Molecular aspects of cellular radiosensitivity in small cell lung carcinoma

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Oncology-Pathology

Sammanfattning: To clarify some important molecular aspects of radiosensitivity (RS) in small cell (SCLC) compared with non small cell (NSCLC) lung carcinoma, five studies focusing on DNA damage repair, cell cycle checkpoints and apoptosis were performed. As repair of DNA double-strand breaks (DNA dsb), a critical DNA lesion, may be an important determinant of cellular RS, induction and rejoining of DNA dsb were studied using pulsed field gel electrophoresis in a radiosensitive SCLC line, U-1285 and a radioresistant NSCLC line, U-1810 (paper I). The induction levels of DNA dsb were of the same magnitude in both cell lines. U-1810 but not U-1285 cells exhibited a fast component of DNA dsb rejoining, suggesting an association between RS and the early DNA-dsb repair phase. In the next study (paper II), RS and rejoining of DNA dsb were analyzed in four SCLC lines and one NSCLC line. RS correlated in these 5 cell lines with the initial phase of DNA-dsb rejoining, confirming the results in the previous study, and with the relative amount unrejoined DNA dsb at 2 hours postirradiation. The content and activity of DNA DIMINUENDOdependent protein kinase (DNA-PK), an important molecular component involved in DNA dsb repair, correlated with RS in this cell system. These results suggest the possibility to use DNA-PK content as a predictive test for RS. Transient delays of cell cycle progression in G1 and G2 may be important factors in the cellular response to DNA damage. Cell cycle flow calculations showed that in radiosensitive U-1285, the G1 -> S-phase transit was accelerated in irradiated compared with untreated cells up to 24 hours postirradiation (paper III). This effect was not detected in radioresistant U-1810 or intermediate sensitive U-1906 cells. The accelerated postirradiation Gl->S transit in U-1285 cells was associated with substantial induction of cyclin E-dependent kinase activity and may account for increased RS in these cells due to complete G1/S checkpoint abrogation. Apoptosis is a common form of radiation-induced death of many types of cells but it is still unclear whether there is a direct qualitative or quantitative correlation between apoptosis and RS. In the first apoptosis study (paper IV), DNA laddering pattern, results of TUNEL assay and detection of poly-(ADP-ribose) polymerase (PARP) cleavage showed that spontaneous apoptosis was detected at a high level in the radiosensitive U-1285, at an intermediate level in U- 1906 and not detected in radioresistant U-1810 cells, suggesting an association between spontaneous apoptosis and RS. Sequencing of all p53 exons disclosed mutations in all three cell lines. Thus, apoptosis was a p53 independent process in this cell system. In the next study (paper V), spontaneous apoptosis was assessed in 15 lung carcinoma cell lines. The TUNEL assay showed significantly higher apoptotic index (AI) in SCLC compared with NSCLC lines. Analysis of Bcl-2, Bax, p53, c-myc and pro-caspase-3 proteins by Western blotting showed no correlation with AL A striking feature observed in SCLC lines was that high Bcl-2 expression did not prevent spontaneous apoptosis.

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