p16 mediates inhibition of both cdk4 and cdk2 in OSp16.1 cells. (A) Inhibition of cdk4 kinase activity. Extracts were prepared from cells cultured with (+) or without (−) TET (1 μg/ml) for 1 to 3 days and were normalized for protein content. cdk4 was immunoprecipitated, and its kinase activity was assayed using gstRB as a substrate. (B) (Left panel) Binding of p16 to cdk4. Extracts were prepared from cells cultured with or without TET for 3 days and were normalized for protein content. p16 complexes were immunoprecipitated and immunoblotted for cdk4 (K4). (Right panel) Inhibition of cyclin D1 binding to cdk4 on p16 induction. Extracts were prepared from cells that were or were not subjected to p16 induction for 3 days. Cyclin D1 complexes were immunoprecipitated (D1). These samples were normalized for cyclin D1 content by immunoblotting (below) and immunoblotted for cdk4 (K4). (C) Inhibition of cyclin E-associated kinase activity. Extracts were prepared from cells that were or were not subjected to p16 induction for 1 to 3 days. Cyclin E complexes were immunoprecipitated and normalized for cdk2 content by immunoblotting (K2) (shown below). Kinase activities were then assayed using histone H1 (HH1) as a substrate. (D) p16 induction prevents S phase entry following cell cycle synchronization with lovastatin and rescue with mevalonic acid. OSp16.1 cells were treated with lovastatin for 40 h in the presence (Uninduced) and absence (Induced) of TET. At the end of this period, a portion of the cells was harvested and assayed for DNA content by flow cytometry (0 h; x axis, DNA content; y axis, cell number, normalized to each G₁ peak). The lovastatin block was rescued in the remaining cells by addition of mevalonic acid for another 36 h. Cells were removed at 12-h intervals for analysis. The S phase fraction at 24 h was 43% in the uninduced cells versus 5% in the induced cells. (E) Inhibition of cyclin E-associated kinase activity by p16 induction in synchronized cells. Extracts were prepared from cells treated as described for panel D and normalized for protein content. Cyclin E complexes were immunoprecipitated, and their kinase activities were assayed using histone H1 (HH1) as a substrate. C, control (employing nonspecific rabbit IgG). The relative signal intensity is given below each lane.

Inhibition of cdk2 by p21 in cells with p16 induction. (A) Increased association of p21 with cdk2 and decreased association with cdk4 following p16 induction. Extracts were prepared from cells cultured with or without p16 induction for 1 and 3 days and were normalized for protein content. cdk2 (K2) or cdk4 (K4) complexes were immunoprecipitated and probed for coprecipitated p21 or p27 by immunoblotting. Un, uninduced; Ind, induced. (B) Inhibition of cdk2 kinase activity in p21-bound complexes. Extracts were prepared from cells with p16 induction for 3 days, and p21 and cdk2 were immunoprecipitated. The immunoprecipitates were normalized for cdk2 protein level and assayed for cdk2 activity with gstRB as a substrate. Quantitation of the relative band intensity is provided below each lane. (C) Immunodepletion of cyclin E bound to p21 following 3 days of p16 induction. Extracts were prepared from cells with (+) or without (−) TET for 3 days and subjected to three successive immunoprecipitations with antibodies directed against p21 or p27. Cyclin E, actin (act; a negative control), p21, and p27 levels in the starting extracts and in the supernatant following the third IP were assessed by immunoblotting. Clearing of a major fraction of cyclin E from the supernatant occurred only following p21 IP, and only in extracts prepared from cells with p16 induction in this and two other experiments (data not shown). Parallel IPs with a negative-control antiserum (anti-hemagglutinin [12CA5]) had no effect on the level of any of these antigens (data not shown). (D) Overexpression of cyclin E or A can prevent the arrest mediated by p16. OSp16.1 cells were cotransfected with a CMV vector expressing β-gal together with an empty expression vector (V) or one expressing cyclin E (E) or cyclin A (A). A portion of the vector-transfected (transf.) cells was replated in the presence of TET as a positive control for BrdU incorporation. p16 was induced in the remaining cells by replating in the absence of TET for 24 h. The cells were then incubated with BrdU for 8 h, fixed, and stained for β-gal and BrdU. One hundred β-gal-positive cells per condition were scored by an observer blinded to the treatment groups, and the results confirmed by a second, similarly blinded observer. The mean percentage of BrdU-positive cells from three experiments is depicted. Error bars indicate standard deviations.

Increase in total cellular and cdk2-associated p21 levels mediated by p16 expression in HCT 116 cells. (A) Induction of p21 by p16 in adenovirus-infected cells. Parental HCT 116 cells (p21^+/+) were mock infected (Un), infected with Ad5lacZ (Z), or infected with Ad5p16 for 24 h. Extracts were prepared and normalized for protein content. p21 was detected by immunoblotting. The same extracts were probed for actin by immunoblotting (below) as a control to assess protein content. An extract prepared from OSp21.1 cells maintained for 3 days in the absence of TET served as a positive control (C). (B) p16 mediates an increase in association of p21 with cdk2 (K2) in adenovirus-infected cells. cdk2 complexes were immunoprecipitated from the extracts described above, and associated p21 was detected by immunoblotting.

Induction of p21 by p16. (A) Lovastatin-synchronized cells. Extracts were prepared from cells synchronized with lovastatin and rescued with mevalonic acid, in parallel with those cells described in the legend to Fig. 1D, and normalized for protein content. The designated proteins (p16, cdk2 [K2], cyclin E [E], cyclin A [A], p27, and p21) were detected by immunoblotting. (B) Unsynchronized cells. Extracts (normalized for protein content) were prepared from cells initially in log-phase growth, then plated with (Ind) or without (Un) p16 induction for 1 or 3 days. p16 and p21 were detected by immunoblotting. (C) p21 mRNA levels are not changed by p16 induction. RNA was prepared from cells that were or were not subjected to p16 induction for 1 and 3 days, and Northern blotting was performed with the p21 cDNA as a probe. The band labeled p21 mRNA was not present in RNA prepared from p21-null HCT 116 cells (data not shown) (see below). As a control for loading and transfer, the blot was rehybridized with a probe to RNase P (labeled “C”). Similar results were obtained in three other experiments (data not shown). (D and E) Increased metabolic labeling of p21 following p16 induction. Cells that were or were not subjected to p16 induction for 3 days were pulse-labeled for 1 hour with [³⁵S]methionine-[³⁵S]cysteine, and extracts were prepared at the end of this period and at 1, 2, and 4 h after addition of an excess of unlabeled amino acids. (D) The extracts were normalized for protein content (we confirmed that this also resulted in normalization for incorporation of label in the pulse phases), and p21 was immunoprecipitated and resolved on an SDS-polyacrylamide gel. A relatively stable protein that appears to cross-react with the antibody (labeled “C”) is shown as a loading control. HCT 116 p21^+/+ and p21^−/− cells (see below) were treated identically to confirm that the band shown corresponds to p21. This band also was not seen in control IPs with nonspecific rabbit IgG and comigrated exactly with p21 detected in the same gels by immunoblotting (data not shown). Similar results were obtained in two other experiments (data not shown). (E) The p21 band intensity was quantitated and graphed (pixels [× 100], on a semilog scale; y axis) versus time (hours; x axis). Open squares represent results from cells with p16 induction (Ind), and closed squares represent results from uninduced (Un) cells. (F) Increased sedimentation of p21 mRNA in polyribosome gradients following p16 induction. Cytoplasmic lysates were prepared from cells that were (right) or were not (left) subjected to 3 days of p16 induction and were fractionated by sucrose gradient density sedimentation. Preliminary Northern blotting studies established that p21 mRNA was largely confined to the bottom half of each gradient (e.g., in contrast to the 36B4 mRNA [data not shown]). For finer fractionation, RNA isolated from the top half of each gradient was pooled as fraction 1 and that from the bottom half of the gradient was further subdivided into four fractions (2 to 5), in order of increasing density. A single major PCR product, of the predicted size, was obtained from each fraction, employing p21- and glyceraldehyde 6-phosphate dehydrogenase (“C”)-specific primers, respectively. Titrations showed that the PCRs were substrate limited under these conditions (data not shown). Similar results were obtained in a second experiment (data not shown).

Inhibition of cyclin E-associated kinase activity by induction of p21 alone to levels induced by P16. (A) OSp21.1 cells were either maintained in medium with suppressive levels of TET (1.5 μg/ml; uninduced [Un]) or treated with concentrations of TET (0.1 and 0.01 μg/ml; Ind) chosen to induce p21 to a level (right) similar to that observed in OSp16.1 cells upon full induction of p16 (left). p21 levels in extracts normalized for protein content were determined by immunoblotting. Protein content was documented by immunoblotting for actin. (B) Inhibition of cyclin E-associated kinase activity by p21 induction in OSp21.1 cells. Extracts were prepared from OSp21.1 cells maintained for 3 days in media containing different concentrations of TET, as described for panel A. Cyclin E was immunoprecipitated. The complexes were normalized for cdk2 (K2) content and assayed for kinase activity with histone H1 as a substrate. cdk4 (K4) activity was also assayed, in a like manner, using gstRB as a substrate (normalization data are not shown). Quantitation of these signals revealed that cyclin E-associated kinase activity was inhibited in OSp21.1 cells by 40 and 75% in 0.1 and 0.01 μg of TET/ml, respectively. Since p21 levels under these conditions were, respectively, slightly lower and higher than that observed with p16 induction, we estimate that 50% inhibition of cyclin E-associated kinase activity can be expected for a comparable level on p21 induction alone. This compares with 75% inhibition of cyclin E-associated kinase activity seen with p16 induction (Fig. 1C).

p16 expression strongly inhibits S phase entry in p21^+/+ cells but not in p21^−/− cells. (A and B) BrdU incorporation. Parental (p21^+/+) and p21^−/− HCT 116 cells were cotransfected with a constant amount of a CMV vector expressing β-gal and either the same empty vector or one expressing p16 or cdk2DN. BrdU was added in several additions 24 to 48 h after transfection, and the cells were fixed and stained for β-gal and BrdU. (A) Quantitative fluorescent micrographs were obtained from a single field of p21^+/+ cells (top) or p21^−/− cells (bottom) transfected with the p16 expression vector. β-Gal is stained red, BrdU is stained green (giving a yellow tinge when combined with red), and nuclear DNA (detected with bisbenzimide) is stained blue. (B) One hundred β-gal-positive cells per transfection condition were scored through direct microscopic examination of random medium-power fields by an observer blinded to the treatment groups. The results were confirmed by a second, similarly blinded observer. The mean percentage of BrdU-positive cells from three experiments is depicted. Error bars indicate standard deviations. (C) Flow cytometry. The respective cells were cotransfected with a constant amount of a CMV vector expressing CD20 and either the same empty vector or one expressing p16. Single, CD20-positive (green fluorescent) cells were identified by flow cytometry 36 h after transfection and analyzed for DNA content. One hundred thousand events were collected for each transfection condition and subjected to identical gating parameters. This analysis yielded a similar number of cells from each transfection condition: 3,812 (p21^+/+; vector), 2,810 (p21^+/+; p16), 2,718 (p21^−/−; vector), 3,503 (p21^−/−; p16), and 49 (mock transfected; data not shown). The S phase fraction is highlighted with cross-hatching and quantitated above each profile. Comparable results were obtained in a second experiment (data not shown).