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Variation in sister chromatid exchange frequencies and cell-cycle kinetics between human whole blood and plasma leukocyte cultures

Marcelo L. Larramendy^I,II; Miguel A. Reigosa^I,III; Carlos Garcia^I; Sonia Soloneski^I; Sakari Knuutila^II

^ILaboratorio de Citogenética y Cátedra de Citología, Facultad de Ciencias Naturales, Universidad Nacional de La Plata, La Plata, Argentina. Send correspondence to M.L.L.
^IIDepartment of Medical Genetics, University of Helsinki, Helsinki, Finland
^IIIInstituto Multidisciplinario de Biología Celular (IMBICE), La Plata, Argentina

ABSTRACT

The effects of bromodeoxyuridine (BUdR) concentration and the length of the synthesis (S) period on the baseline frequency of sister chromatid exchanges (SCEs) and the proliferation rate of human whole blood (WBC) and plasma leukocyte cultures (PLC) were studied. An immunofluorescent method was used to induce sister chromatid differentiation. This method employs a low concentration of BUdR for chromosome labelling during the first cell cycle in vitro (3.3 mM), and fluorescein isothiocyanate (FITC)-conjugated anti-BUdR monoclonal antibody for detection. WBC and PLC were allowed to grow during the first cell cycle (48 h) in the presence of BUdR, and the culture medium was then replaced with BUdR-free culture medium, and cells were cultured for an additional cell cycle (24 h) until harvesting (72 h). Chromosomes were counterstained with propidium iodide and DAPI. PLC showed at least a two-fold increase in SCE frequency over WBC values and cell proliferation was slower in PLC than in parallel WBC, independent of the concentration of BUdR of the culture medium, at least in the range of the base analogue employed (3.3 mM-33.0 mM). Moreover, an estimation of the length of the S period, made by analyzing the proportion of labelled cells that were synthesizing DNA during different harvesting times, revealed that PLC have a markedly lengthened S period compared to WBC. Accordingly, the rate of DNA-fork displacement of lymphocytes in PLC could be slower than in WBC, resulting in an increase in SCE frequency of these cultures.

Keywords: sister chromatid; cell-cycle kinetics; human whole blood; plasma leukocyte cultures.

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