Oncogene Ha ras of Keratinocytes Expressing the v ...

p53 Gene Dosage Modifies Growth and Malignant Progression of Keratinocytes Expressing the v- rasHa Oncogene Wendy C. Weinberg, Christopher G. Azzoli, Namrata Kadiwar, et al. Cancer Res 1994;54:5584-5592. Published online November 1, 1994.

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(CANCERRESEARCH 54.5584-5592,November1, 19941

p53 Gene Dosage Modifies

Growth

and Malignant

Progression

of Keratinocytes

Expressing the v-ras1@Oncogene WendyC. Weinberg,'ChristopherG. Azzoli,2NamrataKadiwar,and StuartH. Yuspa Laboratory of Cellular Carcinogenesis and Tumor Promotion. National Cancer institute. Bethesda. Maryland 20892

ABSTRACT

of epidermal growth and carcinogenesis has beenderived from studies

Epldermal keratinocyte cultures were established from newborn mice expressing

a null mutation

in the p53 gene to explore

the contribution

of

p53 to epidermal growth regulation and neoplasia Keratinocytes were Initiated by transduction with a replication-defective retrovirus encoding

epidermis by dimethylbenzanthracene causes mutations in a cellular ras allele in >90% of resulting tumors (27). Mutations in the p53 gene appear

relatively

late and thus

are associated

with

the malignant

the v@ras@@a oncogene and grafted onto nude mouse hosts. Tumors arising from keratinocytes heterozygous or null for functionalpS3 In the presence

conversion stage of chemically induced mouse epidermal carcinogen

of v-rasâ€•have growth rates approximately 5-fold higher than those derived fromp53(+I+) controls and rapidly form carcinomas, in contrast

replication-defective retrovirus into cultured keratinocytes or dorsal epidermis is sufficient to confer the benign neoplastic phenotype; mouse hosts for such cells will develop skin papillomas (30, 31). Additional genetic changes, such as coexpression of v-fos, can coop crate with v-ras'@ to promote malignant progression (32). The availability of p53-deficient mice with a null mutation in one

to the benign phenotype

observed lnpS3(+/+)Iv-ras'ta

esis (28, 29). Direct introduction

grafts. In vitro,p53-

deficient keratinocytes with and without v-ms@ expression display de @

ofmouse epidermis, both invivo and invitro (26). Initiation of

creased responsiveness to the negative growth regulators transforming growth factors fi@ and I@2 combination with v-ras'@, p53-deficient keratinocytes also exhibit decreased responsiveness to elevated Ca2@. These differences between genotypes cannot be attributed to changes in transforming growth factor @ireceptor types present or altered levels of epidermal growth factor receptor and are independent ofc-myc transcript levels. mRNA expression for the p-53 inducible protein WAF1 correlates

of a mutant v-rasâ€•@oncogene

by a

or bothp53 alleles(33) offers the opportunityto directly explorethe

functional aspects ofp53 within the context of the well-characterized model of mouse skin carcinogenesis. Such mice develop normally but succumb to a variety of malignancies, predominantly sarcomas and with p53 gene dosage, but low levels are still detectable in pS.3(â€”/â€”) lymphomas, beginning as early as 20 weeks of age (33). Recent work keratinocytes. The altered responsiveness ofp53 deficient keratinocytes to by Kemp et a!. (34) has demonstrated that following the classic negative growth regulators may provide a growth advantage to such cells initiation-promotion protocol of skin tumor induction, p53-deficient in vivo and render them more susceptible to genetic alterations and malignant

mice develop reduced numbers of papillomas with a greatly increased

conversion.

frequency of malignant progression. We have been taking a parallel approach by grafting p53-deficient INTRODUCTION keratinocytes to nude mouse hosts. This approach provides the op While loss and/or mutation of the p53 tumor suppressor gene is a portunity to study growth and neoplastic progression in these kerati nocytes without the difficulties encountered due to the increased frequent occurrence in human malignancies (1), a direct role for the susceptibility of p53-deficient mice to other malignancies. Further intact gene product in blocking the progression of the cancer process has not yet been defined. The ability of p53 to bind DNA in a more, this approach offers the advantages of evaluating the relative roles of specific subpopulations mixed in the graft site, as well as in sequence specific manner (2, 3), interact with known transcription vitro analysis of cellular phenotypes. In this study, we have intro factors (4â€”9),and suppress transcription from the promoters of sev duced the v-ras'@ oncogene into epidermal keratinocytes from eral genes (10â€”12)has been demonstrated. It has been proposed that p53-deficient newborn mice to directly test whether loss of func p53 induces expression of a negative regulator or represses transcrip tion of positive signalsfor cell cycle progression(12â€”16); however, tion of p53 cooperates with a mutant ras gene to alter the pheno type of benign papillomas and to explore the biochemistry of such the specific genes targeted in vivo have not yet been identified. Recent reports implicating wild-type p53 in a cell cycle checkpoint (17â€”19) an effect. and pathways for differentiation and apoptosis (20â€”22)support the assumption that loss of this gene product provides a growth advantage MATERIALS AND METHODS and therefore directly contributes to the neoplastic phenotype. While many of the in vitro studies on p53 function have been Cell Preparations@p53-deficient mice and control littermateswere pur helpful in developing hypotheses regarding the contribution of this chased from GenPharm International (Mountain View, CA). Mating pairs were gene product to cancer pathogenesis, few functional studies have been set up between mice heterozygous for a null mutation in the p53 gene (33) and performed on models of multistage carcinogenesis. The accumulation homozygous or control liuermates; in some experiments heterozygote matings of alterations in both oncogenes and tumor suppressor genes during were used. From I to 5 days following birth, pups were sacrificed and the carcinogenic process is well documented (23). The timing and epidermal cells were isolated as described previously (35). Keratinocytes were frequency of p53 mutations vary between organ sites. p53 mutations prepared from epidermis after overnight trypsinization of skin at 4Â°C;in some are observed in 15â€”58%of human epidermal squamous cell carcino experiments each individual epidermis was held in culture medium containing 1.3 mr@i Ca2@for an additional 24 h at 4Â°Cprior to cell preparation to allow mas (24, 25), suggesting that disruption of p53-mediated pathways completion of the genotyping process. All three epidermal cell preparations contributes to this disease. Much understanding of the molecular basis @p53(+/+),

Received 4/27/94; accepted9/1/94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with

18 U.S.C. Section 1734 solely to indicate this fact. I To

whom

requests

for

reprints

should

be

addressed,

at

Building

37,

National Cancer Institute, Bethesda, MD 20892. 2 C.

G.

A.

is

a Howard

Hughes

Medical

Institute-NIH

Research

Scholar.

Room

3B25,

-(+1â€”), and -(â€”Iâ€”)]were handled

identically

within

each exper

iment. Keratinocyte cultures were maintained in Eagle's minimal essential medium (Bio-Whittaker,

Walkersville,

MD) containing 0.05 mM Ca2@, 8%

chelexed fetal bovine serum (UBI, Lake Placid, NY), and penicillin/strepto mycin (GIBCO, Grand Island, NY) as described previously (35). p53 Genotyping. DNA was extractedfromtail snipsof newbornmice with phenol/chloroform/isoamyl

alcohol

using standard

5584


methodology.

DNA (1â€”1.5

@

p53

AND

v.rasH*

EPIDERMAL

P.8)wasamplifiedin a PCR3containingthe DNA sequence gggACAgC CAAgTCFgTFATgTgC as 5' primer, and CFgTCUCCAgATACTCgggA TAC (wild-type specific) and 1TFACggAgCCCFggCgCTCgATgT(mutant specific) as 3' primers.In some experimentsprimerpairsgTglTl'CATTAgT TCCCCACCTFgAC and ATgggAggCTgCCAgTCCFAACCC(wild-type spe cific) and gTgggAgggACAAAAgTFCgAggCC and 1TFACggAgCCCTg gCgCl'CgATgT (mutant specific) were used. Primers were synthesized on a Cyclone Plus DNA synthesizer (Milligen/Biosearch, Burlington, MA). Prod

ucts were analyzed by agarose gel electrophoresis. Because the wild-type product was occasionally lost in multiplex reactions of heterozygous samples, those demonstrating only the mutant reaction product were reanalyzed with individual primer pairs for confirmation. Pooled cell preparations were saved for confinnation

following

the same procedure.

Introduction of v-rasâ€•. Primary keratinocyte cultures derived from each genotype were transduced with a replication-defective

retroviral vector encod

KERATINOCYTES

Hercules, CA). In similar experiments, cells were plated in 60-mm dishes (2.5 X l06/dish), treated as above, and labeled with 5 pCi/ml [3H]thymidine. Following lysis in 1 N NaOH, the DNA was precipitated in perchloric acid and counted or quantified by reaction with diaminobenzoic acid (Aldrich Chemical

Co., Milwaukee,WI) (38). Eachmethodwas repeatedtwice andan additional [3H]thymidine assay was performed by normalizing to cell number, with consistent results. Results shown are from a representative experiment. Receptor Assays. TGF-f3/receptor cross-link assays were performed at 4Â°C as described previously (39). Keratinocyte cultures in 6-well cluster plates (Costar) were washed 3 times with binding buffer [Eagle's minimal essential medium containing 0.05 mM Ca2@, 25 mM 4-(2-hydroxyethyl)-I-pipera

zineethanesulfonic acid (pH 7.4), and 0.1% BSA] and incubated with 25 @M â€˜25I-TGF-@ (Amersham)in bindingbufferfor 2.5 h in the presenceor absence of 10 nM unlabeled competitor TGF-(3 (R&D Systems, Minneapolis, MN). Parallel culture wells were preincubated

on ice for 3 mm in 150 mM NaCl,

ing the v-ras@ gene. Cultures were washed once with Ca2@@Mg2tfreePBS

0.1% acetic acid, and 1 mg/nil BSA to remove prebound ligand (acid-strip

(Bio-Whittaker)

ping). After the incubation with â€˜25I-TGF-@, unbound label was removed by washing 3 times in binding buffer with no BSA and bound ligand was

and incubated in 0.05 mr.i Ca2@ medium with polybrene (4

,.&g/ml;Sigma Chemical Co., St. Louis, MO) and a replication-defective retrovirus encoding v-ra1'@ (30). Fresh culture medium was added after 60â€”90

nan; 48â€”72h later cells were washed with PBS and fresh medium was added. Control cultures were mock-infected by parallel incubation with polybrene alone. In V@ivo Grafting and Tumor Measurements. Cultureswere infected with v-ra1'@-encodingretrovirus 2 days following plating. Four to 6 days following infection, cells were transferred to a grafting chamber on the dorsum of nude mice in combination with 8 X 106dermal cells from wild-type littermates, as described previously (36). The chamber was removed 1 week following grafting; tumors were apparent by 2 weeks. Tumor volume was determined by multiplying width X length x height as measured at the center of each

dimension. Results represent data from 4 independent experiments. Tumor Sample Preparation. Tumor samples were embedded in OCT Compound (Miles, Ellthart, IN) and frozen in a dry ice/methanol bath or fixed in 70% ethanol. Additional portions of each tumor were snap-frozen in liquid nitrogen for isolation of RNA and stored at â€”70Â°C. Immunoetaining. Cryostat sections of OCT embedded tissue were washed twice with PBS, preincubated for 30 min at room temperature with normal goat serum (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD), and reacted for 2 h at 37Â°Cwith polyclonal rabbitanti-p53antibodyCM-l diluted 1:500 (Chemicon International,

Inc., Temecula, CA). Sections were washed 3 times,

5 mm each in PBS, and incubated with fluorescein isothiocyanate-conjugated

swine anti-rabbit immunoglobulins

diluted 1:40 (Dako Corp., Carpinteria, CA)

and then washed 3 times before mounting with a coverslip using Aqua-Poly/ Mount (Polysciences,

Inc., Warrington, PA). Both antibodies were diluted in

12%bovine serumalbuminin PBS. Stainingwas confirmedwith monoclonal antibody supernatant PAbl22 (American Type Culture Collection, Rockville,

MD), using biotinylated anti-mouse linker antibody (Kirkegaard & Perry Laboratories) and streptavidin conjugated Texas red (GIBCO-BRL, Gaithers burg, MD). Bound antibody was visualized through a Nikon Labophot micro

scope containing an epifluorescence attachment (HMXHBO 100 W lamp house) using B2E and G filter blocks. Tumors arising from s.c. injection of A1-5 cells, a rat embryo fibroblast cell line overexpressing a temperature

sensitive mutant ofp53 (37), were used as a positive control. Al-5 cells were the gift of Dr. Arnold Levine. [3HJTh@dine Incorporatlon Keratinocytes were plated in 24-well plates (Costar, Cambridge, MA) at a density of 2â€”3 X l0@cells/well. Cultures were infected with retrovirus 72 h postplating; 5â€”7 days following infection, medium containing either elevated Ca2@,TGF-(31,or TGF-(32was added at concentrations noted for 24 h. [3H]Thymidine(Amersham, Arlington Heights, IL) was added to each well to a final specific activity of 1 @Cilml for the final 2 h. Cultures were washed 3 times with PBS and frozen on dry ice. For protein determinations, cells were washed 4â€”5x on ice with cold 0.2 N perchloric acid until cpm released were lOOcpm and lysed in 1 N NaOH. Aliquots were taken for scintillation counting or protein assay (DC Protein Assay; Bio-Rad,

cross-linked to receptors with 300 @.&M disuccinimidyl suberate (Pierce, Rock ford IL) for 40 mm. Cells were then washed 3 times in 250 mMsucrose-lO mM Tris (pH 7.4)-l mM EDTA and solubilized in 250 pi 1% Triton X-lOO, 10 mM

Tris (pH 7.4), 1 mM EDTA, 0. 1 mM phenylmethylsulfonyl fluoride, and 1 @.tg/mlleupeptin and pepstatin. Lysates were centrifuged at 12,000 X g for 5

mm and supernatants were stored at â€”70Â°C. Equal amounts of soluble protein from each culture were resolved by SDS-polyacrylamide

gel electrophoresis

on 4â€”10%gradient gels (Novex, San Diego, CA), and bound TGF-@ was visualized by autoradiography. Autoradiograms were scanned (Personal Den sitometer; Molecular Dynamics, Sunnyvale, CA) and digitized images were

printed on a Kodak XLT 7720 Digital Continuous Tone Printer. Results shown are representative of duplicate experiments. EGF receptorbindingwas quantitatedas describedpreviously(40). Kera tinocytes were grown in 12-well cluster plates (Costar), washed twice with

cold binding buffer [Dulbecco's modified Eagle's medium (Bio-Whittaker)

containing50 IBMN,N-bis-(2-hydroxyethyl)-2-aminosulfonic acid (pH 6.8) and 1 mg/ml BSA], incubated for 5 h on ice with binding buffer containing 0.1

p@Ci â€˜251-EGF, washed4 timeswith coldbindingbuffer,andlysedin 0.1M Tris, 0.5% SDS, and 1 mMEDTA. Equal volumes of lysates were counted in Ecolite scintillation cocktail (ICN Biomedicals, Costa Mesa, CA). Nonspecific

binding was determined by incubation of parallel wells including 1 @Wmi EGF; an additional well was incubated without radiolabel to determine the total

numberof cells/well.Similarresultswereobtainedin a duplicateexperiment RNA Isolation

and Northern

Blot Hybridization.

mRNA was isolated

directlyfromkeratinocytecultures(41).Cellswerewashed3 timeswithPBS, lysed in lysis buffer [0.2 M NaCl-0.2 M Tris (pH 7.4)-0.15 mM MgCl2-2% SDS-0.2 mg/mi proteinase K], and sheared by sequential passes through 22and 25-gauge needles. Lysates were incubated at 45Â°Cfor 60â€”90mm, adjusted

to 0.5 MNaCl, and incubated with oligo(dT) (InVitrogen, San Diego, CA) at room temperature for 1 h. Oligo(dT)-bound RNA was washed 3 times each in binding buffer [0.5 M NaC1-l0 nmi Tris (pH 7.4)-l mM EDTA] and low salt

buffer [0.1 MNaC1-lOmMThs (pH 7.4)-l mMEDTAJ, transferred to micro centrifuge

spin columns

(InVitrogen),

and eluted in 10 mM Tris (pH 7.4)-I

mM

EDTA. IsolatedRNA was precipitatedand washed once with ethanolbefore separation by formaldehyde/agarose gel electrophoresis overnight at 20 V/gel. RNA was blotted by capillary action and cross-linked (Stratalinker 1800;

Stratagene,La Jolla, CA) onto Nyu-anmembranes(Schleicher & Schuell, Keene,NH). Membraneswereprehybridizedat42Â°Cwith6 X SSC, 5 X Den hardt's reagent, 0.5% SDS, 100 @tg/ml salmon sperm DNA, and 50% form amide, and specific probe was added for 48â€”72h, as described previously (42). Probes were labeled with 32P by random priming (Lofstrand Labs, Ltd.,

Gaithersburg,MD). A 420-base pairPCR fragmentrecognizingWAF1 RNA was generated using primers spanning exon 2 of the mouse gene (13); v-ras and c-myc sequences were detected with v-bas and c-myc plasmid fragments (Lofstrand Labs). Following hybridization for 48â€”72h, blots were washed at room temperature in 2 X SSC-0.2% SDS followed by sequential washings at 65Â°C,lowering SSC to 0.1 X final concentration.Between hybridizations, filters were stripped at 65Â°Cfor 2 h in 5 mr.iTris-HC1(pH 8.0), 0.2 mMEDTA, cmi saline; TOF, transforming growth factor-,BSA, bovine serum albumin; SDS, sodium 0.05% PP@,and 0.1 X Denhardt's reagent. mRNA levels were quantitated by dodecyl sulfate; EUF, epidermal growth factor, SSC, Standard saline-citrate 11 X SSC = 0.15 MNaCI,0.015Msodiumcitrate(pH 7.0)],GAPDH,glyceraldehyde-3-phosphatedensitometry (Personal Densitometer). Hybridization results were normalized dehydrogenase. to GAPDH mRNA levels (43). 5585

3The abbreviations used are: PCR, polymerase chain reaction; PBS, phosphate buff


p53 AND v.ras@@ IN EPIDERMALKERATINOCYTES

mation is supported by the lack of such tumors within pS3(+I+)/vras@ grafts and by the presence of the null allele in malignancies derived from pS3(+/â€”) and pS3(â€”/â€”)keratinocytes, detected by PCR analysis of microdissected foci (data not shown).

( h.... @

It hasbeendemonstratedpreviouslythat high expressionof a ras oncogene can convert benign keratinocyte tumors to carcinoma (44). To confirm that the carcinomas observed were related to p.53 deficiency and not differential expression of v-ras@ among the cell preparations,mRNA was isolated from cell preparationsof each genotype and probed for the v-rasâ€• As shown in Fig. 4, the infected cells of each genotype grown in 0.05 m@iCa2@ expressed similar levels of the v-ras'@ gene transcript. Thus, neither altered growth rates nor increased conversion frequency of tumors is a reflection of differ ences in v@rasF@@@ expression between the cell preparations. To determine whether the wild-type allele is lost in carcinomas derived from pS3(+/â€”) keratinocytes, foci of carcinoma cells were

11

microdissected

I

@

r

@

_

. â€¢4i

Fig. 1. Tumor phenotype in v-rasâ€•@ transducedkeratinocytesis dependenton p53 status. Keratinocyte

cultures from 1â€”4-day-old mice of genotypes noted were infected

with replication defective virus encoding the v-ras'@ oncogene and grafted to nude mouse hosts. A, p53(+/+);

B, p53(+/â€”); C, pS3(â€”/--). A and B, 39 days following grafting; C,

27 days following grafting.

RESULTS @

Tumor Formation from p53-deficient Keratinocytes. Keratino cyte cultures established from p53(+/+), -(+1â€”), and -(â€”Iâ€”)new born

mice

were

grafted

in combination

with pS3(+/+)

fibroblasts

onto nude mice following introduction of the v@ras@@a oncogene. Tumors were apparent within 2 weeks following grafting. By 3 weeks the tumors derived from p53(+/â€”)

and -(+1+)

keratinocytes ap

peared macroscopically to be typical exophytic papillomas, while

@

@

@

tumors derived from p53(â€”/â€”)keratinocytes displayed the less keratinized, ulcerated surface and s.c. growth characteristic of carci nomas (Fig. 1). The growth rates of the resulting tumors differed depending on the p53 status of the originating cells, as shown in Fig. 2. As early as 2 weeks following grafting, tumors derived from pS3(+/â€”) keratinocytes were >5 times larger than control tumors composed of p53(+/+) keratinocytes and remained larger thoughout the time course of these experiments. The tumors derived from pS3(+/â€”) and pS3(â€”/â€”)keratinocytes grew at similar rates. Mice grafted with p53(â€”/â€”)keratinocytes were moribund and were sacri ficed or died before 5 weeks following grafting. At 2 weeks following grafting, histological examination of tumors revealed benign papillo mas from pS3(+/+) cells while pS3(â€”/â€”)tumors exhibited a more dysplastic papilloma phenotype; the latter developed into poorly dii ferentiated carcinomas 3â€”4weeks following grafting (Fig. 3C). After 3â€”5weeks many of the pS3(+/â€”) papillomas displayed foci of

â€”â€˜

and analyzed

by PCR using primer

3000

E E

p53(-/-) p53(+/-)

3

E

2000

>11)

+1

E ..@

1000

U1

carcinoma cells (Fig. 3D; Table 1). There was no evidence of con version to carcinomas within the grafts of wild-type cells during the time course of this study. That the less-differentiated tumors were derived from v-ras'@'-infected keratinocytes and not reactivation of the replication-defective v@rai@avirus resulting in fibroblast transfor

from these tumors

pairs specific for wild-type or null alleles. Both products were de tected, suggesting the wild-type allele is not lost (not shown). To determine whether stabilizing mutations occurred in the wild-type p53 allele, immunostaining was performed with antibodies recognizing both wild-type and mutant p53. No p53 protein was detectable by immunofluorescent analysis (not shown). Interaction of v-rasâ€•@and p53 on Growth Regulation of Kera tinocytes in Vitro. The differences in tumor growth rates reflective of p53 status could be studied further in vitro in primary cultures. Basal keratinocytes proliferate in culture medium containing 0.05 mMCa2@ and cease proliferation and terminally differentiate in response to elevated levels of extracellular Ca2@(35). Basal cell cultures of each genotype were switched to medium containing 1.3 m@Ca2@for 24 h and pulsed with [3H@thymidinefor the terminal 2 h. As shown in Fig. 5, control cells of each genotype responded similarly to elevated calcium, with DNA synthesis declining to 4â€”6% of that of the respective genotype maintained in 0.05 mt@iCa2@. In contrast, the response to elevated Ca2@ of cells transduced with v@ras}@a differed significantly among the three genotypes. Elevated Ca2@ almost complete suppression of DNA synthesis in wild-type cells, while p53(â€”/â€”)/v-ras'@keratinocytes were less suppressed (3% ver sus 17.5% [3H]thymidine incorporated relative to the respective gen otypes maintained in 0.05 m@i Ca2@). Heterozygous cells re sponded in a manner intermediate between p53(+/+) and p53(â€”/â€”)cells, with 7.6% of the [3Hjthymidine incorporated relative to 0.05 mM Ca2@ cultures (Fig. 5).

p5@+/+)

weeks

following

grafting

Fig. 2. p53 status modulates growth rates of tumors developing from v-ras'@ trans.

duced keratinocytes grafted to the backs of nude mice. Keratinocytes from each p53 genotype were infected with retrovirus encoding v-rasâ€•@ and grafted to nude mouse hosts. Tumorswere measuredas describedin â€œMaterials and Methods.â€•

5586


p53 AND v.rasHlIN EPIDERMALKERATINOCYTES

Fig. 3. Histology of tumors developing from v-ms'@ transduced keratinocytes grafted to the

@ @

backs of nude mice. A, p53(+/+), 35 days after grafting; B and D, pS3(+/â€”),35 days after graft ing; C,p53(â€”Iâ€”), 27 days after grafting. A, X 26; B, X30;C,X60;D,

-tÃ§:

..

S /ta

x 53.

@

v.':

@

:-

, -@

@.. * .@ ..@.

@

.

.

. . :

. @;

.,

.-â€˜ @)

@e:@l

@â€˜. -,..

,

â€˜@

â€˜

.

.

.: . .

@

.

-

â€¢@.:â€¢,

â€¢.

.

.

. .

Table1 p53-deficienttumorshaveincreasedpotent/a/formalignantconversion

. @, .,

â€˜. .:.:t,@.

@>L.: â€¢

,t..

is the major positive growth regulator for keratinocytes (45). Levels of

Keratinocyte cultures of each genotype were infected with retrovirus encoding v-ras@@ specific 1@I-EGF binding were uniform across all genotypes in train and grafted in combination with fibroblasts from p53 (+1+) littermates. Samples were fected control cells maintained in 0.05 mrvtCa2@(cpm â€˜@I-EGF bound/ collected for histological evaluation 3-5 weeks grafting.pS3(+/+) following

@

p53(â€”/â€”)papilloma

cells = 1679, 1467, and 1560 for p53(+/+), -(+1â€”),and -(â€”Iâ€”) cultures, respectively). Binding decreased following introduction of v-rasâ€• relative to the mock infected controls. This finding is consistent with down-regulation of EGF receptors due to the v@ras@@a@mediated secretion of the EGF receptor ligand TGF-a observed previously (46).

p53(+/â€”)

papilloma+ carcinoma 7Total carcinoma (poorly differentiated)

8

9

0

0

10

0

0

0

8

19

7

The decreasein EGF receptorsfollowing introductionof v@rasH@@ was

p53 Genotype:

GAPDH

observed in keratinocyte cultures of all genotypes to a similar extent (10â€”12%of mock infected controls). EGF binding levels declined in response to 13 msi @2Â± as previously described (40); levels were too low to assess differences among genotypes in responsiveness to elevated calcium in v-ras'@-infected cells(data not shown). By radioimmunoassay analysis of conditioned medium from each cell type (47), there was no evidence of genotype-dependent changes in secretion of the positive growth regulator TGF-a from v@ra1@akeratinocytes (data not shown). It has been determined previously that 3 distinct TGF-@ receptors are present on the cell surface of epidermal keratinocytes (48). To

(+1+) (+1â€”)(â€”I---)

â€¢ ::

::

determinewhether the differencesin TGF-j3 responsivenessreflect

RelativeExpression: 1.0 0.89 0.80 Fig. 4. v-msâ€• transduced keratinocyte cultures differing in p5.3 gene dose express similar levels of v-ras@ mRNA. Keratinocytes of each genotype were infected with retrovirus encoding the v-ras@ oncogene and cultured for 8 days in 0.05 m@Ca2@before

D

mRNAisolation.Relativelevelsof v@ras@h mRNAnormalizedto GAPDHmRNAare shown below. Similar results were obtained in a duplicate experiment. 00 0

@p53(+/+)

k:::: p53(+/@) U p53(-/-)

Cs .- (V

@

Following introduction of v-ras'@, p53(/) keratinocytes were C also less responsive to growth suppression by TGF-f3. Results from a EE representative experiment are shown in Fig. 6. Decreased responsive â€˜0 ness ofp53(_/_)/v@ra1@a keratinocytes was observed at several doses I of [email protected] response ofp53(+/â€”) cultures was less consistent, in 0 some experiments behaving like pS3(+/+) cells and in others dem 0 onstrating a response intermediate between pS3(+/+) and p53(â€”/â€”) + cells. Similar results were obtained with TGF-f31. The reduced re Fig. 5. p53 gene dose modulates responsiveness of v-ras@ keratinocytes to elevated sponsiveness of p53-deficient polybrene control cells to TGF-@3 [@2+J Results are presented as cpm [3H]thymidine/unit protein in 1.3 [email protected]@ relative shown in Fig. 6 was observed in 3 of 5 experiments. to 0.05 mM @2+ cultures for each genotype. Bars, SEM of triplicate samples. A test for trend to evaluate the relationship between p53 gene dose and Ca2@responsiveness was Analysis of Growth Factor Receptors. To determine if a change perfonned using linear regression. A significant gene dosage effect was observed in in a positive growth effector could contribute to the growth response v-ras@ transduced cells (P 0.003) but not control cultures (P 0.82). Consistent ofeach genotype, EGF receptor modulation was examined. This receptor results were obtained when cpm were normalized to DNA or cell number. 5587


p53 AND v.rasnaIN EPIDERMALKERATINOCYrES control

with 1.3 mM Ca2@ or TGF-f3, regardless of p53 status and/or the presence of an activated ras@ oncogene (Fig. 8; Table 3). Transcript levels for WAF1, the p53-induced growth suppressor protein (13), were also examined to determine whether the altered growth regulation observed in pS3(â€”/â€”)keratinocytes reflects WAF1 levels. Basal levels of expression of this transcript in proliferating keratinocytes, as detected by Northern blot hybridization, correlate with the number of functional p53 alleles present (Fig. 8; Table 2). However, low levels of WAF1 mRNA were present in pS3(â€”/â€”) keratinocytes (Fig. 8; Table 2) in the absence of detectable p53 mRNA (not shown). There was an apparent decrease in GAPDH mRNA following TGF-@3treatment according to p53 gene dose. While den sitometer readings of 0.05 mMCa2@cultures in the experiment shown in Fig. 8 were 630, 728, and 591 in pS3(+/+), -(+1â€”),and -(â€”Iâ€”) keratinocytes, respectively, readings were 663, 699, and 396 follow ing a 24-h incubation with 1.0 ng/ml TGF-@31(Fig. 8). This accounts for the slight increase of WAF1 mRNA levels observed in TGF-13 treated p53 deficient keratinocytes (Table 3). The relative increases in WAF1 mRNA levels following TGF-(3 treatment without normaliza tion to GAPDH mRNA in the 2 experiments shown in Table 3 were 1.28 Â±0.12 (mean Â±range), 1.16 Â±0.08, and 1.35 Â±0.29 in p53(+/+), -(+1â€”), and -(â€”Iâ€”)keratinocytes, respectively. There was no evidence of WAF1 induction following 24 h in 1.3 mM Ca2@ (Table 3).

cells

70

i:i a2ng/ml TO-82

60

@Jl0ng/ml

6C

TGF-82

. 2Osg/ml 10-82

.!@

Sc

so

4c

ic; V x

3c

30

2c

K.

@rL@ p53

genotype

(+/+)

(.1-)

(-I-)

(@/+)

(+/-)

(-I-)

Fig. 6. Keratinocyteresponsivenessto TGF-@is modulatedby p53 gene dose. Results

are presentedas cpm [3H@thymidine/unit proteinin the presenceof T0F-@ relativeto 0.05 msi @2+alone for each genotype. Bars, SEM of triplicate samples. A test for trend betweenp53 gene dose and TGF-@ responsiveness was performed usinglinear regression.

A p53 gene dosage effect was observedin both controlandv-ras@@ transducedkeratino cytes at several doses ofTGF-@. In control cells, P = 0.007, 0.023, and 0.17; in v-ras@ transduced keratinocytes, P = 0.002, 0.007, and 0.010 following treatment with 0.2, 1.0, and 2.0 ng/ml TGF-f32,respectively. Identical experiments using TGF-@,or in which cpm were normalized to DNA or cell number yielded similar results.

changes in expression of these receptor types, TGF-131 bound to cultured keratinocytes was cross-linked to cellular receptors and an alyzed by SDS-polyacrylamide gel electrophoresis. As demonstrated in Fig. 7, all 3 receptors are present in cells regardless of their p53 status. To determine whether the altered TGF-@3responsiveness of p53 deficient keratinocytes is associated with decreased binding to recep tors, specific binding levels were quantitated. No detectable differ ences were observed between genotypes. The binding of 1@I-TGF-@ was similar among all genotypes in cells without the v-ras@ onco gene. TGF-@3binding relative to uninfected controls decreased fol lowing introduction of v@rasHa,as demonstrated previously (39); this appears to be partially due to occupation of the receptors, inasmuch as binding increases following mild acid treatment to strip ligand from occupied receptors. There was no reproducible difference in binding levels of radiolabeled TGF-@3among the 3 genotypes expressing v@rasI@a relative to mock infected control cultures (data not shown). Changes in mRNA Levels Associated with Growth Inhibition. To explore further the pathways involved in the observed p53 de pendent alterations in growth regulation, mRNA was isolated from keratinocytes of each genotype, with and without introduction of the v@rasHaoncogene (Fig. 8). Because c-myc has been implicated in the keratinocyte response to TGF-f3 (49), Northern blots were probed for c-myc mRNA. Densitometric analyses of c-myc transcripts suggest a reduction in c-myc mRNA in the absence of p53 in control, but not v@ras@@a cultures (Table 2). c-myc transcript levels decline in kerati nocytes following TGF-@ exposure (49). In this study, c-myc mRNA levels declined in all cultures to a similar extent following treatment

DISCUSSION In this study we have utilized genetically engineered p53 deficient mice and a nude mouse graft model to explore the role of the p53 tumor suppressor gene product in epidermal growth regulation and malignant conversion. We demonstrate here that decreased p53 gene dosage in keratinocytes cooperates with a mutant ras oncogene, resulting in decreased responsiveness to negative growth regulation in vitro and higher tumor growth rates and malignant conversion in vivo. The increased tendency of pS3(+/â€”) keratinocytes expressing a mutated ras}@ato progress to malignancy is consistent with recent results of Kemp et aL (34) using a chemical carcinogenesis protocol; however, the growth rates of the heterozygous tumors differ between these 2 studies. The increased growth rates reported here were con sistently observed in 4 independent experiments. While the majority of studies characterizing p53 gene alterations in human tumors have focused on identifying p53 mutations, the identification of tumors which are hemizygous for p53 with no evidence of mutation in the remaining allele suggest that p53-deficient cells have a relative growth advantage (50). The discrepancy between the growth rates of p53(+/-) tumors reportedhere and those reportedby Kemp et aL (34) may be due to the interaction of p53 deficiency with the higher expression level of v-ras@ in this study compared to endogenous c-ras mutations. TGF-@3has been implicated in the growth arrest associated with Ca2@-induced terminal differentiation of epidermal keratinocytes, as

control

@ @

Fig. 7. TGF-@3 receptors are expressed in control and v-ras'@'keratinocytes independent of p53 sta uts. Cultures were incubated with @I-TGF-@3 and receptor-ligandcomplexes were cross-linked and

@

sis as described in â€œMaterials and Methods.â€•*, ligand binding was performed in the presence of nonradiolabeled TGF-@ as competitor to demon strate specificity.

resolved by SDS-polyacrylamide

@

p53:

(+1+)

+ v@rasHa

(+1-) *

200

gel electrophore

97

@5:

.@ .

69

(-I-) *

p53:

(+1+)

(-I-)

*

*

200 97

69

46@

(+1-)

*

46

5588


4.

.

@

.â€˜@

@:

p53 AND v-ras0@ INEPIDERMAL KERA11NOCYTES

A p53(+/+)

1 @

2

c-myc

@

p53(+/â€”)

3'

p53(â€”/--)

â€˜1 23

23

1

@4 ,,w

api@@ .@

WAF1

GAPDH Fig. 8. Northern blot analysis of c-myc and

WAF1mRNAin epidermalkeratinocytesdif fetinghsp53 genedose.A,controlcultures;B, v_rasH. transdund keratinocyte cultures. Cells were treated for 24 h with 0.05 nmt Ca2@ (f@

@

1); 1.3 mt.i Ce2@ TGF-@ (Lane3).

2); and 1.0 ng/inl

B p53(+/+)

123 @

c-myc

@

WAF1

-@-

.@

p53(+/â€”) I

I

123

-w

-@-

-

I

p53(â€”/--)

I

123

-

-i..c:@

GAPDH

-w

suggested by the concomitant induction of TGF-@32production and decline in available TGF-f3 receptor binding sites (48). The decreased responsiveness ofp53-deficient cells to TGF-(3 observed in this study

possibility, previous studies (51, 52) have demonstrated a partial abrogation of TGF-@ responsiveness within clonal populations of cells overexpressing mutant p53. Furthermore, wild-type p53 was

is only partial. This in vitro phenotypecould be due to either de-

unableto restoreTGF-(3responsiveness of a squamouscarcinomacell

creased responsiveness of the entire cell population or a total loss of line devoid of functionalp53 (53), indicating that p53 is not sufficient responsiveness of a subpopulation, in which case the resulting tumor for complete regulation of the negative growth response to TGF-g3. phenotypes would likely represent outgrowths of subpopulations deWhile the classic initiation-promotion model of skin carcinogenesis tected by altered growth regulation in vitro. In support of the former generates large numbers of papillomas, the majority of these tumors 5589


p53 AND v.rasHaIN EPIDERMALKERATINOCYTES Table 2 Relative levels ofmRNA for WAFI and c-myc between genotypes

Densitometry of autoradiograms from Northern blot hybridizations, as shown in Fig. 8. Cultureswere maintainedin 0.05 mat Ca2t Results are shown relative to p53 (+1+) cultures and normalized to GAPDH. Values represent mean of duplicate experi ments Â±rangeof samples.p53 genotypeControl

v-ras11@(+1

cells

+ (+1

(â€”Iâ€”)c-myc +) (+/â€”) (â€”Iâ€”) +) (+1â€”) WAF1

1.0

0.80 Â±0.01

0.58 Â±0.08

1.0

1.08 Â±0.34

0.85 Â±0.24

1.0

0.62 Â±0.18

0.24 Â±0.08

1.0

0.98 Â±0.15

0.34 Â±0.01

p53-deficient keratinocytes compared to p53(+/+) keratinocytes under all culture conditions tested. Differences in WAF1 might there fore contribute to the altered growth regulation in this model system, but further studies, including an analysis of protein levels, will be required to address this issue. Whether in vivo selection of malignant p53(@/@)/v-ras'@ kerati nocytes is required for the carcinomas observed is unclear. Two weeks following grafting tumors from p53(@/@)/v-ras'@ keratinocytes are primarily papilloma tissue, although histologically these tumors are considerably more dysplastic than p53(+/+)/v@ras@Ia control tumors. These

results

are consistant

with the increased

grafts of a papilloma cell line overexpressing do not progress to malignancy and many eventually regress. Modifi cation of the promotion protocol elicits tumors with increased fre quency of malignant progression (54). Features of tumors with such a â€œhighriskâ€•profile include early loss of expression of the growth inhibitors TGF-f31 and 1-@2 (55) and a high proliferation rate (56). This suggests that the normal growth inhibitory effects of TGF-(3 may help to maintain the benign phenotype. The increased growth rates in vivo and decreased responsiveness in vitro of p53-deficient/v-ras'@ kera tinocytes suggests that loss of function of p5.3 may provide an alter nate mechanism for circumventing the TGF-f3 mediated pathway of growth regulation. We found no evidence for receptor modulation causing the de creased TGF-f3 responsiveness observed in p53-deficient keratino cytes. Cultures from all three genotypes demonstrated the presence of the same three receptors for TGF-j3, with no consistent change in ligand binding levels detected between genotypes. However, we can not rule out the possibility of alterations in relative amounts of specific receptor types and/or their interactions which could modify the efficiency of ligand presentation or signal transduction. TGF-f3 regulation of epidermal growth is believed to be mediated by the c-myc proto-oncogene (49, 57). c-myc mRNA and protein levels have been shown previously to decline in keratinocytes follow ing treatment with TGF-@3(49), but not elevated Ca2@ (49 58). Decline in c-myc mRNA was observed in this study following both treatments in all genotypes to a similar extent, regardless ofp53 status and/or presence of an activated ras@' oncogene. Thus, the mechanism by which p53-deficient cells escape growth regulation by TGF-f3 appears to be independent of or downstream from the c-myc signal. Recently, several groups have cloned the same cDNA sequence encoding a protein designated WAF1, CIP1, and p21, which is in duced by p53 (13), functions as an inhibitor of cyclin-dependent kinases (59) and cell proliferation (13), and has been found associated with most cellular cyclin-dependent kinase complexes (60). mRNA for this transcript is greatly reduced in p53-deficient fibroblasts (60). The presence of low levels of WAF1 in p53(â€”/â€”)keratinocytes indicates that WAF1 expression in keratinocytes is not entirely regulated by p53. However, WAF1 mRNA levels remain lower in

dysplasia

observed

in

a mutant form of p53

(61).However,therapidphenotypic progression of v-ras'@p53(â€”/â€”) keratinocytes to poorly differentiated carcinomas in the next several weeks indicates that either a rapid multifocal process has occurred when p53 is absent from keratinocytes or the early histological find ings are misleading and result from factors controlling morphogenesis in the grafting system. Previously, malignant clones of human kera tinocytes grafted to nude mice were noted to form tumors indistin guishable from benign clones during the first 2 weeks in vivo (62); only later were the characteristics

of malignant

growth

evident.

While it appears from these results thatp53 gene dosage may affect both in vitro and in vivo growth properties and the neoplastic pheno type, loss of one p53 allele may predispose cells to the loss of the remaining allele. Fibroblast cultures from p53(+/â€”) mice have been shown to lose the wild-type allele during early passages; this loss appears to confer a growth advantage (63). Thus, an alternative explanation for the rapid growth ofp53(+/â€”) tumors observed in this study is the early inactivation or mutation of the remaining wild-type allele in a subpopulation of cells in vitro. Such a loss of the remaining wild-type allele in pS3(+/â€”) epidermal carcinomas has been de scribed by Kemp et a!. (34). PCR analysis of carcinoma foci micro dissected from heterozygous tumors in the present study demonstrated the presence of the wild-type allele, suggesting that the wild-type sequence is not lost. However, the degree of stromal infiltrate makes it difficult to rule out the contribution of a wild-type allele from stromal components. There was no evidence of a stabilizing mutation arising in the remaining wild-type allele of carcinomatous foci within papillomas derived from pS3(+/â€”) cells, as determined by immunostaining. The observation that p53 mutations are late events in the progres sion of many tumor types suggests that loss of functional p53 is involved in the onset or progression of malignancy. While p53 is frequently lost in human cancers, mutant forms of the protein are believed to act in a trans-dominant manner to sequester the wild-type gene product and prevent its function. In addition, recent reports demonstrate that certain mutant forms of p53 can provide a gain-of function advantage (10, 64). Retention of mutant p53 is commonly observed

in human squamous

cell carcinomas

of the skin, presumably

TGF-f3Densitometry Table3 Modulationof c-myc and WAFJnzR.NAlevels associated withgrowtharrest inducedby elevated Ca2'@and of Northern blot hybridizations, as represented in Fig. 8. Results shown are relative to 0.05 [email protected]@ cultures for each genotype and normalized to GAPDH. Values

shown are mean resultsfrom two independentexperimentsÂ±range.T0F-@ dose is 1.0 ng/ml.p53 genotypeControl

v@rasHa(+1+)

cells

(â€”Iâ€”)c-myc:

(+1â€”)

1.3 mp.iCa2@ 0.02w@F1: TGF-@1 1.3 mM Ce2@

0.19k'a TGF-@1

+ (â€”Iâ€”)

0.45Â±0.03 0.41 Â±0.06

0.65Â±0.06 0.44 Â±0.05

0.80Â±0.38 0.56 Â±0.24

0.84 Â±0.24

1.10 Â±0.08

1.03Â± 0.06

1.31Â± 0.12

(+1+)

(+1â€”)

0.59@z 0.42 Â±0.04

0.69Â±0.03 0.32 Â±0.06

1.34 Â±0.04â€•

0.78Â°

0.78 Â±0.07

1.59Â± 001b

1.14Â± 0.10

0.93Â± 0.08

O.68'@ 0.53 Â± 0.78Â°

1.90Â±

Results of a single experiment only. b The

apparent

increase

in

@AFl mRNA

in p53

(-I-)

keratinocytes

is magnified

by aresults.5590 small decrease

in GAPDH

mRNA.

This

does

not significantly


affect

the myc

mRNA

p53 AND v-ras@@ IN EPIDERMAL KERATINOCYTES

induced by UV exposure and in UV or chemically induced murine

13. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M.,

Lin, D., Mercer, W. E., Kinzler, K. W., and Vogeistein, B. WAF1, a potential

epidermal carcinomas (28, 29, 65, 66). The results in this study demonstrate that the oncogenic properties of mutant p53 are not required in combination with v@ra1@afor malignant traniormation in this model; a change in p53 gene dosage is sufficient. Furthermore, this property is inherent to the keratinocyte, inasmuch as epidermal cell preparations from each genotype were grafted with fibroblast preparations

from pS3(+/+)

mice.

p53 is believed to provide a cell cycle checkpoint following DNA damage (18, 67), and lack of functional p53 may predispose cells to replication ofdamaged DNA, resulting in genomic instability (17, 68). While the pathways involved in prevention of cell cycle progression by p53 are just being evaluated, the decreased responsiveness of initiatedp53-deficient keratinocytes to TGF-@3and Ca2@suggest these factors could contribute to genomic instability and accelerated tumor progression

in this cell type. Further

studies

will be needed

to address

this possibility. However, our results imply that the loss of p53 function in keratinocytes undergoing neoplastic transformation would provide a growth advantage to those cells and could explain the clonal selection of cells with p53 mutations in mouse and human skin

1992. 15. Mack,

D. H., Vartikar,

J., Pipas,

J. M., and Laimins,

TATA-mediated but not initiator-mediated (Land.), 363: 281â€”283,1993.

L. A. Specific

transcription

repression

of

by wild-type p53. Nature

16. Mercer, W. E., Shields, M. T., Lin, D., Appella, E., and Ullrich, S. J. Growth suppression induced by wild-type p53 protein is accompanied by selective down regulation of proliferating-cell nuclear antigen expression. Proc. Natl. Aced. Sci. USA, 88: 1958â€”1962,1991. 17. Livingstone, L R., White, A., Sprouse, J., Uvanos, E., Jacks, T., and Tlsty, T. D.

Altered cell cycle arrestand gene amplification potential accompany loss of wild-type p53. Cell, 70: 923â€”935, 1992. 18. Zhan, 0., Carrier, F., and Fornace, A. J., Jr., Induction of cellular p53 activity by

DNA-damaging agents and growth arrest. Mol. Cell. Biol., 13: 4242â€”4250,1993. 19. Rotter, V., Schwartz, D., Almon, E., Goldfinger, N., Kapon, A., Meshorer, A., Donehower, L. A., and Levine, A. J. Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome. Proc. Nail. Acad. Sri. USA, 90: 9075â€”9079,1993.

20. Woodworth, C. D., wang, H., Simpson, S., Alvarez-Salas, L M., and Notario, V. Overexpressionof wild-type p53 altersgrowthand differentiationof normalhuman keratinocytes but not human papillomavirus-expressing cell lines. Cell Growth & Differ., 4: 367â€”376, 1993. 21. Kastan, M. B., Radin, A. I., Kuerbitz, S. J., Onyekwere, 0., Wolkow, C. A., Civin,

C.I.,Stone,K.D.,Woo,T.,Ravindranath, Y.,andCraig,R.W.Levelsofp53protein increase with maturation in human hematopoietic cells. Cancer Res., 51: 4279â€”4286, 1991. 22. Fujiwara, T., Grimm, E. A., Mukhopadhyay, T., Cal, D. %V.,Owen-Schaub, L B., and Roth,J. A. A retroviralwild-typep53 expressionvectorpenetrateshumanlungcancer spheroids and inhibits growth by inducing apoptosis. Cancer Res., 53: 4129â€”4133,

cancers.

ACKNOWLEDGMENTS We thankDavid Morganand SherryLittlesfor assistancewith the grafting procedure and tumor measurements;

mediator of p53 tumor suppression. Cell, 75: 817â€”825, 1993.

14. Vogelstein, B., andKinzler,K. W. p5.3functionanddysfunction.Cell, 70: 523â€”526,

David Morgan, Christina Cheng, and Dr.

Andrzej Dlugosz for assistance collecting skins; Drs. Betty Slagle, Larry Donehower, Allan Bradley, and Terry Timme for recommending the PCR primers for genotyping; Dr. Robert Coffey for performing TGF-a radioimmu

noassays;Dr.AndrzejDlugosz for assistancewith the EGFreceptorassay;Dr. Bill Bennett for advice regarding PCR analysis of microdissected foci and p.53 immunostaining; Dr. Arnold Levine for A1-5 cells; and Gary Best for assis tance generating computerized images. We also acknowledge Dr. Robert

Tarone for assistance with statistical analyses; Donna Gulezian for helpful discumions regarding the p53-deficient

mice; Dr. Brenda Gerwin for critical

reading of the manuscript; and members of the Laboratory of Cellular Carci nogenesis and Tumor Promotion for helpful discussion throughout the course

1993.

23. Vogelstein, B., and Kinzler, K. W. The multistep nature of cancer. Trends Genet., 9: 138â€”141,1993. 24. Urano, Y., Oura, H., Sakaki, A., Nagae, H., Matsumoto, K., Fukuhara, K., Nagae, T., Arase, S., Ninomiya, Y., Nakanishi, H., Shigemi, F., and Takeda, K. Immunological analysis of p53 expression in human skin tumors. J. Dermatol. Sci., 4: 69â€”75,1992. 25. Brash, D. E., Rudolph, J. A., Simon, J. A., Lin, A., McKenna, 0. J., Baden, H. P., Halperin, A. J., and Ponten, J. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc. Nati. Acad. Sci. USA, 88:

10124â€”10128,1991. 26. Yuspa, S. H. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesisâ€”Thirty-ThirdG. H. A. Qowes Memorial Award Lecture. Cancer Res., 54: 1178â€”1187,1994. 27. Quintanilla, M, Haddow, S., Jonas, D., Jaffe, D., Bowden, G. T., and Balmain, A. Comparison of ras activation during epidermal carcinogenesis in vitro and in vivo. Carcinogenesis

(Land.), 12: 1875â€”1881,1991.

28. Ruggeri, B., Caamano, J., Goodrow, T., DiRado, M., Bianchi, A., Trono, D., Conti,

C.J., andKlein-Szanto, A.J. P.Alterationsof thep53 tumorsuppressorgeneduring

of this work.

mouse skin tumor progression. Cancer Res., 51: 6615â€”6621,1991. 29. Burns, P. A., Kemp, C. J., Gannon, J. V., Lane, D. P., Bremner, R., and Balmain, A.

Lossof heterozygosity andmutationalalterationsof thep53genein skintumoursof

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