Reassessing Cell Cycle Specificity Of Anticancer Drugs

Object:Due to technical limitations, there was very few paper published directly exhibiting the drug-induced cell-cycle specific apoptosis by non-synchronous methods. But the cell cycle synchronization has been proved to disturb the cell growing status and might lead to pseudo results. In this part of present research, we will establish a model of exponentially growing molt-4 cells, to test the cell-cycle specificity of chemotherapeutic agent in vitro.Methods: The human acute lymphocyte leukemia cell line molt-4 was cultured to an exponentially growing status. Six commonly used cell-cycle specific agents (CCSA), including camptothecin (CPT), cytarabine (Ara-C), teniposide (VM-26), methotrexate (MTX), vincristine (VCR) and paclitaxel (Taxol), were added into the cell culture at different concentrations and incubated for 4 to 9 hours respectively. The drug-induced cell-cycle specific apoptosis was detected by API assay. For further verification of the API assay, post-sorting laser scan confocal microscopy (PSC) was used.Results: At very low concentration with very short period of incubating time, CCSAs only induced cell cycle blocking effect and very few cells went to apoptosis. At higher concentration with appropriate incubating period, typical cell-cycle specific apoptosis appeared. But at concentration even higher than that or with longer incubating time, cells commited to die in all phases of the cell cycle. 0.2μg/ml CPT, 100μg/ml Ara-C, 50μg/ml VM-26 and 40μg/ml MTX induced S phase specific apoptosis at 4-6 hours after the administration, 1μg/ml Taxol and 1μg/ml VCR induced G2/M phase specific apoptosis at 9 hours. Conclusions: The cell-cycle specific apoptosis induced by CCSAs could be typically detected by the API assay under certain conditions. The cell-cycle specificities of the 6 tested drugs in exponential model were consistent with those of common belief. Objects: Based on the result of the first part of this research, the cell-cycle specificities of chemotherapeutic agents were detected in models of different cell growing status. We wanted to know whether the specificity would be different or not under such condition.Methods: The model of"high-density cultured"molt-4 cells was established and was compared with the exponential model. In order to observe the in vivo effect of CCSAs, the clinical specimens of acute lymphocyte leukemia cells (the ex vivo model) were used and a leukemia patient (the in vivo model) was investigated during her first time chemotherapy.Results: When incubated with high-density cultured molt-4 cells, CPT, Ara-C, MTX, VM-26, VCR and Taxol all induced apoptosis mainly in G0/G1 phase with a small proportion in S phase. In the clinical specimens (ex vivo model), CPT, Ara-C, MTX, VM-26 and VCR again induced apoptosis mainly in G0/G1 phase with a small proportion in S phase, while Taxol only in S phase. During the first day of chemotherapy of the patient, Ara-C induced G0/G1 and a little S phase specific apoptosis. The result of Ara-C plus Taxol together in the second day was almost the same with the first day.Conclusions: The cell-cycle specificity of CCSA might change in different growing status of the target cells. The cell-cycle specific apoptosis induced in high-density cultured molt-4 was similar to that in clinical specimens, and was also consistent with that in vivo. Our observation further confirmed that high-density cultured cell line might be a better model to mimic the in vivo effect than the exponential model. It also suggested that the current classification of cell-cycle specific agent might not suitable for in vivo application and might need to be reassessed.