Research Paper Volume 13, Issue 17 pp 20962—20991

Glutamine promotes escape from therapy-induced senescence in tumor cells

Cells that escape from TIS overexpress the glutamine transporter SLC1A5. (A) Escaped clones were isolated from senescent MCF-7 cells. Expression of SLC1A5 was analyzed by real-time PCR. Data are mean ± S.D. of three independent experiments. (B) Expression of SLC1A5 protein was analyzed in parental MCF-7 cells and in three escaped clones. Filters were stripped and reprobed with anti-actin antibodies as a loading control. SLC1A5 levels, normalized to the relative actin levels, are reported as fold change of parental cells. (C) Expression of NANOG, SNAT1 and SNAT2 was analyzed by real-time PCR. Data are mean ± S.D. of three independent experiments. (D) Dose-dependent effect of GPNA on CD44+/CD24− subpopulation. MCF-7 cells were treated for 72 hours with indicated GPNA concentrations. Expression of CD44 and CD24 was analyzed by flow cytometry. Data are mean ± S.D. of three independent experiments. (E) GPNA treatment abolishes escape from TIS. Doxorubicin-induced senescent MCF-7 cells were grown in Gln-deprived medium (−Gln), or in complete medium (+Gln) in the absence or in the presence of 1 mM or 2 mM GPNA. Colonies that evaded the senescent growth arrest were stained and counted. Data are mean ± S.D. of two independent experiments. (F) GPNA treatment abolishes escape from TIS. Doxorubicin-induced senescent A549 cells were grown in Gln-deprived medium (−Gln), or in complete medium (+Gln) in the absence or in the presence of 0.5 mM or 1 mM GPNA. Colonies that evaded the senescent growth arrest were stained and counted. Data are mean ± S.D. of two independent experiments.

Figure 5. Cells that escape from TIS overexpress the glutamine transporter SLC1A5. (A) Escaped clones were isolated from senescent MCF-7 cells. Expression of SLC1A5 was analyzed by real-time PCR. Data are mean ± S.D. of three independent experiments. (B) Expression of SLC1A5 protein was analyzed in parental MCF-7 cells and in three escaped clones. Filters were stripped and reprobed with anti-actin antibodies as a loading control. SLC1A5 levels, normalized to the relative actin levels, are reported as fold change of parental cells. (C) Expression of NANOG, SNAT1 and SNAT2 was analyzed by real-time PCR. Data are mean ± S.D. of three independent experiments. (D) Dose-dependent effect of GPNA on CD44+/CD24 subpopulation. MCF-7 cells were treated for 72 hours with indicated GPNA concentrations. Expression of CD44 and CD24 was analyzed by flow cytometry. Data are mean ± S.D. of three independent experiments. (E) GPNA treatment abolishes escape from TIS. Doxorubicin-induced senescent MCF-7 cells were grown in Gln-deprived medium (−Gln), or in complete medium (+Gln) in the absence or in the presence of 1 mM or 2 mM GPNA. Colonies that evaded the senescent growth arrest were stained and counted. Data are mean ± S.D. of two independent experiments. (F) GPNA treatment abolishes escape from TIS. Doxorubicin-induced senescent A549 cells were grown in Gln-deprived medium (−Gln), or in complete medium (+Gln) in the absence or in the presence of 0.5 mM or 1 mM GPNA. Colonies that evaded the senescent growth arrest were stained and counted. Data are mean ± S.D. of two independent experiments.