A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

Abstract

Transforming growth factor , (TGF-,B) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the GI-to-S phase transition in mammalian cells. We have studied the effects of TGF-p-mediated growth arrest on GJ/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-j is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-0 inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the Gl-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-j8-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In Go-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses.Control of the eukaryotic cell cycle occurs at key points in G, and at the G2-to-M phase transition (41,80,85,121,122). These transitions are governed by multimeric protein complexes with serine/threonine kinase activity, whose catalytic subunits, proteins of 33 to 34 kDa, are regulated by phosphorylation and by periodic association with positive effector proteins, known as cyclins. Current understanding of cell cycle control derives largely from studies of yeast cells. The Cdc28 kinase in the budding yeast Saccharomyces cerevisiae and its homolog in the fission yeast Schizosaccharomycespombe, Cdc2, appear to regulate both the Gl-to-S and G2-to-M phase transitions (5,81,88,93,95). In S. cerevisiae, association of p34Cdc28 with Gl cyclins, or Clns, is necessary for entry to S phase (12,38,77,98,115), while association with the mitotic or B-type cyclins, the Clbs, promotes entry into mitosis (30, 108).The first human homolog of p34Cdc2/Cdc28 to be identified, Cdc2Hs, could complement p34 deficiency in both S. cerevisiae and S. pombe (16, 61). However, its action appears to be restricted to the G2-to-M phase transition in humans (15,89,97,110).A number of Cdc2-related or cyclin-dependent kinases (Cdks) have subsequently been identified (23,51,61,74,78,86,102,112). These kinases combine with different cyclins to govern different cell cycle transitions. One of these, Cdk2, appears to regulate Gl-and S-phase functions in human cells (62,102,113). The action of Cdk2 in complex with cyclin A is essential for progression through S phase and may play a role in the regulation of DNA synthesis (25,32,33,83,90,123).Several candidate G, cyclins, designated C, D, and E, were identified when human and Drosophila cDNA libraries were screened for sequences that could complement deletion of the S. cerevisiae Cln genes (51,63,65,117). A cyclin D cDNA was

The role of p27Kip1 in gamma interferon-mediated growth arrest of mammary epithelial cells and related defects in mammary carcinoma cells

“…p27 mRNA levels do not change signi®cantly during the time course in which protein levels increase, lending support to the growing body of evidence that post-transcriptional regulation of p27 is a key mechanism in growth arrest (Polyak et al, 1994b;Halevy et al, 1995;Pagano et al, 1995;Hengst and Reed, 1996). Levels of both the G 1 cyclins and cdk's were essentially unchanged within these 24 h, although cdk4 levels began to decrease about 24 h post-treatment and were signi®cantly reduced by 48 h. Similar results have been reported in other systems (Ewen et al, 1993b;Slingerland et al, 1994), implicating down-regulation of cdk4 as a component for the maintenance of long-term growth arrest.…”

“…p21 Cip1 is found in cdk/cyclin complexes throughout G 1 and has been described as a potential modulator of normal cell cycle traverse Li et al, 1994;Noda et al, 1994;Nourse et al, 1994;Sheikh et al, 1994) as well as an e ector of p53-induced growth arrest in response to DNA damage (El-Deiry et al, 1993). p27 is the primary inhibitor of cdk2/cyclin E activity in cells arrested by TGFb (Polyak et al, 1994a; and increased p27 protein levels have been associated with density arrest and growth factor deprivation Polyak et al, 1994b;Slingerland et al, 1994).…”

Deregulated degradation of the cdk inhibitor p27 and malignant transformation

“…Members of the Ink4 family of Ckis (p15, p16, p18, and p20) inhibit the activity of both cdk4 and cdk6, while members of the Kip/Cip family of Ckis (p21, p27, and p57) inhibit specifically the activity of cdk2 by binding both to cyclin E/cdk2 and cyclin A/cdk2 complexes. p27 was first identified as a Cki due to its ability to block the activity of cyclin E/cdk2 and cyclin A/cdk2 in cells arrested in G1 by lovastatin, TGF-␤ and contact inhibition [5][6][7][8].…”

Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma

“…They mediate e ects on cell cycle progression by their ability to bind with cyclin/ cdk complexes and inhibit cdk activity. p27 Kip1 was identi®ed originally as an inhibitory activity upregulated in cells arrested in the G 1 phase by intercellular contact or treatment with TGF-b (Slingerland et al, 1994;Polyak et al, 1994a,b;Ko et al, 1993). An elevated p27 Kip1 protein level has also been associated with a quiescent, G 0 state Reynisdottir et al, 1995;Hengst and Reed, 1996;Pagano et al, 1995) and diminished levels of p27 Kip1 protein have been recently associated with poor prognosis in breast and colon cancers (Catzavelos et al, 1997;Loda et al, 1997;Porter et al, 1997).…”