Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies

Abstract

Calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method to assess structural and numerical chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length chromosomes would decrease the detection sensitivity of numerical and structural chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ( 60 Coc rays; $0.6 Gy/min; 0-30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 AE 2.7 and 9.9 AE 1.6-fold increases in the frequency of G 2 /M-PCC cells with extended length chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G 2 /M-PCC scoring index per PCC in 15-and 30-min treated groups was increased by 1.9 AE 0.2 (P 5 0.001) and 1.8 AE 0.2 (P 5 0.001) compared with the 60-min treated group over 0-20 Gy, respectively. The G 2 /M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable chromosome morphology necessary to assess structural chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.

Lasonolide A, a potent and reversible inducer of chromosome condensation

“…[14][15][16] Calyculin A can induce PCC at 50 nM and achieve 10-20% PCC index after 30 min exposure in both suspension cells and adherent cells. [16][17][18] Okadaic acid is active at 100 nM selectively in unattached suspension cell types. 15,31 By contrast, here we show that LSA including PKC, MAP kinase (p38 and ERK1/2) and AKT, 6 which may account for the cytoplasmic and membranes effects of LSA (see Fig.…”

“…Top2 is essential for mitotic chromosome organization, condensation and segregation. [7][8][9]17,18,30,41 Top2α can be linked to Aurora B, as Top2α is one of the substrates of Aurora B, and Top2α-depletion reduces Aurora kinase B activity. 42,43 Unexpectedly, we found no evidence that MPF is required for LSA-induced PCC.…”

Distinct Responses to Reduplicated Chromosomes Require Distinct Mad2 Responses

“…For colcemid treatment to enrich for metaphase cells tissue was first incubated in colcemid (Sigma, St. Louis, MO) at 50μg/ml for 30min in PBS. For pre-mitotic chromosome spreads with Premature Chromosome Compaction, tissue was incubated in Calyculin A (Cell Signaling Technology, Danvers, MA) at 200nM in PBS for 30min,(Gotoh et al ., 1995; Miura & Blakely, 2011). FISH was performed as in Dej & Spradling, 1999.…”

Cytogenetic biodosimetry for accidental emergency irradiation exposure preparedness, in particular merit of the use of drug-induced premature chromosome condensation (PCC) with calyculin A

“…Drug-induced PCC is now recognized as new tool for cytogenetic biology (Gotoh and Durante, 2006). Drug-induced PCC has now been used in wide area of cytogenetic field (Srebniak et al, 2005;Deckbar et al, 2007;Gotoh, 2007;Kramer et al, 2008;Gotoh, 2009;Gotoh, 2011;Miura and Blakely, 2011). Drug-induced PCC protocol is very simple and described elsewhere (see for example Gotoh, 2009).…”