Anchorage-Dependent Transcription of the Cyclin A Gene

Abstract

NIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G 0 -specific E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27 KIP1 , is stabilized. Ectopic expression of p27 KIP1 blocks cyclin A promoter activity through its E2F binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27 KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.

Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells

“…In human ®broblasts, detachment causes upregulation of the cell cycle inhibitors p21 and p27 and the inhibitors arrest the cell cycle at the G1/S transition by inactivating the cyclin E-cdk2 complex . An increase in the levels of either or both of the inhibitors, accompanied by inactivation of cyclin Dcdk4 or cyclin E/A-cdk2 complexes, has also been observed in other cell types such as NRK, NIH3T3, and Rat1 ®broblasts (Zhu et al, 1996;Schulze et al, 1996). Similar cell cycle inhibition is also seen in smooth muscle cells growth arrested upon loss of anchorage, or as a result of growing on an inhibitory extracellular matrix (Koyama et al, 1996), or disruption of the actin cytoskeleton in normal human ®broblasts .…”

Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

“…When deprived of anchorage, normally adherent cells fail to undergo several G1-speci®c cell cycle-related transitions such as the activation of G1 cdks, the phosphorylation of Rb or the expression of cyclin A (Carstens et al, 1996;Fang et al, 1996;Guadagno et al, 1993;Kang and Krauss, 1996;Schulze et al, 1996;Yang and Cerione, 1997;Zhu et al, 1996). However, many cell type-speci®c di erences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent.…”

“…Several recent reports suggest that the phosphorylation of Rb-related proteins might be dependent upon cell anchorage (Zhu et al, 1996;Kang and Krauss, 1996;Schulze et al, 1996). However, whereas this was observed in NIH3T3 cells, where it can be explained by a strong detachment-induced down regulation of cyclin D1, and therefore of cdk4-cyclin D1 activity, this was not true for example for NRK cells (Kang and Krauss, 1996).…”

“…Many cells, when cultured in suspension, fail to express cyclin A and, as a consequence, do not enter S phase (Guadagno et al, 1993;Zhu et al, 1996;Schulze et al, 1996), a situation which can be overcome by an ectopic expression of cyclin A (Guadagno et al, 1993;Kang and Krauss, 1996). In adherent cells, transcription of the gene coding for cyclin A is cell cycleregulated and the absence of the transcript in quiescent or in early G1 cells results in a large part from the presence of an inhibitory complex on its promoter (Barlat et al, 1993(Barlat et al, , 1995Henglein et al, 1994;Schulze et al, 1995;Zwicker et al, 1995;Huet et al, 1996;Plet et al, 1997;Philips et al, 1998).…”

“…The study of the mechanisms that give rise to the downregulation of cyclin A transcription when normally adherent cells are denied anchorage have also led to con¯icting results. Whereas one group reported the involvement of anchoragedependent E2F-binding to the cyclin A promoter (Schulze et al, 1996), other reports proposed that a new type of CAAT-binding factor might mediate adhesion-dependent cyclin A transcription (Kramer et al, , 1997. There are several reasons for this situation and one of them resides in the fact that most of these studies have been conducted on immortal or transformed cell lines.…”