Dissemination and tissue invasiveness in murine acute leukemia associated with acquisition ofp53 mutation and loss of wild-typep53

Abstract

Approximately 60% of mice treated with split‐dose radiation develop leukemias that disseminate widely through the body, whereas 40% of the treated mice incur leukemias that are contained entirely within the thymus. We studied the status of p53 in non‐cultured samples of thymic leukemias and in cell lines established from these leukemias. In those mice with disseminated disease, primary samples were also obtained from visceral leukemic organs, and cell lines were established from these leukemic organs for further study. Using single‐strand conformation polymorphism (SSCP), nucleic acid sequencing, and immunochemical analysis, we found that mutation of both p53 alleles occurred in leukemic cell lines developed from nine of 10 disseminated leukemias; mutation of one p53 allele with the other remaining wild‐type occurred in one disseminated leukemia. A p53 mutation unique for each mouse was found in all cell lines established from the different leukemic organs of each mouse. The same mutation was also found in the non‐cultured leukemic tissues of each mouse, indicating that the mutations originated in vivo and were clonal. Seven of seven non‐disseminating thymomas possessed wild‐type p53 only. Hence, in vivo dissemination and tissue invasiveness were associated with the loss of wild‐type p53 by mutation of both alleles or by mutation and loss of heterozygosity, as revealed by studies of cell lines established from them. The selective in vivo dissemination of leukemia cells possessing p53 mutations had a parallel in vitro. Leukemia cell lines from mice harboring disseminating leukemia were established more readily (success rate greater than 80%) than lines from mice harboring thymic nondisseminating leukemia (success rate less than 10%). Additionally, while mice with disseminating leukemia harbored a mixture of wild‐type and mutant p53—encoding thymoma cells, only cell lines possessing mutant p53 became established in culture. Mutations found in thymoma cell lines were always detectable by SSCP and sequencing of DNA extracted from non‐cultured thymoma tissue. However, in non‐cultured leukemic tissue of visceral organs, the clonal p53 mutations found in cell lines established from them were often not detectable by SSCP or sequencing but were detectable by immunochemical analysis or polymerase chain reaction amplification. This indicates an unexpected degree of masking of mutant genes by wild‐type genes present in the leukemic tissue. Masking was evident even in leukemic organs that were grossly larger than normal organs. Hence, routine screening of leukemic tissue by SSCP and sequencing may result in a highly significant underestimation of the incidence of p53 mutations. The widely reported discrepancies between the loss of tumor suppressor genes (e.g., p16^INK4) in human tumors compared with the incidence of loss in cell lines established from the tumors may, in part, result from similar detection difficulties.