The absence of Msh2 alters abelson virus pre-B-cell transformation by influencing p53 mutation

Abstract

Defects in DNA mismatch repair predispose cells to the development of several types of malignant disease. The absence of Msh2 or Mlh1, two key molecules that mediate mismatch repair in eukaryotic cells, increases the frequency of mutation and also alters the response of some cells to apoptosis and cell cycle arrest. To understand the way these changes contribute to cancer predisposition, we examined the effects of defective mismatch repair on the multistep process of pre-B-cell transformation by Abelson murine leukemia virus. In this model, primary transformants undergo a prolonged apoptotic crisis followed by the emergence of fully transformed cell lines. The latter event is correlated to a loss of function of the p53 tumor suppressor protein and down-modulation of the p53 regulatory protein p19Arf. Analyses of primary transformants from Msh2 null mice and their wild-type littermates revealed that both types of cells undergo crisis. However, primary transformants from Msh2 null animals recover with accelerated kinetics, a phenomenon that is strongly correlated to the appearance of cells that have lost p53 function. Analysis of the kinetics with which p53 function is lost revealed that this change provides the dominant stimulus for emergence from crisis. Therefore, the absence of mismatch repair alters the molecular mechanisms involved in transformation by affecting a gene that controls apoptosis and cell cycle progression, rather than by affecting these processes directly.

FIG. 1

Some Msh2 null transformants do not express p53. Established transformants from Msh2 null animals and wild-type mice were treated with approximately 1,000 rads of gamma irradiation, and lysates were prepared from these cells (+) and mock-treated cells (−) 4 h later. The proteins were examined by using Western blotting. The blots were cut and one portion was probed with the anti-p53 monoclonal antibody Ab-3 (Oncogene Research Products). The other portion of the blot was probed with the anti-Gag/v-Abl monoclonal antibody H548 (8) to control for protein loading. Null, lysate from L1-2, a p53-negative pre-B-cell line transformed with the Ab-MLV-P120 strain (56); WT, lysate from 38B9, a pre-B-cell line transformed with Ab-MLV-P160 that expresses wild-type p53 (51). Cell lines 10-18, 10-14, and 10-11 express mutant p53; these cell lines and 10-31, 10-2, and the null control do not undergo rapid apoptosis following high-dose gamma irradiation. The WT control, 10-5, and 10-12 cell lines express wild-type p53 and undergo rapid apoptosis following high-dose gamma irradiation (data not shown).

FIG. 2

p53 mutations found in Msh2 null transformants. p53 sequences were amplified from total cellular RNA isolated from five independently derived Msh2 null transformants, and the cDNA was cloned and sequenced. The effect of each mutation is illustrated and reflects the following changes: A135V, GCG to GTG; R246Q, CGA to CAA; frameshift 298, deletion of a C at 1046; frameshift 314, insertion of a C at 1093; and frameshift 379, deletion of A at 1289. Conserved regions of the protein are shaded.

FIG. 3

Primary transformants from Msh2 null mice are established at a high frequency. Primary transformants from Msh2^−/− (open symbols) and wild-type (filled symbols) mice were plated in liquid medium, and their ability to develop into established transformants was monitored. A transformant is considered established when levels of apoptosis are less than 10% and the cells can be subcultured at regular intervals (41, 54). Each point represents the frequency of primary transformants that were established at the day shown. Each line represents data from an independent experiment; a total of 180 transformants from Msh2 null animals and 170 transformants from wild-type animals were examined.

FIG. 4

p19Arf expression is similar in primary transformants from Msh2 null and wild-type mice. Primary transformants from Msh2 null and wild-type mice were explanted from agar, and populations were expanded in liquid culture. Lysates were prepared from representative cells 4 and 8 days postexplantation and analyzed by Western blotting with anti-p19Arf antibodies (Novus Biological). The blot was reprobed with anti-cdk4 antibody to control for protein loading. C, controls with lysates from the Ink4a/Arf null cell line IA-17 (−) and the p19Arf-positive cell line L1-2 (+) (41).

FIG. 5

Primary transformants from Msh2 null and wild-type backgrounds undergo crisis. Primary transformants were plated in liquid medium, and populations were expanded. The plots shown are representative of an established cell line from the Msh2^−/− background (A), an established cell line from the Msh2^+/+ background (B), an Msh2^−/− transformant (18-6) emerging from crisis at 14 days postexplantation (C), an Msh2^−/− transformant (18-46) in crisis at day 14 (D), and an Msh2^+/+ transformant (18-137) in crisis at day 14 (E). Additional cell cycle information can be found in Table 2.

FIG. 6

Loss of p53 function correlates with increased growth and viability. Primary transformants were explanted from agar, and populations were expanded. After several weeks, the cells were counted and 10⁶ viable cells were seeded into 35-mm dishes. The cells were counted daily after staining with trypan blue, and viability was assessed. When the population had doubled, the cells were reseeded at 10⁶ cells per ml. The percentage of viable cells was also calculated. At regular intervals, the frequency of cells lacking functional p53 was assessed by using gamma irradiation; the percentage of cells expressing mutant p53 is indicated in the boxes. The data shown are representative of analyses with seven transformants derived from Msh2 null mice and four transformants derived from wild-type mice.

FIG. 7

Loss of p53 function is strongly selected in primary transformants from both Msh2 null and wild-type backgrounds. Independently derived primary transformants from Msh2 null mice (open circles) and normal mice (filled circles) were expanded and monitored periodically for the appearance of p53 mutations by using a gamma irradiation assay. The fraction of cells resistant to rapid apoptosis following gamma irradiation, a feature that strongly correlates with loss of p53 function in Ab-MLV transformants (51), was calculated at each time point.