Activation of cyclin-dependent kinases by Myc mediates induction of ...

The EMBO Journal vol.15 no.12 pp.3065-3076, 1996

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis

Bettina Rudolph, Rainer Saffrichl, Jdrk Zwicker2, Berthold Henglein3, Rolf Muller2, Wilhelm Ansorgel and Martin Eilers4 Zentrum fur Molekulare Biologie Heidelberg (ZMBH). Im Neuenheimer Feld 282, 69120 Heidelberg, 'Biochemical Instrumentation Programme, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, 2-nstitut fur Molekularbiologie and Tumorforschung, Emil-Mannkopfstrasse 2, 35033 Marburg, Germany and 3Institut Curie, CNRS UMR144, F-75005 Paris, France

4Corresponding author

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin Dl-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G, phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc. Keywords: apoptosis/cell cycle/c-myc/cyclin-dependent kinases

Introduction c-myc was originally identified as the cellular homologue of the transforming oncogene of four chicken retroviruses (for review, see Meichle et al., 1992). c-myc encodes a nuclear phosphoprotein (Myc) that, together with a partner protein termed Max, binds specific DNA elements with the core sequence CAC(G/A)TG (Blackwell et al., 1990; Blackwood and Eisenman, 1991; Kerkhoff et al., 1991; Prendergast et al., 1991; Prendergast and Ziff, 1991; Blackwood et al., 1992). The heterodimeric complex acts as an activator of transcription (Amati et al., 1992; Kretzner et al., 1992; Amin et al., 1993); detailed mutagenesis studies reveal that transcriptional activation and complex formation with Max are prerequisites for virtually all known biological properties of Myc (Stone et al., 1987; K Oxford University Press

Amati et al., 1993a,b). Indeed, several genes have been described that are regulated by Myc in vivo (Eilers et al., 1991; Benvenisty et al., 1992; Bello-Fernandez et al., 1993; Reisman et al., 1993; Wagner et al., 1993a; Gaubatz et al., 1994, 1995; Desbarats et al., 1996). In the absence of Myc, Max can either form homodimers or heterodimers with other partner proteins (termed Mad and Mxi); these complexes lack transcriptional activation domains and repress Myc-mediated transactivation (Amati et al., 1992; Kato et al., 1992; Kretzner et al., 1992; Amin et al., 1993; Ayer et al., 1993, 1995; Zervos et al., 1993; SchreiberAgus et al., 1995). In mammalian cells, deregulated expression of c-myc has been shown to contribute to tumorigenesis (Land et al., 1983), to induce cell proliferation in resting cells (Keath et al., 1984; Eilers et al., 1991), to induce active cell death (apoptosis) (Askew et al., 1991; Evan et al., 1992) and to block terminal differentiation of adipocytes and myeloid cells (Freytag and Geddes, 1992; Steinman et al., 1994). Presumably, therefore, some of the target genes of Myc play critical roles in these biological processes. However, the identity of such critical targets [with the possible exception of ornithine decarboxylase; Packham and Cleveland (1994)] is not known. We have shown previously that induction of Myc in resting cells triggers activation of both cyclin E- and cyclin Dl -dependent kinase activities, suggesting that Myc might act upstream of these kinases in inducing cell proliferation (Steiner et al., 1995). However, several key questions remain open. First, a functional role for cdk2 kinase activity during Myc-induced cell proliferation has not been demonstrated. It is conceivable that active cdk complexes may have little or no role in inducing biological responses to Myc. For example, Myc might act mainly by sequestering pocket proteins like p107 (Beijersbergen et al., 1994; Gu et al., 1994). In this view, induction of cdk activity might occur in response to induction of Myc [e.g. because free p107 can act as a cdk inhibitor (Zhu et al., 1995)], but would have little or no functional role in cellular responses to Myc. Alternatively, Myc might transcriptionally activate genes encoding downstream targets of cdks, such as the E2F family of transcription factors or proteins directly involved in DNA replication. Similar models have been proposed recently for B-myb and for E2F: ectopic expression of either protein restores DNA synthesis in y-irradiated cells (which lack detectable cdk2 kinase activity due to the presence of the cdk inhibitor p21) without inducing cdk kinase activity (Lin et al., 1994; DeGregori et al., 1995). The second question deals with the causal role of cdk activity, if any, in eliciting biological responses to Myc other than proliferation. In particular, Myc-induced apoptosis has been suggested to arise from an inappropriate activation of cyclin A-dependent kinase by Myc in starved 3065

B.Rudolph et al.

RATI cells (Hoang et al., 1994). Similarly, Myc-induced transformation and inhibition of terminal differentiation might be due to its effects on cdk activity (Steinman et al., 1994). In an extreme view, therefore, activation of cdks may be the only biological function of Myc, and all other responses to Myc would be indirect effects of its ability to enhance cdk activity. To address these issues, we decided to test the effect of inhibition of GI cdks on Myc-induced cell proliferation and apoptosis. For a number of reasons, we used the expression of cyclin A as a marker for cell proliferation: (i) cyclin A protein has been shown to be required for cell proliferation during the S phase of the cell cycle (Girard et al., 1991; Pagano et al., 1992); (ii) expression of cyclin A mRNA begins during the late G1 phase of the cell cycle (Henglein et al., 1994) and has been linked to the commitment of cells to enter into a replicative cycle (Dou et al., 1993); (iii) a tight correlation has been observed between expression of c-myc and of cyclin A mRNA (Jansen-Durr et al., 1993; Hoang et al., 1994); and (iv) cyclin A protein has been implicated in Mycinduced apoptosis and in anchorage-independent cell growth, a characteristic feature of transformed cells (Guadagno et al., 1993; Hoang et al., 1994). We now show by a number of different criteria that active GI cyclin/cdk complexes are critical intermediates for induction of cyclin A expression by Myc. In contrast, inhibition of cdk activity had no effect on Myc-induced apoptosis in a variety of experimental settings. Further, Myc was capable of inducing apoptosis in the absence of significant changes in GI cdk activity, strongly suggesting that active cyclin/cdk complexes play little or no role in Myc-induced apoptosis.

Results Activation of conditional alleles of Myc (MycER; Eilers et al., 1989) in density-arrested or serum-starved fibroblasts leads to an 8- to 10-fold increase in cyclin A mRNA levels within 10 h (Jansen-Durr et al., 1993). To identify elements in the cyclin A promoter that might mediate induction by Myc, a series of deletion mutants of the promoter was cloned in front of a luciferase reporter gene. Preliminary experiments in which these reporter plasmids were transfected transiently together with expression vectors encoding Myc did not yield consistent results (not shown). Therefore, stable cell lines expressing each construct were generated by co-transfection of the reporter plasmid with a hygromycin resistance plasmid into RATlA-MycER cells (Eilers et al., 1989). After selection with hygromycin B, resistant colonies were pooled, grown to confluence and re-induced with hydroxytamoxifen (4-OHT) or ethanol as a control. This protocol was used to exclude effects due to variation between individual clones of stably transfected cells. Extracts were prepared 12 h later and the specific luciferase activity was determined; in each experiment, triplicate samples were assayed and at least three independent experiments were performed for each construct (Figure IA). Activation of Myc caused a 2- to 3-fold increase in specific luciferase activity for reporter constructs that contained a cyclin A promoter fragment extending 1 kb upstream from the major start site of transcription (Figure

3066

IA). Similar increases were observed for all reporter constructs with fragments extending beyond 89 bp upstream of the major transcription start site (-89/+6). Time course experiments revealed that promoter activity in these cells closely paralleled the expression of the endogenous cyclin A gene (see Figure 4A). Further deletion of upstream sequences (generating the -32/+11 reporter plasmid) abolished induction by Myc; instead, this construct was somewhat repressed in response to addition of 4-OHT (Figure IA). As a control, stable cell lines were generated that expressed two different cyclin A reporter plasmids in RATlA cells that did not carry a MycER gene; in these cells, addition of 4-OHT did not affect promoter activity (Figure IC). To exclude that an oestrogen-activated transcriptional activation domain located in the ER part of the MycER chimera (Berry et al., 1990) or steroids present in the tissue culture medium contributed to activation, we generated stable cells lines carrying cyclin A promoter constructs in RATI cells carrying a MycERTm allele (Danielian et al., 1993; Littlewood et al., 1995; Solomon et al., 1995). This allele has a mutated hormone binding domain that recognizes only 4-OHT, not oestrogen: addition of 4-OHT to these lines yielded identical results to those obtained from RATlA-MycER cells (Figure ID). We conclude that the response of the cyclin A promoter to activation of Myc is mediated by sequences located between -89 and -32 nucleotides upstream of the major start site of transcription. This region does not contain an E-box nor does it contain a c/EBP recognition element, sequence elements previously shown to interact directly with Myc and/or Max proteins (Hann et al., 1994). Up-regulation of cyclin A expression in response to serum growth factors has been shown to be due to derepression at two critical repressor elements within this region of the promoter, termed CDE ('cell cycle-dependent element') and CHR ('cell cycle genes homology region') (Zwicker et al., 1995). To identify whether these elements are required for induction by Myc, RATlA-MycER and RATI-MycERTM lines were generated that carried promoter fragments with specific mutations at either site (Figure lB and D). The results show first that mutation of either the CDE or the CHR element significantly enhanced the basal activity of the reporter, confirming that both elements exert a repressive effect on promoter activity (Figure ID) (Zwicker et al., 1995). More importantly, mutation of either the CDE or CHR element abolished up-regulation of cyclin A promoter activity by Myc. Taken together, these data show that the response of the cyclin A gene to activation of Myc is mediated by core elements of the cyclin A promoter located between -89 and -32 upstream of the major start site of transcription and that the integrity of both the CDE and CHR elements is critical for this induction. The physiologically important factors binding to either element remain to be identified (Zwicker et al., 1995). Recently, we have shown that the induction of Myc leads to a rapid activation of cdk activities in resting cells (Steiner et al., 1995), and ectopic expression of cyclin Dl has been shown to activate the cyclin A promoter (Schulze et al., 1995). Therefore, we decided to test whether active cdks are required for Myc to up-regulate expression of cyclin A. Two experiments were set up to test this

Induction of apoptosis and proliferation by Myc specific luciferase activity (fold activation relative to control)

A.

-1048

-9L26

+245

\

\\

F

+245 -240

+245

-78

+100

-89

+6 I,,/////z/

-32+11

Y//z////zzX/~

+100

-214

B.

0

CDE

-214 -214 I

+100

CHR+100 M

c

-

+100 +

-78

+100

-214

C.

S RAT1 A-MycER

=

RAT1 A

specific luciferase activity (x 105 light unts/lAg protein)

specific luciferase activity (fold activation relative to control) D.

M

-

-1048

D-

+245

0

\\

-240

CX

0

M

I

0)

+245

-78

+100 "I"

-32+11 Li

+100

-214

-2114

CDE

11,1,11.14A---i

_190 CR+

-21L4

cycdin

-59

A

cyclin0ACOE

CHR

+5

I cydin A CE

cydin A CFR

G |agct Al Ia

Fig. 1. Identification of the Myc-responsive element in the cyclin A promoter. (A) Shown is the specific luciferase activity from RATlA-MycER cell lines carrying the indicated fragments of the human cyclin A promoter. The numbers indicate the position in the cyclin A promoter relative to the major start site of transcription (Henglein et al., 1994). Results are plotted as fold increase in specific luciferase activity relative to uninduced control cells. Shown are average values from a representative experiment performed in triplicate. (B) Mutations in either the CDE or the CHR (indicated by -x-) abolishes up-regulation by Myc. Experiments were performed as described in the text. (C) 4-OHT regulates the cyclin A promoter in RAT1A-MycER cells, but not in RAT1A cells. Stable cell lines expressing the indicated constructs were generated in RAT1A cells and induced as described in the text. (D) Regulation of the cyclin A promoter in RATl-MycERTM cells. Shown is both fold increase in specific luciferase activity by addition of 4-OHT (left panel; measured after 20 h) and the basal specific luciferase activity (right panel) of the indicated constructs after stable integration into RATl-MycERTM cells. (E) Sequence of the cyclin A promoter and the mutations at the CDE and CHR elements (Zwicker et al., 1995).

hypothesis. First, expression plasmids encoding the cdk inhibitors p21, p27 or pl6 were microinjected into serumstarved RATI-MycER cells before 4-OHT was added. Control immunofluorescence experiments documented high levels of expression of each inhibitor in the injected cells (data not shown). Expression of cyclin A was monitored by immunofluorescence 12 h after induction of Myc. In these experiments, activation of Myc induced expression of cyclin A protein in ~80% of the cells within 12 h. In untreated controls, only 10-15% of the cells stained positively with an anti-cyclin A antibody (Figure 2A and B). Injection of any of the three inhibitors abolished induction of cyclin A by Myc (Figure 2B; photographs from representative experiments stained for both cyclin A and injected cells are shown in Figure 2C). Control experiments showed that, at the concentrations used, injection of either control plasmids (not shown) or of cdk2

expression vectors (Figure 2D) did not interfere with Mycinduced expression of cyclin A. Also, microinjection of expression plasmids encoding either pRb or p107 did not affect induction of cyclin A mRNA by Myc (not shown). Inhibition by p16 shows that cdk4 or cdk6 kinase activity is required for Myc-induced cyclin A expression (Figure 2B). Since p21 and p27 inhibit both cdk4/6 and cdk2, these data do not exclude the possibility that only cdk4/6 is required for induction of cyclin A by Myc. To test for a distinct requirement for cdk2 kinase activity, expression plasmids encoding kinase-negative alleles of cdk2 (cdk2 kn; van den Heuvel and Harlow, 1993) were microinjected (Figure 2D). In these experiments, kinase-negative alleles of cdk2 consistently reduced induction of cyclin A by Myc, although the effects were not as strong as with the kinase inhibitors. Staining with specific antibodies revealed that this was not due to lack of expression, because a 3067

B.Rudolph et aL

Fig. 2. Cyclin-dependent kinase activity is required for induction of cyclin A by Myc. (A) Phase-contrast and anti-cyclin A immunofluorescence photographs from serum-starved RAT1-MycER cells either before (upper panel) or 12 h after (lower panel) addition of 200 nM 4-OHT. (B) Quantitation of the percentage of cyclin A-positive cells before (-) and 12 h after addition (+) of 4-OHT for either control cells or cells microinjected with expression plasmids encoding p16, p21 or p27. (C) Immunofluorescence photographs from representative microinjection experiments. Micrographs on the left side show staining for microinjected cells, microinjected with different expression plasmids as indicated on the left side. Micrographs on the right side show the corresponding immunofluorescence for cyclin A. Injected cells are visualized by co-injection of FITC-dextran and indicated by arrows. (D) Quantitation of the percentage of cyclin A-positive cells after microinjection of expression plasmids encoding cdk2 or dominant negative alleles of cdk2 (cdk2 kn) and re-stimulation with 200 nM 4-OHT.

...

ldk.,-2 cdk2 ko,

strong anti-cdk2 immunofluorescence signal was detected in all cells injected with cdk2 kn expression plasmids (not shown). Similar experiments with dominant negative alleles of cdk4 did not give clear results (not shown). To confirm that cdk2 kinase is required for induction of cyclin A mRNA by Myc, we made use of the specific chemical inhibitor of cdk2 kinase, roscovitine. Roscovitine inhibits both cdk2 and cdc2 kinase at low micromolar concentrations (Figure 3A), but has no detectable effect on either cdk4 or cdk6 kinase activity (Glab et al., 1994; Vesely et al., 1994; Meijer, 1995; Abraham et al., 1995). Addition of 25 gM roscovitine to serum-starved RATIMycER cells before addition of 4-OHT abolished induction of cyclin A by Myc without inducing apparent damage to the cells (Figure 3B and C). As inhibition of cdc2 kinase arrests cells in G2, not in GI, these data strongly support a requirement for cdk2 kinase activity during induction of cyclin A mRNA by Myc. Taken together with the results from microinjection experiments, the data show that inhibition of either cdk2 or cdk4/6 kinase interferes with the ability of Myc to activate cyclin A and perhaps other downstream genes in the cell cycle. We wondered whether increases in either cdk2 or

3068

cdk4/6 kinase activity preceded up-regulation of the cyclin A promoter in response to Myc, and therefore performed time course experiments. Specific luciferase activity from two cyclin A promoter reporter plasmids, and cyclin Eand D I -dependent kinase activities were measured in parallel in RATlA-MycER cells after activation of Myc. Induction of a (-214/+100) reporter plasmid was maximal 10 h after activation of Myc, closely paralleling entry into S phase (Eilers et al., 1991). In contrast, a derivative mutated at the CDE element was not up-regulated by Myc at any time point (Figure 4A). Activation of cyclin E/cdk2 kinase preceded induction of the cyclin A promoter by 3 h (Figure 4A). As reported previously (Steiner et al., 1995), increases in cyclin DI/cdk4 kinase activity were significantly slower in response to Myc, both in densityarrested (Figure 4A) and in serum-starved (not shown) rat fibroblasts and did not clearly precede activation of the cyclin A promoter. The data show that only the Mycinduced increase in cyclin E/cdk2, but not cyclin D1/ cdk4/6 activity preceded activation of the cyclin A promoter. To test whether the increase in cyclin E/cdk2 activity might be sufficient for up-regulation of the cyclin A

Induction of apoptosis and proliferation by Myc

C. -

4-OHT

+ 4-OHT

+ 4-OHT + Ro

Fig. 3. The cdk2 kinase inhibitor, roscovitine, blocks transactivation of cyclin A by Myc. (A) Cyclin E-dependent kinase activity is inhibited by micromolar concentrations of roscovitine. Shown is the autoradiogram from immune complex kinase assays using an anti-cyclin E serum carried out in the presence of the indicated concentration of roscovitine. 'Co' denotes immune precipitations with an irrelevant control antibody. (B) Addition of roscovitine blocks induction of cyclin A by Myc in vivo. RATl-MycER cells were serum-starved for 48 h and re-stimulated by addition of 200 nM 4-OHT for 12 h either in the presence or absence of 25 ,uM roscovitine. Cells were fixed and stained with a polyclonal anti-cyclin A antibody. The panel shows a quantitation of three independent expefiments. (C) As above; the panel shows phase-contrast and immunofluorescence photographs of a representative experiment.

promoter by Myc, we asked if ectopic expression of cyclin E was able to stimulate transcription of the cyclin A promoter. Transient transfection experiments revealed that co-transfection of cyclin E together with cdk2 stimulated cyclin A promoter activity and this occurred via core promoter elements (Figure 4B). Similarly to observations made in stable cell lines, activation by cyclin E was largely due to derepression of the CDE and CHR elements, as (i) mutations at either the CDE or CHR element abolished induction by cyclin E and (ii) constructs harbouring such mutations had significantly elevated levels of basal promoter activity (Zwicker et al., 1995) (Figure 4B). Derepression of the cyclin A promoter by cyclin E was inhibited by kinase-negative alleles of cdk2, suggesting that active cyclin E/cdk2 kinase was involved (not shown). Transfection of expression plasmids encoding cyclin A together with cdk2 had no effect on cyclin A promoter activity (not shown). Transactivation by cyclin E/cdk2 was blocked by cotransfection of expression plasmids encoding the kinase inhibitors p21 and p27 (Figure 4C). Surprisingly, cotransfection of p16 (Figure 4C) also inhibited transactivation by cyclin E/cdk2. As p16 does not inhibit cdk2 kinase activity, these data suggest that a basal level of cdk4 (or of cdk6) kinase activity is required for cyclin E to act on the cyclin A promoter. Thus, while ectopic expression of either cyclin Dl or cyclin E is sufficient to induce expression of cyclin A in the absence of cdk inhibitors, a basal activity of both cdk2 and cdk4/6 kinases is required for activation; in this sense, the cyclin A

promoter may 'integrate' signals from both cdk2 and cdk4/6 kinase pathways (see Discussion). Induction by cyclin DI/cdk4 was blocked by p16, p21 and p27, but not by kinase-negative alleles of cdk2 (Figure 4D). Attempts to demonstrate induction of the endogenous cyclin A gene after microinjection of cyclin E expression plasmids failed because few surviving cells could be recovered from these injections (not shown). Taken together, the data show that a Myc-induced increase in cyclin E-dependent kinase activity precedes induction of the cyclin A promoter and is sufficient for its activation. Together with the previous results, the data strongly suggest that induction of cyclin A expression by Myc is mediated by cyclin E/cdk2 kinase. To support this notion further, we analysed a series of cell lines that express mutated alleles of Myc as hormone-inducible chimeras (Steiner et al., 1995). In these cell lines, a close correlation between the ability of alleles of Myc to induce cyclin E-dependent kinase activity and expression of cyclin A protein was observed (Figure 4E). Specifically, mutations that affected the ability to heterodimerize with Max (In 412), to bind to DNA (In 370) (Philipp et al., 1994) or to regulate transcription (A128-143) (Desbarats et al., 1996) all abolished the ability to activate cyclin A expression; in contrast, mutations in Myc box I (A45-63) or in a domain involved in gene repression (In 104, A92106) did not affect the ability to activate cyclin A expression. Importantly, no mutant was identified that activated cyclin E-dependent kinase but failed to activate

3069

B.Rudolph et al

|.rm- !.ir_F-,S .

tr

A