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Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells.

The tumor-homing ability of monocytes renders them a potential cellular delivery system for alternative cancer therapies, although their migratory ability can be impaired following reagent uptake. Approaches that enhance monocyte tumor homing and promote their migration will improve the clinical value of these cells as cellular carriers. Previous studies have shown that irradiation (IR) can promote macrophage aggregation in hypoxic regions. To investigate whether IR enhances the infiltration of bone marrow-derived monocytes (BMDMs) into tumors, the infiltration of BMDMs from GFP-transgenic mice in a murine prostate adenocarcinoma TRAMP-C1 model was examined by fluorescence microscopy. IR did not increase the number of BMDMs that infiltrated initially, but did increase monocyte retention within IR-treated tumors for up to 2 weeks. We also showed that BMDMs can take up various imaging and therapeutic agents, although the mobility of BMDMs decreased with increasing load. When BMDMs were differentiated in IR-treated tumor-conditioned medium (IR-CM) in vitro, the nanoparticle load-mediated inhibition of migration was attenuated. These IR-CM-differentiated BMDMs delivered polymer vesicles encapsulating doxorubicin to radiation therapy (RT)-induced hypoxic tumor regions, and enhanced the efficacy of RT. The prolonged retention of monocytes within irradiated tumor tissues and the ability of IR-CM to enhance the migratory ability of cargo-laden BMDMs suggest that monocytes pre-conditioned by IR-CM can potentially act as cellular carriers for targeted therapy following conventional RT.

In daily life, humans are exposed to the extremely low-frequency electromagnetic fields (ELF-EMFs) generated by electric appliances, and public concern is increasing regarding the biological effects of such exposure. Numerous studies have yielded inconsistent results regarding the biological effects of ELF-EMF exposure. Here we show that ELF-EMFs activate the ATM-Chk2-p21 pathway in HaCaT cells, inhibiting cell proliferation. To present well-founded results, we comprehensively evaluated the biological effects of ELF-EMFs at the transcriptional, protein, and cellular levels. Human HaCaT cells from an immortalized epidermal keratinocyte cell line were exposed to a 1.5 mT, 60 Hz ELF-EMF for 144 h. The ELF-EMF could cause G1 arrest and decrease colony formation. Protein expression experiments revealed that ELF-EMFs induced the activation of the ATM/Chk2 signaling cascades. In addition, the p21 protein, a regulator of cell cycle progression at G1 and G2/M, exhibited a higher level of expression in exposed HaCaT cells compared with the expression of sham-exposed cells. The ELF-EMF-induced G1 arrest was diminished when the CHK2 gene expression (which encodes checkpoint kinase 2; Chk2) was suppressed by specific small interfering RNA (siRNA). These findings indicate that ELF-EMFs activate the ATM-Chk2-p21 pathway in HaCaT cells, resulting in cell cycle arrest at the G1 phase. Based on the precise control of the ELF-EMF exposure and rigorous sham-exposure experiments, all transcriptional, protein, and cellular level experiments consistently supported the conclusion. This is the first study to confirm that a specific pathway is triggered by ELF-EMF exposure.

Radiation therapy for cancer patients works by ionizing damage to nuclear DNA, primarily by creating double-strand breaks (DSB). A major shortcoming of traditional radiation therapy is the set of side effect associated with its long-range interaction with nearby tissues. Low-energy Auger electrons have the advantage of an extremely short effective range, minimizing damage to healthy tissue. Consequently, the isotope ⁹⁹mTc, an Auger electron source, is currently being studied for its beneficial potential in cancer treatment. We examined the dose effect of a pyrene derivative ⁹⁹mTc complex on plasmid DNA by using gel electrophoresis in both aqueous and methanol solutions. In aqueous solutions, the average yield per decay for double-strand breaks is 0.011±0.005 at low dose range, decreasing to 0.0005±0.0003 in the presence of 1 M dimethyl sulfoxide (DMSO). The apparent yield per decay for single-strand breaks (SSB) is 0.04±0.02, decreasing to approximately a fifth with 1 M DMSO. In methanol, the average yield per decay of DSB is 0.54±0.06 and drops to undetectable levels in 2 M DMSO. The SSB yield per decay is 7.2±0.2, changing to 0.4±0.2 in the presence of 2 M DMSO. The 95% decrease in the yield of DSB in DMSO indicates that the main mechanism for DSB formation is through indirect effect, possibly by cooperative binding or clustering of intercalators. In the presence of non-radioactive ligands at a near saturation concentration, where radioactive Tc compounds do not form large clusters, the yield of SSB stays the same while the yield of DSB decreases to the value in DMSO. DSBs generated by ⁹⁹mTc conjugated to intercalators are primarily caused by indirect effects through clustering.

Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations.

The herpes simplex virus thymidine kinase/ganciclovir (HSV-sr39tk/GCV) system is a well-established prodrug system used in cancer gene therapy. However, this system is currently not effective enough to eradicate malignant tumors completely. This study aimed to evaluate whether co-expression of interleukin-3 (IL-3) could enhance the anti-tumor activity of HSV-sr39tk/GCV prodrug gene therapy using a murine TRAMP-C1 prostate tumor model. In vitro results demonstrated that HSV-sr39tk-transfected cells exhibited enhanced sensitivity to the GCV prodrug, which was not affected by co-expression of the mIL-3 gene. However, in vivo studies showed that co-expression of the mIL-3 gene significantly increased the HSV-sr39tk/GCV-induced tumor growth delay and even cured the tumor. The TRAMP-C1-specific immune response of spleen lymphocytes from mice bearing HSV-sr39tk- and IL-3-expressing TRAMP-C1 tumors was measured by ELISA. Results showed that IL-3-activated IL-4-dominant lymphocytes became IFN-γ- dominant lymphocytes after combined HSV-sr39tk/GCV therapy. The efficacy of combined therapies on tumor regression was reduced when macrophages populations were depleted by carrageenan or NO production was inhibited by administration of the iNOS inhibitor, L-NAME. These results suggest that utilizing a bicistronic vector to express HSV-sr39tk and the IL-3 gene induced an enhanced macrophage- or NO-dependent anti-tumor effect.

Radiation therapy (RT) remains the front-line treatment for high-grade gliomas; however, tumor recurrence remains the main obstacle for the clinical success of RT. Using a murine astrocytoma tumor cell line, ALTS1C1, the present study demonstrates that whole brain irradiation prolonged the survival of tumor-bearing mice, although the mice eventually died associated with increased tumor infiltration. Immunohistochemical (IHC) analysis indicated that RT decreased the microvascular density (MVD) of the primary tumor core, but increased the MVD of the tumor invasion front. RT also increased the number of tumor-associated macrophages (TAMs) and the expression of stromal cell-derived factor-1 (SDF-1) and hypoxia-inducible factor-1 (HIF-1) at the tumor invasion front. SDF-1 expression suppressed by siRNA (SDFkd tumors) showed a decrease in RT-enhanced tumor invasiveness, leading to prolonged survival of mice bearing these tumors. The invasion front in SDFkd tumors showed a lower MVD and TAM density than that in the islands of the control or irradiated ALTS1C1 tumors. Our results indicate that tumor-secreted SDF-1 is one key factor in RT-induced tumor invasiveness, and that it exerts its effect likely through macrophage mobilization and tumor revascularization.

Cordyceps sinensis (CS) has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs), in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS), or LPS plus CS (LPS/CS) for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs), were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE) genes of the three different treatments (CS, LPS, and LPS/CS), which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects of a complex compound can be reliably studied by a complex system like cDNA microarray.

The study of biological systems dynamics requires elucidation of the transitions of steady states. A "small perturbation" approach can provide important information on the "steady state" of a biological system. In our experiments, small perturbations were generated by applying a series of repeating small doses of ultraviolet radiation to a human keratinocyte cell line, HaCaT. The biological response was assessed by monitoring the gene expression profiles using cDNA microarrays. Repeated small doses (10 J/m2) of ultraviolet B (UVB) exposure modulated the expression profiles of two groups of genes in opposite directions. The genes that were up-regulated have functions mainly associated with anti-proliferation/anti-mitogenesis/apoptosis, and the genes that were down-regulated were mainly related to proliferation/mitogenesis/anti-apoptosis. For both groups of genes, repetition of the small doses of UVB caused an immediate response followed by relaxation between successive small perturbations. This cyclic pattern was suppressed when large doses (233 or 582.5 J/m2) of UVB were applied. Our method and results contribute to a foundation for computational systems biology, which implicitly uses the concept of steady state.