LKB1 loss is associated with glutathione deficiency under oxidative stress and sensitivity of cancer cells to cytotoxic drugs and γ-irradiation.

The liver kinase B1 (LKB1) gene is a tumor suppressor associated with the hereditary Peutz-Jeghers syndrome and frequently mutated in non-small cell lung cancer and in cervical cancer. Previous studies showed that the LKB1/AMPK axis is involved in regulation of cell death and survival under metabolic stress. By using isogenic pairs of cancer cell lines, we report here that the genetic loss of LKB1 was associated with increased intracellular levels of total choline containing metabolites and, under oxidative stress, it impaired maintenance of glutathione (GSH) levels. This resulted in markedly increased intracellular reactive oxygen species (ROS) levels and sensitivity to ROS-induced cell death. These effects were rescued by re-expression of LKB1 or pre-treatment with the anti-oxidant and GSH replenisher N-acetyl cysteine. This role of LKB1 in response to ROS-inducing agents was largely AMPK-dependent. Finally, we observed that LKB1 defective cells are highly sensitive to cisplatin and γ-irradiation in vitro, suggesting that LKB1 mutated tumors could be targeted by oxidative stress-inducing therapies.

The human T-cell leukemia virus type 1 p13II protein: effects on mitochondrial function and cell growth.

p13(II) of human T-cell leukemia virus type 1 (HTLV-1) is an 87-amino-acid protein that is targeted to the inner mitochondrial membrane. p13(II) alters mitochondrial membrane permeability, producing a rapid, membrane potential-dependent influx of K(+). These changes result in increased mitochondrial matrix volume and fragmentation and may lead to depolarization and alterations in mitochondrial Ca(2+) uptake/retention capacity. At the cellular level, p13(II) has been found to interfere with cell proliferation and transformation and to promote apoptosis induced by ceramide and Fas ligand. Assays carried out in T cells (the major targets of HTLV-1 infection in vivo) demonstrate that p13(II)-mediated sensitization to Fas ligand-induced apoptosis can be blocked by an inhibitor of Ras farnesylation, thus implicating Ras signaling as a downstream target of p13(II) function.

Differential expression of menin in sporadic pituitary adenomas.

Pituitary adenomas represent one of the key features of multiple endocrine neoplasia type 1. The gene involved in this syndrome (MEN1) is a putative tumor suppressor, that codes for a 610-amino acid nuclear protein termed 'menin'. Analyses of sporadic pituitary adenomas have so far failed to reveal MEN1 mutations or defects in MEN1 transcription in these tumors. In the present study we detected menin protein expression in a panel of normal and tumoral pituitary tissues, using a monoclonal antibody against the carboxy-terminus of menin. In the normal human pituitary gland, strong nuclear staining for menin was detectable in the majority of the endocrine cells of the anterior lobe, without a clear association with a particular hormone-producing type. In sporadic pituitary adenomas, menin expression was variable, with a high percentage of cases demonstrating a significant decrease in menin immunoreactivity when compared with the normal pituitary. Interestingly, metastatic tissues derived from one pituitary carcinoma had no detectable menin levels. Altogether, our data provide the first information regarding the status of menin expression in human normal and neoplastic pituitary as determined by immunohistochemistry (IHC).

Expression and functional properties of proteins encoded in the x-II ORF of HTLV-I.

With the aim of identifying viral proteins that contribute to the distinctive properties of HTLV-I biology and pathogenicity, several laboratories have investigated the coding potential of the X region of the genome, which includes five partially overlapping open reading frames (ORFs). We and others have shown that, in addition to the essential regulatory proteins Rex and Tax, a number of accessory proteins encoded in the X region can be produced by alternative splicing and multicistronic translation. One X region ORF, termed X-II, produces two protein isoforms named Tof/p30II and p13II, which are expressed from a doubly- and singly-spliced mRNA, respectively. Initial functional analyses demonstrated that Tof/p30II is a nucleolar/nuclear protein that possesses a region capable of binding to RNA, and p13II is a mitochondrial protein that alters the morphology and function of this organelle. Together with data from other laboratories demonstrating the production of antibodies and CTL against x-II ORF products in HTLV-I infected subjects and the requirement of this ORF for efficient viral replication in vivo, these findings suggest that further characterization of Tof/p30II and p13II will yield insight into remaining undefined aspects of HTLV-I pathogenicity and replication.

Free major histocompatibility complex class I heavy chain is preferentially targeted for degradation by human T-cell leukemia/lymphotropic virus type 1 p12(I) protein.

Human T-cell leukemia virus type 1 (HTLV-1) establishes a persistent infection in the host despite a vigorous virus-specific immune response. Here we demonstrate that an HTLV-1-encoded protein, p12(I), resides in the endoplasmic reticulum (ER) and Golgi and physically binds to the free human major histocompatibility complex class I heavy chains (MHC-I-Hc) encoded by the HLA-A2, -B7, and -Cw4 alleles. As a result of this interaction, the newly synthesized MHC-I-Hc fails to associate with beta(2)-microglobulin and is retrotranslocated to the cytosol, where it is degraded by the proteasome complex. Targeting of the free MHC-I-Hc, and not the MHC-I-Hc-beta(2)-microglobulin complex, by p12(I) represents a novel mechanism of viral interference and disrupts the intracellular trafficking of MHC-I, which results in a significant decrease in surface levels of MHC-I on human T-cells. These findings suggest that the interaction of p12(I) with MHC-1-Hc may interfere with antigen presentation in vivo and facilitate escape of HTLV-1-infected cells from immune recognition.

Identification of a domain in human immunodeficiency virus type 1 rev that is required for functional activity and modulates association with subnuclear compartments containing splicing factor SC35.

The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the DeltaLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.

The p13II protein of HTLV type 1: comparison with mitochondrial proteins coded by other human viruses.

In addition to the essential regulatory proteins Rex and Tax, the HTLV-1 genome encodes several accessory proteins of yet undefined function. One of these "orphan" proteins, named p13(II), was recently shown to be selectively targeted to mitochondria and to induce specific changes in mitochondrial morphology suggestive of altered inner membrane permeability and swelling. This represented the first report of a retroviral gene product targeted to mitochondria, and suggested that p13(II)-induced alterations in the function of this organelle may play a role in HTLV-1 replication and/or pathogenesis. The more recent findings that both Vpr and Tat of HIV-1 are targeted to mitochondria reinforces the proposed relevance of mitochondrial metabolism to the life cycle of retroviruses. Thus, p13(II), Vpr, and Tat can be added to the growing list of mitochondrial proteins produced by clinically important human viruses, including Epstein-Barr virus, human cytomegalovirus, and hepatitis B virus. Mitochondria are known to play a critical role by providing an amplification loop required for the execution of signaling pathways leading to programmed cell death. The functional consequences of the interactions between viral proteins and mitochondria described so far have been attributed to either the positive or negative control of apoptotic responses mediated by this organelle. Further analysis of the effects of p13(II) on mitochondrial function is likely to add to our understanding of the mechanisms underlying the development of HTLV-1-associated diseases.

The MHC class I heavy chain is a common target of the small proteins encoded by the 3' end of HTLV type 1 and HTLV type 2.

Human T cell leukemia/lymphotropic virus types 1 and 2 are two immunologically and phylogenetically related retroviruses that differ in their pathogenicity in vivo. The overall genetic structure of HTLV-1 and -2 is similar. Each contains a unique region at the 3' end of the genome, designated the pX region. p12(I) is a membrane-associated protein encoded by the open reading frame I (ORF I) region of HTLV-1, which lies within the pX region. A corresponding protein, p10(I) is encoded by the ORF I region of HTLV-2 and an additional protein, p11(V), is encoded by ORF V, which overlaps the HTLV-2 ORF I region. As with HTLV-1, the small proteins encoded by the pX region of HTLV-2 appear to be dispensable for viral replication and cellular transformation in vitro. However, the small open reading frames of both viruses are important for viral replication in vivo, which suggests they may play an important role during the viral life cycle. This study was undertaken to investigate and compare the cellular targets of the p10(I), p11(V), and p12(I) putative proteins.

Unique features of HIV-1 Rev protein phosphorylation by protein kinase CK2 ('casein kinase-2').

The HIV-1 Rev transactivator is phosphorylated in vitro by protein kinase CK2 at two residues, Ser-5 and Ser-8; these sites are also phosphorylated in vivo. Here we show that the mechanism by which CK2 phosphorylates Rev is unique in several respects, notably: (i) it is fully dependent on the regulatory, beta-subunit of CK2; (ii) it relies on the integrity of an acidic stretch of CK2 beta which down-regulates the phosphorylation of other substrates; (iii) it is inhibited in a dose-dependent manner by polyamines and other polycationic effectors that normally stimulate CK2 activity. In contrast, a peptide corresponding to the amino-terminal 26 amino acids of Rev, including the phosphoacceptor site, is readily phosphorylated by the catalytic subunit of CK2 even in the absence of the beta-subunit. These data, in conjunction with the observation that two functionally inactive derivatives of Rev with mutations in its helix-loop-helix motif are refractory to phosphorylation, indicate the phosphorylation of Rev by CK2 relies on conformational features of distinct regions that are also required for the transactivator's biological activity.

Unusual CD4+CD8+ phenotype in a greek patient diagnosed with adult T-cell leukemia positive for human T-cell leukemia virus type I (HTLV-I).

We describe the first Greek patient diagnosed with Adult T cell leukemia (ATL) characterized by an expansion of CD4+CD8+ double positive lymphocytes. Low levels of plasma antibodies against HTLV-I Env and Gag proteins were detected. Analysis of the the patient's DNA revealed that she was infected by a cosmopolitan strain of HTLV-I. Since HTLV-I usually leads to the expansion of CD4+ cells, this patient illustrates a rare immunophenotype, which suggests that the HTLV-I-induced proliferative response may occur in a pre-T cell stage.

Influence of Rex and intronic sequences on expression of spliced mRNAs produced by human T cell leukemia virus type I.

Expression of the incompletely spliced HTLV-I mRNAs relies on the viral posttranscriptional activator Rex, whose interaction with the Rex-responsive element (RXRE) overcomes effects of cis-acting repressive sequences (CRSs). Studies based on heterologous reporter plasmids identified an intronic CRS in the 5' LTR and a CRS that overlaps with the RXRE. The present study investigated the effects of these elements in the context of spliced viral mRNAs encoding p21Rex (mRNA 1-3), Tax/Rex (mRNA 1-2-3), and Tof (mRNA 1-2-B). All three mRNAs were inefficiently expressed when transcribed in their mature intronless form, with the p21Rex mRNA showing the weakest expression. In contrast, efficient expression of p21Rex was obtained from a plasmid containing the 5' LTR and 3' portion of the genome that encoded a spliceable RNA. The defective expression of the intronless mRNAs reflected the inhibitory activity of the RXRE and the lack of 5' intronic sequences. Insertion of an intronic 5' LTR segment located upstream of the 5' CRS overcame Rex dependence conferred by the RXRE. The activity of this segment was mapped to the major splice donor and sequences overlapping with, but functionally distinct from, a previously described transcriptional enhancer. The three mRNAs responded differently to Rex and to insertion of the constitutive transport element of simian retrovirus type 1. Taken together, these results suggest that expression of the spliced mRNAs is controlled by the relative influence of positive and negative sequences present on the primary transcript as well as the Rex-RXRE interaction.

Mitochondrial targeting of the p13II protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I).

The X region of the HTLV-I genome contains four major open reading frames (ORFs), two of which, termed x-I and x-II, are of still undefined biological significance. By indirect immunofluorescence and dual labeling with marker proteins, we demonstrate that p13II, an 87-amino acid protein coded by the x-II ORF, is selectively targeted to mitochondria. Mutational analysis revealed that mitochondrial targeting of p13II is directed by an atypical 10-amino acid signal sequence that is not cleaved upon import and is able to target the Green Fluorescent Protein to mitochondria. Expression of p13II results in specific alterations of mitochondrial morphology and distribution from a typical string-like, dispersed network to round-shaped clusters, suggesting that p13II might interfere with processes relying on an intact mitochondrial architecture. Functional studies of mitochondria with the cationic fluorochrome tetramethylrhodamine revealed that a subpopulation of the cells with p13II-positive mitochondria show a disruption in the mitochondrial inner membrane potential (Apsi), an early event observed in cells committed to apoptosis. Taken together, these results suggest novel virus-cell interactions that might be important in HTLV-I replication and/or pathogenicity.

CTL response and protection against P815 tumor challenge in mice immunized with DNA expressing the tumor-specific antigen P815A.

A DNA immunization approach was used to induce an immune response against the tumor-specific antigen P815A in DBA/2 mice. The P1A gene, which encodes the P815A antigen, was modified by the addition of a short sequence coding for a tag epitope recognized by the monoclonal antibody AU1, and cloned into the eukaryotic expression vector pBKCMV, resulting in plasmid pBKCMV-P1A. L1210 cells stably transfected with pBKCMV-P1A expressed P1A mRNA and were lysed by the syngeneic P815A-specific cytotoxic clone CTL-P1:5, thus confirming that the tag-modified P1A protein underwent correct processing and presentation. A single intramuscular injection of 100 microg of pBKCMV-P1A induced the expression of P1A mRNA for at least 4 months. Eighty percent of DBA/2 mice injected three times with 100 microg of pBKCMV-P1A generated cytotoxic T lymphocytes (CTL) that lysed P815 tumor cells, whereas mock-inoculated animals failed to show any cytotoxicity. Moreover, experiments designed to evaluate the protection of pBKCMV-P1A-immunized mice against a lethal challenge with P815 tumor cells showed that 6 of 10 immunized mice rejected the tumor, and 2 mice showed prolonged survival compared to control animals.

Inhibition of human T-cell leukemia virus type 2 Rex function by truncated forms of Rex encoded in alternatively spliced mRNAs.

Three mRNA species encoding the x-III open reading frame are expressed in human T-cell leukemia virus type 2 (HTLV-2)-infected cells. An mRNA composed of exons 1, 2, and 3 produces the essential posttranscriptional regulator Rex; shorter 1-3 and 1-B mRNAs encode a family of x-III proteins of unknown function that represent truncated forms of Rex. This report presents an analysis of the functional interactions between Rex and the x-III proteins, results of which suggest a role for the x-III proteins as negative regulators of Rex function. Cotransfection assays demonstrated that the x-III proteins were able to inhibit the ability of Rex to activate the expression of a Rex-dependent mRNA. Analysis of intracellular compartmentalization in actinomycin D-treated cells showed that coexpression of the x-III proteins resulted in the sequestration of Rex into the nuclear compartment. Subcellular fractionation studies showed that Rex was preferentially localized in the cytoplasmic or nuclear fraction depending on its phosphorylation status and that coexpression of Rex with the x-III proteins changed the phosphorylation pattern of Rex and the intracellular distribution of the x-III proteins. In vitro protein binding assays demonstrated the formation of Rex-Rex homomultimeric complexes; however, mixed Rex/x-III multimers were not detected. These findings indicated a correlation between phosphorylation and intracellular trafficking of Rex and suggested that the mechanism underlying the inhibitory effects of the x-III proteins might result from an interference with these processes.

The human T-cell lymphotropic virus type 1 Tof protein contains a bipartite nuclear localization signal that is able to functionally replace the amino-terminal domain of Rex.

The X region of human T-cell lymphotropic virus type 1 (HTLV-1) encodes two nucleolar/nuclear proteins, the posttranscriptional regulator of mRNA expression Rex and a protein of unknown function named Tof. To gain insight into the possible biological role of Tof, we investigated the mechanism governing its intracellular trafficking and identified its nucleolar/nuclear localization signal (NLS). Mutational analysis of Tof revealed that its NLS was located between amino acids 71 and 98 and contained two arginine-rich domains that functioned in an interdependent manner. Studies of Tof-Rex hybrid proteins showed that the Tof NLS could functionally replace the NLS of Rex at the level of nuclear targeting. As the NLS of Rex is known to mediate its interaction with its RNA target, the Rex-responsive element (RXRE), we tested whether the NLS of Tof could replace that of Rex in mediating activation of a RXRE-containing mRNA. Results showed that the NLS of Tof was indeed able to mediate activation of RXRE-containing mRNAs, suggesting that Tof itself may function as a regulator of RNA expression and utilization. A comparison of their compartmentalization in response to actinomycin D treatment indicated that Tof did not share Rex's shuttling pathway. Expression of Tof from its natural multiply spliced mRNA required the presence of Rex, suggesting that Tof may regulate viral or cellular mRNA expression during the later stages of viral replication.

Phosphorylation of HIV-1 Rev protein: implication of protein kinase CK2 and pro-directed kinases.

HIV-1 Rev transactivator is readily phosphorylated at separate regions by protein kinase CK2 and MAP kinase. Protein kinase CK1 cannot replace CK2 as phosphorylating agent and cdc2 only slowly phosphorylates Rev at one of the two sites affected by MAP kinase. Mutational analysis shows that Ser-8 and, to a lesser extent, Ser-5 are phosphorylated by CK2. In contrast, a mutation (R14TV-->EED) which suppresses Rev activity dramatically enhances Rev phosphorylation either in vitro by CK2 or in vivo, suggesting that phosphorylation by CK2 could play a role in Rev down-regulation.

Coding potential of the X region of human T-cell leukemia/lymphotropic virus type II.

Human T-cell leukemia/lymphotropic viruses type I and type II (HTLV-I and HTLV-II) are complex human retroviruses showing a similar genetic organization but substantially different biologic and pathogenic properties. As in other complex retroviruses, the 3' portion of the HTLV genome contains the peculiar "X region" comprising several partially overlapping open reading frames (ORFs). To search for a possible basis for the pathogenic differences between the two viruses, a number of studies have been carried out to analyze the coding potential of the X region of HTLV-I and, more recently, of HTLV-II. This review focuses on the coding potential of the HTLV-II X region and presents a comparison with that of HTLV-I. Expression of different ORFs present in the X region may be accessed through two expression strategies: alternative splicing and translation of more than one protein from the same mRNA. Initial analyses of the X region proteins indicate that some differ significantly from their HTLV-I homologues, thus providing possible clues to the understanding of the complex life cycle and pathogenicity of the two viruses.

Intracellular trafficking of the human immunodeficiency virus type 1 Rev protein: involvement of continued rRNA synthesis in nuclear retention.

We have explored the mechanism directing the intracellular trafficking and nucleolar accumulation of the human immunodeficiency virus type 1 (HIV-1) Rev protein. Treatment of Rev-expressing cells with mycophenolic acid, an inhibitor of inosine monophosphate dehydrogenase, resulted in a redistribution of Rev from the nucleoli to the nucleoplasm and cytoplasm. In contrast, a Rev effector domain mutant was retained in the nucleus, indicating the involvement of this domain in the protein's nuclear retention/nucleocytoplasmic transport. Identical results were obtained by inhibiting transcription using actinomycin D or 5,6-dichlorobenzimidazole riboside. All three drugs were found to inhibit biosynthetic labeling of ribosomal RNA and to disrupt nucleolar morphology, suggesting a correlation between nucleolar/nuclear retention of Rev, continued ribosomal RNA synthesis, and intact nucleolar architecture. Results of binding/immunofluorescence assays using isolated, permeabilized nuclei and extracts of cells expressing Rev demonstrated that the protein is able to bind to nucleoli in vitro, in the absence of active cellular processes or eukaryotic posttranslational modifications. Rev derived from actinomycin D-treated cells showed equivalent binding, indicating that the inhibitor did not directly interfere with the ability of the protein to interact with nucleolar structures. Rev's interaction with nucleoli was directed by the protein's arginine-rich RNA-binding/nucleolar localization domain, and was abrogated by pretreatment of the nuclei with RNaseA, indicating a requirement for RNA, probably ribosomal RNA.

Expression and characterization of proteins produced by mRNAs spliced into the X region of the human T-cell leukemia/lymphotropic virus type II.

In previous studies we showed that human T-cell leukemia/lymphotropic virus type I (HTLV-I) may produce novel proteins encoded in the X region. To investigate a possible correlation between expression of viral genes and different biologic properties of HTLV-I and HTLV-II, we analyzed expression of HTLV-II in the chronically infected cell line MoT. Reverse transcription-polymerase chain reaction analyses revealed that the virus produces several mRNAs singly or doubly spliced into the X region. Corresponding cDNAs were cloned and transfected into a HeLa cell line; resulting proteins were designated according to their sizes and coding open reading frames (ORFs). p10xI and p11xV were produced by a dicistronic doubly spliced mRNA. p10xI was generated by translation of the first exon of rex linked to the x-I ORF; p11xV was translated from the tax initiation codon linked to the x-V ORF. Two singly spliced polycistronic mRNAs produced p28xII, coded by the x-II ORF, and several isoforms generated by initiation within the x-III ORF. Studies of the proteins' subcellular localization revealed that they exhibited distinct targeting patterns. Comparison of these proteins with their HTLV-I counterparts indicated intriguing differences between these two viruses, suggesting that further study of the X region products may aid in defining genetic determinants of pathogenicity.

Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I.

Novel cytoplasmic mRNA species produced by human T-cell leukemia virus type I (HTLV-I) were cloned by using the polymerase chain reaction technique. Five novel 3' splice sites located in the X region and upstream of the env gene were identified. Splicing to the 3' splice sites in the X region generates mRNAs that express two previously unidentified viral proteins, named Rof and Tof. Tof accumulates in the nucleoli of transfected cells. The other viruses of the HTLV family, such as HTLV-II and bovine leukemia virus, also have a complex splicing pattern and are capable of producing additional proteins encoded in the X region. These results suggest that HTLV-I and other members of the HTLV family produce novel proteins, which may contribute to the biological properties of these viruses.