The data are expressed as the percentage of invasion. The stained nuclei on 6 randomly selected microscopic fields for each sample were counted, and the quantification of migrated cells was done in 3 independent experiments. The cells that migrated to the lower side of the filter were then fixed with 100% methanol for 2 minutes and stained with 0.1% crystal violet. Noninvading or nonmigrating cells were removed from the upper side of the Matrigel chamber by gently but firmly swabbing with cotton tips. The chambers were then incubated for 24 hours at 37☌, in 5% CO 2.
#Superoxide d splitt for free
The same number of cells was also seeded in the control chamber containing an identical polycarbonate membrane filter without Matrigel, for free migration and viability testing. The bottom chamber was filled with MEM supplemented with 10% FBA. Briefly, following transfection, 5 × 10 4 cells were added to the top chamber containing a polycarbonate membrane filter of 8-μm pore size with rehydrated Matrigel. Materials and MethodsĬellular invasion into Matrigel was done with the BD BioCoat Matrigel Invasion Chamber (BD Biosciences). As the tumor progresses and p53 activity is lost, MnSOD levels increase again, creating conditions in which cancer cells can survive under oxidative stress. When a tumor begins to develop, the modulation of stress-sensitive transcription factors, including p53, creates an environment of high oxidative stress achieved by the suppression of MnSOD. The data support a model in which activation of p53 by carcinogens leads to a reduction in MnSOD that precedes the appearance of cancer.
The loss of Sp1 function exacerbates p53 siRNA–mediated cell transformation but not invasion. Using siRNA-knockdown approaches coupled with in vitro tumor invasion assay, we also show that suppression of p53 alone led to increased invasion. Here, we evaluated changes in MnSOD expression during the course of tumorigenesis and show that MnSOD expression was suppressed in early tumorigenic stages but was restored later in advanced carcinoma. p53 is mutated in at least 50% of tumors, and the wild-type p53 tumor suppressor is completely lost in many tumor tissues ( 18, 19). P53 is a transcriptional regulator with tumor suppression capability. These findings also provide strong support for the development of means to reactivate p53 for the prevention of tumor progression. The results identify MnSOD as a p53-regulated gene that switches between early and advanced stages of cancer. We used chromatin immunoprecipitation, electrophoretic mobility shift assay, and both knockdown and overexpression of Sp1 and p53 to verify their roles in the expression of MnSOD at each stage of cancer development. In squamous cell carcinomas, Sp1 binding increased because of the loss of functional p53.
#Superoxide d splitt skin
Exposure to DMBA and TPA activated p53 and decreased MnSOD expression via p53-mediated suppression of Sp1 binding to the MnSOD promoter in normal-appearing skin and benign papillomas. The suppression and subsequent restoration of MnSOD expression were mediated by two transcription-factors, Sp1 and p53. The results show that MnSOD expression was suppressed at a very early stage but increased at late stages of skin carcinogenesis. Here, we generated transgenic mice expressing a luciferase reporter gene under the control of human MnSOD promoter-enhancer elements and investigated the changes of MnSOD transcription using the 7,12-dimethylbenz(α)anthracene (DMBA)/12- O-tetradecanoylphorbol-l3-acetate (TPA) multistage skin carcinogenesis model. However, despite extensive studies in MnSOD regulation and its role in cancer, when and how the alteration of MnSOD expression occurs during the process of tumor development in vivo are unknown. Manganese superoxide dismutase (MnSOD) plays a critical role in the survival of aerobic life, and its aberrant expression has been implicated in carcinogenesis and tumor resistance to therapy.
0 kommentar(er)
