Cyclin D2 Activates Cdk2 in Preference to Cdk4 in Human Breast Epithelial Cells

Cyclin D2 activates Cdk2 in preference to Cdk4 in human breast epithelial cells

Kimberley J Sweeney, Boris Sarcevic, Robert L Sutherland and Elizabeth A Musgrove

Cancer Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, NSW 2010, Australia

To investigate the possibility of diering roles for cyclins Similarly, overexpression of cyclin D2 in myeloid cells

D1 and D2 in breast epithelial cells, we examined the results in a decrease in the duration of G1 and an expression, cell cycle regulation and activity of these two increase in the percentage of cells in S-phase (Ando et

G1 cyclins in both 184 normal breast epithelial cells and al., 1993; Kato and Sherr, 1993). Microinjection or T-47D breast cancer cells. Synchronisation studies in 184 electroporation of cyclin D1 or cyclin D2 antibodies cells demonstrated that cyclin D1 and cyclin D2 were demonstrated that these proteins were not only rate- dierentially regulated during G1, with cyclin D2 limiting but essential for progress through G1 (Baldin abundance increasing by 3.7-fold but only small changes et al., 1993; Quelle et al., 1993; Lukas et al., 1995b). in cyclin D1 abundance observed. The functional These eects are thought to be mediated by activation consequences of increased cyclin D2 expression were of cyclin-dependent kinases (CDKs) and consequent examined in T-47D cells, which express no detectable phosphorylation of the product of the retinoblastoma cyclin D2. Induced expression of cyclin D2 resulted in susceptibility gene, pRB (Hunter and Pines, 1994; increases in cyclin E expression, pRB phosphorylation Sherr, 1994). Phosphorylation of pRB by cyclin/CDK and the percentage of cells in S-phase, while constitutive complexes in late G1 phase relieves its inhibitory role expression resulted in a consistent trend toward reduced by releasing transcription factors, such as E2F, dependence on serum for continued proliferation. Thus, essential for progression through S-phase (Sherr, cyclin D2 is a positive regulator of G1 progression in 1994; Weinberg, 1995). Cyclin D-associated CDKs breast cells analogous to the well-documented eects of are pRB kinases in vitro (Matsushime et al., 1994; cyclin D1. Indeed, equimolar concentrations of inducible Meyerson and Harlow, 1994) and, together with cyclin cyclin D1 and D2 resulted in quantitatively similar cell E/Cdk2, are also important pRB kinases in vivo (Sherr, cycle eects. Marked divergence was found, however, in 1994; Weinberg, 1995). There is clear evidence that the CDKs activated by the two cyclins in breast both cyclin D1 and D2 have a codependent relation- epithelial cells. Cyclin D2 complexes contained a higher ship with pRB: cells that lack functional pRB have Cdk2/Cdk4 ratio than cyclin D1 complexes. The cyclin reduced expression of these D type cyclins (Bates et al., D2-associated kinase activity was largely inhibited by 1994b; Lukas et al., 1995a) and ectopic expression of Cdk2-speci®c inhibitors and could phosphorylate histone either protein can rescue cells from the growth- H1, a substrate for Cdk2 but not for Cdk4 and Cdk6. suppressive eect of pRB (Dowdy et al., 1993; Ewen

Therefore, cyclin D2 preferentially activated Cdk2 in et al., 1993). These similar roles in G1 progression and breast epithelial cells. In contrast, Cdk4 and Cdk6 were interaction with pRB, together with the observed predominantly responsible for cyclin D1-associated sequence homology suggest that cyclins D1 and D2 kinase activity as previously reported. Thus, although have similar functional properties. cyclins D1 and D2 elicited similar eects on breast In spite of these similarities in function, human epithelial cell cycle progression they appeared to achieve cyclin D1 and D2 are more closely related to the this end via activation of dierent CDKs. This is the ®rst corresponding murine cyclins than to each other evidence of cyclin D2 activating Cdk2 in mammalian (490% amino acid identity, compared with 63%). cells thus providing further evidence that D-type cyclins Across a range of human diploid cells and tumor cell are not necessarily redundant. lines of breast, colon, lymphoid and sarcoma origin, cyclin D2 expression was restricted compared to cyclin Keywords: cyclin D2; cyclin D1; breast cancer; CDK D1 (Buckley et al., 1993; Palmero et al., 1993; Lukas et al., 1995b). Cyclin D1 was expressed in most of the cell types examined with the exception of T lymphocytes, which only expressed cyclin D2. Osteosarcomas and Introduction normal breast epithelial cells expressed both cyclins D1 and D2 (Buckley et al., 1993; Lukas et al., 1995b). Cyclins D1 and D2 are closely related proteins that Where both cyclins are expressed they appear to act control cell cycle progression during G1 phase. Induced cooperatively in controlling G1 progression (Bartkova expression of ectopic cyclin D1 in rodent ®broblasts or et al., 1995). This raises the possibility that their human epithelial cells accelerates transit through G1 functions are not wholly redundant. phase, decreases cell size and reduces dependence on Some studies have pointed to functional dierences serum (Jiang et al., 1993; Quelle et al., 1993; Musgrove between cyclins D1 and D2 e.g. overexpression of et al., 1994; Resnitzky et al., 1994; Zwijsen et al., 1996). cyclin D2, but not cyclin D1, in 32D myeloid cells disables the dierentiation pathway (Kato and Sherr, Correspondence: E Musgrove 1993). A basis for functional dierences is suggested by Received 10 September 1996; revised 5 November 1996; accepted 11 dierences in the ability of cyclins D1 and D2 to November 1996 interact with other cellular proteins including CDKs. Cyclin D2 in breast cancer cells KJ Sweeney et al 1330 Although cyclins D1 and D2 bind Cdk2, Cdk4 and Cdk6 in mammalian cells (Matsushime et al., 1992; a Xiong et al., 1992b; Bates et al., 1994a; Meyerson and Harlow, 1994; Lukas et al., 1995b), the cyclin D1-Cdk2 complex appears to be inactive (Dulic et al., 1993; Higashi et al., 1996). While cyclin D1 or cyclin D2 immunoprecipitates phosphorylate pRB in vitro,a number of laboratories have been unable to demonstrate cyclin D1-associated kinase activity using histone H1, the preferred in vitro substrate for Cdk2 (Matsushime et al., 1991, 1994; Ewen et al., 1993; Quelle et al., 1993). Consistent with these observations, cyclin D1 activates Cdk4 and Cdk6, but not Cdk2, when coexpressed in insect cells whereas cyclin D2 activates all three kinases in this system (Matsushime et al., 1992, 1994; Ewen et al., 1993; Meyerson and Harlow, 1994). Although both cyclin D1 and D2 are putative oncogenes and are expressed in at least some breast epithelial cells, only cyclin D1 overexpression has been b implicated in the development and progression of Mitogen Stimulated Controls breast cancer. Cyclin D1 is ubiquitously expressed in epithelial cell lines derived from both normal breast Cyclin D1 and breast cancer (Buckley et al., 1993; Lukas et al., Cyclin D2 1994, 1995b; Tam et al., 1994b) and is frequently overexpressed in breast cancer (Buckley et al., 1993; Cyclin E Bartkova et al., 1994; Gillett et al., 1994; Hui et al., Cdk2 1996). In contrast, cyclin D2 is expressed at very low levels or is absent in most breast cancer cell lines Cdk4

(Buckley et al., 1993; Tam et al., 1994b; Lukas et al., Cdk6 1995b). Cyclin D2 mRNA and protein are abundant in ¨

ppRB some normal breast epithelial cells but very little cyclin ¨ pRB D2 protein was detected in cultured breast luminal 36 91216202461224 epithelial cells, the cell type from which most breast Time (h) cancers evolve (Lukas et al., 1995b). Cyclin D1 is an important cell cycle regulatory gene c in human breast epithelial cells where it is both

necessary and sucient for G1 progression and is intimately involved in the mitogenic response to growth factors and steroids (Sweeney et al., 1996). Furthermore, cyclin D1 is necessary for normal mammary development (Fantl et al., 1995; Sicinski et al., 1995). In contrast, the role of cyclin D2 in these cells remains to be established since there are relatively few published data addressing the functional signifi- cance of cyclin D2 and these studies are mostly con®ned to rodent ®broblasts and haematopoietic cells (Ajchenbaum et al., 1993; Ando et al., 1993; Kato and Sherr, 1993; Quelle et al., 1993; Lukas et al., 1995b). Consequently, to address the potential signi®cance of the dierential expression of cyclins D1 and D2 in normal and neoplastic breast epithelial cells we compared the regulation and function of these cyclins in normal breast epithelial cells expressing both cyclins and in T-47D human breast cancer cells Figure 1 Eects of mitogen stimulation on growth arrested 184 normal breast epithelial cells. 184 breast epithelial cells were expressing ectopic cyclin D2. growth arrested in growth factor-depleted medium then stimulated at 0 h with 0.4% bovine pituitary extract. Replicate ¯asks were harvested at intervals for analysis. (a) Flow cytometric analysis of DNA content. G1 phase (&); S-phase (&); G2/M Results phase (*). (b) Western blot analysis of 184 breast cell lysates following mitogen stimulation. Control cells received fresh growth Cyclin D2 and cyclin D1 are dierentially regulated factor-depleted medium containing only 0.05% bovine pituitary extract. Replicate ®lters were blotted with antibodies speci®c for following growth factor stimulation of normal breast the proteins indicated. (c) Densitometric values from autoradio- epithelial cells graphs of two independent Western blot experiments were obtained and the average fold increase above the average of To determine whether cyclins D1 and D2 were co- controls maintained in growth factor-depleted medium is ordinately regulated during cell cycle progression, the presented. Cyclin D1 (&); cyclin D2 (&); cyclin E (~) Cyclin D2 in breast cancer cells KJ Sweeney et al 1331 a (*22%) (Figure 1a). The percentage of cells in S- Cyclin phase increased markedly between 6 and 12 h after D2 D1 growth factor addition, reaching a maximum of 34% at 16 h. The increase in the percentage of cells in S- Cdk2 phase was followed by an increase in the G2+M fraction, between 12 and 16 h. Thus the mitogen- Cdk4 stimulated cells progressed synchronously through the Cdk6 cell cycle and this was accompanied by the sequential induction of G1 cyclins (Figure 1a and b). Expression IP Iysate IP of cyclin D2 protein began to increase in early G1 phase and continued to increase, reaching a maximum at 16 h (3.7-fold), when cells were in mid-S-phase, after b which the protein expression declined. Cyclins D1 and IP Inhibitor Substrate E showed little change during the timecourse, with at — p21 p16 ros most a 30% increase in cyclin D1 by late G1 (Figure Cyclin D2 GST-pRB 1c). Similarly, there was little change in the expression of Cdk4 or Cdk6. In contrast, there was a fourfold increase in Cdk2 protein beginning as early as 6 h after Cyclin D1 GST-pRB mitogen treatment which was maintained for the 24 h of the experiment (Figure 1b). Thus, while some of the — p21 ros olo changes were modest, the degree of synchrony was sucient to detect changes in the expression of cyclin Cyclin D2 Histone H1 D2 and indicate clear dierences in the pattern of induction of cyclins D1 and D2 during progress through the cell cycle in these breast epithelial cells. c Since pRB is a physiological target for the activities of these cyclins and CDKs, changes in pRB expression and phosphorylation were also examined. In growth factor-depleted cells low levels of pRB were detected, predominantly in the faster migrating, underphosphorylated form (Figure 1b). The total abundance of pRB increased markedly as cells progressed into S- phase at 9 ± 12 h. In addition, the proportion of pRB in the more slowly migrating, phosphorylated form (ppRB) increased and by 9 h this was the predominant form. These data are compatible with growth factor stimulated cell cycle progression being mediated by

increased expression of G1 cyclins, leading to activation of CDKs and pRB phosphorylation as has been previously described in several cell systems. However, perhaps unexpectedly they reveal a potential major role Figure 2 Cyclin D1 and D2 complex formation and kinase for cyclin D2 in cells that also express cyclin D1. activity in 184 normal breast epithelial cells. (a) Cyclins D1 and D2 were immunoprecipitated from exponentially growing 184 breast epithelial cells using antibodies speci®c for each cyclin. The Cyclin D2 associates with Cdk2, Cdk4 and Cdk6 and the immunocomplexes were separated by SDS ± PAGE and blotted cyclin D2/Cdk2 complex is active in 184 cells for the presence of Cdk2, Cdk4 and Cdk6. The lane marked `lysate' shows 184 cell lysate blotted in parallel with the speci®c To identify whether cyclins D1 and D2 display CDK antibodies. (b) Cyclin D2 or cyclin D1 immunocomplexes biochemical dierences re¯ecting their dierential from exponentially growing 184 cells were used in an in vitro kinase assay using recombinant GST-pRB protein or histone H1 regulation in breast epithelial cells, CDK association protein as substrate. These kinase reactions were performed in the and activity were examined in exponentially proliferat- absence or presence of 5 mg GST-p21 or GST-p16 fusion proteins ing 184 cells. Both cyclin D1 and cyclin D2 or 50 mM of the Cdk2-speci®c chemical inhibitors roscovitine (ros) immunoprecipitates from 184 cells contained Cdk2, or olomoucine (olo). (c) The results of three independent cyclin Cdk4 and Cdk6 within the complexes as detected by D1 and cyclin D2 kinase assays, using GST-pRB substrate, are shown as percentage inhibition relative to activity without Western blot analysis but there were marked differ- inhibitor addition ences in the relative abundance of dierent CDKs in the cyclin D1 and D2 complexes (Figure 2a). Whole cell lysate blotted in parallel indicated that the percentage of the total Cdk2 pool immunoprecipitated 184 normal breast epithelial cell line was arrested in with cyclin D2 was greater than the percentage of the growth factor depleted medium and then stimulated to Cdk4 or Cdk6 pools bound to cyclin D2. Indeed Cdk4 re-initiate cell cycle progression by the addition of a was only apparent in cyclin D2 complexes upon growth supplement containing bovine pituitary extract. extended exposure of the autoradiograms in contrast Analysis of cell cycle phase distribution by ¯ow with the readily apparent Cdk4 protein in cyclin D1 cytometry indicated that growth factor-depleted cells complexes. To test if this dierence in complex had a markedly reduced percentage of cells in S-phase composition was re¯ected in substrate speci®city, the (9%) compared with exponentially growing cells dierential activity of cyclin D1 and D2 complexes was Cyclin D2 in breast cancer cells KJ Sweeney et al 1332 examined using antibodies speci®c for the individual cells transfected with the vector only, i.e. T-47D DMT cyclins (Lukas et al., 1995b; Musgrove et al., 1996). cells, were employed as controls. Since preliminary Kinase activity was readily detected in both cyclin D2 experiments indicated that serum-free medium allowed and cyclin D1 immunoprecipitates from 184 cells using maximum stimulation of the transfected gene in this a bacterially expressed truncated GST-pRB fusion system (data not shown), cells were transferred to protein as substrate (Figure 2b). Preincubation of the serum-free medium containing insulin at the time of immune complexes with recombinant GST-p16, a zinc addition. Under these conditions T-47D cells still speci®c Cdk4/Cdk6 inhibitor (Serrano et al., 1993), proliferate exponentially, albeit sub-maximally. Signifi- signi®cantly decreased the pRB kinase activity of the cyclin D1 complexes but had little or no eect on the activity of the cyclin D2 complexes (Figure 2b and c). In contrast, Cdk2-speci®c chemical inhibitors, olomoucine and roscovitine (Glab et al., 1994; Vesely et al., a 1994; Schulze-Gahmen et al., 1995), decreased cyclin Zinc treated control D2-associated kinase activity but had little eect on cyclin D1-associated kinase assays performed in Cyclin D2 parallel (Figure 2b and c). As expected, the general Cyclin D1 CDK-inhibitor p21 (WAF1, CIP1, Harper et al., 1993; Xiong et al., 1993) signi®cantly inhibited both cyclin Cyclin E D1- and D2-associated kinase activity (Figure 2b and Cdk 2 c). Thus the cyclin D1-associated kinase activity in 184 cells was apparently due to Cdk4 and/or Cdk6 while in Cdk 4 contrast much of the cyclin D2-associated kinase activity appeared to be due to Cdk2. To con®rm this Cdk 6 conclusion cyclin D2-associated kinase assays were UT136912 18 24 6 12 24 performed using histone H1 as substrate, since this is a Time (h) preferred substrate for Cdk2 but is not phosphorylated by Cdk4 or Cdk6. These assays demonstrated that b cyclin D2 immunoprecipitates contained histone H1 kinase activity that was inhibited by GST-p21, olomoucine and roscovitine (Figure 2b). As has been reported by others (Dulic et al., 1993; Higashi et al., 1996) cyclin D1 complexes, which contained Cdk2 in breast epithelial cells, did not phosphorylate histone H1 (data not shown).

Induction of cyclin D2 in T-47D breast cancer cells is followed by increases in cyclin E protein expression, pRB phosphorylation and percentage of cells in S-phase c Since the data presented above indicated that cyclins D1 and D2 activated dierent CDKs and therefore might have diering functions in breast epithelial cells, the eects of ectopic cyclin D2 expression were examined in T-47D human breast cancer cells. This cell line was chosen because, unlike the 184 cell strain, it has no detectable cyclin D2 protein. Furthermore, T- 47D cells do not exhibit ampli®cation or overexpres-

sion of other G1 cyclins (Buckley et al., 1993). Following growth factor treatment these cells display sequential induction of cyclin mRNA accompanying Figure 3 Eects of zinc induction of cyclin D2 in T-47D cell cycle progression, consistent with data from other DMTcyclin D2 cells. (a) Western blot analysis of cell-cycle related cell types, indicating that these cells retain at least some proteins after zinc treatment at 0 h of T-47D DMTcyclin D2 cells of the features of cell cycle control present in normal proliferating in serum-free media with insulin. Cells were cells (Musgrove et al., 1993). Furthermore, the eects harvested at the times indicated after 40 mM zinc or vehicle (control) treatment (UT=untreated). Equal amounts of cleared of ectopic cyclin D1 expression in T-47D cells have cell lysate from each time-point were separated by SDS ± PAGE, been well-characterised: cyclin D1 induction is followed transferred to nitrocellulose and blotted for the proteins indicated. by induction of cyclin E, pRB phosphorylation, and (b) Graphical representation of the average relative increase of acceleration through G phase (Musgrove et al., 1994, cyclin D2 protein following zinc treatment of T-47D DMTcyclin 1 D2 cells in two independent experiments. (c) The left panel shows 1996). Hence it was possible to directly compare the the expression of cyclin E and cyclin D1 proteins following zinc eects of the two D-type cyclins in the same cell line. treatment of T-47D DMTcyclin D2 cells relative to the average of T-47D cells were cotransfected with a vector containing vehicle treated controls as the mean of two independent the coding region of cyclin D2 under the control of a experiments. Cyclin E (&); cyclin D1 (*). The panel on the right compares cyclin E protein expression at 0 and 12 h zinc-inducible promoter (pDMTcyclin D2) and following zinc treatment of T-47D DMTcyclin D2 cells (solid pSV2neo, and G418-resistant pools of cells selected to bars) with that in the control, vector-transfected T-47D DMT cells generate T-47D DMTcyclin D2 cells. Similarly selected (hatched bars) Cyclin D2 in breast cancer cells KJ Sweeney et al 1333 cant amounts of cyclin D2 protein were detectable as early as 3 h following zinc treatment and maximal a protein expression occurred at *12 h (Figure 3a and Zinc Control b). To identify the molecular events occurring — ppRB subsequent to the induction of cyclin D2 we examined — pRB the expression of other cyclins and CDKs over the same time-course. Cdk4 and Cdk2 protein levels UT1 3 6 9 12 18 24 6 12 24 Time (h) remained constant for the duration of the experiment while Cdk6 displayed approximately a twofold increase b at 12 to 16 h after zinc treatment (Figure 3a and data not shown). Cyclin D1 protein levels did not change during G1 but declined after 18 h of zinc treatment when a signi®cant proportion of the cells were in S- phase. Cyclin E protein levels began to increase above control levels *3 h after the appearance of cyclin D2 protein and reached maximum levels at *12 h after zinc treatment (Figure 3a and c). This increase in cyclin E expression was associated with cyclin D2 induction rather than zinc treatment since there was little eect of zinc treatment on cyclin E expression in T-47D DMT cells (Figure 3c, right panel). Phosphorylation of pRB has been observed following cyclin D1 induction in T-47D cells (Musgrove et al., 1996). Therefore, pRB phosphoryla- c

tion in vivo was examined following cyclin D2 induction. Zinc induction of cyclin D2 led to ¨ pRB-GST increased pRB abundance and increased phosphorylation (Figure 4a). The ratio of phosphorylated to +p21 underphosphorylated pRB (i.e. ppRB/pRB) relative to IR Ab 0 hour 9 hour that in vehicle-treated control cells increased after 6 h, 12 hour 12 hour reaching a maximum at 18 h (Figure 4B). Little change in pRB phosphorylation occurred within 18 h d of zinc treatment of T-47D DMT cells. Thus, the increase in pRB phosphorylation occurred approximately 3 ± 6 h after cyclin D2 induction which is compatible with activation of endogenous CDKs by ectopic cyclin D2. To con®rm that cyclin D2 induction increased cyclin D2-associated kinase activity, the kinase activity of cyclin D2 immunoprecipitates was measured using GST-pRB substrate. In untreated T-47D DMTcyclin D2 cells only background phosphorylation was detected, similar to that obtained in parallel immunoprecipitates using an irrelevant control antibody (a mouse monoclonal IgG2b isotypic control antibody). However, following 9 or 12 h zinc treatment, when substantial amounts of cyclin D2 protein were present, kinase activity was increased by up to threefold above background phosphorylation at 0 h (Figure 4c). Apparent in this Figure 4 Eect of zinc induction of cyclin D2 on pRB protein ®gure is an unidenti®ed, phosphorylated band that phosphorylation in T-47D DMTcyclin D2 cells. (a) Western blot migrated just above the GST-pRB in vitro substrate. analysis of endogenous pRB protein after zinc treatment or water treatment (controls) of T-47D DMTcyclin D2 cells. The upper band This was a consistent ®nding in zinc-treated cells and represents the hyperphosphorylated ppRB whilst the lower band may represent an endogenous substrate that coimmu- represents the under-phosphorylated form of the protein, pRB. (b) noprecipitates with the cyclin D2 complex. The left panel is a graphical representation of the ratio of CDK activation and pRB phosphorylation were phosphorylated to underphosphorylated pRB protein (i.e. ppRB/ pRB) at various times after zinc treatment of the T-47D DMTcyclin followed by an increase in the number of cells entering D2 transfected cells. The right panel compares the change in pRB S-phase and corresponding decrease in the proportion phosphorylation following zinc treatment of T-47D DMTcyclin D2 of cells in G1 phase upon induced expression of cyclin (solid bars) and vector-transfected T-47D DMT cells (hatched bars). D2 in T-47D cells. This was ®rst apparent at 12 h and (c) Cyclin D2 immunocomplexes from T-47D DMTcyclin D2 cells was maximal at *18 h, when G had decreased from treated with zinc for various times were used in an in vitro kinase 1 assay using recombinant GST-pRB as substrate. IR Ab=lysates 79% to 46% and S-phase had increased from 17% to immunoprecipitated with an irrelevant antibody (mouse IgGb 45% (Figure 4d). Identical experiments in control cells isotypic control antibody). Where indicated (+p21) the kinase transfected with the empty vector demonstrated that reaction was performed in the presence of 5 mg of GST-p21. (d) zinc alone was capable of inducing a modest degree of Eects of zinc induction of cyclin D2 on cell cycle progression. The percentage of cells in S phase was determined by ¯ow cytometry of cell cycle progression. The cyclin D2-expressing cells, T-47D DMTcyclin D2 (&, &) or vector-transfected T-47D DMT however, consistently showed a greater number of cells cells (*) following treatment with zinc (&, *) or vehicle (water, &) Cyclin D2 in breast cancer cells KJ Sweeney et al 1334 progressing into S-phase in parallel experiments con®rming that the cell cycle changes were not solely due to zinc treatment (Figure 4d and data not shown). a Thus, these studies indicated many similarities between the eects of cyclin D1 and cyclin D2 induction in T- 47D cells: induction of either cyclin was followed by induction of cyclin E, pRB phosphorylation and cell cycle progression. CMV Cyclin D2 (A) CMV Cyclin D2 (B) CMV Cyclin D2 (C) CMV Cyclin D2 (D) CMV (A) CMV (B) CMV (C) 184

L Cyclin D2 Ectopic expression of cyclin D2 does not render T-47D cells completely mitogen independent b In view of data which indicated that increased expression of D-type cyclins in breast cancer cells resulted in reduced serum dependence (Musgrove et al., 1994; Zwijsen et al., 1996), the eect of cyclin D2 expression on the response to serum deprivation was examined using T-47D cells transfected with cyclin D2 under the control of the constitutive CMV promoter. Four independent pools of cyclin D2-transfected cells and three control pools transfected with vector alone were generated. Western blotting of lysates (Figure 5A) con®rmed that cyclin D2 was expressed in the T-47D CMVcyclin D2 lines but not in the T-47D CMV control lines. The growth rates of representative cell c lines were then determined and doubling times calculated during exponential growth in dierent serum concentrations (Figure 5b). In each of four independent experiments, the doubling time of vector- transfected cells maintained in medium supplemented with 5% FCS was 1 ± 1.5 days, similar to the doubling time of stock cultures of the parental T-47D cells. The doubling time increased at lower serum concentrations to reach 2.5 ± 3.5 days in the absence of serum, again consistent with data obtained using the parental cell line (Reddel et al., 1985). In four independent experiments there was a consistent trend towards lower doubling times in cyclin D2-transfected cells Figure 5 Cell cycle phase distribution and doubling times of T- compared with vector-transfected control cells over a 47D CMVcyclin D2 cells. (a) Western blot analysis of cyclin D2 range of serum concentrations. In some experiments, expression in lysates from four independent pools of T-47D CMV cyclin D2 cells and three pools of T-47D CMV control lines. 184 such as that shown in Figure 5b, the doubling time of cell lysate (184) served as a positive control for cyclin D2 the cyclin D2-expressing cells in serum-free medium expression. (b) Representative data from one of four independent was lower than that of control cells but such clear experiments showing the doubling times of T-47D CMV+cyclin distinction between doubling times was not consistently D2 cells in medium containing the indicated concentration of seen and the dierences did not always reach statistical FCS. T-47D CMV (open bars); T-47D CMVcyclin D2 (solid bars). (c) The percentage of cells in S-phase after 5 days in serum- signi®cance. In parallel experiments the cell cycle phase free medium or medium supplemented with 5% FCS is shown. T- distribution of cyclin D2-transfected cells was also 47D CMV (open bars); T-47D CMVcyclin D2 (solid bars) examined in a range of serum concentrations. In serum-containing medium there was no dierence in the cell cycle phase distribution between cyclin D2 transfectants and controls (Figure 5c). In serum free of cyclin D2 was not sucient to render the cells medium, however, there was a statistically signi®cant completely mitogen-independent. (P=0.041) increase in the percentage of cells in S-phase in the cyclin D2-expressing cells compared to the Cyclin D2 associates with Cdk2, Cdk4 and Cdk6 when control cells (Figure 5c). Together these data suggest ectopically expressed in T-47D cells and both cyclin D2/ that constitutive expression of cyclin D2 has a similar Cdk2 and cyclin D2/Cdk4/Cdk6 complexes are active eect to constitutive expression of cyclin D1 in breast cancer cells (Zwijsen et al., 1996), i.e. to decrease the The data in Figure 2 raised the possibility that the dependence on serum for proliferation. In the eects of cyclin D1 and D2 in breast cancer cells might experiment shown it appeared that the doubling time be mediated by dierent CDKs. To address this of cyclin D2-expressing cells in 0% FCS diered little possibility exogenous cyclin D2 was immunoprecipi- from the doubling time in 5% FCS (Figure 5b) but tated from T-47D cyclin D2 transfectants and the again this was not a consistent ®nding. In most associated CDKs identi®ed by Western blotting. Cdk2, experiments the doubling time was clearly increased Cdk4 and Cdk6 were all associated with cyclin D2 with reduction of serum concentration in the cells (Figure 6a). Consistent with the data from 184 cells expressing cyclin D2 and hence constitutive expression presented in Figure 2, there was a greater percentage of Cyclin D2 in breast cancer cells KJ Sweeney et al 1335 levels of histone H1 kinase activity were observed a (Figure 6b). While the GST-pRB kinase activity from IP: Cyclin D2 the cyclin D2-expressing cells was inhibited by Cdk2 preincubation with GST-p21 addition of GST-p16 was less eective (data not shown). Together these data indicate that, like the endogenous cyclin D2 in Cdk4 184 cells, ectopically expressed cyclin D2 activated Cdk2 to a greater extent than Cdk4 or Cdk6. Cdk6 IP Iysate Induction of cyclin D2 or cyclin D1 has quantitatively similar eects on T-47D cell cycle progression The eects of induction of cyclin D2 on cell cycle b progression, cyclin E expression and pRB phosphorylation in T-47D cells were qualitatively similar to

L H1 those previously described for cyclin D1 (Musgrove et al., 1994). However, the eects of cyclin D1 on the

+p21 percentage of cells moving into S-phase appeared quantitatively greater than those of cyclin D2. Cyclin D2 Cyclin D2 -47D CMV -47D CMV -47D CMV

T T T Treatment of T-47D DMTcycD1-3 cells with 50 mM zinc resulted in an approximate fourfold increase in the c percentage of cells in S-phase (Musgrove et al., 1994) whilst analogous experiments in cyclin D2 transfectants yielded a less than twofold change when background due to the eects of zinc alone was subtracted (Figure 4d). This could result from a genuine dierence in the function of cyclins D1 and D2, perhaps resulting from the activation of dierent kinases by these cyclins or merely from a dierence in the level of protein expression following activation of the inducible promoter. To address this question, the concentrations of cyclin D1 and cyclin D2 proteins in cell lysates were determined from standard curves generated with recombinant cyclin D1-GST and cyclin D2-GST fusion proteins expressed in insect cells (Figure 7A). These estimates indicated that the maximum levels of cyclin D2 achieved following zinc induction of T-47D DMTcyclin D2 cells were similar to the level of cyclin D2 expression in 184 cells (Figure 7B). The cyclin D1 concentration following treatment of T-47D DMTcycD1-3 cells with 50 mM zinc was approximately Figure 6 Cyclin D2-associated proteins and kinase activity in T- four times greater than the concentration of cyclin D2 47D DMTcyclin D2 and CMVcyclin D2 cells. (a) Cyclin D2 was immunoprecipitated from lysates of exponentially growing T-47D produced following a similar treatment of T-47D DMTcyclin D2 cells treated with zinc for 12 h. The immunocom- DMTcyclin D2 cells. Previous experiments using a plexes were separated by SDS ± PAGE and blotted for the range of zinc concentrations demonstrated a linear presence of Cdk2, Cdk4 and Cdk6 (IP). The control lane marked relationship between induced cyclin D1 abundance and `lysate' shows direct blots of cell lysate from the same experiment. subsequent cell cycle progression (Figure 7C). These (b) Immunoprecipitation of cyclin D2 from exponentially growing T-47D CMV or T-47D CMVcyclin D2 cells. The cyclin D2 data were employed to assess the expected eect on the immunocomplexes were tested for in vitro kinase activity using percentage of cells in S-phase of the increment in D histone H1 substrate. Where indicated (+p21) kinase reactions cyclin levels in T-47D DMTcyclin D2 cells treated with were performed in the presence of 5 mg GST-p21 fusion protein. zinc. An increment of *0.05 ng/ml lysate, i.e. to a total (c) Graphical representation of cyclin D2 kinase activity, using histone H1 substrate, in the absence or presence of GST-p21. of *0.07 ng/ml lysate, would lead to an increase in the Results are shown as the percentage activity above negative percentage of cells in S-phase of between 11 ± 16% control (T-47D CMV cell lysate) (taken from the 95% con®dence intervals for the line of best ®t). This is comparable with the speci®c increase in the percentage of cells in S-phase due to induction of cyclin D2 of 11% at 18 h (Figure 4d). the Cdk2 pool associated with cyclin D2 than either Thus, these data suggest that cyclin D2 led to an eect Cdk4 or Cdk6. Cyclin D2-associated kinase activity on progression into S-phase quantitatively comparable was observed using both histone H1 and GST-pRB with the eect of a similar concentration of cyclin D1. substrates following ectopic expression of cyclin D2 in T-47D cells and this activity was inhibited by preincubation with GST-p21 (Figure 6b and c) and Discussion data not shown). When the same antibody was used to immunoprecipitate proteins from lysates of T-47D cells Our ®ndings indicate divergence of the regulation and transfected with vector alone, only low, background, molecular association of cyclins D1 and D2 in breast Cyclin D2 in breast cancer cells KJ Sweeney et al 1336 a combination of D-type cyclins expressed since lymphocytes, myeloid cells and murine erythroleukemia cells do not express cyclin D1 whilst 184 cells express both cyclins D1 and D2. In normal breast epithelial cells cyclin D2 protein levels were clearly mitogen-regulated

L cyclin D1/GST while little change in cyclin D1 levels was detected.

L cyclin D1 Dierential regulation of the two cyclins in response to A B mitogenic signals has been observed previously in ®broblasts where cyclin D1 but not cyclin D2 mRNA is increased in response to stimulation with both serum and PDGF (Winston and Pledger, 1993). Furthermore, the expression of D type cyclins is also dierentially regulated following induction of apoptosis or differ- b entiation in some cell types (Freeman et al., 1994; Kranenburg et al., 1995). Hence, some growth stimulatory and inhibitory signals preferentially reg- ulate one of these two D-type cyclins. This may explain why some cells apparently require both cyclin D1 and cyclin D2 whilst other cells predominantly express only one. Both endogenous cyclin D2 in 184 cells and exogenous cyclin D2 in T-47D transfectants bound c Cdk2, Cdk4 and Cdk6 (Figures 2 and 6). There was apparently less Cdk4 bound to cyclin D2 than to cyclin D1. This is consistent with a previous observation in which cyclin D2 and D1 proteins were immunoprecipitated from Rat-2 and NIH3T3 cells that had been transfected with the respective cyclin gene (Quelle et al., 1993). Since the cyclin D2 concentration in 184 cells was more than twice that of cyclin D1, the dierence in the amount of coimmunoprecipitated Cdk4 appears unlikely to re¯ect dierences in cyclin abundance but could be due to a dierence in the ability of these cyclins to stably interact with Cdk4 in these cells. In contrast to the ®ndings of Quelle et al. Figure 7 Estimation of cyclin D1 and cyclin D2 protein (1993) in Rat-2 and NIH3T3 cells, where Cdk2 was not concentrations in 184 cells or T-47D cells transfected with cyclin detected in cyclin D2 immunoprecipitates, we found D1 or D2. (A) Dilutions of puri®ed baculovirus expressed GST- cyclin D1 protein were separated by SDS ± PAGE along with cell cyclin D2 bound signi®cant amounts of Cdk2 in breast lysate from T-47D DMTcycD1-3 cells treated with zinc for 12 h epithelial cells. Another study using immunoprecipita- from two independent experiments (marked A and B in the inset). tion followed by Western blotting has shown cyclin D2 After Western blotting for cyclin D1 protein, a standard curve bound to all three CDKs in U-2-OS osteosarcoma cells was established showing ECL response versus puri®ed recombinant cyclin D1 protein concentration. (B) The amounts of cyclin (Lukas et al., 1995b). Whilst this discrepancy may in D1 and cyclin D2 in lysates from the indicated cell lines were part re¯ect dierences in methodology, Cdk2 associa- calculated from the respective standard curves. (C) The tion with cyclin D1 appears to also be a cell type- abundance of cyclin D1 protein in T-47D DMTcycD1-3 cells speci®c phenomenon (Bates et al., 1994a; Tam et al., following treatment with increasing concentrations of zinc is 1994a). Thus cyclin D-CDK association appears not to plotted against the corresponding increase in the percentage of cells in S-phase 18 h after treatment, adapted from (Musgrove et depend solely on the abundance of the cyclin and CDK al., 1994) even in exponentially proliferating cells, where the postulated assembly factors for these complexes might be expected to be present (Matsushime et al., 1994). epithelial cells which is not accompanied by major Results from this study indicate that a major part of divergence of their downstream molecular and cell the cyclin D2-associated kinase activity in breast cycle eects. In synchronised 184 breast epithelial cells epithelial cells expressing either endogenous or cyclin D2 protein expression reached a maximum 3.5- exogenous cyclin D2 is due to Cdk2 (Figures 2 and

fold increase at the G1/S-phase boundary compared to 6). Several lines of evidence support this conclusion:

G0 arrested cells. Maximum levels of cyclin D2 protein the Cdk4/Cdk6-speci®c physical inhibitor p16 (Serrano

have also been observed in late G1 in elutriated et al., 1993) had little or no eect on the cyclin D2- lymphocytes, with a similar increase of three- to associated kinase activity while the Cdk2-speci®c

®vefold compared to cells at the start of G1 (Lukas chemical inhibitors, roscovitine and olomoucine (Glab et al., 1995b). Increases in cyclin D2 mRNA and et al., 1994; Vesely et al., 1994; Schulze-Gahmen et al., protein expression were apparent by 2 h after IL-2 1995) did block the kinase activity. Finally, cyclin D2 stimulation of arrested 32Dc13 murine myeloid cells immunocomplexes phosphorylated histone H1 (Figures but in contrast the level of cyclin D2 protein changed 2b and 5b), a substrate that is not phosphorylated by very little during the cell cycle in murine erythroleuke- Cdk4 or Cdk6 (Matsushime et al., 1994; Meyerson and mia cells (Ando et al., 1993; Kiyokawa et al., 1994). Harlow, 1994). In contrast, histone H1 kinase activity These dierences do not appear to depend on the was not observed in cyclin D2 immunocomplexes from Cyclin D2 in breast cancer cells KJ Sweeney et al 1337 T cells or 32Dcl3 cells (Ajchenbaum et al., 1993; Ando not signi®cantly dierent from control cells despite a and Grin, 1995). Although neither study directly consistent trend toward slightly faster doubling times examined the constituents of the cyclin D2 complexes, in the cyclin D2-expressing cells (Figure 5). MCF-7 the major catalytic partner was thought to be Cdk4 in breast cancer cells engineered to express cyclin D1 these cell types. This conclusion is supported by constitutively also showed similar growth rates to evidence that constitutive overexpression of Cdk4 in parental cells under optimal growth conditions 32Dcl3 cells prevents a TGFb-mediated decrease in (Zwijsen et al., 1996), but under reduced serum cyclin D2-associated kinase activity (Ando and Grin, conditions the percentage of cells in S-phase and the 1995), although an alternative explanation of these proliferation rate were maintained at higher levels than observations is that the overexpressed Cdk4 may act as in control cell lines (Zwijsen et al., 1996). The latter a sink for TGFb-induced CDK inhibitors, thus observation is consistent with results obtained after allowing cyclin D2/Cdk2 complexes to be active. cyclin D1 overexpression in rodent ®broblasts (Quelle When 32D mouse myeloid cells were induced to et al., 1993). In the present experiments, under lowered dierentiate, however, a somewhat greater percentage serum concentrations there was a small but consistent of total cyclin D2 bound to Cdk2. The authors decrease in the doubling times of cyclin D2-over- concluded that the proposed dierentiation-inhibiting expressing cells compared to controls which in some eects of cyclin D2 could be mediated through Cdk2, cases reached statistical signi®cance (Figure 5). although this was not formally demonstrated (Kato However, the doubling time increased in reduced and Sherr, 1993). These results contrast with the serum conditions, indicating that the cells were not consistent ®nding that cyclin D1 cannot activate entirely serum-independent, although in some experi- Cdk2 (see Introduction). Thus, while cyclin D1- ments the increase was small. In contrast to the results dependent kinase activity appears to be solely in serum-containing medium, there was a small but attributable to Cdk4 or Cdk6 (Matsushime et al., consistent increase in the percentage of cells in S-phase 1994; Meyerson and Harlow, 1994), cyclin D2 may in cyclin D2-expressing cells in serum-free medium. preferentially activate dierent CDKs in a cell type- The relatively small magnitude of these eects possibly speci®c fashion. re¯ects the low level of cyclin D2 achieved relative to The activation of dierent CDKs raises the the levels in 184 cells (Figure 7B). Nevertheless, these possibility of diering functions for cyclins D1 and results are consistent with the eects of expression of D2. This was investigated in T-47D cells in which the cyclin D1 in MCF-7 breast cancer cells (Zwijsen et al., function of cyclin D1 was already well-characterised 1996). (Musgrove et al., 1994, 1996). Upon acute cyclin D2 An initial comparison of the magnitude of the cell induction following zinc treatment of asynchronous T- cycle changes following induction of cyclin D1 or D2 47D DMTcyclin D2 cells, an increase in the percentage (Figure 4d and Musgrove et al., 1994) suggested that of cells in S-phase was observed, most likely due to an cyclin D1 may have been more ecient at accelerating acceleration through G1. While zinc treatment had cells through G1 and into S-phase. Indeed, rodent some eect on cells transfected with vector alone, the ®broblasts overexpressing cyclin D2 demonstrate a increase in the percentage of T-47D DMTcyclin D2 smaller reduction of the G0 to S-phase transit time cells in S-phase was consistently greater indicating a compared with cyclin D1-overexpressing cells (Quelle speci®c eect of cyclin D2 (Figure 4). This has been et al., 1993). Both these studies raise the question of observed previously in numerous other studies examin- whether cyclin D2 is a less potent stimulator of G1 ing ectopic expression of G1 cyclins including cyclin progression or whether this re¯ects diering levels of D2 in a range of dierent cell lines (Ando et al., 1993; the two cyclins. Quantitation of the amount of cyclin Ohtsubo and Roberts, 1993; Quelle et al., 1993; D1 and D2 in the T-47D transfected cells (Figure 7) Musgrove et al., 1994; Resnitzky et al., 1994; suggested that equimolar concentrations of the two Rosenberg et al., 1995). Ectopic expression of cyclin cyclins would have very similar eects on the fraction D2 was followed by increased cyclin E protein of cells accelerated into S-phase. Hence, the seemingly abundance and phosphorylation of pRB in vivo greater cell cycle changes observed after induction of responses not observed in vector-transfected cells cyclin D1 are most likely a concentration-dependent treated with zinc (Figures 3, 4a and b). Induction of eect rather than a real indication of functional cyclin E and pRB phosphorylation were also observed dierences between the two molecules. following the induction of cyclin D1 in this cell line In summary, the data presented in this manuscript (Musgrove et al., 1996). There also appears to be an indicate that in cells of breast epithelial origin cyclin increase in the abundance of total pRB protein D1 and cyclin D2 preferentially activated dierent following cyclin D2 induction (Figure 4a). This has CDKs. Since the cyclin D1-associated CDKs and been noted before in cyclin transfection experiments cyclin D2-associated CDKs phosphorylate dierent including overexpression of cyclin D1 in T-47D cells substrates in vitro, they appear likely to target and may indicate that ppRB is more stable (Ewen et dierent substrates in vivo. The observation that they al., 1993; Musgrove et al., 1996). There appears, have quantitatively similar eects on cell cycle therefore, to be a common pattern of molecular events progression is thus perhaps unexpected and raises following induction of cyclin D1 or cyclin D2, possibly the possibility that the pathways activated by Cdk4/ mediated by the release of E2F upon pRB phosphor- Cdk6 and Cdk2 are redundant. However, inhibition of ylation and consequent induction of cyclin E transcrip- Cdk2 function inhibits progression into S-phase in tion via E2F sites in the cyclin E promoter (Ohtani et normal diploid ®broblasts, in which Cdk4/Cdk6 are al., 1995; Botz et al., 1996; Geng et al., 1996). presumably active (Tsai et al., 1993) while inhibition Under optimal growth conditions the doubling time of Cdk4 activity via cyclin D1 antibody microinjection of T-47D cells expressing cyclin D2 constitutively was inhibits progression into S-phase in the presence of Cyclin D2 in breast cancer cells KJ Sweeney et al 1338 active Cdk2 (Baldin et al., 1993; Quelle et al., 1993). the SalIsiteofpDMT (Daly et al., 1991) and the HindIII Furthermore, induction of both cyclins D1 and E site of pRcCMV (Invitrogen), respectively. Parallel ¯asks of T-47D (7-2) were cotransfected with 40 mgof shortens G1 more than induction of either cyclin alone (Resnitsky and Reed, 1995). While these data argue pDMTcyclin D2 (or pDMT) and 4 mgofpSV2neo,using against redundancy of pathways activated by cyclin standard calcium phophate precipitation methods. Simi- larly cells were transfected with 40 mg pCMVcyclin D2 or D1/Cdk4 and cyclin E/Cdk2, it remains possible that pCMV which had the neomycin resistance gene incorpo- cyclin D2/Cdk2 and cyclin E/Cdk2 have dierent rated. Colonies resistant to G418 (600 mg/ml) were pooled targets. This possibility is supported by evidence from after several weeks to produce the following cell lines: T- in vitro studies that demonstrate cyclins E, A and B 47D DMTcyclin D2, T-47D DMT, T-47D CMVcyclin D2 confer distinct substrate speci®cities on CDKs (Peeper and T-47D CMV. et al., 1993; Sarcevic et al., unpublished data). In addition, it is also possible that some of the actions of cyclin D2 are mediated by interactions with proteins Analysis of cell cycle progression other than CDKs or that the dierent cyclin D/CDK Analytical DNA ¯ow cytometry was used to determine cell complexes may have roles in addition to promotion of cycle phase distribution as described previously (Musgrove et al., 1989). Brie¯y, cells were stained overnight with the G1 to S-phase progression. Thus, this study identi®es diculties in assigning a universal function to all D- DNA-speci®c dye combination ethidium bromide and type cyclins, and suggests that they may have subtly mithramycin in the presence of 0.2% Triton to permeabi- lise the cell membrane. Cell cycle phase distribution was dierent modes of action to enhance G progression. 1 calculated by computer analysis of histograms containing 30 000 events.

Western blot analysis, immunoprecipitation and kinase assays Materials and methods For immunoprecipitation or Western blot analysis cell monolayers were washed once in ice-cold PBS and then Cell lines and cell culture scraped into the following lysis buer: 50 mM HEPES The T-47D breast carcinoma cell line from EG and G (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 1% Triton X-

Mason Research Institute (Worcester, MA) was cloned by 100, 1.5 mM MgCl2,1mM EGTA, 10 mg/ml aprotinin, limiting dilution, and one clonal cell line, T-47D (7-2), was 10 mg/ml leupeptin, 1 mM PMSF, 200 mM sodium orthova- selected for transfection studies (Musgrove et al., 1994). T- nadate, 10 mM sodium pyrophosphate, 100 mM NaF. The 47D (7-2) retained the characteristics of the parent line by lysates were incubated on ice for 5 min before clearing cell all the tested criteria - i.e., growth rate under standard debris by centrifugation (14 000 g,5min)andstoredat culture conditions, sensitivity to growth regulation by 7708C until use. Alternatively, cell monolayers were steroids and steroid antagonists (progestin, ORG 2058, harvested as described below for kinase assays. Similar antiestrogen, ICI 164384, and antiprogestin, RU 486), and results were obtained from lysates prepared by either abundance of cyclin D1 and c-myc mRNA. Normal breast method. For use in kinase assays, cell monolayers were epithelial cell strain 184 was obtained from Dr Martha washed once in PBS and scraped into the following lysis Stampfer, University of California, Berkeley, CA, USA. buer, on ice: 50 mM HEPES (pH 7.5), 1 mM DTT, T-47D cells were maintained in RPMI medium supple- 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% mented with 6 mML-glutamine, 14 mM sodium bicarbonate, Tween-20, 10% glycerol, 10 mM b-glycerophosphate, 20 mM HEPES, 10 mg/ml human insulin (CSL-Novo, North 1mM NaF, 0.1 mM orthovanadate, 10 mg/ml leupeptin, Rocks, NSW, Australia) and 10% foetal calf serum (FCS). 10 mg/ml aprotinin and 0.1 mM PMSF. Cells were vortexed 184 cells were maintained in MEGM medium with 0.4% for 15 s every 10 min for 1 h total before centrifugation to bovine pituitary extract (BPE) from Clonetics Corp., San remove cell debris. Diego, CA, USA. 184 cells were synchronised in these studies For Western analyses equal amounts of protein were by growth factor deprivation. Brie¯y, cells were cultured in separated by SDS ± PAGE and transferred to nitrocellulose MEGM (with BPE) for three days, and then the medium was ®lters. Non-speci®c binding was blocked in 5% skim milk replaced with MEBM with only 0.05% BPE. The growth- powder in TBS/Triton (10 mM Tris (pH 7.4), 150 mM NaCl, arrested cells were rescued three days later by replacing this 0.05% Triton X-100), followed by incubation for 2 h at room medium with MEGM containing 0.4% BPE. temperature in primary antibody, typically at a concentration The CellTiter 96 non-radioactive cell proliferation assay of 1 mg/ml in TBS/BSA solution (10 mM Tris (pH 7.4), from Promega was used as per the manufacturers instructions 150 mM NaCl, 5% BSA). Filters were then incubated with to perform the cell growth assays. Cells were seeded into four the appropriate secondary antibody (1 : 2000 in 5% skim milk replicate wells of a 24-well plate (1.56104 cells per well) in powder) for 1 h before enhanced chemiluminescence (ECL) 500 ml of RPMI with 5% FCS. After 48 h the cells were detection (Amersham or Du Pont). Densitometric analysis of washed once in serum-free RPMI before addition of 500 mlof autoradiographs was performed using a Molecular Dynamics fresh serum-free RPMI or RPMI containing 0.5%, 1.0% or (Sunnyvale, CA) Personal Densitometer SI and IP LabGel 5.0% FCS. Cells were assayed every other day for 7 days. analysis software (Signal Analytics, VA). Antibodies used for For each assay replicate wells were stained and solubilised blotting were: cyclin D1 (PRAD1), cyclin D2 (C-17), cyclin E and aliquots were transferred to 96-well plates for measure- (HE12), Cdk2 (M2), Cdk4 (C-22), Cdk6 (C-21), from Santa ment of absorbance. Cruz Biotechnology Inc., Santa Cruz, CA, USA; pRB (G3- 245) from Pharmingen, San Diego, CA, USA; cyclin D1 (DCS6) from Novacastra Laboratories, Newcastle-Upon- Expression vectors and transfection procedures Tyne, UK; mouse monoclonal IgG2b isotypic control The inducible expression vector pDMTcyclin D2 and the antibody from Coulter Immunology, Coulter Corporation, constitutive expression vector pRcCMVcyclin D2 were Hialeah, FL, USA; sheep anti-mouse and donkey anti-rabbit constructed by cloning the human cyclin D2 EcoRI/BglII horseradish peroxidase linked secondary antibodies were cDNA fragment from pD2-P39 provided by Drs Yue from Amersham. Xiong and David Beach, Cold Spring Harbor Laboratory, Proteins were immunoprecipitated using either a rabbit Cold Spring Harbor, NY, USA (Xiong et al., 1992a), into polyclonal anti-cyclin D1 serum raised against a human GST- Cyclin D2 in breast cancer cells KJ Sweeney et al 1339 cyclin D1 fusion protein, (Musgrove et al., 1996), rat tates and incubated for 1 h at 308C prior to the kinase monoclonal anti-cyclin D2 antibody (34B1-3, Santa Cruz) reaction. The bacterially expressed fusion proteins were or a mouse monoclonal anti-cyclin D2 antibody (DCS5, a puri®ed as described elsewhere (Lilischkis et al., 1996). generous gift from Drs Jiri Bartek and Jiri Lukas, Danish GST-p16, GST-p21 or GST alone was added in a ®nal Cancer Society, Copenhagen, Denmark). All cyclin D2 kinase volume of 100 ml. The chemical inhibitors roscovitine and assays were performed using the DCS5 antibody. Lysates olomoucine (a generous gift from Prof Laurent Meijer, were incubated for 3 h at 48C with protein A-sepharose or rat CNRS, Station Biologique, Rosco cedex, France) were IgG agarose beads that had been pre-conjugated with the also added in a ®nal volume of 100 ml in HEPES/DTT giving appropriate antibody. Beads were then washed four times in a ®nal concentration of 50 mM. After the incubation period ice cold lysis buer and once in 50 mM HEPES (pH 7.5), the inhibitors were aspirated o the beads and the beads were 1mM DTT. washed once with HEPES/DTT solution before the addition Samples used to analyse immunoprecipitated proteins were of the substrate and kinase buer. boiled for 3 min in SDS sample buer (60 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 2% SDS, 5% b-mercaptoetha- Production of cyclin GST-D1 and D2 fusion proteins nol) before SDS ± PAGE; visualisation of the immunoprecipitated proteins was as described above for Western analysis. The cyclin GST-cyclin D1 and GST-cyclin D2 fusion Depleted lysate samples were checked for the absence of the proteins were puri®ed using glutathione-agarose from immunoprecipitated protein to con®rm that the amount of lysates of Sf-9 insect cells infected with appropriate antibody used in these experiments was sucient to baculovirus constructs containing the full length ORF of immunoprecipitate all of the protein of interest. Those human cyclins D1 and D2, N-terminally linked to GST. samples to be used in kinase assays had a further two washes in the HEPES/DTT solution before 20 ml of substrate in HEPES/DTT (4.5 mg GST-pRB (Santa Cruz) or 3 mg histone H1 (Sigma Chemicals)) was added along with 10 mlof 36kinase buer giving a ®nal concentration of: 50 mM Acknowledgements HEPES (pH 7.5), 1 mM DTT, 2.5 mM EGTA, 10 mM We thank those members of the Cancer Research Program 32 MgCl2,20mM ATP, 10 mCi [g- P]ATP, 0.1 mM sodium who have in many ways assisted with this study, especially orthovanadate, 1 mM NaF, 10 mM b-glycerophosphate. Catherine Kennedy and Andrea Brady for invaluable help Samples were mixed gently to resuspend beads and with tissue culture. We are grateful to Drs Jiri Bartek and incubated at 308C for 30 min with gentle mixing every Jiri Lukas for providing the DCS5 antibody and in 10 min. The reaction was terminated by the addition of particular for their patient and thorough advice in 36SDS sample buer (®nal concentration: 60 mM Tris-HCl establishing the cyclin D2-associated kinase assay. We (pH 6.8), 10% (v/v) glycerol, 2% SDS, 5% b-mercaptoetha- also thank Prof Laurent Meijer for his generous donation nol). The samples were then incubated at 908C for 3 min of the CDK chemical inhibitors and helpful suggestions. before separation by SDS ± PAGE. Bands were quantitated This work was supported by the National Health and by densitometry after exposure of the dried gel to X-ray ®lm. Medical Research Council (NHMRC) of Australia. If the kinase assay was performed in the presence of Kimberley Sweeney is the recipient of a Dora Lush inhibitors then the inhibitors were added to immunoprecipi- Biomedical Research Scholarship from the NHMRC.

References

Ajchenbaum F, Ando K, DeCaprio JA and Grin JD. FantlV,StampG,AndrewsA,RosewellIandDicksonC. (1993). J. Biol. Chem., 268, 4113 ± 4119. (1995). Genes Dev., 9, 2364 ± 2372. Ando K, Ajchenbaum-Cymbalista F and Grin JD. (1993). Freeman RS, Estus S and Johnson Jr EM. (1994). Neuron, Proc. Natl. Acad. Sci. USA, 90, 9571 ± 9575. 12, 343 ± 355. Ando K and Grin JD. (1995). Oncogene, 10, 751 ± 755. Geng Y, Eaton EN, Picon M, Roberts JM, Lundberg AS, Baldin V, Lukas J, Marcote MJ, Pagano M and Draetta G. Giord A, Sardet C and Weinberg RA. (1996). Oncogene, (1993). Genes Dev., 7, 812 ± 821. 12, 1173 ± 1180. Bartkova J, Lukas J, MuÈ ller H, LuÈ tzhùft D, Strauss M and Gillett C, Fantl V, Smith R, Fisher C, Bartek J, Dickson C, Bartek J. (1994). Int. J. Cancer, 57, 353 ± 361. Barnes D and Peters G. (1994). Cancer Res., 54, 1812 ± Bartkova J, Lukas J, Strauss M and Bartek J. (1995). 1817. Oncogene, 10, 775 ± 778. Glab N, Labidi B, Qin LX, Trehin C, Bergounioux C and Bates S, Bonetta L, MacAllen D, Parry D, Holder A, Meijer L. (1994). FEBS Letts., 353, 207 ± 211. Dickson C and Peters G. (1994a). Oncogene, 9, 71 ± 79. Harper JW, Adami GR, Wei N, Keyomarsi K and Elledge Bates S, Parry D, Bonetta L, Vousden K, Dickson C and SJ. (1993). Cell, 75, 805 ± 816. Peters G. (1994b). Oncogene, 9, 1633 ± 1640. Higashi H, Suzuki-Takahashi I, Saitoh S, Segawa K, Taya Y, Botz J, Zerfass-Thome K, Spitkovsky D, Delius H, Vogt B, Okuyama A, Nishimura S and Kitagawa M. (1996). Eur. Eilers M, Hatzigeorgiou A and Jansen-Durr P. (1996). J. Biochem., 237, 460 ± 467. Mol. Cell. Biol., 16, 3401 ± 3409. Hui R, Cornish AL, McClelland RA, Robertson JFR, Buckley MF, Sweeney KJE, Hamilton JA, Sini RL, Manning Blamey RW, Musgrove EA, Nicholson RI and Suther- DL, Nicholson RI, deFazio A, Watts CKW, Musgrove land RL. (1996). Clin. Cancer Res., 2, 923 ± 928. EA and Sutherland RL. (1993). Oncogene, 8, 2127 ± 2133. Hunter T and Pines J. (1994). Cell, 79, 573 ± 582. Daly RJ, Harris WH, Wang DW and Darbre PD. (1991). Jiang W, Kahn SM, Zhou P, Zhang Y-J, Cacace AM, Infante Cell Growth & Dier., 2, 457 ± 464. AS, Doi S, Santella RM and Weinstein IB. (1993). Dowdy SF, Hinds PW, Louie K, Reed SI, Arnold A and Oncogene, 8, 3447 ± 3457. Weinberg RA. (1993). Cell, 73, 499 ± 511. Kato J-Y and Sherr CJ. (1993). Proc.Natl.Acad.Sci.USA, Dulic V, Drullinger LF, Lees E, Reed SI and Stein GH. 90, 11513 ± 11517. (1993). Proc. Natl. Acad. Sci. USA, 90, 11034 ± 11038. Kiyokawa H, Richon VM, Rifkind RA and Marks PA. EwenME,SlussHK,SherrCJ,MatsushimeH,KatoJand (1994). Mol. Cell. Biol., 14, 7195 ± 7203. Livingston DM. (1993). Cell, 73, 487 ± 497. Cyclin D2 in breast cancer cells KJ Sweeney et al 1340 Kranenburg O, de Groot RP, Van der Eb AJ and Zantema Resnitsky D and Reed SI. (1995). Mol. Cell. Biol., 15, 3463 ± A. (1995). Oncogene, 10, 87 ± 95. 3469. Lilischkis R, Sarcevic B, Kennedy C, Warlters A and Resnitsky D, Gossen M, Bujard H and Reed SI. (1994). Mol. Sutherland RL. (1996). Int. J. Cancer, 66, 249 ± 254. Cell. Biol., 14, 1669 ± 1679. Lukas J, Bartkova J, Rohde M, Strauss M and Bartek J. Rosenberg AR, Zindy F, Le Deist F, Mouly H, Metezeau P, (1995a). Mol. Cell. Biol., 15, 2600 ± 2611. Brechot C and Lamas E. (1995). Oncogene, 10, 1501 ± Lukas J, Bartkova J, Welcker M, Petersen OW, Peters G, 1509. Strauss M and Bartek J. (1995b). Oncogene, 10, 2125 ± Schulze-Gahmen U, Bransden J, Jones HD, Morgan DO, 2134. Meijer L, Vesely J and Kim SH. (1995). Proteins, 22, 378 ± Lukas J, Pagano M, Staskova Z, Draetta G and Bartek J. 391. (1994). Oncogene, 9, 707 ± 718. Serrano M, Hannon GJ and Beach D. (1993). Nature, 366, Matsushime H, Ewen ME, Strom DK, Kato J-Y, Hanks SK, 704 ± 707. Roussel MF and Sherr CJ. (1992). Cell, 71, 323 ± 334. Sherr CJ. (1994). Cell, 79, 551 ± 555. Matsushime H, Quelle DE, Shurtle SA, Shibuya M, Sherr Sicinski P, Liu Donaher J, Parker SB, Li T, Fazeli A, CJ and Kato J-Y. (1994). Mol. Cell. Biol., 14, 2066 ± 2076. Gardner H, Haslam SZ, Bronson RB, Elledge SJ and Matsushime H, Roussel MF, Ashmun RA and Sherr CJ.. Weinberg RA. (1995). Cell, 82, 621 ± 630. (1991). Cell, 65, 701 ± 713. Sweeney KJE, Musgrove EA, Watts CKW and Sutherland Meyerson M and Harlow E. (1994). Mol. Cell. Biol., 14, RL. (1996). Mammary Tumor Cell Cycle, Dierentiation 2077 ± 2086. and Metastasis. Dickson R and Lippman M (eds). Musgrove EA,, Wakeling AE and Sutherland RL. (1989). pp. 141 ± 170. Kluwer Academic Publishers: Boston. Cancer Res., 49, 2398 ± 2404. Tam SW, Shay JW and Pagano M. (1994a). Cancer Res., 54, Musgrove EA, Hamilton JA, Lee CSL, Sweeney KJE, Watts 5816 ± 5820. CKW and Sutherland RL. (1993). Mol. Cell. Biol., 13, Tam SW, Theodoras AM, Shay JW, Draetta GF and Pagano 3577 ± 3587. M. (1994b). Oncogene, 9, 2663 ± 2674. Musgrove EA, Lee CSL, Buckley MF and Sutherland RL. Tsai LH, Lees E, Faha B, Harlow E and Riabowol K. (1993). (1994). Proc. Natl. Acad. Sci. USA, 91, 8022 ± 8026. Oncogene, 8, 1593 ± 1602. Musgrove EA, Sarcevic B and Sutherland RL (1996). J. Cell. Vesely J, Havlicek L, Strnad N, Blow JJ, Donella-Deana A, Biochem., 60, 363 ± 378. Pinna L, Letham DS, Kato J-Y, Detivaud L, Leclerc S and Ohtani K, DeGregori J and Nevins JR. (1995). Proc. Natl. Meijer L. (1994). Eur. J. Biochem., 224, 771 ± 786. Acad. Sci. USA, 92, 12146 ± 12150. Weinberg RA. (1995). Cell, 81, 323 ± 330. Ohtsubo M and Roberts JM. (1993). Science, 259, 1908 ± Winston JT and Pledger WJ. (1993). Mol. Biol. Cell, 4, 1912. 1133 ± 1144. Palmero I, Holder A, Sinclair AJ, Dickson C and Peters G. XiongY,HannonGJ,ZhangH,CassoD,KobayashiRand (1993). Oncogene, 8, 1049 ± 1054. Beach D. (1993). Nature, 366, 701 ± 704. Peeper DS, Parker LL, Ewen ME, Toebes M, Hall FL, Xu Xiong Y, Menninger J, Beach D and Ward DC. (1992a). M, Zantema A, van der Eb A and Piwinca-Worms H. Genomics, 13, 575 ± 584. (1993). EMBO J., 12, 1947 ± 1954. Xiong Y, Zhang H and Beach D. (1992b). Cell, 71, 505 ± 514. Quelle DE, Ashmun RA, Shurtle SA, Kato J-Y, Bar-Sagi Zwijsen RML, Klompmaker R, Wientjens EBHGM, Kristel D, Roussel MF and Sherr CJ. (1993). Genes Dev., 7, PMP, van der Burg B and Michalides RJAM. (1996). Mol. 1559 ± 1571. Cell. Biol., 16, 2554 ± 2560. Reddel RR, Murphy LC and Sutherland RL. (1984). Cancer Res., 44, 2398 ± 2405.