YouBelong Home: A Ugandan Community Mental Health Intervention.

In Uganda, low resources for mental health provision combine with disadvantage and inadequate supports for family and community-based care. Catalysed by the need to reduce overcrowded psychiatric hospital wards and frequent readmissions at Butabika National Referral Mental Hospital (BNRMH) in Kampala, the nongovernment organisation YouBelong Uganda (YBU) developed the YouBelong Home (YBH) intervention. YBH is a theoretically eclectic pre and post hospital discharge intervention. This paper reports on qualitative findings of the project Curtailing Hospital Readmissions for Patients with Severe Mental Illness in Africa (CHaRISMA), which explored how to refine the YBH intervention. The project was funded by a UK Joint Global Health Trials (JGHT) Development Grant. Data was collected through structured interviews with service users and caregivers, reflective practice by the YBH implementing team and a stakeholder focus group. A summary of refinements to the YBH intervention follows the TIDieR format (Template for Intervention Description and Replication).

The sixth vital sign: HIV status assessment and severe illness triage in Uganda.

Setting: Four in-patient health facilities in western Uganda. Objective: To determine the impact of an innovative multi-modal quality improvement program on human immunodeficiency virus (HIV) status assessment and the impact of HIV status on severe illness conditions and mortality. Design: This was a staggered, pre-post quasi-experimental study designed to assess a multi-modal intervention (collaborative improvement meetings, audit and feedback, clinical mentoring) for improving quality of care following formal training in the management of severe illness in low-income settings. Results: From August 2014 to May 2015, 5759 patients were hospitalized, of whom 2451 (42.6%) had their HIV status assessed; 395 (16.1%) were HIV-infected. HIV-infected patients were significantly more likely to meet criteria for shock (27.5% vs. 15.1%, risk ratio [RR] 1.8, 95% confidence interval [CI] 1.7-1.9, P < 0.001) and severe respiratory distress (6.7% vs. 4.3%, RR 1.5, 95%CI 1.2-2.0, P < 0.001), and were significantly more likely to die in hospital (12.0% vs. 2.9%, RR 4.1, 95%CI 3.2-5.4, P < 0.001). There was no evidence of improved HIV status assessment during the intervention period (36.5% vs. 44.8%, +8.3%, 95%CI -8.3 to 24.8, P = 0.33). Conclusions: Hospitalized HIV-infected patients in western Uganda are at high risk for severe illness and death. Novel quality improvement strategies are needed to enhance hospital-based HIV testing in high-burden settings.

Contexte : Quatre structures de santé hospitalières dans l'ouest de l'Ouganda.Objectif : Déterminer l'impact d'un programme innovant multimodal d'amélioration de la qualité sur l'évaluation du statut du virus de l'immunodéficience humaine (VIH) et l'impact du statut VIH sur les états de maladie grave et la mortalité.Schéma : Une étude échelonnée, pré-post et quasi-expérimentale conçue pour évaluer une intervention multimodale (réunions d'amélioration concertée, audit et rétro-information, tutorat clinique) pour améliorer la qualité des soins après la formation initiale sur la prise en charge de maladies graves dans un contexte de faibles ressources.Résultats : Entre août 2014 et mai 2015, 5759 patients ont été hospitalisés : 2451 (42,6%) ont eu une évaluation de leur statut VIH et 395 (16,1%) se sont avérés infectés par le VIH. Ces derniers ont été significativement plus susceptibles de répondre à des critères de choc (27,5% contre 15,1% ; rapport de risque [RR] 1,8 ; intervalle de confiance [IC] 95% 1,7­1,9 ; P < 0,001) et de détresse respiratoire grave (6,7% contre 4,3 ; RR 1,5 ; IC95% 1,2­2,0 ; P < 0,001), et ont été significativement plus susceptibles de décéder à l'hôpital (12,0% contre 2,9% ; RR 4,1 ; IC95% 3,2­5,4 ; P < 0,001). Il n'y a pas eu d'éléments en faveur d'une amélioration de l'évaluation du statut VIH pendant la période d'intervention (36,5% contre 44,8% ; +8,3% ; IC95% −8,3 à 24,8 ; P = 0,33).Conclusions : Les patients infectés par le VIH hospitalisés dans l'ouest de l'Ouganda ont un risque élevé de maladie grave et de décès. De nouvelles stratégies d'amélioration de qualité sont requises afin d'augmenter les tests VIH en hôpital dans les contextes à fardeau élevé de maladie.

Marco de referencia: Cuatro establecimientos hospitalarios en la zona occidental de Uganda.Objetivo: Determinar la repercusión de un programa innovador multimodal de mejora de la calidad sobre la evaluación de la situación frente al virus de la inmunodeficiencia humana (VIH) y la repercusión del estado frente al VIH en materia de enfermedades graves y mortalidad.Método: Se realizó un estudio semi-experimental escalonado pre y post con el fin de evaluar una intervención multimodal (reuniones de colaboración para mejorar de la calidad, auditorías y retroalimentación, tutoría clínica) encaminada a mejorar la calidad de la atención, tras una capacitación formal sobre el manejo de las enfermedades graves en entornos con bajos ingresos.Resultados: De agosto del 2014 a mayo del 2015 se hospitalizaron 5759 pacientes; en 2451 se examinó su situación frente al VIH (42,6%) y 395 presentaban infección por el VIH (16,1%). Los pacientes afectados por el VIH exhibieron una probabilidad significativamente mayor de cumplir con los criterios diagnósticos de choque (27,5% contra 15,1%; cociente de riesgos [RR] 1,8; intervalo de confianza [IC] del 95% 1,7­1,9; P < 0,001) y de insuficiencia respiratoria grave (6,7% contra 4,3%, RR 1,5; IC95% 1,2­2,0; P < 0,001) y la probabilidad de morir en el hospital fue significativamente más alta en estos pacientes (12,0% contra 2,9%, RR 4,1; IC95% 3,2­5,4; P < 0,001). No se encontraron pruebas en favor de una mejor evaluación de la situación frente al VIH durante el período de la intervención (36,5% contra 44,8%; +8,3%; IC95% −8,3 hasta 24,8; P = 0,33).Conclusión: Los pacientes hospitalizados aquejados de infección por el VIH en Uganda occidental son muy susceptibles de sufrir una enfermedad grave o la muerte. Se precisan nuevas estrategias de mejora de la calidad que refuercen la práctica de las pruebas diagnósticas de infección por el VIH en los entornos con alta carga de morbilidad.

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model.

The gene encoding PTPROt (truncated isoform of protein tyrosine phosphatase receptor-type O) is methylated and suppressed in chronic lymphocytic leukemia (CLL). PTPROt exhibits in vitro tumor-suppressor characteristics through the regulation of B-cell receptor (BCR) signaling. Here we generated transgenic (Tg) mice with B-cell-specific expression of PTPROt. Although lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared with the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice, suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt-mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of BCR signaling. These results establish the in vivo tumor-suppressive function of PTPROt and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3.

To identify microRNAs (miRNAs) that may have a causal role in hepatocarcinogenesis, we used an animal model in which C57BL/6 mice fed choline-deficient and amino acid defined (CDAA) diet develop preneoplastic lesions at 65 weeks and hepatocellular carcinomas after 84 weeks. miRNA expression profiling showed significant upregulation of miR-181b and miR-181d in the livers of mice as early as 32 weeks that persisted at preneoplastic stage. The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor (TGF)beta and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented on exposure of hepatic cells to TGFbeta and were significantly reduced by small interference RNA-mediated depletion of Smad4, showing the involvement of TGFbeta signaling pathway in miR-181b expression. Ectopic expression and depletion of miR-181b showed that miR-181b enhanced matrix metallopeptidases (MMP)2 and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of hepatocellular carcinoma (HCC) cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anticancer drug doxorubicin. On the basis of these results, we conclude that upregulation of miR-181b at early stages of feeding CDAA diet promotes hepatocarcinogenesis.

Influenza virus infection induces metallothionein gene expression in the mouse liver and lung by overlapping but distinct molecular mechanisms.

Metallothionein I (MT-I) and MT-II have been implicated in the protection of cells against reactive oxygen species (ROS), heavy metals, and a variety of pathological and environmental stressors. Here, we show a robust increase in MT-I/MT-II mRNA level and MT proteins in the livers and lungs of C57BL/6 mice exposed to the influenza A/PR8 virus that infects the upper respiratory tract and lungs. Interleukin-6 (IL-6) had a pronounced effect on the induction of these genes in the liver but not the lung. Treatment of the animals with RU-486, a glucocorticoid receptor antagonist, inhibited induction of MT-I/MT-II in both liver and lung, revealing a direct role of glucocorticoid that is increased upon infection in this induction process. In vivo genomic footprinting (IVGF) analysis demonstrated involvement of almost all metal response elements, major late transcription factor/antioxidant response element (MLTF/ARE), the STAT3 binding site on the MT-I upstream promoter, and the glucocorticoid responsive element (GRE1), located upstream of the MT-II gene, in the induction process in the liver and lung. In the lung, inducible footprinting was also identified at a unique gamma interferon (IFN-gamma) response element (gamma-IRE) and at Sp1 sites. The mobility shift analysis showed activation of STAT3 and the glucocorticoid receptor in the liver and lung nuclear extracts, which was consistent with the IVGF data. Analysis of the newly synthesized mRNA for cytokines in the infected lung by real-time PCR showed a robust increase in the levels of IL-10 and IFN-gamma mRNA that can activate STAT3 and STAT1, respectively. A STAT1-containing complex that binds to the gamma-IRE in vitro was activated in the infected lung. No major change in MLTF/ARE DNA binding activity in the liver and lung occurred after infection. These results have demonstrated that MT-I and MT-II can be induced robustly in the liver and lung following experimental influenza virus infection by overlapping but distinct molecular mechanisms.

Downregulation of constitutive and heavy metal-induced metallothionein-I expression by nuclear factor I.

Although the existence of repressor protein(s) involved in the regulation of highly inducible metallothionein-I (MT-I) gene expression has been postulated, none has been identified to date. We considered nuclear factor I (NFL) protein as a potential repressor, as three half-sites for NFI binding are present on MT-I promoter and NFI is known to downregulate several cellular gene promoters. Overexpression of all four isoforms of mouse NFI protein (NFI-A, -B, -C, and -X) suppressed both constitutive and heavy metal-induced activation of the MT-I promoter in HepG2 cells. However, unlike other target genes of NFI, direct interaction of NFI with MT-I promoter is not necessary to mediate its repression. Point mutation of the NFI binding sites within the MT-I promoter that abrogates NFI binding in vitro could not alleviate the repression. Similarly, NFI proteins also repress activity of minimal MT-I promoter deficient in the NFI binding sites. Further, an NFI-C deletion mutant lacking the DNA binding domain continued to repress MT-I promoter. Overexpression of MTF-1, the key trails-acting factor involved in MT-I gene transcription, surmounted NFI-mediated repression of the basal and zinc-induced MT-I promoter activity. These data demonstrate that NFI is a repressor of MT-I expression, where its DNA binding activity is not essential to downregulate the MT-I promoter. Interaction of NFI with another protein(s), probably MTF-I, may be involved in this repression.

Regulation of metallothionein gene expression.

The rapid and robust induction of metallothioneins (MT)-I and II by a variety of inducers that include heavy toxic metals, reactive oxygen species, and different types of stress provide a useful system to study the molecular mechanisms of this unique induction process. The specific expression of MT-III in the brain and of MT-IV in the squamous epithelium of skin and tongue offers a unique opportunity to identify and characterize the tissue-specific factors involved in their expression. Studies using transgenic mice that overexpress MTs or MT null mice have revealed the role of MT in the protection of cells against numerous tissue-damaging agents such as reactive oxygen species. The primary physiological function of these proteins, however, remains an enigma. Considerable advances have been made in the identification of the cis-acting elements that are involved in the constitutive and induced expression of MT-I and MT-II. By contrast, only one key trans-activating factor, namely MTF-1, has been extensively characterized. Studies on the epigenetic silencing of MT-I and MT-II by promoter hypermethylation in some cancer cells have posed interesting questions concerning the functional relevance of MT gene silencing, the molecular mechanisms of MT suppression in these cells, particularly chromatin modifications, and the characteristics of the repressors.

Suppression of metallothionein gene expression in a rat hepatoma because of promoter-specific DNA methylation.

Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylated MT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanning MT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors.

Silencing of metallothionein-I gene in mouse lymphosarcoma cells by methylation.

Metallothionein-I (MT-I) gene is silenced by methylation of CpG islands in mouse lymphosarcoma P1798 cells but not in the thymus, the cell type from which the tumor was derived. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides present within -216 bp to +1 bp with respect to transcription start site are methylated in the tumor cell line, but none is methylated in the thymus. The lymphosarcoma cells induced MT-I in response to heavy metals only after demethylation with 5-azacytidine (5-AsaC). The electrophoretic mobility shift assay using specific oligonucleotide probes showed that the key transcription factors regulating MT-I gene (e.g., MTF-1, Sp 1 and MLTF/USF) are active in P1798 cells. In vivo footprinting of the proximal promoter region showed that none of the metal regulatory elements (MREs) or MLTF/USF are occupied in response to heavy metals. Demethylation of the lymphosarcoma cells with 5-AzaC resulted in constitutive footprinting at MLTF/ARE, and zinc-inducible footprinting at MRE-c, MRE-d and MRE-e sites. Demethylation of just 10-20% of the CpG islands was sufficient to render the gene inducible by cadmium or zinc. The MT-I induction persisted in the cancer cells for several generations even after withdrawal of 5-AzaC from the culture medium.

Transcriptional induction of metallothionein-I and -II genes in the livers of Cu,Zn-superoxide dismutase knockout mice.

The levels of metallothionein-I and -II (MT-I and MT-II) mRNAs were elevated (10- to 12-fold), specifically in the livers of mice with homozygous deletion of the gene for Cu,Zn-SOD (Sod1-/-), the enzyme that catalyzes the removal of O(-)(2). The induction of MT mRNA occurred primarily at the level of transcription. In vivo genomic footprinting of the MT-I promoter region revealed distinctive footprinting at MRE-d, MRE-c, and MLTF/ARE sites in the livers of knockout mice. MTF-1, the key factor responsible for the heavy-metal and oxidative stress-induced expression of the MT-I gene, was activated 3-fold in the nuclear extract from the livers of Cu,Zn-SOD null mice. Because metallothioneins are potent scavengers of reactive oxygen species and protect cells from oxidative stress, the apparent normal characteristics of the mice with the disrupted Cu, Zn-SOD gene are probably due to overexpression of MT-I and MT-II in the livers of these animals.

Hypermethylation of metallothionein-I promoter and suppression of its induction in cell lines overexpressing the large subunit of Ku protein.

We have shown previously that the heavy metal-induced metallothionein-I (MT-I) gene expression is specifically repressed in a rat fibroblast cell line (Ku-80) overexpressing the 80-kDa subunit of Ku autoantigen but not in cell lines overexpressing the 70-kDa subunit or Ku heterodimer. Here, we explored the molecular mechanism of silencing of MT-I gene in Ku-80 cells. Genomic footprinting analysis revealed both basal and heavy metal-inducible binding at specific cis elements in the parental cell line (Rat-1). By contrast, MT-I promoter in Ku-80 cells was refractory to any transactivating factors, implying alteration of chromatin structure. Treatment of two clonal lines of Ku-80 cells with 5-azacytidine, a potent DNA demethylating agent, rendered MT-I gene inducible by heavy metals, suggesting that the gene is methylated in these cells. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides in MT-I immediate promoter were methylated in Ku-80 cells, whereas only four CpG dinucleotides were methylated in Rat-1 cells. Almost all methylated CpG dinucleotides were demethylated in Ku-80 cells after 5-azacytidine treatment. To our knowledge, this is the first report that describes hypermethylation of a specific gene promoter and its resultant silencing in response to overexpression of a cellular protein.

Induction of metallothionein by stress and its molecular mechanisms.

This article describes the effect of restraint stress or social reorganization stress on the induction of metallothionein (MT) in the liver, heart, lung, and spleen. Both MT-I and MT-II mRNA were elevated as much as 30-fold following just 12 h (one cycle) of restraint stress. The amount of MT protein also increased following stress. The MT induction was the highest in the liver, followed by the lung, heart, and spleen. MT-I induction was also observed in the fore, mid, and hind regions of the brain whereas the brain-specific MT-III gene was not activated by stress. The increase in MT mRNA correlated well with the rise in stress-induced serum corticosterone. The induction occurred at the transcriptional level and was mediated essentially by the activation of glucocorticoid receptor. The MT mRNA returned to the control level after nine cycles of stress. Exposure of these habituated mice to a different type of stress (treatment with heavy metals such as cadmium or zinc sulfate) led to further MT induction. Because heavy metals induced MT via activation of the factor MTF-1, distinct molecular mechanisms should be responsible for the activation of MT promoter by different inducers.

Metallothionein induction in response to restraint stress. Transcriptional control, adaptation to stress, and role of glucocorticoid.

Metallothioneins (MT) have been implicated in the protection of cells from oxidative stress. We studied the molecular mechanism of induction of MT-I and MT-II in response to restraint stress using a mouse model system in which the animals were restrained in well ventilated polypropylene tubes for 12 h each day (one cycle). Here, we show that MT-I and MT-II mRNA levels were elevated as much as 10-20-fold after just one cycle of this simple stress. Stress-mediated MT induction occurred at the transcriptional level. The level of MT mRNA correlated with the stress-induced increase, and not with the diurnal variation, in the level of serum glucocorticoid. Treatment of the mice with RU 486, a glucocorticoid receptor antagonist, prior to restraint stress inhibited MT induction by at least 50%. Furthermore, the glucocorticoid responsive element-binding activity in the liver nuclear extracts from the stressed mice was significantly higher than that in the control mice. The complex formations between the transcription factor Sp1, MTF1, or MLTF/ARE and the respective specific oligonucleotides were not altered in the liver from the stressed mouse. The MT mRNA levels returned to the basal level at the end of nine cycles of stress, indicating habituation of the animals to restraint stress. At this stage, exposure of the animals to another type of stress, treatment with heavy metals, resulted in further induction of MT. These data indicate that glucocorticoid is the primary physiological factor responsible for MT induction following restraint stress, and the glucocorticoid receptor is the major transcription factor involved in this process.

Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals.

Metallothioneins (MT) are involved in the scavenging of the toxic heavy metals and protection of cells from reactive oxygen intermediates. To investigate the potential role of the protein Ku in the expression of MT, we measured the level of MT-I mRNA in the parental rat fibroblast cell line (Rat 1) and the cell lines that stably and constitutively overexpress the small subunit, the large subunit, and the heterodimer of Ku. Treatment with CdS04 or ZnS04 elevated the MT-I mRNA level 20- to 30-fold in the parental cells and the cells (Ku-70) that overproduce the small subunit or those (Ku-7080) overexpressing the heterodimer. By contrast, the cells (Ku-80) overexpressing the large subunit of Ku failed to induce MT-I. In vitro transcription assay showed that the MT-I promoter activity was suppressed selectively in the nuclear extracts from Ku-80 cells. The specificity of the repressor function was shown by the induction of hsp 70, another Cd-inducible gene, in Ku-80 cells. Addition of the nuclear extract from Ku-80 cells at the start of the transcription reaction abolished the MT-l promoter activity in the Rat 1 cell extract. The transcript once formed in Rat 1 nuclear extract was not degraded by further incubation with the extract from Ku-80 cells. The repressor was sensitive to heat. The DNA-binding activities of at least four transcription factors that control the MT-I promoter activity were not affected in Ku-80 cells. These observations have set the stage for further exploration of the mechanisms by which the Ku subunit mediates suppression of MT induction.

Distinct rat proteins can recognize CCAAT-homologous sequences of the metallothionein promoter and trans-activate this promoter.

We have previously purified and characterized a rat liver protein C'BP-1 that is either identical or closely related to C/EBPdelta (Aniskovitch and Jacob, 1997). The mouse metallothionein-I (MT-I) promoter contains two C'BP-1 binding sites, one of which includes the MRE-c' region (-135 to -110). The C'BP-1 binding activity was detected by EMSA as a major activity for MRE-c' in nonproliferating adult liver cells but not in rat hepatoma cells. In this study, we purified and characterized a factor, C'BP-2, which had a dominant binding activity for MRE-c' in Morris hepatoma 3924A, a poorly differentiated, fast-growing tissue. C'BP-2 is a 28 kDa protein which exists in solution as a monomer. As observed for C'BP-1, affinity-purified C'BP-2 stimulated transcription from the mMT-I gene promoter. DNase I footprinting revealed two C'BP-2 binding sites in the regions that overlap with the CCAAT homologies of the C'BP-1 binding sites on the mMT-I promoter. C'BP-2 made essential contacts with the CCAAT homology and in the region upstream of this sequence. Competition electrophoretic mobility shift assay and methylation interference analysis revealed that C'BP-2 is a protein closely related, but not identical, to CP2. These data suggest that C'BP-1 and C'BP-2 may play a role in hepatocyte proliferation and/or differentiation.

An alternative molecular mechanism of action of 5-fluorouracil, a potent anticancer drug.

It is assumed that the primary mode of action of 5-fluorouracil (5-FUra) is mediated via inhibition of thymidylate synthetase. Persistent inhibition of cellular proliferation after treatment of the 5-FUra-inhibited cells with exogenous thymidine do not support the notion that the anti-proliferitive action of 5-FUra is due exclusively to inhibition of DNA replication. Our studies have revealed an alternative mechanism of action at the level of pre-ribosomal RNA (pre-rRNA) processing. Pre-rRNA processing was inhibited completely in vitro as well as in S-100 extract from the mouse lymphosarcoma P1798 cells that were treated with 5-FUra. Under this condition, the 5-FUra-substituted pre-rRNA substrate was processed efficiently at the primary processing site. This study showed that the activity and/or the synthesis of a factor potentially involved in pre-rRNA processing is blocked in cells treated with the fluoropyrimidine. UV-cross-linking study showed that a 200 kDa polypeptide designated ribosomal RNA binding protein (RRBP) was absent in the S-100 extract from the drug-treated mouse lymphosarcoma cells. Since a polypeptide that cross-links to a processing site on RNA is usually involved in the RNA processing, RRBP may have a direct role in pre-rRNA processing. A key molecular mechanism far the antiproliferative action of 5-FUra may be due to its interference with the activity and/or synthesis of RRBP. Exposure of cells to 5-FUra did not inhibit the interaction between U3 small nucleolar RNA (snoRNA) and pre-rRNA at the primary processing site (a key step in the processing reaction) and the formation of U3 small nucleolar ribonucleoprotein (snoRNP). Treatment of cells with the fluoropyrimidine did not block the 3' end processing of pre-messenger RNA (pre-mRNA). This article also discusses the effects of 5-FUra on pre-mRNA splicing and mRNA translation, and proposes other avenues of research to explore further the mechanism of action of this important pyrimidine analog.

Purification and characterization of a rat liver protein that recognizes CCAAT-homologous sequences of the metallothionein promoter and trans-activates this promoter.

C'BP-1, a protein that binds to the MRE-c' region (-135 to -110) of the mouse metallothionein-I (MT-I) gene in metal-independent manner, was purified from rat liver nuclear extract by ion exchange and affinity chromatography. Analysis by SDS-PAGE, UV cross-linking, and glycerol gradient sedimentation, taken together, showed that C'BP-1 is a dimer of the 34-kDa polypeptides. Affinity-purified C'BP-1 could significantly stimulate transcription from mouse MT-I gene promoter. DNase I footprinting with the purified protein identified two binding sites for C'BP-1 located at positions -135 to -100 and -210 to -175 with respect to the start site of MT-I gene transcription. Both C'BP-1 binding sequences were found to contain imperfect dyad of the CCAAT homology. C'BP-1 was shown to make critical contacts with the CCAAT homology by methylation interference analysis and competition electrophoretic mobility shift assay with mutants harboring alterations in the CCAAT homology. An antibody that specifically recognizes C/EBP delta partially supershifted C'BP-1/MRE-c' complex, suggesting that C'BP-1 is identical to C/EBP delta or is closely related to C/EBP delta.