Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40（SV40） is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine-homopurine duplex DNA（dsDNA） of SV40（4424-4440, gp6） was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5＇-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3＇（HP） containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structttre to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.

Immortalization of Human Precartilaginous Stem Cells by Transfecting SV40Tag

Immortalized human precartilaginous stem cells （1PSCs） were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene （SV40Tag） was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southem blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells （IPSCs）, which were confirmed as fibroblast growth factor receptor-3 （FGFR-3） positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

EXPRESSION OF SV40 Tag AND FORMATION Tag-p53 AND Tag-Rb COMPLEXES IN CHINESE BRAIN TUMORS

Objective: To investigate the expression of SV40 Tag and formation of Tag-p53 and Tag-Rb complexes in Chinese brain tumors. Methods: SV40 large tumor antigen (Tag) were investigated by immunoprecipitation, silver staining and Western blot in 65 cases of Chinese brain tumors and 8 cases of normal brain tissues. Tag-p53 and Tag-Rb complexes were screened by the same way in 20 and 15 Tag positive tumor tissues respectively. Results: Tag was found in all of 8 ependymomas and 2 choroid plexus papillomas, 90% (9/10) of pituitary adenomas, 73% (11/15) of astrocytomas, 70% (7/10) of meningiomas, 50% (4/8) of glioblastoma multiform, 33% (2/6) of medulloblastomas, 5 oligodendrogliomas, 1 pineocytoma and 8 normal brain tissues were negative for Tag. Tag-p53 complex was detected in all of 20 Tag positive tumors as well as Tag-Rb complex in all of 15 Tag positive tumors. Conclusion: SV40 Tag is not only expressed in human brain tumors, but also it can form specific complexes with tumor suppressors p53 and Rb. SV40 is correlated to human brain tumorigenesis. The inactivation of p53 and Rb due to the formation of Tag-p53 and Tag-Rb complexes is possibly an important mechanism in the etiopathogenesis of human brain tumors.

Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.

Roles of dysregulated Notch pathway and small DNA tumor viruses in cancer initiation and progression

Notch pathway is a major determinant of cell fate,and research within the last 30 years has shown dysfunctions within this pathway in the majority of solid tumors and leukemias.The molecular mechanisms causing aberrant expression of Notch in cancer are still partially known.Mesotheliomas,breast,and cervical cancers are among the cancer types for which the dysregulation of Notch has been reported together with the association of simian virus 40(SV40)or human papilloma virus(HPV)infections.In mesotheliomas and cervical cancer,there is clear evidence that these viruses cause and rely on dysregulation of the Notch pathway to promote and sustain cell transformation.The existence of a relationship in tumors between DNA viruses and Notch could have an impact on cancer therapy by implementing Notch inhibition to interfere with the growth of SV40-and HPV-positive cancers.In addition,since Notch links innate and acquired immunity and plays a key role in the regulation of the anti-viral response,targeting Notch in the presence of oncogenic viruses infections may help prevent the onset and progression of cancers associated with the exposure to these viruses.

Construction of an immortalized neural progenitor cell strain and analysis of its immunogenicity

Neural progenitor cells(NPC)are those that are the source of neural cells for cell transplantation and gene therapy.The shortage in quantity and the limited life spans of primary cultured cells limit its widespread use in basic research.Immortalized NPC,which also possess the capacity of self-renewal and can proliferate infinitely,can produce a large number of NPCs with stable phenotype and genotype.Here we report that an immortalized neural progenitor cell strain,which we named as INPC,was suc-cessfully established by gene-transfer of simian virus 40 large T antigen gene mediated by liposomes.The INPC retained the biological characteristics of its original cells and provided a safe and reliable cell platform for the treat-ment of central nervous system diseases and transgenic cell transplantation.INPC could express low levels of MHC antigens which was down-regulated after differentiation.This indicates that INPC possesses poor immunogenicity.The immortalized cells may show good long-term survival and do not elicit an acute immunological response follow-ing transplantation.