Bhj Dontje

The mitochondrial genetic system as a target for chemotherapy: Tetracyclines as cytostatics

The mitochondrial genetic system is indispensable for the biosynthesis of the enzyme complexes involved in aerobic energy generation. Tetracyclines inhibit the expression of only the mitochondrial genes because they specifically block mitochondrial protein synthesis. A salient feature is that this inhibition occurs at the low concentration required for anti-bacterial treatment, provided that this concentration is maintained continuously. Evidence is presented that the growth of carcinogen-induced tumors can be inhibited by tetracyclines. It is further shown that the development in the cheek pouch of the Syrian hamster of a transplantable hypernephroma from human origin can be strongly retarded by tetracyclines as well. Therefore, the mitochondrial genetic system has to be reckoned as a target for chemotherapy and tetracyclines as cytostatic agents.

The antitumour effect of doxycycline on a T-cell leukaemia in the rat

Previous studies showed that T-lymphoid cells are permeable to the tetracyclines, whereas B-lymphoid and erythroid cells are not. The tetracyclines impair mitochondrial protein synthesis if they have access to cells. Inhibition of mitochondrial protein synthesis during a number of cell cycles results, as a consequence, in proliferation arrest. The tetracyclines can therefore be considered as cytostatics. In the present study the effect of prolonged treatment with doxycycline on the growth of a T-cell type leukaemia of the rat was investigated. It is shown that doxycycline treatment inhibits not only tumour cell proliferation, but leads moreover to complete tumour eradication. The way by which the latter is achieved depends on the doxycycline concentration and, surprisingly, on the stage of tumour progression at which doxycycline administration is started. As, because of the permeability barrier, the proliferation of erythroid and B-lymphoid cells is not affected by the tetracyclines, the tetracyclines may provide a tool without serious side-effects in the therapy of T-type tumours.

Induction of human papilloma virus E6/E7-specific cytotoxic T-lymphocyte activity in immune-tolerant, E6/E7-transgenic mice

Despite promising preclinical results of various therapeutic anticancer immunization strategies, these approaches may not be effective enough to eradicate tumors in cancer patients. While most animal models are based on fast-growing transplantable tumors, malignancies in, for example, cervical cancer patients in general develop much more slowly, which may lead to immune suppression and/or immune tolerance. As a consequence, the immunomodulating signal of any therapeutic immunization regimen should be sufficiently potent to overcome this immunocompromised condition. In previous studies, we demonstrated that an experimental vaccine against human papillomavirus (HPV)-induced cervical cancer, based on Semliki Forest virus (SFV), induces robust HPV-specific cellular immune responses in mice. Now we studied whether this strategy is potent enough to also prime a cellular immune response in immune-tolerant HPV transgenic mice, in which CTL activity cannot be induced using protein or DNA vaccines. We demonstrate that, depending on the route of immunization, SFV-expressing HPV16 E6 and E7 indeed has the capacity to induce HPV16 E7-specific cytotoxic T cells in HPV-transgenic mice.

TUMORICIDAL RESPONSE OF LIVER MACROPHAGES ISOLATED FROM RATS BEARING LIVER METASTASES OF COLON ADENOCARCINOMA

Iniraportal Inoculation of CC531 adenocarcinoma cells into syngeneic rats causes an increase of liver macrophage cell number but not of major histocompati bility complex class II antigen expression. On day 1 after inoculation of 105 CC531 cells, a fixed number of isolated liver macrophages lysed significantly more target cells in vitro than did control cells. This effect was still present after 4 weeks, A 10‐fold higher initial tumor dose sig nificantly suppressed the macrophage response during the first 2 weeks. In contrast to tumoricidal activity induced by lipopolysaccharide in vitro, the tumoricidal response following in vivo challenge with tumor cells appeared not closcly related to the production of reactive nitrogen in termediates, as in the latter case it was not abrogated in the presence of nitric oxide synthase inhibitor. Further more, the liver macrophage population appeared not fully activated after tumor inoculation as lipopolysaccharide further increased tumoricidal activity in vitro. The ob served numerical and functional response of liver macro phages to intraportally inoculated tumor cells points at an important role of these cells in aspecific immune reac tivity aimed at the reduction of local tumor growth. Re sults suggest that mechanistic differences exist between macrophage tumoricidal activity induced by tumor cells as compared with lipopolysaccharide. J. Leukoc. Biol. 57: 617–623; 1995.

A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer

Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical cancer or premalignant cervical disease, as induced by high-risk type human papillomaviruses (HPVs). We previously demonstrated that immunization of mice with recombinant replication-defective Semliki Forest virus (rSFV) encoding a fusion protein of HPV16 E6 and -E7 (SFV-eE6,7) induces strong cytotoxic T-lymphocyte (CTL) activity and eradication of established HPV-transformed tumours. In this study, we compared the antitumour efficacy of SFV-eE6,7 with that of a recombinant adenovirus (rAd) type 5 vector, expressing the same antigen construct (Ad-eE6,7). Prime-boosting with SFV-eE6,7 resulted in higher precursor CTL frequencies and CTL activity compared to prime-boosting with Ad-eE6,7 and also in murine tumour treatment experiments SFV-eE6,7 was more effective than Ad-eE6,7. To elicit a therapeutic effect with Ad-eE6,7, 100/1000-fold higher doses were needed compared to SFV-eE6,7. In vivo T-cell depletion experiments demonstrated that these differences could not be explained by the induction of a different type of effector cells, since CD8+ T cells were the main effector cells involved in the protection against tumour growth in both rSFV- and rAd-immunized mice. Also comparable amounts of in vivo transgene expression were found upon immunization with rSFV and rAd encoding the reportor gene luciferase. However, anti-vector responses induced by a single injection with rAd resulted in a more than 3-log decrease in luciferase expression after a second injection of rAd. With rSFV, transgene expression was inhibited by only one to two orders of magnitude in preinjected mice. As an antigen-specific booster immunization strongly increases the level of the CTL response and is essential for efficient induction of immunological memory, it is likely that (part of) the difference in efficacy between rSFV and rAd type 5 can be ascribed to a diminished efficacy of the booster immunization in the case of rAd due to anti-vector antibody responses.

THE EFFECT OF DOXYCYCLINE ON POLYVINYLPYRROLIDONE-INDUCED GRANULOMA-FORMATION IN THE RAT-LIVER

SummaryThe tetracyclines specifically inhibit mitochondrial protein synthesis when present at the same low concentrations as used for their antibacterial action. Inhibition of mitochondrial protein synthesis leads to decrease in the oxidative energy-generating capacity of cells. Therefore, the presence of tetracyclines may result in proliferation arrest.In the present study we show that continuous intravenous administration of polyvinylpyrrolidone (PVP) induces the formation of granulomas in the normal rat liver; the rats usually die within 2 weeks of continuous PVP treatment. Athymic (nude) rats appear to be more résistent to the deleterious effects of PVP as they survivce the treatment for at least 5 weeks. Although the livers of the PVP-treated nude rats are heavily infiltrated with phagocytic cells, they seldom show granulomas. Reconstitution of nude rats with syngenic thymocytes leads, on the other hand, to extensive granuloma formation. Normal rats treated continuously with PVP plus doxycycline, however, all survive, their livers showing only a few very small granulomas and the normal low number of phagocytic cells.We conclude that the formation of granulomas induced by PVP is a process which is mediated by T-lymphocytes. Because doxycycline prevents this kind of granuloma formation it seems likely that doxycycline not only impairs the proliferation and differentiation of T-lymphocytes but also of monocytes and macrophages.

ANTITUMOR REACTIVITY INDUCED BY LIPOSOMAL MTP-PE IN A LIVER METASTASIS MODEL OF COLON-CANCER IN THE RAT

The antitumor effects of muramyl tripeptide phosphatidylethanolamine, incorporated within the lipophilic phase of liposomes (lipMTP-PE) were studied using a model of liver metastasis of colon cancer in the rat. Intravenous immunotherapy with lipMTP-PE, when started 2 days before the inoculation of tumor cells and given twice a week, significantly reduced subsequent tumor growth in the liver. The main effect of treatment appeared to be a substantial local increase in the number of tumoricidal macrophages and lymphocytes. Tumor cell lysis by isolated macrophages in vitro, however, appeared not to be elevated above the level triggered by tumor growth alone. Therefore, the observed therapeutic effect of lipMTP-PE probably results from a combination of (1) an increase in the number of cytotoxic macrophages at the onset of metastatic growth in the liver, thus increasing the probability of lethal contacts between tumoricidal effectors and tumor cells and (2) indirect effects of lipMTP-PE, via the induction of cytokine production by liver macrophages, leading to increased numbers and/or activity of cytotoxic lymphocytes and natural killer cells.

Chemo-immunotherapy of liver metastases; the in vitro and in vivo effects of 5-Fluo-rouracil combi¬ned with liposome-encapsulated muramyl dipeptide.

AbstractWhile investigating the effects of 5-fluorouracil (5FU) on the tumoricidal state of rat liver macrophages activated in vitro by means of liposome-encapsulated muramyl dipeptide (MDP), we observed that 5FU in combination with macrophages produced substantially higher extents of cytolytic activity on tumor cells than 5FU alone. 5FU was able to enhance the cytolytic activity of macrophages activated by liposome-encapsulated MDP. This finding indicates that, rather than inhibiting the activation of macrophages by liposomal MDP, 5FU can act as a stimulator of macrophage activation by itself. This is further supported by the observation that a second treatment of macrophages with the drug, 24 h after the first, fails to produce increased macrophage cytotoxicity. Our results also show that 5FU does not unfavorably influence the induction of cytotoxic activity of the macrophages. Rather, combinations of 5FU and liposomal MDP may result in an additive or synergistic tumoricidal effect. The therapeutic effe...