Biomedical subjects
D Cournoyer
Publications and source records attributed to D Cournoyer.
Gene therapy: a new approach for the treatment of genetic disorders.
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Studies on the control of gene expression of the carcinoembryonic antigen family in human tissue.
The control of expression of genes of the carcinoembryonic antigen family was investigated in 22 specimens of malignant and nonmalignant human colonic tissues. These surgical specimens included seven colonic adenocarcinomas that were compared with normal adjacent colonic mucosal tissues from the same individual. mRNA preparations from all colonic tissues expressed three bands of 3.5, 3.0, and 2.6 kilobases on Northern blots probed with carcinoembryonic antigen (CEA) complementary DNA probe while normal liver and spleen were negative. The major band of 3.0 kilobases was 6 to 10 times more intense in the colon tumor specimens than in the matched normal mucosa. However, the tumor/normal ratios of immunoreactive CEA in these pairs varied from 2- to greater than 100-fold. Furthermore, there was no direct proportionality between mRNA levels and gene product expression, suggesting that the known variations in CEA expression in human colonic tissues result from both transcriptional and posttranscriptional control mechanisms. Southern blots of DNA from these specimens did not reveal any gene rearrangements or amplifications accompanying expression. Finally, Southern blots of DNA digested with methylation-sensitive endonucleases and probed with a genomic DNA fragment upstream of CEA gene coding regions demonstrated that CEA expression is correlated with a decreased level of methylation in the 5' region of the CEA gene.
Transcription of genes of the carcinoembryonic antigen family in malignant and nonmalignant human tissues.
Normal and diseased human tissues were analyzed for the transcription of genes of the carcinoembryonic (CEA) family. Epithelial tissues of colonic origin, whether malignant or normal, all express two closely related mRNA species of 3.0- and 3.5-kilobase mRNA which code for CEA. Only tissues of colonic origin were found to express these CEA-specific transcripts. Colon carcinomas consistently express a 2.6-kilobase mRNA species as well which codes for nonspecific cross-reacting antigen. Nonneoplastic colon mucosas, on the other hand, express lower or nondetectable levels of this transcript. Most breast carcinomas produce only the nonspecific cross-reacting antigen mRNA, whereas leukocytes of chronic myelogeneous leukemia express both nonspecific cross-reacting antigen mRNA and a 2.3-kilobase mRNA corresponding to a yet undefined gene of the CEA family. Thus the multiple CEA-like products reported to be produced by these tissues correspond to only four different mRNA species coding for three different peptides. These data suggest a less complex organization of the CEA family than was previously suspected and point to posttranscriptional modifications, such as variable patterns of glycosylation, as the likely reason for much of the observed complexity in CEA-like glycoproteins.
Hemolytic anemia following intravenous gamma globulin administration.
A young homosexual man with immune thrombocytopenia recently had transient intravascular hemolysis during intravenous gamma globulin treatment. The hemolysis, manifested by a sharp decrease in hemoglobin and by a direct Coombs' test with a positive result, was mediated by anti-A antibody present in the gamma globulin preparation. In view of the increasing use of intravenous gamma globulin in the treatment of patients with immune cytopenia, this problem may result in crossmatching difficulties and should be recognized as a potential complication of therapy.
Trisomy 9 in hematologic disorders: possible association with primary thrombocytosis.
Ten patients with a hematologic disorder and a clone of cells with trisomy 9 in the bone marrow were studied in order to investigate the clinical significance of this chromosome anomaly. In five of the patients, trisomy 9 was the only anomaly; in four, there was also trisomy 8; and in one, a Y chromosome was also lacking. Four patients had a myelodysplastic syndrome, and six had a myeloproliferative disorder. Interestingly, four patients had primary thrombocytosis.
Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.
Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.
Resistance to cytosine arabinoside by retrovirally mediated gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells.
Dose-limiting hematopoietic toxicity produced by the cytosine nucleoside analogue cytosine arabinoside (ARA-C) is one of the major factors that limit its use in the treatment of neoplastic diseases. An interesting approach to overcome this problem would be to insert a gene for drug resistance to ARA-C in normal hematopoietic cells to protect them from drug toxicity. The deamination of ARA-C by cytidine deaminase results in a loss of its antineoplastic activity. The objective of this study was to determine if gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells would confer drug resistance to ARA-C. Retrovirally mediated transfer of the human cytidine deaminase gene into 3T3 fibroblasts resulted in efficient expression of the proviral RNA for this gene and in increased cytidine deaminase activity in cytoplasmic extracts. These cells showed marked resistance to ARA-C as determined by the effects of this drug on colony formation, cell growth, and DNA synthesis. The transfer of the human cytidine deaminase gene into murine bone marrow cells by the retroviral vector conferred a high level of drug resistance to ARA-C in clonogenic assays. These studies indicate that the cytidine deaminase gene could be used in cancer gene therapy by protecting normal hematopoietic cells against the cytotoxic effects of ARA-C and related cytosine nucleoside analogues.