An apoptotic defect in lens differentiation caused by ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6142-6146, June 1995 Genetics

An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele (transgenic mice/dominant negative interference)

TAKAFUMI NAKAMURA*, JosEi G. PICHEL, LISA WILLLAMS-SIMONS, AND HEINER WESTPHAL Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Building 6B, Bethesda, MD 20892

Communicated by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, April 3, 1995

p53 expression on tissue differentiation and homeostasis in

If deprived of wild-type p53 function, the ABSTRACT body loses a guardian that protects against cancer. Restoration of p53 function has, therefore, been proposed as a means of counteracting oncogenesis. This concept of therapy requires prior knowledge with regard to proper balance of p53 function in a given target tissue. We have addressed this problem by targeting expression of the wild-type human p53 gene to the lens, a tissue entirely composed of epithelial cells that differentiate into elongated fiber cells. Transgenic mice expressing wild-type human p53 develop microphthalmia as a result of a defect in fiber formation that sets in shortly after birth. We see apoptotic cells that fail to undergo proper differentiation. In an effort to directly link the observed lens phenotype to the activity of the wild-type human p53 transgene, we also generated mice expressing a mutant human p53 allele that lacks wild-type function. A normal lens phenotype is restored in double transgenic animals that carry both wild-type and mutant human p53 alleles. Our study highlights the difficulties that can arise if p53 levels are improperly balanced in a differentiating tissue.

vivo.

The lens is an attractive tissue for studies of genes that may affect differentiation. Its simple structure is composed of epithelial cells that maintain a life-long program of differentiation into elongated fiber cells (19). Using a lens-specific crystallin promoter sequence, transgenes can be targeted to start their activity in the lens epithelial cells as these convert to fiber cells (20, 21). We purposely chose human p53 transgene sequences to be able to use an antibody that is specific for human p53 and does not recognize the endogenous murine p53 protein and also because human genes would be the ultimate target for gene therapy. As will be shown below, the human gene mimics its murine counterpart in predictable wild-type p53 functions. The human mutant p53 gene that we chose for our studies corresponds to a human colon cancer isolate and contains two missense mutations, a Pro -- Arg change in codon 72 likely to be silent (22) and a Val -) Ala substitution at codon 143 (23, 24). A study of the crystal structure of a p53-DNA complex has revealed that the peptide that carries the codon 143 mutation is one of a group of p53 gene products that cannot bind DNA because of structural defects in the peptide core domain (25). As a result, the ability to activate genes in trans is compromised (26). This dominant negative interference is thought to operate through the formation of mixed wild-type and mutant oligomers (27, 28). We generated several transgenic mouse lines that carry the wild-type and the mutant human p53 transgene, respectively, and selected for further analysis those that expressed the transgene in heterozygous form. We show that each gene generates a distinct lens phenotype and that the genes interact in vivo in a manner entirely consistent with the predicted mechanism of dominant negative interference.

In individual experiments, the nuclear phosphoprotein p53 has been observed to control a G1/S checkpoint in the cell cycle, to trigger apoptosis, and to respond to DNA damage (1-3). Each of these properties is compatible with its action as a negative regulator of malignant growth. Frequently observed mutations inactivate the ability of wild-type p53 to control the growth of malignant cells and can enhance susceptibility to cancer (4-6).

Wild-type p53 is a sequence-specific DNA-binding protein that acts as a positive transcriptional regulator of genes containing p53-binding sites (7). By contrast, it can also inhibit transcription of certain genes by interacting with TATAbinding proteins and thereby interfere with the basal transcriptional machinery (8, 9). Both properties of the protein may come into play during G1 arrest of the cell cycle via activation of growth-arrest genes and repression of cell cycle activators. Potential targets for positive transcriptional regulation in this context include the GADD45, WAFl/Cipl, and MDM2 genes (10-15). The loss of wild-type p53 function, either by gene ablation or via simultaneous over-production of a mutant allele, increases tumor incidence. Twenty percent of transgenic mice over-expressing murine mutant p53 (16) and 74% of mice homozygous for p53 gene ablation (17) develop a variety of tumors. Likewise, Li-Fraumeni patients that express a p53 mutant allele are cancer prone (5, 6). It has been postulated that oncogenesis involving p53-deficient cells may be counteracted by restoring wild-type p53 function (18). In this context, it appears useful to examine the effect of increased wild-type

MATERIALS AND METHODS Hybrid Gene Constructs and Transgenic Mice. The murine lens-specific aA-crystallin promoter and the second intron of the rabbit f3-globin gene were linked to gene sequences of either human wild-type p53 derived from pC53-SN3 (24) or human mutant p53 derived from pC53-SCX3 (24). A Sal I-BamHI fragment derived from pC53-SN3 and pC53-SCX3, respectively, was inserted between an Xba I-Xho I fragment that included the 409-bp aA-crystallin promoter from pMaACrl800 (29) and a 200-bp simian virus 40 poly(A)n signal sequence that is part of the vector plasmid pMSG (Pharmacia). A 3049-bp linearXba I-Sf I restriction fragment was purified and microinjected into zygotes to generate transgenic mice, as described (20, 21). Abbreviations: NT, nontransgenic; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling. *To whom reprint requests should be addressed at: National Institutes of Health, Building 6B, Room 211, Bethesda, MD 20892.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Genetics: Nakamura et al Histological Analysis and Immunohistochemistry of the Eye. The eyes were fixed in 10% formalin/phosphate-buffered

saline, embedded in methacrylate (see Figs. 2 A-D and 3) or paraffin (see Fig. 2 E-H), sectioned vertically parallel to the optical axis, and stained with hematoxylin/eosin. Immunoperoxidase staining of paraffin sections was done (21) by using two different monoclonal antibodies specific for human p53, DO-1 and BpS3-12 (Santa Cruz Biotechnology, Santa Cruz, CA), and the Vectastain ABC visualization kit (Vector Laboratories). Northern Blot Analysis. Total RNA was extracted from adult mouse eyes with RNAzol (Tel-Test, Friendswood, TX) according to the manufacturer's protocol. Total RNA extracted from both eyes of each transgenic mouse or normal mouse was electrophoresed in a formaldehyde/formamide/1% agarose gel, blotted, and hybridized to 32P-labeled probes. The human p53 probe (BamHI fragment of pC53-SN3) detects both wild-type and mutant human, but not mouse, transcripts. Other probes used were as follows: murine p53, Xba IlEcoRI fragment of pECM53; murine WAF1, EcoRI fragment of pCMW35; murine MDM2, BamHI fragment of clone liB cDNA; and murine GADD45, BamHI fragment of pXR45m (Chinese hamster cDNA). Hybridization was done at 65°C, and the final stringent wash was at 68°C in 0.2x standard saline citrate/0.1% SDS. Equality in the level of loading and the integrity of RNA were confirmed by hybridization with a rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe (30). Detection of Apoptosis. For in situ analysis (see Fig. 2 I-L), eyes were fixed with 10% formalin/phosphate-buffered saline, paraffin embedded, and cut in 5-p,m sections. Sections were incubated with terminal deoxynucleotidyltransferase (50 units per slide) and 1 nmol of biotin-16-dUTP (Boehringer Mannheim), as described (31). Biotin incorporation was detected with an avidin-biotin-peroxidase complex reagent (Vector Laboratories), followed by visualization with diaminobenzidine-H202 (Sigma). Sections were counterstained with 1% methyl green solution.

RESULTS Genotypes and Macroscopic Phenotypes of aAhp53wt and aAhp53mt Transgenic Mice. A wild-type and a mutant human p53 transgene were generated (Fig. 1). Both contain the murine aA-crystallin promoter sequence, the second intron of the rabbit 13-globin gene, the human p53 coding sequence, and a simian virus 40 poly(A) 3' end. Sequencing revealed two single nucleotide changes between the wild-type (aAhp53wt) and the mutant (aAhp53mt) construct (data not shown). The mutant allele, originally derived from a human colon cancer

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FIG. 1. Wild-type and mutant human p53 transgene constructs. The mouse lens-specific aA-crystallin promoter (cross-hatched bar) was linked to the second intron of rabbit 83-globin (single line), human p53 DNA sequences (hatched bar), and a simian virus 40 polyadenylylation signal (blank bar). The wild-type p53 gene was derived from pC53-SN3; the mutant gene was derived from pC53-SCX3, together with the rabbit B3-globin intron (24). Single nucleotide exchanges in the mutant allele have generated Tha I (T) and Hha I (H) restriction sites at codons 72 and 143, respectively. Arrows denote the start of transcription. A 3049-bp linear Xba I-Sfi I restriction fragment was used to generate transgenic mice as described (21, 22).

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isolate, contains a C -- G transversion in codon 72 (22) and a T -- C transition in codon 143 (23, 24). These mutations result in a Pro -> Arg change in codon 72 and a Val -* Ala replacement in codon 143. Although the codon 72 mutation may be considered silent (22), a study of the crystal structure of the human p53 core domain bound to a DNA-binding site has revealed that the codon 143 allele is one of a group of p53 mutations that cannot bind DNA because of structural defects in the peptide core domain (25). We generated a number of founders carrying integrated aAhp53wt and aAhp53mt transgenes, respectively, and selected for further analysis two heterozygous lines of either genotype that expressed these transgenes. The integrity of the integrated wild-type (lines Wi and W2) or mutant (lines Mi and M2) human p53 sequences was verified by restriction fragment length polymorphism and by heteroduplex analysis of PCR products (data not shown). Lines Ml and M2 (aAhp53mt) were characterized by faint bilateral lens cataracts, and lines Wi and W2 (aAhp53wt) by severe bilateral lens cataracts that developed after weaning. In adult Wi and W2 progeny, the cataract was accompanied by microphthalmia. Progeny of all four lines had normal life spans, and no lens tumors were ever observed. Lens Histology, p53 Immunostaining, and Terminal Deoxynucleotidyltransferase-Mediated dUTP-Biotin Nick-End Labeling (TUNEL) Staining. The histology of eyes derived from nontransgenic (NT), from aAhp53wt (Wi), from aAhp53mt (Mi), and from Wi/Mi double transgenic progeny is shown in Fig. 2. The antero-posterior eye section of Fig. 2A is derived from a 10-week-old NT animal. The eye is oriented with the anterior structures pointing upward. The transparent lens nucleus (n) is surrounded by a darker staining lens cortex (c). Fig. 2B shows the grossly distorted lens of an age-matched Wi littermate. Remnants of a lens nucleus inside an irregular mass of cortex structure are surrounded by large vacuoles, indicative of a profound disturbance of fiber differentiation. By contrast, no difference is noted at this magnification when comparing the eye section of an age-matched Mi animal (Fig. 2C) with the NT control (Fig. 2A). Wild-type and mutant p53 proteins are known to interact in vitro (28). In an effort to study a possible interaction in the living organism, we crossed aAhp53wt and aAhp53mt mice to obtain progeny that expressed both wild-type and mutant alleles of human p53. An eye section of a double-transgenic Wi/Mi animal closely resembles that of the NT mouse (compare Fig. 2 A and D). The mutant human p53 allele was thus able to rescue an apparently normal lens phenotype, possibly by dominant negative interference with the action of the wild-type allele (27). The observed rescue was not always complete. For example, when a cross was set up between the Mi line of aAhp53mt and the W2 line of aAhp53wt mice that display a more severe lens phenotype, W2/Ml double transgenic progeny resulted that showed only partial recovery of the lens phenotype. Likewise, a more recently obtained M2 allele, expressing noticeably less mutant human p53 RNA than Mi (see Fig. 4), was unable to mount a complete rescue of the lens phenotype in a double transgenic mouse that coexpressed the Wl/M2 allele (data not shown). These findings are suggestive of a dose dependency that is entirely compatible with proposed schemes of dominant negative interference between peptides (27). Unfortunately, much as our model system mimics a true in vivo situation, the complex structure of a developing eye does not lend itself easily to a precise quantitative analysis of wild-type p53 action and its interference by mutant p53 molecules. At birth the lens structure of Wi mice closely resembles that of a NT mouse (compare Fig. 2 E and G). Likewise, sections of Wi, Mi, and NT newborn eyes are indistinguishable with respect to immunostaining of a, ,B, or y crystallins, the main protein constituents of a differentiating mouse lens cell (data

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Proc. Natl. Acad. Sci. USA 92 (1995)

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FIG. 2. (A-D) Gross distortion of lens development visualized in an adult WI transgenic mouse expressing wild-type human p53 and rescue of a normal lens phenotype in an adult Wi/Mi double transgenic animal expressing both wild-type and mutant human p53 alleles. Hematoxylin/eosin-stained anteroposterior sections through methacrylate-embedded eyes of 10-week-old mice are oriented with anterior structures pointing upwards. The lens nucleus (n) and cortex (c) are marked in A. The eyes are derived from nontransgenic (NT, A), Wi (B), Ml (C), and Wi/Mi animals (D), respectively. (Bar = 500 ,um.) (E-H) Morphology and p53 immunostaining of NT and Wi paraffin-embedded newborn eyes. Human p53 was visualized by staining with monoclonal antibodies supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Note the positive Bp53-12 antibody staining of Wi cortical fiber cells in H. (Bar = 200 ,Lm.) (I-L) TUNEL staining for apoptotic lens cell nuclei in newborn eyes. High magnifications of the equatorial region of lens cell differentiation are shown. Note the positive staining of nuclei in the lens of a Wi animal (J). (Bar = 100 ,m.)

not shown). At this stage of lens development, the tissue expands via differentiation of equatorial epithelial cells into cortical fibers. In the newborn Wi eye (Fig. 2H) and the Mi eye (data not shown), we observe strong human p53 immunostaining in these cortical fibers, whereas no staining is seen in the control (Fig. 2F). This result suggests that the newly formed cortical fibers are the site of the deleterious action of wild-type human p53 that leads to a breakdown of lens development in the weeks after birth and that they are also the site of mutant human p53 action. Because cell apoptosis, marked by DNA breakdown and the accumulation of an excess of 3'-OH DNA ends in the cell .nucleus, is a hallmark of p53 action (2, 3), we used TUNEL (31) to visualize apoptotic nuclei in the equatorial zone of cell differentiation in the newborn lens. This area is shown at higher magnification in the newborn lenses of NT (Fig. 21), Wi (Fig. 2J), Mi (Fig. 2K), and Wi/Mi double transgenic mice (Fig. 2L). Staining (brown color) is clearly discernible in cell nuclei depicted in Fig. 2J. Also, a few nuclei stain lightly in Fig. 2I, K, and L, in areas where physiological denucleation of fibers occurs. We conclude that wild-type human p53-mediated apoptosis and, to a much lesser degree, the process of fiber denucleation, as well (32, 33), cause accumulation of 3'-OH ends in the context of DNA breakdown. As differentiating fibers deteriorate in the aAhp53wt lens, little or no additional fiber mass is added to the cortex, the lens structure deteriorates, and microphthalmia ensues (Fig. 2B). A slight retardation of the fiber denucleation process is the only phenotype that we can discern in aAhp53wt animals (Fig. 3). This phenotype has previously been observed in the context of a variety of gene products expressed in the transgenic lens (34, 35). Because of the delay in denucleation, we registered fewer TUNEL-positive nuclei in eye

sections of Mi and Wi/Mi animals than in the NT control (Fig. 2 K and L versus I). Expression of p53 and P53 Target Genes in aAhp53 Eyes. To investigate the pathway used by wild-type human p53 to exert its deleterious action on aAhp53wt lens development, total RNA was extracted from adult eyes of lines Wi and W2 and, by comparison, lines Mi and M2 and subjected to Northern blot analysis (Fig. 4). All transgenic lines express human p53 RNA at levels that are substantially higher than those of endogenous murine p53 RNA (note the difference in film exposure time). However, there is no evidence that the presence of human p53 RNA affects the levels of endogenous p53 transcripts. It is of interest to note that the levels of WAFi/Cipl transcripts are markedly elevated in aAhp53wt but are not elevated in aAhp53mt eyes (Fig. 4). The WAFl/ Cipl gene is regulated by wild-type p53 and encodes a protein that can inhibit cyclin-dependent kinases (13-15). The GADD45 gene may likewise be transcriptionally regulated by wild-type p53 in the DNA-damage response pathway (10). Previous findings indicate that MDM2 is also induced by wild-type p53 and regulates p53 activity (11, 12). We find MDM2 expression in the aAhp53wt lines to be slightly increased, whereas expression of the GADD45 gene appears unaffected by exogenous p53 (Fig. 4). It has been argued that, depending on the cell context, p53 mediates negative regulation of cell growth, either by precipitating apoptosis (2, 3) or by activating cell cycle regulators that halt the cycle at the G1/S transition (1). With respect to wild-type human p53 function in the developing lens, the positive TUNEL staining in the nuclei of differentiating aAhp53wt lens fiber cells (Fig. 2J) would argue in favor of the former possibility; the increase of WAFl/Cipl expression in the aAhp53wt eye (Fig. 4) would support the latter. However, we were able to rule out a connection between an increase in

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FIG. 3. Mutant p53 lens phenotype. Hematoxylin/eosin-stained anteroposterior sections through methacrylate-embedded eyes of 4-week-old animals are oriented with anterior structures pointing upwards. High magnifications of the equatorial region of lens cell differentiation are shown. The eyes are derived from a NT (A) and an aAhp53mt line Ml mouse (B), respectively. Note the delay of the denucleation process in the lens of a Ml animal (arrow in B). The arrowheads in B point to pycnotic or fragmented nuclei. (Bar = 100

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WAFl/Cipl expression and the observed lens phenotype because Wi/Mi double transgenic eyes (Fig. 5), while not undergoing fiber apoptosis (Fig. 2L), still express elevated aAhpS3mt

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levels of WAFl/Cipl (Fig. 5). We have confirmed the presence of elevated levels of WAFl/Cipl in the eyes of several additional Wi and Wi/Mi progeny (data not shown). This result suggests that the mutant p53 allele was able to suppress wild-type p53-mediated lens cell apoptosis but not the induction of a transcriptional activation pathway that connects to cell cycle arrest.

DISCUSSION Our results show that over-expression of wild-type human p53 in the lens of transgenic mice causes apoptosis of differentiating lens cells after birth. The deleterious function of wildtype p53 is related to its level of expression and can result in severe microphthalmia in the adult transgenic mouse. By contrast, eyes of transgenic mice that express excess amounts of the mutant human p53 allele display a faint central cataract caused by the presence of remnants of fiber cell nuclei. In mice that express both the wild-type and the mutant human p53 gene product, the mutant p53 allele can interfere with the apoptotic function of the wild-type product but not with its ability to activate WAFi/Cipl and MDM2. Our result is consistent with recent observations that p53-dependent apoptosis does not require transcriptional activation of a number of known p53 target genes (36, 37). Microphthalmia as a result of excess wild-type p53 action constitutes a drastic example of the power of p53 action. Differentiating lens cells accumulate the transgenic p53 product after birth, their nuclei show biochemical evidence of DNA breakage, and their conversion to elongated fibers is interrupted. Therefore, all available evidence links the breakdown of the epithelial architecture of the lens to the well known ability of wild-type p53 to trigger cell apoptosis. Equally well known is the fact that a subset of p53 mutant alleles can counteract the negative controls exerted by the wild-type product (38) and are often retained during cancer progression (4). This gain of function was initially documented in vitro by the ability of p53 mutants to cooperate with activated ras genes in the transformation of rat embryo fibroblasts (39). These experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type protein and have led to the concept of "transdominant negative" effects of p53 mutations (40). The ability of mutants to decrease the binding to DNA and, thus, the transcriptional activity of the wild-type protein has been interpreted as a dominance of mutant p53 over wild-type p53 function (26, 28). The accumulation of p53 mutations in many

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human tumors may well reflect a selection process favoring the malignant growth of those cells that use mutant alleles to dominantly interfere with the apoptotic function of wild-type

p53 (41).

Our study, together with recent observations on retinoblastoma gene function in the developing lens (42), highlights the importance of apoptosis as a consequence of wild-type p53 action in the living organism. Not only does the protein induce apoptosis as an answer to tissue damage in vivo (43), but our results also suggest that it can do so by itself when produced in excess of a normal endogenous dosage. Although we have used human p53 alleles in our study, the strong functional resemblance of human and murine wild-type p53 products (8, 13, 28) leaves little doubt that increased expression of murine wild-type p53 would have similar effects. Our findings caution against an overly optimistic view with respect to a possible medical application of wild-type p53 as a tumor-suppressing agent. A proportion of tumors could conceivably muster resistance if they express mutant alleles that function like the one tested in this study. And even in tumors that do not contain functional p53 mutant alleles, precise targeting of wild-type p53 to tumor cells would be required to prevent apoptosis of adjacent healthy and essential tissue. We thank Dr. Sam Benchimol, Ontario Cancer Institute, Toronto, for pECM53; Dr. Bert Vogelstein, Johns Hopkins University, Baltimore, for pC53-SN3, pC53-SCX3 and pCMW35; Dr. Albert J. Fornace, Jr., National Institutes of Health, Bethesda, for pXR45m; Dr. Arnold J. Levine, Princeton University, Princeton, for MDM2 clone llB; Ms. Sing Ping Huang, Ms. Kveta Cveklova, Ms. Nicole Newman, and Ms. Mary-Alice Crafford for expert technical assistance; and Dr. Paul Love for his helpful comments on the manuscript. 1. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V. & Kastan, M. B. (1992) Proc. Natl. Acad. Sci. USA 89, 7491-7495. 2. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A. & Jacks, T. (1993) Nature (London) 362, 847-849. 3. Shaw, P., Bovey, R., Trady, S., Sahili, R., Sordat, B. & Costa, J. (1992) Proc. Natl. Acad. Sci. USA 89, 4495-4499. 4. Levine, A. J., Momand, J. & Finlay, C. A. (1991) Nature (Lon-

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