Figure 3.

Defect in a UV-induced G₂/M checkpoint in Gadd45-deficient cells. (A) Flow cytometric analysis of the human colon carcinoma cell line RKO and its GADD45 antisense overexpressing clones in response to IR or MMS treatment. Unsynchronized cells were treated with either 6.3 Gy IR or 25 μg/ml MMS and analyzed by FACScan after a 24-hr incubation. The G₁, S, and G₂/M fractions are indicated, as calculated by modfit analysis. (B) Increased expression of antisense GADD45 allows RKO cells to escape the G₂/M checkpoint arrest induced by UV-induced damage. Parental RKO cells and three antisense GADD45-expressing clones were synchronized with aphidicolin for 24 hr (>95% at G₁) and release from blocking in the presence of BrdUrd. (a–c) Representative profiles of RKO cells subjected to synchronization. The intensities of fluorescein isothiocyanate (anti-BrdUrd signal) and PI (DNA contents) are indicated. Three antisense GADD45 clones show similar synchronization profiles as their parental RKO cells (data not shown). On release from late G₁, cells were treated with 6.3 Gy IR or 5 J/m² UV and incubated for an additional 20 hr in the presence of BrdUrd. The BrdUrd-positive cells were subjected to FACScan and modfit analysis (d). The first solid peaks, the gray area, and the second solid peaks represent the second G₁ (2nd G₁), first S (S) and first G₂/M (1st G₂) fractions, respectively. No second S phase should be observed in this condition. The numbers above the second G₁ peaks are the ratio of G₁/G₂ (percent of G₁ fraction escaped from G₂/M arrest).