Abstract: Objective To explore whether osteosarcoma cells can escape from cell senescence and reproduce, and to evaluate the oncogenic ability of senescent-escaped cells. To explain the mechanism of osteosarcoma recurrence from a new perspective. Methods Doxorubicin (DOX) was used to develop aging model of U2OS and MG63. Cell senescence was detected by SA-Gal staining and senescence-related molecules were detected by Western blot. The cells were replaced with DOX-free medium and cultured for 75 days. Cell proliferation and escape rate were measured by cell counting. Then they were divided into three groups: parent cell,senescent cell,escaped-senescence cell. Agarose cloning assay was used to detect cloning formation and nude mice tumorigenicity test was used to detect tumorigenicity. Results In DOX-induced U2OS or MG63 cells, senescence specific marker SA-β-Gal positive cells emerged 2-day after stimulation and reached the plateau on day 4 with more than 90% SA-β-Gal-positive cells. Effects of DOX on the activities of other SIPS-related regulators in OS cells were also measured at protein level and the expression of senescent regulators p-p53, p-Rb, and p-γ-H2AX were all increased significantly. Senescent OS cells were cultured in medium without DOX. The numbers of cells kept unchanged until day 75 and then began to increase (one-millionth of the escape rate). The reproducing cells gained even higher tumorigenic capability. Conclusions The senescent osteosarcoma cells escape from senescence and gain even higher tumorigenic capability to form new colonies eventually in recurrent tumor.

Key words: osteosarcoma, senescence, recurrent tumor, doxorubicin, chemotherapy