The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing

Abstract

Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.

MTBP inhibits migration and metastasis of hepatocellular carcinoma

“…Conversely, it is possible that MDM2 inhibits MTBP function, since MDM2 overexpression induces cellular phenotypes similar to those induced by MTBP downregulation. These include increased metastatic potential [8, 26], enhanced chromosome instability [22, 27], and inhibited DNA replication origin firing [23, 28]. It would be critical to determine whether MDM2 antagonizes the MTBP functions or if MDM2 induces these cellular phenotypes independent of MTBP.…”

Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication

“…Generation of plasmids, lentiviruses, and stable transfectants expressing GOF p53 or shRNA against GFP or p53 was carried out using pLKO.1 expression vector purchased from Open Biosystem following the supplier's protocols. Lung cells from mice were generated and cultured following standard protocols (28,62). CCNA2 promoter was a gift from Toshio Nikaido (63).…”

“…Replicating nuclei were detected following methods described earlier (28). Briefly, density-arrested cells were replated on coverslips and labeled with 40 μM IdU for 20 minutes at desired times after replating.…”

“…DNA replication origin firing was determined by DNA fiber spreading analysis following published protocols (28)(29)(30)68). Briefly, cells were pulse labeled sequentially with nucleotide analogues IdU (40 μM) and CIdU (100 μM) to track the replication pattern and directionality of fork movement.…”

“…Since GOF p53 mutants activate expression of genes required for DNA replication (27), the ability of these mutants to increase firing of DNA replication origins during S phase entry was determined. To determine whether GOF p53 mutants alter frequency of DNA replication origin firing in otherwise normal cells, lung cells from p53 -/-and p53R172H-KI mice were cultured and the frequency of origin firing during early S phase was determined using fiber analysis of replicating DNA using methods published earlier (28)(29)(30). Cells were partially synchronized by density arrest and replating and sequentially labeled with IdU and chlorodeoxyuridine (CldU) at early S phase.…”