[CANCERRESEARCH53. 4481-4485,October1. 1993]
Advances in Brief
Human Immunodeficiency Virus Type 1 Tat Protein Protects Lymphoid, Epithelial, and Neuronal Cell Lines from Death by Apoptosis Giorgio Zauli, 2 Davide Gibellini, Daniela Milani, Meri Mazzoni, Paola Borgatti, Michele La Placa, and Silvano Capitani Institute of Human Anatomy, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara [G. Z., D. M., M. M., P. B., S. C.], and Institute of Microbiology, University of Bologna, Via Massarenti 9, 40138 Bologna [D. G., M. L. P], Italy
Abstract We report here that the tat gene product of human immunodeficiency virus type 1 was able to protect iymphoblastoid (Jurkat), epithelial (293) and neuronal (PC12) cell lines from apoptotic death induced by serum withdrawal. The rescue from apoptosis by Tat was reflected by an increased expression of Bci-2 protein in tat-positive Jurkat cells with respect to mock-transfected Jurkat cells after 3-6 days of serum-free cultures. We propose that the ability of the regulatory human immunodeficiency virus type 1 Tat protein to suppress apoptosis might have important implications in understanding the pathogenesis of frequent neoplastic disorders observed in human immunodeficiency virus type 1-seropositive individuals. Introduction A p o p t o s i s or p r o g r a m m e d cell death is an a u t o n o m o u s cell suicide m e c h a n i s m , essential in the m a i n t e n a n c e of normal tissue homeostasis (1). Cells often u n d e r g o apoptosis as a response to e n v i r o n m e n t a l information, such as removal o f specific growth factors. This implicates the existence o f physiological m e c h a n i s m s able to prevent apoptosis and the increased cell n u m b e r in neoplastic tissues can be v i e w e d as a violation of normal homeostasis. Since malignant transformation is a multistep process, requiring aberrations in m o r e than a single p a t h w a y (2), an expansion of cell n u m b e r s could result from either increased proliferation or decreased cell death (3, 4). Beside its essential role in p r o m o t i n g viral replication (5), the protein e n c o d e d by the tat gene of HIV-13 can also be actively secreted by infected ceils, acting as an e x o g e n o u s growth factor for uninfected cells (6, 7). In fact, Tat can be rapidly taken up by different cell types in culture, enter the nucleus, and trans-activate both viral and cellular genes (8-10). To find out m o r e about the effect o f HIV-1 Tat on uninfected cells, w e evaluated the influence o f Tat on the survival of three different cell lines under serum-free culture conditions.
Materials and Methods Cell Lines. Jurkat human lymphoblastoid and 293 human kidney epithelial cell lines, transfected with the pRP neo-c Tat/S vector, containing the tat gene of HIV-1 in sense or mock-transfected with the pRP neo-c control vector have been described previously (11, 12). The PC12 rat pheochromocytoma cell line (13) was transfected with the same vector, utilizing the Transfectam lyposome kit (Promega, Madison, WI). Jurkat cells, which grow in suspension, were routinely cultured in 25-cm z tissue culture flasks (Falcon Plastics, Oxnard, CA)
Received 7/22/93; accepted 8/19/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This research was supported by the AIDS project of the Italian Ministry of Health; by CNR Grants ACRO and FATMA, and MURST 40 and 60%. z To whom requests for reprints should be addressed, at Institute of Human Anatomy, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy. 3 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; FCS, fetal calf serum; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline.
in RPMI plus 10% FCS (Gibco, Grand Island, NY). 293 and PC12 cells, which grow as an adherent monolayer, were cultured in 25-cm 2 tissue culture flasks (Falcon) in Dulbecco's modified Eagle's medium plus 10% FCS, and Dulbecco's modified Eagle's medium plus 10% FCS plus 5% horse serum (Gibco), respectively. Both tat-positive and mock-transfected cell lines were kept in the continuous presence of 600 p,g/ml of the aminoglycoside antibiotic G418 (Sigma Chemical Co., St. Louis, MO). In transient cotransfection experiments with the HIV-1 reported plasmid pTZIIICAT, containing the HIV-1 long terminal repeat promoter in front of the bacterial CAT gene, the production of bioactive Tat was evaluated in several tat-positive 293, Jurkat and PC12 cell clones. The CAT assay was then performed as described (14), and the percentage of [~4C]chloramphenicol conversion was calculated as the ratio of the monoacetylate plus biacetylate forms of [14C]chloramphenicol to total [14C]chloramphenicol. Apoptosis Assays. To establish whether Tat was able to rescue Jurkat, PC12, and 293 cells from apoptotic death, cells were examined at different time cultures after serum withdrawal by conventional transmission electron microscopy, DNA gel electrophoresis, and flow cytometry. Once the cell density of adherent PC12 and 293 was near confluence, serum-containing medium was discarded and, after two washings with PBS, replaced with medium alone. Jurkat cells were washed twice with PBS and resuspended in RPMI alone at a concentration of 2 • 105/ml. In some experiments, 2 • l0 s Jurkat cells were pelleted in Eppendorf tubes and supplemented with serial concentrations (from 1 to 100 ng/ml) of full length recombinant Tat protein (American Biotechnologies, Boston, MA) in 50/xl of RPMI plus 1% bovine serum albumin (Sigma) for 2 h at 37~ Jurkat cells were then seeded in 24 multiwell plates in a final volume of 1 ml of RPMI. In parallel, Tat protein was preincubated with a monoclonal (5/xg) or a polyclonal (1:100 dilution) anti-Tat antibody (American Biotechnologies) or a control polyclonal anti-mouse antibody (1:100 dilution) (Dako, Santa Barbara, CA) for 1 h at 37~ before adding to the cells. For the morphological studies, the cells were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer, postfixed with 1% osmium tetroxide, and embedded in Epon according to routine techniques. The thin sections were mounted on nickel grids and examined with an electron microscope, staining with uranyl acetate and lead citrate. For DNA gel electrophoresis experiments, aliquots of 2 • 106 cells were pelleted and resuspended in 20 /xl 10 mM EDTA-50 mM Tris-HC1 (pH 8) containing 0.5% (w/v) sodium lauryl sarcosinate and 0.5 mg/ml proteinase K and incubated at 50~ for 1 h. RNase A (10/xl; 0.5 mg/ml) was added to each sample and samples were incubated at 50~ for 1 h. Samples were heated at 70~ and 10/.d of 10 mM EDTA (pH 8.0) containing 1% (w/v) low-gellingtemperature agarose, 0.25% (w/v) bromophenol blue, and 40% (w/v) sucrose was mixed with each sample before loading into the dry wells of a 2% (w/v) agarose gel containing 0.1/xg/ml ethidium bromide. Electrophoresis was carried out in 2 mM EDTA-800 mM Tris-phosphate (pH 7.8) until the marker dye had migrated 3-4 cm. The evaluation of apoptotic cell death in flow cytometry was performed as described previously (15). Briefly, cells were harvested by centrifugation at 200 • g for 10 min at 4~ The pellets were treated with 0.5 mg RNase (type I-A, Sigma) and resuspended in PBS containing 50/xg/ml propidium iodide. Analysis was performed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA) with the FL2 detector in logarithmic mode, using Lysis II software (Becton-Dickinson).
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SUPPRESSION OF APOPTOSIS BY TAT
Fig. 1. Evaluation of tat-positive and mocktransfected cells after 24 h of serum-free culture by transmission electron microscopy. Most mocktransfected 293 (a), Jurkat (b), and PC12 (c) ceils showed characteristic features of apoptosis, while tat-positive 293 (d), Jurkat (e), and PC12 (f) cells displayed a normal morphology.
Western Blot Analysis of Bci-2 Protein Expression. Western blots were performed as described (16). Briefly, cell lysates from tat-positive or mocktransfected Jurkat cells were obtained by sonicating cells for 2 min and boiling for 5 min in 62.5 mM Tris buffer, pH 6.8, with 2% sodium dodecyl sulfate-5% /3-mercaptoethanol-10% glycerol. Samples containing I • 106 viable cells, containing approximately 100 /xg of proteins, in 30 ~I were separated by discontinuous electrophoresis on a 12.5% acrylamide gel and blotted onto nitrocellulose filters. Blotted filters were blocked for 30 rain using a 3% suspension of dried skimmed milk in PBS. The filter was then incubated at 4~ with a 1:100 dilution of anti-Bcl-2 monoclonal antibody (Dako) or an anti-/3actin monoclonal antibody (Dako) in 3% milk PBS. After overnight incubation, the filter was washed and further incubated for 1 h at room temperature with 1:1500 peroxidase-conjugated anti-mouse IgG (Sigma) in 0.1% bovine serum albumin, washed, and revealed with the enhanced chemiluminescence Western
blotting detection reagent (Amersham Corp., Arlington Heights, IL). Quantitative analysis was performed by scanning densitometry (LKB UltroScan XK). SDS protein molecular weight markers (Sigma) were run with each gel.
Results and Discussion Rescue of 293, Jurkat, and PC12 Cells from Their Entry into Apoptosis b y HIV-1 Tat Protein. 293, Jurkat, and PC12 cell lines w e r e stably transfected with the tat gene o f HIV-1. The tat-positive 293, Jurkat, and PC12 cell clones e x a m i n e d in this study showed a constant production o f bioactive Tat in transient cotransfection experiments with the HIV-1 reporter plasmid pTZIIICAT. The m e a n percentage o f [~4C]chloramphenicol conversion o f three separate ex-
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SUPPRESSION OF APOPTOSIS BY TAT
1
2
3
4
5
6
7
Table 1 Quantitative evaluation of the percentage of apoptotic cell death in serum-free cultures of tat-positive and mock-tran~fected cell lines by flow cytometry a
8
Time (h) of serum-freeculture 24
48
72
293 cells Mock-transfected tat-positive
23_6 4 +_2
32.5__-8 8 +--3
45_+9 11 +-4
Jurkat cells Mock-transfected tat-positive
17_+5 3.5 + 1
23_+8 6.5 ---2
35--+7 8---3.5
PC12 cells Mock-transfected 21.5_+5 33-+7.5 52---8 tat-positive 2---0.5 4_+2.5 5.5---3 a Data are expressed as means -+ SD of five separate experiments performed in duplicate. A statistically significant difference(P < 0.01) in the percentageof apoptotic cells was observedbetweentat-positive and mock-transfected293, Jurkat, and PC12 cells, from 24 h of serum-free culture onwards.
Fig. 2. a, gel electrophoresisanalysis of DNA extracted from 2 • 106 cells after 24 h of serum-freeculturestat-positive 293 (Lane 2), PCI2 (Lane 4), and Jurkat (Lane 6) cells and mock-transfected293 (Lane 3), PCI2 (Lane 5) and Jurkat (Lane 7) cells.Lanes I and 8, molecularsize standards, b, serum-free survival of tat-positive and mock-transfected 293, Jurkat, and PC12 cells, evaluatedcountingviable cells by trypan blue dye exclusion. Data are expressed as means --_ SD (bars) of four separate experiments performed in duplicate.
periments in 1 h of CAT assay was 28 ___ 5 (SD), 35 __+ 7, and 40 ___ 9% in tat-positive 293, Jurkat, and PC12 cell clones, respectively. As early as after 24 h of serum-free culture, ultrastructural analysis of mock-transfected 293, Jurkat, and PC12 cells demonstrated the occurrence of various aspects characteristic of apoptosis, such as chromatin condensation and nuclear fragmentation (Fig. 1, a-c). On the other hand, the majority of tat-positive 293, Jurkat, and PC12 cells
still showed a normal morphology (Fig. 1, d - f ) . Also, a clear ladder of DNA fragmentation, typically observed during the process of apoptosis, was present in mock-transfected cell clones and absent in tatpositive cell clones (Fig. 2a). Consistently, when examined by trypan blue dye exclusion, mock-transfected 293, Jurkat, and PC12 cells died asynchronously over several days (Fig. 2b) whereas tat-transfected cells survived for over 1 week in the complete absence of serum. By day 10, the number of viable tat-positive PC12, 293, and Jurkat cells was still over 50% of the initial cell count (Fig. 2b). To precisely evaluate the percentage of apoptosis at different times after serum deprivation in both mock-transfected and tat-positive cell lines a flow cytometry procedure was used (Fig. 3) (15). After 24-72 h of serum-free cultures, mock-transfected 293, Jurkat, and PC12 cells showed a progressive increase in apoptosis (Table 1), whereas the percentage of apoptosis in tat-positive cells was very low and remained rather constant with time. In the next series of experiments, we investigated whether also recombinant Tat protein, added as an exogenous growth factor, was able to protect mock-transfected Jurkat cells from death by apoptosis. The addition of recombinant Tat to Jurkat cells significantly (P < 0.01) reduced the percentage of apoptosis in serum-deprived cultures at concentrations as low as 10 ng/ml (Table 2). The ability of Tat to promote cell survival was specific since it was completely blocked by either a monoclonal (5 /xg) or a polyclonal (diluted 1:100) anti-Tat antibody, but not by a control polyclonal (diluted 1:100) anti-mouse antibody (Table 2).
Down-regulation of Bcl-2 Protein Expression in Mock-transfected Jurkat Cells but not in tat-Positive Jurkat Cells, under Serum-free Culture Conditions. As shown in Fig. 4o, Bcl-2 protein expression progressively decreased in mock-transfected Jurkat cells incubated under serum-free culture conditions. On the other hand, the level of Bcl-2 expression in tat-positive Jurkat cells did not vary significantly over a period of 6 days (Fig. 4a), suggesting that the
Fig. 3. Evaluation of apoptotic death cells i tat-positive (A) and mock-transfected (B) Jurk~ cells by flow cytometry after 24 h of serum-fr~ culture. Apoptosis appears in flow cytometryas subdiploid peak (arrow). Data were expressed percentage of apoptotic versus nonapoptotic cell regardless of the specific cell cycle phase.
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SUPPRESSION OF APOPTOSIS BY TAT Table 2 Quantitative evaluation of the percentage of apoptotic cell death in Jurkat
cells, supplemented with various concentrations of recombinant Tat in absence or presence of a monoc/onal anti-Tat (5 i~g/ml) or a control antibody (Ab) a
Tat protein concentrations 1 25KD~II~
2
,~,~ .,,~.
5
3
6
7
8
..-._.
24
48
72
0 ng/ml + anti-Tat + control Ab.
16 +- 6 17 +- 5.5 17.5 +- 5
21.5 --- 7 20 + 8 22 ___6.5
38 ___8 40 +- 10 37 +- 9
0.1 ng/ml + anti-Tat + control Ab.
17 ___ 4.5 18 +- 6 19 +- 6.5
24.5 +- 8 22.5 --- 7 25 +- 6.5
36 +- 7 34 +- 9 33 --- 8
1 ng/ml + anti-Tat + control Ab. b 40003500 3000 2500
Time (h) of serum-free culture
12"--5 18 --_ 4 13.5 +- 6
22 _+6.5 23 --- 9 21 +- 4.5
35 +- 7.5 39 _+9 36 --- 7
10 ng/ml + anti-Tat + control Ab.
4 --_ 1.5 17 --- 5 5.5 --- 3
10 _ 3.5 23 ___7 9 +- 4.5
17 +- 5.5 39 --- 8 19 --- 7
20 ng/ml + anti-Tat + control Ab.
5 +- 2 16 --_ 7 6.5 +- 4
6.5 ___3 24 _ 8 5 +- 4.5
14.5 +- 6 37 - 10 16 --- 8.5
100 ng/ml 6 --_ 3 5 ___3 7.5 --- 5 + anti-Tat 15 +- 9 21 _+ 8 41 --- 9 + control Ab. 7.5 +_5 6 +--3.5 8.5 --- 3 a Data are expressed as mean + SD of five separate experiments performed in duplicate. Serum-free cultures supplemented with 10-100 ng/ml of Tat showed a significant (P < 0.05) reduction in the percentage of apoptosis with respect to Jurkat cells supplemented with 0-1 ng/ml of Tat after 24 h of culture. The protective effect of Tat was completely blocked by anti-Tat monoclonal antibody.
2000 1500 1000 500 0 1
Days of
3 serum-free culture
Fig. 4. Down-regulation of Bcl-2 protein expression in mock-transfected Jurkat cells under serum-free culture conditions. In a, immunoblot for the detection of Bcl-2 protein was carried out with cell lysates from 1 • 106 mock-transfected (Lanes 1--4) and tat-transfected (Lanes 5-8) Jurkat cells after 1 (Lanes 1 and 5), 2 (Lanes 2 and 6), 3 (Lanes 3 and 7) and 6 (Lanes 4 and 8) days of serum-free culture. After immunoblotting and hybridization, the autoradiograph was scanned by densitometry (b) A representative of three separate experiments is shown. Data are expressed as units of relative intensity after 1 and 3 days of serum-free culture. (11), mock-transfected Jurkat cells; (lS]), tat-positive Jurkat cells.
rescue f r o m apoptosis by Tat is reflected by a sustained Bcl-2 expression. Quantitative analysis c o n f i r m e d that the level of Bcl-2 protein expression was significantly higher in tat-positive Jurkat cells g r o w n in serum-free cultures (Fig. 4b). A l t h o u g h these data do not prove that HIV-1 Tat protein up-regulates Bcl-2 protein, it is very unlikely that they merely reflect an aspecific loss of protein production in m o c k transfected Jurkat cells, due to the reduction of viable cells. In fact, Bcl-2 expression was m a r k e d l y reduced after 72 h o f culture, w h e n the n u m b e r of viable Jurkat cells was still 4 0 - 5 0 % o f the initial cell n u m b e r (Fig. 2b), and the same n u m b e r (106) of viable tat-positive and mock-transfected Jurkat cells, s h o w i n g a similar a m o u n t of proteins (approximately 100 txg) was used in the different experimental points. Moreover, in one e x p e r i m e n t we observed that while Bcl-2 protein expression declined over time in m o c k - t r a n s f e c t e d Jurkat cells, the level o f / 3 - a c t i n protein was u n m o d i f i e d (data not shown). T h e protein e n c o d e d by the tat gene of HIV-1 plays an essential role in viral replication, acting on the cis-acting sequence responsive T A R element, located at the 5' end of all viral m R N A s (5). A l o n g with this action on viral g e n o m e . Tat protein is able to transactivate genes e n c o d i n g for cytokines, such as t u m o r necrosis factor /3 (9) and transforming g r o w t h factor /31 (10), Moreover, recent findings d e m onstrated that Tat protein can act as an e x o g e n o u s g r o w t h factor, as it can be rapidly taken up by different cell types (8), and displays either
stimulatory (6) or inhibitory (7) effects on cell proliferation, d e p e n d ing on the cell type considered and the concentrations used. D u r i n g the course of HIV-1 infection there is a significant increase o f neoplastic disorders. This is generally ascribed to the i m m u n o d e ficiency status of H I V - l - i n f e c t e d subjects, while the possibility that HIV-1 could play an active role in the pathogenesis of the frequent malignancies o f acquired i m m u n o d e f i c i e n c y virus patients has been excluded thus far, since HIV-1 sequences have never been f o u n d in t u m o r biopsy samples obtained f r o m H I V - l - i n f e c t e d individuals (17, 18). A l t h o u g h our data were obtained in a tissue culture m o d e l utilizing i m m o r t a l i z e d cell lines, the ability of HIV-1 Tat protein to suppress the apoptotic cell death pathway, even w h e n added to cells as an e x o g e n o u s factor, suggests that HIV-1 infection m i g h t play s o m e role in oncogenesis. For instance, bioactive Tat actively secreted by infected cells could p r o m o t e the survival of n e i g h b o r i n g uninfected cells obviating the need for HIV-1 m a i n t e n a n c e in t u m o r tissues. Moreover, the ability of Tat to prevent apoptosis does not s e e m restricted to certain cell types, since similar effects w e r e observed in cell lines of l y m p h o i d , epithelial, and neuronal origin. Since no data are available on the quantitative levels of Tat protein in vivo is still not clear w h e t h e r Tat reaches high e n o u g h levels to exert a general regulatory effect. However, it is conceivable that the highest levels of Tat are present in peripheral l y m p h o i d tissues, w h e r e an active HIV-1 replication is always present (19). Therefore, it is tempting to speculate that a Tat-mediated aberrant cell survival could contribute to the polyclonal follicular lymphoproliferation frequently observed in HIV-l-seropositive individuals, w h i c h often precedes malignant transformation (20).
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Human Immunodeficiency Virus Type 1 Tat Protein Protects Lymphoid, Epithelial, and Neuronal Cell Lines from Death by Apoptosis Giorgio Zauli, Davide Gibellini, Daniela Milani, et al. Cancer Res 1993;53:4481-4485.
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