| Brain tumors account for 1.6% of all primary cancers. The majority of brain tumor are gliomas, and the most malignant form of glioma is grade IV, which is commonly known as glioblastoma multiforme. This highly aggressive tumor develops either from primary gliblastoma multiforme or as the result of the malignant progression from a low grade gliomas. Most importantly, glioblastoma multiforme is characterized by a diffuse tissue-distribution pattern, with extensive dissemination of the tumor cells within the brain that hinders complete surgical resection. Despite intensive multi-modality treatment including tumor resection, post-operative radiotherapy and frequently adjuvant chemotherapy, the prognosis of malignant glioma continues to be poor. Recurrences occur almost exclusively in the brain, most commonly at the site of initial tumor presentation. The median survival when surgical resection, radiotherapy and chemotherapy are combined is 14.6 months.One reason for the lack of clinical advances is ignorance of the cellular origin of this disease, which delays the application of molecular analyses to treatment and impairs the ability to anticipate tumor behavior reliably. To make ture progression in cancer therapy, we need to better understand what derives tumor resistance and mediates tumor recurrence after initial successful therapy. Now there is increasing awareness about which cells may play the critical role in the recurrence after post-operative radiotherapy of gliblastoma multiforme. The previous opinion towards the source cells of recurrence was the residual cancer cells after surgical resection, but now as the cancer stem-cell hypothesis proposes that a minority of transformed stem cells, or progenitors with acquired self-renewal properties, are the source of tumor-cell renewal and thereby determine a tumor's behavior, including proliferation, progression and response to therapy, and recently several groups have discovered that brain cancer stem cells can self-renew under clonal conditions, differentiate into neurons and glia-like cells as well as abnormal cells with mixed phenotypes and CD133 positive cells transplantation at low density could readily form new tumors in immunodeficient mice. So the CD133 positive brain glioma stem cells were considered as a critical cell and should be responsible for the initiation and maintenance of malignant gliomas. Taking the knowledge above as a foundation, in this paper, we hypothesized that CD133 positive cells, representing the small population of brain glioma stem cells, are resistant to current gliblastoma multiforme therapies and play a very important role in tumor recurrence after radiotherapy.In order to verificate this hypothesis and to address the mechanism of brain glioma stem cells showing strong resistance to radiotherapy, we used CD133 as a surface marker to isolate CD133 positive and CD133 negative cells by Magnetic cell sorting from human malignant glioma cell line U251 and the primary glioblastoma multiforme culture cell lines in part 1 of our research and charactered as stem cells identified by stem cells surface markers and differentiated cells surface markers, ultrastructure observing with electron microscope and engrafting to nude mice for tumorigenesis test. Several lines of evidence support that the CD133 positive cells we have isolated from the cell lines above were brain glioma stem cells:(a) The isolated CD133 positive cells can self-renew and proliferate to generate contiguous sub-spheres; (b) The cells keep undifferentiated feature, have multipotency and can differentiate into multi-lineage progenies; (c) The isolated CD133 positive cells can produce brain tumors in nude mice, and the tumors are the phenocopy of the original tumor from which they were derived, however, CD133 negative cells can't form tumors. So the tumorigenicity obviously depend on CD133 positive brain glioma stem cells.To evaluate the radiosensitivity of CD133+ cells which were lived alone in vitro and CD133+ cells which were existed in U251 and the glioblastoma multiforme specimens, 60Co irradiation was carried out to deliver different doses for CD133+ cells lived alone in vitro, and for U251 and the glioblastoma multiforme specimens in part 2 of our research. Flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling technique (TUNEL), Annexin V-FITC staining and nude mice transplantation were employed to examine cell cycle, apoptosis and tumorigenicity for the cells all of the above before and after 60Co irradiation respectively.The results were the following:1 The CD133 positive brain glioma stem cells lived alone in vitro were in active cell cycle, and G0-G1 phase was 51.65±2.75%,G2/M phase was 23.35±1.25%,S phase was 24.15±2.35%。Its'growth and metabolism were vigorous when seeding them in the culture condition of DMEM/F12 combining bFGF, EGF and LIF. The cells were sensitive to 60Coγ- irradiation, exceed 2Gy irradiation could induce apoptosis . Intracranial cell transplantation revealed there was no tumor appear when the cells received exceed 2Gy irradiation.2 As the same actively divided cell lines in vitro, human malignant glioma cell line U251 and the primary glioblastoma multiforme culture cell lines showed the same sensibility to the 60Coγ- irradiation , but there was great difference existed in that they could still form tumors when they received below 14 Gy irradiation.3 After U251 and the glioblastoma multiforme culture cell lines received different dose 60Co irradiation respectively, we utilized Magnetic cell sorting to isolate the CD133+ cells and CD133- cells. Annexin V-FITC staining showed apoptotic rate of CD133- cells obviously increased following irradiation dose increase and there was no tumor appear after intracranial or subcutaneouly transplantation into the nude mice. But towards CD133+ cells, Annexin V-FITC staining showed apoptotic rate slowly increased following irradiation dose increase until the cells received 14 Gy irradiation . The results of CD133+ cells transplantations showed the cells received below 14 Gy irradiation could form tumors respectively within 4 to 10 weeks, and the tumors still be the phenocopy of the original tumor through immunohistochemistry identification.4 After U251 and the glioblastoma multiforme culture cell lines received different dose 60Co irradiation respectively, we utilized Magnetic cell sorting to isolate the CD133+ cells and CD133- cells. From cell cycle distribution we could find the percentage of G0/G1 phase of CD133- cells was 57.35±0.35 %, G2/M phase was 18.17±7.83%, S phase was 24.55±7.45 %. However, the percentage of G0/G1 phase of CD133+ cells was 87.4±4.9 %, G2/M phase was 6.66±3.25%, S phase was 6.97±2.73 %. It indicated that CD133+ cells existed in U251 and the glioblastoma multiforme culture cell lines were in unactive mitotic division status.In our research, we found the most number of CD133 positive cells existed in U251 and the primary glioblastoma multiforme culture cell lines were in G0/G1 phase and were resistant to 60Coγ- irradiation. The maintenance of malignance of human adult gliblastoma multiforme obviously depends on the CD133 positive brain glioma stem cells. Although ionizing radiation could induce a large number of cancer cells (CD133 negative cells) apoptosis or even kill them, but can only show litter influence on CD133 positive cells, and the CD133 positive brain glioma stem cells could still form tumor which recapitulated the phenotypic heterogeneity found in the initial tumor after intracranial or subcutaneouly transplantation. These data suggest that CD133 positive brain glioma stem cells are resistant to current radiotherapy and may represent a cell target for novel malignant glioma therapies.
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