p53 mediates apoptotic crisis in primary Abelson virus-transformed pre-B cells - PubMed (original) (raw)

p53 mediates apoptotic crisis in primary Abelson virus-transformed pre-B cells

I Unnikrishnan et al. Mol Cell Biol. 1999 Jul.

Abstract

Transformation of pre-B cells by Abelson murine leukemia virus (Ab-MLV) involves a balance between positive, growth-stimulatory signals from the v-Abl oncoprotein and negative regulatory cues from cellular genes. This phenomenon is reflected by the clonal selection that occurs during Ab-MLV-mediated transformation in vivo and in vitro. About 50% of all Ab-MLV-transformed pre-B cells express mutant forms of p53 as they emerge from this process, suggesting that this protein may play an important role in the transformation process. Consistent with this idea, expression of p19(Arf), a protein whose function depends on the presence of a functional p53, is required for the apoptotic crisis that characterizes primary Ab-MLV transformants. To test the role of p53 in pre-B-cell transformation directly, we examined the response of Trp53(-/-) mice to Ab-MLV. The absence of p53 shortens the latency of Abelson disease induction but does not affect the frequency of cells susceptible to Ab-MLV-induced transformation. However, primary transformants derived from the null animals bypass the apoptotic crisis that characterizes the transition from primary transformant to fully malignant cell line. These effects do not require p21(Cip-1), a major downstream target of p53; however, consistent with a role of p19(Arf), transformants expressing mutant p53 and abundant p19 retain wild-type p19 sequences.

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Figures

FIG. 1

FIG. 1

Accelerated tumor induction in _Trp53_−/− mice. Age-matched _Trp53_−/− (○), Trp53+/− (□), and Trp53+/+ (●) mice were injected with Ab-MLV-P160 and monitored for tumor development. Animals were sacrificed when tumors were evident; each point represents a single mouse.

FIG. 2

FIG. 2

Trp53+/− transformants lose their remaining Trp53 allele rapidly. (A) DNAs from representative Trp53+/− transformants were amplified with primers specific for the wild-type and targeted alleles, and products were fractionated through an agarose gel containing ethidium bromide. The numbers above each lane identify the cell clone from which the sample was derived. DNAs from Trp53+/− and Trp53+/+ mice and a reaction mix containing no DNA were used as controls. The unmarked lane contains a 100-bp DNA ladder, used as a marker. Arrows denote positions of the wild-type (Wt) and mutant (Mut) specific PCR products. (B) Lysates from the cell lines analyzed in panel A and control cell lines were immunoprecipitated with anti-p53 antibody Ab-4, specific for wild-type p53 (lanes W), or anti-p53 antibody Ab-3, specific for mutant forms of p53, (lanes M) and the immunoprecipitates were analyzed by Western blotting with anti-p53 antibody Ab-7, which recognizes both mutant and wild-type p53 forms on Western blots. The p53 statuses of the control wild-type (204-3-1), mutant (143-2M), and null (L1-2) cell lines were characterized previously (47).

FIG. 3

FIG. 3

Trp53 is required for crisis induction. Primary transformants from Trp53+/+ (●) and _Trp53_−/− (□) mice were assessed for viability by using trypan blue staining when they were removed from agar cultures and at regular intervals thereafter.

FIG. 4

FIG. 4

Trp53+/+ transformants undergo apoptotic crisis during outgrowth. (A) Three independent Trp53+/+ and _Trp53_−/− transformants were stained with MC540 (31) and analyzed by flow cytometry. The percentages of apoptotic cells, represented by black peaks, are noted. The data shown are representative of analyses of more than 10 additional independent transformants from each background. (B) DNA was prepared as described in Materials and Methods from representative Trp53+/+ and _Trp53_−/− transformants and fractionated through an agarose gel containing ethidium bromide. The data shown are representative of analyses of 10 independent transformants from each background. Lane M, 100-bp ladder marker.

FIG. 4

FIG. 4

Trp53+/+ transformants undergo apoptotic crisis during outgrowth. (A) Three independent Trp53+/+ and _Trp53_−/− transformants were stained with MC540 (31) and analyzed by flow cytometry. The percentages of apoptotic cells, represented by black peaks, are noted. The data shown are representative of analyses of more than 10 additional independent transformants from each background. (B) DNA was prepared as described in Materials and Methods from representative Trp53+/+ and _Trp53_−/− transformants and fractionated through an agarose gel containing ethidium bromide. The data shown are representative of analyses of 10 independent transformants from each background. Lane M, 100-bp ladder marker.

FIG. 5

FIG. 5

_p21Cip-1_−/− transformants undergo crisis. Growth and viability of primary transformants from _p21Cip-1_−/− mice were monitored as described in Materials and Methods. The times at which primary transformants succumbed to crisis (●) and at which cell lines that survived crisis and became established (○) are indicated.

FIG. 6

FIG. 6

p19Arf is expressed in transformants from Trp53 null mice. Lysates were prepared from transformants from Trp53 null animals or wild-type (WT) mice and examined by Western blotting for the presence of the p19Arf protein. The cells used were derived with either the P120, P90, or P80 strains of Ab-MLV (24). The transformation properties of these viruses are similar in wild-type and Trp53 null mice (our unpublished data). Lysates from NIH 3T3 (p19Arf-negative) cells and from the p19Arf-positive cell line MEL (29) were used as controls. The blots were also probed with the H548 anti-Gag/v-Abl monoclonal antibody (7) to control for protein loading.

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