Establishment and characterization of a human cell line from paediatric cerebellar glioblastoma multiforme.
Permanent glioma cell lines are invaluable tools in understanding the biology of glioblastomas. The present study reports the establishment of a clonal human cell line, GBM6840, derived from a biopsy of paediatric cerebellar glioblastoma multiforme. GBM6840 had a doubling time of 32 h and grew as a monolayer of large round cells that retained immunopositivity for glial fibrillary acidic protein and vimentin. Karyotypic analysis revealed a modal chromosome number of 68 and polysomies of chromosomes 3, 5 and 20, as well as the presence of 3-4 marker chromosomes. GBM6840 also showed anchorage-independent growth in soft agar and tumour formation in nude mice. The p16(CDKN2A) gene was transcriptionally silenced by hypermethylation, consistent with the lack of protein expression observed in the original tumour and cultured cells. Western blot analysis revealed normal protein expression of pRb and CDK4. It appears that p16 is the major component altered in the cell cycle pathway and may confer these cells unrestrained proliferation potential. Neither EGFR gene amplification nor over-expression of the protein was detected in the cultured cells. Over-expression of the p53 protein was observed in the majority of cells, despite undetectable mutation (exons 5-8) in the gene. One allele of the PTEN gene was found to be mutated during in vitro cultivation. Telomerase activity was demonstrated in the cultured cells but not in the original tumour, supporting the hypothesis that telomerase is required for the in vitro immortalization process.
Di Tomaso
E
E
Department of Anatomical, Cellular Pathology; Department of Chemical Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
Pang
J C
JC
Lam
H K
HK
Tian
X X
XX
Suen
K W
KW
Hui
A B
AB
Hjelm
N M
NM
D000293:Adolescent; D015197:Carcinogenicity Tests; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002528:Cerebellar Neoplasms; D002869:Chromosome Aberrations; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma; D006801:Humans; D007621:Karyotyping; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D017354:Point Mutation; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000032:analysis / Q000235:genetics; D019098:Telomerase / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D025521:Tumor Suppressor Proteins
D019941:Cyclin-Dependent Kinase Inhibitor p16; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D066246:ErbB Receptors; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D019098:Telomerase; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human