Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization

Abstract

Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.

Figure 1

Expression of p19^ARF in early-passage, primary MEF strains. MEFs of the indicated genotypes (left) propagated on a 3T9 protocol were harvested at passage numbers given at the top, lysed, and immunoblotted for p19^ARF protein expression. Equal quantities of protein (200 μg) were loaded per lane.

Figure 2

Expression of ARF, p53, and p53 targets in virus-infected MEFs. (A) Wild-type (WT), ARF-null, or p53-null MEFs (top) were infected with either a control (CD8) or myc-expressing retrovirus. At 48 hr postinfection, total RNA was isolated from infected cells, electrophoretically separated, blotted to filters, and hybridized sequentially with ³²P-labeled probes specific for ARF (exon 1β), INK4a (exon 1α), p53, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Replicate cultures infected with CD8 or myc viruses, or infected with ras or E2F-1 vectors (top) were lysed 48 hr postinfection and immunoblotted using antibodies directed to the proteins indicated at left. Wild-type cells infected with the E2F-1 virus died and could not be analyzed (see text). Because a smaller fraction of cells from other Myc-infected and E2F-1-infected cultures underwent apoptosis, equal quantities of protein were loaded per lane to provide valid comparisons.

Figure 2

Expression of ARF, p53, and p53 targets in virus-infected MEFs. (A) Wild-type (WT), ARF-null, or p53-null MEFs (top) were infected with either a control (CD8) or myc-expressing retrovirus. At 48 hr postinfection, total RNA was isolated from infected cells, electrophoretically separated, blotted to filters, and hybridized sequentially with ³²P-labeled probes specific for ARF (exon 1β), INK4a (exon 1α), p53, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Replicate cultures infected with CD8 or myc viruses, or infected with ras or E2F-1 vectors (top) were lysed 48 hr postinfection and immunoblotted using antibodies directed to the proteins indicated at left. Wild-type cells infected with the E2F-1 virus died and could not be analyzed (see text). Because a smaller fraction of cells from other Myc-infected and E2F-1-infected cultures underwent apoptosis, equal quantities of protein were loaded per lane to provide valid comparisons.

Figure 3

Induction of ARF, p53, mdm2, and p21^Cip1 by Myc–ER. MEFs of the indicated genotypes (top) infected with a myc–ER virus were treated with 4-HT for the indicated intervals (hr), and cell lysates were immunoblotted with antibodies directed to the proteins indicated at left. Levels of Myc–ER expressed in the three cell types were comparable (data not shown).

Figure 4

Myc-induced apoptosis. (A) MEFs of the indicated genotypes infected with myc or CD8 virus for 48 hr (the same populations as in Fig. 2) were cultured for 2 more days and then transferred into serum-free medium for an additional 48 hr. Apoptosis was scored using a propidium iodide-based FACS assay to quantitate cells with subdiploid DNA content 24 and 48 hr after serum starvation. Viruses and times of infection are indicated (top right). All standard deviations were within 10% of the means shown. (B) Cells of the indicated genotypes infected with myc–ER virus and pretreated with 4-HT for 24 hr (the same populations as in Fig. 3) were shifted into serum-free medium (solid symbols), and apoptosis was scored by propidium iodide FACs assay at the indicated times (abscissa). Untreated, viable cells were also shifted into serum-free medium containing 4-HT and scored 24 hr later (open symbols).

Figure 5

Rates of proliferation of virus-infected MEFs. Wild-type (A), ARF-null (B), and p53-null (C) MEFs infected with control CD8 virus were transferred to serum-containing (•) or defined serum-free (○) media 4 days postinfection and counted every day thereafter. Wild-type cells infected with myc virus grew more slowly in serum-containing medium (A, ▪) and died in medium lacking serum (A, ). A significant number of myc-infected ARF-null and p53-null cells survived in serum-free conditions (B, C, ). When reseeded 14 days postinfection, these myc-infected cells grew continuously in serum-free medium (B,C, ▵). All data points represent averages of six to eight determinations using at least three independently derived MEF strains with s.d. less than ±25% of the mean (highly significant on log scale).

Figure 6

Myc-“immortalized” MEFs lose p53 or ARF function. (A) MEFs of the indicated genotype were infected with CD8 or myc retroviruses at passage 5 after explantation and propagated on a 3T9 protocol. Wild-type cells tested 7–10 days after myc virus infection (lanes 2,3) expressed relatively high levels of p19^ARF and wild-type (wt) p53, and were initially sensitive to apoptosis (APO +) when transferred into serum-free medium (see text). However, by 14–21 days postinfection, rapidly growing derivatives were isolated that could grow under serum-free conditions (APO −) and expressed mutant (mut) p53 (lanes 4,5). ARF-null cells infected at passage 5 and transferred 14 days after selection in serum-free medium were resistant to apoptosis but expressed only wild-type p53 (wt) (lanes 7,8). Note that Myc protein levels were significantly higher in ARF-null (lanes 7,8) and p53-null (lane 9) cells than in wild-type MEFs (lanes 2–5). Apoptosis was determined by FACS analysis of propidium iodide- and Hoescht 33342-stained cells. (B) Cells containing a single wild-type ARF allele were infected with myc virus for 4 days and transferred into serum-free medium for 2 days to select for variants resistant to apoptosis. Surviving cells were diluted in microtiter wells and subclones were expanded from single cells in serum-containing medium. Lysates were then blotted for p19^ARF and p53. Results with 13 clones (designated A–M) are compared with those obtained with wild-type (wt) uninfected MEFs.

Figure 7

Model for ARF signaling. ARF is activated via Myc and E2F-1 and acts in turn to trigger p53-dependent cell cycle arrest or apoptosis, depending on the presence of extracellular survival factors. Ras acts through cyclin D-dependent kinases to stimulate pRB phosphorylation, resulting in release of E2F from pRb constraint and activation of E2F-responsive genes. Activation of ARF by MYC and E2F-1 need not be direct, although both transcription factors have been demonstrated to increase ARF mRNA levels (see text). Like Myc, different E2F isoforms are proposed to regulate both cell growth and cell death. In inhibiting cyclin D-dependent kinases, p16^INK4a can modulate certain growth-promoting functions of Ras. Other functions of Myc and Ras are not detailed in the schematic.