Mutual requirement of CDK4 and Myc in malignant transformation ...

Oncogene (1997) 15, 179 ± 192  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Mutual requirement of CDK4 and Myc in malignant transformation: evidence for cyclin D1/CDK4 and p16INK4A as upstream regulators of Myc Kirsten Haas1, Peter Staller2, Christoph Geisen1, Jiri Bartek3, Martin Eilers2 and Tarik MoÈroÈy1 1

Institut fuÈr Zellbiologie (Tumorforschung), IFZ, UniversitaÈtsklinikum Essen, Virchowstrasse 173, D-45122 Essen, Germany; Zentrum fuÈr Molekulare Biologie Heidelberg, ZMBH, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany; and 3Danish Cancer Society, Division for Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen, Denmark

2

We demonstrate in this paper that CDK4 which is a G1 phase speci®c cell cycle regulator and catalytic subunit of D-type cyclins has oncogenic activity similar to Dtype cyclins themselves and is able to provoke focus formation when cotransfected with activated Ha-ras into primary rat embryo ®broblasts. Surprisingly, using two dierent mutants we show that CDK4's ability to bind to p16INK4a and not its kinase activity is important for its transforming potential. In addition, p16INK4a but not a mutant form that is found in human tumours can completely abrogate focus formation by CDK4 suggesting that CDK4 can malignantly transform cells by sequestering p16INK4a or other CKIs. We demonstrate that both cyclin D1 and CDK4 functionally depend on active Myc to exert their potential as oncogenes and vice versa that the transforming ability of Myc requires functional cyclin D/CDK complexes. Moreover, we ®nd that p16INK4a and the Rb related protein p107 which releases Myc after phosphorylation by cyclin D1/CDK4 eciently block Myc's activity as a transcriptional transactivator and as an oncogene. We conclude that both p16INK4a and cyclin D/CDK4 complexes are upstream regulators of Myc and directly govern Myc function in transcriptional transactivation and transformation via the pocket protein p107. Keywords: oncogene cooperation; REF assay; cell cycle regulation; Myc function; CDK4

Introduction Continuously dividing normal and transformed cells progress periodically through a cellular division cycle that consists of the S-phase where the cell replicates DNA, the M-phase where the cell divides and two phases called G1 and G2 that separate S and M. Progression through G1 that precedes S is governed by D- and E-type cyclins and their associated catalytic subunits CDK4, CDK6 and CDK2. In particular, a role of cyclin D1 in G1 progression has been clearly shown in numerous independent studies (Baldin et al., 1993; Kato et al., 1993; Quelle et al., 1993; Jiang et al., 1993). Expression of mammalian D- and E-type cyclins as well as CDK4 can be induced in G1 by mitogens or growth factors and their continuous synthesis depends on the persistence of a mitogenic stimulus (Matsushime et al., 1991; Sewing et al., 1993, 1994). Overexpression of both Correspondence: T MoÈroÈy Received 14 January 1997; revised 24 March 1997; accepted 25 March 1997

D- and E-type cyclins shortens progression through G1 in a number of dierent mammalian cell types tested (Jiang et al., 1993; Kato and Sherr, 1993; Resnitzky et al., 1994). A considerable number of experimental data suggests that the tumour suppressor protein Rb is one of the substrates of the cyclin D1/CDK4 and possibly of the cyclin E/CDK2 kinase (Dowdy et al., 1993; Ewen et al., 1993; Hinds et al., 1994; Kato and Sherr, 1993; Matsushime et al., 1994; Meyerson and Harlow, 1994; Hu et al., 1992; Sherr, 1994). According to this hypothesis, Rb is hyperphosphorylated by either Dtype or E-type cyclin kinases and allows progression to the late G1 phase by releasing transcription factors of the E2F family that prepare the entry into S-phase. A number of other `pocket' proteins that are structurally related to Rb also appear to be substrates for cyclin/ CDK complexes. Among them are p107 and p130 (for review see Lam and LaThange, 1994; Sherr, 1995). More recently, it has been shown that p107 is phosphorylated by cyclin D/CDK4 and by cyclin E/ CDK2 complexes (Xiao et al., 1996; Lees et al., 1992) and that p107 is able to bind E2F (Starostik et al., 1996; Xiao et al., 1996; Zhu et al., 1995; Zamanian and LaThangue, 1993; Schwarz et al., 1993; Lees et al., 1992; Cao et al., 1992) but also the product of the c-Myc proto-oncogene (Beijersbergen et al., 1994, 1995; Gu et al., 1994). A number of inhibitors for cyclin dependent kinases (CKIs) have recently been isolated among them the proteins p16ink4a and p21WAF. Whereas p21WAF can inhibit the activity of dierent cyclin/CDK complexes as well as proliferating cell nuclear antigen (PCNA) that is involved in DNA replication and repair (Waga et al., 1994; Xiong et al., 1993; Chen et al., 1995), p16ink4a is more restricted in its activity and speci®cally inhibits CDK4 and CDK6, both regulatory subunits of D-type cyclins (Serrano et al., 1993). Whereas p21WAF binds to cyclin D1, p16ink4a speci®cally forms complexes with CDK4 and does not bind to D-type cyclins (Hall et al., 1995). Both p16ink4a and p21WAF have been shown to interfere with proper cell proliferation, in particular, p16ink4a can block Myc/Ha-ras mediated transformation and Ha-ras induced cell proliferation (Serrano et al., 1995). Similar to G1 speci®c cyclins the transcription factor c-Myc plays a key role in cellular proliferation (reviewed in Meichle et al., 1992). In serum stimulated cells c-Myc is induced as an immediate early response gene. It has been shown that both micro-injection of cMyc into the nuclei of quiescent cells or the expression of c-Myc through conditional alleles rapidly drives the cells through G1 into the S-phase (Eilers et al., 1991) and that inhibition of Myc function often arrests cells

Interdependence of CDK4 and Myc in malignant transformation K Haas et al

180

in the G1 phase of the cell cycle. Through the work of recent years a functional role of Myc family proteins as regulators of gene expression has emerged (reviewed in Meichle et al., 1992). The Myc transcription factors contain a transactivation domain at the amino terminal end and a basic region directly followed by a helix ± loop ± helix-leucine zipper motif (b-HLHLZip) at its carboxyterminal end. The latter mediates DNA binding and heterodimerization with speci®c partner proteins (Kato et al., 1990; Landschulz et al., 1988; Murre et al., 1989). One of the best studied partners of Myc is the Max protein that also contains a b-HLH-LZip domain (Blackwood and Eisenman, 1991; Prendergast et al., 1991; Wenzel et al., 1991). The Myc/Max heterodimer can speci®cally bind to Ebox motifs with a preference for the sequence CACGTG (Blackwell et al., 1990; Kerkho et al., 1991; Prendergast et al., 1991). Myc/Max heterodimers bind DNA targets with the same sequence speci®city as Max homodimers, but only the heterodimers are capable of transcriptional transactivation (Amati et al., 1992, 1993). Max can dimerize with itself but also with two other proteins that contain b-HLH-LZip structures, namely Mad and Mxi-1, which do not form homodimers (Ayer et al., 1993; Zervos et al., 1993). Both Mad and Mxi-1 bind to Max, they can eciently antagonize the eects of Myc by regulating the availability of Max and thereby the relative abundance of active Myc/Max complexes (Koskinen et al., 1995; VaÈstrik et al., 1995; Schreiber-Agus et al., 1995). Both cyclin D1 and the genes of the Myc family have transforming abilities. While the role of Myc as an oncogene and its implication in the generation of a wide variety of tumours has been exhaustively documented (reviewed in Meichle et al., 1992), the implications of cyclin D1 in the generation of tumours has emerged more recently. While the overexpression of cyclin D1 in a number of malignancies led to the assumption that cyclin D1 may have a functional role in tumour formation, cell transfection experiments and studies with transgenic mice documented this directly also including cyclin D2 (Bodrug et al., 1994; Lovec et al., 1994a,b; Wang et al., 1994; Kerkho and Zi, 1995). Although recent data argue also for the implication of deregulated CDK4 expression in the formation of human tumours (Keyomarsi et al., 1995; Kitahara et al., 1995; Khatib et al., 1993), an oncogenic activity similar to D-type cyclins has so far not been demonstrated for CDK4.

Table 1 Constructs transfected Ras alone Ras+pLTR vector Myc alone cyclin D1 alone Ras+cyclin D1 Myc+CDK4 Ras+CDK4

To investigate the potential of CDK4 to malignantly transform cells, we have employed transfection experiments with primary rat ®broblasts as an experimental system. We show here that CDK4 has oncogenic activity, but that this activity does not correlate with its enzymatic activity but rather with its ability to bind p16INK4a. In addition, we ®nd that CDK4 requires functional Myc protein to exert its function as an oncoprotein and, vice versa, Myc requires cyclin D/ CDK kinase activity to transform cells. Finally, we show that the activity of Myc as a transcriptional transactivator can be blocked by p16INK4a as well as by the Rb related pocket protein p107 suggesting a regulation of Myc activity through cyclin D/CDK complexes and a mode of action for p16INK4a in tumourigenesis.

Results Transforming potential of CDK4 It has previously been shown that coexpression of cyclin D1 or cyclin D2 with activated Ha-ras can induce focus formation and malignant growth in primary rat embryo ®broblasts (REFs) and also in BRK cells (Lovec et al., 1994b; Kerkho and Zi, 1995; Hinds et al., 1994). These three independent studies and further experiments with transgenic mice ®rmly established cyclin D1 and cyclin D2 as bona ®de proto-oncogenes that contribute to malignant transformation when constitutively expressed (Lovec et al., 1994a; Bodrug et al., 1994; Wang et al., 1994). We now ®nd that similar to D-type cyclins, their associated kinase CDK4 is equally able to cooperate with Ha-ras and to generate the outgrowth of foci after transfection of REFs (Table 1). The cotransfection of both activated Ha-ras and HA-tagged CDK4 led to the formation of foci that were morphologically indistinguishable from those generated by cyclin D1 and Ha-ras or by Myc and Ha-ras and expressed the transfected CDK4 at high levels (Figure 1b). However, closer inspection of cells established from foci as continuously growing lines revealed that CDK4/Haras transformed cells appeared somewhat less spindle formed and grew at lower densities than cyclin D1/Haras transformed cells (Figure 1a). As a control, a CDK4 construct without HA-tag was used in a REF cotransfection and yielded similar number of trans-

Oncogenic activity of CDK4

Exp. 1

Exp. 2

2 0 0 0 137 0 93

3 0 0 0 107 0 110

Number of foci Exp. 3 Exp. 4 0 0 0 0 107 ± 74

Exp. 5

Exp. 6

±

±

±

± ± 115 ± 45

± ± 221 ± 147

± ± 145 ± 84

Oncogenic activity of CDK4. Given are the numbers of foci obtained after transfection of REFs with the indicated constructs together with activated Ha-ras (106 cells pers transfection). The modi®ed pLTR vector used for expression of cyclin D1 and CDK4 has been described previously (Lovec et al., 1994b). The data represent six independent experiments


181

CDK4-HA➝

normal REFs

CDK4 + Ha-ras

CDK4 + Ha-ras

b

➝

a

endog. CDK4

Figure 1 (a) CDK4/Ha-ras transformed cells (upper panel) compared to cyclin D1/Ha-ras transformed cells (middle panel) and normal rat embryo ®broblasts (lower panel). (b) Expression of CDK4-HA in two dierent cell lines established from independent foci after cotransfection with Ha-ras and CDK4-HA. CDK4-HA from transfected cells migrates slower than the endogenous rat CDK4

formed foci indicating that the modi®cation through the epitope tagging did not in¯uence the oncogenic activity of CDK4 (data not shown). Next, cells from two independently established CDK4/Ha-ras lines were seeded in soft agar and colonies were counted after 14 days. The experiment showed that CDK4/Ha-ras transformed cells were able to grow and form colonies in soft agar in numbers that were comparable or even higher to those reached with Myc/Ha-ras and cyclin D1/Has-ras transformed cells but were lower compared with cells transformed with SV40 large T-antigen and Ha-ras. No colonies appeared when normal untransformed REFs were seeded (Figure 2). Transforming potential of CDK4 correlates with its ability to bind p16ink4a To investigate which function of CDK4 is responsible for its oncogenic activity, we used two well characterised mutants of CDK4 in REF transformation assays together with activated Ha-ras and scored the number of emerging foci. The ®rst CDK4 mutant contained a methionine at amino acid position 35 instead of the wild type lysine (CDK4 K35M). This mutant is able to bind p16ink4a (Serrano et al., 1995; K Hass and T MoÈroÈy, unpublished) and to relieve the inhibitory eect of p16ink4a on Ha-ras mediated cell

Figure 2 Anchorage independence of REF cell lines established from foci that arose after cotransfections with the indicated constructs and activated Ha-ras. 104 cells were seeded per cell line in semisolid medium and the emerging colonies were counted after 2 weeks. Shown are the results of two independent experiments for each cell line illustrated by darker and lighter shadings of the columns. For CDK4 two dierent cells line established from two dierent foci after cotransfection with Haras were used


Table 2 Correlation between oncogenic activity of CDK4, its catalytical activity and its ability to bind p16 CDK4 and mutants cotransfected with Ras CDK4 wt CDK4 R24C CDK4 K35M

catalytic activity

p16 binding

Exp. 1

+ + 7

+ 7 +

47 1 71

Number of foci Exp. 2 Exp. 3 62 3 49

Exp. 4

31 0 23

25 0 45

Oncogenic activity of dierent CDK4 mutants compared to the activity of wild type CDK4. Given are the number of foci in four dierent REF transfection experiments

proliferation (Serrano et al., 1995) but has lost its catalytic activity due to its inability to bind ATP (Kato et al., 1993). Surprisingly, we ®nd that CDK4 K35M is still able to cotransform REF cells together with activated Ha-ras (Table 2). In contrast, another CDK4 mutant that has a cysteine for arginine replacement in amino acid position 24 (CDK4 R24C) and has lost its ability to bind p16ink4a (WoÈlfel et al., 1995) did not yield foci above background when transfected with Ha-ras in several independent experiments (Table 2) although the construct is functional and directs proper synthesis of the CDK4 mutant (not shown). This could indicate that the transforming potential of CDK4 depends solely on its ability to bind p16ink4a and not on its catalytic activity as a kinase.

a

p16 ➝

3T3

3T3 + p21

NIH3T3 p16*➝

NIH3T3 + p16*

b NIH3T3 + p16

The data presented here (Table 2) support the notion that exogenously expressed CDK4 contributes to malignant transformation by binding and sequestration of endogenous CKIs, notably p16ink4a, that would drive cells into a status of unregulated cycling. If this were true, overexpression of CKIs should in turn abrogate the transforming activity of cyclin D1 and CDK4. To investigate the capacity of the CKIs p21WAF and p16ink4a to directly interfere with the process of malignant transformation, we transfected expression constructs of both p16ink4a and p21WAF along with vectors driving the expression of either c-Myc, cyclin D1 or CDK4 and activated Ha-ras into primary REFs and scored the number of emerging foci. We found that CDK4/Ha-ras, and cyclin D1/Ha-ras and in agreement with earlier ®ndings (Serrano et al., 1995) also Myc/Ha-ras mediated transformation of primary REFs was eciently inhibited by the coexpression of either p16ink4a or p21WAF (Figure 3a). The degree of inhibition correlated directly with the amount of transfected p21WAF and p16ink4a expression construct (data not shown). Interestingly, a p16ink4a mutant found in several independent melanoma lines (Lukas et al., 1995) was inactive and could not inhibit focus formation by CDK4 or cyclin D1 (Figure 3a) although the construct used was functional (Figure 3b) supporting the role of p16ink4a as a tumour suppressor. The p16ink4a mutant carries a leucine instead of a proline at aa position 114 and is unable to associate with CDKs (Lukas et al., 1995). The data demonstrate not only that the oncogenic activity of cyclin D1 and CDK4 can be blocked by CKIs but also show a requirement of functional cyclin/CDK complexes for Myc activity. To verify that the block of focus formation by p21WAF and p16ink4a is not due to a general toxic eect on the REF cells used for transfection, but is indeed

ras + T-Ag + p21

Oncogenic activity of CDK4, cyclin D1 and Myc is blocked by CKIs

NIH3T3

182

p21 ➝

Figure 3 (a) Transformation of REFs by cotransfection of Myc, cyclin D1 and CDK4 expression constructs together with Ha-ras expressing constructs is inhibited by cotransfection of dierent amounts p16ink4a and p21WAF expression vectors, but not by a tumour derived p16ink4a mutant (p16*). Given are the number of foci that are obtained against the cotransfected inhibitor construct. The vectors pX (CMV driven) and pLTR that were used as vehicles to express the inhibitors p21WAF, p16ink4a and the mutant p16ink4a (p16*) had no in¯uence on focus formation when transfected without insert (data not shown, but see also Table 1 for pLTR). The data show average numbers of foci including standard deviations from 3 ± 5 independent experiments. (b) Functionality of the used p21WAF, p16ink4a and p16ink4a* expression constructs. Expression of p21WAF, p16ink4a and p16ink4a* is detected after transient transfection of the constructs into NIH3T3 cells, or, in the case of p21WAF, in cells established from foci that emerged after SV 40 large T-antigen/Ha-ras transformation. Whole cell extracts were loaded and separated on SDS ± PAGE. After transfer onto nitrocellulose membranes the proteins were detected with speci®c antibodies

based on a speci®c inhibition of the transforming abilities of cyclin D1, CDK4 or Myc, control transfections were done with activated Ha-ras and


SV40 large T-antigen expression constructs. Clearly, p21WAF or p16ink4a were no longer eective in inhibiting transformation of REFs and focus formation eciently occurs when SV 40 large T-Antigen is present (Figure 4). As SV 40 large T-Antigen besides other activities binds pocket proteins our result from the REF transfection experiment would be compatible with the notion that the regulation of cyclin/CDK complexes by p21WAF and p16ink4a is only eective in the presence of pocket proteins as for instance Rb or the related protein p107 and would be in agreement with data from Serrano et al. (1995) obtained in REF transfection experiments with E1A and Ha-ras. In these experiments it was also shown that the expression of p16ink4a does not interfere with Ha-ras regulated gene expression which constitutes an important control (Serrano et al., 1995) also for the experiments described here. To malignantly transform cells, cyclin D1 and CDK4 require Myc function One pathway, by which CDK4 contributes to deregulated cell proliferation has been clearly de®ned: CDK4 phosphorylates the retinoblastoma protein and releases free E2F (see Introduction). Our ®nding that sequestration of pocket proteins by SV40 large T-antigen abolishes the requirement for CDK4 in transformation (Figure 4), strongly suggests that this pathway also contributes to transformation by CDK4. However, a second key regulator of G1 progression, Myc, has also been suggested to signal via the cyclin D/CDK4 pathway (Afar et al., 1994, 1995; Roussel et al., 1995). These ®ndings and our own observations that Myc requires functional cyclin/ CDKs for transformation (Figure 3A) prompted us to

test whether, vice versa, cyclin D1 or CDK4 require the activity of Myc to transform REFs in cooperation with activated Ha-ras. To this end, we performed cotransfection experiments using the Myc antagonist Mad as well as a dominant negative Myc (Myc-DN) mutant and as a control a non-functional Myc mutant (Myc DLZ). The dominant negative Myc mutant (Myc-DN) contained a deletion in the aminoterminal transactivation domain (aa position 104 ± 126) but retained its capacity to heterodimerize with Max and to bind DNA (Desbarats et al., 1996). It has been shown that this mutant can eciently interfere with the activity of Myc as a transcriptional transactivator (Desbarats et al., 1996). The non-functional Myc mutant (Myc DLZ) lacks the leucine zipper and is unable to engage in a DNA binding complex with Max (Desbarats et al., 1996). Mad as well Myc-DN and Myc DLZ mutants were used with either Myc itself or with cyclin D1 or CDK4 and Ha-ras in a REF transfection assay. Clearly, CDK4 and cyclin D1 depended on functional Myc in their ability to transform REFs (Figure 5a). The formation of transformed foci in primary REFs cotransfected with a combination of Myc, cyclin D1 or CDK4 and Haras expression constructs was almost completely abrogated when a Mad or Myc DN expression plasmid was included in the transfections (Figure 5a,b) whereas the non-functional Myc mutant (Myc DLZ) had no in¯uence on focus formation (Figure 5a) although the construct was able to correctly drive its expression (Figure 5b). The eect of Mad and the Myc DN mutant on CDK4/Ha-ras and cyclin D1/Haras transformation demonstrates that in order to malignantly transform cells, cyclin D1 as well as CDK4 require a functional Myc protein. In contrast to p21 and p16, coexpression of Mad inhibited focus formation in SV 40 large T-Ag/Ha-ras transfections to about 50% (Figure 4) at the same concentrations that were able to completely abolish focus formation in transfections with cyclin D1 or CDK4 and Ha-ras (Figure 5). Whereas the nonfunctional Myc mutant (Myc DLZ) used as a control had no eect, the dominant negative Myc mutant (Myc DN) inhibited focus formation in SV40 large T-Ag/ Ha-ras transfections similar to Mad to about 50% (data not shown) indicating that a functional Myc protein is still required for cell proliferation and malignant transformation event in the absence of pocket proteins. This suggests that the link of Myc to cyclin D/CDK kinases represents only one of several possible functions of Myc and that all of these functions of Myc are needed for malignant transformation. p107 but not Rb inhibits Myc dependent transcriptional transactivation

Figure 4 Transformation of REFs by cotransfection of Ha-ras and SV40 large T-antigen together with p21WAF, p16ink4a or a Mad expression construct. Given are the number of foci against the transfected constructs. The result of two independent experiments is shown represented by the dierent shading of the columns

Given the results described above we wondered about the molecular mechanism that is responsible for this interdependency between cyclin D1, CDK4 and Myc in transformation. First, it is possible that Myc acts upstream to enhance cyclin D1 or D2 expression. However, published data do not support such a model; in contrast, enforced expression of myc can dampen expression of cyclin D1 in a number of cell lines and in primary rat embryo ®broblasts; Alternatively, CDK4

183


184

Myc DN Myc ∆LZ

NIH3T3 + Mad

ras + T-Ag + Myc DN

ras + Myc

REFs

NIH3T3 ➝

Myc ➝

➝

b

ras + T-Ag + Myc ∆LZ

a

Mad

Figure 5 (a) Transformation of REFs by cotransfection of Myc, cyclin D1 and CDK4 together with Ha-ras expressing constructs is inhibited by the Myc antagonist Mad and by a Myc dominant negative mutant (Myc-DN) that lacks amino acids 104 to 126 within the transactivation domain. A non-functional mutant of Myc that lacks the leucine zipper sequences (Myc DLZ) does not yield foci together with activated Ha-ras and does not inhibit cyclin D1/Ha-ras or CDK4 Ha-ras cotransformation. Given are the number of foci that are obtained against the cotransfected construct. The data show average numbers of foci including standard deviations from 3 ± 5 independent experiments. (b) Functionality of the used expression constructs. Expression of Mad, Myc and the Myc mutants Myc DN and Myc DLZ is detected after transient transfection of the constructs into NIH3T3 cells, or in cells established from foci that emerged after SV 40 large T-antigen/Ha-ras transformation. Whole cell extracts were loaded and separated on SDS ± PAGE. After transfer onto nitrocellulose membranes the proteins were detected with the speci®c anti Myc antibody 9E10

and cyclin D1 might act as upstream regulators of Myc function. Indeed, one substrate of cyclin D1/CDK4 kinase activity, the Rb related protein p107 had previously been shown to bind Myc/Max complexes and to interfere with Myc mediated transcriptional transactivation (Beijersbergen et al., 1994; Gu et al., 1994). This had been tested using a synthetic E box containing reporter gene or with Gal4-Myc fusion proteins and Gal4 dependent reporter gene constructs (Beijersbergen et al., 1994), but it is so far not known if this activity of p107 is also eective in suppressing Myc dependent transactivation from a bona ®de target gene promoter. To test whether CDK4 kinase activity and possibly p107 is directly involved in the regulation of

Myc dependent transactivation, we made use of the recent description of a bona ®de target site of Myc, the E-box located in the ®rst intron of the rat prothymosin-a gene. Transactivation from this distant enhancer site diers signi®cantly from previously published assays using synthetic reporter constructs. Moreover, Myc domains required to activate transcription from this element faithfully re¯ect the regions crucial for transformation (Eilers et al., 1991; Desbarats et al., 1996). Transient transfection assays were used to measure transactivation from this element using prothymosin promoter to drive CAT or luciferase reporter genes (prot-LUC or prot-CAT) in two dierent cell lines: HeLa cells, which lack functional Rb and p107 due to the presence of viral oncoproteins and express only low level cyclin D1 and CDK4 (Lukas et al., 1994) and NIH3T3 cells, which contain both functional pocket proteins and cyclin D/ CDK kinase activity. The prothymosin-a promotor driven reporter constructs comprise sequences 5' of the transcriptional start site, the ®rst exon, the ®rst intron and part of the second exon of the prothymosin-a gene fused to CAT of luciferase open reading frames and provide a bona ®de CACGTG Myc recognition sequence that has been shown to be speci®c for the transregulating activities of c-Myc but not of other E-box binding proteins (Desbarats et al., 1996). Expression of Myc strongly activated expression of a prot-LUC reporter plasmid upon transient transfection in HeLa cells. Strikingly, transactivation by Myc was completely sensitive to expression of p107 (Figure 6a). p107 inhibited neither the basal activity of the prot-LUC reporter construct nor expression of either Myc or Max (Figure 6a,b). A number of controls established that inhibition by p107 was clearly due to interference with Myc function: ®rst, similar results were obtained with a prot-LUC construct in which all sequence elements of the ®rst intron of the prothymosin-a gene with the exception of three base pairs on either side of the E-Box element had been deleted. This construct was still activated by Myc and the activation was sensitive to inhibition by p107 as observed for the wild type construct (not shown). We concluded that p107 does not interfere with the function of proteins binding to neighbouring sequence elements that might aect transactivation by Myc (such as AP-2; Gaubatz et al., 1995). Second, inhibition by p107 was speci®c, as the closely related Rb protein did not inhibit transactivation by Myc in comparison to p107 (Figure 7a). Third, a deletion of sixty amino acid (aa position 45 ± 106) in the p107 binding domain of Myc rendered transactivation essentially resistant to inhibition by p107, suggesting that p107 inhibits Myc activity by binding to it (Figure 7a). Smaller deletions, including mutations of amino acids 58 and 62 as found in tumour-derived alleles of Myc had no discernible eect on inhibition by p107 in this assay (not shown). We next tested whether p16ink4a aected transactivation by Myc in HeLa cells. Coexpression of p16ink4a did not inhibit transactivation by Myc (Figure 7a) not even at higher concentrations (not shown). Further, p16ink4a did not synergize very eciently with p107 in inhibition of Myc (Figure 7b). We concluded that p16ink4a has no discernible eect on Myc function in cells which have low cyclin D1/CDK4 kinase activity and lack functional pocket proteins as


87 —

Myc / Max / p107

Myc / Max

kD 87 —

–

Myc / Max / p107-HA

kD 144 —

Myc / Max

b

–

a

Myc

➝

We repeated the above described experiments in NIH3T3 cells which express cyclin D/CDK complexes

➝

p16ink4a and p107 inhibit Myc's function as a transcriptional transactivator

and contain both functional Rb and p107 with a protCAT reporter gene construct. Together with this plasmid, the Myc expression was transiently transfected into NIH3T3 cells either in the presence or absence of a p16ink4a expression construct. Expression of prot-CAT in these cells yielded a high basal activity, which was stimulated about threefold by ectopic expression of Myc (Figure 8a). Activation by Myc is not as high in these cells as in HeLa cells presumably

➝

for example the HeLa cells used here; in particular, these data also show that p16ink4a does not exert a nonspeci®c toxic eect on transactivation by Myc.

Max

p107-HA 44 —

32.7 — 17.7 —

Figure 6 (a) Transcriptional transactivation of the prot-LUC reporter by Myc and the inhibition of Myc transactivation by p107 in HeLa cells. The use of dierent amounts of cotransfected p107 expression construct shows that this eect is concentration dependent. Shown are average values with standard deviations that resulted from three independent transfections per experiment. Shown is a representative of several independent experiments. (b) Expression control for the transfected Myc and Max expression plasmids as well as for HA-tagged p107

a

b

Figure 7 (a) Comparison of the inhibitory activity (fold repression) of p107, p16ink4a and Rb on the activity of Myc as a transcriptional transactivator of the prot-LUC reporter gene. Shown is also the lack of inhibitory activity of p107 when the Myc mutant is used that lacks the p107 binding region. (b) Eect of p16INK4a on the transactivational properties of Myc in the presence and absence of p107. HeLa cells were transiently transfected with the indicated expression vectors and the prothymosin-LUC reporter (prot-LUC). Shown are average values with standard deviations that resulted from three independent transfections per experiment. Shown is a representative of several independent experiments

185


186

a

b

c

Figure 8 (a) Myc transactivation of a prothymosin-promoter-CAT reporter gene (prot-CAT) is inhibited by p16ink4a but not by a tumour derived mutant of p16ink4a (p16*). As a control Myc transactivation was also inhibited by the transdominant negative Myc mutant that lacks sequences in the transactivation domain (Myc DN). A prothymosin promotor mutant that contains a disrupted Ebox motif (prot*-CAT) was used as control. With this reporter Myc dependent transcriptional transactivation is not seen. Same amounts of the indicated constructs were transiently transfected with the reporter gene construct into NIH3T3 cells. The expression vector pX (CMV promoter) that was the vehicle for expression of p16ink4a and p16ink4a* and the expression vector pSP (SV40 promoter) that was used to drive expression of Myc and the Myc DN mutant did not in¯uence Myc dependent transactivation of the prothymosin-a promotor. The cotransfection of Max expression plasmids did not markedly in¯uence the transactivation of Myc or the inhibition by p16ink4a in this assay which is probably due to appropriate endogenous Max expression (data not shown). (b) Myc mediated transactivation was inhibited by p16ink4a in a concentration dependent manner. The prothymosin reporter (protCAT) was transfected together with Myc expression plasmid and the indicated amounts of p16ink4a expression vector. (c) p16ink4a does not interfere with SV40 promotor driven transcription. A SV40 promoter driven CAT reporter was transfected alone or along with a p16ink4a expression plasmid. In all experiments, relative CAT activity was measured after transient transfections on NIH3T3 cells with the indicated constructs. Shown are average values with standard deviations that resulted from three independent transfections per experiment, except for the SV40 CAT control where duplicate transfections were done. Shown is a representative of several independent experiments. For expression controls see Figures 3, 5 and 6. Numbers represent the amount of plasmid DNA in mg used for transfection

because ectopic expression of Myc induces apoptosis in NIH3T3, but not in HeLa cells. The comparison of relative CAT activities revealed that the transactivating properties of Myc in this assay were clearly inhibited by the coexpression of p16ink4a (Figure 8a). Several controls established that this inhibition of Myc transactivation by p16ink4a was speci®c and actually dependent on its eect on Myc and CDK4: First, inhibition of Myc activity was not found with the tumour derived inactive p16ink4a mutant (Figure 8a, p16*) that contains a leucine at amino acid position 114 and is unable to bind CDK4 (Lukas et al., 1995). Second, the inhibitory eect of p16 ink4a on the transactivational properties of Myc was clearly concentration dependent as transfection assays with varying amounts of p16ink4a expression vector demonstrated (Figure 8b). Third, the Myc DN mutant that could inhibit focus formation (see Figure 5a) was used as a control and was equally eective in blocking the transactivation of wild type Myc in comparison to p16ink4a (Figure 8a). Forth, we used a mutant form of the prothymosine reporter (prot*-CAT) that is not responsive to Myc transcriptional transactivation due to a disruption of the E-box motif (Desbarats et al., 1996). Here, neither the expression of p16ink4a or the Myc DN mutant had any eect (Figure 8a). Fifth, to rule out that the observed block of Myc transactivation

by p16ink4a is due to a block of CAT activity itself, a general eect on transcription or an interference with SV 40 promoter sequences that drive Myc expression in the construct used here, we used as control a CAT reporter construct that is itself under control of SV 40 promoter sequences. Clearly, control transfections with this construct showed that CAT activity itself was not inhibited by p16ink4a in normal NIH3T3 cells (Figure 8c). Further, Myc transactivation was also inhibited by p107 in NIH3T3 cells in a concentration dependent manner (Figure 9a) as was seen in HeLa cells before and this inhibitory eect of p107 on Myc transactivation could be almost completely relieved by the cotransfection of a CMV driven CDK4 expression plasmid (Figure 9b). In contrast to NIH3T3 cells no transcriptional transactivation of the prothymosin reporter by Myc could be detected in primary ®broblasts derived from Rb de®cient mice (data not shown). Although there are alternative explanations this could also be in agreement with a role of p16ink4a as a suppressor of Myc mediated transcriptional transactivation because it has been shown that Rb negative cells express high level endogenous p16ink4a (Parry et al., 1995). A SV40 promoter driven CAT reporter was also used to control that the inhibition of Myc transactivation in Rb negative cells was a speci®c eect on the activity of


187

a

b

Figure 9 Myc mediated transcriptional transactivation of a prothymosin-promotor-CAT reporter gene (prot-CAT) is inhibited by p107 in NIH3T3 cells and this inhibition is relived by CDK4. The prothymosin reporter was transfected together with the Myc expression plasmid and the indicated amounts of p107 expression vector or empty vector control (a) or with the indicated amounts of CDK4 expression plasmid (CMV-CDK4) and empty control vector (b). Shown are average values with standard deviations that resulted from several independent transfections per experiment. Numbers represent the amount of plasmid DNA in mg used for transfection

Myc. CAT activity was found in transfected Rb negative cells and was unaltered by the coexpression of p16ink4a, Myc or a p16ink4a mutant (p16*) demonstrating that the eect seen was not due to a general lack of transcriptional transactivation in Rb negative cells (data not shown). Taken together, the data show that D1/CDK4 kinase activity is required for transactivation of a natural target gene by Myc and would explain the dependency of Myc on D-type kinases for malignant transformation and the inhibitory eect of p16ink4a on Myc transactivation and transformation. As this was found in NIH3T3 (see Figure 8 and 9), but not in HeLa cells, the results would be compatible with a model in which this requirement of D-type kinases is mediated by pocket proteins, possibly the p107 protein (Figure 11). In addition our ®ndings suggest that cyclin D/CDK4 kinase activity ensures the function of c-Myc as a transcriptional transregulator and as a transforming gene. Moreover, they indicate that any interference of this pathway by p16ink4a renders c-Myc unable to act as a transcriptional transactivator and a transforming gene. To further support this model we tested if p107 itself could suppress the oncogenic activity of Myc and if a Myc mutant that is insensitive to p107 would still be sensitive to p16ink4a. To this end we cotransfected the p107 expression construct together with Myc and Ha-

ras into REFs and scored the number of foci. In a parallel experiment we used the p107 insensitive Myc mutant (Myc D45 ± 106) together with Ha-ras to transfect REFs in the presence of p16ink4a. Myc/Haras focus formation was clearly blocked by p107 in a concentration dependent manner (Figure 10) confirming the ®ndings of the transactivation assay. Moreover, the p107 insensitive Myc mutant produced foci together with Ha-ras albeit at a somewhat lower eciency than wild type Myc but its transforming activity was no longer sensitive to the inhibitory eect of p16ink4a (Figure 10), which supports the notion that the eect of p16ink4a on the activity of Myc is mediated through cyclinD/CDK4 complexes and the pocket protein p107 (Figure 11).

Discussion CDK4 is an oncoprotein directly implicated in tumourigenesis The oncogenic activity of D-type cyclins has been well established through a number of independent experimental strategies (Bodrug, et al., 1994; Lovec et al., 1994a,b; Wang et al., 1994; Hinds et al., 1994). We show now that, in addition, the catalytic subunit of D-


188

Figure 10 p107 inhibits focus formation in REFs by Myc and Ha-ras in a concentration dependent manner, but focus formation is no longer blocked by p16ink4a when a p107 insensitive Myc mutant (Myc D45 ± 106) is used together with Ha-ras. The number of foci is given against the transfected inhibitor in two independent experiments represented by the dierent shadings of the columns

type cyclins, CDK4 has itself transforming activities. CDK4 is localised on a region of human chromosome 12q13 which is frequently ampli®ed in a range of human tumours (Sonodat et al., 1995; He et al., 1994, 1995; Ladanyi et al., 1995; Schmidt et al., 1994; Khatib et al., 1993). High level expression of CDK4 was found as a consequence of gene ampli®cation in a number of studies and suggested a direct contribution of CDK4 to the process of tumourigenesis (He et al., 1995; Maelandsmo et al., 1996; Khatib et al., 1993). Moreover, a number of reports describes either the loss or the mutation of the gene encoding the p16ink4a CDK4 inhibitor in human tumours (Nobori et al., 1994; Koh et al., 1995; Lukas et al., 1995; Maelandsmo et al., 1996; Zuo et al., 1996; He et al., 1995; Reymond and Brent 1995; WoÈlfel et al., 1995; Schmidt et al., 1994) suggesting that the deregulation of CDK4 activity or its expression is directly implicated in tumourigenesis. Our data presented here support a notion of CDK4 as an oncoprotein and p16ink4a as a tumour suppressor and demonstrate directly the transforming activity of CDK4 and the inhibitory function of p16INK4a. The transforming activity of CDK4 and the similar already known activity of Dtype cyclins strongly suggest that other and possibly all positive G1 cell cycle regulators are in general oncoproteins and that their deregulated expression leads to a loss of growth control that is incremental in the process of malignant transformation. This would also be in agreement with the recent ®nding that the CDC25A phosphatase has oncogenic activity and can transform mouse ®broblasts in cooperation with activated Ha-ras (Galaktionov et al., 1995b).

Figure 11 Schematic representation illustrating a hypothetical pathway from p16ink4a via the cyclin D/CDK complexes to the pocket protein p107 and to the activation of Myc and the promotion of cell cycle progression. Details see text

The exact molecular mechanism, however, by which the G1 positive cell cycle regulators tested here achieve malignant transformation when expressed in a deregulated manner remains unclear. It is intriguing for example that mutants of cyclin D1 that are unable to bind to Rb are still oncogenically active (Hinds et al., 1994) which is also true for cyclin D2 mutants that lack the Rb binding motif (K Haas and T MoÈroÈy, unpublished). Moreover, as shown here, it is very surprising that a CDK4 mutant that is catalytically inactive (CDK4 K35M) but able to bind p16ink4a still cooperates with activated Ha-ras and can provoke the outgrowth of foci and that a CDK4 mutant (CDK4 R24C) that is unable to bind p16ink4a but retains its catalytic activity is no longer transforming. Thus, one possible mechanism that is responsible for transformation could be at least partly based on the activity of Dtype cyclins or CDK4 to bind and sequester inhibitors like p27KIP, p21WAF and p16ink4a, respectively. According to this model, overexpression of D-type cyclins would titrate p27KIP and p21WAF and release endogenous Dand E-type kinase activities from CKI control. Similarly, CDK4 would transform cells by binding p16ink4a or in a complex with cyclin D1 also p21WAF and p27KIP and liberating endogenous cyclin/CDK kinases from their inhibitors. In support of this model comes our ®nding that excess p21WAF and p16ink4a can in turn very eciently inhibit malignant transformation by cyclin D and CDK4 which strengthens also the arguments for the role of p21WAF and p16ink4a as


tumour suppressors (Lukas et al., 1995; Koh et al., 1995). Indeed, various tumour cell lines have been reported to carry inactive alleles of p16ink4a (Nobori et al., 1994; Kho et al., 1995) and the isolation of a p16ink4a mutant allele from a human tumour line and the inability of this mutant to inhibit malignant transformation REFs as shown here provides more direct evidence that at least p16ink4a can act as a bona ®de tumour suppressor (see also Nobori et al., 1994; Koh et al., 1995; Lukas et al., 1995; Melandsmo et al., 1996; He et al., 1995; Reymond and Brent 1995; Schmidt et al., 1994). The discovery of mutant forms of CDK4 that lack the ability to bind p16INK4a in a number of human melanomas (WoÈlfel et al., 1995; Zuo et al., 1996) fostered the hypothesis that these mutant forms of CDK4 (CDK4 R24C) could act as dominant oncoproteins by securing a constitutive enzymatic activity of cyclin D/CDK4 complexes and continuous cell cycle progression uncontrolled by p16INK4a. According to this, CDK4 R24C should be able to transform REFs in cooperation with activated Ha-ras upon transfection. However, we could not observe such an activity of CDK4 R24C that would allow to de®ne this mutant as a dominant oncogenic form of CDK4, which appears at variance with the hypothesis implicated by the discovery of the CDK4 mutant in melanoma patients (WoÈlfel et al., 1995; Zuo et al., 1996). However, it is possible that the REF assay used here is not well suited to reveal the eect of the CDK4 R24C mutant, or that this mutant acts only under particular circumstances as a dominant oncogene in melanoma. It is also conceivable that although CDK4 R24C/cyclin D complexes have kinase activity (WoÈlfel et al., 1995) they are less ecient to sequester the CKIs p21WAF or p27KIP in vivo compared to CDK4 wild type/ cyclin D complexes. Probably, multiple mechanisms that disrupt the p16INK4a/CDK4 pathway exist and contribute to malignant transformation. Interdependence of cyclin D/CDK4 and Myc in malignant transformation We show here that the transforming ability of cyclin D1 and CDK4 clearly depends on functionally active Myc. Vice versa, we found that CKIs (p21WAF and p16ink4a) very eciently interfere with the transforming activity of cyclin D1 and CDK4 but also with that of c-Myc. Here, the eect of p16ink4a is of particular interest. Due to the speci®city of p16ink4a for D-type cyclin/CDK complexes, this experiment indicates that activity of Myc as an oncogene requires D-type kinase activity. Although other possibilities exist, both ®ndings could be explained by a model in which Myc acts as a downstream eector of cyclin D/CDK complexes. Taking into account recent ®ndings about the activation of the cyclin E/CDK2 kinase by the induction of Myc through conditional alleles (Steiner et al., 1995) one could predict that Myc acts downstream of cyclin D/CDK4, but upstream of cyclin E/CDK2. Additional support for such an interdependence between Myc and D- and E-type kinases comes from an independent series of experiments which investigated signal transduction through the CSF-1 receptor and from studies on the complementation of bcr-abl mutations by Myc and

the signalling of abl through cyclin D1 (Roussel et al., 1995; Afar et al., 1994, 1995). According to our model of Myc as a downstream eector of D-type kinases, a direct activation of Myc by D-type cyclin/CDK complexes would be imperative. Indeed, such an activation of Myc seems possible because we can show here that p16ink4a which is a speci®c CDK4 inhibitor blocks not only malignant transformation by Myc but also Myc's ability to transcriptionally transactivate a bona ®de target promoter. As Myc is not a direct substrate of cyclin D1/CDK4 kinase the link could be provided by the pocket protein p107 that has been shown to interact with Myc in several independent studies (Beijersbergen et al., 1994; Gu et al., 1994). Cyclin D1/CDK4 kinase can phosphorylate p107 and rescue cells from a p107 induced cell cycle block (Beijersbergen et al., 1994); the underlying mechanism being a release of free Myc transcription factor from a p107 complex through phosphorylation of p107 by cyclin D/CDK4 in analogy to the liberation of E2F transcription factors by the phosphorylation of Rb (see Introduction). This, together with our own ®ndings that both p107 and p16ink4a can block Myc function strongly suggests that a sequential pathway p16ink4a/CDK4/p107/Myc exists (Figure 11). Such a pathway would explain the interdependence of CDK4 and Myc in malignant transformation and the eect of p16 INK4 a and p107 on Myc dependent transcriptional transactivation and transformation. Thus, our data argue for a direct requirement for active D1/CDK4 kinase in activation of Myc dependent transcriptional transactivation and transformation. This conclusion is supported by three ®ndings: First, p16ink4a inhibited transactivation by Myc in cells which contain active CDK4 kinase and both functional Rb and p107 (NIH3T3), but not in cells that contain no functional pocket proteins and have low cyclin D/ CDK4 kinase activity (HeLa cells, Lukas et al., 1994). Second, an allele of p16ink4a that is de®cient in binding and inhibition of CDK4 also failed to inhibit transactivation by Myc, demonstrating that p16ink4a exerts its eect on transactivation by Myc via CDK4. Third, a substrate of cyclin D/CDK4 kinase, p107, but not the closely related Rb protein inhibited transactivation by Myc in both NIH3T3 and Hela cells. Fourth, p107 was able to inhibit the activity of Myc as a transcriptional transactivator and as an oncogene. Fifth, a p107 insensitive Myc mutant could still cooperate with Ha-ras and induce focus formation in REFs in the presence of p16ink4a. Nevertheless, these ®ndings present a conundrum as previous work has ®rmly established that the requirement for active cyclin D/CDK4 kinase in cell cycle progression depends on the presence of functional retinoblastoma protein Rb. Therefore, two pathways seem possible: First, inhibition of Myc function by p16ink4a might be mediated by the retinoblastoma protein and the mediated phosphorylation of Rb by D-type kinases may be responsible for Myc function during G1 progression. Second, inhibition of Myc function by p16ink4a is mediated by p107. Our data do not rule out the ®rst possibility, but they are easily compatible with the second pathway in which p16ink4a inhibits Myc function as a transcriptional transactivator through p107. However, other, more indirect possibilities exist. For example, members of the

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190

Brahma family of proteins that associate with Rb may be required for transactivation by Myc from a distal enhancer position. However, based on our data and previously published observations we favour the second pathway involving p107 because no evidence was obtained in our experiments that Rb negatively affects transactivation by Myc. Rather, we ®nd it is more likely that the eect of p16ink4a is mediated by p107, which is a close relative of the retinoblastoma protein. Our data support previously published observations that p107 associates with and inhibits transactivation by Myc (Beijersbergen et al., 1994). They extend these observations by showing that p107 is extremely potent in inhibition of Myc function when assayed on a natural target gene of Myc, prothymosin-a, and in REF transformation assays. Moreover, several independent reports provide clues for Myc as a downstream eector of cyclin D/CDK4 and an upstream activator of cyclin E/CDK2 kinase (Steiner et al., 1995; Roussel et al., 1995; Afar et al., 1994, 1995). Taken together our data suggest a view of Myc function that is strikingly similar to another key regulator of G1 progression, the E2F/DP family of proteins (Figure 11). Similar to Myc, transactivation by E2F/DP proteins is under the control of cyclin D/ CDK4 kinases. One of the critical target genes regulated by E2F during G1 progression is cyclin E (Botz et al., 1996; Geng et al., 1996) and Myc acts as an upstream regulator of cyclin E/CDK2 kinase (Steiner et al., 1995). In contrast to E2F/DP proteins, Myc does not appear to act directly on cyclin E transcription but rather on subsequent activation steps of cyclin E/CDK2 (Steiner et al., 1995). Expression of Myc, for example, blocks the association of the p27KIP inhibitor with cyclin E/CDK2 probably via still unidenti®ed target genes and activates transcription of the CDC25A phosphatase (Galaktionov et al., 1996), which removes inhibitory phosphates from CDK2. This and our own observations reported here suggest that activation of Myc-dependent transcription and its function in malignant transformation may be intimately linked to D-type cyclin/CDK complexes that control progression through the restriction point in G1 in a manner strictly analogous to models proposed for the E2F family of proteins (Figure 11). However, the ®nding that coexpression of the Myc antagonist Mad in SV 40 TAg/Ha-ras transformation assays leads to a partial inhibition of focus formation to the extent of roughly 50% which is in agreement with other reports that describe experiments with the adenovirus oncoprotein E1A and dominant negative Myc mutants (Cerni et al., 1995; Hermeking et al., 1995) clearly points to other roles of Myc that may also be essential for the process of cell cycle progression and malignant transformation and demonstrates that the model outlined here represents one although important aspect of a complicated regulatory network. Materials and methods DNA constructs The coding sequences of D1, CDK4, CDKK35M, CDK4R24C and p21 WAF were in the modi®ed pLTR vector (Lovec et al., 1994b). The CDK4K35M mutant was a gift from D Beach and the CDK4R24C mutant was

generously provided by T WoÈlfel. Coding sequences of Myc, the Myc DN mutant and the Myc DLZ mutant and Mad had been cloned in the expression vector that directs ecient expression by SV40 promotor/enhancer elements. The Mad construct was generously provided by B. LuÈscher. Expression of p16ink4a was driven through an CMV containing expression vector (pX). cDNAS for both p16ink4a and p21WAF was generously provided by D Beach. The SV40 promoter driven CAT expression plasmid was a gift from R MuÈller. Rat embryo ®broblast transformation assay and anchorage independence Pregnant Fisher rats were killed at day 14, the embryos were removed and placed in sterile PBS. Cells from the carcass were dispersed using trypsin/EDTA/1% chicken serum. The cells were separated from debris by low speed centrifugation. The cells were resuspended in DMEM/10% fetal calf serum (FCS) and plated at 1 million cells per 80 cm2 dish. After the cells had reached con¯uency, they were replated at a density of 1 million per dish and used for transfections the following day. Transfections were carried out by the conventional calcium phosphate precipitation method overnight with 30 mg of DNA per dish. This included 10 mg of pEJ6.6 (Land et al., 1983) (containing the activated Ha-ras gene from a human bladder carcinoma), 10 mg of the respectively partner construct and 10 mg of inhibitor expression construct or empty vector DNA, respectively. Sixteen hours after transfection the cells were washed with PBS and fed with fresh medium and another 24 h later were split into four plates and assayed for focus formation. The test for anchorage independence was done by plating 10 4 or 105 REFs, or the same number of cells from Myc/Ha-ras, cyclin D1/Ha-ras, cyclin E/Ha-ras and CDK4/Ha-ras transformed lines established from foci in 0.33% agar 28 cm2 dishes on a 0.5% agar bottom. Colonies were scored after 2 weeks. Transient transfection assays NIH3T3 or HeLa cells were seeded on 3 cm plates (1615 cells per plate) and transfected in medium containing 0.5% FCS with 2 mg prothymosin-CAT or prothymosin Luciferase reporter plasmid (Desbarats et al., 1996) and 1 mg or otherwise indicated amounts of the respective constructs together with 7 ml Lipofectamin (Gibco). Medium was changed 16 h later with 10% FCS and 24 ± 48 h later the cells were harvested in cold PBS and lysed through 3 freeze thaw cycles in 50 ml of CAT buer (15 mM Tris/HCl, pH 8, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.15 mM Spermin, 1 mM DTT, 0.4 mM PMSF). The lysate was cleared by centrifugation and the supernatant was mixed with an equal amount of CAT reaction mix (34.5 ml H2O, 10 ml Tris/HCl pH 8, 5 ml buturyl-CoA (5 mg/ml) 0.5 ml [14C]Chloramphenicol) and incubated at 378C for 1 ± 2 h. Radioactive Chloramphenicol was extracted with xylol/ TMPD and counted in a liquid scintillation counter. Experiments were done in triplicate at least three times. Luciferase activity was determined as described (Desbarats et al., 1996). Immuno-blotting NIH3T3 cells were seeded on a 6 cm plate and transfected transiently with 8 mg total DNA with Lipofectamin. After 2 days cells were harvested in 1 ml PBS, collected by centrifugation, and lysed in 100 ml extraction buer (50 mM HEPES pH 7.8, 20 mM NaF, 1 mM Na-OrthoVanadat, 1 m M Na-Molybdat, 450 m M NaCl, 0.2 m M EDTA, 25% glycerol, 50 mg aprotinin, 50 mg leupeptin,


0.5 mM PMSF, 1 mM DTT, 1% NP40). The lysate was cleared by centrifugation and the supernatant was separated by electrophoresis and transferred onto a nitrocellulose membrane. Untransfected cells were run as control. The membrane was blocked over night in PBS/ 0.1% Tween 20 with 3% skim milk and then incubated in blocking solution containing the respective antibodies for 2 h at 0.3 mg/ml. Blots were developed with appropriate second antibodies and ECL detection reagents (Amersham) as proposed by the manufacturer. Anti-CDK4 and antiMad antibodies stemmed from anity-puri®ed rabbit serum (Santa Cruz) and were used according to the instructions of the supplier. Anti p16 ink4 ascites and anti

p21WAF supernatant have been described earlier (Lukas et al., 1995).

Acknowledgements We thank D Beach and M Serrano for p16ink4a and p21WAF cDNAs and B LuÈscher for the Mad construct. We are also indebted to D Beach and T WoÈlfel for the gift of CDK4 mutants. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG, SFB 215-D10) and the Dr Mildred Scheel Stiftung fuÈ r Krebsforschung to TM.

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