Vol. 2, 661-667, December 1991 Cell Growth & Differentiation 661

3 The abbreviations used are: NIS, nuclear localization signal; cDNA,complementary DNA.

Involvement of Wild�Type p53 Protein in the Cell CycleRequires Nuclear Localization’

Gad Shaulsky, Naomi Goldfinger, Alpha Peled, andVarda Rotter2

Departments of Cell Biology [G. S., N. G., V. R.1 and ChemicalImmunology [A. P.], The Weizmann Institute of Science,Rehovot, Israel 76100

Abstrad

Transfedion of wild-type p53 into a pre-B, p53nonproducer cell line yielded the generation of stableclones. Although constitutively expressing the growth-suppressor wild-type p53 protein, these cellsproliferate continuously in vitro. However, expressionof wild-type p53 in these cells altered their cell cyclepattern and reduced their growth in vivo. When thesame parental cells were transfeded with a plasmidcoding for a wild-type p53 lacking nuclear localizationsignals, a wild-type cytoplasmic p53 protein wasexpressed. Expression of this cytoplasmic p53 produddid not exert any changes in the growth of the parentalcells, suggesting that wild-type p53 affeds the cellcycle only when localized in the nuclear cellcompartment.

Introduction

Wild-type p53 was shown to function as a negativegrowth regulator (1-5). This protein, which acts as agrowth suppressor in normal cells, was found to beinactivated in tumor cells, thus permitting malignant celltransformation (6-i2). Expression of wild-type p53 in-duced growth arrest of a variety of tumor cells (4, 5, 8,13, 14). Overexpression of wild-type p53 in transformedcell lines, such as glioblastoma, colorectal carcinoma, orosteosarcoma, led to reduced levels of DNA replication.This was not the case when the same wild-type codingplasmids were transfected into less transformed cells,such as colorectal adenomas (8).

Association of wild-type p53 with the cell cycle wasshown in several experimental models (1 3-i 7). In anattempt to reveal the function of p53, it was suggestedthat this protein is associated with DNA replication. Wild-type p53 was found to compete with DNA polymerasea in the replication of SV4O DNA regulated by the largeI antigen (18-20) and to localize in the cell nucleus tothe same sites as known DNA replication proteins (2i).Furthermore, it was recently shown that p53 is a targetprotein for the activity of p34cdc.2, a cell cycle-dependent

protein kinase (22, 23). An analysis of subcellular local-ization of the p53 protein indicated that the protein isspatially regulated as the cell progresses through the cellcycle. In the early G0-G1 phase of serum-stimulatedstarved BALB/c-313 cells, p53 is mostly cytoplasmic.Entrance of the cells into the S-phase is accompanied bynuclear localization of p53 (16). As is the case with othernuclear proteins, the p53 protein contains nuclear local-ization signals NLSs3 that are inherent in the primarystructure of the protein (24-26). The COOH-terminalportion of the protein was found to contain three NLSsthat account for nuclear localization of the p53 protein(26). In a series of experiments using a temperature-sensitive mutant p53, it was found that the wild-type p53is nuclear whereas its mutant form is mostly located inthe cytoplasmic compartment (27-29). The fact that thewild-type p53 form, expressed at low temperature, alsoexerts its activity under those same conditions suggestedthat nuclear localization of wild-type p53 is an essentialevent for the activity of the protein. A similar conclusionwas reached when it was found that an NLS-deprivedcytoplasmic form of wild-type p53 lost its suppressiveactivity, as measured in transformation of primary embry-onic fibroblasts (30).

In previous experiments, we found that transfection ofwild-type p53 into a p53 nonproducer pre-B-cell line,designated Li 2 (31, 32), gave rise to stably growing clonesthat constitutively expressed the p53 protein. Expressionof wild-type p53 in these cells modified the cell cyclepattern, giving rise to clones that had an extended G0-G1phase when compared to the parental cell line (33). Ananalysis of the in vivo cell growth of these cell lines,estimated as tumor growth in syngeneic mice, indicatedthat Li2 pre-B-cell lines expressing wild-type p53 exhib-ited a reduced rate of tumor development. These cellsdifferentiated further and, judged by several markers,exhibited a more differentiated phenotype of pre-B-cells(33).

In the experiments reported here, we studied theinterrelationships between nuclear localization of p53and the cell cycle. By using the L12-derived cell lines,we found that NLS-deprived cytoplasmic wild-type p53did not alter the cell cycle pattern, nor did it affect thedevelopment of tumors in mice. These results suggestthat involvement of p53 in the cell cycle requires nuclearlocalization of the protein. When wild-type p53 is unableto enter the cell nucleus, it does not affect the cell cycle.

Results

Received 6/18/91.

1 This work was supported in part by grants from the Leo and Julia

Forchheimer Center for Molecular Genetics and the Rockefeller-Weiz-mann Fund. V. R. holds the Norman and Helen Asher Professional Chairin Cancer Research and a Career Development Award from the IsraelCancer Research Fund.2 To whom requests for reprints should be addressed.

Establishment and Charaderization of Li 2-derived CellLines. In previous experiments, we found that the p53

abed

�_ -

-

662 Cell Cycle and p53 Nuclear localization

L12 L12-p53cDlO L12-p53cD � ‘�‘�“3E L12-p53M8-3A2 L12-p53M8 NLSI. lOB

a b cd a b cd a b cd abed

-

- - 4-p53

Fig. 1. p53 expression in 112-derived cell lines. Equal amounts of trichloroacelic acid-insoluble radioactive cell lysates were immunoprecipitated with

the following antibodies: (a) nonimmune serum, (b( anti-mutant p53 monoclonal PAb-240, (c) anti-wild-type p53 monoclonal PAb-246, and (d) anti-mutant and anti-wild-type murine p53 monoclonal PAh-248.

protein contains three nuclear localization signals, allclustered in the COOH-terminus of the molecule. Site-directed mutagenesis indicated that all three domains actin an additive way (26). Although the most 5’ end do-main, NLSI, is the principal active signal, efficient nuclearlocalization depends on the additive activity of all threedomains. Whereas transfection of p53cD that codes fora wild-type p53 into primary embryonic fibroblasts gaverise to a p53 protein that was mostly expressed in thenucleus, transfections of p53cD�tSh yielded a p53protein that was mostly localized in the cytoplasm (26,30). Recently, we found that overexpression of wild-typep53 in a p53 nonproducer pme-B-ceII line, L12, gave rise

to stably proliferating cells. This system, in which consti-tutive overexpression of wild-type p53 does not totallyarrest cell proliferation (33), permits investigating the roleof this protein in proliferating cells.

To compare the intact wild-type p53 to its cytoplasmicderivatives, we transfected the corresponding plasmidsinto L12 cells. The following representative clones were

compared to the parental L12 cell line: L12-p53cDiO,obtained by transfection of pSVL-p53cD, which encodesthe intact form of wild-type p53; L12�p53cDN�3E,which harbors the NLS-defective form of wild-type p53cDNA; L12-p53M8-3A2, which represents Li2 cells ex-pressing mutant p53; and L12�p53M8r��� lOB, trans-fected with an NLS-defective mutant p53, which is una-ble to enter the cell nucleus.

Characterization of the p53 protein expressed by thesecells was done by specific immunoprecipitation with anti-p53 monoclonal antibodies. Fig. 1 shows that when weused the PAb-248 antibody (34) (Lane d), which mecog-nizes all of the known murine p53 forms, all cell linestransfected with p53 cDNA expressed p53 proteins ofthe expected size. Furthermore, the differential immu-noprecipitation patterns, obtained from the mutant p53

specific antibody PAb-240 (35) (Lane b) and the wild-type p53 specific antibody PAb-246 (35) (Lane c), showedthat all of the proteins carry the expected epitopes,regardless of the NLS structure. Whereas mutant p53forms coprecipitated a protein of about 70 kilodaltons,

which probably was the hsc 70 protein (36), no suchcomplexes were detected when wild-type p53 or NLS-deprived wild-type p53 was studied. This differentialability to complex with hsc 70 agrees with previousreports, showing that wild-type p53 and mutant p53 varyin their affinities to form complexes with hsc 70 (36).Based on the fact that the NLS-deprived protein con-tamed the specific antigenic determinants and on thedifferential complex formation with hsc 70, it was con-cluded that the p53 protein encoded by the NLS-de-prived sequences, although having a modified sequence,still retains its wild-type nature.

Subcellular Localization of p53 Proteins in L12-de-rived Cell Lines. Previously, we reported that complexformation with the large T antigen facilitated nuclearlocalization of NLS-defective wild-type p53 in COS cells(26). This was not the case with mutant p53, which doesnot form complexes with the large T expressed in thesecells. This suggests that, in addition to the presence of

nuclear localization signals inherent in the primary struc-tume of the protein, the actual subcellular localization isaffected by the presence of other cellular or viral proteinsexpressed in the cell (26). Another example of a proteinin which subcellular migration is affected by the specificcellular microenvironment is the v-re! oncogene. In thatcase, it was found that overexpression in chicken embryofibroblasts yielded nuclear localization (37), whereas inearly spleen cells in chicken, the protein was essentiallycytoplasmic (38). It was therefore important to examinewhether subcellulam localization of the various NLS-defective proteins in the L12 pre-B-cells indeed de-pended on the presence of intact NLSs.

Li2 and Li2-demived cell lines were metabolically Ia-

beled and fractionated into two subcellulam fractions con-taming either the nucleus or the cytoplasm. The specificp53-derived proteins were immunoprecipitated, and theresults are shown in Fig. 2. Clearly, the NLS-deficientprotein encoded by p53CDN��t’�h was detected in the

cytoplasmic fraction, without any expression in thecell nucleus. Indeed, cell fractionation of Li2-p53cDNs�u3E or Li 2-p53cD��6B cell lines

- - -

Cell Growth & Differentiation 663

Wild-type p53 is PAb-240 negative and PAb-246 positive.h Mutant p53 is PAh-240 positive and PAb-246 negative. See te’xt for more details.

Li 2-p53cD5B

NI ‘lb C�r Nu

Li 2-p53cD 10

N! Tb Cy Nu

NLSI-.NLSII-

L12-p53cD 3E

NI ‘lb Cy Nu

NLSI-.NLSII-L12-p53cD 6B

NI ‘lb Cy Mi

4- p53

Fig. 2. Subcellular localization of p53 in 112-derived cell lines. tJnfractionated cells (total( and cells fractionated into Triton x-loo-soluble (cytoplasmic(

and insoluble (nuclear) fractions were immunoprecipitated with anti-p53 monoclonal antibodies PAb-242. N!, nonimmune; To, total cell lysate; Cy,cytoplasmic cell fraction; No, nuclear fraction.

showed that most of the p53 protein is localized in thecytoplasmic compartment. Fractionation of Li2-p53cD5B or L12-p53cDiO cell lines expressing the intactwild-type p53 indicated that most of the protein is found

in the nuclear fraction. These results demonstrate that inthe L12 pre-B-cells, subcellulam localization of exoge-nously expressed wild-type p53 is solely dependent onthe presence of intact NLS sequences. The same subcel-lular patterns were also obtained with L12 cell linesexpressing the mutant p53 forms (data not shown). In

agreement with our findings in primary embryonic cells(30), intact mutant p53 was nuclear in its localization,whereas NLS-defective mutant p53 proteins were mostlycytoplasmic.

In the subsequent experiments, we compared celllines expressing wild-type p53 to cell lines expressingNLS-depmived wild-type p53, with respect to the variousin vitro and in vivo growth parameters.

Effect of p53 Localization on in Vitro Growth of 112-derived Cell Lines. To evaluate the doubling times of thevarious cell populations, cells were passaged twice be-

fore each experiment at subconfluent concentrationswithout drug selection. Equal numbers of cells were then

plated in triplicate, and viable cells were counted at 24,48, and 72 h after plating. Cell viability was essentially100% at every time point. Data summarized in Table 1show that the L12-demived clone L12-p53cDlO, which

expresses the intact wild-type p53, have an increaseddoubling time compared to the parental L12 line. How-ever, the L12-demived clones � and L12-cDN���tsu6B, which express the NLS-defective wild-type p53, were essentially similar to the parental L12.The observation that none of the NLS-defective variants

of the wild-type p53 exerted any effect supports the

notion that nuclear localization accounts for the altered

cell cycle patterns.Furthermore, examination ofthe Li 2 cell lines express-

ing mutant p53 (clones L12-M8-3A2 and L12-M8-2C1) ortheir NLS-defective counterparts (clones L12-M8�1PAand L12�M8N�10B) revealed no significant variations in

the doubling time when compared to parental L12 cells.The mean doubling time for all of the p53M8-derived

Table 1 In vitro growth of 112 and its p53-expressing derivative’s

,112-derived e’Il line p53 type

Subcellolarlo alizate)n

t)oublingtinie (h(

Cell ( Y( Ic’ f)h,lses (%(

c -c,, s c .-M

112 None 10.1 32.�6 18.01 29.60

L12-cD1O Wild-type’� Nuclear 15.0 47.81 28.89 23.30L12�cD�SNiS�3E Wild-type Cytoplasmic 1 1.0 .13.45 40.60 25.95L12�cDNLS�h6B Wild-type Cytoplasmic 10.4 31.81 45.48 22.71

112-M8-3A2 Mutanth Nuclear 9.5 34.55 38.06 27.39L12-M8-2C1 Mutant Nucle’ar 10.9 34.20 38.86 26.94

L12-M8�’10B Mutant Cytoplasmic 11.5 (5.01 37.55 27.44

l12-M8’�51PA Mutant Cytoplasmic 10.9 36.42 39.79 23.79

112-Mi i-2 Mutant Nuclear 9.8 35.47 (6. (7 28.16

1i2-Mii-3 Mutant Nudear 10.0 31.38 39.25 29.37

112-Mi 1NsNsh5G Mutant Cytoplasmic 10.6 35.23 16.69 28.08

1i2-Mi 1��s�[sh6B Mutant Ctyoplasmic 10.4 34.98 39.1 3 25.89

100

Fig. 3. Tumorigenicity of p53-ex-pressing 112-derived cell lines.

112-derived cell lines were in-jected s.c. into individual synge-neic, 7-week-old male C57L/Jmice. Tumor development wasmonitored daily, and tumorigenic-ity index was calculated as de-scribed previously (29). #{149},112-in-duced tumors; 0, 112-pS3cDwS�Su 3E-induced tu-moms; �, L12-p53M8-3A2-inducedtumors: #{149},L12-p53cDlO-inducedtumors.

Days after injection

664 Cell Cycle and p53 Nuclear Localization

xw

VC

U

Cwof

0E

I-

Li 2 clones was 10.3 h (SD = 1.0). Another form of mutantp53, encoded by pS3Mll, gave similar results, with amean doubling time of 10.1 h (SD = 0.3). In this case,clones L12-Mi1-2 and L12-M11-3 represented the intactnuclear form of the protein, and the two clones L12-Mi 1N�Si.Ni.SiisG and L12-Mi 1 NiSi.N[Sii6B were the NLS-defective counterparts. Due to an alternative spliced 3’terminus, mutant p53 encoded by p53M8 contains NLSIbut lacks NLSII and NLSIII. However, the encoded pro-tein is capable, although less efficiently, of migrating intothe cell nucleus (26). Mutant p53, encoded by p53M11,contains all three nuclear localization signals and migratesinto the nucleus as efficiently as wild-type p53 proteinencoded by intact wild-type p53cD. The fact that neitherof the mutant p53 forms modified the cell cycle stronglysuggests that involvement in the cell cycle is the functionof the nuclear wild-type p53 only. Mutant p53, althoughcapable of entering the nucleus, is inactive in this assay.

The Effect of p53 Localization on in Vivo Growth of112-derived Cell Lines. The Abelson murine leukemiavirus-transformed lymphoid pre-B-cell line L12 was thefirst example of a p53 nonproducer line (31 , 32). The p53gene of these cells was rearranged by the integration ofa Moloney murine leukemia virus into the first p53 intron(31). Whereas all other Abelson murine leukemia virus-transformed p53 producer cells develop lethal tumors insyngeneic mice, L12 cells develop regressor tumors. Wefound that although reconstitution of mutant p53 in thesecells enhanced their malignant transformation, leading todevelopment of lethal tumors (31, 32), reconstitution ofwild-type p53 expression rendered these cells less malig-

nant, leading to the development of tumors that re-gressed faster than with the parental line (33). In thepresent experiments, we compared the tumorigenicityof the parental Li 2 cell line with that of the derived celllines which expressed wild-type p53, NLS-defective wild-type p53, or mutant p53 forms. Cells were injected, andtumor development was monitored. Fig. 3 shows theprogression of tumors induced by the L12 p53 nonpro-ducer parental cell line, by Li2-p53cDiO, which expressesthe intact wild-type p53, by L12�p53cD�S��u3E,which expresses the NLS-defective wild-type p53, andby L12-p53M8-3A2, which expresses mutant p53. Com-parison of the various cell lines clearly showed thatwhereas wild-type p53 had a suppressive effect on thegrowth of Li2 cells in vivo, manifested as a reducedtumor take, slower tumor progression, smaller overallsize of the tumors, faster regression, and reduced lethal-ity, the cytoplasmic wild-type p53, lacking the ability toenter the cell nucleus, exerted no significant effects onthe tumorigenicity of Li2 cells. In this case, tumor pro-gression curves were indistinguishable from curves of theparental Li 2 cells. In agreement with our previous results,mutant p53 expressed by Li2-p53M8-3A2 enhanced tu-mom progression, whereas NLS-deficient mutant p53 didnot (26). The Li2-p53M8-3A2 tumors were lethal in100% of injected animals.

Modulations in Cell Cycle Patterns of Li 2-derivedClones. In the subsequent experiments, we measuredchanges in cell cycle patterns. To that end, we comparedthe relative numbers of cells at the various cell cyclephases of the individual cell lines. Cells were passaged

L12

- iii �

L12-p53-cDlO

IL12-gpt-3G2

. /‘�A��i�

L12-p53-cD5B

_)L12.p53.cDN1’�i. N1.Sii3E

. - �,1

L12-p53-cDN’-$’ .

_,,_..i�L12-p53-M8-3A2

t.,, l�ji�__J��\V�\lfr�

L12-p53-M8’#{176}-#{176}’ lOB

RI r�j�4�,jr s�

_ _.J�

Cell Growth & Differentiation 665

75

5O

75

75

25.

75�

50

N 2N N 2N

Fig. 4. p53-induced changes in 112 cell cycle. Exponentially growingcells were fixed and stained with propidium iodide. Histograms representthe cellular DNA content of a total of 5000 cells as indicated by propidiumiodide fluorescence intensity. On the linear horizontal axis, N representsthe minimal diploid amount of DNA, and 2N represents the doubledamount of DNA after the S phase. Names of individual cell lines areindicated inside each panel.

in culture as described above, fixed and stained with

propidium iodide, and analyzed by FACS. Fig. 4 is arepresentative comparison between the cell cycle pro-files of L12 and its derivatives as obtained by FACSanalysis, showing the relative cellular DNA content ofthe various cell lines. N represents the normal diploidamount of DNA at the G1-G0 phase of the cell cycle, and2N represents the doubled amount of DNA at the G2-M

phase. Clearly, L12 cells expressing wild-type p53 havea different cell cycle pattern from that of the rest of thecell lines. The wild-type p53-expressing lines, L12-p53cD5B and Li 2-p53cDiO, accumulated significantlymore cells in the G1-G0 phase of the cell cycle than theparental L12 cells. NLS-defective counterparts of wild-type p53, represented by Li2,cD����3E andL12,CDNLS,Sut6B, failed to change the relative fractionof Li2 cells at G1-G0. This observation supports theassumption that the effect of p53 on the cell cycle isdependent upon nuclear localization. Likewise, expres-sion of the gpt selective marker alone in L12-gpt-3G2had no significant effect on the cell cycle pattern. Sinceneither intact nor NLS-defective mutant p53 expressedby p53M1 1 and p53M8 had significant effects on thisgrowth parameter, it is likely that the effect of p53 onthe cell cycle is a unique feature of the wild-type p53.Table 1 summarizes the FACS analyses of L12 and L12-derived cell lines. The arithmetic mean of the G1-G0percentages of all cell lines (except for L12-p53cDiO) is34.08% (SD = 1.63), showing that most of the values arewithin the error margins. L12-p53cDlO, which representscells expressing the intact form of wild-type p53, is sig-nificantly different from the rest of the lines in its in-creased accumulation in G1-G0 (47.8%).

The results presented here support the hypothesis thatnuclear accumulation of p53 is essential for the proteinactivity. Furthermore, these data add more support tothe notion that negative growth regulation is specific towild-type p53, since neither activated mutants of p53,nor the NLS-modified wild-type p53, were able to exertthese activities. In the present study, we described de-tailed analyses of a representative number of clones ofeach of the cell line types. Similar results were obtainedin other clones established independently.

Discussion

Nuclear localization of p53 is dependent on the presenceof nuclear localization signals inherent in the primarystructure of the protein. However, actual localization ofthe protein in the cells may be affected by complexformation with other nuclear proteins expressed in thecell. For example, it was found that NLS-deprived wild-type p53 can migrate into the nucleus by forming acomplex with the large T nuclear protein (26). In the caseof a p53 temperature-sensitive mutant, it was suggestedthat nuclear localization may be affected by complexformation with heat shock proteins (28, 29), or by cyto-plasmic anchorage proteins (27).

In the case of the v-re! oncogene, cell type specificityis an important factor in determination of the ultimatesubcellular localization of the protein. It was shown that,whereas in chicken fibroblasts the v-mel protein is nuclear,in chicken spleen cells the protein is mostly cytoplasmicin its localization (37-39). Similar alterations in patternsof subcellular localization of nuclear proteins which de-pend on cellular factors, rather than just the activity ofNLS, were found with the transcriptional factor NF-KB(40). In the experiments presented here, we show that inthe case of p53, NLS plays a fundamental role in subcel-lular localization and, as in primary embryonic fibroblasts(30), subcellular localization totally depends on the integ-rity of the NLSs.

666 Cell Cycle and p53 Nuclear Localization

In the experimental cell system used in the presentexperiments, there is no endogenous p53 protein thatmay complex with the exogenous protein nor are thereany temperature changes which might induce the expres-sion of HSp. The data obtained are therefore derivedfrom the direct effect of the exogenous protein on thecells. Expression ofwild-type p53 in these cells prolongedthe doubling time, yielding a population with a longerG0-G1 phase and a lower growth rate in vivo. Indeed,when injected into mice, L12 cells expressing the wild-type p53 developed a lower incidence of smaller-sizedtumors than did the parental L12 cells. These growthregulation activities required nuclear localization andwere not expressed when wild-type p53 was deprivedof its NLS sequences. Furthermore, we found thatwhereas nuclear wild-type p53 was actively involved inthe cell cycle, nuclear mutant p53 was inert in vitro. Asimilar pattern was observed in another in vitro system,where transcription activity was measured by a GAL-p53fusion protein; here, too, wild-type p53 was active,whereas mutant p53 was inert (41, 42). It is worth men-tioning, however, that whereas p53 seemed to reducethe development of tumors in mice, mutant p53 had theopposite effect: it enhanced tumor progression. There-fore, it is plausible that in growing tumor cells in vivo,mutant p53 affects parameters in addition to those meas-ured under in vitro conditions.

Li2-derived cell lines are a unique example of stablygrowing clones constitutively expressing wild-type p53.In previous reports, however, it was shown unequivocallythat expression of transfected wild-type p53 in trans-formed cell lines arrested cell proliferation (4, 5, 8, 13,14). This discrepancy could be explained by the fact thatthe ability of wild-type p53 to act as a growth arrest geneis dependent on the neoplastic stage of the transfectedcells. It is possible that L12 cells represent a less trans-formed cell population and, therefore, similar to the caseof the colorectal adenomas (8), they do not undergoterminal growth arrest. Alternatively, growth arrest me-diated by wild-type p53 may be a reflection of a celldifferentiation process that is accelerated by wild-typep53 (33). In an early differentiated cell population suchas L12 pre-B-cells, expression of wild-type p53 mayinduce cell differentiation that is essentially not accom-panied by terminal growth arrest.

In conclusion, the observation that nuclear wild-typep53 increased the number of cells in the G1-G0 phase,coupled with the fact that p53 enters the cell nucleus asthe cell progresses to the S phase (16), suggests that wild-type p53, when in the nucleus, controls the transition ofthe cells from the G1-G0 into the S phase. L12 cells, inwhich no wild-type p53 is expressed, spend less time inthe G1-G0 phase. However, expression of wild-type p53sustains the cells in the G1-G0 phase, thus leading to themanifestation of a cell population with a longer doublingtime.

Materials and Methods

Gene Transfer. p53cD is a cDNA clone isolated from anormal T-cell cDNA library. It consists of the full-lengthwild-type p53 and an additional 95 base pairs containingan SV4O splice acceptor sequence upstream of the cod-ing region, derived from the original pcD-p53 plasmid(43). The p53Mi 1 cDNA clone, isolated from a Meth A

AgtlO cDNA library, codes for a full-length mutant p53protein, and the p53M8 cDNA clone, isolated from thesame library, codes for a mutant p53 protein that has analternatively spliced COOH-terminus (43, 44). The mam-malian expression vector used in these experiments waspSVL (Pharmacia). The recombinant plasmids (20 zg) anda selective marker, pSV2-gpt (5 �g) (45), were introducedinto cells; resistant clones were selected as describedpreviously (26).

Immunoprecipitation. Cells were metabolically Ia-beled for 1 h at 37#{176}Cin methionine-less DMEM, supple-mented with 10% heat-inactivated dialyzed fetal calfserum and 0.125 mCi/mI of[35S]methionine (Amersham).Cells Iysates were immunoprecipitated with the followingspecific anti-p53 antibodies: monoclonal anti-p53 PAb-240 (34), monoclonal anti-p53 PAb-248, and monoclonalanti-p53 PAb-246 (35), as previously described (26).

Cell Fractionation. Cells were labeled as above,washed twice with cold PBS and once with a buffercontaining 50 mM 2-(N-morpholino)ethanesulfonic acid,pH 6.1, 2.5 mM ethyleneglycol bis(fl-ammnoethyl ether)-N,N,N’,N’-tetraacetic acid, 5 mr�i MgCI2, and 1 mr’.iphenylmethylsulfonyl fluoride. Cells were resuspendedin a similar buffer containing 0.1% Triton X-100 andpipetted gently 25 times with a pasteur pipette to releasecytoplasmic proteins. Cell nuclei were pelleted by cen-trifugation at 2000 rpm and washed again with Triton X-100-containing buffer (46). Both fractions were adjustedto 1 50 mM NaCI-0.5% Nonidet P-40 and immunoprecip-itated with PAb-242 anti-p53 monoclonal antibodies (35).

In Vivo Doubling Time. In order to achieve equalexponential growth conditions, cells were plated at 2 xi0� cells/mI and incubated without any selectable drugsat 37#{176}Cfor 24 h. An inoculum of growing cells wasreplated in fresh medium at 2 x i0� cells/mi as above.Twenty-four h later, in vitro growth parameters wereexamined. Viable cell count was determined at 24-hintervals after plating by staining triplicate samples with0.05% Eosin-Y in PBS.

Cell Cycle Analysis. Exponentially growing cells wereobtained as above. Cells were washed twice with PBS,resuspended in a small volume of saline, and fixed in70% ethanol for 30 mm on ice. Fixed cells were washedtwice with PBS and resuspended in PBS containing 50mg/mI propidium iodide (Sigma). At least 5 x i0� cells(as gated by light scatter) were analyzed by FACScan(Becton Dickinson; using the Consort 30 program). DNAhistograms indicating propidium iodide fluorescence in-tensity were generated.

Tumorigenicity in Mice. Approximately 2 x i0� cellswere injected s.c. into individual syngeneic, male CS7L/J mice at the age of 7 weeks. The mice were monitoreddaily as described previously (26).

Acknowledgments

The authors would like to thank Prof. A. Kimchi of the Department ofMolecular Genetics and Virology for fruitful discussions and criticism. M.Baer prepared and edited the manuscript.

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Cell Growth & Differentiation 667

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