A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1.

Angiogenesis inhibitors have shown clinical benefits in patients with advanced cancer, but further therapeutic improvement is needed. We have previously shown that the zinc finger protein 36, C3H type-like 1 (ZFP36L1) enhances vascular endothelial growth factor (VEGF) mRNA decay through its interaction with AU-rich elements within VEGF 3'-untranslated region. In this study, we evaluated the possibility to develop an antiangiogenic and antitumoral strategy using the mRNA-destabilizing activity of ZFP36L1. We engineered a cell-penetrating ZFP36L1, by fusing it to the protein transduction domains (PTDs) TAT derived from HIV, or the polyarginine peptides R7 or R9. PTD-ZFP36L1 fusion proteins were expressed in bacterial cells and affinity-purified to homogeneity. TAT-, R7- and R9-ZFP36L1 were efficiently internalized into living cells and decreased both endogenous VEGF mRNA half-life and VEGF protein levels in vitro. Importantly, a single injection of R9-TIS11b fusion protein into a high-VEGF expressing tissue in vivo (in this study, the mouse adrenal gland) markedly decreased VEGF expression. We further evaluated the effect of R9-ZFP36L1 on tumor growth using Lewis Lung Carcinoma (LL/2) cells implanted subcutaneously into nude mice. Intratumoral injection of R9-ZFP36L1 significantly reduced tumor growth and markedly decreased the expression of multiple angiogenic and inflammatory cytokines, including VEGF, acidic fibroblast growth factor, tumor necrosis factor α, interleukin (IL)-1α and IL-6, with a concomitant obliteration of tumor vascularization. These findings indicate that R9-ZFP36L1 fusion protein may represent a novel antiangiogenic and antitumoral agent, and supports the emerging idea that modulation of mRNA stability represents a promising therapeutic approach to treat cancer.

Inhibition of the hTERT promoter by the proto-oncogenic protein TAL1.

Telomerase activity, which has fundamental roles in development and carcinogenesis, strongly depends on the expression of human telomerase reverse transcriptase (hTERT), its catalytic subunit. In this report, we show that the basic helix-loop-helix factor, TAL1 (T-cell acute lymphoblastic leukemia 1), is a negative regulator of the hTERT promoter. Indeed, TAL1 overexpression leads to a decrease in hTERT mRNA abundance and hence to reduced telomerase activity. Conversely, suppression of TAL1 by RNA interference in Jurkat cells increases hTERT expression. Analysis by chromatin immunoprecipitation assays showed that TAL1 binds to the hTERT proximal promoter and recruits HDAC1. Considering the relationship recently established between TAL1 and the human T-cell leukemia virus type 1 (HTLV-1) Tax protein, which was confirmed in T lymphocyte clones derived from adult T-cell leukemia patients, we analyzed the effect of TAL1 with respect to the earlier characterized effects of Tax and HBZ (HTLV-1 basic leucine zipper) on hTERT expression. TAL1 was observed to reinforce the negative effect of Tax, whereas hTERT transactivation by the HBZ-JunD complex was repressed by TAL1 overexpression. Moreover, HBZ was found to induce proteasome-mediated degradation of TAL1. These observations support a model in which Tax and TAL1 by repressing hTERT would initially favor genomic instability, whereas expression of factors such as HBZ allows at a later stage an increase in hTERT production and consequently in telomerase activity.

Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle.

The mouse int6 gene is a frequent integration site of the mouse mammary tumor virus and INT6 silencing by RNA interference in HeLa cells causes an increased number of cells in the G2/M phases of the cell cycle, along with mitotic defects. In this report, we investigated the functional significance of the interaction between INT6 and MCM7, which was observed in a two-hybrid screen performed with INT6 as bait. It was found that proteasome inhibition strengthens interaction between both proteins and that INT6 stabilizes MCM7. Removal of MCM7 from chromatin as replication proceeds was accelerated in INT6-silenced cells and reduced amounts of protein were transiently observed, followed by a correction resulting from stimulation of mcm7 gene expression. Synchronized cells depleted for either INT6 or MCM7 display a reduction in thymidine incorporation and a reinforced association of RPA and claspin with chromatin. These data show that INT6 stabilizes chromatin-bound MCM7 and that alteration of this effect is associated with replication deficiency.

The human protein HSPC021 interacts with Int-6 and is associated with eukaryotic translation initiation factor 3.

The Int-6 protein has been shown to be a subunit of eukaryotic translation initiation factor 3 (eIF3) and to play a role in the control of cell growth. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Exposure of Jurkat cells to the phosphatase inhibitor H(2)O(2) triggers a marked phosphorylation on tyrosine of HSPC021. Several experiments were performed to evaluate whether this protein is associated with eIF3. It was observed that HSPC021 coelutes with Int-6 and eIF3 in gel filtration, coimmunoprecipitates with eIF3, and is incorporated into eIF3 both in rabbit reticulocyte lysates and in COS7 cells. A direct protein-protein interaction occurs between HSPC021 and Int-6, but the analysis of different mutants of HSPC021 indicated that a larger region of the protein is necessary for incorporation into eIF3 as compared with binding to Int-6. Taken together, our results establish that HSPC021 is tightly associated with the mammalian translation initiation factor eIF3. Analysis of the primary sequence of HSPC021 from different species revealed the presence of a tetratricopeptide repeat, a proteasome-COP9 (constitutive photomorphogenesis 9) signalosome-initiation factor 3 domain along with a Pumilio FBF repeat. These protein motifs are also present in subunits of eIF3, of the lid of the 26 S proteasome, and of the COP9 signalosome.

The PDZ protein TIP-1 interacts with the Rho effector rhotekin and is involved in Rho signaling to the serum response element.

The human T-cell lymphotrophic virus, type 1 Tax protein can interact via its C terminus with various proteins including a PDZ domain. In this work, one of them, TIP-1, is characterized as a cytoplasmic 14-kDa protein mainly corresponding to one PDZ domain. A two-hybrid screen performed with TIP-1 as bait showed that it interacts with the human homologue of rhotekin that was previously identified in mice as a Rho effector. Both human and mouse rhotekins exhibit at their C termini the sequence QSPV-COOH that matches the X(S/T)XV-COOH consensus known for proteins recognizing PDZ domains. Mutation of the serine and valine residues to alanine impairs interaction of rhotekin with TIP-1. Transient expression experiments with a reporter construct including the c-Fos serum response element (SRE) showed that coexpression of TIP-1 with the constitutively active RhoA.V14 mutant and human rhotekin caused a strong activation of the SRE. A negative mutant of Rho, RhoA.N19, was unable to cooperate with TIP-1 and rhotekin. The positive effect of TIP-1 was also lost when the C terminus of rhotekin was mutated. These data show that the complex of active Rho with its effector rhotekin bound to TIP-1 produces in the cytoplasm a signal that triggers strong activation of the SRE.

Identification of functional PDZ domain binding sites in several human proteins.

TIP-15 was previously identified as a cellular protein that can bind to the C-terminal end of the HTLV-1 Tax protein via its two PDZ domains. The sequence of the N-terminal part of TIP-15 is identical to that of the synaptic protein PSD-95. Both proteins are likely to be produced from the same gene by alternative splicing. Whereas expression of the PSD-95 mRNA was detected only with brain RNAs, that of TIP-15 was detected with RNAs from thymus, brain, skeletal muscle and Jurkat cells. The TIP-15 protein exhibits an apparent molecular weight of 40 kD and is weakly expressed in T cell lines. A two-hybrid screen performed with TIP-15 as bait revealed the presence of a PDZ binding site (PDZ-BS) in the following proteins: Lysyl tRNA synthetase, 6-phosphogluconolactonase (6-GPL), Stress-activated protein kinase 3 (SAPK3), NET-1, Diacylglycerol kinase zeta, MTMR1, MCM7, and hSec8. The sequence at the C-terminal ends of these proteins matches the X-S/T-X-V-COOH consensus previously defined for PDZ-BSs, with the exception of 6-GPL and SAPK3 which include a leucine as the C-terminal residue. For Lysyl tRNA synthetase, NET1, MTMR1 and hSec8, binding to TIP-15 was confirmed by co-immunoprecipitation experiments performed with the extracts of transfected COS7 cells. These results show the existence of functional PDZ-BSs in these proteins, but future studies will be necessary to establish whether or not TIP-15 represents a physiological partner. The significance of the presence of a PDZ-BS in these various proteins is discussed with respect to their function.

Interaction between the Ret finger protein and the Int-6 gene product and co-localisation into nuclear bodies.

The mouse int-6 gene was identified in mammary tumors as an integration site for the mouse mammary tumor virus. Its human counterpart encodes a product that interacts with the Tax viral oncoprotein of the human T cell leukaemia virus type 1. This interaction impedes the localisation of over-expressed Int-6 in nuclear bodies containing the promyelocytic leukaemia gene product (PML). In this study, Int-6 is characterised as a 52 kDa protein that is localised within nuclear bodies in primary lymphocytes. Screening of a human B cell cDNA library for proteins that interact with Int-6 led to isolation of four clones coding for the p110 subunit of eIF3, in accordance with previous detection of Int-6 in purified forms of this translation initiation factor. Another clone was interesting with respect to the subcellular localisation of Int-6. It encodes the Ret finger protein (Rfp) which interacts with PML and localises within a subset of PML nuclear bodies. The interaction of Rfp with Int-6 is mediated through a region in Rfp designated 'Rfp domain', distinct from that involved in the interaction with PML. Int-6 and Rfp are co-localised in certain PML nuclear bodies in lymphocytes and transfection studies in HeLa cells strongly suggest that Rfp triggers translocation of Int-6 to nuclear bodies.

Regulation of cdc2 gene expression by the upstream stimulatory factors (USFs).

cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been reported to play a central role, they cannot account for all aspects of the fine modulation of cdc2 gene expression during cell cycle and embryonic development. To characterize the transcription factors that control cdc2 gene expression during nerve cell differentiation in avians, we have previously cloned the quail cdc2 gene promoter region. We had identified an octamer (CAGGTGGC) containing an E-box, which has important activity in regulating cdc2 transcription. Using in vivo genomic footprinting experiments, we show here that this motif, currently named IG, is the target of binding proteins at different stages of neuroretina development, confirming its importance as a regulatory response element for cdc2 gene expression. A subset of Helix-Loop-Helix family of transcription factors, known as Upstream Stimulatory Factors (USFs) specifically bind to this sequence as dimers. Moreover, our results indicate that USFs transactivate the promoter of cdc2 via the IG motif. These data may help to better understand the mechanisms that control cell division in differentiating nerve cells.

The C-terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular proteins.

Infection by HTLV-1 has been correlated with the appearance of various proliferative or degenerative diseases. Some of these disorders have been observed in transgenic mice expressing the Tax protein, which is known to transactivate various viral and cellular promoters through interactions with several transcription factors. In this study we show that the C-terminus of this viral oncoprotein represents a motif permitting binding of Tax to the PDZ domains of several cellular proteins. A two-hybrid screen with Tax as bait indeed yielded complementary DNAs coding for six proteins including PDZ domains. Two of them correspond to truncated forms of the PSD-95 and beta1-syntrophin proteins, another clone codes for a protein homologous to the product of the C. elegans gene lin-7. The other three clones code for new human members of the PDZ family of cellular proteins. The interaction of Tax with the products of these clones was confirmed by immunoprecipitation assays in mammalian cells, and analysis of various mutants of Tax established the importance of the C-terminal amino acids for several of these interactions. These data suggest that Tax could perturb the normal function of targeted cellular proteins by strongly interacting with their PDZ domains.

Biochemical and functional interaction of the human immunodeficiency virus type 1 Tat transactivator with the general transcription factor TFIIB.

Tat strongly stimulates transcription of the human immunodeficiency type 1 (HIV-1) provirus by interacting with various cellular transcription factors, including TFIID. The results presented in this report indicate that the effect exerted by Tat also involves an interaction with TFIIB. A direct protein-protein interaction between Tat and TFIIB was observed in vitro. Detailed analysis of this interaction showed that the cysteine-rich and core domains of Tat bind to the N-terminal moiety of the general transcription factor. The role of the interaction between Tat and TFIIB in the activation of the entire HIV-1 promoter was analysed. Transfection experiments performed using a reporter construct containing the HIV-1 long terminal repeat fused to a reporter gene showed that overexpression of TFIIB progressively suppressed Tat-induced transcription. This effect was weakened by an increase in the intracellular concentration of Tat. A similar consequence of TFIIB overexpression was observed in a HeLa cell line stably transformed with a construct corresponding to the lacZ gene under the control of the HIV-1 promoter. Mutants of TFIIB which differed in their ability to interact with Tat and to function in basal transcription were analysed. The ability of TFIIB mutants defective for basal transcription to inhibit Tat-induced activity of the HIV-1 promoter depended on their capacity to interact with Tat. Mutants of TFIIB functional for basal transcription, but defective for the interaction with Tat, exhibited a dominant negative effect. From these data we propose a model in which interaction between Tat and both general transcription factors TBP and TFIIB maintains the transcriptional initiation complex in an active configuration.

Human TAF(II)28 interacts with the human T cell leukemia virus type I Tax transactivator and promotes its transcriptional activity.

The Tax protein encoded by human T cell leukemia virus type I transactivates the viral promoter by forming a complex with several cellular factors bound to three repeats of a specific upstream regulatory sequence. We have shown that transactivation by Tax was correlated with its ability to interact with the C-terminal moiety of the TATA box-binding protein (TBP). In the present study, the ability of Tax to interact with several human TBP-associated factors (TAF(II)s) was analyzed. We show that Tax interacts selectively with hTAF(II)28 in transfected HeLa cells. A direct interaction between Tax and hTAF(II)28 was also observed in vitro with purified proteins. In transient expression studies we show that overexpression of hTAF(II)28 significantly increased transactivation by Tax, both in the absence and in the presence of overexpressed TBP. The ability of hTAF(II)28 to potentiate transactivation correlated with the ability of Tax to interact with hTAF(II)28 and also with the ability of hTAF(II)28 to interact with TBP. Coexpression of TBP and hTAF(II)28 resulted in an additive increase in transactivation by Tax. From these observations we propose that transcriptional activation by Tax involves multiple interactions with TFIID via its TBP and hTAF(II)28 subunits.

Exclusion of Int-6 from PML nuclear bodies by binding to the HTLV-I Tax oncoprotein.

The Tax transactivator of the human T cell leukemia virus type I (HTLV-I) exhibits oncogenic properties. A screen for proteins interacting with Tax yielded a complementary DNA (cDNA) encoding the human Int-6 protein. In mice, the Int-6 gene can be converted into a putative dominant negative oncogene after retroviral insertion. Here, Int-6 was localized in the cell nucleus to give a speckled staining pattern superposed to that of the promyelocytic leukemia (PML) protein. The binding of Tax to Int-6 caused its redistribution from the nuclear domains to the cytoplasm. Thus, Int-6 is a component of the PML nuclear bodies and Tax disrupts its normal cellular localization by binding to it.

Genetic characterization of transactivation of the human T-cell leukemia virus type 1 promoter: Binding of Tax to Tax-responsive element 1 is mediated by the cyclic AMP-responsive members of the CREB/ATF family of transcription factors.

To achieve a better understanding of the mechanism of transactivation by Tax of human T-cell leukemia virus type 1 Tax-responsive element 1 (TRE-1), we developed a genetic approach with Saccharomyces cerevisiae. We constructed a yeast reporter strain containing the lacZ gene under the control of the CYC1 promoter associated with three copies of TRE-1. Expression of either the cyclic AMP response element-binding protein (CREB) or CREB fused to the GAL4 activation domain (GAD) in this strain did not modify the expression of the reporter gene. Tax alone was also inactive. However, expression of the reporter gene was induced by coexpression of Tax and CREB. This effect was stronger with the GAD-CREB fusion protein. Analysis of different CREB mutants with this genetic system indicated that the C-terminal 92 amino acid residues, which include the basic domain and the leucine zipper, are necessary and sufficient to mediate transactivation by Tax. To identify cellular proteins binding to TRE-1 in a Tax-dependent manner, this strain was also used to screen a library of human cDNAs fused to GAD. Of five positive clones isolated from 0.75 x 10(6) yeast colonies, four were members of the CREB/activating transcription factor (ATF) family: CREB, two isoforms of the cyclic AMP-responsive element modulator (CREM), and ATF-1. Interestingly, these three proteins can be phosphorylated by protein kinase A and thus form a particular subgroup within the CREB/ATF family. Expression of ATF-2 in S. cerevisiae did not activate TRE-1 in the presence of Tax. This shows that in a eukaryotic nucleus, Tax specifically interacts with the basic domain-leucine zipper region of ATF-1, CREB, and CREM. The fifth clone identified in this screening corresponded to the Ku autoantigen p70 subunit. When fused to GAD, the C-terminal region of Ku was able to activate transcription via TRE-1 but this activation was not dependent on Tax.

Effects on NF-kappa B1/p105 processing of the interaction between the HTLV-1 transactivator Tax and the proteasome.

The viral Tax protein, which is encoded by human T-cell leukaemia virus HTLV-I, activates nuclear translocation of the NF-kappa B/Rel transcription factors and relieves cytoplasmic sequestration of RelA and Rel by heterodimerization with NF-kappa B1/p1O5 (refs 1,2). Proteolytic maturation of this precursor protein is performed by the proteasome complex. Here we show that Tax binds specifically to two subunits of the 20S proteasome, HsN3 and HC9. This interaction is weakened with HsN3 and lost for HC9 when a mutant of Tax is substituted that is selectively defective for NF-kappa B activation. Immunoprecipitation shows that p1O5 binds weakly to HC9 and that this interaction is reinforced by Tax. No bridging function of Tax between p1O5 and HsN3 was observed. From these results, we propose that Tax accelerates the proteolytic maturation of P105 by favouring its anchorage to the proteasome.

Characterization of a transcriptional attenuator within the 5' R region of the human T cell leukemia virus type 1.

Several regulatory sequences have been characterized in the HTLV-I promoter. We report here identification of a sequence element downstream of the transcriptional start site within the first 52 nucleotides of the 5' R region, which acts negatively on the activity of the HTLV-I promoter. Determination of the half-lives of the RNAs either including or lacking this sequence element showed that the observed effect intervenes at the transcriptional level. This negative element does not affect basal activity of the HTLV-I TATA box, but down-regulates transcription induced by strong activators. Thus, we propose that this so-called negative regulatory sequence functions as an attenuator of transcription.

Evidence for functional interaction between the HIV-1 Tat transactivator and the TATA box binding protein in vivo.

Tat strongly activates transcription of the HIV-1 provirus by stimulating both initiation and elongation. This transactivator binds to the TAR RNA element, but can also associate with cellular transcription factors, interacting with upstream promoter sequences. To achieve a better understanding of the role of Tat in the assembly of the transcriptional initiation complex in the living cell, we have examined how the activity of this protein is modified when the general transcription factor involved in the first step of this process, TBP, is overexpressed. The activity of Tat, either wild-type or fused to the DNA binding domain of GAL4 (GBTat), was tested using reporter constructs containing GAL4 binding sites upstream of a minimal promoter corresponding to the HIV-1 TATA box, with or without the TAR element. We found that overexpression of TBP led to a dramatic increase in the activity of the GBTat protein. In order to activate GBTat, TBP must be able to interact with the TATA box. Analysis of several Tat mutants indicated that both the cysteine-rich and the core domains of this transactivator are necessary and sufficient to activate transcription when TBP is overexpressed. In vitro experiments showed that Tat binds specifically to TBP. There was a correlation between the ability of different Tat mutants to bind TBP and their capacity to activate transcription in vivo. With the natural HIV-1 promoter, overexpression of TBP first stimulated and then suppressed the Tat-induced activity. This inhibition was abrogated by an increase in the intracellular levels of Tat. These experimental data indicate that Tat stimulates initiation of transcription by interacting with TBP in vivo.

Tax induces nuclear translocation of NF-kappa B through dissociation of cytoplasmic complexes containing p105 or p100 but does not induce degradation of I kappa B alpha/MAD3.

The activity of the NF-kappa B transcription factor is controlled through cytoplasmic retention by either of two types of molecules: the inhibitor I kappa B alpha/MAD3 or the p105 and p100 precursors of the p50 and p52 DNA-binding subunits. Treatment of cells with classical NF-kappa B inducers such as tumor necrosis factor, interleukin-1, phorbol myristate acetate, and lipopolysaccharide results in MAD3 degradation followed by nuclear translocation of NF-kappa B. On the other hand, the mechanisms involved in the dissociation of the cytoplasmic p105/p100-containing complexes are largely unknown. The Tax protein encoded by human T-cell leukemia virus type 1 is a potent activator of viral and cellular gene transcription. It does not bind DNA directly but seems to activate transcription indirectly either by enhancing the activities of the transcription factors that recognize responsive elements located in the promoters of the Tax-responsive genes or by forming ternary complexes with these factors and DNA. It has been previously shown that Tax is able to induce nuclear translocation of NF-kappa B. We demonstrate here that Tax can induce translocation of members of the NF-kappa B family retained in the cytoplasm through their interaction with either p105 or p100. On the other hand, Tax induces no apparent degradation of MAD3, although experiments using cycloheximide indicate that it decreases the half-life of MAD3. However, this activity is shared by a mutant of Tax which is unable to activate NF-kappa B. These results suggest that Tax activates NF-kappa B essentially through the p105/p100 retention pathway.

Functional and biochemical interaction of the HTLV-I Tax1 transactivator with TBP.

The human T-cell leukemia virus type I (HTLV-I) codes for the potent transcriptional activator, Tax1, which induces the enhancer activity of various enhancer elements. In the case of the 21 bp enhancer of the HTLV-I provirus, this induction is correlated with the association of Tax1 with this DNA element via a specific cellular factor. That the indirect association of Tax1 with DNA can lead to transcriptional activation has also been supported by the study of chimeric GAL4-Tax1 proteins. The GAL4-Tax1 stimulatory effect exhibits a strong self-squelching. In order to determine whether Tax1 interacts directly with the general transcription factors or via intermediary molecules, we have analyzed how overexpression of the TATA binding protein (TBP) and TFIIB protein affects the squelching curve of GAL4-Tax1. The data presented here show that overexpression of TBP strongly increases the stimulatory effect of GAL4-Tax1, causes a displacement of the maximum of the squelching curve and partially alleviates the squelching. Under similar conditions TFIIB exhibited little effect. From these results we conclude that Tax1 can increase the recruitment of TBP by directly interacting with this protein. Biochemical experiments with purified proteins produced in bacteria confirmed that Tax1 can interact with TBP but not with TFIIB. Tax1 interacts with the conserved C-terminal part of TBP. Analysis of the ability of different mutants of Tax1 fused to the GAL4 DNA binding domain to activate transcription and to associate with TBP, showed that these activities are correlated. However, since one transcriptionally inactive mutant was able to interact efficiently with TBP in vitro, it would appear that an event other than the Tax1-TBP contact also intervenes in the activation of transcription by Tax1.

Funnel-well SDS-PAGE: a rapid technique for obtaining sufficient quantities of low-abundance proteins for internal sequence analysis.

We report a modified sodium dodecyl sulfate polyacrylamide gel electrophoresis method that permits up to a 60-fold concentration factor, without significant loss of protein. This method leads to very efficient concentration of low-abundance proteins from partially purified fractions or very dilute protein solution. Furthermore, it permits in situ enzymatic digestion and consequently increases the probability of obtaining a suitable internal sequence.

Purification by DNA affinity precipitation of the cellular factors HEB1-p67 and HEB1-p94 which bind specifically to the human T-cell leukemia virus type-I 21 bp enhancer.

Transcription driven by the proviral promoter of the Human T-cell Leukemia Virus type I (HTLV-I) is tightly regulated by the Tax1 transactivator. This viral protein potently induces the enhancer activity of a 21 bp motif repeated three times in the promoter. We have previously shown that this induction results from the binding of Tax1 to this enhancer sequence and that this association is mediated by the cellular factor HEB1. In this paper we report the purification of this factor by chromatography and DNA affinity precipitation. The latter method allowed a rapid and efficient purification which led to the identification of two polypeptides with molecular masses of 94- and 67-kDa, named HEB1-p94 and HEB1-p67, respectively. DMS methylation interference and UV crosslinking experiments indicated that both proteins formed different nucleo-protein complexes, but had the same DNA specificity. Study of the interaction of these two proteins with Tax1 showed that only HEB1-p67 can specifically interact with Tax1.