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Biomedical subjects

C D James

Publications and source records attributed to C D James.

At least 19 recordsLinked to original sources
PubMed1994

Analysis of glioma cell lines for amplification and overexpression of MDM2.

Recently, amplification of the gene encoding a p53 binding protein, MDM2, was determined in 8% of the cases constituting a large series of glioblastomas. Here we have utilized Southern blot analysis to examine 30 cell lines established from such tumors, and our investigation has revealed large increases in MDM2 gene dosage in two cases, one of which showed coamplification of the CDK4 gene that resides in close proximity to MDM2 in chromosomal region 12q13-14. Northern analysis demonstrated overexpression of MDM2 mRNA in the two cell lines with gene amplification, and overexpression of MDM2 protein was evident in each of these by immunohistochemical and Western blot analysis. Analysis of TP53 cDNAs revealed normal TP53 sequences in the cell lines with MDM2 amplification; these results are consistent with those of previous studies suggesting that MDM2 amplification occurs only in tumors expressing wild-type p53. In total, these data suggest that MDM2 amplification in glioblastoma cell lines occurs at a frequency (6.7%) comparable to that determined in primary tumors; occurs in cell lines expressing wild-type p53; and can involve the coamplification of additional genes.

Gene Amplification↗
PubMed1992

Stereoselective high-performance liquid chromatographic assay with fluorometric detection for the isomers of mivacurium in human plasma.

A high-performance liquid chromatographic assay with fluorometric detection was developed for the analysis of the stereoisomers of mivacurium, a new short-acting neuromuscular blocker, in plasma. The isomers were isolated from plasma by solid-phase extraction with C18 and anion-exchange cartridges. The extracts were chromatographed on a LiChrosphere 60 RP Select B column (125 mm x 4.6 mm I.D.) using a mobile phase of acetonitrile-water (4:6, v/v) containing 0.005 M octanesulfonic acid. The fluorescence excitation and emission wavelengths were 202 and 320 nm, respectively. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from true values and the percentage coefficient of variation, respectively, were less than or equal to 10% at all concentrations except for the percentage coefficient of variation at the lower limit of quantitation (5 ng/ml). The assay has been successfully used for the analysis of plasma samples from a pharmacokinetic study in human volunteers.

Chromatography, High Pressure Liquid↗
PubMed1992

Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails.

This study describes genomic rearrangements near the 3' end of the epidermal growth factor receptor (EGFR) gene in eight glioblastomas displaying coamplification and expression of both normal and rearranged EGFR. In four of these cases, it was possible by PCR to amplify tumor EGFR cDNA, which allowed sequence determination of the 3' transcript alterations associated with the rearrangements. Such analysis revealed that the four cases have in common a deletion of 255 bases that encode a portion of the receptor's cytoplasmic domain. The remaining four cases revealed genomic rearrangements in the same region of the gene as those described above and revealed aberrant EGFR transcripts lacking the same 255 bases determined to be missing in the sequenced EGFR cDNAs as well as large regions of contiguous downstream sequences. Therefore, all of the eight cases described here express transcripts that do not encode large C-terminal, intracellular portions of the receptor. In three of the eight cases, the EGFR transcripts displaying a 3' alteration also displayed a 5' inframe deletion of sequences encoding a portion of the extracellular domain, and for one of the corresponding patients it was possible to determine that the two transcript alterations were acquired as separate events. We have now detected the 5' and/or 3' alterations in 21 of 32 cases of glioblastoma with EGFR amplification; no genetic alterations have been detected in glioblastomas without EGFR amplification. In combination with previously published reports, these data suggest the in vivo evolution of EGFR toward an increasingly oncogenic potential through gene amplification with subsequent and successive gene alterations.

Base Sequence↗
PubMed1991

Genes for epidermal growth factor receptor, transforming growth factor alpha, and epidermal growth factor and their expression in human gliomas in vivo.

Anomalies of the epidermal growth factor receptor (EGFR) gene, including amplification, rearrangement, and overexpression, have been reported in malignant human gliomas in vivo. In vitro glioma cell lines coexpress EGFR and at least one of its ligands, transforming growth factor alpha, suggesting the existence of an autocrine growth stimulatory loop. We have studied the tumor tissue from 62 human glioma patients and examined the structure and quantity of the EGFR gene and its transcripts, as well as the quantity of the receptor protein. In addition we have examined the genes and transcripts coding for the pre-pro forms of epidermal growth factor and transforming growth factor alpha, the two endogenous EGFR ligands. EGFR gene amplification was detected in 16 of the 32 malignancy grade IV gliomas (glioblastoma) studied (50%), but only in 1 of 30 gliomas of lesser malignancy grade (I-III). All tumors with an amplified gene overexpressed EGFR mRNA. More than one-half (62.5%) of the glioblastomas with amplified EGFR genes also showed coamplification of rearranged EGFR genes and concomitant expression of aberrant mRNA species. Overexpression, without gene amplification, was observed in some of the low grade gliomas, and aberrant EGFR transcripts were also seen in some cases without gene amplification or detected gene rearrangements. mRNA expression for one or both of the pre-pro forms of the ligands was detected in every tumor studied. Thus, several mechanisms for the activation of the EGFR-mediated growth stimulating pathway are possible in human gliomas in vivo: expression of a structurally altered receptor that may have escaped normal control mechanisms; and/or auto-, juxta-, or paracrine stimulating mechanisms involving coexpression of receptor and ligands, with or without overexpression of the receptor.

Adolescent↗
PubMed1991

Chromosome 9 deletion mapping reveals interferon alpha and interferon beta-1 gene deletions in human glial tumors.

We have applied restriction fragment length polymorphism analysis to a 30-member panel of primary glioma DNAs, which had been previously examined for loss of genetic information (C. D. James, E. Carlbom, J. P. Dumanski, M. Hansen, M. Nordenskjold, V. P. Collins, and W. K. Cavenee, Cancer Res., 48:5546-5551, 1988), to determine the frequency and sublocalization of loss of genetic information from chromosome 9. We have also utilized scanning densitometry for dosage determination of the 9p-localized interferon alpha and interferon beta-1 genes among these same tumors. Our results reveal the following: (a) for those cases in which loss has occurred, the region of common loss lies on the short (p) arm of the chromosome; (b) loss of genetic information from the short arm of chromosome 9 occurs frequently in glial tumors of intermediate (anaplastic, grade III) and high (glioblastoma, grade IV) histological malignancy (10 of 20 cases) but not in tumors of low (grade II) histological malignancy (0 of 10 cases); (c) tumors with 9p deletions are hemi- or nullizygous for interferon beta-1 and the interferon alpha gene cluster; (d) cases of interferon nullizygosity occur exclusively among tumors of highest histological malignancy (glioblastoma). These data, especially the determination of a region of nullizygosity, suggest proximity to or residence within a gene(s) whose function(s) is (are) critical to the suppression of the malignant evolution of glial tumors.

Adolescent↗
PubMed1991

Molecular genetics of human cancer predisposition and progression.

The development of human cancer is generally thought to entail a series of events that cause a progressively more malignant phenotype. Such a hypothesis predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each of the events less the last one and so on. A dissection of the pathway from a normal cell to a fully malignant tumor may thus be viewed as the unraveling of a nested set of aberrations. In experiments designed to elucidate these events we have compared genotypic combinations at genomic loci defined by restriction endonuclease recognition site variation in normal and tumor tissues from patients with various forms and stages of cancer. The first step, inherited predisposition, is best described for retinoblastoma in which a recessive mutation of a locus residing in the 13q14 region of the genome is unmasked by aberrant, but specific, mitotic chromosomal segregation. Similar mechanisms involving the distal short arm of chromosome 17 are apparent in astrocytic tumors and the events are shared by cells in each malignancy state. DNA sequencing indicates that these events accomplish the homozygosis of mutant alleles of the p53 gene. Copy number amplification of the epidermal growth factor receptor gene occurs in intermediate and late-stage tumors whereas loss of heterozygosity for loci on chromosome 10 is restricted to the ultimate stage, glioblastoma multiforme. These results suggest a genetic approach to defining degrees of tumor progression and the locations of genes involved in the pathway as a prelude to their molecular isolation and characterization.

Astrocytoma↗
PubMed1991

Isolation and regional localization of 25 anonymous DNA probes on a chromosome 13 hybrid panel.

Clones were isolated from two flow-sorted chromosome 13 libraries. Twenty-five clones were localized to various regions of chromosome 13, using a well-characterized panel of rodent x human hybrid cell lines. Eight DNA markers were localized to 13q14.2----q22, where the gene for Wilson disease, a recessive disorder of copper metabolism, was previously assigned. The new markers will be useful for the diagnosis of presymptomatic sibs of Wilson disease patients. We isolated six DNA clones proximal to the retinoblastoma gene, a region in which a translocation associated with rhabdomyosarcoma has been observed. Probes for both of these regions will be useful for the cloning of the genes involved in these diseases.

Animals↗
PubMed1990

Loss of genetic information in central nervous system tumors common to children and young adults.

The clonal loss of genetic information as revealed by the comparison of normal and tumor DNA restriction fragment length alleles has permitted the determination of the genomic positions of cancer-recessive mutations. Here we have applied this approach to the analysis of 19 central nervous system tumors that constitute four histologic groups and occur most frequently in children and young adults. The detectable loss of genetic information from cases of medulloblastoma (11 examined) indicates that among such tumors, loss occurs most frequently from the short arm of chromosome 17. For the ependymomas examined (four cases), chromosome 22 was the preferred site for detectable loss. Analysis of pilocytic astrocytomas of the cerebellum (three cases) failed to reveal genetic alterations of any type among such tumors, a finding unique to this histologic group. The single choroid plexus papilloma examined demonstrated loss of genetic information from chromosome 3. Among the 19 tumors, multiple cases of loss were observed from chromosomes 10, 11, 13, and 22, and from the short arm of chromosome 17. Therefore, with regard to the chromosomal locations of implied tumor suppressor genes, these results are consistent with those described for intracranial tumors occurring more commonly in adults of middle to advanced age.

Adult↗
PubMed1990

Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas.

The epidermal growth factor receptor gene has been found to be amplified and rearranged in human glioblastomas in vivo. Here we present the sequence across a splice junction of aberrant epidermal growth factor receptor transcripts derived from corresponding and uniquely rearranged genes that are coamplified and coexpressed with non-rearranged epidermal growth factor receptor genes in six primary human glioblastomas. Each of these six tumors contains aberrant transcripts derived from identical splicing of exon 1 to exon 8 as a consequence of a deletion-rearrangement of the amplified gene, the extent of which is variable among these tumors. In spite of this intertumoral variability, each intragenic rearrangement results in loss of the same 801 coding bases (exons 2-7) and creation of a new codon at the novel splice site in their corresponding transcripts. These rearrangements do not, however, affect the mRNA sequence for the signal peptide, the first five codons, or the reading frame downstream of the rearrangement.

Amino Acid Sequence↗
PubMed1990

Molecular genetic aspects of glial tumour evolution.

Cancer can be viewed as a dynamic process driven by the cellular accumulation of genetic alterations that provide selective growth advantage. Such alterations can be classified as one of two fundamental types: acquired (cancer-affective, dominant) or eliminated (cancer suppressive, recessive). This review considers genetic alterations that occur during the development of the most common type of central nervous system tumour, glioma.

Brain Neoplasms↗
PubMed1989

Loss of genetic information in cancer.

The determination and comparison of genotypic combinations at genomic loci in normal and tumour tissues from patients with various types of cancer have defined the chromosomal locations of loci at which recessive mutations play a role in disease. The predisposing nature of some of these mutant alleles is exemplified in studies of retinoblastoma and osteogenic sarcoma. These two clinically associated diseases share a pathogenetically causal predisposition that maps to chromosome position 13q14. A similar mechanism at 11p15.5 is involved in the development of the embryonal variant of rhabdomyo-sarcoma, Wilms' tumour and hepatoblastoma. Finally, genomic alteration of chromosome 10 is apparent in glioblastomas and mixed tumours of glioblastoma/astrocytoma grade III but not in homogenous astrocytoma grades II or III, suggesting the definition of a locus involved in tumour progression and, perhaps, an approach to molecular genetic staging of tumours.

Blotting, Southern↗
PubMed1989

Mitotic recombination of chromosome 17 in astrocytomas.

Allelic combinations at seven loci on human chromosome 17 defined by restriction fragment length polymorphisms were determined in tumor and normal tissues from 35 patients with gliomas. Loss of constitutional heterozygosity at one or more of these loci was observed in 8 of the 24 tumors displaying astrocytic differentiation and in the single primitive neuroectodermal tumor examined. The astrocytomas showing these losses included examples of each adult malignancy grade of the disease, including glioblastoma (malignancy grade IV), and seven of them demonstrated concurrent maintenance of heterozygosity for at least one chromosome 17 locus. Determination of allele dosage together with the genotypic data indicated that the tumor chromosomes 17 were derived by mitotic recombination in 7 of the 9 cases with shared homozygosity of the region 17p11.2-pter in all cases. In contrast, tumors of oligodendrocytic, ependymal, or mixed cellular differentiation did not exhibit loss of alleles at any of the loci examined. These data suggest that the somatic attainment of homozygosity for loci on chromosome 17p is frequently associated with the oncogenesis of central nervous system tumors, particularly those showing solely astrocytic differentiation, and that mitotic recombination mapping is a useful approach towards the subregional localization of a locus whose rearrangement is involved in this disease.

Astrocytoma↗
PubMed1989

Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines.

To study the cell type specificity of the direction of replication of the human c-myc genes and the relationship of replication polarity to transcriptional activity, we analyzed the directions of replication of the c-myc genes in two Burkitt lymphoma cell lines, CA46 and ST486, and in HeLa cells. On the basis of in vitro runoff replication of forks initiated in intact cells, we found that transcribed c-myc genes in the germ line configuration in HeLa cells were replicated in the direction of transcription from origins in the 5'-flanking DNA, while the repressed, unrearranged c-myc genes of CA46 and ST486 cells were replicated in the antitranscriptional direction. In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.

Burkitt Lymphoma↗
PubMed1988

Clonal genomic alterations in glioma malignancy stages.

Comparison of constitutional and tumor genotypes at chromosomal loci defined by restriction fragment length alleles has proven useful in determining the genomic position and tissue specificity of recessive mutations that predispose to cancer (Hansen, M.F., and Cavenee, W.K. Cancer Res., 47:5518-5527, 1987). Here we have applied this approach to 53 unrelated patients with glial tumors of varying histological malignancy grade. Loss of constitutional heterozygosity for loci on chromosome 10 was observed in 28 of 29 tumors histologically classified as glioblastoma (malignancy grade IV) whereas no similar losses were observed in any of 22 gliomas of lower malignancy grade. Examination of restriction fragment length alleles on other chromosomes revealed that loss of sequences on chromosomes 13, 17, or 22 had occurred at nonrandom frequencies and in at least one instance of each malignancy grade of adult glioma. The tumors in which loss of constitutional heterozygosity was observed were composed of one or a mixture of glial cell subtypes displaying astrocytic, oligodendrocytic, and/or ependymal differentiation. These results demonstrate a close association of the loss of chromosome 10 sequences with the most malignant histological stage of glioma and that glioblastoma arises as the clonal expansion of an earlier staged precursor. Furthermore they suggest that glioblastoma is a common phenotypic and malignancy terminus for glial tumors of various cellular subtypes which is reached through a common molecular pathway. This approach which involves the identification of malignancy stage specific somatic losses of heterozygosity provides a genotypic, rather than phenotypic, analysis of tumor progression.

Adolescent↗
PubMed1986

Polarity of DNA replication through the avian alpha-globin locus.

Toward understanding the controls affecting eucaryotic chromosome replication, we used a runoff replication assay to investigate whether the activity of a gene is related to its use of an upstream or downstream replication origin. When in vivo-initiated DNA polymerases are allowed to complete replication in vitro in the presence of bromodeoxyuridine triphosphate the density label is preferentially incorporated into origin-distal regions of DNA. Isopycnic centrifugation and blot hybridization analysis of the relative bromodeoxyuridine triphosphate incorporation into fragments spanning the chicken alpha-globin locus indicate that this region is replicated from an upstream origin both in chicken lymphocytes and in erythrocytes. Thus the replication polarity of these genes does not change as a function of transcriptional activity, consistent with earlier suggestions that DNA replication in the transcriptional direction may be a necessary but not sufficient condition for gene expression.

Animals↗
PubMed1984

Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair.

In vitro labeling of DNA molecules using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair has been examined as an alternative to existing methods of replacement synthesis labeling. It is shown that exonuclease III is able to act in a common restriction enzyme buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 50 mM NaCl] to produce a population of base-paired primer:template molecules which decrease uniformly in single-strand length with time. After heat inactivation of the exonuclease III and in the presence of radiolabeled deoxynucleotides the polymerase I reaction faithfully resynthesizes full-length molecules, asymmetrically labeled to high specific activity.

Base Composition↗