Ogden A, Rida Personal computer, Knudsen BS, Kucuk O, Aneja R. the observed acceleration of tumor growth was attributable to a protumorigenic environment produced from the co-injected senescent and proliferating malignancy cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was efficiently inhibited by cell immunotherapy using irradiated NOX1 TC-1 cells designed to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for DBeq elimination of the detrimental effects caused by bystander senescent tumor cells such accelerating impact on xenograft tumor cell growth in nude mice was not observed  indicating effect of some still unidentified factors on this trend. Interleukin 12 (IL-12), a cytokine linking innate and adaptive immunity, represents one of the important players in induction of anti-tumor immune response . Produced primarily by antigen showing cells, such as dendritic cells, macrophages, monocytes or B cells upon their activation, IL-12 exerts its effects primarily through induction of IFN, as DBeq well as NK and T cell activation [16, 17]. Antitumor immunotherapy with IL-12 given in different forms, including the usage of irradiated tumor cells generating IL-12, has been analyzed [15, 18, 19]. In several experimental tumor models, including those used in our laboratory, anti-tumor immunogenicity could be enhanced by administration of IL-12 or by gene therapy with tumor cells designed to produce IL-12 (for evaluations, observe [20C22]). This intriguing accumulating data influenced our present operating hypothesis, namely that IL-12-centered immunotherapy might be able to mitigate or entirely eliminate the pro-tumorigenic effects of bystander senescent cells. Indeed, here we document an acceleration of tumor growth, when proliferating TC-1 tumor cells were co-administered into syngeneic mice together with DBeq syngeneic tumor cells that had been subjected to senescence-inducing treatment with docetaxel (DTX). Furthermore, we also document effective treatment of such tumors by cell therapy using irradiated IL-12-generating tumor cells. RESULTS DTX induces senescence in mouse tumor cells TC-1 and TRAMP-C2 First, we evaluated the effect of DTX in terms of senescence induction, using two C57Bl/6 mice-derived tumor cell lines TC-1 and TRAMP-C2 of lung and prostate epithelial source, respectively. Both TC-1 and TRAMP-C2 cells were susceptible to DTX and underwent senescence after a four-day incubation with 7.5 M DTX . After this treatment, the vast majority of TC-1 and TRAMP-C2 cells were alive but senescent, as characterized by the lack of cell proliferation, improved senescence-associated–galactosidase activity, characteristic cell morphology and improved manifestation of p16INK4a and p21waf1 inhibitors of cyclin-dependent kinases. Most of the senescent cells showed persistent DNA damage response, as judged from the presence of DNA damage foci positive for serine 139-phosphorylated histone H2AX (H2AX; Number ?Number1,1, Number ?Number3B).3B). Cessation of DNA replication was verified by incorporation of EdU. Only limited subsets of EdU-positive cells were observed in both TC-1 and TRAMP-C2 cell populations by FACS analysis (Number ?(Figure2A).2A). Such residual EdU positivity can most likely become accounted for by ongoing DNA restoration of the observed DNA damage (H2AX) and/or aberrant endoreduplication uncoupled from cell division (Number ?(Figure2B)2B) once we did not observe any proliferation of cells upon the DTX-treatment (Figure ?(Figure3A).3A). Most importantly for our subsequent experiments, subcutaneous administration of such senescent cells into animals did not lead to DBeq development of tumors (Number ?(Number3C3C). Open in a separate window Number 1 Docetaxel induces senescence in TC-1 and TRAMP-C2 cellsSenescence-associated -galactosidase activity in TC-1 and TRAMP-C2 cells treated with DTX (7.5 M) for 4 days (A). Phase contrast microscopic images of control and DTX-treated (7.5 M) TC-1 and TRAMP-C2 cells at day time 4 after the treatment (B). Immunoblotting detection of mouse p16INK4A (p16) and p21waf1/cip1 (p21) in control and DTX-treated (7.5 M) TC-1 and TRAMP-C2 cells harvested at day time 4 and 6 after the treatment. GAPDH was used as a loading control (C). qRT-PCR quantification of p16 and p21 in control and DTX-treated (7.5 M) TC-1 and TRAMP-C2 cells harvested at day time 4 and 6 after the treatment. Data symbolize means S.D. *< 0.05, **< 0.005 (D). Open in a separate windows Number 2 Analysis of TC-1 and TRAMP-C2.