Transcription Profile of Cells Infected with Human T-Cell Leukemia Virus Type I Compared with Activated Lymphocytes1

[CANCER RESEARCH 62, 3562–3571, June 15, 2002] Transcription Profile of Cells Infected with Human T-cell Leukemia Virus Type I Compared with Activated Lymphocytes1

Cynthia A. Pise-Masison, Michael Radonovich, Renaud Mahieux, Pramita Chatterjee, Craig Whiteford, Janet Duvall, Claire Guillerm, Antoine Gessain, and John N. Brady2 Basic Research Laboratory, Virus Tumor Biology Section, National Cancer Institute, Bethesda, Maryland 20892 [C. A. P-M., M. R., P. C., J. D., C. G., J. N. B.]; Unite d’Epidemiologie et Physiopathologie des Virus Oncogenes, Batiment SIDA-Retrovirus, Institut Pasteur, 75724, Cedex 15, Paris, France [R. M., A. G.]; and Advanced Technology Center, National Institutes of Health, Gaithersburg, Maryland [C. W.]

ABSTRACT vivo for viral infectivity and replication (12–16). In addition, the p12 protein has been implicated in the MHC class I-mediated immune Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent for response and T-cell signaling (16–18). The viral protein p13 has also adult T-cell leukemia and the neurological disorder tropical spastic para- recently been reported to regulate cellular signaling pathways (19). -paresis/HTLV-I-associated myelopathy. CD4؉ T lymphocytes, the pri mary hosts for HTLV-I, undergo a series of changes that lead to T-cell Finally, the p30 protein is reported to play a role in transcriptional activation, immortalization, and transformation. To gain insight into the regulation with the use of Gal4-p30 fusion constructs (20). genetic differences between activated and HTLV-I-infected lymphocytes, DNA microarray technology has facilitated the development of a we performed Affymetrix GeneChip analysis of activated and HTLV-I- more complete and inclusive analysis of gene expression profiles in infected cells. Using the Hu6800 GeneChip, we identified ϳ763 genes that response to many stimuli for a variety of biological systems. Harhaj et had differentially regulated expression in at least three of five HTLV-I cell al. (21) and de La Fuente et al. (22) used Atlas human cDNA arrays lines. Classification of these genes into functional groups including cellular to analyze gene expression patterns in HTLV-I-infected PBMCs com- receptors, kinases, phosphatases, cytokines, signal proteins, and transcrip- pared with uninfected PBMCs or HTLV-I-transformed C8166 cells tion factors provides insight into genes and pathways that are differen- compared with nonvirally transformed CEM cells, respectively. Ng et tially regulated during HTLV-I transformation. al. (23) have used NIH OncoChip cDNA arrays to analyze gene expression patterns of ϳ2000 human genes in Tax-expressing Jurkat INTRODUCTION T lymphocytes. These studies have looked at small numbers of samples and identified a few candidate genes important for HTLV-I- HTLV-I3 is the etiologic agent of an aggressive and fatal disease induced pathogenesis. We have chosen GeneChip microarrays (Af- termed ATL and of the neurodegenerative disease tropical spastic fymetrix, Inc.), containing oligonucleotide hybridization probes paraparesis/HTLV-I-associated myelopathy (1–4). HTLV-I has also representative of Ͼ7000 genes, to perform a more comprehensive been less closely associated with uveitis, arthritis, infectious derma- examination of the expression profiles for HTLV-I-immortalized and titis, and immunosuppression (5–7). The principle target for HTLV-I -transformed cell lines and compared these with the expression profile infection in the lymphoid system are mature CD4ϩ CD45ROϩ T of normal activated PBLs. The results presented here extend earlier lymphocytes, although other cell types of lymphoid origin have been studies by identifying a significant number of new genes that have infected by HTLV-I in vitro, including CD8ϩ T cells, B cells, and altered expression in HTLV-I-transformed cells compared with acti- macrophages (8–11). vated PBLs. We have identified several new response pathways

The mechanism of oncogenic transformation of host T lymphocytes involving G2/M checkpoint control factors, DNA replication and in ATL remains unclear, and to date there is no effective treatment for licensing factors, transcriptional regulators, and kinase/phosphatase this disease. However, as with other cancers, altered gene expression signaling molecules that are deregulated in HTLV-I-infected cells. of networks of genes are linked to ATL initiation and progression. Moreover, we found that by analyzing several HTLV-I cell lines, gene Several studies (8–11) have established that the viral transcriptional expression changes attributable to individual cell types were de- activator protein Tax plays a critical role in cellular transformation. creased. Tax not only activates expression of viral genes via the viral LTR, but has also been reported to affect the expression or activity of several cellular genes. Several of these genes encode proteins involved in cell MATERIALS AND METHODS growth and cell death including proto-oncogenes, growth factors and their receptors, CDKs, and CDK inhibitors (8–11). Cell Cultures. We isolated PBMCs from healthy, HTLV-I-negative donors using Ficoll density gradient centrifugation. After removal of macrophages, In more recent studies, a role for the HTLV-I accessory proteins, cells were stimulated for 18 h with PHA (2 ␮g/ml) and then grown in RPMI p12, p30, and p13 in gene activation and cell signaling have been 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml demonstrated. All three proteins have been shown to play a role in penicillin-streptomycin, 2 mM glutamine, and 50 units/ml IL-2. Activated cells were expanded for 2 weeks and then total RNA was isolated. Resting cells Received 11/15/01; accepted 4/15/02. were expanded for 2 weeks, washed twice in culture medium not containing The costs of publication of this article were defrayed in part by the payment of page PHA and IL-2, and total RNA was isolated 3 days later. HTLV-I-immortalized charges. This article must therefore be hereby marked advertisement in accordance with cells, Champ (ATL), Bes (ATL), and ACH.WT, were grown in complete 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supplementary data for this article is available at Cancer Research Online (http:// medium supplemented with 50 units/ml IL-2. HTLV-I-transformed cells, C81 cancerres.aacrjournals.org). and Hut102, were grown in complete medium. 2 To whom requests for reprints should be addressed, at Basic Research Laboratory, RNA Isolation and Probe Preparation. mRNA was isolated from total Virus Tumor Biology Section, National Cancer Institute, 9000 Rockville Pike, Bethesda, RNA by the RNeasy and Oligotex mRNA isolation procedures as outlined by MD 20892. Phone: (301) 496-0986; Fax: (301) 496-4951; E-mail: [email protected]. 3 The abbreviations used are: HTLV-I, human T-cell lymphotropic virus type-I; ATL, the manufacturer (Qiagen). Experimental procedures for GeneChip were per- adult T-cell leukemia; LTR, long terminal repeat; CDK, cyclin-dependent kinase; PBMC, formed according to the Affymetrix GeneChip Expression Analysis Technical peripheral blood mononuclear cell; PBL, peripheral blood lymphocyte; PHA, phytohem- Manual (Affymetrix). Briefly, double-stranded cDNA was synthesized from ␣ ␣ agglutinin; IL, interleukin; RT-PCR, reverse transcription-PCR; IL-2R , IL-2 receptor ; mRNA with the SuperScript Choice system (Life Technologies, Inc.) and a TNF, tumor necrosis factor; CK1⑀, casein kinase 1 ⑀; MLK, mixed-lineage kinase; DUS, dual-specificity phosphatase; INPP1, inositol phosphate-1-phosphatase; CREB, cAMP- T7-(dT) 24 (GENSET) primer. In vitro transcription was performed on the cDNA responsive element binding protein; ATF, activating transcription factor. to produce biotin-labeled cRNA with an Enzo Transcription Kit (Enzo) as de- 3562

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2002 American Association for Cancer Research. GENE EXPRESSION PROFILES OF HTLV-I-INFECTED CELLS scribed by the manufacturer. The cRNA was linearly amplified with T7 polym- obtained through PBL culture of an ATL patient, fell into a separate erase, the biotinylated cRNA was cleaned with an RNeasy Mini Kit (Qiagen), cluster distinct from the HTLV-I-transformed cells (C81 and Hut102) fragmented to 50–200 nucleotides, and then hybridized to Affymetrix Hu6800 and the HTLV-I-immortalized cells (Bes and ACH.WT). The shading arrays. The arrays were then processed on the Affymetrix fluidics station and across each sample/bar is an indication of the gene-expression levels scanned on an HP GeneArray scanner. The intensity for each probe set of the array for each of the 7300 genes. For each cell sample, a unique expression was captured with Affymetrix GeneChip Software, according to standard Af- pattern emerges (Fig. 1). A section of the tree has been expanded to fymetrix procedures. To determine the quantitative RNA level, the average differences representing the perfectly matched minus the mismatched for each gene- show an example of how individual genes performed. As shown, the specific probe set was calculated. The GeneSpring Software (SiliconGenetics) was genes GATA-2, PON2, and PLAGL2 are all overexpressed in the also used to examine the differential gene expression. HTLV-I-infected cells as compared with the PBL samples. This form RT-PCR. Total cellular RNA was isolated with Rnazol B (Tel-Test) as of clustering allows one to identify groups of genes that are expressed described by the manufacturer. RNA (5 ␮g) was converted to cDNA with either similarly or opposite to the control sample. RETROscript (Ambion) as described by the manufacturer. Samples were then From the expression data, we compiled a list of genes deregulated PCR amplified with SuperTaq Plus polymerase (Ambion) to quantitate indi- an average of 2-fold or greater in at least three of five HTLV-I- vidual genes. The PCR primers for Tax were as follows: 5Ј-TGTTTG- infected cell lines, as compared with activated PBLs (Table 1 and Ј Ј Ј GAGACTGTGTACAAGGCG-3 and 5 -CAGGCTGTCAGCGTGACGG-3 . on-line data supplement1). A 2-fold cutoff was chosen based on The primers for IL-2R␣ were as follows: 5Ј-GGTCCCAGGCAGAGAAT- statistical information provided by Affymetrix. From this initial list, CATA-3Ј and 5Ј-AGAGGGAGAAGGGATGGAGGT-3Ј. PCR products were separated by agarose gel electrophoresis and quantitated with Fluorchem the genes were broken down into functional groups. Because the (Imgen Technologies). Affymetrix array contains a much larger number of genes than pre- Transfections. Jurkat T cells were transiently transfected by electropora- vious analyses, our results provide a more complete and inclusive tion with 4 ␮g of the Cdc25C promoter construct (C290-, C75-, CC51-, and “blueprint” of HTLV-I-induced gene expression. Because of size C75M1-Luc) in the absence or presence of Tax (8 ␮g) as described previously limitations, the list of 763 genes is provided in the on-line data (24). Luciferase activity was measured on a Berthold Luminometer and nor- supplement. Only 84 of the 763 differentially expressed genes have malized to protein concentration. been reported previously (21, 23, 25). These genes are shaded in the on-line data supplement table and the results of individual studies RESULTS from other laboratories are noted. The array analysis by Ng et al. (23) could not be included because the GenBank accession numbers were To investigate the molecular basis of the HTLV-I-induced T-cell not available for this study. The results of the Affymetrix gene- immortalization and transformation, we compared the gene expression expression analysis of several independent genes have been confirmed profiles of normal uninfected PBLs with HTLV-I-infected cells. by quantitative RT-PCR (see below and data not shown). PBMCs from three healthy, HTLV-I-negative donors were cultured in Regulation of Cellular Receptors. One of the hallmark genes the presence of IL-2 and PHA to allow the outgrowth of T lympho- known to be overexpressed in HTLV-I-infected cells is the cytokine cytes. After 1 week of culture, the resulting cell population consisted receptor IL-2R␣. IL-2R␣ plays a pivotal role in the ability of HTLV- of Ͼ95% CD4ϩ cells as determined by fluorescence-activated cell I-transformed cells to proliferate (26–29). Tax has been demonstrated sorting analysis (data not shown). mRNA was isolated from either to activate expression of IL-2R␣ through activation of the nuclear activated or resting PBLs, HTLV-I-immortalized cell lines, Champ, factor ␬B pathway (29). Not unexpectedly therefore, we found that Bes, and ACH.WT (IL-2 dependent), and from the HTLV-I-trans- IL-2R␣ expression was elevated in each of the HTLV-I-infected cells formed cells, C81 and Hut102 (IL-2-independent). mRNA samples (Table 1). Quantitative analysis of microarray hybridization data were then processed to produce biotin-labeled cRNA probes, which demonstrated that the level of IL-2R␣ expression was increased were hybridized to the Affymetrix Hu6800 GeneChip as described in 7–38-fold in the HTLV-I samples as compared with the activated “Materials and Methods.” Arrays were processed with the Affymetrix PBLs (Table 1). The increased expression of IL-2R␣ mRNA in the fluidics station and scanner. Expression analysis files were generated HTLV-I-transformed cells was confirmed by quantitative PCR for each sample, and a database was created. (Fig. 2, B and C). Interestingly, the level of Tax protein as detected by Expression Profiling of Normal and HTLV-I mRNA. Using Western blot analysis (Fig. 2A) did not correlate completely with the GeneSpring Software, we performed a “cluster” or “tree” analysis of level of IL-2R␣ mRNA expression. the cell gene-expression profiles. A hierarchical clustering (Fig. 1), Consistent with recent observations of Mariner et al. (30), we which allowed visualization of a set of samples or genes by organizing observed that the level of IL-15R␣ was elevated 5–10-fold in the them into a mock-phylogenetic tree, was performed for individual HTLV-I-infected cell samples. The level of IL-15R␣ expression was genes (across the top) and for each sample (down the side). In this elevated in four of the various HTLV-I-infected cell samples (Table tree, genes (or samples) having similar gene-expression patterns are 1). Of note, the level of IL-15R␣ expression in the C81 HTLV-I- clustered together. How far across the tree one goes until a subtree is transformed T-cell line was as low as was seen in the activated found containing both genes (samples) is a measure of how closely lymphocyte cells. Thus, the level of IL-15R␣ expression in different correlated those genes (samples) are to each other. As expected, the HTLV-I-infected cells may vary significantly. These results are con- gene-expression profile of the activated PBLs was most closely re- sistent with RNA and protein levels reported previously (30). Of the lated to their unactivated counterparts (Fig. 1, lines A and B) and was microarray reports, ours is the first to demonstrate differential IL- distinct from the HTLV-I-infected cells (Fig. 1, lines C–G). It is 15R␣ expression. To note, the IL-2R␤ chain common to both IL- interesting to note that the overall gene-expression profile of two 15R␣ and IL-2R␣ signaling is also increased in the HTLV-I-infected HTLV-I-transformed cells lines, HUT102 and C81, fell into a closely cells except in the C81 sample (Table 1). related gene-expression cluster. This suggests that they are more Overexpression of Cellular Cytokines or Signal Molecules. In closely related to each other than they are to the HTLV-I-immortal- agreement with the proposed IL-15 paracrine/autocrine loop (30, 31), ized cells. The HTLV-I-immortalized cell lines, Bes, obtained from we observed an increase in the level of IL-15 mRNA in four of five the PBL culture of this ATL patient, and ACH.WT, lymphocytes HTLV-I-transformed cells. These results are consistent with the work immortalized with an infectious clone of HTLV-I, fell into a similar of Azimi et al. (31, 32) who demonstrated that the IL-15 promoter is cluster. Interestingly, the HTLV-I-immortalized cell line Champ, also transactivated by Tax and the level of IL-15 mRNA is increased 3563

Fig. 1. Gene expression cluster analysis of HTLV-I-infected and donor lymphocytes. Using GeneSpring (SiliconGenetics), we generated a gene-expression and experiment tree comparing activated lymphocytes (A), unactivated lymphocytes (B), C81 (C), Hut102 (D), Bes (E), ACH.WT (F), and Champ (G). The branching indicates the relat- edness of each sample. The color scale indicates the expression level ranging from 0.0 (blue, no expression) to 5.0 (red, high expression). The region of the tree indicated by the black lines has been expanded to show the differential expression of the three genes PON2, GATA-2, and PLAGL2.

3–4-fold in HTLV-I-transformed cells lines. C81 cells appear to be an molecules CD58 and CD59 (Table 1). Many cell-surface signaling exception because the level of IL-15 mRNA expression is very low in molecules were also decreased in expression in the HTLV-I-infected this cell line. cells as compared with activated PBLs. These include integrin mol- In addition to the overexpression of cytokines including SCYA1, ecules (ITGAM, ITGAE, and CD11A), the adhesion molecule PE- SCYA17, SCYA22, and P40 T-cell growth factor, there were also CAM1, CD47, CDW52, CD37, CD27, CD7, CD20, CD16, and CD72 several signal pathway gene products that had increased expression (Table 1); all of which play a role in lymphocyte signaling. (Table 1). We have previously shown that Tax activates expression of Overexpression of Kinases/Phosphatases in HTLV-I-infected parathyroid hormone-releasing protein (PTHrP; see Ref. 33). In our Cells. Phosphorylation has been shown to play a key role in regulat- current analysis, we see a 2–5-fold increase in PTHrP gene expression ing protein activity and cellular responses. Several kinases were (Table 1). Our microarray analysis shows that TNF␣ (GenBank ac- overexpressed in the HTLV-I-transformed cells including mitogen- cession no. M16441) expression is elevated in C81, HUT102, and activated protein kinase family members PRKM7 and mitogen-acti- ACH-WT lymphocytes, which are immortalized with an infectious vated protein kinase 3 (MARK3); tyrosine kinase Lyn; cell cycle clone of HTLV-I. In contrast, very low levels of TNF␣ expression regulatory kinases CDK4, CDK7, and CDK2; and CK1⑀. The over- were observed in the Bes and Champ RNA samples. ⑀ We also observed that the expression level of gp34 was elevated in expression of CK1 is of interest because we have recently demon- the HTLV-I-infected cells. This type II membrane protein belongs to strated that the amino terminus of p53 is hyperphosphorylated at the TNF superfamily (TNFSF4) and has been shown to stimulate serine 15 and one other amino acid within amino acids 1–19 (34, 35). T-cell proliferation and cytokine production. At slightly lower levels, Sakaguchi et al. (36) and Dumaz et al. (37) have shown that threonine we observed the elevated expression of TNFSF7 (CD27L), which also 18 can be phosphorylated by CK1⑀. Interestingly, phosphorylation of plays a role in T-cell activation. We saw an increase in the cell surface threonine 18 is dependent on prior phosphorylation of serine 15 (37). 3564

The overexpression of Lyn is consistent with previous reports that Tax creased 3-fold in HTLV-I-transformed cells, whereas the family mem- transactivates the promoter of this src family gene (38). bers IRF1 and IRF3 are decreased compared with PBLs (Table 1). Recently, Ng et al. (23) reported increased levels of the MLK3 This suggests that HTLV-I-infection may impact only a subset of using cDNA arrays to study Tax-regulated genes in transformed IRF-regulated genes. Jurkat T lymphocytes. The studies by de La Fuente et al. (22) Altered Expression of Members of the Cell Cycle Machinery. comparing a Tax/HTLV-I-expressing cell line (C81) with a trans- Several members of the cell cycle machinery have altered expression formed T-cell line (CEM), also noted an increase in MLK3. In in HTLV-I-infected cells compared with control PBLs. The expres- contrast, our results, like those of Harhaj et al. (21) using Atlas sion of the Cdc25C tyrosine phosphatase was increased 3–6-fold (Clontech) filter arrays and Ruckes et al. (25) using subtractive (Table 1) in HTLV-I-transformed cells. This protein functions as a hybridization, do not show differential expression of MLK3 in HTLV- dose-dependent inducer of mitotic control. It is required for progres- I-infected cells compared with activated donor PBLs. The levels of sion of the cell cycle, through dephosphorylating Cdc2, activating MLK3 expression we observed are highlighted in Table 1. One kinase activity (Fig. 3A). The fact that cyclin B and Cdc2 (CDK1) explanation for the difference is the choice of control cells. The first expression are also increased may further indicate a deregulation of studies use transformed cells, whereas our studies and those of Harhaj G2/M cell cycle control. et al. (21) and Ruckes et al. (25) compared the HTLV-I-infected cells The Cdc25C promoter contains nuclear factor Y-binding sites im- with activated PBLs. Perhaps nonviral transformed T-cells have a portant for the regulation of Cdc25C expression (47, 48). We have down-regulation of MLK3 and thus the Tax-expressing cells artifi- previously shown that Tax interacts directly with nuclear factor Y to cially appear to have an increase in MLK3. allow activation of the MHC class II DQ promoter (24). To test Our studies are the first to show that the dual specific phosphatases whether Tax could activate the Cdc25C promoter, we performed DUSP2, DUSP4, and DUSP5 are increased ϳ10-fold in the HTLV- cotransfection experiments in Jurkat T cells. As demonstrated in Fig. I-infected cells as compared with the activated PBLs. These phos- 3(B and C), Tax expression activates the Cdc25C promoter by phatases have been implicated in the mitogenic signaling pathways ϳ13-fold. Deletion of the upstream promoter region to Ϫ75 reduced involving erk1 and erk2 (39–41) and may play an important role in promoter activity, but did not diminish Tax transactivation (Fig. 3C). regulating signaling in the HTLV-I-infected cells. The C75 promoter construct contains four copies of the Y box Likewise, INPP1 is increased ϳ10-fold in the HTLV-I-infected element. That the Y boxes are important for Tax transactivation is cells over activated PBLs. Inositol signaling is an important compo- demonstrated by mutant pCDC25C75mY1, which contains base sub- nent of cellular processes including proliferation, differentiation, and stitutions in the Y1 box. The expression from this plasmid was apoptosis (42–44). INPP1 acts on both Ins(1,4)P2 and Ins(1,3,4)P4, identical in the absence and presence of Tax (Fig. 3, B and C). These which are key intermediary metabolites in several pathways such as results correlate with the increased expression of Cdc25C mRNA seen DNA replication and cell cycle progression (42–44). INPP1 overex- in the microarray analysis. To note, although we consistently observed pression has recently been correlated with human colorectal cancer an increase in Cdc25C mRNA, posttranscriptional regulation is also a (45). factor in the control of Cdc25C at the protein level (data not shown). Overexpression of Transcription Factors. The HTLV-I Tax pro- As with many oncogenic factors, deregulated checkpoint control tein functions as a transcriptional transactivator of the viral LTR and occurs at several stages within the cell cycle. In addition to the several cellular promoters. Transcriptional activation by Tax is attrib- increase in G2/M phase regulatory factors, we see an increase in DNA utable, in part, to protein-to-protein interactions leading to enhanced replication licensing and elongation factors including MCM2, DNA binding or transcriptional activity or increased nuclear accumu- HsMCM6, MCM7, Cdc28 kinase 1 and 2, Cdc18L, RFC3, and RFC4 lation of active transcription factors, causing activation of CREB, (Table 1). As described previously (49–52), we see increased expres- nuclear factor ␬B, and SRF. Tax has also been shown to stabilize the sion of the cell cycle regulators PCNA (2–7-fold), p21 (2–5-fold), indirect binding of coactivator proteins such as CBP to the DNA, CDK2 (2–6-fold), CDK4 (1–7-fold), and thioredoxin (2–7-fold). leading to an increase in transcription initiation and reinitiation. Our Concomitant with an increase in cdk levels, there is a decrease in the microarray data revealed the increased expression of ϳ32 transcrip- cdk inhibitor p19 (Table 1). Together these results suggest that tion regulators in the HTLV-I cells. Not all of these genes are likely HTLV-I infection impacts several steps in cell cycle regulation. to be directly activated by Tax, but may represent “secondary” effects Expression of Genes That Regulate Apoptosis. Experiments of HTLV-I infection because of changes in cell metabolism and cell from numerous laboratories have defined apoptotic pathways and proliferation. gene products that function to inhibit or accelerate cellular apoptosis. In recent reports, Harhaj et al. (21) and Ruckes et al. (25) analyzed For example, HIAP-1 acts to repress apoptosis in mammalian cells, the regulation of transcription factors in HTLV-I positive cells. Tran- presumably by inhibiting the activity of caspases involved in cell scription factors of the Jun family including junB, Jun, and JUND, death (53, 54). Interestingly, HIAP-1 and API1 (55, 56) are overex- B94, Ets2, CDK7 (TFIIH), GTF2A1, Rel, and GATA-2 were over- pressed 5–8-fold in HTLV-I cells. API1 functions in the cell to inhibit expressed in the range of 3–14-fold. The results of our study are the action of specific caspases in the induction of cell death (55, 56). consistent with these reports. All of these transcription factors, with We also found that the apoptosis inhibitor BCL2L1 (Bcl-xL) was the exception of TAFII31 and YB-1, were found to be up-regulated in overexpressed in HTLV-I cells 4–7-fold. Similar to the findings of the HTLV-I cells (Table 1). Also included in our transcription factor Ruckes et al. (25), we also find increased expression of the anti- group were several members of the CREB transcription factor group apoptotic factor I-309. In contrast to the results of Harhaj et al. (21), including ATF3, ATF6, and cAMP-responsive element modulator we did not detect the overexpression of other apoptosis inhibitors such (CREM). As stated above, CREB/ATF plays an important role in as dad1, ddlc1, HSP27, or NKEF in HTLV-I cells compared with HTLV-I-LTR transcription and thus viral expression and replication. activated T-cells. Consistent with the work of Sharma et al. (46), the transcriptional In addition to the overexpression of genes that inhibit apoptosis, we activator IRF4 which binds to the IFN-stimulated response element in also found genes that induce apoptosis were down-regulated. For the immunoglobulin ␭ light chain enhancer and plays a role in example, the expression of caspase-8, a cysteine protease that func- ISRE-targeted signal transduction mechanisms specific to lymphoid tions in the initiation of the apoptotic proteolytic pathway (57, 58) is cells, is up-regulated up to 17-fold. Interestingly, IRF5 is also in- repressed in the HTLV-I cells. Depletion or inactivation of caspase-8 3565

Table 1 A list of genes and their relative expression levels (GeneSpring values) in peripheral blood lymphocytes and HTLV-I-infected cells Cell sampleb

Genea Accession no. PBLc Bes C81 Champ Hut102 ACH.WT Cell cycle and apoptosis Proliferating cell nuclear antigen M15796 0.743 1.565 5.433 1 2.416 3.954 PCNA J05614 0.576 3.197 6.412 2.411 1.207 3.651 cyclin B1 M25753 0.523 1.651 7.109 1 3.037 2.443 (CDC25C) cell division cycle 25C M34065 0.532 0.34 3.325 1.771 1.621 1.384 MCM2 D21063 0.893 1.75 5.898 0.846 2.655 3.156 HsMCM6 D84557 0.816 1.396 3.68 0.89 2.771 2.467 RFC3 L07541 0.685 3.164 14.592 6.325 1 8.891 RFC4 M7339 0.759 1.702 3.626 0.717 1.457 1.465 DP1 L23959 0 13.15 19.014 4.701 0 72.093 (CIP2) associated with cyclin-dependent L25876 1.062 2.133 1.807 1 3.048 3.147 kinase (MAD2L1) MAD2 U65410 0.333 8.588 5.146 3.241 10 12.885 (CDC18L) U77949 0.787 5.236 34.591 3.466 10.92 0.565 (CDC2) cell division cycle 2 X05360 2.047 7.434 25.5 1 18.026 12.102 (CKS1) CDC28 kinase 1 X54941 1.850 0 23.164 0 20.537 11.860 (CKS2) CDC28 kinase 2 X54942 1.640 0 38.653 0 9.783 11.757 (BCL2L1) Bcl-XL Z23115 0.738 2.038 3.219 2.656 0.926 4.391 HIAP-1 U45878 0.816 4.752 0.392 2.843 4.892 3.97 (CDK4) U37022 0.643 1.721 6.747 1.389 2.989 2.014 (API1) apoptosis inhibitor 1 U37546 0.762 3.217 0.578 2.058 4.143 3.751 (API2) apoptosis inhibitor 2 U37547 0.758 1.414 4.07 1.521 1.61 1 p21/CIP1/WAF1 U09579 0.392 4.187 1.42 3.39 3.898 3.75 CDK2 M68520 0.814 3.846 2.204 5.37 2.677 1.905 (CDKN2D) p19 U40343 1.042 .993 .221 .601 .505 1.204 (CASP6) caspase 6, apoptosis-related U20536 1.853 0.794 2.035 0 0.384 0.644 cysteine protease (CASP4) caspase 4, apoptosis-related U28014 1.583 0.568 0 0.164 0.384 1 cysteine protease (TNFRSF1A) TNF receptor superfamily, M58286 1.785 0 0 0.622 0 1.087 member 1A (TNFRSF7) TNF receptor superfamily, M63928 5.18 0 0 0 0.053 0 member 7 (CD27) (CASP8) caspase 8, apoptosis-related X98172 2.157 0 0.276 0 0.895 1 cysteine protease Kinases and phosphatases PRKM7 U29725 0.333 2.183 3.18 2.752 3.923 3.579 CDK7 L20320 0.678 1.489 1.924 1.089 1.665 1.559 (CSNK1E) casein kinase 1, ␧ L37043 0.576 1.197 1.686 1.502 4.13 1.417 MARK3 M80359 0.593 1.195 1.549 1.215 1.645 0.447 DUSP4 U48807 0.516 5.009 4.131 4.287 1.979 2.595 DUSP5 U15932 0.289 14.12 0.857 9.609 3.641 5.07 DUSP2 L11329 0.676 8.742 5.693 9.701 8.799 4.48 INPP1 L08488 0.856 5.959 2.021 2.759 6.483 6.465 (PRKCL2) PRKC-like 2 U33052 1.326 0.535 0.097 0.198 0 0.598 (PRKCO) protein kinase C, ␪ L01087 2.165 0 5.132 0 0 0 (PRKCB1) protein kinase C, ␤1 X06318 9.444 0 0.465 0 1 0.755 PKC␤2 [partial (AA 1–673)] X07109 8.071 0 0 0 0 1 (PRKCL) PKC eta M55284 3.26 0.656 0 0 0 1 (PRKCZ) PKC ␨ Z15108 2.165 0 5.132 0 0 0 MLK3d L32976 0.929 1.351 0.569 0.743 1.02 1.021 Receptors and signaling molecules (TNFSF6) TNF (ligand) superfamily, D38122 0.816 1.197 0.31 0 0.34 0.668 member 6 (SCYA17) small inducible cytokine D43767 0.508 3.129 2.043 25.847 32.269 12.648 subfamily A (Cys-Cys), member 17 (TNFSF4) TNF (ligand) superfamily, D90224 0.651 44.07 23.306 45.258 42.219 14.47 member 4 (Tax-transcriptionally activated glycoprotein 1, 34kD) (IFNG) interferon ␥ J00219 0.601 12.64 0 4.07 0 6.97 (TNFSF7) CD27 ligand L08096 0.294 4.06 6.157 1.682 9.207 7.219 (EBI3) cytokine receptor L08187 0.927 9.363 5.523 6.951 17.2 9.537 TNF M16441 0 0 589.44 0 919.60 253.49 CD33 M23197 0.978 4.498 1 6.143 0.592 23.419 (ICAM1) intercellular adhesion molecule 1 M24283 0.101 1 1.506 2.108 4.393 1.116 (CD54), human rhinovirus receptor (IL2RB) interleukin 2 receptor, ␤ M26062 0.816 2.304 0.197 2.168 2.312 1.907 IL-7 J04156 0.916 1.599 0 3.115 3.284 7.535 IL-6 X04602 1.702 0 103.62 0 309.744 51.228 IL-6R receptor M20566 2.483 1 0 5.88 4.833 7.335 (PTHrP) M26958 0.432 1 1.086 0.747 1.348 2.111 P40 T-cell and mast cell growth factor M30135 0 123.9 0 6020.3 26.45 855.19 (SCYA1) small inducible cytokine A1 M57506 0.143 363.0 14.816 1015.2 13.134 86.712 (I-309, homologous to mouse Tca-3) (A20) TNFAIP1 M59465 0.649 2.307 1.78 2.969 2.043 2.212 CD59 M84349 0.786 6.439 0.231 7.312 5.018 2.763 B94 M92357 0.438 1.061 6.854 3.445 37.005 7.031 IL-15 U14407 0 10.56 0 66.038 307.19 10.383 (IL-15RA) IL-15 receptor, ␣ U31628 0.635 3.378 0.756 6.05 5.819 4.309

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Table 1 Continued Cell sampleb

Genea Accession no. PBLc Bes C81 Champ Hut102 ACH.WT (IL-12RB2) IL-12 receptor, ␤ 2 U64198 0.641 13.41 0 13.011 1 8.95 SCYA22 U83171 0.271 1 3.126 12.88 7.403 2.375 (IL-2RA) IL-2 receptor, ␣ X01057 0.133 1.973 1.495 1 2.291 5.074 TNF ␣ X02910 0.256 2.036 3.009 0.06 3.611 1 14.3.3 protein ␶ X56468 0.626 1.074 1.6 0.567 1.421 1.53 CD58 Y00636 0.345 2.332 3.894 1 1.064 2.189 PON2 L48513 0.152 7.233 9.387 8.149 7.165 10.182 PLAGL2 D83784 0 13.47 15.775 8.752 9.838 14.76 CD7 D00749 1.214 0.332 0.072 0.048 0.201 0.398 CD16 J04162 2.467 0.309 0.330 0 0.396 1 (ITGAM) integrin, alpha M J03925 2.961 1.031 0.392 0 0.41 0.588 (IL-7R) IL-7 receptor M29696 5.375 0.177 0.128 0.155 0.231 0.181 (CD30L) TNSF8 L09753 3.281 0.579 0 0 2.378 0 (ITGAE) integrin ␣ E L25851 1.393 0.311 0.691 0.342 0.54 1 (PECAM1) CD31 L34657 4.964 0.64 0.196 0 0.845 0 (ITGB2) integrin, ␤ 2 [antigen CD18 (p95), M15395 3.294 0.73 0.202 0.117 0.421 0.85 lymphocyte function-associated antigen 1; macrophage antigen 1 (mac-1) ␤ subunit] CD19 M28170 1.901 0 0.308 0.059 0.869 1 granzyme B M28879 1.716 0 0.299 0 0.433 1.202 (IL-7R) IL-7 receptor M29696 5.375 0.177 0.128 0.155 0.231 0.181 (GZMB) granzyme B M36118 2.804 0.923 0.424 1 0.228 0 CD7 M37271 2.86 0.132 0.267 0.09 0.17 1 CD72 M54992 3.035 0.399 0 0.254 1 0.681 (ITGAX) integrin alpha X M81695 1.466 0.657 0.178 0.376 0.458 0.15 (FLT3LG) fms-related tyrosine kinase 3 U03858 1.201 0.17 0.105 0.105 0.853 0.394 ligand (MADH3) SMAD3 U68019 1.57 0.505 0 0.291 0 1 CD20 receptor (AA 1–297) X07203 41.34 0 2.469 0 0 0 (ITGB1) integrin ␤ 1 X07979 1.127 0 0.437 0 2.465 1.348 (CD20) CD20 (AA1–297) X12530 10.82 0 0 0 0.248 1 CD37 X14046 2.281 1 0.635 0.232 0 0.679 (ITGA4) integrin, ␣ 4 (antigen CD49D, ␣ 4 X16983 4.475 01002.534 subunit of VLA-4 receptor) CD6 X60992 1.555 0 0.012 0 0.316 1.568 CDW52 X62466 3.954 0 0.042 0 0.042 1 CD18 X64072 1.455 0.367 0.027 0.201 0.139 0.523 (CD47) CD47 antigen (Rh-related antigen, X69398 1.951 0.496 1.113 0.268 0.149 1 integrin-associated signal transducer) (LPAP) CD45 binding protein X97267 1.572 0.073 0 0.048 0.022 0.787 (ITGAL) integrin ␣ L(CD11A) Y00796 4.174 0 0.571 0 0.07 1 CD69 early activation antigen Z30426 Z30426 2.274 0 0.151 0 0.046 1.583 (ACTB) actin, ␤ X00351 0.708 1.635 1.218 1 1.322 1.551 Transcription (ATF6) CREB binding serum response AF005887 0.635 1.544 0.826 1.915 2.234 1.819 factor (CREM) cAMP responsive element D14826 0.621 3.476 0.402 1.596 0.72 2.009 modulator (REL) v-rel avian reticuloendotheliosis viral X75042 0.982 4.875 3.803 0 2.416 1 oncogene homolog (TXN) thioredoxin X77584 0.795 1.67 4.199 0.598 3.805 4.903 (JUND) jun D proto-oncogene X56681 0.709 1.263 2.084 0.594 1.557 1.199 (GTF2F2) general transcription factor IIF, X16901 0.332 1.03 1.392 1.279 0.153 1 polypeptide 2 (JUNB) jun B proto-oncogene X51345 1.08 0 3.767 0 3.503 2.347 (IRF5) interferon regulatory factor 5 U51127 0.798 1.205 2.94 1.383 2.966 1.488 IRF4 U52682 0.516 0.945 8.838 1.228 3.764 2.185 JunB U20734 0.544 0.637 1.824 1.007 3.867 3.884 B-ATF U15460 0.496 2.426 1.698 1.715 1 2.182 ETS2 J04102 0.255 2.892 5.081 3.144 1.388 1 c-Jun J04111 0.751 2.752 0.137 1.58 5.39 1.81 (ATF3) activating transcription factor 3 L19871 0.399 1 1.864 4.495 16.72 5.373 Lyn M16038 0.739 1.749 1.505 1 8.237 1.232 GTF2A1 U14193 0.602 1.191 2.117 1.607 1.023 1.215 (NFKBIA) IKB ␣ M69043 0.512 4.205 6.125 5.183 3.912 2.758 GATA-2 M77810 0 14.44 19.886 6.604 5.104 18.033 IRF1 L05072 1.228 0.293 0.242 0.387 0.389 1 IRF3 Z56281 1.636 0.510 0.726 0.358 0.262 0.533 YB-1 J03827 1.119 0.887 2.954 0.642 2.073 1.88 a The GenBank accession number and gene identification are listed. b The relative level of gene expression as calculated using GeneSpring (SiliconGenetics) for the indicated cell sample is provided. c The PBL profile represents three independent donor PBL samples. d Expression of the MLK3 gene does not change.

3567

10 structurally related enzymes that have been implicated in a variety of cellular responses. The protein kinase C (PKC) substrate inositol 1,4,5-triphosphate 3-kinase was also down-regulated, further suggest- ing that control of the pathway used in normal T-cell activation has been repressed in HTLV-I-infected cells.

DISCUSSION We have used Affymetrix microarray technology to analyze and compare the gene expression profiles of ϳ7300 genes in activated and HTLV-I-infected lymphocytes. Several new genes that have deregulated expression in HTLV-I-transformed cells have been identified including cellular receptors, cytokines, apoptosis inhibitors, kinases, checkpoint regulators, and transcription factors. Because all of the HTLV-I cells used in these studies are stably infected, it is not possible at this time to distinguish which genes are deregulated during the initial immortalization/transformation events and which genes are induced as the result of secondary effects of HTLV-I infection. In addition, a clear distinction between HTLV-I-immortalized versus -transformed cells cannot be made at this point and requires analysis of more HTLV-I-infected samples. In examining the expression profiles, however, we noted several compelling changes. In particular, several alterations have occurred in the cell cycle/DNA repair pathways. We noted changes in the factors

controlling G2/M progression. Specifically, we have identified for the first time that mRNA expression of the Cdc25C tyrosine phosphatase was increased in HTLV-I-transformed cells. This protein phosphatase functions as a dose-dependent inducer of mitotic control (Fig. 3). Cdc25C is activated by hyperphosphorylation of the N-terminal do- main. Several kinases, including Chk1, phosphorylate Cdc25C at Ser216 (60). Ser216 is phosphorylated throughout interphase, but not in mitosis. Ser216-phosphorylated Cdc25C is recognized and bound by 14-3-3, which may sequester Cdc25C in the cytoplasm, preventing it from interacting with Cdc2. At the appropriate time in the cell cycle, hypo- or unphosphorylated Cdc25C dephosphorylates Cdc2, activating kinase activity. The fact that cyclin B and Cdc2 (CDK1) expres-

sion are also increased may further indicate a deregulation of G2/M cell cycle control. It will be of interest to examine the phosphorylation state of Cdc25C and Cdc2 in the HTLV-I-transformed cells. Fig. 2. Tax expression. A, shown is a Western blot analysis of the Tax protein in PBL1, As noted previously, several changes in the cytokine/cytokine re- PBL2, Bes, C81, Champ, Hut102, and ACH.WT. Cell extracts (50 ␮g) were separated on ceptor signal cascades are altered in HTLV-I-infected cells (for re- a4–20% Tris-glycine gel (Invitrogen), transferred to a nylon membrane, and assayed for Tax protein with an anti-Tax antibody (Tab172). B, RT-PCR analysis was performed on views see Refs. 9–11). We have extended these studies and have total RNA isolated from ACH.WT, Jurkat, C81, MT2, Hut102, Bes, Champ, Molt4, and shown the expression profiles of several cytokines and their receptors ␣ activated PBLs for the presence of Tax and IL-2R . PCR products were separated by including IL-15, IL-15R␣, IL-6, IL-6R, IL-7, and IL-7R, as well as electrophoresis on 2% agarose gels and bands quantitated using Fluorchem (Imgen Technologies). All values were normalized to GAPDH. C, a graph of the fold increase for T-cell signaling molecules. Importantly, kinases and phosphatases, the IL-2R␣ expression in HTLV-I-infected cells over that in activated PBLs (set at 1) is known to play a role in signaling cascades, are also altered in HTLV- shown. The RT-PCR values are shown in open bars. The GeneSpring normalized values are shown in hatched bars. I-infected cells. Further investigation is needed to determine which pathways result in cellular immortalization/transformation and which are a result of immortalization/transformation. in cells is reported to prevent p53 transcription-independent apoptosis The data presented in this study represents the most comprehen- and significantly attenuate overall cell death induced by wild-type p53 sive analysis of gene expression patterns in HTLV-I-transformed (59). Similarly, caspase-4 and -6 expression was down-regulated in cells to date. Of the 7300 genes analyzed in this study, the the HTLV-I cells. expression of ϳ763 genes was deregulated Ͼ2-fold in the HTLV- Repressed Genes. It has been shown that decreases, as well as I-transformed cells. Analysis of the deregulated genes, in terms of increases, in cellular gene expression are important regulatory events. known function, allows several important conclusions. First, there Previous studies (21–23) have identified a limited number of re- is no single regulatory pathway that is solely targeted for activation/ pressed genes. In our study, we have identified 420 genes which are inactivation in the stably transformed cell. Rather, a network of decreased 2-fold or greater in the HTLV-I cells are compared with interrelated pathways including cell proliferation, T-cell signaling, control PBLs (on-line data supplement). Interestingly, several protein and immune regulation are deregulated. Second, there appear to be kinases were down-regulated in expression as compared with acti- multiple alterations in the cell death/survival pathway that have vated PBLs (Table 1). Very striking was the down-regulation of occurred. Several proteins that increase the rate of apoptosis are several protein kinase C isoforms including, ␤1, ␤2, ␪, eta, ␨, and down-regulated in the HTLV-I-transformed cell. Moreover, sev- protein kinase C-like 2 kinase. Protein kinase C is a family of at least eral proteins that inhibit apoptosis were up-regulated. Clearly, the 3568

Fig. 3. The G2/M phase of the cell cycle. A, shown is a diagram of the genes involved in the G2/M phase of the cell cycle. The box by each gene indicates the expression level in HTLV-I- infected cells as compared with activated PBLs (nd indicates not determined). B, Tax transactivation of the Cdc25C promoter in transfected Jurkat cells. Jurkat T cells were transiently transfected by electroporation with 4 ␮gofthe Cdc25C promoter construct (C290-, C75-, CC51-, and C75M1-Luc) in the absence or presence of Tax (8 ␮g). Luciferase activity was measured on a Berthold Luminometer and normalized to protein concentration. C, the fold increase in luciferase activity is shown graphi- cally as fold activation by Tax over the vector control.

overall pattern is to disrupt the cell cycle regulatory points and expression changes within the population await further analysis. It favor survival of the cell. Whether the pattern of gene expression is also clear that the analysis of gene expression in the initial stages is the same in all cells within the transformed cell population or of infection and transformation would be of tremendous value in whether what we are seeing is a global pattern of individual gene defining key transformation pathways. 3569

REFERENCES 26. Morris, J. C., and Waldmann, T. A. Advances in interleukin 2 receptor targeted treatment. Ann. Rheum. Dis., 59 (Suppl. 1): i109–i114, 2000. 1. Gessain, A., Barin, F., Vernant, J. C., Gout, O., Maurs, L., Calender, A., and de The, 27. Nussenblatt, R. B., Fortin, E., Schiffman, R., Rizzo, L., Smith, J., Van Veldhuisen, P., G. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic Sran, P., Yaffe, A., Goldman, C. K., Waldmann, T. A., and Whitcup, S. M. Treatment paraparesis. Lancet, 2: 407–410, 1985. of noninfectious intermediate and posterior uveitis with the humanized anti-Tac mAb: 2. Osame, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M., a phase I/II clinical trial. Proc. Natl. Acad. Sci. USA, 96: 7462–7466, 1999. and Tara, M. HTLV-I associated myelopathy, a new clinical entity. Lancet, 1: 28. Phillips, K. E., Herring, B., Wilson, L. A., Rickford, M. S., Zhang, M., Goldman, 1031–1032, 1986. C. K., Tso, J. Y., and Waldmann, T. A. IL-2R␣-directed monoclonal antibodies 3. Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D., and Gallo, provide effective therapy in a murine model of adult T-cell leukemia by a R. C. Detection and isolation of type C retrovirus particles from fresh and cultured mechanism other than blockade of IL-2/IL-2R␣ interaction. Cancer Res., 60: lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. 6977– 6984, 2000. USA, 77: 7415–7419, 1980. 29. Ruben, S., Poteat, H., Tan, T. H., Kawakami, K., Roeder, R., Haseltine, W., and 4. Yoshida, M., Seiki, M., Yamaguchi, K., and Takatsuki, K. Monoclonal integration of Rosen, C. A. Cellular transcription factors and regulation of IL-2 receptor gene human T-cell leukemia provirus in all primary tumors of adult T-cell leukemia expression by HTLV-I tax gene product. Science (Wash. DC), 241: 89–92, 1988. suggests causative role of human T-cell leukemia virus in the disease. Proc. Natl. 30. Mariner, J. M., Lantz, V., Waldmann, T. A., and Azimi, N. Human T cell lympho- Acad. Sci. USA, 81: 2534–2537, 1984. tropic virus type I Tax activates IL-15R ␣ gene expression through an NF-␬ B site. 5. Ijichi, S., Matsuda, T., Maruyama, I., Izumihara, T., Kojima, K., Niimura, T., J. Immunol., 166: 2602–2609, 2001. Maruyama, Y., Sonoda, S., Yoshida, A., and Osame, M. Arthritis in a human T 31. Azimi, N., Brown, K., Bamford, R. N., Tagaya, Y., Siebenlist, U., and Waldmann, lymphotropic virus type I (HTLV-I) carrier. Ann. Rheum. Dis., 49: 718–721, 1990. T. A. Human T cell lymphotropic virus type I Tax protein trans-activates interleukin 6. LaGrenade, L., Hanchard, B., Fletcher, V., Cranston, B., and Blattner, W. Infective 15 gene transcription through an NF-␬B site. Proc. Natl. Acad. Sci. USA, 95: dermatitis of Jamaican children: a marker for HTLV-I infection [see comments]. 2452–2457, 1998. Lancet, 336: 1345–1347, 1990. 32. Azimi, N., Jacobson, S., Leist, T., and Waldmann, T. A. Involvement of IL-15 in the 7. Sasaki, K., Morooka, I., Inomata, H., Kashio, N., Akamine, T., and Osame, M. pathogenesis of human T lymphotropic virus type I-associated myelopathy/tropical Retinal vasculitis in human T-lymphotropic virus type I associated myelopathy. Br. J. spastic paraparesis: implications for therapy with a monoclonal antibody directed to Ophthalmol., 73: 812–815, 1989. the IL-2/15R ␤ receptor. J. Immunol., 163: 4064–4072, 1999. 8. Bex, F., and Gaynor, R. B. Regulation of gene expression by HTLV-I Tax protein. 33. Dittmer, J., Gegonne, A., Gitlin, S. D., Ghysdael, J., and Brady, J. N. Regulation of Methods, 16: 83–94, 1998. parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an 9. Franchini, G. Molecular mechanisms of human T-cell leukemia/lymphotropic virus inverted CACCC motif and regulates promoter activity in cooperation with Ets1. type I infection. Blood, 86: 3619–3639, 1995. J. Biol. Chem., 269: 21428–21434, 1994. 10. Hollsberg, P. Mechanisms of T-cell activation by human T-cell lymphotropic virus 34. Pise-Masison, C. A., Mahieux, R., Jiang, H., Ashcroft, M., Radonovich, M., Duvall, type I. Microbiol. Mol. Biol. Rev., 63: 308–333, 1999. J., Guillerm, C., and Brady, J. N. Inactivation of p53 by human T-cell lymphotropic 11. Kashanchi, F., Pise-Masison, C. A., and Brady, J. N. Human T cell leukemia virus. virus type 1 Tax requires activation of the NF-␬B pathway and is dependent on p53 In: R. Ahmed and I. Chen (eds.), Persistent Viral Infections, pp. 47–75. New York: phosphorylation. Mol. Cell Biol. 20: 3377–3386, 2000. John Wiley & Sons Ltd., 1999. 35. Pise-Masison, C. A., Radonovich, M., Sakaguchi, K., Appella, E., and Brady, J. N. 12. Bartoe, J. T., Albrecht, B., Collins, N. D., Robek, M. D., Ratner, L., Green, P. L., and Phosphorylation of p53: a novel pathway for p53 inactivation in human T-cell Lairmore, M. D. Functional role of pX open reading frame II of human T-lympho- lymphotropic virus type 1-transformed cells. J. Virol., 72: 6348–6355, 1998. tropic virus type 1 in maintenance of viral loads in vivo. J. Virol., 74: 1094–1100, 36. Sakaguchi, K., Saito, S., Higashimoto, Y., Roy, S., Anderson, C. W., and Appella, E. 2000. Damage-mediated phosphorylation of human p53 threonine 18 through a cascade 13. D’Agostino, D. M., Zotti, L., Ferro, T., Franchini, G., Chieco-Bianchi, L., and mediated by a casein 1-like kinase. Effect on Mdm2 binding. J. Biol. Chem., 275: Ciminale, V. The p13II protein of HTLV type 1: comparison with mitochondrial 9278–9283, 2000. proteins coded by other human viruses. AIDS Res. Hum. Retroviruses, 16: 1765– 37. Dumaz, N., Milne, D. M., and Meek, D. W. Protein kinase CK1 is a p53-threonine 18 1770, 2000. kinase which requires prior phosphorylation of serine 15. FEBS Lett., 463: 312–316, 14. D’Agostino, D. M., Zotti, L., Ferro, T., Cavallori, I., Silic-Benussi, M., Chieco- 1999. Bianchi, L., and Ciminale, V. Expression and functional properties of proteins encoded in the x-II ORF of HTLV-I. Virus Res., 78: 35–43, 2001. 38. Uchiumi, F., Semba, K., Yamanashi, Y., Fujisawa, J., Yoshida, M., Inoue, K., 15. Lairmore, M. D., Albrecht, B., D’Souza, C., Nisbet, J. W., Ding, W., Bartoe, J. T., Toyoshima, K., and Yamamoto, T. Characterization of the promoter region of the src Green, P. L., and Zhang, W. In vitro and in vivo functional analysis of human T cell family gene lyn and its trans activation by human T-cell leukemia virus type lymphotropic virus type 1 pX open reading frames I and II. AIDS Res. Hum. I-encoded p40tax. Mol. Cell. Biol., 12: 3784–3795, 1992. Retroviruses, 16: 1757–1764, 2000. 39. Guan, K. L., and Butch, E. Isolation and characterization of a novel dual specific 16. Nicot, C., Mulloy, J. C., Ferrari, M. G., Johnson, J. M., Fu, K., Fukumoto, R., phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated pro- Trovato, R., Fullen, J., Leonard, W. J., and Franchini, G. HTLV-1 p12(I) protein tein kinase. J. Biol. Chem., 270: 7197–7203, 1995. enhances STAT5 activation and decreases the interleukin-2 requirement for prolifer- 40. Ishibashi, T., Bottaro, D. P., Michieli, P., Kelley, C. A., and Aaronson, S. A. A novel ation of primary human peripheral blood mononuclear cells. Blood, 98: 823–829, dual specificity phosphatase induced by serum stimulation and heat shock. J. Biol. 2001. Chem., 269: 29897–29902, 1994. 17. Ding, W., Albrecht, B., Luo, R., Zhang, W., Stanley, J. R., Newbound, G. C., and 41. Yi, H., Morton, C. C., Weremowicz, S., McBride, O. W., and Kelly, K. Genomic Lairmore, M. D. Endoplasmic reticulum and cis-Golgi localization of human T- organization and chromosomal localization of the DUSP2 gene, encoding a MAP lymphotropic virus type 1 p12(I): association with calreticulin and calnexin. J. Virol., kinase phosphatase, to human 2p11.2-q11. Genomics, 28: 92–96, 1995. 75: 7672–7682, 2001. 42. Auger, K. R., and Cantley, L. C. Novel polyphosphoinositides in cell growth and 18. Johnson, J. M., Nicot, C., Fullen, J., Ciminale, V., Casareto, L., Mulloy, J. C., activation. Cancer Cells, 3: 263–270, 1991. Jacobson, S., and Franchini, G. Free major histocompatibility complex class I heavy 43. Berridge, M. J., and Irvine, R. F. Inositol phosphates and cell signalling. Nature chain is preferentially targeted for degradation by human T-cell leukemia/lympho- (Lond.), 341: 197–205, 1989. tropic virus type 1 p12(I) protein. J. Virol., 75: 6086–6094, 2001. 44. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., 19. Hou, X., Foley, S., Cueto, M., and Robinson, M. A. The human T-cell leukemia virus and Soltoff, S. Oncogenes and signal transduction. Cell, 64: 281–302, 1991. type I (HTLV-I) X region encoded protein p13(II) interacts with cellular proteins. 45. Li, S. R., Gyselman, V. G., Lalude, O., Dorudi, S., and Bustin, S. A. Transcription of Virology, 277: 127–135, 2000. the inositol polyphosphate 1-phosphatase gene (INPP1) is up-regulated in human 20. Zhang, W., Nisbet, J. W., Bartoe, J. T., Ding, W., and Lairmore, M. D. Human colorectal cancer. Mol. Carcinog., 27: 322–329, 2000. T-lymphotropic virus type 1 p30(II) functions as a transcription factor and differen- 46. Sharma, S., Mamane, Y., Grandvaux, N., Bartlett, J., Petropoulos, L., Lin, R., and tially modulates CREB-responsive promoters. J. Virol., 74: 11270–11277, 2000. Hiscott, J. Activation and regulation of interferon regulatory factor 4 in HTLV type 21. Harhaj, E. W., Good, L., Xiao, G., and Sun, S. C. Gene expression profiles in 1-infected T lymphocytes. AIDS Res. Hum. Retroviruses, 16: 1613–1622, 2000. HTLV-I-immortalized T cells: deregulated expression of genes involved in apoptosis 47. Korner, K., and Muller, R. In vivo structure of the cell cycle-regulated human cdc25C regulation. Oncogene, 18: 1341–1349, 1999. promoter. J. Biol. Chem., 275: 18676–18681, 2000. 22. de La Fuente, C., Deng, L., Santiago, F., Arce, L., Wang, L., and Kashanchi, F. Gene 48. Zwicker, J., Gross, C., Lucibello, F. C., Truss, M., Ehlert, F., Engeland, K., and expression array of HTLV type 1-infected T cells: up-regulation of transcription Muller, R. Cell cycle regulation of cdc25C transcription is mediated by the periodic factors and cell cycle genes. AIDS Res. Hum. Retroviruses, 16: 1695–1700, 2000. repression of the glutamine-rich activators NF-Y and Sp1. Nucleic Acids Res., 23: 23. Ng, P. W., Iha, H., Iwanaga, Y., Bittner, M., Chen, Y., Jiang, Y., Gooden, G., Trent, 3822–3830, 1995. J. M., Meltzer, P., Jeang, K. T., and Zeichner, S. L. Genome-wide expression changes 49. de La Fuente, C., Santiago, F., Chong, S. Y., Deng, L., Mayhood, T., Fu, P., Stein, induced by HTLV-1 Tax: evidence for MLK-3 mixed lineage kinase involvement in D., Denny, T., Coffman, F., Azimi, N., Mahieux, R., and Kashanchi, F. Overexpres- Tax-mediated NF-␬B activation. Oncogene, 20: 4484–4496, 2001. sion of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its 24. Pise-Masison, C. A., Dittmer, J., Clemens, K. E., and Brady, J. N. Physical and association with cyclin A/cdk2. J. Virol., 74: 7270–7283, 2000. functional interaction between the human T-cell lymphotropic virus type 1 Tax1 50. Iwanaga, R., Ohtani, K., Hayashi, T., and Nakamura, M. Molecular mechanism of cell protein and the CCAAT binding protein NF-Y. Mol. Cell. Biol., 17: 1236–1243, cycle progression induced by the oncogene product Tax of human T-cell leukemia 1997. virus type I. Oncogene, 20: 2055–2067, 2001. 25. Ruckes, T., Saul, D., Van Snick, J., Hermine, O., and Grassmann, R. Autocrine 51. Masutani, H., Hirota, K., Sasada, T., Ueda-Taniguchi, Y., Taniguchi, Y., Sono, H., antiapoptotic stimulation of cultured adult T-cell leukemia cells by overexpression of and Yodoi, J. Transactivation of an inducible anti-oxidative stress protein, human the chemokine I-309. Blood, 98: 1150–1159, 2001. thioredoxin by HTLV-I Tax. Immunol. Lett., 54: 67–71, 1996. 3570

52. Ressler, S., Morris, G. F., and Marriott, S. J. Human T-cell leukemia virus type 1 Tax 57. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. Involvement of transactivates the human proliferating cell nuclear antigen promoter. J. Virol., 71: MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF 1181–1190, 1997. receptor-induced cell death. Cell, 85: 803–815, 1996. 53. Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. X-linked IAP is a 58. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, direct inhibitor of cell-death proteases. Nature (Lond.), 388: 300–304, 1997. J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, 54. Roy, N., Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. The c-IAP-1 M. E., and Dixit, V. M. FLICE, a novel FADD-homologous ICE/CED-3-like prote- and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J., 16: 6914– ase, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell, 6925, 1997. 85: 817–827, 1996. 55. Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. The 59. Ding, H. F., Lin, Y. L., McGill, G., Juo, P., Zhu, H., Blenis, J., Yuan, J., and Fisher, TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral D. E. Essential role for caspase-8 in transcription-independent apoptosis triggered by inhibitor of apoptosis proteins. Cell, 83: 1243–1252, 1995. p53. J. Biol. Chem., 275: 38905–38911, 2000. 56. Uren, A. G., Pakusch, M., Hawkins, C. J., Puls, K. L., and Vaux, D. L. Cloning and 60. Peng, C. Y., Graves, P. R., Thoma, R. S., Wu, Z., Shaw, A. S., and Piwnica-Worms, expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis H. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by and/or bind tumor necrosis factor receptor-associated factors. Proc. Natl. Acad. Sci. phosphorylation of Cdc25C on serine-216. Science (Wash. DC), 277: 1501–1505, USA, 93: 4974–4978, 1996. 1997.

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Cynthia A. Pise-Masison, Michael Radonovich, Renaud Mahieux, et al.

Cancer Res 2002;62:3562-3571.

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