Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1 (original) (raw)
Proliferation requires the presence of growth factors in p27–/– cells. Bromodeoxyuridine (BrdU) staining, a marker of DNA synthesis, was increased in p27_–/–_ mesangial cells (27 ± 7% vs. 16 ± 5%; P <_ 0.05) and fibroblasts (25 ± 6% vs. 15 ± 4.8%; _P <_ 0.05) compared with p27+/+ cells when exposed to FCS for 24 hours. In contrast, BrdU staining was barely detected in p27_–/–_ mesangial cells (3 ± 0.3% vs. 2 ± 0.4%; _P > 0.05) and fibroblasts (2.1 ± 0.4% vs. 1.9 ± 0.5%; P > 0.05) deprived of growth factors for 24 hours compared with p27+/+ cells. In this study (results not shown) and in a previous report (6), BrdU staining was not detected in rat mesangial cells transfected with p27 antisense in the absence of growth factors (P > 0.05 vs. nontransfected cells; cells transfected with mismatch). Thus, in the absence of growth factors, the loss of p27 was not sufficient to increase DNA synthesis.
Apoptosis is increased in growth factor–deprived p27–/– cells. Mesangial cells and fibroblasts from p27 null (p27_–/–_) and p27 wild-type (p27+/+) mice were grown in the presence or absence of serum, a source of growth and survival factors, for 24 hours, and the percentage of apoptotic cells was determined by TUNEL staining. In the presence of serum, there were very few TUNEL-positive cells, and there was no significant effect of p27 deficiency (p27_–/–_ vs. p27+/+ mesangial cells: 0.6 ± 0.1% vs. 0.8 ± 0.1%, P > 0.05; p27_–/–_ vs. p27+/+ fibroblasts: 0.5 ± .01 vs. 0.75 ± .09%, P > 0.05) (Fig. 1). In contrast, when deprived of growth factors for 24 hours, there was a marked increase in TUNEL staining in p27_–/–_ compared with p27+/+ mesangial cells (35.2 ± 2% vs. 5.0 ± 0.8%; P < 0.001) and p27_–/–_ compared with p27+/+ fibroblasts (30 ± 3% vs. 7.0 ± 1.5%; P < 0.001) (Fig. 1). Similar results were obtained when apoptotic cells were scored by other methods, including H&E and Hoechst staining. At later time points, the increased death of p27-deficient cells could be detected by simple visual inspection. Thus, p27_–/–_ and p27+/+ mesangial cells and fibroblasts were plated at the same confluency, allowed to adhere overnight, and then serum starved for five days. There was a marked decrease in p27_–/–_ mesangial cell confluency compared with p27+/+ cells (Fig. 2), and this was associated with an increase in the number of detached cells, or floaters (not shown).
Quantitation of apoptosis. TUNEL staining was not increased in the presence of growth factors (serum) in p27_–/–_ mesangial cells (a) and p27_–/–_ fibroblasts (b). In contrast, TUNEL staining was increased at 24 h of growth factor deprivation (serum free) in p27_–/–_ cells compared with p27+/+ cells. *P < 0.001 vs. serum; **P < 0.001 vs. p27+/+. TUNEL, deoxynucleotide transferase–mediated nick end-labeling.
Effect of growth factor deprivation on cell confluency. p27+/+ (a) and p27_–/–_ (b) mesangial cells were plated at the same density in FCS. Compared with p27+/+ mesangial cells (c) cell confluency decreased in p27_–/–_ mesangial cells (d) after 5 days of growth factor deprivation.
To determine if p27 also protected cells from other forms of apoptosis, p27+/+ and p27_–/–_ mesangial cells were exposed to the protein synthesis inhibitor cycloheximide for 24 hours. Cycloheximide increased apoptosis in p27_–/–_ mesangial cells compared with control cells (29.2 ± 3.8% vs. 14.19 ± 1.9%, P < 0.05). These results showed that augmented apoptosis in p27-deficient cells may be a generalized phenomenon.
Reconstituting p27 rescues cells from apoptosis. We next determined if p27_–/–_ mesangial cells and p27_–/–_ fibroblasts were protected from apoptosis by transient expression of the p27 protein. Cells transfected with the p27 expression vector were identified by cotransfection with a marker plasmid encoding the GFP plasmid, and apoptosis was induced by serum starvation for 24 hours. In separate experiments, apoptosis was measured in p27_–/–-transfected cells by double-immunostaining for the p27 antigen and TUNEL. Apoptosis (GFP+/Hoechst+) was not detected in 200 p27_–/– mesangial cells and p27_–/–_ fibroblasts transfected with the p27 expression vector when scored 24 hours after serum starvation (Fig. 3). Similar results were obtained using double-staining for p27 and TUNEL (p27+/ TUNEL+) (not shown).
Effect of reconstituting p27 on apoptosis in p27_–/–_ mesangial cells. Transfected p27 mesangial cells were identified by staining with GFP (green nuclei), and apoptosis was measured by Hoechst staining. (a) p27_–/–_ mesangial cells transfected with human p27 plasmid stained green. (b) Apoptosis was not detected in serum-starved transfected p27_–/–_cells transfected with the p27 plasmid (arrows); apoptosis was detected in nontransfected cells (arrowhead). (c) Control p27–/– mesangial cells were transfected with a p27 mutant plasmid that does not bind CDK2 and GFP. (d) p27 mutant plasmid did not prevent apoptosis (arrowhead). (e) In each experiment, 200 cells were quantitated, and the number of cells undergoing apoptosis was expressed as a percentage. *P < 0.001 vs. p27–/– alone (nontransfected cells) and control (p27 mutant plasmid–transfected) p27–/– cells. These data show that p27 protects cells from survival and mitogenic growth factor deprivation–induced apoptosis. CDK2, cyclin-dependent kinase-2; GFP, green fluorescent protein.
To test specificity for the transfection studies with p27 wild-type plasmid discussed above, similar studies were performed in p27_–/–_ cells transfected with a p27 mutant plasmid that cannot bind to CDK2. In contrast to p27 wild-type plasmid, transfecting p27_–/–_ cells with plasmids expressing p27 mutant protein that is incapable of binding CDK2 (Fig. 3), β-galactosidase (not shown) or vector alone (not shown) did not rescue p27_–/–_ cells from apoptosis. Furthermore, the rate of apoptosis was comparable in nontransfected p27_–/–_ cells and p27_–/–_ cells transfected with the p27 mutant plasmid. These results showed that reconstituting p27 reduced apoptosis in p27_–/–_ cells to a level comparable to that of control cells, and also confirmed the specificity of p27 wild-type plasmid in protecting cells from apoptosis.
p27 also protects rat cells from apoptosis. To determine if p27 also protected cells from other species from apoptosis, p27 levels were lowered in rat mesangial cells and rat fibroblasts with antisense oligodeoxynucleotides to p27 (6, 20) and then deprived of growth factors for 6, 10, and 20 hours. Controls included nontransfected cells and cells transfected with mismatch oligonucleotides (Fig. 4). The exclusion of toluidine blue and the absence of released lactate dehydrogenase showed that transfecting these cells did not alter cell viability (results not shown). In the presence of serum, apoptosis was not detected in transfected rat mesangial cells and fibroblasts (Fig. 4). In contrast, apoptosis was significantly increased in serum-starved rat mesangial cells and rat fibroblasts transfected with p27 antisense in comparison to the controls (Fig. 4). These results showed that p27 levels modulate the degree of apoptosis in cells from different species.
(a and b) Western blot analysis for p27. Transfecting rat mesangial cells (a) and rat fibroblasts (b) with antisense oligonucleotides to p27 (AS), but not mismatch oligonucleotides (MS), lowered p27 protein levels compared with nontransfected cells (NT). (c and d) Quantitation of TUNEL staining in rat cells. TUNEL staining was detected at 6 h after growth factor deprivation (serum free) in AS-transfected cells in rat mesangial cells (c) and rat fibroblasts (d), but not in controls, and was increased at each time point compared with controls.
CDK2 activity is increased in growth factor–deprived p27–/– cells. As shown earlier, DNA synthesis as measured by BrdU staining was barely detected in mesangial cells and fibroblasts fromp27_–/–_ and p27+/+ mice afte r withdrawal of growth factors at 24 hours. However, p27 deficiency did have a measurable effect on regulation of CDK2 by mitogens. Fig. 5 shows that CDK2 kinase activity was increased in p27_–/–_ mesangial cells and p27_–/–_ fibroblasts in the presence of growth factors (serum) compared with p27+/+ cells. Eighteen hours of serum starvation reduced CDK2 activity to very low levels in p27+/+ cells. In contrast, CDK2 activity was readily detected in serum-starved p27_–/–_ cells at the same time point (Fig. 5). CDK2 activity was not detected when the primary antibody was omitted or when the primary antibody was absorbed with peptide (results not shown). Furthermore, the increase in CDK2 activity was also shown with a different CDK2 antibody from a different source. In contrast to changes in CDK2 activity, the protein levels for CDK2 remained unchanged in p27_–/–_ and p27+/+ cells in the presence or absence of serum (Fig. 5). These results are likely to represent a delay in the downregulation of CDK2 in the absence of p27, rather than differences in CDK2 protein levels.
CDK2 activity and protein levels. Total cell protein was immunoprecipitated with an antibody to CDK2, and CDK2 activity was detected by H1 kinase assay. CDK2 protein levels were measured by Western blot analysis. In the presence of survival and mitogenic growth factors (FCS), CDK2 activity was increased in p27–/– mesangial cells (a) and p27–/– fibroblasts (c) compared with p27+/+ cells. CDK2 activity remained elevated at 6 h and 18 h after growth factor deprivation (serum free) in p27–/– cells compared with p27+/+ cells. The protein levels for CDK2 did not change in p27–/– and p27+/+ mesangial cells (b) and fibroblasts (d) in the presence or absence of growth factors.
Uncoordinated CDK2 activity in apoptotic cells. To determine if the unconstrained CDK2 activity in growth factor–deprived p27_–/–_ cells represented cyclin E or cyclin A activation, immunoprecipitation studies were performed with antibodies to these cyclins. Fig. 6 shows that in p27_–/–_ cells the presence of growth factors was associated with an increase in cyclin E–CDK2 activity and cyclin A–CDK2 activity measured by 32P incorporation and densitometry. However, in p27_–/–_ mesangial cells deprived of growth factors for six hours, there was a 47% decrease in cyclin E activity (Fig. 6). In contrast, despite the absence of growth factors for six hours, cyclin A–CDK2 activity remained elevated in p27_–/–_ mesangial cells (Fig. 6).
Cyclin E–CDK2 and cyclin A–CDK2 activity in p27–/– and p27+/+ mesangial cells. Total protein was extracted from p27–/– and p27+/+ mesangial cells and immunoprecipitated with antibodies to cyclin E (top) and cyclin A (middle) for histone H1 kinase. Quantitation of CDK2 kinase activity is shown (bottom). In the presence of growth factors (serum), there was an increase in cyclin E–CDK2 and cyclin A–CDK2 activity in p27–/– and p27+/+ cells, which decreased in p27+/+ cells after 6 h of growth factor deprivation (serum free). In p27–/– mesangial cells, cyclin A–CDK2 activity, but not cyclin E–CDK2 activity, remained increased after growth factor deprivation.
To determine if the increase in cyclin A–CDK2 activity (but not cyclin E–CDK2 activity) also occurred in growth factor–deprived p27+/+ mesangial cells, immunoprecipitation studies were performed. There was a 45% decrease in cyclin E activity and a 43% decrease in cyclin A activity after six hours of serum deprivation in p27+/+ cells compared with cells exposed to growth factors (Fig. 6). Furthermore, the decrease in cyclin E–CDK2 activity with growth factor deprivation was similar in p27_–/–_ and p27+/+ mesangial cells. In contrast, there was almost a twofold increase in cyclin A–CDK2 activity in growth factor–deprived p27_–/–_ cells compared with growth factor–deprived p27+/+ cells.
To determine if the difference in CDK2 kinase activity in p27_–/–_ and p27+/+ mesangial cells was due to differences in protein levels for CDK2, cyclin E, or cyclin A, Western blot analyses were performed for these proteins. Fig. 5 shows that the protein levels for CDK2 remained constant in p27_–/–_ and p27+/+ mesangial cells in the presence or absence of growth factors. Furthermore, the protein levels for cyclin E and cyclin A were similar in p27_–/–_ and p27+/+ cells in the presence of serum, and the levels of these cyclins was similar in p27_–/–_ and p27+/+ cells deprived of growth factors for six hours (not shown). These findings show that the increase in cyclin A–CDK2 activity in apoptotic p27_–/–_ cells was not due to the levels of specific cyclins and CDK2, but rather due to the absence of p27.
Thus, when compared with absent CDK2 activity in normal cells, CDK2 activity in p27-deficient cells was transiently elevated after mitogen withdrawal, and this was due to cyclin A–CDK2. However, the elevated CDK activity was accompanied not by DNA synthesis (absent BrdU staining), but rather by cell death.
p27 limits apoptosis by restraining CDK2. Our results suggested that CDK2 was the effector for apoptosis in this model and that p27 protected cells from apoptosis by maintaining CDK2 inactive. To test this hypothesis, p27_–/–_ cells were first exposed to the purine analogue Roscovitine, which reduces CDK2 activity (21, 22). When p27_–/–_ cells were incubated in serum-free media and 12.5 μM Roscovitine for 24 hours, apoptosis was reduced compared with cells exposed to serum-free media and dimethyl sulfoxide (3.5 ± 0.9% vs 34 ± 2%; P < 0.001). Second, p27_–/–_ mesangial cells and p27_–/–_ fibroblasts were cotransfected with a dominant–negative mutant CDK2 plasmid (dnk2) and a plasmid expressing GFP, or the control wild-type CDK2 and GFP, and then deprived of growth factors for 24 hours. The percentage of p27_–/–_ cells undergoing apoptosis after serum starvation was not reduced by overexpression of wild-type CDK2 (Fig. 7) or an irrelevant plasmid (β-galactosidase results not shown). In striking contrast, no apoptosis was detected in cells overexpressing kinase inactive CDK2 (0/200 GFP-positive cells) (Fig. 7). These results show that inhibiting CDK2, either pharmacologically or by overexpression of a kinase inactive mutant CDK2, significantly suppresses apoptosis in p27_–/–_ cells.
Effect of suppressing CDK2 activity on apoptosis in p27–/– fibroblasts in response to serum starvation. (a) p27–/– cells transfected with a dominant–negative mutant CDK2 (dnk2) plasmid were identified by cotransfection with a GFP plasmid (green nuclei). (b) Apoptosis (measured by Hoechst staining) was not detected in dnk2-transfected cells (thick arrow), but was detected in nontransfected cells (thin arrow). (c) p27–/– cells cotransfected with wild-type CDK2 (wtk2) plasmid and GFP were identified as green. (d) wtk2 did not protect p27–/– fibroblasts from apoptosis (arrow). (e) Quantitation of apoptosis. The percentage of apoptotic cells was evaluated in 200 cells. *P < 0.001 vs. nontransfected p27–/– fibroblasts (p27–/– alone) and wtk2-transfected cells. Similar results were obtained in p27–/– mesangial cells.