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DNA damage inducible-gene expression following platinum treatment in human ovarian carcinoma cell lines
D A Delmastro1, J Li, A Vaisman
1Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Purpose:
DNA damage-inducible genes, such as gadd153, gadd45, p21 and c-jun, have previously been shown to be induced by the chemotherapeutic agent cisplatin. One of these genes, gadd153, has previously been reported to be differentially expressed in cisplatin-resistant cell lines and, therefore, to be a potential prognostic indicator for tumor response to cisplatin-based chemotherapy. It is not currently known whether such damage-inducible genes are turned on by the DNA damage itself (e.g. by the formation of Pt-DNA adducts) or by the downstream biological consequences of that damage. It is also not known whether the increased expression of these DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. These experiments were initiated to characterize more fully the nature of the DNA damage-inducible response to cisplatin treatment and to determine whether any of these genes might be useful prognostic indicators of tumor response to cisplatin chemotherapy.
Methods:
The dose-response and time-course for the induction of the DNA damage-inducible genes gadd153, gadd45, p21 and c-jun were examined by Northern analysis in the human ovarian carcinoma cell line 2008 and its resistant subclone C13* following treatment with platinum anticancer agents. The extent of gene expression was correlated with cytotoxicity determined by growth inhibition assay, Pt-DNA adducts determined by atomic absorption spectrometry and inhibition of DNA synthesis determined by 3H-thymidine incorporation.
Results:
All four genes were induced maximally in both sensitive and resistant cell lines at lethal cisplatin doses (> or = ID90). Induction was maximal between 24 and 48 h following exposure to the drug for all genes except c-jun which was induced by 6 h. At 24 h following cisplatin treatment the overall levels of gadd153 were less in the resistant C13* cell line than in the parental 2008 cell line, while those of gadd45 were greater in C13* than in 2008. Maximal expression of p21 and c-jun was not significantly different in the two cell lines. The dose-response of these genes correlated with the cytotoxicity of cisplatin and the inhibition of DNA synthesis by cisplatin, rather than to the actual levels of Pt-DNA adducts. The more cytotoxic platinum analog, ormaplatin, also induced gadd153 and its induction was also based on cytotoxicity.
Conclusion:
These results suggest that the regulation of gadd153 and gadd45 expression occurs thorough separate pathways in the 2008 and C13* cell lines. The DNA damage-inducible gene response for all four damage-inducible genes tested appeared to be more directly correlated with downstream biologic effects of cisplatin damage than with actual Pt-DNA adduct levels. The time-course and dose-response for induction of these genes was more consistent with delayed responses such as apoptosis rather than more immediate responses such as DNA repair. Finally, these results strengthen previous suggestions that the expression of gadd153, and possibly other DNA damage-inducible genes, may be useful indicators of tumor response to cisplatin-based chemotherapy.
Insights
DNA damage-inducible genes like gadd153 are activated by cisplatin chemotherapy, correlating with tumor response rather than DNA adducts. Their induction suggests delayed responses, potentially indicating tumor sensitivity to cisplatin treatment.
Area of Science:
- Molecular biology
- Cancer research
- Chemotherapy
Background:
- DNA damage-inducible genes (e.g., gadd153, gadd45, p21, c-jun) are activated by cisplatin.
- Gadd153 is differentially expressed in cisplatin-resistant cell lines, suggesting prognostic value.
- The precise triggers and downstream effects of DNA damage-inducible gene activation by cisplatin remain unclear.
Purpose of the Study:
- To fully characterize the DNA damage-inducible response to cisplatin.
- To determine if DNA damage-inducible genes are activated by DNA damage itself or its consequences.
- To assess if these genes correlate with immediate (DNA repair) or delayed (apoptosis) cellular responses.
- To evaluate the potential of these genes as prognostic indicators for cisplatin chemotherapy response.
Main Methods:
- Northern analysis was used to examine the dose-response and time-course of gadd153, gadd45, p21, and c-jun induction in sensitive (2008) and resistant (C13*) human ovarian carcinoma cell lines after platinum agent treatment.
- Cytotoxicity was assessed via growth inhibition assays.
- Platinum-DNA adduct levels were quantified using atomic absorption spectrometry.
- DNA synthesis inhibition was measured by 3H-thymidine incorporation.
Main Results:
- All four genes were maximally induced at lethal cisplatin doses in both cell lines.
- Gene induction peaked between 24-48 hours, except for c-jun (6 hours).
- Gadd153 levels were lower in resistant cells, while gadd45 levels were higher.
- Gene induction correlated with cisplatin cytotoxicity and DNA synthesis inhibition, not directly with Pt-DNA adduct levels.
- The cytotoxic analog ormaplatin also induced gadd153 based on cytotoxicity.
Conclusions:
- Gadd153 and gadd45 expression are regulated via distinct pathways in sensitive and resistant cell lines.
- The induction of these genes is more closely linked to downstream biological effects of cisplatin damage than to Pt-DNA adduct levels.
- The observed time-course and dose-response suggest induction is associated with delayed cellular responses like apoptosis.
- These findings support the utility of gadd153 and potentially other DNA damage-inducible genes as indicators of tumor response to cisplatin chemotherapy.