Supplementary Materialsoncotarget-08-9303-s001. with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls. Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment. cultures and to form tumors in NOD/SCID mice. RESULTS Senescent colon cancer HCT116 cells exhibit stem-cell like properties and re-populate culture after chemotherapeutic removal To mimic a regime of chemotherapy in patients, we subjected human colon cancer HCT116 cell cultures to long-term, repeated GNE 2861 treatment with a chemotherapeutic drug. Cells were treated with 100 nM doxorubicin (doxo, D) for 24 hours. Following its removal, the cells were cultured in the drug-free medium for the next 3 days. The cycle was repeated GNE 2861 three times (Figure ?(Figure1A,1A, CHEMO protocol). Subsequently, to mimic a post chemotherapy period, we cultured HCT116 cells in the drug-free medium for additional 14 days, with GNE 2861 the medium changed every four days (Figure ?(Figure1A,1A, AFTER CHEMO protocol). On the 13th day the CHEMO-treated cells Rabbit Polyclonal to RPC8 exhibited several features of senescence: flatten morphology (Figure ?(Figure1B),1B), increased size and granularity (Figure GNE 2861 1B, 1C, Supplementary Figure S1A), augmented SA–gal activity (Figure ?(Figure1D,1D, Supplementary Figure S1B) and polyploidization (Figure ?(Figure1E,1E, Supplementary Figure S1C). Moreover, the elevated expression of DDR proteins: -H2A.X, p-p53, and p21, and geroconversion markers [7]: cyclin D1 and p-S6 (Figure ?(Figure1F)1F) was detectable. In addition, the cells up-regulated secretion of SASP factors: VEGF and IL-8 (Figure ?(Figure1G1G). Open in a separate window Figure 1 Colon cancer HCT116 cells treated with doxorubicin cycles show features of senescence(A) A scheme of the experiment. CHEMO protocol. Cells were subjected to three cycles of doxorubicin (D) as follows: cells were treated with 100 nM of doxorubicin for 24 hours, then the medium was removed and cells were cultured in a drug-free medium for the next 3 days. AFTER CHEMO protocol. After the 3rd doxorubicin cycle HCT116 cells were cultured in a drug-free medium for 14 days. Medium was changed every four days. Cells were examined for senescent markers on the 13th day (CHEMO). (B) Representative photos show morphological alterations in CHEMO-treated cells. Cell nuclei were stained with “type”:”entrez-nucleotide”,”attrs”:”text”:”H33342″,”term_id”:”978759″,”term_text”:”H33342″H33342 (blue). Original magnification 200. Data were acquired with transmitted light and fluorescence microscopy. Scale barC100 m. (C) Percentages of granular cells as determined by FSC/SSC analysis using flow cytometry. (D) Quantification of SA–gal+ cells. Untreated or CHEMO-treated cells were cytospined and cytochemical staining for SA–gal activity was performed. (E) Percentages of polyploid cells. Cell cycle analysis was GNE 2861 performed using PI staining and flow cytometry. (F) The expression of DDR and geroconversion proteins in untreated (un) or CHEMO-treated (ch) cells. Representative blots show levels of -H2A.X, H2A.X, p-p53, p21, cyclin D1 and p-S6 proteins. GAPDH was used as a loading control. (G) Secretion of VEGF and IL-8 in CHEMO-treated cultures. Cytokine levels were determined by colorimetric ELISA in supernatants harvested from untreated and treated cells. Results were normalized to total cell number counted in Brker’s chamber. Each bar represents mean SEM, 3; # 0.05, ## 0.01, ### 0.001 -untreated vs. CHEMO. We observed the six-fold increase in the number of cells within two weeks after doxo removal ( 0.001, Figure ?Figure2A).2A). Using a.