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Experimental Therapeutics |
Department of Internal Medicine [K-H. P., J. Y. S., T-Y. K., C-G. Y., Y. W. K., S. K. H., Y-S. S., C-T. L.], Cancer Research Institute [T-Y. K.], Seoul National University College of Medicine, Lung Institute of Medical Research Center, Seoul National University [C-G. Y., Y. W. K., S. K. H., Y-S. S., C-T. L.], and Gene Therapy Laboratory of Clinical Research Institute, Seoul National University Hospital [K-H. P., J. Y. S., C-T. L.], Seoul 110-744, Korea
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two families of CDKIs are involved in cell cycle arrest. Inhibitors of the INK4 family (p16, p15, p18, and p19) bind Cdk4 and Cdk6 specifically and inhibit cyclin D binding. In contrast, inhibitors of the Cip/Kip family (p21, p27, and p57) act as broad, specific inhibitors of the cyclin D, E, and A complexes. The deregulation of these CDKIs is a common feature in tumor cells and mainly contributes to the disruption of cell cycle control (2) . Since the cloning of the p27 gene (3 , 4) , many other functions have been associated with this cell cycle protein. p27Kip1 (p27) is a CDKI that exerts its inhibitory activity on many steps of the cell cycle. The main inhibitory action of p27 arises from its binding with the cyclin E/Cdk2 complex and its induction of the dephosphorylation of pRb (5) . p27 mRNA levels are constant throughout the cell cycle, and p27 protein levels are regulated both by translational control (6) and by ubiquitin-mediated proteolysis. p27 protein levels and stability are high in quiescent cells and in the majority of other cell types but fall during G1 and reach a minimum in the S-phase (7) . This regulation of the p27 level is linked to the phosphorylation on Thr-187, followed by ubiquitination (8, 9, 10) . In addition, the cyclin E/Cdk2 complex causes the phosphorylation of p27 on Thr-187, which results in the degradation of p27. Therefore, p27 is both an inhibitor and a substrate of cyclin E/Cdk2 (11) . Interestingly, mutation on Thr-187 of p27 to alanine made p27 resistant to cyclin E/Cdk2-induced phosphorylation and induced G1-S arrest resistant to cyclin E/Cdk2 (11) .
In addition to its role as a CDKI, p27 has the role of a putative tumor suppressor gene (12) . p27 knockout mice develop multiorgan hyperplasia and parathyroid tumors (13) , and p27 haplo-insufficient mice are more sensitive to tumor development by radiation and chemical carcinogens (14) . Adenoviral gene transfer of p27 also induced the cell cycle arrest and apoptosis of breast cancer cell lines (15 , 16) . We have shown previously that the transduction of p27 via adenoviral vector to human lung cancer cell lines induced cell growth suppression via G1-S arrest. Furthermore, intratumoral injection of ad-p27 induced growth suppression of established lung cancer xenografts (17) .
On the basis of these two findings of the role of p27 as a tumor suppressor gene and the process of p27 degradation by phosphorylation on Thr-187, we hypothesized that p27mt at Thr-187 would be resistant to degradation and bind to the cyclin E/Cdk2 complex more stably and, consequently, demonstrate a more potent antitumor effect.
To confirm this hypothesis, we constructed two adenoviruses expressing wild p27 (ad-p27wt) and mutant p27 at the Thr-187/Pro phosphorylation site (ad-p27mt) and compared their antitumor effects.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of ad-p27wt and ad-p27mt.
We have described the construction of ad-p27wt (17)
and ad-p16 (18)
previously. These viruses are replication-defective, E1-deleted adenoviruses with CMVie promoter. Briefly, the cDNA of human p27 (kindly provided by J. Massagué, Memorial Sloan Kettering Cancer Center, New York, NY) was subcloned into the KpnI and BamHI sites of the polylinker of the adenoviral shuttle vector, pAC CMV pLpA (kindly provided by Robert Gerard, University of Texas Southwestern Medical Center, Dallas, TX). The resulting pAC CMV-p27wt and pJM17 (also kindly provided by R. Gerard) were cotransfected into 293 cells using the standard calcium phosphate precipitation method. The 293 cells were maintained in RPMI 1640 with 2% fetal bovine serum until the onset of the cytopathic effect. Generating adenovirus was purified three times by plaque assay and confirmed by DNA sequencing of viral DNA and Western blot of p27. A large-scale stock of adenovirus was prepared by the standard CsCl method, and the titer was determined by plaque assay (19)
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p27mt with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) was used for the construction of ad-p27mt. Ad-p27mt was generated by the same procedure as described for ad-p27wt. Recombinant adenovirus expressing ß-galactosidase (ad-lacZ) was used as a control virus in all experiments.
Transduction of Lung Cancer Cells with ad-p27(wt/mt).
Exponentially growing lung cancer cells were transduced with ad-p27(wt or mt) at 20 MOI for 1 h with frequent gentle shaking and then incubated with complete medium for the experiment.
Western Blot for p27 and pRb.
Lung cancer cells (NCI H358) were transduced with 20 MOI of ad-p27(wt/mt) and incubated for 48 h. Western blots of p27 and pRb were performed using the ECL Western blotting system according to its protocol (Amersham). Monoclonal mouse antihuman p27 and pRb antibodies (PharMingen, San Diego, CA) were used as the primary antibodies for p27 and pRb.
Pulse Chase Experiment.
To demonstrate the stability of p27mt produced in ad-p27mt transduced cells, a pulse chase experiment for p27 was performed. Lung cancer cells (NCI H460) were transduced with 20 MOI of ad-p27wt and ad-p27mt. After 24 h, the medium was replaced with serum-free medium without methionine/cysteine for 16 h. Cells were then incubated with complete medium containing 1 µCi of [35S] methionine/cysteine for 4 h (pulse). Proteins were extracted and immunoprecipitated for p27 from cells at 0, 15, 60, and 240 min and 24 h. The stabilities of p27wt and p27mt were compared by 10% SDS-PAGE electrophoresis.
Analysis of the Growth Inhibition on Lung Cancer Cell Lines.
To compare the growth-suppressing effect of ad-p27wt and ad-p27mt, four NSCLC cell lines (NCI H460, large cell carcinoma of the lung; NCI H157, squamous cell carcinoma of the lung; NCI H1264, adenocarcinoma of the lung; and NCI H358, bronchioloalveolar cell carcinoma) were plated in six-well plates (5 x 104/well). After 24 h of incubation, transductions were performed with ad-p27(wt/mt; 20 MOI), ad-lacZ, or PBS for 1 h, and the cells were maintained in complete medium. Cell numbers were counted in triplicate on a daily basis using a hemocytometer.
Cell Cycle Analysis after Transduction with ad-p27wt and ad-p27mt.
Lung cancer cells (NCI H460) were transduced with 20 MOI of ad-p27wt or ad-p27mt. After 24 h incubation, cell cycle alterations were measured using the CycleTest plus kit protocol (Becton Dickinson, San Jose, CA).
Cdk2 Kinase Activity Assay.
Twenty-four h after transduction with ad-p27wt and ad-p27mt, total cell extracts were precipitated with anti-Cdk2. Cdk2 kinase activities of the immunoprecipitates were determined in terms of their ability to phosphorylate histone H1. Reactions were performed for 30 min in 50 µl of H1 kinase buffer with 10 µg of histone H1 and 10 µCi of [
-32P]ATP. Reaction products were resolved by 10% SDS-PAGE gel electrophoresis.
Annexin V Assays for Apoptosis.
To confirm the presence of another p27wt or ad-p27mt growth-suppressing mechanism, we observed the induction of apoptosis by Annexin V apoptosis kit (PharMingen). Briefly, lung cancer cells (NCI H460) were transduced with ad-p27wt or ad-p27mt at 20 MOI. Forty-eight and 96 h after transduction, cells were detached with by a brief trypsin treatment. Cells were then stained with Annexin V and PI, according to the manufacture’s instructions, and sorted using a FACScaliber flow cytometer (Becton Dickinson). Proportions showing early apoptosis (positive for Annexin V and negative for PI) were then measured.
Hoechst Staining.
DNA fragmentation of apoptotic cells were measured by Hoechst 33258 staining. Lung cancer cells (NCI H460) were transduced with ad-p27wt and ad-p27. At 96 h, cells were stained with 1 µl of Hoechst 33258 for 10 min. At least 300 cells were counted under a fluorescence microscope with a 4',6-diamidino-2-phenylindole filter. Cells were defined to be apoptotic if their nuclei showed chromatin condensation, nuclear beading, or fragmentation.
PARP Cleavage Assay.
To confirm apoptotic induction by ad-p27wt and ad-p27mt transduction, we measured the cleavage of PARP. Briefly, a lung cancer cell line (NCI H460) was transduced with ad-p27wt (20 MOI) or ad-p27mt (20 MOI) and incubated for 72 h. The expression and cleavage of PARP were assessed by Western blot assay using an anti-PARP monoclonal antibody (PharMingen).
Soft Agar Clonogenic Assay.
To compare the effect of p27wt or p27mt gene transfer on in vitro tumorigenicity, soft agar clonogenic assay was performed using the method described previously (20)
. Briefly, tumor cells (NCI H358 and NCI H460) were transduced with 20 MOI of ad-p27wt or ad-p27mt and then were detached and plated in 0.2% agarose with a 1% agarose underlay (5 x 103 cells/plate).
Treatment of Established Lung Cancer Xenografts by Intratumoral Injection of ad-p27wt and ad-p27mt.
s.c. xenografts of lung cancer were established in nude mice by injection of 2 x 106 cells (NCI H460) at day 14. After 2 weeks, day 0, when tumors were of moderate size (
350 mm3 in volume), intratumoral injections of ad-p27wt and ad-p27mt (1.0 x 109 plaque-forming units/injection) were initiated and repeated five times every day. Changes in tumor size were measured every other day using the following formula: tumor volume = 1/2 x (length)2 x width.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This finding demonstrated the stability of p27mt, which would support the hypothesis of this experiment.
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). Similar findings were found in other lung cancer cell lines (data not shown). These findings suggest that p27mt protein is a more effective and powerful CDKI, although both p27wt and p27mt are effective.
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. Differences between p27wt and p27mt were not observed in terms of their inhibition of Cdk2 activity. This finding suggests that both p27wt and p27mt are potent CDKIs.
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. Forty-six % of ad-p27mt transduced cells and 31% of ad-p27wt transduced cells showed apoptotic changes (4% in ad-lacZ transduced cells).
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, which could also provide evidence of apoptosis induction by p27wt and p27mt.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we attempted to develop a more potent p27 gene therapy by manipulating the p27 metabolic pathway. As we described, p27 metabolism depends on phosphorylation of Thr-187 and a subsequent ubiquitination. Montagnoli et al. (10) showed that the ubiquitination of p27 did not occur in p27mt with Thr-187 to Ala [p27(T187A)]. Sheaff et al. (11) showed that the transfection of p27(T187A) plasmid causes a G1 block that is both resistant to and not modulated by cyclin E/Cdk2.
On the basis of these observations of p27 metabolism and the nature of the p27 tumor suppressor gene, we tried to use p27mt at Thr-187 for cancer gene therapy, expecting a super-repressor effect on cell cycle arrest. Adenovirus expressing p27mt (Thr-187/Pro-188 to Met-187/Ile-188) was constructed to block cyclin E/CDK2 more stably. 187TPKK190 is a conserved COOH-terminal Cdk target site for phosphorylation (8) . Two amino acids (Thr-187 and Pro-188) were mutated to completely disrupt the conserved COOH-terminal CDK target site (TPKK).
Western blot assay for p27 showed that the transduction of both adenoviruses induced p27 protein overexpression and the dephosphorylation of pRb. The p27 monoclonal antibody was specific for the NH2 portion of p27 that was unchanged in p27mt. The [35S]methionine/cysteine-pulse chase experiment demonstrated that p27mt was resistant to degradation and more stable than p27wt. Contradictory results have been reported of the stability of p27mt. Vlach et al. (8) reported that mutation of Thr-187 to Val of p27k- (p27 deficient in interaction with Cdks) could increase the stability; however, the same mutation in p27wt failed to increase the stability. However, Nguyen et al. (32) reported that mutation of Thr-187 to Ala stabilized p27 in S-phase extracts. Recently, the degradation-resistant novel p27 isoform (p27Kip1R) was cloned. This p27 isoform has no conserved COOH-terminal, Cdk-phosphorylation site (TPKK) and is part of a putative bipartitate nuclear-localization signal (residues 159–169). This p27Kip1R was resistant by in vitro degradation assay and was more effective at inducing apoptosis than p27wt. However, they reported that p27T187A was degraded in a manner similar to p27wt in an in vitro degradation assay (33) . In the present study, we demonstrated that p27mt with double mutation of Thr-187/Pro to Met-187/Ile was resistant to degradation in a pulse chase experiment, in which the levels of mutant p27 were constant for 4 h but markedly decreased at 24 h. This finding suggests some other mechanism of p27 degradation. Actually, Shirane et al. (34) showed that p27 is degraded by ubiquitin-mediated degradation and ubiquitin-independent proteolytic processing.
Ad-p27mt transduction more strongly suppressed the growth of lung cancer cell lines and induced stronger G1-S arrest than ad-p27wt in all cell lines tested. The growth-inhibitory effect of p27 was not related to p53 status. Ad-p27mt showed strong growth inhibition in the p53-deleted cell line (NCI H358, homozygous deletion of p53), the p53 mutated cell lines (NCI H157 and NCI H1264, mutation of codon 298 of p53 from CAG to TAG), and the normal p53 cell line (NCI H460). In this study, we could not determine the effect of Rb on ad-p27, because the four cell lines tested were positive for pRb. Naruse et al. (35) showed recently that growth inhibition by the overexpression of p27 requires the intact expression of pRb.
This growth inhibition in this study could be explained by two mechanisms, cell cycle arrest and induction of apoptosis. Because of the known role of CDKI, we had already expected G1-S arrest by p27mt and p27wt. In four cell lines tested, ad-p27mt induced a more profound G1-S arrest than ad-p27wt. The role of the CDKI of p27wt and p27mt was confirmed again by its suppression of Cdk2 kinase activity.
Conflicting results have been reported of the effect of p27 upon cell susceptibility. Several reports on adenoviral gene transfer of p27 have revealed apoptotic induction by p27 overexpression in several cancer cell lines (24 , 35, 36, 37) . Katayose et al. (24) reported that the overexpression of p27 triggers apoptosis in several cancer cell lines. They demonstrated the induction of apoptosis by terminal deoxynucleotidyltransferase-mediated nick end labeling assay and found that the overexpression of p27 was followed by cleavage of PARP and the degradation of cyclin B1. Naruse et al. (35) also showed that ad-p27 induced apoptosis by terminal deoxynucleotidyltransferase-mediated nick end labeling assay and PI flow cytometry. Wang et al. (36) also reported the induction of apoptosis by ad-p27 by demonstrating DNA fragmentation and PARP cleavage. Schreiber et al. (37) compared several adenoviral vectors expressing CDKIs including p27 and also showed that ad-p27 induced apoptosis in Rb-positive cells after day 3, suggesting an indirect role for p27Kip1. However, some studies have shown that apoptotic stimuli, such as those induced by cytotoxic drugs, induced caspase-mediated p27 cleavage (38) . St. Croix et al. (23) demonstrated that p27 is a regulator of chemoresistance and that tumor-targeted p27 antagonists act as chemosensitizers.
In this report, we clearly confirmed again that ad-p27wt/mt induced apoptosis in lung cancer cell lines by Annexin V assay, Hoechst staining, and PARP cleavage. More interestingly, ad-p27mt is a more potent apoptosis-inducer than ad-p27wt, as confirmed by Annexin V assay and Hoechst staining.
Consistent with previous reports (35 , 37) , apoptosis was not evident at 48 h but became evident at 96 h. From these reports, we think that ectopic overexpression of p27 can induce apoptosis, and high expression of endogenous p27 shows antiapoptotic properties.
This stronger antitumor effect of p27mt was reconfirmed by an in vitro tumorigenicity assay and a treatment model of lung cancer xenograft in mice. Intratumoral injection of ad-p27wt induced modest growth suppression, as we reported previously (17) . As expected from its stronger growth arrest and apoptosis induction, ad-p27mt induced stronger growth suppression in an in vivo animal tumor model than ad-p27wt. For an initial 7 days after ad-p27mt injection, tumors showed regression in size, and tumors completely disappeared in 40% of mice. The remainder of the mice showed delayed tumor growth. When one considers the relatively low transduction efficiency by intratumoral injection, this delayed growth is believed to be related to the low transduction efficiency. If the injection method could be improved for better transduction, results might be improved. The possibility of using mutant p27 for cancer gene therapy has been reported previously. Müller et al. (39) reported upon the transduction of p27T187V via the retroviral vector-inhibited growth of the RAT1-MycER cell line, which was resistant to p27wt.
From the observations described above, we are confident that ad-p27mt at Thr-187 can potentiate p27 gene therapy and that it presents us with a novel, potent, tumor-suppressing gene therapy tool.
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FOOTNOTES |
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1 Supported by the Korea Science and Engineering Foundation through the SRC-Molecular Therapy Research Center. 
2 To whom requests for reprints should be addressed, at Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea. Phone: 82-2-760-2634; Fax: 82-2-762-9662; E-mail: ctlee{at}snu.ac.kr
3 The abbreviations used are: Cdk, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; CMV, cytomegalovirus; MOI, multiplicity of infection; NSCLC, non-small cell lung cancer; PI, propidium iodide; PARP, poly(ADP-ribose) polymerase; ad, adenovirus; wt, wild type; mt, mutant; Rb, retinoblastoma; pRb, Rb protein.
Received 9/29/00. Accepted 6/20/01.
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REFERENCES |
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-tubulin from a transduced lung cancer cell line (NCI H358). Lung cancer cells transduced with ad-p27wt and ad-p27mt produced large amounts of p27 proteins that were indistinguishable by the p27 antibody. The amount of p27 from ad-p27mt transduced cells was slightly higher than that from ad-p27wt transduced cells. ad-p27wt and ad-p27mt transduction induced the dephosphorylation of pRb protein.
