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K Khalili

Publications and source records attributed to K Khalili.

At least 73 records · Page 4Linked to original sources
PubMed1999

Association of HIV-1 Tat with the cellular protein, Puralpha, is mediated by RNA.

The interaction between two regulatory proteins plays a crucial role in the control of several biological events, including gene transcription. In this report, we demonstrate that the interaction between the cellular sequence-specific single-stranded DNA binding protein Puralpha and the HIV type 1 (HIV-1) Tat protein is mediated by specific ribonucleic acids. The region of Tat that is important for its interaction with Puralpha includes the region demonstrated to bind Tat's viral RNA target, TAR. A 10-nucleotide GC-rich consensus sequence identified in RNAs associated with Puralpha derived from human U-87MG cells plays an important role in the Puralpha:Tat interaction as examined by an in vitro reconstitution assay. Furthermore, expression of the Puralpha-associated RNA in these cells enhances transcriptional activation of the HIV-1 promoter by Tat and Puralpha.

Cyclic AMP Response Element-Binding Protein↗
PubMed1999

A 23-bp sequence element from human neurotropic JC virus is responsive to NF-kappa B subunits.

The regulatory region of the human neurotropic JC virus (JCV) is composed of several cis-acting motifs that confer cell type specificity to viral gene transcription and enable the viral promoters to respond to extracellular stimuli. For example, the bidirectional 98-bp tandem repeat sequences, positioned between the JCV early and late genes, were shown to be responsible for basal and activated levels of viral gene transcription in central nervous system (CNS) cells. Additionally, the NF-kappaB site located approximately 75 bp from the repeats on the early side of the viral genome was also found to influence both levels of viral transcription. Recently, we isolated a novel JCV variant, JCV(Phila-1), from a clinical specimen that contains a 23-bp sequence element (23-bpse) within its promoter-enhancer region. Here we demonstrate that this element is responsive to an extracellular stimulatory factor, such as phorbol 12-myristate 13-acetate (PMA), and can augment the basal levels of the viral early and to a lesser degree late promoter activities in cells derived from the CNS. The 23-bpse, by associating with nuclear proteins present in uninduced cells, forms a 40-kDa DNA-protein complex. Although no direct correlation between transcriptional enhancement of the JCV promoter by PMA treatment and the level of the 40-kDa DNA-protein complex was observed, results from site-directed mutagenesis indicated that formation of this complex is critical for the transcriptional activation of the viral promoter by PMA. These observations suggested that transcriptional enhancement of the JCV promoter activity upon PMA treatment may be an indirect event and mediated by an intermediary factor(s). In this respect, we demonstrated that overexpression of the inducible NF-kappaB subunits, p50 and p65, enhanced transcriptional activity of the JCV promoter through the 23-bp region with no evidence for their direct association with the 23-bpse DNA. Of importance, the p50/p65-induced JCV promoter activity requires the nucleotide sequences within the 23-bpse that are critical for the assembly of the 40-kDa DNA-protein complex. Thus, it is likely that the NF-kappaB subunits, by recruiting the cellular factors such as those associated with the 40-kDa DNA-protein complex, influence the basal level of the viral gene transcription. The implications of these findings with respect to regulation of viral and cellular genomes by extracellular stimuli and NF-kappaB pathway are discussed.

Base Composition↗
PubMed1999

Self-association of Puralpha is mediated by RNA.

The 322 amino acid cellular protein, Puralpha, is a sequence-specific single-stranded DNA-binding protein implicated in control of transcription and replication. Previous studies have demonstrated that the interaction between Puralpha and its target DNA sequence results in the formation of multimeric complexes. In this study, we demonstrate that Puralpha can self-associate in the absence of DNA. This self-association, while independent of DNA, is mediated by RNA. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, and the synthetic peptides corresponding to various central regions of Puralpha, the domain which is important for the self-association of Puralpha is localized to acidic leucine-rich repeats. Interestingly, these repeats have previously been shown to interact with the human immunodeficiency virus 1 (HIV-1) Tat protein and in this study we demonstrate that Tat is able to disrupt the self- association of Puralpha. We have recently cloned a Puralpha associated-RNA, PU-RNA, and here we show that PU-RNA can specifically reconstitute the self-association of Puralpha. RNA not only mediates the self-association of Puralpha, but also modulates the ability of Puralpha to interact with its target DNA sequence. Electrophoretic mobility shift assays performed with and without RNase treatment demonstrate that RNA inhibits the interaction between Puralpha and its target DNA sequence. Moreover, we demonstrate that the self-association of Puralpha can be reconstituted by a specific oligonucleotide encompassing the Puralpha binding site. The implications of these findings with respect to Puralpha's role in transcription and replication are discussed.

Amino Acid Sequence↗
PubMed1999

Human neurotropic JC virus early protein deregulates glial cell cycle pathway and impairs cell differentiation.

Progressive multifocal leukoencephalopathy (PML), a human demyelinating disease of the central nervous system (CNS), is induced upon replication of the human neurotropic virus, JCV, in glial cells. Similar to other polyomaviruses, replication of JCV is initiated and orchestrated by the viral early protein, T-antigen, and results in the cytolytic destruction of oligodendrocytes, the subset of glial cells responsible for myelin production, and the appearance of bizarre astrocytic glial cells in affected individuals. Earlier results from studies in transgenic animals have suggested that in the absence of viral replication, expression of JCV T-antigen induces pathology consistent with hypomyelination of the brain. These observations suggest that JCV T-antigen has the ability to deregulate oligodendrocyte and perhaps astrocyte function in the CNS. Here we demonstrate that expression of JCV T-antigen in the bipotential glial cell line, CG-4, severely affects the ability of these cells to differentiate toward oligodendrocyte and astrocyte lineages as evidenced by their distinct morphological changes. Examination of the activity of cell cycle regulatory proteins including cyclins and their associated kinases reveals that in the absence of T-antigen, differentiation of CG-4 cells toward astrocytes and oligodendrocytes is accompanied by a decline in cyclin E, cdk2, cyclin A, and cyclin B activity. In contrast, cdc2 activity increased upon CG-4 differentiation. In T-antigen-producing cells, distinct variations in the activity of several cyclins was observed. For example, while the activity of cdk2 and cyclin E was enhanced in T-antigen expressing astrocytes compared to their levels in control cells, the activity of cdc2 was decreased in this cell type. In oligodendrocytes, expression of T-antigen decreased the activity of several cyclins and cdks including cyclin E and cdc2. On the other hand, the level of expression and activity of cyclin A was increased. Thus, it is evident that JCV T-antigen deregulates several important cell cycle regulators during CG-4 differentiation, and these alterations may contribute to the process of cell growth and differentiation in glial cells. The importance of our findings with regard to the neuropathogenesis of PML is discussed.

Antigens, Polyomavirus Transforming↗
PubMed1999

Human ubiquitous JCV(CY) T-antigen gene induces brain tumors in experimental animals.

JCV is a papovavirus which is widespread in the human population. The prototype Mad-1 variant of JCV induces a fatal demyelinating disease of the central nervous system (CNS) called Progressive Multifocal Leukoencephalopathy (PML) in immunosuppressed individuals. The unique tropism of JCV (Mad-1) to the CNS is attributed to the tissue-specific regulation of the viral early promoter which is responsible for the production of the viral regulatory protein, T-antigen. The archetype form of this virus, JCV(CY), which has been repeatedly isolated from the urine of PML and non-PML individuals, is distinct from JCV(Mad-1) in the structural organization of the regulatory sequence. To characterize the tissue specific expression of JCV(CY) and to investigate its potential in inducing disease, transgenic mice containing the early region of JCV(CY) were generated. Some of these mice between 9-13 months of age exhibited signs of illness as manifested by paralysis of rear limbs, hunched posture, and poor grooming. Neuropathological examination indicated no sign of hypomyelination of the brain, but surprisingly, revealed the presence of primitive tumors originating from the cerebellum and the surrounding brain stem. The tumor masses also infiltrated the surrounding tissue. Results from RNA and protein studies revealed a high level of T-antigen mRNA expression in hindbrains of clinically normal and affected transgenic mice. However, higher levels of T-antigen RNA and protein were detected in brains of the animals exhibiting severe illness. The close resemblance of JCV(CY) induced tumor in transgenic mice to the human medulloblastoma/primitive neuroectodermal tumor (PNETs) in location, histologic appearance, and expression of marker proteins strongly suggests the utility of this novel animal model for the study of human brain tumors.

Animals↗
PubMed1999

Regulation of myelin basic protein gene transcription by Sp1 and Puralpha: evidence for association of Sp1 and Puralpha in brain.

Direct interaction between transcription factors may provide a mechanism for the regulatory function of these proteins on transcription of the responsive genes. These interactions may be facilitated if the target DNA sequences for the participant regulatory proteins are overlapped or positioned in close proximity to each other within the promoter of the responsive genes. In earlier studies, we identified a cellular protein, named Puralpha, which upon binding to the MB1 regulatory DNA sequence of the myelin basic protein (MBP) gene, stimulates its transcription in central nervous system (CNS) cells. Here, we provide evidence for binding of the ubiquitous DNA binding transcription factor, Sp1, to the MB1 DNA motif at the region that partially overlaps with the Puralpha binding site. We demonstrate that binding of Puralpha to its target sequence is enhanced by inclusion of Sp1 in the binding reaction. Under this condition, binding of Sp1 to the MB1 regulatory sequence remained fairly unchanged, and no evidence for the formation of Puralpha:MB1:Sp1 was observed. This observation suggests that transient interaction of Puralpha and Sp1 may result in stable association of Puralpha and the MB1 element. In support of this notion, results from immunoprecipitation/Western blot studies have established association of Puralpha and Sp1 in nuclear extracts from mouse brain. Of interest, Puralpha appears to bind to the phosphorylated form of Sp1 which is developmentally regulated and that coincides with the periods when MBP gene expression is at its maximum level. Results from cotransfection studies revealed that ectopic expression of Puralpha and Sp1 synergistically stimulates MBP promoter activity in CNS cells. The importance of these findings in stage-specific expression of MBP during brain development is discussed.

Animals↗
PubMed1999

Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat.

The broad range of histological lesions associated with HIV-1 are somewhat subtle relative to the clinical manifestations that occur as a result of HIV infection. Although it is clear that HIV has a causative role in CNS disease, dementia appears to be a consequence of the infiltration of inflammatory cells and cytokine dysregulation rather than the amount of virus in CNS. The HIV transregulatory protein Tat plays an important intracellular as well as extracellular role in the dysregulation of cytokines. The cytokines and possibly chemokines that are induced by Tat modify the action of astrocytes such that the survival of neurons is compromised. Pathogenetic alteration induced by Tat involves a series of interactions between circulating monocyte/macrophages, endothelial cells, and astrocytes. Cytokine dysregulation induced by viral infection and extracellular Tat leads to alterations in expression of adhesion molecules and promotes migration of non-infected inflammatory cells into the CNS compartment. We demonstrate here that recombinant HIV-1 Tat protein introduced by stereotaxic injection into mouse brain can induce pathologically relevant alterations including macrophage invasion as well as astrocytosis. The mechanism of destruction of the CNS by Tat appears to involve autocrine and paracrine pathways that depend not only on Tat, but cytokine and chemokine signaling pathways that are altered by viral infection. In this review, we discuss various pathogenic effects of Tat in brain cells and provide experimental evidence for an increased TNF-alpha level in CSF in mice injected intracerebrally with Tat protein.

Animals↗
PubMed1999

Utililization of experimental animal model for correlative multispectral MRI and pathological analysis of brain tumors.

Magnetic resonance imaging is the method of choice for non-invasive detection and evaluation of tumors of the central nervous system. However, discrimination of tumor boundaries from normal tissue, and the evaluation of heterogeneous lesions have proven to be limitations in traditional magnetic resonance imaging. The use of post-image acquisition processing techniques, such as multispectral tissue segmentation analysis, may provide more accurate clinical information. In this report, we have employed an experimental animal model for brain tumors induced by glial cells transformed by the human neurotropic JC virus to examine the utility of multispectral tissue segmentation for tumor cell identification. Six individual tissue types were discriminated by segmentation analysis, including heterogeneous tumor tissue, a clear demarcation of the boundary between tumor and non-tumor tissue, deep and cortical gray matter, and cerebrospinal fluid. Furthermore, the segmentation analysis was confirmed by histopathological evaluation. The use of multispectral tissue segmentation analysis may optimize the non-invasive determination and volumetric analysis of CNS neoplasms, thus providing improved clinical evaluation of tumor growth and evaluation of the effectiveness of therapeutic treatments.

Animals↗
PubMed1999

Suppression of HIV-1 transcription and replication by a Vpr mutant.

Vpr, the 96 amino acid long protein represents one of the auxiliary proteins of human immunodeficiency virus type-1 (HIV-1), which exhibits the ability to increase the rate of replication of the virus in T cells. Structurally, this protein is composed of several regions such as the acidic domain with alpha helix at the amino terminus, leucine-isoleucine-rich domain (LR) near the carboxyl terminus and an arginine-rich domain at the C-terminus. Here, we evaluated the ability of wild-type and a spectrum of Vpr mutants with altered amino acid residues within the three major domains of Vpr to regulate of transcription of the HIV-1 LTR. Our results revealed that alterations of amino acids within the LR domain at position 73 from arginine to serine, renders Vpr defective in stimulating transcription of the viral promoter in human T-lymphocytic and astrocytic cells. Mutations within the N- and C-terminal domains had little or no effect on the transcriptional activity of Vpr. Of interest, ectopic expression of this mutant protein exerts a negative effect on the ability of wild-type Vpr, as well as the viral transactivator, Tat, in augmenting viral gene transcription. Production of the mutant Vpr interferes with the replication of the wild-type and delta Vpr virus in the cells. Accordingly, a Vpr mutant virus containing the transition of arginine to serine at position 73 exhibited an inhibitory effect on the replication of wild-type virus. Our results provide a new avenue for the utilization of the variant of the HIV-1 regulatory protein, Vpr, in suppressing replication of the viral genome in infected cells.

Amino Acid Substitution↗
PubMed1999

Physical and functional interaction between the Y-box binding protein YB-1 and human polyomavirus JC virus large T antigen.

Y-box binding protein YB-1 is a member of a family of DNA and RNA binding proteins which have been shown to affect gene expression at both the transcriptional and translational levels. We have previously shown that YB-1 modulates transcription from the promoters of the ubiquitous human polyomavirus JC virus (JCV). Here we investigate the physical and functional interplay between YB-1 and the viral regulatory protein large T antigen (T-antigen), using JCV as a model system. Results of mobility band shift assays demonstrated that the efficiency of binding of YB-1 to a 23-bp single-stranded viral target sequence was significantly increased when T-antigen was included in the binding reaction mixture. Affinity chromatography and coimmunoprecipitation assays demonstrated that YB-1 and T-antigen physically interact with each other. Additionally, results of transcription studies demonstrated that these two proteins interact functionally on the JCV early and late gene promoters. Whereas ectopic expression of YB-1 and T-antigen results in synergistic transactivation of the viral late promoter, YB-1 alleviates T-antigen-mediated transcriptional suppression of the viral early promoter activity. Furthermore, we have localized, through the use of a series of deletion mutants, the sequences of these proteins which are important for their interaction. The T-antigen-interacting region of YB-1 is located in the cold shock domain of YB-1 and its immediate flanking sequences, and the YB-1-interacting domain of T-antigen maps to the carboxy-terminal half of T-antigen. Results of transient transfection assays with various YB-1 mutants and T-antigen expression constructs confirm the specificity of the functional interaction between YB-1 and T-antigen. Taken together, these data demonstrate that the cellular factor YB-1 and the viral regulatory protein T-antigen interact both physically and functionally and that this interaction modulates transcription from the JCV promoters.

Animals↗
PubMed1999

Association of JC virus large T antigen with myelin basic protein transcription factor (MEF-1/Puralpha) in hypomyelinated brains of mice transgenically expressing T antigen.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by cytolytic destruction of oligodendrocytes, the myelin-producing cells of the central nervous system, by the human neurotropic JC virus (JCV). The early protein of JCV, T antigen, which is produced at the early stage of infection, is important for orchestrating the events leading to viral lytic infection and cytolytic destruction of oligodendrocytes. Results from transgenic mouse studies, however, have revealed that, in the absence of lytic infection, this protein can induce brain hypomyelination and suppression of myelin gene expression. Since expression of the gene encoding myelin basic protein, the major component of myelin, can be regulated by a DNA-binding transcription factor, MEF-1/Puralpha, (Puralpha), we have examined the level of this protein in transgenic mouse brains. Results from immunoprecipitation and Western blots showed that while there was no drastic decrease in the level of MEF-1/Puralpha in transgenic mouse brains, JCV T antigen was found in a complex with MEF-1/Puralpha. Immunohistological studies revealed abnormal oligodendrocytes in white matter, where MEF-1/Puralpha and T antigen were detected. Furthermore, immunogold electron microscopic studies revealed that Puralpha and T antigen are colocalized in the nucleus of the oligodendrocytes and in hippocampal neurons. Interestingly, results from cell culture studies revealed that incubation of oligodendrocytes with JCV led to a drastic decrease in the level of MEF-1/Puralpha protein. These observations provide insight into the molecular pathogenesis of PML and support a model for a dual effect of JCV on inducing hypomyelination by (i) affecting myelin gene expression via interaction of JCV T antigen and the myelin gene transcription factor, MEF-1/Puralpha, and (ii) causing a decline in the level of the host regulatory proteins, including MEF-1/Puralpha, which are involved in myelin gene expression.

Animals↗
PubMed1999

Reciprocal interaction between two cellular proteins, Puralpha and YB-1, modulates transcriptional activity of JCVCY in glial cells.

Cross communication between regulatory proteins is an important event in the control of eukaryotic gene transcription. Here we have examined the structural and functional interaction between two cellular regulatory proteins, YB-1 and Puralpha, on the 23-bp sequence element derived from the enhancer-promoter of the human polyomavirus JCV. YB-1 and Puralpha are single-stranded DNA binding proteins which recognize C/T- and GC/GA-rich sequences, respectively. Results from band shift studies demonstrated that while both proteins interact directly with their DNA target sequences within the 23-bp motif, each protein can regulate the association of the other one with the DNA. Affinity chromatography and coimmunoprecipitation provide evidence for a direct interaction between Puralpha and YB-1 in the absence of the DNA sequence. Ectopic expression of YB-1 and Puralpha in glial cells synergistically stimulated viral promoter activity via the 23-bp sequence element. Results from mutational studies revealed that residues between amino acids 75 and 203 of YB-1 and between amino acids 85 and 215 of Puralpha are important for the interaction between these two proteins. Functional studies with glial cells indicated that the region within Puralpha which mediates its association with YB-1 and binding to the 23-bp sequence is important for the observed activation of the JCV promoter by the Puralpha and YB-1 proteins. The results of this study suggest that the cooperative interaction between YB-1 and Puralpha mediates the synergistic activation of the human polyomavirus JCV genome by these cellular proteins. The importance of these findings for cellular and viral genes which are regulated by Puralpha and YB-1 is discussed.

Binding Sites↗
PubMed1999

Role of HIV-1 Tat and CC chemokine MIP-1alpha in the pathogenesis of HIV associated central nervous system disorders.

Two syndromes affecting cognitive and motor function in the setting of AIDS have been described as HIV encephalopathy (HIVE) and progressive multifocal leukoencephalopathy (PML). HIVE is characterized by the presence of microglial nodules with accompanying astrocytosis. PML is a fatal demyelinating disease of the white matter induced by the human papovavirus JCV which causes cytolytic destruction of glial cells. In addition to the effect of HIV-1 induced immune suppression, HIV may act directly as a co-factor for stimulation of JCV replication in AIDS patients, in part due to Tat-induced activation of JCV gene transcription. Since Tat has been implicated in CNS pathogenesis, we examined its localization in CNS specimens from HIV infected patients with HIVE and PML as well as controls. Based on the observation of CC chemokine induction in monocytes by Tat, we also examined the cellular localization of the CC chemokine Macrophage Inflammatory Protein-1alpha (MIP-1alpha) and its cognate receptor CCR-5 in these samples. In HIVE, Tat was primarily localized in astrocytes and microglia, within the nodular lesions. In PML, a marked increase in the number of Tat positive astrocytes was observed. In both HIVE and PML, prominent expression of MIP-1alpha and CCR-5 was found within areas containing histopathological lesions. CCR-5 positivity of microglia was localized primarily to nodular lesions in HIVE. In PML, increased numbers of cells with monocyte/microglial morphology were observed relative to HIVE. The increased MIP-1 alpha positivity, and potentially other chemokines, may contribute to the pathogenesis of PML in the setting of HIV infection. Tat may play an important role in the pathogenesis of both HIV associated CNS disease states, acting indirectly through cytokine and chemokine dysregulation.

AIDS Dementia Complex↗
PubMed1998

Interaction of the single-stranded DNA-binding protein Puralpha with the human polyomavirus JC virus early protein T-antigen.

Large T-antigen, the major regulatory protein encoded by polyomaviruses, including Simian Virus 40 (SV40) and JC virus (JCV), is a multifunctional phosphoprotein that is involved in many viral and cellular events. In addition to its integral role in viral replication and cellular transformation, T-antigen also regulates transcription of both viral and cellular genes. In particular, the viral late promoter has been used as a model for the analysis of T-antigen-mediated transcriptional activation. Earlier studies have demonstrated that the cellular protein Puralpha is able to attenuate the transcriptional activity of JCV T-antigen. We investigated the mechanism whereby Puralpha affects T-antigen function. Co-immunoprecipitation studies demonstrated that Puralpha and JCV T-antigen associate in vivo, and glutathione S-transferase affinity binding assays revealed that these two proteins interact in vitro. Moreover, we localized the sequences of Puralpha that are important for the interaction between Puralpha and JCV T-antigen. In addition, we demonstrated that Puralpha interacts with the SV40 T-antigen. Transient transfection studies demonstrated that Puralpha and JCV T-antigen interact functionally as well. More specifically, Puralpha and a deletion mutant that interacts with T-antigen attenuated T-antigen-mediated transcriptional activation. A Puralpha deletion mutant that is unable to interact with JCV T-antigen, however, was found to be incapable of abrogating JCV T-antigen transactivation. Taken together, these data demonstrate that Puralpha and T-antigen interact both physically and functionally and that this interaction modulates T-antigen-mediated transcriptional activation. The implication of these findings with respect to the cellular role of Puralpha is discussed.

Antigens, Polyomavirus Transforming↗
PubMed1998

A multi-institutional study confirms the presence and expression of simian virus 40 in human malignant mesotheliomas.

Exposure to the carcinogen asbestos is a major factor in the development of malignant mesothelioma. However, not all mesotheliomas are associated with asbestos exposure, and only a small minority of people exposed to asbestos develop mesothelioma. Therefore, the identification of the cofactors that render certain individuals more susceptible to asbestos or that cause mesothelioma in people not exposed to asbestos has been a major priority of the International Mesothelioma Interest Group. The possible association of SV40 with mesothelioma was recently discussed in a special session at the Fourth International Mesothelioma Interest Group Conference, and it was decided to conduct a multi-institutional study to independently verify the presence of this tumor virus in mesotheliomas. We report the results of this investigation: (a) DNA and protein analyses revealed SV40 sequences and SV40 large T antigen expression in 10 of 12 mesotheliomas tested (83%); and (b) electron microscopy demonstrated variable amounts of asbestos fibers in 5 (71%) of 7 corresponding lung tissues available for analysis. Our results demonstrate that SV40 DNA is frequently present and expressed in mesotheliomas in the United States. Because our data demonstrate that some patients test positive for both SV40 and asbestos, the possibility that these two carcinogens interact should be investigated in future studies.

Asbestos↗
PubMed1998

Retinoblastoma-related protein pRb2/p130 and suppression of tumor growth in vivo.

BACKGROUND: The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T-antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied. METHODS: Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and beta-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored. RESULTS: Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%-73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen. CONCLUSION: Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.

Animals↗
PubMed1998

Association of Puralpha with RNAs homologous to 7 SL determines its binding ability to the myelin basic protein promoter DNA sequence.

Cell type and developmental stage expression of the myelin basic protein (MBP) gene in mouse brain is regulated at the transcriptional level. Earlier studies from our laboratory have led to the identification of a DNA binding protein from mouse brain, named Puralpha, which interacts with the MB1 regulatory motif of the MBP and stimulates its transcription in glial cells. In this report, we demonstrate that a cellular RNA, with significant homology to 7 SL RNA is associated with Puralpha. Results from band shift competition studies indicate that Puralpha-associated RNA (PU-RNA), inhibits the interaction of immunopurified Puralpha with the MB1 DNA sequence. Results from Northern blot studies indicated that PU-RNA is expressed during various stages of brain development. Of interest, this RNA was found in association with Puralpha that was produced in the mouse brain at the early stage of brain development. Results from Northwestern analysis using a PU-RNA probe identified the regions within Puralpha that are important for Puralpha/PU-RNA association. Production of Puralpha at the early stage of brain development and its association with PU-RNA at this stage, when Puralpha exhibits poor binding ability to the MB1 DNA sequence, suggests that PU-RNA may function as a co-factor that negatively regulates Puralpha interaction with the MBP promoter sequence.

Animals↗