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DNA Repair Rate and Etoposide (VP16) Resistance of Tumor Cell Subpopulations derived from a Single Human Small Cell Lung Cancer
Institute of Molecular Pathology, University of Copenhagen.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Section of Radiation Biology, The Finsen Center, University Hospital of Copenhagen.
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2003 (English)In: Lung Cancer, ISSN 0169-5002, Vol. 40, no 2, 157-64 p.Article in journal (Refereed) Published
Abstract [en]

Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated. The tumor was classified as intermediate-type SCLC. The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro. Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs). The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant. In order to explain the VP16 resistant phenotype several mechanisms where considered. The p53 status, P-glycoprotein, MRP, topoisomerase IIα, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance. In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PKcs) and RAD51 was observed. The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PKcs and RAD51 compared with the sensitive 54A subline. We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype.

Place, publisher, year, edition, pages
Elsevier , 2003. Vol. 40, no 2, 157-64 p.
Keyword [en]
DNA-PKcs; RAD51; DNA double-strand break repair; VP16; Etoposide; Ionizing radiation; Drug resistance; Small cell lung cancer
National Category
Biological Sciences
Research subject
URN: urn:nbn:se:su:diva-23260DOI: 10.1016/S0169-5002(03)00026-6OAI: diva2:190949
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-07Bibliographically approved
In thesis
1. Homologous recombination at replication forks in mammalian cells
Open this publication in new window or tab >>Homologous recombination at replication forks in mammalian cells
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accumulating evidence indicates that homologous recombination plays an important role in mammalian cells, primarily in the reactivation of stalled replication forks. During a normal round of replication, stalling of the replication fork occurs frequently when a nick or a lesion, which cannot be by-passed, is encountered. Progression of the replication fork can also be hindered by a protein bound to or secondary structures in the DNA. It is vital for the cell to restart stalled replication forks efficiently since nucleases might otherwise damage the unprotected DNA and since failure to reinitiate replication leads ultimately to cell death. The mechanism by which homologous recombination reactivates stalled replication forks and the substrates that trigger this response in mammalian cells have not yet been elucidated.

The present thesis focuses on the function of homologous recombination as well as on the mechanism for induction of this process in mammalian cells. Our major interest in this context was the repair of lesions encountered during replication and that interfere with replication fork progression. The role of the RAD51 protein in this process was also examined. The main experimental system we employed consisted of Chinese hamster cell lines deficient in different DNA repair pathways, as well as the V79 Chinese hamster SPD8 cell line, which contains an endogenous locus at which homologous recombination events can be monitored.

Our findings demonstrate that homologous recombination can be employed by mammalian cells to repair several different types of lesions encountered during replication. The mechanism underlying such homologous recombinational repair appears to depend on the type of lesion involved. A replication fork encountering a single-strand break was shown to collapse into a double-strand break and subsequently be repaired by homologous recombination involving a mechanism referred to as break-induced replication. A stalled replication fork was shown to induce a substrate for homologous recombination both with and without the induction of a double-strand break. This suggests that homologous recombination could repair lesions other than double-strand breaks during replication. Non-homologous end-joining was shown to play only a minor role in the repair of DNA damage associated with replication, being employed only in situations when the replication fork is processed into a double-strand break.

Experiments concerning the role of RAD51 in repair of lesions encountered during replication, employing a cell line that overexpresses this protein, indicated that RAD51 is rate limiting for the repair of DNA damage associated with the replication fork. The level of RAD51 was also measured in lung cancer cells and found to determine both the survival and the level of double-strand breaks formed in response to etoposide treatment, suggesting RAD51 to be involved in the resistance of tumours to chemotherapeutic drugs.

In summary, our findings demonstrate that RAD51-dependent homologous recombination is an important repair pathway during replication in mammalian cells.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi, 2004. 64 p.
National Category
urn:nbn:se:su:diva-207 (URN)91-7265-922-X (ISBN)
Public defence
2004-09-10, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 13:00
Available from: 2004-08-20 Created: 2004-08-20Bibliographically approved

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