Mutagenesis, Vol. 16, No. 5, 423-430,
September 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor RPR-115135: role of gene mutations
Molecular Pathology Section, Laboratory of Experimental Oncology, National Institute for Research on Cancer, Genova, Italy and 1 Department of Experimental, Environmental and Applied Biology (DIBISAA), University of Genova, Genova, Italy
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Abstract |
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To investigate the relationship between oncogene activation and induction of micronuclei by a new non-peptidic mimetic farnesyltransferase inhibitor, RPR-115135, two isogenic cell lines, human colon cancer line HCT-116, which harbors a K-ras mutation, and spontaneously immortalized human breast epithelial cell line MCF-10A, were utilized. HCT-116 cells were transfected with an empty control pCMV vector (clone CMV-2) or with a dominant negative mutated p53 transgene (clone Mu-p53-2) to disrupt p53 function. In both clones RPR-115135 induced a significant increase in the frequency of micronucleation at concentrations that did not affect cell membrane integrity. RPR-115135 produced a significant increase in the ratio of CREST+ to CREST– micronuclei. MCF-10A cells were stably transfected with either c-Ha-ras or c-erbB-2 or both H-ras + c-erbB-2. No induction of micronuclei was observed. No induction of micronuclei was reported in human lymphocytes and in primary spinal cells obtained from 7-day chick embryos. In conclusion, RPR-115135 acts as an aneugenic agent in a complex manner, dependent upon the complement of mutations in cell regulatory genes in tumour cells and this activity may be independent of ras genotype.
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Introduction |
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Oncogene products or proteins mediating their effects are obvious targets for cancer therapy because, by definition, these proteins are involved in malignant transformation of normal cells (Stass and Mixon, 1997; Gibbs, 2000
). Of the many signal transduction mechanisms which are emerging as potential targets for drug development in cancer, prenylation of Ras family proteins, such as Ras, RhoB and Rab (all members of the Ras superfamily of proteins), is receiving particular attention from both pharmaceutical companies and academic groups (Gibbs and Oliff, 1997; Leonard, 1997; Lerner et al., 1997; Quian et al., 1997; Sebti and Hamilton, 1997; Oliff, 1999; Giraud et al., 2000; Hill et al., 2000). Interest in protein prenylation has increased because of the importance of this modification for the function of Ras proteins, GTP-binding proteins that, when mutated, contribute to the development of different types of cancers (Zachos and Spandidos, 1997). These proteins are localized at the inner surface of the cell membrane and participate in transmitting signals for growth and many other processes from the outside to the inside of the cell. Although several steps are involved in targeting Ras to the plasma membrane (activation), farnesylation by FTase is the only step that is required for Ras transforming activity (Katz and McCormick, 1997).
Single amino acid substitutions in codon 12, 13 or 61 that unmask Ras transforming potential create mutant forms of Ras which have impaired GTPase activity and are insensitive to GAP stimulation. Consequently, these oncogenic mutant Ras proteins are locked in the active, GTP-bound state, leading to constitutive, deregulated activation of Ras function. It has been estimated that 30% of all human tumours contain an activating mutation in Ras (Zachos and Spandidos, 1997). In addition, overexpression of normal Ras, which causes deregulation of wild-type Ras isoforms, is a common feature of human cancers (i.e. glioblastoma and breast cancer; Miyakis et al., 1998; Bredel and Pollak, 1999; Smith et al.2000).
A range of farnesyltransferase (FTase) inhibitors [peptidics, pseudopeptidics, peptidomimetics or farnesyl pyrophosphate (FPP)-competitive non-peptidic inhibitors] have been synthesized or identified. The results achieved are due to the combined efforts of pharmaceutical companies and academic groups (Gibbs and Oliff, 1997; Leonard, 1997; Lerner et al., 1997; Quian et al., 1997; Sebti and Hamilton, 1997; Oliff, 1999; Giraud et al., 2000; Hill et al., 2000).
The first screen for FTase inhibitors involved their evaluation in direct enzyme assays, followed by cell culture assays and subsequent testing in xenographic mice (Gibbs and Oliff, 1997; Leonard, 1997; Lerner et al., 1997; Quian et al., 1997; Sebti and Hamilton, 1997; Oliff, 1999; Giraud et al., 2000; Hill et al., 2000). Recently a number of Phase I trials have demonstrated that these compounds can be administered to cancer patients and multiple daily oral doses have been recommended (Adjei et al., 2000; Zujewski et al., 2000). Although vast resources have been allocated to this specific area of research, it is true to state that the exact mechanisms of action of FTase inhibitors remains unclear. It is possible to conclude that FTase inhibitors affect the cell in a complex manner, their effects varying not only as a function of the ras genotype of the tumour but also being dependent on other characteristics of individual tumours, such as, for example, the complement of mutations in cell cycle regulatory genes (Hill et al., 2000).
Recently, in a preliminary study (Russo et al., 1998, 1999), we have shown that a human colon cancer cell line HCT-116, when treated with a new FPP-competitive non-peptidic mimetic FTase inhibitor, namely RPR-115135, displayed severe morphological alterations (hypertrophic, vacuolated or necrotic, but not apoptotic, cells) 48–72 h after treatment. Treated cells also revealed an increase in micronucleus (MN) induction, suggesting a tendency for RPR-115135 to be an aneugenic agent (Russo et al., 1999).
The aim of this study was to evaluate the ability of RPR-115135 to increase MN induction not only as a function of the ras genotype of the tumour, but also to investigate dependence on other characteristics of each individual tumour, such as, for example, the complement of mutations in cell cycle regulatory genes. To accomplish this we looked at the induction of MN in two different isogenic cell model systems, the human colon cancer line HCT-116, which harbours a K-ras mutation, and a spontaneously immortalized human breast epithelial cell line, MCF-10A. HCT-116 cells were transfected with an empty control pCMV vector or with a dominant negative mutated p53 transgene (248R/W) to disrupt p53 function. MCF-10A cells were stably transfected with c-Ha-ras, c-erbB-2 or H-ras + c-erbB-2. Finally, normal freshly isolated human lymphocytes stimulated with PHA/IL2 and normal primary spinal cells obtained from 7-day chick embryos were analysed.
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Materials and methods |
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Chemical treatments
RPR-115135 (C31H29NO4, mol. wt 479.58) was produced by Aventis Pharma (Centre de Recherches de Vitry, Alfortville, France). It was dissolved as a 1 mM stock solution in dimethyl sulfoxide and aliquots were stored at –20°C until needed. The compound is stable for 6 days at 37°C (Aventis Pharma, personal communication).
Cell culture
Human colon cancer cell line HCT-116 was grown in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum (Gibco BRL) and 2 mM glutamine. Cells transfected with either empty control vector (pCMV) or vector containing a dominant negative mutant p53 transgene (248R/W) (cloned in a pCMV plasmid), to inhibit p53 function, were grown in the same medium. Generation and characterization of the HCT-116 transfectants have been described previously (Fan et al., 1997, 1998). Two clones, CMV-2 and Mu-p53-2, were examined. After 
-irradiation only parental and control transfectant CMV cells but not Mu-p53 cells were able to arrest in the G1 phase of the cell cycle and accumulate p53 or p21cyp1-waf–1 proteins. These experiments clearly showed that the functions of p53 in the cells transfected with mutated p53 are disrupted (Table I). All systems were a kind gift of Dr P.O'Connor (Laboratory of Molecular Pharmacology, NCI–NIH, Bethesda, MD).
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The MCF-10A cells were derived from a population of normal luminal mammary epithelial cells and show characteristics of normal breast epithelium. Different oncogenes (c-Ha-ras activated by a missense mutation, c-erbB-2 or c-Ha-ras in combination with c-erbB-2) were inserted in MCF-10A cells (Ciardiello et al., 1992
). The isogenic cell line system was kindly provided by Dr D.Salomon (Tumour Growth Factor Section, Laboratory Tumour Immunology and Biology, NCI–NIH, Bethesda, MD). Cells were grown in DMEM/F-12 medium (Gibco BRL) supplemented with 5% heat-inactivated fetal bovine serum, 2 mM glutamine, 10 µg/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 0.5 µg/ml hydrocortisone and 0.02 µg/ml epidermal growth factor (Gibco BRL). Human lymphocytes were isolated from heparinized blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation and were resuspended at a density of 1.0x106 cells/ml in complete RPMI 1640 medium and cultured in the presence of 10 µg/ml phytohaemagglutinin. After 48 h incubation cells were treated with different concentrations of RPR-115135 in the presence of 100 IU/ml interleukin-2 for an additional 48 h. Then they were counted and seeded on polylysine-coated microscope slides and immediately fixed.
Primary spinal cells obtained from 7-day chick embryos were a kind gift of the group of Prof. B. Tedesco (DIBE, University of Genova, Italy). Cells were grown on polylysine-coated flasks in Neurobasal Medium supplemented with 3.0% horse serum (Gibco BRL).
Cell counts were determined using a Coulter Counter with Channelyzer attachment to monitor cell size (Coulter Electronics, Hialeah, FL). Cell membrane integrity was determined by trypan blue dye exclusion assay.
Cell cytotoxicity
Cells were plated in log phase into 96-well multiwells plates (250 cells/well) with 190 µl of complete medium for 24 h and then treated with various concentrations (0.1–10 µM) of RPR-115135 (10 µl) for 6 days. At the end of the incubation time (6 days) 40 µl of MTS tetrazolium solution (CellTiter 96 AQueous 1 Solution Cell Proliferation Assay; Promega, Madison, WI) was added for 2 h and then plates were read at 490 nm with a 96-well plate reader. The IC50 was calculated as the drug concentration that inhibits cell growth to 50% of the control cells.
Aliquots of 50 000 cells/2.5 ml were also plated in 6-well Nunc dishes and allowed to attach for 18 h at 37°C, treated with drug for 48 h, then detached and counted.
Micronucleus evaluation
Cells (2700 cells/cm2) were seeded into microscope slide-containing Petri dishes and incubated at 37°C. After 16 h, medium containing different concentrations of RPR-115135 was added for different periods, then the slides were washed twice with phosphate-buffered saline (PBS). Cells were fixed with 4% paraformaldeyde in PBS for 30 min at room temperature and then stained. DAPI staining was performed according to Linn et al. (1977). Briefly, cell culture slides were incubated in a 1:200 solution of 1 µg/ml DAPI in methanol and observed on a Leica microscope (magnification x400), equipped with a UV detector at a wavelength of 
380 nm. DAPI identifies the A-T couples of DNA as blue-white fluorescence. One thousand cells were scored per slide stained with DAPI to evaluate MN induction. Student's t-test was used to assses stastistical significance (not significant, P > 0.05).
The highest tested concentration of RPR-115135 was the maximum tolerated by the cells. Increases, even small, in concentration produced unwanted effects, such as extensive nuclear fragmentation, cell division inhibition and detachment of cells from the slides (Giemsa stained standard preparation).
In a second set of experiments CREST antiserum for kinetochore staining and propidium iodide for nucleus/micronucleus counterstaining were used as previously reported (Nûsse et al., 1989). Generally accepted criteria for identifying MN (Bonatti et al., 1986) were adopted to rule out artefacts. The specificity of propidium iodide staining further guaranteed that the structures scored as MN contained DNA. A minimum of 100 MN were examined for the presence of kinetochore-positive signals.
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Results |
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Cell cytotoxicity
Since we have previously reported IC50 values obtained in the HCT-116 cell line system (Russo et al., 1999
), we began by investigating the sensitivity to RPR-115135, over a 6 day exposure, of MCF-10A parental cells and of MCF-10A-H, MCF-10A-E and MCF-10A-HE cells. Interestingly, all cells appeared sensitive to RPR-115135, however, the most sensitive was MCF-10A-H and the most resistant MCF-10A (~10 times difference) in terms of IC50 values (Table II). Although the untransfected cells (MCF-10A) were 10 times less sensitive to RPR-115135 than MCF-10A-H cells, they displayed a moderate sensitivity to RPR-115135 (Table II; IC50 = 8.0 µM).
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The IC50 value for MCF-10A-H was in the same range of concentrations obtained in the HCT-116 isogenic cell system (0.78 versus 0.55 µM).
When MCF-10-A-H cells were treated with 10 µM RPR-115135 in the MTS assay the percentage of cells surviving was ~8%. In a clonogenic assay no colonies were detected when treated at this concentration (data not shown). Evaluation by Giemsa staining of cells treated with 10 µM RPR-115135 for 48 h showed 100% of cells with a severely altered morphology. From these data a concentration of RPR-115135 of 1.0 µM was chosen to investigate the time course, cell cycle responses and induction of MN in MCF-10A cells. The dose dependence of MN induction was evaluated in HCT-116 cells and in human lymphocytes.
Time course
We have previously demonstrated in time course experiments conducted over a 6 day exposure to 10 µM RPR-115135 that clones HCT-116-CMV-2 and HCT-116-Mu-p53-2 were able to grow for up to 72 h following administration of the drug (Russo et al., 1999), but thereafter, approaching saturation density, a clear growth inhibition was observed.
The same experiments performed in the MCF-10A cell system showed again that the most sensitive clone was MCF-10A-H, while the other cells were moderately sensitive or resistant. The kinetics of the time–growth curve were similar to those obtained in HCT-116 cells (Figure 1A–D; Russo et al., 1999).
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Growth inhibition could not easily be accounted for on the basis of a specific cell cycle arrest phenotype, as assayed by flow cytometry in the two cell systems (Russo et al., 1999
; and data not shown for MCF-10A cells). These observations support the hypothesis that RPR-115135 did not work by inhibiting cell growth at any specific phase of the cell cycle. In addition, no induction of apoptosis was observed (TEM analysis, DAPI and Giemsa staining and flow cytometry; data not shown)
Induction of micronuclei
Since previous observations (Russo et al., 1998, 1999) supported the hypothesis that RPR-115135 did not work by inhibiting cell growth of proliferating cells at any specific phase of the cell cycle and that the growth inhibition effect of RPR-115135 could not be easily ascribed to significant induction of apotosis, we looked at other possible alterations involved in cell cytotoxicity, such as induction of MN.
The frequencies of MN obtained by scoring DAPI stained slides after treatment with different concentrations of RPR-115135 (0.1–10 µM) for 48 h in clones CMV-2 and Mu-p53-2 are reported in Figure 2B. Figure 2A reports cell survival. Only the highest concentrations (10 µM) caused a statistically significant cytotoxicity in both clones. The same cell preparations were used to evaluate the percentage of surviving cells and the frequency of MN. Regarding induction of MN, univariate analysis of variance [multiple comparisons (Dunnett's t-test treating one group as a control and comparing all other groups with it)] revealed that the only statistically significant result was obtained on treating cells with 10 µM RPR-115135 (P = 0.001 for clone CMV-2; P = 0.013 for clone Mu-p53-2).
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In the light of these data, HCT-116 cells were treated with 10 µM RPR-115135 in a time dependence experiment. A significant increase in induction of MN to a similar extent was observed by scoring propidium iodide stained slides of both clone CMV-2 and Mu-p53-2. The increase started 24 h after treatment, when no cytotoxic effect was seen, with the maximum number of MN being reached 2 days after treatment; it remained at a plateau level for an additional 2 days of treatment (Figure 3
), after which, looking at the growth kinetics, a clear growth inhibition induced by RPR-115135 was evident. After 5 or 6 days treatment the MN count became technically difficult because of a tendency of the treated cells to form small compact clusters with highly condensed chromatin (data not shown).
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The evaluation of cell membrane integrity (10 µM RPR-115135 over 6 days) revealed that the percentage of HCT-116 cells able to exclude trypan blue dye was ~85% (cellular debris not considered), suggesting that those cells with MN had substantially intact cell membranes. These data were supported by looking at cell mophology on Giemsa stained slides (data not shown).
Since MN can be formed by chromosomal breakage or chromosome loss (Nûsse et al., 1989), an antibody obtained from serum of patients with the autoimmune disease CREST syndrome was used (Nûsse et al., 1989). Centromeres in MN were immunologically visualized with anti-kinetochore antibodies from CREST patients. The CREST serum contains antibodies to a specific protein of the kinetochore region of chromosomes. Cells with CREST-containing MN are principally induced by aneuploidy-inducing agents (e.g. vincristine), while MN lacking CREST labeling are induced by clastogenic agents (e.g. mitomycin) (Figure 4). Figure 5 shows the frequency (per 1000 cells) of total MN and CREST+ and CREST– MN induced by 10 µM RPR-115135 at different times of treatment in both the CMV-2 and Mu-p53-2 clones. The comparison between total MN and kinetochore-positive MN (CREST+) frequencies showed that RPR-115135 induced predominantly CREST+ MN in both the CMV-2 and Mu-p53-2 clones (Figure 5). With respect to a culture time effect, no statistically significant differences (P > 0.05, Student's t-test) in the percentage of CREST+ MN were observed in relation to incubation time (1–4 days) with RPR-115135 when all data were analysed.
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These data show a tendency for RPR-115135 to be an aneugenic agent.
The frequencies of MN in the MCF-10A cell system were scored after 48 h, when no significant cell death was present after treatment with 1 µM RPR-115135. No induction of MN was observed in MCF-10A cells (Figure 6).
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The evaluation of cell membrane integrity (1 µM RPR-115135 over 6 days) revealed that the percentage of MCF-10A cells able to exclude trypan blue dye was ~79.3%, of MCF-10A-H cells 52.5%, of MCF-10A-E cells 100% and of MCF-10A-EH cells 89% (cellular debris not considered). Only in H-ras transfected cells ~50% did not have intact cell membranes, suggesting massive necrosis and perhaps explaining the absence of MN induction. These data were confirmed by analysing the cell morphology of Giemsa stained cells. About 40% of MCF-10A-H cells showed a typical picture of necrosis.
In human lymphocytes the IC50 concentration of RPR-115135 was 10 µM, while 0.1 and 1.0 µM had no effect on cell proliferation (48 h treatment).
Table III reports the nunber of MN scored; a univariate analysis of variance [multiple comparisons (Dunnett's t-test treating one group as a control and comparing all other groups with it)] revealed that no data were statistically significant (P > 0.05).
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Induction of MN was also studied in primary normal chick spinal cells obtained from 7-day chick embryos. Treatment with 10 µM RPR-115135 over 6 days induced massive cell death, while 1.0 µM was well tolerated over 6 days treatment. After 48 h treatment with 1 µM RPR-115135 cell survival was ~75%. MN were also scored after 48 h treatment with 1.0 µM RPR-115135 (Figure 7
). No induction of MN was observed.
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Discussion |
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The role of RPR-115135, a FPP-competitive FTase inhibitor, in the induction of MN was analysed in cells having different origins (human or chick) and/or carrying different mutations. The aim of the study was not only to evaluate the ability of RPR-115135 to increase MN induction as a function of the ras genotype of the tumour, but also to investigate dependence on other characteristics of each individual tumour, such as, for example, the complement of mutations in cell cycle regulatory genes.
To accomplish this, two well-characterized isogenic cell lines were used. The first model was the HCT-116 human isogenic colon cancer cell line with intact versus disrupted p53 funtion. In this system RPR-115135 is able to induce a significant number of CREST+ MN, suggesting a potential ability of RPR-115135 to act as an aneugenic agent. This ability is independent of p53. In this system evaluation of cell membrane integrity (10 µM RPR-115135 over 6 days) revealed that the percentage of cells able to exclude trypan blue dye was ~85%, suggesting that MN induction was in cells with substantially intact cell membranes. Giemsa staining supported this observation.
However, the parental human colon cancer cell line HCT-116 has several other oncogenic characteristics (Table I
), including loss of ARF normal function. ARF is involved in tumour surveillance (ARF antagonizes Mdm2 to activate p53; Sherr, 2000) and its expression is activated by abnormal mitogenic signals induced by overexpression of oncoproteins such as Myc and Ras (Lloyd, 2000; Sherr, 2000). Loss of the ARF checkpoint subverts this form of cell-autonomous tumour surveillance and allows proteins such as Ras and Myc to function as `pure' proliferation enhancers. Recently, McCormick (2000) reported that the Ras pathway also activates the p14ARF pathway, suggesting that p53 accumulation in response to DNA damage is determined by Ras activity and p14ARF expression. Consistent with this view, loss of ARF makes cells relatively resistant to apoptosis induced by ionizing radiation (Sherr, 2000) and can sensitize cells to polyploidy induced by microtubule inhibitors (Khan et al., 2000). It is possible that RPR-115135 can increase genomic instability in HCT-116 cells (ARF loss, Myc overexpressed, Ras mutated) by inducing MN. Recently it has been reported that two centromere-associated proteins (CENP-E and CEMP-F) are farnesylated (Ashar et al., 2000). Cells treated with FTase inhibitors (SCH66336 or FTI-2153) appeared to have scattered kinetochores (Ashar et al., 2000) or were inhibited in formation of bipolar spindles and chromosome alignment (Crespo et al., 2001). Our preliminary results in treated HCT-116 cells (Diaspro et al., in preparation) showed that RPR-115135 affects the interaction between CENP-E and tubulin. CENP-E localizes to kinetochores during prometaphase and regulates attachment of chromosomes to microtubules (Craig et al., 1999). It is reasonable to propose that MN induction by RPR-115135 in HCT-116 cells may have resulted from interference with this interaction of chromosomes with microtubules.
To further investigate the relationship between oncogene activation and induction of MN by RPR-115135, we used a second isogenic cell system consisting of the MCF-10A cell line, a non-transformed immortalized human breast epithelial line and two transfected lines, one with the c-Ha-ras oncogene (activated by a missense mutation) and one with the c-erbB-2 protooncogene. Since it has been shown that c-Ha-ras and c-erbB-2 have an additive effect on in vitro transformation of the MCF-10A cell line (Ciardiello et al., 1992), we also evaluated the influence, if any, of such co-transfection on MN induction by RPR-115135. All cells were sensitive to the cytotoxic effects of RPR-115135, although the parental cell line was ~10 times more resistant than the c-Ha-ras transfected line. Despite this drug sensitivity, no induction of MN was observed, suggesting that the ability of RPR-115135 to act as an aneugenic agent can be related to other mutations present in cells such as HCT-116. It is also true that in the most sensitive line, MCF-10A-H, ~50% of treated cells were unable to exclude trypan blue dye and after Giemsa staining showed the typical morphology of necrosis, suggesting massive necrosis as a major mechanism of action of RPR-115135 in these cells.
However, in rapidly proliferating human lymphocytes RPR-115135 was cytotoxic at 10–1.0 µM, but did not induce any increase in MN.
Finally, RPR-115135 at concentrations that did not affect cell integrity did not induce MN after 48 h treatment in normal primary spinal cells (obtained from 7-day chick embryos), despite their moderate sensitivity.
In conclusion, RPR-115135 acts as an aneugenic agent in a complex manner, dependent upon the complement of mutations in cell regulatory genes in tumour cells and this activity may be independent of ras genotype.
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Acknowledgments |
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We warmly thank Dr Patrick M.O'Connor (Laboratory of Molecular Pharmacology, NCI–NIH, Bethesda, MD; present address Agouron Pharmaceuticals, San Diego, CA) for his precious guidance during this work. The statistical assistance of Dr Davide Malacarne (Laboratory of Experimental Oncology, Genova, Italy) is very much appreciated. We are also grateful to Dr Francesca Degrassi (Centro Genetica Evoluzionistica, CNR, Roma, Italy) for helpful comments on micronucleus evaluation and, finally, to Prof. Silvio Parodi (Laboratory of Experimental Oncology, Genova, Italy) for helpful discussions. C.O. and A.C. received a fellowship awarded by Fondazione Italiana per la Ricerca sul Cancro, Milano, Italy. This work was partially supported by a grant awarded by Associazione Italiana per la Ricerca sul Cancro (Milano, Italy) and by Tender no. 2000/S 118–076796, Induction of conformational changes in p53 mutants and modulation of sensitivity to selective anti-cancer drugs, awarded by EEC (ISPRA, Italy).
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Notes |
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2 To whom correspondence should be addressed.
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Received on February 1, 2001; accepted on May 3, 2001.
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