Mutagenesis, Vol. 15, No. 4, 341-347,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Clastogenic effects of two tyrosine kinase inhibitors, Tyrphostin 23 and Tyrphostin 46, on a transformed (CHO-K1) and on a primary embryonic Chinese hamster cell line (CHE)
Department of Agrobiology and Agrochemistry, University of Tuscia,Via S. Camillo de Lellis, I-01100 Viterbo and 1 Centre for Evolutionary Genetics, CNR, c/o Department of Genetics and Molecular Biology, University `La Sapienza', Rome, Italy
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Abstract |
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Protein tyrosine kinases (PTKs) play fundamental roles in signal transduction pathways. Many proliferative diseases are characterized by deregulation of PTK activity, therefore PTKs appear as promising targets in the design of anticancer drugs. Tyrphostins are a family of synthetic compounds which efficiently target specific PTKs without competing for ATP and thus are much less cytotoxic with respect to conventional therapeutic agents. We tested two tyrphostin derivatives, Tyrphostin 23 and Tyrphostin 46, on a transformed (CHO-K1) and on a primary embryonic Chinese hamster cell line (CHE) to determine whether these compounds had a genotoxic effect. We found that the tyrphostins increased sister chromatid exchange frequency in both cell lines, but induced chromosomal aberrations only in the transformed CHO-K1 cell line when treatment was in the S phase of the cell cycle, and not in primary CHE cells. Such a result could have important therapeutic implications: it could mean that deregulation of signal transduction pathways in cells which already have a deficit in cell cycle control could cause chromosomal aberrations.
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Introduction |
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Regulation of cell proliferation is essential for the viability of all organisms. The cell cycle represents a focal point of biological research because all aspects of cellular physiology ultimately have a correlation with it. In recent years a great deal of effort has been expended in elucidating the molecular mechanisms at the basis of the cell cycle machinery. The most significant conceptual advance has been the recognition that most, if not all, cell cycle transitions are mediated by protein kinases (van den Heuvel and Harlow, 1993
; Lam and La Thangue, 1994; Morgan, 1995; Nigg, 1995; Pines, 1995; Resnitzky and Reed, 1995; Stillman, 1996). Among these the protein tyrosine kinases (PTKs), a class of enzymes implicated in signal transduction, play a crucial role in the control of normal and abnormal cell proliferation (Schlessinger and Ullrich, 1992; Fantl et al., 1993). A significant number of oncogene products (Collet et al., 1980; Hunter and Sefton, 1980; Levinson et al., 1980) and growth factor receptors (Ullrich et al., 1985; Yaish et al., 1988; Lyall et al., 1989) have PTK activity. Enhanced PTK activity has been observed in many cancers and in some non-malignant proliferative diseases, such as atherosclerosis (Ross, 1989, 1993) and psoriasis (Elder et al., 1989; Cook et al., 1992), and in a large number of inflammatory responses, such as septic shock (Weinstein et al., 1991; Dong et al., 1993). Therefore, PTKs and the signalling pathways in which they are involved appear to be promising targets in the design of drugs directed against proliferative diseases (Levitzki and Gazit, 1995; Klohs et al., 1997; Buolamwini, 1999; Fabbro et al., 1999; Fry, 1999; Traxler and Furet, 1999).
Tyrphostins are a series of low molecular weight synthetic compounds, originally patterned after erbstatin, used as PTK inhibitors (Yaish et al., 1988). They bind with high affinity to the substrate site of the epidermal growth factor receptor (EGF-R), even if different compounds of this family also show affinity for other PTKs and their exact mechanism of action remains to be elucidated.
In this work two tyrphostin derivatives, Tyrphostin 23 (Tyr23) and Tyrphostin 46 (Tyr46) were tested on a transformed and on a primary cell line, namely Chinese hamster ovary cells (CHO-K1) and Chinese hamster primary embryonic cells (CHE). The aim of the present study was to evaluate whether agents acting on cell signalling pathways that do not produce direct lesions in DNA could have a genotoxic effect and cause chromosomal aberrations through an alteration in cellular metabolism.
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Materials and methods |
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Cell lines
Immortalized CHO-K1 cells were cultured in Ham's F10 medium (Gibco) supplemented with 15% newborn calf serum (Gibco), 2% L-glutamine and antibiotics. CHO-K1 cells have a cell cycle length of ~14 h and a chromosome modal number of 21 ± 1.
CHE cells, obtained from Prof A.T.Natarajan (University of Leiden, Leiden, The Netherlands), were established from a 12-week-old embryo and cultured in Ham's F10 medium supplemented with 15% fetal calf serum (HyClone), 2% L-glutamine and antibiotics. CHE have a cell cycle length of ~14 h and a chromosome number of 22. CHE cells were used for the experiments at early passages (passages 6–12).
Both cell lines were incubated at 37°C in a humidified atmosphere of 5% CO2.
Chemicals
Tyr23 and Tyr46 were purchased from Sigma and dissolved in DMSO (Sigma) at a working concentration of 10 mM and prepared fresh every time. The tyrphostin treatment doses were chosen after preliminary toxicological tests in which mitotic index inhibition and proliferation index were taken into account. Doses >300 µM were too toxic for both cell lines.
G2 phase treatments
The experimental protocol for both cell lines is represented in Figure 1. To analyse cell cycle progression a 15 min pulse of 30 µg/ml 5-bromodeoxyuridine (BrdU) (Sigma) was given to the cells immediately before tyrphostin treatment. Then the cells were treated for 1 (Figure 1a) or 2 h (Figure 1b) or for 2 h followed by 2 h of recovery (Figure 1c) before harvesting. Colcemid (0.2 µg/ml) (Sigma) was added to the cultures 1 (Figure 1a) or 2 h (Figure 1b and c) before harvesting. Unlabelled metaphases were considered to correspond to G2 cells at the moment of tyrphostin treatment.
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S phase treatments
The experimental protocol for S phase treatments on both cell lines is represented in Figure 2
. To identify cells which were in the S phase of the cell cycle, a 15 min pulse of 30 µg/ml BrdU was given to the cultures immediately before tyrphostin treatment. In order to recognize early, middle and late replicating cells we used as a criterion the X chromosome BrdU labelling pattern as shown in Figure 3.
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G1 phase treatments
We seeded 25 cm2 flasks with 6x105 CHO-K1 metaphases obtained by shaking off exponentially growing cultures in order to obtain synchronized cells. After 90 min the cultures were treated for 1 h with Tyr23 or Tyr46 according to the experimental schedule of Figure 4
. In the last 2 h before each harvest the cells were treated with 0.2 µg/ml colcemid. Cell synchronization could not be performed on CHE cells because the mitotic shake off technique is not applicable efficiently to these cells. Therefore, we used BrdU to identify CHE cells treated in G1. CHE cells were pulse labelled with 30 µg/ml BrdU and sampled after different periods of recovery (17, 19 and 21 h), as shown in the experimental protocols in Figure 5. Colcemid (0.2 µg/ml) was given to the cells for the last 2 h before harvesting. Unlabelled metaphases were considered to correspond to G1 cells at the moment of tyrphostin treatment.
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Flow cytometric analysis
CHO and CHE cells were treated for 1 h with Tyr23 or Tyr46 and then, after 4, 8, 12 and 16 h recovery, cytometric analysis of cell cycle progression was performed. CHO cells were treated with two different doses of Tyr23 and Tyr46 (100 and 300 µM) while CHE cells received only the higher dose (300 µM).
Analysis of sister chromatid exchange (SCE) induction
In some preliminary experiments we found that the presence of tyrphostins could interfere with BrdU incorporation, therefore BrdU was added to the cultures one cell cycle before tyrphostin treatment, as shown in Figure 6. The tyrphostins at different concentrations were left in the culture medium continuously and therefore lower doses (15, 30 and 50 µM) than those used for induction of chromosomal aberrations had to be used. The tyrphostin concentrations in the medium were chosen after preliminary experiments to find the most suitable doses which produced an inhibition of cell cycle proliferation not greater than 50%. Colcemid (0.2 µg/ml) was given to the cells for the last 2 h before each harvest.
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Cell harvest and slide preparation
The cells were first trypsinized and then given a hypotonic shock (1% Na citrate for CHO-K1 and 75 mM KCl for CHE cells) for 10 min at 37°C. The cells were fixed with methanol/acetic acid (3:1). The cell suspensions were then dropped onto clean slides.
Staining of slides for the study of chromosomal aberrations
Slides corresponding to CHO-K1 and CHE cell treatment with tyrphostins in the G1, S and G2 phases of the cell cycle were processed using an immunocytogenetic technique with anti-BrdU antibodies conjugated with the fluorochrome fluorescein isothiocyanate (FITC). The slides were denatured for 1 min in 10 mM NaOH, 70% ethanol, dehydrated in a 70, 90 and 100% ethanol series and air dried. The slides were than incubated in a moist chamber for 30 min with 100 µl/slide mouse anti-BrdU antibody (Boehringer-Mannheim) diluted 1:100 in immunological buffer [phosphate-buffered saline (PBS), 0.5% bovine serum albumin, 0.5% Tween 20] under a 24x50 coverslip. After incubation the slides were washed three times with PBS and subsequently incubated with 100 µl/slide goat anti-mouse IgG–FITC antibody (Boehringer-Mannheim) diluted 5:100 in immunological buffer for 30 min. After three washes in PBS and dehydration in ethanol the slides were embedded with Vectashield mounting medium (Vector Laboratories) containing 0.3 µg/ml propidium iodide as counterstain.
Analysis of chromosomal aberrations, SCEs and statistical tests
The analysis of chromosome aberrations was carried out on 100 cells for each experimental point using a Zeiss (Axiophot) fluorescence microscope equipped with single and dual bandpass filters for FITC and propidium iodide and a CCD camera (Photometrix) operated by IPLab Spectrum software. Images were captured through the above-mentioned system and analysed on a 21 inch high resolution monitor. The 
2 test was used to check for significant differences in the yield of damaged cells between untreated and treated cells.
Mitotic index was analysed on 1000 cells for each experimental point.
The fluorochrome plus Giemsa (FPG) method (Perry and Wolfe, 1974) was used to analyse SCEs in CHO-K1 and CHE cells. SCE analysis was carried out on 50 well-spread metaphases for each experimental point. Student's t-test was used to check for significant differences in SCE means between untreated and treated cells. Cells with >10 SCEs were considered high frequency cells (HFCs). HFCs were defined by calculating the 95th percentile of the SCE distribution (Carrano and Moore, 1982). Dose-related increases in the frequency of SCEs were evaluated by linear regression and correlation analysis.
Flow cytometric analysis of the cell cycle
In the last 15 min before collecting cells for cytofluorimetric analysis, 45 µM BrdU was added to the cultures. The cells were then trypsinized and fixed in a 1:1 absolute methanol/PBS mixture. After denaturation in 3 N HCl for 30 min, samples were incubated with a mouse monoclonal anti-BrdU antibody (Daco, CA) for a further 30 min, washed twice with PBS, 0.5% Tween 20 and incubated with FITC-conjugated anti-mouse antibody (Vector Laboratories, CA) for 30 min. Finally, samples were stained with 20 µg/ml propidium iodide and analysed on a FACStar Plus flow cytometer (Becton Dickinson) equipped with a 5 W Innova 90 coherent laser with 488 nm wavelength excitation light. DNA histograms were analysed using WinMDI 2.5 software.
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Results |
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The data presented in this work are representative of experiments repeated several times (at least two to four times), all showing the same trend. The results obtained indicate that Tyr23 and Tyr46 cause chromosomal aberrations in the transformed CHO-K1 cell line while such an effect was not observed in CHE primary cells.
G2 phase treatment
At the doses used, in both cell lines a 30–50% reduction in mitotic index (data not shown), depending on the dose, was noticed. Tyr23 was more toxic in comparison with Tyr46, but no chromosomal aberrations were observed. In the case of the 2 h treatments followed by a 2 h recovery time (Figure 1c
), 40–60% of the cells showed a late replicating labelling pattern to signify that the tyrphostin treatments had also hit late S phase cells, however, even in this case no chromosomal damage was observed.
S phase treatment
In the CHE cells no clastogenic effects were noticed. In the CHO cells an increase in chromosomal aberrations related to the dose and recovery time (Table I) was observed, early S phase cells being more sensitive. At longer recovery times there was an induction of heavily damaged cells (with a typical early S phase labelling pattern) characterized by the presence of several chromatid exchanges and breaks (>10).
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G1 phase treatment
In both cell lines no clastogenic effects were observed in the cells treated in G1 (data not shown).
SCE induction
In CHO-K1 (Table II) and CHE (Table III) cells treated with Tyr23 or Tyr46 a statistically significant dose-related increase in SCE with a higher induction at later sampling times was detected. SCE induction, even if not very striking with respect to mean values, is characterized in both cell lines by the appearance of cells with a high frequency of SCE (HFCs).
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Flow cytometric analysis of the cell cycle progression
The flow cytometric analysis showed a different effect on cell cycle progression in the two cell lines. In Figures 7 and 8
the effects of a 1 h treatment with Tyr23 and Tyr46, respectively, on CHO and CHE cells are shown. In CHO cells there was an accumulation of cells in S phase at 4 and 8 h after treatment, which disappeared 12 h after the end of treatment. In CHE cells a block in G1 was evident 4 h after the end of treatment. This block disappeared after 8 h recovery (Figure 8). The successive recovery times (12 and 16 h) showed no difference in comparison with the control.
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Discussion |
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Our data indicate that tyrphostins induce an increase in SCE in both CHO-K1 and CHE cells but they are clastogenic only in CHO-K1 cells, provided that the cells are treated in S phase. These results imply that tyrphostins have a peculiar mechanism of induction of chromosomal aberrations and cannot simply be included in the classical S-dependent or S-independent categories of clastogenic agents (Scott et al., 1991
; Palitti, 1993). They are not known to directly induce DNA lesions so they apparently act indirectly, but their exact mechanism of action remains to be elucidated.
Tyrphostins are PTK inhibitors (Yaish et al., 1988) and consequently have an effect on control of cell cycle regulation. They show a strong affinity for the substrate site of the epidermal growth factor receptor, in contrast to other PTK inhibitors, which compete for ATP. Such a specificity confers on the tyrphostins a better chance of being more selective and non-toxic blockers of PTKs (Levitzki, 1990).
The striking difference in sensitivity between CHO-K1 and CHE cells could be related to the efficiency of cell cycle control, CHE being a primary cell line and CHO-K1 an immortalized cell line with altered p53 function (Hu et al., 1999).
The cytofluorimetric analyses showed that after tyrphostin treatments a G1 block was evident in CHE cells in the first hour of recovery (Figure 8), while CHO-K1 cells did not arrest in the G1 phase of the cell cycle but showed a delayed S phase (Figure 7). On the basis of the clastogenic effect observed one could speculate that PTK activities are differently regulated in the two cell lines and that in the case of CHE cells the tyrphostins have no effect when the cells are in S phase while in CHO-K1 cells the different steps which control PTK activity are less rigorous and consequently can be affected during S phase.
It is known that when cells are delayed in S phase, DNA strand breaks occur with high frequency (Li and Kaminskas, 1984) and some replicons initiate repeatedly (Vassiliev and Russev, 1984). Furthermore, tyrphostins may act not only as direct receptor tyrosine kinase inhibitors but also through downstream inhibition of PTKs controlling DNA synthesis (Sion-Vardy et al., 1995). These facts could explain our results in CHO-K1 cells.
The induction of SCE in both cell lines, although apparently not very striking with respect to the mean value per cell, but with the appearance of HFCs, is an indication that there are some disturbances during S phase, SCE induction being an S-dependent phenomenon. These results confirm that the mechanisms of induction of SCEs and chromosomal aberrations follow different pathways and that SCEs can be caused merely by an alteration in cell metabolism. Eventual differences in the two cell lines concerning apoptosis induction after tyrphostin treatment were also investigated, through analysis of apoptotic bodies, to determine whether the lack of a clastogenic effect in CHE cells could be caused by the elimination of damaged cells by apoptosis. No significant induction of apoptosis was found in either cell line (data not shown) and therefore the difference in sensitivity cannot be due to differential cell death.
If we had tested Tyr23 and Tyr46 only in CHO-K1 cells our conclusion would have been that these compounds are clastogenic, even if with a peculiar mechanism of action. Instead, the lack of induction of chromosomal aberrations in the primary CHE cells and analogous results from preliminary experiments performed on human peripheral blood lymphocytes in vitro (data not shown) indicate that, in the case of agents which alter the mechanism of cell cycle control, the outcome of treatment could be determined by the metabolic set-up of the cell.
Furthermore, the finding that tyrphostins act preferentially on transformed cells must be taken into account in their use as potential anti-proliferative agents (Levitzki and Gazit, 1995; Buolamwini, 1999) as it could have important therapeutic implications: it could mean that deregulation of the signal transduction network in cells, like CHO-K1, which already have a defect in cell cycle control (Hu et al., 1999) can cause chromosomal aberrations and increase genetic variability. This could consequently favour the establishment of clones resistant to therapy.
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Acknowledgments |
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This work was supported by grant 40% MURST.
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Notes |
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2 To whom correspondence should be addressed. Tel: +39 0761 357206; Fax: +39 0761 357242; Email: palitti{at}unitus.it
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References |
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Received on January 20, 2000; accepted on April 3, 2000.
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