The present study demonstrates that treatment of human myeloid cell lines with polar, low molecular weight agentswith differentiation-inducing capacity results in augmented cellular sensitivity to TNF cytostasis/cytotoxicity. The potentiating effect of these agents was found at doses which did not result in inhibition of proliferation but which did not induce any cell death, even upon prolonged incubation. Of several compounds tested, DMSO seemed to be the most potent in enhancing TNF action. DMSO-induced potentiation occurred in those cells that are forced by DMSO to mature towards the monocytic (the myeloblast KG1, the myelomonocytes U937 and THP1, and the monocyte MonoMac6^{27, 49} or the granulocytic phenotype (promyelocyte HL60. ²⁴ In contrast, cells which are described to be unresponsive to DMSO-induced differentiation (erythroleukemia K562²⁴ could not be sensitized to TNF by DMSO treatment.

Several investigators have shown that DMSO can act as a protective agent against TNF cytotoxicity on cell types of diverse origin, including fibrosarcomas and myosarcomas. ^50-52 We also found that DMSO partially protected some fibrosarcoma and carcinoma cells from TNF cytotoxicity. As far as we know, DMSO has not been tested before on cell lines that are highly susceptible to the differentiation-inducing capacity of DMSO, e.g., leukemic cells. This could explain the opposite effect of DMSO on TNF cytotoxicity that we observed in these cells (i.e., potentiation I instead of protection). This effect of DMSO might be related to the mechanism of TNF-induced cell killing. In sarcoma cell lines, TNF induces mainly necrotic cell death, whereas leukemic cell lines are mainly killed by an apoptotic mechanism. ⁵³ The necrotic form of cell death is characterized by lysis of the plasma membrane. In contrast, apoptotic cell death is a process that involves plasma membrane blebbing and internucleosomal genomic DNA fragmentation by nuclear endonucleases, resulting in DNA multimers of about 180 to 200 basepairs. ⁵⁴ However, we gathered evidence that the opposite effects of DMSO on TNF cytostasis/cytolysis in different cells are not related to the way TNF kills these cells.

A trivial explanation of our finding of DMSO-induced potentiation of TNF action could be that DMSO induces a state of inhibition of protein or RNA synthesis, which could then result in enhanced TNF sensitivity as induced by treatment with, for example, actinomycin D or cyclohesimide. ¹¹ Such an explanation would also be suggested by the observation that DMSO treatment resulted in cell growth inhibition. However, we found that DMSO-pretreated U937 cells are still able to be induced by lipopolysaccharide to high production of IL-16,⁵⁵ suggesting that DMSO-induced TNF potentiation cannot simply be explained by a mechanism similar to that described for potentiation induced by protein or RNA synthesis inhibitors.

TNF is believed to initiate its biological activities mainly through specific binding to cell surface TNF receptors. ^28-29 A recent report, however, describes the ability of TNF to insert directly into the hydrocarbon core of phospholipid bilayers without interaction with TNF receptors. ³⁰ Acidification, which resulted in increased TNF insertion, was found by the same authors to increase killing ol' U937 by TNF. They presented evidence that TNF has ion-channel forming capacity in planar Iipid bilayer membranes. ³¹ This was further substantiated by their observation that TNF altered the Na⁺ permeability of U937 cell membranes. As published data suggest that DMSO can stabilize cellular membranes and decrease membrane fluidity,^32-33 a possible explanation for our observation of DMSO-induced potentiation of TNF action could be that DMSO induced changes in membrane characteristics, leading to, for example, a facilitated TNF insertion and enhanced non-receptor-mediated cytotoxicity. Our findings, however, do not favor the latter possibility. First, non-specific binding of [¹²⁵I]TNF was found to be unaltered upon DMSO treatment, indicating that TNF did not become increasingly intercalated at random into the membrane. Moreover, agonistic antibodies to the p55 TNF receptor could mimic the effect of TNF in DMSO-pretreated cells. In addition, TNF mutants with gradually lowered biological activity on the p55 TNF receptor showed a concomitant decrease in bioactivity on DMSO-pretreated U937 cells. The amino acid changes that had been introduced into these TNF mutants did not alter the gross physico-chemical characteristics of the TNF molecule. ³⁵ Moreover, these changes are all located at the outside of the TNF molecule, so that they most probably do not affect the putative ion-channel formed between the subunits of the TNF trimer. Taken together, our data suggest that the action of TNF in DMSO-pretreated cells is largely, presumably exclusively, mediated by the bona fide p55 TNF receptor and not by intercalation of TNF in the cellular membranes. Moreover, DMSO was found even to reduce TNF receptor expression, despite its potentiating effect on TNF cytotoxicity. At present, the relevance of reduced TNF receptor expression, if any, in enhanced TNF sensitivity is unclear. One could speculate that DMSO might potentiate TNF receptor mobility and/or receptor cross-linking by TNF. As cross-linking of TNF receptor by TNF is crucial for TNF activity,^35-36 this phenomenon would result in enhanced signalling by the remaining TNF receptors. However, published data do not support the latter possibility. For example, concanavalin A receptors become immobilized within 36 h during DMSO-induced differentiation of neuroblastoma cells. ⁵⁷

At present, the mechanism of potentiation of TNF action by DMSO is unclear. As this effect seems to be restricted to myelold cell lines, which differentiate with DMSO towards the monocytic or granulocytic phenotype, and as this action of DMSO can be mimicked by other differentiation-inducing agents, it is reasonable to suggest that the TNF-enhancing action of DMSO is related in one way or another to its differentiation-inducing capacity. As in the case of DMSO, TNF has been shown to possess differentiation-inducing potency as well,¹² but it is yet unknown whether TNF and DMSO have a similar mechanism of action in this respect. Several hypotheses have been proposed to explain the mechanism of DMSO-induced cellular differentiation; for example, an alteration of cellular membranes. ³² In this respect, Lyman and co-workers³² measured the temperature of the lipid phase transition from the gel to the liquid-crystalline state in artificial phospholipid membranes, and found that DMSO induced the appearance of a new transition at higher temperatures, implying a stabilization of the membranes and a decrease in membrane fluidity. Concentrations of DMSO needed to induce these altered membrane characteristics correlate well with those needed to induce differentiation. ^{32, 33} However, the sensitization to subsequent TNF-induced cytotoxicity is most probably not directly related to an induction of physico-chemical changes in the membrane bilayer by DMSO. First, there is a need for a prolonged preincubation period with DMSO to augment the cells' susceptibility to TNF. Second, membrane constituents of the different myeloid cell lines tested are expected to be largely similar. Nevertheless, some of these are sensitive to the TNF-potentiating effect of DMSO (U937, THP1, MonoMac6), whereas others (K562) are not. Third, only small differences between U937 and U937·DMSO cells were observed concerning TNF internalization and degradation, suggesting no gross changes in membrane constituents.

An alternative mechanism of DMSO-induced cellular differentiation has been proposed by Tanaka et al.,⁵⁸ who suggest that the freely diffusible DMSO acts on the nucleus by changing the conformation of DNA or DNA-protein complexes, resulting in enhanced transcription of genes regulating differentiation. Indeed, during differentiation, the pattern of cellular gene expression is largely altered, resulting in the induction of some genes (e.g. fos, jun) and the silencing of otliers (e.g. myc, myb). Concomitantly, DMSO could selectively alter the expression of genes regulating the TNF susceptibility of the cells, for example, by partial or total reduction of the expression of TNF protective proteins. This agrees with the requirement for prolonged preincubation periods with DMSO. Alternatively, it is possible that the exposure of the genomic DNA to the cellular transcription machinery also exposes the DNA to cellular endonucleases, rendering it sensitive to degradation. We found that a 24-h treatment with DMSO alone, which results in strong TNF-potentiating effects, did not induce fragmentation of genomic DNA. In addition, Gunji and co-workers⁵⁹ found that DNA fragmentation during DMSO-induced monocytic differentiation of U937 cells only became detectable at later time points (from 72 h on). In contrast to DMSO, a 4-h treatment with TNF alone induced some apotototic DNA degradation, an effect which was found to be drastically enhanced upon DMSO pretreatment. It is at present unclear whether TNF-induced DNA degradation is due to activation of otherwise inactive cellular endonucleases, or to the fact that protected genomic DNA becomes "exposed" to a pre-existing endonuclease by modulation of gene expression by TNF.

The question remains whether other, e.g., non-polar differentiation inducers such as phorbol esters, retinoic acid, or 1,25(dihydroxy)vitamin D₃ can potentiate cells to the cytotoxic effects of TNF. This has not been described in the literature or observed by ourselves. Phorbol esters were shown by Unglaub et al. ⁶⁰ to down-regulate TNF sensitivity in several myeloid cell lines via a down-modulation of TNF receptor expression. A second differentiation inducer, 1,25(dihydroxy)vitamin D₃, similarly induced resistance to TNF-mediated killing in myeloid cells although the underlying mechanism is not completely resolved as yet. ⁶¹ Retinoic acid was found to modulate p75 TNF receptor expression on HL60 cells, but not to modulate cytotoxicity. ⁶²1621. We observed similar effects on TNF cytotoxicity by phorbol esters and retinoic acid on our myelold cells (unpublished data). Thus, the induction of differentiation does not seem to constitute the only prerequisite to potentiation of myelold cell lines. Other, and possibly multiple, effects seem to be required which depend on rnore individual chemical characteristics of the differentiation inducer. In particular, DMSO has been involved in affecting the oxidative metabolism of cells. ⁶³ We have accumulated evidence that points in this direction (manuscript in preparation).

Several polar molecules are being used as differentiating agents in clinical trials for the treatment of acute myelold leukemia. ^{64, 65} Here, we show that DMSO strongly potentiates TNF-induced cytolysis in myclold cells. Possibly, a combinatorial in vivo administration of physiologically acceptable doses of DMSO-related differentiation agents together with TNF might contribute to a more successful treatment of acute myelold leukemias.

We thank Drs. A. Raeymackers, X. Van Ostade, and M. Brockhaus for generously providing cytokines and mAbs, Dr. J. Plum for help in flow cytometry analyses, and P. Hellin and J. Lievens for their artistic contributions. B.V, is a Senior Research Assistant with the NFWO.

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