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PubMed1999

Rapid detection of MYCN gene amplification and telomerase expression in neuroblastoma.

Amplification of the MYCN gene and high telomerase activity predict a poor prognosis for the patients with neuroblastoma. We used PCR techniques for rapid detection of MYCN gene amplification and human telomerase reverse transcriptase (hTERT) expression in neuroblastoma specimens. The detection of MYCN gene amplification is based on differential PCR in which three primer pairs were used to coamplify a 178-bp fragment of target MYCN gene with two reference gene fragments, a 237-bp of p53 exon 7 and a 120-bp of beta-globin exon 3, in a single tube of 40 surgically resected tumor samples. MYCN amplification was identified by this differential PCR in all 10 samples carrying more than 10 copies (already known to have MYCN gene amplification by Southern blot analysis). There were no false-negative or false-positive cases, and the relative intensity of MYCN bands in the differential PCR correlated significantly with the copy number determined by Southern blot analysis (y = 0.99, P<0.0001). This protocol was also applicable in the biopsy or aspirated samples, as well as the paraffin-embedded tissues, and in detecting intratumoral heterogeneity. Using RT-PCR procedures, hTERT mRNA expression was detectable in all 13 tumors with high telomerase activity. These nonradioisotopic PCR-based protocols for detecting MYCN gene amplification and hTERT mRNA expression are rapid and reliable and are likely to be useful to determine the biological behavior of neuroblastoma.

Biomarkers, Tumor↗
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↗
PubMed1996

De novo generation of simple sequence during gene amplification.

Mammalian cells that have undergone gene amplification and/or gene rearrangement have been used as resources to gain insight into the questions of chromosome structure and dynamics. The multidrug resistant murine cell line J7.V2-1 has been shown previously to contain two distinct forms of the highly amplified mdr2 gene, a member of the mouse gene family responsible for the multidrug resistant (MDR) phenotype [Kirschner, L. S. (1995) DNA Cell Biol. 14, 47-59]. Characterization of both forms of the gene revealed that one form corresponded to the wild-type structure of the gene, whereas the other represented a rearrangement. Investigation of this altered gene demonstrated a deletion of 1.6 kb of the wild-type sequence, and replacement of this region with a poly(AT) tract that appears to have been generated de novo. Analysis of the native sequence in this region demonstrated the absence of repetitive elements, but was notable for the presence of two long stretches of polypurine: polypyrimidine strand asymmetry. Analysis of mdr2 transcripts in this cell line revealed that nearly all of the mRNA is transcribed from the rearranged form of the gene. This message is unable to code for a functional mdr2 gene product, owing to a deletion of the fourth exon during this event. Mechanisms of the rearrangement, as well as the significance of this curious effect on transcription, are discussed.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

Breast Neoplasms↗
PubMed1993

MDM2 gene amplification in metastatic osteosarcoma.

The human homologue of the murine double minute 2 gene (MDM2), a p53-binding protein which may act as a regulator of p53 protein function, has recently been cloned. Initial studies of this gene in a variety of human tumors have shown frequent gene amplification in most types of sarcomas, including osteosarcomas. Amplification of the MDM2 gene may produce a functional inactivation of the p53 protein. To examine possible clinical or pathological correlates of MDM2 gene amplification in osteosarcoma, we studied 28 specimens on 26 patients with high grade osteosarcoma (16 primary, 11 metastatic, and 1 local recurrence) for MDM2 gene amplification by Southern blot analysis, using two MDM2 complementary DNA probes isolated by polymerase chain reaction. Four specimens (14%) showed amplification, including 3 metastases and 1 local recurrence. None of the primary osteosarcoma specimens had detectable MDM2 gene amplification. None of the specimens tested showed MDM2 gene rearrangement. In the present series, MDM2 gene amplification was detected significantly more frequently in metastatic or recurrent osteosarcomas than it was in primary osteosarcomas (P = 0.02). Our data suggest that MDM2 gene amplification may be associated with tumor progression and metastasis in osteosarcoma. Further investigation is warranted on the potential clinicopathological correlates of MDM2 gene amplification in osteosarcoma.

Adolescent↗
PubMed1997

Gene amplification and genomic plasticity in prokaryotes.

Gene amplification is a common feature of the genome of prokaryotic organisms. In this review, we analyze different instances of gene amplification in a variety of prokaryotes, including their mechanisms of generation and biological role. Growing evidence supports the concept that gene amplification be considered not as a mutation but rather as a dynamic genomic state related to the adaptation of bacterial populations to changing environmental conditions or biological interactions. In this context, the potentially amplifiable DNA regions impose a defined dynamic structure on the genome. If such structure has indeed been selected during evolution, it is a particularly challenging hypothesis.

Gene Amplification↗
PubMed1998

The MDM2 gene amplification database.

The p53 tumor suppressor gene is inactivated in human tumors by several distinct mechanisms. The best characterized inactivation mechanisms are: (i) gene mutation; (ii) p53 protein association with viral proteins; (iii) p53 protein association with the MDM2 cellular oncoprotein. The MDM2 gene has been shown to be abnormally up-regulated in human tumors and tumor cell lines by gene amplification, increased transcript levels and enhanced translation. This communication presents a brief review of the spectrum of MDM2 abnormalities in human tumors and compares the tissue distribution of MDM2 amplification and p53 mutation frequencies. In this study, 3889 samples from tumors or xenografts from 28 tumor types were examined for MDM2 amplification from previously published sources. The overall frequency of MDM2 amplification in these human tumors was 7%. Gene amplification was observed in 19 tumor types, with the highest frequency observed in soft tissue tumors (20%), osteosarcomas (16%) and esophageal carcinomas (13%). Tumors which showed a higher incidence of MDM2 amplification than p53 mutation were soft tissue tumors, testicular germ cell cancers and neuro-blastomas. Data from studies where both MDM2 amplification and p53 mutations were analyzed within the same samples showed that mutations in these two genes do not generally occur within the same tumor. In these studies, 29 out of a total of 33 MDM2 amplification-positive tumors had wild-type p53. We hypothesize that heretofore uncharacterized carcinogens favor MDM2 amplification over p53 mutations in certain tumor types. A database listing the MDM2 gene amplifications is available on the World Wide Web at http://www. infosci.coh.org/mdm2 . Charts of MDM2 amplification frequencies and comparisons with p53 genetic alterations are also available at this Web site.

Animals↗
PubMed1997

Association of EGFR gene amplification and CDKN2 (p16/MTS1) gene deletion in glioblastoma multiforme.

Glioblastoma multiforme (GBM) can be divided into genetic subsets: approximately one-third of GBM, primarily in older adults, have EGFR amplification; another one-third, primarily in younger adults, have TP53 mutation. The majority of GBM also have homozygous deletions of the CDKN2 (p16/MTS1) gene, resulting in cell cycle deregulation and elevated proliferation indices. We evaluated the relationship between CDKN2 deletions and the GBM subsets as defined by EGFR amplification or TP53 mutation in 70 GBM. Twenty-eight cases (40%) had EGFR amplification, 21 (30%) had TP53 mutation, and 21 (30%) had neither change. CDKN2 deletions were present in 36 (51%) GBM. Of the 28 GBM with EGFR amplification, 20 (71%) had CDKN2 deletion (p = 0.0078). The remaining 16 cases with CDKN2 loss were divided between GBM with TP53 mutations (6 cases) and GBM with neither EGFR amplification nor TP53 mutation (10 cases). Thus, CDKN2 deletions occur twice as commonly in GBM with EGFR amplification (71%) than in GBM with TP53 mutation (29%). CDKN2 deletions occurred in GBM from patients somewhat older than those patients with GBM lacking CDKN2 deletion (mean age 53 vs. 48 years). Specifically among GBM with EGFR amplification, those with CDKN2 deletions also occurred in patients slightly older than those few GBM without CDKN2 deletions (mean age 55 vs. 51 years). The presence of CDKN2 deletions in most GBM with EGFR amplification and in generally older patients may provide one explanation for the potentially more aggressive nature of such tumors.

Carrier Proteins↗
PubMed1997

MDM2 gene amplification and expression in non-small-cell lung cancer: immunohistochemical expression of its protein is a favourable prognostic marker in patients without p53 protein accumulation.

MDM2 is an oncoprotein that inhibits p53 tumour-suppressor protein. Amplification of the MDM2 gene and overexpression of its protein have been observed in some human malignancies, and these abnormalities have a role in tumorigenesis through inactivation of p53 function. To determine the clinicopathological and prognostic value of MDM2 abnormalities in non-small-cell lung cancer (NSCLC), MDM2 gene amplification and its protein expression status were analysed in surgically resected materials. MDM2 gene amplification was detected in only 2 (7%) of the 30 tested patients. MDM2 protein was found immunohistochemically in a total of 48 (24%) of the 201 patients. MDM2 protein was slightly frequently observed in patients with adenocarcinoma, but its presence or absence was not associated with clinicopathological factors such as T-factor, N-factor, stage, tumour size, differentiation or p53 protein status. Overall, MDM2-positive patients tended to have a better prognosis (P = 0.062). In particular, among immunohistochemically p53-negative patients (n = 110), those with positive MDM2 protein expression showed significantly better prognosis (P = 0.039) and, in a multivariate analysis, MDM2 protein status was a favourable prognostic factor (P = 0.037). In contrast, among p53-positive patients (n = 91), there was no difference in prognosis depending on MDM2 protein status. Thus, in the NSCLC patients studied, MDM2 gene amplification was a minor event, but expression of its protein, which was often observed immunohistochemically, was a favourable prognostic marker, especially among patients without p53 protein accumulation. Further study is needed to determine how MDM2 protein expression results in a better prognosis.

Adult↗
PubMed2000

Detection of c-erbB-2 and FGF-3 (INT-2) gene amplification in epithelial ovarian cancer.

We examined gene amplifications of the c-erbB-2 and FGF-3 gene in 48 epithelial ovarian cancers by differential PCR and addressed their association with clinico-pathological features including clinical outcome. Overall, 25.0 and 20.8% of ovarian cancers displayed amplified c-erbB-2 or FGF-3 gene, respectively. Amplification of the c-erbB-2 gene was significantly associated with particular histological cell types, higher histological grade, and low levels of serous CA125. However, there was no correlation between c-erbB-2 gene amplification and patient outcome. No correlation was observed between FGF-3 gene amplification and any clinico-pathological features including overall survival. These findings suggested that c-erbB-2 or FGF-3 gene amplification might be one of the oncogenic events implicated in the development of ovarian cancers, yet is not a useful prognostic marker.

Adult↗
PubMed2002

[Dual gene amplification and selection system with dihydrofolate reductase and glutamine synthetase genes effectively increase the foreign gene expression].

BACKGROUND: To study the effect of gene amplification and selection system with DHFR plus GS and DHFR or GS gene on the foreign gene expression. METHODS: Using the N-terminal truncated hTPO(T184) gene as target gene, two plasmidsre were constructed: pDC- T184 and pGC-T184 where DHFR and GS gene were used respectively as the selective amplification marker. They were cotransfected into CHO dhfr cells to establish dual gene amplification and selection system of DHFR plus GS gen and respectively transfected to establish single gene amplification and selection system of DHFR or GS gene. Three selective methods in dual selective system to compare expression efficiency of hTPO were designed: the first method (DG) was to use drug pressure of MTX, then use MSX; the second method (GD) was reversed; the third method was simultaneously to use MTX and MSX as drug pressure. RESULTS: DHFR+GS dual system had not only higher gene amplification efficiency but also higher level expression. There was no distinct affect in different method of drug pressure. CONCLUSIONS: MTX plus MSX dual drug pressure in dual selection system was an efficient and simple method to increase the expression of foreign gene in mammalian cells.

Animals↗
PubMed1995

C-erbB-2 gene amplification: a molecular marker in recurrent bladder tumors?

C-erbB-2 gene amplification and protein overexpression have been implicated as prognostic markers for patients with recurrent progressive bladder tumors. This event has been investigated as a potential diagnostic indicator in archival samples of transitional cell carcinoma of the bladder. Two hundred thirty-six bladder tumors from 89 patients with recurrent disease (mean follow-up, 4 years), 20 tumors from patients with no evidence of bladder tumor recurrence (mean follow-up, 7 years) and 10 normal bladder controls (patients with no history of transitional cell carcinoma) were studied. A differential PCR was used to provide a semiquantitative estimate of C-erbB-2 gene amplification. Protein overexpression was assessed immunohistochemically. Sixteen of 89 patients with recurrent disease had evidence of C-erbB-2 gene amplification. No C-erbB-2 gene amplification was seen in the nonrecurrent tumors or normal bladder controls. Of the 89 patients with recurrent bladder tumors, 43 had evidence of progressive disease, and of these, 14 patients exhibited C-erbB-2 gene amplification, indicating a strong association with gene amplification and progressive disease (P < 0.0005). Gene amplification in these patients was seen only after disease progression had occurred. Protein overexpression was seen in 50% of patients with recurrent and 45% of patients with nonrecurrent disease. No protein overexpression was seen in normal controls. Protein overexpression could not be linked to disease progression. C-erbB-2 gene amplification and protein overexpression were of predictive value in multivariate analysis for overall bladder cancer death; however stage and grade remained the most important independent prognostic variables. C-erbB-2 gene amplification and protein overexpression were of no value as independent markers for the prediction of disease recurrence or progression. It appears from these results that the role of C-erbB-2 as a diagnostic marker may far outweigh its usefulness as a prognostic indicator.

Aged↗
PubMed2006

Amodiaquine resistance is not related to rare findings of pfmdr1 gene amplifications in Kenya.

OBJECTIVES: Many countries are now adopting artemisinin-based combination therapy (ACT) for treatment of Plasmodium falciparum malaria. In multi-drug resistant areas in South East Asia amplifications of the pfmdr1 gene are frequent and tentatively associated with reduced susceptibility to the common quinoline partner drugs mefloquine and lumefantrine. In Africa where amodiaquine is one of the favoured quinoline partner drugs in ACT, studies on multi-drug resistance associated pfmdr1 gene amplifications are urgent. Our aim was to determine the current prevalence of pfmdr1 gene amplifications and a possible association between pfmdr1 gene copy number and amodiaquine treatment outcome in Kenya. METHODS: Seventy-two children with Plasmodium falciparum infection in Kenya were treated with amodiaquine monotherapy and followed for 21 days. Possible amplification of the pfmdr1 gene was assessed from blood-spotted filterpaper by TaqMan probe based real-time polymerase chain reaction. RESULTS: The recrudescent rate was 14 of 72 (19%). All children had single pfmdr1 copy infections, with the exception of one child who had an infection with two pfmdr1 copies. This child had an adequate treatment response. CONCLUSION: Pfmdr1 amplifications do exist in Kenya but at a very low frequency. Yet, the substantial number of children with recrudescent infections implies that amodiaquine resistance is not related to pfmdr1 gene amplifications in Kenya.

Amodiaquine↗
PubMed2002

Gene amplification and its application in cell and tissue engineering.

Gene amplification means the repeated replication of a certain gene without a proportional increase in the copy number of other genes and is a widespread phenomenon in eukaryotes. It is an important developmental and evolutionary process in many organisms. This article focuses on mammalian gene amplification and its application in cell and tissue engineering. The dhfr gene amplification in Chinese hamster ovary (CHO) cells, the gene amplification mechanism, the selection protocol and the application of gene amplification were reviewed.

Journal Article↗
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↗
PubMed1986

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification.

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G2 chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.

Animals↗
PubMed1995

MDM2 and CDK4 gene amplification in Ewing's sarcoma.

Amplification of the MDM2 gene, which maps to chromosome band 12q13 and encodes a p53-binding protein, may result in functional inactivation of p53 and has been observed in various bone and soft tissue sarcomas. Published studies have included few cases of Ewing's sarcoma (ES) or peripheral neuroectodermal tumour (PNET), a tumour group in which alterations of the p53 pathway have so far not been extensively studied. We examined two ES cell lines, RD-ES and SK-ES-1, and 30 specimens from 27 patients (24 ES, 6 PNET; 19 primary, 4 local recurrence, 7 metastasis) for MDM2 gene amplification by Southern blot analysis. All 30 clinical specimens had been confirmed to contain sufficient ES/PNET DNA by the demonstration of a rearrangement of the t(11;22)-associated EWS gene using an EWS cDNA probe on the same blots. MDM2 gene amplification was detected in 3 of 30 specimens (10 per cent), including two ES and one PNET, but in neither of the cell lines. The three cases with amplification were morphologically typical primary tumours. Two of the three cases also showed co-amplification of the CDK4 gene, which encodes a cyclin-dependent kinase and also maps to band 12q13. Clinically, all three cases had metastatic disease at diagnosis, compared with only 1 of 15 MDM2-negative cases where the primary tumour was studied. The difference was statistically significant (P = 0.005), suggesting an association of MDM2 amplification with advanced stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2002

Evidence for replicative repair of DNA double-strand breaks leading to oncogenic translocation and gene amplification.

Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.

Animals↗