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At least 19 recordsLinked to original sources
PubMed1990

Mammalian drug resistant mutants with multiple gene amplifications: genes encoding the M1 component of ribonucleotide reductase, the M2 component of ribonucleotide reductase, ornithine decarboxylase, p5-8, the H-subunit of ferritin and the L-subunit of ferritin.

Hydroxyurea was used to select two very highly drug resistant cell lines, designated HR-15 and HR-30. Both drug resistant lines contained elevated levels of ribonucleotide reductase activity. Northern and Southern blot analysis indicated that the two drug resistant lines contained increased levels of mRNA for the two components, M1 and M2, of ribonucleotide reductase, and M1 and M2 gene amplifications. Alterations in M1 and M2 protein levels were also evident in Western blot analysis. Further studies of HR-15 and HR-30 cells by Northern and Southern blot analysis showed that the drug resistant cell lines had elevated levels of ornithine decarboxylase mRNA and p5-8 mRNA, as well as increased ornithine decarboxylase and p5-8 gene copy numbers, respectively. Furthermore, characterization of HR-15 and HR-30 drug-resistant cell lines revealed increased mRNA levels for both H- and L-ferritin. Both cell lines exhibited by Southern blot analysis, amplification of the H- and L-ferritin genes. Increases in the cellular levels of H- and L-ferritin subunit proteins were also observed in both HR-15 and HR-30 cells, by Western blot analysis. This is the first description of mutant cell lines containing this complex combination of modified gene expressions and gene amplifications. The alterations exhibited by these lines confirm and extend present models of hydroxyurea resistance, are in agreement with and help substantiate models of ribonucleotide reductase regulation and provide interesting links between the expressions of several cellular activities important in proliferation.

Animals↗
PubMed1991

Gene amplification and gene dosage in cell lines derived from squamous cell carcinoma of the head and neck.

Gene amplification and related alterations in gene dosage were analyzed in a series of 34 cell lines derived from different human head and neck squamous cell carcinomas (SCCHN). INT2 gene amplification was observed in 62%, MYC gene amplification in 24%, and EGFR gene amplification in 21% of the cell lines. There was a strong correlation between EGFR gene amplification and increased copies of the ERBB2 gene on chromosome 17, suggesting a synergistic selection for these two genes either during cancer progression or in culture. Two abnormalities showed a significant correlation with clinical course: MYC gene amplification showed an inverse correlation with tumor recurrence (r = -0.44, p = 0.01), and a small increase in MYCL gene copies on chromosome I correlated with the presence of metastases (r = 0.61, p = 0.001). This altered MYCL gene dosage might represent a chromosome translocation rather than true gene amplification. In addition to gene amplification, 79% of the cell lines had increased copies of chromosome 8. Comparison of the cell lines with several of the corresponding primary tumors demonstrated that most gene amplifications were already present in the primary tumors, although some appeared de novo in cell culture. These studies indicate that gene amplification, especially of INT2, is a prominent abnormality in head and neck squamous cell cancer. Aneuploidy and chromosomal lesions other than gene amplification were also found to alter the dosage of several oncogenes specifically.

Carcinoma, Squamous Cell↗
PubMed1990

Normal diploid human and rodent cells lack a detectable frequency of gene amplification.

Gene amplification is frequently observed in tumors and transformed cell lines. This phenomenon is known to contribute to the generation of drug-resistant tumor cells and quantitation of the event is believed to have prognostic value in several types of neoplasias. To date, most studies of gene amplification have used immortalized cell lines and biopsied tumor samples. In this study I examine the amplification potentials of primary diploid cells, both human and rodent, and quantitatively compare them to the amplification potentials of their transformed counterparts. I have used a strictly defined protocol (i.e., selection at a stringency of 9 X LD50) to measure amplification potential at two loci, the gene for the multifunctional protein containing activities for carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase (CAD) and the gene for dihydrofolate reductase (DHFR). I find that the frequency of amplification in transformed cells is at least four orders of magnitude greater than that in normal cells. Out of 29 cell populations studied, the 7 diploid normal cell populations exhibited no detectable amplification frequency (limit of detection at 10(-8) whereas the 22 transformed cell lines demonstrated amplification frequencies between 10(-3) and 10(-7). These results demonstrate that a dramatic difference exists between primary diploid cell populations and immortalized cell populations in their ability to amplify genomic sequences and suggests a significant difference in genetic stability between these two cell types.

Animals↗
PubMed1992

Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification.

Gene amplification is characteristic of tumors and continuous cell lines but not of primary, normal, diploid, senescing cells. However, the rat cell line REF52, which resembles primary cells in requiring expression of cooperating oncogenes for transformation, is unusual among cell lines as it is not permissive for amplification. REF52 cells did not form colonies in N-(phosphonacetyl)-L-aspartate (PALA), a drug for which the only known mechanism of resistance is amplification of the carbamoylphosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) gene. Colonies did form in a low concentration of methotrexate but did not contain amplified dihydrofolate reductase genes. Expression of two cooperating oncogenes in REF52 cells converted them to a state permissive for amplification. Cells expressing only the 12S E1A mRNA of adenovirus 5 did not give rise to PALA-resistant colonies, but expression of an activated ras gene together with E1A readily allowed the cells to form resistant colonies in which the CAD gene was amplified. Cells expressing E1A plus ras were fully transformed, but expression of simian virus 40 large tumor antigen alone converted REF52 cells to a state permissive for amplification without transforming them fully. The ability to manipulate gene amplification in REF52 cells by expression of oncogenes should contribute to an understanding of the nature of the permissive state.

Adenovirus Early Proteins↗
PubMed1977

Selective amplification of genes on the R plasmid, NR1, in Proteus mirabilis: an example of the induction of selective gene amplification.

The drug-resistance plasmid, NR1, is a 37-micron circular DNA molecule that contains two components: the resistance transfer factor (29 micron) carrying the transfer genes and the genes for tetracycline resistance, and the r-determinant (8 micron) carrying the genes for resistance to several other antibiotics including chloramphenicol (Cm). In Proteus mirabilis, these two components are capable of independent replication, or they may replicate as a composite molecule. When cells of P. mirabilis containing NR1 are cultured in medium containing Cm at 250 microgram/ml a growth lag of 20-35 hr ensues. During this lag, Cm induces the selective amplification of the r-determinant, including the gene for resistance to Cm. The amplification results from the excision of the r-determinant from the R plasmid, the independent replication of the r-determinant to give polymeric as well as monomeric r-determinants, and the eventual reintegration of multiple tandem copies of the r-determinant with the resistance transfer factor to form a new R plasmid with multiple copies of the r-determinant. This mechanism represents a new level of control of gene expression in bacterial systems--namely, the induction of selective gene amplification.

Cell Division↗
PubMed1987

Structure of an inverted duplication formed as a first step in a gene amplification event: implications for a model of gene amplification.

Inverted duplications have been observed to be a common feature of gene amplification in mammalian cells and appear to be generated as a primary event in the amplification process (Ford et al., 1985; Ford and Fried, 1986). The structural features of the amplified inverted duplication, containing the polyoma virus oncogene middle T-antigen, were analysed in transformed 3B rat cells. No unusual sequences such as transposition elements were detected at the site of the inversion. The inversion was generated by a simple illegitimate recombination event in which only a single nucleotide directly at the point of the inversion cannot be accounted for from the sequence of the two parental strands. Possible structural (hairpin formation) and sequence (rich AT) features may have been involved in the illegitimate recombination event at the inversion join. In the cellular DNA near one of its joins with polyoma virus DNA an unusual sequence of 198 bp composed of 99 consecutive purine-pyrimidine pairs has been detected. A model for the generation of amplified DNA containing inverted duplications is proposed.

Animals↗
PubMedDate not supplied

Antiblastic treatment does not affect N-myc gene amplification in neuroblastoma.

Gene amplification has been found in the genome of cells growing in vivo and/or in vitro. In cell lines with acquired multidrug resistance gene amplification has been frequently detected. Moreover, extra-copies of cellular oncogenes have been located in tumor cells in vivo; particularly N-myc gene amplification was discovered in advanced stage of neuroblastoma (NB). Neuroblastoma, a tumor of neural origin, has a high incidence in children. N-myc amplification has been demonstrated in untreated patient and a positive significant correlation with the progression of the disease has been established. In this paper we report on four NB patients treated with a polychemotherapeutic protocol and showing N-myc amplification. One patient examined before and after treatment displayed a slight change in N-myc gene copy numbers. It was shown that N-myc gene amplification is not affected by drug activities and that minimal residual of cells bearing N-myc amplification may remain in the tumor mass. N-myc amplification can also cause advantageous cell growth in the presence of drugs. The implications in the pharmacologic management of NB patient showing N-myc gene amplification is discussed.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Amino Acid Sequence↗
PubMed1991

Inhibition of human immunodeficiency virus gene amplification by heparin.

Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.

Citric Acid↗
PubMed1992

Suppression of gene amplification in human cell hybrids.

Gene amplification, one example of genetic instability, is of prognostic and clinical importance in neoplasia. In tumorigenic cells, gene amplification occurs at a very high frequency, whereas in normal diploid fibroblasts the event is undetectable by the clonogenic assay. To investigate genetic control of gene amplification, amplification frequency was measured in hybrids of tumorigenic cells and normal diploid cells. The ability to amplify an endogenous gene behaved as a recessive genetic trait, and control of gene amplification potential segregated independently of tumorigenicity and immortality.

Aspartate Carbamoyltransferase↗
PubMed1989

[c-myc gene amplification and N-ras transforming gene in two cases of acute myelocytic leukemia with double minute chromosomes].

We report two leukemia patients with double minutes (DMs) chromosomes. Both patients were diagnosed as having acute myelocytic leukemia (AML) FAB M2. Cytogenetic analysis showed normal chromosome karyotype with 1-53 DMs chromosomes in the first patient, and complex chromosome aberrations including deletion of chromosome 8 at 8q24 region and 1-84 DMs chromosomes in the second patient who had a history of extensive radiotherapy for laryngeal cancer 8 years prior to the development of leukemia. Analysis of DNA from the two patients revealed that oncogene of c-myc was amplified about 5 to 10 folds in the leukemic cells. The other fourteen oncogene of c-myc was c-myb, c-abl and N-myc, showed no increases of gene content. Furthermore, a transforming gene, N-ras was detected in the first patient by in vivo selection assay method. This is the second report on AML patients with c-myc gene amplification and DMs chromosomes.

Aged↗
PubMed1989

Multiple populations of deleted mitochondrial DNA detected by a novel gene amplification method.

A gene amplification method for detecting small populations of deleted mitochondrial DNA was used in analysis of skeletal muscle from a patient with ocular myopathy. Multiple populations of differently deleted mtDNA were detected in the patient muscle. The presence of deleted mtDNAs was further confirmed by comparison of the shift in the sizes of the amplified fragments with the shift in the positions of the primers used for the amplification, (the primer shift PCR method). Other methods, namely Southern blotting, enzymic activity measurement, and Western blotting, were inefficient at detecting the mitochondrial abnormality. These findings suggest that the primer shift PCR method could be valuable for accurate diagnosis of ocular myopathy associated with mtDNA deletion.

Blotting, Western↗
PubMed1987

Gene amplification: the Chinese hamster glutamine synthetase gene.

The glutamine analogue, methionine sulphoximine, has been used to develop Chinese hamster ovary cell lines which overproduce the enzyme glutamine synthetase. Southern and Northern blot hybridization studies showed that this overproduction is due to gene amplification of a region of DNA containing the glutamine synthesis gene coding sequences, with a corresponding increase in glutamine synthetase mRNA and protein. This amplification has increased the glutamine synthetase gene copy number to approximately 1000 copies per cell. Genomic DNA sequences from the amplified region have been cloned and the structure of the glutamine synthetase gene within this region is being analysed.

Animals↗
PubMed1992

In vivo gene amplification in non-cancerous cells: cholinesterase genes and oncogenes amplify in thrombocytopenia associated with lupus erythematosus.

The ACHE and BCHE genes, encoding the acetylcholine hydrolysing enzymes acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE), co-amplify with several oncogenes in leukemic patients with platelet deficiency (thrombocytopenia). This and other experiments implicated ACHE and BCHE in the development of bone marrow megakaryocytes, the progenitors of platelets. Therefore, we wished to find out whether cholinesterase gene amplification would also occur in non-cancerous platelet disorders and, if so, whether oncogenes would amplify in such cases as well. The autoimmune disease systemic lupus erythematosus (SLE) presents an appropriate model system for this issue, since patients with SLE may suffer from thrombocytopenia resistant to most treatment modalities. Here, we report a 40-80-fold amplification of genomic sequences from the ACHE and BCHE genes as well as the C-raf, V-sis and C-fes/fps oncogenes in peripheral blood cells from an SLE patient with severe thrombocytopenia. PvuII restriction analysis and DNA blot hybridization of the amplified ACHE and BCHE sequences demonstrated apparent aberrations in both genes, suggesting that malfunctioning of modified, partially amplified cholinesterase genes may be involved in the etiology of thrombocytopenia associated with SLE. These observations imply that cholinergic mechanisms regulate megakaryocytopoiesis, shed new light on the diverse hematologic findings characteristic of SLE, and may become valuable as diagnostic, treatment and prognostic tools in the follow-up of patients suffering from thrombocytopenia associated with SLE. Furthermore, these findings reinforce the notion that cholinesterase gene amplifications are causally related with platelet abnormalities in multiple hemopoietic disorders.

Acetylcholinesterase↗
PubMed1987

The effect of dihydrofolate reductase-mediated gene amplification on the expression of transfected immunoglobulin genes.

Murine/human chimeric gamma 1 and K Ig genes were cloned adjacent to the gene coding for methotrexate-resistant dihydrofolate reductase. These constructs were introduced into myeloma cells, and lines containing stably integrated genes were selected. The integrated Ig genes were then amplified by selection of the cells in increasing concentrations of methotrexate. The extent of gene amplification, mRNA accumulation, and production of Ig was studied in transfectomas containing introduced light chain genes, heavy chain genes, or both. When the light chain gene was introduced alone, it was expressed at low levels, but after selection with methotrexate, light chain expression was increased as much as 63-fold. In contrast, the transfected heavy chain genes were highly expressed, but production of the corresponding protein was increased a maximum of only fourfold by methotrexate treatment. Cellular toxicity of unassembled heavy chain monomer was not observed, even at amounts equivalent to 2% of total cellular protein. Cointroduction of the heavy and light chain constructs with subsequent amplification resulted in as much as 25-fold increase in secretion of intact antibody relative to unamplified cells. The results demonstrate that amplification of Ig genes can induce transfectomas to secrete antibody at nearly the rate of hybridomas.

Drug Resistance↗
PubMed1988

Formation of an inverted duplication can be an initial step in gene amplification.

We have developed a gene transfer approach to facilitate the identification and isolation of chromosomal regions which are prone to high-frequency gene amplification. Such regions are identified by assaying for transformants which show high-frequency resistance to PALA and/or methotrexate by amplification of a vector containing the genes which encode the enzyme targets of these antiproliferative agents. We identified 2 of 47 transformants which displayed high-frequency amplification of the transfected genes, and in this report we describe the analysis of one of them (L46). Molecular analysis of the integration site in transformant L46 revealed that the donated genes were at the center of an inverted duplication which spanned more than 70 kilobase pairs and consisted largely of host DNA. The data suggest that integration of the transfected sequences generates a submicroscopic molecule containing the inverted duplication and at least 750 kilobases of additional sequences. The donated sequences and the host sequences were readily amplified and lost in exponentially growing cultures in the absence of drug selection, which suggests that the extrachromosomal elements are acentric. In contrast to the instability of this region following gene insertion, the preinsertion site was maintained at single copy level under growth conditions which produced copy number heterogeneity in L46. The implications of our results for mechanisms of genetic instability and mammalian gene amplification are discussed.

Animals↗
PubMed1992

Gene amplification as a target for cancer chemotherapy.

Facile gene amplification is one aspect of the genetic instability associated with transformed cells. Amplification of oncogenes and proto-oncogenes contributes to carcinogenesis and tumor progression. Gene amplification is also a common basis for resistance to anticancer drugs. The observation that low level cytotoxic stress can cause rapid loss of amplified genes from cultured cell populations suggests that gene amplification may be a potential target for cancer chemotherapy. Drug-induced loss of amplified genes is seen with a wide variety of extrachromosomally amplified genes, including drug resistance genes and proto-oncogenes. A number of drugs and differentiating agents have been reported to cause rapid loss of unstably amplified genes. An effect on amplified genes or cells carrying amplified genes may contribute to the selective action of drugs presently used for cancer chemotherapy. A better understanding of drug-induced amplified gene loss may lead to new strategies for cancer treatment.

Animals↗