KCa3.1 Channels Confer Radioresistance to Breast Cancer Cells

Figures

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  Figure 1. Activation of KCa3.1 channels by irradiation in MMTV-PyMT breast cancer cells. (A) Representative macroscopic on-cell current tracings recorded with KCl pipette and NaCl bath solution from an unirradiated (left) and a 2 Gy-irradiated (right) MMTV-PyMT WT cell. Currents of the irradiated cell (right) were obtained before (control), during, and after (wash-out) bath application of TRAM-34 (2 µM). (B) Dependence of the mean (±SE, n = 5) TRAM-34-inhibited current macroscopic on-cell current fraction on voltage recorded as in (A) in MMTV-PyMT WT cells 180 ± 34 min post-IR with 2 Gy. (C) Dependence of the mean (±SE, n = 6–20) macroscopic on-cell current fraction on voltage recorded as in (A) in unirradiated (open circles, left) and 2 Gy-irradiated (156 ± 12 and 151 ± 6 min post-IR, respectively, closed triangles, right) MMTV-PyMT WT (black) and KCa3.1 KO (red) cells. (D) Mean (±SE, n = 6–20) KCa3.1-dependent current fraction in unirradiated (open diamonds) and 2 Gy-irradiated (closed diamonds) cells as calculated from the data in (C) by subtracting the KCa3.1 currents from those of the WT cells. (E) Data of (C) replotted to illustrate the IR effect on macroscopic on-cell currents in…

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  Figure 2. IR-induced [Ca2+]i signals in MMTV-PyMT breast tumor cells depend on KCa3.1 channels (A,B) Acute Ca2+ signals of MMTV-PyMT mammary tumor cells evoked by Ca2+ (1.8 mM) superfusion only (gray and light red lines) or Ca2+ (1.8 mM) plus 5% fetal calf serum (FCS; black and red lines) after 30 or 180 min of irradiation (2 Gy). Irradiated FURA-2-AM-loaded cells were monitored in Ca2+-free buffer for 10 min prior to the respective treatments as indicated. Arrow indicated buffer replacement. (C,D) The mean peak fluorescence (FL) ratio of the 340/380 nm wavelength was determined relatively to the respective baseline FL ratio for MMTV-PyMT KCa3.1 KO (n = 14–28 cells per condition derived from three different breast tumors) and WT (n = 21–27 cells per condition derived from three different breast tumors) groups. Statistical analysis was performed by two-way analysis of variance (ANOVA) with * p < 0.05, ** p < 0.01, *** p < 0.001 indicating statistical differences between FCS-treated and -untreated groups and ## p < 0.01, ### p < 0.001 for differences between genotypes either at Ca2+ plus FCS or Ca2+ “only” condition.

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  Figure 3. Clonogenic survival of irradiated MMTV-PyMT breast cancer cells in vitro depends on KCa3.1. (A) Light micrographs of colonies formed from MMTV-PyMT WT (upper row) and KCa3.1 KO (lower row) cells upon exposure to 0, 2, 4, or 6 Gy ionizing radiation. (B) Mean (±SE, n = 9 with 6 technical replicates per experiment) survival fractions of irradiated (0–6 Gy) KCa3.1 WT (black) and KO cells (red) MMTV-PyMT breast cancer cells as determined as in (A) by delayed plating colony formation assay with data given semi-logarithmically as survival fraction/radiation dose plot. Survival fractions are given in semi-logarithmic plots because otherwise differences at higher radiation doses cannot be resolved easily by linear plots. (C) Light micrographs of colonies formed from MMTV-PyMT WT cells upon exposure to 0, 2, 4, or 6 Gy ionizing radiation. Radiation and 24 h post-incubation were performed in the absence (solvent) or presence of TRAM-34 (10 µM). (D) Mean (±SE, n = 5 with 6 technical replicates per experiment) survival fractions of irradiated (0–6 Gy) WT MMTV-PyMT breast cancer cells co-treated with solvent (black) or TRAM-34 (red) as determined as in (C) by delayed plating colony formation…

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  Figure 4. KCa3.1 channels promote repair of DNA double-strand breaks. (A) Fluorescence micrographs showing nuclear γH2AX foci as a measure of DNA double-strand breaks 30 min (left) and 24 h (right) after irradiation of MMTV-PyMT WT (upper row) and KCa3.1 KO cells (lower row) with 0 Gy or 2 Gy. (B,C) Mean number (±SE, n = 7) of nuclear γH2AX foci in MMTV-PyMT WT (black) and KCa3.1 KO (red) cells (B) 30 min and (C) 24 h after irradiation. (D) Fluorescence micrographs showing nuclear γH2AX foci 30 min (left) and 24 h (right) after irradiation of MMTV-PyMT WT with 0 Gy or 2 Gy. Irradiation and 24 h post-incubation were either performed in the absence (solvent, upper row) or presence (lower row) of TRAM-34 (10 µM). (E,F) Mean number (±SE, n = 5) of nuclear γH2AX foci in MMTV-PyMT WT cells (E) 30 min and (F) 24 h (red) after irradiation and post-incubation in the absence (black bars) or presence (gray bars) of TRAM-34 (10 µM). γH2AX foci in (A,D) are shown in yellow, DAPI-stained nuclei appear in blue. *, **, and *** indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively, two-way ANOVA with Sidak’s post-hoc test.

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  Figure 5. KCa3.1 channels confer radioresistance to MMTV-PyMT breast cancer in vivo. Computer-aided depiction of the irradiation setup used to locally irradiate the breast tumors. Part (A) represents the radiation beam (yellow gold-colored) produced by a linear accelerator and the lead shieldings. The beam is trimmed by the shielding satellite to a rectangular miniature beam which then reaches the tumor. (B) Beam, mouse holder, and 2nd on-body shielding in higher magnification. (C) Film dosimetry recorded in the coronal plane of a mouse phantom during application of 5 Gy with the setup described in A,B. Drawings of mouse and holder are superimposed. Dose distribution is given in pseudo colors. (D) Kaplan-Meier curves and (E) individual (symbols) and median (line) survival time (n = 7) of FVB/N mice orthotopically transplanted with MMTV-PyMT WT (black/gray) or KCa3.1 KO (red) cells and treated with 5 fractions for a total dose of either 0 Gy (black, dark red) or 12.5 Gy (gray, bright red) starting at a tumor size of 5 mm. Compared to WT 0 Gy or 12.5 Gy, survival time was significantly prolonged in accordingly treated mice bearing KCa3.1 KO tumors with one complete responder (CR) in the…