TY - JOUR
AU - Dipple,, Anthony
AB - Abstract 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 G 1 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 G 1 . This property of these three reactive potent carcinogens, of diverse chemical types, to induce evasion of G 1 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. 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 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 (G 1 arrest) and cell division (G 2 arrest) ( 9 – 14 ). Tumor suppressor proteins such as p53, p21 waf1/cip1 and pRb play crucial roles in the protective cellular response pathway of G 1 arrest ( 15 – 25 ). For example, in the absence of functional p53 the G 1 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 G 1 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 G 1 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 N 2 position of guanine residues and to a small extent with the N 6 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 N 2 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 G 1 arrest following γ-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 G 1 phase. Thus, the stealth property appears to be associated with a wide range of types of potent chemical carcinogens. Materials and methods 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 5 ), or Model Sync Wizard ModFit LT v.2.0, with data as closed graphs (Figures 7 and 8 ). Quantification was based on software programs provided by the manufacturer, as described ( 31 ). 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 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 1–3 . 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 1 ). In the absence of BaPDE and nocodazole treatment (Figure 2a ), the majority of cells were in the G 1 phase. Exposure to nocodazole (a mitotic inhibitor) for 18 h resulted, as expected, in the majority of cells accumulating in G 2 /M (Figures 2b and 3c ; 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 G 1 phase of the cell cycle, as expected, with 41 ± 3% in G 1 compared with 4 ± 0.3% for nocodazole alone (Figures 2c and 3a ). BaPDE at 0.1 μM did not affect cell cycle progression (Figures 2 and 3 ). At doses of 0.2 and 0.3 μM, the percentages of cells in G 1 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 3a ). Arrest in S phase was marked at these doses (Figures 2e and f and 3b ), confirming DNA damage; since many of these cells would survive (Figure 1 ), perpetuation of this damage would occur. At 0.4 and 0.5 μM BaPDE the higher percentages in G 1 and lower in S phase (Figures 2g and h and 3a and b ) reflect severe damage preventing progression from G 1 to S; these cells were essentially locked in the DNA content distribution applicable to untreated cells ( 35 ). Average percentages in G 2 /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 G 1 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 b ). At concentrations of 0.01 and 0.05 μM, the BaPDE (+) enantiomer had little effect (Figure 4c and d ), but at 0.1 and 0.2 μM led to marked S phase cell accumulation, with G 1 percentages still much lower than for the actinomycin D-treated cells (Figure 4e and f ). 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 6b ). At all doses the percentage G 1 cells was much lower than after actinomycin D (Figures 5l and m 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 G 1 percentage (Figure 6a and b ). 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 ). 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 7 ). Substantial effects were apparent at 5 and 10 μM concentrations of MNNG (Figure 7i and j ). A slight toxicity was apparent at 3 μM AcOAAF and a clear-cut effect was seen with 5 μM AcOAAF (Figure 7n and o ). In neither situation did the percentages of cells in G 1 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 7 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 G 1 (Figure 8f ). After 8 μM MNNG an increase in the percentage of G 1 cells and a decrease in cells in both S and G 2 /M phase in the absence of nocodazole suggests that some of the MNNG-damaged cells continued to G 1 of the next cycle (Figure 8c and g ). 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 G 1 and G 2 /M phase cells, indicating that at least some of those in G 2 /M proceeded to the next G 1 (Figure 8d and h ). When the data for MNNG and AcOAAF were pooled, including duplicate experiments for each, the average percentage in G 0 /G 1 without nocodazole block was 26 ± 1.3 versus 16 ± 0.25 for those with nocodazole ( P = 0.03), while the corresponding percentages for G 2 /M were 32 ± 4 and 50 ± 4 ( P = 0.019). Discussion 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 G 1 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 G 1 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 G 1 arrest ( 50 ). The tumor promoter phenobarbital attentuated the G 1 checkpoint seen in murine hepatocytes after bleomycin treatment ( 51 ). On the other hand, BP did appear to cause G 1 arrest in 3T3 fibroblasts ( 52 ) and treatment of rats with 2-acetylaminofluorene (AAF) resulted in primary cultures of hepatocytes with characteristics of G 1 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 p21 waf1/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 G 1 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 G 1 growth arrest ( 55 ). Also, the two cell lines showing the stealth effect were both neoplastic or preneoplastic. It is possible that insensitivity to G 1 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 p21 waf1/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 G 1 arrest when DNA is damaged may relate to amounts and activities of regulatory proteins such as p53, p21 waf1/cip1 , pRb, etc. Available information is limited but suggests that the answer may not be simple. In mouse hepatocytes, p53, but not p21 waf1/cip1 , appears to be involved in the G 1 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 p21 waf1/cip1 is involved in AAF-induced G 1 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 p21 waf1/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 G 1 . Kaspin and Baird observed substantially increased levels of p21 waf1/cip1 and cell cycle arrest in the G 2 /M phase at longer time points (48–96 h) ( 58 ). For a low dose of (+)- syn -dibenzo[ a , l ]pyrene, p21 waf1/cip1 was induced in the absence of a p53 increase ( 60 ). However, it remains to be established whether the long-term induction of p21 waf1/cip1 after formation of hydrocarbon carcinogen–DNA adducts may lead to G 2 /M arrest. In spite of these response capabilities, even with ionizing radiation only a small fraction of MCF-7 cells were arrested in G 1 ( 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 G 1 checkpoint after certain types of genotoxic damage. Fig. 1. Open in new tabDownload slide Survival of MCF-7 cells exposed to the indicated concentrations of BaPDE as a percentage of untreated cells. Fig. 1. Open in new tabDownload slide Survival of MCF-7 cells exposed to the indicated concentrations of BaPDE as a percentage of untreated cells. Fig. 2. Open in new tabDownload slide 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 3 . Fig. 2. Open in new tabDownload slide 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 3 . Fig. 3. Open in new tabDownload slide Percentages in G 0 /G 1 ( a ), in S phase ( b ) and in G 2 /M phase ( c ) in three or four separate experiments (means ± SE). The following are significantly different ( P < 0.05) by ANOVA. For G 0 /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 G 2 /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. Fig. 3. Open in new tabDownload slide Percentages in G 0 /G 1 ( a ), in S phase ( b ) and in G 2 /M phase ( c ) in three or four separate experiments (means ± SE). The following are significantly different ( P < 0.05) by ANOVA. For G 0 /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 G 2 /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. Fig. 4. Open in new tabDownload slide 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 G 0 /G 1 , S and G 2 /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. Fig. 4. Open in new tabDownload slide 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 G 0 /G 1 , S and G 2 /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. Fig. 5. Open in new tabDownload slide 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 6 . Fig. 5. Open in new tabDownload slide 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 6 . Fig. 6. Open in new tabDownload slide Average percentages of cells (± SE) in G 0 /G 1 ( a ), in S phase ( b ) and in G 2 /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. Fig. 6. Open in new tabDownload slide Average percentages of cells (± SE) in G 0 /G 1 ( a ), in S phase ( b ) and in G 2 /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. Fig. 7. Open in new tabDownload slide 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 G 0 /G 1 , S and G 2 /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. Fig. 7. Open in new tabDownload slide 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 G 0 /G 1 , S and G 2 /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. Fig. 8. Open in new tabDownload slide 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. Fig. 8. Open in new tabDownload slide 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. 2 " To whom correspondence should be addressed Email: khanq@ncifcrf.gov 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 1 Yamagiwa,K. and Ichikawa,K. ( 1915 ) Experimentelle Studie über die Pathogenese der Epithelialgeschwülste. Mitt. Med. Fak. Tokio , 15 , 295 –344. 2 Searle,C.E. (1984) Chemical Carcinogens , 2nd Edn. ACS, Washington, DC. 3 Miller,J.A. and Miller,E.C. ( 1971 ) Chemical carcinogenesis: mechanism and approaches to its control. J. Natl Cancer Inst. , 47 , V–XIV . 4 Dipple,A. ( 1995 ) DNA adducts of chemical carcinogens. Carcinogenesis , 16 , 437 –441. 5 Brown,K., Buchmann,A. and Balmain,A. ( 1990 ) Carcinogen-induced mutations in the mouse c-Ha- ras gene provide evidence of multiple pathways for tumor progression. Proc. Natl Acad. Sci. USA , 87 , 538 –542. 6 Shimokado,K., Watanabe,H., Sumii,M., Miyagawa,K., Kamiya,K., Dohi,K. and Niwa,O. ( 1998 ) p53 gene mutation and loss of heterozygosity of chromosome 11 in methylcholanthrene-induced mouse sarcomas. Jpn J. Cancer Res. , 89 , 269 –277. 7 Denissenko,M.F., Pao,A., Tang,M. and Pfeifer,G.P. ( 1996 ) Preferential formation of benzo[ a ]pyrene adducts at lung cancer mutational hotspots in p53. Science , 274 , 430 –432. 8 Sancar,A. ( 1995 ) DNA repair in human cells. Annu. Rev. Genet. , 29 , 69 –105. 9 Hartwell,L.H. and Weinert,T.A. ( 1989 ) Checkpoints: controls that ensure the order of cell cycle events. Science , 246 , 629 –634. 10 Kastan,M.B., Onyekwere,O., Sidransky,D., Vogelstein,B. and Craig,R.W. ( 1991 ) Participation of p53 protein in the cellular response to DNA damage. Cancer Res. , 51 , 6304 –6311. 11 Kastan,M.B., Zhan,Q., El-Deiry,W.F., Carrier,F., Jacks,T., Walsh,W.V., Plunkett,B.S., Vogelstein,B. and Fornace,A.J. ( 1992 ) A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in Ataxia-Telangiectasia. Cell , 71 , 587 –597. 12 Dulic,V., Kaufmann,W.K., Wilson,S.J., Tlsty,T.D., Lees,E., Harper,J.W., Elledge,S.J. and Reed,S.I. ( 1994 ) p53-Dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell , 76 , 1013 –1023. 13 Namba,H., Hara,T., Tukazaki,T., Migita,K., Ishikawa,N., Ito,K., Nagataki,S. and Yamashita,S. ( 1995 ) Radiation-induced G1 arrest is selectively mediated by the p53-WAF1/Cip1 pathway in human thyroid cells. Cancer Res. , 55 , 2075 –2080. 14 Shackelford,R.E., Kaufmann,W.K. and Paules,R.S. ( 1999 ) Cell cycle control, checkpoint mechanisms and genotoxic stress. Environ. Health Perspect. , 107 , 5 –24. 15 Di Leonardo,A., Linke,S.P., Clarkin,K. and Wahl,G.M. ( 1994 ) DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev. , 8 , 2540 –2551. 16 Kuerbitz,S.J., Plunkett,B.S., Walsh,W.V. and Kastan,M.B. ( 1992 ) Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl Acad. Sci. USA , 89 , 7491 –7495. 17 Nelson,W.G. and Kastan,M.B. ( 1994 ) DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol. Cell. Biol. , 14 , 1815 –1823. 18 Lane,D.P. ( 1992 ) p53, guardian of the genome. Nature , 358 , 15 –16. 19 Husgafvel-Pursianen,K., Ridanpaa,M., Anttila,S. and Vainio,H. ( 1995 ) P53 and ras gene mutations in lung cancer: implications for smoking and occupational exposures. J. Occup. Environ. Med. , 37 , 69 –76. 20 Slack,R.S. and Miller,F.D. ( 1996 ) Retinoblastoma gene in mouse neural development. Dev. Genet. , 18 , 81 –91. 21 Adams,P.D. and Kaelin,W.G.Jr ( 1996 ) The cellular effects of E2F overexpression. Curr. Top. Microbiol. Immunol. , 208 , 79 –93. 22 Levine,A.J. ( 1997 ) p53, the cellular gatekeeper for growth and division. Cell , 88 , 323 –331. 23 Kubbutat,M.H.G. and Vousden,K.H. ( 1998 ) Keeping an old friend under control: regulation of p53 stability. Mol. Med. Today , 4 , 250 –256. 24 Levine,A.J. ( 1995 ) Tumor suppressor genes. Sci. Am. Sci. Med. , 2 , 28 –36. 25 Weinberg,R.A. ( 1995 ) The retinoblastoma protein and cell cycle control. Cell , 81 , 323 –330. 26 Wright,J.A., Smith,H.S., Watt,F.M., Hancock,M.C., Hudson,D.L. and Stark,G.R. ( 1990 ) DNA amplification is rare in normal human cells. Proc. Natl Acad. Sci. USA , 87 , 1791 –1795. 27 Tlsty,T.D. ( 1990 ) Normal diploid human and rodent cells lack a detectable frequency of gene amplification. Proc. Natl Acad. Sci. USA , 87 , 3132 –3136. 28 Livingstone,L.R., White,A., Sprouse,J., Livanos,E., Jacks,T. and Tlsty,T.D. ( 1992 ) Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell , 70 , 923 –935. 29 Yin,Y., Tainsky,M.A., Bischoff,F.Z., Strong,L.C. and Wahl,G.M. ( 1992 ) Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles . Cell , 70 , 937 –948. 30 Donehower,L.A. ( 1996 ) The p53-deficient mouse: a model for basic and applied cancer studies. Semin. Cancer Biol. , 7 , 269 –278. 31 Khan,Q.A., Vousden,K.H. and Dipple,A. ( 1997 ) Cellular response to DNA damage from a potent carcinogen involves stabilization of p53 without induction of p21 waf1/cip1 . Carcinogenesis , 18 , 2313 –2318. 32 Khan,Q.A., Agarwal,R., Seidel,A., Frank,H., Vousden,K.H. and Dipple,A. ( 1998 ) DNA adduct levels associated with p53 induction and delay of MCF-7 cells in S-phase after exposure to benzo[ g ]chrysene dihydrodiol epoxide enantiomers. Mol. Carcinog. , 23 , 115 –120. 33 Dipple,A., Khan,Q.A., Page,J.E., Pontén,I. and Szeliga,J. ( 1999 ) DNA reactions, mutagenic action and stealth properties of polycyclic aromatic hydrocarbon carcinogens. Int. J. Oncol. , 14 , 103 –111. 34 Khan,Q.A., Vousden,K.H. and Dipple,A. ( 1999 ) Lack of p53-mediated G1 arrest in response to an environmental carcinogen. Oncology , 57 , 258 –264. 35 Khan,Q.A., Vousden,K.H. and Dipple,A. (1999) `Stealth properties' contribute to the potent action of polycyclic aromatic hydrocarbon carcinogens. Polycyclic Aromatic Compounds . 36 Cook,J.W., Hewett,C.L. and Hieger,I. (1933) The isolation of a cancer-producing hydrocarbon from coal tar. Parts I, II and III. J. Chem. Soc. , 395–405. 37 Hecht,S.S., Carmella,S.G., Murphy,S.E., Foiles,P.G. and Chung,F.L. ( 1993 ) Carcinogen biomarkers related to smoking and upper aerodigestive tract cancer. J. Cell. Biochem. , 17F (suppl.) , 27 –35. 38 Nakanishi,K., Kasai,H., Cho,H., Harvey,R.G., Jeffrey,A.M., Jennette,K.W. and Weinstein,I.B. ( 1977 ) Absolute configuration of a ribonucleic acid adduct formed in vivo by metabolism of benzo[ a ]pyrene. J. Am. Chem. Soc. , 99 , 258 –260. 39 Jeffrey,A.M., Grzeskowiak,K., Weinstein,I.B., Nakanishi,K., Roller,H. and Harvey,R.G. ( 1979 ) Benzo[ a ]pyrene-7,8-dihydrodiol 9,10-oxide adenosine and deoxyadenosine adducts: structure and stereochemistry. Science , 206 , 1309 –1311. 40 Koreeda,M., Moore,P.D., Yagi,H., Yeh,H.J.C. and Jerina,D.M. ( 1976 ) Alkylation of polyguanylic acid at the 2-amino group and phosphate by the potent mutagen (±)-7b,8a-dihydroxy-9b,10b-epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene. J. Am. Chem. Soc. , 98 , 6720 –6722. 41 Cheng,S.C., Hilton,B.D., Roman,J.M. and Dipple,A. ( 1989 ) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[ a ]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. , 2 , 334 –340. 42 Miller,J.A. ( 1970 ) Carcinogenesis by chemicals: an overview—G. H.A. Clowes memorial lecture. Cancer Res. , 30 , 559 –576. 43 Miller,J.A. and Miller,E.C. ( 1969 ) The metabolic activation of carcinogenic aromatic amines and amides. Prog. Exp. Tumor Res. , 11 , 273 –301. 44 Kriek,E., Miller,J.A., Juhl,V. and Miller,E.C. ( 1967 ) 8-( N -2-Fluorenylacetamido)guanosine an arylamidation reaction product of guanosine and the carcinogen N -acetoxy- N -2-fluorenylacetamide in neutral solution. Biochemistry , 6 , 177 –182. 45 Westra,J.G., Kriek,E. and Hittenhausen,H. ( 1976 ) Identification of the persistently bound form of the carcinogen N -acetyl-2-aminofluorene to rat liver DNA in vivo. Chem. Biol., Interact. , 15 , 149–164. 46 Preussmann,R. and Stewart,B.W. (1984) N -nitroso carcinogens. In Searle,C.E. (ed.) Chemical Carcinogens , 2nd Edn. American Chemical Society, Washington, DC, pp. 643–828. 47 Lawley,P.D. and Thatcher,C.J. ( 1970 ) Methylation of deoxyribonucleic acid in cultured mammalian cells by N -methyl- N ′-nitro- N -nitrosoguanindine. Biochem. J. , 116 , 693 –707. 48 Zieve,G.W., Turnbull,D., Mullins,J.M. and McIntosh,J.R. ( 1980 ) Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. Exp. Cell Res. , 126 , 397 –405. 49 Buening,M.K., Wislocki,P.G., Levin,W., Yagi,H., Thakker,D.R., Akagi,H., Koreeda,M., Jerina,D.M. and Conney,A.H. ( 1978 ) Tumorigenicity of the optical enantiomers of the diastereomeric benzo[ a ]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7b,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene. Proc. Natl Acad. Sci. USA , 75 , 5358 –5361. 50 Black,K.A., McFarland,R.D., Grisham,J.W. and Smith,G.J. ( 1989 ) S-phase block and cell death in human lymphoblasts exposed to benzo[a]pyrene diol epoxide or N-acetoxy-2-acetylaminofluorene. Toxicol. Appl. Pharmacol. , 97 , 463 –472. 51 Zaher,H., Yang,T.J., Gelboin,H.V., Fernandez-Salgeuro,P. and Gonzalez,F.J. ( 1998 ) Effect of phenobarbital on hepatic CYP1A1 and CYP1A2 in the Ahr-null mouse. Biochem. Pharmacol. , 55 , 235 –238. 52 Vaziri,C. and Faller,D.V. ( 1997 ) A benzo[a]pyrene induced cell cycle checkpoint resulting in p53 independent G1 arrest in 3T3 fibroblasts. J. Biol. Chem. , 272 , 2762 –2769. 53 Lindeman,B., Skarpen,E., Thoresen,G.H., Christofferson,T., Wierod,L., Madshus,I.H. and Huitfeldt,H.S. ( 1999 ) Alteration of G1 cell cycle protein expression and induction of p53 but not p21Waf1 by the DNA-modifying carcinogen 2-acetylaminofluorene in growth stimulated hepatocytes in vitro . Mol. Carcinog. , 24 , 36 –46. 54 Weinberg,W.C., Fernandez-Salas,E., Morgan,D.L., Shalizi,A., Mirosh,E., Stanulis,E., Deng,C., Hennings,H. and Yuspa,S.H. ( 1999 ) Genetic deletion of p21WAF1 enhances papilloma formation but not malignant conversion in experimental mouse skin carcinogenesis. Cancer Res. , 59 , 2050 –2054. 55 Gausauge,S., Gausauge,F., Gause,H., Poch,B., Shoenberg,M.H. and Berger,H.G. ( 1997 ) The induction of apoptosis in proliferating human fibroblasts by oxygen radicals is associated with a p53- and p21WAF1CIP1 induction. FEBS Lett. , 404 , 6 –10. 56 Courtemanche,C. and Anderson,A. ( 1999 ) Multiple mutations in a shuttle vector modified by ultraviolet irradiations, (+/–)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and aflatoxin B(1) have different properties than single mutations that may be generated during translesion synthesis. Mutat. Res. , 430 , 23 –36. 57 Ramet,M., Castren,K., Jarvinen,K., Pekkala,K., Turpeenniemi-Hujanen,T., Soini,Y., Paakko,P. and Vahakangas,K. ( 1995 ) P53 protein expression is correlated with benzo[ a ]pyrene–DNA adducts in carcinoma cell lines. Carcinogenesis , 16 , 2117 –2124. 58 Kaspin,L.C. and Baird,W.M. ( 1996 ) Anti- benzo[a]pyrene-7,8,-diol-9,10-epoxide treatment increases levels of the proteins p53 and p21 waf1 in the human mammary carcinoma cell line MCF-7. Polycyclic Aromatic Compounds , 10 , 299 –306. 59 Venkatachalam,S., Denissenko,M.F. and Wani,A.A. ( 1997 ) Modulation of (±)- anti -BaPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase. Oncogene , 14 , 801 –809. 60 Luch,A., Kudla,K., Seidel,A., Doemer,J., Greim,H. and Baird,W.M. ( 1999 ) The level of DNA modification by (+)- syn -(11 S ,12 R ,13 S ,14 R )- and (–)- anti -(11 R ,12 S ,13 S ,14 R )-dihydrodiolepoxides of benzo[ a , l ]pyrene determined the effect on the proteins p53 and p21WAF1in the human mammary carcinoma cell line MCF-7. Carcinogenesis , 20 , 859 –865. 61 Nagasawa,H., Keng,P., Maki,C., Yu,Y. and Little,B. ( 1998 ) Absence of a radiation induced first cycle G1-S arrest in p53+ human tumor cells synchronized by mitotic selection. Cancer Res. , 58 , 2036 –2041. © Oxford University Press
TI -  Diverse chemical carcinogens fail to induce G 1 arrest in MCF-7 cells 
JO - Carcinogenesis
DO - 10.1093/carcin/21.8.1611
DA - 2000-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/diverse-chemical-carcinogens-fail-to-induce-g-1-arrest-in-mcf-7-cells-h24TcQ7MH2
SP - 1611
VL - 21
IS - 8
DP - DeepDyve
ER -