Radiation Resistance Of Glioblastoma: The Preliminary Study In The Role Of Brain Tumor Stem And Progenitor Cells

Objective: Today, the research of glioma Stem cell cells has already moved form stem cells cloning in vitro to experimental therapy research targeting the stem cells. Radiotherapy for glioma research on the case, first of all, is to prove whether it is the most radioresistant cells and the mechanism of the radioresistance. This study was designed to prove whether tumor stem cells from cell lines in vitro or in the solid tumor has a special radioresistant characteristics and clarify the relationship between this characteristic and the priority of DNA damage repairing, preparing for further research on molecular machine of the radioresistance.Methods: Human glioma cell lines SHG-44 and U251 were cultured in DMEM supplemented with 10% calf serum (CS). Human glioma cell lines SHG-44s, U251s and SU-2 were cultured in serum-free DMEM/F 12 containing N2, bFGF and EGF. Brain tumor stem cells were detected before and after radiation with flow cytometry and the immunohistochemistrical staining with Hoechst33342 and antibodies against CD133, Nestin, NSE and GFAP. Clone forming assay was applied to examine the growth inhibition of human glioma cell line SHG-44 in six dose groups of 0, 1, 2, 4, 6, 8Gy. The survival curves were generated and mathematically analyzed by using the L-Q model to obtain the radiosensitivity parameters, SF₂ (the surviving fraction at 2Gy) and SF₈ (the surviving fraction at 8Gy). RT-PCR method was used to determine the mRNA levels of MGMT gene in U251, U251s and SU-2 before and after radiation.Results: (1) SHG44, U251 and SU-2 could expanse unlimited into CD133⁺ and/or Nestin⁺ spherical with growth factor. If recultured in DMEM containing serum, they can grow into glioma like neurons and astrocytes and they can express neurons markers NSE and astrocytes markers GFAP, which suggest successful cloning of glioma stem and progenitor cells from the cell lines and Solid Tumor. (2) Using Flow cytometry, CD133 positive cells accounted for 0.4% of SHG-44 cells and 0.1% of U251 cells when cultured in DMEM supplemented with 10% CS, while in serum-free condition, percentage of SHG-44s, U251s and SU-2 cells expressing CD133 reached 1.0%, 1.4% and 4.6% respectively, which showed that tumor stem cells ball contained much more CD133⁺ stem cells than those of adherent cell lines. (3) The SF2 and SF8 of different glioma cell lines increased with the proportion of CD133+ cells, which suggest there is intrinsic relationship between the GSC and radiosensitivity of different glioma cell lines. After radiotherapy, optical microscopy and transmission electron microscopy revealed obviously damage and slow growth in U251 cell line, but not in SU-2 cell line, which is much more radioresistant. (4) Hochest33342 stain was applied in SHG-44 and U251 cell lines before and after radiotherapy using flow cytometry. As the radiation dose accumulated, the proportion of Hochest33342 cells increased. After the radiation of 5Gy×3 times, the proportion of CD133⁺ cells raised from 0.3% to 6.51% in SHG-44, and from 4.6% to 13.7% in S U-2. (5) The mRNA expression of MGMT was detected in glioma cell lines U251s and SU-2 before and after radiation, but not in U251 cell lines, which suggest there is intrinsic relationship between the escalated DNA repaire ability of GSC and radiosensitivity of different glioma cell lines.Conclusion: (1) Glioma stem cells cloned from cultured glioma cells and glioma surgery specimens can be used to study the radioresistance of glioma. (2) There is intrinsic relationship between the GSC and radiosensitivity of different glioma cell lines. After irradiation the glioma cell lines increased the proportion of stem cells and there is no obvious damage to the stem and progenitor cells, which is direct signs of radioresistance in stem and progenitor cells and can be used in further study of radioresistance in glioma stem and progenitor cells. (3) DNA repair gene MGMT expressed in glioma stem and progenitor cells but not in the corresponding cell lines. This is one of the molecular mechanisms of damaged DNA repairing. There are much more need to be address such as signal transduction, apoptosis, and other molecular mechanisms.