The Effects Of Aclarubicin On The Formation Of Hela Cell Chromosome And Apoptosis

Objective : To investigate the effects of aclarubicin on the fonnation of Hela cell chromosome and its relationship with apoptosis., as for a further study on die mechanism of topoisomerase II inhibitors.Method : Hela cells were cultured in die environment of 37 , 5% CO2for several generations, then digested and transferred to a 48-well cultural plate and incubated over night. MTT assay was used to detect die half-inhibition concentration (IC50,) of aclarubicin on Hela cells. Then, the cells were treated with aclarubicin in different concentrations (0.1 u g/ml and 10 u g/ml) for 24 hours. The cells were harvested and used for die analysis of die following items: (1) chromosome analysis: four hours before harvesting the cell, appropriate concentration of colclncine was added to the medium, cliromosomes were made according to die regular mediod and stained by Giemsa and observed by microscope. (2) DNA electrophoresis: the cell DNA were extracted, DNA fragments were separated on die 1.5% agarose gel, stained by Ediidium Bromide and observed under UV lamp. (3): western blot: Cell protein was extracted and separated by SDS-PAGE. The protein bands were transferred to a NC membrane. A monoclone antibody was used to detect the expression of capase-3 in the cells. All the items detectedabove were contrasted with the control group Which was not treated by the drug.Results : IC^ was identified as 6.83 u g/ml. After 24 hours of incubation with acalarubicin, chromosome analysis showed that cell mitosis was active in die control group and formed normal chromosomes, the nuclei had the intermediate phase characteristics. There \vere not normal chromosome formation both in the 0.1 u g/ml group and 10 u g/ml group. Contrasted with the control group, there were mainly two types of nucleus in the test groups: Type one, the nucleus became larger and stained unevenly but its membrane was intact, chromatin condensed in the central part of the nucleus, and some of them had chromosome shape. Type two, the chromosomes were thin and nuclear membrane had broken in this cell.. Taking together, the nuclei in the drug group had the characteristics of cell cycle G2/M phase, normal fonnation of cliromosome was blocked and chromatin condensed disorderly contrasted with that of control group. Along with the increased drug concentration, both the nucleus of type one and type two became increased, but the chromatin content of type one cell reduced apparently. DNA electrophoresis indicated that a large number of DNA fragments appeared in the 10 u g/ml aclarubicin group while they were not apparent in die 0.1 u g/ml group contrasted widi the control sample. Wester-blot showed tiiat Caspase-3 was not detected in die control one, a slight increase in the 0.1 u g/ml group while a striking increase in diel 0 u g/ml groupConclusion : Aclarubicin might restrain the normal fonnationand separation of chromosome of Hela cell and block them at the cell cycle G2/M phase, then induces apoptosis. Caspase-3 may plays an important role in aclarubicin induced apoptosis...