[CANCER RESEARCH 59, 4984–4989, October 1, 1999]

Kaposi’s Sarcoma-associated Herpesvirus-encoded v-Cyclin Triggers Apoptosis inCells with High Levels of Cyclin-dependent Kinase 61

Paivi M. Ojala,2 Marianne Tiainen, Petri Salven, Tanja Veikkola, Esmeralda Castanos-Velez, Ronit Sarid,3

Peter Biberfeld, and Tomi P. MakelaCell Cycle Laboratory [P. M. O., M. T., T. P. M.] and Molecular/Cancer Biology Laboratory [T. V.], Haartman Institute, 00014 University of Helsinki, Helsinki, Finland;Department of Oncology, University of Helsinki, and Helsinki University Central Hospital, 00029 HYKS, Helsinki, Finland [P. S.]; Immunopathology Laboratory, Institute forPathology and Oncology, Karolinska Institute/Hospital, S-171 76 Stockholm, Sweden [E. C-V., P. B.]; and Department of Pathology and Division of Epidemiology, ColumbiaUniversity College of Physicians and Surgeons, New York, New York 10032 [R. S.]

ABSTRACT

Kaposi’s sarcoma-associated herpesvirus (KSHV) has a key etiologicalrole in development of Kaposi’s sarcoma (KS). v-Cyclin is a KSHV-encoded homologue to D-type cyclins that associates with cellular cyclin-dependent kinase 6 (CDK6). v-Cyclin promotes S-phase entry of quiescentcells and has been suggested to execute functions of both D- and E-typecyclins. In this study, expression of v-cyclin in cells with elevated levels ofCDK6 led to apoptotic cell death after the cells entered S phase. The celldeath required the kinase activity of CDK6 because cells expressing akinase-deficient form of CDK6 did not undergo apoptosis upon v-cyclinexpression. Studies on the mechanisms involved in this caspase-3-medi-ated apoptosis indicated that it was independent of cellular p53 or pRbstatus, and it was not suppressed by Bcl-2. In contrast, the KSHV-encodedv-Bcl-2 efficiently suppressed v-cyclin-/CDK6-induced apoptosis, demon-strating a marked difference in the antiapoptotic properties of c-Bcl-2 andv-Bcl-2. In KS lesions, high CDK6 expression was confined to a subset ofcells, some of which displayed signs of apoptosis. These results suggest thatv-cyclin may exert both growth-promoting and apoptotic functions in KS,depending on factors regulating CDK6 and v-Bcl-2 levels.

INTRODUCTION

Several oncogenic viruses encode both growth-promoting and an-tiapoptotic proteins with a potential role in tumor induction (reviewedin Ref. 1). The KSHV4/human herpesvirus 8 (HHV8) (2) is consis-tently found in spindle cells of all forms of KS lesions, suggesting akey etiological role in the development of KS (reviewed in Ref. 3).The majority of the KS spindle cells are latently infected with KSHV(4–6), but lytic infection is detected only in a small fraction (0.5–1%)of cells (6, 7). The KSHV genome contains homologues to severalcellular genes, includingbcl-2, FLIP, and cyclin D, which maymodulate apoptosis and cell proliferation (reviewed in Ref. 8).

The KSHV-encoded v-cyclin (9), also termed ORF72, vCYC,KSHV-cyclin, v-cyclin D, and K-cyclin (8), is expressed both duringlatency and the lytic viral replication cycle (10, 11). It is closelyrelated to cellular D-type cyclins, which regulate progression throughthe G1 phase of the cell cycle in complex with CDK4 or CDK6(reviewed in Ref. 12). The primary function of cyclin D complexes isapparently inactivation of the retinoblastoma protein (pRb). In malig-

nancies, cell cycle progression is often deregulated by mutations inthese pRb G1 checkpoint pathway genes.

v-Cyclin associates specifically with CDK6 to form a functionalkinase complex when it is transfected in COS or U2OS cells (13–16)and also in insect cell lysates (13–17). Intriguingly, thein vitrosubstrate specificity of the v-cyclin/CDK6 complex is extended fromcellular cyclin D/CDK6 complexes and includes not only pRb (glu-tathioneS-transferase-pRb) but also histone H1, cdc25a, Id-2, and p27(13–17). Ectopic expression of v-cyclin induces several growth-pro-moting effects, including pRb phosphorylation (9); evasion of a G1

arrest by the cellular CDK inhibitors p16, p21, and p27 (17); andinduction of S phase entry in quiescent NIH 3T3 cells (17). Theevasion of p27 arrest is mediated by phosphorylation of p27 by thev-cyclin/CDK6 complex, which triggers p27 degradation (15, 16).

v-Cyclin can apparently overcome normal cell cycle control mech-anisms by executing functions of both D and E cyclin complexes and,therefore, could be implicated in oncogenic properties of KSHV (18).Because v-cyclin apparently requires cellular CDK6 to exert thesefunctions, here we investigated the role of CDK6 in v-cyclin inducedderegulation of cell cycle progression. Expression of v-cyclin in cellswith elevated levels of CDK6 accelerated entry into S phase but alsoled to apoptotic cell death, suggesting that v-cyclin may exert bothgrowth-promoting and apoptotic functions in KS.

MATERIALS AND METHODS

Expression Vectors.The previously described expression vectors used inthis study were: Rc-cycD1-HA (19), pCMV-CDK6-HA and pCMV-CDK4-HA (20), pSG5-v-Bcl-2-HA (Ref. 21; kindly provided by Dr. J. M.Hardwick Johns Hopkins University, Baltimore, MD), pCMV-HA-E2F-1 (22),and pCMV-HA-DP-1 (23).

Nontagged versions of CDK6, CDK6DN, and c-Bcl-2 were expressed frompCMV-CDK6, pCMV-CDK6DN, and pcDNA3-Bcl-2 (Ref. 24; kindly pro-vided by Dr. L. C. Andersson Haartman Institute, University of Helsinki,Finland) respectively.

The cDNA of a myc-tagged viral cyclin (kindly provided by Drs. R. Saridand P. Moore, Columbia University, NY, NY), was subcloned into pCI-neo(Promega, Madison, WI). The viral FLIP cDNA (kindly provided by Dr. ChrisBoshoff, Institute Cancer Research, London, United Kingdom) sequence con-tained a T3C difference to the published sequence at position 20 (GenBankaccession no. U90534; Ref. 25). Following PCR mutagenesis to change thisposition to a T, the cDNA was subcloned into pCI-neo with an NH2-terminalHA tag (pAHC-v-FLIP).

Antibodies and Reagents.Mouse monoclonal antibodies recognizing themyc epitope (9E10) or HA epitope (12CA5) were from Babco Inc. (Berkeley,CA), a-CDK6 rabbit polyclonal (C-21) was from Santa Cruz BiotechnologyInc. (Santa Cruz, CA),a-b-galactosidase rabbit polyclonal serum was fromChemicon International Inc. (Temecula, CA), anda-Bcl-2 mouse monoclonalantibody was from PharMingen (San Diego, CA). Bisbenzimide Hoechst33342 was from Sigma Chemical Co. (St. Louis, MO), and staurosporine,Ac-DEVD-pNA, colorimetric CPP32 substrate, and DEVD-CHO CPP32 in-hibitor were all products of Biomol Research Laboratories, Inc. (PlymouthMeeting, PA).

Cell Culture and Transfections. Human osteosarcoma cell lines U2OSand SaOS-2, mouse myoblast cells C2C12, human embryonic 293 cells, and

Received 4/8/99; accepted 8/4/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the Academy of Finland, the University ofHelsinki, the Finnish Cancer Society, the Finnish Cancer Institute, the Sigrid JuseliusFoundation, and the BIOMED-2 Concerted Action on the “Pathogenesis of AIDS Kapo-si’s Sarcoma” (Contract BMH4-97-2302).

2 To whom requests for reprints should be addressed, at Haartman Institute, P.O. Box21, 00014 University of Helsinki, Finland. Phone: 358 9 191 26439; Fax: 358 9 19126700; E-mail: Paivi.Ojala@helsinki.fi.

3 Present address: Faculty of Life Sciences, Bar Ilan University, Ramat-Gan 52900,Israel.

4 The abbreviations used are: KSHV, Kaposi’s sarcoma-associated herpesvirus; KS,Kaposi’s sarcoma; CDK, cyclin-dependent kinase; HA, hemagglutinin; BrdUrd, 5-bromo-29-deoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end la-beling.

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COS-7 were maintained in DMEM supplied with 10% (w/v) FCS, 100 units/mlpenicillin, 100mg/ml streptomycin, and 2 mM glutamine. Transient transfec-tions were performed using calcium phosphate coprecipitation according tostandard protocols (26) with a total DNA amount of 40mg including 2.5mg ofpCMV-b-gal DNA. DNA precipitates were washed at 20 h, and the cells wereplaced in fresh medium. Cells were analyzed 12–52 h later. Transfectionefficiency was monitored byb-galactosidase staining or activity assay by usingchlorophenol red-b-D-galactopyranoside (Roche, Basel, Switzerland) as thesubstrate according to manufacturer and measured by absorbance at 574 nm.Transfection efficiency was between 10 and 30%.

Western Blotting. The cells were lysed into 1% NP40 lysis buffer [20 mM

NaPO4 (pH 7.4), 1% NP-40, 250 mM NaCl, 5 mM EDTA, 5 mM DTT, 1 mM

phenylmethylsulfonyl fluoride, 2mg/ml leupeptin, and 1.5mg/ml aprotinin].Forty mg of total proteins were analyzed by 12% SDS-PAGE and blottedaccording to standard protocols.

Indirect Immunofluorescence and Immunohistochemistry. Transfectedcells on coverslips were fixed with 3.5% (w/v) paraformaldehyde, permeabi-

lized with 0.1% TX-100 for 5 min, and labeled as described elsewhere (27).DNA was stained with Hoechst 33342 (0.5mg/ml) for 5 min, and the cover-slips were mounted in 50% glycerol in PBS on glass slides and evaluated undera fluorescence microscope. Tissue sections from two paraffin-embedded andtwo frozen, acetone-fixed KS lesions were analyzed by immunohistochemistryessentially as described previously (28) using a rabbit polyclonala-CDK6antibody (C-21; Santa Cruz Biotechnology).

BrdUrd Assay. At desired times after transfection, 30mM BrdUrd (Sigma)was added in the growth medium and incubated for 2 (see Fig. 1A) or 6 h (seeFig. 1B). The cells were fixed with 99% (w/v) ethanol plus 5% (w/v) aceticacid for 20 min and treated with 2M HCl for 10 min. Subsequently incorpo-rated BrdUrd was detected with a monoclonala-BrdUrd (DAKO, Glostrup,Denmark) in a double labeling witha-b-Gal (see above). At least 200 trans-fected (b-galactosidase positive) cells were scored for BrdUrd incorporation.

Apoptosis Assays.For the quantitative apoptosis assay, percentages ofnuclei that displayed apoptotic morphology in Hoechst-staining were deter-mined visually, and the maximal apoptosis (obtained in the v-cyclin-/CDK6-expressing cells) was given a value of 100 (see Fig. 3C); other values arerelative to this. At least 500 nuclei were scored in a double-blind count forapoptotic morphology. In the case in which apoptosis of transfected cells wasdetermined (see Fig. 4), at least 200 transfected cells were scored. For theCPP32 assay, 52 h after the removal of the DNA precipitates, the detached andadherent cells were pooled and used for the CPP32 assays essentially asdescribed previously (29) using 60mM of the CPP32 inhibitor DEVD-CHO inindicated experiments. TUNEL was performed on frozen, paraformaldehyde-fixed KS sections to detect apoptotic cells using anin situ cell death detectionkit according to the manufacturer’s instructions (Roche).

RESULTS

Coexpression of CDK6 Enhances v-Cyclin-induced S-Phase En-try. To analyze whether high levels of CDK6 would affect v-cyclininduced cell cycle promotion, we analyzed S-phase entry of U2OScells transfected with the v-cyclin alone or v-cyclin with CDK6 byBrdUrd incorporation (Fig. 1A). In accordance with previous studies,v-cyclin expression alone promoted S-phase entry of cycling U2OScells (17), presumably by shortening G1. v-cyclin was found to bemore efficient than cyclin D1 in this function (Fig. 1A). Becausev-cyclin associates specifically with CDK6, the increase in S-phasecells was presumed to result from v-cyclin associating with limitingamounts of endogenous CDK6 in U2OS cells (Fig. 1C; Ref. 30).Accordingly, ectopic CDK6 expression significantly increased thenumber of BrdUrd-positive cells when transfected together with eitherv-cyclin or cyclin D1, but the effect of CDK6 on v-cyclin was slightlymore efficient (Fig. 1A). These results indicate that both v-cyclin andcyclin D1 are capable of promoting S-phase entry when complexedwith CDK6.

Fig. 1. Induction of S phase by the v-cyclin/CDK6 complex.A, fractions of BrdUrd-positive U2OS cells transfected with the expression vectors indicated on therightanalyzed at 14 h posttransfection. The BrdUrd pulse was for the last 2 h prior to fixation.Columns, means of triplicate samples;bars, SD.B, U2OS cells transfected with v-cyclin(v-cyc) or v-cyclin and CDK6 (v-cyc1 CDK6) were analyzed at 28 h posttransfection byimmunofluorescence with anti-BrdUrd staining (left). The BrdUrd pulse was for 6 h at10–16 h posttransfection. DNA was stained with Hoechst 33342 (middle).Right, phasecontrast micrographs.Arrows, cells with altered morphology.C, Western blotting analysisof U2OS cells at 28 h posttransfection. Cells were transfected with v-cyclin alone,v-cyclin and CDK6, or cyclinD1 and CDK6. Total lysates of transfected cells wereresolved by SDS-PAGE (12%) and immunoblotted witha-CDK6 antibodies.

Fig. 2. Cells expressing both v-cyclin and CDK6 undergo apoptosis. Caspase-3(CPP32) activity from U2OS cell lysates transfected with expression vectors indicated onthe right was determined based on the ability to cleave Ac-DEVD-pNA. DEVD-CHOpeptide at 60mM was used as a specificity control in the indicated samples.Columns,means of triplicate samples;bars, SD.

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Expression of v-Cyclin in Cells with High Levels of CDK6Triggers Apoptosis. A significant number of BrdUrd-positive cellsin the v-cyclin/CDK6 transfection displayed striking morphologicalalterations consistent with apoptosis (Fig. 1B). Induction of apoptosiswas substantiated by an increased caspase-3 (CPP32) activity inv-cyclin-/CDK6-transfected cell lysates (Fig. 2) and TUNEL staining(data not shown). The caspase-3 activity was comparable with stau-rosporine-induced apoptosis (31, 32) of the U2OS cells (Fig. 2) andwas inhibited by the specific peptide inhibitor, DEVD-CHO. Induc-

tion of CPP32 protease activity is a key early event in apoptosis (33),implying that the v-cyclin-/CDK6-complex activates a common mam-malian cell death pathway. The cell morphology and the activation ofCPP32 indicate that ectopic expression of v-cyclin with CDK6 inU2OS cells results in apoptosis after S-phase entry.

v-Cyclin-induced Apoptosis Is Dependent on CDK6 KinaseActivity. Apoptosis of v-cyclin-/CDK6-transfected cells increasedwith time following transfection. It was first detected 12 h posttrans-fection concomitantly with detectable v-cyclin expression (data notshown), and at 52 h, the chromatin of virtually all v-cyclin/CDK6double-positive cells appeared apoptotic with a single bright sphericalclump (Fig. 3A, arrowhead) or as several condensed fragments(Fig. 3A,arrow).

v-Cyclin-mediated cell death was clearly dependent on high levelsof CDK6 (Fig. 1C) because it was not detected when v-cyclin wastransfected alone (Fig. 3B). In contrast, virtually all v-cyclin-positivecells in the v-cyclin/CDK6 transfection were apoptotic, suggestingthat even low levels of v-cyclin drive cells with an excess of CDK6 toapoptosis. The apoptosis was specific for the viral cyclin D homo-logue because it was not detected in cells transfected with cyclinD1/CDK6 (Fig. 3,B andC).

The inability of a kinase-deficient mutant of CDK6 (CDK6DN;Ref. 20) to induce apoptosis in combination with v-cyclin (Fig. 3,BandC) indicates that the kinase activity of CDK6 was required. WhenCDK4 was cotransfected with v-cyclin, a slight but reproducibleincrease in the proportion of apoptotic cells was observed (Fig. 3C),suggesting that CDK4 also did form functional complexes with v-cyclin when overexpressed in U2OS cells but markedly less effi-ciently than CDK6, in accordance with previous studies (13, 14).

Fig. 3. v-cyclin induced apoptosis is dependent on kinase activity of CDK6.A, U2OScells cotransfected with Myc-v-cyclin and CDK6 expression vectors were analyzed bydouble-label immunofluorescence witha-Myc or a-CDK6 antibodies at 52 h posttrans-fection. DNA was visualized by Hoechst staining (right). Apoptotic cells appeared as asingle bright spherical bead (arrowhead) or as several fragments (arrow). B, U2OS cellstransfected with expression vectors indicated on the left side were analyzed by double-label immunofluorescence as inA with antibodies shown in the panels at 35 h posttrans-fection.C, quantitation of apoptosis from U2OS cells transfected with plasmids indicatedon the right at 35 h posttransfection, as detailed in “Materials and Methods.”Columns,means of triplicate samples;bars, SD.

Fig. 4. v-Cyclin/CDK6 apoptosis occurs in high serum and is p53 and pRb independ-ent.A, percentages of apoptotic cells of U2OS or C2C12 mouse myoblast cells cotrans-fected with v-cyclin and CDK6 plasmids (v-cyc1 CDK6;f), E2F-1 and DP-1 plasmids(M), or an empty vector (o) in low (0.5%) or high (10%) FCS.B, SaOS-2 osteosarcomacells with mutant p53 and pRb cotransfected with Myc-v-cyclin and CDK6 expressionvectors were analyzed for expression of Myc-tagged v-cyclin (a-Myc), CDK6 (a-CDK6),and apoptotic morphology by Hoechst staining (Hoechst). Condensed and fragmentednuclei were limited to v-cyclin-/CDK6-positive cells.

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v-Cyclin-/CDK6-mediated Apoptosis Occurs in High Serumand Is p53 and pRb Independent.Apoptosis resulting from dereg-ulated expression of E1A or Myc only occurs when E1A or Myc isinduced in the presence of limiting growth factors (34, 35). On theother hand, expression of E2F-1, especially together with DP-1 (36),induces apoptosis, even in the presence of a full complement of serumgrowth factors, although less efficiently (37, 38). When cell deathinduced by v-cyclin/CDK6 was compared with E2F-1/DP-1 apopto-sis, cell death by v-cyclin/CDK6 was significantly higher in bothU2OS and C2C12 mouse myoblast cells (Fig. 4A). Moreover, apop-tosis induced by v-cyclin/CDK6 in U2OS cells was unaffected byserum levels, although a slight decrease in apoptosis was seen inC2C12 cells at high serum concentration (Fig. 4A, filled bars).

To address the role of pRb and p53 in the v-cyclin/CDK6-inducedapoptosis, we tested SaOS-2 osteosarcoma cells (with mutant p53 andpRb), COS-7 cells (p53 and pRb inactivated by the SV40 largeT-antigen), and C2C12 mouse myoblasts (wild-type p53 and pRb asin U2OS cells). We also transfected human embryonic 293 cells and,

as in all of the above cell types, the active complex of v-cyclin andCDK6 induced cell death (shown for SaOS-2 in Fig. 4B), whereasv-cyclin/CDK6DN or cyclinD1/CDK6 did not. Thus, apoptosis trig-gered by v-cyclin and CDK6 occurs in various cell types, independentof either p53 or pRb status.

Antiapoptotic Activity of v-Bcl-2 in v-Cyclin-/CDK6-inducedApoptosis. To test for the ability of v-Bcl-2 and v-FLIP as well ascellular Bcl-2 to protect U2OS cells from the v-cyclin/CDK6-inducedapoptosis, we included HA-taggedv-bcl-2,bcl-2, or v-FLIPin trans-fections with v-cyclin/CDK6. The expression of c-Bcl-2 and v-Bcl-2was confirmed by Western blotting analysis usinga-Bcl-2 anda-HAantibodies, respectively (Fig. 5C). Double labeling witha-HA anda-CDK6 antibodies identified cells coexpressing v-Bcl-2 and CDK6(Fig. 5A). Parallel experiments demonstrated that.80% of cellsexpressing CKD6 also contained v-cyclin at levels similar to thoseshown in Fig. 3A. Most double-positive cells did not display anapoptotic morphology (Fig. 5A, arrowheads) demonstrating that ex-pression of v-Bcl-2 suppressed apoptosis by v-cyclin/CDK6 signifi-cantly (quantitated in Fig. 5B), although some apoptotic cells werealso seen (Fig. 5A,arrow). Interestingly, transfection of c-bcl-2orHA-v-FLIP constructs did not inhibit v-cyclin/CDK6 apoptosis

Fig. 5. Suppression of v-cyclin-/CDK6-induced cell death.A, v-cyclin/CDK6 apoptosisis suppressed by v-Bcl-2. U2OS cells transfected with v-cyclin/CDK6 and HA-v-bcl-2were analyzed 35 h posttransfection using double immunofluorescence witha-HA anda-CDK6 antibodies.Arrowheads, double-positive cells not displaying an apoptotic mor-phology; arrow, an apoptotic, CDK6-positive cell.B, v-cyclin/CDK6 apoptosis is notsuppressed by coexpression ofbcl-2 or v-FLIP plasmids. Quantitation of apoptosis ofindicated transfections performed as inA. Columns, means of triplicate samples;bars, SD.C, Western blotting analysis of U2OS cells at 35 h posttransfection. Cells were transfectedwith an empty vector (vector),c-bcl-2, or HA-v-bcl-2. Total lysates of transfected cellswere resolved by SDS-PAGE (12%) and immunoblotted witha-Bcl-2 anda-HA anti-bodies.

Fig. 6. A subset of Kaposi’s sarcoma cells with high CDK6 expression displaymorphological signs of apoptosis.A, a-CDK6 immunostaining of a section from a KSlesion demonstrating reactivity in the tumor area but not the surrounding tissue (bottomright of micrograph; original magnification,320).B, a section adjacent to the one shownin A was stained as a control by omitting the primary antibody. Original magnification,320.C, a-CDK6 immunostaining of a section from a KS lesion demonstrating a low butspecific overall expression. Several cells with high levels of CDK6 display signs ofapoptosis (arrows), but morphologically unaltered cells can also be found (arrowhead).Original magnification,3400. D, TUNEL staining showing apoptotic cells in the KSlesion stained inC andE. Arrows, TUNEL-positive cells. Original magnification,3400.E, staining as inC from a separate field of the KS lesion at a higher magnification.Arrow,a cell with high CDK6 associated with morphological signs of apoptosis. Originalmagnification,31000.F, staining as inD from a separate field of the KS lesion at a highermagnification.Arrow, a TUNEL-positive, apoptotic cell. Original magnification,31000.

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(Fig. 5B), indicating that v-cyclin/CDK6 apoptosis is specificallysuppressed by the KSHV-encoded v-Bcl-2.

A Subset of KS Cells with High CDK6 Expression DisplayMorphological Signs of Apoptosis.The cell death mediated byv-cyclin/CDK6 required high levels of CDK6. To investigate CDK6expression in KS lesions, sections from four KSHV-positive KSnodules were immunostained for CDK6. A weak but specific overallCDK6 staining was observed in KS lesions (Fig. 6,A andC), but notin the surrounding uninvolved tissue (Fig. 6A). Moreover, a subset ofcells in the lesion (;1%) expressed high levels of CDK6 (Fig. 6C),comparable with that seen in cells transfected with CDK6 (data notshown). The high CDK6 signal was most prominent in highly vascu-larized areas of the lesion containing infiltrating lymphoid cells.Interestingly, a significant proportion of high CDK6 expressing cellsdisplayed morphological signs of apoptosis (Fig. 6,C andE, arrows).TUNEL staining of the same lesion indicated that apoptotic cells (Fig.6, D andF, arrows) were also mostly confined to the highly vascu-larized areas containing infiltrating lymphoid cells as reported.5 Theseresults suggest that high CDK6 expression in a subset of cells in KSlesions is associated with apoptosis.

DISCUSSION

These results demonstrate that the efficiency of KSHV v-cyclin topromote S-phase entry is dependent on CDK6 levels. CDK6 alsoenhanced the ability of cyclin D1 to promote S phase, and indeed,v-cyclin was indistinguishable from cyclin D1 in this respect. Incontrast, only cells expressing v-cyclin/CDK6 underwent massiveapoptosis, which was never detected in cyclin D1 transfections, dem-onstrating clearly distinct functions for these cyclins and suggestingthat the v-cyclin/CDK6 complex deregulates the cell cycle by takingover functions of both D- and E-type cyclin complexes (17).

The difference between v-cyclin and cyclin D1 in inducing apop-tosis in our system is unlikely to be due to different expression levelsbecause v-cyclin was expressed at low levels and was barely detect-able in Western blots (data not shown), whereas cyclin D1 wasproduced at high levels. Another possibility for the inability of cyclinD1/CDK6 complexes to induce apoptosis would be inhibiting levelsof CDK inhibitors such as p21 and p27, which would not affectv-cyclin/CDK6 complexes (15–17). This would imply that endoge-nous levels of these inhibitors would be sufficient to inhibit thetransfected cyclin D1/CDK6 complexes. Previously, cyclin D1 hasbeen reported to induce apoptosis in certain cell lines (39) and interminally differentiated neurons (40). The apparent discrepancy ofthese reports and our results could be due to cell type specificity andalso to the observation that cyclin D1-induced apoptosis generallyrequires limiting growth factors, unlike v-cyclin-/CDK6-inducedapoptosis.

Cell death by v-cyclin/CDK6 was, in several ways, similar toapoptosis induced by E2F-1 (37, 41). It occurred after S-phase entry,suggesting that the v-cyclin/CDK6 complex drives the cells into Sphase inappropriately, and this could initiate the apoptotic signal. Itwas also insensitive to genetic backgrounds (p53,pRb), indicating thatit is at least partly mediated via a p19ARF-p53 independent pathwaysimilarly to E2F-1 (38, 42, 43). The occurrence of cell death inRb-negative cells, together with the requirement of CDK6 kinaseactivity, indicates that the substrate of v-cyclin/CDK6 critical forapoptosis was other than pRb.

The only factor that countered apoptosis by v-cyclin/CDK6 was theKSHV-derived v-Bcl-2; cellular Bcl-2 did not. This suggests that

either this apoptosis is independent of Bcl-2 and on a differentpathway (reviewed in Ref. 44) or that v-cyclin/CDK6 can selectivelyinactivate cellular Bcl-2. The antiapoptotic activity of Bcl-2 andv-Bcl-2 was indistinguishable in other systems,6 supporting the lattermodel. In this model, v-Bcl-2 would be insensitive to this inactivation.

The role of apoptosis in KSHV infection was recently demonstratedin vitro in 293 cells using viral isolates from primary KS lesions (45).Interestingly, this apoptosis was also protected by v-Bcl-2 similarly tothe v-cyclin-/CDK6-induced apoptosis. Apoptosis in KS lesions islimited predominantly to tumor-infiltrating lymphoid cells.5

Previous studies implicating CDK6 as the partner for v-cyclin(13–17) have not investigated the expression levels of CDK6 in KSlesions. This study in a limited material indicates that CDK6 isdetected in KS spindle cells throughout the lesion. Interestingly, someof the predominantly lymphoid cells with high CDK6 expressiondisplayed morphological signs of apoptosis. Using TUNEL staining,we detected apoptosis in KS lesions in similar cells, as have others.5

Moreover, lymphoid cells in KS lesions express KSHV v-cyclin (46)and lytic markers of KSHV (6, 7). Taken together, these resultsindicate that v-cyclin may elicit either growth promoting or apoptoticsignals in KS, depending on factors in the cellular microenvironmentregulating CDK6 and v-Bcl-2 levels.

ACKNOWLEDGMENTS

We thank Drs. Patrick Moore and Yuan Chang, Marie Hardwick, ChrisBoshoff, Erwin Tschachler, and Paivi Miettinen for reagents; Marja Ja¨attelaand Kari Alitalo for fruitful discussions and critical reading of this manuscript;Leif Andersson and the Makela laboratory for many helpful suggestions;Birgitta Tjader for excellent technical assistance; and Antti Huittinen fordigital imaging.

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1999;59:4984-4989. Cancer Res   Päivi M. Ojala, Marianne Tiainen, Petri Salven, et al.   Cyclin-dependent Kinase 6Triggers Apoptosis in Cells with High Levels of Kaposi's Sarcoma-associated Herpesvirus-encoded v-Cyclin

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