Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the ...
Oncogene (1998) 16, 1373 ± 1381 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
The retinoblastoma protein is essential for cyclin A repression in quiescent cells Alexandre Philips*, Xavier Huet*, Ariane Plet, Laurent Le Cam, Annick Vie and Jean Marie Blanchard Institut de GeÂneÂtique MoleÂculaire, CNRS, UMR 5535, 1919 route de Mende, 34293 Montpellier cedex 5, France
Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this eect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been dicult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be eective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary ®broblasts from pRb(7/7) embryos and not in ®broblasts from p130(7/7), p107(7/7) or even p130(7/7)/p107(7/7) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of speci®c genes in G0, providing thus the ®rst example of non-overlapping functions achieved by the dierent pocket proteins. Keywords: cyclin A; transcription; pocket proteins
Introduction Cyclin A, as a regulatory component of cdk2 and cdc2 kinases, plays an essential role in the progression through the S phase of the cell division cycle (reviewed by Heichman and Roberts, 1994; Sherr, 1996). Its mRNA and protein start accumulating at the end of the G1 phase, and microinjection of antibodies directed at cyclin A or antisense cyclin A expression vectors prevent DNA replication (Girard et al., 1991; Pagano et al., 1992; Zindy et al., 1992). Cyclin A protein has even been reported to be associated in vitro with replicating SV40 DNA (Fotedar and Roberts, 1991) and in vivo with sites of ongoing DNA synthesis (Cardoso et al., 1993). More recently, cyclin A has also been proposed to participate in the cascade which
Correspondence: JM Blanchard * The ®rst two authors have contributed equally to this work Received 26 August 1997; revised 20 October 1997; accepted 20 October 1997
controls the coordinated execution of S phase and mitosis (Guadagno and Newport, 1996). Moreover, complexed to cdk2 and in cooperation with cyclin E, it has been shown to replace an S phase nuclear extract in triggering the entry into S phase of in vitro incubated G1 nuclei from HeLa cells (Krude et al., 1997). Cyclin A mRNA accumulation results from a transcriptional activation of its gene at the G1/S border (Barlat et al., 1993, 1995; Henglein et al., 1994; Schulze et al., 1995; Zwicker et al., 1995; Huet et al., 1996; Plet et al., 1997). The promoter sequences responsible for this periodic activity have been delineated through both in vitro mutagenesis and in vivo footprinting analysis (Figure 1). A DNA element, named the Cell Cycle Responsive Element (CCRE; Huet et al., 1996) or the Cell cycle Dependent Element (CDE, Zwicker et al., 1995) and located within the region containing the major transcription initiation sites, has been shown to be periodically occupied in vivo by a repressor-type of complex. Consistent with this, mutation of the CCRE/ CDE resulted in a complete loss of the cell cycle regulation of the cyclin A promoter. The nature of the protein(s) binding to the CCRE/CDE is still elusive. Several observations point to E2F/DP (for a recent review see Sardet et al., 1997) as a putative eector of cyclin A down regulation. Cyclin A mRNA accumulation is increased in adenovirus E1A- (Buchou et al., 1993) or polyoma- (Barlat et al., 1993) transformed rodent cell lines, as well as in quiescent human ®broblasts infected by adenovirus type 5 (Zerfass et al., 1996). Moreover, expression of human papillomavirus type 16 E7 transforming protein in established murine ®broblasts leads to constitutive expression of cyclin A in the absence of external growth factors (Zerfass et al., 1995). However, whereas some reports, based on in vitro gel shift experiments, are clearly in favour of E2F/DP mediating the cyclic repression of cyclin A promoter activity in the G1 phase of the cell cycle (Schulze et al., 1995), others do not reach a ®rm conclusion on this issue (Zwicker et al., 1995; Huet et al., 1996; Plet et al., 1997). This leaves open the possibility of the presence of either a loosely bound member of the E2F family, harboring a very low in vitro anity for the cyclin A CCRE, or a yet unknown factor. Whatever the nature of the factors directly involved, overexpression of proteins such as adenovirus E1A or papillomavirus E7 is known to displace proteins related to the retinoblastoma tumor suppressor gene product (pRb) from transcriptional complexes (for a review see Cress and Nevins, 1996). pRb is a member of a protein family that includes p107 and p130 (Ewen et al., 1991; Hannon et al., 1993; Li et al., 1993; Mayol et al., 1993). The three proteins share structural and functional properties through a con-
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Figure 1 Schematic representation of the central portion of mouse cyclin A promoter. ATF, NF-Y and CCRE/CDE (Cell Cycle Responsive Element) correspond to previously identi®ed in vivo footprints (Huet et al., 1996). HS1 and HS2 refer to the two variable hypersensitive sites described in this work and present on the lower DNA strand
served protein domain, referred to as the `pocket', involved in the binding of DNA tumor virus oncoproteins and cellular proteins such as members of the E2F/DP family of transcription factors (for a review see Sardet et al., 1997). The biological importance of the role of pRb is strengthened by its inactivation in a wide range of human tumors (for a review see Weinberg, 1995). These observations provide thus a strong support to the role of such pocket proteins in linking cyclin A transcription to cell cycle regulation. However, because of the existence of related proteins which might exert overlapping functions, the precise role of each individual pocket protein has been dicult to assess in cultured cell lines. Moreover, experiments designed to study their functions have for the vast majority been carried out within transformed cell lines where many unknown genetic alterations have accumulated and which could interfere with the interpretation of the results. The obtention of mice where the genes coding for the various pocket proteins have been inactivated through homologous recombination (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992, 1996; Cobrinik et al., 1996), will allow to circumvent this problem. We report here the analysis of cyclin A gene regulation in primary ®broblasts prepared from pRb(7/7), p107(7/7), p130(7/7) and p107(7/7)/p130(7/7) mice.
Results Cyclin A transcription is deregulated in pRb(7/7) primary mouse ®broblasts The possibility to derive ®broblasts from embryos whose pRb, p107 or p130 genes had been targeted
through homologous recombination (Herrera et al., 1996; Cobrinik et al., 1996) has thus prompted us to analyse the transcription of cyclin A in these cells. Whereas p107(7/7) or p130(7/7) ®broblasts do not show any modi®cation of their cell cycles parameters (Cobrinik et al., 1996), pRb(7/7) cells exhibit a shorter G1 phase and a smaller size than wild type cells (Herrera et al., 1996). When thymidine incorporation was monitored in serum starved pRb(7/7) fibroblasts, we observed a progressive loss of serum dependence when the number of passages increased. This was not the case for p107(7/7) or p130(7/7) ®broblasts which progressively stopped growing, probably entering a senescent state. A representative example is shown in Figure 2a, where pRb(7/7) cells at passage 7 were starved for 36 h either in the absence or in the presence of 0.5% calf serum, and then restimulated with 10% serum for the indicated times. It had previously been shown that cyclin E was expressed earlier in pRb(7/7) cells, with a 6 h shift compared to wild type cells (Herrera et al., 1996). Cyclin A is also deregulated and expressed both at the RNA (Figure 2b) and the protein (Herrera et al., 1996) (Figure 5) levels from the very beginning of the restimulation period, even in resting cells. In pRb(+/+) cells, transcription from the cyclin A promoter is active in proliferating cells and inhibited in quiescent cells through a negative regulatory element (Cell Cycle Responsive Element, CCRE/Cell cycle Dependent Element, CDE) whose mutation renders the promoter constitutively active (Huet et al., 1996; Schulze et al., 1995; Zwicker et al., 1995; Plet et al., 1997). The wild type and the mutated (mCCRE) versions of the cyclin A promoter linked to a luciferase reporter (pCycA-luc) were then transfected into wild type and pRb(7/7) cells. The CCRE-dependent repression of
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Figure 2 Deregulation of cyclin A expression in pRb(7/7) ®broblasts after serum restimulation following a 36 h period of serum starvation. (a) 3H-thymidine incorporation into acid-insoluble material after serum refeeding late passage cells starved either in the absence (&) or in the presence of 0.5% (*, *) serum. Open symbols: pRb(+/+) cells, closed symbols: pRb(7/7) cells. 2.105 cells were labeled for 15 min in 200 ml of medium containing 10 mCi 3H-thymidine (Amersham, 5 Ci/mMole). (b) Northern blot analysis of cyclin A mRNA accumulation following serum restimulation. 20 mg total RNA was prepared from pRb(+/+) and pRb(7/7) cells at each indicated time post-stimulation with 10% serum. Hybridization was performed sequentially with radiolabeled full length human cyclin A and murine GAPDH cDNA probes. (c) pRb(+/+) and pRb(7/7) cells were transfected with a luciferase reporter driven by the murine cyclin A promoter and luciferase activity monitored at the indicated times after serum restimulation. Open symbols: pRb(+/+) cells, closed symbols: pRb(7/7) cells. Wild type promoter: &,&; promoter mutated within the CCRE: *. (d) Wild type (WT) and mutated (mCCRE) cyclin A promoter activity in quiescent pRb(7/7) cells maintained in serum-free medium, which were cotransfected with a phosphorylation-insensitive dominant pRb expression vector (®lled bars), or with a defective mutant version harboring a carboxy terminal deletion of amino acids 830 to 882 (hatched bars). The open bars correspond to the reporters transfected with the empty expression vectors. (e) Wild type (WT) and mutated (mCCRE) cyclin A promoter activity in quiescent pRb(+/+) cells maintained in serum-free medium, which were cotransfected with an adenovirus 12S E1A expression vector (®lled bars), or with a defective deletion mutant of constant region CR2 (hatched bars). As indicated in (d), open bars correspond to the reporters transfected with the empty expression vectors
cyclin A transcription was lost in quiescent pRb(7/7) cells (Figure 2c). A constitutive high activity was monitored whether cells were starved in low serum or in its total absence, or restimulated. This situation was mimicked in pRb(+/+) cells by the mutation of the CCRE, thus functionally linking pRb to CCREmediated repression. We therefore cotransfected an pRB-expressing vector together with pCycA-luc into quiescent mutant cells. This resulted into a 50% inhibition of cyclin A promoter activity (data not shown). Moreover, a phosphorylation-insensitive dominant form of pRb (Hamel et al., 1992) almost completely (70%) restored the inhibition of the cyclin A promoter, whereas a pRb-defective form harboring a deletion in the carboxy terminal domain (Qian et al., 1992) failed to do so (Figure 2d). Cyclin A transcription has been shown to be activated upon infection of quiescent rodent ®broblasts by adenovirus (Buchou et al., 1993; Zerfass et al., 1996). The expression of viral E1A gene products is sucient to induce DNA synthesis in the absence of serum (Braithwaite et al., 1983; Stabel et al., 1983) and is able to cooperate with an activated ras oncogene in the transformation of these cells (Ruley, 1983). Many
transformation-de®cient E1A molecules contain mutations in a region conserved between the various serotypes (conserved region 2, CR2) which mediates the binding of E1A to proteins of the pRb family (reviewed by Dyson et al., 1992). Expressing the 12S version of adenovirus E1A into quiescent pRb(+/+) cells gave rise to a strong stimulation of cyclin A promoter activity. This was not the case for a mutant in conserved region CR2 which is no longer able to interact with pocket proteins (Figure 2e). In contrast, deregulation was not observed when the kinetics of induction by serum of the mRNA coding for cyclin D1, the regulatory subunit of cdk4 and cdk6 kinases, was compared between pRb(+/+) and pRb(7/7) cells (Herrera et al., 1996) (Figure 3). This result is consistent with the absence of change in the activity of a luciferase reporter driven by the promoter of cyclin D1 (Herber et al., 1994) in pRb(7/ 7) cells compared to wild type cells (our unpublished data). Moreover, it is also consistent with recent data showing that many other immediate early events such as ras signaling, upon which cyclin D1 induction depends, are not perturbed (Peeper et al., 1997). Because a functional pRb is necessary to observe a proper CCRE-mediated down regulation of cyclin A,
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we next performed an in vivo genomic footprint analysis of the region surrounding this element in both pRb(+/ +) and pRb(7/7) cells. As previously described, in wild type cells the CCRE is quantitatively occupied only in G0-early G1 cells (Zwicker et al., 1995; Huet et al., 1996; Plet et al., 1997). The CCRE exhibits then a weak protection ¯anked at its 3' border by a strong hypersensitive site (HS1) which vanishes progressively with the protection as cells transit through G1/S (Figure 4a). Interestingly, 35 bp downstream of it, another hypersensitive site (HS2) was found and which in contrast, was weak in resting cells and strong in proliferating cells. A quantitative densitometric analysis is detailed in Figure 4b. The relative intensity of these two hypersensitive sites represents thus a highly sensitive index of chromatin changes preceding cyclin A promoter activation. Cells were either serum-starved for 48 h (quiescent), serum-refed for 18 h (stimulated) or grown to con¯uence for several days (con¯uent) and processed for in vivo footprint analysis. Whereas pRb(+/+) cells exhibit a strong signal for HS2 only when stimulated, this is clearly not the case for pRb(7/ 7) cells where the signal is high even in cells arrested by con¯uence, with a concomitant weakening of the footprint on the CCRE (Figure 4c). Cyclin A expression is not deregulated when p107 or p130 are mutated in primary mouse ®broblasts The upper results are consistent with a role of pRb in the transcriptional inhibition of cyclin A promoter in resting cells. However, they are surprising since a functional distinction has been proposed for the speci®c role of p130-containing complexes between resting cells in G0 and cells which transit through G1 (Chittenden et al., 1993; Cobrinik et al., 1993; Smith et al., 1996). Our data could be interpreted by the existence of a coordinated regulatory link between the various pocket proteins. We therefore checked for a possible absence of p130 in resting pRb(7/7) cells which could explain the lack of transcriptional inhibition of cyclin A. Figure 5 shows a Western blot analysis of extracts prepared from wild type,
WT hour
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pRb(7/7) and p130(7/7) cells. p130 level is high in quiescent cells and shows a decrease associated to a mobility shift in serum-stimulated cells whatever the status of pRb. Conversely, p107 level is low in quiescent cells and high in stimulated cells. Interestingly, an upregulation of p107 can even be seen in quiescent pRb(7/7) cells which is not the case for p130(7/7) cells. Furthermore, in pRb(7/7) cells these proteins can be found associated into functional complexes as de®ned by in vitro band shifting experiments (data not shown). As far as pRb is concerned, it is present mostly under its hypophosphorylated form in quiescent pRb(+/+) and p130(7/ 7) cells and harbors a change in mobility upon serum-induced hyperphosphorylation as expected (Figure 5). These data, as well as the speci®c role proposed for p130 in quiescent cells (Chittenden et al., 1993; Cobrinik et al., 1993; Smith et al., 1996), led us then to assay for the functional repression of cyclin A activity in p130(7/7) cells. When wild type and p130(7/7) cells were compared, their kinetics of S phase reentry following serum withdrawal, as well as their distribution into the cell cycle, were found to be indentical (Cobrinik et al., 1996). Such is also the case for cyclin A mRNA (data not shown) and protein which are absent in quiescent p130(7/7) cells (Figure 6a). Likewise, cyclin D1 mRNA is induced as the same level as it is in wild type cells (Figure 3). When the wild type cyclin A promoter was transfected into p130(7/7) cells, its activity was clearly repressed in quiescent cells and activated upon serum addition (Figure 6b). These data exclude therefore that deregulation of cyclin A transcription could result from a down regulation of p130 in pRb(7/7) cells. Similarly, cyclin A protein (Figure 6a) and mRNA (not shown) induction by serum were not perturbed in p107(7/7) as well as in p107(7/7)/p130(7/7) double mutant cells. Consistent with this, and as just described for p130(7/7) cells, a transfected wild type cyclin A promoter was repressed after serum withdrawal and activated following serum exposure in both types of cells (Figure 6b). In the same experiments, the CCRE mutated promoter harbored
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Figure 3 Northern blot analysis of cyclin D1 mRNA accumulation following serum restimulation. 20 mg total RNA was prepared from wild type (WT), pRb(7/7) and p130(7/7) cells at each indicated time post-stimulation with 10% serum. Q stands for quiescent cells. Hybridizaton was performed sequentially with radiolabeled full length human cyclin D1 and murine GAPDH cDNA probes
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Discussion
a constitutive high activity (data not shown). Finally, these results are also consistent with a recent report published while this work was submitted (Hurford et al., 1997) and which extends the analysis of pRb(7/ 7) cells (Herrera et al., 1996) to cells de®cient for the other pocket proteins. Whereas con®rming that pRb has a distinct role from p107 and p130, it showed that among the genes analysed so far only B-myb is overtly deregulated in p107(7/7)/p130(7/7) double mutant cells.
Cyclin A gene expression is linked to the cell division cycle with a maximum level of transcription in S/G2, whereas its transcription is clearly inhibited in early G1 and in quiescent cells (Barlat et al., 1993; Henglein et al., 1994; Schulze et al., 1995; Zwicker et al., 1995; Huet et al., 1996). The CCRE/CDE, an inhibitory element located within the region of transcriptional initiation, has been shown to be instrumental in this
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Figure 4 Chromatin modi®cations of pRb(7/7) cells at the level of cyclin A transcription initiation sites. (a) Exponentially growing pRb(+/+) ®broblasts were sorted according to their DNA content and processed for in vivo footprinting analysis as described. Asterisks indicate the CCRE proximal (HS1) and distal (HS2) hypersensitive sites. `In vitro' stands for naked DNA methylated in vitro. (b) Quantitative densitometric analysis of the HS1 (open bars) and HS2 (®lled bars) sites when cells transit through the cell cycle (average of three independent experiments). (c) In vivo methylation sensitivity of DNA prepared from wild type and pRb(7/7) cells, either serum-starved (quiescent), serum-restimulated for 18 h (stimulated) or arrested after reaching con¯uency (con¯uent)
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repression of cyclin A promoter activity before the entry into S phase. While the exact nature of the proteins directly binding in vivo to this element is still unknown, numerous studies point to the pocket proteins as putative regulators of cyclin A promoter activity. The precise role of each of these proteins has been dicult to sort out in cell lines maintained in culture for long periods of time. The existence of hidden mutations, whose roles are therefore dicult to assess, could alter the regulatory loops involving the interactions of the various pocket proteins with their eectors, limiting thus the interpretation of the data. However, the possibility to derive primary cells from mice where the individual genes, coding for each pocket protein has been targeted by homologous recombination, brings this problem to an issue (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992, 1996; Cobrinik et al., 1996). Using primary ®broblasts from either pRb(7/7), p107(7/7), p130(7/7) or p107(7/7)/p130(7/7) mice, we show in this work that mutation of the pRb gene has the more profound eect on cyclin A transcription. Cyclin A is expressed early in G1 in pRb(7/7) cells, at a level which is comparable to that found in cells reaching the G1/S boundary. This is due to a relief of a transcriptional block present in quiescent cells, which is not observed in p107(7/7), p130(7/7) or p107(7/7)/p130(7/7) mutant cells. Interestingly, a comparable eect is observed in transformed cell lines with mutations within the pRb locus (our unpublished observation). Data obtained in
single mutant cells are consistent with those of two recent reports (Herrera et al., 1996; Hurford et al., 1997). However, they are at variance with the results published in the latter report on double mutant cells, which showed a weak deregulation of cyclin A. Nevertheless, while no data based on FACS and/or thymidine incorporation were presented which could give an estimate of the percentage of cells actually arrested in G0, the presence of cyclin A mRNA, already detectable in their quiescent matched controls, is a a fortiori likely to be due to contaminating cycling cells in the mutant setting. Such a situation is encountered when cells are either serum-starved for too short a period or kept into culture for too long to be considered primary. Our data are also in contradiction with many reports which seem to exclude a role of pRb in the control of cyclin A expression. However, all of them deal with established cell lines and arti®cial settings based on in vitro data and/or overproduction experiments. For instance, p107 and not pRb has been detected in mobility shift assays using an oligonucleotide with a CCRE/CDE sequence (Schulze et al., 1995). Whereas this result is surprising due to the low amount of p107 in extracts from quiescent cells, it does not exclude the possibility that the anities of proteins for the CCRE are quite dierent whether the sequence is
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GAPDH Figure 5 p130 and p107 pocket proteins are present in pRb(7/ 7) cells. Total cell extracts from either quiescent or serum-refed pRb(+/+) and pRb(7/7) cells were fractionated by SDSpolyacrylamide gel electrophoresis, and subjected to Western blotting with speci®c antibodies as indicated. Q and S stand respectively for quiescent and serum-restimulated cells
Figure 6 Cyclin A is expressed at wild type levels in quiescent cells and remains inducible by serum in 130(7/7), p107(7/7), and p107(7/7)/p130(7/7) primary ®broblasts. The experiment was as described in Figure 4. (a) Total cell extracts were fractionated by SDS-polyacrylamide gel electrophoresis, and subjected to Western blotting with speci®c antibodies as indicated. (b) Transient transfections with the wild type cyclin A construct were performed into the various mutant cells which were either quiescent (open bars) or serum-restimulated (®lled bars)
Rb-mediated cyclin A inhibition A Philips et al
isolated or surrounded by other elements within chromatin. Such a situation is not unprecedented and our recent data suggest that the NF-Y site next to the CCRE could organize the binding of the nearby factors (Plet et al., 1997). Finally, as we mentioned it earlier, this work does not address the mechanisms of action of pRb whose eect on cyclin A down regulation could well be totally indirect. The prevalence of pRb is re¯ected by the phenotypes of the mutant animals. pRb(7/7) embryos die in utero between day 13.5 and 15.5 of gestation, whereas p107(7/7) or p130(7/7) embryos and mice have no overt abnormalities. Nevertheless, p107(7/7)/ p130(7/7) double mutant animals die within hours following birth, indicating an overlap in the function of these two proteins. Interestingly, a de®ciency in p107 has also a enhancing eect in the development of abnormalities induced by a mutation in pRb. Our data are also consistent with the spectrum of mutations observed in transformed cells. In humans, inactivation of pRb has been observed in osteosarcomas, carcinoid tumors and small-cell lung cancers, whereas no loss of function mutations for p107 or p130 have been detected in cancer cells (for reviews see Weinberg, 1995; Sherr, 1996). This speci®city in cell type is only apparent since a disruption of genes coding for regulators of pRb activity is actually a recurrent theme in cancer. Passage through G1 to S is believed to be controlled by the phosphorylation of speci®c proteins by the cdk4 and cdk6 kinases associated with D-type cyclins. One such target is pRb. Speci®c inhibitors, the INK4 proteins, can block the activity of cyclin D-activated kinases and arrest cells in G1. Interestingly, mutations have been found in most of these components (reviewed in Sherr, 1996). For example, on the one hand cyclin D1 and cdk4 are overexpressed in many cancers (Hall and Peters, 1996) following an ampli®cation or a mutation of their genes. On the other hand, mutations within the INK4a locus are frequently encountered (Kamb et al., 1994; Nobori et al., 1994), functionally equivalent in that case to a cyclin D1 or a cdk4 overexpression. As a consequence, the physiological outcome is the same in the two settings: pRb is inactivated. Accordingly, tumor cells overexpressing cyclin D1 or loosing the INK4 gene products frequently harbor a pRb(+/+) phenotype, while those with a pRb(7/7) phenotype present no speci®c alteration in cyclin D1 or p16 levels. Although the precise mechanism underlying cyclin A transcriptional inhibition by pocket proteins is not addressed here, our data clearly point to pRb as an essential element in the down-regulation of cyclin A in resting cells. Moreover, this provides thus the ®rst example of the existence of non-overlapping functions achieved by the dierent pocket proteins. They are also consistent with our previous ®nding of a deregulation of cyclin A in transformed cells, more particularly in cells where pRb had been inactivated by the transforming proteins of Adenovirus, SV40, Polyoma or papillomaviruses (Barlat et al., 1993, 1995; Buchou et al., 1993; Zerfass et al., 1995, 1996). This strengthens the central role of pRb in the control of both G0 exit and transit through G1. Accordingly, pRb was recently proposed to functionally link ras to passage through the G1 phase of the cell cycle (Peeper et al., 1997). In the latter case, invalidating ras in
pRb(7/7) cells was much less eective in blocking the entry into S phase than when performed in pRb(+/+) cells. Interestingly also, our work suggests the existence of at least two sets of regulators controlling cell cycle progression. On the one hand, proteins like cyclin D1, whose expression is a direct consequence of the activation of the ras signaling pathway and on the other hand, proteins like cyclin A which are secondary response eectors. As a result, growth factor stimulation leads to a transcriptional activation of the former set, while the transcription of the latter set is under the control of a repressor whose eect is alleviated after triggering the ras cascade. The status of pRb thus dictates whether cells continue their progression through the cell cycle when ras is mutated, probably by allowing the uncontrolled expression of critical genes like cyclin A. Unravelling this pathway is of primary importance since mutations within both pRb, and p16, which are phenotypically equivalent, as well as in ras, are frequently encountered during oncogenic processes.
Materials and methods Cell culture and transient transfection experiments Early primary ®broblasts from either wild type, Rb(7/7), p107(7/7) or p130(7/7) mice were grown in DMEM containing 10% heat-inactivated fetal calf serum. Cells were transfected using the calcium phosphate procedure 18 h prior to serum starvation, and then grown for 48 h in the absence or in low serum as indicated in the ®gure legends. Cells were then released from mitogen deprivation by addition of complete medium and luciferase activity was monitored on duplicates 18 h later as previously described. Normalization was performed using the dual-luciferase reporter assay system from Promega. The following plasmids were used: pCycA-luc is a pGL2Basic vector containing cyclin A promoter sequences spanning from nucleotides 7177 to +100 relative to the 3' most transcription initiation site (Huet et al., 1996). D1D-973pXP2 is a pXP2 based vector containing a 1112 bp DNA fragment of the human cyclin D1 promoter linked to the ®re¯y luciferase gene (generous gift from MuÈller). pRbDp34 and pRbD830-882 (generous gifts from Hamel) are pECE-based pRb expression vectors corresponding respectively to the dominant form with all phosphorylation sites contained within p34 kinase consensus sequences mutated, and a deletion mutant spanning from amino acids 830 ± 882, unable to bind E2F (Hamel et al., 1992; Qian et al., 1992). pCMV-12S and pCMV12SDCR2 (generous gifts from Dyson) are pCMVneo/ Bam-based adenovirus E1A 12S expression vectors corresponding respectively to the wild type and a deletion mutant lacking amino acids 121 ± 129, unable to interact with pocket proteins. 5 mg total DNA was used for 105 cells per 35 cm diameter petri dishes (1 mg pCycA-luc, 0.1 mg pRL-TK, when required, 1 mg expression vector as indicated and pBluescript SKII+ qsp 5 mg). Purity and Progression through the cell cycle were estimated by monitoring 3H-thymidine incorporation and by quantitative ¯uorescence analysis after staining the cells with propidium iodide. In vivo genomic footprinting In vivo genomic footprinting was carried out on cells from a 9 cm petri dish essentially as described (Barlat et al., 1995; Huet et al., 1996). When required, cells were treated
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for 2 min at room temperature with 1 ml of DMS (99% purity, Aldrich) per ml, and then with 2% b-mercaptoethanol prior to cell sorting. The radioactive elongated DNA molecules were separated on a 6% polyacrylamide sequencing gel and the signals were scanned and analysed with the Image Quant program from Molecular Dynamics. Protein and RNA blot analysis Total cell extracts were prepared for Western blot analysis by directly lyzing cells in the Laemmli loading buer, and fractioned into 7 or 12.5% polyacrylamide SDS-containing gels prior to transfer onto nitrocellulose ®lters. ECL detection was performed with the following primary antibodies: pRb mouse monoclonal mAb245 (BIOMOL Research labs), p130 C-20 and p107 C-18 rabbit polyclonal IgG (SantaCruz); cyclin A CY-A1 mouse monoclonal (Sigma); GAPDH rabbit home-made polyclonal IgG.
RNA was prepared with a standard sodium isothyocyanate-based method and processed for Northern blot analysis as described (Barlat et al., 1993; 1995). The full length human cyclins A and D1, and mouse GAPDH cDNAs were used as probes. Acknowledgements A Ph is supported by a contract from the BIOMED2 program and XH by a fellowship from the Ligue Nationale contre le Cancer. They both contributed equally to this work. We thank R Hipskind, C Sardet and ML Vignais for discussions and review of the manuscript. We acknowledge the generous gift of the various mutant ®broblasts by T Jacks, N Dyson and D Cobrinik. We also thank N Dyson for communicating us data prior to publication. This work was supported by grants from the CNRS, the BIOMED2 program, the ARC and the Ligue Nationale contre le Cancer.
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