Explore the vast range of titles available.

Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3′-phosphatase and DNA 5′-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.

The clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), which is the active metabolite of irinotecan, has been hampered because of its practical water-insolubility. In this study, we successfully synthesized two self-associating SN-38-polymer drug conjugates to improve the water-solubility of SN-38, while retaining its anticancer activity. The polymeric micellar SN-38 conjugates were composed of either methoxy-poly(ethylene oxide)-block-poly(α-benzyl carboxylate-ε-caprolactone) conjugated to SN-38 at the PBCL end (mPEO-b-PBCL/SN-38) or mPEO-block-poly(α-carboxyl-ε-caprolactone) attached to SN-38 from the pendent-free carboxyl site (mPEO-b-PCCL/SN-38). The chemical structure of block copolymers was confirmed by 1H NMR. The physicochemical characterizations of their self-assembled structures including size, surface charge, polydispersity, critical micellar concentration, conjugation content and efficiency, morphology, kinetic stability, and in vitro release of SN-38 were compared between the two formulations. In vitro anticancer activities were evaluated by measuring cellular cytotoxicity and caspase activation by MTS and Caspase-Glo 3/7 assays, respectively. The hemolytic activity of both micellar structures against rat red blood cells was also measured. The results showed the formation of SN-38-polymeric micellar conjugates at diameters < 50 nm with a narrow size distribution and sustained release of SN-38 for both structures. The loading content of SN-38 in mPEO-b-PBCL and mPEO-b-PCCL were 11.47 ± 0.10 and 12.03 ± 0.17 (% w/w), respectively. The mPEO-b-PBCL/SN-38, end-capped micelles were kinetically more stable than mPEO-b-PCCL/SN-38. The self-assembled mPEO-b-PBCL/SN-38 and mPEO-b-PCCL/SN-38 micelles resulted in significantly higher cytotoxic effects than irinotecan against human colorectal cancer cell lines HCT116, HT-29, and SW20. The CRC cells were found to be 70-fold to 330-fold more sensitive to micellar SN-38 than irinotecan, on average. Both SN-38-incorporated micelles showed two-fold higher caspase-3/7 activation levels than irinotecan. The mPEO-b-PBCL/SN-38 micelles were not hemolytic, but mPEO-b-PCCL/SN-38 showed some hemolysis. The overall results from this study uphold mPEO-b-PBCL/SN-38 over mPEO-b-PCCL/SN-38 micellar formulation as an effective delivery system of SN-38 that warrants further preclinical investigation.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

Cellular adaptations to hypoxia promote resistance to ionizing radiation (IR). This presents a challenge for treatment of head and neck cancer (HNC) that relies heavily on radiotherapy. Standard radiosensitizers often fail to reach diffusion-restricted hypoxic cells, whereas nitroimidazoles (NIs) [such as iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA)] can preferentially accumulate in hypoxic tumours. Here, we explored if the hypoxia-selective uptake of IAZA and FAZA could be harnessed to make HNC cells (FaDu) susceptible to radiation therapy. Cellular response to treatment was assessed through clonogenic survival assays and by monitoring DNA damage (immunofluorescence staining of DNA damage markers, γ-H2AX and p-53BP1, and by alkaline comet assay). The effects of reoxygenation were studied using the following assays: estimation of nucleoside incorporation to assess DNA synthesis rates, immunofluorescent imaging of chromatin-associated replication protein A as a marker of replication stress, and quantification of reactive oxygen species (ROS). Both IAZA and FAZA sensitized hypoxic HNC cells to IR, albeit the former is a better radiosensitizer. Radiosensitization by these compounds was restricted only to hypoxic cells, with no visible effects under normoxia. IAZA and FAZA impaired cellular adaptation to reoxygenation; high levels of ROS, reduced DNA synthesis capacity, and signs of replication stress were observed in reoxygenated cells. Overall, our data highlight the therapeutic potentials of IAZA and FAZA for targeting hypoxic HNC cells and provide rationale for future preclinical studies.

Human polynucleotide kinase (hPNK) is a 57.1-kDa monomeric protein with conserved motifs associated with phosphatase and kinase activities. hPNK catalyzes phosphorylation of 5′-DNA termini and dephosphorylation of 3′-DNA termini. Previous studies, employing cell-free systems, have suggested that hPNK participates in the repair of DNA strand breaks. To better define the cellular function of hPNK, a double-stranded small-interfering RNA molecule designed to stably target hPNK transcription was introduced into A549 human lung adenocarcinoma cells. The small-interfering RNA suppressed hPNK gene expression by at least 80-90%. These cells exhibited a 7-fold higher spontaneous mutation frequency based on the development of resistance to ouabain; elevated sensitivity to a broad range of genotoxic agents including γ-radiation, UVC radiation, methyl methanesulfonate, hydrogen peroxide, and camptothecin; and slower repair of radiation-induced DNA strand breaks. These findings underscore the importance of hPNK in the maintenance of DNA integrity after damage induced by endogenous and exogenous agents.

Radiation therapy (RT) is frequently used to locally treat tumors. One of the major issues in RT is normal tissue toxicity; thus, it is necessary to limit dose escalation for enhanced local control in patients that have locally advanced tumors. Integrating radiosensitizing agents such as gold nanoparticles (GNPs) into RT has been shown to greatly increase the cure rate of solid tumors. The objective of this study was to explore the repurposing of an antimalarial drug, pyronaridine (PYD), as a DNA repair inhibitor to further enhance RT/GNP-induced DNA damage in cancerous cell lines. We were able to achieve inhibitory effects of DNA repair due to PYD at 500 nM concentration. Our results show a significant enhancement in DNA double-strand breaks of 42% in HeLa cells treated with PYD/GNP/RT in comparison to GNP/RT alone when irradiated with a dose of 2 Gy. Furthermore, there was a significant reduction in cellular proliferation for both HeLa and HCT-116 irradiated cells with the combined treatment of PYD/GNP/RT. Therefore, the emergence of promising novel concepts introduced in this study could lay the foundation for the transition of this treatment modality into clinical environments.

Chemotherapeutic drugs that are used in anti-cancer treatments often cause the death of both cancerous and noncancerous cells. This non-selective toxicity is the root cause of untoward side effects that limits the effectiveness of therapy. In order to improve chemotherapeutic options for cancer patients, there is a need to identify novel compounds with higher discrimination for cancer cells. In the past, methine dyes that increase the sensitivity of photographic emulsions have been investigated for anti-cancer properties. In the 1970's, Kodak Laboratories initiated a screen of approximately 7000 dye structural variants for selective toxicity. Among these, D112 was identified as a promising compound with elevated toxicity against a colon cancer cell line in comparison to a non-transformed cell line. Despite these results changing industry priorities led to a halt in further studies on D112. We decided to revive investigations on D112 and have further characterized D112-induced cellular toxicity. We identified that in response to D112 treatment, the T-cell leukemia cell line Jurkat showed caspase activation, mitochondrial depolarization, and phosphatidylserine externalization, all of which are hallmarks of apoptosis. Chemical inhibition of caspase enzymatic activity and blockade of the mitochondrial pathway through Bcl-2 expression inhibited D112-induced apoptosis. At lower concentrations, D112 induced growth arrest. To gain insight into the molecular mechanism of D112 induced mitochondrial dysfunction, we analyzed the intracellular localization of D112, and found that D112 associated with mitochondria. Interestingly, in the cell lines that we tested, D112 showed increased toxicity toward transformed versus non-transformed cells. Results from this work identify D112 as a potentially interesting molecule warranting further investigation.

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Förster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates (\"bubble\" DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA₂B and contact of the β-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.

Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo . RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo , but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3σ) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3σ in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3σ protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3σ mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3σ mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3σ were unable to express this factor. Interestingly, the level of 14-3-3σ mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3σ at the levels of mRNA and protein. But the releasable form of 14-3-3σ protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3σ protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.