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1.
Neuropathol Appl Neurobiol ; 26(2): 172-6, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10840280

ABSTRACT

Human tumour xenografts maintained in nude mice are a valuable research tool. The passaging and maintenance of human tumour xenografts in immune-deficient animals are expensive and labour-intensive. This study presents a protocol that permits long-term cryopreservation of viable glioblastoma xenograft tissue pieces in liquid nitrogen. Twenty different human glioblastoma xenografts that have been successfully transplanted and repeatedly passaged in nude mice were cryopreserved to validate the method. Different passages were cryopreserved for up to 40 months. On retransplantation of the tumours, all cases except one grew successfully. In order to ensure that the individual tumours did grow (not induced mouse tumours) restriction fragment length polymorphism analysis with variable number of tandem repeats (VNTR) probes was carried out. The method permits the long-term storage of viable, retransplantable glioblastoma xenografts. It minimizes animal use for the maintenance of xenografts and permits cryo-back-up of valuable tumours, thus markedly reducing the cost and increasing the accessibility of human tumour xenografts as a research tool in biology and genetics.


Subject(s)
Brain Neoplasms/pathology , Cryopreservation , Glioblastoma/pathology , Neoplasm Transplantation/methods , Animals , Blotting, Southern , Brain Neoplasms/genetics , Cell Division , Cell Survival , DNA, Neoplasm/analysis , DNA, Neoplasm/isolation & purification , Glioblastoma/genetics , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Transplantation, Heterologous
2.
J Neuropathol Exp Neurol ; 59(12): 1087-93, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11138928

ABSTRACT

Approximately 30%-35% of human glioblastomas have epidermal growth factor receptor (EGFR) gene amplification. Amplicons containing the EGFR gene frequently include several unidentified adjacent genes. Amplification of adjacent genes in the absence of EGFR amplification has been documented. To define the region in detail, we produced a YAC contig map, determining the orientation of the EGFR gene and the general order of 11 STS and EST markers. Seventy-six tumors with amplification of the region were found in a series of 246 human astrocytic gliomas. The amplicons showed amplification of contiguous or noncontiguous loci both telomeric and centromeric to the EGFR gene. Six percent (12/190) of the glioblastomas and 1 anaplastic astrocytomas had amplicons excluding the EGFR gene. These amplicons commonly contain amplified loci telomeric to the EGFR gene. Some of the amplified loci were found to be consistently overexpressed. The findings suggest that there may be other gene target(s) for amplification in the 7p12 region in addition to EGFR.


Subject(s)
Astrocytoma/genetics , Central Nervous System Neoplasms/genetics , Gene Amplification/genetics , Chromosome Mapping , Gene Expression , Gene Frequency , Genes, erbB-1/genetics , Humans
3.
J Neuropathol Exp Neurol ; 58(7): 697-701, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10411339

ABSTRACT

Amplification of the epidermal growth factor receptor (EGFR) occurs in about 40% of human glioblastomas. In half of these cases, rearrangements of the amplified gene result in aberrant transcripts and proteins. The most frequent rearrangement affects the external domain of the receptor and results in nonbinding of ligand and constitutive activity. Less frequent rearrangements involve changes resulting in the loss of cytoplasmic amino acid sequences necessary for downregulation of the receptor following ligand binding. Here we report the development and selection for a rearranged amplified EGF receptor, which lacks cytoplasmic amino acid sequences in a human glioblastoma xenograft. An identical aberration has previously been reported in glioblastoma tissue. The patient tumor material, as well as the first passages of the xenograft showed amplification of the EGFR gene, but no evidence of gene rearrangement or an aberrant transcript. Interphase FISH data show the amplified gene on double minutes. Between passages 3 and 16, the growth rate of the xenograft almost doubled, the rearranged amplicon became dominant, as did the aberrant transcript, indicating selection under these conditions.


Subject(s)
ErbB Receptors/genetics , Gene Amplification , Gene Rearrangement , Glioblastoma/genetics , Neoplasm Transplantation , Transplantation, Heterologous , Animals , Base Sequence/genetics , Gene Amplification/genetics , Gene Rearrangement/genetics , Humans , In Situ Hybridization, Fluorescence , Mice , Mice, Inbred C57BL , Mice, Nude , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
4.
J Neuropathol Exp Neurol ; 57(12): 1138-45, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9862636

ABSTRACT

The first gene found to be amplified in human glioblastomas was EGFR at 7p12. More recently the MET gene at 7q31 was also reported amplified. We have studied chromosome 7 in a series of 47 glioblastomas by FISH, RFLP and microsatellite analysis. Four per cent (2/47) had 1 centromere, 26% (12/47) 2, 32% (15/47) 3, 4% (2/47) 4, and 34% (16/47) had subpopulations with variable numbers of chromosome 7 centromeres. In 25 of the 47 tumors (53%) the pattern of allelic imbalance observed at each informative locus was similar and in accord with the FISH data, indicating loss or gain of complete chromosome copies. In 32% of tumors (15/47) varying allelic imbalance was seen at different loci along the chromosome indicative of loss or gain of parts of chromosome 7 on a background of disomy, trisomy, tetrasomy, or polysomy. Amplification was studied in an extended series of 121 glioblastomas, and was seen at the 7p12 region in 47 tumors (39%). Forty-two tumors showed amplification of EGFR and 12 of these had extensive amplicons including a number of adjacent loci, always involving only 1 allele. The amplicons of 5 tumors (11%) did not include EGFR, indicating that other unidentified genes in the region are targeted for amplification. Amplification of MET was not found. The findings show that copy number changes of chromosome 7 are common and that a number of genes may be targeted for amplification at 7p12 in glioblastomas.


Subject(s)
Chromosomes, Human, Pair 7 , Gene Rearrangement , Genes, erbB-1 , Glioblastoma/genetics , Receptor Protein-Tyrosine Kinases/genetics , Alleles , Centromere/physiology , Chromosomes, Human, Pair 8 , Humans , In Situ Hybridization, Fluorescence , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
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