Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes.

Bladder cancer, the fourth most common noncutaneous malignancy in the United States, is characterized by high recurrence rate, with a subset of these cancers progressing to a deadly muscle invasive form of disease. Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA, thus potentially modulating signaling pathways in recipient cells. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has been implicated in the initiation of metastasis for cancer progression. We investigated the ability of bladder cancer-shed exosomes to induce EMT in urothelial cells. Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media or from patient urine or bladder barbotage samples. Exosomes were then added to the urothelial cells and EMT was assessed. Urothelial cells treated with bladder cancer exosomes showed an increased expression in several mesenchymal markers, including α-smooth muscle actin, S100A4 and snail, as compared with phosphate-buffered saline (PBS)-treated cells. Moreover, treatment of urothelial cells with bladder cancer exosomes resulted in decreased expression of epithelial markers E-cadherin and ß-catenin, as compared with the control, PBS-treated cells. Bladder cancer exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pretreatment. We further showed that exosomes isolated from patient urine and bladder barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. In conclusion, the research presented here represents both a new insight into the role of exosomes in transition of bladder cancer into invasive disease, as well as an introduction to a new platform for exosome research in urothelial cells.

Killing of sarcoma cells by proapoptotic Bcl-X(S): role of the BH3 domain and regulation by Bcl-X(L).

Kaposi's sarcoma (KS) is the most common tumor affecting AIDS patients with over 20% of these patients afflicted by this disease. Previous studies have demonstrated that KS tumor cells predominantly express the prosurvival protein Bcl-X(L) compared with Bcl-2. In the current study, we have used an adenoviral vector that expresses Bcl-X(S), a functional inhibitor of Bcl-X(L), to study the significance of Bcl-X(L) expression in the KS cell line (SLK) or KS primary cultures. The results demonstrate that 75% to 80% of SLK or KS primary cells were killed by the Bcl-X(S) containing adenovirus whereas KS cells infected with control adenovirus showed no significant cell death or growth inhibition. Overexpression of Bcl-X(L), but not Bcl-2, in SLK cells attenuated apoptosis induced by adenovirus Bcl-X(S). Immunoprecipitation experiments revealed that adenoviral Bcl-X(S) associated with Bcl-X(L), but not with Bcl-2. Mutational analysis showed that the alpha 2 helical region of Bcl-X(S) containing the BH3 motif was critical for killing activity and interaction with Bcl-X(L). These results suggest that Bcl-X(S) is a direct killer and Bcl-X(L) may act by interacting with and sequestering Bcl-X(S.) These studies also suggest that targeting Bcl-X(L) may be of therapeutic benefit for the treatment of tumors that are characterized by inappropriate expression of Bcl-X(L).

Injection of human herpesvirus-8 in human skin engrafted on SCID mice induces Kaposi's sarcoma-like lesions.

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) has been implicated in the development of Kaposi's sarcoma (KS) and several B-cell lymphoproliferative diseases. Serologic and molecular genetic association data has implicated HHV-8 as the causal agent of KS, but its role in the development of KS lesions is not understood. To examine the etiology of KS, HHV-8 was injected into normal human skin transplanted onto SCID mice. Injection of HHV-8 induced lesion formation that is morphologically and phenotypically consistent with KS, including the presence of angiogenesis and spindle-shaped cells latently infected with HHV-8. These findings suggest that HHV-8 is indeed the etiologic agent of KS, and that the virus plays an important role in initiation of this disease.

Induction of human immunodeficiency virus 1 replication by human herpesvirus 8.

BACKGROUND: Human immunodeficiency virus 1 (HIV-1)-infected individuals are commonly infected with herpesviruses, including cytomegalovirus, herpes simplex virus, varicella-zoster virus, and human herpesvirus 8 (HHV-8, also known as Kaposi sarcoma-associated herpesvirus [KSHV]). Previous studies have demonstrated that coinfection with herpesviruses can modulate HIV-1 replication. This can occur either through direct interaction between the 2 viruses or through secondary effects resulting from the release of cellular factors in response to infection. OBJECTIVE: To investigate HIV-1 replication in the presence and absence of HHV-8. DESIGN AND METHODS: HIV-1 replication was analyzed following culture of HIV-1-infected CD4(+) T cells in the presence of HHV-8 infected B-cell lines or control, uninfected B-cell lines. To confirm and extend the results of these in vitro studies, HIV-1-infected T cells were injected into human skin transplanted onto severe combined immunodeficient mice. The human skin was also injected with purified HHV-8 or phosphate-buffered saline as a control and HIV replication measured in biopsy specimens taken 5 to 8 days later. RESULTS AND CONCLUSIONS: The results demonstrated a significant increase in HIV-1 replication in the presence of HHV-8 in both the in vitro and in vivo model systems. Although the mechanism responsible for HHV-8 induction of HIV-1 replication remains to be identified, the results indicate that these 2 viruses may interact at the molecular level in coinfected patients, resulting in increased HIV-1 viral load.

Replicative response, immunophenotype, and functional activity of monocyte-derived versus CD34(+)-derived dendritic cells following exposure to various expansion and maturational stimuli.

Dendritic cells (DCs), generated ex vivo from blood mononuclear cells (PBMC) or CD34(+) stem cells, are being used to develop novel immunotherapies. To establish optimal DC generation, a direct comparison of the optimal cell source, culture conditions, and maturation stimuli was performed, utilizing phenotypic and functional assays as end points. Plastic adherent monocytes from PBMC were expanded in a serum-free medium (X-Vivo 10) for 7 days using GM-CSF/IL-4; CD34(+) cells were expanded for 14 days using GM-CSF/IL-4/ Flt3L, in either X-Vivo 10 alone or with albumin or autologous plasma. Expanded DC from both cell sources were matured for 7 days with CD40L or IFN-alpha/TNF-alpha. Starting from 2 x 10(7) monocytes, the optimal expansion/maturation process yielded 1.73 +/- 0.52 x 10(6) CD86(+) DC. Optimal expansion of CD34(+) cells (83.9 +/- 25.0-fold) was achieved using X-Vivo 10 with 5% plasma, matured with CD40L, and yielded 10.68 +/- 2.72 x 10(6) CD86(+) DC from 1 x 10(6) CD34(+) cells. Mature DC from PBMC or CD34(+) cells had similar enhanced expression of MHC class II HLA-DR, CD80, CD83, and CD86 and were potent stimulators of mixed lymphocyte reactions. Prior to maturation, all groups of DC actively phagocytosed apoptotic melanoma cells (approximately 50% of HLA-DR(+)). CD34(+) DC matured with CD40L or IFN-alpha/TNF-alpha had reduced phagocytic capability (34 and 31% of HLA-DR(+) DC, respectively). Similar expansion and functional activity was found using cryopreserved DC precursors, cultured in gas permeable bags. We conclude that both cell lineages produce potent mature DC, permitting exploration of the optimal clinical strategy to trigger anti-tumor immune responses in patients with malignancies.

Induction of HHV-8 lytic cycle replication by inflammatory cytokines produced by HIV-1-infected T cells.

Human herpesvirus 8 (HHV-8) is a gamma2-herpesvirus consistently identified in Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. Although HHV-8 infection appears to be necessary, it may not be sufficient for development of KS without the involvement of other cofactors. One potentially important cofactor is HIV-1. HIV-1-infected cells produce HIV-1-related proteins and cytokines, both of which have been shown to promote growth of KS cells in vitro. Though HIV-1 is not absolutely necessary for KS development, KS is the most frequent neoplasm in AIDS patients, and AIDS-KS is recognized as a particularly aggressive form of the disease. To determine whether HIV-1 could participate in the pathogenesis of KS by modulating HHV-8 replication (rather than by inducing immunodeficiency), HIV-1-infected T cells were cocultured with the HHV-8-infected cell line, BCBL-1. The results demonstrate soluble factors produced by or in response to HIV-1-infected T cells induced HHV-8 replication, as determined by production of lytic phase mRNA transcripts, viral proteins, and detection of progeny virions. By focusing on cytokines produced in the coculture system, several cytokines known to be important in growth and proliferation of KS cells in vitro, particularly Oncostatin M, hepatocyte growth factor/scatter factor, and interferon-gamma, were found to induce HHV-8 lytic replication when added individually to BCBL-1 cells. These results suggest specific cytokines can play an important role in the initiation and progression of KS through reactivation of HHV-8. Thus, HIV-1 may participate more directly than previously recognized in KS by promoting HHV-8 replication and, hence, increasing local HHV-8 viral load.

Caspase inhibitor blocks human immunodeficiency virus 1-induced T-cell death without enhancement of HIV-1 replication and dimethyl sulfoxide increases HIV-1 replication without influencing T-cell survival.

OBJECTIVES: To determine the relationship, if any, between reagents that modulate survival of T-cells and replication of human immunodeficiency virus 1 (HIV-1) and to determine the effects of the solvent dimethyl sulfoxide (DMSO) and drugs such as cyclosporin A and all-trans retinoic acid on HIV-1 replication. DESIGN: To first establish the direct effects of solvent alone (ie, DMSO) at various concentrations on HIV-1 replication, followed by the ability of various compounds such as the caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethylketone (z-VAD-fmk), cyclosporin A, and all-trans retinoic acid on HIV-1 replication. Next, to determine if HIV-1 induces T-cell apoptosis using TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assays and DNA fragmentation and poly-(ADP-ribose)-polymerase (PARP) cleavage, and then to examine how the various compounds influence T-cell survival after HIV-1 exposure. METHODS: The human T-cell line, CEM cells, were exposed to HIV(IIIB) and viral replication monitored using reverse transcription assays at 3, 6, and 9 days following infection. Cells were pretreated with various compounds dissolved in DMSO over a wide range of concentrations, and DMSO itself was also examined. T-cell death and apoptosis were assessed using TUNEL staining to detect 3'-OH DNA strand breaks and agarose gel electrophoresis to detect DNA fragmentation (laddering). Furthermore, PARP cleavage implicated in the apoptotic process was also examined. RESULTS: At very low levels, such as 0.002%, DMSO itself appears to enhance HIV-1 replication at 6 and 9 days after infection. At low levels of cyclosporin A, such as 0.01 microgram/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 microgram/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 microgram/mL did not influence HIV-1 replication. In addition, a discrepancy was noted in that HIV-1-infected T-cells were TUNEL positive, indicating DNA strand breaks; however, more complete DNA fragmentation was not detected nor was PARP cleavage identified. The induction of TUNEL positivity was blocked by the caspase inhibitor z-VAD-fmk but not by DMSO or cyclosporin A. Even though z-VAD-fmk blocked the appearance of TUNEL-positive T-cells, there was not a consistently observed increase in HIV-1 replication. CONCLUSION: Low levels of DMSO and cyclosporin A can enhance HIV-1 replication in CEM cells. At higher levels, cyclosporin A inhibits HIV-1 replication with no significant effects by all-trans retinoic acid. No evidence for classic apoptosis was detected in CEM cells after HIV-1 infection, although DNA strand breaks may be present as revealed by TUNEL positivity. There was no correlation between levels of HIV-1 replication and T-cell survival or death. The mechanism of T-cell death after HIV-1 infection requires further study, and investigators who add compounds dissolved in DMSO must include controls to carefully examine the direct effects of even trace levels of this solvent on HIV-1 replication.

Expression of costimulatory molecules CD80 and/or CD86 by a Kaposi's sarcoma tumor cell line induces differential T-cell activation and proliferation.

During physiological stimulation of resting T-cells, at least two activation signals by antigen presenting cells are required. Besides the first antigen-specific signal, the second costimulatory signal involves CD80 and CD86 expressed by the antigen presenting cell. These costimulatory molecules have been suggested to be of clinical relevance in many different autoimmune and malignant disease processes. We previously observed that tumor cells in Kaposi's sarcoma (a common AIDS-related cutaneous neoplasm) completely lack both CD80 and CD86, and these tumor cells fail to stimulate T-cell proliferation. In this study, using a Kaposi's sarcoma tumor cell line designated SLK, various stable transfected cell lines were produced. Tumor cells that were either singly positive for either CD80 or CD86, as well as a double-positive cell line, were examined for their ability to induce T-cell activation, T-cell proliferation, and cytokine production profiles. Compared to the parental double-negative tumor cell line, the CD80-positive cells, but not the CD86-positive tumor cells, induced significant T-cell activation and proliferation. Tumor cells expressing both CD80 and CD86 also induced T-cell activation. After stimulation by the transfected tumor cells, T-cells produced a Th-1 type cytokine production profile with increased IL-2 and IFN-gamma levels. These results demonstrate that Kaposi's sarcoma tumor cells lacking co-stimulatory molecules cannot induce T-cell activation; however, if they express CD80, they can induce peripheral blood T-cell proliferation, and there is a differential response as expression of CD86 did not have the same immunostimulatory effect.

Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewisx moieties.

The complement inhibitor soluble complement receptor type 1 (sCR1) and a truncated form of sCR1, sCR1[desLHR-A], have been generated with expression of the selectin-reactive oligosaccharide moiety, sialyl Lewisx (sLex), as N-linked oligosaccharide adducts. These modified proteins, sCR1sLex and sCR1[desLHR-A]sLex, were assessed in the L-selectin- and P-selectin-dependent rat model of lung injury following systemic activation of complement by cobra venom factor and in the L-selectin-, P-selectin-, and E-selectin-dependent model of lung injury following intrapulmonary deposition of IgG immune complexes. In the cobra venom factor model, sCR1sLex and sCR1[desLHR-A]sLex caused substantially greater reductions in neutrophil accumulation and in albumin extravasation in lung when compared with the non-sLex-decorated forms. In this model, increased lung vascular binding of sCR1sLex and sCR1[desLHR-A]sLex occurred in a P-selectin-dependent manner, in contrast to the absence of any increased binding of sCR1 or sCR1[desLHR-A]. In the IgG immune complex model, sCR1[desLHR-A]sLex possessed greater protective effects relative to sCR1[desLHR-A], based on albumin extravasation and neutrophil accumulation. Enhanced protective effects correlated with greater lung vascular binding of sCR1[desLHR-A]sLex as compared with the non-sLex-decorated form. In TNF-alpha-activated HUVEC, substantial in vitro binding occurred with sCR1[desLHR-A]sLex (but not with sCR1[desLHR-A]). This endothelial cell binding was blocked by anti-E-selectin but not by anti-P-selectin. These data suggest that sLex-decorated complement inhibitors have enhanced antiinflammatory effects and appear to have enhanced ability to localize to the activated vascular endothelium.

High-level variability in the ORF-K1 membrane protein gene at the left end of the Kaposi's sarcoma-associated herpesvirus genome defines four major virus subtypes and multiple variants or clades in different human populations.

Infection with Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is common in certain parts of Africa, the Middle East, and the Mediterranean, but is rare elsewhere, except in AIDS patients. Nevertheless, HHV8 DNA is found consistently in nearly all classical, endemic, transplant and AIDS-associated KS lesions as well as in some rare AIDS-associated lymphomas. The concept that HHV8 genomes fall into several distinct subgroups has been confirmed and refined by PCR DNA sequence analysis of the ORF-K1 gene encoding a highly variable glycoprotein related to the immunoglobulin receptor family that maps at the extreme left-hand end of the HHV-8 genome. Among more than 60 different tumor samples from the United States, central Africa, Saudi Arabia, Taiwan, and New Zealand, amino acid substitutions were found at a total of 62% of the 289 amino acid positions. These variations defined four major subtypes and 13 distinct variants or clades similar to those found for the HIV ENV protein. The B and D subtype ORF-K1 proteins differ from the A and C subtypes by 30 and 24%, respectively, whereas A and C differ from each other by 15%. In all cases tested, multiple samples from the same patient were identical. Examples of the B subtype were found almost exclusively in KS patients from Africa or of African heritage, whereas the rare D subtypes were found only in KS patients of Pacific Island heritage. In contrast, C subtypes were found predominantly in classic KS and in iatrogenic and AIDS KS in the Middle East and Asia, whereas U.S. AIDS KS samples were primarily A1, A4, and C3 variants. We conclude that this unusually high diversity, in which 85% of the nucleotide changes lead to amino acid changes, reflects some unknown powerful biological selection process that has been acting preferentially on this early lytic cycle membrane signalling protein. Two distinct levels of ORF-K1 variability are recognizable. Subtype-specific variability indicative of long-term evolutionary divergence is both spread throughout the protein as well as concentrated within two 40-amino-acid extracellular domain variable regions (VR1 and VR2), whereas intratypic variability localizes predominantly within a single 25-amino-acid hypervariable Cys bridge loop and apparently represents much more recent changes that have occurred even within specific clades. In contrast, numerous extracellular domain glycosylation sites and Cys bridge residues as well as the ITAM motif in the cytoplasmic domain are fully conserved. Overall, we suggest that rather than being a newly acquired human pathogen, HHV8 is an ancient human virus that is preferentially transmitted in a familial fashion and is difficult to transmit horizontally in the absence of immunosuppression. The division into the four major HHV8 subgroups is probably the result of isolation and founder effects associated with the history of migration of modern human populations out of Africa over the past 35,000 to 60,000 years.

Distinct biology of Kaposi's sarcoma-associated herpesvirus from primary lesions and body cavity lymphomas.

The DNA sequence for Kaposi's sarcoma-associated herpesvirus was originally detected in Kaposi's sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi's sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi's sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt's B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi's sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi's sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi's sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi's sarcoma-associated herpesvirus isolates from primary Kaposi's sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi's sarcoma.

Geographically distinct HHV-8 DNA sequences in Saudi Arabian Iatrogenic Kaposi's sarcoma lesions.

A new member of the gamma-herpesvirus family, HHV-8 (also known as Kaposi's sarcoma (KS)-associated herpesvirus), has been linked to KS and body cavity-based lymphoma. Other members of this family, eg, Epstein-Barr virus, were originally thought to have only one strain, but subsequent analysis revealed different strains correlating to cellular patterns of infectivity and geographical location. To determine whether multiple strains of HHV-8 exist, we compared DNA sequences among KS and body cavity-based lymphoma-derived HHV-8 and examined differences in HHV-8 subgroups between American and Saudi Arabian iatrogenic KS patients. Samples were analyzed by polymerase chain reaction using multiple primer sets to five different open reading frames from HHV-8, and DNA sequencing was performed. HHV-8 DNA was present in all of our KS and body cavity-based lymphoma samples by polymerase chain reaction. HHV-8 DNA was detected in each body cavity-based lymphoma sample using a majority of the primers, whereas only two primer sets consistently amplified HHV-8 DNA derived from KS lesions. DNA sequencing within open reading frames 26 and 27 indicate the existence of at least three variants of HHV-8, with the majority of iatrogenic KS patients in Saudi Arabia containing unique nucleotide changes that may define a distinct, previously unidentified subgroup we term SA, whereas those from America were of Group A or B. Thus, although the sequencing data within open reading frames 26 and 27 did not permit discrimination between patients with lymphoma versus KS disease processes, HHV-8 derived from Saudi Arabian KS lesions were shown to have a distinct nucleotide sequence not seen in any of the other clinical samples examined.

Molecular and pathologic characterization of an AIDS-related body cavity-based lymphoma, including ultrastructural demonstration of human herpesvirus-8: a case report.

Body cavity-based lymphoma, also known as primary effusion lymphoma, is a newly recognized acquired immunodeficiency syndrome (AIDS)-related lymphoma that has been linked to the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). To date, direct visualization of the virus in a clinical sample has not been demonstrated. We have performed an extensive clinical, histologic, immunophenotypic, ultrastructural, and molecular genetic correlative study on multiple tissue samples obtained premortem and at autopsy from an patient with AIDS with Kaposi's sarcoma and body cavity-based lymphomas. We demonstrate the presence of human herpesvirus-8 in a primary clinical sample at the ultrastructural and molecular level, as well as document multiple lymphomatous tumor masses at autopsy.

In situ polymerase chain reaction-based localization studies support role of human herpesvirus-8 as the cause of two AIDS-related neoplasms: Kaposi's sarcoma and body cavity lymphoma.

Several lines of investigation point to a new herpesvirus, human herpesvirus-8 (HHV-8), as the cause of two different neoplasms seen in AIDS patients-Kaposi's sarcoma (KS) and body cavity B cell lymphoma. If this virus is the etiological agent, rather than another opportunistic infectious agent, it should be present in the earliest detectable clinical lesions on a temporal basis, and localize to specific target cells in a spatial pattern consistent with tumorigenic pathways. In this study, we take advantage of the clinical accessibility to biopsy early (patch stage) skin lesions of KS to address the temporal issue, combined with in situ PCR and dual immunostaining using a marker identifying malignant cells, to address the spatial localization issue. 21 different tissue samples were subjected to PCR analysis and in situ PCR with and without simultaneous immunostaining. In normal skin from healthy individuals, no HHV-8 DNA was detected by PCR or in situ PCR. However, in all PCR-positive tissues, distinct and specific in situ PCR staining was observed. In four different patch stage KS lesions, in situ PCR staining localized to nuclei of endothelial cells and perivascular spindle-shaped tumor cells. Later stage KS lesions (plaques and nodules) revealed additional positive cells, including epidermal keratinocytes (four of five), and eccrine epithelia (two of four). These patterns were nonrestricted to skin, as pulmonary KS also revealed HHV-8-specific infection of endothelial cells and KS tumor cells, as well as epithelioid pneumocytes (two of two). In body cavity B cell lymphoma by dual staining, HHV-8 was present in malignant tumor cells (EMA immunostained positive) and not in reactive lymphocytes. These results reveal an early temporal onset and nonrandom tissue and cellular distribution pattern for HHV-8 infection that is consistent with a causal link between this DNA virus and two AIDS-related neoplasms.

Apoptosis in keratinocytes is not dependent on induction of differentiation.

During embryological development and throughout life, regulation of the thickness of skin is likely to involve modulation of keratinocyte proliferation, differentiation, and cell death. One major mechanism of cell death is apoptosis; but the precise relationship between apoptosis and differentiation has not been well-defined. In this report, we demonstrate that when cultured undifferentiated keratinocytes have their adhesive interactions interrupted by either enzymatic treatment (ie, trypsin) and suspension in a semi-solid methyl cellulose medium, or exposure to antibodies against beta 1 integrins and E-cadherin, induction of differentiation occurs (expression of involucrin), as well as apoptosis (positive terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP-biotin nick end labeling (TUNEL) assay and DNA fragmentation). However, these events are not directly interdependent processes, as determined by two-color immunofluorescence staining. Thus, apoptosis can occur without evidence of differentiation and vice versa. The process o apoptosis in keratinocytes was dissected at the molecular level and found to be correlated with increased expression of Bax and decreased levels of Bcl-XL, with no role for either Bcl-2 or Bcl-XS. We conclude that keratinocytes do not need to undergo differentiation before undergoing apoptosis.

Propagation of a human herpesvirus from AIDS-associated Kaposi's sarcoma.

BACKGROUND: Although unique DNA sequences related to gammaherpesviruses have been found in Kaposi's sarcoma lesions, it is uncertain whether this DNA encodes a virus that is able to reproduce. METHODS: We isolated and propagated a filterable agent whose DNA sequences were found to be identical to those of the Kaposi's sarcoma-associated herpesvirus (KSHV). We obtained early-passage spindle cells from skin lesions of patients with the acquired immunodeficiency syndrome (AIDS) who had Kaposi's sarcoma and cultured them with cells of the human embryonal-kidney epithelial-cell line 293. We characterized the virus according to its effects on cellular morphology and viral replication and its appearance on electron microscopy. RESULTS: KSHV was cytotoxic to 293 cells and was detected by the polymerase chain reaction (PCR) in infected cells but not uninfected ones. Cytotoxicity and positive PCR signals were consistently maintained with viral titers of 1 million per milliliter, for about 20 serial infections of 293 cells. The viral copy number was relatively low (1 to 10 copies per cell). Viral replication was confirmed by Southern blot analysis of DNA isolated from the enriched nuclear fraction of infected cells and by a semiquantitative PCR using dilutions of the lysates of infected cells to detect the 233-bp viral DNA fragment originally described in association with Kaposi's lesions. Electron microscopy revealed herpesvirus-like particles in about 1 percent of cells from infected cultures, as compared with none in cells from uninfected cultures. CONCLUSIONS: A herpesvirus with DNA sequences identical to those of KSHV can be propagated from skin lesions of patients with AIDS-associated Kaposi's sarcoma.

Phenotype and proliferation characteristics of cultured spindle-shaped cells obtained from normal human skin and lesions of dermatofibroma, Kaposi's sarcoma, and dermatofibrosarcoma protuberans: a comparison with fibroblast and endothelial cells of the dermis.

Normal human dermis contains mesenchymal cells that are generally referred to as fibroblasts. However the relationships between fibroblasts and endothelial cells with respect to the types of spindle-shaped cells that are present in cultures obtained from tumor bearing-skin is unclear. To explore the potential heterogeneity amongst dermal-derived cells that grow in culture with a spindle-shaped morphology, we compared the immunophenotype and growth characteristics of several types of cells. Besides dermal fibroblasts and microvascular endothelial cells derived from normal adult skin, we also studied large vessel-derived endothelial cells, and spindle-shaped cells derived from three different tumor-bearing dermal-based neoplasms. Kaposi's sarcoma (KS), dermatofibroma (DF), and dermatofibrosarcoma protuberans (DFSP). A broad panel of eight different antibodies were used to immunophenotype the multi-passaged cultured cells. Spindle-shaped cells from all three neoplasms could be distinguished from the normal skin derived fibroblasts by their constitutive expression of factor XIIIa, and the gamma-interferon induced expression of VCAM-1. All seven types of cultured cells stained positive for s-actin and proline-4-hydroxylase, and none of the cells expressed CD34. Both large and small-vessel derived endothelial cells expressed factor VIII, ELAM-1, and VCAM-1. Using two different types of growth media, significant differences were also observed amongst these cultured cell types. Spindle-shaped cells from DFSP did not grow in DMEM containing 10% fetal bovine serum (DMEM-FBS); but they proliferated in KS cell growth medium (KSGM). Spindle-shaped cells from DF grew best in KSGM, but not in DMEM-FBS. KS tumor cells grew well in KSGM, but not in DMEM-FBS. Fibroblasts proliferated in DMEM-FBS, but failed to grow in KSGM; and even when pre-treated with conditioned medium from a transformed KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that even though dermal-derived cells can have an identical spindle-shape by light microscopy, significant heterogeneity can be defined amongst such cells from normal and tumor-bearing human skin. Having established culture conditions to propagate these different cell types and phenotypic criteria to distinguish them from one another, will provide new research opportunities to explore the function and ontogeny of the diverse mesenchymal cells that take on a spindle-shaped morphology in culture.

Kaposi's sarcoma tumor cells preferentially express Bcl-xL.

Several recently identified proteins such as Bcl-2 and Bcl-x have been found to regulate programmed cell death (i.e., apoptosis). In this report, we examined the levels of expression of proteins that can either prevent apoptosis (i.e., Bcl-2 or the long form of Bcl-x, designated Bcl-x1) or promote apoptosis (i.e., Bax or the short form of Bcl-x, designated Bcl-xs) in proliferating benign and malignant endothelial cells (ECs). In normal skin with quiescent ECs, no detection by immunohistochemical staining was observed for Bcl-xL, Bcl-xs, or Bcl-2. However, in diseased skin samples that feature a prominent angiogenic response such as in psoriasis or pyogenic granulomas, the proliferating ECs markedly overexpressed Bcl-xL, with little to no Bcl-2. In an acquired-immune-deficiency-syndrome-related neoplasm, Kaposi's sarcoma, the spindle-shaped tumor cells also overexpressed Bcl-xL compared with Bcl-2. These in vivo studies were extended in vitro using cultured ECs and Kaposi's sarcoma tumor cells that were examined by flow cytometry and immunoblot analysis. Both cultured ECs and Kaposi's sarcoma tumor cells express significantly higher levels of Bcl-xL than Bcl-2. Such overexpression of cell survival gene products may contribute to prolonging the longevity of EC-derived cells in several different benign and neoplastic skin disorders that are characterized by a prominent angiogenic tissue response.