Induction of senescent cell-derived inhibitor of DNA synthesis gene, SDI1, in hepatoblastoma (HepG2) cells arrested in the G2-phase of the cell cycle by 9-nitrocamptothecin.
BACKGROUND
Recent studies have demonstrated that the plant-derived alkaloid camptothecin (CPT) and its derivative, 9-nitro-CPT (9NC), are cytotoxic in tumorigenic cells but cytostatic in nontumorigenic cells in vitro and in vivo. Also, CPT induces differentiation of human leukemia cells in vitro along specific lineages. In this study, we have investigated the effects of 9NC on nontumorigenic HepG2 cells derived from human hepatoblastoma. A newly discovered senescent cell-derived inhibitor (SDI1) plays a critical role in the cell cycle, so we evaluated the effect of 9NC on the expression of the SDI1 gene.


METHODS
The effects of 9NC on HepG2 cells were evaluated by monitoring DNA synthesis, morphologic and ultrastructural changes of cells, and perturbation 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. Flow cytometric analysis of DNA content showed that 9NC-treated HepG2 cells are arrested in the G2-phase of the cell cycle. Light and electron microscopic examination revealed that 9NC at low concentrations induces morphologic and growth features that resemble properties highly differentiated or senescent cells, i.e., increased cell size and decreased nuclear/cytoplasmic ratio, as well as enlarged numbers of lysosomes, mitochondria, and lipid in the cytoplasm. No significant alteration in the p53 protein level was noted in 9NC-treated cells. In contrast to untreated, logarithmically grown HepG2 cells, 9NC-treated cells arrested at the G2-phase of the cell cycle and contained increased levels of SDI1 mRNA. Kinetic studies revealed gradual increases in SDI1 mRNA synthesis.


CONCLUSIONS
Induction of SDI1 mRNA by 9NC represents the first documentation that the SDI1 gene can be overexpressed in the G2-phase of the cell cycle and provides a valuable cell culture system to dissect the events controlling the G2 checkpoint. In addition, this finding corroborates the hypothesis that genes up-regulated in senescent cells can also be induced in tumor-derived immortalized cells.
Khaoustov
V I
VI
Department of Medicine, Veterans Affairs Medical Center, Houston, Texas, USA.
Ozer
A
A
Smith
J R
JR
Noda
A
A
Mearns
M
M
Krishnan
B
B
Slagle
B L
BL
Yoffe
B
B
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis*; D016195:G2 Phase; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D004247:DNA; C079905:rubitecan; D002166:Camptothecin