Chemoresistance to temozolomide in human glioma cell line U251 is associated with increased activity of O6-methylguanine-DNA methyltransferase and can be overcome by metronomic temozolomide regimen.

Temozolomide (TMZ) is a novel cytotoxic alkylating agent for chemotherapy of malignant gliomas. However, intrinsic or acquired resistance to TMZ often defines poor efficacy of chemotherapy in malignant gliomas. A growing number of studies indicate that expression of O(6)-methylguanine-DNA methyltransferase (MGMT) is one of the principal mechanisms responsible for this chemoresistance. In the present study, we evaluated the relationship between expression of MGMT and resistance to TMZ. We generated a TMZ-resistant cell line, U251/TR, by stepwise (8 months) exposure of parental U251 cells to TMZ. The resistance to TMZ was quantified using SRB assay. MGMT expression was evaluated at mRNA (RT-PCR) and protein (Western blot) levels. U251/TR cells showed increased (~ sevenfold) resistance to TMZ. The MGMT expression (both mRNA and protein) was significantly (P < 0.01) increased in U251/TR cells compared with parental U251 cells. Further, MGMT expression fluctuated during exposure of U251/TR cells to TMZ. The resistance of U251/TR cells to TMZ could be overcome by application of elevated doses of TMZ when MGMT expression was at the lowest level. In conclusion, our results demonstrate that the primary mechanism responsible for resistance of U251/TR cells to TMZ is associated with increased expression of MGMT. Resistance of malignant gliomas to TMZ can be overcome by synchronizing metronomic TMZ regimen with MGMT expression.

[Correlation between the distribution of CD133-positive cells and the proliferation of microvessels in glioblastoma multiforme].

OBJECTIVE: To investigate the expression of CD133 and CD34 in different parts of glioblastoma and its margin and explore the invasive path of glioma stem cells within the tumor and surrounding tissue. METHODS: The surgical specimens were collected from the core of mass, junctional zones between tumor and peritumoral edematous areas and edematous areas in 52 patients with glioblastoma. Immunohistochemical cell staining and Western blot were employed to evaluate the expression of CD133 in different specimens while immunohistochemistry was used to detect the CD34-microvessel postforming. A correlation analysis was performed between them. RESULTS: The expression of CD133 was not detected in the control groups while the scores were 7.3 ± 1.4, 5.2 ± 1.1, 2.7 ± 1.0 in junctional zones, tumor cores and edematous areas with immunohistochemistry and 0.79 ± 0.03, 0.38 ± 0.01, 0.20 ± 0.04 with Western blot respectively. There were significant differences between different groups (P < 0.05). Under light microscope, the CD133-positive cells frequently forming perivascular pseudorosettes were dense in junctional zones and mostly clustered near the microvessels in tumor cores and scattered in edematous areas. At a high magnification (200×), the CD34-MVD (/HP) values were 31.3 ± 4.0, 21.8 ± 2.6, 15.3 ± 2.4, 4.7 ± 1.5 respectively in junctional zones, tumor cores, edematous areas and control tissues. Significant differences were also found in these groups (P < 0.05). The expression level of CD133-positive cell was positively correlated with the distribution of CD34-microvessels (r = 0.948, P < 0.05). CONCLUSION: Glioma stem cells tend to assemble in the junctional zones where the microvessels are enriched. There is probably an intimate nourishing relationship with the microvessels. The distribution of glioma stem cells may be related with the invasiveness within glioblastoma.

Expression and Distribution Characteristics of Human Ortholog of Mammalian Enabled (hMena) in Glioma.

OBJECTIVE: To investigate the utility of hMena, a family of enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), we sought to characterize the expression profile and distribution characteristics of hMena in a large panel of glioma samples and determine whether hMena expression levels might correlate with the pathological grade of glioma. METHODS: Sixty-five specimens of glioma with different pathological grades and five control brain tissues were collected. In 6 of the 21 glioblastoma patients, multi-specimens were obtained respectively from the main tumor mass, the junction zone between the tumor and the normal tissue, and adjacent brain tissue 1.5 cm away from the tumor boundary under assistance of neuronavigation system during the operation. Immunohistochemistry was used to detect the expression and distribution characteristics of hMena. hMena expression was analyzed by Western blot in 20 specimens. RESULTS: The hMena expression was negative in control brain tissue but positive in different grades of glioma. The expression rate of hMena was positively correlated with the increasing grade of the World Health Orgnization (WHO) classification (r(s)=0.682, P=0.000). hMena was located in cytoplasm. Positive cells only distributed around the vessels within the tumor mass in low grade glioma, while in high grade glioma, these cells were able to be detected not only in the tumor but also in the boundary zone and adjacent brain parenchyma. In the tumor mass, hMena expressed highly and diffusedly. In the junction zone, hMena positive cells formed radiolitic pattern around the vessels. In adjacent brain parenchyma, single positive cell was scattered. hMena expression was markedly elevated in Grade III and IV glioma compared with Grade II and I. CONCLUSION: Our data suggested that the expression of hMena is closely related to malignant grade of glioma. hMena can label the migrating cells, and indicate the migrating path of glioma cells from the tumor to adjacent tissue along with the vascular basement membranes and tracts of white matter.