Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The alpha-amylase gene is known to be regulated by the plant hormone gibberellin (GA) in cereal aleurone cells. The accumulation of the mRNA corresponding to a rice high pI alpha-amylase gene, OSamy-c, was stimulated 20-fold by exogenous GA3 in half-seeds lacking embryos. Regulatory regions in the promoter of this high pI sub-family were analyzed. The OSamy-c 5' flanking sequence, spanning positions -231 to +29, was fused upstream of the beta-glucuronidase (GUS) gene coding region. The delivery of this plasmid into rice aleurone cells by the biolistic method resulted in a GA-stimulated synthesis of GUS. Gel retardation assays were performed to study protein-DNA interactions between putative regulatory sequences of OSamy-c and partially purified rice seed extracts. We identified multiple seed-specific protein factors that bind to proximal regions of the OSamy-c promoter between positions -231 and -162. Five different proteins were distinguished based on competitive binding studies. Three protein binding regions were located by footprinting analyses, one of which is located in the conserved sequence also found upstream of other GA-inducible genes. Two protein factors in rice aleurone cells that interact with the putative regulatory sequence do not require GA induction.
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PMID:Regulation and interaction of multiple protein factors with the proximal promoter regions of a rice high pI alpha-amylase gene. 137 14

We have isolated three HSP90-family genes from Arabidopsis: HSP81-1 which is heat-inducible, and HSP81-2 and -3 which are highly expressed under normal growth temperatures. Northern blot analysis and RNase protection analysis, using gene specific probes, showed that HSP81-2 and -3 mRNA were present in all tissues and abundant in roots, floral bud clusters, and flowers at 22 degrees C. A small amount of HSP81-1 mRNA was detected only in roots. In situ hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with an HSP81 promoter region fused to a beta-glucuronidase (GUS) gene, confirmed these results. At 22 degrees C, high GUS activity was observed in the root apical meristems, pollen and tapeta in HSP81-2::GUS and HSP81-3::GUS transgenic plants, while only branches of the root in HSP81-1::GUS transgenic plants expressed high GUS activity. After 2 hours of 35 degrees C treatment, extensively high GUS activity was observed in all tissues in HSP81-1::GUS transgenic plants, while elevated but tissue specific expression was observed in HSP81-2 and -3 transgenic plants. Exogenous application of various chemicals such as ABA, GA3, kinetin, IAA, NaCl, and mannitol revealed that 10 mM IAA and 0.1 M NaCl significantly enhanced the accumulation of HSP81-2 and -3 transcripts. Only a slight response to IAA was observed in HSP81-1 mRNA accumulation at 22 degrees C; the increase was possibly caused by a novel pathway other than heat-shock-response pathway.
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PMID:Analysis of tissue-specific expression of Arabidopsis thaliana HSP90-family gene HSP81. 769 94

Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a prodrug by an enzyme-immunoconjugate localized in tumor tissue. The use of an enzyme of human origin is preferable in ADEPT because it might not be immunogenic when administered to patients. In the case of human beta-glucuronidase, prodrugs should be designed that are rapidly and completely activated at a neutral pH. Four new daunorubicin glucuronides were synthesized by coupling a glucuronide group to daunorubicin via an aliphatic (GA1 and GB1) or an aromatic (GA3, GB6) carbamate spacer, to be released by electron shift (A-type) or by ring closure (B-type). These prodrugs were characterized in vitro for their usefulness in ADEPT and were compared with the previously described prodrugs epirubicin-glucuronide and doxorubicin-nitrophenyl-glucuronide. The four new prodrugs were stable in serum, hydrophilic when compared to the lipophilic daunorubicin, and at least 20-fold less toxic than the parent compound. The hydrolysis rate at clinically relevant enzyme and prodrug concentrations (1 microgram/mL human beta-glucuronidase, 100 microM prodrug) at pH 6.8 were similar for GA3 (T1/2 160 min) and higher for GB6 (T1/2 40 min) when compared to that of doxorubicin-nitrophenyl-glucuronide (T1/2 170 min). Epirubicin-glucuronide, GA1, and GB1 showed a low hydrolysis rate (T1/2 > 400 min). GA1 and GA3, but not GB1 or GB6, were activated to the parent compound. Complete activation was confirmed in OVCAR-3 cells pretreated with a specific antibody-human beta-glucuronidase conjugate, where GA3 had similar antiproliferative effects to those of daunorubicin.
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PMID:Characterization of novel anthracycline prodrugs activated by human beta-glucuronidase for use in antibody-directed enzyme prodrug therapy. 868

The prodrug N-[4-(daunorubicin-N-carbonyl-oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) was synthesized for specific activation by human beta-glucuronidase, released in necrotic areas of tumour lesions. In vitro, DNR-GA3 was 18 times less toxic than daunorubicin (DNR) and the prodrug was completely activated to the parent drug by human beta-glucuronidase. The maximum tolerated dose of DNR-GA3 in nude mice bearing s.c. human ovarian cancer xenografts was 6-10 times higher than that of DNR. The prodrug was cleared more rapidly from the circulation (elimination t1/2 = 20 min) than the parent drug (elimination t1/2 = 720 min). The anti-tumour effects of DNR-GA3 and DNR were investigated in four different human ovarian cancer xenografts OVCAR-3, FMa, A2780 and MRI-H-207 at a mean tumour size between 100 and 200 mm3. In three out of four of these tumour lines, the prodrug given i.v. at the maximum tolerated dose ranging from 150 to 250 mg kg(-1) resulted in a maximum tumour growth inhibition from 82% to 95%. The standard treatment with DNR at a dose of 8 mg kg(-1) given i.v. weekly x 2 resulted only in a maximum tumour growth inhibition from 40% to 47%. Tumour line FMa did not respond to DNR, nor to DNR-GA3. Treatment with DNR-GA3 was also given to mice with larger tumours that would contain more necrosis (mean size 300-950 mm3). The specific growth delay by DNR-GA3 was extended from 2.1 to 4.4 in OVCAR-3 xenografts and from 4.4 to 6.0 in MRI-H-207 xenografts. Our data indicate that DNR-GA3 is more effective than DNR and may be especially of use for treatment of tumours with areas of necrosis.
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PMID:The efficacy of the anthracycline prodrug daunorubicin-GA3 in human ovarian cancer xenografts. 986 70

N-[4-daunorubicin-N-carbonyl (oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) is a glucuronide prodrug of daunorubicin (DNR) which induced a better tumor growth delay than DNR when studied at equitoxic doses in three human ovarian cancer xenografts. These results suggested that the prodrug DNR-GA3 was selectively activated by human beta-glucuronidase present in tumor tissue. We determined the pharmacokinetics and distribution of DNR-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3, FMa, A2780, and MRI-H-207). Administration of DNR at 10 mg/kg i.v. (maximum tolerated dose) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 12.18 microM (t = 1 min). DNR-GA3 at 100 mg/kg i.v. (approximately 50% of the maximum tolerated dose [MTD]) resulted in a peak plasma concentration of DNR that was 28-fold lower than that after DNR itself; in normal tissues, prodrug injection resulted in 5- to 23-fold lower DNR concentrations. DNR showed a relatively poor uptake into OVCAR-3 tumors with a peak concentration of 2.05 nmol x g(-1) after injection. In the same xenograft, DNR-GA3 resulted in a significantly higher DNR peak concentration of 3.45 nmol x g(-1) (P < 0.05). The higher area under the curve of DNR in tumor tissue after DNR-GA3 than after DNR itself would be the result of prodrug activation by beta-glucuronidase. In this respect, a considerably higher beta-glucuronidase activity was found in tumor tissue when compared to plasma. The specific activation of DNR-GA3 by beta-glucuronidase at the tumor site relative to normal organs leads to a more tumor-selective therapy, resulting in greater efficacy without increased toxicity.
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PMID:Distribution and pharmacokinetics of the prodrug daunorubicin-GA3 in nude mice bearing human ovarian cancer xenografts. 1003 53

The doxorubicin (DOX) prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) was synthesised for specific activation by human beta-glucuronidase, which is released in necrotic areas of tumour lesions. This novel prodrug was completely activated to the parent drug by human beta-glucuronidase with V(max)= 25.0 micromol x min(-1) x mg(-1) and K(m) = 1100 microM. The pharmacokinetics and distribution of DOX-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3) were determined and compared with DOX. Administration of DOX at 8 mg x kg(-1) i.v. (maximum tolerated dose, MTD) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 16.4 microM (t = 1 min). A 7.6-times lower peak plasma concentration of DOX was measured after injection of DOX-GA3 at 250 mg x kg(-1) i.v. (50% of MTD). In normal tissues the prodrug showed peak DOX concentrations that were up to 5-fold (heart) lower than those found after DOX administration. DOX-GA3 activation by beta-glucuronidase in the tumour yielded an almost 5-fold higher DOX peak concentration of 9.57 nmol x g(-1) (P< 0.05) than the peak concentration of only 2.14 nmol x g(-1) observed after DOX. As a consequence, the area under the curve of DOX calculated in tumour tissue after DOX-GA3 (13.1 micromol x min(-1) x g(-1)) was 10-fold higher than after DOX (1.31 micromol x min(-1) x g(-1)). The anti-tumour effects of DOX-GA3 and DOX were compared at equitoxic doses in OVCAR-3 xenografts at a mean tumour size of 125 mm(3). The prodrug given i.v. at 500 mg x kg(-1) weekly x 2 resulted in a maximum tumour growth inhibition of 87%, while the standard treatment with DOX at a dose of 8 mg x kg(-1) i.v. weekly x 2 resulted in a maximum tumour growth inhibition of only 56%. Treatment with DOX-GA3 was also given to mice with larger tumours containing more necrosis. For tumours with a mean size of 400 mm(3) the specific growth delay by DOX-GA3 increased from 2.7 to 3.9. Our data indicate that DOX-GA3 is more effective than DOX and suggest that the prodrug will be specifically advantageous for treatment of advanced disease.
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PMID:A novel doxorubicin-glucuronide prodrug DOX-GA3 for tumour-selective chemotherapy: distribution and efficacy in experimental human ovarian cancer. 1120 53

A glucuronide doxorubicin prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) has been developed to improve the antitumor effects of doxorubicin (DOX). The prodrug was originally designed to be activated into drug by human beta-glucuronidase (GUS) released from tumor cells in necrotic areas of tumor lesions. The aim of this study was to further improve the antitumor effects of DOX-GA3 by means of antibody-directed enzyme prodrug therapy (ADEPT). We thus investigated if the administration of an enzyme-immunoconjugate prepared from the pancarcinoma Ep-CAM specific monoclonal antibody (MAb) 323/A3 and beta-glucuronidase would result in improved antitumor effects because of additional enzyme localization in tumor tissue. In vitro, the prodrug DOX-GA3 was found to be 12-times less toxic than the parent drug DOX in a human ovarian cancer cell line. Immunospecific and complete activation of the prodrug took place when the cells were pretreated with 323/A3-beta-glucuronidase conjugate. In nude mice bearing s.c. human ovarian cancer xenografts (FMa) the maximum tolerated dose (MTD) of DOX-GA3 (500 mg/kg weekly x 2) was much higher when compared with that of DOX (8 mg/kg weekly x 2). In mice bearing well-established FMa xenografts, the standard treatment of DOX at the MTD (8 mg/kg weekly x 2) resulted in a tumor growth inhibition of 67%. Treatment with DOX-GA3 at a single dose of 500 mg/kg resulted in a better tumor growth inhibition of 87%. The combination of DOX-GA3 (500 mg/kg) with 323/A3-mGUS conjugate and anti-GUS MAb 105, to clear circulating conjugate, improved the antitumor effect even further to 98%. At the lower dose of 250 mg/kg DOX-GA3 tumor growth inhibition (34%) was not better than that of DOX. The combination, however, of DOX-GA3 at 250 mg/kg and 323/A3-mGUS conjugate plus MAb 105 again greatly improved the antitumor effect (growth inhibition of 93%). DOX given at 8 mg/kg weekly x 2 did not result in tumor regressions. As a result of ADEPT, the number of regressions of tumors improved from 0 out of 12 to 9 out of 11 at a dose of 250 mg/kg DOX-GA3. At the higher prodrug dose (500 mg/kg) the number of regressions improved from 2 out of 12 to 9 out of 10 as a result from the addition of enzyme-immunoconjugate. Our studies show that the efficacy of the widely used anti-cancer agent DOX may be improved by using the prodrug DOX-GA3, in combination with the tumor-specific enzyme-immunoconjugate 323/A3-mGUS and a conjugate clearing antibody.
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PMID:Pronounced antitumor efficacy of doxorubicin when given as the prodrug DOX-GA3 in combination with a monoclonal antibody beta-glucuronidase conjugate. 1125 80

Tumor-specific activation of the glucuronide prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate (DOX-GA3), by beta-glucuronidase present in necrotic tumor areas might be improved after transduction of tumor cells to secrete a targeted form of beta-glucuronidase. To that end, we constructed an adenovirus vector, designated Ad/C28-GUSh, encoding human beta-glucuronidase fused to a human single-chain Fv (scFv) against the epithelial cell adhesion molecule (EpCAM), C28, and preceded by a signal sequence for secretion. Antibody specificity and enzyme activity were retained in the fusion protein secreted by tumor cells infected with Ad/C28-GUSh. Diffusion of fusion protein from transduced tumor cells within MCF-7 multicellular spheroids was visualized by immunohistochemistry. Treatment of spheroids with Ad/C28-GUSh and DOX-GA3 resulted in growth inhibition comparable to treatment with doxorubicin alone. Treatment of well-established FMa human ovarian cancer xenografts with intravenous injection of DOX-GA3 (500 mg/kg) resulted in a tumor volume-doubling time of 23.8 days compared to 8.0 days for phosphate-buffered saline (PBS)-treated mice. Intratumoral administration of Ad/C28-GUSh before DOX-GA3 enhanced the growth inhibition and increased the tumor volume-doubling time to 43.1 days (p < 0.01), while virus alone had no effect. Thus, we have successfully shown that an adenovirus vector encoding a secreted, targeted form of human beta-glucuronidase can further improve DOX-GA3 monotherapy.
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PMID:Pronounced antitumor efficacy by extracellular activation of a doxorubicin-glucuronide prodrug after adenoviral vector-mediated expression of a human antibody-enzyme fusion protein. 1501 32

The glucuronide prodrug of doxorubicin, DOX-GA3, can be selectively activated in tumors by extracellular human beta-glucuronidase, resulting in a better therapeutic index than doxorubicin. DOX-GA3, however, is rapidly excreted by the kidney. We hypothesized that slow release of DOX-GA3 from its methylester, DOX-mGA3, by esterase activity in blood would result in improved circulation half-life (t(1/2)) of DOX-GA3. DOX-mGA3 was synthesized more efficiently with an overall yield of 60% as compared to 37% in the case of DOX-GA3. We showed that DOX-mGA3 was enzymatically converted to DOX-GA3 with a t(1/2) of approximately 0.5 min in mouse plasma to 2.5 h in human plasma, which was in agreement with differences in esterase activity between species. DOX-mGA3, similar to DOX-GA3, was at least 37-fold less potent than the parent drug doxorubicin in growth inhibition of four different human malignant cell lines in vitro. Incubation of OVCAR-3 cells with DOX-mGA3 in combination with an excess of human beta-glucuronidase (0.05 U mL(-1)) resulted in a similar growth inhibition to that of doxorubicin. Intravenous administration of DOX-mGA3 in FMa-bearing mice resulted in an area under the concentration versus time curve (AUC) of DOX-GA3 in tumor and most normal tissues that was 2.5- to 3-fold higher than after the same dose of DOX-GA3 itself. In tumor tissue, this was accompanied by a 2.7-fold increase in the AUC of doxorubicin from DOX-mGA3 than from DOX-GA3. In conclusion, an advantage of DOX-mGA3 over DOX-GA3 is that this prodrug can be produced with a higher yield. Another important advantage is the improved pharmacokinetics of the lipophilic DOX-mGA3 as compared to that of the hydrophilic DOX-GA3. This effect may even be more pronounced in man, because of the lower plasma esterase activity than measured in mice.
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PMID:A methylester of the glucuronide prodrug DOX-GA3 for improvement of tumor-selective chemotherapy. 1549 17

Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC) that plays a key role in intermediary metabolism. OsPDK1 was identified as a gibberellin-up-regulated gene using a cDNA microarray. The full-length cDNA for OsPDK1 was 1498 bp and encoded a predicted polypeptide of 363 amino acids. Genomic DNA analysis showed the presence of another isoform of PDK, OsPDK2, in rice. Reverse transcriptase-PCR analysis revealed differential expression of the two isoforms. OsPDK1 was expressed in leaf blade and leaf sheath but not in callus and root, while OsPDK2 was expressed constitutively in all tissues examined. Maximum expression of OsPDK1 in leaf sheath was detected by Northern blot analysis when seedlings were treated with 5 microM GA3 for 24 h. OsPDK1 expression was up-regulated by GA3, and there was little effect of other plant hormones. Mitochondrial pyruvate dehydrogenase (PDH) activity was reduced compared with control plants in 2-week-old seedlings treated with GA3. The beta-glucuronidase (GUS) reporter gene, driven by a 2,067 bp OsPDK1 promoter region fragment, was mainly expressed in the aleurone layer of germinating seed and leaf sheath. Transgenic rice expressing PDK1 RNAi had altered vegetative growth with reduced accumulation of vegetative tissues. These results suggest that gibberellin modulates the activity of mtPDC by regulating OsPDK1 expression and subsequently controlling plant growth and development.
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PMID:Gibberellin regulates mitochondrial pyruvate dehydrogenase activity in rice. 1635 97


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