Transgenic mouse model for studying the transcriptional activity of the p53 protein: age- and tissue-dependent changes in radiation-induced activation during embryogenesis
1997; Springer Nature; Volume: 16; Issue: 6 Linguagem: Inglês
10.1093/emboj/16.6.1381
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle15 March 1997free access Transgenic mouse model for studying the transcriptional activity of the p53 protein: age- and tissue-dependent changes in radiation-induced activation during embryogenesis Eyal Gottlieb Eyal Gottlieb Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Rebecca Haffner Rebecca Haffner Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Ayala King Ayala King Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Gad Asher Gad Asher Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Peter Gruss Peter Gruss Max Planck Institute of Biophysical Chemistry, POB 2841, D-37018 Gottingen, Germany Search for more papers by this author Peter Lonai Peter Lonai Department of Molecular Genetics,The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Moshe Oren Corresponding Author Moshe Oren Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Eyal Gottlieb Eyal Gottlieb Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Rebecca Haffner Rebecca Haffner Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Ayala King Ayala King Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Gad Asher Gad Asher Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Peter Gruss Peter Gruss Max Planck Institute of Biophysical Chemistry, POB 2841, D-37018 Gottingen, Germany Search for more papers by this author Peter Lonai Peter Lonai Department of Molecular Genetics,The Weizmann Institute of Science, Rehovot, 76100 Israel Search for more papers by this author Moshe Oren Corresponding Author Moshe Oren Department of Molecular Cell Biology, Rehovot, 76100 Israel Search for more papers by this author Author Information Eyal Gottlieb1, Rebecca Haffner1, Ayala King1, Gad Asher1, Peter Gruss2, Peter Lonai3 and Moshe Oren 1 1Department of Molecular Cell Biology, Rehovot, 76100 Israel 2Max Planck Institute of Biophysical Chemistry, POB 2841, D-37018 Gottingen, Germany 3Department of Molecular Genetics,The Weizmann Institute of Science, Rehovot, 76100 Israel The EMBO Journal (1997)16:1381-1390https://doi.org/10.1093/emboj/16.6.1381 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The p53 tumor suppressor protein is a sequence-specific transcriptional activator of target genes. Exposure of cells to DNA damage results in accumulation of biochemically active p53, with consequent activation of p53-responsive promoters. In order to study how the transcriptional activity of the p53 protein is regulated in vivo, a transgenic mouse strain was generated. These mice harbor the p53-dependent promoter of the mdm2 gene, fused to a lacZ reporter gene. Induction of lacZ activity by DNA damage (ionizing radiation) was monitored in embryos of different p53 genotypes. The transgenic promoter was substantially activated in vivo following irradiation; activation required functional p53. The activation pattern became more restricted with increasing embryo age, as well as with the state of differentiation of a given tissue. Generally, maximal p53 activation occurred in rapidly proliferating, relatively less differentiated cells. A striking extent of haploinsufficiency was revealed—induction of promoter activity was far less efficient in mice carrying only one wild-type p53 allele. This suggests that normal levels of cellular p53 are limiting, and any further reduction already compromises the p53 response significantly. Thus, the activation potential of p53 is tightly controlled in vivo, both spatially and temporally, and an important element in this control is the presence of limiting basal levels of activatable p53. Introduction The p53 protein is the product of a tumor suppressor gene, frequently mutated in human cancer (for recent reviews on p53, see Haffner and Oren, 1995; Gottlieb and Oren, 1996; Jacks and Weinberg, 1996; Ko and Prives, 1996). These mutations are believed to inactivate one or more biochemical functions of the wild-type (wt) p53 protein, functions which otherwise may interfere with tumor development. The best characterized, and probably most important, biochemical function of wt p53 is the sequence-specific transactivation (SST) of target genes. The p53 protein can bind to specific sites within DNA and induce the transcription of genes residing in the vicinity of such p53-binding sites. A 20 bp consensus sequence for p53 binding has been defined (El-Deiry et al., 1992; Funk et al., 1992). Typical natural p53 binding sites, which drive p53-dependent expression of physiological target genes, usually diverge from this consensus by one or a few nucleotides. In normal cells, p53 is believed to be biochemically latent (Hupp et al., 1995). In addition, p53 is a very labile protein (Oren et al., 1981; Rogel et al., 1985) and, consequently, its steady-state levels are usually extremely low. However, in response to appropriate signals, p53 can become both stabilized and biochemically activated, resulting in a prominent increase in overall cellular levels of active p53. The best known inducer of p53 is DNA damage (Kastan et al., 1991, 1992; Lu and Lane, 1993; Zhan et al., 1993; Hupp et al., 1995), although other signals, including hypoxia (Graeber et al., 1996) and ribonucleotide depletion (Linke et al., 1996), can also have similar effects. Activation of p53 in response to DNA damage is believed to prevent the propagation of cells with potentially dangerous genetic lesions (Lane, 1992). This preventive effect of p53 can be exerted through arresting cell cycle progression transiently, so that the damaged DNA can be repaired properly before the cell resumes proliferation. Alternatively, p53 induction may permanently remove damaged cells from the replicative pool, either through triggering apoptotic cell death or through imposing a permanent G1 arrest. The involvement of p53 in developmental processes has been studied by several complementary approaches. Mice lacking p53 (‘p53 knock-out’) appear to undergo perfectly normal development, although eventually succumbing to a variety of malignancies at a relatively early age (Donehower et al., 1992). However, more recent work has identified a spectrum of developmental defects in a fraction of p53 null mice; these range from a failure in neural tube closure, resulting in exencephaly and embryonic death, to various less severe abnormalities (Armstrong et al., 1995; Sah et al., 1995). Thus, p53 may indeed have a role in normal development (Rotter et al., 1994), but other genes may often cover up for its absence. The observed defects might reflect rare situations where p53 function cannot be fully substituted, as may be the case during spermatogenesis (Rotter et al., 1993). Alternatively, p53 may be important only in embryos undergoing accidental or induced damage. Indeed, p53 can suppress radiation-induced teratogenesis, via a mechanism involving p53-mediated apoptosis (Norimura et al., 1996). In situ hybridization analysis has revealed p53 mRNA expression in all cells of the embryo up to embryonic day 10.5 (E10.5) (Schmid et al., 1991). Later in development, expression of p53 mRNA becomes more heterogeneous, but high levels are still seen in many tissues. Yet, since p53 is subject to elaborate post-translational regulation, abundant mRNA does not necessarily imply abundant protein, and certainly not high p53 activity. This is exemplified by embryonal carcinoma (EC) cells. These cells, very similar to those of the early normal embryo, express ample p53 mRNA and protein (Oren et al., 1982; Reich et al., 1983); moreover, that p53 is wild-type by sequence (Lutzker and Levine, 1996; Pennica et al., 1984). However, this p53 is practically devoid of SST activity (Lutzker and Levine, 1996), becoming active only when EC cells are either induced to differentiate in vitro or subjected to DNA damage (Lutzker and Levine, 1996). Hence, quantitative analysis of p53 mRNA, or even protein, is not a reliable measure of p53 activity. To study the in vivo regulation of p53 function, particularly in response to stress signals activating the p53 pathway, we generated a transgenic mouse strain carrying a lacZ reporter gene under a p53-responsive promoter. We used the intronic promoter of the mouse mdm2 gene which is a physiological target for transcriptional activation by wt p53. We describe here the effects of radiation on the activity of this promoter during embryogenesis. The data imply that the ability to mount an effective p53 SST response is greatly dependent on tissue type and developmental stage. In general, it becomes gradually more restricted with embryo maturation, and in cells relatively more advanced in differentiation. In addition, at least for the mdm2 promoter, there is a striking in vivo p53 haploinsufficiency, resulting in a much weaker induction of p53-specific SST in embryos containing only one wt p53 allele. Different tissues exhibit non-identical degrees of haploinsufficiency. Hence, critical thresholds for activation of p53 target genes may vary with cell type. Results Generation of transgenic mice carrying an mdm2 promoter–lacZ fusion In order to study the in vivo regulation of p53-mediated SST, a transgenic mouse model was developed. To that end, the p53-dependent intronic promoter of the murine mdm2 gene (P2; Barak et al., 1994) was placed in front of the bacterial β-galactosidase (lacZ) gene, acting as a reporter for transcriptional activity of the promoter. This particular promoter was chosen because it represents a well-characterized physiological target for transactivation by p53 (Juven et al., 1993; Wu et al., 1993; Barak et al., 1994). Importantly, transcription from this promoter in at least three different cell types, fibroblasts and myeloid cells harboring a temperature-sensitive p53, and wt p53-expressing lymphoma cells exposed to ionizing radiation (IR), was shown by RNase protection to be strictly dependent on the presence of activated p53 (Barak et al., 1994). This is unlike the p21 promoter, which can exhibit significant activity even in the absence of functional p53 (Michieli et al., 1994; El-Deiry et al., 1995; Halevy et al., 1995; Parker et al., 1995; Zhang et al., 1995; Tanaka et al., 1996), and unlike the constitutive P1 promoter of mdm2, which is essentially p53 independent (Barak et al., 1994; Montes de Oca Luna et al., 1996). The design of the reporter construct, containing the mdm2 P2 promoter within a 0.4 kb ApaI–NsiI fragment (Juven et al., 1993), is shown in Figure 1. This construct was microinjected into fertilized oocytes to generate transgenic mice. Positive founders were identified by Southern blot analysis. Each founder was then mated to a non-transgenic mouse, and expression of the lacZ transgene was monitored in irradiated E10.5 F1 embryos. Of six founders which transmitted the transgene, the progeny of two (#9 and #20) expressed detectable lacZ activity. The staining patterns of irradiated E10.5 embryos were essentially identical in both strains, except that staining intensity was substantially stronger in #20. Southern blot hybridization revealed that founders #20 and #9 harbored ∼20 and five copies of the transgene, respectively (data not shown). Strain #20 was subjected to detailed analysis, as described below. Figure 1.Schematic diagram of the murine mdm2 gene region including the intronic P2 promoter, and of the DNA fragment used for oocyte microinjection. The latter DNA fragment contains the indicated ApaI–NsiI segment of the mdm2 gene as well as a lacZ gene derived from plasmid pPD46.21 (see Materials and methods). Abbreviations: P1, p53-independent (constitutive) mdm2 promoter; P2, p53-dependent intronic promoter (Juven et al., 1993); p53RE, p53-responsive elements, represented by black boxes; NLS, nuclear localization signal, derived from SV40 large T antigen; pA, polyadenylation signal, derived from the SV40 early region. Exon numbers refer to transcripts initiated at the constitutive P1 promoter (Montes de Oca Luna et al., 1996). Actual transcription start sites for P1 and P2 are indicated by arrows (see Barak et al., 1994). Download figure Download PowerPoint Activation of a p53-responsive promoter by DNA damage is temporally and spatially regulated during embryogenesis A transgenic male was mated with non-transgenic females. The females were sacrificed at different times post-coitum (p.c.), and embryos were subjected to whole mount staining with X-Gal substrate to visualize lacZ activity. Weak positive staining could be observed in E8.5, E10.5 and E12.5 transgenic embryos (Figure 2A, C and E, respectively). At E8.5, there was staining in the branchial arches, in the tail bud and in the periphery of the somites; analysis of sectioned material revealed that the staining in the tail bud is superficial (data not shown). In E10.5 embryos, the clearest staining was seen in the branchial arches, the tips of the limb buds, the midbrain–hindbrain boundary, a small additional zone within the midbrain and the anterior part of the olfactory bulbs (Figure 2C). At E12.5, the pattern became more complex; significant staining could be observed also in the forebrain (in the cortex) and in the hair follicles of the sensory vibrissae (Figure 2E). It is of note that positive staining in the brain and hair follicles of unirradiated embryos, starting at E12, was also observed in transgenic mice in which lacZ expression is driven by a different, chimeric p53-responsive promoter (see accompanying paper by Komarova et al.). Detectable lacZ activity in our mice was dependent on the presence of the transgene; no staining could be detected in non-transgenic E8.5 and E10.5 embryos, and while some staining was observed at E12.5, this did not coincide with any of the major sites of lacZ activity shown in Figure 2E (data not shown). Figure 2.Whole mount lacZ staining of embryos at different days of gestation. (A) and (B) E8.5, (C) and (D) E10.5 and (E) and (F) E12.5. Staining was performed either with (B, D and F) or without (A, C and E) prior exposure to 5 Gy of γ radiation. Incubation with the X-Gal substrate was for 4 h. The embryos presented in this figure were obtained from non-transgenic CB6/F1 females that had been mated with a transgenic male. Abbreviations: BA, branchial arches; HB, hindbrain; Ht, heart; LB, limb bud; MB, midbrain; OB, olfactory bulbs; S, somites; SV, sensory vibrissae; TB, tail bud. Download figure Download PowerPoint The SST activity of p53 is known to be stimulated by DNA damage (Kastan et al., 1992; Lu and Lane, 1993; Zhan et al., 1993, 1994; Barak et al., 1994; Dulic et al., 1994; Gottlieb et al., 1996). To determine whether this could also be demonstrated in vivo in our transgenic mice, pregnant females were subjected to whole body IR (5 Gy) 3 h prior to embryo isolation and staining. As can be clearly seen in Figure 2B, D and F, lacZ expression increased dramatically in these embryos following radiation exposure. This implies that the mdm2 P2 promoter retains its p53 responsiveness in the transgenic mice, a conclusion further corroborated by the analysis of p53 null lacZ transgenic embryos (see below). These mice can thus be utilized for studying the pattern of transcriptional activity of endogenous p53 in animals exposed to DNA damage. Intensive lacZ staining following irradiation could be seen at sites displaying constitutive basal expression in the untreated controls, as well as sites where no basal expression was detectable. In sites of the first type, radiation exposure caused a further increase in staining. Induction of lacZ staining, albeit to sub-maximal levels, could already be seen in E10.5 embryos as early as 1 h after irradiation (data not shown). This rapid induction is consistent with the notion that high SST activity is mediated by the activation of latent p53 protein, rather than the triggering of transcription from the p53 gene. The data in Figure 2 demonstrate that the p53 response to DNA damage varies greatly among different tissues, as well as between different stages of development. In general, the older the embryo, the weaker and more tissue type restricted is the responsiveness of p53 to DNA damage. At E8.5, the entire embryo turns blue after irradiation, the only visible exception being the primitive heart (Figure 2B). On the other hand, at E10.5 the pattern of induction becomes more selective (Figure 2D). All three brain vesicles display positive staining at this stage. The branchial arches, as well as the maxillary areas, are strongly stained, and so are the limb buds. The somites and the tail bud display a gradient of staining, increasing towards the posterior end. In contrast, in the anterior portion of the trunk, lacZ activity is conspicuously weaker. Finally, similarly to E8.5, no staining is seen in the heart, suggesting that this tissue either makes no p53 protein, or contains latent protein which cannot be activated by radiation-induced DNA damage. It is of note that although p53 protein can be detected by immunohistochemistry in the heart of irradiated embryos, this tissue does not undergo any radiation-induced apoptosis (Wubah et al., 1996; D.MacCallum and P.Hall, personal communication). One of the best examples for age-dependent restriction of p53 SST activation in the embryo is the developing neural tube, which is uniformly and intensely stained in irradiated E8.5 embryos (Figure 3A), while staining becomes confined to a limited subset of cells at E10.5 (Figure 3B). Moreover, a strip lacking detectable lacZ induction is visible in the midbrain, within the roof of the third ventricle, at E10.5 (Figures 2D and 3C). The tissue-specific differences in p53 SST induction are also evident in an eosin-stained paraffin section (Figure 3D), which in addition shows clear positive expression of the transgene in the Rathke's pouch, and in contrast no detectable expression in parts of the midbrain, and particularly the ventral midbrain (small arrow in Figure 3D). Thus, at this stage, some neuronal tissues are already selectively losing their ability to activate p53 SST function in response to DNA damage. Figure 3.Histological analysis of lacZ staining in irradiated embryos. Embryos were isolated 3 h after exposure of the pregnant female to 5 Gy of γ radiation, and then subjected to whole mount staining with X-Gal followed by further analysis by different sectioning procedures. (A) Vibratome cross-section of an E8.5 embryo. (B) Vibratome cross-section of an E10.5 embryo. (C) Vibratome sagittal section of an E10.5 embryo. (D) Eosin-stained sagittal paraffin section of an E10.5 embryo. Abbreviations: BA, branchial arches; HB, hindbrain; Ht, heart; MB, midbrain; NT, neural tube; RP, Rathke's pouch. Download figure Download PowerPoint At E12.5, there is a further increase in selectivity of p53 SST induction (Figure 2F). Strong expression of lacZ is evident in the eye, the sensory vibrissae and the tail; notably, the developing eye is a site of marked p53 protein accumulation and apoptosis in irradiated embryos (Wubah et al., 1996; D.MacCallum and P.Hall, personal communication). The signal in the limb buds becomes more specialized, and is evident mainly in the apical ectodermal ridge and in the prospective fingers and toes. Here too, an anterior–posterior gradient is visible along the tail, with the most intense staining seen in the posterior part. It is well established that the axial structures differentiate in an anterior to posterior succession, such that the posterior paraxial mesoderm may still be in an unsegmented state at the time when the thoracic somites have already differentiated to their mature components. We see strong induction of p53 SST activity in the posterior, less differentiated portion of the axis, as well as in the limb buds and eyes, organs which undergo extensive differentiation and morphogenesis during the stages studied here. It therefore follows from our observations that maximal induction of p53 SST activity is seen preferentially in faster-dividing, less-differentiated elements. In conclusion, the ability of endogenous p53 to be activated as a transcription factor appears to be inversely correlated to the stage of maturation of the embryo and the extent of differentiation of the particular tissue. Maximal promoter activation requires both wt p53 alleles: evidence for haploinsufficiency in vivo The strong radiation inducibility of the transgenic mdm2 promoter indicated that its activity is p53 dependent in this system. We wished to confirm formally the p53 dependence of this induction, and to assess the p53 dependence of the lacZ expression observed in the uninduced state. Therefore, mdm2 P2–lacZ transgenic mice were mated to p53+/− mice. A resultant lacZ transgenic male, heterozygous for the p53 null allele, was mated to a p53+/− female not carrying the lacZ transgene. The pregnant female was irradiated at day 10.5 p.c. and sacrificed 3 h later. Individual embryos from the same litter were isolated separately. The yolk sac of each embryo was used for DNA extraction and PCR analysis. The embryos were stained for lacZ. In parallel, the p53 and lacZ genotype of each embryo was determined by PCR analysis using two primer pairs which distinguish between the wt p53 allele and the knock-out p53 allele, and a third pair of primers specific for the lacZ transgene. Representative p53+/+, p53+/− and p53−/− embryos are displayed in Figure 4A–C); the corresponding PCR patterns are shown in Figure 4D. Figure 4.Induction of lacZ activity by radiation in transgenic E10.5 embryos of different p53 genotypes. A p53+/− female was mated to a male heterozygous for the p53 null allele and for the mdm2 P2–lacZ transgene. Embryos of a single litter (a total of 11 embryos) were isolated 3 h after γ irradiation and stained for lacZ activity. Incubation with the X-Gal substrate was for 16 h. In parallel, DNA was extracted from the yolk sac of each embryo and subjected to PCR analysis in order to monitor for the presence of the lacZ transgene, as well as the wt and the null (neor) p53 alleles. One representative lacZ-positive embryo of each p53 genotype is shown (A–C); the p53 genotype is indicated on the upper right side of each panel. The corresponding PCR products for each of the three embryos are shown in (D). The weak wt p53 band in the reaction with embryo C DNA is probably due to a minor contamination of the yolk sac sample with cells of maternal origin. Download figure Download PowerPoint Our results confirm that the strong induction of lacZ activity by IR is indeed p53 dependent, as it does not occur in p53 null mutant embryos (compare Figure 4A and C). On the other hand, the pattern of lacZ expression in p53 null mutant embryos (Figure 4C) was practically indistinguishable from that seen in unirradiated p53+/+ embryos of the same age (compare with Figure 2C). The basal pattern of expression, seen in the absence of DNA damage, therefore appears to be p53 independent. Whether this basal pattern represents physiological sites of p53-independent activity of the mdm2 P2 promoter is presently unclear. Importantly, Figure 4 reveals a striking dependence of lacZ induction on wt p53 gene dosage. While p53-dependent activation of the reporter is clearly seen in irradiated p53+/− embryos (compare Figure 4B and C), the intensity of this staining is far below that obtained in p53+/+ embryos (compare Figure 4A and B). In fact, the picture shown underestimates the true difference between the homozygous and heterozygous wt p53 genotypes: in order to maximize staining intensity in p53−/− and p53+/− embryos, incubation of all embryos with the lacZ substrate was carried out for 16 h. Consequently, the staining in Figure 4A has already reached saturation. Conspicuous staining was already seen in the p53+/+ embryos after 4 h of incubation (see Figure 2D), while staining in p53−/− and p53+/− littermates was still very faint at that time (data not shown). Hence, the actual difference in the extent of lacZ expression between embryos containing one wt p53 allele and those containing two such alleles is even greater than implied by Figure 4. These profound differences between p53+/+ and p53+/− embryos were consistent within the same litter, as well as across litters (data not shown). The data also suggest that the threshold for activation of the p53 SST response by DNA damage varies among individual tissues. Consequently, the presence of a single wt p53 allele is sufficient for at least partial induction of the transgenic mdm2 promoter in some cell types, whereas in others no induction can take place unless both wt p53 alleles are retained. For instance, while the hind limbs, visceral arches and ventral parts of the olfactory bulbs display a substantial p53 response to IR even in p53+/− embryos, the hindbrain and midbrain show only minimal induction, unlike the prominent activation visible at these sites in p53+/+ littermates. Taken together, our findings argue strongly that, at least for the mdm2 P2 promoter, a full complement of wt p53 genes is required for optimal SST response to DNA damage. Retention of a single wt p53 allele results in an incomplete response. The extent of haploinsufficiency varies from tissue to tissue, being partial in some tissues and more severe in others. Thus, relatively moderate differences in basal levels of p53 can already dictate whether or not a particular cell, when exposed to a stress signal, will accumulate enough p53 to drive the efficient activation of downstream target genes. Discussion We describe here the generation of a transgenic mouse model for studying the biochemical activation of p53 in vivo in response to genotoxic stress. The mice carry a reporter gene, β-galactosidase (lacZ), under control of a physiologically relevant p53-dependent promoter derived from the mdm2 gene (mdm2 P2). Induction of lacZ expression monitors the extent of SST by p53—considered the main, though not only, biochemical function through which p53 mediates its various biological effects (Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996). The present analysis is confined to the induction of p53 SST in the developing mouse embryo, following exposure to IR. Two main conclusions can be drawn from this study. First, the potential for induction of p53 SST by DNA damage is not intrinsic to every cell in the embryo; rather, it is spatially and temporally restricted. The spectrum of sites which exhibit detectable activation after exposure to IR varies with developmental age, and also between different compartments of the developing embryo and between different zones of the same compartment. In general, maximal inducibility of p53 SST correlates with a less differentiated, more highly proliferative state. This is perhaps illustrated most vividly by the gradient of induction seen along the anterior–posterior axis at E10.5 (Figure 2D). At E10.5, all cells of the embryo express high levels of p53 mRNA (Schmid et al., 1991). Hence regulation of p53 function, at least in response to DNA damage, is not at the transcriptional level. Presumably p53 mRNA is translated in all tissues but the protein is degraded rapidly and thus does not accumulate to appreciable levels. However, these labile p53 molecules may serve as a constantly available pool for the efficient accumulation of functional protein, once they become stabilized in response to appropriate stress signals. In addition, the constitutive reservoir of p53 mRNA may give rise to more protein through DNA damage-induced relief of inhibitory translational control (Mosner et al., 1995). Our data imply that tissue- and cell type-specific factors can determine whether or not this potential for rapid overproduction and accumulation of functional wt p53 is actually materialized. At later stages of development, further restrictions may be imposed on the p53 response by limiting p53 mRNA expression to only a subset of cells and tissue types (Rogel et al., 1985; Schmid et al., 1991). There is generally a good correlation between extensive accumulation of p53 after DNA damage and the likelihood of apoptosis (Komarova et al., 1997, accompanying paper; D.MacCallum and P.Hall, personal communication). Induction of p53-mediated apoptosis may well require higher levels of p53 protein than for other biological activities of p53. Consequently, even quantitative differences in levels of DNA damage-induced p53 activation may suffice to dictate which cells will undergo apoptosis and which will fail to do so. Moreover, the distinction between apoptosis and viable growth arrest may be a function of the extent of DNA damage (Gottlieb et al., 1996). Even though this distinction is not necessarily always dependent on SST (Caelles et al., 1994; Haupt et al., 1995), it does suggest that there are defined thresholds of DNA damage below which some p53 effects do not take place. It is quite conceivable that higher doses of IR than employed by us might have resulted in induction of SST, as well as of apoptosis, at sites where no prominent induction is seen at 5 Gy of IR. In the adult mouse, tissue-specific restriction of the p53 response is already well documented (Clarke et al., 1994; Merritt et al., 1994; Midgley et al., 1995). This
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