BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

AIM: To study the effect of Qingqi Liangying Injection on febrile rats induced by zymonsa and on content of PGE_2 as well as cAMP in rat hypothalamus. METHODS: The rat febrific model induced by 10% zymonsa solution was used to observe QingqiLiangying Injection's effect on the rat temperature, and the radioimmunoassay(RIA) was used to determin the content of PGE_2 and cAMP in hypothalamus of rats. RESULTS: Qingqi Liangying Injection had an obvious antipyretic effect on febrile rats; Qingqi Liangying Injection could significantly reduce the content of PGE_2 and cAMP. CONCLUSION: Qingqi Liangying Injection can obviously reduce the rat body temperature and the content of PGE_2 and cAMP in hypothalamus of rats.

Objective To investigate the role of TLR9 in the glomerulonephritis through observing the changes of Toll-like receptor 9 in the glomerulo nephritis kidney tissue with or without CpG-ODN stimulation .Methods Wistar male rats were randomly divided into normal group(N),nephritis model group(M),CpG-ODN group(CpG) and GpC-ODN group(GpC).The urine samples were collected at 2,4,6 and 8 weeks after treatment,respectively.Blood samples were collected at the end of the last urine sample was collected,and the kidney tissue was collected,then the rats were killed.24 h urine protein was measured by Coomassie light blue technique.Serum album and renal function were determined by serological method.The pathologic changes of kidney were observed by light microscope and NF-?B p65 expression was detected using immunohistochemystry,RT-PCR was performed to detect the expressions of TLR9,INF-? and IL-6 mRNA.Results The expression of TLR9 was lighter in group M,and significantly increased after CpG-ODN stimulation compared with group M.Furthermore,24 h urine protein excretion was markedly increased,serum album was markedly decreased.The histopathologic changes of kidney were more severe.The mesangial cells(MCs) proliferated diffusifully in midrange and wide range,some of the glomeruli formatted cellularity crescent,micrangium loop was limitted,mononuclear macrophile cells were seen in the mesangial region.Conclusion Inflammatory factors mediated by TLR9 can deteriorate the biochemical and histopathologic changes.The immunologic reaction mediated by TLR9 is one of the mechanisms for the glomerulonephtitis' progression.