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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.
References1 . Hollstein, M., Sidransky, D., Vogelstein, B., and
Harris, C. C. p53-muta-(ions in human cancers. Science (Washington
DC), 253: 49-53, 1991.
2. Lane, D. P., and Benchimol, S. p53: oncogene or
anti-oncogene? GenesDev., 4: 1-8, 1990.
3. Lane, D. P., and Crawford, L. V. T antigen is bound to a host
protein inSV4O-transformed cells. Nature (Lond.), 278: 261-263,
1979.
4. Sarnow, P., Ho, Y. S., Williams, J., and Levine, A. J.
Adenovirus El B-S8Kdtumor antigen and SV4O large tumor antigen are
physically associated withthe same S4Kd cellular protein in
transformed cells. Cell, 28: 387-394,
1982.
S. Werness, B. A., Levine, A. J., and Howley, P. M. Association
of human
papillomavirus types 1 6 and 1 8 E6 proteins with p53. Science
(washingtonDC), 248: 76-79, 1990.
6. Momand, J., Zambetti, G. P., Olson, D. C., George, D., and
Levine, A. j.The mdm-2 oncogene product forms a complex with the
p53 protein andinhibits pS3-mediated transactivation. Cell, 69:
1237-1245, 1992.
7. Moll, U. M., Riou, G., and Levine, A. j. Two distinct
mechanisms alter p53in breast cancer: mutation and nuclear
exclusion. Proc. NatI. Acad. Sci. USA,89: 7262-7266, 1992.
8. Mercer, W. E., Shields, M. T., Amin, M., Sauve, G. J.,
Appella, E.,Romano, J. W., and Ullrich, S. I. Negative growth
regulation in a glioblas-toma tumor cell line that conditionally
expresses human wild-type p53. Proc.
NatI. Acad. Sci. USA, 87: 61 66-61 70, 1990.
9. Baker, S. j., Markowitz, S., Fearon, E. R., Willson, I. K.
V., and Vogelstein,B. Suppression of human colorectal carcinoma
cell growth by wild-type p53.Science (Washington DC), 249: 91 2-91
5, 1990.
10. Diller, L., Kassel, J., Nelson, C. E., Gryka, M. A., Litwak,
G., Gebhardt,
M., Bressac, B., Ozturk, M., Baker, S. j., Vogelstein, B., and
Friend, S. H. p53functions as a cell cycle control protein in
osteosarcomas. Mol. Cell. Biol.,10: 5772-5781, 1990.
1 1 . Johnson, P., Gray, D., Mowat, M., and Benchimol, S.
Expression ofwild-type p53 is not compatible with continued growth
of p53-negative
tumorcells. Mol. Cell. Biol., 11: 1-11, 1991.
1 2. Kastan, M. B., Onyekwere, 0., Sidransky, D., Vogelstein,
B., and Craig,R. W. Participation of p53 protein in the cellular
response to DNA-damage.CancerRes., 51:6304-6311, 1991.
1 3. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan,
M. B. Wild-typep53 is a cell cycle checkpoint determinant following
irradiation. Proc. NatI.Acad. Sci. USA, 89: 7491-7495, 1992.
14. Kastan, M. B., Zhan, Q., El-Deiry, W. S., Carrier, F.,
Jacks, T., Walsh, W.V., Plunkett, B. S., Vogelstein, B., and
Fornace, A. j. A mammalian cell cyclecheckpoint pathway utilizing
p53 and GADD4S is defective in ataxia-
telangiectasia. Cell, 71: 587-597, 1992.
15. El-Deity, W. S., Tokino, T., Velculescu, V. E., Levy, D. B.,
Parsons, R.,Trent, j. M., Lin, D., Mercer, W. E., Kinzler, K. W.,
and Vogelstein, B. WAF1,
a potential mediator of p53 tumor suppression. Cell, 75: 81
7-825, 1993.
16. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and
Elledge, S. j.The p21 cdk-interacting protein Cipi is a potent
inhibitor of G1
cyclin-dependant kinases. Cell, 75: 805-81 6, 1993.
17. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L.,
Kimchi, A., and
Oren, M. Wild-type p53 induces apoptosis of myeloid leukaemic
cells thatis inhibited by interleukin-6. Nature (Lond.), 352:
345-347, 1 991.
18. Yonish-Rouach, E., Grunwald, D., Wilder, S., Kimchi, A.,
May, E.,Lawrence, i-i., May, P., and Oren, M. p53-mediated
cell-death: relationship
to cell cycle control. Mol. Cell. Biol., 13: 1 41 5-1 423,
1993.
1 9. Shaulsky, G., Goldfinger, N., Peled, A., and Rotter, V.
Involvement of
wild-type p53 in pre-B-cell differentiation in vitro. Proc.
NatI. Acad. Sci.USA, 88:8982-8986, 1991.
20. Feinstein, E., Gale, R. P., Reed, j., and Canaani, E.
Expression of normalp53 gene induces differentiation of KS62 cells.
Oncogene, 7: 18S3-l 857,1992.
21 . Johnson, P., Chung, S., and Benchimol, S. Growth
suppression of Friendvirus-transformed erythroleukemia cells by p53
protein is accompanied byhemoglobin production and is sensitive to
erythropoietin. Mol. Cell. Biol.,
13: 1456-1463, 1993.
22. Soddu, S., Blandino, G., Citro, G., Scardigli, R., Piaggio,
G., Ferber, A.,Calabretta, B., and Sacchi, A. Wild-type p53 gene
expression inducesgranulocytic differentiation of HL-60 cells.
Blood, 83: 2230-2237, 1994.
23. Brenner, L., Mu#{241}oz-Antonia, T., Vellucci, V. F., Zhou,
Z., and Reiss, M.Wild-type p53 tumor suppressor gene restores
differentiation of human
squamous carcinoma cells but not the response to transforming
growth factor13.Cell Growth & Differ., 4: 993-1004, 1993.24.
Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J.,
Montgom-
ery, C. j., Butel, j. S., and Bradley, A. Mice deficient for p53
are develop-mentally normal but susceptible to spontaneous tumors.
Nature (Lond.), 356:215-221, 1992.
25. Ahuja, H., Bar Eli, E., Arlin Z., Advani, S., Allen, S. L.,
Goldman, J.,Snyder, D., Foti, A., and Cline, M. The spectrum of
molecular alterations in
the evolution of chronic myelocytic leukemia. I. Clin. Invest.,
87:2042-2047, 1991.
26. Fenaux, P., jonveaux, P., Quiquandon, I., L#{228}i,J. L.,
Pignon, j. M.,Loucheux-Lefebvre, M. H., Bauters, F., Berger, R.,
and Kerckaert, J. P. p53gene mutations in acute myeloid leukemia
with 1 7p monosomy. Blood, 78:
1652-1657, 1991.
-
Cell Growth & Differentiation 17
27. Nakai, H., Misawa, S., Togushida, J., Yandell, D. W., and
Ishizaki, K.Frequent p53 gene mutations in blast crisis of chronic
myelogenous leuke-mia, especially in myeloid crisis harboring loss
of a chromosome 1 7p.
CancerRes., 52:6588-6593, 1992.
28. Galdano, G., Ballerini, P., Gong, J. Z., lnghirami, G.,
Neri, A.,Newcomb, E. W., Magrath, I. T., Knowles, D. M., and
Dalla-Favera, R.
p53 mutations in human lymphoid malignancies: association with
Burkittlymphoma and chronic lymphocytic leukemia. Proc. NatI. Acad.
Sci.
USA, 88:5413-5417, 1991.
29. Jonveaux, P., Fenaux, P., Quiquandon, I., Pignon, J. M.,
LaI, J. L.,Loucheux-Lefebvre, M. H., Goossens, M., Bauters, F., and
Berger, R. Muta-
tions in the p53 gene in myelodysplastic syndromes. Oncogene,
6:2243-2247, 1991.
30. Preudhomme, C., Quesnel, B., Vachee, A., Lepelley, P.,
Collyn-D’Hooge, M., Wattel, E., Fenaux, P. Absence of amplification
of MDM2gene, a regulator of p53 function, in myelodysplastic
syndromes. Leukemia,
7: 1291-1293, 1993.
31 . Sucai, B., Hughes, T., Bungey, J., Chase, A., de Fabritiis,
P., and Gold-man, J. M. p53 in chronic myeloid leukemia cell lines.
Leukemia, 6:
839-842, 1992.
32. Sugimoto, K., Toyoshima, H., Sakai, R., Miyagawa, K.,
Hagiwara, K.,
Ishikawa, F., Takaku, F., Yazaki, Y., and Hirai, H. Frequent
mutations in the
p53 gene in human myeloid leukemia cell lines. Blood, 79:
2378-2383,1992.
33. Andersson, L. C., Jokinen, M., and Gahmberg, C. G. Induction
of ery-throid differentiation in the human leukaemia cell line
K562. Nature (Lond.),
278: 364-365, 1979.
34. Huang, M. E., Ye, Y. C., Chen, S. R., Chai, J. R., Lu, j.
x., Zhoa, L., Gu,L. j., and Wang, Z. Y. Use of all-trans retinoic
acid in the treatment of acute
promyelocyte leukemia. Blood, 72: 567-572, 1988.
35. Peetre, C., Gullberg, U., Nilsson, E., and Olsson, I.
Effects of recombi-nant tumor necrosis factor on proliferation and
differentiation of leukemic
and normal hemopoietic cells in vitro. J. Clin. Invest., 78: 1
694-1 700, 1986.
36. Lozzio, C. B., and Lozzio, B. B. Human chronic myelogenous
leukemia
cell-line with positive Philadelphia chromosome. Blood, 45:
321-324, 1975.
37. Kerr, J. F. R., and Harmon, B. V. Apoptosis: The Molecular
Basis of CellDeath, pp. 5-29. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory,
1 991.
38. Luftig, R. B., Conscience, (-F., Skoultchi, A., McMillan,
P., Revel, M.,and Ruddle, F. H. Effect of interferon on dimethyl
sulfoxide-stimulated Friend
erythroleukemic cells. Ultrastructural and biochemical study. J.
Virol., 23:
799-810, 1977.
39. Milner, J., Cook, A., and Sheldon, M. A new anti-p53
monoclonalantibody, previously reported to be directed against the
large T antigen onSimian virus 40. Oncogene, 1: 453-455, 1987.
40. Zambetti, G. P., and Levine, A. J. A comparison of the
biologicalactivities of wild-type and mutant p53. FASEB j., 7:
855-865, 1993.
41 . Shaw, P., Bovey, R., Tardy, S., Sahli, R., Sordat, B., and
Costa, J.Induction ofapoptosis by wild-type p53 in a human colon
tumor-derived cell
line. Proc. NatI. Acad. Sci. USA, 89: 4495-4499, 1992.
42. Ryan, I. I., Danish, R., Gottlieb, C. A., and Clarke, M. F.
Cell cycleanalysis of p53-induced cell death in murine
erythroleukemia cells. Mol.
Cell. Biol., 13:711-719, 1993.
43, Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R.
G., Bird, C. C.,Hooper, M. L., and Wyllie, A. H. Thymocyte
apoptosis induced by p53-
dependent and independent pathways. Nature (Lond.), 362:
849-852,1993.
44. Laster, S. M., Wood, J. G., and Gooding, L. R. Tumor
necrosis factor caninduce both apoptotic and necrotic forms of cell
lysis. I. Immunol., 141:
2629-2634, 1988.
45. Rubin, B. Y., Smith, L. J., Hellermann, G. R., Lunn, R. M.,
Richardson,N. K., and Anderson, S. L. Correlation between the
anticellular and DNA
fragmenting activities oftumor necrosis factor. Cancer Res., 48:
6006-601 0,
1988.
46. Schmid, D. S., Hornung, R., McGrath, K. M., Paul, N., and
Ruddle, N.H. Target cell DNA fragmentation is mediated by
lymphotoxin and tumor
necrosis factor. Lymphokine Res., 6: 195-202, 1987.
47. Flieger, D., Riethmuller, G., and Ziegler-Heitbrock, H. W.
Zn� inhibits
both tumor necrosis factor-mediated DNA fragmentation and
cytolysis. Int. j.Cancer, 44: 31 5-31 9, 1989.
48. Kizaki, M., Sakashita, A., Karmakar, A., Lin, C. W., and
Koeffler, H. P.Regulation of manganese superoxide dismutase and
other antioxidant genesin normal and leukemic hematopoietic cells
and their relationship tocytotoxicity by tumor necrosis factor.
Blood, 82: 1 1 42-1 1 50, 1993.
49. Trinchieri, G., Rosen, M., and Perussia, B. Induction of
differentiation ofhuman myeloid cell lines by tumor necrosis factor
in cooperation with 1 -a,25-dihydroxyvitamin D3. Cancer Res., 47:
2236-2242, 1987.
So. Jelinek, D. F., and Lipsky, P. E. Enhancement of human B
cell prolifer-ation and differentiation by tumor necrosis factor-a
and interleukin 1 . J.tmmunol., 139:2970-2976, 1987.
51 . Murphy, M., Perussia, B., and Trinchieri, G. Effects of
recombinanttumor necrosis factor, lymphotoxin, and immune
interferon on proliferation
and differentiation of enriched hematopoietic precursor cells.
Exp. Hematol.,
16:131-138, 1988.