Effect of recombinant human erythropoietin on anticancer drug-induced anaemia

Anaemia was induced in rats with fluorouracil (5-FU) or cisplatin (CDDP) and the mechanisms of anaemia induction were analysed. Furthermore, the therapeutic effects of recombinant human erythropoietin (rHu Epo) on these anticancer drug-induced anaemias were investigated. In 5-FU-induced anaemia, marked serum erythropoietin (Epo) elevation was observed in inverse correlation to blood Hb concentration and Hb concentration rapidly recovered to normal levels. On the other hand, in CDDP-induced anaemia, serum Epo elevation was modest and the lowered Hb concentration persisted longer. Treatment with rHu Epo significantly improved both anticancer drug-induced anaemias but rHu Epo was more effective on CDDP-induced anaemia. These results suggest that rHu Epo might be useful for the therapy of anaemia associated with anticancer chemotherapy.     

Antitumor activity of a new platinum complex (R)-(-)-2-aminomethylpyrrolidine (1,1-cyclobutane dicarboxylato) platinum (II) (DWA2114R) by its serial administration

Antitumor activity of a newly synthesized platinum complex, DWA2114R, by the serial administration was examined and compared with that of cis-diammine-1,1-cyclobutane dicarboxylato platinum (II) (CBDCA). In mice transplanted s.c. with tumor, the serial i.p. administration resulted in the increases of both maximal tolerated dose (MTD) and growth inhibitory ratio (GIR) of DWA2114R than single administration. Such increases in MTD and GIR were also shown by CBDCA, but the degree of these increases, such as the ratio of MTD or GIR by the serial administration compared to that at the single administration, was higher in DWA2114R than CBDCA. GIR of DWA2114R by the serial administration was higher than that of CBDCA at the doses to induce the same toxicity which was estimated by body weight loss. In addition, in the experiment using ascites tumor-bearing mice, better antitumor activity of DWA2114R was shown by the elongation of survival time. These results indicate that the cumulative toxicity of DWA2114R is lower than that of CBDCA, which causes the therapeutic advantages of DWA2114R in the serial administration.     

Simple, rapid, quantitative, and sensitive detection of telomere repeats in cell lysate by a hybridization protection assay.

BACKGROUND: Detection of telomere repeats by Southern hybridization of genomic DNA is time consuming, and the reading of a mean terminal restriction fragment (TRF) length from a smear pattern of an autoradiogram can be inaccurate. We developed a hybridization protection assay (HPA) for telomere repeats. METHODS: We heated 5 microL of DNA solution or 10 microL of cell or tissue lysate at 95 degrees C for 5 min, mixed it with 100 microL of hybridization solution containing 3 x 10(6) relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 degrees C. We then added 300 microL of selection buffer and incubated the mixture for 10 min at 60 degrees C to differentially hydrolyze unhybridized probe. Chemiluminescence was measured for 2 s per tube. RESULTS: The amount of telomere repeats was assayed by HPA within linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to 100 000 cell equivalents of lysate. To normalize the amount of DNA in lysate, the amount of Alu sequence was measured by HPA. A ratio of telomere to Alu (TA ratio) = 0.01 corresponded to approximately 2 kbp of mean TRF length determined by Southern blotting in cultured fibroblast and colorectal tissue samples. The TA ratio decreased from 0.06 to 0.02 with increasing division age from 30 to 90 population doubling levels of cultured human fetal fibroblasts. The assay required approximately 45 min from collection of cell or tissue samples. CONCLUSIONS: The amount of telomere repeats was quantitatively measured by HPA in 10 ng of sheared genomic DNA or in the lysate of 1000 cells. This method is simple, rapid, quantitative, sensitive, and applicable to the measurement of telomere repeats in clinical samples such as needle biopsy specimen or as few as 1000 cells in body fluid or washings.     

Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA.

Seven hundred fifty-eight processed sputum sediments received for the diagnosis of tuberculosis or other mycobacterial infections were tested by utilizing a rRNA target amplification assay and traditional culture techniques. The results from the rRNA target amplification assay (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test), available in 5 h, were compared with the results from standard culture techniques held for 6 weeks. A total of 119 specimens (16%) were culture positive for Mycobacterium tuberculosis. Overall sensitivity, specificity, positive predictive value, and negative predictive value were 82, 99, 97, and 96%, respectively, for the Gen-Probe assay; 88, 100, 100, and 97%, respectively, for culture; and 53, 99.8, 99.6, and 91%, respectively, for fluorochrome stain. The Gen-Probe assay employs the isothermal enzymatic amplification of M. tuberculosis complex rRNA followed by detection of the amplicon with an acridinium ester-labeled DNA probe. This assay has the potential of reducing the time for diagnosis of tuberculosis to 1 day.     

Antitumor Activity and Cellular Accumulation of a New Platinum Complex, (—)-(R)-2-Aminomethylpyrrolidine(1,l-cyclobutanedicarboxylato)platinum(II) Monohydrate, in Cisplatin-sensitive and -resistant Murine P388 Leukemia Cells

We have examined the cytotoxicity and accumulation of (—)-(U)-2-aminomethylpyrrolidine(1,l-cyclobutanedicarboxylato)platinum(II) monohydrate (DWA2114R) in parent and cisplatin-resistant mouse P388 leukemia cells (P388 and P388/DDP), in comparison with those of cisplatin (CDDP) and carboplatin (CBDCA). The degrees of resistance to CDDP and CBDCA, expressed as the ratio of IC₅₀for P388/DDP celts to IC₅₀ for P388 cells, were 75–33 and 100-27, respectively, under the conditions of 2–24 h exposure to each drug at a density of 10⁶ cells/ml. The corresponding values (25–7) for DWA2114R were relatively low. Accumulations of CDDP and CBDCA were reduced in P388/DDP cells; however, no reduction in accumulation of DWA2114R was observed at various exposure periods and concentrations of the drugs. The accumulations of CDDP in P388 and P388/DDP cells at drug concentrations corresponding to the IC₅₀ values for drug exposure periods of 2–24 h were 0.41–0.97 and 13.1–33.7 ng Pt/10⁷ cells, respectively, suggesting that an intracellular mechanism of resistance against CDDP could be activated in P388/DDP cells. P388/DDP cells also showed relatively low resistance to DWA2114R via this mechanism in comparison with CDDP and CBDCA. From the relationship between structure and activity of several Pt-complexes, these different properties of DWA2114R compared with CDDP and CBDCA could be due not only to the differences in carrier ligand structure but also to the properties of the whole molecule associated with the carrier ligand and leaving group.     

Antitumor activity and cellular accumulation of a new platinum complex, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato)platinum( II) monohydrate, in cisplatin-sensitive and -resistant murine P388 leukemia cells.

We have examined the cytotoxicity and accumulation of (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato++ +)platinum(II) monohydrate (DWA2114R) in parent and cisplatin-resistant mouse P388 leukemia cells (P388 and P388/DDP), in comparison with those of cisplatin (CDDP) and carboplatin (CBDCA). The degrees of resistance to CDDP and CBDCA, expressed as the ratio of IC50 for P388/DDP cells to IC50 for P388 cells, were 75-33 and 100-27, respectively, under the conditions of 2-24 h exposure to each drug at a density of 10(6) cells/ml. The corresponding values (25-7) for DWA2114R were relatively low. Accumulations of CDDP and CBDCA were reduced in P388/DDP cells; however, no reduction in accumulation of DWA2114R was observed at various exposure periods and concentrations of the drugs. The accumulations of CDDP in P388 and P388/DDP cells at drug concentrations corresponding to the IC50 values for drug exposure periods of 2-24 h were 0.41-0.97 and 13.1-33.7 ng Pt/10(7) cells, respectively, suggesting that an intracellular mechanism of resistance against CDDP could be activated in P388/DDP cells. P388/DDP cells also showed relatively low resistance to DWA2114R via this mechanism in comparison with CDDP and CBDCA. From the relationship between structure and activity of several Pt-complexes, these different properties of DWA2114R compared with CDDP and CBDCA could be due not only to the differences in carrier ligand structure but also to the properties of the whole molecule associated with the carrier ligand and leaving group.     

Adjuvant activity of purified peptidoglycan of Listeria monocytogenes in mice and guinea pigs.

The immunological properties of peptidoglycan (L-PG) purified from the cell wall skeleton (L-CWS) of Listeria monocytogenes strain EGD were investigated and compared with the properties of L-CWS. L-PG consisted of alanine, glutamic acid, alpha, epsilon-diaminopimelic acid, muramic acid, and glucosamine. L-PG showed potent adjuvant activities for circulating antibody formation and development of delayed-type hypersensitivity to bacterial alpha-amylase in vivo and for the primary immune response to sheep erythrocytes in vitro, as well as L-CWS. Both L-PG and L-CWS enhanced the generation of cell-mediated cytotoxicity in allogeneic mice and activated thioglycolate-elicited peritoneal macrophages and macrophage cell line RAW 264 to kill tumor target cells in vitro. We also found that L-PG acted on normal spleen cells as a mitogen. Both L-PG and L-CWS had tumor (Meth A)-suppressive and -regressive activities in syngeneic mice. Our results suggest that the L-PG moiety retains the adjuvant and antitumor activities of L-CWS.     

Adjuvant Activity of N-Acetyl Muramyl Dipeptides for the Induction of Delayed-Type Hypersensitivity to Azobenzenearsonate-N-Acetyl-l-Tyrosine in Guinea Pigs

The adjuvant activity of 23 kinds of synthetic N-acetyl muramyl dipeptide analogs on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine was examined in guinea pigs.     

Development of a hypersensitive detection method for human parvovirus B19 DNA

A new detection method for human parvovirus B19 DNA was established using PCR coupled with a hybridization protection assay. The amplified product was detected using acridinium ester-labeled DNA probes. By this method, a few copies of B19 DNA were detected in human serum albumin.