Retinoblastoma Protein and mRNA Levels Change Significantly in Human HL-60 Cells Prior to Aphidicolin-Induced Apoptosis

Abstract

The retinoblastoma tumor suppressor gene product, pRb protein, is multifunctional and plays an important role in determining the fate of normal and most cancer cells, whether they are undergoing continuous cell division, differentiation or apoptosis. In this study, the pRb protein level including its phosphorylation state and Rb mRNA level in human leukemic HL-60 cells was monitored during treatment with aphidicolin - a DNA synthesis inhibitor. Various approaches, including monitoring of cell viability, Western blots, flow cytometry and quantitative real-time RT-PCR were used. The present results indicate that the RB gene is not only a tumor suppressor gene that restricts progress through the cell cycle, but also is involved significantly in the induction of apoptosis by anti-cancer drugs. In response to aphidicolin, cells first decrease their active pRb level to counteract aphidicolin's DNA synthesis inhibition effect, but later increase their active pRb level to save themselves from apoptosis. In order to confirm this hypothesis, Rb antisense oligonucleotides were used to block Rb mRNA translation before aphidicolin treatment. The results show that cells that are pre-treated with Rb antisense are more sensitive to aphidicolin, although further selection of more effective Rb antisense oligos may be still needed. We conclude that the pRb protein may contribute significantly to chemoresistance of cancer cells and therefore removal of the active form of pRb during apoptosis induction may be a novel cancer treatment strategy.