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1.
Brain Res ; 1034(1-2): 51-61, 2005 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-15713259

RESUMO

GADD34, a stress response protein associated with cell rescue, DNA repair and apoptosis, is expressed in the ischaemic brain. The C-terminal region of GADD34 has homology with the Herpes Simplex Virus protein, ICP34.5, which overcomes the protein synthesis block after viral infection by actively dephosphorylating eukaryotic translation initiation factor 2alpha (eIF2alpha). The carboxy terminus of GADD34 is also capable of dephosphorylating eIF2alpha and therefore has the capacity to restore the protein synthesis shutoff associated with ischaemia. This study examines the distribution and time course of GADD34 expression after focal cerebral ischaemia. Focal ischaemia or sham procedure was carried out on Sprague-Dawley rats with survival times of 4, 12, 24 h, 7 and 30 days. Brains were processed for histology and immunohistochemistry. Ischaemic damage was mapped onto line diagrams and GADD34 positive cells counted in selected regions of cortex and caudate. GADD34 immunopositive cells (mainly neurones), expressed as cells/mm2, were present in ischaemic brains at 4 h (e.g., peri-infarct cortex 20 +/- 5; contralateral cortex 3 +/- 1, P < 0.05). Of the time points examined, numbers of GADD34 positive cells were highest 24 h after ischaemia (peri-infarct cortex 31 +/- 7.3, contralateral cortex 0.1 +/- 0.1, P < 0.05). Immunopositive cells, following a similar time course, were identified within the peri-infarct zone in the caudate nucleus and in ipsilateral cingulate cortex (possibly as a consequence of cortical spreading depression). GADD34 positive cells did not co-localise with a marker of irreversible cell death (TUNEL). Taken together, GADD34 positive cells in key neuroanatomical locations pertinent to the evolving ischaemic lesion, the lack of co-localisation with TUNEL and the protein's known effects on restoring protein synthesis, repairing DNA and involvement in ischaemic pre-conditioning suggests that it has the potential to influence cell survival in ischaemically compromised tissue.


Assuntos
Isquemia Encefálica/enzimologia , Encéfalo/enzimologia , Infarto Cerebral/enzimologia , Degeneração Neural/enzimologia , Neurônios/enzimologia , Proteínas/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Mapeamento Encefálico , Núcleo Caudado/enzimologia , Núcleo Caudado/patologia , Núcleo Caudado/fisiopatologia , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Reparo do DNA/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Giro do Cíngulo/enzimologia , Giro do Cíngulo/patologia , Giro do Cíngulo/fisiopatologia , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Neocórtex/enzimologia , Neocórtex/patologia , Neocórtex/fisiopatologia , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Regulação para Cima/fisiologia
2.
J Gene Med ; 5(8): 681-9, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12898637

RESUMO

BACKGROUND: The neurotropic herpes simplex virus mutant HSV1716 lacks the gene encoding the virulence factor ICP34.5 and cannot replicate in non-dividing cells where proliferating cell nuclear antigen (PCNA) is not actively engaged in cellular DNA synthesis. In the brain, tumoral expression of PCNA therefore confers on it oncolytic specificity and may determine its efficacy. Three phase I trials in glioma patients and one in metastatic melanoma patients have established that HSV1716 is safe and replicates selectively in tumour tissue. Here we examine the in situ PCNA profiles of common human metastatic brain tumours and determine their in vitro permissivity for HSV1716 replication to ascertain their suitability for HSV1716 therapy. METHODS: Histological sections of tumour biopsies obtained from patients undergoing craniotomies were stained for PCNA expression. The replicative ability of HSV wild-type (17(+)) and mutant (1716) viruses was assessed in tissue cultures of the same tumour biopsies and in cancer cell lines by plaque assay. RESULTS: Biopsies of all 10 metastatic tumours (3 melanoma, 4 carcinoma and 3 adenocarcinoma) as well as 4 glioblastoma multiforme were positive for PCNA immunoexpression and supported the replication of HSV1716. The PCNA-positive cells in the metastatic tumours were distributed comparatively in larger and more contiguous areas than in glioblastoma (1.69 +/- 1.61 mm(2) vs. 0.73 +/- 0.77 mm(2)) and numbered 29.0 +/- 12.4 and 12.6 +/- 4.7%, respectively. CONCLUSIONS: The results show that human cerebral metastatic tumours have generally larger and more contiguous proliferative areas, support efficient HSV1716 replication, and are thus potential candidates for such oncolytic viral therapy.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Simplexvirus/fisiologia , Replicação Viral/fisiologia , Adulto , Idoso , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Feminino , Terapia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
J Neurovirol ; 9(4): 477-88, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12907392

RESUMO

The replicative ability of ICP34.5-null herpes simplex virus (HSV) is cell type and state dependent. In certain cells, ICP34.5 interacts with protein phosphatase 1 to preclude host cell protein synthesis shutoff by dephosphorylation of the eukaryotic initiation factor eIF-2alpha. However, host cell shutoff is not induced by ICP34.5-null HSV in most cells, irrespective of type and state. In general, dividing cells support replication of ICP34.5-null HSV; nondividing cells cannot. Previously the authors showed that ICP34.5 binds to proliferating cell nuclear antigen (PCNA), a protein necessary for cellular DNA replication and repair. Here the authors demonstrate that (1) the interaction between ICP34.5 and PCNA involves two regions of the virus protein; (2) ICP34.5 forms a complex with HSV replication proteins that is DNA binding; (3) at early times in infection, ICP34.5 colocalizes with PCNA and HSV replication proteins in cell nuclei, before accumulating in the cytoplasm; and (4) ICP34.5 is a virion protein. In light of ongoing clinical trials assessing the safety and efficacy of ICP34.5-null HSV, it is vital that the roles of ICP34.5 in HSV replication are understood. The authors propose that in nondividing cells, ICP34.5 is required to switch PCNA from repair to replication mode, a prerequisite for the initiation of HSV replication.


Assuntos
Herpes Simples/virologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Simplexvirus/crescimento & desenvolvimento , Simplexvirus/metabolismo , Proteínas Virais/metabolismo , Animais , Anticorpos , Western Blotting , Núcleo Celular/virologia , Células Cultivadas , Cricetinae , Citoplasma/virologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Rim/citologia , Simplexvirus/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Vírion/metabolismo , Replicação Viral/fisiologia
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