Biomedical subjects

Y Yen

Publications and source records attributed to Y Yen.

At least 19 recordsLinked to original sources

PubMed2006

Ribonucleotide reductase inhibitors and future drug design.

Ribonucleotide reductase (RR) is a multisubunit enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides, which are building blocks for DNA replication and repair. The key role of RR in DNA synthesis and cell growth control has made it an important target for anticancer therapy. Increased RR activity has been associated with malignant transformation and tumor cell growth. Efforts for new RR inhibitors have been made in basic and translational research. In recent years, several RR inhibitors, including Triapine, Gemcitabine, and GTI-2040, have entered clinical trial or application. Furthermore, the discovery of p53R2, a p53-inducible form of the small subunit of RR, raises the interest to develop subunit-specific RR inhibitors for cancer treatment. This review compiles recent studies on (1) the structure, function, and regulation of two forms of RR; (2) the role in tumorigenesis of RR and the effect of RR inhibition in cancer treatment; (3) the classification, mechanisms of action, antitumor activity, and clinical trial and application of new RR inhibitors that have been used in clinical cancer chemotherapy or are being evaluated in clinical trials; (4) novel approaches for future RR inhibitor discovery.

Animals↗

PubMed2004

A study of radiotherapy modalities combined with continuous 5-FU infusion for locally advanced gastrointestinal malignancies.

AIM: We describe the feasibility of combining infusional 5-fluorouracil (5-FU) with intraoperative radiation therapy (IORT). METHODS: Patients with surgically resectable locally advanced gastrointestinal cancers were treated concurrently during surgery with IORT and a 72 h infusion of 5-FU. Patients without previous external beam radiation therapy (EBRT) were subsequently treated with EBRT (40-50Gy) concurrent with a 21-day continuous infusion of 5-FU. Pancreatic, gastric, duodenal, ampullary, recurrent colorectal, and recurrent anal cancer were included. RESULTS: During IORT/5-FU, no chemotherapy-related grade III or IV hematologic or gastrointestinal toxicity was noted. Post-surgical recovery or wound healing was not affected. One of nine patients who received post-operative radiation required a treatment break. During follow-up, there were more complications in patients with pelvic tumours, especially those with previous radiation. Nine patients have had local and/or local regional recurrences, two of these in the IORT field. CONCLUSIONS: Treatment with a combination of IORT and 5-FU followed by EBRT and 5-FU is feasible. However, long-term complications may be increased in previously irradiated recurrent pelvic tumours.

Adenocarcinoma↗

PubMed2001

High-dose paclitaxel in combination with doxorubicin, cyclophosphamide and peripheral blood progenitor cell rescue in patients with high-risk primary and responding metastatic breast carcinoma: toxicity profile, relationship to paclitaxel pharmacokinetics and short-term outcome.

We assessed the feasibility and pharmacokinetics of high-dose infusional paclitaxel in combination with doxorubicin, cyclophosphamide, and peripheral blood progenitor cell rescue. Between October 1995 and June 1998, 63 patients with high-risk primary [stage II with >or= 10 axillary nodes involved, stage IIIA or stage IIIB inflammatory carcinoma (n = 53)] or with stage IV responsive breast cancer (n = 10) received paclitaxel 150-775 mg/m(2)infused over 24 hours, doxorubicin 165 mg/m(2)as a continuous infusion over 96 hours, and cyclophosphamide 100 mg kg(-1). There were no treatment-related deaths. Dose-limiting toxicity was reversible, predominantly sensory neuropathy following administration of paclitaxel at the 775 mg/m(2) dose level. Paclitaxel pharmacokinetics were non-linear at higher dose levels; higher paclitaxel dose level, AUC, and peak concentrations were associated with increased incidence of paraesthesias. No correlation between stomatitis, haematopoietic toxicities, and paclitaxel dose or pharmacokinetics was found. Kaplan-Meier estimates of 30-month event-free and overall survival for patients with primary breast carcinoma are 65% (95% CI; 51-83%) and 77% (95% CI; 64-93%). Paclitaxel up to 725 mg/m(2) infused over 24 hours in combination with with doxorubicin 165 mg/m(2) and cyclophosphamide 100 mg kg(-1) is tolerable. A randomized study testing this regimen against high-dose carboplatin, thiotepa and cyclophosphamide (STAMP V) is currently ongoing.

Adult↗

PubMed2001

Phase II trial of high-dose intravenous doxorubicin, etoposide, and cyclophosphamide with autologous stem cell support in patients with residual or responding recurrent ovarian cancer.

This study was performed in order to evaluate the toxicities, progression-free and overall survival of patients with responsive residual or recurrent ovarian cancer treated with high-dose chemotherapy. Twenty-seven patients were treated. Doxorubicin, 165 mg/m(2) over 96 h (days -12 to -8), etoposide 700 mg/m(2) every day x3 (days -6 to -4), and cyclophosphamide 4.2 g/m(2) on d -3 was followed by stem cells and granulocyte colony-stimulating factor. The median days of granulocyte count <500/microl was 14 (range 10-42) and platelets <20,000/microl was 13 (range 2-80). Median numbers of red cell and platelet transfusions were 15 (5-16) and 14 (4-103). Toxicity included mucositis requiring narcotic analgesia in all patients. Asymptomatic decreases in ejection fraction to values <50% were observed in four patients. No clinical congestive heart failure was observed. One death due to sepsis was observed. Median progression-free survival is 7.5 months (1.0-56 months); five patients remain alive, two of whom remain progression-free at 19.5 and 24.5 months post transplant. Median overall survival is 14.0 months (1-68 months). We conclude that high-dose anthracyclines may be safely administered to ovarian cancer patients. The short overall and progression-free survivals observed in our population suggest that this combination is not optimal.

Adult↗

PubMed2001

Tandem-cycle high-dose melphalan and cisplatin with peripheral blood progenitor cell support in patients with breast cancer and other malignancies.

We evaluated the feasibility of tandem-cycle high-dose chemotherapy (HDCT) with cisplatin, melphalan, and peripheral blood progenitor cells (PBPCs). Fifty patients with high-risk primary (n = 17) or stage IV breast cancer (n = 29) or other malignancies (n = 4) received 2 cycles of intravenous melphalan, 20 to 151.8 mg/m2, and cisplatin, 200 mg/m2, followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF. Starting at 40 mg/m2 of melphalan, patients also received PBPCs. Delayed platelet recovery defined the maximum tolerated dose (MTD) for melphalan at 101.2 mg/m2 per cycle. There were no treatment-related deaths. Cycle 2 was delivered at a median of 1.7 months after cycle 1; 72% of patients treated at the MTD received both cycles. Cycle 2 was omitted when patients refused it or had disease progression or toxicities, primarily prolonged thrombocytopenia. Complete response rates in stage IV breast cancer patients increased from 28% pre-HDCT to 55% after cycle 2. At a median follow-up of 4.6 years (range, 1.5-8.1 years), 11 of 29 patients with stage IV breast carcinoma were alive with 5-year projected progression-free and overall survival rates of 19% (95% confidence interval [CI], 7%-41%) and 39% (95% CI, 20%-62%), respectively. Five-year projected progression-free and overall survival rates for patients with stage IV breast cancer in complete response following HDCT versus all others were 35% (95% CI, 15%-70%) versus 0% (P = .01) and 61% (95% CI, 35%-91%) versus 10% (95% CI, 2%-60%) (P = .003; log-rank test), respectively. Estrogen-receptor positivity was predictive of reduced risk of progression (relative risk [RR], 0.25; 95% CI, 0.10-0.65; P = .003) and death (RR, 0.27; 95% CI, 0.10-0.72; P = .009) after adjusting for response status. Five-year projected relapse-free and overall survival rates were 71% (95% CI, 43%-96%) and 82% (95% CI, 56%-100%), respectively, for the 17 patients with high-risk primary breast cancer. Tandem-cycle high-dose melphalan and cisplatin with PBPCs is feasible. Preliminary data suggest significant activity in selected patients with stage IV responding breast carcinoma.

Adult↗

PubMed2001

Human ribonucleotide reductase M2 subunit gene amplification and transcriptional regulation in a homogeneous staining chromosome region responsible for the mechanism of drug resistance.

In our previous publication it was shown that a Gemcitabine-resistant KBGem clone derived from step-wise exposure to Gemcitabine resulted in overexpression of the human Ribonucleotide Reductase M2 subunit (hRRM2) mRNA and protein (Goan et al., 1999). In this study we confirm these results and show that the hRRM2 gene amplification arises in a homogeneous staining region (hsr) derived from chromosome translocation. The hydroxyurea-resistant clone (KBHURs) was studied as a comparison. PCR analysis of the hRRM2 gene promoter confirmed the amplification. Northern and Western blots were further employed to confirm the gene amplification and hRRM2 mRNA and protein expression were compatible with the level of drug resistance. Cells synchronized by serum starvation and then returned to serum-containing growth conditions showed a rapid induction of high levels of transcription of the hRRM2 gene. To clarify whether expression of hRRM2 mRNA was regulated at a transcriptional level, several transcription factors, including AP-1, Sp1, AP-2, CREB, NF-kappa B, and OCT1, were examined by gel-shift assay. Interestingly, the KBGem clone was regulated by different transcription factors than the KBHURs clone. Compared to the wild-type KB cells (KBwt), the KBGem clone exhibited a different binding pattern for Sp1 and NF-kappa B. The KBHURs clone, however, demonstrated a unique binding pattern with AP-1 and CREB, different from the KBwt control as well as the KBGem clone. Therefore, we conclude that the drug-resistant phenotype is associated with human RRM2 gene amplification from a homogeneous staining chromosome region and altered transcription regulation. Each clone demonstrated a unique pattern of transcription factor binding that may play a vital role in the mechanism of drug resistance.

Chromosome Banding↗

PubMed2001

Characterization of the human ribonucleotide reductase M2 subunit gene; genomic structure and promoter analyses.

Ribonucleotide Reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair consisting of two subunits, M1 and M2. RRM2 plays a role in cell proliferation, tumorgenicity, metastasis and drug resistance. To better understand the regulation of RRM2, we have sequenced the entire human RRM2 gene. Approximately 10.3 kb of genomic DNA was sequenced and deposited to GenBank (accession number AY032750). Intron/exon junctions were identified and the gene was found to consist of ten exons. Two transcription initiation sites were identified and correspond to mRNA transcripts of 3.4 kb and 1.65 kb. Deletion analysis of the 5'-flanking region showed that there was promoter activity consistent with the presence of two separate promoters driving expression of the two RRM2 transcripts. A thorough analysis of the genomic sequence and promoter regions of the RRM2 gene will provide insight into the processes involved in tumor transformation and drug resistance.

Base Sequence↗

PubMed2001

Phase I trial of 96-hour continuous infusion of dexrazoxane in patients with advanced malignancies.

Dexrazoxane is a bidentate chelator of divalent cations. Pretreatment with short infusions of dexrazoxane prior to bolus doxorubicin has been shown to lessen the incidence and severity of anthracycline-associated cardiac toxicity. However, because of rapid, diffusion-mediated cellular uptake and the short plasma half-life of dexrazoxane, combined with prolonged cellular retention of doxorubicin, dexrazoxane may be more effective when administered as a continuous infusion. Thus, a Phase I pharmacokinetic trial of a 96-h infusion of dexrazoxane was performed. Dexrazoxane doses were escalated in cohorts of 3 to 6 patients per dose level. All patients received granulocyte-colony stimulating factor at a dose of 5 microg/kg/day starting 24 h after completion of the dexrazoxane infusion. Plasma samples were collected and analyzed for dexrazoxane by high-performance liquid chromatography. Urine collections were performed at baseline and during the infusion to determine the renal clearance of dexrazoxane and the excretion rate of divalent cations. Twenty-two patients were enrolled at doses ranging from 125 to 250 mg/m(2)/day. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 2 patients treated at 250 mg/m(2)/day, grade 3 thrombocytopenia and grade 4 nausea and vomiting in 1 patient treated at 221 mg/m(2)/day, grade 4 diarrhea and grade 3 nausea and vomiting in 1 patient treated at 221 mg/m(2)/day, and grade 3 hypertension in 1 patient treated at 166.25 mg/m(2)/day. Steady-state dexrazoxane levels ranged from 496 microg/l (2.2 microM) to 1639 microg/l (7.4 microM). Dexrazoxane plasma CL(ss) and elimination t(1/2) were 7.2 +/- 1.6 l/h/m(2) and 2.0 +/- 0.8 h, respectively. The mean percentage of administered dexrazoxane recovered in the urine at steady state was 30% (range, 10-66%). Urinary iron and zinc excretion during the dexrazoxane infusion increased in 12 of 18 and 19 of 19 patients by a median of 3.7- and 2.4-fold, respectively. These results suggest that dexrazoxane as a 96-h infusion can be safely administered with granulocyte-colony stimulating factor at doses that achieve plasma levels that have been demonstrated previously to inhibit topoisomerase II activity and to induce apoptosis in vitro. Additional studies will be required to determine whether the combination of continuous infusions of dexrazoxane and doxorubicin would provide enhanced cardioprotection compared with the currently recommended bolus or short infusion schedules.

Aged↗

PubMed2000

A phase I study of carboplatin and etoposide administered in conjunction with dipyridamole, prochlorperazine and cyclosporine A.

PURPOSE: In recognition of the variety of available chemotherapeutic modulating agents and their potential to enhance the efficacy of platinum-based therapy, we embarked upon a phase I study to investigate the feasibility of combining fixed doses of carboplatinum (CBDCA) and etoposide (VP-16) with 24-h concurrent infusions of dipyridamole (DP), prochlorperazine (PCZ) and cyclosporine A (CSA) administered in escalating doses. METHODS: Patients received intravenous VP-16 (200 mg/m2) and CBDCA (300 mg/m2), each over 30 min, starting at hour 6 of the modulator infusions. Resistance modulators were escalated sequentially to determine their respective maximally tolerated doses (MTDs). The pharmacokinetics (PK) of VP-16, CBDCA, and the three drug resistance (DR) modifiers were studied in eight patients. RESULTS: A total of 59 patients were entered on study. The MTD was established at DP 5 mg/kg per day, PCZ 24 mg/h, and CSA 9.5 mg/kg per day. Dose-limiting toxicities included hypotension and severe sedation, presumably related to PCZ. No objective responses were seen. PK studies were performed when PCZ and DP doses were 24 mg/h and 3.3 mg/kg, and the CSA dose was either 8.5 mg/kg (five patients) or 9.5 mg/kg (three patients). The median clearance of VP-16 was 0.96 l/h per m2 (range 0.8-1.5 l/h per m2), which is lower than for VP-16 alone and similar to previously reported effects of CSA on VP-16 elimination. The median measured CBDCA AUC was 3.0 mg/ml x min (range 2.4-4.8 mg/ml x min). CBDCA AUC predicted by the Calvert formula using measured creatinine clearance underestimated the actual AUC in seven of the eight patients, in one case by as much as twofold. The median end of infusion PCZ and total DP plasma concentrations were 1.2 microM (range 0.5-2.2 microM) and 4.4 microM (range 1.3-5.9 microM), respectively, consistent with in vitro resistance modulatory levels. However, free DP was only 0.02 microM (range 0.004-0.04 microM). The median CSA level at 24 h of 1450 microg/l (range 1075-1640 microg/l) is in agreement with concentrations required for partial DR reversal in vitro, although it is much lower than levels achieved in our previous phase I study of CBDCA + CSA alone using similar doses of CSA. The CSA dose on the current trial was escalated beyond the MTD for the previous phase I study, suggesting that there may be an interaction between CSA and one of the other modulators. CONCLUSION: These results demonstrate that in vitro DR-reversing levels of two of the three agents used in this study can be achieved in vivo, and that this combination of DR modulators has significant effects on the pharmacokinetics of VP-16.

Adult↗

PubMed2000

Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA.

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2. It is known that the M2 subunit plays a role in tumorgenicity and metastasis. In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418. The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG). However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by 14CDP reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG. M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours. Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.

Blotting, Western↗

PubMed1999

Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line.

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.

Antimetabolites, Antineoplastic↗

PubMed1999

Recombinant human thrombopoietin in combination with granulocyte colony-stimulating factor enhances mobilization of peripheral blood progenitor cells, increases peripheral blood platelet concentration, and accelerates hematopoietic recovery following high-dose chemotherapy.

Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 micrograms/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 micrograms/kg on days -3, -1, and 1, or 0.6 micrograms/kg on days -1 and 1. G-CSF, 5 micrograms/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34(+) cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 x 10(6)/kg (range, 1.3 to 17.6) versus 0.8 x 10(6)/ kg (range, 0.3 to 4.2), P =.0003. The targeted minimum yield of 3 x 10(6) CD34(+) cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF-mobilized group versus 10% of G-CSF-mobilized patients (P =.001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P =.0001) and platelet recovery (day 9 v 10, P =.07) were accelerated, and fewer erythrocyte (3 v 4, P =.02) and platelet (4 v 5, P =.02) transfusions were needed compared with G-CSF-mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P <.0001) with the greatest effect seen with repeated dosing of rhTPO at 0.6 microgram/kg. rhTPO is safe and well tolerated as a mobilizing agent before PBPC collection. Mobilization with rhTPO and G-CSF, in comparison to a comparable, nonrandomized G-CSF-mobilized group of patients, decreases the number of apheresis procedures required, may accelerate hematopoietic recovery, and may reduce the number of transfusions required following high-dose chemotherapy for breast cancer.

Adult↗

PubMed1999

Ethnic differences in the behavior of hepatocellular carcinoma.

BACKGROUND: The purpose of this study was to examine the clinical presentation, prognostic factors, and survival rates of patients with hepatocellular carcinoma (HCC) and to examine differences between Asian and non-Asian patients with HCC. METHODS: A review of the clinical characteristics and laboratory evaluations for 76 patients in two different broad ethnic groups (Asians [Group 1] and non-Asians [Group 2]) who underwent treatment for HCC from 1977-1995 was performed. Chi-square and Cox regression analyses were performed to assess factor interaction and association with survival. RESULTS: A total of 24 patients in Group 1 and 52 patients in Group 2 were reviewed. Of the clinical variables examined, a higher rate of a history of hepatitis B positivity was observed in Group 1 compared with Group 2 (32% vs. 6%; P=0.001). Among the 76 patients with HCC, a 1-year survival estimate of 41.4% was found. There was a borderline significant difference in survival between Group 1 and Group 2 with a 1-year survival estimate of 29.5% versus 46.9%, respectively (P=0.08). Better overall survival was found in patients who had tumors that were resectable (P=0.0001), had an alpha-fetoprotein level <10 ng/mL (P=0.02), or were a younger age at the time of diagnosis (P=0.01). There was a trend for Asian race (P=0.08) to be associated with poorer survival. When these risk factors were entered into a multivariate analysis, tumor resectability and non-Asian race were most predictive of improved survival (model P value = 0.007). When controlling for the multiple variables most often reported to be associated with HCC, Asians had a significantly lower survival than non-Asians (P<0.01). CONCLUSIONS: In this study it appears that the outcome for Asian patients with hepatoma is worse than for non-Asian patients, even when controlling for factors commonly associated with HCC. Biologic or social factors that are not appreciated currently may be involved in Asian patients with HCC, contributing to a poorer clinical outcome.

Adolescent↗

PubMed1999

Electrochemical treatment of human KB cells in vitro.

Electrochemical treatment (ECT) of cancer is a promising new method by which direct current is delivered into tumor tissue to induce tumor regression. The purpose of this study is to evaluate the effectiveness of ECT on human cancer cells and to investigate the factors that affect ECT. The biological mechanisms of ECT in cancer treatment were also explored. Using human KB cells, ECT was found to delay cell growth by using 0.3 coulombs (C)/ml (1.5 C in 5 ml of culture medium; 3 V, 400 microA for 62.5 min). From the results of a colony-forming assay, it was clearly demonstrated that increasing the ECT dose decreases tumor cell survival. A cytotoxicity study, in which a methylene blue assay was used, determined that, for 2.5 x 10(5) cells in culture, the 1D50 was 0.68 C/ml. For a fixed dose of 0.6 C/ml (3 C in 5 ml), using higher current and shorter treatment time resulted in better cell survival. Time, therefore, is an important factor. When cell concentration was altered, the survival was higher for increased cell concentrations. A thymidine incorporation assay indicated that the amount of [3H]thymidine incorporated into DNA decreased as the ECT dose increased. After treatment with 1 C/ml (5 C in 5 ml; 3 V, 400 microA for 208.4 min), pH at the anode decreased to 4.53 and at the cathode increased to 10.46. These results indicate that ECT is effective for killing human KB cells in vitro and that the toxicity effect is related to charge, current, and treatment time. The effect of pH alteration on cells is one of the mechanisms of ECT.

Cell Division↗

PubMed1998

Role of the M2 subunit of ribonucleotide reductase in regulation by hydroxyurea of the activity of the anti-HIV-1 agent 2',3'-dideoxyinosine.

The ribonucleotide reductase inhibitor hydroxyurea exhibits potent synergism, even at low, non-cytotoxic concentrations, with the anti-HIV-1 dideoxynucleoside 2',3'-dideoxyinosine, bringing about failure of HIV DNA synthesis and, thus, of HIV replication. To elucidate the incompletely defined role of hydroxyurea in the hydroxyurea/dideoxyinosine interaction and, in particular, to identify the reasons for the unusual selective inhibitory action of the combination on retroviral rather than on cellular DNA synthesis, we prepared specific cDNA probes to determine the effects of low-level hydroxyurea on mammalian cell ribonucleotide reductase M1 and M2 subunit mRNA, while simultaneously quantitating the effects of the drug on cell cycle and on deoxynucleoside triphosphate pools. While dTTP, dCTP, and dGTP pools changed little or even increased in the presence of low-level hydroxyurea, there took place a rapid and specific inhibition of M2-subunit-catalyzed generation of dATP, with consequent slowing of cellular DNA synthesis and prolongation of S phase. However, the latter effect, in turn, resulted in increased M2 subunit mRNA transcription (a process blocked in Go/G1-phase cells, with full-length functional M2 transcripts being generated only during S phase) and, hence, in a return to normal levels of dATP and to a normal rate of cellular DNA synthesis. Because of this self-regulating mechanism, hydroxyurea-induced host-cell toxicity was obviated under conditions where HIV DNA synthesis, a process sensitive to both dATP depletion and the chain-terminating properties of the other inhibitory component of the combination (ddATP derived from dideoxyinosine), was unable to recover.

Anti-HIV Agents↗

PubMed1998

Phase I trial of intraperitoneal iododeoxyuridine with and without intravenous high-dose folinic acid in the treatment of advanced malignancies primarily confined to the peritoneal cavity: flow cytometric and pharmacokinetic analysis.

In this Phase I study, the maximally tolerated doses (MTDs) of i.p. iododeoxyuridine (IdUrd) alone and in combination with i.v. calcium leucovorin (LV) were determined. The pharmacokinetics and pharmacological advantage of IdUrd were evaluated, and flow cytometric analysis allowed examination of the extent of incorporation of IdUrd into tumor cells with and without the addition of i.v. LV. Thirty-nine patients with advanced neoplasms primarily confined to the peritoneal space were enrolled in a dose-escalation trial using 4-h dwells of IdUrd administered i.p. daily for 4 days with and without an i.v. infusion of LV 500 mg/m2/day for 4.5 days. Twenty-three patients received single-agent therapy, and 13 patients received i.p. IdUrd in combination with i.v. LV. The MTD of single-agent IdUrd administered on this schedule was 4125 mg/m2/day for 4 days; and that of the IdUrd in combination was 3438 mg/m2/day. Dose-limiting toxicities were myelosuppression and stomatitis. During the period of the dwell, the peritoneal AUC (area under the curve) of IdUrd exceeded the plasma AUC of IdUrd by one or two orders of magnitude in all patients at all doses tested; there was a possible effect of LV on peritoneal AUC. The geometric mean pharmacological advantage (AUCperitoneal/ AUCplasma) was 181 at 625 mg/m2/day and 90 at 4538 mg/m2/day. Flow cytometric analysis suggests saturation of IdUrd measured in DNA at the 2500-3125 mg/m2 dose level, without an increase after the addition of LV. Twelve patients received 4-12 courses of therapy. One patient with recurrent ovarian cancer who received 16 courses of therapy experienced complete resolution of her ascites, near normalization of CA-125 levels, and improved quality of life; two patients with high-risk tumors receiving "adjuvant" therapy are disease-free at 3 and 6 years after treatment; other patients experienced transient clearing of ascites. The recommended Phase II dose of i.p. IdUrd using a 4-h dwell daily for 4 days is 3750 mg/m2/day alone or 3125 mg/m2/day in combination with continuous i.v. LV at 500 mg/m2/day for 4.5 days. Although flow cytometric data suggest that DNA incorporation of IdUrd is not affected by the addition of LV, the cytotoxicity of the combination regimen may be increased due to LV-enhanced, IdUrd-related inhibition of thymidylate synthase. For this reason, we recommend that efficacy studies of the combination continue in parallel with studies of IdUrd alone.

Adult↗

PubMed1998

Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells.

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective increases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1-D clone demonstrated a significant resistance to dNTP inhibition of RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.

Clone Cells↗

PubMed1998

Reversal of hydroquinone-mediated suppression of T cell proliferation by transfection of the M2 subunit of ribonucleotide reductase.

Hydroquinone (HQ) is a benzene derivative that is found in large quantities in cigarette tar as a result of the pyrolysis of tobacco flavinoids. HQ is a potent inhibitor of T cell proliferation, causing an immediate and complete cessation of DNA synthesis in IL-2-dependent human T lymphoblasts and Jurkat T cells without loss of cell viability. Previous studies from our laboratory demonstrated that the antiproliferative effects of RQ could be partially reversed by the addition of deoxyribonucleosides, but not by the corresponding ribonucleosides, suggesting that HQ might inhibit ribonucleotide reductase. In the present study, the molecular mechanism behind this observation was further investigated. Jurkat T cells were stably transfected with a pMEP4 expression vector containing the gene for the M2 subunit of ribonucleotide reductase under transcriptional control of the human metallothionein IIA promoter. M2-transfected Jurkat T cells exhibited a greater than three-fold increase in resistance to HQ compared to untransfected cells or cells transfected with the M2 gene in the reverse orientation. HQ resistance was associated with an increased level of M2 protein detected by Western blot. These results suggest that the benzene derivative inhibits lymphocyte proliferation by inhibiting ribonucleotide reductase.

Blotting, Western↗