p16(Ink4a) interferes with Abelson virus transformation by enhancing apoptosis

Abstract

Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19(Arf), a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19(Arf) and a second tumor suppressor, p16(Ink4a), that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16(Ink4a) plays a role in Ab-MLV transformation, primary transformants derived from Arf(-/-) and p16(Ink4a(-/-)) mice were compared. A fraction of those derived from Arf(-/-) animals underwent crisis, and even though all p16(Ink4a(-/-)) primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf(-/-) cells infected with a virus that expresses both v-Abl and p16(Ink4a) revealed that p16(Ink4a) expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16(Ink4a) can suppress transformation by inducing apoptosis.

FIG. 1.

Arf^−/− primary transformants exhibit crisis. Primary transformants derived by infecting bone marrow from Ink4a/Arf^−/− (•), Arf^−/− (⋄), Ink4a/Arf^+/+ (▪), and Arf^+/+ (▴) mice with Ab-MLV were monitored for establishment. The Arf^+/+ animals are wild-type mice from our Arf^−/− breeding colony. Cells were considered established when they displayed <10% apoptotic cells and grew regularly (15, 29). These data represent analyses of at least 70 primary transformants per genotype. The P value represents comparison of the curves obtained for cells from Ink4a/Arf^−/− and Arf^−/− mice using a log-rank test.

FIG. 2.

p16^Ink4a expression correlates with crisis in Arf^−/− transformants. (A) Primary transformants from Arf^−/− bone marrow cells were analyzed during the outgrowth period by Western blotting using anti-p16^Ink4a and anti-β-actin antibodies. Representative samples are shown. The designations above the lanes indicate the presence (+) or absence (−) of crisis. C, L1-2, a fully established Ab-MLV transformant that expresses abundant p16^Ink4a (29). (B) Densitometry was used to compare levels of p16^Ink4a in primary transformants undergoing crisis to that in those that did not display crisis. Each point represents an individual cell line; the P value was obtained by using an unpaired, two-tailed t test.

FIG. 3.

p16^Ink4a−/− colonies become established more readily than +/+ colonies. (A) Primary transformants from p16^Ink4a−/− (□) and +/+ littermates (○) were monitored for establishment. The data shown represent analysis of 213 and 123 primary transformants from −/− and +/+ mice, respectively. The boxed area identifies transformants that became established within the first 40 days and is illustrated in an enlarged form in panel B. The P values represent comparison of each of the curves using a log-rank test.

FIG. 4.

p19^Arf is expressed normally in p16^Ink4a null transformants. Lysates were prepared from different cell populations and analyzed by Western blotting using anti-p19^Arf and anti-β-actin antibodies. (A) Analysis of lysates from p16^Ink4a−/− and a control p16^Ink4a+/+ primary transformant prepared 8 days post-explant from agar, near the onset of crisis. (B) Analysis of lysates from p16^Ink4a−/− and p16^Ink4a+/+ transformants prepared at the end of the crisis period. In both panels, an established cell line expressing abundant p19^Arf (+) and an established cell line from a p19^Arf null animal (−) are included as controls. The experiments shown are representative of analyses of 14 null and 6 wild-type transformants at crisis onset and 17 null and 15 wild-type transformants at the end of crisis. α-p19^Arf, anti-p19^Arf antibody; α-β-actin, anti-β-actin antibody.

FIG. 5.

p16^Ink4a-v-Abl transforms cells. Ink4a/Arf^−/− bone marrow was infected with a virus expressing either v-Abl alone or both p16^Ink4a and v-Abl and plated in liquid medium. Dishes were scored as transformed when they contained more than 2 × 10⁶ pre-B cells per ml. (A) The day of transformation is indicated for p16^Ink4a-v-Abl- and v-Abl-transformed cells. The asterisk indicates the average day of transformation. All 18 cultures infected with the v-Abl virus and 17 of 18 cultures infected with the p16^Ink4a-v-Abl virus became transformed. (B) Lysates of primary transformants were analyzed by Western blotting with anti-p16^Ink4a, anti-Gag/v-Abl (39), and anti-β-actin antibodies. The experiment shown is representative of experiments in which a total of 17 v-Abl-infected transformants and 18 v-Abl-p16-infected transformants were tested.

FIG. 6.

Expression of p16^Ink4a in primary transformants leads to apoptosis. (A) Ink4a/Arf^−/− cells infected with p16^Ink4a-v-Abl (open bar) or v-Abl (filled bar) were harvested 24 to 72 h after transformation, stained with propidium iodide, and analyzed by flow cytometry to determine the percentage of cells in the G₁, G₂/M, and S phases of the cell cycle. The data are averages obtained from analyses of 18 independent cultures infected with v-Abl and 17 independent cultures infected with v-Abl-p16; the error bars represent standard deviations. (B) The percentage of apoptotic cells in the cultures analyzed in panel A and cultures transformed with either v-Abl or v-Abl-p16 derived from p53^−/− mice were stained with propidium iodide and analyzed for the frequency of apoptotic cells by flow cytometry. Each point represents an individual clone derived from one of three independent experiments. The asterisk indicates the mean; the P values were obtained by using an unpaired, two-tailed t test.