Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Increased Resistance Against Oxidative Stress Is Observed During A Short Period of Renal Reperfusion After A Temporal Ischaemia

Reperfusion of rat kidney submitted to temporal ischaemia induces a decrease in glutathione content. Lipid peroxidation is not detected in kidney homogenates but microsomes obtained after periods of reperfusion longer than 60 minutes show increased malondialdehyde values correlated with high oxygen consumption and superoxide free radical generation. Microsomes obtained from kidneys submitted to 15 or 60 minutes of reperfusion are resistant to NADPH-induced lipid peroxidation but after 120 minutes of reperfusion an increased lipid peroxidative response is observed. Although the mechanism of the protection found in microsomes against the induction of oxidative stress in the first 60 minutes of reperfusion is unknown, it is postulated that this subcellular fraction plays an important role in the oxidative stress observed after longer periods of reperfusion.     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996     

The control of shoot tip necrosis in Pistacia vera L. in vitro

The effect of increased concentrations of calcium (Ca) (3–24 mM) and boron (B) (100–800 M) in the medium was studied on the occurrence of shoot tip necrosis (STN) in cultures of Pistacia vera L. STN was significantly reduced by application of Ca or B, however media with more than 200 M boron had reduced shoot multiplication. Ca (12–24 mM) supplied as calcium chloride reduced STN without any adverse effect on shoot multiplication or elongation, whereas calcium acetate reduced elongation. It is concluded that STN is a physiological mineral disorder associated with Ca and/or B deficiency in the meristematic regions of actively growing shoots. Application of Ca (up to 24 mM) as calcium chloride to the medium was the best treatment for the control of STN. Reduction of humidity or increased aeration in the culture jars did not have any significant effect on the occurrence of STN.Abbreviations MS Murashige and Skoog medium - STN Shoot tip necrosis     

Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells

Abstract: We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte-derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO₂ production. However, addition of either tumor necrosis factor-a (TNF-α) or interferon-γ (IFN-γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC₅₀ values of 39 ng/ml of TNF-α and 9.4 U/ml of IFN-γ), and the effect of TNF-α could be further potentiated (twofold) by the presence of interleukin-1β. The simultaneous presence of TNF-α and IFN-γ yielded a greater response than either cytokine alone; however, the respective EC₅₀ values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca²⁺-independent conversion of l -arginine to l - citrulline, with an apparent K _m of 51.2 n M , and this activity could be blocked by l -arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Effect of interferon γ and tumour necrosis factor α on sensitivity to cisplatin in ovarian carcinoma cell lines

This study aimed to investigate whether the biological response modifiers (BRM) interferon (IFN) and tumour necrosis factor (TNF) could enhance the cytotoxic action of cisplatin on ovarian tumour cells in vitro. The sensitivity of four cell lines (OAW42, GG, JAM and PE01) to drugs and drug combinations was tested by a radiolabelled-thymidine incorporation assay. Cell lines demonstrated a range of sensitivity to cisplatin and the innate cytotoxic effect of each of the BRM. When IFN was used in combination with cisplatin, a significant enhancement of cisplatin toxicity occurred in three of four cell lines. TNF demonstrated such an effect in two cell lines but diminished the toxicity of cisplatin in one cell line. A purely additive effect of the agents may explain the enhanced toxicity of cisplatin in some of these cases. However, in one cell line at least (PEO1), both TNF and IFN demonstrated a clearly synergistic effect with cisplatin. These BRM used in conjunction with cisplatin may provide better antitumour regimen than cisplatin alone in some patients with ovarian cancer, but the response is likely to be heterogeneous between patients.