Figure 6.

Regression of B220^low tumor cells in blood depends on the DNA damage response. (A) BC2 or Eμ-M1, cell lines derived from Eμ-myc mice, were treated with DMSO (fine line) or 10 μM Ara-C (bold line), a DNA-damaging agent, for 16 hours and stained for CD112 and CD155 expression. Some cells were pretreated with the 7.7 mM of the ATM/ATR inhibiting drug caffeine for 1 hour before treatment with Ara-C (dashed line) or DMSO (dotted line) for 16 hours. Filled histogram represents Ara-C–treated cells stained with isotype control. (B) Immunoblot analysis of purified B220^low cells (>98% purity) from Eμ-myc mice at 33 and 68 ± 2 days of age or WT mice probed with antibodies for indicated DNA damage response markers. Levels were quantitated and normalized to WT levels (right panel). Shown is 1 out of 3 representative experiments. (C-E) Eμ-myc mice were injected intraperitoneally with 5 mg/kg KU55933 (n = 9) or vehicle (n = 8) at 37, 39, 47, 49, and 54 days of age. Percentage of B220^low cells in the blood of an individual Eμ-myc mouse was determined at 33, 44, and 58 days of age by flow cytometry (C). KU55933-treated (open circles) or vehicle-treated (filled circles) mice also received 1 mg BrdU at 57 days of age. B220^low tumor cells were stained for incorporation of BrdU and the percentage of BrdU⁺ B220^low tumor cells was determined by flow cytometry at 58 days of age (D). Thirty hours after first injection of KU55933 (squares) or vehicle (circles), B220^low cells were stained for CD155 and IgM expression. Mean fluorescence intensity of CD155 expression on IgM⁻B220^low (filled symbols) and IgM⁺B220^low (open symbols) cells is depicted (E). Statistical significance is indicated. Ctrl, control; d, day; MFI, mean fluorescence intensity.