Mutagenesis, Vol. 15, No. 4, 361-366,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Induction of apoptosis and inhibition of signalling pathways by alkylated purines
1 IST, National Institute for Cancer Research, Genova, 2 CNR Institute of Mutagenesis and Differentiation, Pisa and 3 Department of Oncology, Biology and Genetics, University of Genova, Genova, Italy
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Addition of growth factors such as EGF and insulin to serum-starved G0 Chinese hamster fibroblast cells results in activation of the phosphatidylinositol 3-kinase (PI3-K)/p70 S6 kinase (p70S6K) pathway and the ras-raf mitogen-activated kinase (MAPK) pathway. Activation of these pathways is usually associated with protection of cells from apoptosis. We have studied the effect of three alkylpurines, O6-methylguanine (O6meG), O6-ethylguanine (O6etG) and 6-dimethylaminopurine (6DMAP) on two particular steps of these pathways, namely activation of p70S6K and of MAPK. Under the same experimental conditions we studied the ability of these alkylpurines to induce apoptosis. Our results show that the three alkylpurines induced apoptosis with increasing efficiency from O6meG to 6DMAP to O6etG. The induction of apoptosis was phase specific, with the G0/G1 phase being most sensitive. A reduced apoptotic response was observed in cells with abnormal nuclear accumulation of mutant or wild-type p53, suggesting that functional p53 was required for the induction of apoptosis. At concentrations inducing apoptosis the three alkylpurines inhibited p70S6K activity, while they had the opposite effect on MAPK. Rapamycin, a specific inhibitor of the p70S6K pathway, did not induce apoptosis at doses inhibiting p70S6K activity, suggesting that p70S6K is not directly involved in apoptosis. As expected, and in line with results reported by others, wortmannin, an upstream inhibitor of the p70S6K pathway, did induce apoptosis. We propose that activation of the MAPK pathway and simultaneous inhibition of the p70S6K pathway induce an apoptotic response in the cell.
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Introduction |
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Apoptosis is a ubiquitous process by which multicellular organisms are able to mantain a balance between cell proliferation and death. Although activation of the specific molecular pathways leading to apoptosis appears to depend on cell type, cell phase and external stimuli, it is generally agreed that a disturbance of cell cycle control may elicit apoptotic pathways (Jarpe et al., 1998
). It has also been speculated that there may be critical steps in common between apoptotic and mitotic pathways (King and Cidlowski, 1995); indeed it has been found that activation of cyclin A-dependent protein kinases is an important event in apoptosis induction by the protein kinase inhibitor 6-dimethylaminopurine (6DMAP) (Meikrantz et al., 1994).
We have shown that alkylated purines such as O6-methylguanine (O6meG), O6-ethylguanine (O6etG) and 6DMAP affect mitotic chromosome segregation in Chinese hamster embryonic fibroblast (CHEF/18) cells (Bonatti et al., 1996). Chromosome malsegregation and partial inhibition of DNA synthesis were induced in these cells with kinetics which imply that the targets are mainly present in early G1 (Simili et al., 1995). These effects were coincident with the ability of 6DMAP to inhibit the p70 S6 kinase (p70S6K) but not the MAP kinase (MAPK) pathway, suggesting that this substance is not a broadly aspecific kinase inhibitor in vivo (Bonatti et al., 1996), as it appears to be in vitro (Vesely et al., 1994).
p70S6K is a mitogen-activated kinase involved in the G0–G1 transition, via phosphorylation of S6 ribosomal protein and possibly the initiation factors eIF4B and eIF4G (Vesely et al., 1994; Brown and Schreiber, 1996). Activation of this kinase appears to be necessary for translation of a specific set of mRNAs, namely those with a 5'-terminal oligopyrimidine tract (5'-TOP), encoding ribosomal proteins and translation elongation factors (Sonenberg and Gingras, 1998). In certain cell types it has been found that expression of other genes important for cell cycle progression, such as myc and cyclin D may be controlled by the p70S6K pathway (Jefferies et al., 1994; West et al., 1998). These findings could in part explain the reason why in certain cells inhibition of this pathway induces complete block of the cell cycle while in others there is only partial inhibition (Lane et al., 1993; Hashemolhosseini et al., 1998). Controversial indications also exist about the role that p70S6K may play in cell survival and apoptosis. In many cell types it has been found that inhibition of this kinase induced by rapamycin does not lead to apoptosis (Yao and Cooper, 1996; Canicio et al., 1998). However, induction of apoptosis by rapamycin has recently been reported in human rhabdomyosarcoma cells (Hosoi et al., 1999).
6DMAP has been shown to induce apoptosis in Hela cells treated in G1/S (Steinmann et al., 1991) and we found that concentrations >1 mM of this alkylpurine block DNA synthesis induced by growth factors and provoke a dramatic change in cell morphology similar to that seen in apoptotic cells.
In this paper we have investigated the apoptotic effects of high doses of alkylpurines in relation to p70S6K inhibition. The results indicate that all three alkylpurines induce p53-dependent apoptosis. Induction of apoptosis appears to be associated with the simultaneous inhibition of p70S6K and a sustained activation of the MAPK pathway.
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Materials and methods |
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Cell cultures
Diploid Chinese hamster embryonic fibroblasts, CHEF/18, kindly provided by Professor R.Sager (Dana Farber Cancer Institute, Boston, MA) were routinely grown at 37°C in a 10% CO2 incubator, at a density of 2x104 cells/cm2 in
-MEM (Gibco) supplemented with 10% fetal calf serum (FCS) (ICN Laboratories). All experiments were performed with CHEF/18 cells at early passages (passages 10–20) in order to minimize the variability of the cell response to growth factors (Cherrington and Pardee, 1980).
Growth factors and other chemicals
Epidermal growth factor (EGF) was obtained from Boheringer (Mannheim, Germany); insulin, 6DMAP, thymidine and O6meG were from Sigma (Milano, Italy). O6etG was from Chemsyn Science Laboratories (Lanexa, USA). Rapamycin was from ICN (Milano, Italy); [
-32P]ATP was obtained from Farmacia-Amersham (Milano, Italy).
DNA extraction and agarose gel electrophoresis
Cellular DNA was isolated from cells (untreated and treated with the chemicals) by proteinase K and RNase A digestion of cell lysates followed by phenol/chloroform extraction and ethanol precipitation following standard protocols. Pelleted DNA was resuspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA) (Donaldson et al., 1994) and quantified by measuring A260. Aliquots were applied to a 1.3% agarose gel in TBE buffer (0.089 M Tris base, 0.089 M boric acid, 0.002 M EDTA) and resolved at 20 mA constant current. The resultant DNA was visualized by ethidium bromide staining.
Protein determination
The protein concentration of cell extracts was measured using a Bio-Rad protein assay kit (Segrate, Milano) with bovine serum albumin as the standard.
Determination of p70S6K and MAPK phosphorylation
Cell extracts were prepared as described by Dennis et al. (1996) for p70S6K and as described by Yin et al. (1992) for MAP kinase. After elecrophoresis, proteins were blotted onto Hybond nitrocellulose (Amersham). p70S6K was detected with a polyclonal antibody (Santa Cruz Laboratories, Heidelberg, Germany) while MAP kinase was detected by a monoclonal antibody (ICN Laboratories) which recognizes the two isoforms of 42 and 44 kDa. For time course experiments, a monoclonal antibody (Santa Cruz Laboratories) recognizing the 42 kDa isoform was used. To amplify the signal, the enhanced chemiluminescence method of Amersham was used.
In vitro S6 kinase assay
Cell extracts and 40S ribosomal subunits were prepared as described (Yin et al., 1992). Cell extracts diluted 1:10 (6 mg/assay) were mixed with an equal volume of S6 kinase buffer, 40S ribosomal subunits (2 mg/ml) and 0.5 mM protein kinase inhibitor peptide (Sigma) in 0.005% bovine serum albumin, 0.3 ml [-32P]ATP (20 mCi/ml; Amersham) at 37°C for 30 min as previously described (Han et al., 1995). The reaction was stopped with Laemmli sample buffer heated to 95°C and extracts were centrifuged and loaded onto SDS–PAGE gels. S6 phosphorylation was visualized by autoradiography as previously described (Bonatti et al., 1996).
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Results |
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Alkylated purines induce apoptosis in a cell cycle-dependent manner
It has previously been shown that O6meG, O6etG and 6DMAP inhibit entry into S phase (DNA synthesis) when added to quiescent CHEF/18 cells stimulated with growth factors and that this effect is accompanied by inhibition of S6 ribosomal protein phosphorylation. These effects were maximum when cells were treated in G0/G1 (Simili et al., 1995
; Bonatti et al., 1996). Concentrations of the alkylpurines >1 mM induced dramatic changes in cell morphology, such as rounding up of the cells, chromatin condensation and nuclear fragmentation, similar to those observed in cells undergoing apoptosis (Kerr and Harmon, 1991). To investigate whether these alkylpurines indeed induced apoptosis in our system, CHEF/18 cells were made quiescent by serum starvation and the test compounds were added 30 min before growth factor stimulation. Apoptosis was determined both at the cytological level, by microscopic examination of Giemsa stained cells (Figure 1a and b), and at the DNA level, by determining the typical pattern of DNA degradation (Figure 2). Apoptotic cells were visible starting from 8 h after treatment, with a maximum of apoptotic cells at 16–20 h, after which most of the cells started to detach.
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The results shown in Table I
indicate that all three alkylpurines induce apoptosis at concentrations >1 mM, with O6etG being the most effective and O6meG the least effective. The efficacy of alkylpurines in inducing apoptosis parallels the efficay in inducing chromosome malsegregation and inhibition of DNA synthesis (Simili et al., 1995; Bonatti et al., 1996). Thymidine, known to induce a nucleotide pool imbalance, is a poor inducer of apoptosis in these cells, suggesting that pool imbalance is not responsible for apoptosis induced by alkylpurines.
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To determine whether the effect of the alkylpurines was phase specific, cells were treated with 6DMAP in different phases of the cell cycle (Figure 3
). Cells were serum starved and either treated in G0/G1 by adding 6DMAP along with growth factors or treated in G2/M by adding 6DMAP 16 h after mitogenic stimulation. To treat cells during S phase, 50 mM hydroxyurea was added to proliferating cells for 3 h, after which the medium was changed and 6DMAP added for a further 8 h. Cells were also treated during asynchronous growth. The results show that the most sensitive phase of the cell cycle to apoptosis induction is the G0/G1 transition, while proliferating cells were the least sensitive.
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Apoptosis induction is dependent on p53 status
To assess the p53 dependence of apoptosis induced by alkylpurines, Chinese hamster fibroblast cell lines with a different p53 status were treated with 6DMAP. These included CHEF/18 cells at late passages, presenting a transformed phenotype (Sager and Kovac, 1978
) and nuclear accumulation of p53 (Moro et al., 1995) but no detectable point mutations in the gene (Rainaldi et al., 1998), and the tumorigenic V79 cell line, presenting nuclear accumulation of non-functional p53 carrying point mutations in the gene coding region (Chaung et al., 1997; Lee et al., 1997).
The induction of apoptosis by 6DMAP is reported in Table II as the ratio of the percentage of apoptotic cells observed in normal CHEF/18 cells to that observed in cells showing nuclear accumulation of wild-type or mutant p53. The results indicate that apoptosis induced by 6DMAP is higher in cells with normal p53 expression, while the presence of either point mutations or abnormal nuclear accumulation reduces the apoptotic response of the cells.
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Alkylated purines inhibit growth factor-induced p70S6K activation
We have shown that 6DMAP specifically inhibits S6 ribosomal protein phosphorylation in CHEF/18 cells stimulated with growth factors, without affecting MAPK activation (Simili et al., 1995
). In most cell lines tested the protein kinase responsible for S6 ribosomal protein phosphorylation is the phosphorylated form of p70S6K (Brown and Schreiber, 1996). We then tested the effect of the three alkylated purines on p70S6K by determining both the phosphorylation state of the kinase and its in vitro activity. Rapamycin and wortmannin, specific inhibitors of the p70S6K pathway, were also included as controls. Quiescent CHEF/18 cells were treated with the alkylpurines before and during growth factor addition. The results, shown in Figure 4, indicate that p70S6K activity was enhanced soon after addition of growth factors. Concentrations of the three alkylpurines >0.5 mM inhibited p70S6K activity to the same extent as rapamycin and wortmannin, used at concentrations known to be inhibitory in rodent cells (Petritsch et al., 1995; Jefferies et al., 1997). This effect was accompanied by inhibition of p70S6K phosphorylation (Figure 5), indicating that either an upstream protein kinase was inhibited or a phosphatase was activated.
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To assess the role of p70S6K in apoptosis induction, cells were treated in G0/G1 with different concentrations of rapamycin or wortmannin. The two inhibitors block the pathway at different levels, with rapamycin blocking mTOR, a major kinase of p70S6K, and wortmannin inhibiting the pathway upstream, at the level of phosphatidylinositol 3-kinase (PI3-K). The results (Table III
) show that wortmannin, but not rapamycin, induces apoptosis. These results suggest that inhibition of p70S6K is not causatively related to the apoptotic response.
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Alkylated purines enhance growth factor-induced MAPK phosphorylation
We have previously shown that 6DMAP at 0.5 mM did not prevent growth factor-induced phosphorylation of MAPK, determined as mobility shift of the 42 and 44 kDa isoforms (Simili et al., 1995
). Pretreatment of quiescent CHEF/18 cells with concentrations of the alkylpurines >1 mM followed by stimulation with growth factors did not inhibit and even enhanced phosphorylation of MAPK (Figure 6). Substantial phosphorylation of the 42 kDa isoform was induced by the same concentrations of the alkylpurines even in the absence of growth factors (Figure 6). Wortmannin, at concentrations that induce apoptosis (400 nM), also induced significant phosphorylation of MAPK, while rapamycin did not induce any detectable phosphorylation up to 600 nM (Figure 7).
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It has been found that prolonged activation of MAPK is associated with either a block of the cell cycle (Pumiglia and Dekker, 1997
) or apoptosis (Lee-Kwon et al., 1998) in different cell lines. We then measured the time course of MAPK activation either after growth factor stimulation or after treatment with the alkylated purines. The results (Figure 8) indicate that phosphorylation elicited by growth factors was transient, reaching a maximum at 30 min and starting to decline after 1h. In contrast, phosphorylation elicited by alkylated purines lasted up to 4–6 h.
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Discussion |
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The control of apoptosis is of clinical importance in the therapy of cancer, neurodegenerative diseases and immune disorders. Apoptosis is associated with the activation of several genes. These genes, such as the Bcl-2 family (Williams and Smith, 1993
) and the caspases (Nagata, 1997), are responsible for events that are proximal to actual death of the cells. Upstream of these apoptosis-controlling genes lie the signal transduction pathways that ensure the transition from quiescence to proliferative growth, among which both pro-apoptotic and anti-apoptotic pathways have been identified (Jarpe et al., 1998). Indeed, in certain cell types it has been found that the apoptotic process resembles events typical of the G0/G1 transition (King and Cidlowski, 1995). In the present work we have studied the induction of apoptosis in immortalized, non-tumorigenic Chinese hamster fibroblast cells, CHEF/18, by compounds that interfere with growth factor-dependent signalling pathways important for the G0/G1 transition. Addition of growth factors such as EGF and insulin results in activation of the PI3-K/p70S6K and ras-raf MAPK pathways (O'Brien and Granner, 1996), activation of which usually protects cells from apoptosis (Tateishi and Yamaizumi, 1997; Lee-Kwon et al., 1998). In this paper two particular steps of these pathways, activation of p70S6K and MAPK, have been studied in connection with apoptosis induced by alkylpurines in quiescent cells stimulated by growth factors. Our results show that the three alkylpurines, O6meG, O6etG and 6DMAP, induce apoptosis, with the most efficient being O6etG, followed by 6DMAP and O6meG. The induction of apoptosis was phase specific as cells were more sensitive when treated in the G0/G1 phase than in other phases of the cell cycle, while the lowest sensitivity was shown by cells treated during rapid proliferation. Moreover, both serum and EGF + insulin had a protective effect against apoptosis, as reported in other murine cells (Lee-Kwon et al., 1998).
A reduced apoptotic response was observed in cells with abnormal nuclear accumulation of mutant or wild-type p53, suggesting that functional p53 was required for apoptosis induction. It should be noted in this context that alkylpurines do not damage DNA (Bonatti et al., 1995), thus it is unlikely that the apoptotic response is due to the p53-dependent DNA damage-stimulated pathway (Williams and Smith, 1993).
In our system the three alkylated purines at low concentrations (0.5 mM) inhibited phosphorylation of the S6 ribosomal protein elicited by growth factors, while the MAPK pathway was not affected (Simili et al., 1995; Bonatti et al., 1996). At concentrations inducing apoptosis (>1 mM) the three alkylpurines inhibited p70S6K activity to an extent similar to the inhibition induced by rapamycin and wortmannin, two powerful and specific inhibitors of this pathway. This effect appears to be due to decreased p70S6K phosphorylation, which impairs full activity of the kinase, possibly due either to inhibition of an upstream kinase or to activation of a phosphatase. In our system wortmannin, but not rapamycin, was able to induce apoptosis, as already reported in other cell lines (Yao and Cooper, 1996). This different effect could be due to the fact that the two compounds inhibit the PI3-K pathway at different levels: wortmannin inhibits PI3-K, while rapamycin blocks the pathway at the level of mTOR, downstream of PI3-K (Thomas and Hall, 1997). In different cell types inhibition of PI3-K or C-AKT protein kinase has been associated with apoptosis induction (de Groot et al., 1994; Grammer et al., 1996), while inhibition of mTOR usually leads to a partial or total block of the cell cycle. At variance with these results, induction of apoptosis by rapamycin through inhibition of mTOR has recently been reported in rhabdomyosarcoma cells with mutant p53 (Hosoi et al., 1999). Moreover, a rapamycin-sensitive, anti-apoptotic pathway involving p70S6K has been suggested in hemopoietic cells (Kinoshita et al., 1997). The results obtained with rapamycin in CHEF/18 cells indicate that p70S6K is important for cell cycle progression but is not directly involved in apoptosis. Other base analogs, such as SQ2006 and theophylline, have been shown to inhibit PI3-K in vitro (Grammer et al., 1996); if this kinase was also the target of the purine derivatives tested by us, its inactivation could explain the effects that we have observed in vivo.
Activation of MAPK, which in murine fibroblasts appears to be dependent on the activation of ras/raf rather than PI3-K and therefore to be distinct from the p70S6K pathway (de Groot et al., 1994), usually has an anti-apoptotic role (Williams and Smith, 1993). At variance with these results, in CHEF/18 fibroblasts MAPK phosphorylation was augmened by apoptotic doses of alkylpurines and wortmannin, either alone or in combination with growth factors. However, phosphorylation appeared to be more prolonged than that elicited by growth factors alone (it lasted up to 6 h), suggesting abnormal activation of the ras/raf pathway. Many reports exist about the pro-apoptotic role of ras in apoptosis induced by stress. In the cases where it has been studied in detail, it has been found that the downstream effectors were raf and MAPK. At present we do not know how the alkylpurines tested enhance MAPK phosphorylation. As these compounds are protein kinase inhibitors, it is possible that they either inhibit an inhibitory kinase of the pathway or activate it by an unknown mechanism(s). In any case, it is possible that abnormal activation of this pathway and simultaneous inhibition of the PI3-K pathway induce an apoptotic response in the cell. Apoptosis might then be the result of contrasting signalling (Yonish-Rouach et al., 1991).
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Acknowledgments |
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This work was partially supported by MURST.
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Notes |
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4 To whom correspondence should be addressed at: IST, National Institute for Cancer Research, Largo R. Benzi 10, 16132 Genova, Italy. Fax: +39 010 560 0992; Email: abbondan{at}hp380.ist.unige.it
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References |
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Bonatti,S., Aprile,A., Arena,G., Cavalieri,Z., Pellerano,P., Rocco,M., Seiler,K., Viaggi,S. and Abbondandolo,A. (1995) Induction of kinetocore-containing micronuclei by exogenous O6-methylguanine requires conversion of the methylated base to a nucleotide. Environ. Mol. Mutagen., 26, 226–233.[ISI][Medline]
Bonatti,S., Simili,M., Pellerano,P., Tavosanis,G. and Abbondandolo,A. (1996) Cellular targets for the aneugenic action of alkylating agents. In Abbondandolo,A., Vig,B.K. and Roi,R. (eds) Chromosome Segregation and Aneuploidy. IST, Genova, Italy, pp. 265–273.
Brown,E.J. and Schreiber,S.L. (1996) A signalling pathway to translational control. Cell, 86, 517–520.[ISI][Medline]
Canicio,J., Gallardo,E., Illa,I., Testar,X., Palacin,M., Zorzano,A. and Kaliman,P. (1998) p70 S6 kinase activation is not required for insulin like growth factor-induced differentiation of rat, mouse, or human skeletal muscle cells. Endocrinology, 139, 5042–5049.
Chaung,W., Mi,L.J. and Boorstein,R.J. (1997) The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair. Nucleic Acids Res., 25, 992–994.
Cherrington,P.V. and Pardee,A.B. (1980) Synergic effects of epidermal growth factor and thrombine on the growth stimulation of diploid Chinese hamster fibroblast. J. Cell. Physiol., 105, 25–32.[ISI][Medline]
de Groot,R.P., Ballou,L.M. and Sassone-Corsi,P. (1994) Positive regulation of the cAMP-responsive activator CREM by the p70 S6 kinase: an alternative route to mitogen-induced gene expression. Cell, 79, 81–91.[ISI][Medline]
Dennis,P.B., Pullen,N., Kozma,S.C. and Thomas,G. (1996) The principal rapamycin-sensitive p70S6K phosphorylation sites, T-229 and T-389 are differentially regulated by rapamycin-insensitive kinase kinases. Mol. Cell. Biol., 16, 6242–6251.[Abstract]
Donaldson,K.L., Goolsby,G.L., Kiener,P.A. and Wahl,A.F. (1994) Activation of p34 cdc2 coincident with taxol-induced apoptosis. Cell Growth Differ., 5, 1041–1050.[Abstract]
Grammer,T.C., Cheatham,L., Chou,M.M. and Blenis,J. (1996) The p70S6K signalling pathway: a novel signalling system involved in growth regulation. Cancer Surv., 27, 271–292.[ISI][Medline]
Han,J.W., Pearson,R.B., Dennis,P.B. and Thomas,G. (1995) Rapamycin, wortmannin and the methylxanthine SQ2006 inactivate p70S6K by inducing dephosphorylation of the same subset of sites. J. Biol. Chem., 270, 21396–21403.
Hashemolhosseini,S., Nagamine,Y., Morley,S.J., Descrivieres,S., Mercep,L. and Ferrari,S. (1998) Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J. Biol. Chem., 273, 14424–14429.
Hosoi,H., Dilling,N.B., Shikata,T., Lin,L.N., Ashmun,R.A., Germain,G.S., Abraham,R.T. and Houghton,P.J. (1999) Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells. Cancer Res., 59, 886–894.
Jarpe,M.B., Widmann,C., Knall,C., Schlesinger,T.K., Gibson,S., Yujiri,T., Fanger,R.G., Erwin,W.G. and Jhonson,G. (1998) Anti-apoptotic versus pro-apoptotic signal transduction: checkpoints and stop signs along the road to death. Oncogene, 17, 1475–1482.[ISI][Medline]
Jefferies,H.B.J., Reinhard,C., Kozma,S.C. and Thomas,G. (1994) Rapamycin selectively represses translation of the `polypyrimidine tract' mRNA family. Proc. Natl Acad. Sci. USA, 91, 4441–4445.
Jefferies,H.B.J., Fumagalli,S., Dennis,P.B., Rainhard,C., Pearson,R.B. and Thomas,G. (1997) Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70S6K. EMBO J., 16, 3693–3704.[ISI][Medline]
Kerr,J.F. and Harmon,B.V. (1991) Definition and incidence of apoptosis: an historical perspective. In Tomei,L.D. and Cope,F.O. (eds) Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 5–29.
King,K.L. and Cidlowski,J.A. (1995) Cell cycle and apoptosis: common pathways to life and death. J. Cell. Biochem., 58, 175–180.[ISI][Medline]
Kinoshita,T., Shirouzu,M., Kamiya,A., Hashimoto,K., Yokoyama,S. and Miyajima,A. (1997) Ras/MAPK and rapamycin-sensitive pathways mediate the anti-apoptotic function of p21 Ras in IL-3-dependent hematopoietic cells. Oncogene, 15, 619–627.[ISI][Medline]
Lane,H.A., Fernandez,A., Lamb,J.C. and Thomas,G. (1993) p70S6K function is essential for G1 progression. Nature, 363, 170–172.[Medline]
Lee,H., Larner,J.M. and Hamlin,J.L. (1997) Cloning and characterization of Chinese hamster p53 cDNA. Gene, 184, 177–183.[ISI][Medline]
Lee-Kwon,W., Park,D., Baskar,P.V., Kole,S. and Bernier,M. (1998) Antiapoptotic signalling by insulin receptor in Chinese hamster ovary cells. Biochemistry, 37, 15747–15757.[Medline]
Meikrantz,W., Gisselbrech,S., Tam,S.W. and Schlegel,R. (1994) Activation of cyclin A-dependent protein kinases during apoptosis. Proc. Natl Acad. Sci. USA, 91, 3754–3758.
Moro,F., Ottaggio,L., Bonatti,S., Simili,M., Miele,M., Bozzo,S. and Abbondandolo,A. (1995) p53 expression in normal versus transformed mammalian cells. Carcinogenesis, 16, 2435–2440.
Nagata,S. (1997) Apoptosis by death factor. Cell, 88, 355–365.[ISI][Medline]
O'Brien,R.M. and Granner,D.K. (1996) Regulation of gene expression by insulin. Physiol. Rev., 76, 1109–1161.
Petritsch,C., Woscholski,R., Eldmann,H.M. and Ballou,L.M. (1995) Activation of p70 S6 kinase and erk-encoded mitogen-activated protein kinases is resistant to high cyclic nucleotide levels in swiss 3T3 fibroblasts. J. Biol. Chem., 270, 26619–26625.
Pumiglia,K.M. and Dekker,S.J. (1997) Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc. Natl Acad. Sci. USA, 94, 448–452.
Rainaldi,G., Marchetti,S., Capecchi,B., Meneveri,R., Piras,A. and Leuzzi,R. (1998) Absence of mutations in the highest mutability region of the p53 gene in tumour-derived CHEF18 Chinese hamster cells. Mutagenesis, 13, 153–155.
Sager,R. and Kovac,P. (1978) Genetic analysis of tumorogenesis: I. Expression of tumour forming ability in hamster hybrid cell lines. Somat. Cell Genet., 4, 375–392.
Simili,M., Pellerano,P., Tavosanis,G., Arena,G., Bonatti,S. and Abbondandolo,A. (1995) The induction of aneuploidy by alkylated purines: effects on early and late cell cycle events. Mutagenesis, 10, 105–111.
Sonenberg,N. and Gingras,A.C. (1998) The mRNA 5' cap-binding protein efFAE and control of cell growth. Curr. Opin. Cell, Biol., 10, 268–275[ISI][Medline]
Steinmann,K.E., Belinsky,G.S., Lee,D. and Schlegel,R. (1991) Chemically induced premature mitosis: differential response in rodent and human cells and the relatioship to cyclin B synthesis and p34 cdc2/cyclin B complex formation. Proc. Natl Acad. Sci. USA, 88, 6843–6847.
Tateishi,S. and Yamaizumi,M. (1997) Cell cycle control is aberrant in Chinese hamster ovary cell mutants exhibiting apoptosis after serum deprivation. Somat. Cell Mol. Genet., 23, 313–323.[ISI][Medline]
Thomas,G. and Hall,M.N. (1997) TOR signalling and control of cell growth. Curr. Opin. Cell, Biol., 9, 782–787.[ISI][Medline]
Vesely,J., Havlicek,L., Stround,M., Blow,J.J., Donella-Deanna,A., Pinna,L., Letham,D.S., Kato,J., Detivaud,L., Leclerc,S. and Meuer,L. (1994) Inhibition of cyclin-dependent kinases by purine analogues. Eur. J. Biochem., 224, 771–786.[ISI][Medline]
West,M.J., Stoneley,M. and Willis,A.E. (1998) Translational induction of the c-myc oncogene via activation of the FRAP/TOR signalling pathway. Oncogene, 17, 769–780.[ISI][Medline]
Williams,G.T. and Smith,C.A. (1993) Molecular regulation of apoptosis: genetic controls on cell death. Cell, 74, 777–779.[ISI][Medline]
Yao,J. and Cooper,M.G. (1996) Growth factor-dependent survival of rodent fibroblasts requires phosphatidylinositol 3-kinase but is independent of p70S6K activity. Oncogene, 13, 343–351.[ISI][Medline]
Yin,J., Miyazawa,K. and Yang,J.C. (1992) Characterization of interleukin-11 receptor and protein tyrosine phosphorylation induced by interleukin-11 in mouse 3T3-L1 cells. J. Biol. Chem., 267, 8347–8351.
Yonish-Rouach,E., Resnitzky,D., Lotem,J., Sachs,L., Kimchi,A. and Oren,M. (1991) Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature, 352, 345–347.[Medline]
Received on February 10, 2000; accepted on April 7, 2000.
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 M. Campagna, P. Beffy, R. Del Carratore, L. Hauri, S. Simi, S. Bonatti, and M. Simili Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents Mutagenesis, September 1, 2003; 18(5): 405 - 410. [Abstract] [Full Text] [PDF]
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 Y. Cai, S. M. Ludeman, L. R. Wilson, A. B. Chung, and M. E. Dolan Effect of O6-Benzylguanine on Nitrogen Mustard-induced Toxicity, Apoptosis, and Mutagenicity in Chinese Hamster Ovary Cells Mol. Cancer Ther., November 1, 2001; 1(1): 21 - 28. [Abstract] [Full Text] [PDF]
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