Oncogene (1998) 16, 2075 ± 2086  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Interferon-a-induced G1 phase arrest through up-regulated expression of CDK inhibitors, p19Ink4D and p21Cip1 in mouse macrophages

Masaaki Matsuoka, Kenzaburo Tani and Shigetaka Asano

Department of Hematology and Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan

The mechanism of cell cycle arrest induced by interferon-a dependent kinases or cdks (reviewed by Sherr, 1993). (IFN-a) was analysed using a mouse macrophage cell line, D-type cyclins (Lew et al., 1991; Matsushime et al., 1991) BAC1.2F5A. IFN-a added in media before mid-G1 and cyclin E (Ko€ et al., 1991; Lew et al., 1991) govern the prohibited cells from entering S phase. The blockage of G1/S transition in association with their proper G1/S transition was associated with diminuition of both physiological partners, cdk4 (Matsushime et al., 1992) cyclin D1/cdk4- and cyclin E/cdk2-associated kinase or cdk6 (Meyerson and Harlow, 1994), and cdk2 (Dulic et activities. G1 cyclin-associated kinase activities were al., 1992; Ko€ et al., 1992), respectively. When cells enter down-regulated quickly after the addition of IFN-a. Cells G1 phase with mitogenic stimuli, the synthesis and the treated with IFN-a contained excess amounts of cdk active complex formation of D-type cyclins and cdk4 are inhibitors which down-regulated G1 cyclin/cdk-associated induced in early to middle G1 phase, and their complex kinase activities in the proliferating cells and this action formation and activities reach maximal levels at late G1 was counteracted by exogenously-supplied recombinant (Matsushime et al., 1992, 1994). Enforced overexpression cyclin D2/cdk4 complexes. In parallel, accumulation of of D-type cyclins shortened G1 phase in mammalian p19Ink4D and p21Cip1, and their attachment to cdks were up- ®broblasts (Quelle et al., 1993). Reciprocally, the regulated quickly after the addition of IFN-a. Expression microinjection of anti-cyclin D antibodies caused G1 of p19Ink4D and p21Cip1 was potentiated transcriptionally. arrest, indicating that D-type cyclins are rate-limiting for We concluded that increased attachment of upregulated G1/S transition (Quelle et al., 1993). The expression of cdk inhibitors including p19Ink4D and p21Cip1 to G1 cyclin/ cyclin E is induced later in G1 phase than D-type cyclins cdk complexes contributed to diminuition of G1 cyclin/ (Dulic et al., 1992; Ko€ et al., 1992) and is also cdk-associated kinase activities and resulting G1 phase responsible for the transition from G1 to S in arrest during the early phase of treatment with IFN-a. mammalian cells (Ohtsubo and Roberts, 1993). For cells to complete the G1/S transition, the activities of both Keywords: p19Ink4D;p21Cip1; interferon-a D-type cyclins and cyclin E are essential (Hatakeyama et al., 1994; Resnitzky and Reed, 1995; Resnitzky et al., 1994; van Den Heuvel and Harlow, 1993). In contrast to growth-promoting mitogenic signals, Introduction antimitogenic factors including transforming growth factor-beta or TGF-b (Polyak et al., 1994a), cAMP Interferons (IFNs) are a family of secreted polypeptides (Kato et al., 1994b), rapamycin (Nourse et al., 1994) which include three distinct classes, IFN-a, IFN-b and and prostagrandin A2 (Gorospe et al., 1996) induced IFN-g with a variety of biological functions (Pestka et cell growth arrest by inhibiting cell cycle progression al., 1987). Their most important functions are the from G1 to S phase. The discovery of several cdk antiviral and the antiproliferative activities in mamma- inhibitors has facilitated the elucidation of these lian cells, which have been utilized clinically for mechanisms. Cdk inhibitors brake cell cycle progres- treatments of B- or C-type viral hepatitis and several sion by negatively and stoichiometrically regulating neoplastic disorders. The post-receptor signal transduc- cyclin/cdk activities (Sherr and Roberts, 1995) and are tion pathways of IFNs as far as the transcriptional level the targets of various extracellular growth-modi®ers. have been intensively investigated over the past several Two families of mammalian cdk inhibitors have been years following the identi®cation of JAK kinases, IFN- identi®ed. One includes p21Cip1 (El-Deiry et al., 1993; stimulated gene response elements (ISRE/GAS) and the Harper et al., 1993; Noda et al., 1994; Xiong et al., transcription factors (ISGF-3/STATS) (Darnell et al., 1993), p27Kip1 (Polyak et al., 1994b; Toyoshima and 1994; Ihle and Kerr, 1995). Two possible mechanisms Hunter, 1994) and p57Kip2 (Lee et al., 1995; Matsuoka for the antimitogenic activities of IFNs have been et al., 1995). These inhibitors have similar N-terminal proposed; blockage of growth factor-stimulated path- regions which are responsible for the blockage of cdk ways (Xu et al., 1995), and stimulation or inhibition of kinases. They can inhibit any G1 cyclin/cdk activity. independent pathways such as those through JAK The other family of cdk inhibitors includes INK4 cdk kinases and various transcription factors. G1 arrest is inhibitors (p16Ink4A, p15Ink4B, p18Ink4C and p19Ink4D) induced by IFNs as the ®nal common result of their which inhibit cdk4 and cdk6 kinases but do not antimitogenic activities in mammalian cells. inhibit cdk2 activity (Chan et al., 1995; Guan et al., The cell cycle progression is controlled by a family of 1994; Hirai et al., 1995). p15, p18 and p19 are cyclins and their speci®c catalytic partners, cyclin- expressed ubiquitously while p16 is expressed in a limited range of normal tissues and in several cell lines. Overexpression of any one of these proteins Correspondence: M Matsuoka Received 12 August 1997; revised 21 November 1997; accepted 24 results in G1 phase arrest of mammalian cells (Guan November 1997 et al., 1994; Hirai et al., 1995; Quelle et al., 1995a). Interferon a-induced G1 arrest M Matsuoka et al 2076 During normal cell cycle progression, p18 and p19 Results expression are dominantly induced after cells enter S and G2/M phase in a mouse macrophage cell line Interferon-a blocked G1/S progression; the critical point (Hirai et al., 1995) and the true function of p18 and was around mid-G1 p19 remains to be elucidated. The cDNA of another small protein p19ARF are generated by alternate BAC1.2F5A macrophages proliferated with about 18 ± splicing from the p16 gene. Overexpression of p19ARF 21 h of doubling time and were distributed randomly induced cell cycle arrest (Quelle et al., 1995b). The throughout the cell cycle in media containing CSF-1 genes of p16 or p19ARF and p15 exist contiguously on (Figure 1a, left panel); 52.3% were within G1 phase, human chromosome 9p21 and are deleted or mutated 41.2% within S phase and 6.5% within G2/M phase at in some malignancies (Hall and Peters, 1996; Kamb et 50% cell con¯uency. Preliminary experiments indicated al., 1994; Nobori et al., 1994), supporting the idea that interferon (IFN)-a treatment blocked G1/S that cdk inhibitors can act as tumor suppressors. Cdk transition. To determine the optimal IFN-a concentra- inhibitors also served as the common mediators of tion which blocked G1/S transition completely, the various biological processes including DNA-damages (Dulic et al., 1994), cell di€erentiation (Liu et al., 1996; Parker et al., 1995), cell to cell contact inhibition (Polyak et al., 1994a) and cell senescence (Hara et al., 1996; Noda et al., 1994). Recent studies a indicated that oncogene signals converge to cyclin/cdk and cdk inhibitor (Lloyd et al., 1997). Several mechanisms have been suggested by a series of studies for TGF-b-induced inhibition of cell cycle progression, including the suppression of cdk4 synth- esis for Mv1Lu mink lung epithelial cells (Ewen et al., 1993), inhibition of cyclin E/cdk2-associated kinases by p27Kip1 (Ko€ et al., 1993; Polyak et al., 1994a), and the INK4b increased expression of cdk inhibitors p15 for b human keratinocytes (Hannon and Beach, 1994). A comprehensive study (Reynisdottir et al., 1995) indicated that p27Kip1, p21Cip1 and p15Ink4B cooperated to induce cell cycle arrest in association with the subsequently occurring down-regulation of cdk4 levels. Expression of p15Ink4B mRNA and protein were induced primarily. Up-regulated p15 itself down-regulated cdk4-associated kinase activities while it competed with p27 for binding to cyclin D/cdk4. P27 released from cyclin D/cdk4 complexes attached to cdk2 and down-regulated cdk2-associated kinase activities (Rey- nisdottir and Massague, 1997). Down-regulation of c cdc25A phosphatase by TGF-b was another cause reducing cdk4 and cdk6 activities in the p15-minus cell line (Lavarone and Massague, 1997). cAMP analogues and cAMP inducers inhibited cell growth and caused G1 phase arrest in mouse macrophages by increasing the accumulation of p27 and reducing the activities of CAK, cdk-activating kinase, indirectly (Kato et al., 1994b). Rapamycin induced G1 arrest by inhibiting elimination of p27 by IL-2 in T cells (Nourse et al., 1994). Prostaglandin A2 suppressed the expression of cyclin D and cdk4 and enhanced accumulation of p21 capable to inhibit cyclin E/cdk2 activities in human breast cancer cells (Gorospe et al., 1996). The relationship between IFN-g and cell cycle arrest has been characterized. In many cells, IFN-r upregu- Figure 1 Cell cycle arrest at mid G1 by IFN-a.(a) Exponentially Cip1 growing BAC1.2F5A cells were incubated with or without IFN-a lates p21 gene expression. Chin et al. (1996) (2000 U/ml) for 20 h, and their cell cycle distribution was demonstrated that IFN-r enhanced p21Cip1 gene analysed by ¯ow cytometric analysis of DNA contents. (b) Cells expression through the IFN-r-Stat1 pathway. IFN-a were synchronized in early G1 phase by growth factor starvation induced cell cycle arrest by down-regulating the for 18 h and stimulated with CSF-1 in the presence (lower panel) expression of cyclin D3 and cdc 25A in Daudi cells, or absence (upper panel) of 2000 U/ml IFN-a. Cell cycle distribution of cells harvested at the indicated time points after Burkitt lymphoma cells (Tiefenbrun et al., 1996). stimulation with CSF-1 were analysed. (c) Synchronized cells were In this study, we demonstrated that up-regulated stimulated with CSF-1, and IFN-a (2000 U/ml) was added at accumulation of cdk inhibitors including p19 and p21 indicated hours thereafter. Cells were harvested and cell cycle contributes to the induction of G1 phase arrest during analyses were performed at 15 h after CSF-1 stimulation. Percentages of cells within S and G2/M phase were calculated early-phase IFN-a treatment in a mouse macrophage with FACS analysis and compared to that of control cells treated cell line. with CSF-1 without IFN-a, which was adjusted as 100% Interferon a-induced G1 arrest M Matsuoka et al 2077 proliferating cells were cultured in the presence of treated with IFN-a. Similarily to cAMP or rapamycin various concentrations of IFN-a for 18 h, then treatment (Kato et al., 1994b), when added within 5 h, subjected to the cell cycle analysis. The percentage of IFN-a prevented the majority of cells from entering S cells within G1 phase increased gradually in proportion phase. When added thereafter, cells gradually became to the increase of added IFN-a (data not shown). More resistant to IFN-a treatment and entered S phase. IFN- than 1000 units/ml of IFN-a inhibited G1/S progression a added at more than 8 h after CSF-1 stimulation did almost completely. We used 2000 units/ml of IFN-a for not block G1/S transition e€ectively (Figure 1c). This the following experiments. When we cultured cells in the suggested that the critical period for IFN-a was around presence of 2000 units/ml of IFN-a for 20 h, 88.6% of the mid G1 when G1 cyclin-associated kinase activities cells distributed in G1 phase (Figure 1a, right panel), were essential. We therefore focused on the activities of indicating that IFN-a blocked G1 phase progression. G1 cyclins, D-type cyclins and cyclin E. Macrophages deprived of CSF-1 for 18 h were arrested in early G1 phase (Tushinski and Stanley, Inhibition of both cyclin D1/cdk4- and cyclin E/cdk2- 1985). They were restimulated with CSF-1 in the dependent kinase activities accompanied G1 arrest absence or the presence of 2000 U/ml IFN-a and synchronized cell cycle progression was monitored at Among D-type cyclins the most abundant is cyclin D1, various time points (Figure 1b). Normal G1/S and cdk4 is its major catalytic subunit in BAC1.2F5A transition began to take place after 10 ± 12 h and the cells (Kato et al., 1994b; Matsushime et al., 1994). majority of cells were in S phase after 15 h (Figure 1b, Therefore, we analysed both cyclin D1- and cdk4- upper panel). In the presence of IFN-a, however, associated kinase activities as representatives of D-type almost all cells remained in G1 phase even 15 h after cyclin-dependent kinases. For screening experiments, stimulation with CSF-1, con®rming that IFN-a we used the cells synchronized by CSF-1 starvation induced G1 phase arrest (Figure 1b, lower panel). and restimulated with CSF-1 together with or without IFN-a was added at various time points after CSF-1 IFN-a for 12 h. When the lysates from cells stimulated stimulation to determine which period in G1 phase was with CSF-1 were immunoprecipitated with anti-cyclin critical for induction of G1 arrest with IFN-a. In this D1 antibodies or anti-cdk4 antibodies, the precipitates study, we calculated percentages of cells within both S contained pRb-kinase activities (Figure 2a and b, lane and G2/M 15 h after stimulation with CSF-1 by 2) as compared with those with nonimmune rabbit sera FACScan ¯ow cytometrical analysis of DNA content (Figure 2a and b, lane 1). The addition of IFN-a to determine the percentages of cells entering S phase, induced the marked reduction of kinase activities and compared these values with the values in cells not (Figure 2a and b, lane 3).

a c E D1 D1 E E α α α IP IP α α IFN NRS IFN IFN Lysate Lysate IFN NRS IFN IFN (–) (–) (+)

(–) (–) (+) cAMP

pRb pRb

1234

123

d CDK2 CDK2 CDK2 α IP α α b CDK4 CDK4 IFN NRS IFN IFN IP α α Lysate

(–) (–) (+) cAMP

Lysate IFN NRS IFN IFN (–) (–) (+)

pRb pRb

123 1234 Figure 2 E€ects of IFN-a on G1 cyclin- and cdk-associated kinase activities. (a, b, c and d) BAC1.2F5A cells deprived of CSF-1 for 18 h were stimulated with CSF-1 alone ((7)-IFN-a, lanes 1 and 2), or together with IFN-a((+)-IFN-a, lane 3) or 8 Br-cAMP (cAMP, lane 4). After 12 h, cells were harvested and the lysates were immunoprecipitated with nonimmune rabbit sera (NRS), and antibodies to cyclin D1 (a D1), cdk4 (a cdk4), cyclin E (a E) or cdk2 (acdk2) and assayed for pRb kinase activities. For pRb substrates, recombinant GST-fused Rb large pocket protein (a and c) or recombinant GST-fused Rb C-terminal protein (b and d) were used Interferon a-induced G1 arrest M Matsuoka et al 2078 Another G1 cyclin, cyclin E, and its major catalytic needs 8 h or more to down-regulate cdk2-associated partner cdk2 work at G1 phase, slightly later than D- kinase activities to half the normal levels (Reynisdottir et type cyclins. The immunoprecipitates of lysates from al., 1995). We focused on the mechanisms down- macrophages stimulated with CSF-1 with antibodies to regulating G1-cyclin activities during the early phase of cyclin E and to cdk2 contained Rb kinase activities IFN-a treatment within 9 h. (Figure 2c and d, lane 2). The anti-cdk2 immunopre- cipitates of cell lysates from mammalian cells around Cells incubated with IFN-a contained inhibitors which the G1/S boundary contained cyclin E/cdk2-associated were able to reduce G1 cyclin/cdk-associated kinase kinases as well as cyclin A/cdk2-associated kinase activities in the proliferating cells activities (Dulic et al., 1992; Ko€ et al., 1992). Addition of IFN-a reduced both cyclin E- and cdk2- One possible cause of the reduction in cdk4- or cdk2- associated kinase activities markedly (Figure 2c and d, associated Rb kinase activities was supposed to be up- lane 3). As another control, cell lysates treated with the regulation of cdk inhibitors which act stoichiometri- cAMP analogue, 8 Br-cAMP were also analysed for cally and are heat-stable (Sherr and Roberts, 1995). To cyclin E- or cdk2-associated kinase activities. As test this possibility, we performed mixing experiments expected from a previous study (Kato et al., 1994b), to determine whether the lysates from cells incubated cAMP down-regulated cyclin E- or cdk2-associated with IFN-a contained excess amounts of cdk inhibitors kinase activities (Figure 2c and d, lane 4). For cells to which could down-regulate the G1 cyclin/cdk-asso- complete the progression from G1 to S phase, both ciated kinase activities in the lysates from the cyclin D/cdk4- and cyclin E/cdk2-associated kinase proliferating cells not treated with IFN-a. Immune activities, targets of which are supposed to be distinct complex cdk2-associated kinase assays were performed from each other, are necessary (Hatakeyama et al., under the condition of linear correlation between the 1994; Resnitzky and Reed, 1995). Therefore, the amounts of added cell lysates and immune complex reduction of both cyclin D1/cdk4- and cyclin E/cdk2- kinase activities (Figure 4a). Nearly equal amounts of associated kinase activities cooperated to induce G1 lysates from cells incubated with IFN-a for 9 h could phase arrest. inhibit cdk2 kinase activities in the lysates from control cells (Figure 4a). Similar experiments assessing the activities inhibiting cdk4-associated kinase gave similar IFN-a reduced G1 cyclin/cdk activities quickly results (Figure 4b), indicating that there were sucient To determine how quick IFN-a down-regulated G1 cdk inhibitors in the lysates from cells treated with cyclin/cdk activities, asynchronously-growing subcon- IFN-a to down-regulate not only cdk2- but cdk4- ¯uent BAC12F5A cells were incubated with new media associated kinase activities in the equivalent amounts containing fresh CSF-1 together with or without IFN-a. of normal cell lysates. After the splitting of con¯uent BAC12F5A cells by a one ®fteenth ratio, it took 3 or 4 days for us to acquire 80% cell con¯uence, in which state 60% of cells were in the G1 phase (Figure 3c). When we started to incubate the cells a with IFN-a, we replaced the old media with small P 1 2 3 4 5 6 7 volumes of new media containing fresh CSF-1 with IFN- IFN-α – – – + – + – + a. To be precise, we needed cells which were fed with Time (hr) 0 0 3 3 6 6 9 9 fresh CSF-1 without IFN-a as controls because the expression of various cell cycle components and G1 cyclin/cdks-associated kinase activities were minimally a€ected by the fresh CSF-1 in the new media (see below). IP: α cdk4 pRb The addition of only fresh CSF-1 gradually reduced the percentage of cells within G1 phase and reciprocally increased cells within S and G2/M phase by potentiating G1 phase progression (Figure 3c, lanes 1, 2, 4, 6). In the b presence of IFN-a together with fresh CSF-1, however, the percentage of cells within G1 phase increased gradually after the addition of IFN-a, re¯ecting IP: α cdk2 pRb blockage of G1/S transition by IFN-a (lanes 3, 5, 7). The variation of cdk4- or cdk2-associated Rb kinase activities in the asynchronously-growing cells during treatment with IFN-a was determined with this system c (Figure 3a and b). Re¯ecting mitogenic stimulus of fresh % GI 60 60 59 56 63 45 77 CSF-1 in new media, cdk4-associated kinase activities % S 30 29 30 39 30 44 15 increased to one and a half fold the original level at 6 h in % G2/M 10 11 11 5 7 11 8 the control cells (Figure 3a, lane 4). Cdk2-associated kinase activities were not in¯uenced by media change Figure 3 IFN-a down-regulated both cdk4- and cdk2-associated kinase activities quickly. Asynchronously-growing subcon¯uent (Figure 3b). In the presence of IFN-a, cdk4- or cdk2- BAC1.2F5A cells were cultured in new media containing fresh associated kinase activities fell to less than one-®fth the CSF-1 with or without IFN-a (indicated + or 7) for the control levels within 3 h after addition of IFN-a (Figure indicated periods. Cell lysates were assayed for immune complex 3a and b, lanes for IFN-a+). The rapidity of IFN-a's cdk4- (a) or cdk2- (b) associated kinase activities with substrates of pRb and 16105 cells were analysed for cell cycle distribution e€ect on G1 cyclin activities was in contrast to that of the (c). Lane P indicates the lane for immunoprecipitation with other antimitogenic agents including TGF-b, which preimmune rabbit sera Interferon a-induced G1 arrest M Matsuoka et al 2079 a IP: α cyclin E P 1 2 3 4 Lysate IFN IFN — IFN IFN (–) (–) (–) (+) Cyclin E + – + + +

pRb

b (Ig)

cdk2

Figure 4 The cells treated with IFN-a contained excess inhibitors Figure 5 The kinase activities derived from exogenously-added which down-regulated cdk2- or cdk4-associated kinase activities. human cyclin E complexed with endogenous mouse cdk2 from The asynchronously-growing subcon¯uent cells were co-cultured BAC1.2F5A cells were suppressed by excess inhibitors residing in with new media containing fresh CSF-1 with or without IFN-a the cells treated with IFN-a. Insect cell lysates containing or not for 9 h, subsequently harvested and lysed in the H1 kinase bu€er containing human cyclin E generated baculovirally were mixed containing protease and phosphatase inhibitors. Indicated and incubated for 30 min at 238C with or without 150 mg of the amounts of cleared supernatants from the two types of cell cell lysates in the H1 kinase bu€er from asynchronously-growing (IFN-a, 7 or +) were mixed and incubated for 30 min at 238C cells treated with or without IFN-a (IFN-a,+or7) for 9 h, before immune complexes cdk2- (a) or cdk4- (b) associated kinase then immune complex human cyclin E-associated kinase assays reaction was performed with pRb proteins as substrates. The ®nal were conducted (a) or the mixed lysates were precipitated with volume of each reaction mixture was adjusted with HI kinase antibodies to human cyclin E or preimmune rabbit sera (indicated bu€er. 32P radioisotope counts of phosphorylated pRb were as P) and the precipitates were immunoblotted with antibodies to monitored with Bioimage analyzer BAS-2000II (Fuji Photo Film cdk2 (b). The amounts of added human cyclin E were estimated Co, Ltd, Shizuoka, Japan). The presented results are representa- to be about threefold the physiological level of cyclin E tive of three independent experiments

a Exogenously-added recombinant cyclin D2/cdk4 D2 D2 counteracted inhibitory activities of the lysates from cells Insect – /k4 – D2 K4 /k4 treated with IFN-a IFN-α – – + + + + Human cyclin E generated baculovirally in the insect cells was mixed and incubated with cell lysates for 30 min. Immune complex human cyclin E-associated kinase activities were determined with these lysates as HI described (Polyak et al., 1994a). Human cyclin E bound to endogenous mouse cdk2 derived from the control cell lysates gave rise to kinase activities (Figure 5a, lane 3) while neither single cyclin E or single cell lysate, nor the immunoprecipitates with preimmune b D2/cdk4 rabbit sera did (lane P, 1, 2). We could get only one- 0 0 0.1 0.3 1.0 ®fth the kinase activities of the controls from cells (µg) treated with IFN-a for 9 h (lane 4). To examine the IFN-α – + + + + complex formation between human cyclin E and endogenous mouse cdk2, the mixed cell lysates were immunoprecipitated with antibodies to human cyclin E and the precipitates were electrophoresed on SDS ± HI PAGE gel, then immunoblotted with antibodies to cdk2 (Figure 5b). The alternatively-spliced larger form Figure 6 Excess recombinant cyclin D2/cdk4 complexes counter- of cdk2, 42 kd cdk2 was expressed in the mouse cells in acted the inhibitory action in the IFN-a-treated cells. (a) Insect addition to the usual form, 34 kd cdk2 (Kato and cell lysates containing human cyclin E were mixed and incubated Sherr, 1993). In both the control cells and cells treated for 30 min at 238C with 150 mg of the lysates from asynchro- nously-growing cells treated with or without IFN-a (IFN-a,+or with IFN-a, similar levels of human cyclin E/cdk2 7) for 9 h in the presence of the insect cell lysates not infected complexes existed (lanes 3, 4). Thus, we con®rmed that (indicated as 7) or those containing cyclin D2, cdk4 or cyclin the IFN-a-treated cell lysates contained activities D2/cdk4, after which immune complex human cyclin E-associated negatively modifying cyclin E/cdk2 complexes. kinase assays were conducted with histone H1 as substrate. The added amounts of cyclin D2, cdk4 or cyclin D2/cdk4 were Based on the hypothesis that stoichiometrically- estimated as about threefold the physiological levels. (b) Insect acting cdk inhibitors down-regulated kinase activities, cell lysates containing human cyclin E were mixed and incubated which could be neutralized and trapped by excess for 30 min at 238C with 150 mg of the lysates from asynchro- amounts of cyclin/cdk complexes (Polyak et al., 1994a), nously-growing cells treated with or without IFN-a (IFN-a,+or we added the baculovirally-produced cyclin D2/cdk4 in 7) for 9 h in the presence of the insect cell lysates containing indicated amounts of cyclin D2/cdk4, then immune complex addition to human cyclin E to the above lysates before human cyclin E-associated kinase assays were conducted with assays of human cyclin E-associated kinase activities histone H1 as substrate Interferon a-induced G1 arrest M Matsuoka et al 2080 (Figure 6a). Cyclin D2/cdk4 but not single cyclin D2 for the control lanes from cells shifted to the new nor single cdk4 restored the kinase activities to the media containing only fresh CSF-1 (IFN-a, 7) because control level. This was because cyclin D2/cdk4 fresh CSF-1 slightly changed cell cycle distribution complexes trapped cdk inhibitors more eciently than (Figure 3c) and the expression of various cell cycle cyclin E/cdk2 complexes or single cyclin D2 or single components including cyclins, cdks and cdk inhibitors. cdk4. Furthermore, cyclin D2/cdk4 complexes restored We did not conduct the same experiments with cyclin E activities in a dose-dependent manner (Figure antibodies to p15Ink4A or p15Ink4B as the genes for these 6b). All these results indicated that cells treated with proteins were deleted in this cell line (Quelle et al., IFN-a contained excess cdk inhibitors which were 1995a). neutralized when enough cyclin D2/cdk4 was supplied The levels of total p19 and p21 were up-regulated exogenously. fourfold the control within 3 h (Figure 7a, p19 and Thus we concluded that up-regulated accumulation p21, lanes 1, 3), and remained elevated at 6 ± 9 h (lanes of cdk inhibitors contributed to the decrease of G1 5, 7) after the addition of IFN-a. Fresh CSF-1 without cyclin activities in the cells treated wtih IFN-a. IFN-a increased the relative amounts of total p19 and p21 (Figure 7a, p19 and p21, lanes 1, 2, 4, 6) possibly because it increased the relative percentage of cells The protein levels of p19 and p21 were quickly up- within S and G2/M phases, and such cells contain regulated after addition of IFN-a relatively larger amounts of p19 and p21 (Figure 3c) To see which cdk inhibitors are responsible for (Hirai et al., 1995; Li et al., 1944). The levels of total diminuition of G1 cyclin activities, we determined p27 decreased to about one third the original level by temporal pro®les of known cdk inhibitor levels in replacement of the old media with new media contrast to those of cyclin levels as positive regulators containing fresh CSF-1, irrespective of IFN-a (Figure and cdk levels during the early phase of treatment with 7a, p27). The levels of total cyclin D1 and cyclin E IFN-a (Figure 7). Immunoblot analyses were per- were basically invariant during treatment with IFN-a formed with the antibodies to cdk inhibitors including (Figure 7a D1 and E). p19Ink4D, p21Cip1 and p27Kip1 and those to cyclin D1 and To determine the amount of the various cdk cyclin E using whole cell lysates from cells before inhibitors and cyclins attached to cdks during treatment with IFN-a (indicated as 0) and those treatment with IFN-a, sequential immunoprecipita- cultured in new media with fresh CSF-1 together with tion-immunoblotting was performed. The same series or without IFN-a (IFN-a,+or7) for the indicated of cell lysates used in the kinase assays described above times (3 ± 9 h) (Figure 7a). We again made the lysates (Figure 3) were immunoprecipitated with the anti-

a b c Lysate IP: α cdk4 IP: α cdk2 1 2 3 4 5 6 7 P 1 2 3 4 5 6 7 P 1 2 3 4 5 6 7 IFN-α – – + – + – + IFN-α – – – + – + – + IFN-α – – – + – + – + Time (hr.) 0 3 3 6 6 9 9 Time (hr.) 0 0 3 3 6 6 9 9 Time (hr.) 0 0 3 3 6 6 9 9 (Ig)

p19 cdk4 cdk2

p21 (Ig)

D1

p27 E

p19

p21

D1 p21 cdk2

(Ig) E p27

p27

Figure 7 IFN-a treatment up-regulated the levels of p19 and p21 quickly. Asynchronously-growing subcon¯uent BAC1.2F5A cells were cultured in new media containing fresh CSF-1 with or without IFN-a (indicated + or 7) for the indicated periods. (a) The cell lysates (100 mg) prepared in NP40 lysis bu€er were immunoblotted with antibodies to p19, p21, p27, cyclin D1 and cyclin E. (b) The cell lysates (400 mg) prepared in EBC bu€er were precipitated with antibodies to cdk4 and the precipitates were immunoblotted with indicated antibodies. The lane p indicates the lanes for immunoprecipitation with preimmune rabbit sera. (c) The cell lysates (200 mg) prepared in Tween 20 lysis bu€er were precipitated with antibodies to cdk2 and the precipitates were sequentially immunoblotted with indicated antibodies. The blotting with anti-p27 antibodies was performed with the un-erased membrane previously blotted with anti-cdk2 antibodies Interferon a-induced G1 arrest M Matsuoka et al 2081 bodies to cdk4 (Figure 7b) or cdk2 (Figure 7c), and the the presence of 1000 U/ml IFN-a (Figure 8a, lanes 1, precipitates were electrophoresed, fractionated on the 2), and about four- or ®vefold in the presence of SDS ± PAGE gels and immunoblotted with antibodies 2000 U/ml or 4000 U/ml IFN-a (lanes 3 and 4). to cdk2, cdk4 or a series of antibodies to various However, the levels of cyclin D1 protein were almost proteins bound to cdk4 or cdk2 (Figure 7b and c). constant irrespective of IFN-a concentration (Figure The protein levels of total cdk4 and cyclin D1 bound 8b). This observation indicated that gene expression of to cdk4 were constant after new media with fresh CSF- p19 was induced by IFN-a in a dose-responsive fashion 1 were added irrespective of IFN-a (Figure 7b, cdk4 in this cell line. and D1). The levels of p19 bound to cdk4 were gradually and slightly up-regulated by fresh CSF-1 IFN-a up-regulated mRNA levels of p19 and p21 without IFN-a (Figure 7b, p19, lanes 1, 2, 4, 6) again because fresh CSF-1 transiently potentiated G1 phase Northern blot analysis was performed to see the action progression and increased the non-G1 cell population. point of IFN-a on gene expression of p19 and p21. p19 In the presence of IFN-a, they were up-regulated and p21 mRNA levels increased at 3 h, 6 h and 9 h or sixfold the original level within 3 h (Figure 7b, p19, 12 h after addition of IFN-a (Figure 9a and b). Up- lane 3), and remained elevated at 6 ± 9 h (lanes 5, 7). regulation of steady-state p19 and p21 mRNA levels by The levels of p21 bound to cdk4 were apparently up- IFN-a is about three times the controls and seems to regulated at 3 h by treatment with IFN-a (Figure 7b, be a little smaller than that of p19 and p21 protein cdk4, D1 and p21, lanes 2, 3), but decreased thereafter levels by IFN-a (Figure 7a). We speculate that the in proportion to the levels of cyclin D1/cdk4 stability of those proteins also increased during complexes. The levels of p27 bound to cdk4 gradually treatment with IFN-a. increased after the addition of fresh CSF-1, irrespective of IFN-a (Figure 7b, p27). Note that up-regulated p19 The activities of cdk2 CAK and the protein levels of did not seem to compete with p27 for binding to cdk4 cdc25A were not down-regulated by IFN-a during treatment with IFN-a. Up-regulated p15 did compete with p27 for binding to cdk4 during treatment With the results shown above, we could not completely with TGF-b (Reynisdottir et al., 1995; Reynisdottir exclude the possibility that other post-translational and Massague, 1996). modi®cations contributed to down-regulation of G1 The levels of total cdk2 and cyclin E bound to cdk2 cyclin activities. An important post-translational were not a€ected by replacement of media (Figure 7c, modi®cation of cyclin/cdk is mediated by the cdk- cdk2 and cyclin E). Up-regulation of p21 bound to activating kinases, CAKs, which phosphorylate cyclin- cdk2 by IFN-a was observed at 3 h (Figure 7c, p21, bound cdks on a single threonine residue (Thr 160 in lanes 1, 2, 3). The levels increased ®vefold the control cdk2 and Thr 172 in cdk4) (Fisher and Morgan, 1994; levels at 6 ± 9 h (lanes 4, 5, 6, 7). Note that p21 levels Kato et al., 1994a; Matsuoka et al., 1994; Morgan, decreased to two thirds or less the original level after 1995). These modi®cations are essential for cyclin-cdks transfer of cells to new media containing only fresh to become active holoenzymes and might themselves be CSF-1 (lanes 1, 2, 4, 6). p21 levels were always higher the targets of antiproliferative factors. With the lysates during treatment with IFN-a than their original level from synchronized cells cultured with or without IFN- (lanes 1, 3, 5, 7). p27 attached to cdk2 was up- a or cAMP for 12 h, we performed immune complex regulated about threefold the control level at 9 h cdk2 CAK assays (Figure 10a) (Matsuoka et al., 1994). (Figure 7c, p27, lanes 6 and 7) by treatment with IFN- The substrates were the bacterially-generated Histidine- a. The levels of p27 bound to cdk2 were reduced to a half or less the original level only by replacement of media with those containing fresh CSF-1 (Figure 7c, p27, lanes 1, 2, 4, 6). Therefore, up-regulation of p27 a bound to cdk2 by IFN-a was relatively mild compared 1 2 3 4 (kd) with the original level. 26 Collectively, up-regulation of total p19 and p19 bound to cdk4, and minimally that of total p21 and p19 p21 bound to cdk2 is supposed to be responsible for the down-regulation of cyclin D1/cdk4-associated kinase activities during early phase treatment of IFN- 14 a. We could also conclude that upregulated p21 participated in down-regulation of cyclin E/cdk2- b associated kinase activities. 48 p19 levels were up-regulated by IFN-a in a dose- D1 responsive fashion 32 We examined variation of the p19 protein levels in the lysates from asynchronously-growing cells treated with Figure 8 p19 levels were up-regulated during treatment with several di€erent concentrations of IFN-a for 12 h to IFN-a in a dose-dependent manner. Subcon¯uent cells were co- see whether or not they increased in parallel to the cultured with new media containing fresh CSF-1 without or with concentration of IFN-a (Figure 8a). The internal various concentrations of IFN-a (lane 1-0 U/ml, lane 2-1000 U/ ml, lane 3-2000 U/ml, lane 4-4000 U/ml) for 12 h. Cell lysates controls were the levels of cyclin D1 proteins (Figure (100 mg) in NP40 lysis bu€er were immunoblotted with antibodies 8b). The p19 levels were up-regulated about twofold in to p19 (a) and cyclin D1 (b) Interferon a-induced G1 arrest M Matsuoka et al 2082 tagged cdk2 and the antibodies were against cdk7 tion of cdc25A activities was the primary cause for (Poon et al., 1993). Cdk2 CAK activities were not cdk2-associated kinase activities in the mouse macro- a€ected by the presence of IFN-a or cAMP. We phage cell lines without determining phosphatase obtained similar results with the lysates from activities of cdc25A. asynchronously-growing cells treated or not treated with IFN-a for 9 h (data not shown). Another possible candidate, the activities of which Discussion might be down-regulated during treatment with IFN-a, was cdc25A, which was found to regulate G1/S Among the various biological activities of IFNs, progression of mammalian cells (Gu et al., 1992; antimitogenic activity has attracted a great deal of Jinno et al., 1994; Terada et al., 1995). The expression attention and has been utilized for treatment of of cdc25A was down-regulated by IFN-a in Daudi cells neoplasms. The basic mechanism by which IFNs (Tieferbrun et al., 1996). Therefore, the protein levels block cell cycle progression is now being elucidated. of double 65 and 80 kd mouse cdc25A in BAC1.2F5 Murine BAC1.2F5A is a macrophage cell line cells were assessed with immunoprecipitation and suitable for analysis of the e€ects of IFNs on the cell immunoblotting (Figure 10b). To assure that signals cycle because cells proliferate only in the presence of really indicated cdc25A, in the last lane the immunizing CSF-1, are easily synchronized at early G1 phase by peptides were added at the immunoprecipitation step deprivation of CSF-1, and are arrested in G1 phase to cancel out the signals (Figure 10b, lane 8). with IFN-a treatment. CSF-1 is required until cells Irrespective of IFN-a, the protein levels of cdc25A reach the late G1 restriction point, after which they were constant for at least 12 h after the addition of progress through the remainder of the cell cycle IFN-a in the asynchronously-growing cells. Although automatically. Addition of IFN-a prior to the mid unlikely from above experiments, we could not G1 phase prevented cells from passing the G1/S completely rule out the possibility that down-regula- boundary (Figure 1c). We therefore focused on the activities of G1 cyclins, D-type cyclins/cdk4 and cyclin E/cdk2. Using several antibodies, immune complex a kinase assays were performed. As shown above, in 1 2 3 4 5 6 asynchronously-growing and synchronized cells treated Time (hr.) 3 3 6 6 12 12 INF-α – + – + – +

a p19 CDK7 CDK7 CDK7 α α IP α

Lysate (–) IFN NRS (–) IFN (+) IFN cAMP

β-actin CDK2

1 2 3 4

b 1 2 3 4 5 6 b α Time (hr.) 3 3 6 6 9 9 IP: cdc25A INF-α – + – + – + 1 2 3 4 5 6 7 8 peptide – – – – – – – + INF-α – – + – + – + – p21 Time (hr.) 0 6 6 9 9 1212 0 — 84 kd Blot: cdc25A — 62 kd

Figure 10 Neither the cdk2 CAK activities nor the protein levels of cdc25A were down-regulated during treatment with IFN-a.(a) EF-1 α Subcon¯uent BAC1.2F5A cells were synchronized at early G1 by starvation of CSF-1 and restimulated with media containing fresh CSF-1 with or without IFN-a or with cAMP ((7)-IFN, (+)-IFN or (+)-cAMP) for 12 h. Cell lysates (200 mg) in the H1 kinase bu€er were immunoprecipitated with the non-immune rabbit sera Figure 9 IFN-a increased mRNA levels of p19 and p21. or the antibodies to cdk7 and the precipitates were assayed for Asynchronously-growing subcon¯uent BAC1.2F5A cells were cdk2 CAK activities with 3 mg of bacterially-produced His-tagged cultured in new media containing fresh CSF-1 with or without cdk2 as substrate. (b) Asynchronously-growing subcon¯uent IFN-a (indicated + or 7) for the indicated periods. Fractionated BAC1.2F5A cells were cultured in new media containing fresh mRNAs transferred onto nitrocellulose ®lters were hybridized with CSF-1 with or without IFN-a (indicated + or 7) for the a-32P-dCTP-labeled cDNA of p19 (a) or p21 (b). The same indicated periods. Cell lysates in Tween 20 lysis bu€er were nitrocellulose ®lters were rehybridized with a-32P-dCTP-labeled precipitated with antibodies to cdc25A and the precipitates were human b-actin (a) or human EF-1a (b) to indicate the applied immunoblotted with the same antibodies. The immunizing amounts of mRNA peptides (1 mg) were added to the lysates for the last lane Interferon a-induced G1 arrest M Matsuoka et al 2083 with CSF-1 plus IFN-a, both cyclin E/cdk2 and cyclin et al., 1995). During treatment with IFN-a, however, D1/cdk4 activities were reduced markedly and very the expression of p18 mRNA was not enhanced (M quickly (Figures 2 and 3). The temporal pro®le of G1- Matsuoka and S Asano, unpublished observation) cyclin-associated activities during treatment with IFN-a while that of p19 mRNA was potentiated (Figure indicated that IFN-a down-regulated them within 3 h. 9a), suggesting that p18 does not seem to behave like Therefore searches for mechanisms down-regulating p19 during treatment with IFN-a. cyclins/cdks activities during early-phase IFN-a treat- Regulation of cell cycle components by IFN-a was ment were performed. distinct from that by TGF-b. The mechanism of TGF- A possible mechanism blocking the activities of G1 b-mediated G1 phase arrest has been extensively cyclin D1/cdk was the increased attachment of various analysed (Reynisdottir et al., 1995; Reynisdottir and cdk inhibitors to cdks which act stoichiometrically and Massague, 1997; Lavarone and Massague, 1997). are heat-stable (Sherr and Roberts, 1995). Screening p27Kip1,p21Cip1 and p15Ink4B cooperated to induce cell kinase assays using boiled lysates as the source of cycle arrest. Expression of p15Ink4B mRNA and protein inhibitors indicated that cells treated with IFN-a were induced primarily. Up-regulated p15Ink4B itself contained more heat-stable cdk2 inhibitors than down-regulated cdk4-associated kinase activities while control cells (data not shown). Known cdk inhibitors it competed with p27 for binding to cyclin D/cdk4. P27 act stoichiometrically and should be abundant enough released from cyclin D/cdk4 complexes attached to to down-regulate G1 cyclin/cdk activities. We exam- cdk2 and down-regulated cdk2-associated kinase ined whether or not excess cdk inhibitors existed in the activities. IFN-a similarily up-regulated p19, a family cells treated with IFN-a. The mixing experiments member of Ink4 type cyclin inhibitors. However, up- revealed that the lysates from cells treated with IFN- regulated p19 did not seem to compete with p27 for a contained enough inhibitory activities to down- binding to cyclin D/cdk4. Instead, quickly up-regulated regulate G1 cyclin/cdk-associated kinase contained in levels of total p21 and p21 bound to cdk2 reduced the equivalent amounts of lysates from control cells cdk2-associated kinase activities during treatment with (Figure 4). Furthermore, sucient amounts of IFN-a. exogenously-added baculovirally-generated cyclin D2/ It remains to be clari®ed whether other post- cdk4 complexes counteracted and neutralized these translational modi®cations than cdk inhibitors con- inhibitory activities residing in IFN-a-treated cell tributes to down-regulation of G1 cyclin/cdks activities lysates (Figures 5 and 6). All these observations during treatment with IFN-a. Two types of positive substantiated the idea that cells treated with IFN-a cdk modi®ers have been well characterized, one of contained excess amounts of cdk inhibitors which which is mediated by the cdk-activating kinases, CAKs down-regulated G1 cyclin-associated kinase activities (Fisher and Morgan, 1994; Kato et al., 1994a; stoichiometrically. Matsuoka et al., 1994; Morgan, 1995). CAKs might To assess the actual mediators of the IFN-a e€ect on themselves be the targets of antiproliferative factors the cell cycle, we next determined time courses of the (Kato et al., 1994b). The phosphatase cdc25A levels of known cdk inhibitors as well as the levels of functions as a positive regulator of G1/S transition cdk inhibitors bound to cdks in the cells during (Jinno et al., 1994) by potentiating G1 cyclin/cdk treatment with IFN-a. The levels of total p19 or p21 activities by dephosphorylating tyrosine 15 of cdk2 or and the levels of p19 or p21 bound to cdks were tyrosine 17 of cdk4 (Gu et al., 1992; Terada et al., quickly up-regulated during treatment with IFN-a in 1995), and may be another target of antimitogenic parallel to down-regulation of G1 cyclin activities signals. In Daudi cells, the mRNA and protein levels of (Figures 3 and 7), indicating that up-regulated p19 or cdc25A were quickly down-regulated by treatment with p21 could down-regulate cyclin D/cdk4- or cyclin E/ IFN-a (Tiefenbrun et al., 1996). In MCF7 cells in cdk2-associated kinase activities, respectively, during which p15 gene was deleted but which were still the early phase of IFN-a treatment. mRNA levels of sensitive to antiproliferative action of TGF-b, down- p19 and p21 were upregulated similarily during regulation of cdc25A phosphatase gene expression by treatment with IFN-a (Figure 9) while increase of TGF-b contributed to inactivation of cdk4 and cdk6 protein stability was also supposed to contribute to up- function (Lavarone and Massague, 1997). In BAC12F5 regulation of these protein levels. cells treated with IFN-a, however, the cdk2 CAK Although the precise function of p19 still remains to activities were intact and the protein levels of cdc25A be clari®ed, it is natural for us to think that up- were constant, suggesting that neither cdk2 CAK nor regulated accumulation of p19 reduced cyclin D1/cdk4 cdc25A are the mediators of IFN-a during early-phase activities during treatment with IFN-a. The previous treatment (Figure 9). To be exact, we need to experiments indicated that recombinant p19 selectively determine the dephosphorylating activities of cdc25A inhibited the activities of D type cyclins/cdk in vitro in during treatment with IFN-a to completely rule out a a dose-responsive fashion and enforced overexpression role for cdc25A in IFN-a-induced G1 phase arrest. of p19 induced G1 phase arrest as well as the Furthermore, we did not exclude the possibility that disappearance of cyclin D kinase activities in the another unknown post-translational modi®cation was mammalian cells (Hirai et al., 1995). Currently, we responsible for the IFN-a-mediated down-regulation of cannot completely rule out the possibility that other G1 cyclin/cdk-associated kinase activities especially in cdk inhibitors such as p18INK4C or p57KIP2 participate in the later phase of IFN-a treatment. the inhibition of G1 cyclin/cdk-associated kinase We have few similar evidences supporting the activities during treatment with IFN-a. Northern blot generality of the phenomenon that p19 or p21 is analysis had indicated that the expression of p18 upregulated during treatment with IFN-a. It was mRNA oscillated like that of p19 mRNA during demonstrated that p21 levels were up-regulated in a normal cell cycle progression of BAC1.2F5 cells (Hirai prostatic cancer cell line and Daudi Burkitt's Interferon a-induced G1 arrest M Matsuoka et al 2084 lymphoma cells during treatment with IFN-a (Hobeika and were used for human cyclin E-associated kinase assays, et al., 1997). The gene expression of p19 was not and the polyclonal anti-cdc25A antibodies were also from analysed there. We ourselves did not observe that IFN- SantaCruzInc. a up-regulated the expression of p21 nor p19 gene in Daudi cells (data not shown). In 32D cells, mouse Protein analyses myeloblastic cells which are partially sensitive to IFN- BAC1.2F5A cells (56106) were lysed for 1 h at 48Cwith a, p19 and p21 protein levels were only minimally up- 1mlofEBCbu€er(50mM Tris-HCl [pH 8.0], 120 mM regulated (data not shown). We speculate that p19 and NaCl,0.5%NP-40,1mMEDTA and 1 mM dithiothreitol p21 gene expression is induced by IFN-a especially in [DTT] or Tween 20 lysis bu€er (50 mM HEPES [pH 7.5], myelomonocytoid cells. 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, In conclusion, down-regulation of cyclin D1/cdk4- 0.1% Tween 20) containing 5 mg/ml of aprotinin, 0.2 mM and cyclin E/cdk2-associated kinase activities and phenylmethylsulfonyl ¯uoride (PMSF), 10 mM b-glycero- resulting G1 phase arrest during treatment with IFN- phosphate, 2.5 mg/ml leupeptin, 2.5 mM EGTA and 0.1 mM a were at least partially mediated by quick up- sodium orthovanadate (protease and phosphatase inhibi- regulation of cdk inhibitors including p19 and p21 in tors from Sigma). Cleared cell lysates (100 ± 400 mg) were immunoprecipitated with saturated amounts of antibodies, macrophage cells. separated on denaturing gels, transferred onto nitrocellu- lose ®lters, and blotted with indicated antibodies. Detec- tion was with the ECL system (Amersham, Arlington Materials and methods Heights, Ill). For direct immunoblotting, cells were lysed in NP-40 lysis bu€er (20 mM Tris-HCl [pH 7.5], 150 mM Cell culture and cell cycle analyses NaCl, 1% NP-40, 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors. Aliquots (50 ± 100 mg) Murine BAC1.2F5A macrophages were grown in Dulbec- of clear lysates were loaded in each lane. Densitometric co's modi®ed Eagle's medium (DMEM) supplemented with estimation of chemiluminescence-labeled protein amounts 20% fetal bovine serum (FBS), 100 U of penicillin G per was performed with Quantity One software from PDI Inc. ml and 100 mg of streptomycin per ml and 25% L cell- (Huntington, NY). Several ®lms with di€erent exposures conditioned medium, as described (Matsushime et al., were prepared for comparison within the linear correlation 1991). These cells are dependent on CSF-1 for prolifera- range of protein amount and signal strength. tion and survival. Macrophages were arrested in early G1 by deprivation of CSF-1 for 18 h and stimulated with CSF- 1 to reenter the cell cycle in a synchronized fashion. About Immune complex kinase assays 80% con¯uent (subcon¯uent) cells were used both for IFN- a treatment of asynchronously-growing cells and for CSF-1 Kinase assays were performed as described previously deprivation followed by restimulation with CSF-1 together (Matsushime et al., 1994). Brie¯y, cells were suspended at 6 with IFN-a of synchronous cells unless otherwise cited. 4610 /ml in Tween-20 lysis bu€er containing protease and Usually, 3 ml of media with CSF-1 with or without phosphatase inhibitors, and lysed by sonication. Cyclin 2000 U/ml IFN-a was added to 10 cm dishes. IFN-a was D1-, cdk4-, cyclin E- and cdk2-associated kinase activities kindly supplied by Nippon Roche Inc. and Sumitomo were immunoprecipitated from cleared cell lysates (100 ± Pharmaceutical Inc. or purchased from GIBCO BRL 500 mg) with protein A-Sepharose beads precoated with (Gaitherburg, MD) and 8-bromoadenosine 3',5'-cyclic saturated amounts of antibodies to cyclin D1, cdk4, cyclin monophosphate (8Br-cAMP) was purchased from Sigma E and cdk2, and assayed for pRb kinase activities or Chemicals (St Louis, MO). histone H1. The beads were washed three times with 0.5 ml For cell cycle analyses, cell nuclei were prepared and Tween 20 lysis bu€er and three times with kinase bu€er (50 mM HEPES [pH 7.5}, 10 mM MgCl ,1mM DTT), stained with propidium iodide using CycleTEST (Becton 2 Dickinson, San Jose, CA) according to the manufacturer's suspended and incubated for 1 h at 308Cin20mlofthe instructions. DNA ¯uorescences were measured with a kinase bu€er containing 1 mg glutathione S-transferase- FACScan ¯ow cytometer (Becton Dickinson, San Jose, pRb fusion protein or 1 mg histone H1 as substrates in CA), and the percentages of cells within G1, S and G2/M association with protease and phosphatase inhibitors, 32 phases were determined (Matsushime et al., 1991). supplemented with 25 mM ATP, 10 mCi of [r- P]ATP (6000 Ci/mmol, New England Nuclear). For preparation of pRb substrates, pGEX-Rb-C (C-terminal polypeptide of Antibodies pRb) or pGEX-Rb-Large Pocket (379 ± 928) was used. The The mouse monoclonal antibody D1-72-13G to mouse D1 precise isolation procedures were as described (Matsushime and polyclonal rabbit antibodies to mouse cdk7 or MO15 et al., 1994). Relatively higher background signals were were as described (Matsushime et al., 1994; Matsuoka et observed with GST-Rb-C polypeptides as substrates than al., 1994). The polyvalent rabbit antisera to the C-terminal with GST-Rb-Large Pocket. The signal strength increased peptide of the mouse cdk4 (C-22), the C-terminal peptide in parallel with the amounts of cell lysates (100 ± 500 mg) 32 of the mouse cdk2 (M2), p19 (M-20) (to the epitope under this assay condition. P radioisotope counts of corresponding to amino acids 135 ± 154 of mouse p19), p21 phosphorylated pRb were monitored with Bioimage (C-19) (to the epitope corresponding to amino acids 146 ± Analyzer BAS-2000II (Fuji Photo Film Co, Ltd, Shizuo- 164 of mouse p21) and p27 (N-20) (to the epitope ka, Japan). corresponding to amino acids 2 ± 21 of human p27 which can also recognize mouse p27), were purchased from Santa Kinase inhibition assays Cruz Biotechnology (Santa Cruz, CA). The latter three antibodies recognized mouse p19, p21 or p27 and did not Immune complex cdk2- or cdk4-associated kinase assays cross-react with each other. The polyclonal antibodies to were done after normal cell lysates (200 mg or 400 mg) were cyclin E (the antibody to the epitope corresponding to a mixed with lysates containing cdk inhibitors and incubated peptide, GVLTPPHSSKKQSSEQETE, mapping at the for 30 ± 60 min at 238C. The lysis bu€ers were H1 kinase carbocyxterminus of rat cyclin E which can recognize bu€er (80 mM Na-b-glycerophosphate, pH 7.3-15 mM

mouse cyclin E), the monoclonal antibodies to human MgCl2-20 mM EGTA {ethylene glycol-bis(b-aminoethyl cyclin E (HE111) which recognized only human cyclin E ether)-N,N,N',N'-tetraacetic acid}-5 mM DTT) containing Interferon a-induced G1 arrest M Matsuoka et al 2085 protease and phosphatase inhibitors. The immunoprecipi- Cdk2 CAK assays tates were washed three times with Tween 20 lysis bu€ers Cells were suspended at 46106/ml in H1 kinase bu€er and three times with H1 kinase bu€ers before the kinase containing protease and phosphatase inhibitors and reaction was performed with the substrates of bacterially- sonicated at 48C. The cleared supernatants were precipi- produced pRb proteins or histone H1. tated for 2 h at 48C with the antibodies to mouse cdk7 and the immunoprecipitates were washed three times with Preparation of baculovirally-produced proteins Tween 20 lysis bu€er and three times with H1 kinase bu€er. The beads were suspended in 20 mlofH1kinase Cyclin E, cyclin D2 and cdk4 were prepared by the method bu€er containing 5 mg of bacterially-produced His-tagged of Matsushime et al. (1992). Brie¯y, 60 mm plates of cdk2 as the CAK substrates supplemented with 50 mM ATP con¯uent TN5 cells were infected with the appropriate and 10 mCi [g-32P]ATP, and then incubated at 308Cfor baculovirus. The cells were collected 48 ± 72 h after virus 60 min. His-tagged cdk2 protein was isolated as described infection and lysed in H1 kinase bu€er for 1 h at 48C. The (Matsuoka et al., 1994). cleared lysates were directly used for cdk2 activation assays. Northern blot analysis Cdk2 activation assays Total RNA was extracted from cells using the AGPC Endogenous cdk2 activation in the cell lysates by method. Northern blot hybridization was performed as exogenously-added cyclin E was as described by Polyak described (Matsushime et al., 1991). K et al. (1994a). Brie¯y, indicated amounts of baculovi- rally-generated human cyclin E were mixed with 150 mgof cell lysate in the H1 kinase bu€er containing protease and phosphatase inhibitors and incubated for 30 min at 238C Acknowledgements before the reaction was immunoprecipitated with the cyclin We thank Sumitomo Pharmaceutical Inc. and Nippon E monoclonal antibody. The immunoprecipitates were Roche Inc. for providing mouse interferon-a,DrsCharlesJ washed and assayed for pRb or histone H1 kinase Sherr and Hitoshi Matsushime for the baculoviruses and activities as described in the immune complex kinase antibodies and Dr Jun-ya Kato for critical advice on the assays. In some experiments, indicated amounts of manuscript. We are also grateful to Drs Satoshi Takahashi baculovirally-synthesized cyclin D2, cdk4 or cyclin D2/ and Arinobu Tojo for various supports. Daniel Murozek cdk4 complexes were added to the mixed lysates. helped us to correct English.

References

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