Vol. 9, 639-650, August 1998 Cell Growth & Differentiation 639

Differential Modulation of G1-S-Phase Cyclin-dependentKinase 2/Cyclin Complexes Occurs during theAcquisition of a Polyploid DNA Content1

Nabanita S. Datta, J. Lynne Williams, andMichael W. Long2

Department of Pediatrics [N. S. D., J. L W., M. W. U and theComprehensive Cancer Center [M. W. L], University of Michigan, AnnArbor, Michigan 48109

Abstract

Despite a growing understanding of the biochemical

mechanisms controlling the cell cycle, information

regarding the temporal ordering of S phase and Mphase remains scarce. Polyploid cells represent auseful model for examining S- and M-phase control,

because their cell cycle machinery must be modulated

to retain high levels of DNA content (ploidy) within asingle nucleus. To evaluate the mechanisms of S-

phase control during the process of polyploidization,we investigated the modulations that occur in cyclin-dependent kinase (CDK) complexes during the

induction of megakaryocyte differentiation in human

erythroleukemia cells. We report that duringpolyploidization, megakaryocytic humanerythroleukemia cells undergo a dramatic modulationin the subunit composition of G1-associated and S

phase-associated CDK complexes and a markedincrease in their specific activities. This, in turn, is

facilitated by a differential loss of the p21 or p27 CDK-inhibitory protein/kinase-inhibitory proteins (CIP/KIP)

bound to specific cyclin/CDK complexes. The datashow that the loss of S- and M-phase control inpolyploid cells occurs within the context of an up-regulated function in those CDK complexes associated

with both G1-S-phase transit and S-phase progression.

Additional studies regarding the regulation of thesecomplex CDK interactions will be important tounderstand cell cycle control in such diverseprocesses as megakaryocyte differentiation or thetypes of genomic instability that occur in cancer cells.

IntroductionThe coordination of cell proliferation occurs at specific cellcycle checkpoints that, in turn, require the assembly and

Received 10/22/97; revised 4/24/98; accepted 6/9/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 1 8 U.S.C. Section 1734 solely to mdi-cate this fact.1 Supported in part by Grant HL51459 (to M. W. L).2 To whom requests for reprints should be addressed, at Comprehensive

Cancer Center, Room 3570B, MSRB-Il, Box 0688, University of Michigan,1 150 West Medical Center Drive, Ann Arbor, MI 48109. Phone: (313)647-2897; Fax: (313) 763-9512.

disassembly of heteromeric protein kinase complexes.These complexes are comprised of two essential compo-nents: (a) the CDKs;3 and (b) the cyclins [named for theirperiodic synthesis and destruction (for reviews, see Refs. 1and 2)]. The CDKs are the apoenzymes of these complexes,whereas cyclin binding is a regulatory event required forkinase activity (3, 4), and other proteins within the CDKcomplex fine-tune its kinase activity (see “Results”). Amongthe CDK proteins, CDK2 plays an important role in regulating

both G1-S-phase transit and S-phase progression. A numberof regulatory cyclins are complexed with CDK2: cyclin Eplays a role in G1 progression and in G1-S-phase transit; andcyclin A is essential for both S phase and the initiation of DNAreplication (5-9). Another family of cyclins, the G1 cyclins(D-type cyclins in mammals and the analogous CIn regula-

tors in yeast), together with CDK4 or CDK6, are important intiming G1 progression, as well as in G1-S-phase transit (10,1 1). The D-type cyclins are partially cell type specific, andmost cells express cyclin D3 and either cyclin Dl or D2 (12).Interestingly, the D-type cyclins also bind with CDK2 (13).

Given the central role of CDKs in cell cycle progression,alterations in their kinase activities are a key feature of cellcycle regulation. Two families of proteins function to inhibitCDK activity: (a) the lNK4 proteins (inhibitors of CDK4); and(b) the CIP/KIP inhibitors (CDK inhibitory proteins/kinase in-

hibitory proteins). The INK proteins are comprised of a num-ber of related inhibitory molecules exemplified by p1 6INK4a

and p1 5INK4b (1 4). INK-type inhibitors respond to exogenous

growth factor levels and associate with CDK4 or CDK6 com-plexes (for reviews, see Refs. 1 and 15). The CIP/KIP familyof inhibitors (p21CIP1 and p27KIPl) responds to a diverse set

of signals, such as growth factor depletion, contact inhibi-tion, and DNA damage, thus regulating G1 progression, G1-

S-phase transit, and S-phase progression (16-1 8). p21 CIP1 is

a potent, universal CDKI (19) that is indirectly sensitive tochanges in DNA integrity by virtue of p53 binding elements inits promoter region (1 9) and seems to play a role in coordi-nating 5- and M-phase events (20). The p27’<� protein alsoassociates with most CDK complexes and functions as auniversal CDKI and, like p21 , is a stoichiometric inhibitor ofCDKs.

Despite a growing understanding of the biochemicalmechanisms regulating G1 progression and mitosis, informa-tion regarding the link between S-phase control and DNAreplication remains scarce, as does information on the tem-poral ordering of S phase and M phase. Therefore, the iden-

3 The abbreviations used are: CDK, cyclin-dependent kinase; CDKI, CDKinhibitor; PMA, 4�3-phorbol 12-myristate 13-acetate; HEL human eryth-roleukemia; ECL enhanced chemiluminescence; pRb, retinoblastomaprotein.

640 CDK2/Cyclin Complexes in Endomitosis

4 N. S. Datta and M. W. Long, unpublished observations.

tification of a cell phenotype in which an inducible modula-

tion in cell cycle results in an altered DNA content wouldprovide a useful model for examining S- and M-phase con-trol. Polyploid cells represent such a phenotype, becausetheir cell cycle machinery must be modulated to retain highlevels of DNA content (ploidy) within a single nucleus. Inter-estingly, the bone marrow megakaryocyte, the cell respon-sible for platelet formation, undergoes polyploidization aspart of its normal developmental program (21 , 22). Amongmammalian marrow cells, megakaryocytes are unique in thatthey do not remain diploid (2C) during differentiation butrather synthesize 4-64 times the normal DNA content (within

a single nucleus) as part of their maturational processes (21,22). Nonetheless, the process of polyploidization is itselfregulated, because the entire DNA content is duplicatedduring each replicative event. As a result, megakaryocytescontain 2-fold multiples of the normal diploid DNA content(i.e. , they are 4C, 8C, 1 6C, and so forth, where 2C is the DNAcontent of a normal cell in G0-G1). Bone marrow megakaryo-cytes therefore serve as a model system in which mitosis isdisassociated from the completion of S phase, but globalcontrol regarding the replication of DNA is retained. Theprocess of polyploidization is not, however, unique tomegakaryocytes, because certain neoplastic cells, plantcells, and insect cells also contain polyploid nuclei (23, 24).

The existence of polyploid cells demonstrates that undercertain circumstances, the normal ordering of S- and M-phase progression can be relaxed. It further implies that cellcycle control must be modulated such that mitosis is abro-gated (to keep the cell uninucleate), and that (subsequently)S phase reoccurs to allow the cell to acquire increased levelsof DNA content. A number of studies have shown that spe-cific modifications of the mitotic CDK complex (e.g., CDC2/cyclin B) result in cells with a polyploid phenotype. Thus,mutations in genes essential for mitosis, such as CDC2,p56�’�3, or ts4l , result in polyploid cells (25-27). We re-cently demonstrated that megakaryocytic cells undergoingpolyploidization fail to assemble functional CDC2/cyclin Bcomplexes (28). Likewise, Grafi and Larkins (29) demon-strated that an active inhibition of CDK2/cyclin B kinaseactivity occurs in polyploid maize endosperm. These studiesdemonstrate that the temporal dependency of M phase onthe completion of S phase is lost in polyploid cells, but theyfail to address the mechanism of this loss of S- and M-phasecontrol or to distinguish such 5- and M-phase control fromthe control over rereplication of DNA sequences (for a review,see Ref. 30). However, these investigations do suggest thatmitotic kinase activity inhibits S-phase progression andshow that the inhibition (or abrogation) of M phase allows thereinitiation of S phase without an intervening mitosis.

To evaluate the mechanisms of S-phase control during theprocess of polyploidization, we investigated the modulationsoccurring in CDK complexes during the induction ofmegakaryocyte differentiation in HEL cells. This cell line faith-fully mimics the megakaryocyte differentiation process, be-coming polyploid and expressing megakaryocyte-/platelet-specific markers in response to tumor-promoting phorboldiesters (28, 31). We recently demonstrated that part of thepolyploidization process in HEL cells results in a loss of

CDK2/cyclin B kinase activity (28). We now report that duringpolyploidization, megakaryocytic HEL cells undergo a dra-matic modulation of G1- and S-phase CDK subunit compo-sition and a marked increase in their specific activities. This,in turn, is facilitated by a differential loss of the p21 or p27CIP/KIP inhibitors bound to the cyclin/CDK complexes. Thedata thus show that the loss of M-phase control in polyploidcells occurs within the context of an up-regulated function inthose CDK complexes associated with both G1-S-phasetransit and S-phase progression.

ResultsEndomitotic Cell Cycle Control Involves Complexes ofCDK2, Cyclin D3, and Cyclin E. Evaluation of polyploidmegakaryocytes showed that these cells underwent a mi-totic arrest somewhere in metaphase and did not reachand/or complete anaphase (for details, see Ref. 28).Megakaryocytes (and megakaryocytic HEL cells) thereforebest fit the description of endomitotic cells, in which thereplication of nuclear elements is not followed by chromo-some movement or cytokinesis (see Ref. 28). To investigatethe nature of the protein kinase complexes involved in theendomitotic S phase, we focused our attention on thosecyclins and CDK-related proteins implicated in both G1-S-phase transition and S-phase control. Cyclins A, D, and E areinvolved in G1 progression (cyclin D), G,-S-phase transit

(cyclins A and E), or S-phase control (cyclins A and E; Refs.6, 9, and 32-34). We first examined control and polyploidHEL cells for alterations in CDK2 protein using antibodies toits COOH-terminal domain. CDK2 levels underwent cell cy-do-dependent alterations in relative abundance in both con-trol and PMA� (PMA-resistant HEL cell subclone) cells (Fig.1A, left and middle panels), being somewhat reduced duringmitosis (G2-M phase), elevated in G0-G1, and maximal inS-phase cells. This observed periodicity is consistent withobservations in other blood cells (e.g., T cells) that show cellcycle-regulated changes in CDK2 levels (35). In contrast withcontrol cells, elutriated polyploid HEL cells expressed rela-tively constant concentrations of CDK2, although a slightelevation in relative abundance was noted in 4C cells (Fig.1A, right panel).

We next analyzed the relative protein levels of cyclins Dl,D2, D3, E, and A by ECL-based Western analysis. Cyclin Dlwas not detectable in HEL cells, whereas cyclin 02 waspresent in relatively low levels.4 However, cyclin D3 wasdetected at low levels in control uninduced cells and PMA�cells (Fig. 1B, top row, left and middle panels), whereas inpolyploid cells, the relative abundance of cyclin D3 increased8-10-fold (as determined by a densitometric analysis of un-fractionated cells; Fig. 1B, top row, rightpanel). Thus, cyclinD3 was differentially modulated during endomitosis, be-cause it was markedly increased in polyploid cells and waselevated in each ploidy class.

We next examined the relative abundance of cyclin E, aprotein that is associated with both G1-S-phase transition

Cycling PMA-Resistant Endomitotic

U 2C 4C 8C

Endomitotic

� -. �

U 2C 4C 8C

� ‘� .#{149}U 2C 4C 8C

* -

U 2C 4C 8C

� � �

U 2C 4C 8C

A

B Cycling PMA-Resistant

Cyclin D3 ‘�‘ . �

U G1 S G2 U G1 S

Cyclin E #{149}� O � . . � �- � �d

U G1 S G2 U G1 S G2

Cyclin A � � � � �

U G1 S G2 U G1 S G2

HSP 70 � � �

U G1 S G2

C � �#{149} antibody. cyclin 03 cyclin E cyclin A�

CDK2- ‘ #{149} � ..� #{149}1’�� #{149}�I*4

PMA - + - + - +

I P anhbody. cyclin D3 cyclin D3

Cell Growth & Differentiation 641

Fig. 1 . Relative protein abun-dance and physical associationof CDK2 and cyclins D3 and E inendomitotic cells. Western anal-ysis of equal protein loads fromwhole cell lysates was performedas described in “Materials andMethods,” and transferred pro-teins were visualized by ECL-based Western analysis. U, un-fractionated cells; G1, S, and G2,cell cycle phases; 2C, 4C, and8C, ploidy classes. A, Westemanalysis of CDK2. B, Westemanalysis of cyclins D3, E, and A.HSP 70, whole cell lysatesprobed with antibodies to theheat shock protein 70 as a pro-tein-loading control. C, physicalassociation of cyclins D3, E, andA with CDKs. Control (-) and en-domitotic (+) cells were immu-noprecipitated with either cyclinD3, cyclin E, or cyclin A andprobed with antibodies to CDK2,CDK4, or CDK6 as indicated. +

and - , the presence or absence,respectively, of the endomitotic-inducing agent PMA. 3T3, con-trol NIH-3T3 cells immunopre-cipitated with antibody to cyclinD3 and probed with the antibodyto CDK4.

CDK2 � � � � �. � -U G1 S G2 U G1 S G2

CDK6-CDK4-

PMA - +

and S-phase events (8, 36). Consistent with a role in G1 -5-phase transit, Western analysis of uninduced cells showedthat the relative abundance of cyclin E was maximal in G0-G1

and in S phase and was lowest in G2-M-phase cell popula-tions (Fig. 1B, second row, left and middle panels). Therelative abundance of cyclin E was reduced in 2C endomi-totic cells, elevated in 4C cells, and further elevated in 8C

endomitotic cells (Fig. 1B, second row, right panel). How-ever, this apparent ploidy class-related increase was due to

the reduction in 2C endomitotic cells compared with con-

trols, because densitometric analysis showed that the rela-

tive abundance of cyclin E in 4C and 8C endomitotic cells

actually reached levels approximately equivalent to those

observed in S-phase uninduced cells.

As mentioned, cyclin A plays a role in G1-S-phase transit,its presence is necessary throughout S phase, and it isrequired for the initiation of DNA replication. Consistent with

these observations, the relative abundance of cyclin A pro-tein was reduced in the G0-G1 phase of control uninducedcells and increased as cells progressed through the S phase

- + 3T3

and G2-M phase of the cell cycle (Fig. 1B, third row, left

panel). In sharp contrast, the relative abundance of cyclin A

was markedly lower in polyploid cells, being reduced 65-75% in unfractionated cells compared with that of controlcells. Nonetheless, an approximate 2-fold increase in cyclinA relative abundance was observed in 4C and 8C cells (Fig.

1B, third row, right panel) compared with that of the 2Cendomitotic cells.

The mechanism underlying the alterations in the relativeprotein abundance of cyclins D3, E, and A was analyzed by

evaluating both cyclin mRNA levels and the protein half-lifeof these regulators. Cyclin D3 mRNA in endomitotic cells wastransiently reduced for 1-3 h after induction. Its message

levels then increased sharply, reaching an approximate

7-fold elevation over uninduced levels, with the greatest

change occurring between days 1 and 3 (Fig. 2, top row, left

panel). In control cells, cyclin D3 mRNA levels steadily de-creased (by approximately 50%) over the same period. Ex-amination of cyclin D3 mRNA in elutriated endomitotic cells

indicated an approximate 12-fold increase in 2C cells com-

7

6

5

4

3

2

1

001 5 G2

. I . I . I . I . I

2C4C8C

1.5

1.0

0 30 60 90 120 150 180

7-

�

5-

4 - 00

3-

�

0 30 60 90 120 150 180

3.0 -

2.5 -

2.0

I .5

1.0

3

2

1

0I . I . I . I . I . I .1

0 30 60 90 120 150 180

Times post-induction (hours)

G15G2 2C4C8C

DNA Content

642 CDK2/Cyclin Complexes in Endomitosis

8

7

<6z

E5Cl,4

.� 30>“

0

0

zccEwC

0>“

0

zccE

C

0>“

0

Fig. 2. Expression of cyclin D3,cyclmn E, and cyclmn A mRNA inendomitotic HEL cells. Cellswere harvested at various timesafter induction as indicated (leftpanels), mRNA was extracted,and equal amounts were evalu-ated by Northem analysis as do-scnbed in “Materials and Moth-ods.” Values are arbitrarydensitometry units normalized tothe expression of glyceralde-hyde-3-phosphate dehydrogen-ase. The right panels representelutriated fractions from controlcells (G1, S, and G2) and fromelutriated endomitotic cells (2C,4C, and 8C) 5 days after the in-duction of endomitosis (left pan-els). Solid lines/closed symbols,endomitotic cells; dashed linealopen symbols, control cells.These data are a single repre-sentative experiment of two sop-arate experiments.

pared with that of control 2C cells (i.e. , cells in G0-G1 ; Fig. 2,top row, right panel). The relative abundance of cyclin 03mRNA decreased with increasing DNA content, but none-theless, it remained elevated in comparison with that ofcontrol cells (Fig. 2, top row, right panel). Confirming theobservations of cyclin E protein levels, cyclin E mRNA anal-ysis demonstrated that its relative abundance stayed con-stant during polyploidization (Fig. 2, middle row, left panel).

Interestingly, control cells showed a transient peak of cyclinE transcription (presumably due to the reduction in serumconcentration), followed by steadily decreasing mRNA 1ev-els. Isolated populations of endomitotic 2C, 4C, and 8C cellshad cyclin E mRNA levels essentially equivalent to that ofG0-G1 control cells.

Message levels for cyclin A in endomitotic cells showed atransient peak early after induction (1-3 h; Fig. 2, bottom row,

A 100

C

s-, 80o�

octiC>a) .�

-Ce2w

0�0.c��E 40

0

0 5 10 15 20 25 30

0

B

CwC�j”

0j�C>

.� .�

-CeO�

0�0.� 40.� C.)

ow

0�

�‘ ....o\o

. \.

0 10 20 30 40 50

Chase Period (hours)

precipitations performed using antibodies to each cyclin.Analysis of immunoprecipitates from whole cell lysates dem-

onstrated that cyclins A, D3, and E each physically associ-ated with CDK2 in endomitotic cells (Fig. 1C, upper panels).

As predicted from Western analysis, cyclin D3/CDK2 com-plexes were observed in polyploid cells, whereas relativelylittle complex was observed in uninduced cells (Fig. 1C,

upperleft panel). Moreover, CDK2/cyclin E and CDK2/cyclinA complexes were detected in both control (S-phase) cellsand in polyploid cells (Fig. 1C, middle panel and right panel,

respectively). These physical interactions were confirmedwith reciprocal immunoprecipitations that demonstrated that

CDK2 immunoprecipitates contained cyclins A, D3, and E.4Interestingly, with overexposure of the ECL-based autora-diographs, CDK2 protein was detected in anti-cyclin 03 rn-munoprecipitates from control cells. This weak signal mostlikely represents a bona fide interaction between cyclin D3

and CDK2, because cyclin D3 was detectable, albeit at lowlevels, in unfractionated control cells (see Fig. 1B). Moreover,immunoprecipitation-based pulse-chase experiments of cy-

din D3 also demonstrated the presence of this protein incontrol HEL cells (see Fig. 3A).

Given the role of D-type cyclins in G1 progression (1 1 , 32,33, 37), we also evaluated whether cyclin 03 might physicallyinteract with CDK4 or CDK6. Both control and endomitoticHEL cells expressed CDK4 and CDK6 (as indicated by West-em analysis of total cell lysates).4 However, no physicalassociation between CDK4 and cyclin 03 was detected ineither control or endomitotic HEL cells (Fig. 1C, lower left

panel), even after prolonged exposure of the autoradiograph.In contrast, a physical association of cyclin D3 with CDK4was seen in NIH 3T3 cells, indicating that the lack of acomplex in HEL cells was not due to an antibody failure todetect or precipitate these proteins. The possibility that, inthese circumstances, cyclin D3 might preferentially interactwith CDK6 was excluded by probing anti-cyclin 03 immu-noprecipitates with anti-CDK6 antibody. These data indicatethat cyclin 03 is physically associated with CDK6 in control

cells, but that this interaction is significantly reduced in en-domitotic cells (Fig. 1C, lower right panel). This disassocia-tion of cyclin D3 from CDK6 complexes was not a result of

changes in the INK family inhibitors p1 5 or p1 6, because theirrelative abundance and CDK6 complex association wereunaltered in polyploid cells.4

The unique physical association of CDK2 and cyclin D3suggested that a functional kinase complex existed in thesecells. To address this, cyclin 03-associated kinase activitywas evaluated using Hi histone as a substrate (Fig. 4A).These data show that cyclin D3-associated histone kinaseactivity in endomitotic cells increased within 3-6 h of induc-tion, followed by a transient reduction at 1 2 h, and thereafter

(at 24 h) sharply increased and continued to increasethroughout the induction period. Although equal amounts ofprotein from each cell type were immunoprecipitated, varia-tions in immunoprecipitation efficiency and antibody affinityrequired that these data be normalized. Therefore, the kinet-ics of CDK2/cyclin protein kinase complex function was do-termined using in vitro Hi histone kinase assays, normalizing

the data for the amount of protein immunoprecipitated (Fig.

Fig. 3. Pulse-chase analysis of the cyclin D3 and cyclmn A protein half-life. Control and endomitotic cells were metabolically labeled with[�5Sjmethionine as described in “Materials and Methods” and chased forvarying periods by immunoprecipitation with the respective antibodiesand visualization by autoradiography. Autoradiograms (with the linearregion of the film) were quantitated by densitometry and normalized forthe amount of protein precipitated. A, pulse-chase analysis of cyclin D3;B, analysis of cyclin A. Solid llneslclosed symbols, endomitotic cells;dashed lines/open symbols, control cells. These data are a single repre-sentative experiment of three separate experiments.

left panel). They then increased slightly but steadily over thenext 5 days. Elutnated populations of endomitotic cellsshowed cyclin A mRNA levels similar to that of control cells,except for a moderate increase in 4C endomitotic cells (Fig.2, bottom row, right panel). We next examined cyclin D3, E,and A protein stability by pulse-chase analysis. Analysis ofcyclin 03 protein half-life showed that this protein was sta-bilized in polyploid cells, with its half-life changing from 3 h in

control cells to “1 1 h in endomitotic cells (Fig. 3A). In con-

trast, the cyclin A protein half-life decreased from 26 h incontrol cells to 14 h in endomitotic cells (Fig. 3B), although abiphasic curve in which half of the cyclin A degraded and halfstabilized cannot be excluded in this analysis. Pulse-chaseanalysis of the cyclin E protein half-life showed equivalentprotein stability in control and endomitotic cells.4

Functional Evaluation of CDK2 Protein Kinase Corn-plexes. To determine whether functional interactions exist

between cyclins 03, E, or A and CDK2, we first examined

their physical association. Protein complex formation wasassessed by measuring the presence of CDK2 in immuno-

Cell Growth & Differentiation 643

20

0

100

80

60

20

0 J

EndomitoticA Cycling

Hi Histone- . �,, .�4 �

Time(hrs.): 3 6 12 24 72 96 168

B l.P. Antibody: Cyclin D3

HiHistone- � ��-GSTRb

C l.P. Antibody: Cyclin 03

H1 Histone- � � - + -GST Rb

Hi Histone- i�T�:’�’� � -GST RbPre Post Pre Post

l.P. Antibody: Cyclin D3

� �- . Cdk6 - #{149}��:

Cdk2- Cdk4-

Lys. ID Lys. ID Lys. ID

644 CDK2/Cyclmn Complexes in Endomitosis

Hi Histone- ______

Time(hrs.): 3 6 12 24 72 96 168

l.P. Antibody: Cyclin 03

Fig. 4. Cyclmn D3-associated kinase activities. Control uninduced and endomitotic cells were evaluated for temporal changes, substrate specificity, andCDK complex subunit composition. A, kinetics of cyclin D3-associated Hi histone kinase activity. Equal amounts of protein from normally cycling controlcells (left panels) and endomitotic cells (right panels) taken at the indicated times were immunoprecipitated using antibodies to cyclmn D3. B, cyclinD3-associated kinase substrate specificity. Cyclmn D3-immunoprecipitated kinase activity was evaluated using Hi histone or pRb as substrates (left panels).The CDK associated with Hi histone kinase activity (i.e., CDK2) was determined by the loss of activity after immune depletion of CDK2. C, analysis of Hihistone kinase activity in CDK4/CDK6 immunoprecipitations. Cell lysates were immune-depleted for CDK4 and CDK6 and analyzed for Hi histone andretinoblastoma kinase activity (left panels). Finally, the presence of CDK2 in these CDK4/CDK6 immune depletions was determined by Western analysis(right panels). Lys. , whole cell lysate used as a positive control; ID, after immune depletion.

5). These data indicate that the specific activity of eachCDK2/cyclin complex (i.e. , CDK2/cyclin 03, CDK2/cyclin E,and CDK2/cyclin A) is elevated in endomitotic cells. More-over, the temporal expression of peak kinase activity differedfor each CDK complex. As suggested by the autoradiograms

(Fig. 4A), the normalized kinase activity of CDK2/cyclin D3 inboth control and endomitotic cells showed an early peak at

3 h (Fig. 5). However, polyploid cells continued to increasetheir cyclin D3-associated kinase activity, showing an ap-proximate 10-fold increase in specific activity over days 2-7(Fig. 5A, left panel). In contrast to cyclin 03, which has its

peak activity in the later phases of endomitosis, the specificactivity of cyclin E-associated kinase activity increased rap-

idly after induction (“7-fold over the first 24 h) and thereafter

gradually fell to a level of approximately twice that of prein-duction values (Fig. 5B, left panel). Likewise, cyclin A, after atransient reduction, showed a rapid increase in specific ac-tivity, which increased 3-4-fold by 24 h. However, unlikecyclin D3, cyclin A-associated kinase specific activity re-mained elevated (5-fold higher than that of controls) until day5; thereafter, it fell abruptly (Fig. 5C, left panel). Interestingly,sequential activation of cyclin 0, E, and A-associated kinaseactivities occurred in control cells. This is consistent with ourobservations that control cells undergo proliferation, albeit ata reduced rate, in the low-serum (0.5%) conditions used forthe induction of megakaryocyte differentiation (31).

Earlier reports have shown that cyclin A can associate withboth CDC2 and CDK2 (6, 9, 38). To confirm that the increase

in cyclin A-associated activity in polyploid cells was due to

the CDK2/cyclin A complex, we immune-depleted CDK2from control and polyploid HEL cell lysates with anti-CDK2

antibody, performed an immunoprecipitation with anti-cyClinA, and evaluated the residual cyclin A-associated kinaseactivity. These results show equivalent Hi histone kinase

activity in both control and induced cells. We therefore con-dude that the increased cyclin A-associated kinase activityin polyploid cells was due to CDK2/cyclin A.4

Analysis of various CDK2/cyclin kinase activities (normal-ized for the amount of protein precipitated) within each ploidyclass of endomitotic cells also revealed distinct patterns ofcyclin-specific activity (Fig. 5, A-C, right panels; ploidy classkinase activity evaluated on day 5). Consistent with a role in

G1 progression, cyclin 03-associated kinase activity (Fig. 5A,

right panel) was 5-8-fold higher in G0-G1 control cells ascompared with that of cells in S phase or G2-M phase. Incontrast, all endomitotic cells (i.e. , 2C, 4C, and 8C) containedcyclin 03-associated kinase activity at levels that wereslightly elevated over that seen in G0-G1 control cells. CyclinE-associated kinase activity in endomitotic 2C cells wasreduced compared with that of control G0-G1 cells butshowed an approximate doubling in specific activity witheach subsequent doubling of DNA content (Fig. 5B, right

panel). Cyclin A kinase activity showed a unique pattern ofactivation; it was equivalent to control levels in 2C and 8Cendomitotic cells but was elevated 4-fold (versus controls) in4C endomitotic cells (Fig. 5C, right panel).

To confirm whether the CDK2/cyclin 03 complex is afunctional CDK complex, we performed the following exper-iments. The relative substrate specificity of cyclin 03 was

determined in immunoprecipitates, comparing both Hi his-tone and pRb as substrates (Fig. 4B, left panels). These datashowed a change in cyclin D3-associated kinase substratepreference during endomitosis. Thus, uninduced cellsshowed low Hi histone kinase activity and high pRb activity.In sharp contrast, PMA-induced polyploid cells markedlyincreased their Hi histone kinase activity but showed re-duced pRb phosphorylation. To determine whether cyclin03-associated histone kinase activity was due to a CDK2/

60-

50-

40

30

20

10

0 11l1l1l,l1l1lGi S G2 2C4C8C

- 400

- 350

- 300

- 250

- 200

- 150

- 100

- 50

.ll�l�ll�l o

0 30 60 90 120 150 180 Gi S G2 2C4C8C

210

180

150

120

90

60

30

0

500 -

400 -

300 -

200 -

100-

0 30 60 90 120 150 180

Times post-induction (hours)

G1502 2C4C8C

Cell Growth & Differentiation 645

A7o-

>

0

Cl)

C

.�

Cl)

C

0

0 30 60 90 120150180

60

50

40

30

20

10

0

Fig. 5. Histone Hi kinase activ-ities of CDK2/cyclmn complexes.Cyclin D3-CDK2 �4), cyclmnE-CDK2 (B), and cyclmn A-CDK2(C) kinase activities in controland endomitotic cell populationsare shown. Cells were evaluatedfor temporal changes in kinaseactivity (left panels) or for ploidyclass-dependent alterations(right panels). Cell lysates wereprepared and precipitated withanti-cyclmn antibodies as do-scnbed in “Materials and Moth-ods.” G7, 5, and G2, cell cyclephases; 2C, 4C, and 8C, ploidyclasses. Values were normalizedfor the amount of protean precip-itated. All values are in arbitraryunits. Solid lines/closed symbols,endomitotic cells; dashed linealopen symbols, control cells.These data are a single repro-sentative experiment of two sep-arate experiments.

B 240

.�i 210

>:� 180

C�0) 150Cl)

C 120.�

w 90

C

>

0

0)U)CoC

C

0

30

0

cyclin 03 complex, we performed CDK2 immune depletions(Fig. 4B, rightpanels). These studies showed that in polyploidcells, CDK2 immune depletion removed >90% of detectablecyclin 03-associated Hi histone kinase activity but did notremove pRb kinase activity. Consistent with this, the com-bined immune depletion of CDK4 and CDK6 removed all

DNA Content

detectable cyclin 03-associated pRb kinase activity withoutaltering its Hi histone activity (Fig. 4C, leftpanels). Finally, weconfirmed that the cyclin 03-associated histone kinase ac-tivity in CDK4/CDK6 immune depletions involved CDK2 byshowing that CDK2 was present, whereas CDK4 and CDK6were absent (Fig. 4C, right panels). We concluded that

�‘ ,�:- __

Id�

646 CDK2/Cyclin Complexes in Endomitosis

5 Unpublished observations.

p ,� i � �

,� �

#{149}#1

,.� �

Fig. 6. Immunocytochemical localization ofcyclin D3 and cyclin A in endomitotic cells.Immunocytochemical localization was per-formed as described in “Materials and Meth-ods.” Localization with control antibodies(MOPC; see “Materials and Methods”) failsto detect any nonspecific reactivity in controland endomitotic cells (unpublished observa-tions). A, cyclin D3; B, cyclin E.

polyploid cells contained a bona fide CDK2/cyclin D3 kinase

that used Hi histone as a substrate.

Taken as a whole, the aforementioned data indicate thatdistinct changes occur in the function of G1- and/or S-phaseCDK proteins and protein kinase complexes in endomitotic

cells. Thus, cyclin D3 protein levels markedly increase,whereas those of cyclins E and A increase within each ploidy

class. Importantly, the resultant changes in CDK protein

kinase activity may reflect temporally distinct roles of each

CDK complex. Thus, CDK2/cyclin D3 seemingly plays a role

in the latter phases of endomitosis, whereas cyclins E and A

functioned earlier, but sustained activity throughout the en-

domitotic cell cycle was required.

Nuclear Localization of Cyclins D3 and E in Endorni-

totic Cells. The D-type cyclins, cyclin E, and cyclin A are

localized to predominantly nuclear regions of the cell (39, 40).

In endomitotic cells, increased amounts of cyclin 03 were

detected by immunocytochemistry within the nucleus (Fig.

6A), whereas the amount of cyclin 03 (and cyclin E) in controlcells was below the limits of detection using this procedure.5

The distribution of nuclear cyclin D3 in endomitotic cells was

also unique in that it was present in some, but not all, of the

lobes of a given nucleus. Likewise, detectable cyclin E nu-clear localization was seen to increase in endomitotic cells

(Fig. 6B). However, unlike cyclin 03, cyclin E showed a

diffuse and somewhat homogenous nuclear distribution. Fi-

nally, the use of an inappropriate antibody failed to detectany nonspecific cytoplasmic or nuclear staining (data not

shown).

ACycling

p21

U G1 S G2

p27 �

U G1 S G2

B

l.P. antibody: cyclin D3

p27- �-�W)

Endomitotic

U 2C 4C 8C

U 2C 4C 8C

cyclin E cyclin A

� E::��� ;�

Cell Growth & Differentiation 647

Fig. 7. Relative protein abun-dance and physical associationof CIP/KIP proteins with CDKcomplexes. The p21 and p27protein association with cyclinsD3, E, and A and CDK2 in en-domitotic and control cells isshown. A, Western analysis ofp21 and p27 expression in equalamounts of control and endomi-totic whole cell lysates. B, phys-ical association of p21 and p27with CDK complexes immuno-precipitated with antibodies tocyclmns D3, E, orA. U, unfraction-ated cells; G1, 5, and G2, cellcycle phases; 2C, 4C, and BC,ploidy classes.

p21- T::::

PMA: - + - + +

Modulation of CDK-inhibitory Proteins p21 and p27during the Endomitotic Cell Cycle. In recent years, theimportance of CDKIs in the control of cyclin/CDK activity hasbeen elucidated. To address the mechanism underlying theobserved increase in the specific activity of CDK2/cyclinprotein kinases in polyploid cells, we first determined therelative abundance of p21 ci�i and p27K� in control andendomitotic HEL cells. Western analysis of control cell ly-sates demonstrated that the relative abundance of p21 incontrol cells was elevated in G0-G1 cells and reduced duringS phase and G2-M phase (Fig. 7A, upper left panel). Corn-pared with control G0-G1 (i.e. , 2C) cells, endomitotic 2C cellscontained reduced levels of p21 protein. p21 expression in4C and 8C populations was also reduced compared with thatof G0-G1 cells but was slightly elevated compared with thatof 2C endomitotic cells (Fig. 7A, upper right panel). Consist-ent with reports that transit into S phase is associated withsuppression of the levels of CDKIs (41 , 42), the p271<�protein was expressed in high levels in control G0-G1 cellsand was dramatically reduced in S phase and G2-M phase(Fig. 7A, lower left panel). In sharp contrast to 2C controlcells, polyploid cells contained reduced levels of p27 protein,but its relative abundance seemed to increase slightly in 4Cand 8C endomitotic cells (Fig. 7A, lower right panel). None-theless, the relative abundance of p27 in all endomitotic cellpopulations remained less than that of control G0-G1 cells.

We next examined whether the physical association of theCDK-inhibitory proteins with CDK/cyclin complexes differedin control and endomitotic HEL cells. Given that p27 is sig-nificantly elevated in normally cycling cells, we examined itsphysical association with different cyclin/CDK2 complexes.The association of p27 with CDK2/cyclin 03 in the endomi-totic cells was found to be reduced compared with that of

control cells, as was its association with CDK2/cyclin E andCDK2/cyclin A complexes (Fig. 7B, upper panels). Densito-metric analysis normalized for the amount of immunoprecipi-tated cyclin showed a differential reduction in the amount ofp27 associated with each CDK complex. Thus, the amount ofp27 associated with cyclin 03 complexes was markedlyreduced (by 98%), whereas that associated with cyclin E andcyclin A CDK complexes were reduced 54 and 79%, respec-tively. These data imply that as cells enter into endomitosis,there is a disassociation of p27KI� from all CDK2/cyclincomplexes, but the degree of this dissociation varies. West-em analysis of cyclin D3 immunoprecipitates revealed thatthe amount of p21 associated with the CDK2/cyclin 03 corn-plex in endomitotic cells was markedly reduced comparedwith that of control cells (Fig. 7B, lowerleftpanel). In contrast,no significant variation of the level of p21 was observed inCDK2/cyclin E complexes, and evaluation of its association

with CDK2/cyclin A complexes failed to detect p21 �IP1�

These data indicate that p2i and p27 bound to each of thecyclin D3, E, or A/CDK2 complexes in control cells, but theirassociation was decreased during endomitosis. Presumably,this lack of CDKI association contributed to the enhanced

CDK2/cyclin protein kinase activities during the process ofpolyploidization.

DiscussionThe process of polyploidization represents a unique oppor-tunity to evaluate the interrelationship of cell cycle controland DNA replication. To become truly polyploid (as corn-pared with a multinucleate cell), we hypothesize that the cellcycle must be abolished to prevent mitosis, and that S- andM-phase control must be relaxed such that S phase occurs

648 CDK2/Cyclin Complexes in Endomitosis

without an intervening mitosis. We recently demonstratedthat the CDC2/cyclin B complexes do not form in endomi-

totic HEL cells (28). We report here that CDK complexesassociated with G1-S-phase transit and/or S-phase progres-sion are differentially modulated during the endomitotic Sphase. Thus, CDK2/cyclin 03 kinase-specific activity pro-gressively increases during endomitosis, whereas CDK2/cy-din E complexes function at their highest levels early in thisprocess and continue to be active throughout the inductionperiod. Complexes of CDK2/cyclin A are uniquely modified inpolyploid cells as the relative abundance of cyclin A de-creases, whereas the specific activity of the CDK2/cyclin Akinase complex is markedly increased. These alterations inCDK kinase activity are facilitated by both a reduction in therelative abundance of the CIP/KIP family of CDKIs (i.e., p21and p27) and a differential change in the stoichiometry oftheir physical association with specific CDK/cyclin corn-plexes.

The function of cyclin 03 in polyploid cells seems to beunique. Cyclin 03 protein levels are increased (due to both achange in cyclin 03 mRNA levels and a stabilization of its

half-life), and there is a marked increase in CDK2/cyclin 03Hi histone kinase activity throughout the period of endomi-tosis. These observations contrast sharply with the knownrole of D-type cyclins in timing G1 progression (for reviews,

see Refs. 1 , 1 1 , and 43). Moreover, the G1 function of D-typecyclins is usually mediated by their interaction with CDK4and/or CDK6 (1 1). We observed that cyclin 03 is bound toCDK6 in uninduced HEL cells, but that the CDK6/cyclin 03complexes are greatly reduced during polyploidization. Inaddition, no CDK4/cyclin 03 complexes are evident in unin-duced or polyploid HEL cells. This implies that the majorcyclin 03 kinase complex operative in endomitotic cells isCDK2/cyclin D3. This represents the first report of a func-tional CDK2/cyclin D3 kinase complex in mammalian cells,although ample precedent exists for this observation. Thephysical association of D-type cyclins with CDK2 in humandiploid fibroblasts was shown by Xiong etal. (1 3) and by Katoand Sherr (44) while studying murine hematopoietic (32D)cells, although this study used overexpressed cyclin 02 orcyclin 03. More importantly, Ewen et aL (45) and Sweeney etaL (46) showed that functional interactions exist between

cyclin 02 or cyclin D3 and CDK2. Coupled with its increasedlocalization in the nucleus of polyploid cells and the progres-sive increase in its kinase activity, the data strongly suggestthat cyclin 03 has an important role in the endomitotic proc-ess. This is consistent with the observations of Wang et aL

(47) showing that antisense oligonucleotides abrogatedmegakaryocyte development (although ploidy of the

megakaryocytes was not examined).

The rapid increase in CDK2/cyclin E kinase activity inpolyploid cells is consistent with a role for this complex inmoving cells into or through the endornitotic S phase. Like-

wise, its sustained (albeit progressively lower) high activityand the 2-fold increase in cyclin E-associated kinase activityobserved in each ploidy class strongly suggest that it isimportant in multiple aspects of polyploidization. These dataare consistent with the known role of cyclin E in mediatingG1-S-phase transit (8, 38, 48) and early S-phase events (5,

36, 38). They differ in that we detect no periodic destructionof cyclin E protein (or changes in its kinase activity) inpolyploid cells. The reason for this lack of destruction isunexplained, but it is consistent with our lack of detection ofapparent changes in cyclin E mRNA or protein half-life. Thus,it seems that cyclin E synthesis (i.e., protein/rnRNA abun-dance) is “on” and remains at steady-state (S-phase) levelsthroughout the endomitotic cycle, although CDK2/cyclin Ekinase activity increases within each ploidy class.

The lowering of the relative abundance of cyclin A protein,together with a marked increase in the specific activity of theCDK2/cyclin A kinase complex, suggests a unique require-

ment for this protein during the acquisition of a polyploidnucleus. It is felt that CDK2Icyclin A complexes are importantfor sustaining S phase in normally proliferating cells. Thus,antibodies to cyclin A block both G1-S-phase transit and theinitiation of DNA replication (6, 7, 9). However, cyclin A or itskinase activity may also be responsible for inhibiting therereplication of newly synthesized DNA. Therefore, in en-domitotic cells, unbound cyclin A protein may need to belowered to a critical threshold to allow the cell to “reset” itselffor another round of S phase, whereas the CDK2/cyclin Akinase specific activity needs to be elevated to drive in-creased DNA synthesis.

The increased specific activity observed in endomitoticCDK/cyclin complexes is augmented by reductions in therelative abundance of COK-inhibitory proteins. Thus, during

endomitosis, both p21 and p27 disassociate from the CDK2/cyclin D3 complex but seemingly have little role in cyclinA-associated kinase activity. In contrast, the binding of

p27kII’l primarily influences cyclin E-associated kinase ac-tivity. The disassociation of p21 from CDK2/cyclin 03 corn-plexes is interesting in that p21 is felt to play a role incoordinating S- and M-phase events (20). Our observationssuggest that CDK2/cyclin 03 may be the target for p21 in thisregard; therefore, CDK2/cyclin D3 might mediate the coor-

dination of S- and M-phase events in control cells. Suchregulation is absent in polyploid cells, and this contributes tothe loss of temporal S- and M-phase progression.

The exact mechanism of the loss of S- and M-phasecoordination in polyploid cells is unknown. However, studies

in other systems indicate that this coordination is due, in

part, to the loss of mitotic CDK activity. Thus, rnutations inyeast CIb genes allow the repetition of S phase, as do alter-ations in genes such as ts4l or yeast CDC2 that result in thedestruction of this control point (25-27). Consistent with this,our data and that of others show that polyploid cells lackfunctional CDC2/cyclin B complexes (28, 29, 49, 50). Wefurther demonstrated that the unique mechanism of prevent-ing mitotic kinase complex formation in polyploid HEL cellsinvolves the specific proteolysis of CDC2 as well as a mod-ulation(s) in cyclin B that prevent its interaction with CDC2(28). However, these two modifications (in cyclin B andCDC2), alone, do not seem to be sufficient to establish thepolyploid phenotype, because this study demonstrates thatalterations in G1-S-phase-associated proteins also occur

during this process.It is not clear whether polyploid cells such as megakaryo-

cytes also lose control over DNA rereplication. The observa�

Cell Growth & Differentiation 649

tions that these cells and megakaryocytic HEL cells have2-fold multiples of a G0-G1 DNA content (i.e. , 4C, 8C, 16C,and so forth; Refs. 22, 28, 31 , and 51), coupled with the dataon the loss of mitotic kinase complexes, demonstrate thatthese cells undergo repetitive S phases without interveningmitosis. However, recent work by Heichrnan and Robertsindicates that loss of rereplication control results in polyploidphenotypes similar to those of megakaryocytes. Thus, bothrepetitive S phases and rereplicative S phase yield a log-normal rather than a linear DNA distribution. This implies thatanother checkpoint must exist that regulates the complete-ness of DNA replication, irrespective of whether it occurswithin a single cell cycle or as repetitive S phases.

We conclude that the process of polyploidization involvesextensive rearrangement of the cell cycle machinery. Mitosis isabrOgated after the modulation of CDC2 and/or cyclin B func-t�ion (28). Likewise, those cyclins associated with G1-S-phasetransit (cyclin 0 and cyclin E) as well as S-phase control and the

initiation of DNA replication (cyclin A) are altered to allow repet-itive and/or sustained S phase(s). Whereas these changes ar-gue persuasively for a distinct role for CDK2/cyclin 03, CDK2/cyclin E, or CDK2/cyclin A complexes in endomitosis, they donot, of course, show that these changes cause polyploidy.

Nonetheless, we believe that the unique modulations of thecomplexes described herein play an important role in endomi-tosis, because these afteratkns presumably subserve the dis-association of S- and M-phases in these cells. Mditional stud-ies regarding the master control switches regulating thesecomplex CDK interactions will be important to understandingcell cycle control in such diverse processes as megakaryocytedifferentiation or the types of genomk� instability that occur incancer cells.

Materials and Methods

Induction of Endomltosls, Cell Isolation, and Flow Cytometric Anal-

ysis. HEL cells were passaged, and mogakaryocyte differentiation wasinduced as described previously (28). Briefly, cells were induced in RPMI1640 contaIning 0.5% FCS and 2 n� PMA controls consisted of unin-duced HEL cells and a PMA-resistant HEL cell subclone, PMA,. that isresistant to 100-fold increases In PMA concentration (28). HEL cells wereseparated into cell cycle-specific phases (G0-G1, S phase, and G2-Mphase for control cells and 2C, 4C, and 8C populations for polyploid cells)by countercurrent centrifugal elutriation using a Beckman JE6B elutriationrotoras described previously(28). Unless otherwise stated, polyploid cellswere isolated and elutriated 5 days after PMA induction. Uninduced cells(referred to herein as controls) and PMA-roslstant cells were similarlyisolated. All elutriation fractions were collected on ice, and aliquots woreremoved for analysis of DNA content as described previously (28). DNA

content of polyploid and control coils was measured on 20,000-40,000isolated nuclei (28).

AntibodIes, SDS-PAGE, Western Analysis, lmmunoprecipltatlon,

and Kinase Assays. Cell extracts wore prepared as described previously

�28)elther5daysafterPMA induction orattho indicated lime periods. Briefly,cellswerowashed twotothreotimeswith cold PBS and lysed for30-60 mm

at 4#{176}Cin tysis buffer containing protease inhibitors. Lysateswero centrifugedat 14,000 x gfor45 mm, and an aIIqUOt ofsupematantswasquantffied using

the Bradford protomnassay, using BSAasaprotomn standard. SOS-PAGE wasperformed In iO-i5% polyacrylamido gels (53) using equal amounts ofproteln (50-80 g�g)from the cell lysates ofeach cell typo �e., cycling, PMA,.and polyploid cells). SDS-resolved proteins worotransferred to nitrocollulosomembranes using asomidrytransforapparatus (HOOfer), and the membranes

were blocked by incubating them overnight in TBST frris-bufferod saline (pH

7.6)containings% nonfatdry milkand0.05% Twoen20].The blotswerethenrinsed three times in TBSTand incubated with primaryantibody for2 h. After

washing, the nitrocellulose was incubated with a 2#{176}antibody conjugated tohorseradlsh peroxidasefor20-30 mm. Finally, the proteinswerevlsualized byECL-based autoradiography (Aniersham). When comparing uninducod con-trols and polyploid cells, all samples for a given antibody were run on the

samogel and thus had idenfical ECLexposures. Antibodiostocyclins 02 andD3 were generous gifts of Dr. James Griffin (Dana-Farber Cancer Institute,

Boston, MA), and antibodies to cyclin A were a gift of Dr. Tim Hunt (imperialCancerResoarch, United Kingdom). Monocional antibodiesto cyclins D3andE and polyclonal antibody to p21 were Obtained from PharMingen (San

Diego, CA), monoclonal antibOdies to p27 and antibodies to cyclin A werepurchased from Santa Cruz BiotechnOlOgy (Santa Cruz, � and CDK2antibody was obtained from Upstate Biotechnology (Lake Placid, NY). The

full-length cyclin E cDNA clone was a generous gift from Dr. James Roberts

(Fred Hutchinson Cancer Center, Seattle, WA�. Immunocytochomical local-�ation was performed as described previously (54). The specificity of theimmunocytochemical localization was determined using an inappropriate

primary anthody (MOPC; Sigma Chemical Co., St. Louis, MO).Autoradiographs of Western blots, pulse-chase experiments, and kinaso

assays were exposed within the linear range of the film, scanned, and

quantitated using a densitometer (Melecular Dynamics). For immunoprec�p�tations, equal amounts of cell lysato protein (100-200 �) from all samples(uninducod controls and polypioid cells) were run on the same gel, allowingidentical exposure during aUdioradiographiC analysis. lOnaso activities werenormalized for the amount of protein precipitated, and specific activity wasdetermined by calculating the total amount of kinaso actMty divided by thetotal amount of protein immunoprecipitated (all performed as described inRef. 28). Kinase substrates were Hi histono (Boehringer Mannhelm, Indian-

apolia, IN) and a glutathione S-transferase-retinobostoma fusion protein

(Santa Cruz BiotechnOlogy). Proteln halt-life (ie., pulse-chase) experiments

used HELcOIIS(cOntrOl cells or endOmitOtiC cells)that were pulse-labeled for1 h in methiOnine-free RPMI 1640 containing 100 pCVml Trans-label (ICN,Costa Mesa, C�, as reported previously(28). The media were then replacedwith complete RPMI 1640 containing 0.5% FCS, and the cells we’s har-vested at the indicated times (chase).

RNA Preparation and Northern Blots. Total RNA extractions andNorthern blots were performed as described previously (28). Polyadeny-

lated mRNA was isolated from total RNA using oligodooxythymidylic acidcolumns or the Micro Fast-Tract mRNA isolation kit (Invitrogen, SanDiego, CA). Samples containing equal amounts of polyadonylated mRNA

(2 �g) were subjected to 1% 4-morpholmnopropanosulfonic acid/formal-dehydo agarose gel electrophoresis and blotted onto nitrocellulose morn-branes. The blots were hybridized with cDNA probes labeled with

1�2PIdCW by the hoxanucleotide primer technique. The autoradiogramswere quantitated by densitornotry, and the RNA content was normalizedby stripping the blot and rehybridizing it with a cDNA probe for glyceral-dohyde-3-phosphate dohydrogenase.

AcknowledgmentsWe are indebted to R. Hauko for the careful preparation of the manuscript.

References1 . Shorr, C. J. G1 phase progression: cycling on cue. Cell, 79: 551-555,1994.

2. Pines, J. Protein kinasos and cell cycle control. Somin. Cell Biol., 5:399-408, 1994.

3. Moyerson, M., Enders, G. H., Wu, C-L, Su, LK., Gorka, C., Nelson, C.,Harlow, E., and Tsai, L H. A family of human cdc2-related protein kinases.EMBO J., 11: 2909-2917, i992.

4. Hunter, T., and Pines, J. Cyclins and cancer. Cell, 66: 1071-1074,1991.

5. Ohtsubo, M., Thoodoras, A. M., Schumacher, J., Roberts, J. M., andPagano, M. Human cyclin E, a nuclear protein essential for the G1-to-Sphase transition. Mol. Cell. Biol., 15: 2612-2624, 1995.

6. Pagano, M., Pepporkok, R., Verde, F., Ansorge, W., and Draetta, G.Cyclmn A is required at two points in the human cell cycle. EMBO J., 11:961-971, 1992.

7. Dou, 0. P., Levin, A. H., Zhao, S., and Pardee, A. B. Cyclin E and cyclinA as candidates for the restriction point protein. Cancer Res., 53: i 493-1497, 1993.

CDK2/Cyclin Complexes in Endomitosis

54. Long, M. W., and Hoffner, C. H. Detection of human megakaryocyte

antigonsbysolid-phasoradioimmunoassay. Exp. HematOl, 16:62-70, 1988.

8. Dulic, V., Lees, E., and Rood, S. I. Association of human cyclin E with

a periodic G1-S phase protein kinase. Science (Washington DC), 257:1958-1961, i992.

9. Girard, F., Strausfeld, U., Fernandez, A., and Lamb, N. J. Cyclin A isrequired for the onset of DNA replication in mammalian fibroblasts. Cell,

67: 1169-1179, 1991.

10. Hadwiger, J. A., Wittenberg, C., Richardson, H. E., do Barros Lopes,M., and Reed, S. A family of cyclin homologues that controls the G1 phase

in yeast. Proc. NatI. Acad. Sci. USA, 86: 6225-6259, 1989.

ii. Matsushime, H., Ewen, M. E., Strom, D. K., Kato, J. Y., Hanks, S. K.,Roussel, M. F., and Sherr, C. J. Identification and properties of an atypicalcatalytic subunit (p34 psk-j3/cdk4) for mammalian D type G1 cyclins. Cell,

71: 323-334, 1992.

12. Hunter, T., and Pines, J. Cyclins and cancer. II. Cyclin D and CDKinhibitors come of age. CoIl, 79: 573-582, 1994.

13. Xiong, V., Zhang, H., and Beach, 0. D-type cyclins associate withmultiple protein kinases and the DNA replication and repair factor PCNA.

Cell, 71: 505-514, 1992.

14. Hannon, G. J., and Beach, D. pi5���4B is a potential effector ofTGF-�3-induced cell cycle arrest. Nature (Lond.), 371: 257-21 6, 1994.

15. Hirama, T., and Kooffler, H. P. The role of cyclin-dependent kinaseinhibitors in the development of cancer. Blood, 86: 841-854, 1995.

i6. Sauer, K., Knoblich, J. A., Richardson, H., and Lehner, C. F. Distinctmodes of cyclin E/cdc2c kinase regulation and S-phase control in mitoticand endoreduplication cycles of Drosophila embryogonesis. Genes Dov.,

9: 1327-1339, 1995.

17. Polyak, K., Lee, M. H., Erdjument-Bromage, H., Koff, A., Roberts, J.M., Tempst, P., and Massagu#{233},J. Cloning of p27’�Pl, a cyclmn-depondentkinase inhibitor and a potential mediator of extracellular antimitogenic

signals. Cell, 78: 59-66, 1994.

18. Reynisdottir, I., Polyak, K., lavarone, A., and Massague, J. Kip/Cipand lnk4 Cdk inhibitors ccoperate to induce cell cycle arrest in responseto TGF-f3. Genes Dev., 9: 1831-1945, 1995.

19. Xiong, V., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., andBeach, 0. p21 is a universal inhibitorofcyclin kinases. Nature(Lond.), 366:70i-704, 1993.

20. Waldman, T., Lengauer, C., Kinzler, K. W., and Vogelstein, B. Uncou-

pling of S phase and mitosis induced by anticancer agents in cells lacking

p21 . Nature (Lond.), 381: 713-716, 1996.

21 . Odell, T. T., Jackson, C. W., and Friday, T. J. Megakaryocytopoiosisin rats with special reference to polyploidy. Blood, 35: 775-782, 1970.

22. Ebbe, S. Biology of mogakaryocytes. Prog. Hernostasis Thromb., 3:211-229, 1976.

23. Bennett, M. D. Nuclear characters in plants. Brcokhaven Syrnp. Biol.,25: 344-366, i973.

24. Therman, E., Sarto, G. E., and Stubblefiold, P. A. Endomitosis: areappraisal. Hum. Genot., 63: 13-18, i983.

25. Brook, D., Bariett, R., Crawford, K., and Nurse, P. Involvement ofp34cdc2 in establishing the dependency of S phase on mitosis. Nature

(Lond.), 349: 388-393, 1995.

26. Handoli, S., and Weintraub, H. The ts4l mutation in Chinese hamstercells leads to successive S phases in the absence of intervening G2, M,and G1. Cell, 71: 599-6i1, 1992.

27. Hayles, J., Fisher, D., Wcollard, A., and Nurse, P. Temporal order ofS phase and mitosis in fission yeast is determined by the state of thep34CdC2mitotic B cyclin complex. Cell, 78: 813-822, 1994.

28. Dafta, N., Williams, J. L, and Long, M. W. Alterations in cyclin-dependent kinase complex formation during the acquisition of a polyploidDNA content. Mol. Biol. Cell, 7: 209-223, 1996.

29. Grafi, G., and Larkins, B. A. Endoreduplication in maize ondosporm:involvement of M phase-promoting factor inhibition and induction of S

phase-related kinases. Science (Washington DC), 269: 1262-1264, 1995.

30. Heichman, K. A., and Roberts, J. M. Rules to replicate by. Cell, 79:557-562, 1994.

31 . Long, M. W., Heffnor, C. H., Williams, J. L, Peters, C., andProchownik, E. V. Regulation of megakaryocyte potential in human eryth-

roleukemia cells. J. Clin. Investig., 85: 1072-1084, 1990.

32. Xiong, V., Connolty, T., Futcher, B., and Beach, D. Human D-typecyclin. Cell, 65: 691-699, 1991.

33. Xiong, V., Minwinger, J., Beach, 0., and Ward, D. C. Molecular cloning

and chromosomal mapping of CCND genes encoding human 0-typecyclins. Genomics, 13: 575-584, 1992.

34. Ajonbaum, F., Ando, K., Docaprie, J., and Griffin, J. 0. Independentregulation of human 0-type cyclin expression during G1 phase in primary

human T lymphocytes. J. Biol. Chom., 268: 41 13-41 19, 1993.

35. Lucas, J. J., Terada, N., Szepesi, A., and Golfand, E. W. Regulation of

synthesis of �cdc2 and its homologenes and their relationship top1 iORb phosphorylation during cell cycle progression of normal human Tcells. J. Immunol., 148: i804-l8ii, 1992.

36. Koff, A., Griordino, A., Dosai, 0., et al. Formulation and activation ofa cyclin E-cdk2 complex during G1 phase of the human cell cycle. Science(Washington DC), 257: i689-i694, 1992.

37. Kato, J. V., Matsuoka, M., Strom, 0. K., and Shorr, C. J. Regulation ofcyclmn 0-dependent kinaso 4 (odk4) by odk4-activating kinaso. Mol. Cell.

Biol., 14: 2713-2721, 1995.

38. Lees, E., Faha, B., Dulic, V., Rood, S. I., and Harlow, E. Cyclin E/cdk2and cyclin Ncdk2 kinasos associate with p107 and E2F in a temporallydistinct manner. Genes 0ev., 6: 1874-1885, 1992.

39. Pines, J., and Hunter, T. Human cyclins A and Bi are differentiallylocated in the cell and undergo cell cycle-dependent nuclear transport.J. Cell. Biochem., 115: 1-17, 1991.

40. Pines, J., and Hunter, T. The differential localization of human cyclinsA and B is due to a cytoplasmic retention signal in cyclin B. EMBO J., 13:

3272-3781, 1994.

41 . Hengst, L, and Reed, S. I. Translational control of p27� accumulationduring the cell cycle. Science (Washington DC), 271: 1861-1864, 1996.

42. Nourso, J., Firpo, E., Flanagan, W. M., Coats, S., Polyak, K., Lee, M.H., Massague, J., Crabtree, G. R., and Roberts, J. M. Interloukin-2-mediated elimination of the p27’�Pl cyclin-dependent kinaso inhibitorprevented by rapamycin. Nature (Lend.), 372: 570-573, 1994.

43. Freeman, M., Nussloin-Volhard, C., and Glover, 0. The dissociation ofnuclear and centrosomal division in gnu, a mutation causing giant nuclei

in Drosophila. Cell, 46: 457-468, 1986.

44. Kato, J., and Sherr, C. J. Inhibition of granulocyte differentiation by G1

cyclins 02 and 03 but not Dl . Proc. NatI. Aced. Sci. USA, 90: 11513-1i517, 1993.

45. Ewen, M. E., Stuss, H. K., Sherr, C. J., Matsushimo, H., Kate, J., andLivingston, 0. Functional interaction of the retinoblastoma protein with the

mammalian 0-type proteins. Cell, 73: 487-497, 1993.

46. Sweeney, K. J. E., Sariban, E., Sutherland, R. L, and Musgrove, E. A.Cyclin 02 activates cdk2 in preference to cdk4 in human breast epitheliaJ

cells. Oncogono, 14: 1329-1340, 1997.

47. Wang, Z., Zhang, V., Kamen, 0., Lees, E., and Ravid, K. Cyclmn 03 isessential for megakaryocytopoiesis. Blood, 86: 3783-3788, 1995.

48. Tsai, L H., Lees, E., Faha, B., Harlow, E., and Riabowol, K The odk2kinase is required for the G1-to-S transition in mammalian cells. Onco-gone, 8: 1593-1602, 1993.

49. Garcia, P., and Cales, C. Endoreplication in mogakaryoblastic celllines is accompanied by sustained expression of G1-S cyclmns and down-regulation of cdc25C. Oncogone, 13: 695-703, 1996.

50. Zhang, Y., Wang, Z., and Ravid, K. The cell cycle in polyploidmegakaryocytes is associated with reduced activity of cyclin B1 -depend-

ent cdc2 kinase. J. Biol. Chem., 271: 4266-4272, 1996.

51 . Odoll, T. T., Jr., and Jackson, C. W. Polyploidy and maturation of rat

megakaryocytes. Blood, 32: 102-1 10, 1968.

52. Heichman, K A., and Roberts, J. M. Thoyoast CDCi6and CDC27 genes

restrict DNA replication to once per cell cycle. Cell, 85: 39-48, 1996.

53. Laemmli, U. K. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.