Oncogene (1997) 15, 2743 ± 2747 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 SHORT REPORT Dierences in the mechanisms of growth control in contact-inhibited and serum-deprived human ®broblasts Cornelia Dietrich, Katja Wallenfang, Franz Oesch and Raimund Wieser Institute of Toxicology, Johannes-Gutenberg University, 55131 Mainz, Germany In the present work we studied mechanisms of growth small inhibitory proteins, known as p15, p16, p18, p19, control in contact-inhibited and serum-deprived human p21, p27 and p57 (for review see Sherr, 1994, 1996; diploid ®broblasts. The observation that the eects on Sherr and Roberts, 1995). [3H]thymidine incorporation and reduction of retinoblas- The activating pathway of mitogenic signals such as toma gene product-phosphorylation were additive when serum, growth factors or mitogenic hormones has contact-inhibition and serum-deprivation were combined been well established in the last few years. As a led us to the conclusion that the underlying mechanisms delayed early response, growth factors induce the might be dierent. Both contact-inhibition and serum- expression and synthesis of D-type cyclins (D1, D2, deprivation led to a strong decrease of cdk4-kinase- D3) which are dierentially and combinatorially activity and cdk2-phosphorylation at Thr 160, while the expressed in mammalian cells (Won et al., 1992; total amounts of cdk4 and cdk2 remained constant. In Matsuchime et al., 1991; Xiong et al., 1993). In contact-inhibited cells, we revealed a strong protein complex with their major catalytic partner cdk4, they accumulation of the cdk2-inhibitor p27 and a slight, but phosphorylate pRB in mid to late G1 (Matsushime et signi®cant increase of the cdk4-inhibitor p16. In serum- al., 1994; Xiong et al., 1993) which induces cyclin E deprived cells, the protein levels in p27 and p16 remained expression. At G1/S-transition, cyclin E accumulates low. In contrast, we detected a rapid decrease of cyclin which in association with cdk2 also phosphorylates D1 and cyclin D3 which did not occur in contact- pRB and, in addition, still unknown substrates inhibited cells. These results indicate that serum- (Hatakeyama et al., 1994). deprivation and contact-inhibition have dierent mechan- In contrast, less is known about signal transduction isms although they aect the same pathway cyclin D ± pathways due to inhibiting signals such as contact- cdk4, pRB, cyclin E ± cdk2. inhibition and serum-deprivation. Polyak and co- workers (1994a) as well as Hengst and coworkers Keywords: Cyclin-dependent kinase regulation; contact- (Hengst et al., 1994; Hengst and Reed, 1996) could inhibition; serum-deprivation show an accumulation of the cdk-inhibitor p27. In contrast, ®broblasts from p27-knock out mice still show full contact-inhibition (Nakayama et al., 1996) which suggest the involvement of additional, still The decision of mammalian cells wether to replicate unknown mechanism(s). DNA and proliferate or to withdraw from cell cycle In general, no mechanistic discrimination is made and go into quiescence is taken in late G1 phase. This between quiescence due to high density or mitogen G1 checkpoint referred to as restriction (R) point has withdrawal. Since in vivo contact-inhibition, and not been ®rst described by Pardee (1989). It is now serum-deprivation, plays the fundamental role in generally believed that the retinoblastoma gene proliferation control (i.e. wound-healing), we postu- product (pRB) plays a crucial role in regulating the lated dierences in the mechanisms of contact- restriction point. In its hypophosphorylated form pRB inhibition and serum-deprivation. In addition, in binds to the transcription factor E2F hence inhibiting vitro, dierences between contact-inhibition and ser- transcription. (Hyper)phosphorylation of pRB leads to um-deprivation have been described (Del Sal et al., its functional inactivation resulting in the loss of 1992; Gustincich and Schneider, 1993; Moreton et al, binding to E2F thus permitting entry into S-phase 1995a). In the present work we studied the eects of (Mittnach and Weinberg, 1991; DeCaprio et al., 1989). contact-inhibition and serum-deprivation on closely pRB is phosphorylated by a speci®c family of serine/ connected upstream regulators of pRB in human threonine kinases, the cyclin-dependent kinases (cdks). diploid ®broblasts. Several mechanisms are employed to regulate cdk- Exponentially growing FH109 human embryonal activity: activation of a cdk, the catalytic subunit, is lung ®broblasts (Wieser et al., 1985) were seeded either dependent on the association with a cyclin, the sparsely or to high density in CG-medium (Vitromex) regulatory subunit. In addition, activation of the supplemented with 0.5% fetal calf serum (Gibco). catalytic subunit requires phosphorylation at a FH109 cells have been shown to highly sensitive to conserved Thr residue by cdk-activating kinase and cell-cell contacts with respect to growth-inhibition dephosphorylation at Thr14 and Tyr15. The activity of (Wieser et al., 1985, 1990; Wieser and Oesch, 1986). the complex is further modulated by the association of After 6 h the cells adhered to the plates (t=0), and the medium of sparsley seeded cells was changed to CG- medium without fetal calf serum for serum-deprivation Correspondence: R Wieser experiments. The cells were harvested after 24, 48 and Received 9 April 1997; revised 18 July 1997; accepted 21 July 1997 72 h. Growth control by contact-inhibition and serum-deprivation C Dietrich et al 2744 We ®rst investigated the eect of contact-inhibition clear shift to faster electrophoretic mobility. Phosphor- and serum-deprivation on proliferation of FH109 cells. ylation of pRB was even less when contact-inhibition and Proliferation was measured by [3H]thymidine incorpora- serum-deprivation were combined. The fact that the tion and pRB-phosphorylation. Phosphorylation of pRB antiproliferative eects of contact-inhibition and serum- from mid to late G1-phase is believed to be the critical deprivation were additive led to the assumption that step for G1/S-transition and hence re¯ects cell-cycle dierent inhibitory pathways might be involved. progression in G1. After 24 h, both contact-inhibition Since phosphorylation of pRB is belived to be the and serum-deprivation reduced [3H]thymidine incor- ®nal control point for G1/S-transition we next poration to 20% compared to exponentially growing examined the upstream regulators of pRB-phosphor- cells whereas the combination resulted in a decrease to ylation, namely regulation of cdk4 and cdk2. The 5% (Figure 1a). [3H]Thymidine incorporation did not kinase cdk4 is activated by association of D-cyclins, decrease further after 48 h or 72 h. The inhibition of cdk2 is activated by cyclin E. While p16 speci®cally proliferation was also re¯ected by a reduction of pRB- blocks cdk4 (Serrano et al., 1993), p27 inhibits phosphorylation (Figure 1b). Using Western blot although not selectively but preferentially cdk2 analysis, we predominantly detected the slower migrat- (Harper et al., 1995; Soos et al., 1996). We ®rst ing species of pRB in proliferating cells which determined protein levels of cdk4 and cdk2, cyclin D1 corresponds to the hyperphosphorylated, inactive pRB and D3 as well as p16 and p27. Protein levels of cdk4 (DeCaprio et al., 1989). In contact-inhibited or serum- were not aected, neither in response to serum- deprived cells, phosphorylation of pRB was strongly deprivation nor to contact-inhibition, and protein reduced and, concomitantly, we revealed an increase of levels of cdk2 also remained constant after contact- the hypophosphorylated, active species of pRB with a inhibition and for 48 h after serum-deprivation (Figure 2a). However, serum-deprivation resulted in a marked decrease of the cyclins D1 and D3 whereas they remained unaected in contact-inhibited ®broblasts a (Figure 2b). This result is in accordance with the work of Won et al. (1992) who detected a 70%- a C SD CI kD 34 — cdk4 35 — cdk2 1 2 3 4 5 6 7 b C SD CI b kD 34 — D3 KD 117 — pRB* 105 — pRB 34 — D1 C SD CI CI + SD 1 2 3 4 5 6 7 Figure 1 Proliferation of exponentially growing (C), serum- deprived (SD), contact-inhibited (CI), or serum-deprived and contact-inhibited (CI+SD) FH109 cells. Proliferation was determined by [3H]thymidine incorporation (a) and phosphoryla- c tion of pRB by Western blot analysis (b). (a)56103 (sparsley) or C SD CI 56104 (high density) cells were seeded into microtiter plates, kD treated and cultured as mentioned. 24 h, 48 h and 72 h after cell 16 — p16 adhesion, the cells were labeled with 0.25 mCi/well of [3H]thymidine for 4 h. Incorporated radioactivity was determined by liquid scintillation spectrometry. (b)56105 (sparsley) or 3.56106 (high density) cells (60 mm-plates) were cultured as 27 — p27 described and solubilized 24 h after cell adhesion in 500 mlof 1 2 3 4 5 6 7 boiling SDS-sample buer (Laemmli, 1970). Protein determina- tion was performed according to (Smith et al., 1985), proteins Figure 2 Protein levels of cdk4 and cdk2 (a), cyclin D1 and D3 (20 mg) precipitated as described by (Wessel and FluÈ gge, 1984) (b), and p16 and p27 (c) in exponentially growing (c), serum- and solubilized in 20 ml of SDS-sample buer. After SDS- deprived (SD) or contact-inhibited (CI) FH109 cells. Total cell polyacrylamide gel electrophoresis (12.5%), proteins were extracts were harvested 24 h (lanes 1, 2, 5), 48 h (lanes 3 and 6) transferred onto Immobilon membrane (Millipore). Immuno- and 72 h (lanes 4 and 7) after cell adhesion followed by Western detection with anti-pRB-antibodies (0.1 mg/ml, Santa Cruz) was blot analysis as described in Figure 1 except that 50 mg of protein performed as previously described (Dietrich et al., 1996). was precipitated for p16-detection. The position of the molecular pRB*=(hyperphosphorylated) pRB weight marker proteins is shown on the left Growth control by contact-inhibition and serum-deprivation C Dietrich et al 2745 decrease of cyclin D1 and D3 mRNA in human diploid after 24 h which was more pronounced after 48 h and ®broblasts within 7 h after serum-depletion.