Oncogene (1997) 15, 1295 ± 1302 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Genomic instability and apoptosis are frequent in p53 de®cient young mice Kenji Fukasawa1,2, Francis Wiener3,4, George F Vande Woude1 and Sabine Mai4 1ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702-1201, USA; 2Department of Cell Biology, Neurobiology & Anatomy, University of Cincinnati College of Medicine, P.O. Box 670521, Cincinnati, OH, USA; 3Microbiology and Tumor Biology Center, Karolinska Institute, 17177 Stockholm, Sweden; 4Manitoba Institute of Cell Biology, Manitoba Cancer Treatment and Research Foundation, and Department of Physiology, University of Manitoba, 100 Olivia Street, Winnipeg, MB, R3E 0V9, Canada The loss of p53 tumor suppressor functions results in at the G1/S (reviewed in Hartwell, 1992; Weinert and genetic instability, characteristically associated with Lydall, 1993; Hartwell and Kastan, 1994; Kastan et al., changes in chromosome ploidy and gene ampli®cation. 1995) and G2/M transitions (Stewart et al., 1995; Cross In vivo, we ®nd that cells from various organs of 4 to 6- et al., 1995; Guillouf et al., 1995; Orren et al., 1995; week old p53-nullizygous (p537/7) mice display aneu- Wahl et al., 1996) of the cell cycle. Thus, the absence of ploidy and frequent gene ampli®cation as well as p53 results in loss of checkpoint control, leading to evidence for apoptosis. Regardless of tissue types, many genome destabilization. p537/7 cells contain multiple centrosomes and abnor- p53 has recently been shown to be involved in the mally formed mitotic spindles. Thus, chromosome regulation of the centrosome duplication cycle; in instability in vivo may be associated with abnormal embryonic ®broblasts derived from p53-nullizygous centrosome ampli®cation. Moreover, we observed a mice (p537/7 mice), abnormal ampli®cation of signi®cant increase in the number of cells overexpressing centrosomes is frequently observed (Fukasawa et al., c-Myc in p537/7 mice. Consistent with previous studies 1996). During mitosis, centrosomes become spindle showing that c-Myc overexpression is associated with poles and play a vital role in bipolar spindle gene ampli®cation in vitro, many of the p537/7 cells formation and accurate chromosomal segregation. In exhibited, in the same cell, c-Myc overexpression and the presence of an abnormal number of centrosomes, ampli®ed c-myc, dihydrofolate reductase (DHFR), and chromosome instability occurs frequently due to carbamoyl-phosphate synthetase-aspartate transcarba- multipolar mitosis. Consistent with the proposed role moyl-dihydroorotase (CAD) genes. Furthermore, apop- of p53 in genome stability and that of genomic tosis was frequently observed in cells isolated from instability in tumorigenesis, p537/7 mice develop a p537/7 mice. The apoptotic cells contained abnormally variety of tumors early in life (Donehower et al., 1992; ampli®ed centrosomes, displayed aneuploidy, high levels Jacks et al., 1994). of c-Myc expression, as well as gene ampli®cation. These In addition to abnormal centrosome duplication, results indicate that a high number of aberrant cells is recent studies have shown that in the absence of p53, eliminated by p53-independent pathways in vivo. gene ampli®cation occurs at higher frequencies (Yin et al., 1992; Livingstone et al., 1992). Gene ampli®cation Keywords: p53; genomic instability; gene ampli®cation; aects several genomic loci, among them are oncogenes centrosome; Myc; apoptosis (Alitalo, 1985; Yokota et al., 1986; Van der Bliek et al., 1986; Stark, 1993; Feo et al., 1994). Gene ampli®cation is a dynamic process, and is often accompanied by the generation of double minutes (Wahl, 1989). Introduction The c-Myc protooncogene is frequently deregulated in tumors; translocation and ampli®cation of c-myc as The term genomic instability summarizes a variety of well as increased half life and overexpression of the genomic alterations which include the loss or gain of oncoprotein are observed in many tumors (for a chromosomes as well as genetic changes at the level of review, see Marcu et al., 1992; Bishop, 1995). More- single genes, such as rearrangements, translocations, over, c-Myc overexpression has been implicated in gene ampli®cations, deletions and point mutations, and has ampli®cation (Denis et al., 1991; Mai, 1994; Mai et al., been considered to be a major driving force of 1996). multistep carcinogenesis (Nowell, 1976; Pienta et al., Here, we have studied genomic instability in vivo in 1989; Temin, 1988; Solomon et al., 1991). Genomic dierent organs of p537/7 mice (4 ± 6 weeks old), with integrity is maintained by checkpoint mechanisms; age-matched p53 homozygous (p53+/+) mice as when cells suer damage imposed by exposure to controls. We found that, in all p537/7 tissues genotoxic drugs or microtubule toxins, the cell cycle is examined, a substantial percentage of cells contained halted until the damage is repaired or apoptosis is abnormal numbers of centrosomes and displayed initiated (for reviews, see Hartwell, 1992; Weinert and aneuploidy. Moreover, c-Myc overexpression was Lydall, 1993; Hartwell and Kastan, 1994). The p53 observed in 5 ± 15% of p537/7 cells. In these cells, tumor suppressor protein exerts checkpoint functions dihydrofolate reductase (DHFR), carbamoyl-phosphate synthetase-aspartate transcarbamoyl-dihydroorotase (CAD) and c-myc genes exhibited gene ampli®cation. Correspondence: S Mai Apoptosis of cells, which displayed abnormal numbers Received 29 May 1997; revised 4 August 1997; accepted 4 August of centrosomes, aneuploidy, gene ampli®cation and c- 1997 Myc overexpression, was frequently observed. Genomic instability and apoptosis in p537/7 mice K Fukasawa et al 1296 1994), and the number of centrosomes per cell was Results scored. The numerical distribution of centrosomes in various organs of p53+/+ and p537/7 mice is Aneuploidy in p537/7 mice summarized in Figure 2a. More than 99% of the We examined genomic instability in dierent organs of interphase p53+/+ cells contained one or two centro- clinically healthy p537/7 mice (4 ± 6 weeks old) by somes, most likely depending on their duplication cytogenetically assessing chromosome ploidy. Age- cycle, while 20 ± 30% of the interphase p537/7 cells matched parental p53+/+ mice were used as controls. contained 42 centrosomes. A typical example of We characterized spleen-, thymus-, and bone marrow- normal vs aberrant centrosome duplication is shown derived cells, as well as skin- and spleen-derived in Figure 2b. Thus, as previously observed in vitro in ®broblasts. These analyses revealed aneuploidy; hyper- p537/7 MEFs, we found now in vivo that mitotic diploid, hypo-, hypertetraploid, and polyploid meta- p537/7 cells frequently displayed abberant spindles, phase plates were present in all organs examined organized by multiple copies of centrosomes. However, (Figure 1). The percentage of aneuploid mitotic plates as we have reported (Fukasawa et al., 1996) in some varied between individual mice; the mean frequencies mitotic p537/7 cells, abnormally ampli®ed centrosomes were 25.6% in the thymus, 34.8% in ®broblasts, 10% sequestered to the poles to form bipolarity (data not in the bone marrow, and 20% in the spleen (Figure 1). shown). These results imply that abnormal amplifica- No aneuploidy was observed in p53+/+ mice (Figure 1). tion of centrosomes occurs in vivo, and this may lead to Cytogenetic studies also revealed that ®ve percent of chromosome instability in p537/7 mice. all metaphases present in the p537/7 fetal liver hematopoietic cells were aneuploid (Figure 1). In Gene ampli®cation in p537/7 mice contrast, there was no evidence of aneuploidy in age- matched p53+/+ fetal liver hematopoietic cells. To evaluate the genomic (in)stability of single genes in p537/7 mice in vivo, we performed ¯uorescent in situ Abnormal centrosome ampli®cation in vivo in p537/7 mice a p53 has been implicated in the regulation of centrosome duplication, and multiple centrosomes are generated in p537/7 embryonic ®broblasts (MEFs) during a single cell cycle (Fukasawa et al., 1996). In the present work, we tested for the ®rst time whether the chromosome instability observed in p537/7 mice was associated with multiple centrosomes per cell in vivo. Spleen-, thymus-, and bone marrow-derived cells were immunostained with anti-g-tubulin antibody to identify centrosomes (reviewed in Oakley, 1992; Joshi, b Centrosome DNA p53 +/+ P53 –/– Figure 2 Abnormal ampli®cation of centrosomes in p537/7 mice. (a) Number of centrosomes detected by immunostaining in bone marrow, spleen, and thymic cells isolated from p53+/+ and p537/7 mice. N1: one centromere; N2: two centromeres; N53: three or more centromeres (see text). (b) Representative picture of Figure 1 Genomic instability in p537/7 mice. Cytogenetic normal and aberrant centrosome numbers. The picture illustrates analysis of bone marrow, spleen, thymus-derived cells as well as the normal number of centrosomes as found in all organs of of ®broblasts (passage 0) isolated from ®ve p537/7 and ®ve p53+/+ mice (top panel) as well as aberrant numbers of parental p53+/+ (C57B1/6) mice, and of p537/7 and p53+/+ centrosomes as observed in all organs p537/7 mice in vivo fetal liver hematopoietic cells of 16 day old embryos (bottom panel) Genomic instability and apoptosis in p537/7 mice K Fukasawa et al 1297 hybridization (FISH). We chose to analyse the c-myc but not of the R1 gene within the same individual gene, which is frequently translocated and/or ampli®ed p537/7 bone marrow-, thymus-, spleen-derived cells as in tumors (for review, see Marcu et al., 1992; Bishop, in primary ®broblasts (Figure 4, panels a-a'', b-b''). 1995), and the DHFR gene, whose genomic instability Thus, locus-speci®c gene ampli®cation in p537/7 mice is altered following either drug selection (Stark, 1993), appears to be accompanied by c-Myc overexpression. growth factor (Huang and Wright, 1994) or c-Myc overexpression (Denis et al., 1991; Mai, 1994; Mai et Apoptosis of genomically altered cells in p537/7 mice al., 1996).