Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0002871 (anemia)
 52,094 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.
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PMID:Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells. 204 39

We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA. A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined. Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene. Intermolecular recombination was assessed by two methods after cotransfection. In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria. In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form. By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay. The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination. Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection. Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination.
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PMID:Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects. 255 Sep 80

As a first step to the cloning of the Fanconi anemia (FA) gene, we have attempted to correct the sensitivity of FA cells to DNA crosslinking agents by the introduction of wild-type DNA. The protocol involved the introduction of both genomic and pRSVneo DNA, selection for G418-resistant colonies and the subsequent selection of mitomycin C-resistant cells from the latter. Preliminary experiments indicated that untransformed FA cells were not suitable recipients for the introduction of foreign DNA, so all experiments were performed with an SV40-transformed FA cell line. Approximately 40,000 G418-resistant colonies were obtained in 5 separate experiments at an overall frequency of about 5 X 10(-4). These were then selected in mitomycin C and 15 colonies were recovered. Colonies were obtained with wild-type DNA (both human and rodent) and with FA DNA at about the same frequency of 2 X 10(-7). Colonies were isolated and shown to have a stable, partial (from 25 to 90% of wild-type) resistance to mitomycin C. One colony was also shown to be partially resistant to two other DNA crosslinking agents, diepoxybutane and nitrogen mustard. This clone also had an intermediate level of spontaneous and MMC-induced chromosome aberrations. pRSVneo, but not rodent, DNA could be demonstrated in the high molecular weight fraction of several colonies. Thus, it is likely that these colonies represent partial revertants rather than transfectants. These mitomycin C-resistant FA cells should be useful for the biochemical analysis of the FA mutation.
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PMID:Studies of gene transfer and reversion to mitomycin C resistance in Fanconi anemia cells. 311 27

Although PDGF is not a primary hematopoietic cytokine, effects in hematopoietic cell cultures have been reported. We recently described responses of multilineage hematopoietic precursors to PDGF. The effects were shown to be neutralized by antibody to IL-1 beta and mediated by marrow macrophages that expressed PDGF receptor RNA and responded to PDGF by upregulation of IL-1 RNA. The present study was performed to determine whether constitutive expression of PDGF by hematopoietic cells would have hematopoietic consequences in vivo. Retroviral vectors containing a PDGF-B gene were constructed and infected into normal marrow cells. Irradiated mice reconstituted with infected cells consistently developed a lethal myeloproliferative syndrome with anemia, neutrophilia and monocytosis, declining hematopoiesis in marrow with shift to the spleen, and extensive infiltration of immature hematopoietic cells into the parenchymal organs and connective tissues. In addition to PDGF, the retroviral constructs expressed a neo resistance marker. Phenotypic expression patterns in fibroblasts and in hematopoietic colony-forming cells in vitro were consistent with a significant degree of interaction between the two expressed inserts. Moreover, selection of infected cells for G418 resistance significantly reduced not only the number of infected reconstituting cells but also the intensity of the evoked syndrome in vivo. The observations have important implications for projected gene therapy protocols, and identify a novel potential pathway to myeloproliferative disease.
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PMID:Overexpression of PDGF-B in murine hematopoietic cells induces a lethal myeloproliferative syndrome in vivo. 830 75

Retroviral vectors are effective shuttle systems by introducing therapeutically relevant genes stably into the genome of proliferating cells. The majority of vectors applied for research or clinical applications use neomycin for cell selection and identification. To circumvent the time consuming and potentially toxic G418 selection process in transduction studies we constructed a novel marker vector using I-NGFR as a cell surface marker to identify DNA repair defective Fanconi anemia cells complemented with the FAC gene. The new vector constructed is based on a MoMLV backbone, a signal peptide-deleted I-NGFR receptor gene under control of a LTR promoter and the therapeutically relevant FAC gene placed downstream of a SV40 promoter. Supernatants containing high titers of amphotropic viruses from FACS cloned cell cultures were obtained and tested for primary transduction rates, rapid detection of transduced cells within 48 h and correction of mitomycin C-induced cell cycle G2 phase accumulation in a single assay using multiparameter, dual laser flow cytometry. Primary transduction efficiency detected via (I-NGFR) antibody was between 5% and 30% with Fanconi cell lines, 5% with CD34+ cells and 15% with PBLs. MMC-induced G2 phase cell cycle disturbances were fully complemented in Fanconi anemia B cell lines of complementation group C but not in B cell lines of another FA complementation group (D). In addition to the normalization of the G2 phase arrest, induction of cell death in the FAC cell line was also decreased three to 10-fold at different MMC concentrations.
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PMID:A novel, membrane receptor-based retroviral vector for Fanconi anemia group C gene therapy. 917 20