Molecular Determinants of Cellular Response to Anticancer Agents Treatment

Broggini, Massimo (2003). Molecular Determinants of Cellular Response to Anticancer Agents Treatment. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f562

Abstract

The response of cancer cells to treatment with anticancer agents is mediated by several factors, among which the functionality of proteins participating in the control of cell cycle progression and genomic integrity is an important one. The studies reported here were aimed at understanding the role of crucial proteins, participating in key processes in normal cells, in determining cellular sensitivity towards the cytotoxcity of anticancer agents. Four different proteins have been investigated: p53, p73 and the two cell cycle checkpoint proteins CHKl and CHK2.
P53 has been selected because of its undisputed role in tumor formation and development, for its high prevalence of mutations in human cancer and for its role in normal cells in response to different stimuli. P73 is a structural homologue of p53 which shares functions with p53 but in addition has other very distinct functions. The two checkpoint proteins CHKl and CHK2 control cell cycle progression subsequent to DNA damage, particularly during the S and G2 phases of the cell cycle, and their importance is currently under intensive elucidation.
The studies have been conducted in isogenic cell systems, to minimize as much as possible interference by other alterations invariably present when two different cell types are considered. Using two widely used anticancer agents, cisplatinum (DDP) and taxol, it was shown that for both drugs the presence of functional p53 was associated with resistance to drug-induced cytotoxicity. These results have been obtained in different experimental systems of human cancer cells of epithelial origin such as the colon carcinoma cell line HCT116 and the ovarian cancer cell line A2780. With respect to p73, the work took advantage of the availability in the laboratory of two subclones overexpressing p73 derived from a human ovarian cancer cell line by transfection with the p73 alpha cDNA. These two clones over-express DNA repair genes, particularly those participating in the nucleotide excision repair (NER). These cells are prone to repair lesions recognized by NER. Due to this fact, the p73- overexpressing clones were less susceptible to treatment with DDP and UV irradiation, as both lesions are recognized by NER.
Other drugs, such as doxorubicin and topotecan inducing damage which is not repaired by NER, exhibited similar activities in parental and p73-overexpressing clones. For both CHKl and CHK2, experiments which were performed using clones transfected with dominant negative mutants failed to show differences between transfected clones and parental cells in the cytotoxicity of DNA-damaging agents. However the use of inhibitors of these kinases resulted in increased activity of DDP, suggesting that both CHKl and CHK2 may play a role in determining sensitivity of cancer cells to drugs, but that the dominant negative mutants in some way masked these effects.
The potential involvement of CHKl in response to stress is underlined by evidence of a link between p53 and CHKl, implying that they mutually regulate each other in a way which controls both the activation and the repression of checkpoint response following DNA damage.

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