| Background and aims: The tumor suppressor gene p53 plays a critical role in apoptosis pathway known as membrane apoptosis, mitochondrion apoptosis, and is a regulator of many molecules related to apoptosis in the nuclear. This genomic safeguard exerts protection as the cell encounters emergency stress signals like UV-radiation, ionizing radiation, cytotoxic agents, environment injury. ASPP(Apoptosis Stimulating Protein of p53 or Ankyrin repeat , SH3 domain , Proline-rich domain containing Proteins) family, a novel protein family comprised of three members named as ASPP1, ASPP2 and iASPP, can specifically regulate apoptosis mediated by p53. ASPP1 and ASPP2 stimulate the proapoptotic function of p53, p63 and p73, iASPP inhibits p53 function in cell cycle and apoptosis. In this studyβ-elemene, pirarubicin and cisplatin were selected to treat myeloid leukemia cell line K562 cell which contains homozygously deleted p53, respectively. Alternations in morphology, cell cycle and mRNA expression of ASPP1 and ASPP2 are recorded. These works aimed at observing the molecular changes of ASPP family in chemotherapeutic reaction in p53 null type cell for further developing the mechanism in chemotherapeutic responsibility.Methods: K562 was exposed toβ-elemene, pirarubicin or cisplatin respectively. The proliferation inhibition rate of K562 cells was measured with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Morphologic alternations were observed by electron microscope. Cell cycle and apoptosis rate were detected by flow cytometry (FCM). RT-PCR is adopted to manifest the expression level of ASPP1 and ASPP2 mRNA after the treatment.Results:1.β-elemene, pirarubicin and cisplatin had an inhibitory effect on the proliferation of K562 cells in a dose-dependent manner. Apoptotic and necrotic cells are distinguished by TEM (transmission electron microscopy).The characteristic chromatin massive rearrangement and margination and cytosol condensation appears in pirarubicin and cisplatin treated cells. Cup-shaped masses are formed in cytoplasm. The good preservation of membrane and organelles can be seen. No blebbing phenomenon also known as apoptotic bodies has been observed.Afterβ-elemene was administrated for 24 hours, the cell membrane loses its integrity, organelles disrupt, and karyorrhexis appears, suggesting cells undergo necrosis.2. FCM assays showed thatβ-elemene block cell cycle at S phase and the apoptosis rates are 5.66%,4.58% at different concentration of 50μg/ml,25μg/ml. Pirarubicin ( 1.25μmol/l , 2.5μmol/l ) induced apoptosis and arrested cell cycle at S phase with apoptotic rate at 11.64%,12.11%,respectively. CDDP(1.5μg/ml,3.0μg/ml)induced apoptosis and arrested cell cycle at G1 stage, and apoptotic rate are 8.83%,11.45%,respectively.3. ASPP1, ASPP2 expression shows no significant difference between control group and agents group by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). ASPP1 mRNA displays a different electrophoresis strip at 1159bp inβ-elemene group in agarose gel electrophoresis, while ASPP1 mRNA shows 580bp in other groups. The ASPP2 mRNA base pair is 389 bp in all groups.Conclusion:1 Pirarubicin and cisplatin can induce p53-independent apoptosis, while obvious necrosis was seen in K562 cells treated withβ-elemene.2 Cisplatin arrests cell cycle in G1 phase, while pirarubicin andβ-elemene blocks cell cycle in S phase.3 No significant difference was detected between different agents groups in p53 null type cell K562.This indicates that the expression of ASPP family may be regulated by p53 at its transcription levels.
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