In addition to its well-established function in controlling cell survival, the Bcl-2 family (1
) has been found to influence the cell cycle. Although Bcl-2 and its prosurvival relatives do not affect the growth rate in proliferating cultures, they can both accelerate withdrawal from the cycle (46
) and retard reentry (4
). Conversely, a shortened G1
is found in lymphocytes that lack Bcl-2 (29
) or express the Bcl-2 antagonist Bax (4
). Thus, Bcl-2 appears to have a physiological role in influencing the transition between the quiescent and cycling states. That this ability is separate from its role in cell survival (46
) is most clearly shown by mutations of Bcl-2 that eliminate its cell cycle activity but spare its antiapoptotic function (17
The cell cycle control function of Bcl-2 has ramifications for cellular homeostasis. Cycling cells are often more vulnerable to apoptosis, perhaps because, under conditions unfavorable for proliferation, certain cell cycle effectors promote apoptosis (11
). Hence, promoting quiescence under conditions of stress may provide Bcl-2 with an additional, albeit indirect, means to enhance cell survival (30
). Interference with Bcl-2's cell cycle effect may also augment its oncogenic role (see Discussion).
Progression through the cell cycle requires the action of cyclin-dependent kinases (Cdks) (38
). As cells enter the cycle, newly synthesized D-type cyclins associate with and activate their Cdk-4/6 catalytic partners in mid- to late G1
phase, while cyclin E appears later in G1
and activates its Cdk-2 kinase subunit near the G1
/S boundary. Opposing their activity are Cdk inhibitors (Cki) of two classes: INK4 proteins, such as p16, specifically inhibit D-cyclin kinases, whereas Cip/Kip proteins, such as p21 and p27, also inhibit Cdk-2 (38
). Other key negative regulators include the best-known Cdk substrates, the retinoblastoma (RB) family of nuclear “pocket” proteins: pRB itself, p130, and p107 (10
). They are thought to act by forming repressive complexes with E2F transcription factors, which control the expression of genes essential for cell cycle progression. Phosphorylation of the pocket protein by Cdks frees the E2F and thereby derepresses or activates E2F target genes.
-S-phase transition is thought to be controlled by pRB, which binds E2Fs 1 to 3. Hence, p107 or p130, both of which bind E2F4 or E2F5 and probably regulate a distinct subset of E2F target genes (18
), might govern the poorly understood exit from G0
. p107 is unlikely, because its expression is restricted to cycling cells and it associates with E2F4 only late in G1
, but p130 is prominent during quiescence, and p130-E2F4 complexes appear almost exclusively during G0
). Hence, regulation of E2F4 activity by p130 has been implicated in control of the G0
transition, but no antiproliferative pathway is yet known to rely on p130.
How Bcl-2 favors the quiescent state is largely unknown. Neither p53 nor p16 can be essential, because Bcl-2 promoted quiescence in cells lacking those genes (35
). In bcl-2
transgenic cells, delayed cycle entry correlated with increased expression of p27 (4
), hypophosphorylated pRB (30
), and increased p130 (28
), but those alterations might simply be an indirect consequence of the increased proportion of noncycling cells.
To establish the negative regulators through which Bcl-2 acts, we have used cells derived from mice deficient in pRB, p130, and p27. We report that pRB is dispensable for the inhibitory effect but that both p27 and p130 are essential, and we present evidence that p130 may act through E2F4 to control the level of E2F1. These findings thus establish the framework through which Bcl-2 impacts the cell cycle. They also identify the first antiproliferative pathway requiring p130 and provide the first functional evidence that p130 plays an essential role in a pathway controlling G0 exit.