Critical requirement for cell cycle inhibitors in sustaining nonproliferative states

Abstract

In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference-mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D-cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle-driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.

Figure 1.

Identification of relevant CKIs in myotubes and effects of their suppression. (A) Electrophoretic gel separation of proteins immunoprecipitated with an anti-cdk4 (α-cdk4) antibody or control Igs (IgG) from C2C12 myotubes. A silver-stained gel is shown here. Mass spectrometry was performed on bands excised from a Coomassie-stained gel loaded with 50 times as much total protein. Bands that appeared to be uniquely present in the cdk4 lane were processed for mass spectrometry identification. Corresponding portions of the control lane were processed identically. Bands containing p21 and cyclin D3 are indicated. (B) Western blot analysis of p21 in whole-cell lysates from C2C12 myotubes 48 h after transfection with a pool of four p21 siRNAs or control siRNA. (C) Immunofluorescence staining of C2C12 myotubes for BrdU (green) and MyHC (red). (D and E) Percentages of BrdU-positive C2C12 (D) and MSC (E) myotubes 48 h after interference for single or multiple CKIs as indicated. Data are presented as means and SDs (error bars) of up to five experiments. (F) Primary human myotubes were triple transfected with siRNAs for p21, p27, and p18 and were double immunostained for BrdU (green) and MyHC (red) 48 h later. Ctr, control; β-tub, β-tubulin, which was used as a loading control. Bars, 50 μm.

Figure 2.

cdk4-associated kinase activity in CKI-interfered C2C12 myotubes. (A) C2C12 myotubes were infected with an adenovirus carrying dnK4 or a control virus (Ad-ctr) and/or were transfected 6 h later with siRNAs for the indicated molecules. (top) Western blot (WB) analyses of the indicated proteins in whole-cell lysates prepared 24 h after transfection. Proliferating myoblasts are shown for reference. (bottom) cdk4-associated kinase activity measured using GST-Rb as the substrate in immunoprecipitates obtained with an anti-cdk4 antibody from the same lysates as in the top panel. cdk4 was quantitated by Western blotting in the immunoprecipitates as a control for precipitation efficiency. (B) C2C12 myotubes (Mt) were transfected with p21 siRNA #4 (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200608109/DC1) at the indicated concentrations in the presence or absence of 5 nM siRNA to cyclin D3. An identical culture was transfected with control siRNA. (top) Western blot analysis of p21 in whole-cell lysates collected 48 h after transfection. l ctr, loading control. (bottom) Percentages of BrdU-positive myotubes.

Figure 3.

Cyclin D1– and cyclin D3–associated kinase activities in p21-interfered myotubes. C2C12 myotubes were transfected with control or p21 siRNA, and immunoprecipitations for both cyclins were performed side by side 22 h later. Proliferating myoblasts synchronized in mid-G1 phase (see Materials and methods) are shown for reference. (A, top) Cyclin D1–associated kinase activity was measured using recombinant GST-Rb as the substrate. Western blot analysis of cyclin D1 in whole-cell lysates and immunoprecipitations are shown. (bottom) Cyclin D3–associated kinase activity measured as in the top panel. IgG, immunoprecipitation of myotube lysates with normal Igs. β-tubulin loading controls for whole-cell lysates are shown in Fig. S2 (A and B, available at http://www.jcb.org/cgi/content/full/jcb.200608109/DC1). (B) Densitometry-assisted quantitation of cyclin D1– and cyclin D3–associated kinase activities shown in A. See Materials and methods for details. Ctr, control.

Figure 4.

Kinase activity and CKIs associated with cyclin E in CKI-interfered C2C12 myotubes. (A) Western blot analysis of specific proteins in C2C12 myotubes subjected to single or multiple RNAi. Cells were transfected with siRNAs for the indicated molecules, and whole-cell lysates were prepared 30 h later. These lysates are the same as those used to immunoprecipitate cyclin E and measure cyclin E–associated kinase activity in B. Proliferating myoblasts are shown for reference. β-tubulin (β-tub) is shown as a loading control in each of two separate blots. (B) C2C12 myotubes were transfected with siRNAs for the indicated molecules. Cyclin E–associated kinase activity on histone H1 was measured 30 h after transfection in anti–cyclin E immunoprecipitates. Immunoprecipitates were analyzed for p21 and cdk2. (C) C2C12 myotubes were treated as in A, and Western blot analyses of the whole-cell lysates and immunoprecipitates were performed. β-tubulin is shown as a loading control; cdk2 verifies immunoprecipitation efficiency. IgG, immunoprecipitation performed on myotube lysates with normal Igs.

Figure 5.

CKIs can substitute for one another in preserving the postmitotic state. MEF-Mts p21^−/− were transfected with siRNAs for the indicated CKIs. (A) Western blot analysis of p57 performed 48 h after transfection with p57 siRNA. l ctr, loading control. (B) Percentages of BrdU-positive MEF-Mt p21^−/− measured 48 h after interference for single or multiple CKIs as indicated.

Figure 6.

Effects of CKI RNAi in serum-starved primary human foreskin fibroblasts. (A) Western blot analyses of p21, p27, p16, and p18 in cells transfected with siRNAs for the indicated CKIs. Lysates prepared 48 h after transfection. (B) BrdU-positive cells in cultures subjected to RNAi for the indicated molecules. BrdU was added to culture medium 24 h after transfection, and the cells were fixed and stained 24 h later. Values are shown as means of up to three independent experiments with SDs (error bars) and are expressed relative to 10% serum-reactivated cells, which was set to 100. (C) Growth curves of cells subjected to p21 interference. Cells were transfected with siRNA to p21 or were mock-transfected and counted at the indicated times after RNAi. Ctr, control siRNA; serum, cells re-fed with 10% FBS.

Figure 7.

Effects of CKI RNAi in senescent HEK cells and human foreskin fibroblasts. (A) Acidic β-galactosidase staining of young and senescent HEK cells. (B) Western blot analyses of p21 and p16 in HEK cells transfected with siRNAs for the indicated CKIs. Lysates prepared 48 h after RNAi. (C) Senescent HEK cells were subjected to RNAi for the indicated molecules. BrdU incorporation assay as in Fig. 6 B. Percentages of BrdU-positive cells are shown after the subtraction of background. Error bars represent SD. (D) BrdU incorporation in senescent fibroblasts. Assay performed as in Fig. 6 B. (E) Mitotic figures in senescent HEK cells transfected with p21 siRNA. Arrows point to mitotic figures. (F) Growth curves of HEK cell populations transfected with siRNAs for the indicated molecules. The cells were transfected twice, at 0 and 48 h. (G and H) Percentages of TUNEL-positive HEK cells and human fibroblasts in cultures transfected with p21 siRNA. Ctr, control siRNA. Bars, 50 μm.