Vol. 5, 71 1-721, July 1994 Cell Growth & Differentiation 711 Preferential Overexpression of a 1 72’”’ Leu Mutant p53 in the Mammary Gland of Transgenic Mice Results in Altered Lobuloalveolar Development1 Baohin Li, Norman Greenberg, 1. Clifton Stephens, Raymond Meyn, Daniel Medina, and Jeffrey M. Rosen2 Department of Cell Biology, Baylor College of Medicine lB. L., N. G., D. M., I. M. RI, and Department of Urology IN. G.l, and Departments of Veterinary Medicine and Surgery IC. 5.1 and Experimental Radiotherapy IR. Ml, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Abstract Regulatory sequences derived from the rat whey acidic protein gene have been used to preferentially overexpress a murine 1 72’ LC’U mutant p.53 in the mammary gland of transgenic mice. Several different lines of mice expressing the 1 72’’ mutant p.5.3 displayed an impaired ability to lactate, and the mice expressing the highest levels of mutant p.5.3 were unable to nurse their young. This failure was related to the inhibition of normal lobuloalveolar development that occurred during late pregnancy and a marked decrease in milk protein gene expression at early lactation. Interestingly, immunohistochemical analysis revealed that the mutant p53 was localized predominantly in the cytoplasm of alveolar cells. Ductal development was not overtly impaired in these mice. Expression of the 1 72Arg-.Leu mutant p53 resulted in radiation-induced apoptosis, and transactivation or repression of the expression of a number of genes, including mdm-2 and proliferating cell nuclear antigen, known properties of wild-type p53. The availability of lines of mice preferentially expressing specific p53 mutants in the mammary gland should facilitate evaluation of the roles of other factors, such as hormones, oncogenes and chemical carcinogens, in the etiology of breast cancer. Introduction Mutations in the p53 gene have been observed in about 50% of frequently observed human cancers and are the most common genetic alterations detected to date in all human cancers (1-3). Alterations in four highly conserved regions of p53 account for most of the observed mutations (4). In general, these mutations appear to abrogate the normal function and extend the half-life of the p53 protein. Some p53 mutations can act in a dominant-negative man- ner such that specific mutations at a single allele can block normal p53 function (3). Some specific p53 mutations result in a gain of function (5), and tumors with mutant missense p53 proteins may be more aggressive than those without any p53 protein. This is not unexpected since different domains of the p53 protein have been identified that are important for transcriptional transactivation (6, 7), nuclear translocation (8, 9), and in- teraction with a variety of other proteins such as mdm-2 (1 0), SV4O T-antigen (1 1 ), TATA-binding protein (1 2), hsp70 (13, 14), and the DNA replication factor, replication protein A (1 5). As defined primarily by cell transfection experiments, the various mutant alleles of p53 have been demonstrated to exhibit markedly different biological and biochemical properties (16). Expression of mutant p53 protein has been associated with high tumor proliferation rates, early disease recur- rence, and early death in node-negative breast cancer (17, 1 8). Two distinct mechanisms appear to account for the altered forms of p53 observed in breast cancer: mutation and nuclear exclusion (1 ). Missense mutations are by far the most common p53 mutations observed in breast cancer. Usi ng immunoh istochemical methods, abnormal p53 pro- tein has been detected in primary breast cancer, and it has been estimated that approximately 50% of tumors contain altered p53 protein (3, 4, 19-22). Recent studies have demonstrated that the effects of p53 mutants defined in vitro and in transfected cells may differ from those observed in situ in the whole animal (23). Evi- dence that overexpression of a mutant p53 gene contributes to the oncogenic process in vivo has been obtained in only one case for a temperature-sensitive p53 mutant (tsi 35; mouse p53) and resulted in lung, bone, and lymphoid tumors (24). Mice deficient for one or both p53 alleles are developmentally normal but susceptible to spontaneous development of different types of tumors, particularly lym- phomas and sarcomas (25). Heterozygous and homozygous p53 knockout mice have also been used to study the role of p53 in apoptosis (26) and in progression of chemically induced skin tumors to the malignant state (27). These studies have shown that, although not required for normal mouse development, p53 is an important regulator of the cellular response to DNA-damaging agents. The p53 knockout mice, while of enormous utility, present some limitations for the study of breast cancer. When homozy- gous p53 knockout mice were administered the chemical carcinogen DMBA,3 the mice die from lymphomas and sar- comas before they developed mammary tumors.4 Further- more, heterozygous p53 mice and wild-type littermate con- trols exhibited no difference in susceptibility to induction of mammary tumors by oral administration of DMBA, and mu- tations in the wild-type p53 allele in the carcinogen-induced tumors were infrequent (28). Since the loss of a normal p53 allele is sufficient to predispose an animal to many types of Received 2/17/94; revised 4/25/94; accepted 5/5/94. This work was supported by Grants CAl 6303 (to J. M. R.), CA06294 (to R. E. Ml, CA58028 (to N. M. G.), and CA25215 (to D. M.) from the NIH. 2 To whom requests for reprints should be addressed, at Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. 3 The abbreviations used are: DMBA, dimethylbenzlalanthracene; cDNA, complementary DNA; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; UTR, untranslated region; poly(A) , polyadenylated; PCNA, proliferating cell nuclear antigen; bp, base pair(s). 4 D. Medina and L. Donehower, personal communication.
Preferential Overexpression of a 1 72’�”�’ Leu Mutant p53in the Mammary Gland of Transgenic Mice Resultsin Altered Lobuloalveolar Development1
Baohin Li, Norman Greenberg, 1. Clifton Stephens,Raymond Meyn, Daniel Medina, and Jeffrey M. Rosen2
Department of Cell Biology, Baylor College of Medicine lB. L., N. G.,
D. M., I. M. RI, and Department of Urology IN. G.l, and Departments ofVeterinary Medicine and Surgery IC. 5.1 and Experimental Radiotherapy
IR. Ml, The University of Texas M. D. Anderson Cancer Center, Houston,Texas 77030
AbstractRegulatory sequences derived from the rat whey acidicprotein gene have been used to preferentiallyoverexpress a murine 1 72�’ LC’U mutant p.53 in themammary gland of transgenic mice. Several differentlines of mice expressing the 1 72’�’ mutant p.5.3displayed an impaired ability to lactate, and the miceexpressing the highest levels of mutant p.5.3 were unableto nurse their young. This failure was related to theinhibition of normal lobuloalveolar development thatoccurred during late pregnancy and a marked decreasein milk protein gene expression at early lactation.Interestingly, immunohistochemical analysis revealedthat the mutant p53 was localized predominantly in thecytoplasm of alveolar cells. Ductal development was notovertly impaired in these mice. Expression of the1 72Arg-.Leu mutant p53 resulted in radiation-inducedapoptosis, and transactivation or repression of theexpression of a number of genes, including mdm-2 andproliferating cell nuclear antigen, known properties ofwild-type p53. The availability of lines of micepreferentially expressing specific p53 mutants in themammary gland should facilitate evaluation of the rolesof other factors, such as hormones, oncogenes andchemical carcinogens, in the etiology of breast cancer.
Introduction
Mutations in the p53 gene have been observed in about50% of frequently observed human cancers and are themost common genetic alterations detected to date in allhuman cancers (1-3). Alterations in four highly conservedregions of p53 account for most of the observed mutations(4). In general, these mutations appear to abrogate thenormal function and extend the half-life of the p53 protein.Some p53 mutations can act in a dominant-negative man-ner such that specific mutations at a single allele can blocknormal p53 function (3).
Some specific p53 mutations result in a gain of function(5), and tumors with mutant missense p53 proteins may bemore aggressive than those without any p53 protein. This is
not unexpected since different domains of the p53 proteinhave been identified that are important for transcriptionaltransactivation (6, 7), nuclear translocation (8, 9), and in-teraction with a variety of other proteins such as mdm-2(1 0), SV4O T-antigen (1 1 ), TATA-binding protein (1 2),hsp70 (13, 14), and the DNA replication factor, replicationprotein A (1 5). As defined primarily by cell transfectionexperiments, the various mutant alleles of p53 have beendemonstrated to exhibit markedly different biological andbiochemical properties (16).
Expression of mutant p53 protein has been associatedwith high tumor proliferation rates, early disease recur-rence, and early death in node-negative breast cancer (17,1 8). Two distinct mechanisms appear to account for thealtered forms of p53 observed in breast cancer: mutationand nuclear exclusion (1 ). Missense mutations are by far themost common p53 mutations observed in breast cancer.Usi ng immunoh istochemical methods, abnormal p53 pro-tein has been detected in primary breast cancer, and it hasbeen estimated that approximately 50% of tumors containaltered p53 protein (3, 4, 19-22).
Recent studies have demonstrated that the effects of p53mutants defined in vitro and in transfected cells may differfrom those observed in situ in the whole animal (23). Evi-dence that overexpression of a mutant p53 gene contributesto the oncogenic process in vivo has been obtained in onlyone case for a temperature-sensitive p53 mutant (tsi 35;mouse p53) and resulted in lung, bone, and lymphoidtumors (24). Mice deficient for one or both p53 alleles aredevelopmentally normal but susceptible to spontaneousdevelopment of different types of tumors, particularly lym-phomas and sarcomas (25). Heterozygous and homozygousp53 knockout mice have also been used to study the role ofp53 in apoptosis (26) and in progression of chemicallyinduced skin tumors to the malignant state (27). Thesestudies have shown that, although not required for normalmouse development, p53 is an important regulator of thecellular response to DNA-damaging agents.
The p53 knockout mice, while of enormous utility, presentsome limitations for the study of breast cancer. When homozy-gous p53 knockout mice were administered the chemicalcarcinogen DMBA,3 the mice die from lymphomas and sar-comas before they developed mammary tumors.4 Further-more, heterozygous p53 mice and wild-type littermate con-trols exhibited no difference in susceptibility to induction ofmammary tumors by oral administration of DMBA, and mu-tations in the wild-type p53 allele in the carcinogen-inducedtumors were infrequent (28). Since the loss of a normal p53allele is sufficient to predispose an animal to many types of
Received 2/17/94; revised 4/25/94; accepted 5/5/94.� This work was supported by Grants CAl 6303 (to J. M. R.), CA06294 (to R.
E. Ml, CA58028 (to N. M. G.), and CA25215 (to D. M.) from the NIH.2 To whom requests for reprints should be addressed, at Department of CellBiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
3 The abbreviations used are: DMBA, dimethylbenzlalanthracene; cDNA,
contains the WAP promoter-WAP5’ UTR, the wild-type murine p53
genomic DNA (containing exons2-1 1 and introns 2-9)-WAP 3’UTR, and polyadenylation sig-
nals. The codon 1 72 is the wild-
type CGC, Arg. 2. The mutant p53minigene construct with the WAP
3’ RNA processing signals is iden-tical to that shown in 1., except it
contains the codon 1 72, CTC
(Leu) mutation. 3. The mutant p53minigene construct with the SV4O
RNA processing signals is identi-
cal to that shown in 2., except thatthe WAP RNA processing signalshave been replaced by those from5V40 as described in “Materials
and Methods.”
712 p53 Transgenic Mouse Model
5 M. Osbun, personal communication.
tumors, an oncogenic mutant form of p53 protein may not benecessary for tumorigenesis (25). However, there is increasingevidence to support the hypothesis that specific p53 mutationsmay represent a gain-of-function and confer a selective ad-vantage upon specific target cells to facilitate their progressionto a more aggressive phenotype.
Since a heterologous gene can be targeted preferentiallyto the mammary gland using regulatory sequences charac-terized both in our laboratory and by others (29-31), it isnow possible to study the effects of overexpression of mdi-
vidual p53 mutants against a wild-type p53 background intransgenic mice to determine whether a specifically mu-tated p53 allele plays a causative role in either the initiationor progression of breast cancer. Of the missense mutationsthat have been reported in human breast tumors, mutationsat amino acid 1 75 are among the most frequent and ac-count for more than 8% of the total missense mutationsdetected to date (2).� Accordingly, in this study we haveexamined the effects on mammary gland development oftargeted overexpression of a specific Arg- Leu mutation atamino acid i 72 in the mouse p53 gene (equivalent toamino acid 175 in human pS3), a result of a G-*T trans-version identified in several breast cancer biopsies (2). Sur-prisingly, the 1 72Arg-. Leu p53 mutant exhibited severalproperties characteristic ofwild-type p53 protein, and over-expression ofthe mutant resulted in the inhibition of normallobuloalveolar development in the mammary gland. Theavailability of lines of mice preferentially expressing spe-cific p53 mutants in the mammary gland should facilitateevaluation of the roles of other factors such as hormones,oncogenes, and chemical carcinogens in the etiology ofbreast cancer.
Results
Construction of WAP-p53 Minigene Constructs ContainingSpecific Mutations. In order to generate mutant p53 con-structs that could be expressed preferentially in the mam-mary gland in transgenic mice, we used regulatory elements
from the rat whey acidic protein ( WAP) gene that have beendemonstrated to be capable of directing high level, tissue-specific expression of heterologous transgenes (30-32). TheWAP regulatory elements were chosen because of theirmore restricted pattern and reproducibly higher levels ofexpression than those derived from �3-casein in transgenicmice. In previous studies, rat �-casein-SV40 Tag constructshad resulted in lymphomas but no breast carcinomas intransgenic mice, most likely due to expression in thymiccytotoxic 1-lymphocytes (33).
To facilitate efficient transgene expression, a genomicminigene construct was used in these studies rather that acDNA-based construct, because it has been demonstratedthat introns are important for efficient expression, as well asappropriate transgene architecture (34, 35). It had beendemonstrated previously that a murine p53 minigene lack-ing introns 1 and iO could be expressed in transgenic mice(34); therefore, this minigene was used as the starting pointfor the construction of the WAP-p53 expression vector.However, the available mouse p53 minigene contained the
tsl 35 Ala-s Val mutation present in the original p53 clone;therefore, it was necessary to first generate a wild-type p53construct (Fig. 1 , 1.). This was accomplished using a doublerecombinant PCR method as described in “Materials andMethods.” The wild-type construct was then used to gen-erate the Arg-e Leu mutation at amino acid 1 72. In addition,WAP-p53 constructs were generated that contained eitherthe SV4O or WAP 3’UTRs and polyadenylation signals.
These constructs are illustrated in Fig. 1 . The constructdesignated pBLi 03D carries the WAP promoter sequencesand 5’ UTR, the murine p53 minigene with the codon 172Arg-s Leu mutation, and the SV4O poly(A)� signals (Fig. 1,3.). The construct designated pRL4 is identical to pBLi 03D,except that it carries the WAP 3’ UTR and poly(A)� signals(Fig. 1 , 2.). Both of these constructs were used to generatetransgenic mice, and a total of seven founders carrying theWAP-mtp53-SV4O and eight founders carrying theWAP-mtp53-WAP 3’ UTR transgenes were identified byPCR. Southern blot analysis of the positive founder miceindicated that all transgenes were integrated in a head to tailarrangement (data not shown; Fig. 3).
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Fij,’. .2. Expression ot mutant vS I in the mammary gland of transgenic mi( #{128}‘.
A. RNA blot analysis of total RNA isolated from the mammary gland at (lay
11) 01 lactation eX(’l’t)t for line 449 1 in which biopsies were pertorme(linlnle(Iiat(’ly after birth. Line 1. F , oifspring 01 line 449 1 ; Lane 2, F,, of line
449 1 ; Lant’ 1, F,, 01 line 4�)22; Lane 4, F,) of line 4562; Lane 5, F,, of line
4687; 1 eu (,, FVB m)ntransgeni( (ontn)l. Lanes 2- 6 contain 20 pg o totalRNA, exposure time 01 18 h; line I contains 5 pg ol RNA, exposure 1mw 5mm. 13, qUantitative RT-PCR .tnalysis of p53 expression. Total RNA ) 1 . i 0,
and 11)1) rig) was used br reverse trans( ription as clescrilx’(l in “Materials and
Methods The’ PCR l)rimers used were P1 and P2 shown in Fig. 1 . Cyclelim(’s were I 1, 2 1 . and 2 3 as illustrated on the figure for each RNA input. Thehybridiiation prolx’ was a ‘‘t’-kinase-lal)eled oligonucleotide from exon 2,
.111(1 tli#{128}exposure tinie was 5 nun. A2, A4, A5, and A3 refer to the same RNA
sanlpl(”. .inalyied in A, L,i,ies .2. 4. 5. and 1, respectively.
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Expression of Mutant p53 in the Mammary Gland ofTransgenic Mice. All of the positive founder transgenicmice were screened initially for the expression of mutantp53 using RT-PCR. Two of the lines containing thepBLiO3D construct (lines 449i and 4562) and two contain-ing the pRL4 construct (lines 4922 and 4687) displayeddetectable levels of pS3 expression in RNA samples isolatedfrom the mammary glands of transgenic mice at i 0 days oflactation (Fig. 2B). Sequence analysis of the PCR productsindicated that the p53 transcripts contained the appropriatemutation at codon 1 72 (data not shown). The use of differ-ent PCR primer sets established that the majority of thetranscripts in all but line 4687 were correctly spliced andcontained the expected 5’ and 3’ termini (Fig. 28). Thesmaller transcript detected in line 4687 indicated the exis-tence of an alternatively spliced p53 transcript (Fig. 2B).Quantitation of the RT-PCR results (shown in Fig. 28) mdi-cated that lines 4491 and 4922 expressed the highest levelsof p5.;1 RNA.
Northern l)lot analysis confirmed that the RT-PCR data forthe relative abundance of the mutant p53 transcripts in thefour founder lines and that expression was the highest inlines 4491 and 4922 (Fig. 2A). Mutant p53 mRNA wasreadily detected in 20 pg of total RNA after an 1 8-h expo-sure (Fig. 2A, Lanes 2 and 3, respectively). Under theseconditions, mutant p53 transcripts were not detected inmammary gland RNA from lines 4562 and 4687 (Fig. 2A,Lanes 4 and 5). Furthermore, no wild-type p53 transcriptswere detected in RNA isolated from the mammary gland ofa lactating nontransgenic littermate (Fig. 2A, Lane 6). Thetranscripts were slightly different sizes due to the presence
I 2 3 .4 5 () 7 5 () �O
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Fig. 3. Southern blot analysis of line 4491 . Genomic DNA (10 pg) wascligeste(l l)y B.iriHI, and the Southern blots were hybridized with a probespanning exon 2 and intron 3 of the murine pSi gene. The 2. 3-kb internal
tragment derived from the transgen( as well as a 5.6-kb fragment derived
froni the endogenous p5 J gene. Lane 1. F,, of line 4491 ; Lines 2, 4. 6, and
9are DNA from F offspring of line 4491 mice 5071 , 5074, 5075, and 6094,respectively; Line 5, an otfspring of mouse 5074; Lanes 7 and 8, two
otfspring of mouse 5075; Lanes .1 and 10, FVB nontransgeni( controls.
othe WAPor SV4O 3’ sequences (Fig. 2A). Both high andlow level expressing lines of mice were generated from both
constructs independent of the presence of the WAP 3’ UTRand 3’ processing or SV4O poly(A)* signals.
In all cases, the founders transmitted the transgenes totheir offspring, and WAP-p53 transgene expression was
detected in RNA isolated from the mammary glands of theoffspring. However, for line 449i , a several hundred-foldhigher level of transgene expression was observed in RNAisolated from the mammary gland of one of the four positivefemale F offspring (mouse 507i ; Fig. 2A, Lane 1; 5 pg oftotal RNA; 30-mm exposure time) at day 1 of lactation ascompared to founder 449i at day iO of lactation. In addi-tion, ofthe four female offspring ofline 4491 , only two wereable to successfully nurse their offspring. The litters of theother two F females (mouse 5071 and 5074) died shortlyafter birth, and after several attempts, one litter was main-tamed by foster nursing the pups. A male offspring was usedto carry the line.
To characterize these lines further, quantitative Southernanalysis was performed on each of the four founder linesand on the F1 and F offspring of line 4491 (Fig. 3). Com-parison of the RNA and DNA analyses indicated that thelevel of p53 expression was not copy-number dependentfor either construct. Copy number analysis for line 4491(Fig. 3) indicated that the offspring that failed to nurse theirpups contained approximately three times the number oftransgene copies as the founder (45 versus 1 5 copies) ortheir sisters that were able to successfully nurse their young.The F1 and F, female offspring that contained the higherCopy numbers of the transgene also expressed a corre-spondingly higher level of the mutant p53 mRNA.
These results suggested that the founder female of line4491 may have been mosaic and carried transgenes inte-grated at more than a single chromosomal site. This wasdifficult to confirm by Southern blot analysis because ofmultiple transgene copies and incomplete restrictionenzyme digestions with the limited number of suitable re-striction endonucleases with a unique cleave site in the
714 p53 Transgenic Mouse Model
Fig. 4. Preferential overexpression of mutant p53 in the mammary gland.RNA blot analysis (20 rig) of p53 expression in different tissues of the F,
offspring of line 4491 (A and B) and from a nontransgenic FVB mouse of thesame age (C), both at 2 days of lactation. The tissues assayed are: ma,
mammary gland; br, brain; lu, lung; sk, skin; he, heart; in, intestine; ki,kidney; sp, spleen; Ii, liver; ov, ovary; and th, thymus.
transgene. However, fluorescence in situ hybridizationanalysis confirmed the presence of two independent trans-gene integration sites on separate chromosomes that weredifferent from the location of the wild-type p53 alleles.Based upon the hybridization intensity, one of these sitesappeared to contain a several-fold higher copy-numberthan the other site, consistent with the Southern blot results(data not shown).
Preferential Overexpression of Mutant p.5.3 in the Mam-mary Gland. To determine the tissue distribution pattern
and relative levels of mutant and wild-type p53 expressionin these mice, RNA was extracted from the mammary glandand a variety of other organs at 2 days of lactation fromtransgenic mice and nontransgenic littermates and ana-lyzed by Northern blotting. As shown in Fig. 4, the WAP-mutant p53 minigene was preferentially expressed in themammary gland during lactation, but expression was alsodetected in the lung, spleen, and ovary. Previous results hadsuggested that intragenic sequences in the p53 gene mightregulate its pattern of tissue-specific expression, and, spe-cifically, that the fourth intron carried an enhancer respon-sible for the high level of expression normally observed inthe spleen (34). However, direct quantitation of the North-em blots showed that the expression in the mammary glandwas approximately 25-fold greater than in any of the othertissues. Furthermore, the mutant p53 transcripts were ex-pressed at least at 100-fold greater levels than endogenouswild-type p53 in the mammary gland (Fig. 4, B and C).
Analysis of mutant p53 expression in the mammary glandat different stages of development detected a low level oftransgene expression in the virgin mammary gland andduring early pregnancy. As expected from previous studieswith WAP constructs, precocious transgene expression (ascompared to the endogenous WAP gene) occurred duringmid-pregnancy (day 10) with a further increase during latepregnancy (day 1 5-i 9) and at 2 days of lactation (data notshown). Thus, the regulatory sequences in the WAP pro-moter fragment were sufficient to preferentially target p53
expression to the mammary gland despite the fourth intronsequences.
Consequences of Mutant p.53 Overexpression. In addi-tion to the inability to lactate by the high copy-numberoffspring of line 4491 , it appeared that there may have beena general reduction in the lactation capacity of all four lines.This was confirmed by comparing the pups’ weight at dif-ferent times during lactation of all four litters of the trans-genic lines as compared to nontransgenic control mice. Asillustrated in Fig. 5, there was a highly significant reductionin the pup weight of 20-60#{176}k (P < 0.01) in the differenttransgenic lines independent of the litter sizes.
In order to determine the basis for the decreased lactationcapacity observed in several of the transgenic lines and thecomplete failure to lactate in the high copy-number off-
spring of line 4491 , whole mount and histological analysesof the mammary glands were performed at different stagesof development. Compared to nontransgenic littermates,only minor differences in all cases were observed in themammary gland morphology at different stages of develop-ment, except for the high copy-number offspring of line4491 . In the latter case, a slight abnormality in ductalmorphology was observed as a nonuniform ductal diameterin the virgin gland, and a slight decrease in the extent oflobuloalveolar development was observed in whole mountsduring mid-pregnancy (data not shown). The histogical ex-amination revealed little difference in the extent of lobu-loalveolar development at day 15 of pregnancy (Fig. 6, Aand C); however, a striking difference in the histology of thegland was observed during lactation (Fig. 6, B and E).Instead of the extensive lobuloalveolar development thatusually fills the mammary fat pad, the high copy-numberoffspring of line 4491 displayed incomplete developmentwith few normal lobuloalveolar structures (Fig. 6E). Thisphenotype was seen to a lesser extent in the other highexpressing lines.
It appeared from these studies that there might have beenan incomplete penetrance of transgene expression in themammary gland of line 4491 . Direct confirmation of thishypothesis was obtained using immunohistochemical anal-ysis to examine mutant p53 expression directly in the mam-mary gland at different stages of development (Fig. 6). Usingthe polyclonal antibody CM5, intense cytoplasmic stainingfor mutant p53 was observed in the majority of alveolarcells at day 1 5 of pregnancy (Fig. 6C, arrow 1; 6D, arrow 1).Little positive staining was observed in ductal cells (Fig. 6C,
arrow 2), and some alveolar cells were clearly negative aswell (Fig. 6D, arrow 2). Analysis of mutant p53 expressionin the mammary gland samples obtained immediately afterbirth revealed intense cytoplasmic staining in all cells in the
collapsed alveoli that also appeared to be non-secretory(Fig. 6, F-H, arrow 1). In contrast, the large, apparentlynormal lobuloalveolar structure (seen in Fig. 6, E and F,arrow 1) did not express detectable p53. These resultssuggest that the alveolar cells that overexpress mutant p53
48
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NON-TRANSGENIC MUTANT
Cell Growth & Differentiation 715
f. D. Creenhalgh, D. Roop, B. Li, and I. Rosen, unpublished observations.
Fit,’. 5. Mutant p53 expression in the mammary gland results in an impairment of lactation. The weight of the pups determined at 10 (lays of lactation is shownbr sever,tl different transgenic lines and two nontransgenic FVB age-matched controls. The liner sizes are shown above the bus. The solid bars are line 4922;
the horizontal hat(’he(l b.irs are line 4687; the gray bars are line 4491 ; and the slanted hatched bars are line 4562. t, death of the PUpS and failure to lactate.
during pregnancy fail to proliferate and do not generatenormal lobuloalveolar structures, while the small percent-age of cells that fail to express mutant p53 during pregnancymay continue to develop normal alveoli in early lactation.Similar immunohistochemical results were obtained for theother lines expressing the higher levels of mutant p53, butthe intensity of the staining and the phenotypic effects onmammary gland development were less pronounced (datanot shown). Thus, the failure to express the mutant p53transgene in all the alveolar cells, and the requirement for acritical threshold of overexpression relative to the wild-type, may account for the differences in the severity of thephenotype observed during lactation.
In general, there were few other phenotypic conse-quences of overexpression of mutant p53 in these lines ofmice. No spontaneous tumors have been observed in fourdifferent female mice of the high, and two of the lowexpressing lines, even after multiple breedings over a periodof more than i 2 months. In several cases, enlarged lymphnodes have been observed in mammary glands during thepreparation of mammary whole mounts from the lower
copy-number offspring of line 449i , but these did not ex-hihit any pathological abnormalities. Skin lesions havebeen observed in the hind quarters of several of the highcopy-number offspring of line 4491 . These lesions wereexamined histologically and found to represent a folliculardilatation and comedo formation, most likely due to alteredsebaceous gland differentiation.”
Overexpression of the 1 72At� Leu Mutant p.5.3 Results inApoptosis. The inhibition of normal mammary gland de-velopment and the failure to lactate observed in the lineexpressing the highest level of mutant p53 suggested that
overexpression of this mutant may have lead to apoptosis
because wild-type p53 expression has been reported to bean essential component of the pathway leading from DNA
damage to apoptosis (26, 36). One characteristic of apop-tosis is a progressive degradation of DNA, yielding a ladderof DNA fragments corresponding to multimers of nucleo-
somal-sized DNA. In initial studies, such a pattern of deg-radation was observed for DNA isolated from the mammaryglands of mice carrying the mutant p53 transgene at day 1 5of pregnancy, and this was not observed in DNA isolatedfrom nontransgenic controls, but it was difficult to quanti-tate these results (data not shown). Therefore, a more quan-titative apoptosis assay was used (37) in which the number
of apoptotic cells could be directly measured prior to andfollowing whole body -y-irradiation (Table 1 ). Prior to y-ir-radiation of i 5-day pregnant, nontransgenic control mice
and mice bearing the mutant p53 transgene, only i .2-1 .27% of the cells were apoptotic. However, following-y-irradiation, there was approximately a 2-fold increase inthe presence of apoptotic cells in the mice bearing themutant p53 transgene (7.97 versus 4.i 3%). No differencesin the frequency of apoptotic cells in y-irradiated or un-treated mice were observed in the spleen, thymus, lymphnodes, or intestinal crypts of these mice. These results areconsistent with the hypothesis that overexpression of themutant p53 transgene results in a selective increase in-y-irradiation-induced apoptosis in the mammary gland.
The 1 72Arg-’ Leu Mutant p53 Exhibits Trans-ActivationProperties Characteristic of Wild-type p53. The p53 pro-tein displays sequence-specific DNA binding and containsa potent trans-activation domain (7). It can both activateand repress the transcription of a number of cellular genesvia mechanisms that involve both protein-DNA and pro-tein-protein interactions (iO, i2, 38). The previous experi-ments have suggested that the i72� .Ieu mutant p53
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716 p53 Transgenic Mouse Model
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C, F, and C, 20 X 2.5; 0, 20 X 3.3;
E, 10 x 2.5; H, 40 x 2.5 magniui-
cation, respectively. A. B. and Fare stained with hematoxylin and
eosin; C, 0, F, C, and H are stained
with a polyclonal antibody CM5specific br mutant p513. A, 1 5 (lays
of pregnancy from a nontransgenic
FVB mouse. B, 2 days of lactationfrom a nontransgenic mouse. C
and 0, 1 5 days of pregnancy fromtransgenic line 4491. F. F, C. andH, 2 days of lactation from trans-
genic line 4491 . p53 expression is
detected in some but not all cells ofthe mammary gland at 1 .5 days of
pregnancy in the lobuloalveolar
structures ( C, arrow 1 ; 0, arrow 2).Note the absence of expression in
the duct (C. ,irrow 2). The lack oflobuloalveolar development in the
mammary gland of transgenic line4491 (F, F, C, and H) compared to
the nontransgenic control (B).Note the incompletely filledstroma. The p53 staining is cyto-
plasmic (H, arrow(. The cells
expressing mutant p5.3 display in-
complete lobu)oalveolar organiza-lion and decreased secretion (F, ,ir-row 2). Compare the region not
expressingp5ioiEand F(arrow!)that shows an apparently normalsecretory alveolar structure con-
taming lipid droplets with the lackof same in the cells expressing mu-
tant pS3 (F. arrow 2).
Table 1 Apoptosis Morphometry in Mice Carrying the Mutant p53Transgene and Controls at Day 1 5 of Pregnancy.
Mice GroupNo.
glands
No. cells
counted% apoptosis”
wt control
irrad.
3
6
i500
3000
1.27 ± .31”
4.13 � .64”
nit control
irrad.
6
6
3000
3000
1 .20 ± .42”
7.97 ± .76’
.‘ Apoptosis, mean ± SD.Groups with different superscripts are significantly different from control
wild-type. p < 0.05.
exhibits some properties characteristic ofthe wild-type p53.Therefore, to characterize the trans-activation properties ofthe i 72�g . Leu mutant p53 in vivo, the expression of sev-eral genes that have been reported to be induced by wild-type p53 and repressed by several p53 mutants was com-pared (Fig. 7). The high level of expression of mutant p53
transcripts is shown in Fig. 7A. Under these conditions, no
wild-type p53 mRNA was detected in RNA isolated fromthe mammary gland of nontransgenic littermates (see Fig.
7A, alternating lanes). The RNA blots were then probed for
the expression of mdm-2 mRNA, a gene known to bepositively regulated by wild-type p53 in an autoregulatory
loop (Fig. 7B; Ref. 10). Despite the reduction in lobuloal-veolar development observed in the mice expressing the
mutant p53 transgene, there was an increased expression of
the mdm-2 mRNA detected in mammary gland RNA. In
contrast, the expression of proliferating cell nuclear antigen(PCNA) mRNA levels was markedly reduced in RNA iso-
lated from mammary glands of the transgenic as compared
to the non-transgenic controls (Fig. 7C). Unexpectedly, twoother genes whose expression is increased during cell pro-liferation, histone H4 and c-myc, displayed slightlyelevated mRNA levels in the mutant p53 mice (Fig. 7, DandE, respectively) as compared to the controls. Consistent withthe failure of the transgenic mice to lactate, the levels of themRNAs encoding �-casein and WAP milk proteins were
(elI Growth ,5, l)ifferentiation 717
: T. Soussi, personal communi( ation.
I 2 3 4 5 6 7 S
A.
B.
C.p)
D.
#{149}1.�
E.
F.
*
“t�i�’t ,-�
p53
�4DM-2
PCNA
c-rslYc
HISTONE
H4
J3.CA S FIN
G MOUSE. 4 . , . . �- WA P
RNA (pg) 20 20 10 10 5 5 2.5 2.5
H � 2345678
�:�i[� 11.IJ�.�L�J L.J L��J
20 10 5 2.5 (pg)
Fig. 7. RNA blot analysis ot t 72”- ‘‘‘“ mutant p53 mRNA and related p53
p,ithw.iy gene expression. A titration of decreasing amounts of RNA (as
showii isolated from an F offspring of line 449 t I Lanes 1, 3. 5, and 7) anda nontransgenic control ( Lint’s 2. 4. 6. and (0 at 2 days of lactation. A. B. C.
D. F, F. and C sv(’re hybridized with p53. mdm-2, PCNA, c-myc, histone H4,mouse �-(,i5(’i!i, md riioi.is#{128}’t’V�\P cDNA prol)es, respectively. H is the
elhidiuni bromide-stained RNA gel.
markedly inhibited (82 and >90%, respectively) in the mice
expressing the mutant p53 transgene as compared to thenon-transgenic controls (Fig. 7, Fand G). The integrity of theRNA is illustrated by the ethidium bromide stained gelprofile shown in Fig. 7H. Thus, overexpression of the1 72Arg . I vu mutant p53 in vivo resulted in irradiation-in-duced apoptosis, and an increase in mdm-2 and a decreasein PCNA mRNA levels, consistent with several of the prop-erties expected for the wild-type p53.
Discussion
In this study, we have successfully targeted the expressionof 1 72Arg . I �‘ti mutant p53 to the mammary gland of trans-genic mice. In several different independent lines of mice,expression of the 1 72”� �‘ � mutant p53 was correlatedwith an impaired ability to lactate, and the mice expressingthe highest levels of mutant p53 were unable to nurse theiryoung. It appeared that this was related to the inhibition ofnormal lobuloalveolar development during late pregnancy,and, as a consequence, a marked decrease in milk proteingene expression in early lactation, consistent with the tim-ing of expression of the WAP promoter-driven transgeneduring mammary gland development (30). A similar phe-notype has been reported for mice bearing a WAP-TGF-f31
transgene (32). In these WAP-TGF-f31 mice, the mammaryglands of normal and transgenic mice were morphologi-cally indistinguishable up to day 1 3 of pregnancy, but themice expressing the WAP-TGF-)3 1 transgene were also un-able to lactate and exhibited an inhibition of normal lobu-loalveolar development. Consistent with our results, ductaldevelopment was not overtly impaired in the WAP-TGF-f3 1mice. The presence of some normal lobuloalveolar struc-tures in the lactating mammary gland may be explained bythe nonuniform penetrance as observed for the WAP-mutant p53 transgene in the mammary alveolar cells duringlate pregnancy (Fig. 6) that has also been reported for anumber of other transgenes introduced into mice (39).
Expression of the 172Ar�� .tt’ii mutant p53 resulted inradiation-induced apoptosis and trans-activation or repres-
sion of a number of genes known to be regulated by wild-type p53. However, immunohistochemical analysis mdi-cated that the mutant p53 protein was exclusively localizedin the cytoplasm (Fig. 6) and not in the nucleus as would beexpected for wild-type p53. While nuclear localization wasoriginally reported to be essential for the activity of p53
(40), there are number of recent reports where nucleartranslocation-defective p53 proteins still retain suppressorand trans-activation functions (8, 41 ). In our experiments, itis conceivable that a small amount of mutant pS3 waspresent in the nucleus but was not detected by immunocy-tochemistry or that the effects of p53 on gene activation orrepression were mediated by an indirect mechanism inde-pendent of specific DNA-p53 protein interactions (42). It isof interest that a predominance of cytoplasmic staining hasbeen reported for wild-type pS3 in both normal lactatingtissue and in approximately 37% of human breast cancersusing monoclonal antibody PAbi8Oi (1). It has also beensuggested that cytoplasmic sequestration of p53 may pro-vide a mechanism to promote transient cell proliferation (1).Hence, despite the location of the i 72� � Lt�i mutation inone of the known “hot spots” in the p53 protein (4) and thenatural occurrence of this mutation in a number of breastand other cancers (2), this mutation appears to result in ap53 protein that has pseudo-wild-type properties. This re-suIt was unexpected since the more commonly occurringArg-� His mutation at amino acid 1 72 (human p53 aminoacid 175) had been demonstrated to encode a dominant-negative p53 (3). This mutant p53 has been reported to bea dominant oncogene that acts cooperatively with ras in celltransformation assays and is incapable of trans-activationand DNA binding (3, 7, 43).
Recent analysis of a number of human p53 mutants atposition 1 75 have indicated that several of these p53 mutantsdo not encode an altered p53 protein as assayed by a numberof different in vitro and cell transfection criteria and that the1 75Arg . vu mutation in human p53 encodes a protein withsome properties of both wild-type and mutant p53 proteins.7Studies are in progress in our laboratory to determine theeffects of overexpression of the 1 72� � H�. dominant-negativep53 mutant on mammary gland development and carcino-genesis using the WAP-p53 expression constructs described.
Prior to initiating our studies in transgenic mice, wecompared the properties of the 1 72� � (‘0 ,�53 mutant tothose of the wild-type p53 in their ability to generate stabletransfectants following cotransfection of CMV-driven p53
718 p53 Transgenic Mouse Model
minigene and pSV2neo constructs into both the HC1 1mammary epithehial cell line and NIH 3T3 cells, followedby G4i 8 selection. More recently, we have also comparedthese results to those obtained with a CMV-driveni 72Arg-* His p53 mutant. In these studies, numerous stabletransfectants were generated with both mutants, but as ex-pected, few or no colonies were obtained with the wild-type p53 construct. In addition, when equal amounts of thewild-type and 1 72Arg-� Leu p53 mutant were transfected intocells, the mutant construct was unable to overcome thesuppressive function of the wild-type p53, and no stabletransfectants were obtained. This provides another examplein which it is difficult to correlate the effects of specific p53mutations in cell culture systems with their phenotypes insitu (23).
At first glance, it might seem unlikely that single aminoacid changes such as an Arg to Leu or His at position 172could result in such dramatically different effects on thefunction of the p53 protein. However, recent studies havedemonstrated that conformational shifts in the p53 proteincan exert a major influence on its ability to ohigomerize andto bind DNA in a sequence-specific manner (43, 44). Mark-edly different effects of specific point mutations on p53conformation have been demonstrated using differentmonoclonal antibodies that recognize specific p53 epitopesand by using proteases to determine the susceptibility ofvarious domains in the p53 protein to digestion (43). Con-sidering the multiplicity of proteins identified to date thatcan interact with p53, it is, therefore, not surprising thatchanges in p53 conformation can exert such dramatic ef-fects on its function (45). Furthermore, various autocrineand paracrine factors regulated in part by stromal-epithehialinteractions may influence these protein interactions withp53 via an increasingly complex network of signal trans-duction pathways. Thus, in hindsight it is not surprising thatan increasing number of differences are now appearingbetween the properties of p53 and p53 mutants in trans-fected cells and those observed in situ.
The development of transgenic mouse models that over-express specific p53 mutants preferentially in the mammarygland should now permit a better evaluation of the role ofother factors such as hormones, oncogenes, chemical car-cinogens, and other DNA-damaging agents in the initiationand progression of breast cancer. In particular, it should bepossible to test the hypothesis that different biochemicaland biological properties of p53 mutants may confer aselective advantage upon specific mammary epithelial celltypes to facilitate their progression to a more aggressivephenotype. This hypothesis is based upon the clinical ob-servations that mutant p53 expression correlates with moreaggressive metastatic breast cancer and a poorer prognosisfor survival (i 7, 1 8). Thus, these transgenic mice may pro-vide a more sensitive in vivo carcinogenesis model. In thisregard, we have initiated studies to determine the effects oftreatment of mice expressing the 1 72Arg-. Leu p53 mutanttransgene with DMBA on mammary carcinogenesis. Pre-himinary results suggest that mice expressing the i 72’���’ Leu
p53 mutant transgene are refractory to DMBA-inducedcarci nogenesis.
Materials and Methods
Construction of WAP-p53 Expression Vectors. The major-ity of techniques used for subcloning were performed asdescribed in Sambrook et a!. (46). Restriction enzymes and
DNA higase were purchased from Pharmacia-LKB. The mu-rifle p53 genomic minigene, containing an Ala-s Val pointmutation at codon 1 35 ( ps3cgg; Ref. 34), was kindly pro-vided by Dr. G. G. Lozano at M. D. Anderson CancerInstitute. It was converted to the wild-type p53 allele and
subsequently mutated at codon 1 72 (from Arg to Leu) usinga recombinant PCR mutagenesis and recombination proto-col (47). Briefly, two separate PCR products were synthe-sized containing overlapping sequences with a single basepair mutation located in the middle of the overlap. ThesePCR products were denatured and allowed to reanneal toproduce a heteroduplex. The recessed 3’ termini of theheteroduplex were extended using the Klenow fragment ofDNA polymerase I to produce the sum of the two overlap-ping products. A second PCR was then performed usingonly the distal 5’ and 3’ primers to produce the full-lengthproduct with the desired mutation. This fragment was sub-sequently inserted into the p53 minigene construct. Tointroduce the Arg-s Leu mutation in exon V, the forward 5’primer derived from the exon IV/intron 4 junction was5 ‘-GTTATGTGCACGGTGAGTGGGCCC-3 ‘. The reverse3’ primer derived from the intron Vh/exon VII junction was5 ‘-CTCAGAGCCGGCCTGGGGGAAGAC-3 ‘ . The twomiddle, overlapping primers from exon V containing thei-base mutations were reverse 5’-GCTCATGGT-GGGGGCAGAGTCTCACGACCTCCGTC-3 ‘ and forward5 ‘-GACGGAGGTCGTGAGACTCTGCCCCCACCATGA-GC-3’. To convert the Val codon at amino acid 1 35 in exonV in the p53cgg minigene to the Ala codon present in thewild-type p53, the same 5’ and 3’ primers were used withthe middle primers forward 5 ‘-GCTATTCTGCCAGCTGG�
1 and reverse 5 ‘-GCA-CAGGGCACGTCTTCGCCAGCTGGCAGAATAGC-3 ‘ . Bi-
directional sequencing of all exons and exon/intronjunctions of the PCR-derived p53 minigene fragments re-vealed that the desired mutation was obtained and that allother sequences were wild type.
The original murine p53 minigene designated p53cggcontained the entire murine p53 gene lacking introns 1 and10 and a 3’ SV4O polyadenylation signal (34). To place thep53 minigene under the control of WAP regulatory se-quences, the rat WAP promoter fragment from -949 to +1and the WAP5’ UTR (from +1 until the ATG at +34) wereamplified by PCR using primers forward 5’-CCGTCGACG-GCCACAGTGAAGACCTCCGGCCAG-3’ and reverse 5’-CTGAGGAUCGGTGTCGGCGGCGGCAGGCAAGTG-3’.
The amplified, blunt-ended WAP fragment was ligated tothe 5’ terminus of the murine mutant p53-SV4O poly(A)�fragment that was excised from p53cgg with BamHl andsubcloned into the pBluescriptll SK� vector (Stratagene).This construct was designated pBL1O3D (Fig. 1, 3.). TheWAP 3’ UTR (from the stop codon to +70 bp 3’ to thepoly(Ai� cleavage site) was amplified by PCR using primersforward 5’-GTGCTCGAGGAAGCCTGCCCTGGGATC-CCTGCC-3’ and reverse 5 ‘-CTCGGTACCATCCTGCCCTC-CCTCCCTTCTGTG-3’. The SV4O poly(AY’ signal was ex-cised from pBL1 03D by digestion with SaclI and BamHI andthen inserted into pSKll� and designated pBLiO5. pBLiO5was then digested with Pstl to remove the SV4O poly(AY�signal, and the remaining fragment was ligated to the WAP3’ UTR 3’ to the Sacll-Pstl fragment in pSKlI�, designatedpBLiO7. Finally, the SacII-WAP 3’ UTR fragment frompBLiO7 was used to replace the Sacll-BamHl fragment ofpBL1 03D to yield the WAP-p53-WAP 3’ UTR construct,pRL4 (Fig. 1 : 1., wild-type; 2., mutant).
Cell Growth & Differentiation 719
Generation and Screening of p5.3 Transgenic Mice.Linear WAP-mtp53-5V40 or WAP-mtp53-WAP 3’ UTRDNA fragments were excised by BssHll digestion, purifiedby preparative electrophoresis through agarose gels, andrecovered by adsorption to glass beads (Bioi 01 , Inc., LaJolla, CA). Transgenic mice were generated by microinjec-tion of linear DNA into FVB mouse embryos. Mouse tailDNA was isolated as described previously (48). Mice car-rying the WAP-p53 transgenes were identified by multiplexPCR. The screening primers (forward on the WAP promoter5 , -CCGTCGACGGCCACAGTGAAGACCTCCGGCC-
AG-3’, reverse on exon 2 of murine p53 5’-GCCTGAAAAT-GTCTCCTGGCTCAGAGGG-3’) yielding a 1 .2-kb product.Mouse �3-casein exon 7 forward 5’-GATGTGCTCCAG-GCTAAAGTT-3 ‘ and reverse 5 ‘-AGAAACGGAATGTTGT-GGAGT-3’ primers were included as internal controls,yielding a 0.5-kb product. The PCR conditions used were2.0 msi MgCl2 and 1 .5 pg template DNA at 94#{176}C1 mm,64#{176}C2 mm, and 72#{176}C3 mm for 30 cycles. Copy numberswere determined by Southern blot analysis. The tailgenomic DNA was digested with BamHI, separated byelectrophoresis on a 0.6% agarose gel, denatured in 1 .5 M
NaCI2-0.5 M NaOH twice for 30 mm, renatured in 1 M
NH4OAC, 0.02 N NaOH twice for 30 mm, and transferredto Zetoprobe GT membranes (Bio-Rad) in renaturating so-lution. A Xhol-ApaLl fragment excised from the mouse p53gene was labeled with [a-32PIdCTP using random primingand the Klenow fragment of DNA polymerase I and used asthe probe. Hybridization and washing of the blots was asdescribed by the manufacturer, and the results were quan-titated using a Betascope 603 blot analyzer (�3etagen).
RNA Extraction and Analysis. Mammary gland biopsieswere performed under anesthesia (Avertin, 0.25 ml/g, i.p.)
at either day 2 or i 0 of lactation as described previously(48). RNA was isolated by homogenization of frozen tissuewith a Polytron (Brinkmann) homogenizer using the RNazolB protocol as described by the manufacturer (2 mI/i 00 mgmammary gland; BioTecx Labs, Houston, TX). RNA wasfractionated by electrophoresis in a 1 .2% agarose gel con-taming 0.66 M formaldehyde with 1 x 4-morpholinepro-panesulfonic acid buffer, then transferred to Zetoprobemembranes with 1 0 x SSC (1 X SSC = 0.1 5 M NaCl-0.Oi 5M sodium citrate) and hybridized as described above forSouthern analysis.
RT-PCR analysis: Total RNA (1 pg) isolated from themammary glands of transgenic mice at day 1 0 of lactationwas annealed to 100 ng ofoligo(dT)12_18 (Pharmacia-LKB),and cDNA was prepared in a 2O-pl reaction containing 50
mM KCI, 10 mM Tris-HCI (pH 8.4), 3 msi MgCl2, 1 msideoxynucleotide triphosphate, 5 units reverse transcriptase(GIBCO-BRL), and 1 6 units RNAsin (Promega) for 1 h at37#{176}C.The reactions were heated at 90#{176}Cfor 5 mm, andthen 80 p1 ofthe PCR reaction buffer containing 50 mtvi KCI,iO mM Tris (pH 8.4), 2 mrvi MgCl2, 0.625 �M forward andreverse primers, and 1 .25 units Taq polymerase (Promega)were added. The PCR conditions were 94#{176}Cfor i mm, 62#{176}Cfor 2 mm, and 72#{176}Cfor 3 mm for 30 cycles. For quantitativePCR analysis, 19, 21, and 23 cycles were used. To amplifythe full-length p53 cDNA, primer 1 forward 5’-ATCAGT-CATCACTTGCCTGCCGCCGCCGACACC-3 ‘ located in theWAP 5’ UTR and primer 4 (reverse for the pRL4 construct)5 , -AGCAGCGGTTAGAAAGCATTATGTTCTCTCTGG-3’
located at the exact end of the WAP poly(A)� signal wereused (see Fig. i ), yielding a 1 .35-kb product. Alternatively,primer 5 (5’-CCGGGAAGCTAGAGTAAGTAGTTCGC-
CAGT-3’) located in the SV4O poly(Ai’ signal was used asthe reverse primer for the pBLi 03D construct, yielding a1 .40-kb product. To amplify the 5’-end of the p53 cDNA,the forward primer was primer 1 , and the reverse primer 2was 5 ‘ -GCTCATGGTGGGGGCAGAGTCTCACGAC-CTCCGTC-3’ located in the mutant p53 exon 5, resulting ina 570-bp product. To amplify the 3’-end of the p53 cDNA,primer 3 5’-GACGGAGGTCGTGAGACTCTGCCCCCAC-CATGAGC-3’ located in mutant p53 exon 5 was the for-ward primer, and the reverse primers were either primer 4or 5, resulting in 780- or 830-bp products, respectively.
The following cDNA clones were used to hybridize RNAblots: pKS-mdm2 and mdm-2, kindly provided by Dr. A.Levine (Depts. of Molecular Biology, Princeton University,Princeton, NJ; Ref. iO); pCG4 and PCNA, kindly providedby Dr. R. Baserga (Dept. of Microbiology, Jefferson CancerInstitute, Philadelphia, PA; Ref. 49); and mouse histone H4and mouse c-myc cDNAs were kindly provided by Dr.Li-yuan Yu-Lee (Depts. of Medicine and Cell Biology, Bay-or College of Medicine, Houston, TX; Ref. 50).
Mammary Gland Whole Mounts. The entire fourth (in-guinal) mammary glands were removed under anesthesia ateither 5 weeks of age (virgin) or at 10 or 1 5 days followingthe initiation of pregnancy. The glands were placed inhistology cassettes in Telly’s fixative overnight at room tem-
perature. The whole mounts were then defatted in acetone,stained with hematoxylin, dehydrated, and stored in 100%methyl salicylate.
Immunohistochemistry and Histology. Mammary glandbiopsies were placed in 1 0% neutral buffered formalin for 6h, rinsed with water, and placed in 70% ethanol untilprocessed. These tissues were embedded in paraffin, and5-pm sections were placed on Probe-on Plus slides (FisherScientific). The sections were processed for immunohisto-chemical staining using the capillary gap method (Si). Thesections were incubated for 12 mm with CM5 antiserumdiluted 1 :200. This antiserum was produced essentially asdescribed by Midgley et a!. (52), except that the antigen wasmurine p53 expressed in bacteria. Specific immune com-plexes were detected by the avidin-biotin-peroxidase com-plex method (53) using Vectastain ELITE kits (Vector Labo-ratories). Incubation times for the secondary antibody andavidin-biotin-peroxidase complex were 10 mm each. Im-mune complexes were visualized using diaminobenzidineand were counterstained with 1 % methyl green. The regularhistology slides were stained with hematoxylin and eosin.
Apoptosis Analysis. The quantitation of the number ofapoptotic cells in sections of mammary glands was per-formed essentially as described (37). Briefly, FVB wild-typeand transgenic mice were exposed to whole body y-irradi-ation given as a single dose of 5 Gy. Irradiation was deliv-ered from a 137Cs unit to unanesthetized mice immobilizedin a jig. Following euthanasia, the mammary glands fromboth the thoracic and inguinal areas were collected andprocessed for embedding in paraffin blocks; sections werecut at 4 pm and stained with hematoxylin and eosin. Codedslides were scored blindly by one of us (L. C. S.) for thepresence of apoptotic cells using microscopic examinationof the stained tissue sections at a magnification of X400.Multiple fields of nonnecrotic areas were selected in eachspecimen, and 1 00 nuclei in each field were categorized asnormal, mitotic, or apoptotic. The apoptotic nuclei werecharacterized as condensed, homogeneous basophilic bod-ies most frequently, but not invariably, of round or crescentshape. Infrequently, several small apoptotic fragments were
720 p53 Transgenic Mouse Model
encountered in close proximity and considered to representthe remains of a single cell and counted as one apoptoticnucleus.
AcknowledgmentsWe thank Drs. G. G. Lozano and Larry Donehower for their helpful advice
and critical comments prior to initiating this project, as well as for providingthe p53 minigene and unpublished sequence information for the mouse p53gene; Dr. A. Baldini for performing the fluorescence in situ hybridizationanalysis; and Drs. David Greenhalgh and Dennis Roop for a pathological
description of the skin lesions. We also thank Dr. Franco DeMayo and LuAnn
Stanley for generation of the transgenic mice; Shu-wen Sun for mousebreeding and preparation of tail DNA; Dr. Susanne Krnacik for help withmammary gland whole mounts; and Liz Hopkins and Anne White for per-
forming the immunohistochemical analyses. In addition, we also thank Drs.
Larry Donehower and Janet Butel for their critical reading of this manuscript.
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