Chun-Sik Kwak

Target site search and effective inhibition of leukaemic cell growth by a covalently closed multiple anti-sense oligonucleotide to c-myb.

Systematic secondary structure simulation of a target mRNA sequence is shown to be effective for locating a good anti-sense target site. Multiple selected anti-sense sequences were placed in a single molecule. The anti-sense oligonucleotide (oligo) was covalently closed to avoid exonuclease activities and was designated CMAS (covalently closed multiple anti-sense)-oligo. CMAS-oligo was found to be stable, largely preserving its structural integrity after 24 h of incubation in the presence of either exonuclease III or serum. When human c-myb mRNA was targeted by the c-myb CMAS-oligo, expression of the gene was completely abolished. Further, tumour cell growth was inhibited by 82+/-3% as determined by an MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and by 90+/-1% by [(3)H]thymidine incorporation. When a leukaemic cell line K562 was treated with CMAS-oligo, colony formation on soft agarose was also decreased by 93%. In contrast, treatment with a scrambled control oligo did not significantly inhibit leukaemic cell growth. These results suggest that a rational target site search is possible for an anti-sense oligo and that CMAS-oligo can be employed as an effective anti-sense agent with enhanced stability.

CYANATE AS A HEMOLYTIC FACTOR

During advanced renal failure, and particularly in patients with end-stage renal disease, proteins are carbamylated as a result of a reaction with cyanate. If the carbamylation of proteins adversely alters their biologic activities and structures, then urea must be viewed as an uremic toxin, rather than a surrogate. Therefore, we studied in this paper the role of cyanate as a hemolytic factor of erythrocytes to explain anemia observed in patients with high blood urea levels due to inadequate dialysis. Cyanate was added to make the final concentration 150, 300 and 600 nmol to each test tube containing the final concentration of 140×106 with human erythrocytes per mL of phosphate buffered saline solution. And they were incubated at 37°C for 24, 48 and 72 hours. The extent of hemolysis and carbamylation was monitored. The levels of hemolysis and carbamylated erythrocytes increase as the time of exposure to cyanate increased from 24 hours to 72 hours. Furthermore, those increased as cyanate concentration in the incubation media rose from 150 nmol to 600 nmol. Cyanate can induce hemolysis by carbamylation of erythrocytes. Urea, through cyanate, may contribute to hemolysis. If one extrapolates these results to patients with end-stage renal disease, it may help explain one of the reasons for the anemia in patients with high levels of BUN due to inadequate dialysis.