INDUCTION OF SENESCENT CELL-DERIVED INHIBITOR OF DNA-SYNTHESIS GENE, SDI1, IN HEPATOBLASTOMA (HEPG2) CELLS ARRESTED IN THE G(2)-PHASE OF THE CELL-CYCLE BY 9-NITROCAMPTOTHECIN

BACKGROUND: Recent studies have demonstrated that the plant-derived al kaloid camptothecin (CPT) and its derivative, 9-nitro-CPT (9NC), are c ytotoxic in tumorigenic cells but cytostatic in nontumorigenic cells i n vitro and in vivo. Also, CPT induces differentiation of human leukem ia cells in vitro along specific lineages. In this study, we have inve stigated the effects of 9NC on nontumorigenic HepG2 cells derived from human hepatoblastoma. A newly discovered senescent cell-derived inhib itor (SDI1) plays a critical role in the cell cycle, so we evaluated t he effect of 9NC on the expression of the SDI1 gene. EXPERIMENTAL, DES IGN: The effects of 9NC on HepG2 cells were evaluated by monitoring DN A synthesis, morphologic and ultrastructural changes of cells, and per turbation in the cell cycle and by assessing the levels of p53 protein and SDI1 mRNA. RESULTS: Treatment of HepG2 cells with 9NC results in a dose-dependent inhibition of cell proliferation and DNA synthesis. F low cytometric analysis of DNA content showed that SNC-treated HepG2 c ells are arrested in the G(2)-phase of the cell cycle. Light and elect ron microscopic examination revealed that 9NC at low concentrations in duces morphologic and growth features that resemble properties highly differentiated or senescent cells, i.e., increased cell size and decre ased nuclear/cytoplasmic ratio, as well as enlarged numbers of lysosom es, mitochondria, and Lipid in the cytoplasm. No significant alteratio n in the p53 protein level was noted in SNC-treated cells. In contrast to untreated, logarithmically grown HepG2 cells, SNC-treated cells ar rested at the G(2)-phase of the cell cycle and contained increased lev els of SDI1 mRNA. Kinetic studies revealed gradual increases in SDI1 m RNA synthesis. CONCLUSIONS: Induction of SDI1 mRNA by 9NC represents t he first documentation that the SDI1 gene can be overexpressed in the G(2)-phase of the cell cycle and provides a valuable cell culture syst em to dissect the events controlling the G(2) checkpoint. In addition, this finding corroborates the hypothesis that genes up-regulated in s enescent cells can also be induced in tumor-derived immortalized cells .