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Polar Agents With Differentiation Inducing Capacity Potentiate Tumor Necrosis Factor-Mediated Cytotoxicity in Human Myeloid Cell Lines, Part 2
This article was received February 28, 1994; accepted August 29, 1994.

Results

Characterization of growth arrest and cell death of U937 cells following combined DMSO/TNF treatment

Several investigators have demonstrated that treatment of human leukemic cells with DMSO is associated with growth inhibition and induction of differentiation. 2427 Incubation of our U937 cell line in the presence of DMSO induced a time- and concentration-dependent reduction of the cells' proliferative capacity: over an assay period of 72 h and at 210 mM DMSO, cells were fully growth-inhibited (Fig. 1A). As inhibition of thymidine incorporation can reflect both cytostatic as well as cytotoxic effects, we also investigated cell viability by means of trypan blue exclusion and the colorimetric MTT assay. Fig. 1B shows that treatment with 210 mM DMSO, a dose inducing nearly complete growth inihibition, did not induce any cytotoxicity: total cell viability as measured by trypan blue, remained constant over an assay period of 72 h. Furthermore, at this time point, over 85% of the cells in culture could be stained with MTT (data not shown). Higher DMSO doses (280 mM) became cytotoxic. In further experiments, concentrations of DMSO used were maximally 180 mM.

Figure 1

Thymidine incorporation Number of viable cells
Figure 1. Effect of DMSO on cell growth of &937 cells. U937 were seeded at 105 cells/ml in culture medium without (green) or with DMSO at 70 mM (red), 140 mM (blue), 18  mM (yellow), 210 mM (brown), or 280 mM (pink). At indicated time points, the number of viable cells was determined by trypan blue exclusion (B) or cells were washed free of DMSO and reincubated at 5 x 102 cells/ml in culture medium for 1 h in the presence of [³H]thymidine (A). Incorporated radioactivity was determined as described in Materials and Methods. Data represent the average of three determinations (coefficient of variance <10%).

Treatment of U937 with 50 pg/ml TNF for 24 h did not induce growth inhibition or cell death (Fig. 2A). However, upon 24-h pretreatment with increasing doses of DMSO, TNF-induced growth inhibition and cell death became apparent within a 24 h period (Fig. 2A). The observed TNF activity could be blocked completely by neutralizing antiserum to human TNF (data not shown).

Figure 2

Number of viable cells  Thymidine incorporation


Figure 2. Cytotoxic and antiproliferative activity of TNF on U937 cells pretreated with DMSO. A: U937 cells were seeded in the presence of the indicated concentrations of DMSO. Twenty-four h later, DMSO was washed away and the cells were reseeded at 2 x 105 cells/ml with (green) or without (red) 50 pg/ml TNF for 24 h, after which cell viability was determined by trypan blue exclusion. Initial cell concentration=2. Bars represent mean ± SD. B: Cells were seeded in culture medium without (red) or with DMSO at 70 mM (green), 140 mM (blue) or 180 mM (yellow) in the presence of 50 pg/ml TNF for 20 h, after which [³H]thymidine incorporation was measured during a 4-h period. Data are presented a % of control, 100% being the radioactivity incorporated by the respective DMSO-treated cells subsequently cultured in the absence of TNF [e.g., for 24 h DMSO pretreatment, 100% = 90,000 cpm (red), 85,000 cpm (green), 70,000 cpm (blue), or 60,000 cpm (yellow)]. Data represent the average of two experiments (coefficient of variance <10%).

Besides a DMSO concentration dependency, there was also a need for a prolonged preincubation period: restricted DMSO pretreatment times (less than 7 h) were not sufficient to render the cells susceptible to TNF (Fig. 2B). At 180 mM DMSO, priming of the U937 cells was optimal after a 24-h continuous incubation with DMSO, a time point at which antiproliferative effects of DMSO alone were not yet apparent (Fig. 1A). In further experiments, U937 cells, pretreated for 24 h with 180 mM DMSO, will be designated as U937·DMSO cells.

Potentiation of TNF cytotoxicity by DMSO in myeloid cell lines with differing degrees of maturation

We investigated whether DMSO also altered the TNF sensitivity of established human myeloid cell lines other than U937. These included (classified by increasing degree of maturity): erythroleukemic K562, myeloblastic KG1, promyelocytic HL60, monoblastic (myelomonocytic) THP1, and monocytic MonoMac6 cell lines.

As for U937 cells, treatment with 180 mM DMSO for 72 h induced inhibition of cell growth, as measured by the MTT colorimetric assay, in KG1, HL60, and THP1 without loss of cell viability (Table 1, data for KG1 not shown). The MonoMac6 cell line, 180 mM, but not 90 mM, was cytotoxic. In K562 cells, only modest antiproliferative effects were observed, even following 72 h of culture in the presence of 180 mM DMSO.

Sensitivity to TNF alone of the indicated cell lines largely differed, at least when assayed over long periods. After 72 h of incubation in the presence of 10 ng/ml TNF, all three monoblastic/monocytic cell lines (U937, THP1, and MonoMac6; Table 1) as well as the KG1 cell line (data not shown) showed diminished cell growth, in contrast to K562 and HL60 cell lines. The absence of a TNF effect on both K562 and HL60 cells was not due to the absence of p55 or p75 TNF receptors, as both receptor types were detected on these cell lines by flow cytometry analysis (data not shown).

Finally, we tested whether DMSO pretreatment and/or cotreatment influenced the TNF sensitivity of these cell lines. In pretreatment experiments, cells were incubated for 24 h with 180 mM DMSO, followed by 24 h incubation with TNF (Table 2). DMSO only up-regulated the responsiveness of cell lines which already displayed TNF susceptibility as such in long term assays (KG1, U937, THP1, and MonoMac6). The potentiating effect was most obvious in the case of U937 and THP1 cells. Similar observations were made when cell viability was measured by trypan blue exclusion (data not shown). We also tested U937-derived cell lines which were resistant to TNF cytotoxicity. This resistance was not due to the absence of p55 or p75 receptors (whose expression was not significantly different from parental U937 cells as detected by flow cytometric analysis; data not shown), nor to the absence of TNF internalization and degradation (data not shown). As expected, U937r cells did not show any growth inhibition when cultured in the presence of 10 ng/mi TNF for 72 h (data not shown). However, these U937r cells could be sensitized to TNF cytotoxicity by DMSO pretreatment (Table 2). In contrast to U937 cells, however, about 100 times higher TNF doses were needed to obtain this effect.

Table 1

Effect of DMSO Cotreatment on TNF Sensitivity of Various Human Myloid Cell Linesa

TNF (ng/ml)
Cell line Time oftreatment DMSO (mM) 10 1 0.1 0
K562 72 h     0 96 (96)   91 (91)  NTb 100          
120 72 (85)   75 (88)   NT     85 (100)
180 70 (88)   75 (94)   NT     80 (100)
HL60 72 h     0 95 (95) 104 (104) NT   100          
120 59 (62)   89 (93)   NT     96 (100)
180 14 (32)   35 (79)   NT     45 (100)
U937 24 h     0 79 (79)   87 (87)     95 (95)   100          
120 50 (49)   63 (61)     82 (80)   102 (100)
180 29 (28)   42 (40)     70 (68)   103 (100)
72 h     0 47 (47)   67 (67)     74 (74)   100          
120   8 (8)     21 (22)     39 (42)     93 (190)
180   2 (5)       3 (6)       11 (22)     49 (100)
thP1 24 h     0 43 (43)   53 (53)     68 (68)   100          
120 28 (28)   36 (36)     54 (54)   100 (100)
180 17 (19)   24 (26)     44 (49)     90 (100)
72 h     0 30 (30)   42 (42)     51 (51)   100          
120   4 (5)       7 (8)       21 (23)     88 (100)
180   2 (4)       2 (4)         7 (14)       50 (100)
MonoMac6 24 h     0 88 (88)   93 (93)   101 (101) 100          
  60 80 (90)   83 (95)     88 (100)   88 (100)
  90 73 (91)   78 (97)     84 (105)   80 (100)
72 h     0 70 (70)   92 (92)     89 (89)   100          
  60 27 (34)   40 (51)     70 (88)     79 (100)
  90 15 (30)   24 (46)     43 (82)     52 (100)
aHuman myeloid cell lines were grown for 24 and/or 72 h in the presence of DMSO together with TNF, at the concentrations indicated, after which cell viability was estimated by MTT colorimetric assay. Data are presented as % of control, 100% being the number of viable cells grown in the absence of both DMSO and TNF, or, for the data between brackets, 100% being the number of viable cells grown in absence of TNF. the results are representative of two experiments.

bNot tested.

Table 2

Effect of DMSO Pretreatment on TNF Sensitivity of Various Human Myelold Cell Linesa

% [?H]thymidine incorporation

TNF (ng/ml)
Cell line DMSO pretreatment 100 1 0.01 0 Reference (cpm)
K562 - 103 103 104 100 145,000
+ 101 98 102 100 135,000
KG1 - 85 89 93 100 40,000
+ 49 72 98 100 25,000
HL60 - 85 89 105 100 100,000
+ 85 93 102 100 80,000
U937 - 81 83 98 100 90,000
+ 9 17 56 100 60,000
THP1 - 76 79 92 100 100,000
+ 5 9 45 100 70,000
MonoMac6 - 83 86 95 100 110,000
+ 40 49 75 100 70,000
U937r - 97 102 101 100 150,000
+ 28 45 105 100 125,000
aHuman myeloid cell lines were grown for 24 h with or without 180 mM DMSO (90 mM in case of MonoMac6), after which they were washed free of DMSO and incubated with or without TNF. Twenty h later, [³H]thymidine incorporation during 4 h was measured. Data are presented as % of control cpm, 100% being the radioactivity incorporated by DMSO-pretreated or untreated cells grown in the absence of TNF for 24 h. The results are representative of three experiments.

With cotreatment with DMSO and TNF, we obtained potentiating effects of DMSO on TNF-induced cytostasis/cytotoxicity in U937, THP1, and MonoMac6 cells, whereas the K562 cell line remained unsusceptible to TNF, even after 72 h (Table 1). Furthermore, in contrast with pretreatment results, cotreatment of the HL60 cell line with DMSO and high doses of TNF (10 ng/ml) for 72 h resulted in the induction of TNF sensitivity. Finally, it is important to note that in all cell lines tested, except for K562, DMSO could also induce or potentiate TNF-induced cytostasis/cytotoxicity at a concentration which, on its own, had no antiproliferative effect, even after 72 h (120 mM, Table 1).

Potentiation of TNF cytotoxicity by DMSO is restricted to myeloid cell lines

Table 3 summarizes the results of a representative experiment in which the TNF sensitivity of several non-myeloid cell lines was tested, either in the absence or presence of DMSO, in a 72-h MTT viability assay. First, DMSO alone reduced the amount of cells in all cell lines in comparison with control cult9A6, MCF-7, PC60 TR55/75) TNF sensitivity. Potentiation of TNF cytostasis/cytotoxicity by DMSO was not found in any of the non-myeloid cell types tested and thus, DMSO-induced potentiation was restricted to myelold cell lines.

Table 3

Effect of DMSO Cotreatment on TNF Sensitivity of Non-Myelold Cells


  Type TNFa (ng/ml) DMSO (mM) Effect of DMSO on TNF cytotoxicityb TNF-induced DNA fragmentationc
180 120 0
NIH3T3 murine 0 73 (100) 80 (100) 100 (100) none NTd
fibroblast 100 77 (105) 83 (103) 108 (108)    
24T2-5 murine 0 57 (100) 76 (100) 100 (100) none +
fibrosarcoma 0.1 32 (56) 39 (51) 55 (55)    
1.0 15 (26) 18 (23) 23 (23)    
WEHI 164 cl13 murine 0 82 (100) 92 (100) 100 (100) inhibition +
fibrosarcoma 0.1 43 (52) 37 (40) 32 (32)    
KYM39A6 human 0 62 (100) 75 (100) 100 (100) none +
rhabdomyosarcoma 0.1 32 (51) 36 (48) 45 (45)    
1 4 (6) 2 (3) 2 (2)    
ME-180 human 0 55 (100) 56 (100) 100 (100) inhibition -
cervix carcinoma 1 52 (94) 56 (100) 74 (74)    
10 32 (58) 33 (59) 34 (34)    
MCF-7 human 0 57 (100) 73 (100) 100 (100) none -
breast carcinoma 1 32 (57) 39 (54) 50 (50)    
10 22 (39) 24 (33) 31 (31)    
PC60 TR55/75 rat/mouse 0 89 (100) 98 (100) 100 (100) none +
thymoma 100 50 (54) 47 (48) 54 (54)    

Cells were grown in the presence of TNF and/or DMSO, both at the concentrations indicated, for 72 h after which cell viability was estimated by MTT calorimetric assay. Data are presented as % of control, 100% being the number of viable cells grown in the absence of both DMSO and TNF, or, for the data in brackets, 100% being the number of viable cells grown in absence of TNF. The results are representative of three experiments.

bSummary of the effect of DMSO cotreatment on TNF sensitivity of each cell line investigated, on the basis of the data shown in a

cDNA fragmentation was estimated by agarose gel electrophoresis of total cellular DNA of TNF-treated cell lines ( + , TNF-induced DNA fragmentation; - , no DNA fragmentation upon TNF treatment).

dNot tested.

Myeloid cell line-specific potentiation of TNF-induced DNA fragmentation

It has been reported that TNF can cause DNA degradation before cell lysis. 45 Agarose gel electrophoresis of total cellular DNA revealed that 24-h treatment of U937 cells with 180 mM DMSO did not induce DNA degradation, in contrast to a 4-h treatment with 10,000 pg/ml TNF which induced some apoptotic DNA degradation (Fig. 3). Apoptosis or programmed cell death is characterized by internucleosomal cleavage of the DNA resulting in the appearance of oligonucleosome fragments consisting of 200 basepairs or multimers of it. It is remarkable that 10,000 pg/ml TNF induced some apoptotic DNA degradation in U937 cells within 4 h while this TNF dose only induced small cytostatic/cytotoxic effects within 24 h (Table 1). DMSO pretreatment was found to potentiate TNF-induced DNA fragmentation in U937, resulting in almost complete DNA degradation at 10,000 pg/ml TNF. Enhancement of TNF-induced DNA fragmentation by DMSO was also observed in THP1 and HL60 cell lines (data not shown). In the TNF-resistant U937r cell line, TNF induced DNA fragmentation only upon pretreatment with DMSO (Fig. 3). Thus, in myeloid cells there was a good correlation between the potentiation of TNF action by DMSO and the occurrence of TNF-induced fragmentation. This observation probably indicates that TNF potentiation by DMSO was restricted to cells with a propensity for apoptotic cell death. However, a TNF-induced ladder pattern of DNA digestion was also observed in some non-myelold cells (24T2·5, WEHI 164 cl13, KYM39A6, PC60 TR55/75), whose TNF sensitivity was either inhibited or remained unaltered upon DMSO cotreatment (see summary in Table 2). Thus, apoptotic DNA digestion does not predict a propensity for DMSO to potentiate TNF cytotoxicity.

Figure 3

Effect of DMSO on TNF-induced DNA degradation in U937 cells

Figure 3. Effect of DMSO on TNF-induced DNA degradation in U937 cells. Agarose gel electrophoresis of DNA isolated from U937, U937·DMSO. U937r or U937r-DMSO cells, treated for 4 h with the indicated concentrations of TNF.