Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex–binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response.

Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53R175, one of the most frequent ‘hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53R175 mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53R175 mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53R175 mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53R175 and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

Werner syndrome (WS) is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi Anemia (FA), a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2−/− cells, NSC 617145 acted synergistically with very low concentrations of Mitomycin C (MMC) to inhibit proliferation in a WRN-dependent manner, and induce double strand breaks (DSBs) and chromosomal abnormalities. Under these conditions, ATM activation and accumulation of DNA-PKcs pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2−/− cells exposed to NSC 617145 and MMC, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination (HR) at ICL-induced DSBs. Thus, when the FA pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of FA-deficient tumors by their sensitization to DNA cross-linking agents is discussed.

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Elongated styloid process is a relatively common cause of facial and neck pain, but it is often misdiagnosed due to its varied clinical presentation. Since an elongated styloid process is often confirmed by radiological means, it would be helpful to find a more accurate mode of depicting the styloid process. In this prospective study, 39 cases were evaluated. A three-dimensional computed tomography (3D CT) reconstruction of the styloid process was performed in 18 cases. In these patients, we compared the length and medial angulation of the symptomatic styloid process as viewed on an orthopantomogram and a 3D CT reconstruction. It was noted that a 3D CT reconstruction was more accurate in depicting the styloid process. This investigation can be considered as the 'gold standard' in the radiological diagnosis of an elongated styloid process.

Background We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53 R175H mutant in vitro and in SK-BR-3 (p53 R175H ) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. Methods Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. Results We demonstrated that PEITC inhibits the growth of prostate cancer cells with different “hotspot” p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53 R248W contact (VCaP) and p53 R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53 P223L (structural) and p53 V274F (contact) mutants by targeting p53 P223L mutant selectively, but not p53 V274F . The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53 R175H and p53 P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. Conclusion Our studies provide the first evidence that PEITC’s anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent. Electronic supplementary material The online version of this article (10.1186/s13046-019-1267-z) contains supplementary material, which is available to a...

Designing strategies for anti-cancer therapy have posed a significant challenge. One approach has been to inhibit specific DNA repair proteins and their respective pathways to enhance chemotherapy and radiation therapy used to treat cancer patients. Synthetic lethality represents an approach that exploits pre-existing DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated repair deficiency. Since helicases play critical roles in the DNA damage response and DNA repair, particularly in actively dividing and replicating cells, it is proposed that the identification and characterization of synthetic lethal relationships of DNA helicases will be of value in developing improved anti-cancer treatment strategies. In this review, we discuss this hypothesis and current evidence for synthetic lethal interactions of eukaryotic DNA helicases in model systems.Keywords synthetic lethality; helicase; cancer therapy; DNA repair; anti-cancer drug Cells exposed to DNA damage have multiple pathways in order to cope with the stressed condition. DNA repair is believed to represent a good target for enhancing cancer treatment strategies that are based on chemotherapy drugs or radiation that induce death of rapidly proliferating cells. There is a growing interest in synthetic lethality of compensating DNA repair pathways as a strategy to target cancer therapies based on the genetic background of the tumor. Since helicases play an integral role in the DNA damage response, it seems reasonable that this particular class of proteins may be one of the optimal bull's eye targets for cancer therapy based on synthetic lethality (Fig. 1). Specifically, compensatory repair pathways mediated by DNA helicases are a priority for further studies. Research in the basic sciences has demonstrated and characterized synthetic lethal interactions of eukaryotic DNA helicases. This work is likely to serve as a foundation for the development of anti-cancer therapies that utilize small molecule helicase inhibitors or antagonists to helicase gene expression such as RNA interference.

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.