Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation.
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PMID:Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F. 822 41

The differential activation of cyclin and cyclin-dependent kinase genes by the adenovirus E1A gene product (E1A) or serum factors was studied with a rat 3Y1 derivative cell line, g12-21, in which the E1A12S cDNA can be expressed in response to dexamethasone (dex). The induction of DNA synthesis in quiescent g12-21 cells occurred within 12 h after serum stimulation, while it occurred within 8 h after treatment with dex. The expression of cyclin D1 and E genes in the serum-stimulated cells was induced in mid G1 and mid to late G1, respectively, while that of the cyclin D1 gene was not induced and the induction of the cyclin E gene was shifted to the G1/S boundary in the dex-treated cells. The cdk2 gene was induced in late G1 and cdc2 and cyclin A genes at the G1/S boundary in both serum-stimulated and dex-treated cells. These results suggest that E1A skips cell cycle events which normally occur in early to mid G1 and may directly activate late-response genes. Analysis of the transcription factor E2F complexes formed in the promoter regions of cdc2 and dihydrofolate reductase genes showed that the amount of complexes formed is maximal at the G1/S boundary, but decreases in S phase when these genes are transcribed extensively.
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PMID:Differential activation of cyclin and cyclin-dependent kinase genes by adenovirus E1A12S cDNA product. 837 70

p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. We show that p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppressed the activity of E2F-responsive promoters (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) were unaffected. Moreover, the simian virus 40 early promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppressed growth and E2F activity in cells lacking a functional pRb. Moreover, p21 protein (wild type but not mutant) specifically disrupted an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription.
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PMID:Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein. 864 10

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (-381 to -17) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that 1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetectable in both early passage and senescent cells; and 3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human fibroblasts. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells.
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PMID:Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence. 881 12

The product of the c-mos proto-oncogene is a protein kinase that is normally expressed in germ cells and functions during oocyte maturation. It has been shown, however, that inappropriate expression of either the viral or cellular mos gene can induce neoplastic progression in somatic cells. Furthermore, v-mos-transformed NIH3T3 cells will undergo arrest of proliferation in early G1 upon serum withdrawal but are unable to appropriately down-regulate cell cycle regulatory proteins, such as cyclin and cdc2 proteins, that normally are down-regulated in quiescent, untransformed NIH3T3 cells. Since the levels of these proteins are partially transcriptionally controlled, we investigated whether there were alterations in the expression of E2F and AP-1 transcription factor complexes. Indeed, the putative G0/G1-specific p130-E2F complex that is normally observed during low serum-induced cell cycle arrest in NIH3T3 cells is not present in serum starved v-mos-transformed cells. Instead, G1-phase arrested v-mos-transformed cells stably express two E2F protein complexes that are normally observed only during S-phase in untransformed cells. The elevation of these complexes in arrested v-mos-transformed cells may be the cause of the transcriptional activation of the E2F-regulated genes cdc2, DHFR, cyclin A, and E2F1 seen in serum starved v-mos-transformed cells. In addition, there are high levels of AP-1 DNA binding activity in serum starved v-mos-transformed cells compared to very low amounts in nontransformed cells. This altered regulation of transcription factor complexes and cell cycle control proteins upon serum withdrawal may provide a mechanism for the uncontrolled cell growth associated with neoplastic transformation induced by certain proto-oncogenes.
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PMID:Deregulation of specific E2F complexes by the v-mos oncogene. 922 66

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
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PMID:p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes. 930 76

Histone acetylation is emerging as a major regulatory mechanism thought to modulate gene expression by altering the accessibility of transcription factors to DNA. In this study, treatment of human tumor cells with the histone deacetylase inhibitor, trapoxin (TPX), resulted in selective changes in genes that control the cell cycle. TPX activated p21(waf1) transcription that led to elevated p21(waf1) protein levels in three human tumor cell lines without altering the protein levels of cdk2, cdk4, or cyclin B. In addition, TPX increased cyclin E transcription without increasing the levels of Rb, E2F, dihydrofolate reductase, or glyceraldehyde-3-phosphate dehydrogenase. The elevated levels of p21(waf1) protein led to decreased Rb phosphorylation and cdk2 activity. These effects resulted in G(1) and G(2) cell cycle arrest in H1299 human lung and MDA-MB-435 breast carcinoma cells and apoptosis in A549 lung carcinoma cells. Chromatin immunoprecipitation assays revealed that TPX increased the level of chromatin acetylation associated with histone H3 in the trapoxin-responsive region of the p21(waf1) promoter. This study demonstrates that inhibition of HDAC by TPX increases acetylation of H3-associated chromatin and alters gene expression with marked selectivity.
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PMID:Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. 1057 69

RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D11 cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.
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PMID:RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F. 1096 46

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

Cancer cells are characterized by limitless proliferative autonomy and immunity to inhibitory and apoptotic signals, thus ensuring growth and metastasis [1]. Epidemiological studies have long implicated human papillomavirus (HPV) as a pathogenic agent in cervical cancer. Progress in cancer research now provides an understanding of how these characteristics are achieved by the interaction of HPV proteins with the cell cycle machinery. Expression of oncoproteins E7 and E6 induces immortalization of cells through their inhibitory effects on tumor suppressor proteins pRb and p53, respectively. Undermining of pRb's growth-inhibitory role with release of E2F transcription factors renders the cells independent of mitogenic stimuli. The abundance of growth transcription factors grants limitless proliferative potential by allowing expression of products such as cyclins A, E, and B, dihydrofolate reductase, and DNA polymerase which fuel the various stages of the cell cycle. There is subsequent disruption of both the G1-S and G2-M cell cycle checkpoints. Overexpression of cyclin E results in chromosomal instability and possible unmasking of genetic mutations, allowing disease progression. Cyclin A grants anchorage-independent growth, facilitating tissue invasion and tumor spread. Apoptotic and growth-inhibitory mechanisms are also evaded. p53 is degraded by E6 and its own downstream protein mdm2. Its other downstream protein, p21 is rendered ineffective against cyclin-cyclin-dependent kinase units by E7, as is p27. The understanding of the molecular pathology of disease will provide us with the ability to prognosticate and treat patients more effectively.
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PMID:Cell cycle aberrations in the pathogenesis of squamous cell carcinoma of the uterine cervix. 1153 Dec 73


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