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Inhibition of DNA synthesis in senescent-proliferating human cybrids is mediated by endogenous proteins
Abstract:
Cytoplasts were prepared from senescent human diploid fibroblasts. Brief treatments of the senescent cells with cycloheximide or puromycin prior to or after enucleation eliminated the ability of senescent cytoplasts to block initiation of DNA synthesis in senescent-young cybrids. Senescent cells treated with cycloheximide, enucleated and allowed to recover in complete medium without cycloheximide, regained the ability to block initiation of DNA synthesis in senescent-young cybrids. These results support the hypothesis that senescent cells synthesize an inhibitor of DNA synthesis which is either a protein(s) or its activity is mediated by a protein(s) found in the cytoplasm of the senescent cell.
Insights
Senescent cells contain a cytoplasmic factor that inhibits DNA synthesis initiation. This inhibitor is likely a protein, as its activity is lost with protein synthesis inhibitors and regained upon recovery.
Area of Science:
- Cellular senescence
- Molecular biology
- Biochemistry
Background:
- Cellular senescence is a state of irreversible growth arrest.
- The mechanisms underlying senescence-induced cell cycle arrest are not fully understood.
- Cytoplasmic factors in senescent cells may play a role in maintaining cell cycle arrest.
Purpose of the Study:
- To investigate the role of cytoplasmic factors in senescent cells.
- To determine if senescent cells produce an inhibitor of DNA synthesis.
- To identify the nature of this potential inhibitor.
Main Methods:
- Preparation of cytoplasts from senescent human diploid fibroblasts.
- Treatment of senescent cells with protein synthesis inhibitors (cycloheximide, puromycin) before or after enucleation.
- Fusion of senescent cytoplasts with young cell components to form cybrids.
- Assay of DNA synthesis initiation in senescent-young cybrids.
Main Results:
- Brief treatment with cycloheximide or puromycin abolished the ability of senescent cytoplasts to block DNA synthesis initiation.
- Senescent cells, when treated with cycloheximide, enucleated, and allowed to recover, regained the ability to inhibit DNA synthesis.
- These findings indicate the presence of a labile inhibitor in senescent cytoplasts.
Conclusions:
- Senescent cells synthesize an inhibitor of DNA synthesis initiation.
- This inhibitor is likely protein-based or requires protein activity for its function.
- The inhibitor resides in the cytoplasm of senescent cells.