Trevisan, Marta (2008) Analisi dell'effetto dell'infezione da citomegalovirus umano su cellule corticosurrenaliche. [Ph.D. thesis]
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Aim of the study: HCMV has been reported to involve the adrenal gland and cause adrenalitis in immunosuppressed patients, but information in literature about the effect of human cytomegalovirus (HCMV) infection on adrenal function and its potential role in adrenal tumorigenesis is lacking. Aim of this study is to investigate the presence of HCMV in a large series of normal and tumor adrenal samples and to analyze whether HCMV infects and replicates in adrenocortical cells in vitro and the effects of HCMV infection on adrenocortical steroidogenesis, cell growth, and gene expression profile.
Materials and Methods: The clinical investigation was performed on a total of 127 human adrenal tissues, which were examined by real-time PCR for the presence of HCMV genome sequences. Adrenal samples included 20 normal adrenal tissues, 36 non-functioning adrenocortical adenomas, 16 cortisol producing adenomas, 12 aldosteronomas, 3 androgen producing adenomas, 16 adrenocortical carcinomas (ACCs), and 24 medullary tumors.
The in vitro study was performed on the human ACC cell lines NCI-H295R and SW-13 and in primary ACC cell cultures, which were infected with clinical isolates of HCMV and with the HCMV strain AD169. Expression of the HCMV early pp72 and late pp65 genes was evaluated in infected cells by immunofluorescence. Kinetics of viral replication in ACC cells was investigated by quantitative real-time PCR and plaque assay. Steroid hormone production in the supernatant of infected cells was measured by EIA. Evaluation of the impact of HCMV infection on host cell machinery was investigated in terms of cytopathic effect (microscope exhamination), cell cycle (analyzed by propidium iodide staining), apoptosis (through annexin-V test), and cellular proliferation (by BromoDeossiUridine test). Cellular gene expression profile after AD169 infection was assessed by quantitative RT-PCR and DNA microarray analysis in timecourse experiments.
Results: Investigation of human adrenal tumors demonstrated the presence of HCMV genome sequences in 19% cortical adenomas, 12% pheochromocytomas and 1 normal tissue, but not in malignant tumors. Moreover, HCMV sequences were significantly more frequently detected and at higher titre in functioning than in nonfunctioning adrenocortical tumors. In these tumors, expression of both early (pp72)and late (pp65) HCMV genes was demonstrated by immunohistochemistry.
The in vitro study showed that both clinical isolates and laboratory strains of HCMV could replicate in ACC cells, as demonstrated by expression of early and late viral antigens and efficient production of infectious viral particles in infected adrenocortical cells, even if at lower levels than in fibroblasts. HCMV had a cytopathic effect on SW13 and NCI-H295R ACC cells, outlined by the presence of apoptotic and vacuolized cells. Furthermore, it slightly: increased cellular S phase, inhibited cell proliferation, and induced apoptosis. The proapoptotic effect was markedly increased by co-treatment with etoposide, a topoisomerase II inhibitor.
AD169 infection of NCI-H295R cells, which are able to produce all adrenal steroids, significantly increased production of cortisol and 17?-estradiol, but not of aldosterone. Conceivably, this effect was due to viral replication and/or viral gene expression rather than interaction of the virion with the host cell, since UVinactivated HCMV particles had not effect on steroidogenesis. Also, AD169 infection of NCI-H295R cells, markedly induced CYP11B1 and CYP19 expression, e.g., the genes encoding the key enzymes for cortisol and estrogens synthesis, and modulated hormone receptors DAX-1 and SF-1, that are involved in the transcriptional regulation of steroidogenic enzymes. Thus, the effect of HCMV infection on adrenal steroidogenesis seem to be mediated by induction of steroidogenic enzyme expression. Microarray analysis was employed to further elucidate the mechanism of induction of steroid hormone production. Interestingly, besides inducing genes promoting cell proliferation and tumor invasiveness and repressing tumor suppressor genes and pro-angiogenetic genes, in agreement with its oncomodulatory effects, HCMV infection markedly induced a cluster of 12 genes in the first hours post-infection. Bayesian Gaussian Network analysis identified two crucial genes in the Bayesian Network structure, i.e., the genes encoding the purinergic receptors P2Y11 and P2X4, which play the role of dam genes controlling the modulation of the others. Since purinergic receptors are known to mediate induction of corticol production, upregulation of these two genes could be a key event responsible of the effects of HCMV infection on adrenal steroidogenesis.
To investigate the effects of steroid hormones on HCMV replication, fibroblasts and ACC cells were treated during infection with hydrocortisone and with the inhibitor of steroidogenic enzymes aminoglutethimide. Hydrocortisone increased HCMV replication and yield in both fibroblasts and ACC cells, whereas aminoglutethimide enhanced viral release for infected cells.
Conclusions: The results of this study conducted with clinical specimens suggests that the adrenal gland could be a reservoir of HCMV and that hormone overproduction by the adrenal gland could represent a trigger for virus reactivation. These results of the in vitro study demonstrate for the first time that HCMV infects and replicates in human adrenocortical cells. HCMV infection stimulates adrenal cortisol and estrogen production by modulation of steroidogenic enzyme expression. These data represent the basis for further investigation on the implication of HCMV infection on adrenal diseases.
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