[Quality of products of three panoramic units in clinic and practice].
Explore the source record for details and available documents.
Radiography, Panoramic↗
Biomedical subjects
C Benz
Publications and source records attributed to C Benz.
Chemoendocrine therapy with prolonged estrogen priming in advanced breast cancer: endocrine pharmacokinetics and toxicity.
Recent efforts to improve response rates in advanced breast cancer have used short, alternating courses of antiestrogen therapy followed by estrogen priming to cytokinetically enhance tumor cell sensitivity to antimetabolites. Based on recent in vitro and in vivo studies, we have introduced a chemoendocrine regimen that uses prolonged courses of estrogen priming. The present protocol consists of alternating monthly cycles of tamoxifen (TAM) and estradiol during which sequential (24-hr) methotrexate, 5-fluorouracil, and leucovorin are administered at 2-week intervals. Twenty-five patients with metastatic breast cancer received greater than 80 endocrine cycles and greater than 300 courses of chemotherapy by this protocol; two-thirds of these patients had previously failed other endocrine or chemotherapy regimens. Most patients experienced grade 1 or 2 myelosuppression or gastrointestinal symptoms during at least one treatment cycle; however, overall toxicity was considered to be mild and therapy was very well tolerated. Serum levels of estradiol, estrone, TAM, and TAM metabolites were measured during all phases of the endocrine cycle in five patients on chronic therapy. Concentrations of TAM and its more abundant metabolite, N-desmethyltamoxifen (N-desTAM), rose twofold during antiestrogen therapy and fell during estrogen priming, with mean levels persisting greater than 100 ng/ml throughout the priming interval. Mean estradiol (E2) and estrone (E1) levels rose during priming and were sustained fivefold and tenfold above the basal postmenopausal levels measured during antiestrogen treatment. Calculating the serum molar ratios of [TAM + N-desTAM]/[E2 + E1] during each phase of the treatment cycle confirmed that estrogen priming was achieved pharmacologically by this endocrine schedule. Clinical remissions were observed in this small patient sample with seven of 18 patients achieving either complete (28%) or partial (11%) responses, and an additional 39% obtaining disease stabilization. Further clinical study is necessary to evaluate the optimal response rate of this regimen and to determine whether cyclic estrogen priming by this schedule results in enhanced tumor cell proliferation in vivo.
Adult↗
Effects of cis- and trans-tamoxifen isomers on RNA incorporation of human breast cancer cells.
Cultured T47-D human breast cancer cells were used to investigate growth-inhibiting effects of the antiestrogen, trans-tamoxifen, on [3H]Cyd incorporation into specific classes of nuclear and cytoplasmic RNA. The steroid agonist, 17 beta-estradiol, and the inactive cis isomer of tamoxifen were used as treatment controls to compare antiestrogen-induced changes in RNA metabolism, independent of estrogen receptor-binding properties. Using a 24-hr labeling interval, trans-tamoxifen produced a 1.4- to 4-fold enhanced incorporation into pre-rRNA species (20S, 32-45S), with slight reduction in mature 18S rRNA incorporation, and a 2- to 3-fold increased incorporation into 5S and 5.8S rRNA and 4-4.5S tRNA. Most notable, trans-tamoxifen enhanced incorporation into the less abundant low molecular weight U1, U3, and 7S RNA species by 3- to 6-fold. These findings were associated with an apparent reduction in pre-rRNA content and little change in U1, U3, or 7S RNA levels in antiestrogen-treated cells, suggesting that trans-tamoxifen independently regulates RNA transcription and turnover. The present study provides new rationale for the choice of molecular probes to study trans-tamoxifen effects on synthesis and turnover of specific nuclear and cytoplasmic RNA species.
Breast Neoplasms↗
Noninvasive spectroscopic analysis of fluoropyrimidine metabolism in cultured tumor cells.
Nuclear magnetic resonance spectroscopy is a technique that may be used noninvasively to follow the intracellular metabolism of fluorinated antimetabolites such as 5-fluorouracil (FUra) and 5-fluorouridine. Intracellular 19F spectral peaks are assigned by comparison with the pH-dependent chemical shifts measured for eight commercially available fluoropyrimidine metabolites as well as by comparison with the literature recorded values of five known catabolites of FUra. Five murine and human tumor cell lines (N1S1, Sarcoma 180, L1210, HL-60, and Mia-PaCa) were exposed in vitro for 24 h to cytostatic doses of FUra or 5-fluorouridine. Treated cells were harvested and analyzed immediately or following a subsequent incubation under either nutrient-rich or nutrient-poor conditions. A major narrow component peak at 4.6-4.9 ppm was observed in all cell samples analyzed immediately after treatment. This peak was identified as intracellular FUra nucleotides, and its T1 value was approximately 800 ms. No fluoropyrimidine catabolites were detectable in any of the treated cell lines. Free FUra could be measured in cells only after subsequent incubation under nutrient-poor conditions, and this was associated with a decline in the prominent FUra nucleotide peak. In treated cells chased with drug-free media containing 1 microM thymidine, spectra revealed a broad component signal underlying and downfield from the narrow nucleotide-containing peak. By biochemically fractionating treated cells into an acid-soluble fraction and phenol-purified cytoplasmic and nuclear RNA extracts, we were able to completely separate the nucleotide peak from the broad component signal resulting from FUra incorporation into RNA. Thymidine produced a marked enhancement of this 19F signal into both cytoplasmic and nuclear RNA without affecting the nucleotide signal from the acid-soluble fraction. The present ability of nuclear magnetic resonance to monitor the metabolic channeling of fluoropyrimidines in intact tumor cells suggests that future spectroscopic imaging of patients treated with fluorinated antimetabolites may provide clinically important information about tumor biochemistry and drug sensitivity.
Animals↗
Endocrine-responsive pancreatic carcinoma: steroid binding and cytotoxicity studies in human tumor cell lines.
We have begun to investigate the steroid responsiveness of pancreatic cancer by comparing human (MiaPaCa, Colo-357, RWP-1, RWP-2) and rodent (AR42j) pancreatic tumor cell lines with cultured estrogen receptor-positive breast cancer cells (MCF-7, T47-D). The four human pancreatic tumors contain measurable levels of specific estradiol binding sites with dissociation constants (Kd) that range from 1 to 9 nM, in contrast to the higher-affinity binding sites measured in the breast cancer cells (Kd less than or equal to 1 nM). Growth of one pancreatic tumor line (MiaPaCa) is stimulated 40% above control by exposure to nanomolar concentrations of estradiol, suggesting that the estrogen receptor in these cells is functioning like that in MCF-7 and T47-D cells. Glucocorticoids (dexamethasone, hydrocortisone) and androgen (fluoxymesterone) stimulate proliferation of Colo-357 cells by as much as 30%. Paradoxically, glucocorticoids inhibit AR42j cells to less than 50% of control growth. Micromolar exposures of estrogen (17 beta-estradiol), antiestrogen (tamoxifen), antiandrogen (dehydroxyflutamide), progestins (progesterone, R5020, medroxyprogesterone acetate), and inhibitors of steroid-metabolizing enzymes (17 beta-N,N-diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, danazol) impair growth of these pancreatic tumors to varying degrees, and with little relationship to estrogen receptor content. In general, progestins are slightly more growth inhibiting to these pancreatic tumor lines than the other endocrine agents tested, including tamoxifen. Only the RWP-2 cells appear completely resistant to steroidal therapy, showing less than 25% growth inhibition with exposure to therapeutic concentrations (less than or equal to 2.5 microM) of these agents. Colo-357, MiaPaCa, and AR42j cells are most responsive to these endocrine agents, and their overall pattern of sensitivity suggests that the steroid-dependent growth-inhibitory mechanisms of some pancreatic carcinomas may involve both receptor antagonism and direct inhibition of steroidal oxidoreductases. 17 beta-N,N-Diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, a potent inhibitor of 5 alpha-reductase with minimal affinity for androgen receptor, inhibits growth of Colo-357 cells to less than 40% of control and also inhibits AR42j and MiaPaCa cells. Dehydroxyflutamide, a potent androgen receptor antagonist with no direct influence on 5 alpha-reductase activity, inhibits growth of MiaPaCa and AR42j cells but has no affect on Colo-357 growth.(ABSTRACT TRUNCATED AT 400 WORDS)
Aminoglutethimide↗
Flow cytometric analysis of fluorescein-conjugated estradiol (E-BSA-FITC) binding in breast cancer suspensions.
With greater utilization of histochemical methods for detecting estrogen binding (ER) in tumor cells, there is an increasing need to quantitate objectively these fluorescently stained cells. This study utilizes flow cytometry (FCM) to examine the binding specificity and kinetics of 17 beta-estradiol-6-CMO-BSA-FITC(E-BSA-FITC) in two human mammary carcinoma cell lines, MCF-7 and 47-DN. Cells are rendered permeable to this ligand by freeze-thawing, a process analogous to the routine staining of frozen tumor sections with E-BSA-FITC for the clinical detection of ER. FCM quantification of E-BSA-FITC binding intensity demonstrates a saturable dose-response that is specifically reduced in the presence of diethylstilbestrol (DES) in doses known to saturate Type I ER. Scatchard analysis suggests that E-BSA-FITC binding occurs with receptors of varying affinities (Kd). Lineweaver-Burk plots show that the DES inhibition is competitive for a high-affinity receptor binding to E-BSA-FITC with a Kd of approximately 50 nM. This report also compares both FCM and biochemical methods of quantitating ER under two conditions of tumor cell growth potentially encountered in clinical specimens: quiescent versus actively proliferating cells, and cells pretreated with the antiestrogen tamoxifen. By FCM analysis, the cells with greater proliferating activity contain tenfold more specifically bound E-BSA-FITC, and tamoxifen pretreatment reduces this specific binding by 50%. These FCM measurements correlate well with biochemical results and suggest that this new methodology may supplement the detection of E-BSA-FITC binding by fluorescence microscopy.
Binding, Competitive↗
Inhibition of growth of leukemic cells by inhibitors of calmodulin: phenothiazines and melittin.
Calmodulin, a ubiquitous calcium-binding protein, has recently been shown to play an important role in cellular proliferation. The calmodulin inhibitors melittin, trifluoperazine, and chlorpromazine inhibited the growth and clonogenicity of human and murine leukemic cells, and their potency reflected their activity as inhibitors of calmodulin. Melittin, which is a far more potent inhibitor of calmodulin activity, was also a more potent inhibitor of cell growth and clonogenicity. The less active phenothiazine metabolite, chlorpromazine sulfoxide, had much less potent cytotoxic activity.
Animals↗
Sequential infusions of methotrexate and 5-fluorouracil in advanced cancer: pharmacology, toxicity, and response.
Preclinical studies have suggested that synergistic antitumor toxicity occurs when methotrexate (MTX) is administered prior to 5-fluorouracil (FUra). A protocol of sequenced, overlapping infusions of MTX and FUra was designed to achieve 5 microM MTX serum levels lasting 36 h and 1 to 5 microM FUra levels lasting 24 h, with leucovorin started at the end of the MTX infusion. Thirty-nine patients with metastatic neoplasms received a total of 127 treatment courses; two-thirds of the patients had received prior treatment with radiation therapy or chemotherapy; most of the latter treatment regimens included MTX or FUra. In three patients, the duration of FUra infusion was prolonged up to 72 h to determine the toxic limits of therapy. Blood samples were collected during treatment courses to estimate the half-lives and total-body clearances of MTX and FUra. The initial serum half-lives and total-body clearances of both MTX and FUra appeared within the range of reported normal values. The terminal half-life of MTX appeared less than previously reported values, and there appeared to be a substantial delay in achieving a FUra steady-state concentration; these two differences may have resulted from either the prolonged intervals of drug infusion or from metabolic interaction between the two drugs. During the 127 courses of treatment, nearly one-half of the patients experienced mild toxicity occurring after at least one treatment, but this toxicity was predominantly Grade I mucositis and/or diarrhea. Of the three patients who received extended intervals of FUra infusion, none was able to tolerate more than 48 h of FUra without developing mucositis. Thirty-four patients were evaluable for response; no one experienced a complete response, but 11 (32%) patients had either a partial or minimal response. Adenocarcinomas as a group, arising from the lung, gut, breast, and unknown site, appeared to respond best. Sequenced MTX-FUra infusion by this schedule is a generally well-tolerated regimen that deserves further clinical assessment.
Adult↗
Thymidine enhancement of methotrexate and 5-fluorouracil toxicity in cultured human colon carcinoma.
Cultures of human colon carcinoma, HCT-8, were treated with millimolar concentrations of thymidine by different schedules designed to cytokinetically and biochemically modulate methotrexate (MTX) and 5-fluorouracil (FUra) toxicity. Thymidine (dThd)-synchronized HCT-8 cells monitored by flow cytofluorometry showed increased sensitivity to MTX and synergistic cytotoxicity to the combination MTX-FUra. FUra toxicity in synchronized cells showed no significant phase specificity overall, but a pattern of relative G2/M resistance was correlated with decreased intracellular FUra accumulation, incorporation into RNA, and formation of FdUMP. In asynchronous cultures dThd reduced MTX toxicity when given within the first 12 h of a 24-h MTX exposure, and also appeared to reduce the MTX-induced synergistic enhancement of FUra toxicity. When dThd was administered with FUra alone in asynchronous cultures, progressive, and synergistic enhancement of FUra toxicity was observed only after 6 h dThd pretreatment. Unlike MTX-FUra synergy, this schedule-dependent synergism between dThd and FUra did not correlate with intracellular FUra accumulation or specific incorporation into total cellular RNA. These results suggest that less well studied mechanisms of dThd modulation, other than enhanced deoxynucleotide formation or total RNA incorporation, may biochemically enhance FUra toxicity in HCT-8 cells.
Cells, Cultured↗
Cytotoxicity of several chemotherapeutic agents in a human pancreatic cancer cell line (Colo-357).
Seven commonly available chemotherapeutic drugs were used to determine the concentration and time of exposure necessary to kill the newly available human pancreatic cancer cell line, Colo-357. The exposure periods were 2, 6, 12, and 24 hours. The most active drugs were methotrexate, mitomycin, and doxorubicin, each with an ID50 less than 0.1 microM for both a 12- and a 24-hour exposure. Cisplatin was intermediate, with an ID50 of approximately 0.7 microM at 12 and 24 hours. 5-FU, carmustine, and streptozocin were the least effective agents, with ID50 values greater than 50 microM after a 12-hour exposure and greater than 25 microM after a 24-hour exposure.
Antineoplastic Agents↗
Tamoxifen and 5-fluorouracil in breast cancer: cytotoxic synergism in vitro.
The cytokinetic and cytotoxic interactions involved in combining tamoxifen, methotrexate, and 5-fluorouracil were studied in two hormone-dependent human breast cancer cell lines, 47-DN and MCF-7. These cells had measurable cytosol and nuclear estrogen receptor and cytosol progesterone receptor. Growth of the MCF-7 cells in medium containing gelding serum was stimulated maximally by addition of 10 pM estradiol. Both MCF-7 and 47-DN cells showed dose-dependent in vitro growth inhibition on exposure to tamoxifen, and toxicity from tamoxifen at concentrations up to 10 microM could be prevented by 1 nM estradiol. After exposure of 47-DN cells to 10 microM tamoxifen, cytosol progesterone and nuclear estrogen receptor levels were still detectable at 30 and 60% of control values. With this same concentration of tamoxifen, 47-DN cells in S phase declined 50% in association with a buildup of G0-1 cells. By clonogenic assay, tamoxifen enhanced 47-DN and MCF-7 cytotoxicity to 5-fluorouracil and 5-fluorouridine, but not to methotrexate alone. When given either concurrently or using a pretreatment-synchronizing schedule, tamoxifen enhanced markedly the growth inhibition of sequentially combined methotrexate and 5-fluorouracil. Isobologram analysis was used to prove that the cytotoxic interaction between tamoxifen and 5-fluorouracil was synergistic.
Breast Neoplasms↗
Tamoxifen and 5-fluorouracil in breast cancer: modulation of cellular RNA.
Biochemical studies were undertaken with the human breast carcinoma cell line, 47-DN, to explore the mechanisms underlying cytotoxic synergy between tamoxifen (TAM) and the fluoropyrimidines, 5-fluorouracil (FUra) and 5-fluorouridine (FUrd). The influence of TAM pretreatment was measured on intracellular FUra accumulation, FUra nucleotide formation, and incorporation of fluoropyrimidines into cellular RNA. Unlike other modulators of FUra metabolism and toxicity. TAM decreased intracellular FUra accumulation and total RNA incorporation by 20 to 60%. Cells treated with TAM contained 10 to 20% less cellular RNA and showed reduced transcription and altered RNA turnover, independent of fluoropyrimidine treatment. Newly synthesized RNA from control and TAM-treated cells was fractionated by sucrose gradient centrifugation. The specific incorporation of FUrd (2 hr) was compared to that of FUra (6 hr) and labeled uridine incorporation into controls. Compared to its effect on uridine incorporation, TAM produced nearly twice as much FUra incorporation and 3 times as much FUra incorporation into 32 to 45S RNA. Since accumulation of this high-molecular-weight RNA has been associated with fluoropyrimidine toxicity and impaired ribosomal RNA processing, it is believed that TAM enriched the RNA-mediated toxicity of FUra and FUrd in this breast carcinoma cell line.
Breast Neoplasms↗
Use of high-dose oral methotrexate sequenced at 24 hours with 5-FU: a clinical toxicity study.
Twenty-three patients with advanced carcinoma were treated with 131 courses of high-dose oral methotrexate (MTX), sequenced at 24 hours with 5-FU iv and subsequent leucovorin rescue. The 30% incidence of toxicity was predominantly mild to moderate mucositis and myelosuppression. Trough and peak serum MTX levels demonstrated that micromolar concentrations were sustained greater than 24 hours. Toxicity correlated with shorter re-treatment intervals and not with serum MTX levels. This regimen can be safely and conveniently administered to an outpatient population and deserves further assessment in phase II trials.
Breast Neoplasms↗
Optimal schedule of methotrexate and 5-fluorouracil in human breast cancer.
We have shown previously that methotrexate pretreatment of murine leukemia and human colon carcinoma cell cultures results in augmented intracellular accumulation of 5-fluorouracil metabolites. Both of these drugs are commonly used for the treatment of women with breast cancer; thus, sequencing of methotrexate before 5-fluorouracil was evaluated in vitro using a human mammary carcinoma cell line, 47-DN. Intracellular 5-fluorouracil accumulation was maximally increased 4-fold in cultures pretreated with 10 microM methotrexate for 24 hr. This enhancement of 5-fluorouracil metabolism was associated with increased intracellular levels of 5-phosphoribosyl 1-pyrophosphate, resulting from the antipurine effect of methotrexate. Brief exposure to exogenous hypoxanthine at physiological concentrations reversed the biochemical synergism between methotrexate and 5-fluorouracil. Other antimetabolites associated with elevations of 5-phosphoribosyl 1-pyrophosphate enhanced intracellular accumulation of 5-fluorouracil up to 2.5-fold. In cloning assays, 18 hr of methotrexate pretreatment followed by 5-fluorouracil resulted in optimal synergistic cytotoxicity, which could be prevented if high concentrations of leucovorin were given between methotrexate and 5-fluorouracil administration. Since these results indicated that optimal breast tumor toxicity in vitro was achieved by 18- to 24-hr sequencing of methotrexate and 5-fluorouracil, clinical toxicity study was carried out to assess whether this drug schedule could be tolerated. Seven patients with advanced cancer were treated with 21 courses of sequential therapy. No toxicity occurred with 38% of treatment courses; mild to moderate leukopenia and mucositis occurred with 29 and 38% of courses respectively. Toxicity was related to treatment interval and not cumulative drug dose or elevated serum methotrexate levels. These clinical results suggest that Phase II studies evaluating 24-hr-sequenced methotrexate and 5-fluorouracil in breast cancer are warranted.
Breast Neoplasms↗
The influence of methotrexate pretreatment on 5-fluorouracil metabolism in L1210 cells.
Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 microM methotrexate. The nucleotide derivatives of FUra, including a 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly 5-fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 microM and 10 microM. Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (N5-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de novo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-2'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of intracellular FUra accumulation. These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intracellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.
Animals↗
Biochemical alterations during unperturbed suspension growth of L1210 cells.
The following parameters were evaluated at several points throughout unperturbed suspension culture growth of L1210 cells: cell volume; DNA histograms; the mean content of cellular DNA, RNA, and protein; ribonucleoside and deoxyribonucleoside triphosphate pools; phosphoribosyl pyrophosphate; and the incorporation of glycine into purine bases. The cell volume, the incorporation of glycine into purine bases, and the intracellular pools of phosphoribosyl pyrophosphate and dexoyribunucleotides began to decrease significantly during the midportion of logarithmic cell growth. However, there was no significant change in the DNA content per cell during culture growth. The RNA, protein content, and ribonucleotides all demonstrated a biphasic pattern with the highest values obtained during the midportion of logarithmic growth followed by rapid decline as the culture approached plateau growth. These intracellular fluctuations in de novo synthesis and precursor pools were correlated with the variable intracellular accumulation of three fluoropyrimidines (5-fluorouracil, 5-fluorouridine, and 5-fluorodeoxyuridine) and their active metabolites (5-fluorouridine triphosphate and 5-fluorodeoxyuridylate). These studies were performed to demonstrate that multiple biochemical alterations occur during logarithmically growing suspension cell cultures and could result in misleading conclusions of experiments with antimetabolites unless these factors are considered in the context of the performed studies.
Animals↗
Modulation of 5-fluorouracil metabolism and cytotoxicity by antimetabolite pretreatment in human colorectal adenocarcinoma HCT-8.
The modulation of 5-fluorouracil (FUra) metabolism by methotrexate (MTX) pretreatment in monolayer cultures of human colorectal adenocarcinoma. HCT-8, was examined and correlated to clonal growth of this cell line. There was a gradual and nearly linear total intracellular accumulation and incorporation into RNA of FUra for 30 hr in control cells. A 12-hr 10 microM MTX pretreatment before adding 100 microM FUra resulted in approximately a 3-fold increase in total FUra accumulation, 59% of which was fluorouridine triphosphate. Soluble fluorodeoxyuridine monophosphate was increased 5-fold following MTX pretreatment; however, [3H]deoxyuridine incorporation into the acid-precipitable fraction of cells pretreated with MTX was no more than that observed when FUra was given alone. There was also an increase in 5-phosphoribosyl 1-pyrophosphate pools following MTX which was associated with the enhanced FUra metabolism. The maximum synergistic inhibition of clonal growth occurred when FUra was given during the last 6 hr of a 24-hr MTX exposure period. Other antimetabolites associated with elevations of 5-phosphoribosyl 1-pyrophosphate also resulted in an enhanced total intracellular accumulation of FUra.
Adenocarcinoma↗
Uridine and cytidine metabolism following inhibition of de novo pyrimidine synthesis by pyrazofurin.
Pyrazofurin, an inhibitor of orotidylate decarboxylase, imposes an absolute nutritional requirement for exogenous uridine to maintain normal growth of L5178Y, P388, L1210, W256 and S180 cells in vitro. The amount of uridine necessary for cell division when de novo uridine nucleotide synthesis is inhibited by pyrazofurin is: L5178Y, 30.5; P388, 39.7; L1210, 53.3: W256, 70.6; and S180, 886 fmol/cell. Cytidine, which can be deaminated to uridine, will substitute for uridine to maintain normal cell growth in the presence of growth-inhibitory concentrations of pyrazofurin (5 microM). The requirements for cytidine and uridine are identical. If cytidine deamination is prevented by tetrahydrouridine (100 microM), cytidine can no longer support growth in the presence of pyrazofurin. Cytidine and uridine, as expected, are additive in their effect to permit normal growth of pyrazofurin treated cells. Tetrahydrouridine does not alter this additive effect, indicating that when both nucleotides are added to pyrazofurin treated cells each nucleotide replenishes their respective nucleotide pools and cytidine deamination is unnecessary to allow cell growth. Incorporation of [14C]uridine into the acid insoluble cell fraction of L5178Y cells was 25 fmol/cell at 48 h and remained constant during the remaining growth of the pyrazofurin treated cell suspension. The [14C]uridine acid soluble pool of 4 fmol/cell also was maximum at 48 h but declined during the subsequent growth of the suspension culture to approx. 2 fmol/cell at 96 h. This decline in the acid soluble pool is correlated with a 42% decrease in modal cell volume during this phase of cell growth which would maintain a constant specific activity of uridine in this pool. This may explain the decline in the acid soluble pool while the acid insoluble pool remains constant during growth of suspension cultures of L51878Y cells. The block in pyrimidine synthesis de novo induced by pyrazofurin provides a useful and quick method for the evaluation of uridine and cytidine metabolism of tumor cell specimens.
Amides↗