Interleukin-1α gene expression and localization of interleukin-1α protein during tumor promotion

Treatment of the dorsal epidermis of SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and dose-dependent stimulation of interleukin-1α (IL-1α) gene expression. Levels of IL-1α mRNA were elevated as early as 15 min and peaked at 3–4 h after a single application of TPA (2 μg or 10 μg). IL-1α gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent twice-weekly application of 2 μg TPA. IL-1α-immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA-TPA for 1–22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL-1α-immunoreactive protein. DMBA treatment alone did not induce IL-1α gene expression. Injection of IL-1α-specific antibodies (50 μg) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 μg or 10 μg) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti—IL-1α antibodies inhibited incorporation of [methyl-³H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL-1α is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL-1α primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL-1α may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.     

Bioactive tumour necrosis factor alpha but not granulocyte- macrophage colony-stimulating factor correlates inversely with Langerhans' cell numbers in skin tumours

Langerhans' cells (LCs) are thought to play an important role in presentation of tumour antigens for the induction of anti-tumour immunity. Epidermis overlying some transplanted murine skin tumours contains increased numbers of LCs; however, alterations in LC numbers are not related to tumour antigenicity or host immunity, suggesting that another factor(s), such as tumour-produced cytokines, influences LC density. It has been postulated that dendritic epidermal T cells (DETCs) play a role in immunosurveillance within the normal epidermis. Two cytokines which potentially alter LC numbers or function include granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α). GM-CSF maintains LC viability in culture, and there are reports that it can increase LC density. There is evidence that TNF-α induces LC to migrate from the epidermis. In the present study, LC densities in regressor and non-regressor murine skin tumours and overlying epidermis were enumerated, and bioactive GM-CSF and TNF-α present in the tumours were measured. We found significantly increased epidermal LC numbers above non-regressor, but not regressor, tumours. DETC numbers were significantly increased above some tumours. Although all tumour types produced TNF-α, the regressors, which did not increase LC numbers, produced the most TNF-α. In contrast, tumour production of GM-CSF did not correlate with any pattern of alteration of LC density or tumour growth. Tumour production of neither cytokine nor tumour growth correlated with DETC numbers overlying tumours. Our results suggest that TNF-α may be associated with skin tumour regression and may prevent LC accumulation by tumours. Int. J. Cancer 75:210–216, 1998. © 1998 Wiley-Liss, Inc.     

Effects of chronic low-dose ultraviolet B radiation on DNA damage and repair in mouse skin.

Chronic exposure to sunlight causes skin cancer in humans, yet little is known about how habitual exposure to low doses of ultraviolet B radiation (UVB) affects DNA damage in the skin. We treated Skh-1 hairless mice with daily doses of suberythemal UVB for 40 days and analyzed the amount and distribution of DNA photodamage using RIAs and immunofluorescence micrography. We found that DNA damage accumulated in mouse skin as a result of chronic irradiation and that this damage persisted in the dermis and epidermis for several weeks after the chronic treatment was terminated. Although the persistent damage was evenly distributed throughout the dermis, it remained in the epidermis as a small number of heavily damaged cells at the dermal-epidermal boundary. Rates of DNA damage induction and repair were determined at different times over the course of chronic treatment in response to a higher challenge dose of UVB light. The amount of damage induced by the challenge dose increased in response to chronic exposure, and excision repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone dimers was significantly reduced. The sensitization of mouse epidermal DNA to photoproduct induction, the reduction in excision repair, and the accumulation of nonrepairable DNA damage in the dermis and epidermis suggest that chronic low-dose exposure to sunlight may significantly enhance the predisposition of mammalian skin to sunlight-induced carcinogenesis.     

Time course for early adaptive responses to ultraviolet B light in the epidermis of SKH-1 mice.

Hairless SKH-1 mice were exposed once to UVB light (180 mJ/cm2), and mechanistically important early adaptive responses in the epidermis were evaluated by immunohistochemical and morphological methods. Interrelationships in the time course for these UVB-induced responses were examined. The number of epidermal cells with DNA strand breaks (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells) or with thymine dimers increased to maximal levels within 30 min after UVB. The number of cells with DNA strand breaks located specifically in the basal layer of the epidermis was increased substantially by 3-30 min after UVB and gradually increased further over the next 5.5 hours. DNA strand breaks specifically in the basal layer of the epidermis were increased maximally at 6 h after UVB. The number of epidermal cells with DNA strand breaks or thymine dimers decreased markedly between 12 and 36 h. Pyrimidine (6-4) pyrimidone photodimers (6-4 photoproducts) in isolated epidermal DNA were increased immediately after irradiation of the mice with UVB and decreased markedly during the next 6 h. Exposure to UVB caused a rapid 8-fold increase in the number of epidermal cells with the DNA mismatch repair protein, MSH2 (within 30-60 min), and the level of MSH2-positive cells remained elevated for at least 48 h. These observations suggest a possible role of MSH2 in the repair of UVB-induced DNA damage. The number of epidermal cells with wild-type p53 protein started to increase at 1 h after UVB exposure and reached maximal levels by 8-12 h. The number of p53-positive cells fell markedly between 24 and 48 h. The time course for UVB-induced increases in the number of p53-positive cells was paralleled very closely by the time course for UVB-induced increases in the number of cells with p21(WAF1/CIP1), increases in morphologically distinct apoptotic sunburn cells, and decreases in the number of epidermal cells with bromodeoxyuridine (BrdUrd) incorporation into DNA. Although the start of UVB-induced increases in the number of p21(WAF1/CIP1)-positive cells was similar to that for the increase in p53-positive cells and very high levels of p21(WAF1/CIP1)-positive cells were observed at 8-12 h, maximal increases in p21(WAF1/CIP1)-positive cells were not achieved until 24 h after UVB irradiation (approximately 12 h after the peak value for p53). Myeloperoxidase-positive epidermal cells started to increase by 30 min after UVB exposure, and maximal numbers of myeloperoxidase-positive epidermal cells were observed at 2 h after UVB (18-fold higher than in nonirradiated control mice). An increased level of epidermal peroxidase enzyme activity in the epidermis was also observed from 1 to 24 h after exposure of the mice to UVB. Although neutrophil infiltration into the epidermis was not seen after exposure to UVB, neutrophil infiltration into the dermis (inflammatory response) was observed from 4 to 144 h after UVB exposure. In contrast to the marked inhibitory effect of UVB on BrdUrd incorporation into the DNA of epidermal cells observed at 8-12 h after UVB irradiation (>90% inhibition), BrdUrd incorporation into the DNA of epidermal cells was markedly increased (approximately 30-fold increase in the number of BrdUrd-positive cells) at 48 h after UVB exposure, and increases in epidermal cell layers and epidermal thickness (hyperplasia) were also observed. These later effects were associated with regeneration of the damaged epidermis.     

Biodistribution and tumor localization of stealth liposomal tumor necrosis factor-α in soft tissue sarcoma bearing rats

The blood residence half-life and organ distribution of recombinant human tumor necrosis factor-α (TNF-α) encapsulated in sterically stabilized liposomes, were investigated in rats bearing a soft tissue sarcoma in the hind leg. We studied the decay in blood concentration of “empty” liposomes using the aqueous marker ⁶⁷gallium-desferal, as well as the blood concentration of soluble TNF-α and liposome encapsulated TNF-α using ¹²⁵I. Encapsulation efficacy of TNF-α was 24%. The pharmacokinetics of TNF-α were markedly altered after encapsulation in liposomes, with a 33-fold increase in mean residence time of TNF-α in the blood, and a concomitant 14-fold increase in the area under the plasma concentration vs. time curve for liposomal TNF-α. Although the liposomes exhibit Stealth characteristics, uptake by mononuclear phagocyte-rich organs (e.g., liver and spleen) was noticeable, especially at later time points. Encapsulation of TNF-α in sterically stabilized liposomes resulted in a marked increase in localization of the cytokine in tumor measured as total uptake over time. However, peak TNF-α concentration levels in tumor were not significantly enhanced compared with free TNF-α. Besides the augmented localization of TNF-α after encapsulation in sterically stabilized liposomes, a diminished toxicity was observed. Int. J. Cancer 77:901–906, 1998.© 1998 Wiley-Liss, Inc.     

Increased tumorigenicity of human keratinocytes harboring human papillomavirus type 16 is associated with resistance to endogenous tumor necrosis factor-α-mediated growth limitation

The aim of this study was to evaluate the relationship between tumorigenicity of cell sublines derived from weakly tumorigenic SKv-e and SKv-I keratinocytes harboring human papillomavirus type 16 (HPV 16) and their susceptibility to autocrine growth limitation mediated by tumor necrosis factor-α (TNF-α). These sublines displayed different in vitro proliferative potential which correlated with tumorigenicity in nu/nu mice. Recombinant TNF-α inhibited in vitro growth of weakly tumorigenic parental SKv cell lines while it did not affect proliferation of their respective highly tumorigenic sublines. Resistance to TNF-α correlated with both increased in vitro proliferation and tumorigenicity. Anti-TNF-α antibodies (Ab) significantly increased in vitro proliferation of weakly tumorigenic parental SKv cells up to the levels of their highly tumorigenic sublines. Growth of highly tumorigenic SKv cells was not affected. On the other hand, proliferation of SKv cells was affected neither by transforming growth factor-β (TGF-β) nor by anti-TGF-β Ab. All SKv cell sublines tested spontaneously released TNF-α, as evaluated by a specific radioimmunoassay; however, the levels of the endogenous cytokine were not related to their proliferative potential and tumorigenicity. An increased resistance to the anti-proliferative effect of TNF-α may be associated with decreased expression of TNF-α receptors (TNF-αR) inasmuch as evaluation of ¹²⁵ I-TNF-α binding and Northern-blot analysis of TNF-αR-specific mRNA showed that highly tumorigenic SKv cell sublines expressed significantly lower numbers of TNF-αR than their respective parental cells. These results show that an increased tumorigenicity of HPV 16-harboring SKv keratinocytes may be, at least partially, due to escape from autocrine TNF-α-mediated growth limitation.     

Disaccharide Esters Screened for Inhibition of Tumor Necrosis Factor-α Release Are New Anti-cancer Agents

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine playing a part in various pathological states. Non-toxic inhibitors of TNF-α release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF-α release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF-α release with synthetic disaccharide esters, such as 6,6′-di-0-alkanoyl-α,α-trehaloses (6,6′-diester-trehaloses), 4,4′-di-0-alkanoyl-α,α-trehaloses (4,4′-diester-trehaloses) and 6,6′-diamino-6,6′-dideoxy-N, N′-dial-kanoyl-α,α-trehaloses (6,6′-diamide-trehaloses) bearing fatty acids of various chain lengths, and n- dodecyl-β-D-maltoside as a disaccharide monoester. 6,6′-Diester-trehaloses and 4,4′-diester-treha-loses of C₈ to C₁₄ fatty acids, 6,6′-diamide-trehaloses of C₈ to C₁₄ fatty acids, and n-dodecyl-β-D-maltoside all inhibited TNF-α release in a dose-dependent manner. The IC₅₀ values are 7.4-14.8 μM for 6,6′-diester-trehaloses (C₈ to C₁₂), 14.6-21.6 μM 4,4′-diester-trehaloses (C₈ to C₁₂), 2.9-15.0 μM for 6,6′-diamide-trehaloses (C₈ to C₁₄) and 23 μM for dodecyl-β-D-maltoside. Both 6,6′-di-O-octanoyl-α,α-trehalose (C₈, designated as SS555) and n-dodecyl-β-D-maltoside (C₁₂) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor-bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n-dodecyl-β-D-maltoside. Moreover, SS555 inhibited TNF-α gene expression mediated through inhibition of AP-1 activation, but not NF-αB activation. This paper reports that diester-trehaloses of C₈ to C₁₂ fatty acids and mimics of disaccharide monoesters such as n-dodecyl-β-D-maltoside appear to be potential cancer-preventive agents of a new type.     

Febrile-range hyperthermia augments reversible TNF-α-induced hyperpermeability in human microvascular lung endothelial cells

Fever commonly occurs in acute lung injury (ALI) and ALI occurs in 25% of victims of heat stroke. We have shown in mouse models of ALI that exposure to febrile-range hyperthermia (FRH), 39.5°C, increases non-cardiogenic pulmonary oedema. In this study we studied the direct effects of FRH on endothelial barrier integrity using human microvascular endothelial cells (HMVEC-Ls). We analysed the effect of exposure to culture temperatures between 38.5° and 41°C with and without tumour necrosis factor-α (TNF-α) up to 250?U/mL for 6–24?h. We found that exposure to 2.5–250?U/mL TNF-α increased HMVEC-L permeability by 4.1–15.8-fold at 37°C. Exposure to 39.5°C alone caused variable, modest, lot-specific increases in HMVEC-L permeability, however raising culture temperature to 39.5°C in the presence of TNF-α increased permeability an additional 1.6–4.5-fold compared with cells incubated with the same TNF-α concentration at 37°C. Permeability occurred without measurable cytotoxicity and was reversible upon removal of TNF-α and reduction in temperature to 37°C. Exposure to 39.5°C or TNF-α each stimulated rapid activation of p38 and ERK but the effects were not additive. Treatment with inhibitors of ERK (U0126) or p38 (SB203580) each reduced TNF-α-induced permeability in 39.5°C monolayers to levels in 37°C cells, but did not alter TNF-α-induced permeability in the 37°C cells. These results demonstrate that FRH directly increases paracellular pathway opening through a process that requires ERK and p38 MAPKs. A better understanding of this mechanism may provide new understanding about how fever may contribute to the pathogenesis of ALI and provide new therapeutic targets to improve clinical outcomes.     

Tumor suppressor function of a dominant negative retinoic acid receptor mutant

Mutations in receptors for the vitamin A metabolite retinoic acid (RAR) that repress retinoic acid (RA)-responsive gene expression have been identified and characterized. We previously reported an absence of target gene response to RA in all but one of a series of transformed human epithelial cell lines. To elucidate the mechanisms of this unresponsiveness, we created stable transfectants that expressed an RARα mutant (RARα403) previously shown to have dominant negative activity due to a C-terminal truncation. All clones exhibited repressed RA-responsive gene expression. These cells grew slowly and demonstrated greater growth inhibition by RA. Pretreatment of both control and experimental groups with RA enhanced epidermal growth factor–induced proliferation despite RA-dependent downregulation of epidermal growth factor receptor expression. In addition, clones expressing the mutant RARα were 60% less invasive in an in vitro assay. This reduced invasiveness correlated with decreased gelatinase activity in these cells. We showed for the first time that a dominant negative mutation in RARα can function as a tumor suppressor in transformed epithelial cells. Mol. Carcinog. 22:26–33, 1998. © 1998 Wiley-Liss, Inc.     

Apoptotic and necrotic effects of tumour necrosis factor-alpha potentiated with hyperthermia on L929 and tumour necrosis factor-alpha-resistant L929

Purpose: The cytotoxic effect of the combination treatment of TNF-α and hyperthermia on L929 and TNF-α-resistant L929 (rL929) cells was investigated.

Materials and methods: L929 cells were treated with TNF-α (5 ng/mL), heating at 43°C or the combination of TNF-α and heating. The cells were harvested at different time within the 24-hour period. The viability and the type of cell death of the harvested cells were examined.

Results: When L929 cells were treated with a combination of TNF-α and heating the cells died quickly and apoptosis increased to an overwhelming extent, especially in the group pre-treated with TNF-α for 1 h prior to heating. Although rL929 cells were resistant to TNF-α alone, the cells became sensitive to TNF-α treatment when combined with heating. Similar to the L929 cell, the cells also died rapidly and exhibited apoptosis to a higher extent. Using an Annexin-V-FITC kit and flow cytometer, we found that both necrosis and apoptosis occurred. Agarose gel electrophoresis of DNA extracted from treated cells showed that the DNA fragments were multiples of approximately 200 bp. Furthermore, by studying the kinetics of cell death and apoptosis, we found that the loss of cell membrane integrity preceded the DNA fragmentation in both L929 and rL929 cells.

Conclusion: The results suggested that hyperthermia may enhance the necrotic and apoptotic effects of TNF-α on some tumour cells and overcome the resistance of some tumour cells to TNF-α.     

Therapeutic effects of monoclonal antibody g250, interferons and tumor necrosis factor,in mice with renal-cell carcinoma xenografts

Because renal-cell carcinoma (RCC) is considered relatively resistant to radio-and chemotherapy, RCC patients may benefit from new treatment modalities, e.g. immunotherapy. In vitro and in vivo studies suggest that combinations of cytokines such as interferon γ or interferon a (IFN-γ, IFN-α) and tumor necrosis factor a (TNF-α) may act synergistically. In this study we tested whether a monoclonal antibody (MAb) G250, reactive with a RCC-associated antigen, showed anti-tumor effects in vivoin nude mice with established s.c. human RCC xenografts, and also whether this MAb could enhance the anti-tumor effect of combinations of IFNs and TNF-α. Treatment with combinations of IFN-α/TNF-α or IFN-γ/TNF-α, or with MAb G250 alone, resulted in a significant inhibition of tumor growth. Treatment with MAb G250, in combination with IFN-γ/TNF-α, did not result in an improve anti-tumor effect as compared to that of either treatment alone. In contrast, MAb G250 combined with IFN-α/TNF-α resulted in a significantly enhanced anti-tumor response. In one experiment, 3 out of 10 mice showed complete tumor regression, with no recurrence after 90 days. Large numbers of infiltrating macrophages were found surrounding viable and necrotic tumor tissue after treatment with G250 combined with IFN-α/TNF-α. These results suggest that combination therapy, consisting of IFN-α, TNF-α and MAbs, may have therapeutic value in the treatment of RCC.     

Activation of the insulin‐like growth factor‐1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation

The ultraviolet B (UVB) component of sunlight causes non‐melanoma skin cancers due to the damage it inflicts on genomic DNA. The response of epidermal keratinocytes to sunlight depends on the dose of UVB received and the severity of the damage to the DNA. Mild DNA damage typically induces DNA‐repair pathways and cell survival, while severe DNA damage provokes apoptosis. Primary human keratinocytes grown in serum‐free media respond in a similar manner to UVB irradiation. However, we observed that keratinocytes are exquisitely more susceptible to UVB‐induced apoptosis if the growth medium is depleted of exogenous growth factors. Therefore, an exogenous growth factor could provide protection from UVB‐induced apoptosis. We found that the only growth factor that provided protection from UVB‐induced apoptosis was insulin and that the protective effect elicited by insulin was not due to binding the insulin receptor but, rather, to activation of the insulin‐like growth factor‐1 (IGF‐1) receptor. Additionally, activation of the IGF‐1 receptor in combination with UVB irradiation induced keratinocytes to become post‐mitotic. This survival function of the IGF‐1 receptor in response to UVB irradiation was influenced by activation of phosphatidylinositol‐3 kinase and MAP kinase. Prior to UVB irradiation, insulin or IGF‐1 had little to no effect on cell growth or viability. Therefore, activation of the IGF‐1 receptor in conjunction with UVB irradiation promotes keratinocyte survival at the expense of cell proliferation. Int. J. Cancer 80:431–438, 1999. © 1999 Wiley‐Liss, Inc.     

Azithromycin Reduces Tumor Necrosis Factor-Alpha Production in Lipopolysaccharide-Stimulated THP-1 Monocytic Cells by Modification of Stress Response and p38 MAPK Pathway

Abstract

Macrolide antibiotics are known to have a variety of immunomodulatory effects in addition to antimicrobial activity, but the mechanisms of immunomodulation are still unclear. We investigated in vitro the effect of azithromycin on tumor necrosis factor alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated THP-1 cells, a human monocytic cell line, and compared the results with those for other macrolides, minocycline and ofloxacin. In the presence of LPS, treatment with azithromycin (AZM) resulted in a significant decrease in LPS-induced TNF-α production compared to that with other antimicrobial agents. The results of phosphorylation of three MAPKs, ERK, JNK and p38, indicated that the phospho-p38 level was reduced by AZM. IκB-α, an inhibitor of NFκB, was not disrupted by the antibiotics. LPS-induced TNF-α release from THP-1 cells was inhibited in the presence of KNK437, a potent 70-kDa heat shock protein (HSp-70) inhibitor. Interestingly, the induction of HSp-70 by LPS was attenuated with the concurrent addition of AZM in the cells. AZM was found to restrain TNF-α production by monocytes at least in part by modifying the HSP-70 and p38 related signaling pathways to LPS stimulation.     

UVB-induced (6-4) photoproducts in hairless mouse epidermis studied by quantitative immunohistochemistry

DNA photoproducts in epidermal basal cells caused by in vivoexposure to various wavelengths of ultraviolet B (UVB) lightwere studied by immunohistochemical and microfluorometric methods.Hairless mice were irradiated with UVB doses ranging between6.3 and 100 mJ/cm² at 280, 290, 297, 302 and 313 nm. A doseof 25 mJ/cm² at 297 nm corresponded to a minimal erythema dosein the Hr/Hr mice. Immediately after the exposure, frozen skinsections were stained in situ with affinity-purified antiserumhaving a major specificity against UV-induced (6–4) photoproducts.microfluorometric measurements of nuclear immunofluorescenceintensities were then performed to quantitate the DNA lesionsin epidermal basal cell nuclei. All wavelengths except 313 nmproduced DNA photoproducts, and the induction was both doseand wavelength dependent. The most effective wavelength was290 nm, followed by 297, 280 and 302 nm. The quantitative determinationsindicated variations in the DNA photoproduct content betweennuclei exposed to the same dose and wavelength. This may bedue to shielding by other nuclei or to differences in the sensitivityto UVB irradiation among basal cells, resulting in non-randomdistribution of DNA-damaged cells. Hence, measurable amountsof UVB-induced (6–4) photoproducts are produced hi hairlessmouse epidermis following in vivo exposure with biologicallyrelevant doses of narrow-banded UVB light.     

Potentiation of the anti-tumor effect of actinomycin D by tumor necrosis factor α in mice: Correlation between in vitro and in vivo results

The anti-tumor effects of actinomycin D (Act D) and recombinant human tumor necrosis factor (TNF)-α have been studied on 4 established murine tumor cell lines: MmB16 melanoma, Lewis lung (LL/2) carcinoma, L1 sarcoma and L1210 leukemia. During short-term incubation (24 hr) Act D produced dose-dependent cytostatic/cytotoxic effects against MmB16, LL/2 and L1 tumor cells but did not reduce the viability of these cells even at high concentration (10 μg/ml), below a threshold of 30–60%. However, L1210 leukemic cells were highly susceptible to Act D, and no viable cells were detected in cultures incubated with 1 μg/ml of Act D. TNF-α alone, when used under the same culture conditions, had only a negligible effect on all cell lines tested. However, the combination of this cytokine with Act D produced synergistic cytotoxic effects against MmB16, LL/2 and L1 cells but not against L1210 leukemia cells. In an in vivo model of regional therapy in which tumor-bearing mice were treated with Act D and TNF-α, a correlation with in vitro results was observed. In mice bearing MmB16 melanoma, LL/2 carcinoma and L1 sarcoma, the most potent anti-tumor effects were observed in mice treated with Act D and TNF-α together. This treatment led to a delay of tumor growth and induced complete tumor regression in some cases. On the contrary, TNF-α did not enhance the effect of Act D in mice injected with L1210 leukemia cells. Our results show that TNF-α can potentiate the anti-tumor effects of Act D against tumors weakly susceptible to Act D and may be a useful adjuvant to chemotherapy in the local treatment of neoplasia. © 1996 Wiley-Liss, Inc.