High-quality Si3N4 circuits as a platform for graphene-based nanophotonic devices.

Hybrid circuits combining traditional nanophotonic components with carbon-based materials are emerging as a promising platform for optoelectronic devices. We demonstrate such circuits by integrating single-layer graphene films with silicon nitride waveguides as a new architecture for broadband optical operation. Using high-quality microring resonators and Mach-Zehnder interferometers with extinction ratios beyond 40 dB we realize flexible circuits for phase-sensitive detection on chip. Hybrid graphene-photonic devices are fabricated via mechanical transfer and lithographic structuring, allowing for prolonged light-matter interactions. Our approach holds promise for studying optical processes in low-dimensional physical systems and for realizing electrically tunable photonic circuits.

Comprehensive microRNA expression profiling of the hematopoietic hierarchy.

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

Is the transnasal access for esophagogastroduodenoscopy in routine use equal to the transoral route? A prospective, randomized trial.

BACKGROUND AND STUDY AIMS: Routine esophagogastroduodenoscopy (EGD) is increasingly performed without sedation. Transoral (TO) and transnasal (TN) EGD offer different patient comfort and complications. PATIENTS AND METHODS: For a controlled, randomized, clinical trial comparing TN-EGD with TO-EGD without sedation, patients were assigned to TN-EGD using a thin endoscope (group 1, 93 patients), or TO-EGD using a standard endoscope (group 2, 90 patients). Physician-rated procedural time and complications as well as patient-rated side effects and preferences were compared. In group 3, patients (118) who had previously undergone TO-EGD, now underwent TN-EGD. RESULTS: Between group 1 and 2 there was no significant difference for procedural time. Nausea (p = 0.047) and epistaxis (p < 0.001) were significantly more frequent for TN-EGD. Conversion rate from TN- to TO-EGD was low with 4.3 %. For TN-EGD, patients' tolerance was better (p < 0.001), gagging was less (p < 0.001). In case of a future EGD, patients who know both procedures (group 3), strongly vote for TN-EGD (80 %). All groups vote against sedation for future procedures (90 %/90 %/89 %). CONCLUSIONS: Epistaxis can be relevant after TN-EGD, but can mostly be managed conservatively. TN-EGD is superior to TO-EGD regarding subjective and objective gagging as well as procedural tolerance. Patients who experienced both access routes, prefer TN-EGD. TN-EGD without sedation should be aspired for patient comfort and is recommended for routine use.

[What kind of school do children need?]. / Welche Schule brauchen Kinder?

At every age abilities mature and are internalized through corresponding experience. Every child wants to develop and learn, but at his own pace. In this process the child is not only active, but also selective, that is, it seeks experiences that correspond to its stage of development. The task of the school consists of making it possible for the child to gain learning experience that suits its stage of development in the respective areas of competence. Not only does the cognitive competence of children vary, but they also have differing needs as to emotional security and social experience. If it is possible to create an optimal balance in each of the three areas, learning experience, emotional security and socialization, between the needs and the individual development of the child and its environment, then the child can develop in the best possible way.

Morphological and mechanical characterisation of three-dimensional gyroid structures fabricated by electron beam melting for the use as a porous biomaterial.

Additive manufactured porous biomaterials based on triply periodic minimal surfaces (TPMS) are a highly discussed topic in the literature. With their unique properties in terms of open porosity, large surface area and surface curvature, they are considered to have bone mimicking properties and remarkable osteogenic potential. In this study, scaffolds of gyroid unit cells of different sizes consisting of a Ti6Al4V alloy were manufactured additively by electron beam melting (EBM). The scaffolds were analysed by micro-computed tomography (micro-CT) to determine their morphological characteristics and, subsequently, subjected to mechanical tests to investigate their quasi-static compressive properties and fatigue resistance. All scaffolds showed an average open porosity of 71-81%, with an average pore size of 0.64-1.41 mm, depending on the investigated design. The design with the smallest unit cell shows the highest quasi-elastic gradient (QEG) as well as the highest compressive offset stress and compression strength. Furthermore, the fatigue resistance of all unit cell size (UCS) variations showed promising results. In detail, the smallest unit cells achieved fatigue strength at 106 cycles at 45% of their compressive offset stress, which is comparatively good for additively manufactured porous biomaterials. In summary, it is demonstrated that the mechanical properties can be significantly modified by varying the unit cell size, thus enabling the scaffolds to be specifically tailored to avoid stress shielding and ensure implant safety. Together with the morphological properties of the gyroid unit cells, the fabricated scaffolds represent a promising approach for use as a bone substitute material.

Alterations in the mechanical, chemical and biocompatibility properties of low-cost polyethylene and polyester meshes after steam sterilization.

INTRODUCTION: In Africa and other Low Resource Settings (LRS), the guideline-based and thus in most cases mesh-based treatment of inguinal hernias is only feasible to a very limited extent. This has led to an increased use of low cost meshes (LCMs, mostly mosquito meshes) for patients in LRS. Most of the LCMs used are made of polyethylene or polyester, which must be sterilized before use. The aim of our investigations was to determine changes in the biocompatibility of fibroblasts as well as mechanical and chemical properties of LCMs after steam sterilization. MATERIAL AND METHODS: Two large-pored LCMs made of polyester and polyethylene in a size of 11 x 6 cm were cut and steam sterilized at 100, 121 and 134 °C. These probes and non-sterile meshes were then subjected to mechanical tensile tests in vertical and horizontal tension, chemical analyses and biocompatibility tests with human fibroblasts. All meshes were examined by stereomicroscopy, scanning electron microscopy (SEM), LDH (cytotoxicity) measurement, viability testing, pH, lactate and glycolysis determination. RESULTS: Even macroscopically, polyethylene LCMs showed massive shrinkage after steam sterilization, especially at 121 and 134 °C. While polyester meshes showed no significant changes after sterilization with regard to deformation and damage as well as tensile force and stiffness, only the unsterile polyethylene mesh and the mesh sterilized at 100 °C could be tested mechanically due to the shrinkage of the other specimen. For these meshes the tensile forces were about four times higher than for polyester LCMs. Chemical analysis showed that the typical melting point of polyester LCMs was between 254 and 269 °C. Contrary to the specifications, the polyethylene LCM did not consist of low-density polyethylene, but rather high-density polyethylene and therefore had a melting point of 137 °C, so that the marked shrinkage described above occurred. Stereomicroscopy confirmed the shrinkage of polyethylene LCMs already after sterilization at 100 °C in contrast to polyester LCMs. Surprisingly, cytotoxicity (LDH measurement) was lowest for both non-sterile LCMs, while polyethylene LCMs sterilized at 100 and 121 °C in particular showed a significant increase in cytotoxicity 48 hours after incubation with fibroblasts. Glucose metabolism showed no significant changes between sterile and non-sterile polyethylene and polyester LCMs. CONCLUSION: The process of steam sterilization significantly alters mechanical and structural properties of synthetic hernia mesh implants. Our findings do not support a use of low-cost meshes because of their unpredictable properties after steam sterilization.

Schedule-dependent cytotoxicity of methotrexate and 5-fluorouracil in human colon and breast tumor cell lines.

The effect of sequential methotrexate and 5-fluorouracil on the clonal growth of the human colon adenocarcinoma cell, HCT-8, and the hormone-dependent human breast carcinoma cell, 47-DN, was examined. In both cell lines, when 5-fluorouracil was given during the last 6 h of a 24 h methotrexate exposure period, there was marked synergistic inhibition of clonal growth. Shorter intervals or the reverse sequence of drugs were either additive or antagonistic. These results indicate the importance of the drug sequence and time interval between drug administration for optimal cytotoxicity in these human cell lines. This information suggests that the administration of methotrexate 18 h before 5-fluorouracil may have potential application in the design of clinical trials for these malignancies.

Lactic dehydrogenase isozymes, 31P magnetic resonance spectroscopy, and in vitro antimitochondrial tumor toxicity with gossypol and rhodamine-123.

Three compounds that share specific antimitochondrial properties are gossypol, rhodamine-123, and lonidamine. We compare the antiproliferative activities of these drugs against six human cell lines derived from breast (T47-D), pancreas (MiaPaCa, RWP-2), prostate (DU-145), colon (HCT-8), and cervix (HeLa) carcinomas. Tumor cells enriched in cathodal LDH isozymes (LDH4 and LDH5) are significantly more sensitive to gossypol and rhodamine-123. When compared for ability to inhibit growth of human marrow in soft agar, 10 microM gossypol shows little effect on colony formation whereas 10 microM rhodamine-123 completely prevents stem cell growth, suggesting that gossypol may have the most favorable therapeutic index. Within 24 h of drug administration, there is a relative increase in intracellular inorganic phosphate pools and a marked decline in soluble high-energy phosphates in sensitive tumor cells, as measured by 31P magnetic resonance spectroscopy. These studies suggest that specific antimitochondrial agents might be selectively administered on the basis of tumor LDH isozyme content and noninvasively monitored for antiproliferative activity by 31P spectroscopy.

Truncated forms of DNA-binding estrogen receptors in human breast cancer.

The likelihood a breast cancer will respond to antiestrogen therapy depends on the tumor content of immunoreactive or ligand-binding estrogen receptor (ER). To investigate the failure of many ER-positive breast cancers to respond to antiestrogen therapy, we examined by gel-shift assay the ability of tumor ER to bind its cognate estrogen response element (ERE). Analysis of 38 primary breast cancers showed that some tumors containing abundant immunoreactive ER failed to demonstrate DNA binding ER. In many other ER-positive tumors, the fraction of DNA binding ER was low and consisted primarily of truncated receptor forms, which on Western analysis were revealed to be 50 kD homodimers and 67-50 kD ER heterodimers. The use of protease inhibitors during tumor extraction and the demonstration of nuclear-localizing ER and ERE-binding COUP (chicken ovalbumin upstream promoter) protein in these tumors indicated that the truncated forms of ER were likely present in vivo. The presence of intact DNA binding ER correlated with higher tumor content of immunoreactive sex steroid receptors (ER and/or PR), standard predictors of tumor responsiveness to antiestrogen, suggesting that loss or truncation of DNA binding ER may be an important prognostic parameter accounting for some forms of clinical resistance to antiestrogen therapy.

A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes.

In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cyclin-dependent kinase inhibitor protein p21 (Cip-1/WAF1/mda6) (p21). This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. Prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in DNA synthesis. MAPK signaling increased the phosphorylation of the transcription factors Ets2, C/EBPalpha, and C/EBPbeta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Eight hours after MAPK activation, loss of C/EBPbeta or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21 protein expression. At this time, MAPK signaling increased both p21 mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either C/EBPbeta or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21 protein expression. In contrast, loss of C/EBPalpha function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase protein levels of p21. This data suggested that prolonged enhancement of p21 protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple transcription factors, which alone were insufficient for a robust prolonged increase in p21 protein levels in primary hepatocytes, and that to increase p21 protein levels also required enhanced stabilization of p21 mRNA and p21 protein. Collectively, these data suggest that loss of transcription factor and mRNA/protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.

Efficacy of intraoral topical anesthetics in children.

OBJECTIVES: The literature reports mixed results regarding the efficacy of intraoral topical anesthetics. Only a few studies have been performed in children. Some non-placebo controlled studies have been carried out to compare the efficacy in reducing children's injection pain between different topical anesthetics METHODS: In a randomized, double blind, placebo controlled study with split-mouth design 104 children were enrolled to evaluate the efficacy of four topical anesthetics (Gingicain Spray, Gingicaine Topical Anesthetic, Legecain-Solution, EMLA Crème) when used prior to buccal injections within the conservative treatment of carious upper primary molars. The heart rate change and a Face Pain Scale were used as primary variables. The Visual Analog Scale, the modified Children's Hospital Pain Scale and the Sound-Eyes-Motor Scale were also evaluated. RESULTS: There was no significant difference between the placebo and any corresponding topical anesthetic with regard to the primary variables (HRC and FPS). A significant difference was found in favour of Gingicain Spray and Gingicaine Topical Anesthetic according to secondary variables (VAS, S(E)MS). CONCLUSION: While the secondary variables point to a benefit of the topical anesthetics Gingicain Spray and Gingicaine Topical Anesthetic compared to placebo, the results of the primary variables showed no differences in effectiveness of topical anesthetics and their corresponding placebos.

Expression of c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes in normal and malignant human breast epithelial cells.

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Stress response protein (srp-27) determination in primary human breast carcinomas: clinical, histologic, and prognostic correlations.

Expression of an estrogen-regulated protein known as the 27,000-d heat-shock or stress-response protein (srp-27) was evaluated in human breast carcinomas and established breast cancer cell lines. Results obtained by Northern and Western blot analyses and immunohistochemical methods were concordant. Immunohistochemical assessment of srp-27 expression in 300 breast carcinomas (with median patient follow-up of 8 years) was performed. Twenty-six percent of lymph node-negative and 45% of lymph node-positive tumors were overexpressors. Univariate analysis demonstrated significant correlations between srp-27 overexpression and estrogen receptor (ER) content, pS2 protein expression, nodal metastases, advanced T stage, lymphatic/vascular invasion, and a shorter disease-free survival period (but not a shorter overall survival) for the study population as a whole. Regression tree analysis showed that srp-27 expression was an independent prognostic indicator for disease-free survival only in patients with one to three positive lymph nodes. The Cox proportional hazards model confirmed the independent prognostic significance of nodal involvement, T stage, and ER content but failed to recognize srp-27 overexpression as a significant independent parameter predictive of patient outcome in the patient population as a whole. The observed associations between srp-27 overexpression and more aggressive tumors suggest a biologic role for srp-27 in human breast carcinomas.

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.

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.

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)

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.

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.

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.

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.