Victor Umansky

Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients

Previous studies with animal tumors showed that bone marrow (BM) is a privileged site where potentially lethal tumor cells are controlled in a dormant state by the immune system. Here, we investigated BM of breast cancer patients with respect to tumor cell content, immune activation status and memory T‐cell content. BM‐derived cells from primary operated breast cancer patients (n = 90) were compared with those from healthy donors (n = 10) and also with cells from respective blood samples. Cytokeratin 19‐positive tumor cells were detected by nested polymerase chain reaction. Three‐color flow cytometry was used to identify numbers and activation state of T cells, natural killer (NK) cells, monocytes/macrophages and subsets by a panel of monoclonal antibodies (mAbs). The proportion of memory T cells among the CD4 and CD8 T cells was much higher in BM of cancer patients than in healthy donors (p < 0.001). The extent of memory T‐cell increase was related to the size of the primary tumor. Patient‐derived BM memory CD8 T cells could be shown to contain specific HLA‐A2/Her‐2/neu369–377 tetramer binding cells. Patients with disseminated tumor cells in their BM had more memory CD4 T cells and more CD56+ CD8+ cells than patients with tumor cell‐negative BM. Only some of the immunological changes seen in BM samples of cancer patients were also detectable in peripheral blood samples. Our hypothesis that BM is a special compartment for immunological memory and tumor dormancy is supported by the above findings. The overall results reveal that BM is a valuable additional compartment for immune diagnosis in pathological conditions and possibly for follow‐up treatment strategies.

Co-stimulatory effect of nitric oxide on endothelial NF-κB implies a physiological self-amplifying mechanism

Here we investigated the effects of the second messenger molecule NO at various concentrations on the activation of transcription factor NF‐κB, IκB‐α kinase (IKK‐α), Jun N‐terminal kinase (JNK) and apoptosis in murine endothelial cells. Low concentrations of NO alone failed to activate NF‐κB, IKK‐α and JNK. When NF‐κB was prestimulated by TNF‐α or phorbol 12‐myristate 13‐acetate, the addition of NO at low concentrations enhanced the activation of NF‐κB. This provides a mechanism for a self‐amplifying signal in the inflammatory response, since the inducible NO synthase in endothelial cells is regulated by NF‐κB. The co‐stimulatory effect of NO on NF‐κB activation was also evident from IKK‐α kinase assays and reporter gene experiments in endothelial cells. High doses of NO impaired the TNF‐α‐induced DNA‐binding activity of NF‐κB. Accordingly, these high amounts of NO also repressed the TNF‐α‐induced transactivation by NF‐κB as efficient as dexamethasone. The doses of NO required for the inhibition of NF‐κB are not cytotoxic for the endothelial cells, enabling the establishment of an autoregulatory loop for NF‐κB signaling.

Nitric oxide-induced apoptosis in tumor cells.

Nitric oxide (NO), an important molecule involved in neurotransmission, vascular homeostasis, immune regulation, and host defense, is generated from a guanido nitrogen of L-arginine by the family of NO synthase enzymes. Large amounts of NO produced for relatively long periods of time (days to weeks) by inducible NO synthase in macrophages and vascular endothelial cells after challenge with lipopolysaccharide or cytokines (such as interferons, tumor necrosis factor-alpha, and interleukin-1), are cytotoxic for various pathogens and tumor cells. This cytotoxic effect against tumor cells was found to be associated with apoptosis (programmed cell death). The mechanism of NO-mediated apoptosis involves accumulation of the tumor suppressor protein p53, damage of different mitochondrial functions, alterations in the expression of members of the Bcl-2 family, activation of the caspase cascade, and DNA fragmentation. Depending on the amount, duration, and the site of NO production, this molecule may not only mediate apoptosis in target cells but also protect cells from apoptosis induced by other apoptotic stimuli. In this review, we will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells and discuss the mechanisms of NO-mediated apoptosis.

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAOhigh and NAOlow, respectively). Sorted NAOhigh cells were found to survive in culture whereas sorted NAOlow cells died. Moreover, NAOhigh cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long‐term culture. These cells showed a similar cardiolipin content and expressed the same level of anti‐apoptotic proteins Bcl‐2 and Bcl‐xL as APO‐S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine‐sulfoximine (BSO) correlated with a significant stimulation of NO‐mediated apoptosis whereas the exposure of NO‐sensitive APO‐S cells to the glutathione precursor N‐acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO‐induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role. J. Cell. Biochem. 78:578–587, 2000.

New insights into tumor-host interactions in lymphoma metastasis

The metastatic process is characterized by a complex series of sequential steps involving constant interactions (mutual “cross-talks”) of metastasized tumor cells with their microenvironment (lymphocyte, macrophages, endothelial cells, etc.) in target organs. These interactions determine the outcome of metastasis (either the eradication of metastatic cells or their increased proliferation and invasion). Recently developed methods of tumor and host cell analysis at the molecular level allow better elucidation of molecular mechanisms of metastasis and of immune mechanisms involved in antitumor responses. Direct modulation of these processes will probably increase the success of clinical cancer treatment. Here we review data (a) on the expression of some costimulatory (MHC class II, CD80, sialoadhesin) and adhesion (LFA1, ICAM-1, VLA-4) molecules on both metastasized tumor cells and host cells and (b) on the production of a cytotoxic molecule, nitric oxide, by in situ activated Kupffer and endothelial cells in the process of liver metastasis. This study was performed with well-characterized murine ESbL T lymphoma cells transduced with the bacterial lacZ gene, which allows detection and quantification of metastases at the single cell level throughout lymphoma growth and metastasis. Experimental results are discussed in the context of recent literature.

Liver endothelial cells: participation in host response to lymphoma metastasis

Interactions between metastasizing tumor cells and host cells in target organs determine the outcome of metastasis. This review discusses the dual role of activated host endothelial cells in the metastatic process. On one hand, the upregulation of the expression of particular adhesion molecules leads to increased tumor cell binding, and the stimulation of angiogenesis provides the vascular support for the growth of already established metastases. On the other hand, endothelial cells can contribute to host anti-metastatic responses, e.g. by production of the cytotoxic molecule nitric oxide (NO) from arginine with the help of the inducible nitric oxide synthase (iNOS). Using a well-characterized ESbL-lacZ mouse T lymphoma model with a typical three phasic growth profile, we showed during the period of growth retardation a stimulation of NO production by ex vivo isolated liver sinusoidal endothelial cells. The induction of NO synthesis in liver endothelial cells did not require the presence of Kupffer cells and appeared to be stimulated by and dependent on mature T lymphocytes. A breakdown of this NO synthesis coincided with the second tumor expansion phase.

Immunotherapy of Metastases

Many aspects of tumor growth and metastasis are dependent on tumor cell-host cell interactions. The capacity of tumor cells to elicit or suppress various host tissue responses seems as important for development of solid tumors as for tumor cell proliferation in different organ microenvironments. The process of tumor angiogenesis, the consequences of which extend far beyond nutrient supply, can serve as an illustrative and important example (Rak et al. 1993). The importance of tumor-host interactions and of signals from the microenvironment for development of cancer metastases was pointed out as early as 1980 (Schirrmacher 1980), but has only recently attracted much attention and been studied at the molecular level (Schirrmacher 1994; Nicolson 1988, 1991; Fidler 1990; Kerbel 1990; Gullino 1991; Heppner 1989). The way in which tumor cells react to signals from the microenvironment is determined by their reception via specific receptors, by the way these signals are transduced, and by the way these transduced signals are translated in the cellular response. Cellular calcium homeostasis is the result and the regulator of many signal transduction pathways and plays a central role of regulation of cell proliferation, invasion, and metastatic potential (Liotta et al. 1991). While the interaction of tumor cells with host endothelial cells and extracellular matrix (Ingber 1991; Ingber and Folkman 1989; Jain 1990) is being discussed by experts contributing to this series of articles, the interaction with cells of the immune system, in particular with antigen-specific immune mechanisms, will be the focus of this review.

Conference overview: Cancer Immunotherapy and Immunomonitoring (CITIM): Moving forward

The immune system is a critical element involved in the control of tumor development and progression. While professionals have learned how to manipulate the immune system to generate tumor-specific immune responses, cancer immunotherapy has not yet delivered substantial clinical benefits. It has become increasingly clear that tumor-induced abnormalities in the immune system not only hamper tumor immunosurveillance, but also limit the efficacy of cancer immunotherapy. Meanwhile, the results of recent studies allow the belief that one is on the edge of a real breakthrough in this promising direction in cancer therapy. The 2nd International Conference ‘Cancer Immunotherapy and Immunomonitoring (CITIM)’ was the second meeting in Eastern Europe to specifically focus on the issue of immune regulation in the tumor environment, cancer immunotherapy, and immunomonitoring of immunotherapeutic clinical trials. This CITIM Conference held in Budapest, Hungary, was comprised from 12 plenary sessions, Best Abstract Award session, Poster session, and four Keynote lectures. Outstanding presentations and numerous productive discussions summarized the current place of the field and opened new directions for improving monitoring and therapy for patients with cancer.

Cancer Immunotherapy and Immunomonitoring (CITIM): Redefining Cancer Therapy

The immune system is a critical element involved in the control of tumor development and progression. While we have learned how to manipulate the immune system to generate tumor-specific immune responses, cancer immunotherapy has not yet delivered substantial clinical benefits. It has become increasingly clear that tumor-induced abnormalities in the immune system not only hamper natural tumor immune surveillance, but also limit the effect of cancer immunotherapy. If the results of recent studies are of any indication, then we are on the verge of a real breakthrough in our understanding of the immunobiology of tumor-host interactions and of ways to manipulate it. This 1st International Conference on “Cancer Immunotherapy and Immunomonitoring (CITIM)” was the first meeting in Eastern Europe to specifically focus on the issue of immune regulation in the tumor environment, cancer immunotherapy, and immunomonitoring of immunotherapeutic clinical trials. This CITIM Conference held in Kiev, Ukraine, was comprised from eight plenary sessions and two special selected poster presentation sessions. Selected contributions from the participants of the Conference are presented in this issue of the Journal of Immunotoxicology.

Dissection of tumor and host cells from metastasized organs for testing gene expression directly ex vivo.

The interaction between tumor and host cells determines to a large extent the outcome, namely tumor growth and progression toward metastases or tumor arrest, dormancy, or rejection. Most of the studies published so far on interactions of tumor cells and host cells were made in vitro and dealt with aspects such as cell adhesion, proliferation, invasiveness, cytotoxicity, or cytokine production. As the microenvironment in tissue culture differs in many respects from that in vivo, new approaches for in vivo studies of tumor-host cell interactions is of utmost importance in cancer research. To elucidate the metastatic phenotype, approaches have been made to relate, for instance, cell surface molecules expressed on the tumor cell lines from tissue culture to their propensity to generate metastases in vivo (1). Several authors have reported that certain steps of the metastatic cascade are rate limiting (2-6). To produce metastases, tumor cells must complete each of the sequential steps in the pathogenesis of cancer metastasis. Each discrete step appears to depend on the interaction between tumor cells and multiple host factors (i.e., the microenvironment of the tumor) and to be regulated by transient or permanent changes in multiple genes at the level of DNA, RNA, or protein. On this background, the need for comprehensive in vivo/ex vivo studies on tumor-host interactions and their kinetics in relevant model systems becomes obvious.