Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a.

Copyright

q

1997, American Society for Microbiology

Human T-Cell Leukemia Virus Type 1 Tax Releases Cell

Cycle Arrest Induced by p16

INK4a

KENNETH G. LOW,

1

_{* LYDIA F. DORNER,}

1

_{DINALI B. FERNANDO,}

1

_{JOSEPH GROSSMAN,}

1

KUAN-TEH JEANG,

2_AND

_{MICHAEL J. COMB}

1

Laboratory of Cell Signaling, New England Biolabs, Beverly, Massachusetts 01915,

1

_{and Laboratory of Molecular}

Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892

2

Received 13 August 1996/Accepted 27 November 1996

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein causes cellular transformation by

de-regulating important cellular processes such as DNA repair, transcription, signal transduction, proliferation,

and growth. Although it is clear that normal cell cycle control is deregulated during HTLV-1-induced cellular

transformation, the effects of Tax on cell cycle control are not well understood. Flow cytometric analyses of

human T cells indicate that cell cycle arrest in late G

1

, at or before the G

1

/S restriction point, by p16

INK4a

is

relieved by Tax. Furthermore, Tax-dependent stimulation of 5-bromo-2

*

-deoxyuridine incorporation and

tran-scriptional activation is inhibited by p16

INK4a

_{. This result suggests that p16}

INK4a

_{is able to block Tax-dependent}

stimulation of DNA synthesis and cell cycle progression into S phase. In vitro binding assays with recombinant

glutathione

S

-transferase fusion proteins and [

35

_{S]methionine-labeled proteins indicate that Tax binds}

spe-cifically with p16

INK4a

_{but not with either p21}

cip1

_{or p27}

kip1

_{. Furthermore, sequential immunoprecipitation}

assays with specific antisera and [

35

_{S]methionine-labeled cell lysates subsequent to coexpression with Tax and}

p16

INK4a

_{indicate that the two proteins form complexes in vivo. Immunocomplex kinase assays with}

cyclin-dependent kinase 4 antiserum indicate that Tax blocks the inhibition of cdk4 kinase activity by p16

INK4a

_{. This}

study identifies p16

INK4a

_{as a novel cellular target for Tax and suggests that the inactivation of p16}

INK4a

function is a mechanism of cell cycle deregulation by Tax.

Adult T-cell leukemia/lymphoma is caused by human T-cell

leukemia virus type 1 (HTLV-1) and is characterized by

mul-tistage transformation and immortalization of CD4

1

_T

lym-phocytes, which includes a long latent period following

infec-tion (6, 13). Oncogenic transformainfec-tion of T lymphocytes by

HTLV-1 is due primarily to the expression of the 40-kDa Tax

retrovirus oncoprotein, which deregulates a variety of

impor-tant cellular processes (6, 13). Tax has previously been shown

to directly interact and interfere with the ability of specific

cellular proteins to perform pivotal and critical roles in

tran-scriptional activation and growth factor signal transduction (6,

13). For example, Tax transactivates the expression of a variety

of cellular genes by increasing the DNA binding of cellular

transcription factors such as NF-

k

B (4, 22, 32), serum response

factor (12), AP-1 proteins, cyclic AMP response element

bind-ing protein (CREB), cyclic AMP response element modulatbind-ing

proteins, and ATF proteins (11, 23, 40, 45). The increased

DNA binding has been shown to be due to enhanced

transcrip-tion factor dimerizatranscrip-tion (2, 5, 44) or increased transcriptranscrip-tion

factor nuclear translocation resulting from the degradation of

cytoplasmic inhibitors (19). Tax can also transactivate gene

expression by interacting directly with components of the basal

transcriptional complex such as the TATA-binding protein (7)

or with transcriptional coactivators such as CREB-binding

pro-tein which mediate interactions between transcription factors

and the basal transcriptional complex (21). Recent studies also

suggest that Tax may interact with and activate specific

com-ponents of growth factor signal transduction pathways such as

the Ras/mitogen-activated protein kinase pathway (31, 43) and

the protein kinase A (PKA) and PKC pathways (18).

How-ever the immortalization of CD4

1

_{T lymphocytes by HTLV-1}

Tax requires other deregulatory events since its effects on

transcription or signal transduction alone have not been

dem-onstrated to be sufficient for oncogenic transformation. It is

clear that normal cell cycle control is deregulated during

HTLV-1 leukemogenesis; however, the molecular mechanism

through which this occurs is not well understood.

Normal cell cycle progression is intricately regulated at

dif-ferent checkpoints by a family of cyclin-dependent kinases

(cdks) which are activated by complex formation with cyclins

and phosphorylation (15, 27, 30, 36, 37). Recent studies have

identified a novel family of mammalian cdk inhibitors which

can be divided into two subfamilies based on amino acid

se-quence homology, functionally distinct modes of action, and

range of specificities for cdks (15, 27, 30, 36, 37). Members of

one subfamily of cdk inhibitors, which includes p21

cip1

_{, p27}

kip1

_,

and p57

kip2

_{, have significant amino acid sequence homology}

and inhibit a broad range of cdks by binding cdk-cyclin

com-plexes as multimers, whereas members of the other subfamily,

which includes p16

INK4a

_{, p15}

INK4b

_{, p18}

INK4c

_{, and p19}

INK4d

_,

in-duce cell cycle arrest by specifically inhibiting cdk4 and cdk6

kinase activities through the displacement of cyclin Ds from

the cdk-cyclin complexes and are composed of four ankyrin

motifs (15, 27, 30, 36, 37). Interestingly, Tax has previously

been demonstrated to interact specifically with the ankyrin

motifs of I

k

B

g

(14) and a three-amino-acid residue AAR motif

on CREB (1) which is also present in p16

INK4a

_{(35). Since}

p16

INK4a

_{serves a critical role in regulating cell cycle}

progres-sion into S phase and can suppress cellular transformation (20,

24, 34), we hypothesized that Tax deregulates normal cell cycle

control by interacting with p16

INK4a

_{and interfering with its}

ability to repress cdk4 kinase activity and cellular

transforma-tion.

In this study, we demonstrate that although p16

INK4a

over-expression alone induces cell cycle arrest in late G

1

, at or

* Corresponding author. Mailing address: New England Biolabs, 32

Tozer Rd., Beverly, MA 01915. Phone: (508) 927-5054, ext. 335. Fax:

(508) 922-7069. E-mail: [email protected].

1956

on November 9, 2019 by guest

http://jvi.asm.org/

before the G

1

/S restriction point, it is unable to do so in the

presence of Tax. In addition, the stimulation of DNA synthesis

and cell cycle progression into S phase by Tax alone or with

oncogenic Ras is inhibited by p16

INK4a

_{overexpression.}

Tax-dependent transactivation of proenkephalin gene transcription

is also inhibited by p16

INK4a

_{overexpression. Furthermore, Tax}

blocks the p16

INK4a

_{-dependent inhibition of cdk4 kinase}

activ-ity, suggesting that Tax and p16

INK4a

_{might interact directly. In}

vitro binding assays indicate that Tax binds p16

INK4a

_{but not}

p21

cip1

_{or p27}

kip1

_{and forms complexes with p16}

INK4a

_{in vivo.}

These studies identify a novel cellular target for Tax and

sug-gest a mechanism for cell cycle deregulation by HTLV-1.

MATERIALS AND METHODS

Materials.All reagents, horse serum, and fetal calf serum (FCS) were from Sigma Chemical Company (St. Louis, Mo.) unless otherwise stated. Cell culture media were from GIBCO/BRL (Gaithersburg, Md.). [3_{H]chloramphenicol (37.9}

Ci/mmol), [g-32_{P]ATP (3,000 Ci/mmol), and [}35_{S]methionine (1,175 Ci/mmol)}

were from DuPont New England Nuclear (Boston, Mass.).

Antibodies.Antiserum against p16INK4a_{was from PharMingen (San Diego,}

Calif.), antiserum against Tax was from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (Bethesda, Md.) (originally contributed by B. R. Cullen), and antisera against cdk4 and cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, Calif.). Fluorescein-conjugated CD8 antiserum was from Becton Dickinson (San Jose, Calif.), and antiserum against 5-bromo-29-deoxyuridine (BrdU) was from Amersham (Arlington Heights, Ill.).

DNA plasmids.DNA plasmids for the in vitro transcription and translation reactions, the expression of recombinant glutathioneS-transferase (GST) fusion proteins in bacteria, and the expression of recombinant proteins in mammalian cell lines were generous gifts from C. Z. Giam (Case Western University School of Medicine, Cleveland, Ohio) (pET-11d-TaxH6), F. Kashanchi and J. N. Brady

(National Cancer Institute, Bethesda, Md.) (pGEX-2T-Tax), B. R. Cullen (Duke University Medical Center, Durham, N.C.) (pCMV-Tax), M. Serrano and D. Beach (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) (pBluescript II SK[2]-p16INK4a_{, pGEX-KG-p16}INK4a_{, and pCMV-cdk4), W. S. El-Deiry and}

B. Vogelstein (Johns Hopkins University School of Medicine, Baltimore, Md.) (pGEX-2T-WAF1 and pCEP4-WAF1), H. Toyoshima and T. Hunter (Salk In-stitute, La Jolla, Calif.) (pBluescript II SK[1]-p27kip1_{and pGEX-KG-p27}kip1_{), P.}

Hinds (Harvard Medical School, Boston, Mass.) (pCMV-cyclin D1), M. Meyer-son and E. Harlow (Massachusetts General Hospital Cancer Center, Charles-town) (pGEX-30X-Rb773-928), and R. A. Weinberg (Massachusetts Institute of

Technology, Cambridge) (pEJ 6.6). pBluescript II SK-WAF-1 was con-structed by subcloning a 2.1-kbHindIII/XhoI fragment encoding the p21cip1

cDNA from pCEP4-WAF1 into pBluescript II SK (Stratagene, La Jolla, Calif.). pTM3-p16INK4a_{was constructed by in-frame subcloning of a 475-bp}

PCR-am-plified fragment containing the p16INK4a_{cDNA intoNcoI andXhoI restriction}

endonuclease sites of pTM3 (9, 28). This fragment was amplified as previously described (23), using 18 cycles of PCR with 0.25 mM deoxynucleoside triphos-phates and 400 ng each of pBluescript II SK(2)-p16INK4a_{as the template and}

primers 59-GAAACGGATCCATGGAGCCTTCGGCTGACTGG-39and 59-G AATCGGATCCCTCGAGTCAATCGGGGATGTCTGAGGG-39 per 100-ml reaction volume. The DNA sequence of the PCR-amplified fragment was veri-fied by sequencing. pTM3-TaxH6was constructed by in-frame subcloning of an NcoI/BamHI fragment containing the full-length Tax coding sequence with a carboxyl-terminal His6tag from pET-11d-TaxH6into pTM3. pCMVbwas from

Clontech Laboratories (Palo Alto, Calif.).

Culture of cell lines and vaccinia virus strains.All cell lines and vaccinia virus strains were from the American Type Culture Collection (Rockville, Md.) unless otherwise stated. The JPX-9 cell line was a gift from K. Sugamura (Tohoku University School of Medicine, Sendai, Japan) and is a stably transfected deriv-ative of human Jurkat-derived T-lymphocyte cells (H33HJ-JA1) which express Tax under regulation by the murine metallothionein promoter (29). The treat-ment of JPX-9 cells with 30mM CdCl2for 14 h stimulates Tax expression in

greater than 90% of the cells to a level approximately equivalent to that observed in the HTLV-1-transformed human T-cell line MT-2 (reference 29 and unpub-lished observations). JPX-9 and H33HJ-JA1 cells were cultured in RPMI 1640 containing 10% (vol/vol) FCS. JEG-3 human choriocarcinoma, IMR-90 human diploid fetal lung, and BS-C-1 African green monkey kidney cells were cultured in minimal essential medium containing 10% (vol/vol) FCS. Hs27 human fore-skin fibroblast cells were cultured in Dulbecco modified Eagle medium contain-ing 10% (vol/vol) FCS. HeLa S3 human cervical carcinoma epithelial cells were cultured in S-minimal essential medium containing 5% (vol/vol) HS. Vaccinia virus strain WR recombinant vTF7-3 stocks were amplified in HeLa S3 cells, and titers were determined in BS-C-1 cells by plaque assays (8). Cells were infected at a multiplicity of infection of;10 for protein expression as previously described (10).

Transient transfections.JPX-9 cells were transiently transfected by electro-poration (300 V, 960mF, single pulse), and H33HJ-JA1 cells were transiently transfected by the DEAE-dextran protocol as previously described (23). JEG-3, IMR-90, and Hs27 cells were transiently transfected by a modification of the calcium phosphate protocol as previously described (23).

Propidium iodide labeling and flow cytometry.JPX-9 cells (53105_{) were}

synchronized in G0/G1phase by serum starvation for 18 h, incubated with

fluorescein-conjugated CD8 antiserum for 30 min at 48C, washed twice with phosphate-buffered saline (PBS), fixed in 0.25% (wt/vol) paraformaldehyde in PBS for 30 min at 48C, permeabilized with 0.2% (vol/vol) Tween 20 in PBS for 15 min at 378C, labeled with propidium iodide (50mg/ml), and treated with RNase A (50mg/ml) in PBS for 1 h at 378C. CD81_{cells were then analyzed for} DNA content as indicated by propidium iodide labeling.

BrdU labeling and immunohistochemistry.DNA synthesis in JEG-3 cells was determined by BrdU incorporation, using a cell proliferation assay kit (Amer-sham, Arlington Heights, Ill.). Cells were labeled with BrdU–5-fluoro-29 -deoxy-uridine (10:1), and BrdU-positive cells were identified by immunohistochemistry, using a cell proliferation assay kit (Amersham).

CAT andb-galactosidase assays.Following transient transfections, cells were harvested and lysed, and chloramphenicol acetyltransferase (CAT) activity in cell lysates was determined as previously described, with [3_{H]chloramphenicol as a}

substrate and butyryl coenzyme A as an acyl donor (23).b-Galactosidase activity in cell lysates was determined as previously described, usingo-nitrophenyl-b-D

-galactopyranoside as a substrate, and used to normalize for differences in the efficiency of transfection and protein expression between the samples (23). His-tochemical detection ofb-galactosidase activity was determined by fixing cells in 2% (wt/vol) paraformaldehyde and 0.2% (vol/vol) glutaraldehyde for 10 min and using 0.1% (wt/vol) 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal)

in reaction buffer [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 5%

(wt/vol)N,N-dimethylformamide] as a substrate.

In vitro transcription and translation.[35_{S]methionine-labeled proteins were}

synthesized as previously described (23), using the TNT T7-T3 coupled reticu-locyte lysate system (Promega Corporation, Madison, Wis.) with either pBlue-script II SK[2]-p16INK4a_{, pBluescript II SK-WAF-1, pBluescript II SK[1}

]-p27Kip1_{, or pET-11d-TaxH} 6.

Bacterial expression and purification of recombinant GST fusion proteins. Escherichia coliDK-1 was transformed with either pGEX-KG-p16INK4a_,

pGEX-2T-WAF1, pGEX-KG-p27Kip1_{, pGEX-2T-Tax, or pGEX-30X-Rb}

773-928.

Recom-binant GST fusion proteins were expressed and immobilized on glutathione-agarose beads, and the amount of protein was estimated as previously described (23, 33, 39).

In vitro binding assays.Five microliters of each [35_{S]methionine-labeled}

pro-tein was added to equal amounts of different agarose-immobilized GST fusion proteins in 100ml of binding buffer (0.25 M NaCl, 50 mM Tris/HCl [pH 7.4], 1% [vol/vol] Nonidet P-40 [NP-40], 10 mM EDTA, 2 mM EGTA, 0.02% [wt/vol] sodium azide, 0.25 mM phenylmethylsulfonyl fluoride [PMSF], 1mg each of aprotinin, leupeptin, and pepstatin A per ml) for 45 min at 48C. The agarose-immobilized complexed proteins were washed five times with binding buffer and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.

[35_{S]methionine in vivo cell labeling and immunoprecipitations.Cells were}

cultured in methionine-free medium for 1 h and then in the same medium containing 0.25 mCi of [35_{S]methionine/ml for 4 h, harvested, and lysed by}

scraping in lysis buffer (0.15 M NaCl, 50 mM Tris-HCl [pH 7.4], 0.5% [vol/vol] NP-40) containing 0.1 mM PMSF and 2.5mg each of aprotinin, leupeptin, and pepstatin A per ml for 10 min at 48C. Cell debris was pelleted at 14,0003gmax

for 10 min at 48C and discarded. Cell extracts were incubated with polyclonal antiserum against either cdk4 (1:100 dilution), cyclin D1 (1:100 dilution), p16INK4a_{(1:100 to 1:500 dilution), or Tax (1:100 to 1:500 dilution) for 18 h and}

then with 25ml of 50% (vol/vol) protein A-Sepharose in lysis buffer for 4 h. Protein A-Sepharose protein complexes were washed three times with 1 ml of lysis buffer and analyzed immediately by SDS-PAGE followed by autoradiogra-phy. In vivo protein complexes were detected by subsequent denaturation of the protein A-Sepharose protein complexes at 958C for 5 min in 0.1 ml of denatur-ation buffer (50 mM Tris-HCl pH 7.5, 0.5% [vol/vol] SDS, 70 mMb -mercapto-ethanol) and incubation with specific polyclonal antiserum against either p16INK4a_{(1:100 to 1:500 dilution) or Tax (1:100 to 1:500 dilution) in}

radioim-munoprecipitation assay (RIPA) buffer (minus SDS) (0.15 M NaCl, 10 mM Tris-HCl [pH 7.5], 1% [vol/vol] NP-40, 1% [wt/vol] deoxycholate, and 0.25 mM PMSF) for 18 h and then with 25ml of 50% (vol/vol) protein A-Sepharose in RIPA buffer (minus SDS) for 4 h. Protein A-Sepharose protein complexes were washed three times with 1 ml of RIPA buffer (minus SDS) and analyzed by SDS-PAGE followed by autoradiography.

Immunocomplex kinase assays.Cells were lysed by sonication at 48C in im-munoprecipitation buffer (0.15 M NaCl, 50 mM HEPES [pH 7.5], 1 mM EDTA, 2.5 mM EGTA, 1 mM dithiothreitol [DTT], 0.1% [vol/vol] Tween 20, 10% [vol/vol] glycerol, 0.1 mM PMSF, 10 mMb-glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4, 10mg each of aprotinin, leupeptin, and pepstatin A per ml). Cell

debris was pelleted at 14,0003gmax for 10 min at 48C and discarded. Cell

extracts were incubated with polyclonal antiserum against cdk4 (1:100 dilution) for 18 h and then with 25ml of 50% (vol/vol) protein A-Sepharose in immuno-precipitation buffer for 4 h. Protein A-Sepharose protein complexes were washed

on November 9, 2019 by guest

http://jvi.asm.org/

four times with 1 ml of immunoprecipitation buffer and twice with 1 ml of 50 mM HEPES (pH 7.5) containing 1 mM DTT. Kinase assays were performed as previously described (26) for 30 min at 308C in a final volume of 20ml (includes protein A-Sepharose bead volume) in kinase buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 25mM ATP, 10mCi of [g-32P]ATP)

containing 200 ng of an;45-kDa GST fusion protein containing the carboxyl-terminal portion of the retinoblastoma protein (GST-RbC) as a substrate. Then 200ml of immunoprecipitation buffer at 48C was added to stop the kinase reaction, and supernatants were removed to fresh tubes and incubated with 25ml of 50% (vol/vol) glutathione-agarose in immunoprecipitation buffer for 2 h at 48C. Glutathione-agarose beads were washed three times with 1.0 ml of immu-noprecipitation buffer, and phosphorylated GST-RbC proteins were separated by SDS-PAGE and visualized by autoradiography.

Digitization of autoradiographs and preparation of figures.Autoradiographs were digitized and prepared for the figures as previously described (23). Briefly, autoradiographs were digitized with a Microtek (Torrance, Calif.) Scanmaker III and an Apple (Cupertino, Calif.) Power Macintosh 8100/80, using Photoshop version 3.0 (Adobe Systems, Mountain View, Calif.). Digitized images were saved as tagged image file format files and exported into Pagemaker version 5.0 (Aldus Corp., Seattle, Wash.), where the images were cropped, text was added, and documents were printed at 2,540 dots per inch and a line screen of 150 lines per inch, using a Varityper 5300E (PageWorks, Cambridge, Mass.).

RESULTS

Tax releases JPX-9 cells from cell cycle arrest induced by

p16

INK4a

_.

_{The effects of p16}

INK4a

_{overexpression on cell cycle}

progression in the absence and presence of Tax were examined

in synchronous JPX-9 human T-cells (Fig. 1). Serum starvation

of JPX-9 cells for 18 h resulted in cell cycle arrest and the

synchronization of

.

95% of the total population of cells in

G

0

/G

1

phase (data not shown). Subsequent culture of JPX-9

cells in serum-containing medium stimulated reentry into the

cell cycle, resulting in the progression of 30% of the cells into

S phase and the retention of 70% of the cells in G

0

/G

1

phase

after 14 h (Fig. 1A). The induction of Tax expression in JPX-9

cells with 30

m

M CdCl

2

(see Materials and Methods for the

source and a complete description of JPX-9 cells) over the

same time period did not significantly alter the distribution of

cells in G

0

/G

1

and S phases (68 and 32%, respectively (Fig.

1B). In contrast, the overexpression of p16

INK4a

_{in JPX-9 cells}

by transient transfection significantly altered the distribution of

cells, with 84% accumulating in G

0

/G

1

phase and only 16%

progressing into S phase (Fig. 1C). This result suggests that

p16

INK4a

_{overexpression arrests cell cycle progression in late}

G

1

, at or before the G

1

/S restriction point. However, in the

presence of Tax, p16

INK4a

_{was unable to arrest cells from}

progression into S phase (Fig. 1D), with the distribution of

cells in G

0

/G

1

and S phases being not significantly different

from that of control cells (Fig. 1A and D). These results

sug-gest that Tax can potentially deregulate the normal restriction

of cell cycle progression in late G

1

/S mediated through

p16

INK4a

_{by directly binding and inactivating it.}

p16

INK4a

_{overexpression blocks Tax-dependent stimulation}

of cell cycle progression into S phase and transcriptional

ac-tivation.

Since Tax can reverse the effects of p16

INK4a

_{on cell}

cycle arrest in vivo, it was hypothesized that p16

INK4a

_might

also interfere with Tax function. Previous studies have

dem-onstrated that Tax can induce cellular transformation either

alone or synergistically with oncogenic Ras (31, 42). Thus, the

effects of Tax on BrdU incorporation of JEG-3 cells were

examined in the absence and presence of p16

INK4a

overexpres-sion (Fig. 2). The effects of Tax on BrdU incorporation are

most likely due to an increase in DNA synthesis and not an

increase in DNA repair since Tax is known to decrease

expres-sion of the DNA repair enzyme, DNA polymerase

b

(16).

Although the overexpression of p16

INK4a

_{alone did not have a}

significant effect on DNA synthesis (Fig. 2A and B), the

ex-pression of Tax alone (Fig. 2A) or with oncogenic Ras (38)

(Fig. 2B) stimulated DNA synthesis of JEG-3 cells to fivefold

compared to control cells in which neither Tax nor oncogenic

Ras was expressed. In contrast to the expression of Tax by

using the vaccinia virus-T7 system, expression using

cytomeg-alovirus vectors did not result in the stimulation of DNA

syn-thesis in JEG-3 cells (Fig. 2A and B). This probably results

from a difference in the levels of Tax expression achieved by

the two different expression systems. Coexpression with

p16

INK4a

_{completely blocked the ability of Tax alone or with}

oncogenic Ras to stimulate DNA synthesis in JEG-3 cells (Fig.

2A and B). Since DNA synthesis occurs only during the S

phase of the cell cycle, these results suggest that the

stimula-tion of cell cycle progression into S phase by Tax alone or with

oncogenic Ras can be completely blocked by p16

INK4a

overex-pression.

To further determine whether p16

INK4a

_{might also regulate}

Tax-dependent transcriptional activation of cellular gene

ex-pression, p16

INK4a

_{was overexpressed relative to Tax in human}

Jurkat T lymphocytes (Fig. 2C). Previous studies have clearly

demonstrated that Tax is a very potent transcriptional activator

and that it greatly stimulates the expression of a number of

different of cellular and viral genes (6, 13), including the

hu-man proenkephalin gene (17, 23). Expression of Tax alone

stimulated human proenkephalin gene expression fourfold

over basal levels (data not shown) (23). The coexpression of

Tax with increasing amounts of p16

INK4a

_{in human Jurkat T}

lymphocytes inhibited Tax-dependent transactivation of the

human proenkephalin gene expression in a dose-dependent

manner (Fig. 2C). Although the mechanism by which p16

INK4a

overexpression inhibits Tax-dependent transactivation of

pro-enkephalin gene transcription is not completely defined by this

experiment, the results are consistent with direct interference

of Tax function by p16

INK4a

_{through formation of functionally}

inactive complexes in vivo.

Tax and p16

INK4a

_{interact directly in vitro and form}

com-plexes in vivo.

The release of human T lymphocytes from

[image:3.612.61.297.68.253.2]

p16

INK4a

_{-mediated cell cycle arrest by Tax and the inhibition of}

FIG. 1. Tax releases human T cells from cell cycle arrest induced by p16INK4a

overexpression. The DNA content of synchronous JPX-9 cells was analyzed by propidium iodide labeling and flow cytometry in control cells (neither Tax nor p16INK4a_{was expressed) (A) or in cells subsequent to expression of Tax (B),}

p16INK4a_{(C), and p16}INK4a_{/Tax (D) by electroporation. JPX-9 cells were}

tran-siently transfected by electroporation with 20mg of pSV-CD8 in the absence or presence of 10mg of pCMV-p16INK4a_{(300 V, 960mF, single pulse) and then}

cultured for 14 h in the absence or presence of 30mM CdCl2(Tax expression in

JPX-9 cells is maximally induced with 30mM CdCl2by 8 h [data not shown]).

Hatch marks on either axis denote approximately equivalent DNA content or cell number.

on November 9, 2019 by guest

http://jvi.asm.org/

Tax-dependent cell cycle progression and transcriptional

acti-vation by p16

INK4a

_{suggest direct protein-protein interactions}

between Tax and p16

INK4a

_{. Therefore, the potential}

interac-tions between Tax and p16

INK4a

_{or other cdk inhibitors such as}

p21

cip1

_{and p27}

kip1

_{were examined by in vitro binding assays}

with equal amounts of recombinant GST fusion proteins

im-mobilized on glutathione-agarose beads and in

vitro-tran-scribed and -translated [

35

_{S]methionine-labeled proteins (Fig.}

3A to E). Approximately 10% of the total input p16

INK4a

_was

retained by GST-Tax but not by GST alone (compare Fig. 3A,

lane 2, with Fig. 3E, lane 1; compare lanes 1 and 2 in Fig. 3A).

This result indicates that p16

INK4a

_{binds specifically to the Tax}

portion of the recombinant GST-Tax fusion protein. In

con-trast, neither p21

cip1

_{nor p27}

kip1

_{was retained by GST-Tax (Fig.}

3B and C, lanes 2). Conversely, Tax was retained only by

GST-p16

INK4a

_{but not by either GST alone, GST-p21}

cip1

_{, or}

GST-p27

kip1

_{(Fig. 3D). Approximately 20% of the total input}

Tax was retained by GST-p16

INK4a

_{(compare Fig. 3D, lane 2, to}

Fig. 3E, lane 4). These results indicate that Tax specifically

interacts with p16

INK4a

_{but not p21}

cip1

_{or p27}

kip1

_{in vitro.}

To determine whether Tax and p16

INK4a

_{form complexes in}

vivo, both proteins were expressed in JEG-3 human

choriocar-cinoma cells by using the vaccinia virus-T7 RNA polymerase

hybrid system, and the cells were subsequently labeled with

[

35

_{S]methionine (Fig. 3F). Protein complexes were}

coimmuno-precipitated under native conditions (primary

immunoprecipi-tation) and subsequently disrupted by denaturation prior to

the identification of specific components of the complexes by

immunoprecipitation under denaturing conditions (secondary

immunoprecipitation). Although Tax and p16

INK4a

_{are not}

ex-pressed at detectable levels in JEG-3 cells (Fig. 3F, lanes 1 and

2), the expression of either protein by using the vaccinia

vi-rus-T7 RNA polymerase hybrid system resulted in significant

levels of detectable protein (Fig. 3F, lanes 3, 4, and 6). The

coexpression of Tax with p16

INK4a

_{resulted in a significant}

amount of p16

INK4a

_{coimmunoprecipitating with Tax in vivo}

(Fig. 3F; compare lane 4 or 6 with lane 7). The polyclonal

antiserum against Tax does not cross-react with p16

INK4a

because p16

INK4a

_{was not immunoprecipitated in the absence}

of Tax expression (Fig. 3F, lane 5). The results of the converse

experiment to determine whether Tax coimmunoprecipitates

with p16

INK4a

_{were inconclusive because the polyclonal}

anti-serum against p16

INK4a

_{cross-reacts with Tax (data not shown).}

These results suggest that Tax and p16

INK4a

_interact

specifi-cally in vitro and form stable complexes in vivo. It is also likely

that the formation of these complexes is dependent on ankyrin

or AAR motifs present in p16

INK4a

_{which are absent in either}

p21

cip1

_{or p27}

kip1

_{(15, 27, 30, 36, 37).}

Tax blocks the inhibition of cdk4 kinase activity by p16

INK4a

in vivo.

The formation of stable complexes with p16

INK4a

_in

vivo and the release of human T lymphocytes from p16

INK4a

-induced cell cycle arrest in late G

1

phase suggest that Tax

might also interfere with the ability of p16

INK4a

_{to inhibit cdk4}

kinase activity. To analyze the functional consequence of the

interactions between Tax and p16

INK4a

_{on cdk4 kinase activity}

in vivo, both proteins were either expressed alone or

coex-pressed in IMR-90 human diploid fetal lung or Hs27 human

foreskin fibroblast cells (Fig. 4A and B, panels i and ii). Both

cell lines were selected because they do not express detectable

levels of either protein (Fig. 4A and B, panels i and ii, lane 1)

but do express detectable albeit low levels of cdk4 kinase

ac-tivity (Fig. 4A and B, panel iii, lane 1). The expression of Tax

(Fig. 4A and B, panel i, lanes 3 and 4) and p16

INK4a

_{(Fig. 4A}

and B, panel ii, lanes 2 and 4) by using the vaccinia virus-T7

RNA polymerase hybrid system resulted in significant levels of

detectable protein. Immunocomplex kinase assays using

cdk4-specific antisera and the recombinant GST-RbC fusion protein

as a substrate revealed that both cell lines possess active

cdk4-cyclin D complexes and low levels cdk4 kinase activity (Fig. 4A

and B, panel iii, lane 1) which are inhibited by p16

INK4a

ex-pression as expected (Fig. 4A and B, panel iii; compare lanes

1 and 2). Although Tax expression alone did not significantly

FIG. 2. (A) Stimulation of DNA synthesis and cell cycle progression into S phase by Tax is inhibited by p16INK4a_{in JEG-3 cells. JEG-3 (0.2310}6_{) cells were}

transiently transfected with 8mg of pCMVbplus pTM3-p16INK4a_{and/or pTM3-TaxH}

6for 20 h. Cells were subsequently infected with 23106PFU of vaccinia virus

strain WR recombinant vTF7-3 for 4 h, washed, cultured for 18 h, and labeled with BrdU for 2 h. BrdU-positive transfected cells were identified by colocalization of immunohistochemical staining for BrdU and histochemical staining forb-galactosidase activity. The histogram depicts the mean6standard error of a representative experiment from three different experiments. †, statistically significant difference from the control (transfected with pTM3 and pCMVbonly) and other treatment groups (P,0.001;n53; unpaired Student’sttest). (B) Stimulation of DNA synthesis and cell cycle progression into S phase by Tax and Ras is inhibited by p16INK4a

in JEG-3 cells. JEG-3 cells (0.23106_{) were transiently transfected with 6mg of pCMVbplus pCMV-p16}INK4a_{, pCMV-Tax, and/or pEJ6.6 (oncogenic Ras) (38),}

cultured for 42 h, and labeled with BrdU for 2 h. BrdU-positive transfected cells were identified as described above. The histogram depicts the mean6standard error of a representative experiment from three different experiments. †, statistically significant difference from the control (transfected with pGEM3 and pCMVbonly) and other treatment groups (P,0.04;n53; unpaired Student’sttest). (C) p16INK4a_{overexpression inhibits Tax-dependent transcriptional activation of the human}

proenkephalin gene enhancer in a dose-dependent manner in Jurkat human T cells. Jurkat cells (107_{) were transiently cotransfected with 10mg of pENKAT-12, 10mg}

of pCMV-Tax, 5mg of pCMVb, and increasing amounts (10, 20, and 30mg) of pCMV-p16INK4a_{. Cells were then cultured for 48 h posttransfection before harvesting}

and lysis for CAT andb-galactosidase assays. The histogram depicts the mean6standard error of a representative experiment from three different experiments. †, statistically significant difference from the control (transfected with pGEM3 and pCMVbonly) (P,0.0003) and each other (P,0.03) (n53; unpaired Student’st

test).

on November 9, 2019 by guest

http://jvi.asm.org/

[image:4.612.75.542.70.217.2]

affect cdk4 kinase activity in either cell line (Fig. 4A and B,

panel iii; compare lanes 1 and 3), coexpression of Tax with

p16

INK4a

_{blocked the inhibition of cdk4 kinase activity by}

p16

INK4a

_{in both cell lines in the absence of a significant}

dif-ference in the levels of p16

INK4a

_{expression (Fig. 4A and B,}

panels ii and iii; compare lanes 2 and 4).

To better analyze the functional consequence of the

inter-actions between p16

INK4a

_{and Tax in the presence of higher}

levels of cdk4 kinase activity, both proteins were either

ex-pressed alone or coexex-pressed with cdk4 and cyclin D1 in JEG-3

cells (Fig. 4C, panels i, ii, iv, and v). The coexpression of cdk4

and cyclin D1 in JEG-3 resulted in consistently equivalent

levels of protein expression (Fig. 4C, panels iv and v; compare

lanes 1 to 4). Although Tax and p16

INK4a

_{are not expressed at}

detectable levels in JEG-3 cells (Fig. 4C, panels i and ii, lane

1), the expression of both proteins by using the vaccinia

vi-rus-T7 RNA polymerase hybrid system resulted in significant

and approximately equivalent levels of detectable protein (Fig.

4C, panels i and ii; compare lanes 2 or 3 to lane 4).

Immuno-complex kinase assays using cdk4-specific antiserum and

re-combinant GST-RbC fusion protein as substrates revealed

sig-nificant levels of cdk4 kinase activity which were dramatically

inhibited by p16

INK4a

_{expression (Fig. 4C, panel iii, compare}

lanes 1 and 2). Although Tax expression alone had no

signif-icant effects on cdk4 kinase activity (Fig. 4C, panel iii, compare

lanes 1 and 3), the coexpression of Tax with p16

INK4a

_blocked

the inhibition of cdk4 kinase activity by p16

INK4a

_{(Fig. 4C,}

panel iii; compare lanes 2 and 4). These results suggest that

Tax can directly interact with p16

INK4a

_{and interfere with the}

ability of p16

INK4a

_{to inhibit cdk4 kinase activity in vivo.}

DISCUSSION

A model describing the functional consequence of

interac-tions between Tax and p16

INK4a

_{that is consistent with}

pub-lished data and our results is depicted in Fig. 5. Previous

studies (15, 27, 30, 36, 37) indicate that active complexes of

cdk4 and cyclin D1 are disrupted in the presence of p16

INK4a

_,

resulting in the preferential formation of inactive complexes of

cdk4 and p16

INK4a

_{(Fig. 5). Our results and other recent}

stud-ies (41) clearly demonstrate that Tax interacts and forms in

vivo complexes directly with p16

INK4a

_{(Fig. 3) and that this}

interaction blocks the inhibition of cdk4 kinase activity by

p16

INK4a

_{(Fig. 4). This model suggests that in the presence of}

Tax, complexes consisting of cdk4 and p16

INK4a

_would

de-crease concurrently with an inde-crease in the formation of

com-plexes consisting of both Tax and p16

INK4a

_{as well as active}

complexes consisting of cdk4 and cyclin D1 (Fig. 5). Complexes

of cdk4 and p16

INK4a

_{have indeed been observed to be}

de-creased in the presence of Tax (41). Furthermore, cdk4 kinase

activity is observed to be increased in the presence of Tax

despite the presence of p16

INK4a

_{(Fig. 4) (41). Both of these}

observations provide further support for this model (Fig. 5).

The critical role served by cdk4 activation in regulating cell

cycle progression from G

1

phase into S phase is thought to be

dependent on its phosphorylation of the retinoblastoma

pro-tein (pRb) and subsequent release of transcription factors such

as E2F capable of activating the expression of S-phase genes

(37). Thus, the derepression of cdk4 kinase activity by Tax

through interactions with p16

INK4a

_{would result in an increase}

in the progression of cells from G

1

to S phase. The

observa-tions that Tax releases T lymphocytes from p16

INK4a

_-mediated

cell cycle arrest in late G

1

, at or prior to the G

1

/S restriction

point, and that p16

INK4a

_{inhibits a Tax-dependent stimulation}

of DNA synthesis are both consistent with a direct role for Tax

[image:5.612.73.285.89.539.2]

in deregulating cell cycle progression from G

1

phase into S

FIG. 3. Tax interacts specifically with p16INK4a_{but not p21}cip1_{or p27}kip1_in

vitro. Interactions between agarose-immobilized recombinant GST or GST-Tax fusion proteins and in vitro-transcribed and -translated35_S-p16INK4a_(A),35

S-p21cip1_{(B), or}35_S-p27kip1_{(C) were examined by in vitro binding assays. (D)}

Interactions between agarose-immobilized recombinant GST, GST-p16INK4a_,

GST-p21cip1_{, or GST-p27}kip1_{and in vitro-transcribed and -translated}35_S-Tax

were also examined by similar in vitro binding assays. (E) Amounts of input

35_S-p16INK4a_,35_S-p21cip1_,35_S-p27kip1_{, and}35_{S-Tax. The amount of [}35

S]methi-onine incorporated was different for each protein, although efforts were made to use equal amounts of in vitro-transcribed and -translated [35

S]methionine-la-beled protein. Results are representative of at least three different experiments. (F) Tax comimmunoprecipitates with p16INK4a_{in vivo. Each lane represents}

0.53106_{JEG-3 cells transiently transfected with 7.5 mg of pTM3-p16}INK4a

(lanes 4 to 7) and 7.5mg of pTM3-TaxH6(lanes 3, 6, and 7), infected with 53

106_{PFU of vaccinia virus strain WR recombinant vTF7-3 for 18 h, and}

subse-quently in vivo labeled with [35_{S]methionine. Protein complexes were identified}

by sequential immunoprecipitations under native and then denaturing conditions with polyclonal antiserum against either p16INK4a_{(ap16) or Tax (aTax). Results}

are representative of at least two different experiments. Autoradiographs were digitized and the figures were prepared as described in Materials and Methods.

on November 9, 2019 by guest

http://jvi.asm.org/

phase. Although this study clearly demonstrates that cell cycle

progression from G

1

phase to S phase can be deregulated

by Tax in a p16

INK4a

_{-dependent manner, it is unclear}

wheth-er Tax can also stimulate progression through the G

1

/S

re-striction point in a p16

INK4a

_{-independent manner. For}

exam-ple, it is possible that Tax also interacts with and activates

downstream targets of cdk4 such as the pRb tumor

suppres-sor or transactivates S-phase gene expression through

inter-actions with transcription factors such as E2F. However,

pre-liminary studies using in vitro binding assays indicate that

Tax does not interact with pRb directly. Although potential

direct interactions between Tax and other cdks or cyclins

are currently under investigation, it is unclear whether Tax

deregulates progression through other cell cycle checkpoints.

However, there are no detectable in vitro interactions

be-tween Tax and the non-ankyrin motif-containing cdk

inhib-itors such as p21

cip1

_{or p27}

kip1

_{which have broader}

specific-ities and are involved in regulating cell cycle progression

through other cell cycle checkpoints (Fig. 4) (15, 27, 30, 36,

37).

Recently the adenovirus E1A oncoprotein has been

demon-strated to interfere with transforming growth factor

b

-medi-ated growth arrest in G

1

phase of the cell cycle in a p27

kip1

-dependent manner (25), suggesting that derepression of cdks

may represent a common mechanism for cell cycle

deregula-tion by viral oncoproteins. The repression of p16

INK4a

_{by Tax is}

similar to the interaction between Tax and another ankyrin

motif-containing cytoplasmic inhibitor, I

k

B (14). I

k

B represses

transcriptional activation by NF-

k

B through the binding and

retention of NF-

k

B dimers as inactive cytoplasmic complexes

in unstimulated cells (3). The phosphorylation of I

k

B in

re-sponse to activation by phorbol esters or PKC results in the

release and nuclear translocation of active NF-

k

B dimers

which can stimulate transcription (3). Tax interacts directly

with the ankyrin motifs of I

k

B, resulting in the release and

nuclear translocation of NF-

k

B dimers which are subsequently

[image:6.612.79.534.71.311.2]

capable of transcriptional activation (14). Although Tax is

pri-marily known to activate cellular processes directly (6, 13), it is

clear that is can also activate cellular processes indirectly, by

repressing the effects of inhibitory cellular proteins. Although

most likely required, it is unclear whether the deregulation of

cell cycle progression by Tax it is sufficient for complete

cellu-lar transformation of HTLV-1-infected T lymphocytes.

Trans-formation by HTLV-1 probably requires the concurrent

Tax-dependent deregulation of a number of other critical cellular

processes such as transcription and signal transduction in

ad-dition to a deregulation of cell cycle control.

FIG. 4. Overexpression of p16INK4a_{with Tax in vivo blocks the ability of p16}INK4a_{to inhibit cdk4 kinase activity. p16}INK4a_{and/or Tax were expressed in IMR-90}

human diploid fetal lung cells (A), Hs27 human foreskin fibroblast cells (B), and JEG-3 cells (C). Each lane represents 0.53106_{to 4.0310}6_{IMR-90, Hs27, or JEG-3}

cells transiently transfected with 7.5mg of pTM3-p16INK4a_{(lanes 2 and 4) and pTM3-TaxH}

6(lanes 3 and 4) and subsequently infected with 53106to 403106PFU

of vaccinia virus strain WR recombinant vTF7-3 for 18 h. JEG-3 cells were also transiently cotransfected with 20mg of pCMV-cdk4 and 20mg of pCMV-cyclin D1 (lanes 1 to 4). Cells were in vivo labeled with [35_{S]methionine and lysed, and expressed proteins were immunoprecipitated under native conditions with polyclonal antiserum}

against either Tax (panel i), p16INK4a_{(panel ii), cdk4 (panels iii and iv), or cyclin D1 (panel v). Immunocomplex kinase assays were used to determine cdk4-dependent}

kinase activities in duplicate sample lysates from a single experiment (panel iii). All results are representative of at least three different experiments. Autoradiographs were digitized and the figures were prepared as described in Materials and Methods.

FIG. 5. Interactions between Tax and p16INK4a_{result in a derepression of}

cdk4 kinase activity. Previous studies indicate that active complexes of cdk4 and cyclin D1 that form in the absence p16INK4a_{are disrupted in the presence of}

p16INK4a_{, resulting in the formation of inactive complexes of cdk4 and p16}INK4a

(15, 27, 30, 36, 37). Our results indicate that Tax can form complexes with p16INK4a_{and relieve the inhibition of cdk4 kinase activity p16}INK4a_.

on November 9, 2019 by guest

http://jvi.asm.org/

[image:6.612.319.558.619.677.2]

ACKNOWLEDGMENTS

We thank M. Melner, S. Reeves, and P. Green for helpful

discus-sions and critical reading of the manuscript.

K.G.L. is supported by postdoctoral fellowship PF-3847 from the

American Cancer Society. This work was supported in part by NIH

grants DA00151, DA05706, and DA07745 to M.J.C.

REFERENCES

1.Adya, N., L. J. Zhao, W. Huang, I. Boros, and C. Z. Giam.1994. Expansion of CREB’s DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB. Proc. Natl. Acad. Sci. USA91:5642–5646.

2.Anderson, M. G., and W. S. Dynan.1994. Quantitative studies of the effect of HTLV-I Tax protein on CREB protein-DNA binding. Nucleic Acids Res.

22:3194–3201.

3.Baeuerle, P. A., and D. Baltimore.1988. IkB: a specific inhibitor of the NF-kB transcription factor. Science242:540–546.

4.Ballard, D. W., E. Bohnlein, J. W. Lowenthal, Y. Wano, B. R. Franza, and W. C. Greene.1988. HTLV-I Tax induces cellular proteins that activate the kB element in the IL-2 receptoragene. Science241:1652–1655. 5.Baranger, A. M., C. R. Palmer, M. K. Hamm, H. A. Giebler, A. Brauweiler,

J. K. Nyborg, and A. Schepartz.1995. Mechanism of DNA-binding enhance-ment by the human T-cell leukaemia virus transactivator Tax. Nature376:

606–608.

6.Cann, A. J., and I. S.-Y. Chen.1990. Human T-cell leukemia virus types I and II, p. 1501–1527.InB. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman (ed.), Virology, 2nd ed, vol. 2. Raven Press, New York, N.Y.

7.Caron, C., R. Rousset, C. Be´raud, V. Moncollin, J. M. Egly, and P. Jalinot.

1993. Functional and biochemical interaction of the HTLV-I Tax1 transac-tivator with TBP. EMBO J.12:4269–4278.

8.Earl, P. L., N. Cooper, and B. Moss.1991. Preparation of cell cultures and vaccinia virus stocks, p. 16.16.1–16.16.7.InF. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. Greene Publishing Associates and Wiley-Interscience, New York, N.Y.

9.Elroy-Stein, O., T. R. Fuerst, and B. Moss.1989. Cap-independent transla-tion of mRNA conferred by encephalomyocarditis virus 59sequence im-proves the performance of the vaccinia virus/bacteriophage T7 hybrid ex-pression system. Proc. Natl. Acad. Sci. USA86:6126–6130.

10. Elroy-Stein, O., and B. Moss.1991. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system, p. 16.19.1–16.19.9.InF. M. Aus-ubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. Greene Publishing Associates and Wiley-Interscience, New York, N.Y.

11. Franklin, A. A., M. F. Kubik, M. N. Uittenbogaard, A. Brauweiler, P. Utais-incharoen, M. A. Matthews, W. S. Dynan, J. P. Hoeffler, and J. K. Nyborg.

1993. Transactivation by the human T-cell leukemia virus Tax protein is mediated through enhanced binding of activating transcription factor-2 (ATF-2) response and cAMP element-binding protein (CREB). J. Biol. Chem.268:21225–21231.

12. Fujii, M., H. Tsuchiya, T. Chuhjo, T. Akizawa, and M. Seiki.1992. Interac-tion of HTLV-1 Tax1 with p67SRF_{causes the aberrant induction of cellular}

immediate early genes through CArG boxes. Genes Dev.6:2066–2076. 13. Gitlin, S. D., J. Dittmer, R. L. Reid, and J. N. Brady.1993. The molecular

biology of human T-cell leukemia viruses, p. 159–192.InB. R. Cullen (ed.), Human retroviruses. Oxford University Press, New York, N.Y.

14. Hirai, H., T. Suzuki, J. I. Fujisawa, J. I. Inoue, and M. Yoshida.1994. Tax protein of human T-cell leukemia virus type I binds to the ankyrin motifs of inhibitory factorkB and induces nuclear translocation of transcription factor NF-kB proteins for transcriptional activation. Proc. Natl. Acad. Sci. USA

91:3584–3588.

15. Hunter, T., and J. Pines.1994. Cyclins and cancer II: cyclin D and cdk inhibitors come of age. Cell79:573–582.

16. Jeang, K. T., S. G. Widen, O. J. Semmes, and S. H. Wilson.1990. HTLV-I trans-activator protein, Tax, is a trans-repressor of the humanb-polymerase gene. Science247:1082–1084.

17. Joshi, J. B., and H. P. G. Dave.1992. Transactivation of the proenkephalin gene promoter by the Tax1protein of human T-cell lymphotropic virus type

I. Proc. Natl. Acad. Sci. USA89:1006–1010.

18. Kadison, P., H. T. Poteat, K. M. Klein, and D. V. Faller.1990. Role of protein kinase A intaxtransactivation of the human T-cell leukemia virus type I long terminal repeat. J. Virol.64:2141–2148.

19. Kanno, T., K. Brown, G. Franzoso, and U. Siebenlist.1994. Kinetic analysis of human T-cell leukemia virus type I Tax-mediated activation of NF-kB. Mol. Cell. Biol.14:6443–6451.

20. Koh, J., G. H. Enders, B. D. Dynlacht, and E. Harlow.1995. Tumour-derived

p16 alleles encoding proteins defective in cell-cycle inhibition. Nature375:

506–510.

21. Kwok, R. P. S., M. E. Laurance, J. R. Lundblad, P. S. Goldman, H. M. Shih, L. M. Connor, S. J. Marriott, and R. H. Goodman.1996. Control of cAMP-regulated enhancers by the viral transactivator Tax through CREB and the co-activator CBP. Nature380:642–646.

22. Leung, K., and G. J. Nabel.1988. HTLV-1 transactivator induces interleu-kin-2 receptor expression through an NF-kB-like factor. Nature333:776– 778.

23. Low, K. G., H. M. Chu, P. S. Schwartz, G. M. Daniels, M. H. Melner, and M. J. Comb.1994. Novel interactions between human T-cell leukemia virus Tax and activating transcription factor 3 at a cyclic AMP-responsive element. Mol. Cell. Biol.14:4958–4974.

24. Lukas, J., D. Parry, L. Aagaard, D. J. Mann, J. Bartkova, M. Strauss, G. Peters, and J. Bartek.1995. Retinoblastoma-protein-dependent cell-cycle inhibition by tumour suppressor p16. Nature375:503–506.

25. Mal, A., R. Y. C. Poon, P. H. Howe, H. Toyoshima, T. Hunter, and M. L. Marter.1996. Inactivation of p27Kip1_{by the viral E1A oncoprotein in TGFb}

-treated cells. Nature380:262–265.

26. Matsushime, H., D. E. Quelle, S. A. Shurtleff, M. Shibuya, C. J. Sherr, and J. Y. Kato.1994. D-type cyclin-dependent kinase activity in mammalian cells. Mol. Cell. Biol.14:2066–2076.

27. Morgan, D. O.1995. Principles of CDK regulation. Nature374:131–134. 28. Moss, B., O. Elroy-Stein, T. Mizukami, W. A. Alexander, and T. R. Fuerst.

1990. New mammalian expression vectors. Nature348:91–92.

29. Nagata, K., K. Ohtani, M. Nakamura, and K. Sugamura.1989. Activation of endogenous c-fosproto-oncogene expression by human T-cell leukemia virus type I-encoded p40tax_{protein in the human T-cell line, Jurkat. J. Virol.63:}

3220–3226.

30. Peter, M., and I. Herskowitz.1994. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell79:181–184.

31. Pozzatti, R., J. Vogel, and G. Jay.1990. The human T-lymphotropic virus type Itaxgene can cooperate with therasoncogene to induce neoplastic transformation of cells. Mol. Cell. Biol.10:413–417.

32. Ruben, S., H. Poteat, T.-H. Tan, K. Kawakami, R. Roeder, W. Haseltine, and C. A. Rosen.1988. Cellular transcription factors and regulation of IL-2 receptor gene expression by HTLV-1taxgene product. Science241:89–92. 33. Sasse, J., and S. R. Gallagher.1991. Detection of proteins, p. 10.6.1–10.6.8.

InF. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. Greene Publishing Associates and Wiley-Interscience, New York, N.Y. 34. Serrano, M., E. Go´mez-Lahoz, R. A. DePinho, D. Beach, and D. Bar-Sagi.

1995. Inhibition of ras-induced proliferation and cellular transformation by p16INK4a_{. Science267:249–252.}

35. Serrano, M., G. J. Hannon, and D. Beach.1993. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature366:

704–707.

36. Sherr, C.1994. G1 phase progression: cycling on cue. Cell79:551–555. 37. Sherr, C., and J. M. Roberts.1995. Inhibitors of mammalian G1

cyclin-dependent kinases. Genes Dev.9:1149–1163.

38. Shih, C., and R. A. Weinberg.1982. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell29:161–169.

39. Smith, J. A.1991. Quantitation of proteins, p. 10.1.1–10.1.3.InF. M. Aus-ubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. Greene Publishing Associates and Wiley-Interscience, New York.

40. Suzuki, T., J. Fujisawa, M. Toita, and M. Yoshida.1993. The trans-activator Tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1. Proc. Natl. Acad. Sci. USA90:610– 614.

41. Suzuki, T., S. Kitao, H. Matsushime, and M. Yoshida.1996. HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4a_and

coun-teracts its inhibitory activity towards CDK4. EMBO J.15:1607–1614. 42. Tanaka, A., C. Takahashi, S. Yamaoka, T. Nosaka, M. Maki, and M.

Ha-tanaka.1990. Oncogenic transformation by thetaxgene of human T-cell leukemia virus type I in vitro. Proc. Natl. Acad. Sci. USA87:1071–1075. 43. Uchiumi, F., K. Semba, Y. Yamanashi, J. I. Fujisawa, M. Yoshida, K. Inoue,

K. Toyoshima, and T. Yamamoto.1992. Characterization of the promoter region of thesrcfamily genelynand itstransactivation by the human T-cell leukemia virus type I-encoded p40tax_{. Mol. Cell. Biol.12:3784–3795.}

44. Wagner, S., and M. R. Green.1993. HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization. Science262:395– 399.

45. Zhao, L. J., and C. Z. Giam.1992. Human T-cell lymphotropic virus type I (HTLV-I) transcriptional activator, Tax, enhances CREB binding to HTLV-I 21-base-pair repeat by protein-protein interactions. Proc. Natl. Acad. Sci. USA89:7070–7074.

on November 9, 2019 by guest

http://jvi.asm.org/