Oncogene (1997) 14, 1329 ± 1340 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Cyclin D2 activates Cdk2 in preference to Cdk4 in human breast epithelial cells Kimberley J Sweeney, Boris Sarcevic, Robert L Sutherland and Elizabeth A Musgrove Cancer Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, NSW 2010, Australia To investigate the possibility of diering roles for cyclins Similarly, overexpression of cyclin D2 in myeloid cells D1 and D2 in breast epithelial cells, we examined the results in a decrease in the duration of G1 and an expression, cell cycle regulation and activity of these two increase in the percentage of cells in S-phase (Ando et G1 cyclins in both 184 normal breast epithelial cells and al., 1993; Kato and Sherr, 1993). Microinjection or T-47D breast cancer cells. Synchronisation studies in 184 electroporation of cyclin D1 or cyclin D2 antibodies cells demonstrated that cyclin D1 and cyclin D2 were demonstrated that these proteins were not only rate- dierentially regulated during G1, with cyclin D2 limiting but essential for progress through G1 (Baldin abundance increasing by 3.7-fold but only small changes et al., 1993; Quelle et al., 1993; Lukas et al., 1995b). in cyclin D1 abundance observed. The functional These eects are thought to be mediated by activation consequences of increased cyclin D2 expression were of cyclin-dependent kinases (CDKs) and consequent examined in T-47D cells, which express no detectable phosphorylation of the product of the retinoblastoma cyclin D2. Induced expression of cyclin D2 resulted in susceptibility gene, pRB (Hunter and Pines, 1994; increases in cyclin E expression, pRB phosphorylation Sherr, 1994). Phosphorylation of pRB by cyclin/CDK and the percentage of cells in S-phase, while constitutive complexes in late G1 phase relieves its inhibitory role expression resulted in a consistent trend toward reduced by releasing transcription factors, such as E2F, dependence on serum for continued proliferation. Thus, essential for progression through S-phase (Sherr, cyclin D2 is a positive regulator of G1 progression in 1994; Weinberg, 1995). Cyclin D-associated CDKs breast cells analogous to the well-documented eects of are pRB kinases in vitro (Matsushime et al., 1994; cyclin D1. Indeed, equimolar concentrations of inducible Meyerson and Harlow, 1994) and, together with cyclin cyclin D1 and D2 resulted in quantitatively similar cell E/Cdk2, are also important pRB kinases in vivo (Sherr, cycle eects. Marked divergence was found, however, in 1994; Weinberg, 1995). There is clear evidence that the CDKs activated by the two cyclins in breast both cyclin D1 and D2 have a codependent relation- epithelial cells. Cyclin D2 complexes contained a higher ship with pRB: cells that lack functional pRB have Cdk2/Cdk4 ratio than cyclin D1 complexes. The cyclin reduced expression of these D type cyclins (Bates et al., D2-associated kinase activity was largely inhibited by 1994b; Lukas et al., 1995a) and ectopic expression of Cdk2-speci®c inhibitors and could phosphorylate histone either protein can rescue cells from the growth- H1, a substrate for Cdk2 but not for Cdk4 and Cdk6. suppressive eect of pRB (Dowdy et al., 1993; Ewen Therefore, cyclin D2 preferentially activated Cdk2 in et al., 1993). These similar roles in G1 progression and breast epithelial cells. In contrast, Cdk4 and Cdk6 were interaction with pRB, together with the observed predominantly responsible for cyclin D1-associated sequence homology suggest that cyclins D1 and D2 kinase activity as previously reported. Thus, although have similar functional properties. cyclins D1 and D2 elicited similar eects on breast In spite of these similarities in function, human epithelial cell cycle progression they appeared to achieve cyclin D1 and D2 are more closely related to the this end via activation of dierent CDKs. This is the ®rst corresponding murine cyclins than to each other evidence of cyclin D2 activating Cdk2 in mammalian (490% amino acid identity, compared with 63%). cells thus providing further evidence that D-type cyclins Across a range of human diploid cells and tumor cell are not necessarily redundant. lines of breast, colon, lymphoid and sarcoma origin, cyclin D2 expression was restricted compared to cyclin Keywords: cyclin D2; cyclin D1; breast cancer; CDK D1 (Buckley et al., 1993; Palmero et al., 1993; Lukas et al., 1995b). Cyclin D1 was expressed in most of the cell types examined with the exception of T lymphocytes, which only expressed cyclin D2. Osteosarcomas and Introduction normal breast epithelial cells expressed both cyclins D1 and D2 (Buckley et al., 1993; Lukas et al., 1995b). Cyclins D1 and D2 are closely related proteins that Where both cyclins are expressed they appear to act control cell cycle progression during G1 phase. Induced cooperatively in controlling G1 progression (Bartkova expression of ectopic cyclin D1 in rodent ®broblasts or et al., 1995). This raises the possibility that their human epithelial cells accelerates transit through G1 functions are not wholly redundant. phase, decreases cell size and reduces dependence on Some studies have pointed to functional dierences serum (Jiang et al., 1993; Quelle et al., 1993; Musgrove between cyclins D1 and D2 e.g. overexpression of et al., 1994; Resnitzky et al., 1994; Zwijsen et al., 1996). cyclin D2, but not cyclin D1, in 32D myeloid cells disables the dierentiation pathway (Kato and Sherr, Correspondence: E Musgrove 1993). A basis for functional dierences is suggested by Received 10 September 1996; revised 5 November 1996; accepted 11 dierences in the ability of cyclins D1 and D2 to November 1996 interact with other cellular proteins including CDKs. Cyclin D2 in breast cancer cells KJ Sweeney et al 1330 Although cyclins D1 and D2 bind Cdk2, Cdk4 and Cdk6 in mammalian cells (Matsushime et al., 1992; a Xiong et al., 1992b; Bates et al., 1994a; Meyerson and Harlow, 1994; Lukas et al., 1995b), the cyclin D1-Cdk2 complex appears to be inactive (Dulic et al., 1993; Higashi et al., 1996). While cyclin D1 or cyclin D2 immunoprecipitates phosphorylate pRB in vitro,a number of laboratories have been unable to demon- strate cyclin D1-associated kinase activity using histone H1, the preferred in vitro substrate for Cdk2 (Matsushime et al., 1991, 1994; Ewen et al., 1993; Quelle et al., 1993). Consistent with these observations, cyclin D1 activates Cdk4 and Cdk6, but not Cdk2, when coexpressed in insect cells whereas cyclin D2 activates all three kinases in this system (Matsushime et al., 1992, 1994; Ewen et al., 1993; Meyerson and Harlow, 1994). Although both cyclin D1 and D2 are putative oncogenes and are expressed in at least some breast epithelial cells, only cyclin D1 overexpression has been b implicated in the development and progression of Mitogen Stimulated Controls breast cancer. Cyclin D1 is ubiquitously expressed in epithelial cell lines derived from both normal breast Cyclin D1 and breast cancer (Buckley et al., 1993; Lukas et al., Cyclin D2 1994, 1995b; Tam et al., 1994b) and is frequently overexpressed in breast cancer (Buckley et al., 1993; Cyclin E Bartkova et al., 1994; Gillett et al., 1994; Hui et al., Cdk2 1996). In contrast, cyclin D2 is expressed at very low levels or is absent in most breast cancer cell lines Cdk4 (Buckley et al., 1993; Tam et al., 1994b; Lukas et al., Cdk6 1995b). Cyclin D2 mRNA and protein are abundant in ¨ ppRB some normal breast epithelial cells but very little cyclin ¨ pRB D2 protein was detected in cultured breast luminal 36 91216202461224 epithelial cells, the cell type from which most breast Time (h) cancers evolve (Lukas et al., 1995b). Cyclin D1 is an important cell cycle regulatory gene c in human breast epithelial cells where it is both necessary and sucient for G1 progression and is intimately involved in the mitogenic response to growth factors and steroids (Sweeney et al., 1996). Furthermore, cyclin D1 is necessary for normal mammary development (Fantl et al., 1995; Sicinski et al., 1995). In contrast, the role of cyclin D2 in these cells remains to be established since there are relatively few published data addressing the functional signifi- cance of cyclin D2 and these studies are mostly con®ned to rodent ®broblasts and haematopoietic cells (Ajchenbaum et al., 1993; Ando et al., 1993; Kato and Sherr, 1993; Quelle et al., 1993; Lukas et al., 1995b). Consequently, to address the potential sig- ni®cance of the dierential expression of cyclins D1 and D2 in normal and neoplastic breast epithelial cells we compared the regulation and function of these cyclins in normal breast epithelial cells expressing both cyclins and in T-47D human breast cancer cells Figure 1 Eects of mitogen stimulation on growth arrested 184 normal breast epithelial cells. 184 breast epithelial cells were expressing ectopic cyclin D2. growth arrested in growth factor-depleted medium then stimulated at 0 h with 0.4% bovine pituitary extract. Replicate ¯asks were harvested at intervals for analysis. (a) Flow cytometric analysis of DNA content. G1 phase (&); S-phase (&); G2/M Results phase (*). (b) Western blot analysis of 184 breast cell lysates following mitogen stimulation. Control cells received fresh growth Cyclin D2 and cyclin D1 are dierentially regulated factor-depleted medium containing only 0.05% bovine pituitary extract. Replicate ®lters were blotted with antibodies speci®c for following growth factor stimulation of normal breast the proteins indicated. (c) Densitometric values from autoradio- epithelial cells graphs of two independent Western blot experiments were obtained and the average fold increase above the average of To determine whether cyclins D1 and D2 were co- controls maintained in growth factor-depleted medium is ordinately regulated during cell cycle progression, the presented. Cyclin D1 (&); cyclin D2 (&); cyclin E (~) Cyclin D2 in breast cancer cells KJ Sweeney et al 1331 a (*22%) (Figure 1a).