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Keywords:

  • cyclin D1;
  • cyclin D2;
  • human T-cell leukemia virus type I;
  • Tax;
  • NF-κB

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Our aim was to examine the involvement of G1 cell-cycle regulators in cell growth dysregulation induced by HTLV-I. Compared to uninfected cells, higher expression levels of cyclin D1 and D2 mRNA were detected in HTLV-I–infected T-cell lines, which were at least in part mediated by the viral transforming protein Tax since Tax activated both cyclin D1 and D2 promoters in the human T-cell line Jurkat. A Tax mutant that did not activate NF-κB failed to activate cyclin D1 and D2 promoters. Inhibitors of NF-κB (dominant negative IκBs mutants) suppressed Tax-dependent activation of cyclin D1 and D2 promoters, indicating that Tax-induced activation was mediated by NF-κB. Wild-type and mutant Tax capable of activating NF-κB, but not Tax mutant incapable of activating NF-κB, converted cell growth of a T-cell line from being IL-2–dependent to being IL-2–independent; and this conversion was associated with IL-2–independent induction of cyclins D1 and D2. Our data suggest that induction of cyclins D1 and D2 by Tax is involved in IL-2–independent cell-cycle progression as well as IL-2–independent transformation of primary human T cells by HTLV-I. High expression levels of cyclin D1 and D2 mRNAs were also detected in some patients with ATL. Our findings link HTLV-I infection to changes in cellular D-type cyclin gene expression, transformation of T cells and subsequent development of T-cell leukemia. © 2002 Wiley-Liss, Inc.

HTLV-I is a retrovirus etiologically associated with ATL.1, 2, 3 HTLV-I activates and immortalizes primary human T lymphocytes in vitro, resulting in polyclonal proliferation of infected cells, followed by oligoclonal or monoclonal growth. The viral protein Tax is thought to play crucial roles in the development of ATL since it can immortalize primary human T cells,4, 5 transform rodent fibroblastic cell lines6, 7 and induce tumors and leukemia in transgenic mice.8, 9 However, the exact mechanisms through which Tax exerts its oncogenic effects are not fully understood. Tax was originally identified as a transcriptional activator of HTLV-I LTR,10 but subsequent studies identified numerous other activities, including modulation of the expression of several cellular genes involved in cellular proliferation. For example, Tax upregulates the expression of IL-2 receptor, c-fos, c-jun and egr-1.11, 12 Tax can also repress the expression of β-polymerase and the proapoptotic gene bax.13, 14 In addition, Tax inhibits the functions of p53 tumor-suppressor protein and Smads, signaling mediators of transforming growth factor-β.15, 16

Cell-cycle dysregulation is the hallmark of transformed cells. Transition of the cell cycle from one phase to the next is regulated in part by CDKs assembled with partner cyclins. CDKs can also be inactivated through physical binding with CKIs, e.g., p16, p21 and p27.17, 18 One of the key roles of cyclin D–CDK complexes in cell-cycle progression is phosphorylation of the tumor-suppressor pRb, consequently releasing the transcription factor E2F.19 E2F then activates the expression of genes that are critical for S-phase events. Thus, regulation of pRb phosphorylation by cyclin D–CDK complexes and CKIs is critical in the control of cell-cycle progression as well as cell proliferation.

Tax stimulates cell-cycle progression under various experimental conditions. Two reports demonstrated that Tax stimulates the G1-to-S-phase transition of the Tax-inducible T-cell line JPX-9 and the IL-2–dependent T-cell line Kit-225.20, 21 One possible explanation for this effect is dissociation of the pRb–E2F complex by direct binding of Tax to pRb, similar to other viral oncoproteins, such as adenovirus E1A protein and simian virus 40 large T antigen. However, direct binding of Tax with pRb is not detected in in vitro binding assays.22 Thus, the exact mechanism by which Tax stimulates cell-cycle progression has not been elucidated.

To define the events related to dysregulated cell-cycle progression induced by HTLV-I, we analyzed the expression of D-type cyclins in HTLV-I–infected T-cell lines. Our results demonstrated high expression levels of cyclins D1 and D2 in T-cell lines infected with HTLV-I and that Tax was responsible for such overexpression. Furthermore, the observed induction of cyclins D1 and D2 correlated well with Tax-induced IL-2–independent growth of a mouse T-cell line. These data suggest that induction of D-type cyclins is the underlying event in Tax-induced stimulation of cell-cycle progression and abnormal proliferation of HTLV-I–infected T lymphocytes.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Cell Lines

Human leukemic T-cell lines (Jurkat, MOLT-4 and CCRF-CEM) and HTLV-I–infected T-cell lines (MT-2,23 MT-4,24 C5/MJ,25 SLB-126 and HUT-1021) (for further details of these cell lines, see Mori et al.27) were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS (Hyclone, Logan, UT), 100 U/ml penicillin and 100 μg/ml streptomycin. JPX-9 and JPX/M (kindly provided by Dr. M. Nakamura, Tokyo Medical and Dental University, Tokyo, Japan) are subclones of Jurkat cells expressing Tax and nonfunctional Tax mutant under the control of the metallothionein promoter.28, 29 CTLL is a mouse IL-2–dependent T-cell line; however, upon transfection and selection of the tax gene, these cells become IL-2–independent.30

Clinical Samples

The diagnosis of ATL was based on clinical features, hematologic findings and serum anti-HTLV-I antibodies. Peripheral blood samples were obtained from patients with acute-type and chronic-type ATL. Integration of monoclonal HTLV-I provirus into the DNA of leukemic cells was confirmed by Southern blot hybridization in all cases (data not shown). Mononuclear cells from the peripheral blood of healthy volunteers, asymptomatic seropositive carriers and patients with ATL were purified by Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden) and washed with PBS. Leukemic cells constituted >90% of blood cells in each patient at the time of analysis. All samples were taken after obtaining informed consent.

Plasmids and Transfections

A series of expression vectors based on the human β-actin promoter for HTLV-I Tax (pβMT-2Tax) and its mutants (TaxM22 and Tax703) were described previously.7, 31 IκBαΔN32 and IκBβ ΔN33 (kindly provided by Dr. D.W. Ballard, Vanderbilt University School of Medicine, Nashville, TN) are deletion mutants of IκBα and IκBβ lacking the NH2-terminal 36 amino acids and 23 amino acids, respectively. We used a luciferase reporter construct for the human cyclin D1 promoter, pD1luc.34 The luciferase reporter plasmids D1-κB1M, D1-κB2M, D1-κB1/2M, pGL2HIV and pGL2HIVD1κB2 were described previously.34 The luciferase reporter plasmid containing the human cyclin D2 promoter (–1624 to –1), D2–1624,35 was a kind gift from Dr. P.G. Milner (CV Therapeutics, Palo Alto, CA). We also used a reporter construct for the HTLV-I LTR and an NF-κB reporter construct. Transient transfections were performed in Jurkat cells by electroporation using 5 × 106 cells, 1 μg of appropriate reporter, 5 μg of effector plasmids and 1 μg of pRL-TK (renilla) to control transfection efficiency. After 24 hr, transfected cells were collected by centrifugation, washed with PBS, lysed in reporter lysis buffer (Promega, Madison, WI) and assayed using the Dual Luciferase Assay system (Promega). Luciferase activity, measured with a Berthold (Bad Wildbad, Germany) luminometer, was normalized for transfection efficiency, and the SD was calculated from 3 independent transfections.

Northern Blot Analysis

Total cellular RNA was extracted with Trizol (Life Technologies, Gaithersburg, MD) according to the protocol provided by the manufacturer. Total RNA (20 μg) was electrophoresed through a formaldehyde-agarose gel and transferred onto a nylon filter. Filters were prehybridized (in 0.5 M sodium phosphate, 0.1% BSA, 7% SDS, 100 μg/ml salmon testis DNA and 100 μg/ml yeast RNA) for 2 hr at 65°C and then hybridized overnight with the following [α-32P]-radiolabeled probes: cDNA of HTLV-I Tax; human cyclin D1; mouse cyclins D1, D2 and D3 (kindly provided by Dr. H. Matsushime, Yamanouchi Pharmaceutical, Tsukuba, Japan); and GAPDH. Radiolabeled probes were generated using the Megaprime DNA Labeling system (Amersham, Arlington Heights, IL).

Western Blotting

Cells were collected by centrifugation, washed with PBS and lysed in RIPA buffer (0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 66 μg/ml aprotinin, 100 μg/ml PMSF and 1 mM sodium orthovanadate). Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts (50 μg) of protein were separated by SDS-PAGE on a 10% polyacrylamide gel and transferred onto PVDF membranes (Millipore, San Diego, CA). Following the transfer, membranes were blocked with TRIS-buffered saline–Tween (0.05%) with 3% nonfat dry milk overnight at 4°C and then blotted for 45 min with rabbit polyclonal anticyclin D1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anticyclin D2 antibody (Santa Cruz Biotechnology), rabbit polyclonal anticyclin D3 antibody (Santa Cruz Biotechnology) or mouse monoclonal anti-Tax antibody (Lt-436). Membranes were washed with TRIS-buffered saline–Tween and incubated with a 1:1,000 dilution of horseradish peroxidase–conjugated secondary antibody (Amersham). Thereafter, proteins recognized by the antibodies were visualized using the ECL Western blotting detection system (Amersham).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Expression of Cyclins D1, D2 and D3 in HTLV-I–infected T-cell Lines

To determine whether any D-type cyclins were dysregulated in the HTLV-I–infected cell lines, we performed Northern blot analysis with total cellular RNA from both infected and uninfected human T-cell lines (Fig. 1). Both cyclin D1 and D2 mRNAs were expressed at markedly higher levels in all HTLV-I–infected T-cell lines than in any uninfected lines. In contrast, expression of cyclin D3 mRNA was not detected in any HTLV-I–infected T-cell lines, while high levels of the mRNA were detected in all HTLV-I-negative T-cell lines. The HTLV-I–encoded transactivator protein Tax activates or represses the expression of a number of cellular genes. Thus, we next measured the expression of Tax in these T-cell lines. Tax-related RNAs (8.5, 4.2 and 2.1 kb) were expressed highly in 4 HTLV-I–infected T-cell lines (MT-2, MT-4, SLB-1 and HUT-102) and weakly in 1 (C5/MJ). Hybridization with the GAPDH probe showed that comparable amounts of RNAs were loaded in the lanes of these T-cell lines. These results indicated that HTLV-I Tax might modulate expression of D-type cyclins in a positive or negative manner.

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Figure 1. Northern blot analysis of D-type cyclins and HTLV-I in T-cell lines. Total cellular RNA (20 μg) was subjected to Northern blot analysis. Jurkat, MOLT-4 and CCRF-CEM (lanes 6–8) are HTLV-I–uninfected T-cell lines; MT-2, MT-4, C5/MJ, SLB-1 and HUT-102 (lanes 1–5) are HTLV-I–infected T-cell lines. Predominant 2.1, 4.2 and 8.5 kb viral mRNA species were detected in MT-2, MT-4, C5/MJ, SLB-1 and HUT-102 cell lines (lanes 1–5). The amount and integrity of RNA were demonstrated by rehybridizing the blot with a GAPDH probe, as shown in the bottom panel.

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Differential Transactivation of D-type Cyclins by Tax in T Cells

To examine whether Tax indeed alters the expression of D-type cyclins in HTLV-I–infected T-cell lines, we used a Tax-transfected human T-cell line (JPX-9), which expresses Tax only after addition of CdCl2.28, 29 Expression of Tax and D-type cyclins in these cells was determined by Western blot analysis. As shown in Figure 2, JPX-9 cells expressed Tax 5 hr after addition of CdCl2 (20 μM) to the culture medium, and the expression persisted until 72 hr after treatment. Expression of cyclin D1 and D2 proteins concomitantly increased in JPX-9 cells at 48 hr and 5 hr, respectively, after treatment with CdCl2. In contrast, expression of Tax was not associated with any concomitant change in cyclin D3 expression. Induction of cyclin D1 and D2 proteins was attributed to Tax rather than CdCl2 since it was not observed in JPX/M, which expressed a nonfunctional Tax protein after treatment with CdCl2 (data not shown). These results indicated that Tax could increase the expression of cyclin D1 and D2 proteins in a human T-cell line, though the kinetics of cyclin D1 induction completely differed from that of cyclin D2.

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Figure 2. Induction kinetics of D-type cyclins in JPX-9 cells treated with CdCl2. Whole-cell lysates were prepared from CdCl2-treated JPX-9 cells at the indicated time points. Whole-cell lysate corresponding to 50 μg of protein was immunoblotted with anti-Tax, anticyclin D1, anticyclin D2 or anticyclin D3 antibody.

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Transactivation of Cyclin D2 Promoter by Tax

We then assessed whether Tax can enhance human cyclin D2 promoter activity in Jurkat cells by transient transfection assay. A cyclin D2 promoter fragment linked to the luciferase reporter gene was transfected into Jurkat cells together with Tax expression plasmid. Tax increased the expression of luciferase under the control of cyclin D2 promoter by >3-fold (Fig. 3a, bottom panel). Tax can stimulate such transcription through distinct transcription factors, including CREB and NF-κB. Two additional Tax mutants that selectively retain the ability to activate the CRE site within the HTLV-I LTR (M22) or NF-κB (703) (Fig. 3a, top and middle panels) were tested for cyclin D2 promoter transactivation. Neither Tax mutant showed full transactivation of the cyclin D2 promoter (Fig. 3a, bottom panel). The promoter activity in Jurkat cells transfected with the M22 mutant was almost equivalent to that in control cells. The 703 mutant activated cyclin D2 promoter, but its activity was less than that of wild-type Tax. Cotransfection with 2 mutants (M22 and 703) restored full transactivation with 4-fold induction, indicating that both the CREB and NF-κB pathways are required for full Tax-induced activation of the cyclin D2 promoter. It is possible that the M22 mutant complements the impaired function of the 703 mutant, while the 703 mutant complements the impaired function of the M22 mutant.

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Figure 3. Transcriptional regulation of the cyclin D2 promoter by Tax. (a) Mutation of Tax influences its transactivation function. Jurkat cells were transfected with HTLV-I Tax (WT), M22, 703 or empty vector and HTLV-I LTR, NF-κB or cyclin D2 (D2–1624) luciferase reporter construct. (b) Induction of the cyclin D2 promoter by Tax is reduced by IκBα or IκBβ mutant expression. Jurkat cells were transfected with Tax, IκBα mutant IκBαΔN or IκBβ mutant IκBβΔN and HTLV-I LTR, NF-κB or cyclin D2 (D2–1624) luciferase reporter construct. Results represent means ± SD of 3 individual transfections and are expressed as fold induction relative to the basal level measured in cells transfected with empty vector.

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We then examined whether blocking of the NF-κB pathway influences Tax-induced transactivation of the cyclin D2 promoter using super-repressor forms of IκBα (IκBαΔN) and IκBβ (IκBβΔN). As shown in Figure 3b, the Tax-induced elevation of promoter activity of the cyclin D2 reporter was markedly suppressed by cotransfection with IκBαΔN and IκBβΔN. The promoter activity of NF-κB reporter plasmid was also suppressed by IκBαΔN and IκBβΔN, whereas that of CREB reporter (HTLV-I LTR) was not affected, indicating the specificity of NF-κB inhibitors. Taken together, these results confirm that the NF-κB pathway is necessary but not sufficient for Tax-induced transactivation of the cyclin D2 promoter.

Transactivation of Cyclin D1 Promoter by Tax

We also examined the effect of Tax on cyclin D1 promoter activity. The human cyclin D1 promoter reporter construct linked to the luciferase gene was transfected into Jurkat cells along with Tax expression plasmid. Tax increased expression of the luciferase gene from the cyclin D1 promoter by >8-fold (Fig. 4b). While the 703 mutant, which could activate NF-κB, also stimulated the activity of the cyclin D1 promoter, no significant activation was observed with the M22 mutant (Fig. 4b). Consistent with these findings, the Tax-induced increase in promoter activity was markedly suppressed by cotransfection with IκBαΔN and IκBβΔN (Fig. 4c). The human cyclin D1 promoter contains 2 putative NF-κB binding sites, termed D1-κB1 and D1-κB2 (Fig. 4a). We tested promoter constructs containing point mutations of the D1-κB1 and D1-κB2 sites (Fig. 4d). Mutation of the distal NF-κB binding site (D1-κB1M) did not interfere with Tax activation of cyclin D1 promoter, whereas mutation of the proximal NF-κB binding site (D1-κB2M) or of both caused significant reduction in Tax-induced activation. We further investigated the activity of Tax for the D1-κB2 sequence in a heterologous promoter context. For this purpose, the D1-κB2 sequence was inserted into a luciferase expression construct that contained only the HIV core promoter (pGL2HIV) (Fig. 4e). The core promoter containing a single copy of the D1-κB2 sequence was activated 6-fold by Tax, indicating that this NF-κB element is responsive to Tax in a different promoter context. Taken together, these results suggest that Tax activates transcription of the cyclin D1 promoter primarily through the proximal NF-κB binding site D1-κB2.

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Figure 4. Transcriptional regulation of the cyclin D1 promoter by Tax. (a) Schematic map of the cyclin D1 promoter. Sequences of NF-κB binding sites are shown, designated D1-κB1 and D1-κB2. (b) Mutation of Tax influences the transactivation function of Tax. Jurkat cells were transfected with HTLV-I Tax (WT), M22, 703 or empty vector and cyclin D1 (pD1luc) luciferase reporter construct. (c) Induction of the cyclin D1 promoter by Tax is reduced by IκBαΔN or IκBβΔN expression. Jurkat cells were transfected with Tax, IκBαΔN or IκBβΔN and cyclin D1 (pD1luc) luciferase reporter construct. (d) Reporter gene activation of wild-type (pD1luc) and mutant (D1-κB1M, D1-κB2M or D1-κB1/2M) constructs by Tax was determined in Jurkat cells. (e) Activation of a heterologous promoter through the D1-κB2 sequence. A single copy of a cyclin D1 promoter fragment containing the D1-κB2 site was cloned in front of an HIV core promoter luciferase construct (pGL2HIV). Results represent means ± SD of 3 individual transfections and are expressed as fold induction relative to the basal level measured in cells transfected with empty vector.

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Induction of Cyclins D1 and D2 in Mouse T-cell Lines Stably Transfected with Tax

To examine the role of cyclins D1 and D2 in Tax functions, we used a mouse T-cell line, CTLL-2, that had been transfected with the wild-type or mutant Tax plasmids characterized above. The growth of parental CTLL-2 is strictly dependent on IL-2, but CTLL-2 expressing wild-type Tax (CTLL-2/WT) can continuously grow in the absence of IL-2 for several years.30 Like CTLL-2/WT, CTLL-2 expressing Tax mutant 703 (CTLL-2/703), which can activate NF-κB, also grew in the absence of IL-2, while CTLL-2 expressing Tax mutant M22 (CTLL-2/M22), which cannot activate NF-κB, grew only in the presence of IL-2, like parental CTLL-2.30 In the next series of experiments, we examined whether Tax in a stable foreign setting could still activate endogenous D-type cyclin genes. All CTLL-2 clones stably transfected with various Tax plasmids expressed Tax protein, as detected in Western blot assays (Fig. 5a). The mRNA level of cyclin D2 in CTLL-2/Vector cells was markedly decreased after 48 hr of IL-2 deprivation (Fig. 5b). In contrast, 3 CTLL-2/WT (WT-7, WT-14 and WT-21) and 3 CTLL-2/703 (703-2, 703-3 and 703-7) cell lines showed constitutive expression of cyclin D2 mRNA even in the absence of IL-2. However, expression of cyclin D2 mRNA in CTLL-2/M22 cells (M22-5) was strongly dependent on the presence of IL-2. Similar results were obtained for cyclin D1 by Northern blot analysis (Fig. 5b). Cyclin D3 showed no induction by Tax in these cells (Fig. 5b). These results reinforced the notion that wild-type Tax and mutant 703 allow overexpression of cyclins D1 and D2 in T cells and suggested that the mechanism of IL-2 independence by Tax in CTLL-2 cells may, at least in part, be due to induction of cyclins D1 and D2.

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Figure 5. Constitutive expression of cyclin D1 and D2 mRNAs in CTLL-2/Tax transfectants in the absence of IL-2. (a) Expression of Tax proteins in CTLL-2/Tax transfectants. (b) Northern blot analyses for D-type cyclins were performed using samples extracted from CTLL-2/Tax transfectants after 48 hr of culture in the presence (+) or absence (–) of IL-2.

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Expression of D-type Cyclins in Leukemic Cells of ATL Patients

Finally, we assessed the relevance of our findings in T-cell lines to ATL cells in vivo. For this purpose, we analyzed the levels of D-type cyclin mRNA in uncultured leukemic cells from ATL patients. Northern blotting showed that peripheral blood mononuclear cells from normal healthy volunteers and asymptomatic seropositive carriers expressed very low levels of cyclin D1 mRNA and undetectable levels of cyclin D2 mRNA (Fig. 6). In contrast, high expression levels of cyclin D1 and D2 mRNAs were detected in 4 of 11 ATL samples tested (patients 1, 3, 6 and 8; Fig. 6). Expression of cyclin D1 mRNA in ATL samples almost paralleled that of cyclin D2.

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Figure 6. Northern blot analysis of cyclins D1 and D2 in primary ATL cells. Total cellular RNA (20 μg) was subjected to Northern blot analysis. A, acute-type; C, chronic-type.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Previous studies37 and the present data demonstrate that the viral transforming protein Tax induces expression of cyclin D2 in T cells (Figs. 2, 5). In addition to cyclin D2, we showed here that Tax induces expression of cyclin D1 in T cells. All HTLV-I–infected T-cell lines examined in our study expressed high levels of cyclin D1 and D2 mRNAs relative to uninfected ones. Cyclins D1 and D2 may be involved in the development of neoplastic diseases. For instance, increased cyclin D1 protein expression by gene translocation has been described in a subset of parathyroid adenomas38 and B-cell lymphomas.39 Overexpression or amplification of cyclin D2 was also reported in various cancers, such as chronic B-cell malignancies.40 Furthermore, overexpression of D-type cyclins is oncogenic in several experimental systems. For example, expression of the cyclin D1 gene can complement a defective adenovirus E1A oncogene to transform primary neonatal rat kidney cells.41 Overexpression of cyclin D1 or D2 in transgenic animals resulted in hyperplasia and carcinoma.42, 43, 44 Thus, our data suggest that the Tax-induced expression of 2 D-type cyclin genes may play a role in the transformation of primary T cells in HTLV-I infection.

We previously showed that Tax can abrogate the requirement for IL-2 in the mouse T-cell line CTLL-2.30 Expression of cyclins D1 and D2 in CTLL-2 was strictly dependent on IL-2, but Tax induced their expression even in the absence of IL-2. We also showed here that CTLL-2 cells expressing Tax 703, but not M22, acquired IL-2–independent growth and that this acquisition correlated with IL-2–independent expression of cyclins D1 and D2. These data suggested that the induction of cyclins D1 and D2 by Tax is involved in IL-2–independent growth of CTLL-2. Consistently, all IL-2–independent, HTLV-I–transformed cell lines constitutively expressed high levels of cyclins D1 and D2.

Another major finding of the present study was that Tax activated cyclin D1 and D2 promoters mainly through NF-κB. The cyclin D1 promoter contains 2 NF-κB binding sites. The proximal NF-κB binding site was the main contributor to the activation by Tax since mutation of this element completely abrogated Tax activation. Furthermore, a series of 5′-deletion mutants of the cyclin D2 promoter showed that the Tax-inducible enhancer was within the promoter fragment from –345 to –1 nt relative to the RNA start site (data not shown). This 345 nt cyclin D2 promoter fragment contains putative DNA-binding sites for NF-κB and CREB. Experiments using Tax mutants indicated that both NF-κB and CREB pathways are required for the full activation of cyclin D2 promoter by Tax, though the requirement for the CREB pathway was less. We are currently defining the contribution of NF-κB and CRE sites within the cyclin D2 promoter. All HTLV-I–infected T-cell lines investigated in the present study expressed high NF-κB activity, measured by transcriptional assay and nuclear NF-κB DNA binding assay.45

Several studies have suggested that Tax-induced activation of NF-κB is essential for the transformation of rodent cells and primary human T lymphocytes.7, 46–49 For instance, experiments conducted with infectious molecular clones of HTLV-I DNA indicated that activation of NF-κB by Tax is critical for the immortalization of primary human T cells by HTLV-I.50 Studies from our laboratories also suggested that Tax-induced activation of NF-κB is important for IL-2–independent growth of CTLL-2.30 Therefore, NF-κB–induced expression of cyclins D1 and D2 may contribute to the transformation of primary human T cells by HTLV-I infection. The direct link between Tax-induced activation of NF-κB and overexpression of early G1 cyclins presented here advances our understanding of how NF-κB dysregulation by Tax may result in cell transformation and leukemogenesis.

However, it is questionable whether Tax alone can explain the expression of cyclins D1 and D2 in T cells infected with HTLV-I. We also stress the possible role of Tax-independent mechanisms in the constitutive expression of cyclin D1 and D2 genes in HTLV-I–infected T cells because of the following observations. First, Tax expression level does not correlate with the expression of cyclins D1 and D2 in T-cell lines (Figs. 1,5). Second, Tax transactivates the cyclin D2 promoter by only 3-fold (Fig. 3). Third, the relevant difference between the induction kinetics of cyclins D1 and D2 is that cyclin D1 expression displays relatively delayed induction compared with cyclin D2 activated by Tax, indicating that the mechanism involved in the induction of cyclin D1 expression by Tax is distinct from that for cyclin D2. Fourth, despite the apparent overexpression of cyclins D1 and D2 in fresh ATL cells from some patients (Fig. 6), ATL cells usually express Tax at low levels, if at all (data not shown but see Furukawa et al.51). It is thus reasonable to assume that transactivation by Tax is not the only mechanism underlying overexpression of cyclins D1 and D2 in T cells infected with HTLV-I.

We detected overexpression of cyclins D1 and D2 in leukemic cells from 4 of 9 patients with acute-type ATL. However, we could not detect such overexpression in 2 patients with chronic-type ATL. Our results suggest that the increased expression of cyclins D1 and D2 may be associated with acute but not chronic ATL and may be related to progression from chronic to acute ATL. However, we could not define these relationships because of the small number of patients examined in the present study. We are currently investigating whether expression of cyclins D1 and D2 contributes to disease progression and clinical outcome in ATL.

NF-κB is constitutively activated in all primary ATL cells.45 However, we could not detect overexpression of cyclins D1 and D2 in 7 of 11 ATL patients. Hypermethylation of the CpG island within the promoter region is one of the possible mechanisms by which cellular genes are inactivated, resulting in lack of expression of these methylated genes. Methylation of the cell cycle control genes has been reported in ATL.52 One of the reasons for the low expression of cyclins D1 and D2 but high expression of NF-κB observed in ATL patients may be methylation of these genes.

In summary, expression levels of cyclins D1 and D2 are deregulated in all HTLV-I–infected T-cell lines and primary ATL cells from some ATL patients. Overexpression of both genes is thought to play a critical role, presumably by collaborating with other oncogenic events, including abrogation of the inhibitory function of CKIs, in the promotion of cell cycle progression. Further studies are required to understand the mechanism of induction of cyclins D1 and D2 in ATL as well as the significance of the process in the pathogenesis and/or progression of ATL.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We are deeply indebted to the ATL patients and control subjects who donated blood for these studies. We thank Drs. K. Matsumoto and D.W. Ballard for providing expression plasmids; Drs. P.G. Milner, J. Fujisawa and I. Futsuki for providing reporter plasmids; and Dr. H. Matsushime for providing cDNA of mouse D-type cyclins. We also thank Dr. M. Nakamura for providing JPX-9 and JPX/M cells and Fujisaki Cell Center, Hayashibara Biomedical Laboratories (Okayama, Japan) for providing Jurkat, HUT-102 and C5/MJ cell lines. Recombinant human IL-2 was kindly provided by Takeda Chemical Industries (Osaka, Japan). We are grateful to Ms. M. Yamamoto and Ms. M. Sasaki for their excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci USA 1980;77: 74159.
  • 2
    Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto T, Kinoshita K, Shirakawa S, Miyoshi I. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc Natl Acad Sci USA 1981;78: 647680.
  • 3
    Yoshida M, Miyoshi I, Hinuma Y. Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease. Proc Natl Acad Sci USA 1982;79: 20315.
  • 4
    Grassmann R, Berchtold S, Radant I, Alt M, Fleckenstein B, Sodroski JG, Haseltine WA, Ramstedt U. Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture. J Virol 1992;66: 45705.
  • 5
    Akagi T, Ono H, Shimotohno K. Characterization of T cells immortalized by Tax1 of human T-cell leukemia virus type 1. Blood 1995;86: 42439.
  • 6
    Tanaka A, Takahashi C, Yamaoka S, Nosaka T, Maki M, Hatanaka M. Oncogenic transformation by the tax gene of human T-cell leukemia virus type I in vitro. Proc Natl Acad Sci USA 1990;87: 10715.
  • 7
    Matsumoto K, Shibata H, Fujisawa J, Inoue H, Hakura A, Tsukahara T, Fujii M. Human T-cell leukemia virus type 1 Tax protein transforms rat fibroblasts via two distinct pathways. J Virol 1997;71: 444551.
  • 8
    Nerenberg M, Hinrichs SH, Reynolds RK, Khoury G, Jay G. The tat gene of human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic mice. Science 1987;237: 13249.
  • 9
    Grossman WJ, Kimata JT, Wong F-H, Zutter M, Ley TJ, Ratner L. Development of leukemia in mice transgenic for the tax gene of human T-cell leukemia virus type I. Proc Natl Acad Sci USA 1995;92: 105761.
  • 10
    Sodroski J, Rosen C, Goh WC, Haseltine W. A transcriptional activator protein encoded by the x-lor region of the human T-cell leukemia virus. Science 1985;228: 14304.
  • 11
    Siekevitz M, Feinberg MB, Holbrook N, Wong-Staal F, Greene WC. Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus type I. Proc Natl Acad Sci USA 1987;84: 538993.
  • 12
    Fujii M, Niki T, Mori T, Matsuda T, Matsui M, Nomura N, Seiki M. 1991. HTLV-1 Tax induces expression of various immediate early serum responsive genes. Oncogene 1991;6: 10239.
  • 13
    Jeang K-T, Widen SG, Semmes OJ IV, Wilson SH. 1990. HTLV-I trans-activator protein, Tax, is a trans-repressor of the human β-polymerase gene. Science 1990;247: 10824.
  • 14
    Brauweiler A, Garrus JE, Reed JC, Nyborg JK. 1997. Repression of bax gene expression by the HTLV-I Tax protein: implications for suppression of apoptosis in virally infected cells. Virology 1997;231: 13540.
  • 15
    Mulloy JC, Kislyakova T, Cereseto A, Casareto L, LoMonico A, Fullen J, Lorenzi MV, Cara A, Nicot C, Giam C-Z, Franchini G. Human T-cell lymphotropic/leukemia virus type 1 Tax abrogates p53-induced cell cycle arrest and apoptosis through its CREB/ATF functional domain. J Virol 1998;72: 885260.
  • 16
    Mori N, Morishita M, Tsukazaki T, Giam C-Z, Kumatori A, Tanaka Y, Yamamoto N. Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor β signaling through interaction with CREB-binding protein/p300. Blood 2001;97: 213744.
  • 17
    Sherr CJ. G1 phase progression: cycling on cue. Cell 1994;79: 5515.
  • 18
    Morgan DO. Principles of CDK regulation. Nature 1995;374: 1314.
  • 19
    Nevins JR. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science 1992;258: 4249.
  • 20
    Neuveut C, Low KG, Maldarelli F, Schmitt I, Majone F, Grassmann R, Jeang K-T. Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb. Mol Cell Biol 1998;18: 362032.
  • 21
    Ohtani K, Iwanaga R, Arai N, Huang Y, Matsumura Y, Nakamura M. Cell type-specific E2F activation and cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I. J Biol Chem 2000;275: 1115463.
  • 22
    Low KG, Dorner LF, Fernando DB, Grossman J, Jeang K-T, Comb MJ. Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a. J Virol 1997;71: 195662.
  • 23
    Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, Nagata K, Hinuma Y. Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature 1981;294: 7701.
  • 24
    Yamamoto N, Okada M, Koyanagi Y, Kannagi M, Hinuma Y. Transformation of human leukocytes by cocultivation with an adult T cell leukemia virus producer cell line. Science 1982;217: 7379.
  • 25
    Popovic M, Sarin PS, Robert-Gurroff M, Kalyanaraman VS, Mann D, Minowada J, Gallo RC. Isolation and transmission of human retrovirus (human T-cell leukemia virus). Science 1983;219: 8569.
  • 26
    Koeffler HP, Chen ISY, Golde DW. 1984. Characterization of a novel HTLV-infected cell line. Blood 1984;64: 48290.
  • 27
    Mori N, Fujii M, Iwai K, Ikeda S, Yamasaki Y, Hata T, Yamada Y, Tanaka Y, Tomonaga M, Yamamoto N. Constitutive activation of transcription factor AP-1 in primary adult T-cell leukemia cells. Blood 2000;95: 391521.
  • 28
    Nagata K, Ohtani K, Nakamura M, Sugamura K. Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I–encoded p40tax protein in the human T-cell line, Jurkat. J Virol 1989;63: 32206.
  • 29
    Ohtani K, Nakamura M, Saito S, Nagata K, Sugamura K, Hinuma Y. Electroporation: application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I. Nucleic Acids Res 1989;17: 1589604.
  • 30
    Iwanaga Y, Tsukahara T, Ohashi T, Tanaka Y, Arai M, Nakamura M, Ohtani K, Koya T, Kannagi M, Yamamoto N, Fujii M. Human T-cell leukemia virus type 1 Tax protein abrogates interleukin-2 dependence in a mouse T-cell line. J Virol 1999;73: 12717.
  • 31
    Smith MR, Greene WC. 1990. Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes. Genes Dev 1990;4: 187585.
  • 32
    Brockman JA, Scherer DC, McKinsey TA, Hall SM, Qi X, Lee WY, Ballard DW. Coupling of a signal response domain in IκBα to multiple pathways for NF-κB activation. Mol Cell Biol 1995;15: 280918.
  • 33
    McKinsey TA, Brockman JA, Scherer DC, Al-Murrani SW, Green PL, Ballard DW. Inactivation of IκBβ by the Tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-κB. Mol Cell Biol 1996;16: 208390.
  • 34
    Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M. NF-κB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol Cell Biol 1999;19: 26908.
  • 35
    Brooks AR, Shiffman D, Chan CS, Brooks EE, Milner PG. Functional analysis of the human cyclin D2 and cyclin D3 promoters. J Biol Chem 1996;271: 90909.
  • 36
    Tanaka Y, Yoshida A, Takayama Y, Tsujimoto H, Tsujimoto A, Hayami M, Tozawa H. Heterogeneity of antigen molecules recognized by anti-tax1 monoclonal antibody Lt-4 in cell lines bearing human T cell leukemia virus type I and related retroviruses. Jpn J Cancer Res 1990;81: 22531.
  • 37
    Akagi T, Ono H, Shimotohno K. 1996. Expression of cell-cycle regulatory genes in HTLV-I infected T-cell lines: possible involvement of Tax1 in the altered expression of cyclin D2, p18Ink4 and p21Waf1/Cip1/Sdi1. Oncogene 1996;12: 164552.
  • 38
    Arnold A, Kim HG, Gaz RD, Eddy RL, Fukushima Y, Byers MG, Shows TB, Kronenberg HM. Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest 1989;83: 203440.
  • 39
    Komatsu H, Iida S, Yamamoto K, Mikuni C, Nitta M, Takahashi T, Ueda R, Seto M. A variant chromosome translocation at 11q13 identifying PRAD1/cyclin D1 as the BCL-1 gene. Blood 1994;84: 122631.
  • 40
    Delmer A, Ajchenbaum-Cymbalista F, Tang R, Ramond S, Faussat A-M, Marie J-P, Zittoun R. Overexpression of cyclin D2 in chronic B-cell malignancies. Blood 1995;85: 28706.
  • 41
    Hinds PW, Dowdy SF, Eaton EN, Arnold A, Weinberg RA. Function of a human cyclin gene as an oncogene. Proc Natl Acad Sci USA 1994;91: 70913.
  • 42
    Wang TC, Cardiff RD, Zukerberg L, Lees F, Arnold A, Schmidt EV. 1994. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 1994;369: 66971.
  • 43
    Deane NG, Parker MA, Aramandla R, Diehl L, Lee W-J, Washington MK, Nanney LB, Shyr Y, Beauchamp RD. Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice. Cancer Res 2001;61: 538995.
  • 44
    Rodriguez-Puebla ML, LaCava M, Miliani de Marval PL, Jorcano JL, Richie ER, Conti CJ. Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia. Am J Pathol 2000;157: 103950.
  • 45
    Mori N, Fujii M, Ikeda S, Yamada Y, Tomonaga M, Ballard DW, Yamamoto N. Constitutive activation of NF-κB in primary adult T-cell leukemia cells. Blood 1999;93: 23608.
  • 46
    Kitajima I, Shinohara T, Bilakovics J, Brown DA, Xu X, Nerenberg M. 1992. Ablation of transplanted HTLV-1 Tax-transformed tumors in mice by antisense inhibition of NF-κB. Science 1992;258: 17925.
  • 47
    Yamaoka S, Inoue H, Sakurai M, Sugiyama T, Hazama M, Yamada T, Hatanaka M. Constitutive activation of NF-κB is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein. EMBO J 1996;15: 87387.
  • 48
    Coscoy L, Gonzalez-Dunia D, Tangy F, Syan S, Brahic M, Ozden S. Molecular mechanism of tumorigenesis in mice transgenic for the human T cell leukemia virus Tax gene. Virology 1998;248: 33241.
  • 49
    Akagi T, Ono H, Nyunoya H, Shimotohno K. 1997. Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes. Oncogene 1997;14: 20718.
  • 50
    Robek MD, Ratner L. Immortalization of CD4+ and CD8+ T lymphocytes by human T-cell leukemia virus type 1 Tax mutants expressed in a functional molecular clone. J Virol 1999;73: 485665.
  • 51
    Furukawa Y, Osame M, Kubota R, Tara M, Yoshida M. 1995. Human T-cell leukemia virus type-1 (HTLV-1) Tax is expressed at the same level in infected cells of HTLV-1-associated myelopathy or tropical spastic paraparesis patients as in asymptomatic carriers but at a lower level in adult T-cell leukemia cells. Blood 1995;85: 186570.
  • 52
    Hofmann W-K, Tsukasaki K, Takeuchi N, Takeuchi S, Koeffler HP. Methylation analysis of cell cycle control genes in adult T-cell leukemia/lymphoma. Leuk Lymphoma 2001;42: 11079.