% for 0.1 µM (+)-anti-BaPDE and 32, 59 and 9% for 0.2 µM (+)-anti-BaPDE.

 


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  		Fig. 5. Representative flow cytometry analyses of MCF-7 cells grown in the presence of nocodazole for 18 h after a 3 h treatment with: (a) vehicle; (b) 5 nM actinomycin D; (c–m) (–)-anti-BaPDE at the indicated concentrations. Data collected from triplicate experiments with some of the (–)-anti-BaPDE concentrations are shown in Figure 6Go.

 


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  		Fig. 7. Flow cytometry analyses of MCF-7 cells grown in the presence of nocodazole for 18 h after a 3 h treatment with: (a and k) vehicle; (b and l) 5 nM actinomycin D; (c–j) MNNG at the indicated concentrations; (m–q) AcOAAF at the indicated concentrations. Values from a duplicate experiment for G0/G1, S and G2/M were 4, 10 and 86% for the MNNG study control, 43, 7 and 50% for actinomycin D, 3, 4 and 93% for 0.1 µM MNNG, 4, 2 and 94% for 0.5 µM MNNG, 4, 24 and 72% for 1.0 µM MNNG, 3, 1 and 96% for 2.0 µM MNNG, 5, 4 and 91% for 3.0 µM MNNG, 7, 17 and 76% for 4.0 µM MNNG, 20, 15 and 65% for 5.0 µM MNNG, 25, 56 and 18% for 10.0 µM MNNG, 7, 9 and 84% for the AcOAAF study control, 39, 10 and 51% for 5 nM actinomycin D, 7, 14 and 79% for 1 µM AcOAAF, 9, 39 and 52% for 3 µM AcOAAF, 13, 55 and 32% for 5 µM AcOAAF, 14, 64 and 22% for 8 µM AcOAAF and 27, 56 and 17% for 10 µM AcOAAF.

 


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  		Fig. 8. Flow cytometry analyses of MCF-7 cells grown in the presence of nocodazole for 18 h after a 3 h treatment with: (a) vehicle; (b) 5 nM actinomycin D; (c) 8 µM MNNG; (d) 5 µM AcOAAF. Analyses of MCF-7 cells grown in the absence of nocodazole for 18 h after a 3 h treatment with: (e) untreated; (f) 5 nM actinomycin D; (g) 8 µM MNNG, (h); 5 µM AcOAAF.

 
Cell survival assay
For cell survival analyses, 200 or 400 cells were placed in each well (35 mm diameter) of a 6-well plate and grown for 24 h. Fresh medium was then supplied and carcinogen was added to each well. Medium was replaced with fresh medium after 3 h and the cells were allowed to grow for 5 days (5 mitotic cycles). Thereafter, medium was removed and plates were washed with PBS followed by PBS/methanol (1:1) and then with methanol to fix the cells before staining with crystal violet (1%). After washing, colonies were then counted (35).

Statistical analysis
Most experiments were repeated at least twice and many three or four times. For experiments with replicates, the data for the experiments not illustrated are given in the figure legends. Data from experiments with three or four repeats were analyzed by analysis of variance (ANOVA) or the Kruskal–Wallis non-parametric test, as appropriate, using software from Instat.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The effects of exposure to BaPDE at doses of 0.1–1.0 µM on the survival of MCF-7 cells and their progress through the cell cycle are summarized in Figures 1–3GoGoGo. The lower BaPDE doses, 0.1–0.3 µM, permitted survival of 40–70% of the cells, whereas at the three highest concentrations >80% of the cells were killed (Figure 1Go).



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  		Fig. 1. Survival of MCF-7 cells exposed to the indicated concentrations of BaPDE as a percentage of untreated cells.

 


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  		Fig. 3. Percentages in G0/G1 (a), in S phase (b) and in G2/M phase (c) in three or four separate experiments (means ± SE). The following are significantly different (P < 0.05) by ANOVA. For G0/G1: control versus actinomycin D and versus 0.4 and 0.5 µM BaPDE; actinomycin D versus 0.1, 0.2 and 0.3 µM BaPDE; 0.1 µM versus 0.4 and 0.5 µM BaPDE. For S phase: control and actinomycin D versus 0.2, 0.3, 0.4 and 0.5 µM BaPDE; 0.1 µM versus 0.2, 0.3, 0.4 and 0.5 µM BaPDE. The decrease in percentage in S phase with dose in the range 0.2–0.5 µM (P = 0.065): for G2/M, control versus actinomycin D and versus 0.2, 0.3, 0.4 and 0.5 µM BaPDE; actinomycin D versus all BaPDE doses; 0.1 µM versus 0.2, 0.3, 0.4 and 0.5 µM BaPDE.

 
In the absence of BaPDE and nocodazole treatment (Figure 2aGo), the majority of cells were in the G1 phase. Exposure to nocodazole (a mitotic inhibitor) for 18 h resulted, as expected, in the majority of cells accumulating in G2/M (Figures 2b and 3cGoGo; 48). Actinomycin D, used as a positive control and known to activate p53 and cause cell cycle arrest in MCF-7 cells (17), induced cell cycle arrest in the G1 phase of the cell cycle, as expected, with 41 ± 3% in G1 compared with 4 ± 0.3% for nocodazole alone (Figures 2c and 3aGoGo). BaPDE at 0.1 µM did not affect cell cycle progression (Figures 2 and 3GoGo). At doses of 0.2 and 0.3 µM, the percentages of cells in G1 were significantly lower than after actinomycin treatment and, while higher than in controls, these increases were not of statistical significance (Figures 2e and f and 3aGoGo). Arrest in S phase was marked at these doses (Figures 2e and f and 3bGoGo), confirming DNA damage; since many of these cells would survive (Figure 1Go), perpetuation of this damage would occur. At 0.4 and 0.5 µM BaPDE the higher percentages in G1 and lower in S phase (Figures 2g and h and 3a and bGoGo) reflect severe damage preventing progression from G1 to S; these cells were essentially locked in the DNA content distribution applicable to untreated cells (35). Average percentages in G2/M were low after all of the effective doses of BaPDE (>=0.2 µM). These studies demonstrate that at concentrations allowing substantial cell survival, BaPDE exposure failed to induce G1 arrest effectively and, consequently, cells began replication on the damaged DNA and accumulated in S phase.

The cellular response to a range of concentrations of each of the optically active components of racemic BaPDE, i.e. the (+) and (–) enantiomers, was also investigated. The negative (nocodazole only) and positive (5 nM actinomycin D) controls were essentially as before (Figures 4a and b and 5a and bGoGo). At concentrations of 0.01 and 0.05 µM, the BaPDE (+) enantiomer had little effect (Figure 4c and dGo), but at 0.1 and 0.2 µM led to marked S phase cell accumulation, with G1 percentages still much lower than for the actinomycin D-treated cells (Figure 4e and fGo). The BaPDE (–) enantiomer had no effect except for an increase in the percentage of S phase cells at high concentrations, indicating toxic damage (Figures 5 and 6bGoGo). At all doses the percentage G1 cells was much lower than after actinomycin D (Figures 5l and mGo and 6a). For doses where experimental replicates permitted statistical analysis, an increase in cells in S phase at 0.3 µM chemical was not accompanied by any increase in G1 percentage (Figure 6a and bGo). These flow cytometry results are consistent with previous reports that the (+) enantiomer of BaPDE reacts far more extensively with DNA than the (–) enantiomer (38) and is correspondingly much more carcinogenic than the (–) enantiomer (49).



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  		Fig. 6. Average percentages of cells (± SE) in G0/G1 (a), in S phase (b) and in G2/M (c) in three experiments utilizing (–)-anti-BaPDE. Single control and actinomycin D treatment values from one experiment are given for comparison. The only significant difference among the carcinogen exposure groups was in the increased percentage of S phase cells after 0.3 µM compared with 0.05 µM (–)-anti-BaPDE.

 
To determine if other classes of chemical carcinogen might also exhibit stealth properties, we investigated the effects of MNNG (a directly reactive alkylating agent) and AcOAAF (the reactive form of an aromatic amide) on MCF-7 cells. For each of these carcinogens cells were exposed to a range of concentrations (0.1–10 µM) in order to determine doses of each agent that generated a toxic response detectable as an increase in percentage S phase cells (Figure 7Go). Substantial effects were apparent at 5 and 10 µM concentrations of MNNG (Figure 7i and jGo). A slight toxicity was apparent at 3 µM AcOAAF and a clear-cut effect was seen with 5 µM AcOAAF (Figure 7n and oGo). In neither situation did the percentages of cells in G1 approach that seen after actinomycin D.

To confirm the ability of damaged cells to continue through the cell cycle after MNNG or AcOAAF, experiments similar to those shown in Figure 7Go were carried out in the presence or absence of nocodazole with selected concentrations of chemical carcinogen. In the absence of a nocodazole block (48), the majority of the cells treated with actinomycin D were held in G1 (Figure 8fGo). After 8 µM MNNG an increase in the percentage of G1 cells and a decrease in cells in both S and G2/M phase in the absence of nocodazole suggests that some of the MNNG-damaged cells continued to G1 of the next cycle (Figure 8c and gGo). Similarly, after 5 µM AcOAAF the percentage of S phase cells was similar in the presence and absence of nocodazole, whereas reciprocal changes were noted for G1 and G2/M phase cells, indicating that at least some of those in G2/M proceeded to the next G1 (Figure 8d and hGo). When the data for MNNG and AcOAAF were pooled, including duplicate experiments for each, the average percentage in G0/G1 without nocodazole block was 26 ± 1.3 versus 16 ± 0.25 for those with nocodazole (P = 0.03), while the corresponding percentages for G2/M were 32 ± 4 and 50 ± 4 (P = 0.019).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
A general conclusion arising from the experiments described herein is that neither of the reactive polycyclic aromatic hydrocarbon metabolites studied so far nor the reactive AcOAAF and MNNG, despite their differences in structure and in their sites of reaction on DNA, are able to effectively arrest cells in G1 in MCF-7 cells. Consequently, cells attempt to replicate their DNA on a damaged template and this presumably enhances the mutation frequency, which could eventually lead to the transformation of cells to a malignant form, even if all cells do not manage to complete the cell division process.

Induction of G1 arrest in cells has been widely studied for agents that interfere with DNA transcription and replication, such as ionizing radiation, which causes DNA strand breaks, and actinomycin D, an intercalating agent. However, few studies have been reported for chemical carcinogens. Results similar to ours were reported for BPDE and AcOAAF treatment of human lymphoblasts, with S phase arrest due to DNA damage but little G1 arrest (50). The tumor promoter phenobarbital attentuated the G1 checkpoint seen in murine hepatocytes after bleomycin treatment (51). On the other hand, BP did appear to cause G1 arrest in 3T3 fibroblasts (52) and treatment of rats with 2-acetylaminofluorene (AAF) resulted in primary cultures of hepatocytes with characteristics of G1 arrest (53). An important role for cell cycle control in susceptibility to 7,12-dimethylbenz[a]anthracene-initiated skin papillomas was implied by the higher incidence of these in mice lacking the p21waf1/cip1 gene (54).

These diverse results indicate that, while we have obtained consistent evidence for the stealth effect for a variety of types of carcinogens in MCF7 mammary tumor cells, it may not be a universal property of all chemical carcinogen–cell interaction situations. It is therefore a variable that could well be a determining factor for susceptibility to mutation. It is not clear from the limited data available at present whether the different responses relate to the cell type or to the experimental models. The experiments showing G1 arrest all used parent carcinogens requiring metabolic activation, whereas the two studies illustrating the stealth property utilized activated carcinogens. It is possible that release of reactive oxygen, associated with metabolism of the relatively high doses of parent carcinogen, played a role, as reactive oxygen species are effective in inducing G1 growth arrest (55).

Also, the two cell lines showing the stealth effect were both neoplastic or preneoplastic. It is possible that insensitivity to G1 growth control after DNA alteration is a property acquired as part of the neoplastic progression process. If so, then the stealth property may have particular relevance for tumor promotion and progression, as suggested by the results with phenobarbital cited above. It is of particular interest that in the study with p21waf1/cip1-deficient mice, while the number of papillomas was enhanced compared with normal, the rate of malignant conversion of these tumors was not affected (54). Obviously there is also relevance in the context of resistance of cancers to therapy based on DNA-damaging agents.

Further systematic study of the stealth phenomenon and discovery of its underlying cellular and molecular mechanisms will be required to resolve these questions. The reason for the failure of some cells to undergo G1 arrest when DNA is damaged may relate to amounts and activities of regulatory proteins such as p53, p21waf1/cip1, pRb, etc. Available information is limited but suggests that the answer may not be simple. In mouse hepatocytes, p53, but not p21waf1/cip1, appears to be involved in the G1 arrest induced by bleomycin and attenuated by phenobarbital (51). Similarly, in the rat hepatocyte model (53), an increase in nuclear p53 but not up-regulation of p21waf1/cip1 is involved in AAF-induced G1 arrest; cyclin D1/cdk4 complex and nuclear translocation of cdk2 were reduced. In normal or p53-deficient mouse fibroblasts assayed for mutations in a shuttle vector, the absence of p53 greatly enhanced the number of mutations for a high but not a low dose of BaPDE (56).

In the cell line utilized in our present work, MCF-7, p53 is wild-type and its induction by anti-benzo[g]chrysene dihydrodiol epoxide, BP, BaPDE and dibenzo[a,l]pyrene has been demonstrated (31,57–60), whereas increases in p21waf1/cip1 were more variably seen and with a longer lag time. A delayed response for some genes may be part of the reason for the failure to arrest in G1. Kaspin and Baird observed substantially increased levels of p21waf1/cip1 and cell cycle arrest in the G2/M phase at longer time points (48–96 h) (58). For a low dose of (+)-syn-dibenzo[a,l]pyrene, p21waf1/cip1 was induced in the absence of a p53 increase (60). However, it remains to be established whether the long-term induction of p21waf1/cip1 after formation of hydrocarbon carcinogen–DNA adducts may lead to G2/M arrest. In spite of these response capabilities, even with ionizing radiation only a small fraction of MCF-7 cells were arrested in G1 (61), confirming our observations. More detailed work is needed to discover the complete mechanism for failure of cells such as MCF-7 to establish a G1 checkpoint after certain types of genotoxic damage.


   Notes
 
2 To whom correspondence should be addressed Email: khanq{at}ncifcrf.gov Back


   Acknowledgments
 
We would like to thank Dr Lucy M.Anderson and Dr Yih-Horng Shiao for critical input and advice, Dr Dominic A.Scudiero for providing MCF-7 cells and Kathleen Noer for flow cytometric analyses. This research was supported by a National Cancer Institute, DHHS, contract with ABL.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 29, 2000; revised May 15, 2000; accepted May 17, 2000.


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