| Popis: |
A method for rapid synchronization of neuroblastoma cells was developed using the thymidine block to arrest cells in the G1-S boundary. Following release from the thymidine block, cells traversed to G2-M in 7-8 h with 85% cell synchrony. Determination of the steady-state level of proliferating cell nuclear antigen (PCNA) mRNA and protein by Northern and Western blots revealed an accumulation of the PCNA messenger RNA transcripts and PCNA protein at G1-S and a rapid decrease when cells entered S phase. The level of both the messenger RNA transcripts and protein increased as the cells moved to late-S and G2-M. Similarly, the steady-state level of c-myc and N-myc messenger RNA transcripts and proteins increased during the G1-S block, decreased when the cells entered S, and increased as the cells moved through S phase to G2-M. However, immunofluorescence staining for PCNA and myc protein indicated a low level of staining for all three proteins at G1-S and a significant increase in staining intensity during S phase. Similarly, immunoelectron microscopy revealed low levels of N-myc and c-myc staining during G1-S and increased staining during mid-S and late S phase of the cell cycle. These results suggest differential cell cycle-dependent accessibility of myc protein and PCNA to staining in the intact cells compared to the whole cell extract. Furthermore, using immunofluorescence staining, confocal microscopy, and immunoelectron microscopy, we demonstrate for the first time that myc proteins are associated with the chromosomes during mitosis. |
