Affiliations of authors: O. Prakash, Z.-Y. Tang, Y. He, M. S. Ali, R. Coleman (Laboratory of Molecular Oncology), J. Gill, G. Farr (Department of Pathology), Alton Ochsner Medical Foundation, New Orleans, LA; F. Samaniego, Institute of Human Virology and Greenbaum Cancer Center, University of Maryland, Baltimore.
Correspondence to: Om Prakash, Ph.D., Laboratory of Molecular Oncology, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, LA 70121 (e-mail: oprakash{at}ochsner.org).
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ABSTRACT |
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INTRODUCTION |
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The HIV-1 Tat is an 86101 amino-acid protein encoded by two exons [reviewed in (5)]. The product of the first exon (72 amino acids) is sufficient for transactivation of the HIV-1 gene expression from the viral promoter. However, the C-terminal (7386/101 amino acids) encoded by the second exon contains the arginineglycineaspartate (RGD) sequence that is believed to function primarily as a cell-attachment domain and binds cell-surface integrin receptors (6,7). Studies (8,9) have shown that Tat protein released into the extracellular medium by HIV-1 infection of T cells stimulates the growth of AIDSKS-derived spindle cells. Tat also stimulates the growth of and induces spindle morphology in normal endothelial cells that have been pretreated with inflammatory cytokines (10). Tat-induced migration, invasion, proliferation, and collagenase IV expression in KS spindle cells and in cytokine-activated endothelial cells in culture indicated that Tat can support angiogenesis (10,11). The in vivo angiogenic response of Tat has been demonstrated by subcutaneous injection of purified Tat in mice (11,12) and in transgenic mice expressing full-length Tat protein (1315). The angiogenic properties of Tat have been mapped to its RGD sequence (6,7,11) and to the basic amino-acid region that is similar to that of angiogenic growth factors and binds heparin complexes (16,17). Basic fibroblast growth factor (bFGF) and heparin have been shown to significantly enhance the in vivo angiogenic effects of Tat (11,16). bFGF and Tat are present in AIDSKS lesions, and integrin receptors that bind Tat are highly expressed by vessels and spindle cells.
Histologically, KS lesions consist of a heterogeneous population of cells, including characteristic aggregates of spindle-shaped cells, fibroblasts, dendritic cells, and a prominent infiltrate of leukocytes and other inflammatory cells (18). A number of studies [(18); reviewed in (19)] suggest that infiltrating leukocytes in AIDSKS lesions could be a possible source of HIV-1 Tat, inflammatory cytokines, and HHV8, thereby contributing to the aggressive and rapid growth of tumors. HIV-1 infection of leukocytes is also known to induce synthesis and secretion of matrix metalloproteinases (MMPs), the enzymes that degrade components of the extracellular matrix, and aid cells in traversing the tissues (2022). More recently, HIV-1 Tat protein alone has been shown to increase the production of MMP-9 (92-kd MMP) in monocytes and also to increase their adhesion to endothelial cells (23), causing them to extravasate into lesions.
In this study, we used transgenic mice expressing two-exon-encoded Tat (Tat86) and one-exon-encoded Tat (Tat72) to investigate whether Tat expression will promote the growth and tumorigenic potential of human KS SLK cells subcutaneously injected into these mice. These findings may be relevant for the understanding of the aggressive and rapid course of KS in HIV-1-infected individuals and for the development of targeted anti-AIDSKS therapies.
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MATERIALS AND METHODS |
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Transgenic mice expressing one-exon-encoded Tat protein (Tat72) and two-exon-encoded Tat protein (Tat86) under the transcriptional control of simian virus 40 promoter were generated as previously described (24). The heterozygous lines of mice established from the transgenic founders expressed Tat protein in several tissues, including bone marrow cells (24). The Tat protein was also biologically active as determined by the transactivation of the HIV-1 long terminal repeat (LTR) in doubly transgenic mice carrying both Tat and chloramphenicol acetyltransferase (CAT) reporter gene under the transcription control of the LTR (LTRCAT) (24). Tat transgenic mice were routinely identified by the Southern blot analysis of the tail DNA.
Injection of Human KS Cells in Mice
The KS SLK cell line established from a KS lesion of gingiva of an HIV-1-negative immunosuppressed renal transplant recipient (25) was maintained in gelatin-coated flasks as previously described (26). The SLK cell line is of endothelial origin and, when inoculated in nude mice, induces tumors with endothelial characteristics (27). Approximately 1 x 106 KS SLK cells were mixed with liquid Matrigel (Collaborative Biomedical Products, Bedford, MA) in a final volume of 0.5 mL and injected subcutaneously near the abdominal midline of immunosuppressed Tat transgenic and nontransgenic mice. For activation experiments, KS SLK cells were treated with 10 ng/mL of phorbol 12-myristate 13-acetate (PMA) for 1 hour in culture followed by incubation in medium without PMA for 4 hours before injection in mice. PMA (10 ng/mL) was incorporated in the Matrigel before injection, where indicated. Immunosuppression in mice was induced by weekly intraperitoneal injections of 0.3 mg of GK1.5 (American Type Culture Collection, Manassas, VA) antibody, a rat monoclonal antibody targeting the CD4 molecule (28). The antibody was partially purified from ascites fluid by the ammonium sulfate precipitation procedure described previously (29). Flow cytometric analysis of splenic cells with fluorescein isothiocyanate-conjugated anti-CD4 antibody (Pharmingen, San Diego, CA) showed more than a 85% loss of CD4+ T cells 57 days after anti-CD4 antibody treatment. Mice were killed to recover Matrigel implants, when necessary, for histologic examination or other procedures.
Gelatin Zymography
For zymography, tumor and tissue extracts were prepared by homogenization and sonication in 0.01 M Tris buffer (pH 7.0) containing 0.01% Triton X-100 followed by centrifugation at 10000 rpm for 10 minutes at 4°C. The protein content of cell-free supernatants was determined by the Bio-Rad protein assay method (Bio-Rad Laboratories, Hercules, CA) (30). Gelatinase activity was assayed by electrophoresis of tissue or cell extracts containing 20 µg protein on 10% polyacrylamide gels with 0.1% gelatin (NOVEX, San Diego, CA), according to the supplier's instructions. After electrophoresis, the gels were incubated overnight in developing buffer and stained with Coomassie Blue. Proteolytic activity was detected as unstained bands against a blue background. To characterize these bands as MMPs, gels were incubated with developing buffer containing 20 mM EDTA, where indicated.
Isolation of Mouse Mononuclear Cells and Polymorphonuclear Leukocytes
Mononuclear cells (MNCs) and polymorphonuclear leukocytes (PMNs) from mouse blood and bone marrow were isolated by use of a two-component step gradient procedure (Cardinal Associates, Inc., Santa Fe, NM). A light microscopic examination revealed that the isolated PMN and MNC fractions from blood contained 70% and 4% segmented neutrophils, respectively. On the other hand, the PMN and MNC fractions from bone marrow contained 54% and 80% neutrophils, respectively.
RNA Extraction and Reverse TranscriptionPolymerase Chain Reaction for Quantitation of Tat and Cytokine Messenger RNAs From Tumor Tissues
Total cellular RNA from liquid nitrogen-frozen tissues was extracted by use of the Rneasy Mini Kit (Qiagen, Valencia, CA), according to the supplier's instructions. To eliminate DNA contamination, extracted RNA was treated with ribonuclease-free deoxyribonuclease. The final preparation was quantitated spectrophotometrically, examined for the presence of 18S and 28S ribosomal RNA bands, and stored at -70°C until use.
Reverse transcription (RT) of the RNA (500 ng) was performed in a final volume of 20 µL containing 50 mM TrisHCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 µM each of the deoxynucleotide triphosphates, 200 ng random primers (Promega Corp., Madison, WI), and 200 U of reverse transcriptase (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). The reaction mixture was incubated at 42°C for 1 hour and then heated at 94°C for 10 minutes to inactivate the enzyme. One microliter of this reverse transcribed mixture was used for polymerase chain reaction (PCR) amplification of the complementary DNA in a 50-µL reaction volume.
The primers for the PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), human nuclear factor-kappa B (p50) (NF-B [p50]) and all of the human cytokines, except bFGF, and their positive-control PCR products were obtained from Clontech Laboratories, Inc. (Palo Alto, CA). The primer and oligoprobe sequences for bFGF, Tat86, and Tat72 were obtained from the published sequence and were as follows: bFGF (GenBank, Accession No. EO2544), 5'-GGCTTCTTCTTCCTGCGCATCCAC-3', 5'-CTGCCCAGTTCGTTTCAGTGC-3', and 5'-TGGCTTCTAAATGTGTTACGGATGAGTGTT-3'; Tat86 (31), 5'-CCTACCTGCCATGGAACCAGTCGACCCTA-3', 5'-GATCCTTACTATTCCTTAGGACCTGTCG-3', and 5'-GGCTTTGGGTATCTCCTACGG-3'; and Tat72 (32), 5'-TCGACCCGCGTCTGGAACCATTGGAAAC-3', 5'-AGAGAAACCTGGTGGGTCTGGGA-3', and 5'-CGGCCGTAAAAAACGTCGTCA-3'. The PCR amplification was carried out for 30 cycles by use of Perkin-Elmer (The Perkin-Elmer Corp., Foster City, CA) PCR reagents in a 50-µL volume and a DNA Thermal Cycler Model 9700 according to the PCR protocol provided by Clontech Laboratories, Inc. To ascertain that equal amounts of tumor RNAs were used in each RTPCR reaction, the same RT mixture was used for concurrent amplification of GAPDH.
The amplified RTPCR products were detected by Southern blot analysis of 25 µL (5 µL for Tat86 and GAPDH) of the final reaction mixture by use of the 32P-labeled positive internal PCR products or oligoprobes. The intensity of the resultant bands on the autoradiograms was quantitated by use of the Molecular Dynamics PhoshoImager Model 840 (Molecular Dynamics Corp., Sunnyvale, CA).
Statistical Analysis
Student's t test was applied to estimate the statistical significance of difference (33) in the tumor weights from nontransgenic mice, Tat72-, and Tat86-transgenic mice. All P values were two-sided.
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RESULTS |
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Earlier studies (24) have shown that the transgenic mice carrying the one-exon Tat gene (Tat72) and the two-exon Tat gene (Tat86) express detectable levels of Tat protein in several tissues. Tat is also biologically active in these mice, as seen by the transactivation of the HIV-1 LTR in doubly transgenic mice carrying both Tat and LTRCAT transgenes (24). To determine whether HIV-1 Tat expression influences the tumorigenic potential of human KS cells, we compared the tumorigenicity of KS SLK cells in nontransgenic and Tat-transgenic mice. All nontransgenic and Tat-transgenic mice developed tumors in 34 weeks after inoculation with KS SLK cells. However, the tumor growth response in the three lines of mice was different. In general, the tumor growth in Tat86 mice was considerably higher than that in nontransgenic animals, whereas an intermediate growth response was seen in Tat72 mice (Fig. 1). A statistical analysis performed on the weight differences in tumors obtained from each of the lines of mice revealed a sixfold to sevenfold higher tumor growth in Tat86 mice (~600 mg) compared with nontransgenic mice (~100 mg) and a twofold to threefold higher tumor growth in Tat72 mice (~200 mg) (Fig. 2
). The average tumor weight in Tat86 mice was statistically significantly greater than that in the other two groups (P<.005). The observed difference between the Tat72 and the nontransgenic groups was not statistically significant (P = .1). These observations clearly indicated that Tat86 and, to a lesser extent, Tat72 promote the growth of KS SLK cell-derived tumors in Tat transgenic mice.
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Numerous studies [reviewed in (4)] have suggested that inflammatory cytokines that are elevated in HIV-1-infected individuals and cytokines produced by AIDSKS cells cooperate with HIV-1 Tat in the development of AIDSKS. To test the possibility that Tat and certain inflammatory cytokines might play a role in the enhanced tumor growth in Tat-transgenic mice, we measured the expression of a number of cytokines, such as interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1 (IL-1
), interleukin 1ß (IL-1ß), tumor necrosis factor-
(TNF-
), interferon gamma (IFN
), granulocytemacrophage colony-stimulating factor (GMCSF), and bFGF. Since the two-exon-encoded Tat is the predominant form of Tat produced during HIV-1 infection and the observed difference between the tumors in one-exon-encoded Tat (Tat72) and the nontransgenic group was not statistically significant, we randomly selected four tumors from nontransgenic and Tat86-transgenic mice for analysis by the semiquantitative RTPCR method as described in the "Materials and Methods" section. As shown in Fig. 4
, all of the tumors from Tat-transgenic mice showed the expression of Tat mRNA, suggesting that the infiltrating mouse cells actively expressed Tat. The comparative profile of cytokine expression in tumors from the Tat-transgenic and nontransgenic mice showed substantial differences, although GAPDH expression in all of the tumors was comparable. Four of four tumors from Tat-transgenic mice showed expression of TNF-
, IL-6, IL-8, and bFGF compared with only two tumors from the nontransgenic group. The level of mRNA expression was also higher in the tumors from Tat mice than in the tumors from nontransgenic mice. Of interest, three of the four tumors from Tat mice showed expression of GMCSF, but no expression was detectable in the tumors from nontransgenic animals. The expression of IL-2, IL-4, IL-1ß, and IFN
mRNAs was undetectable in all tumors. The transcription factor NF-
B is known to play a key role in the expression of a number of genes involved in inflammatory responses and cellular growth regulation (34,35). The most abundant form of NF-
B is a complex composed of p50p65 heterodimer belonging to the Rel family. In most cells, NF-
B/Rel proteins are sequestered in the cytoplasm by any of several inhibitor proteins I
B. Activation of NF-
B involves I
B degradation and subsequent translocation of NF-
B/Rel proteins to the cell nucleus. Earlier reports (3638) have indicated that extracellular Tat can induce the activation of NF-
B. In this study, we found that NF-
B p50-related transcripts in three of the four tumors from Tat mice were substantially elevated, whereas negligible transcription was observed in the tumors from nontransgenic animals. The results of cytokine mRNA analysis suggested that exposure of KS SLK cells to extracellular Tat and to certain inflammatory cytokines might play a role in the enhanced growth of KS SLK cell-derived tumors in Tat mice.
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The KS-derived cells in culture have been shown to constitutively release MMPs into the culture supernatants, suggesting that these cells constitutively have an invasive and metastasizing potential (39). To determine whether tumors from Tat-transgenic mice produced increased levels of MMPs that contributed to more aggressive tumor growth, we performed gelatin zymography. As shown in Fig. 5, AD, only one of 10 tumors in nontransgenic mice showed a 105-kd MMP band (panel A), whereas all of the tumors from Tat86 mice showed an intense band of 105-kd (panel B). A similar band was observed in four of 10 tumors from Tat72 mice (panel C). All members of the MMP family contain a zinc ion at the catalytic site, require Ca2+ for full activity, and are inhibited by chelating agents [reviewed in (40)]. Inhibition with 20 mM EDTA (panel D) showed that the 105-kd band was associated with MMP activity. Blankaert et al. (39) have reported that human KS cells produce a 92-kd MMP activity. We also found a similar activity produced by the KS SLK cells (not shown), suggesting that the 105-kd specific activity in our study originated from infiltrating mouse cells in the tumors. We next screened mouse tissues to determine the source of the 105-kd MMP activity. As shown in Fig. 6
, A, of all the tissues examined, abundant activity was detectable only in the bone marrow. For the determination of the cell type specificity of the 105-kd MMP, bone marrow cells were fractionated into MNCs and PMNs by gradient centrifugation, and the fractions were analyzed by gelatin zymography. We also determined if a similar activity was present in MNC and PMN fractions collected from mouse blood. As shown in Fig. 6
, B (lanes 14), the 105-kd MMP activity was detectable almost entirely in the blood-derived PMN fraction (lane 2); only a negligible activity was seen in the MNC fraction (lane 1). In contrast, in the bone marrow, substantial activity was observed in both fractions (lanes 3 and 4). A light microscopic examination (Fig. 7
, AD) of the MNC and PMN fractions from blood and bone marrow from tumor-bearing and control mice revealed that PMN fractions (Fig. 7
, A and C) were mainly (>75%) composed of segmented neutrophils, suggesting that these neutrophils might be the source of 105-kd MMP activity. The abundance of MMP activity in the MNC fraction obtained from bone marrow (Fig. 7
, D) might also be attributed to segmented neutrophils, since these cells were abundantly present in this fraction. On the other hand, a limited presence (~4%) of segmented neutrophils in the MNC fraction from blood (Fig. 7
, B) was associated with the negligible 105-kd MMP activity. In conclusion, the results of these experiments suggested that the 105-kd MMP activity predominantly observed in the tumors from Tat86-transgenic mice might be associated with the infiltrating host neutrophils.
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The KS lesions contain a prominent infiltrate of blood cells. These cells in HIV-1-infected individuals could contribute to tumor growth through the release of inflammatory cytokines and Tat protein. To determine whether the KS SLK cells attract and bind circulating leukocytes that secrete 105-kd MMP, we inoculated KS SLK cells mixed with Matrigel into nontransgenic mice and assessed the extent of leukocyte infiltration. Subcutaneously injected Matrigel alone was used as a control. As shown in Fig. 8 (lanes 14), the KS SLK cell pellet showed substantially higher gelatinase activity than the Matrigel pellet (compare lanes 3 and 1). The activity was further increased substantially (lane 4) by pretreatment of the KS cells in culture with PMA, whereas PMA alone in Matrigel had a minimal effect (lane 2). The changes in the MMP activity (lanes 3 and 4) were associated with the extravasation of leukocytes (not shown) into the SLK cell pellets. Since PMA, lipopolysaccharides, and certain cytokines, such as TNF-
, IL-1ß, and IL-8, are known to increase the chemotaxis and adhesion of leukocytes to primary endothelial cells and KS cells (4143), our observations suggested that activation of KS SLK cells increased their adhesion for leukocytes that could, in turn, stimulate a local inflammatory response for the development of KS.
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DISCUSSION |
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In addition to other sources of inflammatory cytokine production in HIV-1-infected individuals [reviewed in (4)], Tat itself has been shown to modulate the expression of a number of cytokines. Lafrenie et al. (46) have shown that treatment of monocytes with purified Tat in culture enhances the production of IL-1ß, IL-6, IL-8, and TNF- and their respective mRNAs. In other studies, induced cellular expression of Tat in cells has been shown to increase the expression of IL-2 (47), IL-6 (48), and TNF-ß (49,50). Tat expression in transgenic mice has been shown to increase the expression of TNF-ß and TGF-ß (51) as well as IL-6 and IL-10 (52,53). In our study, the HIV-1 Tat protein could promote the growth of SLK cell-derived tumors in Tat-transgenic mice in a number of ways. Since the tumors contain a prominent infiltrate of mouse blood cells, these cells could provide exogenous Tat. As shown in Fig. 4
, Tat mRNA is abundantly expressed in the tumors from Tat86 mice. We have previously reported that these mice also show readily detectable levels of Tat protein in most tissues, including the bone marrow cells (24). In addition to bFGF expression, these tumors also show substantial increases in the mRNA levels for TNF-
, IL-6, IL-8, and GMCSF. These are some of the cytokines that have an autocrine/paracrine role in the proliferative activity of AIDSKS, and their activity is further augmented in the presence of Tat protein [reviewed in (4)].
Several reports (36,37,54) have demonstrated that extracellular Tat is associated with activation of transcription factor NF-B. The major form of NF-
B is composed of a p50 and p65 heterodimer. In this study, we found that the expression of p50 transcripts is significantly increased in tumors from Tat mice but not in tumors from nontransgenic mice. Although we have not examined the expression of the p65 subunit, its stochiometric appearance with p50 (55) suggests that the induction of the native NF-
B complex might be increased as well in the tumors from Tat mice. NF-
B is involved in the transcriptional regulation of a number of inflammatory cytokines [reviewed in (56,57)]. Aberrant NF-
B activity is also associated with enhanced cell proliferation and oncogenesis. For example, constitutive NF-
B activation is required for proliferation and survival of many cells, including breast cancer cells (58) and Hodgkin's lymphoma cells (59). Consistent with these observations, it is conceivable that NF-kB might participate in tumor progression in Tat transgenic mice.
Tat contains several functional domains. Previous studies (7,15) have shown that binding of extracellular Tat to the cell surface is mediated by two distinct domains: a basic domain (amino acids 4264) and a C-terminal domain (amino acids 6580) containing the RGD sequence that binds cell-surface intergrin receptors (6). The same intergrins are highly expressed by KS spindle cells and are inducible by inflammatory cytokines on endothelial cells (7). The basic domain of Tat is similar to that of several growth factors [reviewed in (60)] and mediates binding of Tat to Flk-1/KDR, a receptor for vascular endothelial growth factor-A, which is a potent growth factor for AIDSKS cells (61). Interaction of extracellular Tat with these receptors can induce the growth and migration of endothelial and KS cells (7,11,17,60). In our study, the selective presence of RGD in Tat86 might explain the enhanced tumor growth in Tat86 mice over that in Tat72 mice.
Numerous studies (2022) have suggested that, in addition to providing inflammatory cytokines and viral Tat, the infiltrating HIV-1-infected leukocytes in KS lesions could also be a source of MMPs, which are essential for tumor invasion and metastasis. MMP secretion has been shown to be increased in HIV-1-infected T lymphocytes, monocytes, and macrophages (2022). In addition, treatment of monocytes with soluble Tat in culture has been shown to increase the production of MMP-9 (23). In this study, we found that the mouse neutrophil-associated 105-kd gelatinase activity was significantly increased in all of the tumors from Tat86 mice. Although the mechanism of its activation remains to be explored, our observations suggest that this MMP might also play a role in the accelerated growth of KS SLK cell-derived tumors in Tat86 mice.
In conclusion, our in vivo study strengthens the role of extracellular Tat and inflammatory cytokines on the pathogenesis of AIDSKS and may provide one of the explanations for why KS is a more aggressive disease in HIV-infected individuals. Our transgenic mouse model may be useful to study the efficacy of the anti-KS therapies, especially those targeting Tat activity.
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NOTES |
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Supported in part by a grant from the AIDS Crisis Trust (O. Prakash).
We thank John and Mary Lou Ochsner and Marguerite Littman for supporting this study. We also thank Peng Zhou for technical assistance, Nicholas Lanson for assistance in the phosphoimaging analysis of the reverse transcriptionpolymerase chain reaction data, and Robert Gallo for his encouragement in this work and for his careful reading of the manuscript.
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REFERENCES |
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---|
1 Beral V, Peterman TA, Berkelman RL, Jaffe HW. Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet 1990;335:1238.[Medline]
2
Gallo RC. The enigmas of Kaposi's sarcoma. Science 1998;282:18379.
3 Vogel J, Hinrichs SH, Reynolds RK, Luciw PA, Jay G. The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice. Nature 1988;335:60611.[Medline]
4 Samaniego F, Gallo RC. Immunopathogenesis of Kaposi's sarcoma. In: Gupta S, editor. Immunology of HIV infection. New York (NY). Plenum Press; 1996. p. 43750.
5 Jones KA, Peterlin MB. Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem 1994,63:71743.[Medline]
6 Brake DA, Debouck C, Biesecker G. Identification of an ArgGlyAsp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat. J Cell Biol 1990;111:127581.[Abstract]
7
Barillari G, Gendelman R, Gallo RC, Ensoli B. The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence. Proc Natl Acad Sci U S A 1993;90:79415.
8 Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F. Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature 1990;345:846.[Medline]
9 Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Morgan RA, et al. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol 1993;67:27787.[Abstract]
10
Barillari G, Buonaguro L, Fiorelli V, Hoffman J, Michaels F, Gallo RC, et al. Effects of cytokines from activated immune cells on vascular cell growth and HIV-1 gene expression. Implications of AIDSKaposi's sarcoma pathogenesis. J Immunol 1992;149:372734.
11 Ensoli B, Gendelman R, Markham P, Fiorelli V, Colombini S, Raffeld M, et al. Synergy between basic fibroblast growth factor and HIV-1 Tat protein in induction of Kaposi's sarcoma. Nature 1994;371:67480.[Medline]
12 Albini A, Fontanini G, Masiello L, Tacchetti C, Bigini D, Luzzi P, et al. Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2. AIDS 1994;8:123744.[Medline]
13 Corallini A, Altavilla G, Pozzi L, Bignozzi F, Negrini M, Rimessi P, et al. Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes. Cancer Res 1993;53:556975.[Abstract]
14 Corallini A, Campioni D, Rossi C, Albini A, Possati L, Rusnati M, et al. Promotion of tumour metastases and induction of angiogenesis by native HIV-1 Tat protein from BK virus/tat transgenic mice. AIDS 1996;10:70110.[Medline]
15 Albini A, Benelli R, Presta M, Rusnati M, Ziche M, Rubartelli A, et al. HIV-tat protein is a heparin-binding angiogenic growth factor. Oncogene 1996;12:28997.[Medline]
16 Chang HC, Samaniego F, Nair BC, Buonaguro L, Ensoli B. HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 1997;11:142131.[Medline]
17 Albini A, Soldi R, Giunciuglio D, Giraudo E, Benelli R, Primo L, et al. The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells. Nat Med 1996;2:13715.[Medline]
18 Galea P, Frances V, Dou-Dameche L, Sampol J, Chermann JC. Role of Kaposi's sarcoma cells in recruitment of circulating leukocytes: implications in pathogenesis. J Hum Virol 1998;1:27381.[Medline]
19 Weeks BS. The role of HIV-1 activated leukocyte adhesion mechanisms and matrix metalloproteinase secretion in AIDS pathogenesis. Int J Mol Med 1998;1:3616.[Medline]
20 Weeks BS, Klotman ME, Holloway E, Stetler-Stevenson WG, Kleinman HK, Klotman PE. HIV-1 infection stimulates T cell invasiveness and synthesis of the 92-kDa type IV collagenase. AIDS Res Hum Retroviruses 1993;9:5138.[Medline]
21 Chapel C, Camara V, Clayette P, Salvat S, Mabondzo A, Leblond V, et al. Modulations of 92 kDa gelatinase B and its inhibitors are associated with HIV-1 infection in human macrophage cultures. Biochem Biophys Res Commun 1994;204:12728.[Medline]
22
Dhawan S, Weeks BS, Soderland C, Schnaper HW, Toro LA, Asthana SP, et al. HIV-1 infection alters monocyte interactions with human microvascular endothelial cells. J Immunol 1995;154:42232.
23 Lafrenie RM, Wahl LM, Epstein JS, Hewlett IK, Yamada K, Dhawan S. HIV-1-Tat modulates the function of monocytes and alters their interactions with microvessel endothelial cells. A mechanism of HIV pathogenesis. J Immunol 1996;156:163845.[Abstract]
24 Prakash O, Teng S, Ali M, Zhu X, Coleman R, Dabdoub RA, et al. The human immunodeficiency virus type 1 Tat protein potentiates zidovudine-induced cellular toxicity in transgenic mice. Arch Biochem Biophys 1997;343:17380.[Medline]
25 Siegal B, Levinton-Kriss S, Schiffer A, Sayar J, Engelberg I, Vonsover A, et al. Kaposi's sarcoma in immunosuppression. Possibly the result of a dual viral infection. Cancer 1990;65:4928.[Medline]
26
Popescu NC, Zimonjic DB, Leventon-Kriss S, Bryant JL, Lunardi-Iskandar Y, Gallo RC. Deletion and translocation involving chromosome 3 (p14) in two tumorigenic Kaposi's sarcoma cell lines. J Natl Cancer Inst 1996;88:4505.
27 Herndier BG, Werner A, Arnstein P, Abbey NW, Demartis F, Cohen RL, et al. Characterization of human Kaposi's sarcoma cell line that induces angiogenic tumors in animals. AIDS 1994;8:57581.[Medline]
28
Dialynas DP, Quan ZS, Wall KA, Pierres A, Quintans J, Loken MR, et al. Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule. J Immunol 1983;131:244551.
29 Araujo FG. Depletion of L3T4+ (CD4+) T lymphocytes prevents development of resistance to Toxoplasma gondii in mice. Infect Immun 1991;59:16149.[Medline]
30 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72: 24854.[Medline]
31 Arya SK, Guo C, Josephs SF, Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 1985;229:6973.[Medline]
32 Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988;55:118993.[Medline]
33 Daniel WW. Comparison of means. In: Biostatistics: a foundation for analysis in the health sciences. New York (NY): John Wiley & Sons; 1978. p. 12059.
34
Baeuerle PA, Baltimore D. NF-B: ten years after. Cell 1996;87:1320.[Medline]
35 Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:22560.[Medline]
36 Conant K, Ma M, Nath A, Major EO. Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes. J Virol 1996;70:13849.[Abstract]
37 Ramazzotti E, Vignoli M, Re MC, Furlini G, La Placa M. Enhanced nuclear factor-kappa B activation induced by tumour necrosis factor-alpha in stably tat-transfected cells is associated with the presence of cell-surface-bound Tat protein. AIDS 1996;10:45561.[Medline]
38 Kelly GD, Ensoli B, Gunthel CJ, Offermann MK. Purified Tat induces inflammatory response genes in Kaposi's sarcoma cells. AIDS 1998;12:175361.[Medline]
39 Blankaert D, Simonart T, Van Vooren JP, Parent D, Liesnard C, Farber CM, et al. Constitutive release of metalloproteinase-9 (92-kd type IV collagenase) by Kaposi's sarcoma cells. J Acquir Immune Defic Syndr Hum Retrovirol 1998;18:2039.[Medline]
40 Jeffrey JJ. Interstitial collagenases. In: Parks WC, Mecham RP, editors. Matrix metalloproteinases. San Diego (CA): Academic Press; 1998. p. 1542.
41 Galea P, Lebranchu Y, Thibault G, Bardos P. Interleukin 4 and tumour necrosis factor alpha induce different adhesion pathways in endothelial cells for the binding of peripheral blood lymphocytes. Scand J Immunol 1992;36:57585.[Medline]
42
Yang J, Xu Y, Zhu C, Hagan MK, Lawley T, Offermann MK. Regulation of adhesion molecule expression in Kaposi's sarcoma cells. J Immunol 1994;152:36173.
43 Taub DD. Chemokineleukocyte interactions. The voodoo that they do so well. Cytokine Growth Factor Rev 1996;7:35576.[Medline]
44 Ensoli B, Barillari G, Buonaguro L, Gallo RC. Molecular mechanisms in the pathogenesis of AIDS-associated Kaposi's sarcoma. Adv Exp Med Biol 1991;303:2738.[Medline]
45
Samaniego F, Markham PD, Gallo RC, Ensoli B. Inflammatory cytokines induce AIDSKaposi's sarcoma-derived spindle cells to produce and release basic fibroblast growth factor and enhance Kaposi's sarcoma-like lesion formation in nude mice. J Immunol 1995;154:358292.
46 Lafrenie RM, Wahl LM, Epstein JS, Yamada KM, Dhawan S. Activation of monocytes by HIV-Tat treatment is mediated by cytokine expression. J Immunol 1997;159:407783.[Abstract]
47 Westendorp MO, Li-Weber M, Frank RW, Krammer PH. Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells. J Virol 1994;68:417785.[Abstract]
48 Scala G, Ruocco MR, Ambrosino C, Mallardo M, Giordano V, Baldassarre F, et al. The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein. J Exp Med 1994;179:96171.[Abstract]
49
Sastry KJ, Reddy HR, Pandita R, Totpal K, Aggarwal BB. HIV-1 tat gene induces tumor necrosis factor-beta (lymphotoxin) in a human B-lymphoblastoid cell line. J Biol Chem 1990;265:200913.
50 Buonaguro L, Barillari G, Chang HK, Bohan CA, Kao V, Morgan R, et al. Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines. J Virol 1992;66:715967.[Abstract]
51 Brady HJ, Abraham DJ, Pennington DJ, Miles CG, Jenkins S, Dzierzak EA. Altered cytokine expression in T lymphocytes from human immunodeficiency virus Tat transgenic mice. J Virol 1995;69:76229.[Abstract]
52 Garza HH Jr, Prakash O, Carr DJ. Aberrant regulation of cytokines in HIV-1 TAT72-transgenic mice. J Immunol 1996;156:36317.[Abstract]
53
Kundu RK, Sangiorgi F, Wu LY, Pattengale PK, Hinton DR, Gill PS, et al. Expression of the human immunodeficiency virus-Tat gene in lymphoid tissues of transgenic mice is associated with B-cell lymphoma. Blood 1999;94:27582.
54 Westendorp MO, Shatrov VA, Schulze-Osthoff K, Frank R, Kraft M, Los M, et al. HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state. EMBO J 1995;14:54654.[Abstract]
55 Baeuerle PA, Baltimore D. A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. Genes Dev 1989;3:168998.[Abstract]
56 Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994;12:14179.[Medline]
57 Wulczyn FG, Krappmann D, Scheidereit C. The NF-kappa B/Rel and I kappa B gene families: mediators of immune response and inflammation. J Mol Med 1996;74:74969.[Medline]
58
Sovak MA, Bellas RE, Kim DW, Zanieski GJ, Rogers AE, Traish AM, et al. Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J Clin Invest 1997;100:295260.
59
Bargou RC, Emmerich F, Krappmann D, Bommert K, Mapara MY, Arnold W, et al. Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. J Clin Invest 1997;100:29619.
60
Ganju RK, Munshi N, Nair BC, Liu ZY, Gill P, Groopman JE. Human immunodeficiency virus tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells. J Virol 1998;72:61317.
61
Masood R, Cai J, Zheng T, Smith DL, Naidu Y, Gill PS. Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDSKaposi sarcoma. Proc Natl Acad Sci U S A 1997;94:97984.
Manuscript received November 16, 1999; revised January 27, 2000; accepted February 13, 2000.
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