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CD44 is a molecular marker associated with cancer stem cell populations and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting glioma cells high in CD44.Glioma-initiating cell lines were derived from fresh surgical glioblastoma samples. Expression of tissue transglutaminase 2 (TGM2) was attenuated through lentivirus-mediated short hairpin RNA knockdown. MTT assay [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to evaluate the growth inhibition induced by TGM2 inhibitor. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling was used to evaluate cell apoptosis following TGM2 inhibition. CD44(+) glioma stem cells were sorted by flow cytometry. A nude mice orthotopic xenograft model was used to evaluate the in vivo effect of TGM2 inhibitor.TGM2 was highly expressed in CD44-high glioblastoma tissues and tumor-derived glioma-initiating cell lines. TGM2 knockdown impaired cell proliferation and induced apoptosis in CD44-high glioma-initiating cell lines. Further studies indicated that expression of inhibitor of DNA binding 1 protein (ID1) is regulated by TGM2 and might be an important mediator for TGM2-regulated cell proliferation in CD44-high glioma-initiating cell lines. TGM2 inhibitor reduces ID1 expression, suppresses cell proliferation, and induces apoptosis in CD44-high glioma-initiating cell lines. Furthermore, TGM2 is highly expressed in CD44(+) glioma stem cells, while pharmacological inhibition of TGM2 activity preferentially eliminates CD44(+) glioma stem cells. Consistently, TGM2 inhibitor treatment reduced ID1 expression and induced apoptosis in our orthotopic mice xenograft model, which can be translated into prolonged median survival in tumor-bearing mice.TGM2 regulates ID1 expression in glioma-initiating cell lines high in CD44. Targeting TGM2 could be an effective strategy to treat gliomas with high CD44 expression.

Despite the increasing number of clinical applications of circulating Epstein–Barr virus (EBV) DNA analysis for the detection (1)(2), monitoring(3)(4), and prognostication(5)(6) of nasopharyngeal carcinoma (NPC), several fundamental questions concerning plasma EBV DNA, including its tissue origin and relationship with tumor mass, remain unanswered. In the first part of this study, we investigated the relative contributions of tumor cells and other latently EBV-infected lymphoid tissues to the pool of circulating EBV DNA. Previous studies have shown that different EBV genotypes are harbored by latently EBV-infected lymphoid tissues and blood cells (7)(8)(9)(10) in healthy persons and patients with malignancies. On the other hand, it is well established that the EBV is monoclonal in NPC tumor tissues and other EBV-associated cancers(11)(12)(13)(14). Therefore, investigation of the genotype of plasma EBV DNA should reveal the relative contributions of tumor cells and other latently EBV-infected lymphoid tissues to the pool of circulating EBV DNA in NPC patients. For this study, 25 patients with newly diagnosed NPC, 15 patients with systemic lupus erythematosus, and 13 renal transplant recipients were recruited and gave informed consent. This study was approved by the ethics committee of the Prince of Wales Hospital, Hong Kong. Plasma samples were collected from all patients, and tumor specimens for microdissection were obtained from 14 of the 25 NPC patients. DNA extracted from the plasma samples and the microdissected tumor tissues was used for the amplification of EBV DNA, targeting a region encoding the carboxy terminus of latent membrane protein (LMP-1), using the primers 5′-ATGGTAATGCCTAGAAGTAAAGAAAGG-3′ (LMP1f) and 5′-CATAGCCCTAGCGACTCTGCTG-5′ (R2b). The PCR products were ligated to the pGEM®-T Easy Vectors (Promega), and the ligation products were transformed into competent Escherichia coli cells. Twelve bacterial clones were …

Current evidence suggests that epigenetic changes play an important role in the evolution of human cancers. In this study, we evaluated whether hypermethylation of CpG islands at the gene promotor regions of several tumor-related genes is involved in the carcinogenesis of oligodendroglial tumors. We examined the methylation status of 11 genes in a series of 43 oligodendroglial tumors (19 oligodendrogliomas, 13 anaplastic oligodendrogliomas, 9 oligoastrocytomas, and 2 anaplastic oligoastrocytomas) by methylation-specific polymerase chain reaction. Our results showed that hypermethylation of CpG islands was detectable in 8 of 11 genes studied and 74% of tumors were hypermethylated in at least 1 gene. Promotor hypermethylations were detected in O6-methylguanine-DNA methyltransferase (MGMT), RB1, estrogen receptor, p73, p16INK4a, death-associated protein kinase, p15INK4b, and p14ARF at 60%, 34%, 30%, 16%, 12%, 10%, 7%, and 2%, respectively. No hypermethylation was detected in the promotors of glutathione-S-transferase P1, von Hippel-Lindau or the DNA mismatch repair (hMLH1) genes. Statistical analysis revealed that concordant hypermethylation of at least 2 genes, p16INK4a and p15INK4b were significantly associated with anaplastic oligodendroglial tumors, and hypermethylation of MGMT was significantly associated with loss of chromosome 19q and with combined loss of chromosomes 1p and 19q. More importantly, several candidate tumor suppressor genes such as p16INK4a, p15INK4b, and p73 that were previously reported as unmutated in oligodendroglial tumors were found to be hypermethylated in their CpG islands. Taken together, we conclude that hypermethylation of CpG islands is a common epigenetic event that is associated with the development of oligodendroglial tumors.

Glioblastoma multiforme (GBM) often displays morphological heterogeneity in that low-grade (LG) area with well-differentiated cells are commonly found adjacent to high-grade (HG) area with poorly-differentiated cells. This heterogeneity may cause difficulty in obtaining representative tumor samples. Nevertheless, the genetic composition of these cells has only been occasionally examined. In the present study, we examined 29 de novo glioblastomas in which distinct LG and HG areas of sufficient volumes could be identified. These areas were microdissected from paraffin-embedded tissues and analyzed for genetic alterations: p53 mutations and immunohistochemistry; allelic losses at 17p13.1, 9p21, and 10q23-25; and amplification of the epidermal growth factor receptor (EGFR) gene and immunohistochemistry. We also examined 14 paired astrocytic tumors, in which a primary Grade II astrocytoma progressed over a period of time to a Grade III or Grade IV tumor. Our findings showed that the LG areas of the de novo glioblastomas exhibited numerous genetic aberrations, the proportion of which was increased in the HG areas. Genetic abnormalities seen in the LG areas were conserved in the HG areas suggesting that these morphologically different cellular subsets were derived from a common transformed clone. Also, the LG areas were genetically different from Grade II astrocytomas of the paired tumor group, in spite of their morphological similarity. In particular, the LG areas had more deletions on 10q23-25 (75% vs 20%, p = 0.04), but fewer p53 mutations (24% vs 71%, p = 0.003) and less p53 protein labeling (45% vs 79%, p = 0.04). These differences suggest that LG and HG areas in de novo glioblastoma are genetically closer to each other compared with paired low- and high-grade tumors that have progressed over time. Moreover, only a small proportion (17%) of our de novo glioblastomas exhibited EGFR amplification while a high proportion (62%) showed either p53 mutations or allelic loss of 17p13.1. We speculate that some de novo GBMs with copious LG areas may constitute a separate group with rapid progression from Grade II astrocytomas.

A polymerase chain reaction (PCR) method has been validated for the quantitation of retinoic acid (RA) induction of cellular retinoic acid-binding protein II (CRABP-II) RNA from cultured human skin fibroblasts and human skin biopsies. The method utilizes reverse transcription and PCR (RT-PCR) to compare cellular CRABP-II RNA with a known amount of added internal standard RNA generated from a modified CRABP-II cDNA containing a 42 bp deletion. Thus, after RT-PCR of cellular and standard CRABP-II RNA in the same tube, the resulting DNA bands could be distinguished by size on ethidium bromide-stained, nondenaturing polyacrylamide gel. Serial dilutions of cellular RNA were co-amplified with a fixed amount of internal standard CRABP-II RNA, and the ratio of intensities of the two DNA bands was determined by computerized image analysis of the gel photograph. A linear relationship was found between the logs of this ratio and the input RNA. Absolute quantitation of cellular CRABP-II RNA was determined from the 'equivalence point', the dilution at which band intensities from cellular and standard RNAs were identical. Using this quantitative assay, the amount of CRABP-II RNA in cultured fibroblasts was 24 attomoles per microgram total RNA. A 4.2-fold increase in CRABP-II RNA was seen following 24 hours treatment with 10(-6) M RA. CRABP-II RNA content in skin biopsies taken from 3 human subjects ranged from 16 to 25 attomole/micrograms RNA. Topical treatment with 0.1% RA cream resulted in induction ranging from 3.9- to 12-fold over vehicle treatment. The method described here offers a rapid, sensitive and quantitative assay of specific RNAs, and should be especially useful for the measurement of RNA levels from small solid-tissue biopsies.