0022-1767/87/1388-2711902,00/0
`THE JOURNAL OF IMMUNOLOGY
`Copyright © 1987 by The American Association of Immunologists
`
`Vol. 138, 2711-2717, No. 8, April 15, 1987
`Printed in U_S.A.
`
`CYTOSTATIC AND CYTOTOXIC ACTIVITY OF TUMOR NECROSIS FACTOR ON
`HUMAN CANCER CELLS’
`
`VITO RUGGIERO,? KATHRYN LATHAM, anp CORRADO BAGLIONI
`
`From the Departmentof Biological Sciences, State University of New York at Albany, Albany, NY 12222
`
`The cytostatic and cytotoxic activity of human
`recombinant tumor necrosis factor (rTNF) was as-
`sayed on different tumorcell lines. Human BT-20
`breast and ME-180 cervix cancer cells were growth-
`inhibited by rTNF, whereastwoothercell lines were
`not significantly inhibited. However, when protein
`synthesis wasinhibited by cycloheximide, rTNF was
`cytotoxic for these cells but not for BT-20 cells. This
`finding suggested that different mechanisms are
`responsible for the cytostatic and cytotoxic activity
`of rTNF. The sensitivity of different cell lines to
`rTNF could not be correlated with a high number or
`affinity of rTNF receptors. Occupancyof only a few
`receptors was sufficient for rTNA cytotoxicity, but
`an increase in receptor number after treatment with
`interferon-y, or a decrease after pretreatment with
`cycloheximide, correspondingly enhanced or de-
`pressed the cytotoxicity of rTNF. It seemed possible
`that some cells could be protected from this effect
`of rTNF by synthesizing “protective” proteins. While
`searching for such proteins, we observed that rTNF
`induced the synthesis of two polypeptides in SK-
`MEL-109 melanoma cells, but not in other cancer
`cells. Actinomycin D added with rTNF abolished
`synthesis of these polypeptides, suggesting that
`rTNF induced transcription of the corresponding
`mRNAs. Surprisingly, rTNF stimulated growth of
`SK-MEL-109 cells cultured in medium with low se-
`rum.
`
`mediator of cachexia in chronically infected animals, and
`was designated cachectin (2) before discovering that its
`amino acid sequenceis identical to that of TNF(8, 9).
`TNF showsdiversebiological effects on different cells.
`For example, TNF suppresses lipoprotein lipase activity
`(10), enhances prostaglandin E, andcollagenase produc-
`tion by human synovial cells and dermalfibroblasts (11),
`and stimulates bone resorption by osteoclasts (12) and
`production of a procoagulant activity by vascular endo-
`thelial cells (13), Furthermore, TNF is a mediator of the
`cytocidal activity of natural cytotoxic cells (14). These
`activities of TNF follow its binding to high affinity recep-
`tors, which were initially identified in murine cells (7).
`TNFreceptors with a Kp of about 2 x 107'° M have been
`detected in human fibroblasts and tumorcells (15-19).
`It was reported that TNF is cytotoxic for some human
`tumorcell lines and cytostatic for other cells, but that it
`shows no activity on several tumor cells (20, 21). There
`is at present no explanation for this diverse response to
`TNF. The present study was aimed at an understanding
`of the different activities of TNF on human tumorcells.
`For this purpose, cell lines with a wide rangeof sensitivity
`to the cytostatic action of TNF were studied. Further-
`more, we examined the activity of TNF on these cells
`treated with cycloheximide (CHX), because TNF cytotox-
`icity is greatly enhanced byinhibitors of protein and RNA
`synthesis (22).
`
`MATERIALS AND METHODS
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`Cells, rTNF, and rIFN-y. Colon adenocarcinoma HT-29cells were
`A protein found in the serum of primed, endotoxin-
`cultured as described (23) in Dulbecco's medium with 10% fetal calf
`treated animals elicits hemorrhagic necrosis of some mu-
`serum (FCS). A375 melanoma cells were cultured in RPMI 1640
`medium with 5% FCS. Breast tumor BT-20, cervix carcinoma ME-
`rine sarcomas(1) and is designated tumor necrosis factor
`180, and other melanoma cell
`lines were cultured in F-12 and
`(TNF).° Priming promotes proliferation of macrophages,
`minimal Eagle's medium (1/1) with 8% FCS. The cells were resus-
`which are stimulated to secrete TNF by endotoxin lipo-
`pended from monolayers with phosphate-buffered saline (PBS) con-
`polysaccharide (2). The human TNFsecreted by myelo-
`taining 1 mM EDTA. rTNF wasa gift of Dr. T. Nishihara of the
`Suntory Institute for Biomedical Research, Osaka, Japan; rIFN-y
`monocytic cells has been purified to homogeneity, se-
`(1.7 x 10° U/mg) wasagift from Biogen (Cambridge, MA).
`quenced, and produced by recombinant DNA technology
`Assays for antiproliferative and cytotoxic activity of rTNF.
`(S-—6). Human recombinant TNF (rTNF) shows the same
`Growth inhibition was measured by seeding 1 to 4 x 10* cells/well
`biological activities as natural TNF (3-6). A factor se-
`in cluster plates; the cells were stained with crystal violet after 4
`days of treatment (23). Cytotoxicity was measured by treating 0.8 to
`creted by macrophages suppresses lipoprotein lipase ac-
`2 X 10° cells/well for 18 hr with rTNF and 0.1 mg/ml of CHX. The
`tivity (7). This factor has been proposed as an endogenous
`monolayers were washed twice with PBS to remove dead cells and
`
`were stained with crystal violet. This dye was eluted with 33% acetic
`acid and the As4) was measured in a microdensitometer. The assays
`were carried out in quadruplicate and gave a standard error of <5%.,
`Cell labeling and protein analysis. Confluent monolayers were
`incubated for 2 hr with 25 wCi/ml of (*°S|methionine in methionine-
`free medium. After this incubation, the cells were washed twice with
`PBS; 0.2 ml of 0.1 mg/ml leupeptin and 4 mM phenylmethylsulfony1
`fluoride in 10 mM NaCl, 1.5 mM Mg(OAc}, and 10 mM Tris-HCl, pH
`7.4, were added for 10 min. The cells were then lysed by adding
`sodium deoxycholate and Brij-58 to a final concentration of 0.3%.
`The lysates were centrifuged for 10 min at 30,000 x G, and aliquots
`of the supernatants (200,000 cpm) were fractionated by SDS-PAGE
`on 10% gels. The autoradiographs were scanned in an LKB Ultroscan
`2711
`
`Received for publication October 28, 1986.
`Accepted for publication January 6, 1987.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-
`cate this fact.
`' This investigation was supported by US Public Health Service Grant
`CA29895, awarded by the National CancerInstitute, DHHS.
`? Present address: Istituto di Virologia, Universita’ di Roma, Viale di
`Porta Tiburtina 28, 00185 Roma, Italy.
`* Abbreviations used in this paper: TNF, tumor necrosis factor; rTNF,
`recombinant tumor necrosis factor; CHX, cycloheximide.
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`2712
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`ACTIVITIES OF TUMOR NECROSIS FACTOR
`
`laser densitometer with peaks integrator. The absorbancy of pro-
`teins of interest was normalized to a reference band in each track
`of the autoradiographs which was unchangedin intensity after the
`treatment with rTNF.
`
`RESULTS
`
`Cytostatic and cytotoxic activity of TNF on human
`cancercells. The cytostatic activity of rTNF was assayed
`on cells seeded at low density to allow exponential growth
`for several days. After 4 days of treatment with different
`concentrations of rTNF, HT-29 and SK-MEL-109 cells
`wereslightly growth-inhibited, whereas ME-180 and BT-
`20 cells were 50% inhibited by ~500 and ~20 pM rTNF,
`respectively (Fig. 1). These cell lines showed a wide spec-
`trum of sensitivity to the cytostatic activity of rTNF. The
`following experiment was aimed at establishing whether
`these cells were sensitive to rTNF cytotoxicity. Nearly
`confluent cultures were treated for 18 hr with different
`concentrations of rTNF and 0.1 mg/ml of CHX, which
`enhances TNFcytotoxicity (22). All of the cell lines were
`extremely sensitive to this treatment, with the exception
`of BT-20 (Fig. 2). The LDsp was between 0.1 nM rTNFfor
`HT-29 cells and ~50 fM rTNF for ME-180 cells. The
`latter cells were sensitive in the cytotoxicity assay to
`rTNF concentrations 10*-fold lower than those inhibitory
`in the cytostatic assay. However, BT-20 cells wereresist-
`ant to cytotoxicity and, conversely, SK-MEL-109 cells
`were highly sensitive to rTNF cytotoxicity but fairly re-
`sistant to its cytostatic activity. This finding suggested
`that some cells could only respond either to the cytostatic
`
`100
`
`= 80
`
`60
`
`83
`
`=3
`
`a
`
`40
`
`20
`
`:
`0.001
`
`0.01
`
`td
`Ol
`|
`nM rTNF
`
`10
`
`Figure 1. Dose dependencyof the antiproliferative activity of rTNF on
`four human tumorcell lines. The cells were seeded in wells of cluster
`plates and were treated for 4 days with the rTNF concentration indicated
`in the abscissa. Growth inhibition was measured as indicated in Mate-
`rials and Methods; the Agao relative to control untreated cells is shown.
`
`
`
`
`or to the cytotoxic activity of rTNF.
`The onsetof cell death after treatment with rTNF and
`CHX varied amongthecell lines examined (Fig. 3). Cyto-
`toxicity was assayed eitherafter an initial treatment and
`incubation in fresh medium or after continuous incuba-
`tion with rTNF and CHX.In this way, we determined the
`duration of a treatment which caused irreversible dam-
`age and the onsetof cell death. A 16-hr treatment was
`required to observe maximal cytotoxicity in HT-29 cells,
`which were insensitive to the cytostatic activity of rTNF,
`whereas 8 hr were required for highly sensitive ME-180
`cells (Fig. 3). The onset of cell death after continuous
`treatment with rTNF and CHX was about 8 hr for SK-
`MEL-109 cells and 2 hr for ME-180 cells (Fig. 3). There-
`fore, each cell line showed a different time course of
`cytotoxic response. These experiments were carried out
`with a high concentration of rTNF (14.3 nM), but the
`different sensitivity of the variouscell lines was reflected
`by the length of the treatment required forcell killing.
`Role of TNF receptors in cytostatic and cytotoxic
`activities. BT-20 cells were growth-inhibited by pM con-
`centrations of rTNF, whereas ME-180 cells were killed
`by fM concentrations in the presence of CHX (Figs. 1 and
`2). It was not clear whether the sensitivity to such low
`rTNF concentrations was mediated by its binding to a
`single class of receptors. It was reported previously that
`HT-29 and SK-MEL-109 cells have, respectively, 800 and
`9000 receptorspercell (23) and ME-180 cells have 2000
`receptors per cell (19). The Kp of these receptors is ~2 x
`107-'° M (19, 23). We measured the binding of !*°I-rTNF
`to BT-20 cells at 4°C, as described (23), and found 900
`receptors per cell with a Kp of about 1 x 107'° M. There-
`fore, the sensitivity of these cells to the activity of rTNF
`cannot be explained by a different number or binding
`affinity of these receptors.
`
`A 50% occupancyof receptors by 0.2 nM rTNF (on the
`
`4
`
`8
`Hours
`
`12
`
`16
`
`Figure 3. Time course of cytotoxicity in cells treated with 0.1 mg/ml
`of CHX and 14.3 nM rTNF. Thecells were either continuously treated (@)
`and stained with crystal violet at the time indicated in the abscissa, or
`were treated for the time indicated in the abscissa, were washed four
`times, were incubated with fresh medium up to 20 hr, and were then
`stained (A). The Asao relative to cultures treated with CHX alone is shown.
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`sao(%ofcontrol) 8
`40—20
`
`al
`
`. ~~
`, ME-180
`l
`0
`pM rTNF
`
`K-MEL -109
`
`
`1000
`
`100
`
`Figure 2. Dose dependencyof the cytotoxic activity of rTNF on four
`human tumorcell lines. The cells were seeded in cluster plates and were
`treated for 18 hr with 0.1 mg/ml of CHX and the rTNF concentration
`indicated in the abscissa, The Asqo relative to cells treated with CHX alone
`is shown.
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`ACTIVITIES OF TUMOR NECROSIS FACTOR
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`NMoO Hours
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`8
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`8
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`a.oO%Cyototoxicity
`
`
`
`%"1-rTNFbinding
`
`basis of the Kp measured at 4°C) resulted in maximal
`growth inhibition of BT-20 cells (Fig. 1), but only very
`few TNF receptors could be occupied by 50 fM rTNF,
`which wascytotoxic for ME-180 in the presence of CHX
`(Fig. 2). However, the following experiment showed that
`the relative number of TNF receptors within a cell line
`determinedits sensitivity to cytotoxicity. We took advan-
`tage of the observation that the TNF receptors of HT-29
`cells are increased up to threefold by treatment with IFN-
`y. but the Kp of these receptors is unchanged(23). This
`is a specific response to IFN-y, because no increase in
`TNFreceptorsis detected in HT-29 cells treated with IFN-
`«2 (23). Cytotoxicity was measured in HT-29 cells pre-
`treated for 5 hr with IFN-y; these cells were killed by
`rTNF concentrations threefold lower than those required
`to kill control cells (Fig. 4). This experiment indicated
`that the TNF receptors induced by IFN-y may be involved
`in the cytotoxic response.
`Further evidence that the cytotoxic response wasre-
`lated to the numberof receptors was obtained by treating
`ME-180 cells with CHX before '**I-rTNF binding. As
`shownin Figure 5A, inhibition of protein synthesis re-
`sulted in a drastic reduction in TNF binding. Few TNF
`receptors were detected after 2 hr, but the receptors
`reappeared whentheinhibition of protein synthesis was
`reversed by incubating the cells in fresh medium (Fig.
`5A). This indicated that TNF receptors turned over with
`a very short half-life. The rTNF cytotoxicity decreased by
`about 50% when the numberof receptors was reduced
`by 90%, and was almost abolished after ME-180 cells
`were preincubatedfor 2 hr with CHX (Fig. 5B). Therefore,
`the cytotoxic response was inhibited when the rTNF
`receptors decreased below a critical number. This, and
`similar experiments carried out on HeLa S2 cells (data
`not shown), showed that the rTNF receptors turned over
`with a half-life of ~30 min when protein synthesis was
`inhibited.
`Induction of the synthesis of new proteins in TNF-
`treated cells. A possible explanation for the high sensi-
`tivity to rTNF of cells treated with CHX is an inhibition
`of the synthesis of proteins which may be induced by
`rTNF and may protect the cells from cytotoxicity. This
`
`
`
`Asao(%ofcontrol)
`
`nNoO
`£oaoOooOOooO
`
`pM
`Figure 4. Effect of pretreatment with IFN-y on rTNF cytotoxicity. HT-
`29 cells (2 x 10°) were seeded in each well of a cluster plate and were
`treated for 5 hr with 25 ng/ml of IFN-y (A) or were kept as controls (@).
`The cells were then washed and were incubated for 18 hr with fresh
`medium containing 0.1 mg/ml of CHX and the rTNF concentration indi-
`cated in the abscissa. The Asap relative to cultures treated with CHX alone
`is shown.
`
`Figure 5. Binding of '**!-rTNF (A) and cytotoxicity of rTNF (B) for ME-
`180 cells treated with CHX. A, Confluent ME-180 monolayers in 9.6-cm?
`plates (~1.3 x 10° cells/plate) were incubated with 0.1 mg/ml cyclohexi-
`mide for the time indicated in the abscissa, or after 2 hr incubation
`(arrow) the cells were washed and were incubated in fresh medium.
`Binding of 0.15 nM '**I-rTNF was measured after 3 hr at 4°C. Nonspecific
`binding was determined by adding a 100-fold excess of unlabeled rTNF,
`and was on the average 14% of total binding. Nonspecific binding is
`subtracted from the data shown. The binding is expressed as a percentage
`of that of untreated ME-180 cells (1610 cpm). B, ME-180 cells seeded in
`cluster plates were treated with 0.1 mg/ml of CHX for the time indicated
`in the abscissa before the addition of 1 pMrTNF. The cells were incubated
`for a further 18 hr before staining with crystal violet. The percent
`cytotoxicity was calculated from the formula I — a/b x 100, where a and
`b are the Ago of cells treated with rTNF plus CHX andof cells treated
`with CHX alone.
`
`explanation has been suggested by the observation that
`relatively short incubations with TNF alone protect some
`cells from the cytotoxicity of a subsequent combined
`treatment with TNFand cycloheximide (24). In the follow-
`ing experiments, we observed that rTNF induced the
`synthesis of two proteins in SK-MEL-109 cells, but we
`could not detect any proteins synthesized in response to
`rTNFin othercell lines. This finding argues against a
`protective role for the TNF-inducedproteins.
`SK-MEL-109 cells were treated for different times and
`with different concentrations of rTNF and were labeled
`with [**S]methionine. The proteins were examined by gel
`electrophoresis and autoradiography(Fig. 6). The synthe-
`sis of two proteins of M, 42,000 and 36,000 (p42 and
`p36) was detectable in SK-MEL-109cells labeled from 2
`to 4 hr after the addition of 10 ng/ml rTNF(Fig. 6A) and
`in cells treated for 18 hr with 0.1 ng/ml of rTNF and
`labeled for the last 2 hr (Fig. 6B). Induction of p42 was
`quite evident, because there waslittle background in the
`correspondingposition of the gel track of untreated cells.
`However, p36 was clearly separated from other bandsin
`some gels (Fig. 6A), but was incompletely separated in
`othergels (Fig. 6B). Small changes in the composition or
`time of polymerization of the gels appeared to be respon-
`sible for this variability.
`The autoradiographsof the experiments shownin Fig-
`ure 6 and of other similar experiments were scanned in
`a microdensitometer to measure the amount of p42 and
`p36relative to a reference band(Fig. 7). The induction of
`p36 and p42 wascorrelated with the rTNF concentration
`(Fig. 7A). The time course of induction of p36 showed a
`peakat 3 hr and a decline in the synthesis of this protein
`afterwards, whereasthe synthesis of p42 increased with
`the time of rTNF treatment up to 8 hr andthen leveled
`off (Fig. 7B). Synthesis of these proteins could not be
`detected in the other cell lines examined for sensitivity
`to rTNF (Fig. 6C and data not shown). It seemed possible
`that this response to TNF could be characteristic of mel-
`anomacells. Therefore, the proteins synthesized by the
`melanoma cell lines A375, SK-MEL-13 (Fig. 6C), SK-
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`2714
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`ACTIVITIES OF TUMOR NECROSIS FACTOR
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`HT-29
`
`A375
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`SK-I3
`—=
`ee FUNE
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`[-—— Hours
`M. x 1073 O
`4
`6
`8
`16
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`r
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`r—ng/ml r1NF—-
`The position of M, markers and of the two proteins induced by rfNF in SK-MEL-109cells ts indicated.
`
`A
`
`Figure 6. Proteins synthesized by SK-MEL-109 cells treated for increasing times with 10 ng/ml of rfNF(A) or treated for 18 hr with increasing
`concentrations of rTNF (B). and byothercell lines (C) treated for 18 hr with 10 ng/ml of rTNF(+). or untreated (—). The cells were labeled with [*°S]
`methionine during the final 2 hr of incubation with rTNF. Proteins were separated by electrophoresis on 10° gels and the autoradiographs are shown.
`
`and p42. This finding suggested that transcription of the
`mRNAfor these proteins was induced by rTNF. An ex-
`periment was performed to establish whether this was a
`primary induction or whether synthesis of other proteins
`was required to induce transcription of p36 and p42
`mRNA, SK-MEL-109 cells were treated with rTNF in the
`presence of CHX, were washed, and were then labeled
`with [°°S]methionine. Both p36 and p42 were synthesized
`by cells treated in this way (Fig. 8). The synthesis of p36
`appeared to be superinduced, suggesting that the mRNA
`for this protein accumulated when protein synthesis was
`blocked. This finding agreed with the observation that
`maximal synthesis of p36 was an early response to rTNF
`and that its synthesis declined after a few hours(Fig. 7).
`This result suggested that transcription of mRNAfor p36
`and p42 was a primary event, which did not require
`ongoing protein synthesis.
`The stability of p42 was examined in an experiment in
`which SK-MEL-109cells labeled during treatment with
`rTNF were washed and were incubated for increasing
`times in fresh medium before SDS-PAGE analysis. The
`p42 band was measured by densitometry of the autora-
`diographsanddid not decrease during a 6-hr chase(Fig.
`9). The decay of the synthesis of p42 on removal of rTNF
`was examined by labeling SK-MEL-109cells after incu-
`bation in fresh medium (Fig. 9). The synthesis of p42
`declined ~50% in 6 hr, suggesting that p42 mRNA de-
`cayed with a similar half-life when rTNF was removed.
`However, alternative explanations for the decline in p42
`synthesis, such as a translational regulatory mechanism,
`cannotbe ruled out. Similar studies on the decay of p36
`synthesis could not be carried out because of the incom-
`plete resolution of this protein from other bands.
`A preliminary characterization of p42 and p36 has
`been carried out. These proteins are not membrane-
`bound, because they can be extracted by homogenizing
`SK-MEL-109 cells in low ionic strength buffer without
`added detergents. By labeling rTNF-treated cells with
`inorganic “*P and analyzing phosphoproteins bygel elec-
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`nM
`
`Hours
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`Figure 7. Dose response (A) and time course (B) of the induction of
`p36 and p42 by rTNF in SK-MEL-109 cells. Autoradiographs like those
`shownin Figure 5 were scanned in an LKB laser microdensitometer with
`peak area integrator. The absorbancyof the p36 and p42 bands normal-
`ized to that of a protein band which did not change with the rT™NF
`treatment is shown in arbitrary units.
`
`MEL-28, and DX-2 (data not shown) were examined be-
`fore and after treatment with rTNF. Thesecells did not
`show induction of p36 and p42, although they were
`sensitive to the cytostatic activity of rTNF (0.6 nM rTNF
`inhibited A375 and SK-MEL-13 cells 50 and 60%, re-
`spectively, in the assay described in Fig. 1, whereasit
`inhibited SK-MEL-28 and DX-2 cells 30 and 10%, re-
`spectively). Furthermore, HeLa cells, human osteosar-
`coma cells, and lung adenocarcinoma (A549) cells were
`examined before and after treatment with rTNF, but
`failed to show induction of p36 and p42 (data not shown).
`The only other cells which synthesize two new proteins
`in response to rTNF, in addition to SK-MEL-109, are
`humanfibroblasts (25). When a sampleof labeled protein
`obtained from rTNF-treated fibroblasts was run along-
`side that from SK-MEL-109 cells, the proteins induced
`by rTNF co-migrated. This showed that rTNF induced
`synthesis of presumably identical proteins in fibroblasts
`and SK-MEL-109cells.
`The addition of 1 ug/ml actinomycin D to SK-MEL-109
`cells together with rT'NF abolished the induction of p36
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`ACTIVITIES OF TUMOR NECROSIS FACTOR
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`ab Cc
`
`d
`
`M, x 107?
`200—
`
`<—p42
`
`<—p36 rTNF
`9 a
`
`CHX
`
`-
`
`-
`
`+
`
`+
`
`Figure 8. Synthesis of p36 and p42 by SK-MEL-109cells treated with
`rTNF in the presence of CHX. Cells seeded in cluster plates were treated
`for 3 hr with 10 ng/ml of rTNF or/and with 0.1 mg/ml of CHX, as indicated
`in the figure. The cells were then washed and werelabeled for 2 hr with
`(S]methionine. Proteins were fractionated by gel electrophoresis, and
`the autoradiograph is shown. The position of M, markers of p36 and p42
`is indicated.
`
`Absorbancy
`
`2
`
`a
`Hours
`
`6
`
`Figure 9. Stability of p42 and decay of its synthesis in SK-MEL-109
`cells after removalof rTNF. Cells, 8 x 10*, were seeded per well of cluster
`plates and were grown overnight: then 10 ng/ml of r!NF were added for
`19 hr. The stability of p42 was examined by labeling the cells with [*°S]
`methionine during the final 2 hr of rTNF treatment, washing the cells,
`and adding fresh medium for the time indicated before analyzing cell
`proteins by gel electrophoresis. Autoradiographs were scanned in a mi-
`crodensitometer, and the absorbancy of the p42 band before the chase
`(®) or after the chase(*) is indicated in arbitrary units. The decay of the
`synthesis of p42 was examined by washingthe cells after the treatment
`with rTNF and incubating the cells in fresh medium. The cells were
`labeled from 0 to 2, 2 to 4. and 4 to 6 hr after rTNF removal (@). Samples
`were processed and were analyzed as described in Materials and Meth-
`ods.
`
`trophoresis, we could exclude that p42 is phosphorylated.
`A phosphorylated polypeptide corresponding in M,to p36
`increased with the time of rTNF treatment (data not
`
`2715
`
`
`
`Figure 10, Growth-stimulatoryactivity of rTNF on SK-MEL-109cells.
`Cells. 2 x 10*. were seeded per well of cluster plates, were incubated
`overnight, were washed three times with medium minus serum, and were
`cultured in medium supplemented either with 0.2% FCS or with 0.5%
`FCS. After 3 days, 0.1 ng/ml of rTNF were added to some wells (+TNF).
`whereas others were kept as controls (C). On days subsequent to r™NF
`addition, individual wells were stained and the Agao Was measured as
`described in Materials and Methods.
`
`shown). This polypeptide may correspond to p36, but
`further experiments will be required to establish that p36
`is indeed a phosphoprotein.
`Growth-stimulatory activity of TNF. It was reported
`previously that rTNF can stimulate humanfibroblasts to
`divide (21, 26, 27). Because of the similarity between the
`response of SK-MEL-109 cells and fibroblasts to rTNF,
`shownby the induction of p36 and p42, we investigated
`whether these melanoma cells were also stimulated to
`divide by rTNF. No difference in growth rate was observed
`between control and rTNF-treated cells grown in 7.5%
`serum (data not shown). However, when the serum con-
`centration was decreased to 0.5 or 0.2%, rTNF stimulated
`proliferation of SK-MEL-109 cells about 20% (Fig. 10).
`This effect of r!NF on cell growth was not observed in
`experiments with HT-29 cells (data not shown). These
`findings indicated that rTNF can paradoxically stimulate
`the growth of some cancercells.
`
`DISCUSSION
`
`Some cancer cells, such as BT-20, are particularly
`sensitive to the cytostatic activity of TNF, whereas other
`cells are insensitive. We observed that rTNF is cytotoxic
`for these cells, but not for BT-20 cells. when protein
`synthesis is inhibited. This finding suggests that the
`cytostatic and cytotoxic activities of rTNF may be medi-
`ated by separate mechanisms which are poorly under-
`stood. The rTNFalone doesnot appearto be significantly
`cytotoxic for the cancer cell lines studied. This result
`differs from a previous report that a partially purified
`TNF produced by LukII lymphoblastoid cells is by itself
`cytotoxic for BT-20 cells (20). Possible explanations for
`this discrepancy are the presence of other cytotoxins in
`the TNF(LukIl) preparation andthe different cytotoxicity
`assay used by Williamsonetal. (20).
`The extraordinary sensitivity of ME-180 cells to the
`cytotoxic effect of TNF in the presence of CHX has not,
`to our knowledge, been reported previously. The use of
`ME-180 cells as a target
`in the TNF bioassay might
`represent a significant methodological advance over the
`commonly used assay with L929cells. The cytostatic or
`cytotoxic response to rTNF cannot be correlated in gen-
`eral with a different numberoraffinity of cellular recep-
`tors, but a lack of receptors may account for the finding
`that human B lymphoblastoid cell lines are insensitive to
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`8107‘€Z[dyuojsonsAq/S10°jounumT!MMmM//:dyYWopopeojuMog
`
`2716
`
`ACTIVITIES OF TUMOR NECROSIS FACTOR
`
`rTNFcytostatic activity (17). Accordingly, a loss of recep-
`tors in CHX-treated ME-180 cells abolishes rTNF cytotox-
`icity, but an increase in receptors in HT-29 cells treated
`with IFN-y enhances rTNFcytotoxicity (Fig. 4). The rapid
`loss of rTNF receptors in CHX-treated cells (Fig. 5) shows
`that these receptors turn over with a half-life of ~30 min.
`This is a very shorthalf-life for the receptor of a mediator
`of cell growth and differentiation such as TNF. This
`finding may have some implications for the mechanism
`of action of rTNF. In ME-180cells treated with CHX and
`rTNF, the events which lead to cytotoxicity must take
`place within a relatively short time of the binding of rTNF
`to receptors. At the rTNF concentration causing 50%
`cytotoxicity of ME-180 cells (~50 fM), only about onecell
`in two will have a TNF molecule boundat any giventime.
`This calculation is based on the Kp value and receptor
`number calculated for TNF binding at 4°C. However,
`receptor occupancy maybeinitially greater at 37°C, but
`in the presence of CHX the numberof receptors drops
`very rapidly. It therefore seemspossible that a single “hit”
`maysuffice to kill ME-180 cells in a relatively short time.
`Interestingly, a model proposed by Ruff and Gifford (28)
`is based on a less-than-first-order kinetics of the dose
`response to the cytocidalactivity of TNF, and may explain
`the apparentsensitivity to a single hit of extremely sen-
`sitive cells such as ME-180. However, it is now known
`whya long treatment with rTNF is required to kill rela-
`tively insensitive cells such as HT-29. Continuous bind-
`ing of rTNFto residual receptors or a slower turnoverof
`these receptors may explain this finding.
`A possible explanation for the variousactivities of TNF
`is a different cell response to signals from TNF-receptor
`complexes, but as yet there is no available information
`on their mechanism of signal transduction. We can gain
`some insight on the events which follow the binding of
`rTNFto receptors by examining the cellular response to
`this factor. The finding that most cells are only sensitive
`to the cytotoxic activity of TNF when protein synthesis
`is inhibited suggests that these cells counteractthe activ-
`ity of TNF by synthesizing some hypothetical “protective”
`protein. Failure to synthesize such protein mayresult in
`irreversible damage to TNF-treated cells. The ability of
`TNFto induce synthesis of new proteins is shown by the
`present finding that SK-MEL-109 cells synthesize p36
`and p42 after a few hoursof treatment with rTNF. The
`possible role of these proteins is unknown, but they are
`not synthesized in appreciable amounts by other cancer
`cells sensitive to TNF cytotoxicity. Therefore, it seems
`unlikely that these proteins play some role in counter-
`acting this cytotoxicity and that “new” proteins need to
`be synthesized by TNF-treated cells to block its cytotoxic
`activity, as suggested by the observation that a short
`pretreatment with TNF alone protects somelines after
`the subsequent addition of CHX (24, 25). Furthermore,
`the cell lines examinedin the present investigation were
`not protected from cytotoxicity by pretreatment with
`rTNF alone (our unpublished observations).
`Synthesis of p36 and p42 represents a novel biological
`activity of TNF, which can be added to the longlist of
`activities elicited by this pleiotropic factor in different
`cells. The synthesis of these polypeptides is not observed
`when fibroblasts are incubated with rTNF in the pres-
`ence of CHX, are washed, and are then labeled (25). In
`contrast, p36 and p42 are synthesized by SK-MEL-109
`
`cells treated in the same way(Fig. 8). The synthesis of
`the mRNAfor these proteins is a “primary” response in
`SK-MEL-109 cells, but apparently not in fibroblasts (25).
`However,it seems possible that the same basic regulatory
`mechanism exists in both cell types, but that metabolic
`differences between melanomacells and fibroblasts re-
`sult in altered stability of the protein intermediates in-
`volved in initiating p36 and p42 synthesis.
`It is surprising that rTNF induces synthesis of p36 and
`p42 in only one of the melanomacell lines tested. These
`SK-MEL-109 cells respond to rTNF similarly to fibro-
`blasts, and synthesize these proteins at an enhancedrate
`as long as rTNFis present in the culture medium (25).
`However, we have observed that the synthesis of p42
`decays in SK-MEL-109 cells when rTNF is removed, in-
`dicating that the mRNAfor this protein turns over with
`a half-life of 6 hr. Surprisingly, in medium with low
`serum, rTNF stimulates the proliferation of SK-MEL-109
`cells. This observation suggests that TNF has a rather
`unique activity on SK-MEL-109 cells and that it can
`promote growthof these cancercells. A likely explanation
`for these findings is that SK-MEL-109 cells possess some
`features of the response to TNF which are characteristic
`of humanfibroblasts.
`Thecytotoxic activity of TNF may explain somebiolog-
`ically relevant phenomena, because it has been reported
`recently that TNF is a mediator of the cytocidal activity
`of activated macrophages (29). Furthermore, the hypoth-
`esis that TNF elicits cytostatic and cytotoxic responses
`by separate mechanisms may have some bearing onits
`pro