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Herbal medicines are attractive agents for human care. In this study, we found that the alcohol extract of Ajuga taiwanensis (ATE) screened from a chemical bank exhibited potent capacity for suppressing senescence associated biomarkers, including SA-β-gal and up-regulated p53 in old human dermal fibroblasts (HDFs) without induction of significant cytotoxicity up to 100 µg/ml. Concomitantly, cells re-entered the cell cycle by reducing G1 phase arrest and increasing cell growth rate. The ATE was further partitioned to obtain the sub-fractions of n-butanol (BuOH), ethyl acetate (EA) and water. The BuOH and water sub-fractions exhibited less effects on prohibition of cell growth than the EA sub-fraction. All of these sub-fractions exhibited the ability on suppressing SA-β-gal and p53 of old HDFs as low as 5–10 µg/ml. Under the activity guided fractionation and isolation, a major active constituent named AT-1 was isolated. The AT-1 was further identified as 8-O-acetylharpagide by structural analysis, and it could suppress SA-β-gal and p53 of old HDFs below 10 µM. In addition, the intracellular reactive oxygen species (ROS) levels of old HDFs were suppressed by ATE, the sub-fractions of BuOH and water, and AT-1. However, the EA sub-fraction showed little ability on suppression of ROS. Furthermore, we performed an in vivo study using aging mice to be fed with ATE and the sub-fractions followed by immunohistochemical (IHC) staining. The expression of p53 and SA-β-gal was significantly reduced in several tissue sections, including skin, liver, kidney, and spleen. Taken together, current data demonstrated that A. taiwanensis could suppress cellular senescence in HDFs, and might be used for health care.

Radiotherapy is routinely used for the treatment of head and neck squamous cell carcinoma (HNSCC). However, the therapeutic efficacy is usually reduced by acquired radioresistance and locoregional recurrence. In this study, The Cancer Genome Atlas (TCGA) analysis showed that radiotherapy upregulated the miR-182/96/183 cluster and that miR-182 was the most significantly upregulated. Overexpression of miR-182-5p enhanced the radiosensitivity of HNSCC cells by increasing intracellular reactive oxygen species (ROS) levels, suggesting that expression of the miR-182 family is beneficial for radiotherapy. By intersecting the gene targeting results from three microRNA target prediction databases, we noticed that sestrin2 (SESN2), a molecule resistant to oxidative stress, was involved in 91 genes predicted in all three databases to be directly recognized by miR-182-5p. Knockdown of SESN2 enhanced radiation-induced ROS and cytotoxicity in HNSCC cells. In addition, the radiation-induced expression of SESN2 was repressed by overexpression of miR-182-5p. Reciprocal expression of the miR-182-5p and SESN2 genes was also analyzed in the TCGA database, and a high expression of miR-182-5p combined with a low expression of SESN2 was associated with a better survival rate in patients receiving radiotherapy. Taken together, the current data suggest that miR-182-5p may regulate radiation-induced antioxidant effects and mediate the efficacy of radiotherapy.

(1) Destabilization of microtubule dynamics is a primary strategy to inhibit fast growing tumor cells. The low cytotoxic derivative of microtubule inhibitor D-24851, named BPR0C261 exhibits antitumor activity via oral administration. In this study, we investigated if BPR0C261 could modulate the radiation response of human non-small cell lung cancer (NSCLC) cells with or without p53 expression. (2) Different doses of BPR0C261 was used to treat human NSCLC A549 (p53+/+) cells and H1299 (p53−/−) cells. The cytotoxicity, radiosensitivity, cell cycle distribution, DNA damage, and protein expression were evaluated using an MTT assay, a colony formation assay, flow cytometry, a comet assay, and an immunoblotting analysis, respectively. (3) BPR0C261 showed a dose-dependent cytotoxicity on A549 cells and H1299 cells with IC50 at 0.38 μM and 0.86 μM, respectively. BPR0C261 also induced maximum G2/M phase arrest and apoptosis in both cell lines after 24 h of treatment with a dose-dependent manner. The colony formation analysis demonstrated that a combination of low concentration of BPR0C261 and X-rays caused a synergistic radiosensitizing effect on NSCLC cells. Additionally, we found that a low concentration of BPR0C261 was sufficient to induce DNA damage in these cells, and it increased the level of DNA damage induced by a fractionation radiation dose (2 Gy) of conventional radiotherapy. Furthermore, the p53 protein level of A549 cell line was upregulated by BPR0C261. On the other hand, the expression of PTEN tumor suppressor was found to be upregulated in H1299 cells but not in A549 cells under the same treatment. Although radiation could not induce PTEN in H1299 cells, a combination of low concentration of BPR0C261 and radiation could reverse this situation. (4) BPR0C261 exhibits specific anticancer effects on NSCLC cells by the enhancement of DNA damage and radiosensitivity with p53-dependent and p53-independent/PTEN-dependent manners. The combination of radiation and BPR0C261 may provide an important strategy for the improvement of radiotherapeutic treatment.

Radiation is a widely used therapeutic method for treating breast cancer. N-dihydrogalactochitosan (GC), a biocompatible immunostimulant, is known to enhance the effects of various treatment modalities in different tumor types. However, whether GC can enhance the radiosensitivity of cancer cells remains to be explored. In this study, triple-negative murine 4T1 breast cancer cells transduced with multi-reporter genes were implanted in immunocompetent Balb/C mice to track, dissect, and identify liver-metastatic 4T1 cells. These cells expressed cancer stem cell (CSC) -related characteristics, including the ability to form spheroids, the expression of the CD44 marker, and the increase of protein stability. We then ex vivo investigated the potential effect of GC on the radiosensitivity of the liver-metastatic 4T1 breast cancer cells and compared the results to those of parental 4T1 cells subjected to the same treatment. The cells were irradiated with increased doses of X-rays with or without GC treatment. Colony formation assays were then performed to determine the survival fractions and radiosensitivity of these cells. We found that GC preferably increased the radiosensitivity of liver-metastatic 4T1 breast cancer cells rather than that of the parental cells. Additionally, the single-cell DNA electrophoresis assay (SCDEA) and &gamma, H2AX foci assay were performed to assess the level of double-stranded DNA breaks (DSBs). Compared to the parental cells, DNA damage was significantly increased in liver-metastatic 4T1 cells after they were treated with GC plus radiation. Further studies on apoptosis showed that this combination treatment increased the sub-G1 population of cells, but not caspase-3 cleavage, in liver-metastatic breast cancer cells. Taken together, the current data suggest that the synergistic effects of GC and irradiation might be used to enhance the efficacy of radiotherapy in treating metastatic tumors.

Colorectal cancer is one of the major causes of cancer-related death in Taiwan and worldwide. Patients with peritoneal metastasis from colorectal cancer have reduced overall survival and poor prognosis. Hybrid protein-inorganic nanoparticle systems have displayed multifunctional applications in solid cancer theranostics. In this study, a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP), which is a hybrid protein-inorganic nanoparticle, and its radioactive surrogate 111In-labeled Au@HSANP (111In-Au@HSANP), were developed and their biological behaviors were investigated in a tumor/ascites mouse model. 111In-Au@HSANP was injected either intravenously (iv) or intraperitoneally (ip) in CT-26 tumor/ascites-bearing mice. After ip injection, a remarkable and sustained radioactivity retention in the abdomen was noticed, based on microSPECT images. After iv injection, however, most of the radioactivity was accumulated in the mononuclear phagocyte system. The results of biodistribution indicated that ip administration was significantly more effective in increasing intraperitoneal concentration and tumor accumulation than iv administration. The ratios of area under the curve (AUC) of the ascites and tumors in the ip-injected group to those in the iv-injected group was 93 and 20, respectively. This study demonstrated that the ip injection route would be a better approach than iv injections for applying gold-albumin nanoparticle in peritoneal metastasis treatment.

The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy.

Despite standard treatment, about 70% of ovarian cancer will recur. Cancer stem cells (CSCs) have been implicated in the drug-resistance mechanism. Several drug resistance mechanisms have been proposed, and among these, autophagy plays a crucial role for the maintenance and tumorigenicity of CSCs. Compared to their differentiated counterparts, CSCs have been demonstrated to display a significantly higher level of autophagy flux. Moreover, mitophagy, a specific type of autophagy that selectively degrades excessive or damaged mitochondria, is shown to contribute to cancer progression and recurrence in several types of tumors. Nanomedicine has been shown to tackle the CSCs problem by overcoming drug resistance. In this work, we developed a nanomedicine, 188Re-liposome, which was demonstrated to target autophagy and mitophagy in the tumor microenvironment. Of note, the inhibition of autophagy and mitophagy could lead to significant tumor inhibition in two xenograft animal models. Lastly, we presented two cases of recurrent ovarian cancer, both in drug resistance status that received a level I dose from a phase I clinical trial. Both cases developing drug resistance showed drug sensitivity to 188Re-liposome. These results suggest that inhibition of autophagy and mitophagy by a nanomedicine may be a novel strategy to overcome drug resistance in ovarian cancer.

Ionizing radiation induces cellular senescence to suppress cancer cell proliferation. However, it also induces deleterious bystander effects in the unirradiated neighboring cells through the release of senescence-associated secretory phenotypes (SASPs) that promote tumor progression. Although autophagy has been reported to promote senescence, its role is still unclear. We previously showed that radiation induces senescence in PTTG1-depleted cancer cells. In this study, we found that autophagy was required for the radiation-induced senescence in PTTG1-depleted breast cancer cells. Inhibition of autophagy caused the cells to switch from radiation-induced senescence to apoptosis. Senescent cancer cells exerted bystander effects by promoting the invasion and migration of unirradiated cells through the release of CSF2 and the subsequently activation of the JAK2-STAT3 and AKT pathways. However, the radiation-induced bystander effects were correlated with the inhibition of endogenous autophagy in bystander cells, which also resulted from the activation of the CSF2-JAK2 pathway. The induction of autophagy by rapamycin reduced the radiation-induced bystander effects. This study reveals, for the first time, the dual role of autophagy in radiation-induced senescence and bystander effects.

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin upregulated in G(1) phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G(1) cells. To investigate the role of cofilin in the control of G(1) phase progression, a tet-on gene expression system was introduced to overexpress different concentrations of cofilin in cells. The results showed that G(1) phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G(1) phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27(kip1) was the primary molecule induced by overexpressed cofilin in a time and dose dependent manner. Upregulated p27(kip1) repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27(kip1) expression in the cofilin overexpressing cells released the G(1) phase arrest. Furthermore, we found that overexpression of cofilin led to induction of p27(kip1) gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27(kip1) mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27(kip1) protein for ubiquitinyl-proteasomal mediated degradation was also involved in upregulation of p27(kip1). These data suggest that cofilin expression and its regulation of p27(kip1) expression is important for the control of G(1) phase progression.

While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells.To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene.In these in vitro models, we showed that amelogenin can activate beta-catenin signaling.Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.

A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity.Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632.When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1.CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.

Tumor growth is dependent on a kinetic model that is based on the progression of cell proliferation and cell loss. The parameters for cell proliferation during tumor progression include the cell-cycle time (Tc), growth fraction (GF), and potential tumor-doubling time (Tpot). In contrast, the cell-loss factor is determined by Tpot and the actual time for doubling of the tumor volume (Td).1 The causes of cell loss include malnutrition and lack of oxygen caused by rapid proliferation, necrosis and apoptosis, immunological attack, escape from the primary site, and exfoliation.2 These conditions can be regarded as stresses for cells residing in a rapidly growing tumor. Whether cells escaping from these stresses inherit or obtain resistance abilities is unknown. The tracking and characterization of living cells in a tumor are important for cancer treatment. Reporter-gene imaging is an indirect approach to labeling cells for image-based in vivo tracking and targeting by different modalities.3 This method is especially important for tracking cell viability in vivo because gene transcription and translation occur only in living cells.4 In addition, the transmission of genes to progeny is in principle not diminished or diluted if the reporter genes can replicate within the genomes of host cells.5 Firefly luciferase and fluorescent proteins are canonical reporter genes used for bioluminescent imaging and optical imaging, respectively. For radionuclide-based reporter-gene imaging, herpes simplex virus type-1 thymidine kinase (HSV1-tk) is commonly used because it can uptake a broad range of radiolabeled nucleoside analogues by substrate phosphorylation for imaging the target cells in vivo.6 Expression of the HSV1-tk reporter gene can be used for living cell tracking by positron emission tomography (PET) or single-photon emission computed tomography (SPECT), depending on the types of radionuclide-labeled substrates. For instance, iodine-123-labeled 5-iodo-2′-fluoro-1-beta-𝒟-arabinofuranosyluracil (123I-FIAU) is the most reliable radiolabeled nucleoside analogues for SPECT imaging of HSV1-tk gene expression because it exhibits high tumor/background ratio in vivo.7, 8 As described above, the sensitivity and specificity of 123I-FIAU on cancer detection is based on the expression of HSV1-tk reporter gene in the transduced cancer cells. These cancer cells can be distinguished from the non-cancer cells without HSV1-tk genes using the radionuclide imaging modality. Technically, cultured cancer cells are stably transduced with HSV1-tk genes and implanted into animals for tumor formation. Using SPECT imaging, this cancer population can be distinguished from non-cancer portion in vivo by injecting 123I-FIAU as a radiotracer that are only accumulated in HSV1-tk gene expressing cancer cells.7 Multimodality reporter-gene imaging using coexpressed luciferase/fluorescent proteins and HSV1-tk has been reported to be a powerful tool for basic biological and preclinical research.9, 10 In addition, PET and SPECT can be merged with computed tomography (CT) to obtain functional/anatomic imaging with high sensitivity and spatial resolution. Although reporter-gene imaging is widely used for functional studies in vivo, concerns regarding the safety and transduction pathways of exogenous genes hamper the clinical application of this approach. Viral-mediated methods are commonly used for the transduction of reporter genes, but the uncontrolled infection and random genomic integration of genes of interest currently limit the clinical application of these methods.11 In contrast, nonviral gene delivery is more acceptable in the clinic because biocompatible materials can be used, and the safety of these approaches is relatively easily assessed by pharmacokinetic and pharmacodynamic studies.12 The piggyBac transposon system has recently attracted a great deal of attention because the system is a nonviral gene-delivery approach that uses DNA-transposition ability for the stable expression of exogenous genes via a ‘cut-and-paste' mechanism.13 The beauty of this system includes its known integration sites at ‘TTAA' sequences and intron-preferred positions, mammalian compatibility, large cargo capacity, and its ability to be removed from the integration site without changing the DNA sequence.14 In practice, this system requires a helper plasmid that encodes the piggyBac transposase gene to facilitate the transposition of exogenous gene.15 The piggyBac transposon system has been applied in induced pluripotent stem cells, cancer research, and immunotherapy.16, 17 However, the use of this system has been less frequently reported in multimodality reporter-gene imaging of tumor progression. Cancer stem cells (CSCs, or tumor-initiating cells) belong to the hierarchy model that a subset of rare cell population inherits stem cell-like characteristics, including self-renewal and generation of non-tumorigenic progeny.18 This theory has intrigued many researchers in recent years because CSCs are resistant to chemoradiotherapy and are likely to be the cause of tumor recurrence and metastasis.19 However, the identification of CSCs in vivo remains a challenge because of the lack of suitable markers for this purpose. If CSCs naturally resist environmental stresses, it would be speculated that this population may also escape from cell loss during tumor progression. More evidence is required to support this hypothesis. The percentage of cell loss during tumor progression is approximately 40–90%, depending on the cancer type.1 The remnant viable cells may be important for promoting tumor growth and metastasis. Reporter-gene imaging should be ideal to track these living cells for further investigation of their characteristics. In this study, we established a syngeneic tumor model derived from 4T1 murine breast carcinomas transduced with monomeric red fluorescent protein (mRFP)/HSV1-tk dual reporter genes using the piggyBac transposon system. A combination of optical imaging and SPECT/CT fusion imaging using 123I-FIAU as a probe was exploited to track the remnant living cells in late-stage primary tumors. Furthermore, ex vivo studies showed that the surviving cells exhibited CSC-like characteristics. These findings may contribute to therapeutic designs for cancer treatment.

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.

Arsenic exerts its cytotoxicity via the generation of reactive oxygen species and inhibition of DNA repair. How arsenic disturbs oxidative DNA damage repair is, however, unclear. We found that arsenic trioxide (ATO), like ultraviolet (UV) irradiation, induced the expression of xeroderma pigmentosum group C (XPC) but not of xeroderma pigmentosum A in a human glioma cell line, U87. To explore the role of XPC in the toxic effects of ATO, small interfering RNA was used to silence XPC (siXPC) in U87 cells. siXPC cells were more susceptible to UV irradiation and ATO-induced cell death than control cells. Increased siXPC cell death induced by ATO was accompanied by increased senescence and autophagy. Because increased DNA strand breaks in siXPC cells were observed only when cells were concomitantly treated with ATO and DNA repair inhibitors, XPC silencing apparently did not interfere with repair of ATO-induced DNA damage. Although intracellular ROS levels were not significantly enhanced in siXPC cells, ATO treatment did result in increased 8-hydroxy-2'-deoxyguanosine and hyperoxidized peroxiredoxin. Enhanced superoxide production and autophagy by ATO in siXPC cells were suppressed by co-incubation with N-acetylcysteine (NAC). Furthermore, XPC silencing caused decreased glutathione levels and increased catalase and Mn-superoxide dismutase activities. Increased catalase activity in siXPC cells was suppressed by ATO treatment. XPC silencing also enhanced reporter activity of activator protein-1, whereas enhanced activity was suppressed by NAC. Taken together, our results indicate that XPC silencing causes increased ATO susceptibility by disturbing redox homeostasis rather than reducing DNA repair.

The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein–protein interactions in translational molecular–genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Methods: Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP:dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP:dMODC or TKGFP and determined by optical imaging and PET. Results: Unlike TKGFP, NESTKGFP:dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP:dMODC occurred in a 26S proteasome–dependent manner. Furthermore, NESTKGFP:dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP:dMODC expression showed that NESTKGFP:dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. Conclusion: In translational molecular–genetic imaging, NESTKGFP:dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.

The tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.

Herpes simplex virus type 1 thymidine kinase (HSV1-TK) is a widely used reporter for in vivo noninvasive monitoring of therapeutic gene expression, immune cell trafficking, and protein‐ protein interactions in various animal systems. However, the stability of HSV1-TK limits its application in studies that require rapid turnover of the reporter. The purpose of this study was to create a destabilized HSV1-TK as a transcription reporter that allows for dynamic studies of short-time-scale gene expression events. Methods: A destabilized HSV1-TK was created by targeting inactivating mutations in the nuclear localization signal of HSV1-TK and fusing the degradation domain of mouse ornithine decarboxylase to the C-terminal end. The protein or enzyme stability was determined by Western blot analysis and HSV1-TK enzyme activity assay, respectively. The proteasome inhibition assay was used to test whether the rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome‐ dependent manner. The suitability of destabilized HSV1-TK as a transcription reporter was tested by linking it to a tetracyclineturnoff‐expressing system. The dynamic transcriptional events mediating a series of doxycycline inductions were monitored by destabilized HSV1-TK or by native HSV1-TK and were determined by an in vitro HSV1-TK enzyme activity assay and in vivo small-animal PET imaging. Results: The destabilized HSV1TK, unlike wild-type HSV1-TK, was unstable in the presence of cycloheximideandhadashorthalf-lifeofproteinandenzymeactivity. The rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome‐dependent manner. Furthermore, thedestabilizedHSV1-TKhadlowcytotoxicitywhenitwashighly expressed in living cells. The results of dynamic gene expression studies invitro and invivo showed that thedestabilized HSV1-TK is an optimal reporter for monitoring short-time-scale dynamic transcriptional events mediating a series of doxycycline inductions, whereas the wild-type HSV1-TK is not optimal to achieve this purpose. Conclusion: The use of destabilized HSV1-TK as a transcription reporter together with a molecular probe, which has a short physical and biologic half-life, allows more direct monitoring of transcription induction and easier monitoring of its coincidence with other biochemical changes.

The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Although several stromal cells and molecular pathways have been identified, the microenvironment of the stem cell niche remains largely unclear. Recent evidence suggests that stem cells are localized in areas with low oxygen. We have hypothesized that hypoxia maintains the undifferentiated phenotype of stem/precursor cells. In this report, we demonstrate that hypoxia reversibly arrests preadipocytes in an undifferentiated state. Consistent with this observation, hypoxia maintains the expression of pref-1, a key stem/precursor cell gene that negatively regulates adipogenic differentiation. We further demonstrate that the hypoxia-inducible factor-1 (HIF-1) constitutes an important mechanism for the inhibition of adipogenic differentiation by hypoxia. Our findings suggest that hypoxia in the stem cell niche is critical for the maintenance of the undifferentiated stem or precursor cell phenotype.

The significance of actin cytoskeleton on cell growth was historically studied using toxic drugs, such as cytochalasin. However, it is possible that unpredictable effects of these agents may have influenced the reported observations. In our study, we have established a drug-free system using cofilin overexpression to investigate the relationship between actin filaments and cell cycle progression. Cofilin is a member of the actin depolymerization factor (ADF)/cofilin family, cofilin cDNA was cloned to a tetracycline-inducible gene expression vector and stably transfected to human lung cancer H1299 epithelial cells. Destabilization of actin filaments and morphological change was detected in cofilin overexpressing cells by actin analysis and microscopy, respectively. Measurements of growth rates showed that cell proliferation was retarded in cells with overexpressed cofilin. Also, cell cycle analysis showed that approx 90% of cofilin overexpressing cells were arrested in G1 phase, which is consistent with previous reports that drug-mediated disruption of actin filaments can cause G1 phase arrest. Taken together, cofilin overexpression cell model provides evidence that the effects of actin cytoskeletal destabilization on cell cycle progression can be studied using molecular approach instead of drug.

D-Amino-acid oxidase (DAO2; EC 1.4.3.3) catalyses the oxidative deamination of D-amino acids to alpha-keto acids and ammonia. The purified DAO protein from Rhodosporidium toruloides was used to determine its amino acid sequence. Three internal peptide sequences, YCQYLARELQ, IAGIDDQAAEPIR and RCTMDSSDP, were obtained and used to synthesize four fully degenerated oligonucleotides for cloning of the DAO gene. Both cDNA and genomic DNA encoding R. toruloides DAO were cloned and sequenced. Comparison of these two DNA sequences revealed that the DAO gene contains six exons and five introns. The gene encodes a polypeptide of 368 amino acids with a calculated molecular mass of 40,079 Da. Using an Escherichia coli protein expression system, the DAO protein of R. toruloides can easily be produced in an active form and purified in a large quantity.

The sublethal dose radiation may induce cellular senescence and promote secondary maligancy during radiotherapy. However, the molecular mechanisms are unclear. It has been reported that a variety of early response oncogenes can be up-regulated by the low dose radiation. Because oncogene-induced senescence (OIS) has been considered important for tumor development, we are interested in investigating whether the sublethal dose radiation also raises OIS to affect the malignancy. To this end, human diploid fibroblasts WI-38 cells and human non-small lung cancer H1299 cells were exposed to less than 2Gy of X-rays delivered by the RS2000 X-rays machine. Both cell types exhibited senescent phenotypes within a dose-dependent manner using the senescence-associated β-galactosidase (SA-β-gal) staining. Subsequently, we investigated the expression of c-Myc and c-Ras oncogenes, and the results showed that sublethal dose radiation could induce c-Myc, but not c-Ras, in both transcriptional and translational levels. Additionally, up-regulated c-Myc exhibited transcriptional activity as demonstrated by the promoter-based reporter gene assys. Although the sublethal dose radiation caused cellular senescence and growth delay in exposed cells, the collected culture medium could increase the motility of non-irradiated H1299 cancer cells using the wound healing assay. Interestingly, the culture medium obtained from irradiated H1299 cancer cells caused higher motility in non-irradiated cells than that from irradiated WI-38 fibroblasts. Furthermore, the sublethal dose radiation induced senescent phenotype was suppressed in cells treated with siRNA against c-Myc. Meanwhile, the motility of non-irradiated cells was not increased by the cultured medium obtained from irradiated c-Myc silenced cells. Taken together, the current data suggest that the sublethal dose radiation causes a c-Myc oncogene induced senescence, and this event may promote the bystandard effects that influence the motility of non-irradiated cancer cells. Citation Format: Chia-Chien Lo, Yen-Ting Chou, Su-Jun Chiu, Jeng-Jung Hwang, Yi-Jang Lee. The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 440. doi:10.1158/1538-7445.AM2013-440 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Metallothionein ( MT ) cDNAs were cloned and sequenced from two genera of ducks, Muscovy ( Cairina muschata ) and Tsai ya ( Anas platyrhynchos ). The two cDNAs show an extremely high sequence homology and contain an open reading frame encoding 63 amino acids. MT mRNA expressions were studied after metal induction using the cloned cDNA as a probe. Cadmium and copper induced MT gene efficiently, whereas zinc showed a markedly less effect. In addition, the MT mRNA accumulations in various developmental stages were also investigated. The result reveals a different pattern of expression from that of mammals. The discrepancy in MT gene between Tsai ya and Muscovy was further explored by examining genomic DNA structures. The duck MT showed three exons and two introns. The most significant variation of the genes occurs at intron II in which Tsai ya MT has 24 bases more than Muscovy MT . Moreover, MT expressions in the hybrids of Muscovy and Tsai ya were investigated using a reverse transcriptase-polymerase chain reaction. Those results demonstrated that both parental MT genes are expressed in the hybrids after metal induction.

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Sestrin 2 (SESN2) is a stress-inducible protein required for maintaining redox homeostasis. However, its mode of action remains to be clarified. In this study, we found that SESN2 is induced in human glioblastoma U87 cells following ionizing radiation (IR). SESN2 silencing not only results in increased oxidative stress but also sensitizes U87 cells to IR. Intriguingly, we found SESN2 silencing significantly increases the expression of platelet-derived growth factor receptor β (PDGFRβ). Using a double knockdown technique, we showed that inhibition of PDGFRβ accumulation in SESN2-silencing cells protects the cells from the deleterious effects induced by SESN2 silencing. Taken together, we revealed that PDGFRβ accumulation is associated with increased oxidative stress and cellular damage in SESN2 silenced human glioblastoma U87 cells.