Molecular pathways of senescence regulate placental structure and function.

The placenta is an autonomous organ that maintains fetal growth and development. Its multinucleated syncytiotrophoblast layer, providing fetal nourishment during gestation, exhibits characteristics of cellular senescence. We show that in human placentas from pregnancies with intrauterine growth restriction, these characteristics are decreased. To elucidate the functions of pathways regulating senescence in syncytiotrophoblast, we used dynamic contrast-enhanced MRI in mice with attenuated senescence programs. This approach revealed an altered dynamics in placentas of p53-/- , Cdkn2a-/- , and Cdkn2a-/- ;p53-/- mice, accompanied by histopathological changes in placental labyrinths. Human primary syncytiotrophoblast upregulated senescence markers and molecular pathways associated with cell-cycle inhibition and senescence-associated secretory phenotype. The pathways and components of the secretory phenotype were compromised in mouse placentas with attenuated senescence and in human placentas from pregnancies with intrauterine growth restriction. We propose that molecular mediators of senescence regulate placental structure and function, through both cell-autonomous and non-autonomous mechanisms.

Senescent cells communicate via intercellular protein transfer.

Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell-cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell-cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.

p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling.

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age-related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage-induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)-κB kinase, leading to decreased cell survival. NF-κB activation induced TNF-α secretion and JNK activation to mediate death of senescent cells in a caspase- and JNK-dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.

mRNA-transfected Dendritic Cells Expressing Polypeptides That Link MHC-I Presentation to Constitutive TLR4 Activation Confer Tumor Immunity.

Recently, we have developed a novel genetic platform for improving dendritic cell (DC) induction of peptide-specific CD8 T cells, based on membrane-anchored ß2-microglobulin (ß2m) linked to a selected antigenic peptide at its N-terminus and to the cytosolic domain of toll-like receptor (TLR)4 C-terminally. In vitro transcribed mRNA transfection of antigen presenting cells resulted in polypeptides that efficiently coupled peptide presentation to cellular activation. In the present study, we evaluated the immunogenicity of such constructs in mRNA-transfected immature murine bone marrow-derived DCs. We show that the encoded peptide ß2m-TLR4 products were expressed at the cell surface up to 72 hours and stimulated the maturation of DCs. In vivo, these DCs prompted efficient peptide-specific T-cell activation and target cell killing which were superior to those induced by peptide-loaded, LPS-stimulated DCs. This superiority was also evident in the ability to protect mice from tumor progression following the administration of B16F10.9 melanoma cells and to inhibit the development of pre-established B16F10.9 tumors. Our results provide evidence that the products of two recombinant genes encoding for peptide-hß2m-TLR4 and peptide-hß2m-K(b) expressed from exogenous mRNA can cooperate to couple Major Histocompatibility Complex (MHC-I) peptide presentation to TLR-mediated signaling, offering a safe, economical and highly versatile genetic platform for a novel category of CTL-inducing vaccines.

Cryoimmunotherapy with local co-administration of ex vivo generated dendritic cells and CpG-ODN immune adjuvant, elicits a specific antitumor immunity.

Cryoablation is a low-invasive surgical procedure for management of malignant tumors. Tissue destruction is obtained by repeated deep freezing and thawing and results in coagulative necrosis and in apoptosis. This procedure induces the release of tumor-associated antigens and proinflammatory factors into the microenvironment. Local administration of immature dendritic cells (DCs) potentiates the immune response induced by cryoablation. To further augment the induction of long-lasting antitumor immunity, we investigated the clinical value of combining cryoimmunotherapy consisting of cryoablation and inoculation of immature DCs with administration of the immune adjuvant, CpG oligodeoxynucleotides. Injection of the murine Lewis lung carcinoma, D122-luc-5.5 that expresses the luciferase gene, results in spontaneous metastases, which can be easily monitored in vivo. The local tumor was treated by the combined treatment. The clinical outcome was assessed by monitoring tumor growth, metastasis in distant organs, overall survival, and protection from tumor recurrence. The nature of the induced T cell responses was analyzed. Combined cryoimmunotherapy results in reduced tumor growth, low metastasis and significantly prolonged survival. Moreover, this treatment induces antitumor memory that protected mice from rechallenge. The underlying suggested mechanisms are the generation of tumor-specific type 1 T cell responses, subsequent induction of cytotoxic T lymphocytes, and generation of systemic memory. Our data highlight the combined cryoimmunotherapy as a novel antitumor vaccine with promising preclinical results. Adjustment of this technique into practice will provide the therapeutic benefits of both, ablation of the primary tumor and induction of robust antitumor and antimetastatic immunity.

Impaired immune surveillance accelerates accumulation of senescent cells and aging.

Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1-/- mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.

p53 in Bronchial Club Cells Facilitates Chronic Lung Inflammation by Promoting Senescence.

The tumor suppressor p53 limits tumorigenesis by inducing apoptosis, cell cycle arrest, and senescence. Although p53 is known to limit inflammation during tumor development, its role in regulating chronic lung inflammation is less well understood. To elucidate the function of airway epithelial p53 in such inflammation, we subjected genetically modified mice, whose bronchial epithelial club cells lack p53, to repetitive inhalations of lipopolysaccharide (LPS), an exposure that leads to severe chronic bronchitis and airway senescence in wild-type mice. Surprisingly, the club cell p53 knockout mice exhibited reduced airway senescence and bronchitis in response to chronic LPS exposure and were significantly protected from global lung destruction. Furthermore, pharmacological elimination of senescent cells also protected wild-type mice from chronic LPS-induced bronchitis. Our results implicate p53 in induction of club-cell senescence and correlate epithelial cell senescence of chronic airway inflammation and lung destruction.

Human CTL epitopes prostatic acid phosphatase-3 and six-transmembrane epithelial antigen of prostate-3 as candidates for prostate cancer immunotherapy.

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer-associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.

Quantitative identification of senescent cells in aging and disease.

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single-cell basis. The method combines a senescence-associated beta-galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high-content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.

Combined dendritic cell cryotherapy of tumor induces systemic antimetastatic immunity.

PURPOSE: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. EXPERIMENTAL DESIGNS: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumin-transfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. RESULTS: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapyprotected mice that had survived primary MO5 tumors from rechallenge with parental tumors. CONCLUSIONS: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.

Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

NKG2D ligands mediate immunosurveillance of senescent cells.

Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis. Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis.

In vivo rejection of tumor cells dependent on CD8 cells that kill independently of perforin and FasL.

Perforin/granzyme B- and Fas/FasL-mediated killing pathways are the main effector mechanisms of CTL and NK cells in antitumor immune responses. In this study, we investigated the relative role of these two lytic mechanisms in protection of the host from tumor progression, as well as spontaneous metastasis, using the D122 Lewis lung carcinoma and its gene-modified cells. Utilizing perforin knockout mice (B6-PKO) and Fas and FasL mutant (B6-MRL and B6-Smn) mice, we found that perforin expression in the host plays a crucial function in the prevention of metastasis. However, local tumor rejection of an H-2K(b) and B7-1 transfectant, 39.5-B7 cells, was not dependent either on perforin or Fas/FasL expression in vivo. In addition, CTL lysis of 39.5-B7 cells was independent of perforin and Fas/FasL interactions in 18-hour in vitro assays. We also confirmed that CD8 T-cells were responsible for rejecting 39.5-B7 local tumors, yet cytokines, TNF-alpha and gammaIFN were not involved in tumor rejection in vivo. Furthermore, blocking assays using caspase inhibitors (zVAD-fmk, zLETD-fmk and zLEHD-fmk) showed that, whereas caspase activation was partially required to induce 39.5-B7 lysis mediated by the perforin-dependent pathway, 39.5-B7 lysis by CTLs through the perforin-independent mechanism required caspase activation. Thus, these results suggested that perforin, Fas/FasL, gammaIFN and TNF-alpha independent lytic mechanisms, mediated by CD8 T cells, have a crucial role in rejection of 39.5-B7 cells in vivo. Caspase activation is a pre requisite for apoptosis of targets by CTLs.

Expression of FasL by tumor cells does not abrogate anti-tumor CTL function.

The effects of Fas-ligand (FasL) expression by tumor cells on their tumorigenicity and immunogenicity have been reported as opposite, contradictory results. In some systems the killing of Fas positive cytotoxic T-cells (CTL) by FasL expressing tumors resulted in increased tumorigenicity while in other systems tumors expressing FasL were eliminated by neutrophil mediated inflammation. In the present study, we investigated how FasL expression influences the low immunogenic Lewis lung carcinoma clone D122 and its highly immunogenic MHC I (H-2Kb) and B7-1 (CD80) transfectant 39.5-B7, by transfecting the human FasL (FasL) gene into these cells. Despite the fact that FasL-expressing cells kill effectively appropriate target cells (L1210-fas) compared to parental cells (D122) and low expressors (DFasL-33), these tumor cells were completely rejected in syngeneic mice (C57BL/6), but not in Fas mutant B6-MRL mice, suggesting that functional Fas receptor expression in the host was required to induce an anti-tumor mechanism. In addition, although FasL-expressing immunogenic tumor cells (39.5-B7-FasL 7) kill effectively target cells in vitro, both the transfectant and the mock transfectant (39.5-B7-pBabe) were rejected in syngenic mice. The sensitivity of FasL expressing tumor cells to lysis by CTLs was similar to that of FasL non-expressors. Therefore, these results indicate that FasL expression on immunogenic tumor cells does not affect their immunogenicity in vivo, as well as CTL functions in vitro.

A systems immunology approach to the host-tumor interaction: large-scale patterns of natural autoantibodies distinguish healthy and tumor-bearing mice.

Traditionally, immunology has considered a meaningful antibody response to be marked by large amounts of high-affinity antibodies reactive with the specific inciting antigen; the detection of small amounts of low-affinity antibodies binding to seemingly unrelated antigens has been considered to be beneath the threshold of immunological meaning. A systems-biology approach to immunology, however, suggests that large-scale patterns in the antibody repertoire might also reflect the functional state of the immune system. To investigate such global patterns of antibodies, we have used an antigen-microarray device combined with informatic analysis. Here we asked whether antibody-repertoire patterns might reflect the state of an implanted tumor. We studied the serum antibodies of inbred C57BL/6 mice before and after implantation of syngeneic 3LL tumor cells of either metastatic or non-metastatic clones. We analyzed patterns of IgG and IgM autoantibodies binding to over 300 self-antigens arrayed on slides using support vector machines and genetic algorithm techniques. We now report that antibody patterns, but not single antibodies, were informative: 1) mice, even before tumor implantation, manifest both individual and common patterns of low-titer natural autoantibodies; 2) the patterns of these autoantibodies respond to the growth of the tumor cells, and can distinguish between metastatic and non-metastatic tumor clones; and 3) curative tumor resection induces dynamic changes in these low-titer autoantibody patterns. The informative patterns included autoantibodies binding to self-molecules not known to be tumor-associated antigens (including insulin, DNA, myosin, fibrinogen) as well as to known tumor-associated antigens (including p53, cytokeratin, carbonic anhydrases, tyrosinase). Thus, low-titer autoantibodies that are not the direct products of tumor-specific immunization can still generate an immune biomarker of the body-tumor interaction. System-wide profiling of autoantibody repertoires can be informative.

Amelioration of brain pathology and behavioral dysfunction in mice with lupus following treatment with a tolerogenic peptide.

OBJECTIVE: Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZBxNZW)F1 (NZB/NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations. METHODS: Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test. RESULTS: Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3+ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1beta (IL-1beta), IL-6, IL-10, interferon-gamma, transforming growth factor beta, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-xL gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests. CONCLUSION: Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.

Exuberated numbers of tumor-specific T cells result in tumor escape.

Cytotoxic T cells (CTL) play a major role in tumor rejection. Expansion of CTLs, either by immunization or adoptive transfer, is a prominent goal in current immunotherapy. The antigen-specific nature of these expansion processes inevitably initiates a clonotypic attack on the tumor. By injecting an Ovalbumin-expressing melanoma into OT-I mice, in which >90% of CTLs recognize an Ovalbumin peptide, we show that an increased number of tumor-specific CTLs causes emergence of escape variants. We show that these escape variants are a result of antigen silencing via a yet undetermined epigenetic mechanism, which occurs frequently and is spontaneously reversible. We further show that an increase in the time of tumor onset in OT-I compared with C57BL/6J is a result of immune selection.

Preventive and therapeutic vaccination with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice.

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201-PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.

Cu/Zn superoxide dismutase plays a role in angiogenesis.

Endothelial cells produce oxygen radicals spontaneously and this process is augmented by hypoxia/reoxygenation. Cu/Zn superoxide dismutase (SOD-1) is an important enzyme in cellular oxygen metabolism. To determine whether alterations in SOD-1 activity affect angiogenesis we used transgenic SOD-1 (Tg-SOD) mice with elevated level of SOD-1. Angiogenesis induced subcutaneously by bFGF in Tg-SOD mice was 3-fold higher than in control non-transgenic (ntg) mice. Oral administration of disulfiram (DSF), an inhibitor of SOD-1, inhibited angiogenesis in Tg-SOD mice as well as in CD1 nude mice. Effects of DSF on cultured cells were also tested. Application of DSF to cultured bovine capillary endothelial (BCE) cells caused inhibition of DNA synthesis and induction of apoptosis. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. DSF also reduced the level of glutathione and the production of H(2)O(2) in BCE cells. Moreover, PC12-SOD cells with elevated SOD-1 were less sensitive to DSF treatment then control cells. These data indicate that the effects of DSF are mediated by inhibition of SOD-1 activity. Tumor development is known to largely depend on angiogenesis. We found that oral administration of DSF to mice caused significant inhibition of C6 glioma tumor development and marked reduction (by 10-19-fold) in metastatic growth of Lewis lung carcinoma. The data suggest a role for SOD-1 in angiogenesis, establish DSF as a potential inhibitor of angiogenesis and raise the possibility that attenuating SOD-1 activity may be important in treatment of angiogenesis-dependent pathologies.