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

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.
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PMID:DNA contents of replication without DNA density labeling. 219 30

DC-3F/FA3 cells (FA3) were derived from antifolate-sensitive CHL cells by selection for growth in folate-free media containing 15 pM [6S]-5CHOFH4. These cells undergo a 30-fold decrease in intracellular folates, overexpress folate receptor alpha (FR alpha) and metallothionein II, and display increased sensitivity to the dihydrofolate reductase (DHFR) targeted anti-folates methotrexate (MTX) and trimetrexate (TMTX), which can be attributed primarily to the folate pool status. Upon folate repletion by growth in 15 nM [6S]-5CHOFH4, they display a 5- and 10-fold increase in resistance to both drugs, respectively, even though folate pools are restored by only 43%. Enforced overexpression of FR alpha in transfectants cultured in nanomolar folate did not confer resistance to MTX but did support a modest 2-fold increase in resistance to TMTX. Enforced overexpression of MTII had a similar effect, but when both were overexpressed together no increase in resistance beyond that conferred by each one separately was noted, suggesting that both confer resistance to TMTX through a common downstream mechanism. Analysis of three independent low folate selected clones, FA3, FA7, and FA14, showed that each had a 5- to 6-fold increase in DHFR activity accompanied by a similar increase in DHFR protein level. However, no differences were detected in the DHFR gene copy number or in the steady-state amount of DHFR mRNA, suggesting that a posttranscriptional mechanism was responsible for the increase in DHFR expression.
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PMID:Severe folate restriction results in depletion of and alteration in the composition of the intracellular folate pool, moderate sensitization to methotrexate and trimetrexate, upregulation of endogenous DHFR activity, and overexpression of metallothionein II and folate receptor alpha that, upon folate repletion, confer drug resistance to CHL cells. 1241 30