Gene-targeting and the p53 tumor-suppressor gene

Fundamental and Molecular Mechanisms of Mutagenesis

Mutation Research 307 (1994) 557-572

ELSEVIER

Gene-targeting and the p53 tumor-suppressor gene Arthur

S a n d s a, L a w r e n c e

A. Donehower

b, A l l a n B r a d l e y

a,c,.

a Department of Human and Molecular Genetics, b Division of Molecular Virology and c Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA

(Received 7 July 1993; revision received 21 September 1993; accepted 23 September 1993)

Abstract

Gene-targeting techniques are now frequently applied to embryonic stem (ES) cells to introduce mutations of endogenous genes in mice. Modifications introduced into tumor-suppressor genes by this technology have produced mice and cell lines with unique tumorigenic and growth characteristics, respectively. A number of strategies have been developed to enhance the efficiency of homologous recombination between targeting vectors and endogenous genes. This review describes recent advances in the techniques used to construct mice with a variety of genetic alterations. In addition, an application of gene-targeting is illustrated in the study of a class of genes with tumor-suppressor function. Recent findings from experiments using gene targeted mice to study the p53 tumor-suppressor gene are discussed and the potential of gene-targeting for the discovery and study of novel tumor-suppressor genes are explored. Key words: p53; Homologous recombination; Embryonic stem cells; Gene targeting; Tumor suppressor

I. Introduction

The recombination event that results in a genetic exchange between vector D N A introduced into a cell and its homologous chromosomal target is known as gene-targeting. The application of gene-targeting techniques to mammalian cells is a relatively recent advance (Lin et al., 1985; Smithies et al., 1985; Thomas et al., 1986) which was followed rapidly with successful application of the techniques to embryonic stem cells (Thomas and Capecchi, 1987). These ceils derive from the pluripotential cell population of the inner cell mass (ICM) of pre-implantation blastocysts and

* Corresponding author. Tel. 713 798 3533; Fax 713 798 6521.

may be maintained as large populations of cultured cell lines (Evans and Kaufman, 1981), thereby making possible the generation and selection of genetic modifications that occur very infrequently, such as those associated with homologous recombination at a targeted genomic locus. Most importantly, the fact that embryonic stem cell lines are capable of recolonizing embryos and contributing to the germ-line (Bradley et al., 1984), allowed for the development and phenotypic analysis of mouse strains containing gene-targeted mutant alleles (Zijlstra et al., 1990). The application of these techniques to any cloned gene is theoretically possible and constitutes a very powerful genetic system for the creation of novel mutations. Successful targeting of the p53 gene is one example of the application of these

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techniques to the study of tumor-suppressor gene function that has yielded significant information regarding the consequences of the null allele in vivo and in vitro. The scope of phenotypic analysis of various genetic modifications may be broadened by subsequent crosses of animals containing different mutant alleles in order to obtain more than one genetic alteration in a single animal, or by crosses of animals with mutant alleles to those containing transgenes, thereby allowing for the in vivo evaluation of synergistic effects of genetic modifications. While desired homologous gene recombination events may be obtained by the techniques of gene-targeting, it is generally a low frequency event as compared to the competing pathway of random chromosomal integration. Recent studies have demonstrated efficient strategies to achieve site-directed mutagenesis of nonselectable genes (Davis et al., 1992; Hasty et al., 1991a). This review describes current strategies to achieve vector-chromosome homologous recombination in embryonic stem cells and reviews the application of these strategies to the targeting of the p53 tumor-suppressor gene. In addition, recent findings from studies using p53-deficient mice and cells derived from them are discussed with particular attention to hypotheses relating p53 function and DNA damage.

2. Embryonic stem cells

The preimplantation blastocyst and early post-implantation murine embryo contain a population of uncommitted pluripotential ICM cells. Although these cells ultimately become committed to specific developmental pathways, at early stages individual cells have potent proliferative and differentiative capacities and are capable of contributing to tissues derived from ectodermal, endodermal and mesodermal layers as well as the germ-line when transferred to host blastocyst stage embryos (Gardner and Lyon, 1971; Gardner and Rossant, 1979). Undifferentiated cell lines that derive from these early cells have been established either directly from the embryo (embryonic stem cells, ES) or indirectly from teratocarcinoma

tumors (embryonal carcinoma cells, EC). With several years experience characterizing EC culture conditions, cell morphology and biochemical properties (Evans, 1981), the successful isolation of ES cells was achieved by Evans and Kaufman (1981) and Martin (1981). Both groups chose to use murine blastocysts as the starting material based on a variety of studies that suggested some equivalence between ICM cells and EC cells including the ability of EC ceils to contribute to somatic tissues of chimeric mice when injected into blastocysts. A variety of growth conditions have been used to achieve the goal of perpetual proliferation of ES cells in vitro without differentiation. Classically, feeder layers of mitotically inactivated permanent STO cell lines (Evans, 1981) or embryonic fibroblasts (Doetschman et al., 1985) are used to appropriately condition the media for ES cell culture. Other approaches include the use of media conditioned by BRL (Buffalo rat liver) cells in high proportions in the absence of feeder cells (Smith and Hooper, 1987) or media conditioned by established EC cells (Martin, 1981). The heterogeneity associated with apparently morphologically normal ES cells has been reported by single cell cloning and blastocyst injection (Joyner et al., 1989; Zimmer and Gruss, 1989). The ability of an embryo to select normal from abnormal cells has ensured germ-line transmission in experiments where populations of cells were injected into the embryo (Bradley et al., 1984; Robertson et al., 1986). Cloning of ES cell populations, a necessary procedure with most gene-targeting experiments, has revealed the rapid progression of ES cell lines to a state in which only a relatively minor fraction of cells can contribute extensively to the somatic and germ lineages of chimeric mice. In fact, the majority of injected clones show low levels of somatic mosaicism and are generally excluded from the germ-line (Fig. 1). The provision of components that facilitate the proliferation of the totipotent fraction in an ES cell population can eliminate much of the clonal heterogeneity that is currently observed (McMahon and Bradley, 1989; Soriano and Friedrich, 1991). Of course, one of the most important and

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3. Strategies of gene-targeting

Primary ES line

Targeting frequency: general considerations

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widespread uses of the developmental potential of ES cells is to genetically manipulate the germline of mice (Cappechi, 1989). As tissue culture lines, ES cells clearly have potent recombination machinery which has been widely used to mutate many genes by the insertion of a selectable marker. Many of these mutated alleles have been established in the germ-line where the consequences of the mutation can be studied in the homozygous or heterozygous state (DeChira et al., 1990; McMahon and Bradley, 1989; Zijlstra et al., 1990). Remaining challenges include the refinement of techniques to create different types of mutations by gene-targeting, such as the insertion of subtle mutations, while maintaining targeted ES cells in conditions that do not compromise in vivo differentiation and allow routine germ-line transmission of these specific genetic traits.

Integration of a targeting vector into the genome of an embryonic stem cell after transfection is most likely to occur at a random chromosomal locus, while recombination at the homologous site in the genome occurs at a frequency that can be lower by several orders of magnitude (Mansour et al., 1988; Smithies et al., 1985; Thomas and Capecchi, 1987; Thomas et al., 1986). Considering the size and complexity of the mammalian genome, the alignment of homologous sequences was thought to be a significant ratelimiting constraint on targeted recombination. It was hypothesized, therefore, that the targeting frequency would be elevated by increasing the number of copies of either the vector or the target in the genome. Surprisingly, however, there was no change in the frequencies of targeting when the copy number of the vector was increased (Rommerskirch et al., 1988; Thomas et al., 1986). Furthermore, targeting experiments performed under identical conditions with two cell lines, one of which had an increased target site copy number, showed no significant difference in targeting frequency between the cell lines (Zheng and Wilson, 1990). Thus, the search for homology is probably not a rate-limiting step in homologous recombination after transfection. In contrast to copy number, the length of homologous sequence present in a vector has a well documented effect on the recombination frequency in both extrachromosomal (Rubnitz and Subramani, 1984) and targeting experiments (Hasty et al., 1991b; Shulman et al., 1990; Thomas and Capecchi, 1987). The targeting frequency increases as more homologous sequences are added to the vector, although there is some discrepancy between the exact mathematical relationship between these two parameters. Whether the effect of the additional homology occurs at the level of a homology search, in the formation of complexes, or in the resolution of the recombination complex is not currently known. Insertion and replacement vectors

The relatively low abundance of targeted clones in populations of transfected mammalian

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cells has encouraged the development of targeting schema that allow for the in vitro selection of desired recombinant clones. In general, enhancement of isolating correctly targeted clones may be achieved by constructs that incorporate positively selectable genes for identification of all integration events and negatively selectable genes for exclusion of random integration events (Mansour et al., 1988). The configuration of the positive and negative selection cassettes within recombi-

nation vectors and the placement of a doublestranded break by restriction enzyme digest determines the type of gene interruption that is likely to occur. These vectors may be broadly classified as insertion vectors or replacement vectors. Insertion vectors are designed to use a double-stranded break in a homologous region of the targeted gene to facilitate a single reciprocal exchange event which results in the addition of the whole plasmid vector including a selectable

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Fig. 2. Configuration of targeting vectors. (A) Insertion vectors. These vectors integrate via a single reciprocal exchange stimulated by a double-strand break in homology. This class of integration event incorporates the entire vector. (B) Replacement vector. These vectors integrate via a double reciprocal exchange or gene conversion replacing the host sequences. Sequences external to the homologous sequences are lost during this type of genetic exchange. (C) Positive/negative selection for targeted events. Homologous recombination results in the integration of the neo gene and the loss of the HSVtk gene. The desired recombinant event may be selected for as indicated.

A. Sands et al. / Mutation Research 307 (1994) 557-572

marker such as the ned gene usually located in the plasmid backbone (Fig. 2A). The replacement vector, meanwhile, contains a positively selectable gene nested within homologous sequences and requires a double reciprocal exchange (or gene conversion) resulting in replacement of chromosomal sequences with the engineered cassette and simultaneous loss of plasmid vector backbone sequences (Fig. 2B). For replacement vectors, the location of the doublestranded break should be at the periphery of homologous sequences to avoid the insertional

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pathway (Lin et al., 1990; Siedman, 1987; Wake et al., 1985). Hasty et al. have found that insertion vectors target the hprt and other loci in embryonic stem cells more efficiently than replacement vectors (Bradley et al., 1992; Hasty et al., 1991b,c), while others have reported that the two types of vectors target at approximately equal efficiencies (Deng and Capecchi, 1992; Thomas and Capecchi, 1987). This discrepancy may be partly related to the exact choice of the DNA sequences used for the construction of targeting constructs which has

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Fig. 3. Selection schemes using endogenous regulatory elements to enrich transfected populations for targeted events. (A) Promoter traps use homologous sequences to place a promoterless selectable marker gene under transcriptional control of the endogenous promoter of the target gene. This enrichment scheme requires that the promoter is functional in ES cells. (B) Polyadenylation traps use homologous sequences to place a polyA-less selectable marker gene in proximity to transcription termination/polyadenylation signals to enhance expression of targeted events relative to random integration events.

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been demonstrated to affect the efficiency of homologous recombination events (te Riele et al., 1992). Most recently, the relative targeting efficiencies of insertion versus replacement vectors using isogenic D N A has been demonstrated to also be affected by specific D N A sequences or the immediate chromosomal environment (Hasty et al., 1993). It appears that some chromosomal loci may be targeted very efficiently (Hasty et al., 1993; Tybulewicz et al., 1991), while others demonstrate a substantially lower frequency of targeted recombination (Hasty et al., 1993). In some situations, the preferential homologous integration pathway has been documented to be an insertion-type mechanism, even when the vector is configured to undergo a replacement event (Hasty et al., 1991b,c; Thomas and Capecchi, 1990). Enrichment of the desired replacement events relative to random integration events has been demonstrated by the inclusion of a negatively selectable gene such as HSVtk peripheral to the region of homology (Fig. 2C). Since most random integration events are multiple copy integration events, they will usually contain the HSVtk gene and may be selected against with FIAU, l(2-deoxy-2-fluoro-/3-D-arabinofuranosyl)-5-iodouracil. Thus, homologous recombinant clones produced by a double reciprocal exchange event will survive selection against the HSVtk gene which is lost with the rest of the plasmid backbone. Enrichment for homologous recombinant clones provided by this positive/negative selection scheme are most commonly in the range of 2-30-fold (DeChira et al., 1990; McMahon and Bradley, 1989; Zijlstra et al., 1990). Some additional level of enrichment of targeted clones may be achieved by inclusion of two negatively selectable cassettes at both ends of the homologous sequences in a replacement vector. However, this strategy requires that the opportunity for extrachromosomal, intermolecular homologous recombination be minimized by using two different negatively selectable genes or genes which have diverged at the sequence level such as HSVltk and HSV2tk (reviewed by Hasty and Bradley, 1993). Permutations of these basic elements of targeting vectors have been used in a number of strategies to

enhance the frequency of homologous recombination events or to select for the placement of subtle mutations in targeted genes. Promoter or polyadenylation capture Some of the strongest in vitro selection techniques for gene-targeting have utilized vectors which are designed to juxtapose specific transcriptional elements from the target locus with selectable genes from the vector. For instance, vectors have been used in which the positive selection marker lacks a promoter (Charron et al., 1990; Schwartzberg et al., 1990; Stanton et al., 1990; te Riele et al., 1990) or transcriptional t e r m i n a t i o n / p o l y a d e n y l a t i o n signal elements (Donehower et al., 1992) (Fig. 3). The technique of promoter capture is based on targeting a selectable marker so that its expression is driven by the promoter elements of a gene that is normally expressed in ES cells, thereby facilitating the in vitro selection of the targeted clones. The integration of such a vector into a random location in the genome will not, in most cases, result in the efficient transcription of the selectable marker. Thus, there will be a preferential survival of targeted clones under selective conditions specific for the positive selection marker cloned into the vector. Promoter capture of specific genetic targets has also been demonstrated with lacZ as the reporter gene; the expression pattern of the target gene may then be revealed by staining the appropriate tissue with X-gal (Le-Mouellic et al., 1990; Mansour et al., 1990). The targeting of many different genes to the cis elements of heterologous loci will probably become an important experimental system because the expression pattern of the target gene may be very precisely replicated. The efficiency of isolation of successfully targeted clones by the above method may be enhanced by inclusion of a negatively selectable marker external to the region of homology. This technique is discussed in greater detail below. Subtle mutations created by gene-targeting Most of the mutations introduced into mammalian cells by homologous recombination have been designed to generate null alleles, usually by interruption a n d / o r deletion of coding se-

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Fig. 4. Strategies of generating subtle alterations by gene-targeting. (A) Co-elcctroporation, isolation o f the desired targeted mutation requrics another random integration event of the selectable marker. (B) Modifications by linkage, the selcctable marker is placed in a location that is theoretically non-essential for regulation o f the target gene so that effects of a subtle mutation (m) introduced in homologous coding exon may be analyzed. (C) Hit-and-Run, step one is a targeted single reciprocal recombination event resulting in insertion of the entire targeting vector; step two, intrachromosomal recombination, is selected for with F I A U and resultant colonies are screened for the desired mutation; (D) Double Hit Gene Replacement, step one is a targeted double reciprocal recombination using a replacement vector strategy; step two requires a second double reciprocal recombination with a second targeting vector carrying a subtle mutation. Step two is se|ected by the loss of the hprt gene (use o f the hprt marker gene requires an hprt-negatJve ES cell line).

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quences with the insertion of a selectable marker. While such an approach produces a subject for the study of the effects of ablating a gene product, many experiments require the introduction of subtle mutations at the target locus. For instance, the study of regulatory regions of genes at their normal chromosomal location requires the generation of a variety of mutations involving changes of just a few nucleotides. Furthermore, certain loci may contain several genes in close proximity or may be subject to alternative splicing or developmental rearrangements (such as at the immunoglobulin loci). Introduction of large deletions or insertions to such complex loci may produce phenotypes that are not just a consequence of the mutation in the target gene but may in fact also reflect alterations on surrounding genes. Analysis of a complex locus, therefore, may best be achieved by the introduction of small deletions or specific, small mutations. Subtle mutations introduced into the majority of genes are unlikely to be directly selectable in tissue culture. Reported techniques for targeting non-selectable genes have depended on either: (1) treatment of a few cells with highly efficient DNA-transfer methods and identification of transfected clones; or (2) treatment of millions of cells with inefficient DNA-transfer techniques but highly efficient identification of transfected clones using a selectable marker gene. The first technique developed to make subtle mutations in the genome relied on very efficient D N A delivery and identification of transfected clones using direct microinjection of ES cells (Zimmer and Gruss, 1989). However, this method is technically difficult and there have not been any subsequent reports of the successful use of this strategy, nor has it been shown that targeted ES cells generated by microinjection can be transmitted through the germ-line. The second experimental design uses the relatively inefficient DNA-transfer technique of electroporation and depends on the identification of the rare transfected cell by a positive selectable marker that either does not integrate into the target locus or may subsequently be removed by a secondary recombination event. Four recombination strategies have been demonstrated using electroporation and are

summarized in Fig. 4. One method for introducing a degree of selectability to a subtle mutation generated by homologous recombination is by co-electroporating a selection cassette with the targeting vector (Fig. 4A). The rationale behind this technique is simply that the positive selectable marker will purify transfected cells (both targeted and random integrants) from the population of non-transfected cells. Co-electroporation has been used to successfully target a 4base-pair frame shift mutation to the selectable hprt locus at a modest frequency (Davis et al., 1992), although the use of this technique at a non-selectable locus has not been demonstrated. Another method of selecting for a targeted subtle mutation is by linkage with a positive selectable marker introduced into a non-coding region of the targeted locus (Fig. 4B). While this strategy has been used successfully to introduce a mutation at the human /3-globin locus in M E L cells (Shesely et al., 1991), there remains the considerable uncertainty as to the effect the positive selectable marker may exert upon the regulation of the locus. Notably, there are numerous examples of the phenotypic consequences of aberrant expression patterns resulting from the insertion of genetic elements close to or within the introns of genes (Nusse, 1986; Schnieke et al., 1983). Probably the most efficient way to make minor changes to the genome is to modify the target locus by selecting for successive recombination events. This may be accomplished by using two rounds of single reciprocal recombination described as the Hit-and-Run Strategy (Hasty et al., 1991a) (Fig. 4C), or by using two rounds of double reciprocal recombination known as the Double Hit Strategy (Bradley et al., 1992) (Fig. 4D). The rationale behind these double recombination events is to make the primary targeted clone relatively easy to isolate by conventional genetargeting selection and screening techniques. Clones bearing this allele then serve as a substrate for the very inefficient (10 -3 to 10 7) secondary recombinant event. Although these secondary events occur at a very low frequency, the recombinant clones may be readily isolated because they can be designed to exclude a negatively selectable marker included in the locus by

A. Sands et al. / Mutation Research 307 (1994) 557-572

the first recombination step. The best characterized double recombination strategy to date is the Hit-and-Run (Hasty et al., 1991a) which is also called the In and Out strategy (Valancius and Smithies, 1991). The first step in this technique is an insertional recombination of the vector into the target locus facilitated by a double-strand break within the region of homology. This insertion event generates a duplication of the target homology separated by the plasmid and the positive/negative selectable cassette. Once the primary colonies have been isolated, the proliferation of these cells in the absence of selection allows the direct repeat caused by the insertion event to recombine by intrachromosomal recombination or unequal sister-chromatid exchange to excise the vector. Although the frequency of vector excision is low, a subset of the revertant clones may be selected since they will also have lost the negatively selectable gene. The Hit-andRun strategy has been demonstrated to be effective at both selectable (Hasty et al., 1991a; Valancius and Smithies, 1991) and non-selectable loci (Hasty et al., 1991a). The basis of the Double Hit gene replacement strategy is the introduction of a negatively selectable marker into the target site of interest. This mutated allele becomes the target for a second recombination event with another vector carrying the desired subtle mutation. The second recombination event is directly selectable because the negative marker would be removed by gene replacement or gene conversion (Fig. 4D). While negative selection is a very powerful means of selecting for the loss of gene activity at the target locus, the background number of cells that survive selection must not be above 10 -6 to 10 -7 per treated population since the secondary recombination events with the transfected vector are likely to be generated at this frequency. Negatively selectable genes have been targeted to both the H o x b 4 and c - m y c loci and, after secondary transfection with subsequent negative selection, a high background number of surviving clones were detected (Bradley et al., 1992). The relatively high frequency of loss of the negative marker without integration of the new mutant vector could be explained by gene conversion or

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mitotic recombination with sister chromatids, or by chromosome loss followed by reduplication. Recently, this strategy (called Tag-and-Exchange) was reported to work with an acceptable frequency of the second event to introduce point mutations into the a2 isoform of the Na,KATPase homolog in mouse ES ceils (Askew et al., 1993). In principle, Double Hit gene replacement is an attractive strategy for targeting subtle mutations because once an allele is obtained containing a negatively selectable marker, it could be used repeatedly as a substrate for generating a series of targeted alleles.

4. Gene-targeting of p53 Although inherited mutations in tumor-suppressor genes behave as autosomal dominant genetic traits in humans, it is established that the mutations are functionally recessive in that both copies of the gene are found to be mutated in tumor cells. The recessive nature of a single, null tumor-suppressor allele is a confounding problem in the identification of novel tumor-suppressor genes as compared to dominant proto-oncogene mutations. Using gene-targeting techniques, however, tumor-suppressor function may be studied in the context of both normal development and tumor formation by creating mutations in this class of genes in vivo. It appears that such mutations alter normal cellular growth control and thereby allow proliferation of tumor cells (Fig. 5). Gene-targeting has been used to study a number of tumor-suppressor genes in mice including the p53 gene (Donehower et al., 1992), retinoblastoma gene (Lee et al., 1992) and to identify novel tumor suppressors such as a-inhibin (Matzuk et al., 1992). The mechanisms by which these genes act are diverse and production of mice deficient in different aspects of tumor-suppressor function provides valuable subjects for the study of different pathways of growth control. There are several advantages to using gene-targeting for the study of tumor-suppressor function (or any other gene for that matter): (a) the targeted mutation is well defined; (b) it may potentially be examined in a heterozygous and homozygous state; (c) the phe-

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Positive and Negative Growth Control

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Fig. 5. Schematic of cellular growth control. Dysfunction of regulatory mechanisms (presumably as a result of mutation) controlling either growth promotion or growth inhibition can lead to a proliferative response and ultimately tumor formation.

notype of heterozygous and homozygous embryos may be determined in developmental studies and if not lethal, adult animals may be studied as well; and (d) cell lines may be generated with single defined defects which are considerably less heterogeneous than existing tumor cell lines which contain many undefined genetic alterations in a number of growth regulatory genes in addition to the gene under study. p53 is one of the best characterized tumorsuppressor genes to date and mice deficient in p53 have proven to be a relevant model for the L i - F r a u m e n i tumor susceptibility syndrome (Donehower et al., 1992). In humans, p53 gene alterations have come to be recognized as the most commonly known genetic defect associated with cancers (Vogelstein and Kinzler, 1992). For example, a chromosomal region containing the p53 gene, located on the short arm of human chromosome 17, was found to be deleted in 77% of 58 colon carcinoma specimens (Baker et al., 1989). p53 mutations have also been associated with cancers of lung, brain, breast, esophagus, liver, ovary, brain and hematopoietic cells (Hollstein et al., 1991). Point mutations of the p53 gene associated with human cancers cluster in four "hot spots" which are localized to the four most highly conserved regions of the gene (Nigro et al., 1989). Mutation of p53 is also associated with L i - F r a u m e n i syndrome, an autosomal domi-

nant syndrome characterized by a high incidence of cancer development in a variety of different tissues. Of six affected families examined, all were found to have p53 mutations clustered in a highly conserved region of the gene. Inclividuals in these families have a 50% incidence of cancer by age 30 and a 90% incidence by age 70 (Malkin et al., 1990). Another means of p53 inactivation has recently been hypothesized that involves the amplification of the human MDM2 gene. This gene was recently cloned and found to be amplified in over one-third of 47 human sarcomas studied (Oliner et al., 1992). Furthermore, the protein product of the MDM2 gene was demonstrated to bind to p53 (Oliner et al., 1992) and inhibit p53-mediated transactivation (Momand et al., 1992). Generation of mice with p53 null alleles by homologous recombination A null allele of the p53 tumor-suppressor gene has been created in mice using a replacement vector strategy with positive/negative selection. Specifically, 450 base pairs of the p53 gene were deleted with simultaneous insertion of a neo selection cassette (Donehower et al., 1992). Analysis of p53 gene expression was performed on wild-type ( + / + ) animals, mice heterozygous for the p53 null mutation (p53 + / ) and mice homozygous for the p53 null mutation (p53 - / ). No intact p53 m R N A was produced in homozygotes. Further confirmation of a successful null mutation was obtained by immunoblot assay which demonstrated that only wild-type and heterozygote mice produced a normal size p53 protein while the homozygotes produced no detectable protein (Donehower et al., 1992). Both homozygous and heterozygous offspring were completely normal in appearance with no gross developmental abnormalities noted. In addition, there was no significant bias against embryonic and fetal survival of homozygotes analyzed at 4 weeks post partum (Donehower et al., 1992). As has been reported, the most notable phenotype of these p53-deficient animals is their propensity for tumor formation. Of 35 animals homozygous for the p53 mutation, 26 (74%) developed obvious tumors by 6 months of age. The mean time to

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tumor for this group of 35 homozygous animals was 4.5 months. The tumors most frequently observed were malignant lymphomas which affected 20 of 26 homozygous animals, with 9 of the 26 animals manifesting more than one type of primary tumor. Non-lymphoid neoplasms included undifferentiated sarcomas, hemangiosarcomas, hemangiomas, osteosarcomas and germ-cell tumors. 95 wild-type littermates of the p53-deficient mice failed to develop any tumors or illness. Of the heterozygous animals, approximately 50% develop tumors by 18 months of age and the predominant types of tumors in these animals are osteosarcomas and soft tissue sarcomas (Harvey et al., 1993b). In addition, the genetic background of p 5 3 ( - / - ) mice has been demonstrated to affect the spectrum and timing of tumor formation. Pure 129/Sv mice with two p53 null alleles developed tumors sooner than the mixed genetic background p53-deficient animals (mix of 75% C 5 7 B L / 6 and 25% 129/Sv) (Harvey et al., 1993a). The pure 129/Sv p53-deficient mice also showed a greatly increased incidence of aggressive teratocarcinomas as compared to the mixed background mice. Thus, genetic background plays a role in both the rate and spectrum of tumor development in p53-deficient mice (Harvey et al., 1993a). Cell-cycle control, D N A damage and p53

While early work had demonstrated that p53 binds to the SV40 large T antigen, it was several years later that p53 was shown to compete with D N A polymerase a for SV40 T antigen binding (Gannon and Lane, 1987) and block SV40 origindependent D N A replication in SV40-transformed monkey cells (Braithwaite et al., 1987). Later, p53 was demonstrated to block in vitro SV40 D N A replication by inhibiting the initiation functions of SV40 large T antigen whereas mutant p53 failed to exert that effect (Wang et al., 1989). It has been hypothesized, therefore, that wild-type p53 interacts with a cellular homologue of the large T antigen to exert a negative affect on assembly of the D N A replication-initiation complex. Recently, the product of the MDM2 gene has been proposed as a candidate for cellular mediator of p53 function (Oliner et al., 1992). Meanwhile, it

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Fig. 6. Cell-cycletime comparisons of p53-deficientand wildtype embryonic fibroblasts. Embryonic fibroblasts were isolated from 13.5 day embryos and cultured in DMEM, 15% fetal calf serum. Early passage cells (passage 2) were plated at low density (150000 cells per 100 mm dish) and cell counts performed on triplicate plates at 24, 48, 72 and 96 h. Cell-cycle times were calculated during log phase growth.

has been known for some time that mutant p53 may bind wild-type p53 to form an inactive oligomeric complex and thereby constitute a dominant loss-of-function mutation (Eliyahu et al., 1988). Another line of investigation has focused on evidence that p53 binds D N A and can act as a transactivator of gene transcription (Fields and Jang, 1990; Raycroft et al., 1990), perhaps affecting the regulation of a set of genes involved in controlling passage from late G1 to S phase of the cycle. Indeed, cell-cycle perturbations due to p53 deficiency are clearly manifested in vitro by early passage, p 5 3 ( + / - ) and 1 9 5 3 ( - / - ) embryonic fibroblasts which demonstrate a markedly reduced cell-cycle time (Fig. 6). Any interpretation of p53 function based on in vitro studies must be made in light of the fact that mice homozygous for the p53 null allele develop normally. This fact indicates that p53 is not essential for normal cell division, cell differentiation, or embryonic growth and development (a conclusion that was not expected when the gene was originally targeted). The increased susceptibility of p53-deficient mice to formation of a wide variety of tumors indicates some sort of global protective function, perhaps related to cell-cycle control, that prevents a cell from entering a tumorigenic pathway. An early observation regarding the potential role of p53 was made in 1984, when it was demonstrated that mouse fi-

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broblast lines treated with ultraviolet light or UV-mimetic drugs rapidly increased p53 protein levels which was due in large part to increased stability of the protein (Maltzman and Czyzyk, 1984). Recently, an analogous result was reported in vivo with induction of p53 following irradiation of intact human skin (Hall et al., 1993). In addition, normal bone marrow myeloid progenitor cells and ML-1 myeloblastic leukemia cells have demonstrated similar increases in p53 protein levels after exposure to DNA-damaging agents such as 7-radiation and actinomycin D (Kastan et al., 1991). Notably, cells with wild-type p53 but not with mutant p53 displayed a temporary arrest in G1 of the cell cycle after exposure to the DNA-damaging agents and transfection of wildtype p53 into malignant cells lacking functional p53 resulted in a restoration of G1 arrest following ionizing radiation (Kastan et al., 1991). Corroborating results have been obtained using fibroblasts derived from p 5 3 ( - / - ) mice which failed to arrest in G1 after exposure to ionizing radiation (Kastan et al., 1992). The above results imply a role of p53 in cell-cycle control in response to DNA damage. Further evidence that p53 may be a part of a DNA-damage response pathway has been described by Kastan et al. using cells derived from patients with the radiation-sensitive, cancer-prone disease ataxia-telangiectasia (AT). These cells, like p53-deficient cells, did not arrest in G1 after exposure to ionizing radiation and, notably, did not demonstrate increased levels of p53. In addition, GADD45 (growth arrest and damage-inducible gene) was shown not to be upregulated in p53-deficient cells (Kastan et al., 1992). The above results have led the authors to propose a pathway of response to DNA damage in which radiationinduced DNA-damage results in an increase in p53 protein levels via AT gene products with subsequent induction of expression of GADD45 and other effector genes resulting in arrest of the cell in G1 until DNA damage may be repaired. While mediating G1 arrest to allow repair of DNA damage may be one function of p53 in the cell, another potential effect, apoptosis or programmed cell death, has been illustrated when wild-type p53 is added to certain types of tumor

cells. Two studies have demonstrated that the addition of wild-type p53 to myeloid leukemia cells (Yonish-Rouach et al., 1991) and a colon carcinoma cell line (Shaw et al., 1992) results in the induction of apoptosis. Importantly, apoptosis was significantly reduced in thymocytes of p53deficient mice after exposure to ionizing radiation (Clarke et al., 1993; Lowe et al., 1993). The accumulation of data linking p53 to a G1 arrest, DNA damage response and apoptosis has supported the formulation of models as stated by Kastan et al. (1992) and Lane (1992) in which p53 was visualized as a sort of molecular guardian for genomic integrity. In this framework, certain types of DNA damage induce or stabilize p53 which results in either (a) arrest of the cell cycle in G1 so as to allow for repair of the damage; or (b) destruction of the cell via apoptosis if DNA damage is beyond repair capabilities. The fate of cells that lack the p53 monitoring mechanism was speculated to be accumulation of mutations a n d / or chromosomal rearrangments after DNA damage which would allow for the selection of other oncogene and tumor-suppressor gene alterations which confer some growth advantage to the cell and ultimately result in tumor formation. Thus, neoplastic transformation and malignant progression would occur at a greater frequency in cells lacking p53. This model has recently been supported by in vivo evidence of increased rate of tumor formation after exposure of p53-deficient mice to DNA-damaging protocols. In a liver carcinogenesis protocol, p53( + / - ) mice exposed to low doses of dimethyl nitrosamine, showed a more rapid development of liver hemangiosarcomas that treated wild-type animals and significantly decreased survival time (mean 29 weeks vs. 42 weeks, Fig. 7) (Harvey et al., 1993b). Additionally, in a skin carcinogenesis protocol, treatment of p53-deficient mice with an initiator dimethylbenzanthracene (DMBA) and a promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) showed a more rapid rate of malignant progression as compared to wild-type mice (Kemp et al., 1993). Although tumor formation is an unambiguous endpoint for assaying effects of p53 deficiency in the whole animal, identifying intermediary effects on DNA and the genomic structure is consider-

A. Sands et al. / Mutation Research 307 (1994) 557-572

100

r~ e,

o E

40

20-

2o

40

60

TiME (WEEKS)

Fig. 7. Effects of dimethyl nitrosamine (DMN) on p53-deficient, heterozygous mice 1053(+ / - ) and normal mice. Twenty hetrozygous and 22 wild-type mice were treated with 0.0005% DMN in their drinking water continuously from the age of 8 weeks. Since mice on this regimen did not develop overt tumors, the endpoint of the study was spontaneous death of the mice. Liver and lung tissues were then examined grossly and by histologic section.

ably more difficult. There is some evidence that without p53 the integrity of the genome is indeed compromised under certain conditions. For example, after exposure to a uridine biosynthesis inhibitor, PALA, p53-deficient cells not only proceed into S phase more readily than cells with normal p53, but also demonstrate gene amplification (Livingstone et al., 1992; Yin et al., 1992). In addition, early passage embryonic fibroblasts derived from p53-deficient embryos ( p 5 3 - / - ) were capable of gene amplification in contrast to wild-type embryonic fibroblasts. These same p53-deficient fibroblasts became highly aneuploid in culture as compared to wild-type cells (Harvey et al., 1993c; Livingstone et al., 1992), suggesting that the loss of p53 contributes to chromosome breakage and genomic instability.

5. Future directions

Mice generated by homologous recombination that are p53-deficient have provided valuable in vivo evidence of the tumorigenic endpoint of the

569

p53-deficient state. These mice have also provided a source of ceils with well defned mutations as subjects for a variety of in vitro studies. Thus far two major endpoints of a model of p53 activity have been supported by in vivo and in vitro studies, specifically, the tumor formation (Donehower et al., 1992) and apoptosis (Clarke et al., 1993; Lowe et al., 1993). In addition, in vitro evidence of perturbations in cellular DNA organization such as gene amplification (Livingstone et al., 1992; Yin et al., 1992) and hyperdiploid DNA content have been reported (Harvey et al., 1993c). Still, an intermediate step on the proposed pathway, "survival with mutation" (Hall et al., 1993) has yet to be supported by direct experimental evidence. Such evidence may be obtained only by applying novel techniques that directly measure rates of mutation. As there is a large body of evidence that loss of function mutations of tumor-suppressor genes are major contributors to a tumorigenic state, the application of gene-targeting deletion strategies to study this class of genes promises to be a productive experimental paradigm. Beyond applications to tumor-suppressor genes, embryonic stem cells offer, of course, many experimental opportunities in genetics since virtually any genetic change can be introduced in vitro and established in a mouse line in vivo. Genetic alterations include large and small deletions, subtle nucleotide changes, or directed expression of a transgene by targeting it to the cis elements of an endogenous gene. There is considerable variability in vector design, depending on the exact experimental goals, but all share the characteristic of being able to direct specific changes in the target locus at its normal chromosomal location. Creating insertions within a n d / o r deletions of genes is by far the most common mutation introduced by gene-targeting strategies and may be readily accomplished by either insertion or replacement vectors. The Hit-and-Run strategy has been demonstrated to be an effective means of introducing more subtle mutations into the germ-line of mice. A strategy with potential to be the most versatile for introduction of mutations is double-hit gene replacement. While adequate efficiencies have yet to be demonstrated using this

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methodology, virtually any genetic change including deletions, insertions, exchange of control regions and exchange of coding sequences could theoretically be made and isolated by selection.

Acknowledgements We would like to thank our colleague Larry Donehower for his comments on the manuscript. Work in the Laboratory is supported by grants from the National Institutes of Health and The Howard Hughes Medical Institute. A.S. is a Fellow of the American Cancer Society. A.B. is an Associate Investigator with the Howard Hughes Medical Institute.

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