Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

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Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weedAmaranthus palmeri

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719354115 ◽

2018 ◽

Vol 115 (13) ◽

pp. 3332-3337 ◽

Cited By ~ 56

Author(s):

Dal-Hoe Koo ◽

William T. Molin ◽

Christopher A. Saski ◽

Jiming Jiang ◽

Karthik Putta ◽

...

Keyword(s):

Gene Amplification ◽

Herbicide Resistance ◽

Germ Cells ◽

Gene Copy Number ◽

Gene Copy ◽

Circular Dna ◽

Dna Structures ◽

Meiotic Chromosomes ◽

Extrachromosomal Elements ◽

Mitosis And Meiosis

Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs).Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplifiedEPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplifiedEPSPScopies in glyphosate-resistant (GR)A. palmeriare present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock’s postulated innate systems [McClintock B (1978)Stadler Genetics Symposium] that can rapidly produce soma variation, amplifyEPSPSgenes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.

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Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group study.

Journal of Clinical Oncology ◽

10.1200/jco.1991.9.4.581 ◽

1991 ◽

Vol 9 (4) ◽

pp. 581-591 ◽

Cited By ~ 413

Author(s):

A T Look ◽

F A Hayes ◽

J J Shuster ◽

E C Douglass ◽

R P Castleberry ◽

...

Keyword(s):

Tumor Cell ◽

Gene Amplification ◽

Gene Copy Number ◽

Disease Free Survival ◽

Gene Copy ◽

Early Treatment Failure ◽

Free Survival ◽

Myc Gene ◽

Disease Free

We assessed tumor cell DNA content (ploidy) and N-myc gene copy number as predictors of long-term disease-free survival in 298 children with neuroblastoma. Diploid tumor stem lines were identified in 101 patients (34%), clonal hyperdiploid abnormalities in 194 (65%), and hypodiploid stem lines in three (1%). In children with widely disseminated tumors at diagnosis (stage D), ploidy had a highly age-dependent influence on prognosis. Among infants (less than 12 months) treated with cyclophosphamide-doxorubicin, hyperdiploidy was closely associated with long-term disease-free survival (greater than 90% of cases), while diploidy invariably predicted early treatment failure (P less than .001). Similarly, in children 12 to 24 months of age who were treated with cisplatin-teniposide and cyclophosphamide-doxorubicin, diploidy uniformly predicted early failure, whereas half of the children with hyperdiploidy achieved long-term disease-free survival (P less than .001). There was no relationship between ploidy and treatment outcome in children older than 24 months with stage D tumors who had a very low probability of long-term disease-free survival (less than 10%). N-myc gene amplification was detected in 37 (25%) of the 147 tumors tested, with the remainder showing single-copy levels of the gene. N-myc gene amplification was more frequent in diploid than in hyperdiploid tumors (23 of 57 v 14 of 87, P = .001) and predicted a high likelihood of early treatment failure. In children younger than 2 years with disseminated neuroblastoma, tumor cell ploidy and N-myc gene copy number provide complementary prognostic information that will distinguish patients who can be cured on current regimens from those who require new treatment strategies.

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MET Gene High Copy Number (Amplification/Polysomy) Identified in Melanoma for Potential Targeted Therapy

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab171 ◽

2021 ◽

Author(s):

Nisha S Ramani ◽

Ajaykumar C Morani ◽

Shengle Zhang

Keyword(s):

Targeted Therapy ◽

Gene Amplification ◽

Copy Number ◽

Kinase Inhibitors ◽

Gene Copy Number ◽

Tissue Microarrays ◽

High Copy Number ◽

Gene Copy ◽

Aberrant Expression ◽

Mesenchymal Epithelial Transition Factor

Abstract Objectives Aberrant expression of the mesenchymal epithelial transition factor (MET) gene has been observed in several malignancies, and drugs targeting the MET gene have been implicated in clinical trials with promising results. Hence, MET is a potentially targetable oncogenic driver. We explored the frequency of MET gene high copy number in melanomas and carcinomas. Methods The study group included 135 patients. Tissue microarrays were constructed with 19 melanomas and 116 carcinomas diagnosed from 2010 to 2012. We screened MET gene copy number by fluorescence in situ hybridization analysis using probes for MET gene and CEP7 as control. Results We found MET gene amplification in 2 (11%) of 19 melanoma cases, whereas 5 (26%) of 19 melanoma cases showed polysomy. For carcinomas, there was no MET gene amplification identified. However, 8 (7%) of 116 cases showed polysomy. Conclusions In our study, MET gene amplification was identified in 11% of melanomas and is relatively concordant with few reported studies. However, about 26% of the additional melanoma cases showed MET gene polysomy, which has not been reported as per our knowledge. If these results are validated with further orthogonal studies, more of the melanoma cases could potentially benefit from targeted therapy with MET tyrosine kinase inhibitors.

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Mathematical models of gene amplification with applications to cellular drug resistance and tumorigenicity.

Genetics ◽

10.1093/genetics/125.3.633 ◽

1990 ◽

Vol 125 (3) ◽

pp. 633-644

Author(s):

M Kimmel ◽

D E Axelrod

Keyword(s):

Mathematical Models ◽

Cell Lines ◽

Gene Amplification ◽

Dihydrofolate Reductase ◽

Gene Copy Number ◽

Gene Copy ◽

Published Data ◽

Reductase Gene ◽

Double Minute ◽

Double Minute Chromosomes

Abstract An increased number of copies of specific genes may offer an advantage to cells when they grow in restrictive conditions such as in the presence of toxic drugs, or in a tumor. Three mathematical models of gene amplification and deamplification are proposed to describe the kinetics of unstable phenotypes of cells with amplified genes. The models differ in details but all assume probabilistic mechanisms of increase and decrease in gene copy number per cell (gene amplification/deamplification). Analysis of the models indicates that a stable distribution of numbers of copies of genes per cell, observed experimentally, exists only if the probability of deamplification exceeds the probability of amplification. The models are fitted to published data on the loss of methotrexate resistance in cultured cell lines, due to the loss of amplified dihydrofolate reductase gene. For two mouse cell lines unstably resistant to methotrexate the probabilities of amplification and deamplification of the dihydrofolate reductase gene on double minute chromosomes are estimated to be approximately 2% and 10%, respectively. These probabilities are much higher than widely presumed. The models explain the gradual disappearance of the resistant phenotype when selective pressure is withdrawn, by postulating that the rate of deamplification exceeds the rate of amplification. Thus it is not necessary to invoke a growth advantage of nonresistant cells which has been the standard explanation. For another analogous process, the loss of double minute chromosomes containing the myc oncogene from SEWA tumor cells, the growth advantage model does seem to be superior to the amplification and deamplification model. In a more theoretical section of the paper, it is demonstrated that gene amplification/deamplification can result in reduction to homozygosity, such as is observed in some tumors. Other applications are discussed.

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Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani

Nepal Journal of Biotechnology ◽

10.3126/njb.v3i1.14225 ◽

2015 ◽

Vol 3 (1) ◽

pp. 22-28 ◽

Cited By ~ 4

Author(s):

Mahendra Maharjan ◽

Rentala Madhubala

Keyword(s):

Leishmania Donovani ◽

Gene Copy Number ◽

Clinical Isolates ◽

Gene Copy ◽

Hsp70 Expression ◽

First Line ◽

Over Expression ◽

Hsp70 Protein ◽

Pentavalent Antimony ◽

Different Parts

Pentavalent antimonials have long been the first line of defence against leishmaniasis, but resistance has been reported in different parts of the world. Pentavalent antimony is reduced into trivalent form in the cells and is a potential inducer of HSP70 in L. donovani. Expression profile of HSP70 in antimony susceptible and resistant L. donovani isolates were characterized by Southern blot, Northern blot and western blot analysis. HSP70 gene copy number, gene expression and HSP70 protein expression was found uniform in both antimony sensitive and resistant clinical isolates. In laboratory condition, Leishmania cells respond to antimonial drug stress by three fold over expression of the HSP70 protein. The observed results indicated that HSP70 play important role in stress tolerance against antimonial drug without differential expression in antimony sensitive and resistance clinical isolates of L. donovani.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 22-28

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Characterization of Human Pregnancy Specific Glycoprotein (PSG) Gene Copy Number Variations in Pre-eclampsia Patients

Advances in Experimental Medicine and Biology - Circulating Nucleic Acids in Serum and Plasma – CNAPS IX ◽

10.1007/978-3-319-42044-8_12 ◽

2016 ◽

pp. 63-65

Author(s):

Chia Lin Chang ◽

Chia Yu Chang ◽

Da Xian Lee ◽

Po Jen Cheng

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Copy Number Variations ◽

Gene Copy ◽

Human Pregnancy

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Resistance to methotrexate due to gene amplification in a patient with acute leukemia.

Journal of Clinical Oncology ◽

10.1200/jco.1984.2.1.16 ◽

1984 ◽

Vol 2 (1) ◽

pp. 16-20 ◽

Cited By ~ 92

Author(s):

M D Carman ◽

J H Schornagel ◽

R S Rivest ◽

S Srimatkandada ◽

C S Portlock ◽

...

Keyword(s):

Gene Amplification ◽

Copy Number ◽

Cdna Probe ◽

Leukemia Cell ◽

Gene Copy Number ◽

Leukemia Cell Line ◽

Gene Copy ◽

Acute Myelocytic Leukemia ◽

Human Leukemia ◽

Dot Blot

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.

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