Mutated ras genes are frequently found in human cancer. However, it has been shown that oncogenic ras inhibits growth of primary cells, through pathways involving p53 and the cell cycle inhibitors p16INK4a and p19ARF. We have analysed the effect of the ectopic expression of the three mammalian ras genes on the proliferation of K562 leukemia cells, which are deficient for p53, p16INK4a, p15INK4b and p19ARF genes. We have found that high expression levels of both wild-type and oncogenic H-, K- and N-ras inhibit the clonogenic growth of K562 cells. Induction of H-rasV12 expression in K562 transfectants retards growth and this effect is accompanied with an increase of p21WAF1 mRNA and protein levels. Furthermore, p21WAF1 promoter is activated potently by oncogenic ras and less pronounced by wild-type ras. This induction is p53-independent since a p21WAF1 promoter devoid of the p53 responsive elements is still activated by Ras. Finally, inhibition of p21WAF1 expression by an antisense construct partially overcomes the growth inhibitory action of oncogenic H-ras. Altogether, these results indicate that the antiproliferative effect of ras in myeloid leukemia cells is associated to the induction of p21WAF1 expression and suggest the existence of p19ARF and p16INK4a-independent pathways for ras-mediated growth inhibition.
The mammalian ras gene family is composed of three members, H-, K-, and N-ras that encode closely related 21 kDa proteins. Mutated ras genes carrying point mutations mainly in codons 12, 13 or 61 are commonly found in human neoplasias and in experimental cancer (reviewed in Barbacid, 1989; León and Pellicer, 1993). ras genes harboring such mutations are capable of transforming immortalized cell lines as NIH3T3 fibroblast in vitro. However, they induce growth inhibition and premature senescence in murine and human primary fibroblasts (Serrano et al., 1997; Lin et al., 1998). The antiproliferative effect of oncogenic Ras in primary cells is achieved at least through two pathways. By the first one, H-ras (Serrano et al., 1997; Lin et al., 1998) and the Ras effector, Raf (Zhu et al., 1998) up-regulate the cyclin-dependent kinases (CDKs) inhibitor p16INK4a thus blocking Rb phosphorylation and G1 progression. By the other pathway, H-ras (Serrano et al., 1997; Lin et al., 1998) and Raf (Lloyd et al., 1997), up-regulate p53 and subsequently p21WAF1, an inhibitor of CDKs and a transcriptional target of p53 (El-Deiry et al., 1993). This second pathway impinges in the induction of p19ARF (p14ARF in human cells) (Palmero et al., 1998). p19ARF in turn has been shown to inactivate Mdm2, resulting in high levels of p53 (reviewed in Sharpless and DePinho, 1999).
Thus, in murine fibroblasts devoid of either p53 or p16INK4a, ras oncogenes provoke cell transformation instead of growth arrest or senescence. On the other hand, in human primary fibroblasts the inactivation of both the p16INK4a/Rb and p19ARF/p53 pathways is required for Ras transformation (for reviews see Lloyd, 1998; Malumbres and Pellicer, 1998). So far, the antiproliferative response induced by activated Ras has been mainly studied in primary cells and its relevance to human cancer and especially to hematological malignancies, remains unknown.
The involvement of ras in myeloid cell proliferation is suggested by the high frequency of N-ras activation in acute myeloid leukemia (AML) and myelodysplasic syndromes (20 to 45% of cases). However, ras mutations are practically absent in chronic myeloid leukemia (CML). Moreover, there is no correlation between the presence of activating ras mutations and the malignant progression of AML (reviewed in Beaupre and Kurzrock, 1999), casting doubts so as to the role of Ras oncoprotein in the processes underlying in myeloid cell malignant transformation.
In an effort to understand Ras function in CML we have studied the effect of the three ras genes in a biologically relevant model such as K562 cells. K562 cells do not carry mutated ras genes (Todd and Iland, 1991) and the three ras genes are expressed at similar levels as we have previously shown (Delgado et al., 1992). Here we show that H-, K- and N-ras oncogenes induce growth arrest, activation of p21WAF1 promoter and up-regulation of its mRNA and protein levels. The same effect, albeit much less pronounced, was exerted by wild-type ras. This is a striking finding in view of the lack of expression of p53 (Law et al., 1993), p15INK4b (Otsuki et al., 1995), p16INK4a (Ogawa et al., 1994; Otsuki et al., 1995) and p19ARF (Vonlanthen et al., 1998; and this study) in K562 cells, indicating that ras-mediated growth inhibition in myeloid cells takes place by a p19ARF and p16INK4a independent mechanism. Even more so, our data could help explain the absence of activating ras mutations observed in human CML.
Ras genes inhibit clonogenic growth of K562 cells
In order to accurately compare the effect of the three ras genes on K562 cell growth, we subcloned the three wild-type ras genes and its corresponding oncogenic mutants in the same vector (pCEFL). In these constructs, high constitutive expression of ras is achieved by the elongation factor 1α promoter. They were transiently transfected into COS cells and the level of Ras proteins was analysed by immunoblot with specific antibodies. As shown in Figure 1, H-, K- and N-Ras proteins were markedly overexpressed, and the expression levels of the wild-type proteins and their mutated counterparts were similar. To assess that all these constructs yielded similar expression levels in vivo, we tested the biological activity by a standard transformation assay in NIH3T3 cells. While the wild-type genes produced only a small number of transformed foci (15–25 foci/μg of transfected DNA), codon 12 mutants potently transformed NIH3T3 and the three genes showed similar transformation potencies (3800–4500 foci/μg DNA) (data not shown).
We then assessed the effect of ras oncogenes on clonogenic growth of myeloid leukemia cells. For this purpose we used the K562-derived cell line K526/S, capable of growing attached onto tissue culture plates. Cells were electroporated with equivalent amount (10 μg) of the ras-encoding expression vectors, as well as empty vector. Interestingly, the number of K562/S colonies was drastically reduced upon transfection with either H-, K- or N-ras oncogenes (Figure 2), only 2–10% of the number of colonies obtained after transfection with the empty vector. A similar effect was observed upon transfection with the wild-types. Although the number of colonies obtained was higher than with the mutated versions, the reduction was still remarkable (20–55%) when compared to the empty vector (Figure 2a). Among the three ras genes, H-ras was the most potent inhibitor of cell growth (inset in Figure 2a). A representative view of the K562/S colony densities appearing after transfection is shown in Figure 2b. The aforementioned results could also be due to ras genes inhibiting the adhesion of K562/S cells rather than inhibiting clonogenic growth. To rule out this possibility, we tested the effect of H-ras V12 on the growth on soft agar of parental K562 cells as shown in (Figure 2c). This resulted in a dramatic reduction of agar-grown K562 colonies, indicating that ras inhibited K562 growth rather than adhesion to surfaces.
Similar results were obtained using different ras expression vectors such as pBabe-Puro-H-ras V12 (not shown). We also transfected murine wild-type N-ras and N-ras K61 genes in inducible expression vectors under the control of the MMTV promoter, inducible by dexamethasone (Quincoces et al., 1997). Upon selection in G418 and dexamethasone, a pronounced decrease in the number of colonies was found after transfection with oncogenic N-ras (89%), and to a lesser extent with normal N-ras (42%), as compared to the empty vector.
Ras oncogene retards growth of stable transfectants
In order to study the effect of H-ras on K562 growth rates, we generated sublines in which H-rasV12 was constitutively expressed. In view of the ras-mediated growth inhibition, we aimed for relatively low H-Ras oncoprotein expression levels so we avoided the use of pCEFL and other commonly used expression vectors, that carry potent promoters. Therefore, we used plasmid pT24c, which harbors the entire human H-rasV12 gene driven by its own, relatively weak, TATA-less promoter (Malumbres and Pellicer, 1998). We co-electroporated pT24c and pSV2neo and selected transfectants G418-resistant. Several clones were isolated and the presence of exogenous human H-ras gene was verified by Southern analysis. We chose a clone termed KT24, which contained exogenous human H-ras sequences for further studies (Figure 3a). In these cells, Ras expression levels were barely 2–3-fold over those found in parental cells, as assayed by immunoblot (Figure 3b). KT24 cells growth rate exhibited no significant differences with parental cells under standard growth conditions. However, when cells were cultured in low serum (RPMI-0.5% FCS), the growth rate of KT24 was markedly lower than that of parental cells (Figure 3c).
As a parallel approach, we stably transfected K562 with the inducible H-rasV12 vector pDxHras. We selected the subline termed KDxHT9 which showed a clear induction of H-rasV12 mRNA upon addition of dexamethasone, as shown by Northern analysis (Figure 4a). A second subline KDxHT18 was selected, in which the expression of the exogenous ras gene was lower than that in KDxHT9. The growth rates of K562, KDxHT9 and KDxHT18 were monitored in the presence or absence of 1 μM dexamethasone. In the presence of dexamethasone, KDxHT9 showed the slowest growth rate, in consistence with the highest expression of H-ras oncogene (Figure 4b). KDxHT18 grew faster than KDxHT9 but slower than parental cells, again in agreement with their relative H-ras expression levels, further confirming that the expression of activated ras genes impairs K562 cells growth.
Ras oncogenes do not induce differentiation or apoptosis in K562
K562 cells can be induced to differentiate into erythroid or myelomonocytic phenotypes, a process associated to growth arrest (Delgado et al., 1992). Thus it would be conceivable that Ras-mediated growth arrest of K562 cells is a consequence of the induction of a differentiation program. We tested this possibility by analysing changes in the presence of erythroid markers (positive benzidine reaction and expression of ε-globin) or myeloid markers (reduction of nitroblue tetrazolium and expression of vimentin) upon the induction of ras expression. However, we did not detect any significant difference in differentiation markers in the ras-expressing cells as compared to the parental line (data not shown).
In view of previous reports describing that ras oncogenes induce apoptosis in some models (reviewed in Downward, 1998), we next explored whether Ras could impair cell growth by inducing apoptosis. However, we did not observe any increase in apoptosis in dexamethasone-stimulated KDxHT9 cells as determined by DNA laddering (Figure 5a) and DAPI staining of the nuclei (not shown). To further confirm this finding, we investigated whether Bcl-2 could rescue the inhibition of clonogenic growth brought about by oncogenic Ras. As shown in Figure 5b, co-transfection of H-ras V12 with an excess of bcl-2 did not modify Ras-mediated growth inhibition. Altogether these results suggest that the Ras-mediated growth inhibition on K562 is not due to the induction of apoptosis.
Ras genes induce the expression of p21WAF1
To explore possible mechanisms for the Ras-mediated growth inhibition of K562, we analysed whether Ras could up-regulate inhibitors of cyclin-dependent kinases. The antiproliferative effects of ras oncogenes in human fibroblasts require the presence of either p19ARF or p16INK4a (see Introduction). However, K562 cells are deficient in p16INK4a as well as p15INK4b and p53, while there are conflicting reports as to the expression of p19ARF (Della Valle et al., 1997; Vonlanthen et al., 1998). To clear this point we performed Southern blot analysis of K562 DNA and demonstrated that the p19ARF gene is homozygously deleted in this cell line (not shown). Therefore, an involvement of p15INK4b, p16INK4a, and p19ARF in Ras-mediated inhibition of K562 growth was ruled out. Thus, it was conceivable that ras effects could be explained by the induction of the CDK inhibitor p21WAF1. Northern hybridization demonstrated that p21WAF1 mRNA expression is up-regulated in KDxHT9 cells upon induction of ras expression by dexamethasone (Figure 6a). As a control we determined the up-regulation of p21WAF1 mediated by wild-type p53, using a K562 subline transfected with a termosensitive p53 mutant (A1 in Figure 6a). A clear increment of p21WAF1 protein was also observed 12 to 16 h after dexamethasone addition (Figure 6b). The up-regulation of p21WAF1 mediated by Ras however was smaller than that mediated by wild-type p53 (Figure 6b, lane A1). We also found that the expression of p27KIP1, the other CDK inhibitor expressed in K562, did not change upon induction of ras expression (results not shown).
To determine whether the increase in p21WAF1 levels induced by Ras is due to a transcriptional up-regulation we assayed the activity of p21WAF1 promoter. The results in Figure 6c show that wild-type ras activated p21WAF1 promoter (5–14-fold), while stronger up-regulation (25–32-fold) was found with the three oncogenic ras versions. Other less potent ras expression vectors induced weaker p21WAF1 transactivation (data not shown). As an additional control, p53 induced a very strong activation of the p21WAF1 promoter (Figure 6c).
The p21WAF1 reporter plasmid used in the previous experiments contains a 2.3 kb promoter fragment. To more precisely define the Ras-responsive region we constructed and tested a series of promoter deletions. The Figure 7 shows the results with two of them: p21ΔSac-Luc, which only lacks the 5′ p53-responsive element (containing 2.2 kb of the promoter) and p21ΔPst-Luc, which contains the proximal 0.21 kb of the promoter (Figure 7a). H-RasV12 transactivated to a similar extent the three reporters. The effect of H-, K- and N-Ras oncoproteins were compared on the shorter p21ΔPst-Luc reporter, and we found no significant differences (Figure 7b). Thus, this 0.21 kb region must contain the Ras-responsive element(s).
To investigate whether p21WAF1 up-regulation was responsible for Ras-mediated growth inhibition, we transfected K562/S cells with a p21WAF1 expression vector. p21WAF1 caused inhibition of colony formation (Figure 8a) although this inhibition was smaller than that induced by H-rasV12. We also cotransfected K562/S cells with H-rasV12 and an expression vector carrying an antisense p21WAF1 (pPuro-αsp21). It was found that the suppression of p21WAF1 by the antisense vector was capable of partially overcoming the growth inhibitory effects of H-rasV12 (Figure 8b). Altogether, the results suggest that the antiproliferative effect of ras in these cells is at least partially mediated by the induction of p21WAF1 expression.
In this work we describe that ras genes induce cell growth inhibition of K562 leukemia cells by a novel mechanism independent of p53, p19ARF, p15INK4b and p16INK4a, that may be related to p21WAF1 induction.
We show that high expression levels of oncogenic mutated versions of H-, K- and N-ras inhibited K562 clonogenic growth. Likewise, overexpression of wild-type ras genes also impaired clonogenic growth but their effect was weaker as compared to their mutated counterparts. This growth arrest does not seem to be associated with the induction of differentiation or the triggering of apoptotic processes. Although growth inhibition mediated by H-ras is not unprecedented, this is to our knowledge the first report showing a comparison of the antiproliferative effects of the three ras genes and oncogenes. Interestingly, there are differences in the growth inhibitory capacity of the three ras on K562 cells, H-ras being the most potent inhibitor. It is noteworthy that there is a differential expression of the three ras genes during differentiation of K562, in which H-ras but not K- or N-ras is down-regulated (Delgado et al., 1992).
It has been an enigmatic finding that ras mutations are frequent in AML but they are absent in CML, even in the blastic phase of the disease (Watzinger et al., 1994) even though CML in blast crisis share many morphological and biological properties with AML cells. The results presented here, showing that activated ras have growth inhibitory effects in K562, may help to explain the absence of ras mutations in CML. Interestingly, in AML the frequencies of ras mutations depend on the particular member of the family involved. As such, mutations in N-ras are the most prevalent, while those in K-ras are less frequently found and mutations in H-ras are very rare (Beaupre and Kurzrock, 1999). These frequencies are consistent with our results in K562, where H-ras oncogene is the most potent growth inhibitor, followed by K- and N-ras.
An antiproliferative effect of ras oncogenes has been previously reported for murine and human primary fibroblasts and shown to be dependent on the presence of p19ARF, p16INK4a and p53. In murine cells lacking one of these genes, the antiproliferative or senescence response to oncogenic ras is abolished and ras induces cell transformation instead (reviewed in Malumbres and Pellicer, 1998). In human fibroblasts, by contrast, the disruption of both p19ARF/p53 and the p16INK4a/Rb pathways is required to prevent ras-mediated growth arrest (Serrano et al., 1997). Likewise, transfer of mutated ras genes into bone marrow-derived cells did not lead to cell proliferation but promotes myeloid cell differentiation or growth retardation (Pierce and Aaronson, 1985; Hawley et al., 1995). In contrast to primary fibroblasts and bone marrow cells, K562 are devoid of p53, p15INK4b, p16INK4a and p19ARF. To our knowledge this is the first report of an antiproliferative effect of oncogenic ras in cells lacking these four genes, demonstrating that ras oncogenes can block cellular growth by mechanisms other than those so far described. H-ras oncogenes also impairs growth of human monocytic U937 cells (Maher et al., 1996). However U937, unlike K562, express p16INK4a (Juan et al., 1998) which may explain the antiproliferative effect of Ras in U937 through the p16INK4a/Rb pathway.
How does Ras inhibit K562 proliferation in spite of the absence of p19ARF and p16INK4a activities? The most likely explanation involves the CDKs inhibitor p21WAF1, which expression is up-regulated in K562 upon the induction of ras. In this line, our data indicate that the p21WAF1 promoter is potently activated by ras. The idea that p21WAF1 is responsible for Ras-mediated growth inhibition is supported by the abrogation of Ras growth-inhibitory effect by anti-sense p21WAF1.
It has been reported that ras induces p21WAF1 expression in primary fibroblasts (Serrano et al., 1997; Lin et al., 1998) and Raf does so in primary Schwann cells (Lloyd et al., 1997). However, in both cases p21WAF1 up-regulation was p53-dependent. This is consistent with p21WAF1 being a target gene of p53 (El-Deiry et al., 1993), and ras up-regulating p53 (Serrano et al., 1997; Lin et al., 1998). In contrast, in K562 the elevation of p21WAF1 proceeds through a p53-independent pathway, because (a) K562 cells are p53-deficient and (b) a p21WAF1 promoter devoid of the p53 responsive elements is still activated by Ras in K562 (this study). A number of responsive elements have been described in p21WAF1 human promoter (Gartel and Tyner, 1999). We have found that Ras up-regulation depends on a promoter fragment of only 0.21 kb. Further work is aimed to precisely define the Ras-responsive sequences. It has also been described that high-intensity Raf signals up-regulates p21WAF1 in a p53-independent fashion in murine primary fibroblasts (Sewing et al., 1997; Woods et al., 1997), although its dependence from p15INK4b, p16INK4a or p19ARF was not explored. It is reported that Ras-mediated up-regulation of p21WAF1 is blocked by Rho (Olson et al., 1998). However, we previously showed that K562 express Rho and that Rho ectopic expression induces apoptosis in these cells (Esteve et al., 1998).
Altogether our results strongly suggest that oncogenic Ras impair growth of CML-derived cells through the elevation of p21WAF1. Other mechanisms could operate subsequently to p21WAF1 mediated growth arrest, as autophagic degeneration (Chi et al., 1999) or cellular senescence (Serrano et al., 1997; Fang et al., 1999) although in murine fibroblasts p21WAF1 is not essential for Ras-mediated senescence (Pantoja and Serrano, 1999). In conclusion, we propose a new pathway to explain the growth arrest mediated by oncogenic Ras, as schematized in Figure 9. In some cell types Ras inhibits growth through mechanisms dependent on p19ARF (pathways A and B in Figure 9) or on p16INK4a (pathway C). In other cells as CML-derived cells, Ras would lead to p21WAF1 up-regulation in a way independent from p16INK4a, p19ARF and p53 (pathway D). Moreover our results in K562 suggest that this effect is, to some extent, p21WAF1-independent (E).
Materials and methods
Cell lines and cell culture
K562 cell line derives from a chronic myeloid leukemia and was purchased from American Type Culture Collection. K562/S is a K562-derived cell line able to grow attached to a plastic surface. The line was selected after multiple passages on tissue culture plates, removing the non-adherent cells. Cells were grown in RPMI-8% fetal calf serum (FCS). Clonogenic assays on soft agar of K562 were carried out using Iscove's medium supplemented with 15% FCS and 0.3% agar (Difco). NIH3T3 cells were grown in DMEM-10% newborn calf serum and COS cells in DMEM-10% FCS.
cDNAs corresponding to human H-, K- and N-ras proto-oncogenes and activated oncogenes were cloned into the pCEFL vector (Teramoto et al., 1996). This is a derivative of pcDNA3 where the expression is driven by the human elongation factor 1α promoter. cDNAs encoding human wild-type H-, K- and N-ras (provided by R Weinberg, Massachusetts Institute of Technology, Boston, MA, USA), human K-rasV12 (K-ras4B isoform) (provided by R Weinberg) and N-rasE12 (provided by J de Gunzburg, Institute Curie, Paris, France) were isolated as 0.6 kb BamHI-NotI fragments. The human cDNA encoding for mutant H-rasV12 (provided by M Serrano, Centro Nacional de Biotecnología, Madrid, Spain) was isolated as a 0.6 kb EcoRI-BamHI fragment. pcDNA3-H-rasV12 has been described (Crespo et al., 1994a). pBabe-Puro H-rasV12 was provided by M Serrano and carries human H-rasV12 cDNA in the pBabe-Puro vector (Morgenstern and Land, 1990). The entire human H-rasV12 oncogene, including its promoter (plasmid pT24-c) was provided by M Barbacid (Instituto Carlos III, Madrid, Spain). pDxH-ras have been previously described (Quincoces et al., 1997), pLTR-bcl2 was provided by P Koskinen (Turku Center for Biotechnology, Turku, Turkey) and carries human bcl2 cDNA in the pLTR poly vector (Koskinen et al., 1995). The expression vector for antisense-p21 (pPuro-αsp21) was provided by A Carnero (Institute of Child Health, London, UK).
Transfections and clonogenicity assays
For clonogenicity assays K562 or K562/S cells (2×106) in exponential growth were resuspended in 0.8 ml of RPMI-8% FCS containing the appropriate amount of the indicated plasmids and electroporated at 260 V and 1 mFa with a Bio-Rad electroporator. The plasmids were prepared by the Qiagen method. Forty-eight hours after electroporation, G418 (500 μg/ml) or puromycin (1 μg/ml) was added and the colonies counted 12 days after transfection. For stable transfection, 107 cells were electroporated with the indicated plasmids, selected with G418 as above and cell clones were isolated by limiting dilution. COS cells were transfected with DEAE-dextran (Crespo et al., 1994a). Transformation assays of ras genes in NIH3T3 cells were carried out as described (Crespo et al., 1994b).
Assessment of cell growth, differentiation and apoptosis
Growth was determined by cell counting in hemocytometer and by the determination of metabolic activity by the reduction of the tetrazolium salt WST-1 (Roche Molecular Biochemicals). Morphological differentiation was monitored by examining fixed slide preparations stained with May-Grünwald Giemsa and assessed using established cytological criteria. Erythroid differentiation was also assessed by the fraction of hemoglobin-producing cells scored by benzidine staining and by expression of ε-globin as described (Delgado et al., 1992). Monocytic differentiation was also assessed by the fraction of cells positive for the nitroblue tetrazolium dye reduction and the expression of vimentin (Delgado et al., 1992). The presence of internucleosomal fragmentation was assayed as described (Lerga et al., 1995).
Northern and Southern analysis
Total RNA was isolated from cells by the acid guanidine thiocyanate method and Northern blots were prepared according to standard procedures. The probes for human H-ras, ε-globin and vimentin were as described (Delgado et al., 1992). The probe for human p21WAF1 was a 0.5 kb BamHI-EcoRI from plasmid pWZL-Hygro-hp21 (provided by M Serrano). For Southern, 10 μg of genomic DNA were digested with restriction enzymes as indicated, electrophoresed, blotted onto nitrocellulose and hybridized to 32P-labeled probes.
The probe for human p19ARF was a 190 bp fragment of the exon 1β obtained by PCR (forward primer: 5′-TGGTGCGCAGGTTCTTGGTGA-3′, reverse primer: 5′-GTCTTCTAGGAAGCGGCTGCT-3′).
Cell lysates were prepared as described (Serrano et al., 1997). 30–50 μg of protein per lane were separated on SDS–PAGE gels and transferred to PVDF membranes by standard procedures. p21WAF1 was detected by the WAF-1 Ab-1 monoclonal antibody (Oncogene Research, Calbiochem). Anti-K and N-Ras antibodies were from Oncogene Research. Anti-H-Ras (F235 monoclonal antibody) and anti-p27KIP1 were from Santa Cruz. Immunoblots were revealed by chemiluminescency (ECL, Amersham).
K562 cells (4×106) were transfected by electroporation at 260 V and 1 mFa with 430 nmol (corresponding to 1.4 to 2 μg) of reporter plasmids and 2 to 10 μg of pCEFL-derived ras expression vectors of human p53 wild-type expression vector (pLPC-hp53wt, provided by M Serrano) as indicated. The reporter plasmids used were: pGL3-Waf1-Luc (provided for M Oren, Weizmann Institute, Rehovot) which contains a 2.3 kb genomic p21WAF1 DNA fragment (El-Deiry et al., 1993) cloned into the pGL3-basic vector (Promega) and two deletions derived from this plasmid. The construct termed p21ΔSac-Luc was prepared by digestion with SacI and recircularization. The construct termed p21ΔPst-Luc, was prepared by digestion with BglII and PstI, blunting with Klenow enzyme and religation. Constructs were confirmed by sequencing. Transfected cells were seeded in duplicated plates and 36 h after transfection cells were lysed with 80 μl of passive lysis buffer (Dual Luciferase Reporter System, Promega) and the firefly and Renilla luciferase activities were measured in a Turner luminometer. To normalize for transfection efficiencies, 1 μg of the pRL-TK plasmid (Promega) was co-transfected in each case. The co-transfection with ras expression vectors did not modify significantly the Renilla luciferase activity values. Promoter activity was defined as the ratio between the light units generated by the firefly and the Renilla luciferases.
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We thank Rosa Blanco for expert technical assistance and Mariano Barbacid, Amancio Carnero, Jean de Gunzburg, Päivi Koskinen, Moshe Oren, Manuel Serrano and Robert Weinberg for plasmids. We are also indebted to Manuel Serrano for helpful discussions. This work has been supported by grants PM98-0109 from Spanish Ministry of Education and Culture and Biomed 96-3532 from European Community (J León) and from Fundacion Marcelino Botin (P Crespo).
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Delgado, M., Vaqué, J., Arozarena, I. et al. H-, K- and N-Ras inhibit myeloid leukemia cell proliferation by a p21WAF1-dependent mechanism. Oncogene 19, 783–790 (2000). https://doi.org/10.1038/sj.onc.1203384
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