Retinoblastoma Susceptibility Gene Product (pRb) and p107 Functionally Separate the Requirements for Serum and Anchorage in the Cell Cycle G 1 -phase*

Growth factors and cell anchorage are both required for cell cycle G 1 -phase progression, but it is unclear whether their function is mediated through the same set of cell cycle components and whether they are both required during the same periods of time. We separately analyzed the requirements of serum and anchorage during G 1 -phase progression and found that human dermal fibroblasts as well as wild type, pRb (cid:1) / (cid:1) , and p107 (cid:1) / (cid:1) mouse embryonic fibroblasts needed serum (growth factors) until mid-G 1 -phase but required cell anchorage until late G 1 -phase to be competent for S-phase entry. Importantly, however, pRb/p107 double-null mouse embryonic fibroblasts lacked serum requirement in mid-G 1 -phase but still required cell anchorage until late G 1 - phase to enter S-phase. Our results indicate that pRb and p107 do not constitute the last control point for extracellular factors during G 1 -phase progression, and they functionally separate the requirements for serum and cell anchorage in terms of involved cell cycle components. total protein was immunoprecipitated by using anti-cyclin D1/D2 mAb 5D4 (a kind gift from Dr. Jiri Bartek) or anti-cyclin E mAb M-20 (Santa Cruz Biotechnology), followed by an in vitro kinase assay using GST-pRb (sc4112, Santa Cruz Biotechnology) (cyclin D) or histone H1 (Calbiochem) (cyclin E) as substrates. The kinase assays were otherwise performed as described (24) and analyzed and quantified by using a Cyclone PhosphorImager (Packard).

Normal tissue cells need extracellular stimuli by growth factors and by integrin-mediated cell anchorage to the extracellular matrix to proliferate, whereas loss of these requirements is a hallmark of tumor progression. Cells that lack growth factors and/or cell anchorage arrest in the G 1 -phase of the cell cycle. The G 1 -phase is controlled by several cyclin-dependent kinases (Cdk), 1 of which the cyclin D-Cdk4/6 complex is the first to be present (1)(2)(3). The cyclin D-Cdk4/6 complex phosphorylates, and thereby inactivates, members of the retinoblastoma susceptibility gene product (pRb) family of pocket proteins, consisting of pRb, p107, and p130 (4). Active pRb blocks G 1 -phase progression by binding and inhibiting E2F transcription factors (5). The inhibitory phosphorylation of pRb by cyclin D-Cdk4/6 results in the activation of E2F and subsequent expression of proteins promoting cell cycle progression, including cyclin E and cyclin A. The subsequent cyclin E-Cdk2 activity might phosphorylate pRb on additional phosphorylation sites, resulting in a complete inhibition of pRb (3,6,7). Inactivation of pRb has also been suggested as the event in the G 1 -phase that corresponds to passage through the restriction point, a point after which cells are independent of extracellular mitogenic stimuli to enter S-phase (2,8,9). Growth factors and cell anchorage jointly stimulate cell proliferation by regulation of cell cycle G 1 -phase progression (10 -12). Signals from various growth factors and from cell anchorage converge on cyclin D-Cdk4/6 activity (13,14), mainly mediated by the regulation of levels and timing of increased cyclin D1 protein that constitute the rate-limiting step in cyclin D-Cdk4/6 complex formation (14,15). Cells that are denied either growth factors or cell anchorage fail to induce and to keep sustained levels of cyclin D1 and thereby get arrested in early to mid G 1 -phase. Taken together with reports demonstrating that signals from both growth factors and cell anchorage also regulate Cdk2-inhibitor protein levels and cyclin E-Cdk2 activity (16 -21), these facts support the hypothesis that signals from growth factors and cell anchorage jointly control the cell cycle G 1 -phase progression. According to this model, growth factors and anchorage stimulate G 1 -phase progression jointly through the same downstream cell cycle targets, including through inactivation of pRb and the restriction point (10,14). However, although many cell cycle G 1 -phase components can be regulated by both growth factors and cell anchorage, it is unclear whether growth factors and cell anchorage functionally regulate the G 1 -phase through an identical set of cell cycle components or whether there are distinct sets of functional cell cycle targets. Therefore, we tested whether the requirements of growth factors (serum) and cell anchorage may be separate during the G 1 -phase progression in primary fibroblasts.
We found that the requirements of serum and cell anchorage can be functionally separated both in time and with regard to involved cell cycle components during the G 1 -phase of primary human and mouse fibroblasts, where serum was required until mid-G 1 -phase, whereas cell anchorage was needed until late G 1 -phase. Importantly, the final anchorage-dependent G 1phase control was intact in pRb/p107 double-null mouse embryonic fibroblasts (MEFs), whereas these cells lacked the normal serum requirement. We conclude that the final functional G 1 -phase control by cell anchorage is distinct from the serum control, in that it occurs after and independent of normal serum control, normal cyclin D-associated kinase activity, pRb, and p107.

EXPERIMENTAL PROCEDURES
Cells and Cell Cycle Experiment-Primary normal human dermal fibroblasts (NHDF) (BioWhittaker) from two different donors, primary human neonatal foreskin BJ-fibroblasts (kindly provided by Dr. J. W. Shay), NIH 3T3 mouse fibroblasts (American Type Culture Collection), wild-type, pRb-null, p107-null, and pRb/p107 double-null primary MEFs were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) with 10% fetal calf serum (FCS) (BioWhittaker). 50% confluent NHDF at passage 4, or wild-type, pRb-null, p107-null, or pRb/ p107 double-null MEFs at passage 3-4 were synchronized by a 48-h incubation in starvation media (DMEM with 5 g/ml insulin (Sigma), 2 g/ml transferrin (Invitrogen), 4 ng/ml Na-Selenite (Sigma), 1 mg/ml bovine serum albumin (BSA) (Sigma), trypsinized and released from G 0 by reseeding in DMEM (Invitrogen) containing 10% FCS and 30 M BrdUrd (Sigma). At different time points, the serum-containing medium was removed and replaced with starvation medium containing 30 M BrdUrd. In parallel dishes at the same time points, cells were denied adhesion by gently scraping them off the culture dish in DMEM containing 10% FCS and 0.5% BSA. The cells were collected and kept in suspension by using non-tissue culture-treated plastic plates blocked with 1% heat-denatured BSA combined with very gentle shaking for the remaining time in the presence of 10% FCS. 30 h after the initial G 0 -release, cells were harvested and fixed in 70% ice-cold EtOH. To monitor the time of S-phase entry, G 0 -released cells were harvested at different time points after release and fixed as above. The cell cycle distribution was determined by using anti-BrdUrd mAb G3G4 (Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa, IA) and propidium iodide double-staining, followed by flow cytometry analysis as described previously (22). To facilitate comparisons between different experiments and cell types, values of cell proliferation after depletion of serum or anchorage were calculated as the percentage of cells dependent on serum and anchorage to enter S-phase within 30 h. A portion of MEFs deficient in pRb and p107 grew independently of both serum and adhesion, which is consistent with our previous observations (23). However, the fraction of pRb/p107-null MEFs regulated by both serum and anchorage in our assay was similar in size with those of wild-type, pRb Ϫ/Ϫ , and p107 Ϫ/Ϫ MEFs (data not shown).
Immunoprecipitation and Kinase Assays-Cell lysates were obtained as above, and 50 -100 g of total protein was immunoprecipitated by using anti-cyclin D1/D2 mAb 5D4 (a kind gift from Dr. Jiri Bartek) or anti-cyclin E mAb M-20 (Santa Cruz Biotechnology), followed by an in vitro kinase assay using GST-pRb (sc4112, Santa Cruz Biotechnology) (cyclin D) or histone H1 (Calbiochem) (cyclin E) as substrates. The kinase assays were otherwise performed as described (24) and analyzed and quantified by using a Cyclone PhosphorImager (Packard).

Temporal Separation of the Requirements for Serum and
Anchorage in the Fibroblast Cell Cycle G 1 -phase-Although several critical cell cycle components, including cyclin D and cyclin E are regulated both by growth factors and by cell anchorage, it is not yet clear whether the entire regulation of the G 1 -phase by extracellular factors occurs jointly or if the requirements for growth factors and cell anchorage can be separated. To circumvent the fact that late G 1 -phase control depends on early G 1 -phase signaling, we used the model of the restriction point as a tool to examine whether the final requirements for growth factors and cell anchorage occurred jointly or could be separated in time. We compared the period of time after G 0 -release that NHDFs needed cell anchorage and serum FIG. 1. Temporal separation of the requirements for serum and anchorage in the fibroblast cell cycle G 1phase. a, quiescent, primary NHDF were released from G 0 -phase into G 1 -phase. The time until serum (छ) or anchorage (Ⅺ) was needed to achieve competence for S-phase entry (left axis) as well as the time of S-phase entry (E) (right axis) were examined and calculated as described under "Experimental Procedures." The graph is representative of five independent experiments. b-e, G 0 -synchronized NHDF were restarted and allowed to progress through the G 1 -phase in the presence (b) or absence (c) of cell anchorage. Cells were harvested at the indicated time points and subjected to Western blot analysis of cyclin D1, cyclin E, pRb, and cyclin A, as described under "Experimental Procedures." ␤-actin was used as a control for equal loading. d, quiescent NHDF were restarted, and cyclin D1/D2 (upper panel) and cyclin E (lower panel) were immunoprecipitated followed by in vitro kinase assays analyzing associated Cdk-activities. The displayed gels are representative among three independent experiments. e, quantification of the cyclin D-associated (छ) and cyclin E-associated (Ⅺ) kinase activities shown in d.
pRb/p107 Separate Serum and Anchorage Dependence to be able to enter S-phase. NHDF cells were synchronized in G 0 by starvation and then released into the G 1 -phase by reseeding in serum-containing media. At different time points, serum or attachment was then removed from the cells, and the number of cells that had entered S-phase after a total of 30 h was quantified (Fig. 1a). Previous studies have shown that fibroblasts need growth factors for ϳ10 h after release from G 0 to be able to progress into S-phase (25,26). Consistent with these results, NHDF in our experiments required serum for ϳ10 -14 h after release from G 0 to achieve competence for S-phase entry for most cells. However, surprisingly, NHDF needed cell anchorage for a longer time, ϳ14 -18 h, to be able to proceed into S-phase. This suggests the existence of a functional cell anchorage-dependent control in the G 1 -phase that is temporally separated from the serum-dependent control in primary human fibroblasts. Similar to NHDF, primary human adult dermal fibroblasts of a different origin, BJ primary human neonatal foreskin fibroblasts and mouse NIH 3T3 fibroblasts, all required cell anchorage for a longer time than serum to enter S-phase (data not shown). This finding demonstrates that fibroblasts of various origins are regulated by serum and cell anchorage in a similar manner. Because cells were denied cell anchorage by gently scraping them off the substratum, an alternative possibility could be that the scraping as such might affect the capability or timing for S-phase entry. To control for potential effects of the cell scraping, cells were scraped off at different times during the G 1 -phase and were then allowed to re-adhere and continue G 1 -progression. These cells entered S-phase within 30 h to the same degree as equivalent cells that were allowed to adhere undisturbed during the entire period (data not shown), demonstrating that the scraping as such did not affect the ability of cells to enter S-phase within our system.
Regulation of Cell Cycle Components during G 1 -phase Progression of NHDF-To identify essential cell cycle components active at the critical time points of serum and cell anchoragemediated regulation in our system, and to make sure that the NHDF displayed a normal G 1 -phase progression, we analyzed cyclins, cyclin-dependent kinase activities, and the phosphorylation status of pRb in these cells after release from quiescence, with or without cell anchorage. As shown in Fig. 1, b and c, cyclin D1, cyclin E, cyclin A, and pRb-phosphorylation were regulated by cell anchorage in our NHDF and by kinetics similar to those previously reported for various fibroblasts (2,18,27). Furthermore, the increased levels of cyclin D-associated kinase activity at 10 -14 h occurred at the time point of final control by serum, whereas the peak in cyclin E-associated kinase activity at 14 -18 h occurred during the final control by cell anchorage (Fig. 1, a, d, and e).
Genetic Targeting of pRb and p107 Functionally Separates the Requirements for Serum and Anchorage-Members of the retinoblastoma susceptibility gene product (pRb) family of pocket proteins are central for G 1 -phase control, and inactivation of these proteins is regulated both by growth factors and anchorage (14). To examine whether inactivation of the pRbfamily of pocket proteins influenced the requirements for serum and/or anchorage, we analyzed for what length of time serum and anchorage were needed to enter S-phase for primary MEFs isolated from mice where pRb and/or p107 were genetically targeted. As shown in Fig. 2, wild-type, pRb Ϫ/Ϫ , and p107 Ϫ/Ϫ MEFs all required anchorage for a longer time than serum to enter S-phase; therefore, we concluded that the functional control for serum and anchorage in the G 1 -phase were FIG. 2. Genetic targeting of pRb and p107 functionally separates the requirements for serum and anchorage. G 0 -synchronized wild-type (a), pRbnull (b), p107-null (c), or pRb/p107 doublenull (d) primary MEFs at passage 3-4 were released, and the time until serum (छ) or anchorage (Ⅺ) were required to achieve competence for S-phase entry (left axis) as well as the time of S-phase entry (E) (right axis) were examined; values were calculated as described under "Experimental Procedures." The displayed graphs for each cell type are representative among three independent experiments. Wild-type, pRb-null, or pRb/p107 double-null MEFs were released from G 0 into the G 1 -phase with anchorage (A) or in suspension (S). e, 10 h (cyclin D) or 16 h (cyclin E) after release, cells were lysed, and cyclin D1/D2 (upper panel) or cyclin E (lower panel) were immunoprecipitated followed by in vitro kinase assays analyzing the associated Cdk activities. f, 16 h after release from G 0 , wt and pRb/p107 double-null MEFs were lysed and cyclin E, p21 CIP1 , and p27 KIP1 protein levels were analyzed by immunoblot. ␤-actin was detected as a control for equal loading. The displayed gels in e and f are representative of three independent experiments. pRb/p107 Separate Serum and Anchorage Dependence distinct in time in three additional primary fibroblast cells. In contrast, pRb/p107-null MEFs lacked a normal serum requirement in mid-G 1 -phase, because 30 min of serum stimulation after restart was sufficient for these cells to be able to proceed into S-phase (Fig. 2d). Importantly, although the pRb/p107null MEFs lacked the regular control by serum, these cells retained an intact anchorage control in the late G 1 -phase (Fig.  2d). This separates the cell anchorage-dependent control from the serum-dependent control not only in time but also with regards to the mediating cell cycle components.
Cell Anchorage Regulates Cyclin E-associated Kinase Activity Independent of Normal Cyclin D, pRb, and p107-The peak of cyclin E-Cdk2 activity correlated in time to the final requirement for cell anchorage in the late G 1 -phase (Fig. 1). In addition, cyclin E-associated kinase activity was regulated by cell anchorage in fibroblasts after release from quiescence (Fig. 2e), which confirms previous studies (16,18,27). However, given that cyclin D1 levels are tightly regulated by cell anchorage (18,28,29), and that cyclin D-Cdk4/6 activity is a pre-requisite for subsequent cyclin E-Cdk2 activity (1), results obtained so far showing cell anchorage-dependent regulation of cyclin E-Cdk2 activity might just reflect cell anchorage-dependent cyclin D regulation. To determine whether cyclin E-associated kinase activity could be regulated by cell anchorage also independently of the regular induction of cyclin D-associated kinase activity in mid G 1 -phase, we examined cyclin E-coupled activities in the pRb/p107 double-null MEFs that lacked detectable cyclin D-associated kinase activity (Fig. 2e, upper panel). Interestingly, the pRb/p107-null MEFs still required cell anchorage to maintain cyclin E-dependent kinase activity, because we observed a dramatic decrease of cyclin E-dependent kinase activity when these cells were set in suspension (Fig. 2e, lower  panel). The residual cyclin E-associated kinase activity in suspended pRb/p107-null MEFs most likely originated from the fraction of cells progressing through the G 1 -phase independently of cell attachment (data not shown). However, cell anchorage did not regulate cyclin E protein levels in pRb/p107null MEFs (Fig. 2f), indicating that the regulation of cyclin E-Cdk2 activity occurs at another level. Interestingly, we found markedly higher levels of the Cdk2-inhibitor p27 KIP1 in suspended pRb/p107 double-null MEFs compared with attached cells, but no difference for another Cdk2-inhibitor, p21 CIP1 (Fig. 2f). DISCUSSION Our study demonstrates that normal primary human and mouse fibroblasts require cell anchorage for a longer time than serum during the cell cycle G 1 -phase to be able to enter Sphase. Wild-type, pRb Ϫ/Ϫ , and p107 Ϫ/Ϫ MEFs had normal requirements for serum and also required cell anchorage for a longer time than serum. This result confirms previous findings that germline inactivation of pRb alone does not alter the serum-dependent control in MEFs (30). However, a recent study indicates that MEFs with acute loss of pRb can proliferate under low-serum conditions, whereas MEFs with germlineinactivated pRb cannot (31). The difference between germline pRb-null MEFs and MEFs with acute loss of pRb might be explained by a compensation of pRb function by p107 in the germline-targeted pRb-null MEFs (31). Consistent with this finding, we found that although both the pRb-null MEFs and p107-null MEFs had normal periods of serum requirement, pRb/p107 double-null MEFs lacked serum requirement in mid-G 1 -phase, and that only 30 min of serum stimulation after restart from G 0 made these cells competent to enter S-phase. It has been proposed that growth factors are only required during two separate narrow windows in the G 1 -phase; for the G 0 to G 1 -phase transition during the first 30 min, and then during 30 min in mid-G 1 -phase (26). Applied in this model, our results indicate that events involving pRb and p107 may regulate the mid-G 1 -phase window, whereas other factors, such as p130, may control the G 0 to G 1 -phase transition (4).
Importantly, although the pRb/p107-null MEFs lacked a regular serum requirement, they retained an intact anchorage-dependent control until late G 1 -phase. This finding suggests that, whereas the serum control in these cells seems to be dependent on events involving the pocket proteins pRb and p107, the final anchorage-dependent control is located after and independent of these events. These results functionally separate the requirements for serum and cell anchorage in terms of involved cell cycle components. These results also indicate that inactivation of pRb and p107 does not constitute the final point of control in the G 1 -phase. Furthermore, the existence of a distinct cell anchorage-dependent control in the late G 1 -phase, in addition to the mid-G 1 -control, highlights the importance for a cell to ensure that cellular proliferation occurs only in a proper extracellular matrix context.
Cyclin E-Cdk2 activity is considered crucial for S-phase entry (32), although a recent study indicates that it is possible that cyclin A-Cdk2 may compensate for cyclin E in cyclin E-deficient mouse cells (33). Given that pRb/p107-null MEFs fail to induce cyclin E-associated kinase activity in suspension, the G 1 -phase progression in pRb/107-null MEFs must be controlled by anchorage at or before cyclin E-Cdk2 activity. However, we found that this control by cell anchorage seems to be independent of normal cyclin D-associated kinase activity, pRb, and p107, which are implicated in the serum-dependent control (23,31). Considering (i) that cyclin E has been described as emerg- FIG. 3. Hypothetical model of growth factor and cell anchorage control of the cell cycle G 1 -phase. Growth factors, via growth factor receptors and cell anchorage to the extracellular matrix mediated by integrins, jointly induce cyclin D protein (12). The consequential cyclin D-Cdk4/6 kinase activity inactivates pRb and p107 (4). The resulting release of E2F has been demonstrated to lead to transcription of cyclin E and other S-phase-promoting genes (41). We found that primary fibroblasts required serum until mid-G 1 -phase, whereas they depended upon cell anchorage until late G 1 -phase to be able to enter S-phase (indicated by horizontal gray bars). Based on our results and previous studies, we hypothesize that serum and cell anchorage may control cell cycle progression by means of pRb and p107 until mid-G 1phase. Thereafter, cell anchorage is needed until late G 1 -phase. We found that the cell anchorage-dependent control in late G 1 -phase was independent of normal serum control, cyclin D-associated kinase activity, pRb, and p107. Instead, it may include regulation of cyclin E-Cdk2 activity by cell anchorage.
ing after the serum restriction point and as being dispensable for passage through this point (34); (ii) that the peak of cyclin E-associated kinase activity correlated in time with the final anchorage-dependent control but not with the final serum control; and (iii) that cyclin E-Cdk2 activity was regulated by anchorage in pRb/p107 double-null fibroblasts lacking a regular serum-dependent control, we hypothesize that cyclin E-Cdk2 involves into the cell anchorage-dependent control during the G 1 -phase rather than into the serum-dependent control. However, we cannot exclude involvement of other components in cell anchorage-dependent cell cycle control. For example, studies in normal rat kidney cells and in v-H-Ras transformed ratER-1-2fibroblastssuggestthatthesecellshaveananchoragedependent control on cyclin A expression without any anchoragedependent regulation of cyclin D, cyclin E, or pRb-phosphorylation (27,35). However, it is unclear if these results obtained in immortalized or transformed cell lines reflect a cell cycle control that also exists in normal primary cells. A potential role for cyclin E-Cdk2 in the G 1 -phase independent of the pocket proteins is supported by the observation that cyclin E is required regardless of pRb status (32), in contrast to cyclin D1, which is only required in pRb-positive cells (24). In addition, the phosphorylation of pRb might be a rate-limiting event in the G 1phase that is controlled by cyclin D-Cdk4/6 rather than by cyclin E-Cdk2 activity (36,37). Furthermore, overexpression of cyclin E can overcome G 1 -phase arrest imposed by a phosphorylation-deficient pRb mutant (38). However, cell anchorage did not regulate cyclin E protein levels in pRb/p107-null MEFs, suggesting that the adhesion-dependent control of cyclin E-associated kinase activity may act at another level. Interestingly, the Cdk2-inhibitors p21 CIP1 and p27 KIP1 are regulated by cell anchorage (16, 18 -21). To this end, we found markedly higher levels of p27 KIP1 in suspended pRb/p107 double-null MEFs compared with attached cells, indicating that p27 KIP1 might contribute to the anchorage-dependent regulation of cyclin E-Cdk2 activity in these cells. Furthermore, the finding that pRb-null MEFs depend upon anchorage to proliferate, whereas pRb/p21 CIP1 double-null MEFs do not (39), supports the notion of an anchorage-dependent cell cycle control which is independent of pRb and possibly mediated by Cdk2-inhibitors, although it is unclear how the growth-suppressing role of p107 that we observed could be overcome in the pRb/p21 CIP1 double-null MEFs.
It is also unclear if the cell anchorage-dependent G 1 -phase control we observed was mediated by integrin signaling and/or cytoskeletal arrangements. However, recent evidence suggests that the cell anchorage-dependent organization of the actin cytoskeleton into stress fibers is required for the mid-G 1 -phase induction of cyclin D1, but not for other aspects of cyclin E-Cdk2 activity (40). Furthermore, we previously found that integrin signaling through Cdc42 and Rac without any contribution of cell spreading led to proteasomal degradation of the Cdk-2 inhibitors p21 CIP1 and p27 KIP1 (20), indicating that integrin signaling by itself might be involved in cell anchoragedependent control of cyclin E-Cdk2 activity. Taken together with our present result, this may suggest that cytoskeletal rearrangements are important for the joint regulation by growth factors and cell anchorage of the G 1 -phase progression up and until cyclin D-Cdk4/6 activity, and that integrin signaling alone might mediate the functional control by cell anchorage during late G 1 -phase.
We show for the first time that the requirements for serum and cell anchorage can be functionally separated in time and in terms of involved cell cycle components during the G 1 -phase of primary fibroblasts. Although the final requirement for serum corresponds to inactivation of pRb and p107 in mid-G 1 -phase, the requirement for anchorage is maintained in the late G 1phase independently of normal serum control, normal cyclin D-associated kinase activity, pRb, and p107 (Fig. 3). The anchorage-dependent control in the late G 1 -phase may instead involve regulation of cyclin E-Cdk2 activity. These results provide new functional insights into the extracellular control of cell proliferation.