Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.
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PMID:Double-minute chromosomes as megabase cloning vehicles. 162 62

The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 10(6), 2 x 10(6), and 3 x 10(6) base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.
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PMID:X-ray induction of methotrexate resistance due to dhfr gene amplification. 212 27

Altered mouse dihydrofolate reductase gene (DHFRR) was expressed in murine cells using Abelson murine leukemia provirus genome as a prototype vector. A cDNA clone of DHFRR was inserted into a plasmid structure containing retroviral transcriptional as well as packaging signals. The recombinant plasmid was transfected into psi-2 ecotropic cells and the transient virus was used to infect amphotropic PA-12 cells. Recombinant virus (ABL-DHFRR) was detected in the culture medium of transfected PA-12 cells and was free of helper virus. The ABL-DHFRR was capable of conferring methotrexate (MTX) resistance to a variety of cells in culture. The titer of ABL-DHFRR virus was at least tenfold higher than other DHFR retroviruses. The ABL-DHFRR virus titer was increased by selection at increasing concentrations of MTX. The presence of the DHFRR in the virus-infected cells was confirmed by assays which showed reduced inhibition of enzyme activity by MTX. A helper-virus-free, amphotropic, high-titer retrovirus containing the altered DHFR was obtained which may be of use as a dominant selectable marker in infecting hematopoietic progenitor cells.
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PMID:Development of an amphotropic, high-titer retrovirus vector expressing the dihydrofolate reductase gene and conferring methotrexate resistance. 303 Aug 94

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increased oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involved in DM formation, we have developed strategies to map amplified circular DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed by stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofolate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region containing SsII and NotI sites. At least 490 kb of this DM appears to be composed of unrearranged chromosomal DNA.
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PMID:CpG island mapping of a mouse double-minute chromosome. 833 94

The demonstration tha RNA can be cleavaged by cis-ribozyme(catalytic RNAs, RNA enzyme) has potentially important therapeutic implications. Ribozymes are effective for modulation of gene expression because of their simple structure, site-specific cleavage activity and catalytic potential. The targets of ribozyme-mediated gene modulation have ranged from cancer cells to foreign genes that cause infectious diseases. Additional target sites for ribozymes are in initial phases of development and design. Ribozymes have been targeted against myriad genes, including oncogenes (ras, BCR-ABL) and drug resistance genes(MDR-1, c-fos, DHFR). These ribozymes have cleaved the target RNAs in culture system(in vitro) and developed in vivo system. We reported that anti-fos ribozyme has altered the expression of c-fos and DNA repair genes in cisplatin-resistance cancer cells, and reversed the sensitivity to ciaplatin. Furthermore, we have developed high efficiency by the transfer system using an electroporation in vivo.
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PMID:[Circumventing multidrug resistance in human cancer by anti-ribozyme]. 915 62

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ABL gene rearrangement. The only curative therapy is allogeneic transplantation. Although autologous transplants may prolong survival, most patients relapse because of disease persisting in the host and in the graft. Continued administration of chemotherapy after transplant could reduce the incidence of relapse provided that the autograft can be protected by transfer of a drug-resistance gene. However, CML autografts will almost certainly contain malignant stem cells that will also be rendered drug-resistant. The presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CML. We thus hypothesized that transfer of a vector that combines a drug-resistance gene with anti-BCR/ABL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX)-resistant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS sequences directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and MO7e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210(BCR/ABL) protein levels by 6- to 10-fold in most cells. This subsequently led to the restoration of normal function of BCR/ABL cDNA+ cells: they grew significantly slower in the presence of interleukin-3 (IL-3); they underwent apoptotic cell death when cultured without IL-3; and they had restored expression and function of adhesion receptors. These effects were specific, because LasBD-containing AS sequences directed at the b3a2 BCR/ABL breakpoint did not affect p190(BCR/ABL)-containing cells. LasBD also rendered 20% to 30% of primary Ph- and Ph+ CD34(+) cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resistant Ph+ CD34(+) cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitro and for more than 70 days in vivo. Finally, LasBD decreased tumorigenicity of 32DBCR/ABL cells in vivo by 3 to 4 logs. In conclusion, the tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD can suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prove to be an extremely useful adjunct in autologous transplantation for CML.
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PMID:Gene therapy for chronic myelogenous leukemia (CML): a retroviral vector that renders hematopoietic progenitors methotrexate-resistant and CML progenitors functionally normal and nontumorigenic in vivo. 938 83

Double minute chromosomes (DMs) are the principal genetic vehicles for amplifying oncogenes in human tumors and drug resistance genes in cultured mouse cells. Mouse EMT-6 cells resistant to methotrexate (MTX) generally contain circular DMs, approximately 1 megabase (Mb) in size, that amplify the dihydrofolate reductase (DHFR) gene. The 1 Mb DMs generally have CpG islands located 500 kb upstream of the DHFR gene. The purpose of this study was to determine the relationship between CpG islands and chromosomal breakpoints giving rise to the DM. We show that EMT-6 cells growing in very low levels of MTX that do not yet contain the 1 Mb DHFR-amplifying DM, develop a NotI/EagI site 500 kb upstream of the DHFR gene. This NotI site is close to, if not identical with, one of the chromosomal breakpoints giving rise to the DM. We show that 500 kb of DM DNA from upstream of the DHFR gene is derived from 500 kb of chromosomal DNA upstream of the chromosomal DHFR gene. The downstream breakpoint maps to a region approximately 200 kb downstream of the DHFR gene near a chromosomal SstII/EagI site. Therefore, approximately 700 kb of DM DNA was derived from the genomic region surrounding the DHFR gene. To confirm the organization of the DM DNA, we isolated DNA probes from the 1 Mb DM. Using pulsed field gel electrophoresis and Southern hybridization, we determined the approximate location of each probe with respect to the CpG island in both the DM and the chromosome. Approximately 300 kb of chimeric DNA from a region unrelated to the DHFR gene was incorporated during DM formation. Implications for the mechanism of DM formation are discussed.
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PMID:Chromosome breakpoints near CpG islands in double minutes. 975 10

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.
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PMID:Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques. 1458 36

Active site mutations that disrupt drug binding are an important mechanism of drug resistance. Computational methods capable of predicting resistance a priori are poised to become extremely useful tools in the fields of drug discovery and treatment design. In this paper, we describe an approach to predicting drug resistance on the basis of Dead-End Elimination and MM-PBSA that requires no prior knowledge of resistance. Our method utilizes a two-pass search to identify mutations that impair drug binding while maintaining affinity for the native substrate. We use our method to probe resistance in four drug-target systems: isoniazid-enoyl-ACP reductase (tuberculosis), ritonavir-HIV protease (HIV), methotrexate-dihydrofolate reductase (breast cancer and leukemia), and gleevec-ABL kinase (leukemia). We validate our model using clinically known resistance mutations for all four test systems. In all cases, the model correctly predicts the majority of known resistance mutations.
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PMID:Efficient a priori identification of drug resistant mutations using Dead-End Elimination and MM-PBSA. 2265 99

Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher's test p=0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p=0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.
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PMID:Copy number analysis of 24 oncogenes: MDM4 identified as a putative marker for low recurrence risk in non muscle invasive bladder cancer. 2502 75


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