Vol. 6, 9-17, January 1995 Cell Growth & Differentiation 9

3 The abbreviations used are: TNF, tumor necrosis factor; CMv, cytomega-lovirus; FBS, fetal bovine serum.

Involvement of the Tumor Suppressor Gene p53 in TumorNecrosis Factor-induced Differentiation of theLeukemic Cell Line K562’

Mats Ehinger,2 Eva Nilsson, Ann-Maj Persson,Inge Olsson, and Urban Gullberg

Division of Hematology, Department of Medicine, University of Lund,Sweden

Abstract

The cDNA of the human wild-type p53 tumorsuppressor gene was constitutively overexpressed in theleukemic cell line K562 (which lacks detectable amountsof p53 protein) in order to investigate the consequencesfor growth and differentiation. Several stable cloneswere established by transfedion of the expression vectorpc53SN3. Expression of p53 protein was characterizedby biosynthetic labeling and immunoprecipitation withthe monoclonal antibodies pAb 1 801 (reacting withwild-type and mutant human p53), pAb 240 (reactingwith mutant human p53) and pAb 1 620 (reacting withwild-type human p53). All clones which were 1 801 +,240-, 1620- or 180i+, 240-, i620+ were defined as“wild-type-like p53-expressing” clones. Our results showthat expression of p53 protein is compatible withcontinuous proliferation of K562 cells. The growthcharacteristics of wild-type-like p53-expressing clonesdid not differ from that of control clones. However, theformer were more sensitive than p53-negative controlclones to growth inhibition by tumor necrosis factor(TNF), a cytokine with a potential role in growth anddifferentiation of myeloid leukemic cells. In addition, a2- to 4-fold increase of the amount of hemoglobin, amarker of erythroid differentiation, was observed whenwild-type-like pS3 protein-expressing clones wereincubated with TNF. This suggests that differentiation isthe mechanism responsible for the increased TNFsensitivity of these clones. Our results support a role forp53 in mediating growth inhibitory and differentiationinducing signals by TNF.

Introduction

Several lines of evidence indicate that normal function ofthe tumor suppressor gene p53 is important for maintainingthe benign phenotype of mammalian cells. For instance,functional inactivation of p53 (by a variety of mechanisms)is probably of importance for the development of severalcancers (1-7). Moreover, overexpression of wild-type p53protein in various cell lines often leads to reversion of themalignant phenotype (8-1 1).

Received 4/1 5/94; revised 1 0/1 3/94; accepted 10/25/94.

C This work was supported by the Swedish Cancer Society, the Georg

Danielsson Foundation, the Greta and Johan Kocks Foundation, the AlfredOsterlund Foundation, and the Medical Faculty of Lund.

2 To whom requests for reprints should be addressed, at Research Dept. 2,E-blocket, University Hospital, 5-221 85 Lund, Sweden.

The exact biological function of p53 is unclear but itseems to be closely related to cell cycle control. G1 arrestfollowed by certain kinds of DNA damage has been shownto depend upon a normal regulation of wild-type p53 ex-pression (12-14). This allows DNA repair mechanisms tooperate before continued progression into the cell cycleoccurs. A mechanism by which wild-type p53 can induceG1 arrest has recently been clarified (1 5, 16). Wild-type p53also seems to be important for the induction of apoptosis.For example, overexpression of wild-type p53 induces ap-optosis in the myeloid cell-line Ml lacking endogenous p53(1 7, 1 8). G1 arrest and induction of apoptosis may provideimportant defense mechanisms against survival andaccumulation of genetically altered cells.

Besides playing an important role in controlling prolifer-ation and apoptosis, p53 is probably involved in the regu-lation of differentiation. Some evidence supporting this no-tion exists. For example, reintroduction of wild-type p53 ina pre B-cell line leads to partial differentiation (1 9). Simi-larly, overexpression of wild-type p53 protein in leukemicK562 cells, HL-60 cells, or Friend virus-transformed eryth-roleukemic cells induces signs of differentiation (20-22).Yet another example is the appearance of differentiationmarkers in squamous carcinoma cells upon overexpressionof wild-type p53 (23). On the other hand, transgenic micelacking the p53 gene display an apparently normal embry-onic development, thus questioning an important role forp53 in the differentiation process (24).

In leukemia, functional inactivation of p53 does notseem to be a general phenomenon (25-30). However,some immortalized leukemic cell lines (such as the hu-man myeloid Ieukemic cell line K562) have lost theexpression of p53, presumably as a step in the process ofimmortalization (31 , 32). In vitro, it is possible to inducedifferentiation of leukemic cell lines with various agents.Included among such agents are, for example, sodiumbutyrate, (alI-trans) retinoic acid, and cytokines such asTNF3 (33-35). The sensitivity for such agents variesamong different cell lines. For instance, TNF can inducedifferentiation in the leukemic cell line HL-60 but not inthe myeloid leukemic cell line K562 (35).

Would it be possible to restore genetic programs of dif-ferentiation by reintroducing p53 in a leukemic cell linelacking p53 such as K562? To answer this question, wedecided to artificially express p53 in K562 cells bytransfection of wild-type p53 cDNA.

Transient overexpression of wild-type p53 in several tu-mor-derived cell lines leads to a dramatic inhibition ofgrowth (8-1 1 ). We found, however, that constitutive over-expression of wild-type p53 in K562 cells is compatiblewith conti nuous proliferation . Moreover, overexpression ofwild-type p53 in K562 cells resulted in increased sensitivity

All

1,

A 29 A 30 A 55 A 19

10 p53 and TNF-induced Differentiation of Leukemic Cells

/d�Yq/i

11Ua:

0a:0

U

iflfl�

94-.

67-

43-

30-

Fig. 3. Clonogenic growth of p53-transfected and mock-transfected clones.Cells were cultured in soft agar, and the number of colonies (>40 cells) wasdetermined after 15 days as described in “Materials and Methods,” Cellswere plated in duplicate. The growth ofthe mock-transfected clones M1-M6

and ofthe p53-transfected clones Al 9, Al 1 , A29, A30, and A55 is shown asthe percentage of colonies relative to the number of seeded cells (clonoge-nicity). The mean values for each of the six mock transfectants Ml -M6 and

each of the p53 transfectants (all determined from three separate experi-ments) are shown. Bars, SEM. There was no statistically significant differencein clonogenicity between the group of wild-type-like p53-transfectants Al 1,

A29, A30, or ASS and the group of mock transfectants Ml-M6. Nor was

there a significant difference between the mutant transfectant A19 and thegroup of mock transfectants.

Cell Growth & Differentiation 11

Table 1 p53-transfected clones Al 1 , Al 9, A29, A30, A55 and the

mocktransfected clone Ml and their respective reactivity with different

p53-specific antibodies

The pAb 1801 reacts both with wild-type andrnutant human p53. The pAb

240 reacts only with mutant human p53. The pAb 1620 reacts only withwild-type human p53 (although some mutant forms may react).

pAbClone

1801 240 1620

Ml - - -

A19 + + -

All + - -

A29 + - -

A30 + - -

ASS + - +

-�

20- �

Fig. 2. In vitro translation of p53. RNA was transcribed in vitro from

p53BSK- and translated in vitro with I 3’)Slcysteine as described in “Mate-rials and Methods.” The translation product was immunoprecipitated withindicated antibodies and subjected to SDS-PAGE and fluorography. The

translation product prior to immunoprecipitation is also shown as a positivecontrol. Molecular weight markers are indicated to the left (kilodaltons).

p53 is indicated with an arrow to the right.

as the transfected clones Al 1 , A29, and A30. The reactivityof the specific antibody pAb 1 620 is known to be weakcompared to the other specific p53 antibodies (39, 40).Thus, the lack of reactivity with the wild-type-specific an-tibody pAb 1 620 in the transfected clones does not seem toexclude the existence of wild-type p53 protein. The trans-fectants Al 1 , A29, A30, and A55 were defined as “wild-type-like” and the transfectant A19 as “mutant.”

The transfected clone K562/pc53SN3/A4 did not expressany p53 protein, as judged by the lack of reactivity with anyof the specific p53 antibodies (data not shown). Therefore,

Ml M2 M3 M4 MS M6 A19 All A29 A30 MS

“Mutant p53’ “Wild-type-like p53”No p53 (240-positive) (240-negative)

this clone was not used in the subsequent experiments. Theexpression of the p53 protein in the transfected clonesK562/pc53SN3/A50 and A54 was not stable, since no re-producible pattern of immunoprecipitation was found on

different occasions (data not shown). Thus, we were notable to characterize the quality of p53-expression in theseclones. For this reason, they were excluded from furtherexperiments.

Morphological Characteristics of Transfected Cells.Under certain circumstances, the expression of wild-typep53 in some cells is known to cause apoptosis or differen-tiation (1 7-23). For this reason, the p53 transfectants wereexamined for changes in morphology as compared to theparental cell line as described in “Materials and Methods.”In general, the transfectants displayed a somewhat moreheterogeneous picture with more mitoses, more vacuoles,more multinucleated cells, and more giant cells. Therewere, however, no obvious signs of a different phenotype inthe transfectants as compared to the parental cell line. Thus,K562 cells seemed to tolerate p53 expression withoutobvious changes in morphology.

Growth Characteristics of Transfected Cells. Several in-vestigators have demonstrated that transient expression ofwild-type p53 is incompatible with continuous cell prohif-eration (9-1 1 ). In order to determine the effect of stable pS3gene expression on the clonogenic growth properties in softagar, cells were plated as described in “Materials and Meth-ods.” After 15 days of culture, the number of coloniescontaining more than 40 cells was determined. In Fig. 3, theclonogenicity of the wild-type-like p53 transfectants and

150

� 125

� 100

;� 75

�50

� ‘5

-0- Ml

-0-- so - No p53

-0- M4

A19 - “Mutantp53�(240-positive)

i i �

T’ime(days)

Fig. 4. Growth in suspension culture of p53-transfected and mock-trans-fected clones. Cells were grown in suspension culture and counted daily asdescribed in “Materials and Methods.” The growth of the p53-transfected

clones Al 1 , Al 9, A29, A30, and ASS and the mock-transfected clones Ml,M2, and M4 is shown. Results are from one representative experiment.

TNF(M)

A

3

! -L�.--. A19 - “MuCaotpS3’0 (24.0-posItive)

� .-_-- All

I -.--- A29 “Wat-t���.� PS3”.� -.-- A30 (240-negative)

�

U

B

3

§ -0-MI

�

�

il -0- M4

�

TNF(M)

Fig. S. Effects of TNF on clonogenic growth of p53-transfected (A) andmock-transfected (B) clones. Cells were cultured in soft agar, and the number

of colonies (>40 cells) was determined after 1 5 days as described in

“Materials and Methods.” Cells were plated in duplicate. Clonogenic growthis shown as the percentage of the number of colonies in control cultures

without TNF. A, at different concentrations ofTNF (1 0 � 2l 0_ti M), the meanvalues for each of the p53 transfectants Al 9, Al 1 , A29, A30, and ASS(determined from three separate experiments) are shown. B, the mean valuesat different concentrations of TNF for each of the mock-transfectants Ml -M6

(determined from three separate experiments) are shown. Bars, SEM. For

each concentration of TNF used, there was a statistically significant differ-ence (P < 0.001 ) in inhibition of clonogenic growth between the group ofwild-type-like p53-transfectants Al 1 , A29, A30, and ASS and the group of

mock transfectants Ml-M6.

-12 -11 -10 -9 -810 10 10 10 10

12 p53 and TNF-induced Differentiation of Leukemic Cells

All

-.4-- A29- “Wild-type-likep53�

-.--. A30 � (240-negative)

-A- A55 I

that of the mock-transfectants and the mutant transfectantK562/pc53SN3/A19 is shown. The difference in clonoge-nicity between the group of wild-type-like p53 transfectantsand the group of mock transfectants was not statisticallysignificant. Nor could we find any statistically significantdifference between the mutant transfectant K562/pc53SN3/Al 9 and the group of mock transfectants. This indicates thatstable expression of wild-type-like p53 in K562 cells iscompatible with cell proliferation. In order to determine theeffect of p53 expression on the growth rate in suspensionculture, cells were counted daily for 4 days. No differencesin growth rate between the transfected clones and themock-transfected clones could be observed (Fig. 4). Again,this indicates that stable expression of wild-type-like p53 isnot inconsistent with continuous growth of K562 cells.

Effects of TNF, all-trans Retinoic Acid, and Sodium Bu-tyrate on Growth Characteristics of Transfeded Cells.Previous work has shown that several agents such as TNF,all-trans retinoic acid, and sodium butyrate are capable ofinducing differentiation in certain hematopoietic cells(33-35). We were interested in determining if the expres-sion of p53 confers an increased sensitivity to the action ofthese agents. With the intention of determining this, softagar cultures were made and plated with cells as describedin “Materials and Methods” with different concentrations ofTNF, all-trans retinoic acid, or sodium butyrate. As shownin Fig. 5A, rising concentrations of TNF led to dose-depen-dent reduction of clonogenic growth for all p53-transfectedclones. However, the wild-type-like transfectants (K562/pc53SN3/Al 1 , A29, A30, and A55) were clearly more sen-sitive to the action ofTNF than the mock transfectants or themutant transfectant K562/pc535N3/Al 9. In control experi-ments, the cells of the six mock transfectants (K562/SN3/Ml-M6) were plated with TNF in an identical way. Asshown in Fig. SB, the clonogenic growth of the mock trans-fectants was also influenced by TNF in a dose-dependentmanner. The influence of TNF was, however, clearly lesspronounced than that seen in the experiments with thewild-type-like p53 transfectants. For each concentration ofTNF in the range 10� 2l0_8 M, there was a statisticallysignificant difference (P < 0.001) in TNF-induced inhibitionof clonogenic growth between the group of wild-type-likep53 transfectants and the group of mock transfectants. Thisindicates that expression of wild-type-like p53 may lead to

an increased sensitivity to TNF. The sensitive clones werethe ones expressing p53 protein not reacting with the mu-tant-specific antibody pAb 240 [240-negative (wild-type-like); Al 1 , A29, A30, and A55], whereas the clone express-ing a mutant form of p53 [240-positive (mutant); Al9]showed no increased sensitivity to the action ofTNF. Whencells were exposed to all-trans retinoic acid or sodiumbutyrate, a dose-dependent inhibition of clonogenic growthwas observed, but no difference in clonogenic growth be-tween p53 transfectants and mock transfectants could beobserved (data not shown).

In order to determine the effect ofTNF on proliferation insuspension culture, transfected clones were incubated withTNF at different concentrations, then counted daily for 4days as described in “Materials and Methods.” As shown inFig. 6, the wild-type-like transfectants K562/pc53SN3/Al 1,A29, A30, and ASS displayed a dose-dependent inhibitionof growth when exposed to TNF. This effect was mostpronounced for the transfectant ASS. No growth inhibitionof the mutant transfectant K562/pcS3SN3/Al 9 or the mock

A

I

E

z

B

30

.6

B0

z

Time (days)

15

.E

.0

.1 T�C� 5 J I0� T� I

-. TT TT 1#{149}

0

TNF -+ -+ -+ -+ -+ -+ -+ -+

All A29 A30 ASS

-11 .10 -9

10 10 10

TNF(M)

-WiId-type.Iike p53”(240-negative)

Fig. 6. Effects of TNF on growth rate in suspension culture of p53-trans-

fected and mock-transfected clones. The p53-transfected clones Al 1 , Al 9,A29, A30, and ASS and the mock-transfected clones Ml , M2, and M4 weregrown in suspension culture as described in “Materials and Methods.”

Exponentially growing cells were diluted at 0.25 x 106 cells/mI with TNF at0.01 nsi, 0.1 nsi, or 1 n� or without TNF (control) and then counted daily. A,growth curves for the indicated clones when incubated with TNF at 1 ntis. B,number of cells (expressed as the percentage of the number of untreated

control cells) as a function of TNF concentration at a chosen point of time(60 h). Results are from one representative experiment.

Fig. 7. Effects of TNF on the amount of hemoglobin in p53-transfected andmock-transfected clones. Cells were incubated (0.25 X l06/ml) with TNF at0.1 nsi or without TNF for 96 h. The amount of hemoglobin was then

determined as described in “Materials and Methods.” The relative amount ofhemoglobin of the p53-transfected clones Al 1 , Al 9, A29, A30, and ASS and

the mock-transfected clones Ml , M2, and M4 as compared to the mock-

transfected clone Ml is shown without and with TNF. Mean values from 3-4separate experiments. Bars, SEM. A statistically significant difference(P< 0.01) in the increase ofthe amount ofhemoglobin upon TNF incubationwas found between the group of wild-type-like p53 transfectants Al 1 , A29,A30, and ASS and the group of mock transfectants Ml, M2, and M4.

Cell Growth & Differentiation 13

-0-- Ml

-0’-- M2 � NopS3

-0�-

“Mutant p53W

-h--- A19 (240-positive)

-U--- All

-.-- A29 I � �53�� - (240-negative)

-.- A30

-&--- A�

“-0-- MI I

-0’--- M2� - Nop53

-0-- M4“Mutant p5)”

-.�--- A19 (240-positive)

-.-- All

-.- A29 � “Wildtype.tikep53”

(240-negative)-.-- A3O

-A- A55

transfectants K562/SN3/Ml , M2, or M4 could be observedupon exposure to TNF (Fig. 6). Again, this indicates thatexpression of wild-type-like p53 leads to an increased sen-sitivity to the growth inhibitory action of TNF.

Induction of Apoptosis in the Transfected Cells. Theincreased sensitivity to TNF-induced inhibition of growth ofthe wild-type-like p53 transfectants shown above may de-pend on induction of apoptosis. Therefore, we were inter-ested in determining if the differences in TNF sensitivityregarding clonogenic growth and growth rate in suspension

culture between the wild-type-like transfected cells andcontrol cells were due to induction of apoptosis. Cells wereexposed to TNF, and the incidence of apoptosis was deter-mined as described in “Materials and Methods.” No signif-cant difference in the incidence of apoptosis could be

observed between p53-transfected control cells and p53-transfected cells incubated with TNF (data not shown).Thus, apoptosis did not seem to be the mechanism in causefor the increased TNF sensitivity.

Hemoglobin Synthesis in the Transfected Cells. If apop-tosis does not seem to be responsible for the observeddifferences in TNF sensitivity between the wild-type-likep53 transfectants and the mock transfectants, then induc-tion of differentiation could be the mechanism in cause. Inorder to determine the effect of pS3 expression on the

Ml M2 M4 Al9

“Mutant p53”No p53 (240-positive)

differentiation-associated hemoglobin synthesis, cell lysateswere allowed to react with tetramethylbenzidine in thepresence of hydrogen peroxide. An appreciation of the

hemoglobin concentration can then be determined spec-troscopically (38). As shown in Fig. 7, the wild-type-like

transfectants K562/pcS3SN3/Al 1 , A29, A30, and ASSseemed to have more hemoglobin than the mutant trans-fectant KS62/pcS3SN3/Al 9 or the mock transfectants KS62/SN3/Ml , M2, or M4, thus suggesting partial induction ofdifferentiation (as judged by the amount of hemoglobin) bywild-type-like p53. However, although there was a ten-dency for a difference in the amount of hemoglobin be-tween the wild-type-like p53 transfectants and the mocktransfectants or the mutant transfectant, it did not reach

statistical significance. Upon incubation with TNF (0.1 nM)of the wild-type-like transfectants Al 1 , A29, A30, and ASS,but not of the mock transfectants or the mutant transfectantAl 9, a 2- to 4-fold increase of the hemoglobin concentra-tion could be observed (Fig. 7). The difference in increasewas statistically significant (P < 0.01 ) between the group ofwild-type-like p53 transfectants and the group of mocktransfectants. The augmentation of the amount of hemoglo-bin was dose dependent in response to TNF in the range of0.01 nM-0.l nM (data not shown). This indicates that ex-pression of wild-type-like p53 in KS62 cells confers anincreased sensitivity to induction of hemoglobin synthesisby TNF.

14 p53 and TNF-induced Differentiation of Leukemic Cells

Discussion

We are interested in trying to investigate a role of p53 in theapoptotic and differentiation processes of leukemic cells.For this purpose, a cell system overexpressing wild-typep53 was created and assayed for differentiating and apop-totic potential. After having established stable p53 transfec-tants in K562 cells, a major problem we had to face was thatof the quality of the expressed p53 protein. For severalreasons, it is far from evident that the transfectants, althoughoriginally transfected with wild-type p53 cDNA, actually doexpress wild-type p53. Firstly, functional inactivation ofp53 often seems to be an important step in the evolution ofmany tumors (1-7) and probably in the immortalization ofsome cell lines (32). Secondly, transient expression of ex-ogenous wild-type p53 does not seem to be compatiblewith growth in many cases (9-1 1 ). Moreover, in our trans-fection experiments, over four times more clones arose fromcells electroporated with the plasmid SN3 alone as com-pared to cells electroporated with p53 cDNA. These datasuggest that there was a selection pressure against the ex-pression of wild-type p53 of the transfected K562 cells.Thus, it is possible that the transfected cells, in order tosurvive, must have abrogated the effects of high levels ofwild-type p53. One way for the cell to avoid the obviousinconvenience of expressing a protein with antiproliferativeproperties would be if most of the expressed p53 in thetransfected cells actually is in a mutant form no longer

capable of inhibiting growth. To characterize the p53 pro-tein in the transfected clones, monoclonal antibodies withdifferent specific reactivities were used to determine if theconstitutively expressed p53 protein was actually in a wild-type or mutant form.

Our results show that one clone (Al 9) seemed to expressa mutant form of p53 (reacting with the mutant-specificantibody 240), despite the fact that it had been originallytransfected with wild-type p53 cDNA. The other transfectedclones (Al 1 , A29, A30, and ASS) did not express mutantp53 as judged by the lack of immunoreactivity with thisantibody. judging from the reactivity with the wild-type-specific antibody pAb 1 620, only one clone (ASS) seemedto express small amounts of wild-type p53. The fact that theantibody pAb 1 620 failed to detect p53 in all clones exceptASS may indicate that p53 actually was in a mutant form inthese clones. However, the lack of reactivity with pAb 1620was identical for in vitro-translated wild-type p53, thusindicating that the specific antibody supposed to detectwild-type p53 in fact was not sensitive enough to detect thisprotein under these circumstances. Moreover, the reactivityof the specific wild-type antibody is known to be weak (39,40), and it does not seem unreasonable to believe that thedifficulties in precipitating wild-type p53 were due to in-sufficient sensitivity of the pAb 1 620. Therefore, we believethat most of the clones (Al 1 , A29, A30, and A5S) indeedexpress wild-type p53 protein, and only one clone (Al9)may have overcome possible growth inhibitory effects ofwild-type p53 by expressing a mutant form of the protein.

Thus, overexpression of wild-type-like p53 in the KS62leukemic cell line does not seem to be incompatible withcontinuous cell proliferation. This is in contrast to previousfindings where transient overexpression of wild-type p53 intumor cell lines derived from colon cancer, osteosarcoma,and glioblastoma led to a dramatic inhibition of growth(8-1 0). However, in these cases, selection for stable clonesconstitutively expressing wild-type p53 was not made.

Moreover, there does seem to be a selection pressureagainst clones expressing wild-type p53, as judged by therelative difficulties of obtaining clones transfected withwild-type p53 cDNA and the preferential expression ofmutant p53 in one clone. The mechanism(s) for the abilityof K562 cells to successfully cope with wild-type-like p53 isat present unclear.

Our results show that clones stably expressing wild-type-like p53 display an increased sensitivity to TNF-inducedinhibition of growth. The clone expressing a mutant form ofp53 (Al 9) did not seem to be more sensitive than the mocktransfectants to the action of TNF. This was true for theclonogenic growth in soft agar as well as the growth rate insuspension culture. It seems reasonable to believe that themutant clone Al9 displays a relative insensitivity to TNFbecause most of the expressed p53 in this clone is in amutant conformation.

It could be possible that the differences observed regard-ing TNF sensitivity were due to increased induction ofnecrosis or apoptosis by TNF. Overexpression of wild-typep53 can induce apoptosis in myeloid leukemic cells as wellas in other cells (41 , 42). In addition, induction of apoptosisin mice thymocytes upon ionizing radiation has beenshown to depend on expression of wild-type p53 (43). TNF,too, is involved in the process of apoptosis. It is capable ofinducing apoptosis in many kinds of tumor cells includingseveral leukemic cells but not KS62 cells (35, 44-48). Thus,both p53 and TNF may be involved in the induction ofapoptosis. Our results show that TNF did not seem to in-duce apoptosis in the p53-transfected K562 cells, as judgedby their morphological appearance. This suggests that in-duction of apoptosis is not the mechanism responsible forthe reduced clonogenic growth or reduced growth rate insuspension culture of wild-type-like p53 transfectants whenincubated with TNF. It is, however, difficult to completelyrule out quantitative differences in a morphological assay.Moreover, probably only a minority of the cells are clono-genic (i.e., capable of giving rise to several generations ofprogeny). Theoretically, it is possible that TNF specificallyinduces apoptosis in clonogenic cells. This would bedifficult to detect in a quantitative assay.

Another explanation for the observed differences regard-ing TNF-induced inhibition of growth would be if cellsexpressing wild-type-like p53 are more prone to inductionof differentiation by TNF than cells expressing no p53 or amutant form of p53. Some evidence for the involvement ofp53 in the hematopoietic differentiation process exists. Forexample, overexpression of wild-type p53 in leukemicKS62 cells, HL-60 cells, or Friend virus-transformed eryth-roleukemic cells leads to signs of differentiation in all threecases (20-22). Moreover, it is known that TNF could beinvolved in the induction of differentiation in myeloid leu-kemic cells (49-51). Our data suggested signs of partialdifferentiation in the clones expressing wild-type-like p53as compared to the other clones. Although the difference inthe amount of hemoglobin between wild-type-like p53transfectants and mock transfectants did not reach statisticalsignificance, others (20) have shown that wild-type p53induces signs of erythroid differentiation in K562 cells.What is even more interesting, a 2- to 4-fold increase of theamount of hemoglobin could be observed upon incubationwith TNF. Thus, differentiation could be the mechanism incause for the increased sensitivity to TNF of wild-type-likep53-transfected clones. It is possible that reintroduction ofp53 restores parts of genetic programs designed for

Cell Growth & Differentiation 15

differentiation pathways. In conclusion, our results supporta role for p53 in mediating growth inhibitory and differen-tiation-inducing signals by TNF.

Materials and Methods

Vector Constructs. The eukaryotic expression vectorpcS3SN3 containing the complete cDNA for the humantumor suppressor gene p53, driven by a CMV promotor waskindly provided by Dr. Bert Vogelstein (Baltimore MD; Ref.9). The vector carries resistance against neomycin for theselection of transfected clones. Plasmid SN3 was con-structed by removing the entire coding region for p53 bycutting pcS3SN3 with the restriction enzyme BamHl, fol-lowed by religation of the plasmid. Plasmid SN3 was usedas a negative control (mock transfectant) in the experiments.

Antibodies. The monoclonal anti p53-antibodies pAb1 801 , 240, and 1 620 were purchased from Oncogene Sci-ence (Uniondale, NY). The monoclonal antibody anti-ras(used as a negative control antibody) was purchased fromSanta Cruz Biotechnology (Santa Cruz, CA). The pAb 1801reacts both with wild-type and mutant human p53. The pAb240 reacts only with mutant human p53. The pAb 1620reacts only with wild-type human p53 (although some mu-tant forms may react).

Cell Lines. The human myeloid cell line KS62 (36) wascultured in RPMI 1640 (GIBCO-BRL, Gaithersburg, MD)supplemented with 10% heat-inactivated FBS in a 5% CO2atmosphere at 37#{176}C.Exponentially growing cells were usedfor all experiments.

Tumor Necrosis Factor, all-trans Retinoic Acid, and So-dium Butyrate. Recombinant TNF (produced by Genen-tech, Inc., South San Francisco, CA) was kindly supplied byDr. G. Adolf (Ernst Boehringer Institut, Vienna, Austria).All-trans retinoic acid and sodium butyrate were purchasedfrom Sigma Chemical Co., St Louis, MO.

Transfection Procedure. Plasmids pc53SN3 and SN3were linearized with Hino’Ill in order to facilitate integrationof the vectors into the genome of the transfected cells. Cellswere harvested at exponential growth. All subsequent stepswere performed at 4#{176}C.Cells were washed once in ice-coldtransfection buffer [21 mtvi HEPES (pH 7.05), 1 37 mr’�i NaCI,S mM KCI, 0.7 mM Na2PO4, and 6 m�i glucose] and thensuspended in transfection buffer at a concentration of 1 -2 Xl0� cells/mI. The cell suspension (0.8 ml) was incubatedwith 1 6 �ig oflinearized plasmid on ice for 10 mm, followedby electroporation . Electroporation was performed usingthe Bio-Rad gene-pulser (Bio-Rad, Melville, NY) with acapacitance setting of 25 1iF and two alternative voltagesettings of 1 500 and 1600 V, respectively. After electropo-ration, cells were again incubated on ice for 10 mm, thentransferred to fresh culture medium (RPMI + 1 0% FCS) at aconcentration of 0.5 x 1 O� cells/mI and incubated at 3 7#{176}C.After 48-72 h, the electroporated cells as well as negativecontrol cells (not electroporated) were distributed in 96-well plates at a number of 1000 cells/well, and geneticin(Sigma) at a concentration of 1 mg/mI was added for theselection of stably transfected clones. After selection withgeneticin for 3-S weeks, individual clones were expandedto mass cultures and subsequently used in the experiments.

PCR Analysis. PCR analysis was used for determinationof integration of transfected DNA into the genome of thehost cell. PCR primers were chosen from different exons,thereby readily (by different sizes ofthe amplified products)distinguishing transfected p53 cDNA from endogenous

genomic p53. The following primers were chosen for de-tection of p53: upstream primer, 5’-TGTGCAGCTGT-GGGTTGATTC-3’; and downstream primer, S’-GAGAG-GAGCTGGTGTTGTTGG-3’. DNA from cells was isolatedas follows: 1 x iO� cells were washed twice in PBS andthen resuspended in 1 30 �.il of PCR buffer with nonionicdetergents and proteinase K [50 mM KCI, 1 0 mr�.i Tris-HCL(pH 8.3), 2.5 mM MgCl2, 0.1 mg/mI gelatin, 0.45% NP4O,45% Tween 20, and 0.06 �ig proteinase K/mI]. The mixturewas incubated at 55#{176}Cfor 1 hr and then for 1 0 mm at 95#{176}Cto inactivate the protease. Twenty-five pI ofthe mixture wasused as template in a 40-cycles PCR reaction performed ina Perkin Elmer Cetus DNA thermal cycler using the primersdescribed above. The amplified products were analyzed ona 2% agarose gel stained with ethidium bromide.

Biosynthetic Labeling and Immunoprecipitation. Cellswere harvested at exponential growth, washed once withHanks’ balanced salt solution (GIBCO-BRL, Gaithersburg,MD) and then incubated for 30 mm at 37#{176}Cin methionine-and cysteine-free RPMI 1640 supplemented with 1 0% dia-lyzed FBS (GIBCO-BRL) at a concentration of 2 X 10’”cells/mI in order to deplete the intracellular pools of me-thionine and cysteine. Subsequently, the cells (2 x lO’”/ml)were incubated for 60 mm at 37#{176}Cwith identical mediumsupplemented with 7-10 �iCi/ml of [35S]methionine and[35S]cysteine (Dupont-NEN, Wilmington, DE) to obtain Ia-beling of newly synthesized proteins. Following labeling,all steps were performed at 4#{176}C.The cells were resus-pended in a lysis buffer consisting of 50 mt�i Tris HCI (pH8.0), 0.15 M NaCI, S mt�’i EDTA (pH 8.0), 0.5% NP4Oincluding the protease inhibitors aprotinin (1 pg/mI), phe-nylmethylsulfonyl fluoride (100 pg/mI), EDTA (0.5 mM),leupeptin (0.5 pg/mI), and pepstatin (1 pg/mI), followed byincubation on ice for 1 h prior to three sequential 30-sbursts of sonication using a sonicator (Kistner Lab, Stock-holm, Sweden). After lysis, the DNA was removed by cen-trifugation at 37,500 x gfor 1 h at 4#{176}C.The supernatant wasstored frozen at -20#{176}Cuntil immunoprecipitation. Immu-noprecipitation was performed twice. The first immunopre-cipitation (preadsorbtion) was nonspecific, aiming at re-moving from the supernatant proteins bindingnonspecifically to the monoclonal antibodies. For this pur-pose, a polyclonal mouse lgG-agarose was used. The sec-ond immunoprecipitation was specific, aiming at extractingradioactively labeled p53 protein from the supernatant.Preadsorbtion was performed in the following way: 20 �il ofmouse IgG-agarose (Sigma) was added to the supernatant,and immunocomplexes were allowed to form with mouseIgG at 4#{176}Covernight. Next, the solution was centrifuged toremove the lgG-agarose and preadsorbed proteins. The su-pernatant was subjected to specific immu noprecipitationwith the different monoclonal p53 and ras (negative con-trol) antibodies in the same way, except that the immuno-complexes were adsorbed to a mixture of protein A- andprotein G-Sepharose (Sigma). After centrifugation, the pre-cipitate was washed four times with lysis buffer. The im-munoprecipitated proteins were separated on a 7-20%SDS-PAGE. The gel was dried, and Hyperfilm MP (Amer-sham, Amersham, United Kingdom) was exposed for 6 daysat -70#{176}C after fluorographic amplification with Amplify(Amersham).

In Vitro Translation of p53. pS3 cDNA from pcS3SN3was cloned into pBluescript (pBSK-; Stratagene). After lin-earization with SmaI, 1 �ig of pS3BSK- was subjected to invitrotranscription with T3 RNA polymerase using an in vitro

16 p53 and TNF-induced Differentiation of Leukemic Cells

transcription kit (Promega, Madison, WI) according to themanufacturer’s instructions. In vitro-transcribed RNA was invitro-translated using rabbit reticulocyte lysate (Promega)according to the manufacturer’s instructions. [35S]Cysteine(Amersham) was included to obtain labeling ofthe proteins.

Determination of Growth Rate in Suspension Culture.Cells at exponential growth were diluted at a concentrationofO.25 X lO’”/ml in RPMIx1O% FBSand kept in a humified5% CO2 atmosphere at 37#{176}C.Aliquots were removed daily,and the number of cells and viability as judged by Trypanblue exclusion was determined.

Assessment of Clonal Proliferation in Soft Agar. Cells5,000 or 1 0,000 at exponential growth were seeded in 1 mlof 0.3% agar on top of 1 ml of 0.5% agar in McCoy’smedium (GIBCO-BRL) supplemented with 1 5% FBS in35-mm tissue culture dishes. The cells were allowed togrow for 1 5 days in a humified 5% CO2 atmosphere at37#{176}C.The number of colonies Containing more than 40cells was then determined.

Assessment of Differentiation and Apoptosis by Morpho-logical Characterization. Exponentially growing cells wereincubated at 3 X 105/ml in RPMI 1640 with 10% FBSwithout addition (control cells) or with TNF (0.1 nM). After24, 48, and 72 h, aliquots were withdrawn for cytospinpreparation and staining with May-Grunwald-Giemsa formorphological characterization. For determination of theinduction of apoptosis, 400 cells were counted on eachcytospin preparation, and the percentage of cells displayingmorphological criteria for apoptosis (such as chromatincondensation and appearance of membrane protuberancesor apoptotic bodies; Ref. 37) was determined.

Determination of Hemoglobin. The amount of hemoglo-bin was determined as described (38). Briefly, cells at ex-ponential growth were washed twice with PBS, then lysedat a concentration of 1 X 108 cells/mI in the same bufferwith 1% NP4O. After incubation on ice for 1 h, DNA wasremoved by centrifugation at 37,500 X g for 1 h at 4#{176}C.Supernatants were stored frozen at -70#{176}Cuntil hemoglobindetermination. Supernatants of S �il were mixed with 200 �ilof 1% (w/v) tetramethylbenzidine (Sigma) solution in 90%acetic acid and with 200 p1 of freshly prepared 1% (v/v)H2O2. After incubation at room temperature for 20 mm, 2ml of 10% acetic acid was added to stop the reaction, andthe absorbance at 51 5 nm was determined within an hour.From a standard curve made from the lysate of the mock-transfected clone Ml, the relative amount of hemoglobinwas determined.

Statistical Analysis. Clonogenicity (percentage of cob-flies >40 cells relative to number of seeded cells), TNF-induced inhibition of cbonogenic growth, relative amount ofhemoglobin, and the increase of the amount of hemoglobinupon TNF incubation were compared between mock trans-fectants and wild-type-like p53-transfectants usingStudent’s t test.

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