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Selective impairment of p53-mediated cell death in fibroblasts from sporadic Alzheimer's disease patients

¹ Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy
² Department of Pathology, University of Brescia, Brescia, Italy
³ Department of Experimental and Applied Pharmacology, University of Pavia, Pavia, Italy

View larger version (42K): [in a new window]   Fig. 1. Biochemical (A) and morphological (B) evidence for H2O2-induced loss of human skin fibroblasts. Cell viability was determined by measuring LDH activity in conditioned media (A); cells in apoptosis were determined morphologically by Hoechst 33258 staining (B). Cells were exposed to 1 mM H2O2 for 15 minutes, washed and analysed at different times after the lesion, as indicated at the bottom of A and in inserts in B. Bars represent mean±s.e.m. of at least three different experiments and are from three separate cell preparations. Arrows in B indicate cells in apoptosis. *P<0.01 versus 0 time.

View larger version (13K): [in a new window]   Fig. 2. H2O2-induced cell loss of human skin fibroblasts from non-AD and AD patients. Cell viability was determined by measuring LDH activity in the conditioned media 24 hours after H2O2 exposure. Each point represents an individual sample. Values are expressed as a percentage increase over basal values. The insert shows the means±s.e.m. from at least three different experiments for each sample using at least three separate cell preparations. *P<0.01 versus non-AD.

View larger version (34K): [in a new window]   Fig. 3. H2O2-induced DNA base damage of human skin fibroblasts from non-AD and AD patients. DNA damage was evaluated immunocytochemically using a specific antibody against 8OH-dG. A shows representative images of non-AD (1,3) and AD (2,4) fibroblasts in basal conditions (1,2) and 2 hours after H2O2 treatment (3,4). B shows quantitative analysis of 8OH-dG-positive cells in six non-AD and six AD samples. Bars represent basal (open) and experimental (gray) values and are expressed as a percentage of positive cells per field. Values are the mean±s.e.m. of the results from at least six different fields for each sample. *P<0.01 versus the corresponding basal values.

View larger version (13K): [in a new window]   Fig. 4. [3H]Thymidine incorporation in fibroblasts from non-AD and AD patients after H2O2 treatment. [3H]Thymidine was added to the culture media at the times indicated by the white arrows. Cells were exposed to H2O (circles) or 1 mM H2O2 (triangles) for 15 minutes at time 0 as indicated by black arrows, then washed and cultured for additional 6 hours or 24 hours. Data are from six non-AD and six AD different cell lines and are expressed as mean±s.e.m. of at least three separate experiments for each sample.

View larger version (55K): [in a new window]   Fig. 5. Representative patterns of cell cycle phase distribution by flow cytometric analysis of fibroblasts from non-AD and AD sample in resting conditions and at different times after H2O2 treatment. Cells were exposed to 1 mM H2O2 for 15 minutes, washed and cultured for an additional 6 or 20 hours (as indicated by the numbers in the inserts). Time 0 indicates that cells are in the basal condition.

View larger version (27K): [in a new window]   Fig. 6. Expression of p53 and p53-related gene products in fibroblasts from non-AD and AD patients: western blot analysis. (A) Protein extracts from two non-AD (lanes 1-2 and 3-4) and two AD (lanes 5-6 and 7-8) fibroblast cultures in basal conditions (lanes 1, 3, 5 and 7) and 2 hours after the H2O2 pulse (lanes 2, 4, 6 and 8) were processed with specific antibodies against p53, p21, bax, GADD45 and ß-tubulin, as indicated in the method section. (B) Data obtained in fibroblasts from six non-AD and six AD patients in basal conditions (open bars) and after H2O2 pulse (black bars) were calculated by densitometric analysis and normalized as a percentage of basal expression. Values are expressed as means±s.e.m. of at least three separate experiments for each sample. *P<0.01 versus the corresponding basal values.

View larger version (15K): [in a new window]   Fig. 7. Phosphorylation status of p53 in fibroblasts from non-AD and AD patients: an immunoprecipitation study. Protein extracts from one non-AD (lanes 1-2) and three AD (lanes 2-4, 5-6 and 7-8) fibroblast cultures in basal conditions (lane 1, 3, 5 and 7) and 2 hours after the H2O2 pulse (lane 2, 4, 6 and 8) were immunoprecipitated with antibodies against p53 and immunoblotted with specific antibodies against phospho ser15, phospho ser392 or p53, as indicated.

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