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*Substance via MeSH
Medline Plus Health Information
*AIDS
*Kaposi's Sarcoma
The Journal of Immunology, 1998, 161: 1694-1704.
Copyright © 1998 by The American Association of Immunologists

Endogenous Basic Fibroblast Growth Factor Is Essential for Cyclin E-CDK2 Activity in Multiple External Cytokine-Induced Proliferation of AIDS-Associated Kaposi’s Sarcoma Cells: Dual Control of AIDS-Associated Kaposi’s Sarcoma Cell Growth and Cyclin E-CDK2 Activity by Endogenous and External Signals

Kaoru Murakami-Mori1,*, Shunsuke Mori{dagger} and Shuji Nakamura*

* Institute of Molecular Medicine, Huntington Memorial Hospital, Pasadena, CA 91105; and {dagger} Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095-1747


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AIDS-associated Kaposi’s sarcoma (KS) cell, a key element for development of KS lesions, proliferates in response to external cytokines, such as oncostatin M, the soluble IL-6R-IL-6 complex, TNF-{alpha}, and IL-1ß. In addition, the KS cell-produced basic fibroblast growth factor (bFGF) was reported to function as an autocrine growth factor. However, little is known of the exact roles of these external growth factors and endogenous bFGF on proliferation of KS cells, and underlying intracellular events have remained to be defined. We obtained evidence that anti-bFGF Ab abolished growth of KS cells by preventing S phase entry of the cell cycle, even in the presence of the external growth factors. Blockade of the FGF action profoundly inhibited cyclin E expression and cyclin-dependent kinase-2 (CDK2) activity, but not D-type cyclin expression and CDK4 activity. Exogenously added acidic FGF (aFGF), which generated a rapid tyrosine phosphorylation of FGFR1 and FGFR2 on KS cells, reversed the inhibitory effects of anti-bFGF Ab. Thus, FGF actions are essential for cyclin E-CDK2 activity and S phase entry. We also observed that the presence of external growth factors markedly induced cyclin E-CDK2 activity and S phase entrance, while the addition of aFGF or bFGF alone was insufficient to induce these responses. All this evidence shows that integration of the activities of external growth factors and endogenous bFGF is required for full activation of cyclin E-CDK2 activity and KS cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Kaposi’s sarcoma (KS)2 is the most common tumor in persons infected with HIV (AIDS-KS) (1, 2, 3). During the past decade, KS has shifted from an index diagnosis for AIDS to an opportunistic neoplasm occurring later in the course of AIDS and leading to increased morbidity and mortality (4, 5). In addition, development of KS has been noted in association with immunosuppressive therapy following solid organ transplantation, autoimmune disorders, or lymphoproliferative diseases (6, 7). It is commonly accepted that, once initiated, KS progresses in response to various external stimuli, including cytokines and hormones, and can regress spontaneously (8, 9). Thus, KS may not be a genuine malignancy, rather it may represent a reactive and reversible proliferative lesion, at least in the early stages (10, 11). KS is a multicentral, highly vascular tumor consisting of a proliferation of spindle-shaped cells (KS cells), generally considered to be of mesenchymal origin, microvascular endothelial cells, extravasated erythrocytes, and infiltration of mononuclear cells (12, 13). KS cells produce angiogenic factors and inflammatory cytokines that presumably drive the development and progression of KS lesions (14, 15, 16, 17). Indeed, inoculation of KS cells into nude mice induces proliferative vascular lesions that closely resemble early KS seen in patients (18). These lines of evidence suggest that the KS cell is a regulatory element in KS development, aggravation, and resolution.

The growth of KS cells in culture is markedly increased if cytokines such as oncostatin M (OM), the soluble IL-6 receptor (sIL-6R{alpha})/IL-6 complex (sIL-6R{alpha}/IL-6), TNF-{alpha}, and IL-1ß are added exogenously (19, 20, 21, 22, 23, 24). Considering such a functional redundancy of diverse external cytokines as KS cell growth factors, it has to be elucidated whether signaling pathways specific for these growth factors may converge on activation of a common intracellular molecule(s). In addition to these external growth factors, the commitment of KS cell-derived factors to the control of KS cell proliferation has been noted. For example, basic fibroblast growth factor (bFGF) is expressed at high steady state levels in KS cell isolates derived from different patients (14, 17, 25). Ensoli et al. showed that antisense oligonucleotides targeting bFGF can block the basal growth of KS cells by reducing the production of intracellular bFGF molecules (26). However, the molecular mechanism underlying bFGF action as an autonomous growth factor of KS cells is unknown, and the exact role of endogenous bFGF in intracellular events involved in external growth factor-induced proliferation of KS cells has not been defined.

We have now obtained evidence that the autocrine signal of bFGF is required for basal growth and external factor-induced proliferation of KS cells. Block of the endogenous bFGF activity abolished the proliferation of KS cells by preventing entry into S phase of the cell cycle. In addition, we found that FGF-specific action is essential for cyclin E expression and cyclin E-associated cyclin-dependent kinase 2 (CDK2) activity, key regulators of the cell cycle machinery. However, FGF activity alone was not sufficient to induce KS cell proliferation and cyclin E expression, and the presence of external growth factors was required for full KS cell growth and cyclin E-CDK2 activity. Taken together, the KS cell cycle seems to enter S phase, as determined by integration of both signals of the endogenous bFGF and the external growth factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

AIDS-KS22 cells were developed in our laboratory from pleural effusion of lung KS in an HIV-infected patient (22). Lung KS cells (AIDS-KS3) and oral mucosa KS cells (AIDS-KS10B) were developed in the Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health (Bethesda, MD), from tissues obtained from different patients with HIV infection (19). Using PCR amplification with KS 330 primers under conditions established by Chang et al. (27), we confirmed that these KS cells do not contain detectable human herpesvirus-8 (HHV-8) DNA sequences. KS cells were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD), supplemented with 10% FBS (Gemini Bio-Products, Calabasas, CA) and conditioned medium (CM) from human OM-expressing Chinese hamster ovary cells (22), to a final OM concentration of 10 ng/ml. A human prostate adenocarcinoma cell line, LNCaP (CRL-1740) was purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% FBS. Recombinant human bFGF, acidic FGF (aFGF), OM, IL-6, sIL-6R{alpha}, TNF-{alpha}, and IL-1ß were purchased from R&D Systems (Minneapolis, MN). Neutralizing goat anti-human bFGF polyclonal Ab was also purchased from R&D Systems.

AIDS-KS cell growth assays

AIDS-KS cells were incubated in triplicate in 24-well plates (0.5 ml/well) in KS basal medium (RPMI 1640 and 10% FBS), with or without various test factors, at an initial cell density of 3 x 103 cells/well. Culture medium was changed every 3 days, and adherent cells were counted on the sixth day of culture after trypsinization, using a Coulter particle counter (Coulter Electronics, Hialeah, FL). Data are expressed as the mean ± SD of triplicate determinations from two or three separate experiments. Statistical analysis was determined by Student’s t test. Cell viability was determined by trypan blue dye exclusion (Sigma, St. Louis, MO). The criterion that cell death should be <1% was met by all growth assays included in this study, even without KS cell growth factors or even in the presence of anti-bFGF Ab.

Cell cycle analysis

AIDS-KS cells (3 x 105 cells) were cultured in a 75-cm2 flask with or without 2 µg/ml of neutralizing goat anti-human bFGF Ab for 4 days, in the KS basal medium alone or in this medium supplemented with recombinant human OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), or sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml). To restore FGF action on KS cells, aFGF (50 ng/ml) was added to the culture. For DNA staining, 5 x 105 of KS cells were washed twice with PBS, and then incubated with 50 µl of 70% ice-cold ethanol for 20 min. After washing with PBS, 250 µl of PBS containing 0.1% Triton X-100, 0.1 mM EDTA, 50 µg/ml RNase A (Sigma), and 100 µg/ml propidium iodide (Sigma) was added to each sample. After 1-h incubation in the dark, DNA analysis was performed using an EPICS XL flow cytometer (Coulter Electronics).

Western blotting analysis

AIDS-KS cells were cultured for 4 days at an initial density of 3 x 105 cells/75-cm2 culture flask, with and without the neutralizing goat anti-human bFGF Ab (2 µg/ml), in the KS basal medium alone or in this medium supplemented with recombinant human OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), or sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml). For restoration of FGF action, aFGF (50 ng/ml) was added to the culture. Cells were lysed at 4°C in 1 ml of the modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/m aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). Cell lysates (15 µg) were boiled for 3 min and subjected to 10 to 20% gradient SDS-PAGE (Novex, San Diego, CA). Transfer of proteins from gels onto Hybond nitrocellulose membranes (Amersham, Arlington Heights, IL) was performed electrophoretically, using a transblotting cell (Bio-Rad, Hercules, CA). Membranes were blocked by immersion for 1 h at room temperature in 5% nonfat skim milk/PBS and then incubated with mouse anti-human cyclin D1, D2, D3, or E mAb (0.2 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human CDK2 or CDK4 polyclonal Ab (0.2 µg/ml; Santa Cruz Biotechnology), mouse anti-human p21/WAF1/Cip1 or p27/Kip1 mAb (1 µg/ml; Oncogene Research Products, Cambridge, MA), or rabbit anti-human FGFR1 or FGFR2 polyclonal Ab (0.2 µg/ml; Santa Cruz Biotechnology) for 2 h at room temperature. After washing in PBS-0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary Ab (New England Biolabs, Beverly, MA) for 1 h at room temperature, and then developed using an enhanced chemiluminescence detection kit (Amersham).

Tyrosine phosphorylation assays

AIDS-KS cells (1.5 x 106 cells) in a 75-cm2 flask were starved of serum for 48 h. Next, these cells were pretreated for 10 min with 1 mM Na3VO4, and then stimulated with 50 ng/ml bFGF or 50 ng/ml aFGF for various periods at 37°C. The cells were then lysed at 4°C in 1 ml of the modified RIPA buffer. Before immunoprecipitation with specific Abs, the cell lysates (200 µg of proteins in 200 µl) were precleared by incubation for 1 h at 4°C with 1 µg of rabbit IgG together with 20 µl of protein A/G agarose beads (50% suspension; Santa Cruz Biotechnology). The precleared lysates were incubated with rabbit anti-human FGFR1 and FGFR2 Abs (1 µg; Santa Cruz Biotechnology) overnight at 4°C, and then with 20 µl of protein A/G agarose for an additional 1 h. After washing four times with modified RIPA buffer, the immunoprecipitates were suspended in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.025% bromophenol blue, and 2.5% ß-ME). The immunoprecipitated proteins were eluted from the agarose beads by heating at 95°C for 5 min, and then subjected to 8% SDS-PAGE (Novex) and Western blotting analysis. To visualize tyrosine-phosphorylated FGFR1 and FGFR2, membranes were incubated with horseradish peroxidase-conjugated anti-phosphotyrosine mAb (1 µg/ml; Upstate Biotechnology, Lake Placid, NY) for 2 h at room temperature. After washing in PBS-0.1% Tween-20, membranes were developed using an enhanced chemiluminescence detection kit.

In vitro kinase assays

AIDS-KS cells were cultured at an initial density of 3 x 105 cells/75-cm2 culture flask, with and without the neutralizing anti-human bFGF Ab (2 µg/ml) for 4 days, in the KS basal medium alone or in this medium supplemented with recombinant human OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), or sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml). To restore FGF action, aFGF (50 ng/ml) was added to the culture. Cells were lysed at 4°C in 1 ml of lysis buffer for kinase assays (50 mM HEPES (pH 7.0), 0.1% NP40, 250 mM NaCl, 2 mM EGTA, 1 mM PMSF, 1 µg/m aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). The cell lysates (200 µg of proteins in 200 µl) were precleared by incubation for 1 h at 4°C with 1 µg of mouse IgG or rabbit IgG together with 20 µl of protein A/G agarose beads. The lysates were incubated overnight with mouse anti-human cyclin E mAb or with rabbit anti-human CDK2 or CDK4 polyclonal Ab (1 µg; Santa Cruz Biotechnology) at 4°C. The immune complexes were then isolated by incubation with 20 µl of a 50% suspension of protein A/G agarose for an additional 1 h at 4°C. After washing four times with the lysis buffer and twice with the kinase buffer (50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, and 1 mM NaF), the immunoprecipitates were incubated for 30 min at 30°C in 25 µl of the kinase buffer in the presence of 2.5 µg of histone H1 (Boehringer Mannheim, Indianapolis, IN) or 1 µg of glutathione-S-transferase-pRB fusion protein containing the carboxyl-terminal domain of pRB (Santa Cruz Biotechnology) as a substrate, 5 µCi of [{gamma}-32P]ATP (6000 Ci/mmol, Amersham), and 50 µM cold ATP. The reaction was terminated by addition of 2x SDS sample buffer. The samples were boiled for 5 min and analyzed by 12% SDS-PAGE (Bio-Rad) and autoradiography.

RNA preparation and PCR amplification

AIDS-KS cells (3 x 105 cells) were plated in a 75-cm2 culture flask, with and without the neutralizing anti-human bFGF Ab (2 µg/ml), for 4 days in the KS basal medium alone or in this medium supplemented with recombinant human OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), or sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml). To reverse the action of FGF, aFGF (50 ng/ml) was added to the culture. Total RNA was prepared from these cells by guanidine isothiocyanate disruption of cells and centrifugation through 1.51 g/ml cesium trifluoroacetate (Pharmacia Biotech, Uppsala, Sweden). Two micrograms of total RNA were subjected to the cDNA synthesis by incubation for 1 h at 42°C with reverse transcriptase and random hexanucleotides (Life Technologies), followed by cyclin D1-, D2-, D3-, and E-; FGFR1-; and FGFR2-specific PCR amplifications, respectively. The primers used were: 5'-TGC GAG GAG GAG GTC TTC CCG CT-3' and 5'-CCT CAG ATG TCC ACG TCC CGC AC-3' (for cyclin D1); 5'-CTG GAG GTC TGT GAG GAA CAG AA-3' and 5'-CGC ACG TCT GTA GGG GTG CTG GC-3' (for cyclin D2); 5'-GCT TAC TGG ATG CTG GAG GTA TG-3' and 5'-ACA TCT GTA GGA GTG CTG GTC TG-3' (for cyclin D3); 5'-AGG GAG ACC TTT TAC TTG GCA CA-3' and 5'-GGT CAC GCC ATT TCC GGC CCG CT-3' (for cyclin E); 5'-AGA ATT GGA GGC TAC AAG GTC CG-3' and 5'-GAG TTA CCC GCC AAG CAC GTA TA-3' (for FGFR1); and 5'-AAC GGG AAG GAG TTT AAG CAG GA-3' and 5'-AAG GAT ATC CCA ATA GAA TTA CC-3' (for FGFR2). RNA integrity and the efficiency of cDNA synthesis were confirmed by PCR amplification, using a human ß-actin-specific primer (Clontech, Palo Alto, CA). The primers for cyclins D1, D2, D3, and E and ß-actin directed the amplification product corresponding to 673, 665, 680, 749, and 838 bp, respectively. PCR amplification was performed under the following conditions: 30 cycles at 94°C for 1 min, at 58°C for 2 min, and at 72°C for 3 min; and one cycle at 94°C for 1 min, at 58°C for 2 min, and at 72°C for 10 min. The amplification products were electrophoresed on 1.2% agarose gels.

Quantitation of human bFGF in AIDS-KS cell-derived CM and extracellular matrix

AIDS-KS cells were seeded in duplicate wells at an initial density of 1 x 105 cells/well in six-well plates and cultured for 1 day in the KS basal medium. These cells were then incubated in 1.5 ml of the basal medium alone or in this medium supplemented with recombinant human OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), TNF-{alpha} (10 ng/ml), or IL-1ß (10 ng/ml). After 48 h, the CM was collected for quantitation of bFGF. To release and harvest bFGF from extracellular matrix, the remaining cells were washed with RPMI 1640 and then incubated with 1.5 ml of NaCl buffer (20 mM Tris-HCl (pH 7.2) and 2 M NaCl) for 5 min (17). Appropriate dilutions of the CM and extracellular matrix fraction were used to achieve values within the linear range of the standard curve. ELISA was performed in duplicate wells of ELISA kits according to instructions from R&D Systems.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibitory effects of anti-bFGF Ab on basal growth and OM-, sIL-6R{alpha}/IL-6-, TNF-{alpha}-, and IL-1ß-induced proliferation of AIDS-KS cells

AIDS-KS cells produce and release into the extracellular medium and matrix a biologically active form of bFGF, an event that may allow these cells to acquire an autocrine signaling loop (14). To quantify amounts of released bFGF, human bFGF-specific ELISA was performed on CMs and extracellular matrix fractions derived from three different KS cell isolates from lung (KS3), oral mucosa (KS10B), and pleural effusion (KS22). As shown in Table IGo, these KS cells constitutively produced and released a steady state level of bFGF molecules into the CM and extracellular matrix. The treatment of these cells with the established KS cell growth factors OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), TNF-{alpha} (10 ng/ml), and IL-1ß (10 ng/ml) enhanced total amounts of released bFGF compared with the basal level. To determine whether the released endogenous bFGF is critical for basal and external growth factor-induced proliferation of KS cells, the neutralizing anti-bFGF Ab was added to KS cell cultures in the presence or the absence of these growth factors (Fig. 1Go). In the absence of anti-bFGF Ab, KS cells proliferated readily in response to each of the growth factors. After culture for 6 days, the external factor-induced growth was 3 to 5 times higher than the basal growth level observed with the KS basal medium alone. When anti-bFGF Ab was added to the culture, the factor-induced growth was precluded in a dose-dependent manner. Almost complete inhibition was observed with 2 to 5 µg/ml of anti-bFGF Ab. The inhibitory effects of anti-bFGF Ab were also observed in the absence of the external growth factors; when 5 µg/ml of anti-bFGF Ab was added to the culture, the cell number remained at the initial density. Thus, blockade of the endogenous bFGF signal led to a marked inhibition of basal growth and factor-induced proliferation of KS cells, indicating that the existence of a signaling loop of endogenous bFGF is essential for KS cell growth regardless of the presence or the absence of the external KS cell growth factors. The combination of external factors had additive stimulating effects on KS cell growth, which were also completely inhibited by the addition of 5 µg/ml of anti-bFGF Ab (data not shown). Anti-bFGF Ab treatment had no apparent effect on cell viability, as determined by trypan blue dye exclusion. Control Ab (5 µg/ml) did not affect KS cell growth.


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  		Table I. Amounts of bFGF in AIDS-KS cell-derived CM and extracellular matrix1

 


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  		FIGURE 1. Inhibitory effects of anti-bFGF Ab on basal growth and OM-, sIL-6R{alpha}/IL-6-, IL-1ß-, or TNF-{alpha}-induced proliferation of AIDS-KS cells. KS3, KS10B, and KS22 cells (3 x 103 cells) in triplicate wells were incubated on 24-well plates for 6 days in the absence or the presence of various KS cell growth factors, with and without the increased concentrations of the neutralizing anti-bFGF Ab or 5 µg/ml of control Ab. Cell growth was determined using a Coulter particle counter. Data represent the mean ± SD of triplicate measurements in three separate experiments (n = 9). The asterisk indicates a significant decrease compared with KS cell numbers observed in the absence of anti-bFGF Ab, respectively (p < 0.005). Open bars, KS basal medium alone; closed bars OM (10 ng/ml); horizontally lined bars, sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml); hatched bars, TNF-{alpha} (10 ng/ml); dotted bars, IL-1ß (10 ng/ml). C, control goat Ab (5 µg/ml).

 
FGF action alone is insufficient to induce AIDS-KS cell growth

One possible explanation for the growth inhibitory effect of anti-bFGF Ab is that proliferative signals of the external growth factors may be mediated through an increased release of endogenous bFGF molecules; namely, bFGF may be a direct effector to promote KS cell proliferation. Indeed, we found that KS cell growth factors are capable of augmenting the release of endogenous bFGF (Table IGo). To examine this possibility, various amounts of recombinant bFGF were added exogenously to cultures. If this possibility is tenable, exogenous addition of bFGF would mimic the external growth factor-induced mitogenic responses. As shown in Figure 2GoA, addition of bFGF generated only minor stimulation of growth, the extent of which was much lower than that induced by OM (10 ng/ml), TNF-{alpha} (10 ng/ml), or IL-1ß (10 ng/ml). Thus, bFGF action cannot substitute for the growth-promoting activities of external factors. KS cells express at least two major FGF receptors, FGFR1 (the flg gene product) and FGFR2 (the bek gene product), which are transmembrane receptor tyrosine kinases (25, 28). To confirm that the exogenously added bFGF has the potential to activate FGFR on KS cells, we examined tyrosine phosphorylation of FGFR1 and FGFR2, an event that initiates intracellular signaling of bFGF. As shown in Figure 2GoB, bFGF induced a rapid tyrosine phosphorylation of these receptors within 10 min of stimulation, and the phosphorylation increased with time up to 30 min. Even before stimulation, low levels of tyrosine phosphorylation of FGFRs were observed in KS cells, suggesting constitutive activation of these receptors. In addition, we found few differences between the external growth factor-treated and untreated KS cells regarding time course and intense of the bFGF-induced tyrosine phosphorylation of FGFR1 and FGFR2 (data not shown), thereby indicating that activation of these receptors is not affected by external growth factors. No appreciable changes in expression of FGFRs were observed between the external factor-treated and untreated KS cells at mRNA and protein levels (data not shown). Thus, it is unlikely that the proliferative effects of external growth factors are due to the simple augmentation of expression and binding affinities of FGFR. Instead, it seems reasonable to conclude that diverse actions of endogenous bFGF plus external growth factors are required to induce and sustain KS cell growth.



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  		FIGURE 2. A, Effects of exogenously added bFGF on growth of AIDS-KS cells. KS3, KS10B, and KS22 cells (3 x 103 cells) in triplicate wells were incubated on 24-well plates for 6 days in the basal medium alone (open bars) or in this medium supplemented with 10 ng/ml of OM (closed bars), 10 ng/ml of TNF-{alpha} (horizontally lined bars), 10 ng/ml of IL-1ß (hatched bars), or increased concentrations of bFGF (dotted bars). Data are expressed as the mean ± SD of triplicate determinations from three separate experiments (n = 9). The asterisk indicates a significant increase compared with KS cell growth in medium alone (p < 0.005). B, bFGF-induced tyrosine phosphorylation of FGFR1 and FGFR2 in AIDS-KS cells. Subconfluent KS3 cells (2 x 106 cells/75-cm2 culture flask) were deprived of serum for 2 days and then stimulated with 50 ng/ml bFGF for 0, 10, 30, and 60 min at 37°C. The FGFR1 and FGFR2 proteins in the cell lysates (200 µg) were isolated by immunoprecipitation with the respective specific Abs and then subjected to 8% SDS-PAGE and immunoblotting with anti-phosphotyrosine Ab. The respective data are from two separate experiments.

 
Addition of aFGF can reverse anti-bFGF Ab-induced inhibition of AIDS-KS cell growth

Both acidic and basic FGFs share FGFR1 and FGFR2 for intracellular signal transduction (29). Indeed, addition of recombinant aFGF (50 ng/ml) induced similar patterns of tyrosine phosphorylation of these two receptors on KS cells (Fig. 3GoA). Low levels of tyrosine-phosphorylated FGFR1 and FGFR2 were detected before stimulation. To confirm that FGF action is essential as a growth-promoting factor of KS cells, aFGF (50 ng/ml) and neutralizing anti-bFGF Ab (2 µg/ml) were simultaneously added to KS cell cultures. KS cells were reported to express aFGF mRNA, but the level was negligible compared with that of bFGF mRNA (14). As shown in Figure 3GoB, the presence of aFGF rescued KS cells from the anti-bFGF Ab-induced growth suppression in the presence or the absence of external growth factors. In contrast, simple addition of aFGF had no additional enhancing effects on basal and external growth factor-induced KS cell growth. Similar data were obtained with combined uses of the external growth factors; the mitogenic effects of these factors in combination were almost completely inhibited by the anti-bFGF Ab, and this inhibition was reversed in the presence of aFGF (data not shown). Thus, the FGF-specific action is essential for basal growth and for mitogenic responses to external growth factors; however, the requirement of the external mitogens for maximum growth induction cannot be replaced by FGF activity. These findings strongly support the idea that concerted actions between FGF and the external growth factors facilitate KS cell proliferation.



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  		FIGURE 3. A, aFGF-induced tyrosine phosphorylation of FGFR1 and FGFR2 in AIDS-KS cells. KS3 cells (2 x 106 cells/75-cm2 culture flask) were deprived of serum and then stimulated with 50 ng/ml aFGF for 0, 10, 30, and 60 min at 37°C. The FGFR1 and FGFR2 proteins in cell lysates (200 µg) were isolated by immunoprecipitation with the respective specific Abs and then subjected to 8% SDS-PAGE and immunoblotting with anti-phosphotyrosine Ab. The respective data are from two separate experiments. B, Effects of aFGF on anti-bFGF Ab-induced inhibition of AIDS-KS cell growth. KS3, KS10B, and KS22 cells (3 x 103 cells) in triplicate wells were incubated on 24-well plates for 6 days in basal medium alone or in this medium supplemented with OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), IL-1ß (10 ng/ml), or TNF-{alpha} (10 ng/ml) in the presence or the absence of the neutralizing anti-bFGF Ab (2 µg/ml), aFGF (50 ng/ml), or a combination of both. Data are expressed as the mean ± SD of triplicate determinations from three separate experiments (n = 9). The asterisk indicates a significant decrease compared with KS cell number observed in the absence of the anti-bFGF Ab, respectively (p < 0.005). The double asterisk indicates a significant restoration by aFGF compared with KS cell number cultured in the presence of anti-bFGF Ab (p < 0.005). Open bars without aFGF and anti-bFGF Ab; horizontally lined bars with aFGF; hatched bars with anti-bFGF Ab; closed bars with aFGF and anti-bFGF Ab.

 
Blockade of the autocrine signaling loop of bFGF prevents S phase entry of the cell cycle of AIDS-KS cells in the presence of OM, sIL-6R{alpha}/IL-6, TNF-{alpha}, and IL-1ß

Ensoli et al. showed that treatment with bFGF antisense oligonucleotides inhibited the basal growth of KS cells by blocking S phase entry of the KS cell cycle (26). To better understand the role of endogenous bFGF in the multiple external factor-induced growth of KS cells, the effects of anti-bFGF Ab on S phase entry were examined in the presence of external growth factors. KS cells were incubated for 4 days with OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), and sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml) in the presence or the absence of 2 µg/ml of the neutralizing anti-bFGF Ab (Fig. 4Go). Compared with culture in basal medium alone, external growth factors prominently increased the percentage of KS cells in S phase of the cell cycle (Fig. 4Go; ninefold increase for OM, ninefold increase for sIL-6R{alpha}/IL-6, eightfold increase for TNF-{alpha}, and eightfold increase for IL-1ß). In addition, there was a significant inhibition of S phase entry in the anti-bFGF Ab-treated KS cell population compared with that in Ab-untreated cells (OM, 81% inhibition; sIL-6R{alpha}/IL-6, 82% inhibition; TNF-{alpha}, 79% inhibition; IL-1ß, 81% inhibition). The DNA fragmentation pattern was hardly detected (<1%), and this was not affected by anti-bFGF Ab treatment. Control Ab (5 µg/ml) had no obvious effects on S phase entry or the relative distribution of cells among the various cell cycle phases (data not shown). Notably, we found that aFGF (50 ng/ml) reversed the anti-bFGF Ab-induced block of S phase entry, although the simple treatment of aFGF had little effect on the percentage of cells in the S phase. Thus, FGF-specific action is essential for entry into S phase of the KS cell cycle, working in a cooperative fashion with external growth factors.



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  		FIGURE 4. Evaluation of percentages of AIDS-KS cells in S phase. KS22 cells (3 x 105 cells/75-cm2 culture flask) were incubated with and without the neutralizing anti-human bFGF Ab (2 µg/ml) for 4 days in KS basal medium alone or in this medium supplemented with recombinant human OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), TNF-{alpha} (10 ng/ml), or IL-1ß (10 ng/ml). For rescue experiments, aFGF (50 ng/ml) was simultaneously added to the culture. Distribution of cell populations in different phases of the cell cycle was examined using propidium iodide staining and flow cytometric analysis. Data are presented as the percentage of cells in S phase. Values are the mean ± SD of triplicate determinations from two separate experiments (n = 6). The asterisk indicates a significant increase in S phase entry compared with KS cells incubated with medium alone (p < 0.005). The double asterisk indicates a significant inhibitory effect of anti-bFGF Ab compared with the percentage of cells in S phase in the absence of the anti-bFGF Ab, respectively (p < 0.01). The triple asterisk indicates a significant restoration of S phase entry by aFGF compared with that in the anti-bFGF Ab-treated cells, respectively (p < 0.01). Open bars without aFGF and anti-bFGF Ab; horizontally lined bars, with aFGF; hatched bars, with anti-bFGF Ab; closed bars, with aFGF and anti-bFGF Ab.

 
Effects of anti-bFGF Ab on expression of G1 cyclins and CDK inhibitors in AIDS-KS cells

There is now an increasing body of evidence that as positive regulatory subunits of CDKs, G1 cyclins are rate-limiting controllers of G1 progression or entry into S phase of the cell cycle of mammalian cells (30, 31, 32). To better understand the molecular events underlying the anti-bFGF Ab-induced growth arrest of KS cells, the expression of G1 cyclins in KS cells was determined using RT-PCR amplification and immunoblotting analysis. KS3 cells were cultured for 4 days, with and without the neutralizing anti-bFGF Ab (2 µg/ml), in KS basal medium alone or in this medium supplemented with OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), TNF-{alpha} (10 ng/ml), or IL-1ß (10 ng/ml). As shown in Figure 5Go, significant amounts of D cyclins (D1, D2, and D3), at mRNA and protein levels, were detected in KS cells regardless of the presence or the absence of external growth factors. The addition of anti-bFGF Ab to cultures had little effect on the amounts of these molecules. In contrast, the presence of the external growth factors augmented the expression of cyclin E at both mRNA and protein levels compared with its basal expression. Thus, there is a positive correlation between increases in cyclin E expression and factor-induced growth promotion. Notably, blockade of the autocrine bFGF signal using the anti-bFGF Ab had striking inhibitory effects on cyclin E expression in the presence or the absence of external growth factors, indicating that endogenous bFGF action is required for cyclin E expression. Therefore, anti-bFGF Ab-induced growth arrest appears to be due to suppression of cyclin E expression. Inhibitors of cyclin-CDK activities, p21/WAF1/Cip1 and p27/Kip1, act as negative regulators of orderly progression through the cell cycle (33). As shown in Figure 5Go, both inhibitors are expressed at extremely low levels in KS3 cells compared with those in positive control cells. In addition, treatment of these cells with anti-bFGF Ab had no apparent effect on the protein levels of these inhibitors. Essentially similar expression patterns of G1 cyclins and CDK inhibitors were obtained in experiments using KS22 and KS10B cells (data not shown).



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  		FIGURE 5. Effects of anti-bFGF Ab on expression of cyclins D1, D2, D3, and E and CDK inhibitors p21/WAF1/Cip1 and p27/Kip1 in AIDS-KS cells. KS3 cells (3 x 105 cells/75-cm2 culture flask) were cultured for 4 days, with and without 2 µg/ml of the neutralizing anti-bFGF Ab, in the KS basal medium alone or in this medium supplemented with OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), TNF-{alpha} (10 ng/ml), or IL-1ß (10 ng/ml). A, Western blotting: cell lysates (15 µg) were subjected to 10 to 20% SDS-PAGE and immunoblotting with anti-cyclin D1, D2, D3, and E Abs and anti-p21/WAF1/Cip1 and p27/Kip1 Abs. As positive controls of p21/WAF/Cip1 and p27/Kip1, cell lysates from cAMP (1 mM)-treated LNCaP cells were included in immunoblotting assays. The positions of size standards are shown. B, PCR amplification: equal amounts (2 µg) of total RNA were subjected to the cDNA synthesis and cyclin D1-, D2-, D3-, and E-specific PCR amplifications using paired primers, respectively. The PCR products were electrophoresed on 1.2% agarose gels. The positions of size standards are shown. Representative data are from three separate experiments. MD, medium control; C, positive control; M, size standard.

 
Addition of aFGF can reserve anti-bFGF Ab-induced suppression of cyclin E expression and cyclin E-associated CDK2 activity in AIDS-KS cells

We found that the presence of aFGF can mimic autocrine bFGF function to support S phase entry and proliferation of KS cells in response to external growth factors (Figs. 3Go and 4Go). To further determine the specificity of FGF action for the control of S phase entry, we examined the effects of aFGF addition on cyclin E expression in anti-bFGF Ab-treated KS cells (Fig. 6Go). The presence of the neutralizing anti-bFGF Ab (2 µg/ml) strikingly reduced the amount of cyclin E in the presence or the absence of external growth factors such as OM (10 ng/ml), sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml), TNF-{alpha} (10 ng/ml), or IL-1ß (10 ng/ml). When 50 ng/ml of recombinant aFGF was added to cultures, cyclin E expression was completely restored in the anti-bFGF Ab-treated KS cells. Thus, the aFGF signal can rescue the anti-bFGF Ab-treated KS cells from the state of down-regulation of cyclin E. Inhibitory effects of anti-bFGF Ab on cyclin E expression and its restoration by aFGF addition were also observed with the combined use of these external factors. Addition of aFGF alone did not enhance the basal or external growth factor-induced expression of cyclin E (data not shown). In contrast to cyclin E, there were no detectable levels of change in cyclin D1 expression. Thus, the FGF-specific function apparently involves the contribution to cyclin E expression, resulting in S phase entry of the KS cell cycle. Essentially similar expression patterns of cyclins D and E were obtained with KS22 and KS10B cells (data not shown).



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  		FIGURE 6. Effects of the exogenous addition of aFGF on anti-bFGF Ab-induced suppression of cyclin E expression in AIDS-KS cells. KS3 cells (3 x 105 cells/75-cm2 culture flask) were incubated, with and without the neutralizing anti-human bFGF Ab (2 µg/ml) for 4 days, in KS basal medium alone or in this medium supplemented with OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), or sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml). To restore FGF action, aFGF (50 ng/ml) were simultaneously added to the culture. A, Western blotting: cell lysates (15 µg) were subjected to 10 to 20% SDS-PAGE and immunoblotting with anti-cyclin D1 and E Abs. The positions of size standards are shown. B, PCR amplification: equal amounts (2 µg) of total RNA were subjected to cDNA synthesis and cyclin D- and E-specific PCR amplifications using paired primers, respectively. The samples were analyzed by 1.2% agarose gel electrophoresis. The positions of size standards are shown. Representative data are from four separate experiments. MC, medium control; M, size standard.

 
It is now widely accepted that cyclin E binds to and activates CDK2, the kinase activity of which governs the G1 to S phase transition of the mammalian cell cycle (30, 31, 32). We next examined kinase activities of the cyclin E-CDK2 complex in KS cells after immunoprecipitation for cyclin E, CDK2, and CDK4 (Fig. 7GoA). Compared with KS cells cultured in KS basal medium alone, treatment with the external growth factors led to significant increases in the histone H1 kinase activity of cyclin E and CDK2 immunoprecipitates, suggesting that cyclin E-CDK2 kinase activity is indeed associated with external factor-induced KS cell growth. The cyclin E-CDK2 activity was almost completely abolished by the neutralizing anti-bFGF Ab (2 µg/ml) in the presence or the absence of external growth factors, thereby indicating that blockade of endogenous bFGF activity can induce drastic decreases in this kinase activity. When aFGF (50 ng/ml) was exogenously added to Ab-treated cultures, the histone H1 kinase activity of the cyclin E and CDK2 immunoprecipitates reverted to the level observed in Ab-untreated KS cells, a finding that supports the idea that FGF-specific action is required for cyclin E-CDK2 activity in basal growth and external factor-induced proliferation. Anti-bFGF Ab-induced inhibition of cyclin E-CDK2 activity and the reversing effects of aFGF were also obtained when these factors were used in combination (data not shown). Since the progression from G1 to S phase is also thought to be regulated by D cyclins associated with CDK4 (30, 31, 32), we assayed CDK4-associated kinase activity toward pRB (Fig. 7GoA). The CDK4 immunoprecipitate showed a steady state level of pRB kinase activity in KS cells in the presence or the absence of external growth factors, an event not affected by treatment with the anti-bFGF Ab. As shown in Figure 7GoB, expressions of CDK2 and CDK4 protein were not affected by anti-bFGF Ab treatment. Similar data were observed consistently in experiments using KS22 and KS10B (data not shown).



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  		FIGURE 7. In vitro kinase assays of cyclin E-, CDK2-, and CDK4-associated kinases in AIDS-KS cells. KS3 cells (3 x 105 cells) were cultured for 4 days in 75-cm2 culture flasks, with and without neutralizing anti-bFGF Ab (2 µg/ml), in KS basal medium alone or in this medium supplemented with OM (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-1ß (10 ng/ml), or sIL-6R{alpha} (50 ng/ml)/IL-6 (20 ng/ml). For rescue experiments of the anti-bFGF Ab-treated KS cells, aFGF (50 ng/ml) was simultaneously added to the culture. Cell lysates (200 µg) prepared from these cells were subjected to immunoprecipitation with anti-cyclin E, CDK2, and CDK4 Abs. A, In vitro kinase assays: the immunoprecipitates were incubated for 30 min at 30°C in 25 µl of the kinase buffer containing 5 µCi of [{gamma}-32P]ATP (6000 Ci/mmol) and 50 µM cold ATP in the presence of 2.5 µg of histone H1 and 1 µg of glutathione-S-transferase-pRB as a substrate, respectively. The samples were separated by 12% SDS-PAGE and visualized by autoradiography. Representative data are from three separate experiments. B, Western blotting: cell lysates (15 µg) were subjected to 10 to 20% SDS-PAGE and immunoblotting with anti-CDK2 and CDK4 Abs. The positions of size standards are shown. MC, medium control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we obtained evidence that addition of anti-bFGF Ab to cell cultures can inhibit cyclin E expression and cyclin E-CDK2 activity, resulting in accumulation of AIDS-KS cells in the G1-arrested state even in the presence of external KS cell growth factors, such as OM, sIL-6R{alpha}/IL-6, TNF-{alpha}, and IL-1ß. We also found that the exogenous addition of aFGF completely overrode the inhibitory effect of anti-bFGF Ab. Thus, FGF has a special role to sustain cyclin E expression, cyclin E-CDK2 activity, and entry into S phase, and the external growth factors we used cannot replace this activity. Unlike many growth factors, bFGF does not contain a consensus secretory sequence (34); however, we found that KS cells released steady state levels of bFGF into the extracellular matrix and medium. Since 1) addition of anti-bFGF Ab to the medium had direct inhibitory effects on cyclin E-CDK2 activity and KS cell proliferation, and 2) addition of aFGF reversed the inhibitory effects of anti-bFGF Ab, the FGF-specific action is probably exerted through extracellular interactions of FGF with KS cell surface molecules such as FGFRs. Further, we found that addition of aFGF or bFGF generated a rapid tyrosine phosphorylation of FGFR1 and FGFR2. Presumably, after release from KS cells, bFGF binds to and activates cell surface receptors through tyrosine phosphorylation, leading to transduction of FGF-specific signals to intracellular machinery that could be crucial for cyclin E expression in KS cells. Indeed, FGFR1 and FGFR2 were constitutively tyrosine phosphorylated in KS cells even in the absence of external mitogens. Thus, the constitutive activation of FGFRs by endogenous bFGF appears to contribute to the growth of KS cells. In addition to the classical receptor tyrosine kinase pathways for acidic and basic FGFs, data are accumulating to support the idea that there may be alternative signaling pathways important for cell proliferation and that nuclear translocation and signaling are involved (34). Several reports showed that a direct accumulation of endogenously synthesized bFGF from cytoplasm to nucleus stimulated cell proliferation independent of release and interaction with cell surface FGFRs (35, 36). For the reasons mentioned above, it is unlikely that the release-independent mechanism may be functioning in the endogenous FGF action observed in this study, although the possibility that this mechanism may also function in parallel with signal transduction through cell surface receptors in KS cells would need to be ruled out. In contrast, other workers indicated that extracellular FGF molecules interact with cell surface FGFRs and undergo receptor-mediated endocytosis and nuclear accumulation, events required for cell cycle progression (37, 38). Mitogenic signals of released bFGF through FGFRs may occur both at the cell surface and after receptor translocation to the nucleus in KS cells.

Various growth factors have been established as potent mitogens for KS cells in culture, including OM, sIL-6R{alpha}/IL-6, IL-1ß, and TNF-{alpha} (19, 20, 21, 22, 23, 24). As high levels of production of these cytokines are present in sera and culture supernatants of monocytes/macrophages from HIV-infected individuals (39, 40, 41, 42), circulating and/or locally produced KS cell growth factors may contribute to the development and progression of KS lesions by directly acting on the growth of KS cells. In addition, DNA sequences of a novel human herpesvirus, termed HHV-8, have been identified in all epidemiologic forms of KS at a high frequency. The HHV-8 genome encodes an IL-6-like protein that shares functional properties with cellular IL-6 (43, 44, 45). Viral IL-6 is only expressed by a minority of cells in KS lesions; however, in patients with KS, viral IL-6 is abundantly expressed in HHV-8-infected hemopoietic cells and in lymph nodes (43). Further, both circulating and tumor-infiltrating mononuclear cells are productively infected with HHV-8, suggesting that this virus may trigger inflammatory responses and induce the production of various cytokines and growth factors (46, 47, 48). Thus, cellular and viral cytokine-rich environments might favor KS cell proliferation in patients even though HHV-8 could not directly transform precursor cells for KS. Indeed, despite the absence of HHV-8, the cultured KS cells we used maintained many KS-like features, responsiveness to KS cell growth factors, and the capability of inducing KS-like lesions in nude mice. We found here that cyclin E expression and cyclin E-CDK2 kinase activity are much higher in KS cells proliferating in external growth factor-containing medium than in cells cultured in KS basal medium alone. In contrast, levels of D-type cyclin expression and CDK4 kinase activity remained constant in the presence or the absence of these growth factors. Thus, the external factor-induced proliferation of KS cells appears to be related to the up-regulation of cyclin E rather than to that of cyclin D. We noted that the combination of external growth factors generated additive, but not synergistic, growth-stimulating effects on KS cells, and the anti-bFGF Ab almost completely inhibited the individual and combined effects on cell proliferation, cyclin E expression, and cyclin E-CDK2 activation, thereby suggesting that these growth factors independently function together with the endogenous bFGF to promote cyclin E-CDK2 activation and cell proliferation. Thus, the individual factors appear to activate, in parallel, a common intracellular target by which such different external mitogens participate in a growth-promoting cascade in KS cells.

Samaniego et al. (49) showed that the presence of IL-1ß or TNF-{alpha} stimulates the synthesis and release of biologically active bFGF from KS cells. This finding suggests that external factor-enhanced growth is mediated through an increase in the production and release of endogenous bFGF. However, when KS cells were cultured in basal medium with exogenously added acidic or basic FGF, cell growth and cyclin E expression remained at levels similar to those in KS cells cultured in basal medium alone. Thus, FGF cannot compensate for the lack of external growth factors. In addition, the expression and activation of FGFR1 and FGFR2 were not enhanced by treatment with external growth factors; therefore, it is unlikely that the proliferative effects of these factors are due to the simple augmentation of KS cell responsiveness to extracellular FGF molecules. Such a definite requirement for FGF-specific action and external factor-dependent proliferative signal suggests that these two types of factors probably function at different cellular events in KS cells, which ultimately act in concert to regulate cyclin E expression and cyclin E-CDK2 activity. This dual control system may play a central role in the multiple factor-driven proliferation of KS cells. Recent studies revealed that molecular events controlling cell cycle advance are regulated in concert by external mitogenic factors and cell anchorage to the extracellular matrix (50, 51). The HIV-1 tat gene product stimulates KS cell growth by mimicking the extracellular matrix through interactions with integrins, cell surface adhesion receptors (52, 53). We observed that treatment of KS cells with anti-bFGF Ab resulted in a striking decrease in expression of focal adhesion kinase, a cytoplasmic tyrosine kinase activated by integrin (K. Murakami-Mori, unpublished observations). The FGF-specific action, therefore, may be involved in transduction of cell-adhesive signals in KS cells. A possible relationship between the decreased expression of focal adhesion kinase and the down-regulation of cyclin E has yet to be addressed.

Cyclins D1 and E control different events, both rate limiting for the G1 to S phase transition, as evidenced in experiments using microinjection of anti-cyclin E or D Ab and overexpression of cyclin D or E molecules (54, 55, 56, 57). We found that anti-bFGF-induced inhibition of cyclin E expression and cyclin E-CDK2 activity resulted in G1 growth arrest of KS cells, while cyclin D-CDK4 activity remained at a steady state level. Therefore, the cyclin E-CDK2 activity apparently contributes to the G1 to S phase transition of the KS cell cycle, and this role is indispensable even in KS cells containing active forms of cyclin D-CDK4. By blocking the endogenous FGF action, we noted the selective inhibition of cyclin E/CDK2 activity in KS cells. This experimental model may be useful for further investigation of a sequential mechanism governing the G1 progression and S phase entrance of the KS cell cycle. Recent studies show that HHV-8 encodes a cyclin D homologue that is capable of stimulating CDK6 to phosphorylate pRB and histone H1 in in vitro kinase assays (58, 59). The finding that transcripts of viral cyclin are expressed in KS tissues suggests an active role of viral cyclin in KS cell growth (60). Whether ectopic expression of viral cyclin can overcome the bFGF Ab-induced growth arrest of KS cells will be addressed in ongoing investigations.

We obtained evidence of a correlation between KS cell growth and cyclin E-CDK2 activity, events tightly regulated through integration of the external mitogenic signal and the endogenous FGF signal. Blockade of either of these two signals can decrease cyclin E-CDK2 activity, and growth arrest follows. These findings provide support for the concept that KS is a potentially controllable hyperplasia, at least in the early stages. In addition, these data provide new insights into the possibility of preventing the development and progression of KS lesions. Further exploration of the molecular basis of the cyclin E-CDK2 regulation pathway is expected to advance knowledge of the pathogenesis of KS and lead to the development of compounds that specifically inhibit this pathway.


   Acknowledgments
 
We thank Dr. A. Jewett (Dental Research Institute, University of California, Los Angeles Dental School, Los Angeles, CA) for flow cytometric analysis, Dr. W. Marshall (DNA Technology Group, Amgen, Boulder, CO) for PCR primers, and M. Ohara for critical readings of the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Kaoru Murakami-Mori, Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1747. Back

2 Abbreviations used in this paper: KS, Kaposi’s sarcoma; OM, oncostatin M; sIL-6R{alpha}, soluble IL-6R; bFGF, basic fibroblast growth factor; CDK, cyclin-dependent kinase; HHV-8, human herpesvirus-8; aFGF, acidic fibroblast growth factor; CM, conditioned medium; FGFR, fibroblast growth factor receptor. Back

Received for publication January 8, 1998. Accepted for publication April 14, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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