Mutagenesis vol. 18 no. 5 pp. 405-410,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents
International Center for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy and 1Institute of Clinical Physiology, National Research Council, Via Moruzzi 1, I-56124 Pisa, Italy
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It has been shown that the ethylating agent diethylsulphate (DES) induces centromere-containing micronuclei with kinetics suggesting that molecules other than DNA could be targets. In quiescent Chinese hamster fibroblasts CHEF/18, O6-alkylated bases inhibit ribosomal protein S6 kinase (S6K1), the terminal member of a kinase cascade responsible for an increased rate of protein synthesis, but not extracellular signal-activated kinases (ERK1/2) or terminal kinases of a second cascade which activates transcription. The inhibition correlates with the appearance of abnormal metaphases at the following mitosis, suggesting that alkylation of the nucleotide pool and inhibition of S6K1 could be one of the mechanisms leading to chromosome loss by alkylating agents. To clarify the role of protein kinases in chromosome loss induced by alkylating agents, we have studied the effects of DES and methylnitrosourea (MNU) on S6K1 and ERK1/2 activation by growth factors. The alkylating agents were studied in a battery of Chinese hamster fibroblasts (CHEF/18, CHO and ClB) with normal and mutated p53 to control for DNA damage-induced activation of p53, which could indirectly inhibit protein kinases. The role of repair in induction of micronuclei was studied in mismatch repair-proficient CHO and repair-deficient ClB cells. Our results indicate that DES induced micronuclei in a mismatch repair-independent manner, within 8 h of treatment, in agreement with a role for S6K1 inhibition in micronucleus formation. MNU induced centromere-containing micronuclei only in CHO cells, one cell cycle after treatment, without any detectable influences on either kinase cascade, suggesting a role for mismatch repair in chromosome loss.
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Introduction |
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Alkylating agents are known to induce a variety of genetic changes, ranging from point mutations to aneuploidy in mammalian cells (Bautz and Freeze, 1960
; Natarajan et al., 1984). Although alkylation of DNA purines is considered to be responsible for many of the effects exerted by alkylating agents such as induction of point mutations, chromosomal aberrations and sister chromatid exchanges (Kaina et al., 1993), the molecular mechanisms responsible for chromosome loss are still a matter of debate.
Targets other than DNA were proposed by Nusse et al. (1989), who showed that, in the Chinese hamster cell line V79, the ethylating agent diethylsulphate (DES) induces chromosome loss within 4–8 h of treatment (‘early induction’) and before DNA damage undergoes attempted repair. Modified purines such as O6-ethylguanine (O6etG) and O6-methylguanine (O6meG) are aneugenic in mammalian cells in culture, after their conversion to nucleotides (Bonatti et al., 1986, 1995a). Since nucleotides are readily alkylated by methylating and ethylating agents (Snow and Mitra, 1988), it was postulated that in vivo formation of O6-alkylated nucleotides could be responsible for ‘early’ chromosome loss (Bonatti et al., 1995b).
When investigating cell cycle-specific targets in Chinese hamster fibroblasts (CHEF/18), synchronized in G1 phase by serum starvation and exposed to O6-alkylated bases, aberrant metaphases were observed at the next mitosis and the cells were most sensitive to base analogues added before S phase, suggesting that targets were present in G1/S (Simili et al., 1995).
Activation of two main protein kinase cascades takes place in the G1/S transition of the cell cycle: Ras–Raf–ERK1/2 (extracellular signal-regulated kinase), necessary for activation of transcription factors and mRNA synthesis; phosphoinositide 3-kinase (PI3K)–Akt–mTOR–S6K1 (ribosomal S6 protein kinase 1), essential for initiation of protein synthesis (Denhard, 1996). We have shown that in G1 synchronized CHEF/18 cells, O6-alkylated bases inhibit activation of the terminal kinase S6K1 but not ERK1/2 (Bonatti et al., 2000), suggesting that the S6K1 pathway could be involved in chromosome segregation. This concept is supported by the finding that rapamycin, a specific inhibitor of the protein kinase mTOR (mammalian target of rapamycin), an upstream activator of S6K1, is also aneugenic in both mammalian (CHEF/18 and human lymphoblastoid cells) and yeast cells (Bonatti et al., 1998). It was thus interesting to investigate whether alkylating agents with aneugenic effects, such as DES and the methylating agent methylnitrosourea (MNU), also inhibit S6K1 in Chinese hamster fibroblasts.
It is known that DNA damage induced by alkylating agents activates p53, which in turn leads to a rapid block of the cell cycle (Jaiswal and Narayan, 2001). In order to distinguish between a direct inhibition of protein kinases involved in G1/S transition and an indirect effect due to p53 inhibition of the cell cycle, Chinese hamster cell lines with wild-type and mutated p53 were used. The effects of DES and MNU on S6K1 and ERK1/2 were analysed in the following cell lines synchronized in G1: CHEF/18, a diploid cell line non-tumourigenic in nude mice with wild-type p53 (Rainaldi et al., 1998), the tumourigenic cell line CHO with mutated p53 (Hu et al., 1999) and its derived mismatch repair-deficient clone ClB (Aquilina et al., 1988). The time course of micronucleus induction was studied in exponentially growing CHO and ClB cells to elucidate the role of DNA damage repair mechanisms and in particular of mismatch repair in chromosome loss. Our results indicate that the ethylating agent DES inhibits S6K1 but not ERK activation in all cell lines studied independent of p53 activity; moreover, DES induces a significant increase in micronuclei in both CHO and ClB cells 8 h after treatment, in keeping with the hypothesis that targets other than DNA could be involved. The methylating agent MNU did not inhibit S6K1 or ERK1/2 in any of the cell lines and the induction of micronuclei took place in the mismatch repair-proficient cell line CHO 24–48 h after treatment, suggesting that repair of DNA damage is required for chromosome loss induced by this alkylating agent.
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Materials and methods |
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Growth factors, antibodies and chemicals
Epidermal growth factor (EGF) was obtained from Boehringer (Mannheim, Germany), foetal calf serum (FCS) from ICN Laboratories (Amsterdam, The Netherlands) and insulin from Sigma (Milan, Italy). MNU and DES were obtained from Fluka (Germany), rapamycin, wortmannin, O6meG and O6etG from Sigma.
The polyclonal antibody against S6K1 was from Santa Cruz (Heidelberg, Germany), the secondary antibody [peroxidase-conjugated goat anti-rabbit immunoglobulins (IgG)] and the protein kinase inhibitor peptide PKI were from Sigma. ERK1/2 were detected with a monoclonal antibody from ICN laboratories. In some experiments a polyclonal antibody specific for ERK2 from Santa Cruz was used. The secondary antibody (peroxidase-conjugated rabbit anti-mouse IgG) was from DAKO (Glostrup, Denmark). The ECL (Enhanced Chemiluminescence) kit was obtained from Amersham (The Netherlands) and CREST antiserum from Antibodies Inc. (Davis, CA).
Cell culture
Diploid Chinese hamster CHEF/18 fibroblasts were routinely grown at 37°C in a 5% CO2 incubator, seeded at a starting density of 2 x 104 cells/cm2 in 
MEM (Gibco, Grand Island, NY) supplemented with 10% FCS. All experiments were performed with CHEF/18 cells at early passages (10–20) in order to avoid cell transformation, which occurs spontaneously in culture during serial passages. The average cell cycle was 22 h. Chinese hamster ovary fibroblasts (CHO and ClB), kindly provided by Dr M.Bignami (Istituto Superiore della Sanità, Roma, Italy) (Aquilina et al., 1988), were routinely grown at a starting density of 2 x 104 cells/cm2 at 37°C and 5% CO2 in medium composed of 50% MEM and 50% DMEM (Gibco) with 10% FCS. The average cell cycle was 16 h for both CHO and ClB.
Cell survival was evaluated by determining the colony forming ability after treatment. Briefly, cells were seeded at a density of 200 cells/5 cm Petri dishes, allowed to attach and treated with alkylating agents for 3 h. The medium was then changed and the cells allowed to form colonies. The IC50 dose (50% inhibitory concentration) represents the concentration of mutagen which reduces colony formation by 50% relative to controls. The IC50 dose for DES was 6 mM in the three cell lines; for MNU the IC50 doses were 150 µM for CHEF/18 cells, 100 µM for ClB and 20 µM for CHO cells (data not shown), respectively.
Experimental design
To determine the effect of alkylating agents on protein kinase activation by growth factors, cells grown to confluence (CHEF/18) or near confluence (CHO and ClB) were serum starved. Cells arrested in the G0/G1 phase re-enter the cell cycle after addition of growth factors and divide once. Alkylating agents were added to serum-starved cells 1 h before addition of growth factors to allow formation of alkylated nucleotides and extracts made at the indicated times after growth factor stimulation (see Cell extracts). As positive controls for inhibition of the S6K1 pathway, rapamycin and wortmannin, inhibitors respectively of mTOR and PI3K, were added 30 min before growth factors.
Micronucleus induction was studied in CHO and ClB cells treated with alkylating agents during exponential growth (2 days after plating) as it is very difficult to detect micronuclei in serum-starved sub-confluent cells stimulated with growth factors. Cells were treated with the IC50 doses in order to balance possible effects of cell toxicity.
Cell extracts
CHEF/18 cells grown for 5 days, with one medium change on day 3, were serum starved for 20 h. Cells, predominantly in G0/G1, were extracted at the indicated times after addition of new medium containing chemicals and EGF as previously described (Simili et al., 1994).
CHO and ClB cells were grown for 3 days to sub-confluence; new medium containing 10% conditioned medium and 1% FCS was then added for 36 h. At this point about 75% of cells were in G1 and re-entered the cell cycle after addition of 10% FCS (FACS analysis; data not shown). Cell extracts were made at the indicated times after addition of chemicals and FCS.
S6K1 phosphorylation
Extracts were prepared as described elsewhere (Han et al., 1995). Briefly, cells were washed twice with ice-cold buffer containing 120 mM NaCl, 50 mM Tris–HCl pH 8.0, 20 mM NaF, 10 mM pyrophosphate, 5 mM EGTA, 1 mM EDTA, 1 mM benzamidine, 0.1 mM phenylmethylsulphonyl fluoride and then extracted in the same buffer containing 1% Nonidet P-40 (NP40). Cell extracts were centrifuged at 12 000 g and the supernatants were immediately frozen in liquid nitrogen and stored at –70°C. Equal amounts of proteins from extracts were subjected to SDS–PAGE, electrophoretically tranferred onto Hybond nitrocellulose (Amersham) and S6K1 phosphorylation detected with the specific polyclonal anti-S6K1 antibody followed by peroxidase conjugated anti-rabbit IgG. In order to amplify the signal the ECL chemiluminescence method was used.
In vitro S6 kinase 1 assay
Extracts (
6 µg protein/assay) were mixed with kinase buffer containing 50 µM ATP, 5 µC [
-32P]ATP (Amersham), 2 mg/ml 40S ribosomal subunits and 0.5 µM PKI in a total volume of 20 µl and incubated at 37°C for 30 min. The reaction was stopped by addition of Laemmli sample buffer. The extracts were then boiled, centrifuged and the supernatants loaded onto 10% SDS–polyacrylamide gels; the amount of S6 phosphorylation was visualized by autoradiography and quantified with the aid of a PhosphorImager.
Ribosomal S6 phosphorylation in vivo
To determine the phosphorylation of ribosomal protein S6 in vivo quiescent cells were extracted according to previously described procedures (Nielsen et al., 1982). After centrifugation to remove nuclei, the supernatant was layered on a 1 M sucrose cushion and the ribosomes pelleted. The ribosomes were then resuspended in Laemmli sample buffer and run on urea/SDS–acrylamide gels. After western blotting, a monoclonal antibody against human ribosomal protein S6, kindly provided by Dr H.Towbin (Ciba Geigy, Basel Switzerland), was used to detect both the phosphorylated and dephosphorylated forms of the protein. Peroxidase-conjugated rabbit anti-mouse IgG were then used followed by incubation with 4-nitrochloronaphthol and H2O2.
ERK1/2 phosphorylation
ERK1/2 activation was evaluated by determining the phosphorylation of the kinases. To determine ERK1/2 kinase phosphorylation extracts were prepared as described (Meloche, 1995). After electrophoresis proteins were blotted onto Hybond nitrocellulose and ERK1/2 detected with antibodies recognizing either both isoforms (44 and 42 kDa) (ICN) or only the 42 kDa isoform (Santa Cruz). To amplify the signal the ECL chemiluminescence method was used.
Micronucleus assay
Cells were seeded at a density of 1.7 x 104/5 cm Petri dish (on coverslips) and 2 days later during exponential growth were treated with DES and MNU at concentrations corresponding to the respective IC50 doses. Cells were treated for 2 h with the alkylating agent, the medium was changed and 4, 8, 24 and 48 h later cells were fixed in ethanol for micronucleus detection. Each value represents the average of two independent experiments and at least 1500 cells were analysed. To discriminate between micronuclei with or without kinetochores (indicative of the presence of entire chromosomes or acentric fragments, respectively), immunostaining with CREST antiserum was performed as previously described (Nusse et al., 1989).
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Results |
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Effect of DES and MNU on growth factor-induced S6K1 activation in Chinese hamster cells
The finding that O6etG and, to a lesser extent, O6meG specifically inhibit S6K1 but not ERK1/2 in CHEF/18 cells and induce aneuploidy in the following metaphase suggested that early induction of aneuploidy by alkylating agents could be related to inhibition of the S6K1 pathway, probably via nucleotide pool alkylation (Bonatti et al., 1995a
; Simili et al., 1995). To investigate this possibility three cell lines of Chinese hamster cells, CHEF/18, CHO and ClB, were treated while quiescent or partially quiescent (G0 or G1 phase) with concentrations equal to the IC50 doses of DES or MNU and the effects on S6K1 and ERK1/2 activation by growth factors determined.
We have previously found that addition of EGF to quiescent CHEF/18 cells induces a rapid phosphorylation of S6K1, indicative of the protein kinase activation, which can be visualized on western blots as the appearance of slower migrating bands (Bonatti et al., 1998). Wortmannin and rapamycin, respectively inhibitors of PI3K and mTOR, block S6K1 activation by inhibiting upstream elements and preventing full phosphorylation of the protein (Han et al., 1995). In CHEF/18 cells the two agents inhibit the phosphorylation of S6K1, shown by the disappearance of the two slowest migrating bands (Figure 1). DES pre-treatment also causes the disappearance of the slowest migrating band, indicative of a partial inhibition of S6K1 phosphorylation (Figure 2).
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To verify whether the partial inhibition of S6K phosphorylation reflected altered kinase activity, extracts from growth factor-stimulated and treated cells were mixed with 40S ribosomal subunits (source of ribosomal protein S6) and the in vitro kinase activity measured. The results indicated that DES treatment decreases the activity of the kinase by about 40% (Figure 3a,b). S6K1 activity in vivo was also evaluated, by determining the phosphorylation state of ribosomal S6 in extracts from treated cells, and the results show that DES markedly inhibits S6 phosphorylation (Figure 4). These results indicate that inhibition of S6K1 phosphorylation by DES is accompanied by an inhibition of the kinase activity both in vitro and in vivo.
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CHEF/18 cells have wild-type p53 (Rainaldi et al., 1998
) which is known to block the cell cycle following DNA damage by alkylating agents (Jaiswal and Narayan, 2001). To show that S6K1 inhibition was not a consequence of a cell cycle block by p53, the effect of DES on SK1 phosphorylation was also studied in two cell lines with inactive p53, CHO and the derived clone ClB. In serum-starved CHO cells, S6K1 is dephosphorylated, in keeping with the majority of the cells being in G1, and addition of FCS induces a rapid phosphorylation of the kinase with an apparent maximum at 1 h (Figure 5a). DES treatment before FCS addition induced a partial inhibition of S6K1 phosphorylation as observed in CHEF/8 cells (lanes 4 and 5). In serum-starved ClB cells (Figure 5b, lane 1) S6K1 appeared to be phosphorylated, suggesting an incomplete G1 block, however, FCS addition further increased phosphorylation of the protein kinase with disappearance of the lower (non-phosphorylated) band and appearance of two upper (fully phosphorylated) bands (lanes 2 and 5). DES (lane 4) inhibits S6K1 phosphorylation to the same extent as wortmannin (lanes 3 and 6), while rapamycin (lane 8) was a more potent inhibitor. This set of data suggests that DES partially inhibits S6K1 activity in CHO and ClB cells, probably affecting upstream protein kinases.
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The effect of DES on ERK activation following growth factor addition was studied in the three cell lines. In Figure 6 the results obtained with CHO (Figure 6a) and ClB (Figure 6b) are reported, showing that ERK phosphorylation is not inhibited by this agent in CHO cells, with a slight inhibition being seen in ClB cells with 10 mM DES. Interestingly, wortmannin, an inhibitor of PI3K, induces a slight inhibition of ERK1/2 phosphorylation in this cell line, suggesting that either it is not a very specific inhibitor or the two pathways cross-talk.
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In CHEF/18 cells the methylating agent MNU (150 µM) inhibited neither S6K1 (Figure 7a) nor ribosomal protein S6 phosphorylation (Figure 7b) and the same results were obtained in CHO and ClB treated with MNU at the respective IC50 doses of 20 and 100 µM (Figure 8). In summary, these data indicate that DES inhibits S6K1 but not ERK activation, as previously shown for O6etG, while MNU does not induce any significant inhibition of either pathway. To our surprise, both CHO and the tolerant ClB clone appeared to be more sensitive than CHEF/18 to MNU; at present we have no explanation for this different sensitivity other than the presence in CHEF/18 cells of O6-methylguanine methyltransferase activity, which is totally absent in Chinese hamster ovary cells (Kaina et al., 1993
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Induction of micronuclei in CHO and ClB cells by DES and MNU
Early induction of chromosome loss has been observed after DES treatment in different cell lines (Nusse et al., 1989
), however, it is not known whether induction of micronuclei by methylating agents such as MNU follows similar kinetics and/or whether DNA repair mechanisms have a role in chromosome loss. It has been shown that the DNA adduct O6meG, the main lesion induced by methylating agents, contributes to chromosomal aberrations in the second cell cycle after treatment, by attempted correction of O6meG:T mispairs by mismatch repair (Armstrong and Galloway, 1997). To investigate the role of mismatch repair in chromosome loss, the time course of micronucleus (centromere positive and negative) induction was studied in both mismatch repair-proficient (CHO) and repair-deficient (ClB) cell lines. If mismatch repair was responsible for chromosome loss induced by MNU, one would expect an increase in centromere-positive micronuclei only in CHO cells one cell cycle after treatment. The DNA adduct O6etG is not a target for mismatch repair, so the same increase in micronuclei was expected in CHO and ClB (Armstrong and Galloway, 1997). Micronucleus induction was determined in exponentially growing CHO and ClB cells as micronuclei were not detectable in serum-starved sub-confluent cells, due to cell crowding. The results in Table I indicate that DES induces a significant increase (P = 0.046) in total micronuclei in the two cell lines at 8 h (90% of which are CREST+) and a highly significant increase (P > 0.001) 24 and 48 h after treatment. MNU induces no significant increase in micronuclei at 4 and 24 h after treatment in either CHO or ClB cells, while a significant increase (P > 0.001) is observed after 48 h in CHO cells but not in the mismatch repair-deficient cell line. The induction of CREST+ and CREST– micronuclei in CHO and ClB by MNU, reported in more detail in Table II, indicates that the frequency of both types of micronuclei increases significantly 48 h after treatment in CHO while in ClB no significant increase in either type was observed. These findings suggest that mismatch repair of DNA damage might be involved in chromosome loss induced by MNU.
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Discussion |
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We have previously demonstrated that CHEF/18 cells grown in the presence of alkylated purines, O6meG and O6etG, show chromosome displacement at mitosis and aneuploidy (Simili et al., 1995
). In the case of O6etG and, to a lesser extent, O6meG this effect correlated with inhibition of S6K1 (Bonatti et al., 2000), the terminal kinase of one of the major protein kinase cascades (PI3K–Akt–S6K1) activated by growth factors (Denhard et al., 1996). This has led us to investigate the mechanisms of aneuploidy induction by the classic alkylating mutagens DES and NMU. A series of Chinese hamster cell lines was chosen with normal (CHEF/18) and inactive p53 (CHO and ClB), since this transcription factor, activated by damaged DNA, inhibits cell proliferation and could complicate interpretation of the results. CHO and ClB lines, proficient and deficient in mismatch repair, were used to determine the role, if any, of this type of repair in chromosome loss.
Our results show that in CHO and ClB cells DES induces a significant increase in micronuclei within 8 h of treatment, the majority of which are CREST+. The number of micronuclei continues to increase up to 48 h after treatment. This kinetic is similar to that described (Nusse et al., 1989) for V79 cells, in which CREST+ micronuclei reached a plateau at 8 h but CREST– micronuclei continued to accumulate at 24–48 h. It is evident that DES can induce ‘early’ chromosome loss in both CHO and ClB, by mismatch repair-independent mechanisms.
The effect of DES on two of the major protein kinase cascades, in G1 synchronized cells, was measured. The activity of the PI3K–Akt–S6K1 cascade was normally evaluated indirectly as the degree of phosphorylation of S6K1. However, in CHEF/18 cells we have determined both the in vitro and in vivo activity of the kinase by measuring its ability to phosphorylate ribosomal protein S6, and found good agreement between the apparent degree of phosphorylation of the kinase and its activity. DES treatment was found to inhibit, totally or partially, S6K1 phosphorylation in all cell lines tested, both synchronized in G1 and stimulated with growth factors. The phosphorylation of ribosomal protein S6 is needed to up-regulate translation of a family of mRNAs encoding components of the protein synthetic machinery (Stewart and Thomas, 1994) and its inhibition was shown to affect the levels of cell cycle regulating proteins such as cyclin D and p27kip (Pene et al., 2002; Decker et al., 2003); assuming that ‘early’ chromosome loss takes place in cells treated during the transition G1/S, this disturbance could be one of the causative factors of aneuploidy induced by DES. However, we cannot rule out the possibility that attempted repair of DNA damage in G1/S cells might also be responsible for the early induction of chromosome loss.
DES did not affect the activity of the other major kinase cascade, RAS–Raf–ERK1/2, involved in transcriptional regulation. Should DES have produced disruption in cell metabolism, we would have expected both cascades to be affected. Moreover, the inhibition of S6K1 by DES is independent of p53 activity, indicating that the cell cycle block induced by p53 after DNA damage is unlikely to be the cause of this effect. Finally, the effects of DES on micronuclei induction and S6K1 are quite similar to those seen in CHEF/18 cells with O6etG (Simili et al., 1995), in keeping with a kinase-mediated chromosome loss possibly induced by the alkylated nucleotide.
The methylating agent MNU displayed very different characteristics, as CREST+ and CREST– micronuclei were only induced in significant numbers in the mismatch-proficient cells CHO and then at 24 h or later times. At the tested doses (IC50) MNU was without significant effect on either S6K1 or ERK1/2 in any of the cell lines tested. It may be noted that the IC50 for MNU is lower than that of DES, in keeping with the greater cytotoxicity of this methylating agent (Armstrong and Galloway, 1997). However, O6meG was one of the weakest alkylated bases as far as S6K1 inhibition was concerned (Bonatti et al., 2000); it is thus possible that the intracellular concentrations of O6-methylated nucleotides never reached a level great enough to inhibit the protein kinase. In any case, S6K1 inhibition does not play a role in MNU induction of chromosome loss and the main target appears to be DNA itself. A possible mechanism by which chromosome loss could be achieved is the deletion of parts of the chromosomes necessary for chromosome segregation. Chinese hamster chromosomes are rich in interstitial telomeric sequences (TTAGGG)n (Bertoni et al., 1996). As the most important lesion induced by methylating agents is O6meG, these regions represent hot-spots for chromosomal aberrations induced by these agents. Breaks in either interstitial regions or centromeres could lead to deletion of parts of the chromosome (Simi et al., 1998) necessary for chromosome segregation, thus resulting in aneuploidy.
In summary, our results indicate that the classic mutagens DES and NMU induce aneugeny in Chinese hamster cell lines, but they do so by different mechanisms. The former induces rapid formation of CREST+ micronuclei by mechanisms independent of p53 and mismatch repair, possibly via inhibition of the S6K1 protein kinase cascade. The apparent targets are nucleotides, although there is a potential for direct alkylation of protein kinases/phoshatases upstream of S6K1, since another alkylating agent, N--tosyl-L-phenylalanyl-chloromethylketone has been shown to inhibit PDK (3-phosphoinisotide-dependent kinase), a member of the complex family of protein kinases which activate S6K1, by direct alkylation (Ballif et al., 2001). The methylating agent MNU shows a relatively slow induction of CREST+ and CREST– micronuclei, which appears to be mismatch repair-dependent but p53- and S6K1-independent, in keeping with DNA being the main target.
Overall these results indicate that the final mutagenic and cytotoxic effects of alkylating agents reflect not only DNA modification but also alteration of other cellular targets, such as protein kinase cascades, raising the intriguing question of the potential benefits of alkylating agents with multiple targets in chemotherapy. In this sense alkylating agents which inhibit S6K1 activation might be particularly effective in the treatment of tumours with an up-regulated PI3K–Akt–S6K1 pathway (Neshat et al., 2001), as they not only induce DNA damage but also inhibit the translation of proteins necessary for cell proliferation and survival.
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Acknowledgement |
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The authors wish to thank Dr M.A.Minks for his critical reading of the manuscript.
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2To whom correspondence should be addressed. Tel: +39 0503152780; Fax: +39 0503153367; Email: marcella.simili{at}ifc.cnr.it
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References |
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Received on October 7, 2002; revised on April 28, 2003; accepted on May 2, 2003.
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