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`0022-] 1'67367fl 368-271 ISOZDOIO
`Tue JOURNAL or Imuuuomcv
`Copyrlghl Gil [987 by The American Assoclatlon of Immunologists
`
`Vol. 138. 2711—2717. No. 3. Apr" 15. 1937
`Printed in 0.3.21.
`
`CYTOSTATIC AND CYTOTOXIC ACTIVITY OF TUMOR NECROSIS FACTOR ON
`HUMAN CANCER CELLS1
`
`VITO RUGGIERO.2 KATHRYN LATHAM, AND CORRADO BAGLIONI
`
`From the Department of 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 tumor cell lines. Human BT-2O
`breast and ME-lBO cervix cancer cells were growth-
`inhibited by rTNF. whereas two other cell lines were
`not significantly inhibited. However. when protein
`synthesis was inhibited 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. Occupancy of only a few
`receptors was sufficient for rTNA cytotoxicity. but
`an increase in receptor number after treatment with
`interferon-7. 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-IOQ 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-IOQ cells cultured in medium with low se-
`rum.
`
`mediator of cachexla in chronically Infected animals. and
`was designated cachectin (2] before discovering that its
`amino acid sequence is Identical to that of TNF [8. 9].
`TNF shows diverse biological cffects on different cells.
`For example. TNF‘ suppresses lipoprotein lipase activity
`(10]. enhances prostaglandln E2 and collagenase produc-
`tion by human synovial cells and dermal fibroblasts [l l).
`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 activlty of natural cytotoxic cells [14}. These
`activities of TNF follow its binding to high affinity recep-
`tors. which were initially identified in murlne cells (7].
`TNF receptors with a K0 of about 2 X 10"” M have been
`detected in human fibroblasts and tumor cells [15-19].
`It was reported that TNF‘ is cytotoxic for some human
`tumor cell 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 tumor cells.
`
`For this purpose. cell lines with a wide range of 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 by inhibitors of protein and RNA
`synthesis (22).
`
`MATERIALS AND METHODS
`
`A protein found in the serum of primed. endotoxin—
`treated animals elicits hemorrhagic necrosis of some mu-
`rine sarcomas (1] and is designated tumor necrosis factor
`[TNF]."' Priming promotes proliferation of macrophages.
`which are stimulated to secrete ’I‘NF by endotoxin lipo-
`polysaccharide [2]. The human TNF secreted by myelo-
`monoeytlc cells has been purified to homogeneity. se-
`quenced. and produced by recombinant DNA technology
`[3-6]. Human recombinant TNF [rTNF] shows the same
`biological activities as natural TNF‘ (3—6]. A factor se-
`creted by macrophages suppresses lipoproteln lipase ac-
`tivity (7]. This factor has been proposed as an endogenous
`
`Received for publication October 28. 1986.
`Accepted for publication January 6. 198?.
`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 Indl—
`catc this fact.
`' Thls Investigation was supported by US Public Health Service Grant
`CA29895. awarded by the National Cancer Institute. DHHS.
`’ Present address: Istituto di Virologla. Universlta' di Roma. Vlale dl
`Porta 'l‘iburtina 23. 00185 Roma. Italy.
`3 Abbreviations used in this paper: TNF. tumor necrosis factor: rTNF.
`recombinant tumor nacrosls factor: CHX. cyclohexlmlde.
`
`Cells. rTNF. and rl’FN-y. Colon adenocarcinoma HT-29 cells were
`cultured as described [23} in Dulbecco's medium wlth 10% fetal calf
`serum {PCS}. A375 melanoma cells were cultured in RPMI 1640
`medium with 5% FCS. Breast tumor BT-20. cervix carcinoma ME-
`180. and other melanoma cell
`lines were cultured in F~12 and
`minimal Eagle's medium (1/1) with 8% FCS. The cells were resus-
`pended from monolayers with phosphate-buffcred saline [PBS] con-
`taining 1 mM EDTA. rTNF was a gift of Dr. T. lehihara of the
`Suntory Institute for Biomedical Research. Osaka. Japan; rlFN—T
`[1.7 x 107 U/mg] was a gift from Biogen (Cambridge. MA].
`Assays for anttproiiferatiue and cytotoxic activity of r‘l‘NF.
`Growth inhibition was measured by seedng 1 to 4 x 10‘ cells/well
`In cluster plates: the cells were stained with crystal violet after 4
`days of trealment [23]. Cytotoxlcity was measured by treatlng 0.8 to
`2 x 105 cells/well for 18 hr with rTNF and 0.1 mg/ml of CHX. The
`monolayers were washed twice with PBS to remove dead cells and
`were stained with crystal violet. This dye was eluted with 3.3% acetic
`acid and the A5“. was measured In a microdensltometer. The assays
`were carried out in quadruplicate and gave a standard error of <5%.
`Ceii labeling and protein analysis. Confluent monolayers were
`incubated for 2 hr with 25 pCi/ml of [asslmethlonlnc 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 phenylmethylsulfonyl
`fluoride in 10 mM NaCl. 1.5 mM Mg[0Ac]g. and 10 mM Tris-HCI. pH
`7.4. were added for 10 min. The cells were then Iysed 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 SDSFPAGE
`on 10% gels. The autoradlographs were scanned in an LKB Ultroscan
`2711
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`2712
`
`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 unchanged in intensity after the
`treatment with rTNF.
`
`RESULTS
`
`Cytostatic and cytotoxic activity of TNF on human
`cancer cells. 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-lOQ cells
`
`were slightly growth-inhibited. whereas MEI-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 i’I‘NF. The
`following experiment was aimed at establishing whether
`these cells were sensitive to rTNF cytotoxlcity. Nearly
`confluent cultures were treated for 18 hr with different
`concentrations of rTNF and 0.1 rug/m1 of CHX. which
`enhances TNF cytotoxicity (22). All of the cell lines were
`extremely sensitive to this treatment. with the exception
`of BT—20 (Fig. 2]. The LD50 was between 0.1 nM rTNF for
`FIT-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 were resist-
`ant to cytotoxicity and. conversely. SK-MEL-lOQ cells
`were highly sensitive to rTNF cytotoxlcity but fairly re-
`sistant to its cytostatic activity. This finding suggested
`that some cells could only respond either to the cytostatic
`
`88g
`Agut'ltctconlroll Ao
`
`m0
`
`-
`l.__ ._I_
`
`1
`0.00i
`0.0I
`{1|
`nM rTNF
`
`Figure 1. Dose dependency of the antlprollferstive activity of rTNF on
`four human tumor cell 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 More-
`rlals and Methods; the Aug relative to control untreated cells is shown.
`
`
`s9
`
`It- MEL - P09
`
`E-rao
`"""--..._E‘____.
`
`IOOD
`It)
`POO
`pM rTNF
`
`.l
`
`I
`
`Figure 2. DOSE dependency of the cytotoxic activity of i’I‘NF‘ on four
`human tumor cell lines. The cells were seeded in cluster plates and were
`treated for 18 hr with 0.1 mgfml of CHX and the rTNF concentration
`indicated in the abscissa. The A5“ relative to cells treated with CHX alone
`IS shoWn.
`
`or to the cytotoxic activity of r’I‘NF‘.
`The onset of cell death after treatment with rTNF and
`
`CHX varied among the cell lines examined (Fig. 3]. Cyto-
`toxicity was assayed either after 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 onset of cell death. A lS—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 NIB-180
`cells (Fig. 3]. The onset of cell death after continuous
`treatment with rTNF and Ci-IX was about 8 hr for SK-
`
`MEL-lOQ cells and 2 hr for MEI-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 various cell lines was refiected
`by the length of the treatment required for cell killing.
`Role of TNF receptors in cytostatic and cytotoxic
`activities. EST-20 cells were grewth-inhibited by pM con-
`centrations of rTNF, Whereas MIC-180 cells were killed
`by f M concentrations in the presence of CHX (Figs. 1 and
`2]. it was not clear whether the sensitivity to such 10w
`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 receptors per cell (23] and MEI-180 cells have 2000
`receptors per cell (19). The [to of these receptors is ~2 x
`10“0 M (19. 23]. We measured the binding of ”Ed-rTNF
`to BT-2O cells at 4°C. as described (23). and found 900
`receptors per cell with a KB of about 1 X 10—10 M. There-
`fore. the sensitivity of these cells to the activity of r’l‘NF
`cannot be explained by a different number or binding
`affinity of these receptors.
`A 50% occupancy of receptors by 0.2 nM rTNF (on the
`
`
`
`4
`
`3
`
`12
`
`I5
`
`Figure 3. Time course of cytotoxicity in cells treated with 0.] rug/ml
`of CHX and 14.3 nM rTNF. The cells were either continuously treated [I]
`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 [l]. The Am relative to Cultures treated with CHX alone is shown.
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`ACTIVITIES 0F TUMOR NECROSIS FACTOR
`
`IOD
`
`,E' 80
`E
`.L 60
`5
`T 40
`if
`as 20
`
`basis of the [(9 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 mt rTNF.
`which was cytotoxic for ME-lSO in the presence of CHX
`(Fig. 2}. However. the following experiment showed that
`the relative number of TNF receptors within a cell line
`determined its 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—
`7. but the Km of these receptors is unchanged (23]. This
`is a specific response to lFN-y. because no increase in
`TNF receptors is detected in PIT-29 cells treated with lFN-
`a2 (23]. Cytotoxicity was measured in HT-29 cells pre-
`treated for 5 hr with lFN-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-v may be involved
`in the cytotoxic response.
`Further evidence that the cytotoxic response was re-
`lated to the number of receptors was obtained by treating
`ME-180 cells with CHX before ‘251-1’I‘NF binding. As
`shown in 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 when the inhibition 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 number of receptors was reduced
`by 90%. and was almost abolished after MEI—130 cells
`were preincubated for 2 hr with CHX (Fig. 53). 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 Clix is an inhibition
`of the synthesis of proteins which may be induced by
`rTNF and may protect the cells from cytotoxicity. This
`
`Am(93ofcontrol1
`
`I00
`
`43a)a:OO0
`
`NO
`
`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 ngfml of lF‘N-‘y {A} or were kept as controls (0].
`The cells were then washed and were incubated for 18 hr with fresh
`medium containing 0.1 mg/mi of CHX and the rTNF concentration indi—
`cated in the abscissa. The A5...) relative to cultures treated with CHX alone
`ls shown.
`
`20
`
`B
`
`100
`
`so ,,
`:5
`so 5
`‘3
`40 u
`a!
`
`Hours
`Figure 5. Bindingof ”"i-fl‘NF {A} and cytotoxicity of rTNF (B) for ME-
`180 cells treated with CHX. A. Confluent ME-lSO monolayers in ENS-cma
`plates {-4.3 x 10“ cells/plate] were incubated with 0.] mg/‘ml cyclohexi-
`mlde 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 lz’l—r’l‘i'llF‘ was measured after 3 hr at 4°C. Nonspecific
`binding was determined by adding a IOU-fold excess of unlabeled (TNF.
`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-l 30 cells (1610 cpm]. B. MES-180 cells seeded in
`cluster plates were treated with 0.1 mgfrnl of CHX for the time indicated
`in the abscissa before the addition of i phi 1’1“NF. The cells were incubated
`for a further 18 hr before staining with crystal violet. The percent
`cytotoxicity was calculated from the formula I — all) x 100. where a and
`b are the AW of cells treated with rTNF plus CHX and of 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 TNF and 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
`rTNF in other cell lines. This finding argues against a
`protective role for the TNF-induced proteins.
`SK-MEL—lOQ cells were treated for different times and
`with different concentrations of rTNF and were labeled
`
`with [35$]methionine. The proteins were examined by gel
`electrophoresis and autoradiography (Fig. 6]. The synthe-
`sis of two proteins of Mr 42.000 and 36.000 [p42 and
`p36) was detectable in SK-MEL-lOQ cells 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. SB]. Induction of p42 was
`quite evident. because there was little background in the
`corresponding position of the gel track of untreated cells.
`Hawever. p36 was clearly separated from other bands in
`some gels (Fig. 6A]. but was incompletely separated in
`other gels (Fig. SB]. Small changes in the composition or
`time of polymerization of the gels appeared to be respon-
`sible for this variability.
`The autoradiographs of the experiments shawn in Figs
`are 6 and of other similar experiments were scanned in
`a microdensitometer to measure the amount of p42 and
`p36 relative to a reference band (Fig. 7]. The induction of
`p36 and p42 was correlated with the rTNF concentration
`(Fig. 7A]. The time course of induction of p36 showed a
`peak at 3 hr and a decline in the synthesis of this protein
`afterwards. whereas the synthesis of p42 increased with
`the time of rTNF treatment up to 8 hr and then leveled
`off (Fig. 731. 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 me]-
`anoma cells. Therefore. the proteins synthesized by the
`melanoma cell lines A375. SK-MEL-13 (Fig. BC). SK-
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`2714
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`ACTIVITIES OF TUMOR NECROSIS FACTOR
`
`l—Hours—1
`M,xlo"0
`4
`6
`8
`l6
`-
`
`r-ng/rnl rTNF—I
`.9".
`I__
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`
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`+rTNF
`‘
`
`
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`A
`
`Figure 6. Proteins synthesized by SK-MEL-lOQ cells treated for increasing times with It) rig/ml ol' rTNF [A] or treated [or let hr with Increasing
`concentrations of r'i‘NF‘ [BL and by other cell lines [(3) treated for 18 hr with [0 ng/ml of rTNF [+1. or untreated [—|. The cells were labeled with 1"531
`methionine during the final 2 hr of incubation with rTNF. Proteins were separated by clectrophoresison lG‘fE gels and the ailtoratiimzrophs are shown.
`The position of M. markers and of the two proteins induced by rTNF in SK—Ml-Ii.~i09 cells is indicated.
`
`
`
`nM
`
`Hours
`
`Figure 7. Dose response [Al and time course [it] of the induction of
`p36 and p42 by rTNF in SK-MEL-IGQ cells. Autoradiographs like. those
`shown in Figure 5 were scanned in an LKB laser mierodensltometer with
`peak area integrator. The absorbancy of the p36 and 1342 bands normal-
`lzed to that of a protein band which did not change with the rTNF
`treatment is shown in arbitrary units.
`
`MEL-28. and DX-2 (data not shown] were examined be«
`fore and after treatment with rTNF. These cells 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-IS cells 50 and 60%. re-
`
`spectively. in the assay described in Fig. 1. whereas it
`inhibited SK-MEL-ZS and DIX-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-IOQ. are
`human fibroblasts (25]. When a sample of labeled protein
`obtained from rTNF-treated fibroblasts was run along-
`side that from SK-MEL-lOQ cells. the proteins induced
`by rTNF co-mlgrated. This showed that rTNF‘ induced
`synthesis of presumably identical proteins in fibroblasts
`and SK-MEL-IOQ cells.
`
`The addition of l pgfml actinomycin D to SK-MEL-IOQ
`cells together with rTNF abolished the induction of p36
`
`and p42. This finding suggested that transcription of the
`mRNA for 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-IOQ cells were treated with rTNF in the
`
`presence of CHX. were washed. and were then labeled
`with l"5S]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 mRNA for 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-lOQ cells 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 dcnsitometry of the autora-
`diographs and did 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—IOQ cells 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 asa translational reguiatory mechanism.
`Cannot be 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-IOQ cells in low ionic strength buffer without
`added detergents. By labeling rTNF-trcatcd cells with
`inorganic 32P and analyzing phosphopmtcins by gel elec-
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`ACTIVITIES OF TUMOR NECROSIS FACTOR
`
`2715
`
`
`
`Figure it). Growth-stimulatory activity of rTNF on SK—MEL—IOQ cells.
`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% PCS or with 0.5%
`l-‘CS. After 3 days. 0.1 ng/mi of rTNF were added to some wells |+TNFL
`whereas others were kept as controls {C}. On days subsequent to rTNF
`addition. individual wells were stained and the Anna 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 human fibroblasts to
`divide {21. 26. 27]. Because of the similarity between the
`response of SK—MEL-lOQ cells and fibroblasts to rTNF‘.
`shown by 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-iOQ cells about 20% (Fig. 10].
`This effect of rTNF 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 cancer cells.
`
`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 rTNF‘ alone does not appear to 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 TNFiLukII} preparation and the different cytotoxicity
`assay used by Williamson et a1. (20).
`The extraordinary sensitivity of MEI-180 cells to the
`cytotoxic effect of TNF‘ in the presence of CI-IX has not.
`to our knowledge. been reported previously. The use of
`ME-IBO cells as a target
`in the TNF bioassay might
`represent a significant methodological advance over the
`commonly used assay with 1.929 cells. The cytostatic or
`cytotoxic response to rTNF cannot be correlated in gen-
`eral with a different number or affinity of cellular recep-
`tors. but a lack of receptors may account for the finding
`that human B lymphoblastoid cell lines are insensitive to
`|PR2018—00685
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`Celgene Ex. 2031, Page 5
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`o__b
`
`c
`
`d
`
`Mr I I0"3
`200'-
`
`
`
`-
`
`—
`
`+
`
`+
`
`rTNF
`CHx
`
`Figures. Synthesis of p36 and p42 by SK-MEL-IOQ cells treated with
`rTNF In the presence of CHX. Cells seeded In cluster plates were treated
`for 3 hr with 10 ng/ml of rTNF cur/and with 0.] mgfml of CHX. as Indicated
`tn the figure. The cells were then washed and were. labeled for 2 hr with
`|°5S|methionlne. Proteins were fractionated by gel electrophoresis. and
`the autoradiograph is shown. The position of M. markers of p36 and p42
`ls indicated.
`
` 2
`
`6
`
`4
`Hours
`
`Figure 9. Stability of p42 and decay of Its synthesis in SK-MEL- l 09
`cells after removal of rTNF‘. Cells. 3 X 10‘. were seeded per well of cluster
`plates and were grown overnight: then it) ng/ml of rTNF were added for
`19 hr. The stability of p42 was examined by labeling the cells with la’SI
`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. Automdlographs were scanned in a mi-
`crodensitometcr. and the absorbancy of the p42 band before the chase
`[I] or after the chase [x] is indicated In arbitrary units. The decay of the
`synthesis of p42 was examined by washing the cells after the treatment
`with r'i'NF and lncubatlng the cells In fresh medium. The cells were
`labeled from 0 to 2. 2 to 4. and 4 to 6 hr after r'l‘NF‘ removal [I]. Samples
`were processed and were analyzed as described In Materials and Meth-
`ads.
`
`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
`
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`2716
`
`ACTIVITIES OF TUMOR NECROSIS FACTOR
`
`rTNF cytOstatic activity (1 7]. Accordingly. a loss of recep-
`tors in CHX—treated ME- 1 80 cells abolishes rTNF cytotox-
`icity. but an increase in receptors in HT-29 cells treated
`with IFN-y enhances rTNF cytotoxicity (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 short half«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 r’I‘NF. In ME-lBO cells 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 MEI-180 cells (~50 fM]. only about one cell
`in two will have a TNF molecule bound at any given time.
`This calculation is based on the Kg value and receptor
`number calculated for TNF binding at 4°C. However.
`receptor occupancy may be initially greater at 37°C. but
`in the presence of CHX the number of receptors drops
`very rapidly. It therefore seems possible that a single “hit"
`may suffice 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 cytocidal activity of TNF. and may explain
`the apparent sensitivity to a single hit of extremely sen-
`sitive cells such as ME-lSO. However. it is now known
`
`why a long treatment with rTNF is required to kill rela-
`tively insensitive cells such as HT—29. Continuous bind-
`ing of rTNF to residual receptors or a slower turnover of
`these receptors may explain this finding.
`A possible explanation for the various activities 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
`rTNF to 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 counteract the activ-
`ity of TNF by synthesizing some hypothetical "protective"
`protein. Failure to synthesize such protein may result in
`irreversible damage to TNF-treated cells. The ability of
`TNF to induce synthesis of new proteins is shown by the
`present finding that SK-MEL-IOQ cells synthesize p36
`and p42 after a few heurs of 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 some lines after
`the subsequent addition of CHX {24. 25). Furthermore.
`the cell lines examined in 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 long list 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-IOQ
`
`cells treated in the same way (Fig. 8]. The synthesis of
`the mRNA for 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 melanoma cells 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 melanoma cell lines tested. These
`SK-MEL-lOQ cells respond to rTNF similarly to fibro-
`blasts. and synthesize these proteins at an enhanced rate
`as long as rTNF is present in the culture medium [25}.
`However. we have observed that the synthesis of p42
`decays in SK-MEL-IOQ cells when rTNF is removed. in-
`dicating that the mRNA for 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-IOQ cells and that it can
`promote growth of these cancer cells. A likely explanation
`for these findings is that SK-MEL- 1 09 cells possess so