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Biomedical subjects

Y Yen

Publications and source records attributed to Y Yen.

At least 37 records · Page 2Linked to original sources
PubMed1998

A simple and sensitive ribonucleotide reductase assay.

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.

Calibration↗
PubMed1998

Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential.

The ribonucleotide reductase (RR) gene has been associated with malignant transformation and metastatic potential. In this report, the significance of the expression of RR mRNA and enzymatic activity to the invasive potential was examined by Boyden chamber invasion assay. Our results suggest that overexpression of RR M2 mRNA and RR enzymatic activity correlates to an increase in cell invasive potential. The drug-induced HURs clone expressed a higher level RR M2 mRNA and enzyme activity which contributes significantly to the 3-fold increase in invasive potential of the cells observed relative to the KB wild-type control. On the contrary, the HUr revertant clone decreased the RR M2 mRNA level and enzymatic activity, concomitantly decreasing their invasive potential. This phenomenon is most likely due to the return of RR to levels comparable to that of the KB wild-type cells. To confirm that this observation was not of a drug-resistance phenotype associated with multiple gene alterations, the panel of RR transfectants (M1-D transfected M1 subunit cDNA, M2-D transfected M2 subunit cDNA, X-D transfected M1/M2 cDNA) characterized in a previous study were also tested in the invasion assay. The M2-D clone expressed 6-fold higher RR M2 mRNA and RR activity and also demonstrated 6-fold higher invasive potential in vitro than either the parental or vector only transfected cell line (KB-V). The X-D clone demonstrated 3-fold higher M2 mRNA expression and revealed 4-fold higher invasive potential than control cells. The M1-D clone, in contrast, expressed a baseline level of RR M2 mRNA and higher M1 mRNA. In contrast to the X-D and M2-D cells, the invasive potential of M1-D reached an even lower level in the invasive assay than the control. These results, therefore, suggest that RR M2 overexpression plays an important role in a tumor's invasiveness.

Carcinoma↗
PubMed1998

Effects of oxyhemoglobin on vasoconstriction in response to 5-hydroxytryptamine in isolated, perfused canine basilar arteries.

OBJECTIVE: Oxyhemoglobin (OxyHb) is thought to be a critical trigger in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage. We investigated whether extraluminally applied OxyHb influenced vascular responses to intraluminally applied vasoactive agents in isolated, perfused, canine basilar arteries. METHODS: The steel cannula insertion method was used to examine vascular responses to intraluminally applied 5-hydroxytryptamine (5-HT) receptor agonists, i.e., 5-HT, 5-carboxamidotryptamine (selective for 5-HT1 receptors), and alpha-methyl-5-hydroxytryptamine (selective for 5-HT2 receptors), potassium chloride, and acetylcholine, before and after extraluminal treatment with OxyHb. RESULTS: Extraluminal application of 2.5 x 10(-5) mol/L OxyHb immediately induced a transient elevation of the basal perfusion pressure, which gradually decreased and then stabilized at a level slightly higher than the control level. Each 5-HT agonist induced dose-dependent vasoconstriction. The potencies of the agonists were not very different, but the efficacies varied, i.e., alpha-methyl-5-hydroxytryptamine > 5-HT > 5-carboxamidotryptamine. Each response was strongly inhibited by ketanserin (a selective 5-HT2 receptor antagonist), indicating that each agonist induces vasoconstriction mediated by 5-HT2 receptors. The vasoconstriction in response to each 5-HT receptor agonist was consistently potentiated by treatment with OxyHb (2.5 x 10(-5) mol/L). 5-HT receptor agonist-induced constrictions after OxyHb treatment were much more markedly inhibited by ketanserin, compared with those before OxyHb treatment. Acetylcholine-induced constrictions were enhanced by OxyHb, but KCl-induced constrictions were significantly decreased by OxyHb. CONCLUSION: It is suggested that OxyHb enhancement of constrictions in response to 5-HT receptor agonists may be mediated by increased sensitivity of 5-HT2 receptors, in addition to actions in the endothelium, in canine basilar arteries. This potentiated vasoconstrictor mechanism may be partially implicated in cerebral vasospasm after subarachnoid hemorrhage.

Animals↗
PubMed1997

Pharmacokinetics and toxicity of 120-hour continuous-infusion hydroxyurea in patients with advanced solid tumors.

A group of 18 patients with advanced cancer were entered on a phase I study of a 120-h continuous intravenous infusion of hydroxyurea. The dose of hydroxyurea was escalated in cohorts of patients from 1 to 2 to 3.2 g/ m2 per day. The primary dose-limiting toxicity was neutropenia, often accompanied by leukopenia, thrombocytopenia and generalized skin rash. Prophylactic treatment of patients with dexamethasone and diphenhydramine hydrochloride prevented the skin rash, but not the hematopoietic toxicities. The pharmacokinetics of hydroxyurea were studied in all patients. The steady-state concentrations of hydroxyurea were linearly correlated with the dose (R2 = 0.71, n = 18, P < 0.0001). The mean +/- SE concentrations were 93 +/- 16, 230 +/- 6 and 302 +/- 27 microM at 1, 2 and 3.2 g/m2 per day, respectively. The mean +/- SE renal and nonrenal clearances of hydroxyurea were 2.14 +/- 0.18 and 3.39 +/- 0.28 l/h per m2 (n = 16), neither of which correlated with the dose. The concentration of hydroxyurea in plasma decayed monoexponentially with a mean +/- SE half-life of 3.25 +/- 0.18 h (n = 17). The steady-state concentration of hydroxyurea was > 200 microM in all nine patients treated at 2 g/m2 per day, a dose which was well tolerated for 5 days. We recommend this dose for phase II trials in combination with other antineoplastic agents.

Adult↗
PubMed1997

High-dose chemotherapy and stem-cell rescue in the treatment of high-risk breast cancer: prognostic indicators of progression-free and overall survival.

PURPOSE: To examine the predictive value of tumor- and treatment-specific prognostic indicators of relapse-free survival (RFS) and overall survival (OS) in patients with high-risk breast cancer (HRBC) treated with high-dose chemotherapy (HDCT) and stem-cell rescue. PATIENTS AND METHODS: Between June 1989 and September 1994, 114 patients with HRBC (stage II with > or = 10 axillary lymph nodes involved, stage IIIA, and stage IIIB inflammatory carcinoma) received adjuvant chemotherapy followed by HDCT with etoposide, cyclophosphamide, and either doxorubicin (CAVP) or cisplatin (CCVP). Variables analyzed included stage, tumor size, number of axillary nodes involved, grade and receptor status, and types of adjuvant chemotherapy and radiation therapy and HDCT. RESULTS: With a median follow-up time of 46 months (range, 23 to 93), Kaplan-Meier estimates of 3.5-year OS for stage II, IIIA, and IIIB HRBC are 82% (95% confidence interval [CI], 67% to 97%), 79% (95% CI, 67% to 91%), and 72% (95% CI, 53% to 91%); RFS estimates are 71% (95% CI, 56% to 85%), 57% (95% CI, 43% to 72%), and 50% (95% CI, 29% to 71%) irrespective of the HDCT regimen. In univariate analysis, the risk of relapse was lower for patients with progesterone receptor (PR)-positive tumors (risk ratio [RR], 0.43; 95% CI, 0.22 to 0.81; P = .01) and higher for patients with inflammatory carcinoma (RR, 2.20; 95% CI, 1.02 to 4.76; P = .05). OS was better for patients with PR (RR, 0.16; 95% CI, 0.05 to 0.55; P = .003) and estrogen receptor (ER)-positive tumors (RR, 0.42; 95% CI, 0.17 to 1.02; P = .05); OS was worse for patients with high-grade primary tumors (RR, 4.08; 95% CI, 1.21-13.7; P = .02). In multivariate analysis, PR positivity was associated with improved RFS (P = .01) and OS (P = .001). CONCLUSION: HDCT in selected patients with HRBC is safe and warrants further evaluation. Patients with receptor-negative, high-grade, or inflammatory tumors require improvement in their therapeutic options. Better assessment of the role of HDCT awaits completion of ongoing randomized trials.

Adult↗
PubMed1995

Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells.

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis. The enzyme consists of two subunits, M1 and M2. Hydroxyurea (HU) is an M2-specific inhibitor. It has been shown that a HU-resistant clone derived from stepwise exposure to HU overexpresses the M2 mRNA and the RR protein (Y. Yen et al., Cancer Res., 54: 3868-3691, 1994). In this study, we established stable clones by transfecting human KB cells with the cDNA of human wild-type RR in which each subunit was overexpressed by a SV40 promoter. The mammalian cell expression vector ph beta APr-1 was used for constructing M1, M2, and M1/M2 subunit cDNA. The transfected cells were selected with G418. The clones designated M2-D, M1-D, X-D, and KB-V represent transfectant clones which contain M2 cDNA, M1 cDNA, M1/M2 cDNA, and vector alone, respectively. The parental KB cells and clones containing vector plasmid KB-V express equally low amounts of M2 and M1 mRNA from the endogenous genes. The expression of M2 mRNA and M1 mRNA is elevated 2-3 fold in the X-D transfectants. M2-D clone demonstrated a 6-fold higher M2 mRNA level although the M1 mRNA expression remains the same as parental cells. M1-D transfectants have a 3-fold increase in M1 mRNA expression relative to parental cells, but reveal no alteration of M2 mRNA. Southern analysis of genomic DNA suggested the incorporation of the plasmid into the genome. The X-D clone revealed both integration of the M2 and M1 gene while the M2-D clone only showed M2 gene integration. The M1-D clone revealed M1 gene integration relative to the parental cells. The Western blot of M2 protein showed a 3-fold increase in the X-D and M2-D clones whereas the M2 protein level in M1-D was the same as it was in parental cells. The M1 protein was increased 3-fold in X-D and 1.5-fold in M1-D over that of parental cells. However, lower M1 protein levels were identified in the M2-D clone. The specific activity of the RR enzyme from each transfectant showed a 3-fold increase in both the X-D and M2-D clones and slightly increased in M1-D clone over that of parental cells. However, X-D and M2-D both demonstrated a 3-fold increase in resistance to HU as compared to M1-D which showed the same sensitivity as the parental enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Blotting, Southern↗
PubMed1995

Pseudoleukemia after granulocyte colony-stimulating factor therapy.

Therapy with myeloid colony-stimulating factors has been safely and effectively used in a wide variety of situations associated with neutropenia. We present a case of pseudoleukemia occurring in a patient with lymphoma and pancytopenia after 2 days of treatment with granulocyte colony-stimulating factor (G-CSF). Bone marrow aspirate and flow cytometry study results were consistent with acute myelomonocytic leukemia but were normal after G-CSF was discontinued for 4 days. As previous phase I studies of bone marrow morphology after G-CSF use have not described the extreme myeloid immaturity seen in this patient, it seems likely that the action of G-CSF was enhanced by factors associated with the patient's illness. We emphasize the clinical importance of this case in light of the widespread use of G-CSF.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine.

Hydroxyurea (HU) is currently used in the clinic for the treatment of chronic myelogenous leukemia, head and neck carcinoma, and sarcoma. One of its drawbacks, however, is the development of HU resistance. To study this problem, we developed a HU-resistant human KB cell line which exhibits a 15-fold resistance to HU. The characterization of this HU-resistant phenotype revealed a gene amplification of the M2 subunit of ribonucleotide reductase (RR), increased levels of M2 mRNA and protein, and a 3-fold increase of RR activity. This HU-resistant cell line also expressed a "collateral sensitivity" to 6-thioguanine (6-TG), with a 10-fold decrease in the dose inhibiting cell growth by 50% as compared to the KB parental line. The mechanism responsible for this supersensitivity to 6-TG is believed to be related to an increasingly efficient conversion of 6-TG to its triphosphate form, which is subsequently incorporated into DNA. After passage of the resistant cells in the absence of HU, the cell line reverts. The revertant cells lose their resistance to HU and concomitantly their sensitivity to 6-TG. This phenomenon is due to the return of RR to levels comparable to that of the KB parental cell line. These observations and their relevance to cancer chemotherapy will be discussed in this paper. Our results suggest that a clinical protocol could be designed which would allow for a lower dose of 6-TG to be used by taking advantage of the increased RR activity in HU-refractory cancer patients. Two drugs which display collateral sensitivity are known as a "Ying-Yang" pair. Alternate treatment with two different Ying-Yang pairs is the rationale for the "Ying-Yang Ping-Pong" theory in cancer treatment. This rationale allows for effective cancer chemotherapy with reduced toxicity.

Animals↗
PubMed1993

Identification, characterization, and comparison of RNA-degrading enzymes of wheat and barley.

RNA-degrading enzymes play an important role in regulating gene expression, and sequence analyses have revealed significant homology among several plant RNA-degrading enzymes. In this study we surveyed crude extracts of the above-ground part of the common wheat (Triticum aestivum L.) and the cultivated barley (Hordeum vulgare L.) for major RNA-degrading enzymes using a substrate-based SDS-PAGE assay. Fifteen wheat and fourteen barley RNA-degrading enzymes, with apparent molecular masses ranging from 16.3 to 40.1 kD, were identified. These RNA-degrading enzymes were characterized by their response to pH changes and addition of EDTA and ZnCl2 to the preincubation or incubation buffers. The 33.2- to 40.1-kD wheat and barley, 31.7-kD wheat, and 32.0-kD barley enzyme activities were inhibited by both zinc and EDTA and were relatively tolerant to alkaline environment. The 22.7- to 28.2-kD enzymes were inhibited by zinc but stimulated by EDTA. The 18.8-kD enzyme exists in both wheat and barley. It was active in an acid environment, was inhibited by zinc, but was not affected by EDTA. Two enzyme activities (31.0 and 32.0 kD) are unique to the common wheat.

Chlorides↗
PubMed1993

The lupus anticoagulant. High incidence of 'negative' mixing studies in a human immunodeficiency virus-positive population.

We identified 100 patients (51 males and 49 females) as having the lupus anticoagulant. The following diagnoses were found in the patient population: human immunodeficiency virus positivity, 20%; systemic lupus erythematosus, 10%; prolonged preoperative activated partial thromboplastin time (APTT), 10%; procainamide hydrochloride-induced inhibitor, 9%; deep vein thrombosis, 6%; seizure disorders/epilepsy, 5%; and miscellaneous conditions, 40%. Identification was based on a prolonged APTT (> 40 seconds) that normalized with increased phospholipid concentrations and/or a prolonged Russell viper venom clotting time patient-control ratio of 1.20 or greater. In 68 cases (group 1), patient plasma prolonged the APTT of normal plasma in a 1:1 mixing study. However, in 32 cases (group 2), no such prolongation was observed. There was a significant difference between presenting APTTs in patients from group 1 (mean +/- SD, 58.29 +/- 13.30 seconds) compared with that in group 2 (mean +/- SD, 47.93 +/- 5.09 seconds). Furthermore, 66% of group 1 patients had elevated anticardiolipin antibody titers compared with only 41% in group 2. Of the 32 patients in group 2, 16 (50%) were positive for human immunodeficiency virus. We concluded that the investigation of a lupus anticoagulant should not be abandoned because patient plasma does not prolong the APTT of normal plasma in a mixing study, especially in a human immunodeficiency virus-positive population.

Antibodies, Anticardiolipin↗
PubMed1991

Identification and Properties of the Major Ribonucleases of Arabidopsis thaliana.

The profile of major ribonuclease (RNase) activities of Arabidopsis thaliana has been identified and characterized using a substrate-based gel assay. Following sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as many as 16 RNases, varying in size from 9 to 41 kilodaltons can be detected. Most of the RNase activities exhibit a pH optimum of about 6.5; however, the activity of a 22.6-kilodalton RNase is greatly enhanced at low pH. A number of the RNases in the 30- to 41-kilodalton range are sensitive to ethylenediaminetetraacetic acid, and their activities are enhanced by the presence of a low concentration of zinc during renaturation. At least one RNase appears to comigrate with a major DNase activity. The differential accumulation of several RNases in stems versus leaves indicates that some RNases are controlled in an organ-specific manner in A. thaliana.

Journal Article↗
PubMed1990

Genomic analysis of diploid plants.

The numerical analysis of meiosis in the triploid hybrid between an induced autotetraploid and a related diploid provides the basis for investigations of the genomic relationships of diploid species based on chromosome pairing. It is also possible to make legitimate comparisons between different triploid hybrids of this type so that the genomic relationships can be converted into measures of phylogenetic distance for taxa where only diploids are known.

Journal Article↗
PubMed1988

Analysis of pivotal-differential evolutionary patterns.

The numerical analysis of meiosis in hybrids between a wild allotetraploid and an autotetraploid of one of its putative diploid progenitors allows the identification of which genomes are pairing and also the verification of pivotal-differential evolution. This type of analysis should be applicable to all genera in which allopolyploid series exist.

Journal Article↗
PubMed1988

Lectin histochemistry of uterine adenocarcinomas.

Lectin histochemistry was used to compare normal endometrial and endocervical epithelium with adenocarcinomas arising in these locations. Endometrial carcinomas were characterized by much more variable lectin binding than normal endometrial cells. Endocervical carcinomas essentially retained the phenotype of parental epithelium. Type I lectin from Griffonia simplicifolia was the only lectin consistently nonreactive with normal endocervix that reacted with all endocervical carcinomas. In both anatomic sites, malignant transformation was associated with a loss of binding sites for some lectins and an acquisition of binding sites for others.

Adenocarcinoma↗
PubMed1986

Lectin binding sites on human endocervix: a comparison with secretory and proliferative endometrium.

Endocervix and corresponding endometrium of women of reproductive age were studied histochemically with 13 fluorescein isothiocyanate-labeled lectins to delineate the differences between the epithelial cells in two anatomical sites. Lectin from Maclura pomifera (MPA), Ulex europaeus (UEA-I), Glycine max (SBA), and Vicia villosa (VVA) bound only to endocervical epithelium and were the only four lectins that distinguished endocervical from endometrial epithelium. These differences were independent of menstrual cyclic changes and blood group antigen secretion. These data show that lectins can be used to histochemically distinguish endocervical from endometrial glands.

Cell Division↗