Association studies of genetic polymorphism and environmental factors in ischemic stroke with atherosclerotic middle cerebral artery stenosis

Objectives: Intracranial atherosclerosis, especially the middle cerebral artery (MCA), is the commonestvascular lesion for ischemic stroke the Chinese population. We explored the association of geneticpolymorphism and environmental factors in MCA atherosclerosis in the Chinese population. Methods:One hundred fifty-six ischemic stroke patients with MCA stenosis and 181 well-matched ischemicstroke patients without MCA stenosis were examined by polymerase chain reaction (PCR). ThePCR products were analyzed for lipoprotein lipase (LPL) S447X and paraoxonase1 (PON1) Q192Rpolymorphisms by restriction enzyme digestion. Medical history documentation and investigationof biochemical markers were performed for each subject. Results: Univariate analysis showed thatthe levels of systolic blood pressure (SBP) were higher in the MCA stenosis group. There were nosignificant differences in the genotype and allele frequencies of the LPL S447X and PON1 Q192Rpolymorphism observed between the two groups. But, in the patients above 60 years of age with andwithout MCA stenosis, LPL X carriers have higher level of SBP than the LPL SS genotype carriers.Multivariate logistic regression found that SBP was the significant, independent predictor of thepresence of MCA stenosis patients above 60 years of age (P < 0.001, OR=1.206, 95% confidenceintervals: 1.014-1.032).Conclusions: SBP appears to contribute to the pathogenesis of MCA stenosis among Chinese. Thegene polymorphism of LPL S447X may be associated with atherosclerotic MCA stenosis in Chinesepopulation.

Glioblastoma stem cells resistant to temozolomide-induced autophagy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells (GSCs) to temozolomide (TMZ) and to explore the possible molecular mechanisms underlying TMZ resistance.</p><p><b>METHODS</b>Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit. The cytotoxic effect of TMZ on CD133(+) and CD133(-) glioblastoma cells was determined by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Autophagy-related proteins (Beclin-1, LC3 and Atg5) and cleaved caspase-3 (p17) were analyzed by Western blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein (GFAP) and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P < 0.05.</p><p><b>RESULTS</b>CD133(+) glioblastoma cells exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133(-) glioblastoma cells treated with 5 or 50 micromol/L TMZ was significantly higher than that in CD133(+) glioblastoma cells ((14.36 +/- 3.75)% vs (2.54 +/- 1.36)% or (25.95 +/- 5.25)% vs (2.72 +/- 1.84)%, respectively, P < 0.05). Atg5, LC3-II and Beclin-1 levels were significantly lower in CD133(+) glioblastoma cells than those in autologous CD133(-) cells after TMZ treatment (P < 0.05). Caspase-3 was mildly activated only in CD133(-) glioblastoma cells after exposure to TMZ (P < 0.05). Immunofluorescent staining revealed elevated expression of Atg5 in GFAP(+) cells following TMZ treatment.</p><p><b>CONCLUSIONS</b>The GSCs display strong capability of tumor's resistance to TMZ. This resistance is probably contributed by the CD133(+) cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients.</p>

Different hypermethylation status of RASSF1A in medulloblastoma and supratentorial primitive neuroectodermal tumor / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the epigenetic involvement of RASSF1A in intracranial primitive neuroectodermal tumors (PNETs) and compare the methylation patterns between medulloblastoma (MBs) and supratentorial PNETs (SPNETs).</p><p><b>METHODS</b>The methylation status at the promoter regions of RASSF1A was examined by methylation-specific polymerase chain reaction (MSP) in a cohort of 25 primary MBs, 9 primary SPNETs, 3 MB and 2 SPNET cell lines. RASSF1A-deficient PNET cell lines were treated with 5-aza-2'deoxycytidine, a demethylating agent, to explore the relationship between hypermethylation and the gene expression.</p><p><b>RESULTS</b>The results revealed no promoter hypermethylation of RASSF1A in 2 normal cerebellar and 5 normal cerebral tissue specimens examined. In contrast, promoter hypermethylation of RASSF1A was detected in 100% (25/25) of primary MBs, 6/9 of primary SPNETs, and all PNET cell lines. These results demonstrated that such epigenetic alteration was tumor-specific. The frequency of hypermethylation of RASSF1A in SPNETs was also found to be significantly lower than that in MBs (Fisher's exact test, P = 0.014). Treatment of RASSF1A-deficient PNET cell lines with 5-aza-2'deoxycytidine restored RASSF1A expression, providing evidence that promoter hypermethylation contributes to transcriptional silencing.</p><p><b>CONCLUSIONS</b>These results demonstrate that RASSF1A plays an important role in the development of intracranial PNETs. Different hypermethylation status of RASSF1A are found in PNET subtypes suggesting that MBs and SPNETs are epigenetical distinct tumors.</p>

Molecular genetic studies on ganglioglioma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the genetic alterations of ganglioglioma through the entire genome, and to investigate the pathogenesis of this neoplasm.</p><p><b>METHODS</b>Comparative genomic hybridization was used to provide an overview of genetic abnormalities in gangliogliomas.</p><p><b>RESULTS</b>Five cases of gangliogliomas, including 3 males and 2 females, were studied genetically. Loss of genetic materials on the short arm of chromosome 9(9p) was a common genetic alteration found in 3 of 5 cases. Overrepresentation of chromosome 7 was another recurrent chromosomal imbalance, which was further confirmed by fluorescence in situ hybridization. Immunohistochemical analysis was performed on epidermal growth factor receptor (EGFR), which was located on 7p11-p13. All five cases revealed no abnormal expression of EGFR. On the other hand, genetic imbalances were also involved in multiple chromosomes including 2q33-q34, 8q12-q22, 14q21-qter, 15q26-qter and Y.</p><p><b>CONCLUSION</b>Loss of genetic materials on chromosome 9p and gain on chromosome 7 may be associated with the pathogenesis of this neoplasm.</p>

Identification of TPO receptors on central nervous system-a preliminary report / 中国实验血液学杂志

To identify the expression of thrombopoietin (TPO) receptors (c-mpl) on central nervous system (CNS) and to evaluate the role of TPO on neural cell proliferation and protection, immunohistochemical staining, RT-PCR, MTT, and annexin-V methods were used in this study. The results showed the expression of TPO receptor on human CNS and murine neural cells. C-mpl mRNA was identified in human cerebral hemispheres and cerebellum, and mouse neural cell line C17.2 by RT-PCR. C-mpl was also confirmed in human cerebral hemispheres by immunohistostaining with con-focal microscopy. Furthermore, TPO had a stimulating effect on the growth of in vitro neural cell C17.2 by MTT assay. The anti-apoptotic effect of TPO on C17.2 cells was also demonstrated by staining with annexin-V and PI. In conclusion, the first evidence showed the expression of TPO receptor c-mpl in central nervous system. Moreover, the effect of TPO on neural cell proliferation and anti-apoptosis was also demonstrated on in vitro neural cells.

Genome-wide genetic study of medulloblastoma using allelotype analysis / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate global genetic alterations in medulloblastoma, and to localize critical chromosomal loci with allelic imbalances associated with the development of medulloblastoma.</p><p><b>METHODS</b>A high-resolution genome-wide allelotype analysis, including 384 microsatellite markers, was performed in 12 medulloblastomas.</p><p><b>RESULTS</b>An average of 238 (62.3%) allelic imbalances were detected on all 39 autosomal arms. Non-random allelic gains or losses were detected on chromosomes 7q (58.3%), 8p (66.7%), 16q (58.3%), 17p (58.3%) and 17q (66.7%). In addition, chromosomal arms with frequencies of allelic imbalances higher than the mean percentage were identified on 3p (33.3%), 3q (33.3%), 4q (41.7%), 7p (33.3%), 8q (41.7%), 10q (41.7%), 13q (33.3%), 14q (33.3%) and 20q (33.3%). No relationship was found between the frequency of allelic imbalances and the clinical outcome of the patients.</p><p><b>CONCLUSIONS</b>A global view of the genetic alterations in medulloblastoma was provided. The allelic imbalances involving chromosomes 7q, 8p, 16q, 17p and 17q may play an important role in the pathogenesis of medulloblastoma.</p>

Genome-wide allelotype study of primary glioblastoma multiforme / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the molecular genetic pathogenesis of primary glioblastoma multiforme (GBM) and identify which chromosomes or chromosomal regions of the entire genome may harbor tumor suppressor genes (TSGs) associated with GBM.</p><p><b>METHODS</b>A high-resolution allelotype study of 21 cases of primary GBM was performed by PCR-based loss of heterozygosity (LOH) analysis. Three hundred and eighty-two fluorescent dye-labeled microsatellite markers covering all 22 autosomes were applied. The mean genetic distance between two flanking markers was about 10 cM.</p><p><b>RESULTS</b>LOH was observed on all 39 nonacrocentric autosomal arms examined in this study. The LOH frequencies of 10q, 10p, 9p, 17p and 13q were the highest (> 50%). Furthermore, high LOH frequencies were detected in the regions containing known TSGs including PTEN, DMBT1, p16, p15, p53 and RB; the LOH frequencies on 14q, 3q, 22q, 11p, 9q, 19q were also high (> 40.5%). Our study observed the following commonly deleted regions: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q25.2-26.2, 11p12-13, 14q13-31, 14q32.1, 14q11.1-13, 22q13.3, 4q35, 4q31.1-31.2, 6q27 and 6q21-23.3.</p><p><b>CONCLUSIONS</b>The molecular pathogenesis of GBM is very complicated and associated with a variety of genetic abnormalities on many chromosomal arms. The most closely related chromosomal arms to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs including PTEN, DMBT1, p16, p15, p53 and RB, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions detected in the present study.</p>

An allelotype study of primary and corresponding recurrent glioblastoma multiforme / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate molecular genetic alterations associated with primary and corresponding recurrent glioblastoma multiforme(GBM) and to identify which chromosomal regions of the whole genome may be involved in the recurrence of primary GBM.</p><p><b>METHODS</b>A high-resolution allelotyping study of one patient's primary GBM and corresponding recurrent GBM was performed by PCR-based loss of heterozygosity(LOH) analysis with the use of 382 fluorescent dye-labeled polymorphic microsatellite markers covering all 22 autosomes. The mean genetic distance between two flanking markers is 10 cM.</p><p><b>RESULTS</b>LOH at locus D9S157 on 9p21 and at loci D10S537, D10S185, D10S192, D10S597, D10S587, D10S217 on 10q21.3-26.3 was observed in the primary GBM. As for corresponding recurrent tumor, LOH was observed not only in expanded regions on 9p21 and 10q21.3-26.3 but also on multiple other chromosomal arms, including 1q, 7p,7q, 21q, 20p, 20q, 10p, 19p, 19q.</p><p><b>CONCLUSION</b>Chromosome 9p and 10q may be involved in the development of this GBM. Although histopathological diagnoses of the primary and corresponding recurrent tumor are identical, the recurrence of GBM is characterized by an increased involvement of molecular genetic abnormalities and may be accompanied by inactivation of more tumor suppressor genes.</p>

Mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells.</p><p><b>METHODS</b>Glioblastoma U87MG cells, which over-express epidermal growth factor receptor (EGFR), were transfected with antisense-EGFR constructs. Several clones with stable expression of lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR on cell differentiation was studied using morphological evaluation and western blotting analysis of glial fibrillary acidic protein (GFAP) expression. The effect of antisense-EGFR on cell cycle was studied by flow cytometry and immunohistochemical analysis of p53, Rb, p16 and CDK4 expressions. The effect of antisense-EGFR on telomerase activity was studied by telomeric repeat amplification protocol (TRAP) assay.</p><p><b>RESULTS</b>U87MG cells that were transfected with antisense-EGFR constructs had smaller cell bodies and longer processes, and expressed higher level of GFAP compared with that of the control cells. Flow cytometric analysis showed that the proportion of cells in G(0)/G(1) phases of the cell cycle in the antisense EGFR cDNA transfected clones increased significantly when compared with control cells, whereas the proportion of cells in S phase decreased markedly. In addition, immunohistochemical analysis showed that the expression of wild-type p53 was significantly increased in the antisense-EGFR cDNA transfected clones, whereas the expressions of Rb, p16 and CDK4 were not altered. TRAP assay revealed that telomerase activity in the antisense-EGFR clones was significantly decreased.</p><p><b>CONCLUSIONS</b>Antisense-EGFR transfection inhibits U87MG cell growth by inducing cell differentiation and p53 expression, G(1) cell cycle arrest and inhibition of telomerase activity.</p>

Chromosome 14q may harbor multiple tumor suppressor genes in primary glioblastoma multiforme / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate whether deletion of chromosome 14q is involved in the carcinogenesis of primary glioblastoma multiforme and to identify possibly common deletion regions. METHJODS: Fourteen fluorescent dye-labeled polymorphic markers were used and polymerase chain reaction-based microsatellite analysis was employed to investigate loss of heterozygosity (LOH) on chromosome 14q in 20 primary glioblastoma multiforme (GBM).</p><p><b>RESULTS</b>Ten of twenty (50%) GBM displayed LOH at one or more of the markers on chromosome 14q. Five tumors showed either LOH or non-informative on all markers tested. The most frequent LOH was observed at locus D14S65 (57.1%) on 14q32.1, and in the chromosomal region spanning from D14S63 (47.1%) to D14S74 (46.7%) on 14q23-31. None of the informative loci exhibited microsatellite instability.</p><p><b>CONCLUSIONS</b>Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM. Chromosomal regions at locus D14S65 on 14q32.1 and spanning from D14S63 to D14S74 on 14q23-31 may harbor multiple tumor suppressor genes associated with GBM.</p>

An allelotype study of human glioblastoma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To reveal the molecular genetic mechanisms for the pathogenesis of glioblastoma (GBM) and determine which chromosomes or chromosomal regions may play a role in the pathogenesis of GBM or may harbor tumor suppressor genes (TSGs) associated GBM.</p><p><b>METHODS</b>An allelotype study of 21 cases of GBM was performed by polymerase chain reaction and loss of heterozygosity (LOH) analysis. Three hundred and eighty-two microsatellite markers covering all 22 autosomes were used. The mean genetic distance between two flanking markers is about 10 cM. Fluorescent dye-labeled primers and Perkin Elmer 377 DNA Sequencer were applied.</p><p><b>RESULTS</b>LOH was observed on all chromosomal arms examined in this study. The LOH frequencies of 10q, 10p, 13q, 17p and 9p were the highest (>50%), on which high LOH frequencies were detected at the regions resided by the known TSGs including PTEN, DMBT1, p16, p15, p53 and Rb. The following commonly deleted regions were detected: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q24-27, 11p12-13, 14q31-32.3, 14q21-24.1, 22q13.2-13.3, 4q35, 4q31.1-31.2, 6qtel, 6q16.3.</p><p><b>CONCLUSION</b>This study demonstrated that the pathogenesis of GBM is very complicated and associated with various molecular genetic abnormalities on lots of chromosomes. The chromosomal arms most closely relevant to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs, such as PTEN, DMBT1, p16, p15, p53 and Rb, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions observed for the first time in this study.</p>