The role of wild-type p53 in the differentiation of primary hemopoietic and muscle cells

Abstract

Experiments previously performed on 32D and C2C12 cell lines indicated that wild type p53 (wtp53) protein has a role in granulocyte and myotube di erentiation. Since these are immortal cells, we asked whether the inhibition of di erentiation induced by the expression of dominantnegative p53 (dnp53) proteins was dependent on the immortalization-determined microenvironment. Thus, we evaluated the e ects produced by interfering with the endogenous p53 gene in murine primary hemopoietic and muscle cells. Expression of dnp53 protein reduced the di erentiation of bone marrow cells into granulocytes and macrophages. Moreover, p53 activation was measurable during the di erentiation process of primary myoblasts, while interference with this activation led to a consistent slow down of terminal di erentiation. Since the impairment of the di erentiation was not accompanied by alterations in the cell cycle withdrawal and in the rate of apoptosis which are coupled with these types of di erentiation, the data here reported support a speci®c role for p53 in the di erentiation process. However, the di erence in the intensity of inhibition between immortal and primary cells, i.e., complete versus slow down, respectively, suggests that the immortalization process might render the cells more sensitive to the loss of wtp53 activity for the di erentiation process.

p53 is involved in the differentiation but not in the differentiation-associated apoptosis of myoblasts

“…Unstimulated, proliferating cells were chosen as a reference point in these experiments. As expected, proliferating C2-PG 13 CAT cells had a basal chloramphenicol acetyltransferase (CAT) activity comparable to the C2-MG 15 CAT control cells (data not shown). Absence of CAT activity was also found in DAA cells, while a fourfold induction of activity was consistently found in the differentiating population ( Figure 1B).…”

“…PG 13 GFP-transduced C2C12 cells (C2-PG 13 GFP) were selected and checked for response to p53 activation by Adriamycin (ADR)-induced DNA damage. In the presence of differentiation promoting medium, the appearance of green fluorescence in C2-PG 13 GFP cells began after 24 h and reached its maximum after 48 ± 72 h ( Figure 1A). Morphological analysis of C2-PG 13 GFP cells showed that GFP was present in all the differentiated C2C12 cells.…”

“…In the presence of differentiation promoting medium, the appearance of green fluorescence in C2-PG 13 GFP cells began after 24 h and reached its maximum after 48 ± 72 h ( Figure 1A). Morphological analysis of C2-PG 13 GFP cells showed that GFP was present in all the differentiated C2C12 cells. Despite the long half-life of the GFP protein (more than 3 days, data not shown), no fluorescence was ever detectable in the DAA cells, at any time point, in both cell clones.…”

“…Thus, to evaluate p53 activity at single cell level in terminally differentiating and DAA C2C12 cells, we exploited the properties of GFP-based vectors to study the p53-specific transcriptional activity. 7 We cloned the 13 copies of the p53 consensus binding-sequence of the PG 13 CAT reporter vector 8 upstream of the GFP gene (PG 13 GFP). PG 13 GFP-transduced C2C12 cells (C2-PG 13 GFP) were selected and checked for response to p53 activation by Adriamycin (ADR)-induced DNA damage.…”

“…C2-PG 13 CAT cells and C2-MG 15 CAT controls were cultured in a differentiationpromoting medium for 48 h, separated, incubated with PI, and quantified by cytofluorimetric analysis. Unstimulated, proliferating cells were chosen as a reference point in these experiments.…”