Here we describe a pathway involving the E2F1-specific induction of Chk2
expression that links loss of proliferation control to an apoptotic pathway with some similarity to the apoptosis pathway induced by DNA double-strand breaks. Unlike the DNA damage signals that activate p53 in the absence of de novo gene expression (39
), E2F1-mediated apoptosis requires induction of Chk2
expression and possibly other components of apoptosis signaling (62
) to fully activate p53 and kill cells. The results presented here suggest that deregulated E2F1 function induces apoptosis by activation of an Atm/Nbs1/Chk2/p53 pathway following disruptions in the Rb/E2F proliferation pathway (Fig. ).
Model of p53 activation following deregulation of E2F1.
The search for E2F target genes has been complicated by the fact that E2F family members can regulate gene expression by transcriptional activation, promoter repression, and derepression of promoters (27
). In addition to regulating genes involved in cell cycle regulation, DNA replication, and chromatin remodeling, E2F family members are also found at the promoters of genes involved in DNA repair and checkpoint activation (71
). Our finding that E2F1 specifically induces expression of the gene encoding the human checkpoint kinase, Chk2, as a requirement for apoptosis ascribes biological function to the data emerging from E2F gene expression profiles (38
), although Chk2
induction by E2F was not tested in these studies. While Chk2
may not be a direct transcriptional target of E2F1, our data confirm that Chk2
is specifically regulated by E2F1. We speculate that the induction of Chk2
expression by E2F1 in vivo sensitizes cells to undergo apoptosis in the event of DNA damage or loss of proliferation control due to Rb mutation or inactivation. This induction of Chk2
by E2F1 is not a direct consequence of promoting the G1
-to-S-phase transition since both E2F1 and E2F2 are adept at inducing S phase. Similarly, Chk2
induction is not a result of simply increasing cellular E2F transcriptional activity and thus upregulating many E2F responsive genes involved in apoptosis signaling, like Apaf1, caspase 3, and caspase 7 (60
). In fact, we found that neither E2F1 nor E2F2 induces Atm
mRNA in human fibroblasts, which could, in principle, be a simple way to lower the activation threshold of this signaling pathway.
The finding that Atm and Nbs1 are required for apoptosis associated with deregulated E2F1 is similar to the requirement for p53 activation following DNA damage from gamma-irradiation and certain genotoxic agents (9
). How Atm is activated following expression of E2F1, E2F2, or HPV-16 E7 remains unclear. We speculate that expression of E2F proteins or E7 results in chromatin changes associated with induction of S phase or activation of DNA damage response proteins. In the case of HPV-16 E7, activation of Atm may be a result of chromosomal structural changes and DNA breaks that occur following E7 expression (21
). However, since activation of Atm by E2F1 results in apoptosis, we cannot rule out the possibility that the different E2F family members activate Atm by distinct mechanisms.
Activation of Atm can result in phosphorylation of Chk2 at threonine 68 (11
), and this Chk2 modification requires functional Nbs1 (13
). However, the phospho-threonine 68 modification of Chk2 may not be a reliable marker of Chk2 activation due to the complexity of Chk2 regulation and the importance of individual phosphorylation events on Chk2 activation status (3
). Instead, we examined phosphorylation of the serine 20 residue on p53 as a reliable marker for Chk2 activation. While we observed an increase in total p53 levels in normal, AT, and NBS cells, only in normal cells did we observe an increase in the phospho-serine 20 form of p53, a substrate for active Chk2 kinase. Inhibition of Chk2 activity by a dominant negative construct or by siRNA targeting resulted in a failure to phosphorylate the serine 20 residue following expression of either E2F1 or E7, demonstrating the specificity of this modification by Chk2.
Interestingly, Atm was found not to be required for apoptosis resulting from Rb inactivation in murine brain choroid plexus epithelium (50
). While it is not apparent why this apoptosis is Atm independent in the choroid plexus epithelium, p53-dependent apoptosis that is Atm independent has been described in certain cell types (7
). Alternatively, these observations suggest that there may be a species-specific bias for Atm in the E2F1-mediated apoptosis pathway. Indeed, the reduction in apoptosis observed in atm−/−
MEFs is not as dramatic as that seen in human dermal fibroblasts following E2F1 expression. We speculate that in the murine system other signaling pathways such as Atr/Chk1 may compensate for the loss of Atm, whereas a more stringent requirement for Atm in human dermal fibroblasts is observed. Therefore, there may be both cell-type and species-specific requirements for Atm in apoptosis induction, and it is possible that other Atm-related kinases may compensate for the loss of Atm function in some cells.
Although we have defined a pathway linking deregulated E2F1 activity to p53 and apoptosis, integration of E2F1 signaling and activation of the Atm/Chk2/p53 pathway also offer a mechanism for the proposed involvement of E2F1 in apoptosis resulting from DNA damage. Following treatment of cells with DNA-damaging agents, E2F1 protein accumulates (10
) and is phosphorylated at an N-terminal Atm recognition sequence that is unique to E2F1 among the E2F family members (52
). This phosphorylation of E2F1 is largely dependent on Atm and is required for efficient E2F1 stabilization following DNA damage (52
). Chk2 has also been shown to phosphorylate and stabilize E2F1 following DNA damage, and this modification has been shown to be required for E2F1-dependent apoptosis following DNA damage by altering its promoter specificity (81
). Additionally, DNA damage-induced apoptosis is compromised in thymocytes from E2F1−/−
), suggesting that E2F1 has multiple roles in DNA damage signaling. We speculate that activation of E2F1 by DNA damage leads to increased p14ARF
expression, resulting in increased pools of p53 protein. E2F1 is also able to activate Atm kinase activity and induce Chk2
expression, leading to increased p53 activation and E2F1 activity. E2F1 activation following DNA damage would therefore act to amplify DNA damage signals converging at p53 to result in apoptosis.