Expression of c-fos and AP-1 activity in senescent human fibroblasts is not sufficient for DNA synthesis.

Abstract

Abstract, Human fibroblasts have a limited replicative life span when maintained in culture after which they become unresponsive to treatment with mitogens, a phenomenon most commonly called senescence. Experiments indicating that serum does not induce expression of the c-fos proto-oncogene in senescent fibroblasts raised the issue of a potential central role for c-fos in the phenotype of sustained growth arrest. This was directly tested by microinjection of oncogenic c-Ha-ms protein into senescent fibroblasts. While ras injection was found to induce marked nuclear c-fos expression and functional AP-1 transcription activity, this did not lead to DNA synthesis. These results suggest that the senescence phenotype cannot be solely attributed to the absence of c-fos expression and that the proliferative block in these cells is either independent of AP-1 transcriptional activity, downstream of it, or involves multiple molecular mechanisms.

Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300

“…Insulinresponsive Rat-1 fibroblasts were injected with plasmids and CBP-specific or p300-specific antibodies as previously described (25). Approximately 1 hr after injection, the cells were stimulated as indicated with 1000 units͞ml IFN-␥ and analyzed the next day as described (25). In peptide neutralization experiments, the CBP 2-22 peptide was preincubated with the antibody in the same buffer at approximately a 2:1 molar ratio of peptide to antibody.…”

“…Eight copies of the consensus GAS sequence TTCTCGGAA (24) were placed upstream of the minimal prolactin promoter to generate 8 ϫ GAS-luciferase. Insulinresponsive Rat-1 fibroblasts were injected with plasmids and CBP-specific or p300-specific antibodies as previously described (25). Approximately 1 hr after injection, the cells were stimulated as indicated with 1000 units͞ml IFN-␥ and analyzed the next day as described (25).…”

Origin of carcinoma associated fibroblasts

“…[100][101][102][103][104][105] Cancer usurps this barrier by various intracellular mechanisms, but on the level of cancer microenvironment, senescence may also promote cancer. [106][107][108] The linear increase in cancer incidence with age fits the model of cumulative somatic mutations for cancer etiology [109][110][111][112] but other contributions of age are possible.…”

Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP

“…Microinjection of antibodies and reporter genes was done as described 24,25 . The RKIP antiserum was puri®ed over a GST±RKIP af®nity column.…”