Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have established an ultraviolet (UV) resistant Chinese hamster ovary (CHO) cell line B11UVres by repetitive UV exposure of the CHO cell line B11. We have characterized the resistant cell line with respect to growth, sensitivity to various DNA damaging agents, and the repair of UV induced DNA lesions. When examining sensitivity to UV in clonogenic survival studies, we find that the ID50 is increased 2.3-fold in the resistant cell line B11UVres compared to the parental cell line B11. Although the doubling time of the resistant cell line is greater than that of the parental cell line, there is no difference in the rate of replication after UV irradiation. When measuring repair of UV induced DNA lesions in the overall genome we find no significant difference between the two cell lines. However, at early times after UV, there is a significant increase in the rate of repair of cyclobutane pyramidine dimers (CPDs) in the transcribed strand of the dihydrofolate reductase (DHFR) gene in the B11UVres cells compared to the B11 cells. There is a small increase of steady state transcription of the DHFR gene in the UV resistant cells, but hardly enough to account for the repair increase. The UV resistant cell line B11UVres is not cross-resistant to the cross-linking agents mitomycin C or cisplatin, but shows increased sensitivity to these compounds.
Carcinogenesis 1995 Dec
PMID:DNA repair in a UV resistant Chinese hamster ovary cell line. 860 88

Among various 150-nM methotrexate-resistant (MTXr) V79 clones isolated, we found that two near-tetraploid clones as well as a near-diploid clone with amplification in the dihydrofolate reductase (dhfr) gene readily developed resistant to 40 000 nM MTX within 3 months during stepwise increased MTX selection, while two near-diploid clones without gene amplification could not acquire resistance beyond 5000 nM MTX. Then, we studied how the clones increased the resistance to MTX, and compared the propensity for gene amplification among three types of clones. Dot blot analysis showed that the acquisition of the high levels of resistance to MTX observed in two near-tetraploid clones and a near-diploid clone with gene amplification was associated with amplification in the dhfr gene. The amplified dhfr gene was overexpressed at mRNA and protein levels in the clones. Southern blot analysis of Hind III- and Eco RI-digested DNA in the clones at the time when they became resistant to 10 000 nM MTX indicated that they amplified the dhfr gene fragments which existed in low amounts in parental V79 cells, and that no gross rearrangement of the amplified dhfr gene was detected. Furthermore, fluorescence in situ hybridization analysis showed that the amplified dhfr gene was located on one chromosome as cluster(s). On the other hand, two near-diploid clones without gene amplification did not show any amplification of the dhfr gene even at 5000 nM-MTX resistant stage. These combined results suggest that the near-tetraploid clone as well as the near-diploid clone with the dhfr gene amplification have genomic instability with the propensity for gene amplification during stepwise MTX selection, and have a similar process for the development of the dhfr gene amplification.
Carcinogenesis 1996 Mar
PMID:A comparison of the propensity for gene amplification between near-tetraploid and near-diploid V79 clones resistant to 150 nM methotrexate. 863 Nov 21

Development of resistance to cisplatin in previously treatment-responsive malignancies is a major obstacle to successful treatment. Enhanced DNA repair as well as enhanced replicative bypass of DNA adducts have been suggested to play a role in the development of resistance to cisplatin. However, the relative contribution of these mechanisms is unknown. Second generation platinum compounds containing the 1,2-diaminocyclohexane (dach) carrier ligand have been of particular interest in the studies of resistance mechanisms since they have been effective in treatment of cells resistant to cisplatin. We have investigated the formation and repair of interstrand crosslinks (ICL) in the mouse leukemia cell line L1210/0 and its carrier ligand specific resistant derivatives L1210/DDP and L1210/DACH after treatment with ethylenediamine (en)-Pt and diaminocyclohexane (dach)-Pt compounds. ICL in the overall genome were examined using a modification of the alkaline elution assay. A Southern blot technique was employed for the study of ICL in specific regions of the genome. In the overall genome we found decreased formation of ICL with either -en or -dach carrier ligands in the two resistant cell lines without carrier ligand specificity. Some carrier ligand specificity of ICL formation was observed in the dihydrofolate reductase (DHFR) gene, but it did not correlate with the carrier ligand specificity of resistance. At the level of the overall genome there was no difference in repair of ICL between the sensitive and the two resistant cell lines. When measured in the DHFR gene, however, there was enhanced repair of ICL in the two resistant cell lines compared with the sensitive cell line. The enhanced repair at the level of the gene did not display any carrier ligand specificity.
Carcinogenesis 1996 Dec
PMID:Increased gene specific repair of cisplatin induced interstrand crosslinks in cisplatin resistant cell lines, and studies on carrier ligand specificity. 900 94

The mechanisms by which the hepatitis B x protein (HBx) contributes to hepatocarcinogenesis remain unclear. However, interaction with the tumor suppressor gene p53 and inhibition of p53-dependent cellular functions, including nucleotide excision repair, could be central to this process. We studied the levels of global repair (removal of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts) and transcription-coupled repair (removal of CPDs in both strands of the dihydrofolate reductase gene) in primary wild-type and p53-null mouse hepatocytes. We show that global repair of CPDs appears to be more efficient in mouse hepatocytes than in other commonly studied rodent cells and approaches the levels of human cells and that p53 is required for global genomic DNA repair of CPDs but not for transcription-coupled repair. We then investigated the effect of HBx expression on hepatocyte nucleotide excision repair. We demonstrate that HBx expression affects DNA repair in a p53-dependent manner. Transient HBx expression reduces global DNA repair in wild-type cells to the level of p53-null hepatocytes and has no effect on the repair of a transfected damaged plasmid. Therefore, in viral hepatitis, the hepatitis B virus could inhibit the p53-dependent component of global repair leading, over time, to accumulation of genetic defects and fostering carcinogenesis.
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PMID:Hepatitis B x protein inhibits p53-dependent DNA repair in primary mouse hepatocytes. 983 6

Tumour cell growth may be accelerated by protein kinase C (PKC) agonists such as phorbol esters and receptor tyrosine kinases, but receptor tyrosine kinases are in turn desensitized to growth factors by PKC agonists. To clarify this apparent PKC bifunctionality, we have used phosphoantibodies to determine the relationship between PKC-dependent phosphorylation events affecting the ErbB2 oncoprotein in G8/DHFR 3T3 cells. Neither the kinetics nor the extent of phorbol-induced juxtamembrane domain (Thr686) phosphorylation vary directly with C-terminal (Tyr1222) dephosphorylation, with Tyr1222 continuing to be dephosphorylated long after Thr686 phosphorylation has also declined. Platelet-derived growth factor (PDGF) mimics the short-term effects of phorbol on Thr686 and Tyr1222 phosphorylation, and confocal microscopy reveals that both of these PKC agonists induce rapid internalization of PKC-modified ErbB2. Phorbol causes sustained cytoplasmic accumulation of PKC-phosphorylated receptors, however, whereas PDGF triggers the appearance of this ErbB2 subset only briefly. Metabolic labelling and co-precipitation studies fail to implicate heterologous molecules in either the tyrosine dephosphorylation or internalization of PKC-modified ErbB2. Taken in the context of earlier juxtamembrane domain mutagenesis studies, these findings indicate that phorbol-activated PKC may desensitize growth factor receptors to extracellular ligands solely by triggering sustained receptor internalization. We submit that PKC-dependent juxtamembrane domain phosphorylation represents a physiological mechanism for shortening the duration and enhancing the specificity of growth factor signalling by promoting internalization of liganded and unliganded receptors, respectively.
Carcinogenesis 1998 Nov
PMID:The duration of phorbol-inducible ErbB2 tyrosine dephosphorylation parallels that of receptor endocytosis rather than threonine-686 phosphorylation: implications for the physiological role of protein kinase C in growth factor receptor signalling. 985 18

It is thought that recovery of RNA synthesis following UV-irradiation is closely related to the removal of UV-induced lesions from the transcribed strand of active genes. To test this hypothesis, nascent RNA synthesis from three different locations within the DHFR gene in CHO cells was assessed following exposure to UV light (254 nm). Using both in vivo RNA labeling as well as the nuclear run-on technique, it was found that RNA synthesis from the middle and the 3'-end of the gene was inhibited within 20 min by approximately 30 and 70%, respectively, while RNA synthesis from the 5'-end of the DHFR gene was enhanced. RNA synthesis from the middle portion of the gene fully recovered within 30-45 min of post-UV incubation, while recovery was slower from the 3'-end of the gene. Compared with previously published data for the kinetics of removal of UV-induced DNA lesions from the 5'-half of the DHFR gene in these cells, it is concluded that RNA synthesis resumed significantly faster in this region than could be accounted for by the removal of photolesions from the transcribed strand. Thus, although RNA synthesis was initially inhibited by UV-induced photolesions, the results suggest that RNA polymerase II was able to bypass these lesions prior to their removal.
Carcinogenesis 1999 Mar
PMID:Recovery of RNA synthesis from the DHFR gene following UV-irradiation precedes the removal of photolesions from the transcribed strand. 1019 May 52

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.
Carcinogenesis 2000 Sep
PMID:Defective nuclear localization of p53 protein in a Chinese hamster cell line is associated with the formation of stable cytoplasmic protein multimers in cells with gene amplification. 1096 93

Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.
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PMID:Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation. 1598 34

Alternative genetic pathways characterized by specific genetic profiles and exhibiting distinctive biological and clinical features have been proposed in colorectal carcinogenesis. Methotrexate (MTX) is a potent inhibitor of the dihydrofolate reductase (DHFR) enzyme, which is essential for DNA synthesis and cell growth. We have evaluated the association between different genetic features and the capacity to develop MTX resistance in colon cancer cell lines representative of alternative genetic pathways. Three aneuploid cell lines (HT-29, SW480, and SK-CO-1) showed pre-existing amplifications, but only one (HT-29) developed MTX resistance, showing amplification of the DHFR gene at 5q12-14 (>20-fold amplification and presence of extrachromosomal double minutes). Failure to develop resistance was attributed to the absence of two complete chromosomes 5 in SW480 and SK-CO-1 cells. Four near-diploid cell lines (LoVo, HCT116, DLD-1 and KM12C) and two aneuploid KM12C-derived metastases (KM12SM and KM12L4A) developed MTX resistance but none exhibited DHFR amplification. All resistant cells without DHFR gene amplification showed microsatellite instability. We conclude that chemoresistance capacity and the mechanism of chemoresistance are related with the genetic pathway and the karyotypic features of colon cancer cells.
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PMID:Genetic determinants of methotrexate responsiveness and resistance in colon cancer cells. 1600 55

The MSH3 and dihydrofolate reductase (DHFR) genes, located on chromosome 5, share a common promoter but are divergently transcribed. Dysregulation of the mismatch repair (MMR) pathway has been found to occur in cell line models due to co-amplification of MSH3 as a coincident effect of DHFR amplification, acquired as a mechanism generating resistance to methotrexate (MTX). The increased levels of MSH3 perturbed MutSalpha function resulting in hypermutability and increased resistance to thiopurines, drugs whose cytotoxic effects are triggered by MutSalpha. The relevance of this phenomenon in clinical samples is unknown but is extremely pertinent in childhood acute lymphoblastic leukaemia (ALL) in which children are exposed for prolonged periods to both MTX and thiopurines such that a single amplification event involving both the DHFR and the MSH3 genes may cause chemotherapeutic resistance to both agents. Thus, we have generated a leukaemic cell line (PreB697) and a normal human lymphoblastoid cell line (TK6) that are resistant to a pharmacologically relevant dose of MTX and show that while increased DHFR levels result in MTX resistance, the associated increased levels of MSH3 are insufficient to perturb MutSalpha functionality, in terms of MMR capacity or 6-thioguanine sensitivity. In addition, we show that although low-level DHFR amplification occurs alone in a significant number of samples, both at disease onset and relapse, co-amplification of both MSH3 and DHFR is rarely found in primary ALL samples, even after prolonged MTX therapy and is not at a sufficiently high level to perturb MMR function.
Carcinogenesis 2007 Jun
PMID:DHFR and MSH3 co-amplification in childhood acute lymphoblastic leukaemia, in vitro and in vivo. 1714 5


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