In the present work we studied mechanisms of growth control in contact-inhibited and serum-deprived human diploid ®broblasts. The observation that the e ects on [ 3 H]thymidine incorporation and reduction of retinoblastoma gene product-phosphorylation were additive when contact-inhibition and serum-deprivation were combined led us to the conclusion that the underlying mechanisms might be di erent. Both contact-inhibition and serumdeprivation led to a strong decrease of cdk4-kinaseactivity and cdk2-phosphorylation at Thr 160, while the total amounts of cdk4 and cdk2 remained constant. In contact-inhibited cells, we revealed a strong protein accumulation of the cdk2-inhibitor p27 and a slight, but signi®cant increase of the cdk4-inhibitor p16. In serumdeprived cells, the protein levels in p27 and p16 remained low. In contrast, we detected a rapid decrease of cyclin D1 and cyclin D3 which did not occur in contactinhibited cells. These results indicate that serumdeprivation and contact-inhibition have di erent mechanisms although they a ect the same pathway cyclin D ± cdk4, pRB, cyclin E ± cdk2.

Growth of non-transformed cells in vitro is regulated by density-dependent mechanisms via cell-cell contacts, leading to arrest in late G1-phase at con¯uency (contact-inhibition of growth). In the present study it is shown that this results from p16 INK4 -mediated dissociation of the complex cdk4-cyclin D1, which is responsible for the inactivation of the gate keeper of G1-S transition, the retinoblastoma protein pRb. As a consequence of the inactivation of cdk4, downstream the activation of cdk2 and hyperphosphorylation and thus inactivation of pRb was impaired. Direct evidence for the central role of p16 INK4 in growth control comes from the observation that a competitive inhibitor of p16 INK4 repressed contact inhibition of growth. These ®ndings provide an explanation for the high incidence of mutation or loss of INK4 in human tumours.Keywords: contact-inhibition; cdk4; p16 INK4 The cell cycle is regulated by an ordered cascade of phosphorylation reactions by a speci®c family of serine-threonine kinases, the cyclin-dependent kinases (cdks) (Lees, 1995;Sherr, 1996), and speci®c check points ensure that the cell's proper cycle is tightly controlled. In order to determine at which point of the G1-phase cells are arrested by contact-dependent inhibition of growth, time course studies were performed. In FH109 cells, human embryonal diploid lung ®broblasts (Wieser et al., 1985), contactdependent inhibition of growth is exclusively mediated by the interaction of two cell membrane proteins on adjacent cells, i.e. by the glycoprotein, contactinhibin (Wieser et al., 1990), which binds to its receptor referred to as contactinhibin receptor (Gradl et al., 1995).Cell-cell contacts were imitated by the addition of glutaraldehyde-®xed cells (in which contactinhibin remains active, as its growth inhibitory activity is mediated exclusively by its N-glycans not a ected by the ®xation process) to sparsely seeded ®broblasts in the presence of serum. In previous studies we have demonstrated that this system mimics physiological conditions concerning cell proliferation and differentiation which occur in con¯uent cultures (Wieser et al., 1985;Wieser and Oesch, 1986), thus enabling the study of early e ects of cell-cell contact-dependent signals. FH109 cells were sparsely seeded and cultured for 24 h in DMEM/0.5% FCS to achieve partial synchronization. Control cells were stimulated with 10% FCS in DMEM whereas contact-inhibited cells were generated by the addition of ®xed cells in DMEM/10% FCS prepared from con¯uent cultures (Wieser et al., 1985). These experiments revealed that when ®xed cells were added within the ®rst 8 h after serum-stimulation, proliferation was inhibited by 70 ± 80% as determined by [ 3 H]-thymidine incorporation, which correlates with the reduction of growth rate achieved by seeding ®broblasts to con¯uency (Figure 1). The addition of ®xed cells 10 h after serum stimulation no longer resulted in any growth inhibition, indicating that contact-dependent inhibition of growth is limited to a window in the cell ...

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Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclindependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the Gl-phase of cell cycle progression.There is evidence that intracellular proteases play a dominant role in cell cycle control. Accordingly, antiproliferative effects are shown by some cell-permeable protease inhibitors. For example, the lipid soluble derivative of E-64, E-64d, an inhibitor of cathepsins and calpains, has been shown to arrest squamous cell carcinoma cells in metaphase (1). Cathepsins are known as acid cysteine proteases in contrast to the neutral, Ca2+-requiring cysteine proteases, calpains (calpain 1 and calpain 2). Mellgren and coworkers (2) also suggested that calpains participate in cell growth regulation since benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone, which is thought to inhibit calpains, reduced proliferation of human TE2 (simian virus 40-transformed human esophageal epithelial cells) and C-33A cells (human cervical carcinoma cells). In vascular smooth muscle cells, N-acetyl-L-leucinyl-L-leucinyl-Lnorleucinal (LLnL) (calpain inhibitor 1) caused a repression of platelet-derived growth factor-(PDGF), as well as seruminduced cell cycle progression before G1/S transition (3). Unfortunately, these commonly used thiol protease inhibitors are not highly selective, making it difficult to precisely define the intracellular targets. LLnL, for example, is not selective for calpains, but inhibits also cathepsin L (4), as well as the proteasome complex (5). Consequently, the assumption that calpains function in cell cycle control (2, 3), remains to be verified. Furthermore, it is not known which distinct step within the cell cycle is affected by calpain inhibitors resulting in G1 arrest. Interestingly, cyclin B degradation, which requires ubiquitin-conjugation (6), can be inhibited by LLnL (7). In previous studies in which we addressed the question if proteolytic activity is required to allow confluent cultures to mitogenically respond to PDGF, we noticed that LLnL strongly inhibited PDGF-induced proliferation. We therefore studied the effect of LLnL in PDGF-AB-stimulated human fibroblasts to determine (i) the protease(s) that is/are inhibited by LLnL, (ii) the target of the proteas...