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)

The oncoprotein cyclin D1 binds to and activates cyclin-dependent kinase 4 (cdk4), whose activity is inhibited by p16INK4, the product of the putative tumor suppressor gene MTS1. Cyclin D1 controls the timing of S phase onset in mammalian cells. We show that cyclin D1 acts as a positive regulator of the transcription factor E2F. In particular, cyclin D1 overexpression leads to the activation of the dihydrofolate reductase (DHFR) gene promoter. Activation depends on the E2F binding site in the DHFR promoter, known to mediate its activation at the G1/S transition in vivo. Cyclin D1 can also activate the adenovirus E2 promoter via E2F. Both promoters are repressed by p16INK4 and this repression can be released by overexpression of cdk4. The data reported here support a direct role for cyclin D1 and its associated kinase in cell cycle regulation of E2F activity and S phase-specific gene expression. In addition, we show that both E2F sites bind complexes containing the retinoblastoma protein (pRB) and that in RB-deficient cell lines overexpression of cyclin D1 fails to activate E2F-dependent transcription, indicating that pRB may be involved in promoter activation.
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PMID:Activation of the E2F transcription factor by cyclin D1 is blocked by p16INK4, the product of the putative tumor suppressor gene MTS1. 797 Jul 7

The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G(1) cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G(1) cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G(0) quiescent cells and becomes hypophosphorylated (approximately 2 mol of PO(4) to 1 mol of pRB) in early G(1) and hyperphosphorylated (approximately 10 mol of PO(4) to 1 mol of pRB) in late G(1) phase. Here, we report that hypophosphorylated pRB, present in early G(1), represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G(0) and hyperphosphorylated pRB in late G(1) fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT-dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G(1) and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G(1). Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16(INK4a) gene, contained hypophosphorylated pRB that was bound to E2Fs in early G(1) and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G(1) cyclin-Cdk complexes.
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PMID:Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo. 1141 52

We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts methylthioadenosine into adenine which serves as an alternative purine source, if de novo purine biosynthesis is inhibited by antimetabolites (i.e., methotrexate). The idea of the chemoselectivity concept is that tumors with MTAP deletion at chromosome 9p21 are more susceptible to antimetabolites than normal cells without such a deletion. First, we screened 13 T-ALL lines for 9p21 deletions by comparative genomic hybridization. Five cell lines revealed deletions at the short arm of chromosome 9, dim(9p21pter). Further analyses were performed with CEM cells in which the 9p21 deletion was corroborated by fluorescence in situ hybridization. CEM cells were transfected with an MTAP expression vector. A green fluorescent protein (GFP) plasmid was cotransfected, to monitor the transfection efficacy by flow cytometry. The response of MTAP-transfected cells to the antimetabolites methotrexate (MTX), trimetrexate (TMX), and L-alanosine (ALA) was decreased compared to mock control transfectants using growth inhibition assays. The activity of doxorubicin (DOX) which is not involved in DNA biosynthesis was not changed in MTAP transfectants. As the p16(INK4a) tumor suppressor gene resides also at 9p21, we transfected CEM cells with a p16(INK4a) expression vector. These transfectant cells were more resistant to all four drugs indicating that p16(INK4a) did not specifically affect antimetabolites. The chemoselective effect of antimetabolites in MTAP-deleted tumor cells may, however, be compensated by the development of drug resistance. To prove this possibility, we analyzed an MTX-resistant subline, CEM/MTX1500LV, in which the MTX-resistance conferring dihydrofolate reductase (DHFR) gene was amplified. While TMX exhibited considerable cross-resistance in CEM/MTX1500LV cells, ALA did not. Thus, ALA could exhibit chemoselectivity in 9p21/MTAP-deleted cells, even if DHFR amplification occurs. We conclude that ALA may be more suitable than MTX or TMX for MTAP-mediated chemoselective treatment of T-ALL. Pretherapeutical detection of 9p21 and MTAP deletion may be helpful in developing a predictive molecular chemosensitivity test for T-ALL.
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PMID:Methylthioadenosine phosphorylase as target for chemoselective treatment of T-cell acute lymphoblastic leukemic cells. 1198 41

Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing a methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of a LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): Escherichia coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16(INK4a), a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb(3+) with a submicromolar dissociation constant. LRET was measured from the Tb(3+) site on LBT to transition metals bound to the hexa-His tag and to fluorescein methotrexate bound to DHFR. The measured distances were consistent with a molecular model constructed from the known crystal structures of the constituent domains of DAL. The results indicate that the two C-terminal ankyrin domains of p16(INK4a) are stably folded when combined with other protein domains. We found that Tb(3+) binds to DAL in the cytoplasm of live E. coli cells, and thus, DAL is useful as an indicator for studies of metal transport. We also used DAL to measure LRET from Tb(3+) to Cu(2+) in the cytoplasm of live E. coli cells. The rates of Tb(3+) and Cu(2+) transport were not affected by a proton uncoupler or an ATP synthase inhibitor. Reversal of the membrane potential had a small inhibitory effect, and removal of lipopolysaccharide had a small accelerating effect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb(3+) signal, suggesting that the Tb(3+)-LBT interaction is useful as a cytoplasmic pH indicator in the range of approximately pH 5-6.
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PMID:Luminescence resonance energy transfer in the cytoplasm of live Escherichia coli cells. 2173 54