Overexpression of p101 activates PI3Kgamma signaling in T cells and contributes to cell survival.

p101, the regulatory subunit of phosphatidylinositol-3-kinase-gamma (PI3Kgamma), was recently reported as a common site of retroviral insertion in T-cell lymphomas induced in mice by MoFe2-MuLV, a unique recombinant gammaretrovirus. The common interruption of p101 by retroviral integration suggests that the locus encodes an oncogene whose altered expression is related to the induction of T-cell malignancy. To examine a possible role in the malignant process, p101 was overexpressed in human T-cell lines Molt-4 and Jurkat. Transient overexpression of p101 induced apoptosis in recipient cells; however, stable expression could be established in cells that expressed moderate levels of p101. Constitutive p101 overexpression in those cells conferred significant protection against ultraviolet-induced apoptosis. Protection against apoptotic induction was attributed to p101-mediated activation of the Akt pathway. Constitutive overexpression of p101 enhanced the activity of p110gamma and further sensitized it to activation upon stimulation of G protein-coupled receptor. These findings are the first to implicate altered expression of p101 in malignancy, specifically in T-cell lymphoma. The findings further provide insight into the regulation of p110gamma, indicating that the stoichiometry of p110gamma and p101 are important in regulating PI3Kgamma signaling.

Accelerated G(1) phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein.

Tax, the human T cell leukemia virus type I oncoprotein, plays a crucial role in viral transformation and the development of the virally associated disease adult T cell leukemia. Because oncogenesis involves alterations in cell growth, it is important to examine the effects of Tax on cell cycle progression. Using a synchronized cell system, we have found that Tax expression accelerates G(1) phase progression and S phase entry with concomitant DNA replication. This accelerated progression is accompanied by an earlier onset of cdk2 kinase activity. In contrast to the shortening of G(1) phase, the length of S phase is unaffected by Tax expression. As a result of a more rapid cell cycle progression, cells expressing Tax exhibit faster growth kinetics and display an altered cell cycle distribution. Additionally, the decreased time allowed for growth in the presence of Tax results in a decreased cell size. Tax-associated acceleration of cell cycle progression may play a role in the ability of this viral oncoprotein to mediate cellular transformation and promote the development of human T cell leukemia virus type I-associated diseases.

Identification of an initiator-like element within the HTLV-I promoter.

Using the 5' long terminal repeat (LTR) as its only promoter, the HTLV-1 provirus generates a single RNA transcript that undergoes differential splicing to express the various viral proteins. Examination of sequence near the transcription start site revealed an element resembling a transcriptional initiator (Inr) at position -8 to -15 in addition to the canonical TATA box at -25. To elucidate basal control of HTLV-I gene expression, functional traits of this element were examined. It specifically bound a protein complex, the mobility of which was altered by antibody to serum response factor, and independently mediated reporter gene expression. Mutating the Inr in a minimal construct reduced basal transcription, whereas mutation of the element within the context of the complete LTR left basal transcription unaffected. Presence of the element influenced transcription start site choices. Exhibiting many characteristics of an Inr, this element may play an important role in regulating HTLV-I gene expression in vivo, particularly during the long clinical latency period prior to development of HTLV-I-induced disease.

Transcriptional activity of HTLV-I Tax influences the expression of marker genes associated with cellular transformation.

Human T cell leukemia virus type I (HTLV-I) has been identified as the etiologic agent of adult T cell leukemia (ATL). HTLV-I encodes a transcriptional regulatory protein, Tax, which also functions as the viral transforming protein. Through interactions with a number of cellular transcription factors Tax can modulate cellular gene expression. Since the majority of Tax-responsive cellular genes are important regulators of cellular proliferation, the transactivating functions of Tax appear to be necessary for cellular transformation by HTLV-I. Gaining a complete understanding of the broad range of genes regulated by Tax, the temporal pattern of their expression, and their effects on cell function may identify early markers of disease progression mediated by this virus.

p53-independent induction of apoptosis by the HTLV-I tax protein following UV irradiation.

Human T cell leukemia virus type 1 (HTLV-1) encodes a transforming protein, Tax. Tax is a promiscuous viral transactivator involved in both cell growth and death control. We have previously shown that Tax sensitizes cells to apoptosis induced by DNA-damaging agents and this report further characterizes the Tax-mediated apoptosis pathway. We found that Tax-mediated apoptosis in response to UV irradiation was inhibited by Bcl-2 and Bcl-X(L) overexpression and by treatment with the caspase inhibitor z-VAd-FMK. Since Tax has been shown to functionally inactivate the apoptosis regulator p53, the effect of Tax on apoptosis in the absence of p53 was examined. In these studies, Tax sensitized p53-negative cells to apoptose, suggesting that Tax can mediate a p53-independent form of apoptosis. In addition, cells expressing both Tax and p53 displayed higher levels of apoptosis than cells expressing either protein alone, suggesting that the apoptosis-inducing activities of Tax and p53 are not completely overlapping. These observations demonstrate that Tax can utilize a p53-independent mechanism to induce apoptotic cell death following UV irradiation.

Suppression of DNA repair by HTLV type 1 Tax correlates with Tax trans-activation of proliferating cell nuclear antigen gene expression.

The human T cell leukemia virus type 1 (HTLV-1) viral oncoprotein Tax acts as a transcriptional trans-activator affecting viral as well as cellular gene expression. To understand how Tax induces transformation, the consequences of its ability to alter expression of cellular genes must be examined. We have previously demonstrated that Tax activates expression of the cellular gene, proliferating cell nuclear antigen (PCNA), and that Tax suppresses DNA repair. In this study we tested the ability of previously described Tax mutants to activate PCNA gene expression and their ability to interfere with DNA repair. The results revealed a strong correlation between Tax trans-activation of PCNA gene expression and its ability to inhibit DNA repair via the nucleotide excision repair (NER) pathway. Thus, a consequence of activated PCNA gene expression appears to be reduced DNA repair capacity. These effects of Tax are likely to play important roles in its transforming activity.

Suppression of DNA repair by human T cell leukemia virus type 1 Tax is rescued by a functional p53 signaling pathway.

The Tax protein of human T cell leukemia virus type 1 is a viral transactivator and transforming protein. Tax is known to suppress cellular nucleotide excision repair (NER), and this activity has been proposed to play an important role in Tax transformation. In this study we have investigated the mechanism by which Tax suppresses NER with specific focus on the previously characterized ability of Tax to inhibit p53 function. Suppression of NER by Tax was rescued by overexpression of wild-type p53; however, a p53 transactivation-incompetent mutant did not restore NER activity. The cyclin-dependent kinase inhibitor p21, a major transcriptional target of p53, plays an important role in regulating DNA replication and repair. Overexpression of p21 reversed Tax-induced suppression of NER; however, a p21 C-terminal mutant that lacks the proliferating cell nuclear antigen binding domain did not restore NER activity. Thus, p53 and its downstream effector p21 can inhibit Tax-mediated suppression of DNA repair. These results imply that the inactivation of p53 function by Tax contributes to Tax suppression of DNA repair.

Sequences flanking the cAMP responsive core of the HTLV-I tax response elements influence CREB protease sensitivity.

The human T cell leukemia virus type I (HTLV-I) Tax protein activates transcription from the viral long terminal repeat and select cellular promoters by interacting with cellular DNA-binding proteins. The HTLV-I promoter contains three copies of a Tax-responsive element (TRE-1), each of which possesses a core cAMP response element (CRE). The cAMP response element-binding protein, CREB, binds TRE-1 and mediates Tax association with, and transactivation of, the viral promoter. These activities depend on DNA sequences that flank the core CRE. Although CREs are found in a variety of cellular promoters, cellular CREs vary in sequence from TRE-1, especially in the flanking regions, and are generally not Tax responsive. The molecular basis for differential Tax responsiveness of viral and cellular CREs has not been determined. Here we demonstrate that the conformation of CREB is influenced by the nucleotide sequence of its DNA-binding element. CREB showed altered sensitivity to V8, chymotrypsin, and trypsin proteases when bound to the HTLV-I TRE-1 element as compared to the rat somatostatin CRE element. The phosphorylation state of CREB did not influence its protease sensitivity on either element. Sequences flanking the core CRE-binding site in each element were found to specify protease sensitivity. Since the TRE-1-flanking sequences also modulate Tax association with CREB, and Tax transactivation of CREB-dependent LTR transcription, these results suggest that CREB conformation may determine the ability of Tax to bind CREB.

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

Extracellular human T cell leukemia virus type I tax protein stimulates the proliferation of human synovial cells.

OBJECTIVE: The present study was designed to investigate whether the proliferation of normal synovial cells from patients with meniscus injury is stimulated by human T cell leukemia virus type I (HTLV-I) Tax protein. METHODS: The effect of Tax protein on the proliferation of synovial cells was evaluated using a 3H-thymidine incorporation assay. Production of cytokines was determined by enzyme-linked immunosorbent assay. Nuclear factor kappaB (NF-kappaB) DNA binding activity and the transcription of several NF-kappaB-mediated genes was detected by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction. RESULTS: The proliferation of synovial cells, as well as their expression of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-6, was significantly enhanced by extracellular Tax at concentrations of 2.5 pM to 25 nM. In contrast, extracellular bacterial extract did not change the cytokine expression or the proliferation of these cells. Proliferation of synovial cells induced by Tax protein may be due to activated expression of several cytokines and protooncogenes that contain NF-kappaB regulatory sequences. CONCLUSION: Our results suggest that extracellular Tax can regulate the expression of endogenous cellular genes in synovial cells and may contribute to the NF-kappaB-mediated synovial hyperplasia.

Relationship between anti-Tax antibody responses and cocultivatable virus in HTLV-I-infected rabbits.

The presence of anti-Tax antibody responses in human T cell leukemia virus type I (HTLV-I)-infected individuals has been correlated with increased proviral load, increased risk of transmitting infection, and increased risk of developing tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). In this study, a rabbit model of HTLV-I infection was used to determine whether anti-Tax antibody responses could predict the presence of virus with the potential to replicate. Seven of 14 HTLV-I-infected rabbits developed anti-Tax antibody responses. The onset of Tax reactivity was variable, but once detected remained constant throughout the remainder of the 60-week course of the study. All anti-Tax antibody positive rabbits produced virus as measured by p19 expression upon coculture, while p19 was detected in only one of the Tax antibody negative animals. Thus the presence of an anti-Tax antibody response correlates with p19 expression following cocultivation, and may be a useful predictor of virus replication in HTLV-I infected individuals.

Augmentation of human T cell leukaemia virus type I Tax transactivation by octamer binding sites.

The human T cell leukaemia virus type I (HTLV-1) Tax protein is an activator of viral and cellular gene expression. Tax does not bind DNA directly, but does interact with cellular DNA binding proteins. These interactions bring Tax to a specific group of promoters and may help to determine the specificity of Tax transactivation. Previous studies have demonstrated that the activity of Tax, when tethered to a given promoter, is enhanced by the presence of adjacent transcription factor binding sites. To examine the specificity of this augmentation, a series of transcription factor binding sites was tested for the ability to enhance the activity of a Gal-Tax fusion protein. The greatest increase in Gal-Tax activity was observed when an octamer binding site was placed adjacent to the Gal4 binding sites. However, the octamer binding site failed to independently function as a Tax responsive element in the absence of an adjacent Tax-tethering element. Oct-2 was not required for augmentation of Gal-Tax activity as this enhancement was observed in BHK-21 cells, which lack Oct-2. The ability of octamer binding sites to augment transcription was specific for Gal-Tax, as compared to other transactivators. Taken together, these results demonstrate that the degree of Tax transactivation can be influenced by the elemental composition of the target promoter.

Human T-cell leukemia virus type 1 Tax transactivates the human proliferating cell nuclear antigen promoter.

The human T-cell leukemia virus type 1 (HTLV-1) transforming protein, Tax, is a potent transactivator of both viral and cellular gene expression. The ability of Tax to transform cells is believed to depend on its transactivation of cellular-growth-regulatory genes. Expression of proliferating cell nuclear antigen (PCNA) is intimately linked to cell growth and DNA replication and repair. By testing a series of PCNA promoter deletion constructs, we have demonstrated that the PCNA promoter can be transactivated by Tax. The smallest construct that was activated did not include the ATF/CRE binding site at nucleotide -50, and mutations in the ATF/CRE element in the context of a larger promoter were still activated by Tax. In addition, a Tax mutant that is defective for activation of the CRE pathway retained the ability to activate the -397 promoter construct. When a series of linker scanner mutations that span the region from nucleotide -45 to -7 were assayed, mutations in and around a repeat sequence were found to abolish Tax transactivation. Multimerized copies of either half of the repeat were Tax responsive. A single protein complex was shown to bind specifically to the Tax-responsive region, and the binding of this complex was enhanced in the presence of Tax. These results demonstrate that the PCNA promoter contains a Tax-responsive element located between nucleotides -45 and -7 whose sequence is different from those of other, previously identified Tax-responsive elements. The ability of Tax to activate the PCNA promoter may play an important role in cellular transformation by HTLV-1.

Function of the human T-cell leukemia virus type 1 21-base-pair repeats in basal transcription.

The human T-cell leukemia virus type 1 (HTLV-1) promoter contains three copies of an imperfect 21-bp repeat called Tax-responsive element (TRE1). To examine the role of individual TRE1 sequences in basal transcription of the HTLV-1 promoter, site-directed mutations were generated in all possible combinations of one, two, or all three TRE1 elements in the viral long terminal repeat (LTR) and tested in vivo for transcriptional activity. Mutation of the middle TRE1 resulted in the greatest reduction in basal activity. Electrophoretic mobility shift analysis demonstrated that the protein complexes bound to each of the three TRE1 sequences were not identical. The complexes formed with the TATA-distal and middle TRE1s were dependent on the core cyclic AMP response element (CRE) found in all three TRE1s, while the cellular transcription factor Sp1 bound the TATA-proximal TRE1 in a CRE-independent manner. Sp1 binding produced a footprint on the viral LTR which covered the 5' region of the proximal TRE1. Mixing experiments demonstrated that the bindings of CREB and Sp1 to the proximal TRE1 were mutually exclusive. Sp1 was able to activate transcription both from the complete LTR and from the proximal TRE1 alone. These studies demonstrate that the TRE1 elements in the HTLV-1 LTR are functionally nonequivalent and suggest that Sp1 can influence HTLV-1 basal transcription.

Activation of the HTLV-I long terminal repeat by the hepatitis B virus X protein.

The human T-cell leukemia virus type I (HTLV-I) Tax protein and the hepatitis B virus (HBV) X protein have each been shown to activate transcription of their respective viral promoters as well as a subset of cellular gene promoters. Here we show that the HTLV-I long terminal repeat (LTR) is responsive to HBV X transactivation. Maximum levels of X-mediated transactivation of the LTR were 8-fold. An X-responsive-region (XRR) of the LTR is located between nucleotides -355 and -276 and contains an AP-2 binding site, a previously recognized X-responsive element. We demonstrated that Tax and X synergize to activate transcription from the HTLV-I LTR, although the AP-2 binding site was not required for this synergy. These results raise the possibility that the HBV X protein may affect the level of HTLV-I gene expression in co-infected individuals.

Cellular transformation by human T-cell leukemia virus type I.

Human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that was isolated in 1980 from a patient with adult T-cell leukemia. From the numerous experiments using infected patient T-cells, transgenic mice and tissue culture transformation assays, the Tax protein has been determined to be the transforming component of HTLV-I. Tax-mediated transformation is linked to its ability to transcriptionally regulate the expression of cellular genes involved in growth and proliferation. Ultimately, unregulated and continued activation of these important growth modulating genes by Tax leads to transformation.

Control of cAMP-regulated enhancers by the viral transactivator Tax through CREB and the co-activator CBP.

The Tax protein of human T-lymphotropic virus (HTLV)-1 activates expression of the HTLV-1 long terminal repeat through a DNA element that resembles the cellular cyclic AMP-regulated enhancer (CRE). Tax contains a transcriptional activation domain, but its ability to activate gene expression depends on interactions with cellular CRE-binding proteins such as CREB. Whether Tax can activate the expression of cellular CRE-containing genes has been controversial. Here we show that Tax can activate both the HTLV-1 and consensus cellular CREs, and propose that this activation may occur through mechanisms that are differentially dependent on CREB phosphorylation. Tax not only increases the binding of CREB to the viral CRE but also recruits the transcriptional co-activator CBP in a manner independent of CREB phosphorylation. In contrast, association of Tax with the cellular CRE occurs through CBP which, in turn, is recruited only in the presence of phosphorylated CREB.

Twenty-one base pair repeat elements influence the ability of a Gal4-Tax fusion protein to transactivate the HTLV-I long terminal repeat.

The Tax1 protein of the human T-cell leukemia virus (HTLV-I) is a 40-kDa positive transactivator of viral gene expression. Tax1 does not bind directly to DNA, but associates indirectly with DNA via cellular transcription factors. To further investigate the activation of HTLV-I transcription by Tax1, a chimeric protein containing Tax1 fused to the DNA binding domain of Gal4 was created (Gal4-Tax). HTLV-I long terminal repeat (LTR) reporter plasmids were constructed in which specific Tax1 responsive elements were replaced with Gal4 binding sites. Cotransfection of Gal4-Tax or Tax1 with HTLV-I LTR reporter constructs containing Gal4 binding sites demonstrated that Gal4 sequences were necessary but not sufficient for maximal activation of the promoter by Gal4-Tax. Sequences surrounding the Gal4 binding sites were important in determining the level of Gal4-Tax activation. Association of Gal4-Tax with promoters which contained six Gal4 binding sites, but which lacked flanking LTR sequences, were weakly transactivated by Gal4-Tax (sevenfold). In contrast, LTR-CAT reporter constructs containing three Gal4 binding sites flanked by two 21 base pair repeat elements demonstrated a ninefold greater response to Gal4-Tax. These results suggest that cellular transcription factors, which bind the 21 base pair repeat elements, influence the ability of Tax1 to function as a transactivator. Furthermore, this effect is not fully explained by the ability of these factors to physically direct Tax1 to the LTR.

Activation of interleukin-2 receptor alpha expression by extracellular HTLV-I Tax1 protein: a potential role in HTLV-I pathogenesis.

The Tax1 protein of human T cell leukemia/lymphoma virus type I (HTLV-I) has been shown to stimulate the proliferation of human lymphocytes. Here we report that lymphocyte proliferation can be induced at extracellular Tax1 concentrations as low as 25 pM. The proliferative response induced by extracellular Tax1 is accompanied by an activation of endogenous interleukin-2 receptor alpha-chain (IL-2R alpha) expression in human lymphocytes. Functional activation of IL-2R alpha expression in peripheral blood lymphocytes treated with Tax1 was demonstrated using an [125I]IL-2-binding assay. In addition, an enzyme-linked immunosorbent assay demonstrated that soluble IL-2R alpha in the medium of IL-2- and Tax1-treated cells was over 13-fold greater than in the medium of control treated cells. Overexpression of IL-2R alpha is a common clinical feature of some patients with adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myopathy (TSP/HAM). The ability of extracellular Tax1 protein to activate expression of IL-2R alpha in both infected and uninfected lymphocytes may contribute to the abnormal lymphocyte proliferation observed in both ATL and TSP/HAM.

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.