For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancer.

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Cyclin B1 should have some rate limiting function for cell cycle progression. To test this, we measured the effect of siRNA-mediated depletion of cyclin B1 on mitotic entry and timing. We depleted cyclin B1 in HeLa and hTert-RPE1 cells to levels equivalent or below those achieved in the telophase-to-G(1) window. Average cyclin B1/Cdk1 activity was measured in HeLa cells and depleted by approximately 99%. In both cell lines, this caused approximately 20% increase in the G(2) and approximately 20% increase the M traverse time. However, co-depletion of cyclin B1 and B2 induced a profound increase in G(2) cells, a dramatic reduction in mitotic cells, and an increase in a 4C cycling population. We conclude that any residual levels of cyclin B1 were not sufficient to promote stable mitotic entry and transition in absence of normal levels of cyclin B2. Therefore, we conclude that B cyclin is necessary for mitosis but cyclin B1 is not. Nocodazole treated, cyclin B1-depleted HeLa cells arrested but exited that arrest at higher rates than controls, suggesting that the duration of the spindle checkpoint was affected. In B1 depleted cells, population growth was delayed but evidence of cell death was not consistently observed. A strong phenotype of mitotic chromosomal aberration was observed in HeLa cells depleted for either cyclin but not in RPE cells. In B1 or B2 depleted cells, maloriented chromosomes at metaphase were increased 10 fold and one third of affected metaphase cells entered anaphase without congression. Lagging chromosomes at anaphase were dramatically increased. The aggregate evidence from our study and others suggests that the common effect of cyclin B1 depletion is mild cell cycle perturbation. Lack of uniformity in other phenotypes suggest that these are low penetrance effects that are exacerbated or compensated in some systems by other mechanisms.

1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment.