Inhibition of G₁ Cyclin-Dependent Kinase Activity during Growth Arrest of Human Breast Carcinoma Cells by Prostaglandin A₂

Abstract

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.

Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction

“…This result is consistent with previous reports and support the notion that the inhibition of D-type cyclins may be the crucial event responsible for the antiproliferative action of numerous compounds, such as cyclic-AMP, prostaglandin A2 and alpha IFN (Gorospe et al, 1996;L'Allemain et al, 1997;Sewing et al, 1993;Tiefenbrun et al, 1996;Vadiveloo et al, 1996). The role of cyclin D3/ CDK4 in the cell cycle of 7TD1 cells is not clear.…”

“…Growth inhibition and cell cycle arrest in G 1 may be induced by a wide variety of factors such as TGFb (Florenes et al, 1996;Reynisdottir et al, 1995), nerve growth factor (Decker, 1995), alpha interferon (Tiefenbrun et al, 1996), ®brillar collagen (Koyoma et al, 1996), prostaglandin A2 (Gorospe et al, 1996), antiprogestin (Musgrove et al, 1997) or by antiproliferative agents such as cyclic-AMP , staurosporine (Schnier et al, 1996), lovastatin (Hengst et al, 1994). To date, the mechanisms underlying these antiproliferative e ects are a matter of great interest.…”

“…To date, the mechanisms underlying these antiproliferative e ects are a matter of great interest. Several lines of evidence have demonstrated that these molecules inhibit cell progression through di erent mechanisms such as alteration of the CDK expression, modi®cation of cyclin levels (Gorospe et al, 1996;L'Allemain et al, 1997;Poon et al, 1995;Tiefenbrun et al, 1996), regulation of CDK activity through phosphorylation/dephosphorylation (Tiefenbrun et al, 1996) and induction or redistribution of CDK inhibitors (CKIs) (Florenes et al, 1996;Hengst et al, 1994;Koyoma et al, 1996;Poon et al, 1995;Reynisdottir et al, 1995).…”

Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro

“…A progressive loss of cyclin D1 expression was noted with increasing tRA concentration, approaching the limit of detection at 1 mM (Figure 1a). Cyclin D2 was undetectable in MCF-7 cell lysates, in agreement with previously published observations Gorospe et al, 1996). Re-probing of the Western blot with antibody to cyclin D3 revealed a similar trend (Figure 1b).…”

“…For the inhibitors, p21 expression was also decreased by RAs, but was una ected by tamoxifen (Figure 7g). The p16 inhibitor is mutated and undetectable in MCF-7 cells (Gorospe et al, 1996, and data not shown). To determine if this complex series of changes in G 1 cell cycle proteins has functional consequences, assays for Cdk2 and Cdk4 activity were performed.…”