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Cyclin D1 Expression Is a Major Target of the Camp-Induced Inhibition of Cell Cycle Entry in ®Broblasts

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Cyclin D1 Expression Is a Major Target of the Camp-Induced Inhibition of Cell Cycle Entry in ®Broblasts Oncogene (1997) 14, 1981 ± 1990 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Cyclin D1 expression is a major target of the cAMP-induced inhibition of cell cycle entry in ®broblasts Gilles L'Allemain1,*, Jose e N. Lavoie1,*, Nathalie Rivard1,VeÂronique Baldin1,2 and Jacques Pouyssegur1 1Centre de Biochimie, CNRS-UMR134, Faculte des Sciences, Parc Valrose, 06108 NICE Cedex 02, France We previously described in the CCL39 hamster ®broblast of cAMP on growth factor-induced activation of p42/ cell line the inhibition of DNA synthesis reinitiation by p44 MAP kinases was very transient and could not agents that elevate cyclic AMP. Here, we show that 8Br- account for the growth inhibitory action of cAMP cAMP strongly blocks both the growth factor-induced (Kahan et al., 1992; McKenzie and Pouyssegur, 1996). increase in cyclin D1 protein expression and decrease in Therefore, other cAMP targets should be considered p27KIP1 protein levels, leaving untouched the levels of and we looked for target(s) downstream of the p42/p44 cyclin D3, cdk2 and cdk4. To assess the role of cyclin MAP kinase pathway. Progression through the G1 D1 in the cAMP-mediated inhibition of DNA synthesis, phase of the cell cycle is orchestrated by complexes we overexpressed the cyclin D1 gene in CCL39 and containing a G1-speci®c cyclin and a G1-speci®c cyclin- analysed the cAMP response in stable transfectants. We dependent kinase (Cdk): among those, cyclin D family showed that the kinase activities associated to G1 cyclin- members are found associated with either cdk4 or cdk6 cdk complexes are signi®cantly more resistant to cAMP in early G1 phase, and cyclin E with cdk2 in late G1 (see in cyclin D1 transfectants than in their normal counter- for reviews Draetta, 1994; Grana and Reddy, 1995; parts, although the serum-induced p27KIP1 disparition is Reed et al., 1994; Sherr, 1993). Activity of these still cAMP sensitive in cyclin D1 overexpressors. complexes can be inhibited by the presence of a cyclin Interestingly, the mitogen-induced DNA synthesis re- kinase inhibitor (CKI), which belongs to an expanding initiation is also much less inhibited by cAMP in cyclin new family of mammalian cell cycle modulators D1 transfectants than in control cells. These data clearly comprising so far p21Cip1, p27KIP1,p57KIP2 in one sub- INK4A INK4B INK4C INK4D establish that the cAMP-inducible blockade of the G1 family, and p16 , p15 , p18 and p19 in phase of the cell cycle can be partially alleviated by the other (Lees, 1995; Sherr and Roberts, 1995 for overexpression of cyclin D1 in hamster ®broblasts, thus reviews), with p27KIP1 being recently involved in the strongly suggesting that cyclin D1 protein is one of the inhibitory process induced by cAMP in macrophages major targets for cAMP inhibitory action in ®broblasts. (Kato et al., 1994). At the G1-4S phase transition, the tumor suppressor pRb, product of the retinoblastoma Keywords: Cyclin D1; cAMP; cyclin-CDK complexes; gene, is inactivated by hyper-phosphorylation, thus p27KIP1; DNA synthesis leaving free the transcription factors E2Fs to activate E2 site-containing gene promoters (Dyson, 1994 for a review). The inactivation of pRb has recently been shown to be performed in vivo by a series of various G1 Introduction cyclin-kinase complexes (Horton et al., 1995; Kato et al., 1993; Suzuki-Takahashi et al., 1995). Not only pRb Cyclic AMP has been described as a mediator of regulates cyclin D1 expression (Muller et al., 1994), but numerous physiological processes. Large increases in a strict relationship between cyclin D1 and pRb has cAMP concentration inside the cell are generally growth been established: if pRb function is de®cient, the cyclin inhibitory for most cell lines of mesenchymental origin. D1 requirement in the G1-4S progression becomes Accordingly, we previously published (Magnaldo et al., dispensable (Bates et al., 1994; Lukas et al., 1994a, 1989) that any sustained increase in cAMP level was 1995a). detrimental for DNA synthesis reinitiation in the Here, we addressed the question of which, if any, hamster ®broblast cell line CCL39. But the exact cell cycle regulatory protein was targeted by cAMP. A mechanism by which cAMP exerts its inhibitory action previous report from our laboratory (Magnaldo et al., remains largely unknown or somewhat controversial. 1989) targeted the cAMP growth inhibition as taking We subsequently showed that activation of the p42/p44 place in the early G0/G1 phase, more precisely during MAP kinase pathway (ERK pathway) was an the ®rst 6 h of growth factor addition. Interestingly, obligatory step to trigger cell mitogenicity (PageÁ s et addition of cAMP agonists for the ®rst 4 h following al., 1993, 1994). This signalling cascade was indeed a growth factor stimulation delayed S phase entry by potential target for cAMP inhibition. However, we now roughly the same time period (Magnaldo et al., 1989). show that in various ®broblastic cell lines, the inhibition Thus, the cell cycle regulatory protein cyclin D1, that has been described as an essential component of an early G1 checkpoint (Pagano et al., 1994) and as Correspondence: G L'Allemain governing cell cycle exit (Zwijsen et al., 1996), appeared 2Present address: Institut de Pharmacologie et Biologie Structurale, to be a good candidate. Indeed, cyclin D1 over- CNRS, 31077 Toulouse, France expression deregulated cell growth of normal cells (Han *The ®rst two authors contributed equally to this work Received 17 November 1996; revised 8 January 1997; accepted et al., 1995; Jiang et al., 1993; Quelle et al., 1993; 13 January 1997 Resnitzky et al., 1994) whereas antisense to this cyclin cAMP inhibits cell growth via cyclin D1 GL'Allemainet al 1982 inhibited growth of transformed cells (Zhou et al., cyclin D1, cyclin D3, cdk2, cdk4, p27KIP1 or pRb. 1995). Here, we used the 8Br-cAMP, a non-hydro- Upper panel of Figure 1 indicates that cyclin D1 lysable but diusible cAMP analog, and found that protein is not present (or barely detectable) in cyclin D1 protein expression was strongly repressed by quiescent cells but its expression could be detected as such a sustained increase in cAMP level. In order to soon as 6 h after serum stimulation, with the protein establish whether or not cyclin D1 was the major level increasing between 6 and 20 h. Interestingly, protein targeted by the inhibitory pathway triggered by 1mM 8Br-cAMP strongly blocked such an expression cAMP, we then ectopically expressed the human cyclin over all this period of time. Like in other studies D1 gene in the non-transformed hamster ®broblast cell (Lukas et al., 1995a; Matsushime et al., 1994), the line CCL39 and analysed the resulting sensitivity to appearance of a slow migrating form of cyclin D1 cAMP of G1 cyclin-cdk complexes and of DNA correlates with the strongly induced major cyclin D1 synthesis reinitiation. band. Its identity is unknown but cannot correspond to either the cyclin D2 or D3 isoforms. Besides cyclin D1 (Baldin et al., 1993; Lukas et al., 1994b), both cyclin D2 and cyclin D3 have also been Results involved in controlling the G1 phase (Ando et al., 1993; Kiess et al., 1995; Kiyokawa et al., 1994; Lukas et al., cAMP eect on kinetics of expression of various cell 1995b; Skapek et al., 1995) with cyclin D3 mostly cycle regulatory proteins in hamster ®broblasts implicated in cell dierentiation (Kiess et al., 1995; We ®rst examined the eect of a pre-incubation with Kiyokawa et al., 1994; Skapek et al., 1995), a process 1mM 8Br-cAMP on the serum-induced protein inhibited by cyclin D1 (Skapek et al., 1995). We thus expression of dierent cell cycle regulatory proteins in checked for their presence into CCL39 cells. Upper the hamster ®broblast line CCL39. Figure 1 shows a panel of Figure 1 also shows that the cyclin D3 isoform series of immunoblots loaded with CCL39 cell lysates was already present in G0-arrested cells but its and decorated with speci®c antibodies against either expression was only slightly induced by growth factors cycD1 cycD3 FCS 0 6 10 15 20 6 10 15 20 (hours) 8Br-cAMP – + pRb cdk4 cdk2 Kip1 FCS 0610 15 20 0610 15 20 (hours) 8Br-cAMP – + Figure 1 Eect of 8Br-cAMP on the expression levels of various cell cycle regulatory proteins in hamster ®broblasts. G0-arrested CCL39 cells (0) were activated with 10% FCS with (+) or without (7) for the indicated times before cell lysis. All protein lysates (75 mg) were separated by 10% SDS ± PAGE (except 7.5% for pRb), blotted and immunodecorated with speci®c antibodies as indicated cAMP inhibits cell growth via cyclin D1 GL'Allemainet al 1983 and resistant to the 8Br-cAMP treatment, thus D1) exhibited much higher levels of cyclin D1 excluding its putative role in cAMP inhibition. Use (respectively, ®ve and 12 times more as assessed by of four dierent antibodies, monoclonal and poly- densitometry) than their control counterparts trans- clonal, did not allow us to detect cyclin D2 isoform in fected with the empty vector (39-C, PS-C). Interest- CCL39 cells (data not shown), a situation encountered ingly, the PS-D1 clone was expressing 3 ± 4 times more in ®broblasts as well as in other cell types (Tam et al., cyclin D1 than 39-D1. Main panel of Figure 2 shows 1994). the timing of entry into S phase exhibited by these four On the lower panel of Figure 1, we analysed the cell lines after serum deprivation for 24 h followed by hyper-phosphorylation state of pRb which is, in late G1 reactivation by growth factors. Thymidine incorpora- phase, a landmark for normal cells passing the tion in resting cells was very low and essentially restriction point and entering S phase (Grana and identical both in control and D1-transfected clones, Reddy, 1995 for a review).
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