p27Kip1 inhibits the cell cycle through non-canonical G1/S phase-specific gatekeeper mechanism

Abstract

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 has been shown to regulate cellular proliferation via inhibition of CDK activities. It is now recognized that p27Kip1 can regulate cellular processes through non-canonical, CDK-independent mechanisms. We have developed an inducible p27Kip1 model in cultured cells to explore CDK-independent p27Kip1 regulation of biological processes. We present evidence that p27Kip1 can function in a CDK-independent manner to inhibit entry and/or progression of S phase. Even though this p27Kip1 mechanism is non-canonical it does requires the intact cyclin-binding motif in p27Kip1. We suggest a mechanism similar to that proposed in post-mitotic neural cells whereby p27Kip1 functions to coordinate growth arrest and apoptosis. Our hypothesis supports the concept that p27Kip1 is a gatekeeper for the entry and progression of S phase through interaction with specific protein(s) or via binding to specific DNA sequences in a CDK-independent manner.

Keywords: cell cycle; cyclin F; cyclin-dependent kinases; non-canonical; p27Kip1.

Figure 1.

Parental U2OS, U20S-p27WT, and U2OS-p27K cellswere grown in culture. After 24 h, DOX (1µg/ml) was added to one set of cultures of U2OS-p27K cells. After another 48 h, BrdU (30 µM) was added to all cell cultures including induced and non-induced p27K, parental U2OS cells and U2OS-p27WT cells. Cultures were incubated with BrdU for 60 min (Panel A) and 24 h (Panel C). Cells in each condition and at the indicated times were removed from plates and processed for cell cycle analysis and BrdU incorporation. In Panel B annexin V staining (Materials and Methods) for cells isolated at each time and condition are shown.

Figure 2.

Parental, U2OS-p27WT, and U2OS-p27K cells were grown and treated as described in Figure 1 with one set of U2OS –p27K cells receiving DOX (1μg/ml) and then cells were incubated for 72 h. BrdU (30 μM) was added at this time for 1 h. Panel A shows picture of the cell populations at 72 h and Panel B shows the BrdU incorporation into the cells in all populations.

Figure 3.

U2OS cells, U2OS-p27WT cells and U2OS-p27K cells were placed in nocodazole inhibition as described in Material and Methods. Some cultures of U2OS-p27K and U2OS-p27WT cells had Dox (1µg/ml) added for 6 h. Cultures of parental, non-induced U2OS-p27WT, non-induced U2OS-p27K, and induced U2OS-p27K cells were released from the nocodazole block. Panel A shows propidium iodide straining of these cells, as described in Material and Methods, before release and 15 h after release from the mitotic inhibition. Data show 2N and 4N staining. Panel B shows the cell cycle distribution with propidium iodide staining of parental cells, U2OS-p27WT, and U2OS-p27K induced 6 h before mitotic release. Panel C illustrates the induction of p27K by 1 µg/ml of Dox. Panel D shows the analysis of BrdU incorporation in cells treated as in Panel B when released into fresh medium containing 30 μM BrdU.

Figure 4.

Parental, U2OS-p27WT and U2OS-p27K cells where placed in a nocodazole-induced mitotic block as described in Figure 3 and Material and Methods. Dox (1µg/ml) was added to cultures of U2OS-p27K cells 6 h before mitotic release. Cells were washed and placed in fresh medium with Dox. At indicated times cells were collected and extracts prepared. Extracts were analyzed by western blot analysis for cyclin D1, Cyclin E and cyclin A. β-Actin was used as a loading control.

Figure 5.

Parental cells were plated at 1million cells/dish (upper panel) and U2OS-p27K cells were plated at various densities as indicated (lower panel). All cells were put into nocodazole-induced mitotic inhibition as described in Figure 4 and Materials and Methods. Cells were released from inhibition and placed in fresh medium. Cell cycle analysis was performed on cells in mitotic inhibition and 14 h post mitotic release. Pictures show cell density.

Figure 6.

Panel A shows the western blot analysis for cyclin F in parental U2OS cells, U2OS-p27WT cells and in U2OS-p27K cells with induced p27K that were released from a nocodazole-induced mitotic block. The p27K was induced 6 h before release from mitotic inhibition. Cells were collected at the indicated times. Panel B shows cyclin F at indicated times. CTRL is parental cells released from nocodazole inhibition. CHX are cells release in presence of 10 µg/ml cycloheximide added 1 h prior to release. MG132 show cyclin F when cells were released in the presence of 10µM MG132 added 2 h prior to release. Panel C shows cyclin F at the indicated times after parental cells were released from double thymidine block using 2 mM thymidine. Panel D shows cyclin F at the indicated times after parental cells were released from thymidine block (as in Panel C) into the fresh medium with or without nocodazole inhibition. Panel E shows the co-immunoprecipitation of cyclin F with various p27 constructs: wild-type (p27WT), altered cyclin-binding site (p27C), altered CDK-binding site (p27K) and altered cyclin- and CDK- binding sites (p27CK). U2OS-derived p27-inducible cells were grown in medium containing Dox (1µg/ml) for 24 h. Cells were harvested, extracts prepared and HA-tagged p27 proteins were immunoprecipitated and separated by SDS-PAGE gel. Immunoblots were analyzed for cyclin F.

Figure 7.

U2OS cells were placed in a nocodazole-induced mitotic arrest as described in Materials and Methods. Some cultures received cyclin F siRNA (Cyclin F-siRNA), others received a non-targeting siRNA (NT-siRNA) and cells not treated with siRNA (untransfected) were also used (On-TARGETplus SMARTpool siRNAs from Dharmacon were used and knockdown was performed as per manufacturer's instruction using Dharmafect-1). After the cyclin F knockdown with siRNAs,cells were placed in a nocodazole-induced mitotic arrest as described in Materials and Methods. Cells were then rinsed and released into fresh medium. Panel A shows cyclin F at the indicated times following release from the nocodazole arrest. Panel B shows the cell cycle distribution at 0,16, 20, and 24 h after release from the mitotic arrest. Panel C shows the incorporation of BrdU at various times after the release from mitotic arrest for untransfected, cyclin F-siRNA and NT-siRNA treated cells.

Figure 8.

Parental and U2OS-p27K were placed in nocodazole-induced mitotic arrest. Cells were released from the mitotic arrest, p27K had been induced 6 h before release for one culture and at 0, 2, 4, 8, 12 h after release for other cultures. Cells were incubated in medium with BrdU. The percent of cells not entering S phase (DNA synthesis) was determined.

Figure 9.

Parental, U2OS-p27WT, and U2OS-p27K cells were inhibited in a nocodazole-induced mitotic arrest. Dox was added as indicated for 6 h and then cells were washed and placed in fresh medium with or without Dox. At the indicated times cells were harvested and immunoblotted to determine comparative amounts of ORC1 and CDT1.