Takemura Y

Variable expression of the folylpolyglutamate synthetase gene at the level of mRNA transcription in human leukemia cell lines sensitive, or made resistant, to various antifolate drugs.

We investigated the expression of the folylpolyglutamate synthetase (FPGS) gene at the mRNA level in MOLT-3 and K562 human leukemia cell lines sensitive, or made resistant, to methotrexate (MTX) and/or trimetrexate (TMQ), or raltitrexed (ZD1694). Northern blot analysis demonstrated approximately 3-fold higher FPGS mRNA expression in K562 cells than that in MOLT-3 cells, being consistent with graded polyglutamation capacities of these cell lines. A slight increase in the expression of the FPGS gene was observed in the TMQ-resistant MOLT-3 cells (MOLT-3/TMQ800); moreover, sequential development of MTX resistance in the TMQ-resistant cells (MOLT-3/TMQ800-MTX10,000) resulted in a further enhancement of FPGS mRNA expression despite of decreased polyglutamation capacity in this subline. Another MTX-resistant subline with impaired reduced folate carrier (MOLT-3/MTX10,000) also showed overexpression of FPGS mRNA. Conversely, both raltitrexed-resistant sublines (MOLT-3/ZD1694 x C and K562/ZD1694 x C) displayed a moderately decreased expression of FPGS mRNA. These findings did not correspond to the virtual absence of ZD1694 polyglutamates inside the former cells nor to possibly intact polyglutamation capacity in the latter cells. These results indicate that FPGS mRNA expression may predict cellular ability to produce polyglutamate metabolites of antifolate drugs in the sensitive cells, but does not necessarily reflect FPGS function at the enzyme level in the antifolate-resistant tumor cells.

Plasma cell leukemia producing monoclonal immunoglobulin E

A 78-year-old male with lumbar pain and dim consciousness presented the clinical pictures of plasma cell leukemia (PCL) producing a large amount of monoclonal immunoglobulin E (IgE)/kappa protein. Laboratory investigation demonstrated an elevated serum calcium level and renal dysfunction. Systemic bone X-ray survey disclosed only a solitary osteolytic lesion. Circulating plasma cells demonstrated CD19−/CD56− and MPC-1−/CD49e−/CD45+/−, the latter indicating the immature phenotype of the tumor cells. Bone marrow was occupied with immature, atypical plasma cells, of which cytoplasms were positive for IgE by direct immunofluorescence analysis. Chromosomes revealed a translocation of (11;14)(q13;q32), which is concordant with cyclinD1-protein overexpression by immunohistochemistry. He was treated with dexamethasone and vincristine, which somewhat improved the laboratory findings. He died of tumor progression after 4-month admission. The clinical and biological characteristics of IgE-producing PCL, a very rare type of plasma cell dyscrasia, are discussed, reviewing the past literature.

Utilization of common inflammatory markers in new, symptomatic, primary care outpatients based on their cost-effectiveness

Abstract Few studies have demonstrated the optimal usage of common inflammatory markers, alone or in combination, based on the cost-effectiveness. We analyzed the yield and cost of C-reactive protein (CRP), white blood cell count (WBC), erythrocyte sedimentation rate (ESR), sialic acid, and protein fractionation in 177 new primary care outpatients with inflammation-related symptoms. A useful result (UR) was assigned if tests contributed to a change in physicians diagnosis or decision-making. Costs of testing were calculated based on either single or simultaneous measurement. Five inflammatory markers generated 147 URs in 123 patients. CRP showed the best contribution to generation of UR, followed by sialic acid, protein fractionation, WBC, and ESR. CRP demonstrated poor correlation with WBC (r = 0.458), while sialic acid strongly correlated with total absolute amount of α1 and α2 fractions in protein fractionation (r = 0.855) and moderately with ESR (r = 0.651). The combination of CRP and WBC produced the best cost-effectiveness at a cost of ￥ 1169 (US

Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin

9.6 or Euro 9.7)/additional UR against CRP testing alone. Sialic acid, an automated multichannel analyzer-based test, demonstrated the favorable cost-effectiveness over ESR or protein fractionation when combined with CRP (and WBC). Our results indicate that the optimal usage of these inflammatory markers should involve careful cost-effectiveness considerations.

Expression level of MDR1 message in peripheral blood leukocytes from healthy adults: a competitive nucleic acid sequence-based amplification assay for its determination

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high‐dose MTX regimens combined with leucovorin (LV) rescue, we established MTX‐resistant human T‐cell leukemia cell line CCRF‐CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 μM in the presence of 1000 nM LV resulted in establishment of three MTX‐resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40‐, 900‐ and 44 000‐fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC→TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF‐CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport‐mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high‐dose MTX regimens with LV rescue. (Cancer Sci 2003; 94: 210–214)

C-Reactive Protein Kinetics in Newborns: Application of a High-Sensitivity Analytic Method in Its Determination

Abstract Accurate quantification of multidrug resistance-1 gene (MDR1) expression in target cells would be of important therapeutic value in predicting cellular response to anticancer drugs. Because certain normal cells in peripheral blood physiologically express MDR1, increasing the sensitivity of the detection methods might result in confounding low-degree expression in tumor cells with physiologic expression in normal cells. The purpose of this study was to determine MDR1 mRNA expression levels in peripheral blood leukocytes obtained from healthy adult volunteers using a competitive nucleic acid sequence-based amplification (NASBA) assay. We determined the reference intervals of MDR1 mRNA expression in peripheral blood obtained from 98 healthy adults by measuring its expression with the quantitative NASBA assay between 5.50 × 104 copies/µg RNA and 6.76 × 105 copies/µg RNA. The new reference intervals were evaluated using a number of sensitive or resistant cell lines as control; positive or negative MDR1 expression was clearly demonstrated. We also reevaluated MDR1 expression levels in leukemia cells obtained from patient peripheral blood; 18 of 31 samples (58%) exceeded the newly established upper reference limit. The cutoff value established could be used to distinguish significant MDR1 expression in tumor cells from physiologic expression in certain normal cells coexistent in peripheral blood.