The antineoplastic drug hydroxyurea (HU), when used at subtoxic doses, induces prolonged replication stress and centrosome amplification. This causes genomic instability and increases the malignancy of the recurring tumor. The mechanism of centrosome amplification induced by prolonged replication stress, however, is still unclear. Here, we examined the involvement of ataxia telangiectasia, mutated (ATM), ataxia telangiectasia, mutated and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) and found that HU-induced centrosome amplification was inhibited by the depletion of DNA-PKcs, but not ATM and ATR. Inactivation of ATM/ATR in U2OS cells instead caused aneuploidy and cell death. We found DNA-PKcs depletion also abrogated ATM phosphorylation, indicating that ATM activation during prolonged replication stress depends on DNA-PK. Depletion of DNA-PK abrogated checkpoint kinase (Chk)2 activation and partially reduced Chk1 activation. Chk2 depletion blocked HU-induced centrosome amplification, indicating a function of Chk2 in centrosome amplification. We further found that Chk2 was phosphorylated at Thr68 on the mother centriole at late G2 and mitosis when unstressed and on all amplified centrioles induced by HU. In summary, we have elucidated that DNA-PK/Chk2 signaling induces centrosome amplification upon long-term HU treatment, therefore increasing our insight into tumor recurrence after initial chemotherapy.

Pharmacokinetics and residues of oxolinic acid (OXO) in serum, muscle, liver, and kidney were measured in orange-spotted grouper Epinephelus coioides, snubnose pompano Trachinotus blochii and giant seaperch Lates calcarifer. Fish were treated with OXO after a single-dose trial (60 mg/kg) by oral gavages and repeat-doses of 60 mg/kg of body weight once daily in feed for five consecutive days. Analysis of OXO kinetic profiles, when determined by HPLC provided elimination half-life in muscle were 131, 26, and 85 h for 26-27 °C orange-spotted grouper, snubnose pompano, and giant seaperch, respectively. Depletion studies were conducted the time for OXO concentrations to fall below 0.1 µg/g (the current tolerance set by the European Medicines Agency (EMA) among fish species establishing an adequate depletion period). The mean tissue OXO concentration in the edible portion was below maximum residue level (MRL) of snubnose pompano at 6 d postdosing, for orange-spotted grouper and giant seaperch, at 10 d postdosing, respectively. When possible, depletion data were fitted to a one-compartment pharmacokinetic model. For all test species, the longest withdrawal period of 9 days in snubnose pompano and 15 days in orange-spotted grouper and giant seaperch were calculated because of a safety span (corresponds to 50% of the time point when at which residues fall below the MRL added to the depletion time). These results suggest withdrawal times with reference to human consumption of treated fish, to establish policy guidelines and basic principles regarding the use of OXO for fish-farming, and to fish-farmers for the proper handling to ensure safe fish that the consumer will not be at risk.

Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

Histone deacetylase (HDAC) inhibitors are used in treating certain human malignancies. Our laboratories demonstrated their capability in enhancing antitumor effect of DNA vaccine driven by an RNA polymerase II (RNA pol II) promoter. However, it is unknown whether HDAC inhibitors enhance the therapeutic short hairpin RNA (shRNA) expressed by an RNA polymerase III (RNA pol III) promoter. We investigated whether HDAC inhibitors augmented antitumor effect of indoleamine 2,3 dioxygenase (IDO) shRNA. HDAC inhibitor OSU-HDAC42 and suberoylanilide hydroxamic acid enhanced RNA pol III-driven U6 and H1 promoter activity in three different cell types in vitro: 293, NIH3T3 and dendritic cell line DC2.4. Subcutaneous injection of OSU-HDAC42 enhanced U6 and H1 promoter activity on abdominal skin of mice in vivo. Combination of IDO shRNA and OSU-HDAC42 increased antitumor effect of IDO shRNA in MBT-2 murine bladder tumor model. IDO shRNA induced tumor-infiltrating CD8⁺ and CD4⁺ T cells, whereas OSU-HDAC42 treatment induced tumor-infiltrating CD4⁺ T cells. Combination of OSU-HDAC42 and IDO shRNA further induced tumor-infiltrating natural killer cells and enhanced interferon-γ in lymphocytes, but suppressed interleukin (IL)-4 expression of lymphocytes. In addition, OSU-HDAC42 treatment did not alter mRNA expression of IL-12 and tumor necrosis factor-α. In conclusion, HDAC inhibitor OSU-HDAC42 may serve as adjuvant of the therapeutic shRNA expressed by an RNA pol III promoter.