Since methylation selleck screening library of the RASSF1A promoter is described as an early and frequent event in tumorigenesis, it could serve as a useful diagnostic signal in cancer screens. Previous studies suggested that RASSF1A may implicate in various cellular mechanisms including cell cycle arrest, apoptosis, inhibition of cell proliferation in vitro [14–17] as well as repression of tumor formation
in nude mice , however, little is known about the underlying mechanisms of RASSF1A. The most interesting structure feature of RASSF1A proteins is the presence of a Ras association (RA) domain, which determines the role of RASSF1A protein functions as a Ras-effector, and endows RASSF1A the ability to interact with Ras family protein. The Idasanutlin in vitro Ras proteins are intimately involved in the regulation of a wide variety of biological processes by interacting with different downstream effectors. Although it is widely accepted that the Ras functions as an oncoprotein that contribute to cell proliferation through the RAS-MAP-kinase pathway and antiapoptotic effect, more and more studies found that it also induces growth arrest of cells,
such as apoptosis and senescence by interact with specific effectors . RASSF1A, act as a newly discovered downstream negative effector of Ras protein, may interact with Ras protein in a GTP-dependent manner and induce a potent, Ras-mediated apoptosis . In this study, we characterized the hypermethylation status of promoter of RASSF1A in NPC tumor biopsies and normal nasopharyngeal epithelia. Growth inhibition effect including cell cycle arrest, apoptosis and senescence was also observed in CNE-2 cells that were transfected with exogenous RASSF1A gene. Furthermore, we have initiated to figure out whether this tumor suppression effect of RASSF1A could
be enhanced in the presence of activated Ras. Materials and methods NPC cell lines and tissue samples Two NPC cell lines, CNE1 and CNE2 were maintained in RPMI 1640 supplemented with 10% fetal bovine Cell press serum at 37°C. A total of 38 primary tumor biopsies cases were obtained from newly diagnosed and untreated NPC patients with consent and 14 samples of normal nasopharyngeal epithelial tissues were obtained from the suspected patients as normal controls at the department of otolaryngology at the Union Hospital of Tongji Medical College (Wuhan, China). All of the specimens were subjected to histological diagnosis by pathologists according to the WHO selleck chemicals llc classification. Relative data involving age, gender, clinical stage, lymph node metastasis and distance metastasis were collected after the patients visiting. High-molecular weight DNA was extracted from the samples using DNA extract kit (Tiangen) according to the manufacture’s instructions. RT-PCR Total RNAs from cell lines, normal nasopharyngeal epithelia and tumor biopsies was isolated with TriZOL regent (Huashun biotechnology).