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[Cancer Research 59, 5341-5348, October 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 5341-5348, October 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Microinjection of Anti-p21 Antibodies Induces Senescent Hs68 Human Fibroblasts to Synthesize DNA but not To Divide1

Yuliang Ma, Sally A. Prigent, Teresa L. Born, Craig R. Monell, James R. Feramisco and Bonnie L. Bertolaet2

Cancer Center and Departments of Pharmacology and Medicine, University of California, San Diego, La Jolla, California 92093 [Y. M., S. A. P., T. L. B., J. R. F., B. L. B.]; Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 [Y. M.]; Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom [S. A. P.]; Immunex Corporation, Seattle, Washington 98101 [T. L. B.]; PharMingen, Inc., La Jolla, California 92121 [C. R. M.]; and The Scripps Research Institute, La Jolla, California 92037 [B. L. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Replicative senescence is characterized by irreversible growth arrest and has been defined by four genetic complementation groups. One of these groups is associated with the predominance of underphosphorylated, growth-suppressive retinoblastoma tumor suppressor protein (pRb). Although certain members of the cyclin-dependent kinase (cdk)/cyclin family, some of which phosphorylate pRb, are underexpressed in senescent cells, others are expressed but inactive. This lack of cdk activity and arrest in the G1 phase of the cell cycle is likely attributable to the induction upon senescence of the G1-S cdk/cyclin inhibitors p21 (WAF1/CIP1/Sdi) and p16INK4. In fact, in early presenescent normal diploid fibroblasts in which p21 is inactivated, senescence is bypassed or postponed. Moreover, in senescent cells in which p53 function was inhibited, DNA synthesis was reinitiated, an effect likely attributable, in part, to the dependence of p21 expression on p53. We report here that the apparent inactivation of p21 in senescent human fibroblasts through the introduction of inhibitory {alpha}-p21 antibodies causes these cells to reenter the S-phase of the cell cycle. The disruption of p21 activity affects the p21-Rb-E2F pathway in that the expression of genes transcriptionally regulated by E2F, such as cyclin A and cdc2, were found to be up-regulated in injected cells. No evidence of cell division was observed. This suggests that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 activity is insufficient to permit cells to complete the cell cycle.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human diploid fibroblasts have been used as a model system for studying replicative senescence. Normal fibroblasts can only undergo a finite number of cell divisions when passaged in culture. As they approach senescence, cell division slows significantly until the fibroblasts enter an irreversible growth-arrested state (13) . It has been proposed that there is an intrinsic genetic program complete with sensors that responds to the number of divisions that a cell has undergone (possibly monitored by telomere shortening) and turns on the senescent phenotype (14) . mRNA from senescent cells can induce growth arrest when injected into presenescent cells (15) , yet the identity of the elements responsible for the senescence phenotype remain obscure.

A significant alteration in the pattern of the expression of various genes occurs upon cells entering senescence (13 , 16, 17, 18) . Such patterns and activities include the appearance of a senescent-associated {beta}-galactosidase activity (19) , the overexpression of collagenase concomitant with a down-regulation of collagenase inhibitors (20 , 21) , and modified expression of interleukins, to name a few (22) . However, the most remarkable change in senescent cells is their inability to synthesize DNA or divide in response to physiological mitogens. Mitogenic factors can stimulate serum-starved, presenescent cells to undergo DNA synthesis. In contrast, the quiescent state of senescent cells is characterized by an extended growth arrest that is unresponsive to mitogens, much like that which occurs in terminal differentiation. Previous studies have shown that introduction of c-Ha-ras was unable to induce DNA synthesis in senescent fibroblasts (23) , although it could induce c-fos expression (24) , one of the transcripts characteristically repressed in senescent cells (25) . c-fos expression, however, is not enough to initiate progression through the cell cycle.

Fusing senescent cells with cell lines immortalized by SV40, adenovirus, or papillomavirus can lift the block in these cells and can bypass their growth arrest (1 , 26) . Immortalization by these viruses involves binding and inactivation of p53 and/or pRb (27, 28, 29, 30) . It is likely that these viruses disrupt the growth-suppressive block in senescent cells using similar mechanisms (31, 32, 33) . Indeed, senescent fibroblasts transfected with SV40 large T-antigen (34 , 35) have been shown to reenter the cell cycle. Moreover, it has been reported that disabling the function of p53 or one of its downstream effectors, p21WAF1/CIP1/Sdi1 (5) , affects the senescence program of normal fibroblasts. The injection of {alpha}-p53 antibodies into senescent fibroblasts has been shown to induce their reentry into the cell cycle and cause these cells to undergo cell division, suggesting that p53 is required for maintaining growth arrest and preventing mitosis in senescence (11) . Antisense oligonucleotides directed against p21 have been shown to stimulate serum-starved, presenescent fibroblasts to undergo DNA synthesis (36) . Although such antisense experiments have not shown that senescent cells escape senescence (37) , loss of p21 through homologous recombination knock-out in presenescent fibroblasts was sufficient to bypass senescence in these cells (10) . Thus, whether p53 renders its effect through p21WAF1/CIP1/Sdi1 and whether p21WAF1/CIP1/Sdi1 is responsible for maintaining the growth arrest characteristic of senescence is the focus of this report.

The induction of p21 (7) and p16 (8 , 9) in senescent cells helps explain the lack of certain cdk3 /cyclin activity in senescence, despite the fact that various cdks and cyclins are still expressed (4) . Thus, one possible mechanism of growth arrest in senescent cells is the inhibition by p21 of cdk/cyclin kinase activity that phosphorylates and inactivates growth-suppressive cell cycle regulators including pRb (2 , 6 , 38, 39, 40) . E2F transcriptional activity helps drive proliferation, and one way pRb is thought to effect its growth-suppressive function is through its inhibition of E2F function (41) . In addition to its interactions with cdk/cyclin complexes, p21 has been shown to bind PCNA (42, 43, 44) as well as interact with E2F in a complex (45) , which may also contribute to its growth-suppressive function. Our data show that the injection of {alpha}-p21 antibodies into senescent fibroblasts promotes their reentry into the cell cycle and progression through S phase but not through mitosis. Our data further suggest that this process involves, at least in part, the p21-pRb-E2F pathway.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human foreskin fibroblast Hs68 was purchased from American Type Collection Corporation. The cells were propagated in DMEM with 10% FBS. The percentage of cells, either presenescent or senescent, undergoing DNA synthesis was determined by serum starvation of the cells in DMEM plus 0.5% FBS for 24–48 h, followed by serum stimulation in DMEM plus 10% FBS along with the addition of BrdUrd. Cells were fixed and analyzed after 24 h unless otherwise noted. The cells were regarded as senescent when they had a doubling time of greater than 1 month. Senescent cells had passage numbers >60, resulting in their being maintained for over 2 years. When at senescence, cells were transferred to glass coverslips at 50% confluence for microinjection experiments. When serum starvation was required, cells on coverslips were incubated in DMEM plus 0.5% FBS for 24 h to render them quiescent.

Western Blots.
Hs68 fibroblasts were lysed in RIPA buffer [50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS], and the cell lysates were analyzed by electrophoresis using a 12.5% SDS-polyacrylamide gel. Antibodies against p21WAF1/CIP1/Sdi1 (PharMingen, Inc.), CycA (Santa Cruz Biotechnology, Inc.), and PCNA (Santa Cruz Biotechnology, Inc.) were used in immunoblots. Protein bands were visualized using the ECL substrate system (Amersham Pharmacia Biotech UK Ltd.), according to manufacturer’s instructions.

Microinjection.
For cytoplasmic injections with c-Ha-ras, senescent and presenescent Hs68 fibroblasts were grown on coverslips, rendered quiescent, and injected with either c-Ha-ras protein (T24-Ras; 2 mg/ml) plus guinea pig IgG (8 mg/ml) or guinea pig IgG alone (8 mg/ml). One h after injection, BrdUrd was added to the medium, and at 24 h after injection, cells were fixed and immunostained for BrdUrd incorporation. Injected cells were identified by immunostaining for the coinjected guinea pig IgG with an FITC-coupled anti-guinea pig antibody. For nuclear injections, senescent Hs68 fibroblasts were injected with {alpha}-p21 antibodies from two suppliers. Two {alpha}-p21 clones from Oncogene Science, Inc., Ab-1 and Ab-3, were combined to a total concentration of 10 mg/ml and used in experiments relative to those conducted with a control antibody, SEN7 (46) . Ten mg/ml SEN7 was kindly provided by Dr. Rolf A. Stahel, University Hospital, Zurich, Switzerland. Two {alpha}-p21 clones from PharMingen, Inc., 18A10 and 2G12, were combined to a total concentration of 1.2 mg/ml, coinjected with guinea pig IgG (3.6 mg/ml), and used in experiments relative to those conducted with a control antibody, {alpha}-CD3 (clone UCHT1, PharMingen; 2.4 mg/ml) coinjected with guinea pig IgG (3.6 mg/ml). For reporter injections, senescent cells were injected in the nucleus, with either {alpha}-p21 or {alpha}-CD3 antibodies from PharMingen together with guinea pig IgG and a -700 cdc2 promoter-luciferase plasmid (250 µg/ml; Ref. 47 ). At 30 h after injection, cells were fixed and immunostained.

Immunostaining and Photomicroscopy.
Cells were fixed with 4% formaldehyde in PBS for 10 min, washed twice with PBS, and then permeabilized with 0.3% Triton X-100 in PBS for 15 min before staining with different antibodies. Cells were sequentially incubated with primary and secondary antibody for 1 h at 37°C in PBS plus 0.5% NP40 and 5 mg/ml BSA. The cells were washed twice with PBS for 5 min at room temperature after each antibody incubation. Rat {alpha}-BrdUrd (Harlan Sera-Lab Limited) was used at a 1/250 dilution plus 25 units of Dnase I (Boehringer Mannheim Corp.). Rabbit {alpha}-cycA (SC-75; Santa Cruz Biotechnology) and {alpha}-PCNA (Santa Cruz Biotechnology) were used at 1/100 dilution. Rhodamine and FITC {alpha}-rabbit, rat, and mouse IgG secondary antibodies were all from Jackson Immuno Research Laboratories, Inc. and used at a 1/100 dilution in PBS plus 0.5% NP40 and 5 mg/ml BSA. Luciferase expression was detected with a polyclonal antibody to luciferase (Cortex Biochemicals), followed by a Texas Red-conjugated anti-rabbit antibody (Jackson). Injected guinea pig IgG (Sigma Chemical Co.) was detected with a FITC-conjugated donkey {alpha}-guinea pig antibody (Jackson). Hs68 fibroblasts, presenescent and senescent, were immunostained for senescent-associated {beta}-galactosidase activity as described by Dimri et al. (19) . Immunostained cells were photographed using a Zeiss Axiophot fluorescence microscope and a x40 oil immersion lens equipped with a Hamamatsu C5810 digital camera.

In Vitro Cdk2 Kinase Assay.
Active cdk2/cyclin complexes were immunoprecipitated from synchronized, proliferating Hs68 fibroblasts as described previously (48) using 50 µg of total protein and 2.5 µg of {alpha}-cdk2 antibody-agarose conjugate (sc-163AC; Santa Cruz). Precipitates were washed in RIPA buffer and then incubated for 30 min at 30°C in the presence of 40 ng of GST, or GST-p21 (PharMingen) that had been preincubated with 1.2 µg of each of the two PharMingen {alpha}-p21 antibodies. Complexes were washed with RIPA buffer, followed by TBS [50 mM Tris/HCl (pH 7.4) and 150 mM NaCl] containing 0.1% Triton X-100 and then twice with kinase buffer [50 mM Tris/HCl (pH 7.4) and 10 mM MgCl2]. The washed complexes were resuspended in 20 µl of kinase buffer containing 100 mM ATP, 10 mCi [{alpha}-32P]ATP, and 1 µg of Histone H1 (Boehringer Mannheim). The kinase reaction was allowed to continue for 15 min at 30°C before being terminated by addition of SDS-PAGE sample buffer. Reaction products were analyzed by electrophoresis and then autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Senescent Cells.
The senescent human Hs68 fibroblasts used in these experiments were defined to be senescent by the low number of serum-stimulated cells undergoing DNA synthesis, as measured by BrdUrd incorporation (10–15%) after serial passage (25 , 49) . This characterization was further confirmed by the fact that these cells expressed the senescence-associated {beta}-galactosidase activity (19) , whereas presenescent fibroblasts did not (Fig. 1A)Citation . The senescent fibroblasts used in this study also displayed the characteristic alteration in expression levels of p21WAF1/CIP1/Sdi1 (7) , PCNA (17) , and cyclin A (3) genes. That is, in the senescent fibroblasts relative to the presenescent fibroblasts, p21 expression was up-regulated, whereas that of PCNA and cyclin A was down-regulated (Fig. 1B)Citation .



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  		Fig. 1. Characterization of senescent Hs68 human fibroblasts. A, senescent-associated {beta}-galactosidase activity in Hs68 human fibroblasts. Hs68 fibroblasts passaged 17 generations (Pre-Senescent; a) and >50 generations (Senescent; b) were immunostained for senescent-associated {beta}-galactosidase activity as described by Dimri et al. (19) . B, Western blot analysis of protein expression probing with antibodies to either p21, PCNA, or cyclin A. C, induction of DNA synthesis in Hs68 human fibroblasts by microinjection of c-Ha-ras. The ability of c-Ha-ras protein to induce Hs68 fibroblasts to enter S phase was assessed by the number of cells undergoing DNA synthesis (via BrdUrd incorporation) after microinjection into the cytoplasm with either c-Ha-ras protein plus marker IgG or IgG alone. a, c, e, and g, injected cells; b, d, f, and h, cells incorporating BrdUrd. a–d, senescent Hs68 fibroblasts injected in the cytoplasm with either IgG alone (a and b) or c-Ha-ras protein plus IgG (c and d). e–h, presenescent Hs68 fibroblasts injected in the cytoplasm with either IgG alone (e and f) or c-Ha-ras protein plus IgG (g and h). Ab, antibody.

 
The irreversible growth arrest exhibited by these cells, compared with young, presenescent cells rendered quiescent through serum deprivation, was confirmed by their resistance to entry into S phase upon introduction of oncogenic c-Ha-ras (23) . Serum-starved and serum-stimulated senescent Hs68 fibroblasts microinjected with c-Ha-ras failed to incorporate BrdUrd, which was used as a marker for DNA synthesis and S-phase progression, above background (Fig. 1C, a–d)Citation . In contrast, serum-starved, low passage number, presenescent Hs68 fibroblasts microinjected with c-Ha-ras exhibited, on average, a 3-fold increase in BrdUrd incorporation relative to uninjected cells, as expected for nonsenescent cells (Fig. 1C, e–h)Citation . This induction of DNA synthesis in young, presenescent cells was measured after a 24-h labeling period with BrdUrd. Interestingly, in contrast to the short-term effects of ras, other studies have shown that prolonged expression of H-ras V12 (6 days) in young human fibroblasts induces premature senescence in these cells (50) .

Increased Incorporation of BrdUrd upon Microinjection of {alpha}-p21 Antibodies into Senescent Fibroblasts.
Monoclonal antibodies raised against p21WAF1/CIP1/Sdi1 were injected into the nuclei of senescent cells, and the percentage of cells undergoing DNA synthesis was assessed by BrdUrd incorporation measured after injection. Almost 60% of senescent cells injected with {alpha}-p21 antibodies underwent DNA synthesis relative to cells injected with control IgG (Fig. 2Citation and Table 1Citation ). This represented a 4-fold increase in BrdUrd incorporation in cells injected with {alpha}-p21 antibodies. Similar results were obtained using two sets of four different {alpha}-p21 antibodies (Ab-1 and Ab-3; 18A10 and 2G12) and two different control antibodies (SEN7 and {alpha}-CD3, respectively). Serial dilutions of the Ab-1 and Ab-3 antibodies with the SEN7 antibody were made and injected into senescent fibroblasts. This resulted in a concomitant reduction in the amount of apparent DNA synthesis observed in these cells (Fig. 3)Citation , thereby demonstrating that the amount of BrdUrd incorporation correlates with the amount of {alpha}-p21 antibody injected into the senescent nuclei. Furthermore, the percentage of BrdUrd incorporation in {alpha}-p21-injected senescent cells approximates that exhibited by senescent cells injected with expression constructs for the viral oncogenes, SV-40 T-antigen and adenovirus E1A, which yield 60–70% BrdUrd incorporation (data not shown).



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  		Fig. 2. Induction of DNA synthesis by microinjection of {alpha}-p21 monoclonal antibodies. The ability of {alpha}-p21 monoclonal antibodies to induce senescent Hs68 fibroblasts to enter S phase was assessed by comparing the number of cells undergoing DNA synthesis (via BrdUrd incorporation) that had been microinjected in the nucleus with {alpha}-p21 monoclonal antibodies plus marker IgG with those cells injected with control antibodies plus marker IgG. a and b, injected cells; c and d, cells incorporating BrdUrd; e and f, merged images of a plus c and b plus d, respectively.

 

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  		Table 1 Induction of DNA synthesis in Hs68 fibroblasts fixed after 24 h

 


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  		Fig. 3. Correlation of DNA synthesis in Hs68 human fibroblast with the concentration of injected {alpha}-p21 monoclonal antibodies (Ab). The effects of diluting {alpha}-p21 monoclonal antibodies injected into senescent Hs68 fibroblasts on DNA synthesis in the injected cells (via BrdUrd incorporation) were assessed. {alpha}-p21 monoclonal antibodies were diluted 3-, 9-, and 27-fold with a control antibody in equal concentrations (10 mg/ml) to maintain a uniform total protein concentration. Bars, SD.

 
To establish the time frame required for detecting the increase in BrdUrd incorporation, BrdUrd was added to the culture medium, and cells were incubated for various lengths of time after injection. By 4 h of labeling after injection, only trace amounts of senescent cells injected with {alpha}-p21 antibodies incorporated BrdUrd, whereas 14 h of labeling resulted in maximal BrdUrd incorporation, as expected for the time frame of a typical entry into S phase (data not shown). Experiments conducted using FITC-conjugated antibodies to stain for injected cells and rhodamine-conjugated antibodies to stain for BrdUrd incorporation, and conversely, FITC-conjugated antibodies to stain for BrdUrd incorporation and rhodamine-conjugated antibodies to stain for injected cells, yielded the same results. Finally, to eliminate any possibility of "bleed through" of the injected marker antibody fluorescence into the BrdUrd fluorescence channel, injected cells were confined to a defined region on the coverslips and stained for BrdUrd only. Similar amounts of increased BrdUrd incorporation were observed in senescent cells injected with the {alpha}-p21 antibodies as in previous experiments (Table 1)Citation .

{alpha}-p21 Antibodies Block the Inhibition of cdk2 Kinase Activity by p21WAF1/CIP1/Sdi1.
To further interpret the microinjection data obtained with the {alpha}-p21 antibodies, it was important to test these antibodies for their ability to functionally inhibit p21 in in vitro kinase assays. cdk2 kinase activity was measured using cdk2 immunoprecipitated from low passage Hs68 fibroblasts and histone substrate, and it was shown that addition of GST-p21 protein to the kinase reaction reduced cdk2 activity compared with the addition of GST alone. However, when GST-p21 was preincubated with {alpha}-p21 before addition to the kinase assays, the inhibitory effect of p21 was abolished, presumably by means of the {alpha}-p21 monoclonal antibodies inhibiting ternary complex formation between p21 and cdk2/cyclin complexes (Fig. 4)Citation . Having determined this, it was examined whether the {alpha}-p21 antibodies could reactivate cdk2 kinase activity in cdk2/cyclin/p21 complexes by removing p21 from these inactive ternary complexes. Inactive cdk2 complexes were immunoprecipitated from senescent Hs68 fibroblasts with an {alpha}-cdk2 antibody and then preincubated with increasing concentrations of {alpha}-p21 antibodies prior to initiation of the kinase reaction by addition of substrate and ATP. Although some spontaneous activation of cdk2 kinase activity occurred with prolonged incubation at room temperature (presumably due to the dissociation of the cdk2/cyclin/p21 complex), no specific increase in kinase activity toward histone was detected attributable to the presence of {alpha}-p21 antibodies (data not shown). Even antibody concentrations up to 5 mg/ml failed to produce any specific activation of the cdk2/cyclin/p21 complexes. This result indicates that {alpha}-p21 antibodies do not disrupt the cdk2/cyclin/p21 ternary complex. Similar results were obtained using an {alpha}-cyclin E antibody for immunoprecipitation of the cdk/cyclin/p21 complexes (data not shown).



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  		Fig. 4. Neutralizing effect of {alpha}-p21 monoclonal antibodies in an in vitro cdk2 kinase assay. The ability of bacterially expressed p21 fusion protein (p21 fused to glutathione S-transferase, GST) to inhibit the kinase activity of cdk2/cyclin complexes toward histone H1 was assessed in the presence and absence of {alpha}-p21 monoclonal antibodies. As controls, cdk2/cyclin complexes were incubated with histone H1 without further additions, in the presence of GST alone, and with GST plus the {alpha}-p21 monoclonal antibodies.

 
Injection of {alpha}-p21 Antibodies Stimulates the Expression of Cell Proliferation Markers in Senescent Fibroblasts.
Because p21 plays an important role in the regulation of the cdk/cyclin phosphorylation of pRb (6 , 38, 39, 40) , we hypothesized that inhibition of p21 function by the microinjection of the {alpha}-p21 antibodies would facilitate pRb phosphorylation and release of E2F, which could then function to transcriptionally up-regulate the expression of its targets genes. We therefore investigated the expression of two markers of cell proliferation in the injected senescent cells. The first of these, cyclin A, is an S-phase cyclin that is induced upon E2F overexpression (12) and is normally repressed in senescent cells (3) . Indeed, after injection with {alpha}-p21 antibodies, 44% of the senescent fibroblasts expressed cyclin A 24 h after injection (Fig. 5, a and bCitation ; Table 2Citation ). In contrast, uninjected cells and cells injected with control antibody expressed cyclin A at low frequency (<15%; Fig. 5, c and dCitation ; Table 2Citation ). Similarly, cyclin A expression was detected in a small amount of cells when injected with {alpha}-p21 antibodies and then analyzed for cyclin A protein only 2 h after injection.



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  		Fig. 5. Expression of markers of proliferation in senescent Hs68 human fibroblasts microinjected with {alpha}-p21 ({alpha}p21) monoclonal antibodies (Ab). The ability of {alpha}-p21 monoclonal antibodies to induce senescent Hs68 fibroblasts to enter S phase was assessed by the expression of cyclin A ({alpha} cycA) in cells injected with {alpha}-p21 monoclonal antibodies plus marker IgG (a and b) or injected with an {alpha}-CD3 control monoclonal antibody plus marker IgG (c and d), respectively. Expression of a luciferase reporter under the control of the cdc2 promoter was also assessed in cells injected with the same antibodies together with a reporter construct. a, c, e, and g, injected cells; b and d, cells immunostained for the expression of cyclin A; f and h, cells immunostained for luciferase.

 

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  		Table 2 Induction of cyclin A expression and stimulation of cdc2 promoter

 
In addition to looking at the endogenous expression of cyclin A, we examined the induction of an exogenous luciferase reporter under the control of a truncated cdc2 promoter containing the canonical E2F site (47) . A 4-fold difference was observed in the proportion of senescent cells expressing luciferase in response to coinjection of {alpha}-p21 antibodies together with the reporter (16%; Fig. 5, e and fCitation ; Table 2Citation ), as compared with those injected with control antibody together with the reporter (4%; Fig. 5, g and hCitation ; Table 2Citation ). This demonstrates that the cdc2 promoter is also up-regulated by introduction of {alpha}-p21 antibodies. Thus, in addition to promoting DNA synthesis, inhibition of p21 results in the expected increase in expression of genes associated with cell cycle progression into S phase.

To determine whether injection of {alpha}-p21 antibodies induces senescent cells to progress beyond S phase, cells were injected with {alpha}-p21 antibodies, followed by fixation at 24, 36, or 48 h after injection, and then examined for evidence of cell division. The cell density in the area of injected cells for each time point did not increase after injection of the {alpha}-p21 antibodies. In accordance with this finding, when cells were immunostained for the marker antibody representing injected cells, no adjacent pairs of injected cells were visible. This would have indicated partitioning of antibody between injected cells that have undergone mitosis (data not shown). Therefore, although the injection of {alpha}-p21 antibodies into senescent fibroblasts induced these cells to reenter the cell cycle and progress into S phase, it is not sufficient to drive these cells through a full cell cycle.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
p21WAF1/CIP1/Sdi1 is an important regulator of cell cycle progression (51) . It interacts with and inhibits cdk/cyclin activity, thereby maintaining pRb in its underphosphorylated, growth-suppressive form (6 , 38, 39, 40) . This in turn negatively modulates the transcriptional activity of E2F (41) . p21 can also bind E2F in complexes containing pRb/cdk/cyclin in senescent cells (45) as well as bind PCNA and inhibit DNA replication (42, 43, 44) . It is possible that the role of p21 in senescence is simply to inhibit E2F activity directly; however, it has been shown that the overexpression of E2F is not enough to coax cells out of senescence (52) . Thus, a more likely scenario is that p21 exerts its role in senescence via multiple interactions beyond simply that with E2F.

We have shown that the inhibition of p21 through the injection of {alpha}-p21 antibodies into senescent Hs68 fibroblasts promotes DNA synthesis and an increase in expression of genes associated with cell cycle progression through S phase (i.e., those under the control of E2F activity such as cyclin A and cdc2). We have demonstrated that the {alpha}-p21 antibodies we used can neutralize the ability of p21 to inhibit cdk2 in vitro kinase activity when the {alpha}-p21 antibodies and p21 protein are preincubated (Fig. 4)Citation . In contrast, under the conditions we used, these antibodies were not able to stimulate kinase activity from a p21-cdk2/cyclin complex immunoprecipitated from senescent cells. Therefore, it appears that when the {alpha}-p21 antibodies are injected into senescent Hs68 fibroblasts, they bind to free p21, either as it is newly synthesized or as it is released from inactive cdk/cyclin complexes, and can tip the equilibrium from inactive ternary cdk/cyclin/p21 complexes to active cdk/cyclin complexes. In addition, although immunoprecipitations and immunoblots from senescent and low passage number Hs68 fibroblasts show that p21 protein expression is considerably elevated upon senescence (Fig. 1B)Citation , the half-life of p21 either free or in the context of a cdk complex in senescent human fibroblasts is an open question. Given either possible mechanism, introduction of {alpha}-p21 antibodies enables active cdk2 kinase complex to promote limited cell cycle reentry. Therefore, our studies suggest one of the roles of p21 in senescence is to inhibit cdk/cyclin activity, thus down-regulating its downstream effectors and promoting growth arrest.

The neutralization of p21 afforded by the {alpha}-p21 antibodies does not permit progression through a full cell cycle, however. This is in contrast to when {alpha}-p53 antibodies are injected into senescent cells, because such cells not only reenter the cell cycle and proceed through S phase but also undergo mitosis (11) . In those studies, it was shown that the expression of p21 was significantly reduced in senescent cells injected with the {alpha}-p53 antibodies, although it has been suggested that the up-regulation of p21 in senescence is not wholly dependent on p53 (53 , 54) . Our results build on those obtained in the {alpha}-p53 antibody microinjection experiments with senescent fibroblasts and further demonstrate by experiments directed against p21 specifically that p21 alone is not sufficient to impose the complete growth block characteristic of senescence (55) . Studies conducted on rat embryo fibroblasts immortalized with SV40 large T antigen show that these cells undergo senescence upon its inactivation and are growth-arrested in both G1 and G2 phases of the cell cycle (34) . It appears, from the {alpha}-p53 microinjection experiments (11) , that there may be multiple roles for p53-induced growth arrest at different points in the cell cycle in senescence. Our studies show that the inhibition of p21 can alleviate the G1-S growth arrest; however, the {alpha}-p53 microinjection experiments indicate that there are other p53-dependent factors besides p21 involved in preventing senescent cells from entering and completing mitosis. Apart from the reliance on p53, the pathways using such factors may act independently or in a complementary fashion to the p21-imposed G1-S block in senescence.

Indeed, in a recent study, another factor implicated in inhibition of the G1-S transition, i.e., pRb, has been found to display a distinct role in inhibition of S-phase progression in Rat-1 cells (56) . This inhibition of S-phase progression by pRb is proposed to be overcome by continual phosphorylation of pRb by kinase activity that cannot be inhibited by the introduction of exogenous p21 or p27 in S phase. This is postulated to be attributable to either insufficient levels of these inhibitors or to the existence of Rb kinase activity that is insensitive to these inhibitors. Inactivation of such kinase activity could play a part in maintaining the growth-arrested phenotype characteristic of senescent cells because proteins independent of p21 or p53 have been implicated in the progression of fibroblasts from early passage to senescence in Li-Fraumeni cells (57) . Whether pRb or other factors are involved in the maintenance of senescence beyond S- phase entry remains to be investigated. In any case, the experiments reported here show that the disabling of p21 function can partially reverse the quiescent state of senescent fibroblasts by inducing their entry into S phase, indicating that p21 is a principal player in establishing senescence.


   ACKNOWLEDGMENTS
 
We thank Carolan Buckmaster and Rebecca Shakeley for excellent technical assistance. We would also like to thank Drs. Erik Knudsen, Shiyuan Cheng, and Erich Weber for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by grants from the California Tobacco Related Diseases Program and the National Institutes of Aging (to J. R. F.) and a National Cancer Institute/NIH Research Fellowship award (to B. L. B.). The microinjection core is supported by grants from the National Cancer Institute. T. L. B. was the recipient of an NIH Pharmacology training grant, and this work represents partial fulfillment of the Ph.D. degree in the Biomedical Sciences Graduate Program. S. A. P. was the recipient of a North Atlantic Treaty Organization postdoctoral fellowship. Back

2 To whom requests for reprints should be addressed, at The Scripps Research Institute, 10550 North Torrey Pine Road, MB-7, La Jolla, CA 92037. Back

3 The abbreviations used are: cdk, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; FBS, fetal bovine serum; BrdUrd, bromodeoxyuridine. Back

Received 6/ 3/99. Accepted 8/19/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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