KS Cells Research Articles

Kidney organoid derived from renal tissue stem cells is a useful tool for histopathological assessment of nephrotoxicity in a cisplatin-induced acute renal tubular injury model.

Organoids derived from renal tissue stem cells (KS cells) isolated from the S3 segment of adult rat nephrons have previously been developed and evaluated. However, data regarding the histopathological evaluation of these organoids are limited. Therefore, in this study, we performed histopathological examinations of the properties of these organoids and evaluated the nephrotoxicity changes induced by cisplatin treatment. We observe that the tubular structure of the organoids was generally lined by a single layer of cells, in concordance with previous findings. Microvilli were exclusively observed under electron microscopy on the luminal side of this tubular structure. Moreover, the luminal side of the tubular structure was positive for aquaporin-1 (Aqp1), a marker of the proximal renal tubule. Cisplatin treatment induced cell death and degeneration, including cytoplasmic vacuolation, in cells within the tubular structure of the organoids. Cisplatin toxicity is associated with the induction of γ-H2AX (a marker of DNA damage) and the drop of phospho-histone H3 (a marker of cell division) levels. During the nephrotoxicity assessment, the kidney organoids displayed various features similar to those of the natural kidney, suggesting that it is possible to use these organoids in predicting nephrotoxicity. The histological evaluation of the organoids in this study provides insights into the mechanisms underlying nephrotoxicity.

γ-Interferon Produced by CD8+ T Cells Infiltrating Kaposi's Sarcoma Induces Spindle Cells With Angiogenic Phenotype and Synergy With Human Immunodeficiency Virus-1 Tat Protein: An Immune Response to Human Herpesvirus-8 Infection?

AbstractKaposi's sarcoma (KS) is an angioproliferative disease associated with infection by the human herpesvirus-8 (HHV-8). HHV-8 possesses genes including homologs of interleukin-8 (IL-8) receptor, Bcl-2, and cyclin D, which can potentially transform the host cell. However, the expression of these genes in KS tissues is very low or undetectable and HHV-8 does not seem to transform human cells in vitro. In addition, KS may not be a true cancer at least in the early stage. This indicated that besides its transforming potential, HHV-8 may act in KS pathogenesis also through indirect mechanisms. Evidence suggests that KS may start as an inflammatory-angiogenic lesion mediated by cytokines. However, little is known on the nature of the inflammatory cell infiltration present in KS, on the type of cytokines produced and on their role in KS, and whether this correlates with the presence of HHV-8. Here we show that both acquired immunodeficiency syndrome (AIDS)-KS and classical KS (C-KS) lesions are infiltrated by CD8+ T cells and CD14+/CD68+monocytes-macrophages producing high levels of γ-interferon (γIFN) which, in turn, promotes the formation of KS spindle cells with angiogenic phenotype. γIFN, in fact, induces endothelial cells to acquire the same features of KS cells, including the spindle morphology and the pattern of cell marker expression. In addition, endothelial cells activated by γIFN induce angiogenic lesions in nude mice closely resembling early KS. These KS-like lesions are accompanied by production of basic fibroblast growth factor, an angiogenic factor highly expressed in primary lesions that mediates angiogenesis and spindle cell growth. The formation of KS-like lesions is upregulated by the human immunodeficiency virus Tat protein demonstrating its role as a progression factor in AIDS-KS. Finally, γIFN and HLA-DR expression correlate with the presence of HHV-8 in lesional and uninvolved tissues from the same patients. As HHV-8 infects both mononuclear cells infiltrating KS lesions and KS spindle cells, these results suggest that HHV-8 may elicit or participate in a local immune response characterized by infiltration of CD8+ T cells and intense production of γIFN which, in turn, plays a key role in KS development.

Lipoxins exert antiangiogenic and anti-inflammatory effects on Kaposi's sarcoma cells

Lipoxin A4 (LXA4) is an endogenously produced host molecule with anti-inflammatory resolution effects. Previous studies demonstrated it to be involved in anti-vascular endothelial growth factor (VEGF)-mediated angiogenesis and in a possible anticancer role via interaction with its receptor, lipoxin A 4 receptor (ALXR). Here, we examined the effects of LXA4 and its epimer 15-epi-LXA4 in inhibiting proinflammatory and angiogenic functions in a human Kaposi's sarcoma tumor-derived cell line (KS-IMM). KS-IMM cells expressed increased levels of inflammatory cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LO) pathway enzymes when compared with human microvascular dermal endothelial cells (HMVEC-d). KS-IMM cells secreted high levels of prostaglandin E2 (PGE2) and chemotactic leukotriene B4 (LTB4). Treatment with LXA4 or 15-epi-LXA4 effectively reduced the levels of COX-2, 5-LO proteins, and secretion of PGE2 and LTB4 in KS-IMM cells. LXA4 or 15-epi-LXA4 treatment also decreased secretion of proinflammatory interleukin 6 (IL-6) and IL-8 cytokines but induced the secretion of anti-inflammatory IL-10. LXA4 treatment reduced the phosphorylation of VEGF receptor (VEGFR) and ephrin family receptor tyrosine kinases. LXA4 treatment effectively induced dephosphorylation of multiple cellular kinases such as Focal Adhesion Kinase, Protein kinase B, nuclear factor kappa-light-chain-enhancer of activated B cells, and Extracellular signal-regulated kinases (ERK)1/2, and reduced angiogenic factor VEGF-C secretion in KS cells. LX treatment drastically induced the Src-homology 2 domain-containing phosphatase tyrosine (Y542) phosphatase and reduced VEGFR-2 phosphorylation at sites Y1059, Y1175, and Y1212. Treatment of KS-IMM cells with LXA4 resulted in selective localization of VEGFR-2 in nonlipid raft (non-LR) and ALXR to LR fractions. These results demonstrated that LXA4 or 15-epi-LXA4 induce anti-inflammatory and antiangiogenic effects in KS cells and suggest that treatment with LXs is an attractive novel strategy against KS.

Single adult kidney stem/progenitor cells reconstitute three-dimensional nephron structures in vitro.

The kidneys are formed during development from two distinct primordial tissues, the metanephric mesenchyme and the ureteric bud. The metanephric mesenchyme develops into the kidney nephron, the minimal functional unit of the kidney. A nephron consists of several segments and regulates water, electrolyte, and acid-base homeostasis in addition to secreting certain hormones. It has been predicted that the kidney will be among the last organs successfully regenerated in vitro due to its complex structure and multiple functions. Here, we show that adult kidney stem/progenitor cells (KS cells), derived from the S3 segment of adult rat kidney nephrons, can reconstitute a three-dimensional kidney-like structure in vitro. Kidney-like structures were formed when a cluster of KS cells was suspended in an extracellular matrix gel and cultured in the presence of several growth factors. Morphological analyses revealed that these kidney-like structures contained every substructure of the kidney, including glomeruli, proximal tubules, the loop of Henle, distal tubules, and collecting ducts, but no vasculature. Our results demonstrate that a cluster of tissue stem/progenitor cells has the ability to reconstitute the minimum unit of its organ of origin by differentiating into specialized cells in the correct location. This process differs from embryonic kidney development, which requires the mutual induction of two different populations of progenitors, metanephric mesenchymal cells and ureteric bud cells.

Human herpesvirus 8 induces polyfunctional B lymphocytes that drive Kaposi's sarcoma.

ABSTRACTKaposi’s sarcoma (KS) is an unusual neoplasia wherein the tumor consists primarily of endothelial cells infected with human herpesvirus 8 (HHV-8; Kaposi’s sarcoma-associated herpesvirus) that are not fully transformed but are instead driven to excess proliferation by inflammatory and angiogenic factors. This oncogenic process has been postulated but unproven to depend on a paracrine effect of an abnormal excess of host cytokines and chemokines produced by HHV-8-infected B lymphocytes. Using newly developed measures for intracellular detection of lytic cycle proteins and expression of cytokines and chemokines, we show that HHV-8 targets a range of naive B cell, IgM memory B cell, and plasma cell-like populations for infection and induction of interleukin-6, tumor necrosis factor alpha, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, and interleukin-8 in vitro and in the blood of HHV-8/HIV-1-coinfected subjects with KS. These B cell lineage subsets that support HHV-8 infection are highly polyfunctional, producing combinations of 2 to 5 of these cytokines and chemokines, with greater numbers in the blood of subjects with KS than in those without KS. Our study provides a new paradigm of B cell polyfunctionality and supports a key role for B cell-derived cytokines and chemokines produced during HHV-8 infection in the development of KS.

Exposure to HIV-protease inhibitors selects for increased expression of P-glycoprotein (ABCB1) in Kaposi's sarcoma cells

Background:Given that HIV-protease inhibitors (HIV-PIs) are substrates/inhibitors of the multidrug transporter ABCB1, can induce ABCB1 expression, and are used in combination with doxorubicin for AIDS-Kaposi's Sarcoma (KS) treatment, the role that ABCB1 plays in mediating multidrug resistance (MDR) in a fully transformed KS cell line (SLK) was explored.Methods:The KS cells were exposed to both acute and chronic treatments of physiological concentrations of different HIV-PIs (indinavir, nelfinavir, atazanavir, ritonavir, or lopinavir), alone or together with doxorubicin. The ABCB1 mRNA and protein expression levels were then assessed by qRT–PCR and western blotting, flow cytometry, and immunofluorescence.Results:Chronic treatment of SLK cells with one of the five HIV-PIs alone or together resulted in increased resistance to doxorubicin. Co-treatment with one of the HIV-PIs in combination with doxorubicin resulted in a synergistic increase in resistance to doxorubicin, and the degree of resistance was found to correlate with the expression of ABCB1. The SLK cells were also revealed to be cross-resistant to the structurally unrelated drug paclitaxel.Conclusion:These studies suggest that ABCB1 is primarily responsible for mediating MDR in SLK cells selected with either HIV-PIs alone or in combination with doxorubicin. Therefore, the roles that ABCB1 and drug cocktails play in mediating MDR in KS in vivo should be evaluated.

Induction, regulation, and biologic function of Axl receptor tyrosine kinase in Kaposi sarcoma

AbstractAxl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-κB inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations.

The role of Ephs, Ephrins, and growth factors in Kaposi sarcoma and implications of EphrinB2 blockade

AbstractKaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and is dependent on the induction of vascular endothelial growth factors (VEGFs). VEGF regulates genes that provide arterial or venous identity to endothelial cells, such as the induction of EphrinB2, which phenotypically defines arterial endothelial cells and pericytes, and represses EphB4, which defines venous endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine kinases to determine which members are expressed and therefore contribute to KS pathogenesis. We demonstrated limited Eph/Ephrin expression; notably, the only ligand highly expressed is EphrinB2. We next studied the biologic effects of blocking EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin (sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in response to various growth factors. Finally, we determined the biologic effects of combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than the VEGF antibody, and combination of the 2 had at least additive activity. sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and increased hypoxia, with an associated increase in VEGF and DLL4 expression. The combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for further investigation.

Gene Regulation and Functional Alterations Induced by Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORFK13/vFLIP in Endothelial Cells

Kaposi's sarcoma (KS) is an angioproliferative inflammatory disorder induced by endothelial cell infection with the KS-associated herpesvirus (KSHV). ORFK13/vFLIP, one of the KSHV genes expressed in KS, encodes a 188-amino-acid protein which binds to the Ikappab kinase (IKK) complex to activate NF-kappaB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. Retroviral transduction of ORFK13/vFLIP into primary human endothelial cells induces the spindle morphology distinctive of KS cells and promotes the formation of abnormal vascular networks typical of KS vasculature; upregulates the expression of proinflammatory cytokines, chemokines, and interferon-responsive genes; and stimulates the adhesion of inflammatory cells characteristic of KS lesions. Thymidine phosphorylase, a cellular enzyme markedly induced by ORFK13/vFLIP, can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU), a potent thymidine synthase inhibitor, which blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1 to 1 microM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies.

Properties of Hodgkin cell lines. Possible significance for pathophysiology and clinical medicine

In the last three years, five permanently in-vitro growing cell cultures with malignant properties were established from tumour material of patients with histologically confirmed Hodgkin's disease. Four cell lines have been maintained in culture. L 428 had identical characteristics in every respect with Hodgkin and Sternberg-Reed cells, tested in-vivo on biopsy tissue. The other lines--L 538, L 540 and L 591 - had certain characteristics of Hodgkin and Sternberg-Reed cells with a number of markers, but were not fully congruent. All lines reacted with a heterologous antiserum against L 428, which selectively cross-reacted with Hodgkin and Sternberg-Reed cells in fresh biopsies. Two sublines, L 428 KS and L 428 KSA, were established from L 428 by modifying the culture medium. Tests on L 428 KS cells with conventional methods and with monoclonal antibodies demonstrated that this line carried antigens of myeloid cells; however, it could not be definitely placed into any haematopoetic line. Conditioned medium of L 428 and its sublines showed CSF activity (colony-stimulating factor) and suppression of cell-mediated cytolysis.

Expression of estrogen receptor ß-isoforms in breast cancer cell lines with different potentials for mobility and invasiveness

Estrogen receptor (ER) β is involved in physiological and pathological processes in the mammary gland. Different ERβ-isoforms are described in the literature, without providing their exact function. In order to determine the biological function of distinct ERß-isoforms for breast cancer patients it is important to get detailed information about their expressional regulation. For this purpose we have established four classic multiplex- and six real-time RT-PCRs to quantify wild-type ERβ (ERßwt), ERβcx-, ERβδ5-, ERβ5-splice variants. Expression of ERβ-splice variants was examined in seven standard-cultured breast cancer cell lines: Tamoxifen-resistant MCF-7 (MCF-7TR) cell line, its parental MCF-7 cell line, MCF-10A, MDA-MB 231, AR, KS, and BM cells. Invasion- and mobilityassays were performed using cambers coated with matrigel or uncoated, respectively. RT-PCR data indicate that ERßwt is expressed in MCF-7TR cells compared to MCF-7 cells missing expression of ERβwt. On the other hand MCF-7 cells express ERβ5 which could not be detected in MCF-7TR cells. Comparing the ERß isoform expression patterns of all cell lines investigated it can be observed that the expression pattern of ERβ-isoforms is very similar in MCF-7TR, MDA-MB 231 and AR cells with strong signals for ERβcx. In mobility assays MCF-7TR and AR cells like MDA-MB 231 cells are highly mobile. However, in contrast to MCF-7TR and MDA-MB 231 cells, AR cells are not able to pass through matrigel. PCR analysis of two separate fractions of MCF-7TR cells which passed through matrigel and MCF-7TR cells which remained in or on top of it show a different expression pattern of ERβ-isoforms with a signal for ERβcx only in the invasive cells. MCF-7, MCF10A, KS, and BM cells having a different expression pattern of ERβ-isoforms are neither mobile nor invasive. These data suggest that the expression pattern of ERß-isoforms might be correlated with the mobility of cells in breast cancer.

Agonist Met antibodies define the signalling threshold required for a full mitogenic and invasive program of Kaposi’s Sarcoma cells

We previously showed that the Kaposi Sarcoma line KS-IMM express a functional Met tyrosine kinase receptor, which, upon HGF stimulation, activates motogenic, proliferative, and invasive responses. In this study, we investigated the signalling pathways activated by HGF, as well as by Met monoclonal antibodies (Mabs), acting as full or partial agonists. The full agonist Mab mimics HGF in all biological and biochemical aspects. It elicits the whole spectrum of responses, while the partial agonist Mab induces only wound healing. These differences correlated with a more prolonged and sustained tyrosine phosphorylation of the receptor and MAPK evoked by HGF and by the full agonist Mab, relative to the partial agonist Mab. Since Gab1, JNK and PI 3-kinase are activated with same intensity and kinetics by HGF and by the two agonist antibodies, it is concluded that level and duration of MAPK activation by Met receptor are crucial for the induction of a full HGF-dependent mitogenic and invasive program in KS cells.

Interleukin-1 beta increases the BCL-2/BAX ratio in Kaposi's sarcoma cells.

Interleukin-1 (IL-1) is a multifunctional cytokine known to act as a growth factor for AIDS-KS cells. In addition to its mitogenic effects, we found that IL-1 induced the protection of KS cells from apoptotic death induced by serum deprivation in a dose-dependent manner. AIDS-KS cells as well as cells derived from iatrogenic and sporadic KS exhibited a similar response to IL-1, which stresses the key role of this cytokine in the pathogenesis of KS regardless of its epidemiological form. Using both an immunohistochemical and an immunoblot approach, we found that IL-1 increased the expression of Bcl-2 and decreased that of Bax, while having no effect on the expression of Bclx(L), Fas and CD40. The effects of IL-1 were inhibited by IL-1ra, suggesting that imbalance between these two counter-acting cytokines may contribute to the altered accumulation of KS spindle cells. Our findings may provide a link between KS cell escape from apoptosis and the immune dysregulation known to be associated with KS.

Exogenous Fibroblast Growth Factor-2 Induces a Transformed Phenotype in Vascular Kaposi's Sarcoma-like Cells

Vascular TTB cells derive from murine Kaposi's sarcoma-like dermal lesions and share several phenotypic features with AIDS-associated KS spindle cells. We have recently reported that fibroblast growth factor-2 (FGF-2) promotes dramatic cytoskeletal and morphological alterations in TTB cells, concomitant with the induction of an autocrine loop for hepatocyte growth factor and a relocalization of the urokinase receptor. Since all these alterations are hallmarks of cell transformation. we attempted to verify whether FGF-2 induces a transformed phenotype in TTB cells. Our results show that FGF-2-treated TTB cells acquire the ability to grow under anchorage-independent conditions. In addition, FGF-2 markedly reduced the levels of thrombospondin-1, an antiangiogenic and tumor suppressor protein, in TTB cells. Therefore, FGF-2 induces KS-like spindle cells to acquire properties characteristic of transformed cells. This suggests that FGF-2 plays a pathogenetic role in KS not only by promoting angiogenesis, but also by conferring a transformed phenotype upon KS cells. In light of previous reports on Tat-induced release of FGF-2 into the extracellular space, our findings may provide an additional mechanism for the observed synergism between Tat and FGF-2 in the pathogenesis of KS.

HIV-Tat dependent chemotaxis and invasion, key aspects of tat mediated pathogenesis.

Extracellular Tat acts as a pleiotropic molecule inducing several biological effects on different target cells. Tat stimulates the chemotaxis of numerous cell types and it appears to have oncogenic activities, including acting as a co-factor for Kaposi's sarcoma. The Tat protein has been shown to bind integrins through an RGD amino acid motif. Tat is an angiogenic factor able to induce the migration and invasion of endothelial and KS cells through the interaction of its basic domain with the VEGF receptor VEGFR2 (Flk-1/KDR). We have also found that Tat is able to mimic chemokines, activating monocyte migration through the chemokine like' cysteine-core domain. Tat is a chemoattractant for dendritic cells, and both the RGD and basic domains appear to be involved in this response. In a recent study we demonstrated that Tat is chemotactic for PMN and induces Ca2+ mobilization in vitro. Experiments using synthetic peptides showed that Tat activities on PMN are mediated by the chemokine like' region. Finally Tat is also able to induce B cell chemotaxis, while its activity on helper T cells has not yet been clarified. Here we review data on Tat-dependent chemotaxis and discuss the possible implications in Tat mediated pathogenesis.

P53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.

Potential of CD80-transfected human breast carcinoma cells to induce peptide-specific T lymphocytes in an allogeneic human histocompatibility leukocyte antigens (HLA)-A2.1+-matched situation.

Allogeneic human histocompatibility leukocyte antigen (HLA)-matched tumor cell lines that have been made immunogenic by the transfer of genes encoding for costimulatory molecules such as CD80 are considered to be potential vaccines for the induction of systemic immune reactions in cancer patients. We used a human HLA-A2.1+ CD80-transfected breast carcinoma cell line (KS-CD80) and investigated in vitro the efficiency at which antigen (Ag)-specific responses were induced following the stimulation of allogeneic HLA-A2.1-matched T lymphocytes. The influenza matrix protein M1 was used as a model Ag. It was either endogenously expressed or exogenously loaded as a peptide (matrix protein), and the frequency of the generated specific T cells was determined. The expression of CD80 in KS cells was required for an effective activation and expansion of Ag-specific T cells. This response was augmented following the pretreatment of KS-CD80 cells with interferon-gamma and tumor necrosis factor-alpha. Interleukin-4 (IL-4), IL-7, and IL-12 further increased T-cell expansion. IL-7 was best at supporting the generation of T cells with Ag specificity. This investigation demonstrates that allogeneic CD80+ tumor cells can induce Ag-specific, HLA-restricted T lymphocytes at a high frequency. Our study supports the use of allogeneic cell lines for the induction of specific T-cell responses in tumor patients.

CD24 mediates rolling of breast carcinoma cells on P-selectin.

P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.

Human immunodeficiency virus tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells.

Kaposi's sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130(cas). Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.

AIDS-KAPOSI'S SARCOMA CELLS DEMONSTRATE INCREASED SUSCEPTIBILITY TO A REDOX CYCLING DRUG.

29 Background: It is well accepted that cytokines contribute to the pathogenesis of AID-related Kaposi's sarcoma (AIDS-KS). Further, because AIDS-KS cells demonstrate high autologous production of growth promoting cytokines, existing AIDS-KS lesions can function not only to promote their own growth, but also to stimulate development of new tumor foci. Our laboratory is currently developing sustained release drug delivery vehicles to be used in the treatment of patients with early mucocutaneous AIDS-KS. Because data from our laboratory have shown that AIDS-KS cells possess significantly lower levels of oxidant stress protective compounds such as glutathione (GSH), we hypothesized that AIDS-KS cells would demonstrate enhanced cytotoxicity in response to chemotherapeutic agents which promote redox cycling e.g. doxorubicin (dox). Methods: Human cell strains isolated from HIV donors (norm), AIDS-KS cells (KS), and nonlesional cells from the AIDS-KS donors (non) were challenged with dox and assessed for: 1) inhibition of proliferation (BrdU incorporation), 2) alterations in cellular GSH and nucleotide levels (spectrophotometric and HPLC assays, respectively). Concurrent viability studies (trypan blue exclusion) showed cell population specific viability differences after dox exposure (4 μg/ml), with both the norm (p<0.018), and the non (p<0.010) strains demonstrating statistically significantly higher viabilities relative to KS cells. (Kruskal Wallis ANOVA, 2 tailed Mann Whitney U). In addition, BrdU labeling studies indicate that dox is more effective at inhibiting DNA synthesis in KS cells relative to either non or norm cell populations. Ongoing studies to assess the contribution of the thiol redox status in cellular response to redox active concentrations of dox (50 μg/ml) show: 1) Thiol augmentation (addition of 30 mM N-acetylcysteine) is cytoprotective and results in preservation of the cellular energy charge, high energy phosphates and GSH levels., 2) In contrast, GSH depletion prior to dox challenge significantly sensitizes the cells as reflected by marked decreases in cellular viabilities, loss of both high energy phosphates and cellular energy charges, and an oxidation of the redox state. Conclusion: Our findings suggest that the cellular thiol redox status modulates cellular responsiveness to redox cycling agents. Further, our data showing that AIDS-KS cells are more susceptible to the redox active concentrations of dox, provide a biochemical basis for the recognized clinical efficacy of this drug. Therefore, we anticipate that our proposed treatment modality, which will provide high local drug concentrations without the systemic side effects, e.g. cardiotoxicity, is a promising route for early intervention in the treatment of AIDS-KS. NIH support: HL RO-1 48547, DE R-O1 12183, CA U-O1 66351.