[CANCERRESEARCH54,6078-6082,December1,1994] Advances in Brief

Transcriptional Repression of the D-Type -dependent Kinase Inhibitor by the Retinoblastoma Susceptibility Product pRbt

Yan Li, Michael A. Nichols, Jerry W. Shay, and Yue Xione Department of Biochemistry and Biophysics. the Program in Molecular Biology and Biotechnology, Lineberger Comprehensive Center. The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280 (Y. L, M. A. N., 1'.XI, and the Department of Cell Biology and Neurosciences, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9039 Ii. W. S.]

Abstract of by . p2i encodes a potent inhibitor of all cyclin-CDK enzymes that have been tested (6—8).The p21 is Progression of the eukaryotic cycle is regulated by a series positively regulated by the tumor suppressor p53 (6, 9), providing a of structurally related senne/threomne kinases known as cydin critical link between the tumor suppression function of p53 and cell dependent kinases (CDKs). The D-type cydlin-dependent kinases, CDK4 cycle control. and CDK6, have been strongly implicated in the control of G1 progression and the phosphorylation of the , pRb. The forma The involvement of CDK inhibitors in cancer development is don of complexes and enzymatic activity of -CDK4 and cydlin further illustrated by the studies of pi6. p16 was first identified as a D-CDK6 kinases is negatively regulated by p16'@4 (MTS1/CDK4IJ CDK4-specific associated protein in virally transformed cells (5). The CDKN2) via its specific interaction with CDK4 and CDK6 catalytic gene encoding p16, pi61―4 (also known as MTSJ, CDK4J, and subunits. Here we report that the p16 mRNA accumulates to a high level CDKN2) was subsequently isolated by yeast two-hybrid screening and in cells lacking pRb function and ofpl6 is repressed by pRb. shown to form specific binary complexes with CDK4 and CDK6 and Our results provide evidence supporting a feedback regulatory loop inhibit the kinase activity of D-type cyclin-dependent CDK4 (10). pi6 involving pRb, p16, and cydin-dependent kinases. was recently found to be homozygously deleted or mutated at a high frequency not only in a wide variety of human tumor-derived cell Introduction lines (1 1, 12) but also in several specific types of primary tumors The primary control of the eukaryotic is provided by a (13—16),demonstrating that p16 is a bonafide tumor suppressor. The family of serine/threonine protein kinase complexes consisting of a extent to which pi6 is involved in tumorigenesis is currently under catalytic subunit, a CDK3; and a regulatory subunit, a cyclin (1). CDK intensive investigation. subunits by themselves are inactive and binding to a cyclin protein is The growth-suppressing activity of the prototype tumor suppressor, required for their activity as well as their regulation. CDKS are also the retinoblastoma gene product pRb, is known to be regulated by cell regulated intrinsically by activating and inhibitory phosphorylations cycle-dependent phosphorylation and both CDK4 and CDK6 have (reviewed in Refs. 1 and 2). In addition to cyclin activation and been strongly implicated as physiological pRb kinases (see the two subunit phosphorylation, it has become clear recently that CDK ac most recent reviews in Refs. 17 and 18). Elucidation of the mecha tivity is also regulated (predominantly negatively) by a number of nisms that regulate the activities of CDK4 and CDK6 is important to small that physically associate with , CDKS, or their our understanding of both cell cycle control and tumor suppression by complexes. In mammalian cells, the number of small cell cycle pRb. Toward this goal, we have investigated the mechanism regulatory proteins identified by virtue of their ability to physically regulating the expression of CDK4-inhibitory proteins, p16. interact with cyclin or CDK proteins is rapidly increasing. Although these small CDK-interacting proteins were identified only very re Materials and Methods cently, their biochemical and biological properties are being charac terized very quickly. Most importantly, a striking connection between Cell Culture. All cells were culturedin DMEMsupplementedwith 10% some of these newly discovered CDK inhibitors and tumor growth fetal bovine serum in a 37°Cincubator with 5% CO2. IMR-90 is a normal suppression has been discovered. human diploid lung fibroblast strain (American Type Culture Collection, The first evidence that a CDK inhibitor may play an important role CCL186). IMR-90 cells expressing type 16 human papilloma viral E6 and E7 in tumor growth suppression came from the studies of the p21 CDK oncoproteins were as described before (19). HSF43 is a human diploid fibro blast strain derived from neonatal foreskin, and CflO-2C-T1 (CT1O) was inhibitor. p21 (also variously known as WAFJ, CJPJ, SD/i, CAP2O, derived from nontransformed HSF43 by introducing a plasmid containing the and CDKNJ), first discovered in normal human fibroblast cells as a SV4Ovirus large tumor antigen (‘I')linkedto a Rous sarcoma virus promoter. component of cyclin D-CDK quaternary complexes that also contain Saos-2 and U-2 OS are human osteosarcoma cell lines. proliferating cell nuclear antigen (3), was subsequently identified as a Northern Analysis Procedures. Total RNA preparation and Northern universal component of all cyclin-CDK complexes in normal human analysis were carried out as described before (20). All hybridizations were fibroblasts (4, 5). p21 protein was not detected in cyclin-CDK com carried out at 68°Cfor16 h in a solution containing 5 X Denhardt's solution, plexes in cells transformed by a variety of tumor viruses or in 2 x SSC, 0.1% SDS, 100 @g/mldenatured salmon sperm DNA, 25 @MNaPO4 p53-deficient Li-Fraumeni cells (5), suggesting a possible regulation (pH 7.0), and 10% dextran sulfate. A 0.9-kilobase full length human pitS cDNA (10) was labeled by the random priming labeling method and used as a probe for Northern hybridization. Received 10/13/94; accepted 10/28/94. The costs of publication of this article were defrayed in part by the payment of page Antibodies and Immunochemistry Procedures. Procedures for [355] charges. This article must therefore be hereby marked advertisement in accordance with methionine metabolic labeling, immunoprecipitation, and immunoblotting 18 U.S.C. Section 1734 solely to indicate this fact. have been described previously (5). Anti-CDK4 peptide antibody has been 1 This study was supported by a start-up fund to Y. X. from the University of North described in Ref. 5, anti-CDK6 peptide antibody used in this study was Carolina at Chapel Hill. 2 To whom requests for reprints should be addressed. described and provided by Dr. M. Meyerson (21), and anti-p16 antibody was 3 The abbreviations used are: CDK, cyclin-dependent kinase; CMV, cytomegalovirus. described and provided by Drs. M. Serrano and D. Beach (10). 6078

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Geaomic Clones and Cell Transfection. A 500- cDNA fragment corresponding to the coding region of p16 was isolated by PCR using oligo- nucleotide primers derived from p16 cDNA (10) and used as a probe to screen a human placenta genomic library cloned in AFlX I1 (Stratagene, La Jolla, CA). A 1.1-kilobase genomic fragment corresponding to the 5' upstream region of p16 cDNA was determined by DNA sequencing and subcloned into a promot- erless luciferase expression plasmid, pGU-Basic (Pmmega. Madison, W). The resultant plasmid, pl6-Luc, or control pGU-Basic and pSV40-Luc DNA were transfected with lipid reagents (Lipofectamine Reagent; Gibco-BRL, Gaithersburg, MD) into 4 X ld cells cultured on &well dishes (40-602 confluence) that were plated 1 day prior to transfection. In cotransfection experiments shown in Fig. 3, B and C, 1 pg of pl6-Luc DNA was wtrans- fected with an increasing amount of pCMV-RB complemented by canier pGU-Basic DNA to provide equal amounts of a total of 2 pg transfected DNA in each transfection. Twenty-eight h after transfection, cells were lysed with 250 pl Triton lysis buffer (1% Triton X-100/15 mu MgSOJ4 mM [ethylenebii (oxyethylenenitrilo)]tetraacetic acidtl mM DlTt25 mu glycylglycine, pH 7.8) and gently shaken in a cold room for 15 min. Cell lysates were collected into a 1.5-ml tube and clarified by centrifugation for 5 min at 14,000 rpm on a microcentrifuge in the cold room. Twenty-five pl of clarified cell lysate was mixed with 231 pI of 50 mM glycylglycine (pH 7.8) and a 44-4 reaction mixture to give rise to a final volume of 300 pl that contains 15 mu MgSO,, 6 mu ATP, and 0.67 pg/ml BSA. Luciferase activities were measured by integrating light emission over 30 s on a Berthold luminometer (Lumat LB 9501; Wallac, Inc., Gaithersburg, MD) with injection of 100 pl luciferrin (ICN, Irvine, CA) to each reaction. Results and Discussion p16 was initially identified as a CDK4-associated protein in cells transformed by a variety of DNA tumor viruses including SV40, papilloma, and adenovi-s (5). It is present in the CDK4 c&nplexes at a high level in transformed cells as determined by immunoprecipi- tation, but in normal untransformed cells the level of CDK4-associ- ated p16 is extremely low or undetectable. A common property shared by these DNA tumor viruses is their ability to bind to and conse- quently inactivate the function of two tumor suppressors, pRb and p53, suggesting the possibility that pRb andlor p53 may regulate the expression of pl6. To test this hypothesis, we first analyzed the steady state level of p16 mRNA. Total RNA was prepared from a number of tumor-derived cell lines the pRb and/or p53 status of which has been previously characterized. TWOsuch examples are shown in Fig. 1. Saos-2 and U-2 OS cell lines were both derived from human osteosarcomas. Northern analysis revealed a high level of p16 mRNA in Saos-2 cells, but p16 mRNA was undetectable in U-2 OS cells (Fig. 1B). Failure to detect the p16 mRNA in U-2 OS cells is not due to homozygous of the p16 as is observed in many cultured tumor cell lines (11, 12), since we have verified the presence of the p16 gene in both cell lines by PCR (data not shown). Whether the p16 gene in U-20s (and Saos-2) cells contains point mutation(s) in the coding region or promoter sequences has not been determined. The high level of p16 mRNA in Saos-2 cells is correlated with a high level of p16 protein and its association with CDK4 and CDK6, as shown in Fig. 1C. In addition to p16, we also noticed that both CDK4 and CDK6 appear to associate with several additional cellular proteins such as p13, p14, and p15 that do not correspond to any of the recently identified small CDK inhib- itors as judged by their mobility. The specificity and identity of these FI& 1. Adysk dp16mRNA aml prorein in bumwemamma cells with wild-type potential CDK4 and CDK6 interacting proteins are currently un- (U-2 OS) aad deletion of RBI gnc -2). A. anslysis of pub pmteh. [3SS]Methiinine- labeltd all lysattq werc prcpsnd fmm S-2 and U-2 OS dls anrl werc immunopre- cipitatsd with antisera ~paificto pRb protein w7701. pRb memwas not detected in Sac&! atla using this antibody due to a deletion in cxom 11-27 of the RBI (22). 8, cxprcsrion of plh mRNA In Saos-2 and U-2 OS cells. Twenty lrg of total RNA samples plexes. ["SJMethionine-labeled cell lysates were prepared from both Saos-2 and U-2 OS prPparcd fmcach cell line were rcsolved on u 1% rgaKgc gel. Equal amounts of RNA cells and immunopncipitated with antisera specific to CDK4, CDK6, and p16 with or wcrc lolsded 8s delemined by hybridization with a rat glycdehy&-3-phosphate de- without prior incubation with a competing antigen peptide as indicated at the top of each bydmg- probe (data mt shown). Resolved RNA samples werc transferred to a lane and resolved by SDS-PAGE. nK relative position of proteins with established nitrarllulm filter and the blot was hybridized with a pbederived from full length identities and potential CDK4 and CDK6 interacting proteins are marked. kDa molecular hump16 (10) and $121 cDNA (6). C,analysis of CDK4, CDK6. and p16 immunocom- weight in thousands; cyc Dl, cyclin Dl. 60

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TRANSCRIPTIONALREPRESSION OF p16 BY pRb

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Fig. 2. Increased expression of p16 mRNA and association of p16 protein with CDK4 and CDK6 in cells lacking pRb function. A, Northern analysis of p16. The procedures on cell culture, RNA preparation, and Northern blotting were the same as described previously (20). Total RNA was prepared from normal human diploid fibroblasts IMR-90 cells and IMR-90 cells expressing type 16 papilloma viral oncoproteins E6, E7, and E6 together with E7 (19), and from a different strain of normal human fibroblasts (HSF43) and its SV4O large T antigen-transformed derivative [Cf1O (5)]. Twenty @gofeach of the RNA samples were resolved on a 1% agarose gel. Similar amounts of RNA were loaded for each sample as determined by hybridization with a rat g!yceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. Resolved RNA samples were transferred to a nitrocellulose filter and the blot was hybridized with a probe derived from full length human p115cDNA (10). As a comparison, the same blot was also hybridized with a probe derived from full length human p21 cDNA, the expression of which is drastically reduced as the result of inactivation of p53 function by the E6 oncoprotein of type 16 papillomavirus (6, 9, 23). Right ordinaie, positions of pl6,p21 and GAPDH mRNA. The steady state level of p16 mRNA accumulation was determined on a Phospholmager (Molecular Dynamics, ImageQuant software version 3.3). B, analysis of CDK4, CDK6, and p16 immunocomplexes. [35SlMethionine-labeledcell lysates were preparedfrom parentalIMR-90 cells and IMR-90 cells infected with retroviral vectorscontainingthetype16HPVE6(IMR-90/E6),E7(IMR.90/E7),orE6plusE7(IMR-90/E6+E7).Labeledcelllysateswereimmunoprecipitatedwithantiseraspecific to CDK4 (5), CDK6 (21), and p16 (10) with or without prior incubation with a competing antigen peptide as indicated at the top of each lane and resolved by SDS-PAGE. Left ordinate, relative position of proteins with established identities. Cyc DI, cyclin Dl.

known. One of the obvious genetic differences between these two beled cells with antibodies specific to CDK4, CDK6, or p16 (Fig. 2B). osteosarcoma cell lines is their pRb status. While an apparently They furtherdemonstrate that expression of either E6 or E7 oncoprotein normal plO5―@'protein was readily detectable in U-2 OS cells, it was leads to an increased level of p16 mRNA, with E7 being a more potent not detected in Saos-2 cells, apparently due to a deletion in stimulus indicating that both p53 and pRb may negatively regulate the 21—27 of the RB] gene (Ref. 22; Fig. 1A). expression of p16 mRNA. Subsequent experiments therefore were fo Similarly, when total RNA was prepared from normal human cused on the regulation of the level of p16 mRNA by pRb. As a fibroblasts (HSF43) and their SV4O large T antigen-transformed de comparison,the same blot was also hybridizedwith a probe derived from rivative (CF1O) and hybridized with the p16 probe, a 10-fold increase human p21 CDK inhibitor cDNA. While expression of 16E7 protein had in the steady-state level of p16 mRNA was observed in the trans little effect, expression of 16E6 (and 16E6 together with 16E7) dramat formed CT1O cells (Fig. 2A). The same result was also obtained with ically reduced the level of p21 mRNA (Fig. 2A) that is also correlated several additional pairs of normal human fibroblasts and their virally closely with a reduced level ofp2l protein in these cells (data not shown). transformed derivatives (e.g., WI-38 versus VA13; data not shown). These results are entirely consistent with the previous finding that the These findings are consistent with the previously observed higher expression of p21 mRNA is transcriptionallyactivated by p53 (6, 9). level of p16 protein associated with CDK4 and CDK6 in virally We next investigated whether transcriptional activation of p16 transformed cells (5, 10).@Taken together the data suggest that inac contributed to the higher level of p16 mRNA seen in virally trans tivation of function of pRb and/or p53 activates the transcription formed or Rb-deficient cells. Genomic fragments containing p16 and/or induces the stabilization of p16 mRNA. sequences were isolated from a human placenta genomic library and To distinguish which tumor suppressor,pRb or p53, regulates the level a 1.1-kilobase genomic fragment corresponding to the 5' upstream of p16 mRNA, we analyzed the level of p16 mRNA in normal human region of p16 cDNA was subcloned into a promoterless luciferase fibroblast IMR-90 cells and IMR-90 cells expressing type 16 papilloma expression plasmid (jGL2-Basic). The resultant plasmid, p16-Luc, viral oncoprotein E6 (16E6), E7 (16E7), or E6 together with E7 (19). gave rise to considerable luciferase activity as compared to the pa 16E6 and 16E7 bind to and inactivate the functions of p53 and pRb, rental promoterless luciferase construct pGL2-Basic when it was respectively (23, 24). Compared to the parental IMR-90 cells, the level of transiently expressed in several cell lines including Saos-2 and U-2 p16 mRNA was increased by 2.7-fold in IMR-90 cells expressing 16E6, Os (Fig. 3A). To allow comparisonof luciferaseactivity in cells 4.2-fold in IMR-90 cells expressing 16E7, and 4.7-fold in IMR-90 cells transfected with p16-Luc, an SV4O-Luc plasmid was transiently expressing both 16E6 and 16E7 (Fig. 2A). The increased level of p16 expressed in parallel as a control for normalizing the transfection mRNA in cells expressing viral oncoproteins also correlates closely with efficiency between the different cells. In five independent expen an increased level ofpl6 protein and its increased association with CDK4 ments, we have reproducibly observed an average of 10-fold higher and CDK6 as determined by immunoprecipitation in metabolically la luciferase activity in Saos-2 cells than in U-2 OS cells (Fig. 3A), indicating that the higher level of p16 mRNA seen in Saos-2 cells was

4 Unpublished observations. most likely the result of transcriptional activation ofthe p16 promoter. 6080

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Finally, to determine whether pRb can directly repress transcription through the p16 promoter, the p16-Luc reporter plasmid was cotrans fected into Saos-2 cells with wild-type pRb cDNA under the control of the strong immediate-early promoter of CMV [pCMV-Rb (25)]. Ectopic expression of pRb in these Rb-deficient cells reproducibly resulted in an average 40% reduction in luciferase activity from the p16 promoter (Fig. 3B), but had no detectable effect on the SV4O @(@@DK4,6

A e p16 Fig. 4. Schematic presentation of a feedback regulatory loop showing transcriptional I repression of p16 by pRb. A (TF) such as bound to the hypophosphorylated pRb is released after the phosphorylation of pRb by the cyclin D-associated CDK4 or CDK6 and activates the transcription of p16. The increasing amount of p16 protein binds to the catalytic subunit CDK4 and CDK6 and dissociates the c.ll Ua. Sao..2 Ssa-2 S.s.-2 U.2os tJ-2OS U-2OS complexes, thus inhibiting the activity of cyclin D (Cyc D)-CDK4 and CDK6 enzymes. DNA [email protected]@ p16.L@ic ptt-Lac pGU-0@c pt6-Luc p16-too (2i@) (110) (2pm) (2@) (1j@) (2w) promoter (data not shown). Similar results were also obtained in separate experiments using mouse NIH 3T3 cells. Cotransfection of B the p16-Luc plasmid with an increasing amount of pCMV-pRb re peatedly led to a progressive reduction ofluciferase activity up to 40% of the full activity from the p16 promoter (Fig. 3C). These results provide direct evidence for the repression ofthe p16 promoter by pRb. We have been consistently obtaining a maximal 40% reduction of luciferase activity in cotransfection with pCMV-pRb in different cell @ I lines, suggesting that an additional factor(s) that is not controlled by pRb may also regulate the expression of p16. A number of cellular transcriptional factors such as -1 have been identified as the functional targets of pRb (reviewed in Refs. 17 and 26). We have determined more than 3 kilobases of nucleotide upstream sequences p16.L.e(p@) 1 1 I t I pcMv.fts (p5) 0 0.1 0.2 0.5 I of the p!6 gene including the 1.6-kilobase region used in the trans fection assay and found no apparent E2F binding site (26) or conven tional TATA sequences.5 Whether the pitS gene contains a weak E2F C binding site or pRb represses pitS transcription by a mechanism independent of E2F factors are currently under investigation. The function of pRb is known to be down-regulated by cell cycle dependent phosphorylation and both D-type cyclins and their associ A 4 ated kinases (primarily CDK4 and CDK6) have been strongly impli I cated as physiological pRb kinases (see two most recent reviews in ‘a I Refs. 17 and 18). Biochemical analyses have indicated that p16 acts as -I an inhibitor of CDK4 [and likely CDK6 as well (10)]. Our results demonstrate that transcription of p16 is repressed by pRb, providing evidence for a regulatory feedback loop involving pRb, CDKS, p16, and an as yet unidentified transcription factor (Fig. 4). In this model,

plS-L.elMS I I I 1 I phosphorylation of pRb by cyclin D-CDK4 or cyclin D-CDK6 results PCMV-51I8SI 0 0.1 02 0.4 I in the release of a transcription factor (e.g., E2F-1) which is inactive Fig. 3. Transcriptional repression of the p16 promoter by pRb. A, genomic fragments when bound to hypophosphorylated Rb. Dissociation of the pRB containing p16 were isolated from a human placenta genomic library using p16 cDNA as a probeanda 1.1-kilobasegenomicfragmentcorrespondingtothe5' upstreamregionof transcription factor complex would then activate p16 transcription. As p16 eDNA was subcloned into a promoterless luciferase expression plasmid levels of p16 mRNA increase, increased binding of p16 to CDK4 and [pGL2-Baskj. The resultant plasmid, p16-Luc, and parental pGL2-Basic DNA were CDK6 would result in inhibition of the cyclin D-CDK4 and cyclin transiently expressed in both human osteosarcoma Saos-2 and U-2 OS cells. Luciferase activities were measured as relative light units 28 h after transfection. The relative D-CDK6 kinase activities. Ultimately, phosphorylation of pRb would luciferase activity of each transfection was calculated by dividing individual light emis decrease, as would the level of p16 expression. This model is con sion values with the values obtained from a parallel transfection with pSV4O-Luc DNA to sistent with, and provides a plausible explanation for, the absence of normalize the transfection efficiency between two cell populations. Bars, SD of three separate readings of triplicate transfections in a single experiment. B. ectopic expression cyclin D proteins and cyclin D-CDK complexes in cells lacking pRb of pRbsuppressesthep16promoteractivity.Thep16-Lucreporterplasmidwascotrans (27, 28). A high level of p16 in cells lacking pRb function may lead fected with the wild type pRb cDNA under the control of a strong immediate early promoter of CMV [pCMV-Rb (25)] into the Rb-deficient Saos-2 cells. C, repression of p16 promoter activity by the ectopic expression of pCMV-RB in mouse NIH3T3 cells. 3 M. A. Nichols and Y. Xiong, unpublished observations. 6081

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. TRANSCRIPTIONAL REPRESSION OF p16 BY pRb to dissociation of cyclin D from CDK complexes and subsequent potentially involved in genesis of many tumor types. Science (Washington DC), 264: degradation. In addition, an increased level of p16 mRNA in cells 436—440,1994. 12. Nobori, T., Miura, K., Wu, D. J., Lois, A., Takabayashi, K., and Carson, D. A. lacking p53 function [IMR-90 cells expressing 16E6 (Fig. 14)] sug Deletion ofthe cyclin-dependent kinase-4 inhibitor gene in multiple human . gests that the expression of p16 may also be regulated negatively by Nature (Land.), 368: 753—756,1994. p53. Our results also provide a potential pathway by which the 13. Mori, T., Miura, K., Aoki, T., Nishihira, T., Show, N., and Nakamura, Y. Frequent somatic mutation of the MTS1/CDK4I (multiple tumor suppressor/cyclin-dependent expression of p16 is regulated by p53. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. Transcriptional Repression of the D-Type Cyclin-dependent Kinase Inhibitor p16 by the Retinoblastoma Susceptibility Gene Product pRb

Yan Li, Michael A. Nichols, Jerry W. Shay, et al.

Cancer Res 1994;54:6078-6082.

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