Research Article Combined Depletion of Cell Cycle and Transcriptional Cyclin- Dependent Kinase Activities Induces Apoptosis in Cancer Cells Dongpo Cai,1,2 Vaughan M. Latham, Jr.,1,2 Xinxin Zhang,1,2 and Geoffrey I. Shapiro1,2 1Department of Medical Oncology, Dana-Farber Cancer Institute; 2Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts Abstract COOH-terminal domain (CTD) of RNA polymerase II and promote Selective cyclin-dependent kinase (cdk) 2 inhibition is readily initiation and elongation of nascent mRNA transcripts, respec- compensated. However, reduced cdk2 activity may have tively. Some of the cdks are involved in both cellular processes. antiproliferative effects in concert with other family members. For example, cyclin H-cdk7 is a component of cdk-activating Here, inducible RNAinterference was used to codeplete cdk2 kinase and contributes to cell cycle progression as well as and cdk1 from NCI-H1299 non–small cell lung cancer and transcription. Cyclin E-cdk2 and cyclin B-cdk1 have been shown U2OS osteosarcoma cells, and effects were compared with to phosphorylate the CTD in vitro (3, 4) and therefore may play a those mediated by depletion of either cdk alone. Depletion of role in transcriptional regulation at specific cell cycle phases. cdk2 slowed G progression of NCI-H1299 cells and depletion of Cdk2 was initially considered a viable antineoplastic drug 1 target, because several approaches inhibiting its activity in cancer cdk1 slowed G2-M progression in both cell lines, with associated endoreduplication in U2OS cells. However, com- cells have induced perturbations during multiple cell cycle phases pared with the incomplete cell cycle blocks produced by as well as apoptosis. For example, the inducible expression of a individual depletion, combined depletion had substantial dominant-negative cdk2 mutant in U2OS osteosarcoma cells led to G2 arrest after low-level induction and arrest during both S and consequences, with G2-M arrest predominating in NCI-H1299 cells and apoptosis the primary outcome in U2OS cells. In G2 phases when higher levels were induced (5). Effects on G1 U2OS cells, combined depletion affected RNApolymerase II progression also occurred but were weak and only unmasked expression and phosphorylation, causing decreased expression when cells were synchronized and released from a nocodazole- of the antiapoptotic proteins Mcl-1 and X-linked inhibitor of induced mitotic block. In exponentially growing transformed cell apoptosis (XIAP), effects usually mediated by inhibition of the lines, S and G2 cell cycle effects have also been described after ectopic expression of p27Kip1 (6) or introduction of peptides that transcriptional cdk9. These events do not occur after indi- vidual depletion of cdk2 and cdk1, suggesting that reduction inhibit cdk2 activity (7). The latter approaches have also resulted of cdk2, cdk1, and RNApolymerase II activities all contribute in significant cytotoxicity as has the targeted disruption of cyclin to apoptosis in U2OS cells. The limited cell death induced by A (8). combined depletion in NCI-H1299 cells was significantly These profound effects on S and G2 cell cycle progression and increased by codepletion of cdk9 or XIAP or by simultaneous apoptosis need to be reconciled with the absence of similar cell treatment with the cdk9 inhibitor flavopiridol. These results cycle effects or cell death after introduction of antisense or small show the potency of concomitant compromise of cell cycle and interfering RNA (siRNA) targeting cdk2 in cancer cells (9). One transcriptional cdk activities and may guide the selection of possibility is that the former approaches are targeting both cdk1 clinical drug candidates. (Cancer Res 2006; 66(18): 9270-80) and cdk2. For example, expression of the dominant-negative cdk2 mutant resulted in reduced cyclin B–associated kinase activity (5). Ectopic p27Kip1 would be expected to inhibit both cdks. The Introduction reported cdk2-inhibitory peptides were capable of cdk1 inhibition The cyclin-dependent kinases (cdk) comprise a family divided at high concentration, and the proteasomal degradation of cyclin into two groups based on their roles in cell cycle progression and A should affect cyclin A-cdk1 activity as well. In fact, small hairpin transcriptional regulation (1, 2). Members of the first group form RNA (shRNA)–mediated silencing of cdk1 produced greater effects À À the core components of the cell cycle machinery, including cyclin on cellular proliferation in cdk2 / mouse embryo fibroblasts D–dependent kinases 4 and 6, cyclin E-cdk2 complexes, cyclin than in wild-type cells (10), suggesting functional redundancy A–dependent kinases 2 and 1, and cyclin B-cdk1 complexes, which between these cdks. However, whether shRNA-mediated codeple- facilitate the transition between cell cycle phases. The second tion of cdk2 and cdk1 can similarly affect transformed cells or group includes cyclin H–dependent kinase 7 and cyclin induce apoptosis has not yet been clarified. Additionally, the T–dependent kinase 9 (p-TEFb), which phosphorylate the definition of the effects of combined depletion in established cancer cells is critical for the design of small-molecule cdk Note: Supplementary data for this article are available at Cancer Research Online inhibitors. Although compounds highly selective for cdk2 may (http://cancerres.aacrjournals.org/). have limited utility, those targeting both cdks with high potency Corrected online December 20, 2019. may have therapeutic value. D. Cai, V.M. Latham, Jr., and X. Zhang contributed equally to this work. Inhibition of the transcriptional cdks has also attracted intense Present address for X. Zhang: Department of Medicine, University of California, San Diego, CA. interest because the most sensitive transcripts are those with short Requests for reprints: Geoffrey I. Shapiro, Department of Medical Oncology, half-lives that encode cell cycle regulators, mitotic regulatory Dana-Farber Cancer Institute, Dana 810A, 44 Binney Street, Boston, MA 02115. Phone: kinases, nuclear factor-nB–responsive gene transcripts, and 617-632-4942; Fax: 617-632-1977; E-mail: [email protected]. I2006 American Association for Cancer Research. apoptosis regulators, such as Mcl-1 and X-linked inhibitor of doi:10.1158/0008-5472.CAN-06-1758 apoptosis (XIAP; refs. 11, 12). Diminution of levels of these Cancer ResDownloaded 2006; 66: (18). from September cancerres.aacrjournals.org 15, 2006 on October9270 1, 2021. © 2006 American Association forwww.aacrjournals.org Cancer Research. Downloaded from cancerres.aacrjournals.org on November 4, 2019. © 2006 American Association for Cancer Research. Dual Cell Cycle and Transcriptional Cdk Inhibition transcripts and their encoded proteins may produce anticancer cdk1 or cdk2 shRNA vectors as well as a vector encoding a hygromycin activity or augment apoptotic responses. Flavopiridol, the most selection marker at a 10:1 ratio; individual clones were selected in 200 Ag/ potent known inhibitor of cdk9, has recently shown striking mL hygromycin with blasticidin and puromycin and tested for knockdown A activity in chronic lymphocytic leukemia, which may be in part of both cdks after exposure to 5 g/mL doxycycline for 6 days. For cell growth experiments, cells were plated at 3.75 Â 105 per 10-cm dish in the attributable to a drug-induced decrease in Mcl-1 expression absence or presence of doxycycline. One quarter of the medium was (13, 14). The potential role of cdk2 and cdk1 in RNA polymerase replaced every 3 days. II CTD phosphorylation suggests that their inhibition may similarly Lentivirus production and infection. The CalPhos Mammalian deplete transcripts encoding proteins that affect cancer cell Transfection kit (Clontech Laboratories) was used to transfect lentiviral survival. shRNA as well as relevant helper plasmids into 293T cells. A NCI-H1299TR To define the effect of depletion of cdk2 and cdk1 activities in cdk2/cdk1 double shRNA clone was plated for 24 to 48 hours in the established cancer cells of diverse origin, we have engineered absence or presence of doxycycline before application of the appropriate NCI-H1299 non–small cell lung cancer (NSCLC) cells and U2OS lentivirus at a multiplicity of infection of f5. Cells were washed in PBS osteosarcoma cells to inducibly express shRNA targeting cdk2, 24 hours after transduction and maintained in medium in the absence or cdk1, or both together. This allowed us to probe the effects of presence of doxycycline for an additional 6 or 7 days for pLKO-cdk9 and pLVTHM-XIAP infection, respectively. One quarter of the medium was relatively acute depletion of these cdk activities, an appropriate replaced every 3 days. model for assessment of these kinases as drug targets. U2OS Cell synchronization. Cells were cultured in medium with or without cells express wild-type p53 and Rb as well as cyclins D1 and D3, doxycycline for 24 hours and subsequently replated at 6 Â 105 per 10-cm INK4A and lack p16 expression because of promoter hyper- dish and grown for an additional 24 hours before synchronization. Cells methylation (15, 16). They undergo S and G2-M arrest and were treated with 1 mmol/L hydroxyurea for 24 hours or 4 Ag/mL apoptosis in response to cdk2 inhibition by dominant-negative nocodazole (both from Sigma-Aldrich Co., St. Louis, MO) for 16 hours to cdk2 mutant expression (5), inhibitory peptides (7), and targeted achieve a G1-S or mitotic block, respectively. Cells were subsequently cyclin A degradation (8) but have not shown cell cycle washed in PBS and maintained in medium in the absence or presence of alterations in response to antisense-mediated specific depletion doxycycline. of cdk2 (9). NCI-H1299 cells are similarly Rb wild-type and Flavopiridol treatment. Flavopiridol was provided by the Drug p16INK4A methylated but carry a p53 deletion (17, 18). These cell Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. lines show that either G -M arrest or apoptosis is the major 2 A 50 mmol/L stock solution in DMSO was maintained at À20jC.