Thiol redox modulation of tumor necrosis factor-α responsiveness in cultured AIDS-related Kaposi's sarcoma cells

Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). While virtually every cell responds to TNF-α with gene activation, the extent of TNF-α-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-α membrane interaction, are part of this TNF-α-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-α. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-α challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV⁻ normal fibroblasts. In contrast, following TNF-α challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-α-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-α challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-α, suggest a biochemical rationale for the recognized TNF-α AIDS-KS clinical correlation. J. Cell. Biochem. 68:339–354, 1998. © 1998 Wiley-Liss, Inc.     

Pegylated liposomal doxorubicin in the treatment of AIDS-related Kaposi's sarcoma

Kaposi's sarcoma is a vascular tumor of skin and viscera first described in 1872. Prior to the 1980s, this disease was rarely seen in the Western world, but was quite prevalent in Sub-Saharan African countries. Since the onset of the HIV pandemic in the 1980s, the incidence of Kaposi's sarcoma has increased markedly in Africa and continues to be a significant problem in association with AIDS in Western countries. Many therapies have been demonstrated to be effective in the treatment of HIV-related Kaposi's sarcoma, including alitretinoin gel, interferon alpha, and various forms of cytotoxic chemotherapy. Antiretroviral therapy combined with cytotoxic agents has yielded significantly greater efficacy than chemotherapy alone. However, as reviewed in this report, pegylated liposomal doxorubicin has been established as the treatment of choice for patients with AIDS-associated Kaposi's sarcoma in Western countries. Compelling preclinical and clinical evidence, reviewed herein, has demonstrated that the nanoparticle (pegylated liposome) delivery system of this formulation leads to greater tumor localization of doxorubicin and consequent improved efficacy, as well as reduced toxicity.     

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levelsof virus-induced cytokines and, because of a sparing effect on their phagoctic cells, retention of the phagocytic inflammatory response. A component of phygocytic activation is the initiation of the oxidative brust, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess drcreased cytoprotective capabilities. Relativeto either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplement with conditioned medium from MOT, an TLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed in inherent biochemical responsiveness, by increasing catalase activity following exposure to exaogenous H₂(O₂). In contrast, the catalase activity of AIDS-KS cells decreased following (H₂O₂) challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression AIDS-KS.     

Spindle cells and their role in Kaposi's sarcoma

Spindle cells represent the main cell type of the advanced final nodular stage of Kaposi's sarcoma lesions. Despite some clinical and epidemiological differences, the four Kaposi's sarcoma forms (classic, endemic, post-transplant and epidemic) display very similar histopathological features, with the proliferation of spindle cells (considered as the Kaposi's sarcoma tumor cells) associated with inflammation and neo-angiogenesis. Electron-microscopy and immuno-histochemistry studies have led to the consensus that the spindle cells originated from the endothelial lineage. However, only recently, studies that used specific lymphatic immunological markers (such as podoplanin) and molecular features (gene expression microarrays) strongly linked Kaposi's sarcoma spindle cells to the endothelium lymphatic cell lineage. Both hybridization and immuno-histochemistry techniques have demonstrated that human herpesvirus 8 also known as Kaposi's sarcoma associated herpesvirus was present in spindle cells at all stages of the disease (patch, plaque, nodule). Interestingly, while the human herpesvirus 8 latent genes are expressed in nearly all tumor spindle cells, only a small fraction of them expresses markers of viral lytic replication. Recent findings showing that nodular Kaposi's sarcoma lesions display all patterns of human herpesvirus 8 clonality support the model according to which this tumor begins as a polyclonal disease with a subsequent evolution to a mono/oligoclonal process involving infected spindle cells. Spindle cells appear to be the central masterpiece in KS tumorigenesis, however the exact respective role of each human herpesvirus 8 gene, in the initiation and the disease progression is still under investigation and the question of whether or not this tumor is a reactive process or a true malignant proliferation of spindle cells remains yet unclear.     

Emerging targets and novel strategies in the treatment of AIDS-related Kaposi's sarcoma: bidirectional translational science

Through the mentorship process, Dr. Arthur Pardee emphasized the critical importance of bidirectional translational research-not only advancing drug development from bench to bedside, but also bringing back precious clinical material to the laboratory to assess the biologic effects of therapeutic agents on their targets. This mini-review focuses on the signal transduction pathways of Kaposi's sarcoma (KS) and on how the knowledge of such pathways has led to the rational development of molecularly targeted pathogenesis-driven therapies. Acquired immune deficiency syndrome (AIDS) related-KS results from co-infection with human immunodeficiency virus and KS herpesvirus/human herpesvirus-8 (KSHV/HHV8), which leads to the development of an angiogenic-inflammatory state that is critical in the pathogenesis of KS. KS is driven by KSHV/HHV8-specific pathways, which include viral G protein-coupled receptor (vGPCR), viral interleukin-6 (vIL-6), and viral chemokine homologues. In addition, cellular growth/angiogenic pathways, such as vascular endothelial growth factor (VEGF), insulin-like growth factor, platelet-derived growth factor (PDGF), angiopoietin and matrix metalloproteinases (MMPs) are "pirated" by KSHV/HHV8. As a very tangible example of how translational research has led to a marked improvement in patient outcome, the signal transduction inhibitor imatinib (a tyrosine kinase inhibitor of c-kit and PDGF) was administered to patients with KS whose tumors were serially biopsied. Not only did the patients' tumors regress, but also the regression was correlated with the inhibition of PDGF receptor (PDGFR) in the biopsy samples. Recent and future clinical trials of molecularly targeted therapy for the treatment of KS are a prelude to a shift in the paradigm of how KS is managed.     

Measuring inhalant nitrite exposure in gay men: Implications for elucidating the etiology of AIDS-related Kaposi's sarcoma

We reviewed 12 epidemiologic studies conducted among gay men with AIDS to examine the role of potential cofactors in the development of KS. Aspects of the studies reviewed include basic study design, wording of the questionnaires, and published results comparing KS patients with those who developed opportunistic infections indicative of AIDS. The studies included questions about sociodemographics, medical history, use of drugs, travel, and sexual behaviors. Patients were invited to provide blood and/or other specimens for laboratory analysis.The results of the review of epidemiologic studies are inconclusive. Nitrite inhalant use was a variable often associated with KS (five studies). The differences in outcomes of these studies may reflect differences in study designs, sample sizes, timing, quality, and content of interviews regarding nitrites, sexual behaviours and other potential cofactors. Epidemiologic study with careful consideration to content of questionnaires and laboratory testing may yet reveal the causes or cofactors for this tumor.     

Host range of Kaposi's sarcoma-associated herpesvirus in cultured cells

Difficulties in efficiently propagating Kaposi's sarcoma-associated herpesvirus (KSHV) in culture have generated the impression that the virus displays a narrow host range. Here we show that, contrary to expectation, KSHV can establish latent infection in many adherent cell lines, including human and nonhuman cells of epithelial, endothelial, and mesenchymal origin. (Paradoxically, the only lines in which we have not observed successful latent infection are cultured lymphoma cell lines.) In most latently infected lines, spontaneous lytic replication is rare and (with only two exceptions) is not efficiently induced by phorbol ester treatment-a result that explains the failure of most earlier studies to observe efficient serial transfer of infection. However, ectopic expression of the KSHV lytic switch protein RTA from an adenoviral vector leads to the prompt induction of lytic replication in all latently infected lines, with the production of infectious KSHV virions. These results indicate (i) that the host cell receptor(s) and entry machinery for KSHV are widely distributed on cultured adherent cells, (ii) that latency is the default pathway of infection, and (iii) that blocks to lytic induction are frequent and largely reside at or upstream of the expression of KSHV RTA.     

Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells

Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is known to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin cytotoxicity in a variety of cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, LoVo human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, cytotoxicity and mutagenicity of anthracyclines.     

睫状神经营养因子对NO引起海马神经元毒性反应的影响

本研究采用原代培养大鼠海马神经元,观察睫状神经营养因子（ｃｉｌｉａｒｙ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ,ＣＮＴＦ）对ＮＯ引起细胞毒性反应的影响。ＮＯ供体硝普钠与Ｓ－亚硝基－乙酰青霉胺,ＮＯＳ底物Ｌ－Ａｒｇ及钙载体ｉｏｎｏｍｙｃｉｎ,均可引起海马神经元存活率下降,ＬＤＨ漏出增加;提前２４ｈ给予不同浓度ＣＮＴＦ,均能提高神经元的存活率,减少ＬＤＨ漏出,其作用呈剂量依赖性。     

Kaposi's sarcoma in an Eskimo

An Eskimo who had been treated for a nasopharyngeal carcinoma was subsequently found to have a rapidly progressive form of Kaposi''s sarcoma confirmed at biopsy. No objective response was obtained by irradiation treatment of isolated nodules. However, vinblastine sulfate arrested the progression of the disease. Because this neoplasm is most prevalent in tropical climates its presentation in an Eskimo is believed sufficiently unusual to warrant this report.     

Expression of functional tyrosine kinases on immortalized Kaposi's sarcoma cells

Kaposi's sarcoma (KS) is the most frequent malignant lesion in patients with AIDS and is characterized by spindle cell proliferation, inflammatory cell infiltration, angiogenesis, edema, and invasiveness. KS origin is still debated. The complex aspect of this disease is probably supported by multiple concomitant pathogenetic factors, among which growth factors and their cognate tyrosine kinase receptors are deeply involved. Here we have investigated the expression status and functional integrity of KDR and Met receptors, as well as of their ligands, in an immortalized KS cell line (KS-IMM). The MET and KDR genes encode the tyrosine kinase receptors for Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth Factor (VEGF) respectively. Both factors are pleiotropic cytokines controlling growth, survival, motility, invasive migration and differentiation of endothelial cells. We have found that KS-IMM cells, which retain most of the features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice, express the Met receptor, but not its ligand. The receptor, which is basally inactive, is functional, being tyrosine phosphorylated in response to ligand stimulation and mediating the expected HGF-dependent biological responses of motility, invasion and proliferation. Moreover, we report that KS-IMM cells coexpress VEGF and KDR and that KDR is constitutively tyrosine phosphorylated, possibly as a consequence of the establishment of an autocrine loop. The receptor, however, maintains responsiveness to exogenously added ligand, by increasing the level of tyrosine phosphorylation and by responding in biological assays of motility, invasion and proliferation. Finally, we have found that the two growth factors synergize in a motility assay. These data show that HGF and VEGF are growth factors active on KS-IMM cells.     

Growth patterns,inflammatory cells and prognosis in AIDS-associated Kaposi's sarcoma

AIDS-associated cutaneous Kaposi's sarcomas (KS) in 35 patients were the subject of a histopathological and immunohistochemical study. Histologically, the tumours were classified as granulation-tissue-like (Gr, n=10), angioma-like (A, n=11), angiosarcoma-like (AS, n=9) and as spindle cell type (SP, n=5). There was a distinct relationship between the main growth patterns of KS and the CD4+ peripheral blood lymphocyte (PBL) count. A decrease in numbers of CD4+ PBL was correlated with an increase in the frequency of AS and SP growth patterns. There were no correlations between growth pattern and concentrations of protein S100+, MT1+, CD45+ and MAK387+ tumour-associated inflammatory cells. When the GR type was grouped together with the A type, and the AS type with the SP type, statistically significant differences emerged for CD45+ and MT1+ tumour-associated cells, and for survival time, respectively.In all these instances, the GR/A group values were higher than those of the AS/SP group. Additionally, linear correlation analysis revealed an inverse relationship between the concentrations of CD4+ PBL and of protein S100+ tumour-associated inflammatory cells within the GR/A group. In contrast to what is observed with certain tumours of HIV-negative patients, this suggests that a high concentration of protein S100+ tumour-associated cells may indicate a worse prognosis in the GR/A group. The concentration of MAK387+ tumour-associated macrophages did not vary as a function of the growth patterns, even when they were grouped together as described above. It is concluded that the different types of KS fo not vary in the production of tumour-derived chemotactic factors crucial for monocyte/macrophage migration into the stroma of malignant tumours.     

Antibody-dependent lymphocyte mediated cytotoxicity mechanism and modulation by cyclic nucleotides.

The injury to antibody coated thymocytes by the “K cell” among nonsensitized rat splenocytes was modulated by altering the cellular level of cAMP and cGMP. Preincubation of the attacking cell population with 1 μg/ml cholera toxin caused an elevation of cAMP levels of 1–18 pM per 10⁷ cells and was associated with a proportionate reduction in cytotoxicity. Agents which are known to raise cAMP levels including the Prostaglandins (PG) E₁ (10^?7–10^?5 M), PGF_2α (10^?5–10^?3 M), PGA₁ (10^?9–10^?5M), and theophylline (10^?3 M) also produced marked suppression of antibody dependent lymphocyte mediated cytotoxicity. Direct elevation of cellular levels of cGMP by the analog 8-bromo cGMP (5 × 10^?6M) led to augmentation of cytotoxicity. Removal by EDTA and MgEDTA of calcium and magnesium ions from the culture media markedly inhibited cytotoxicity.