Emerging Viruses-Aids & Ebola - By Leanard ... - preterhuman.net

peripheral blood lymphocytes to lymphoblast with a mitogenicagent. Fourth, the polymerase activities of tumour cells aregenerally higher than those of normal adult organs. A muchgreater content of various polymerases would be more likely tolead to a spurious interpretation of a unique polymerase in suchcell types. For this reason, we would expect a better controlledcomparison between normal and nooplastic cells of comparableDNA and RNA polymerase activities.The simple use of peripheral blood leucocytes, which consistprimarily of fully mature non-proliferating granulocylcs andlymphocylcs, cannot be considered as controls for leukaemicblast cells, particularly in view of the fact that these cells haveminimal or no detectable DNA dependent DNA polemeraseactivity. On the other hand, after 72 h of stimulation of normalhunan lymphocytes with phytohaemagglutinin (PHA), DNAsynthesis is maximal. In addition, Loeb 'et al' have reported a 30to 100-fold induction of DNA polymerase at this time, so thatactivities reach levels comparable with neoplastic cells, andHausen 'et al' have reported an induction of RNA polymerase inlymphocytes stimulated with PHA. We have confirmed boththese finding (unpublished results). Fifth, human cells obtaineddirectly from peripheral blood instead of human tissue culturecell lines were chosen for these initial investigations because theyobviously are a more true reflexion of the disease. Furthennore,there is much less chance of contamination with microorganismsor of developing mutations not relevant to leukaemogenesis.The leukaemic cells utilized in this study, therefore wereperipheral blood lymphoblasts obtained from three patients withacute lymphoblastic leukaemia (ALL). In each, the number oflymphoblasts was more than 100,000/mm of blood. Two patientswere untreated and the third received hydroxyurea for one day.Normal lymphocyctes were obtained from the peripheral bloodlymphocytes of forty-eight normal donors.Tho lymphocytes were separated from other blood cells, aspreviously described, except that an additional nylon columnchromatographic step was carried out to obtain more pure cellpopulations (more than 98 per cent lymphocytes). These cellswere incubated with the mitogenic agent and harvested after 72 has previously described. In our conditions, at 72 h the number ofcells transformed to lymphoblasts and the rate of DXA synthesisare maximum. After terminating the incubation, the cells wereextensively washed with 0.15 NaC1 and used for polymeraseassays.RNA dependent DNA Polymerase ActivityNueleic acid from preparations were made by gentle manualhomogenization (Ten.Broeck) of purified lymphoblast pellets in3 volumes of 25 mM Tris-sulphate buffer, pH 8.3; 1mM MgSo; 6mM NaCl; 4 mM dithiothrecitol; and 0.1 mM EDTA. Thesamples were centrifuged at 15,000 r.p.m., and the supernatants