Hepa1-6 Cells Research Articles

Overexpression of gankyrin in mouse hepatocytes induces hemangioma by suppressing factor inhibiting hypoxia-inducible factor-1 (FIH-1) and activating hypoxia-inducible factor-1

Gankyrin (also called p28 or PSMD10) is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It consists of 7 ankyrin repeats and interacts with multiple proteins including Rb, Cdk4, MDM2 and NF-κB. To assess the oncogenic activity in vivo, we produced transgenic mice that overexpress gankyrin specifically in the hepatocytes. Unexpectedly, 5 of 7 F2 transgenic mice overexpressing hepatitis B virus X protein (HBX) promoter-driven gankyrin, and one of 3 founder mice overexpressing serum amyloid P component (SAP) promoter-driven gankyrin developed hepatic vascular neoplasms (hemangioma/hemangiosarcomas) whereas none of the wild-type mice did. Endothelial overgrowth was more frequent in the livers of diethylnitrosamine-treated transgenic mice than wild-type mice. Mouse hepatoma Hepa1-6 cells overexpressing gankyrin formed tumors with more vascularity than parental Hepa1-6 cells in the transplanted mouse skin. We found that gankyrin binds to and sequester factor inhibiting hypoxia-inducible factor-1 (FIH-1), which results in decreased interaction between FIH-1 and hypoxia-inducible factor-1α (HIF-1α) and increased activity of HIF-1 to promote VEGF production. The effects of gankyrin were more prominent under 3% O2 than 1% or 20% O2 conditions. Thus, the present study clarified, at least partly, mechanisms of vascular tumorigenesis, and suggests that gankyrin might play a physiological role in hypoxic responses besides its roles as an oncoprotein.

TGF-β1 Secreted by Hepatocellular Carcinoma Induces the Expression of the Foxp3 Gene and Suppresses Antitumor Immunity in the Tumor Microenvironment

The purpose of this study was to explore the mechanisms of TGF-β1 mediated immunosuppression in tumor stroma. The expression of TGF-β1 was investigated in Huh7, Hep 3B, SGC-7901, Eca-109 and Hepa1-6 cell lines using immunofluorescence. Knocked-down TGF-β1 of the Hepa1-6 cell line was established through lentivirus-based RNA interference. The interference efficiency of the TGF-β1 gene was tested by real-time PCR and ELISA; the expression of Foxp3, IFN-γ and CD83 in CD4(+), CD8(+) or dendritic cells was examined via flow cytometry; and the tumorigenic ability of the cancer cells was investigated in the animal experiments. The diverse digestive cancer cells were found to secrete TGF-β1, mRNA of which was knocked down by 78 % thanks to lentivirus-based interference in Hepa1-6 cells. Flow cytometry showed that CD4(+)CD25(+)Foxp3(+) regulatory T cells significantly increased in hepatocellular carcinoma patients when compared with those in the healthy controls. The supernatant from Hepa1-6 cells and recombinant TGF-β1 significantly induced the expression of Foxp3 gene in vitro, while that from sh TGF-β1 Hepa1-6 cells restored it. Hepa1-6 cells inhibited IFN-γ and CD83 expression in CD8(+) or dendritic cells by secreting TGF-β1. The animal experiments indicated that the knockdown TGF-β1 gene impaired the tumorigenic ability of Hepa1-6 cells. TGF-β1, expressed in cancer cells, might be a potential therapeutic target for cancer treatment.

Nrf2-induced antiapoptotic Bcl-xL protein enhances cell survival and drug resistance

Nuclear transcription factor Nrf2 binds with the antioxidant-response element (ARE) in the promoter regions of cytoprotective genes, leading to their increased expression and cellular protection. In this study, we investigated the role of Nrf2 in the regulation of antiapoptotic Bcl-xL protein and its effect on cellular apoptosis. Treatment of mouse Hepa-1 cells with the antioxidant tert-butylhydroquinone led to the induction of Bcl-xL gene expression. Promoter mutagenesis, transfection, and chromatin immunoprecipitation assays identified an ARE between nucleotides −608 and −600 in the forward strand of the proximal Bcl-xL promoter that bound to Nrf2 and led to increased Bcl-xL gene expression. In addition, short interfering RNA (siRNA) inhibition and overexpression of Nrf2 led to a respective decrease and increase in Bcl-xL gene expression. These results implicated Nrf2 in the regulation of expression and induction of Bcl-xL protein. Nrf2-mediated expression of Bcl-xL protein downregulated Bax and decreased caspase 3/7 activity. SiRNA inhibition of both Nrf2 and Bcl-xL increased the susceptibility of cancer cells to etoposide-mediated cell death and reduced cell survival. Moreover, dysfunctional/mutant INrf2 (inhibitor of Nrf2) in human lung cancer cells failed to degrade Nrf2, resulting in increased Bcl-xL levels and increased cell survival. These data provide the first evidence of Nrf2 in the control of Bcl-xL expression and apoptotic cell death with implications for antioxidant protection, survival of cancer cells, and drug resistance.

TRAIL suppresses tumor growth in mice by inducing tumor-infiltrating CD4(+)CD25 (+) Treg apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) but did not affect CD4(+)CD25(+)Foxp3(+) Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4(+)CD25(+)Foxp3(+) Tregs, but not of CD4(+)CD25(-) T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4(+)CD25(+)Foxp3(+) Tregs, but not on naïve CD4(+)CD25(+)Foxp3(+) Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4(+)CD25(+)Foxp3(+) Tregs but also increased the number of tumor-specific CD8(+) CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.

Lactoferrin and its hydrolysate bind directly to the oleate-activated form of the lipolysis stimulated lipoprotein receptor

The hepatic removal of triglyceride-rich chylomicrons during the postprandial phase represents an important step towards determining the bioavailability of dietary lipids amongst the peripheral tissues. Indeed, elevated postprandial lipemia is often associated with obesity and increased risk of coronary heart disease. The milk protein, lactoferrin, has been shown to inhibit hepatic chylomicron remnant removal by the liver, resulting in increased postprandial lipemia. Despite numerous studies on potential targets for lactoferrin, the molecular mechanisms underlying the effect of lactoferrin remain unclear. We recently demonstrated that the lipolysis stimulated lipoprotein receptor (LSR) contributes to the removal of triglyceride-rich lipoproteins during the postprandial phase. Here, we report that while lactoferrin does not have any significant effect on LSR protein levels in mouse Hepa1-6 cells, this protein colocalizes with LSR in cells but only in the presence of oleate, which is needed to obtain LSR in its active form as lipoprotein receptor. Ligand blotting using purified LSR revealed that lactoferrin binds directly to the receptor in the presence of oleate and prevents the binding of triglyceride-rich lipoproteins. Both C- and N-lobes of lactoferrin as well as a mixture of peptides derived from its hydrolysis retained the ability to bind LSR in its active form. We propose then that the elevated postprandial lipemia observed upon lactoferrin treatment in vivo is mediated in part by its direct interaction with free fatty acid activated LSR, thus preventing clearance of chylomicrons and their remnants through the LSR pathway.

The role of Smad ubiquitination regulatory factor 1 in hepatocellular carcinoma

To determine the expression level of Smad ubiquitination regulatory factor 1 (SMURF1) gene in hepatocellular carcinoma, and to explore its role in liver cancer. With non-neoplastic adjacent normal tissues as controls, real-time PCR and Western blotting were used for measuring the expression of SMURF1 mRNA and protein in 89 samples of hepatocellular carcinoma. Correlations between SMURF1 expression and clinical features were explored. Following transfection of SMURF1--specific small interference RNA(siRNA), the apoptosis and proliferation of hepatic cancer cells Hep G2 were detected using flow cytometry and MTT assays . The expression of SMURF1 mRNA and protein were significantly higher in hepatocellular cancer tissues compared with the paired normal tissues (P< 0.05). The expression of SMURF1 however did not correlate with any clinical features (P> 0.05). Transfection of SMURF1-specific siRNA can promote the apoptosis whilst inhibit the proliferation of Hep G2 cells. The expression of SMURF1 is enhanced in hepatocellular carcinoma, which may have played a role in the disease through affecting apoptosis and proliferation of hepatic cancer cells.

Effect of enhanced expression of COL8A1 on lymphatic metastasis of hepatocellular carcinoma in mice

The present study aimed to investigate the influence of COL8A1 expression on cell invasiveness, drug sensitivity and tumorigenicity of hepatocellular carcinoma Hepa1-6 cells with low metastatic potential. COL8A1-1-pEGFP-N2 and pEGFP-N2 were transfected into experimental and control group cells. The COL8A1 expression in transfected Hepa1-6 cells was analyzed with RT-PCR and western blot analysis. The invasive potential of transfected Hepa1-6 cells was tested in invasion experiments in vitro and the tumorigenic ability of the transfected Hepa1-6 cells was tested in mouse tumors in vivo. Hepa1-6 cell proliferation and D-limonene sensitivity was analyzed using the MTT method. Expression of COL8A1 in the Hepa1-6/COL8A1 group showed a significant increase when compared with the untransfected cells of the Hepa1-6 control group and empty-plasmid transfected cells from the Hepa1-6/mock control group. Enhanced COL8A1 expression increased cell proliferation and matrix adhesion ability via invasion and tumorigenesis in vivo while the sensitivity to D-limonene was concurrently inhibited. The expression of COL8A1 in hepatocarcinoma cells was correlated with increased tumor cell proliferation, invasion, in vivo tumorigenicity and reduced antitumor drug sensitivity, and may provide novel targets for tumor therapy.

Diet-induced obesity and ethanol impair progression of hepatocellular carcinoma in a mouse mesenteric vein injection model

Hepatocellular carcinoma (HCC) is a rapidly increasing cancer whose known risk factors are chronic ethanol abuse, viral hepatitis infection, and aflatoxin exposure. Obesity, an emerging HCC risk factor, is reaching epidemic proportions in developed nations. This study investigated the effects of diet-induced obesity (DIO) and chronic ethanol consumption on HCC progression in mice in vivo. In this study, C57BL/6 DIO mice and lean litter mates were maintained on a 60% (high-fat diet [HFD]) diet or a 10% (control diet [CD]) kcal% fat diet for 7 weeks before they were weaned to 10/20% ([v/v], alternating days) ethanol in drinking water (EtOH) or maintenance on drinking water (H(2)O) alone. Hepatic tumor formation was initiated by intrahepatic Hepa1-6 cell (6 × 10(6) cells) inoculation 6 weeks later via the mesenteric vein. The animals receiving the HFD showed decreased tumor incidence and area of hepatic foci versus the CD animals maintained on H(2)O alone. The action of EtOH suppressed tumor incidence further in both the CD and the HFD mice. Serologic analysis showed no significant differences in liver enzymes among the groups. Protein analysis demonstrated increased P450 2E1 (CYP2E1) in the groups maintained on EtOH, an effect exacerbated by HFD. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated increased tumor necrosis factor-alpha (TNF-α) expression in HFD HCC mice (H(2)O and EtOH) concomitant with decreased transforming growth factor-beta (TGF-β) expression. Although obesity and EtOH consumption are known risk factors for HCC initiation and development, the data in this study suggest that these factors impair progression of established tumors within the liver.

In Vitro–In Vivo Translation of Lipid Nanoparticles for Hepatocellular siRNA Delivery

A significant challenge in the development of clinically viable siRNA delivery systems is a lack of in vitro-in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time associated with generating in vitro-in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examination of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple experimental systems. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro experiments was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when <300 nm) as measured by dynamic light scattering were not. These data provide guiding principles in the development of clinically viable siRNA delivery materials and have the potential to reduce experimental costs while improving the translation of materials into animals.

N-acetylglucosaminyltransferase IVa regulates metastatic potential of mouse hepatocarcinoma cells through glycosylation of CD147

N-acetylglucosaminyltransferase (GnT)-IV a is a key enzyme that catalyzes the formation of the GlcNAC β1-4 branch on the core structure of complex N-Glycans, which is the common substrate for other N-acetylglucosaminyltransferases, such as GnT-III and GnT-V. Our recent study indicates that the expression of GnT-IVa in Hca-F cells was much higher than that in Hepa1-6 cells, these two mouse hepatocarcinoma cell lines have high and no metastatic potential in lymph nodes respectively. To investigate the effects of GnT-IVa on the metastasis of hepatocarcinoma, exogenous GnT-IVa was introduced into Hepa1-6 cells, and on the other hand, the expression of GnT-IVa was down-regulated in Hca-F cells. The engineered overexpression of GnT-IVa in Hepa1-6 cells increased the antennary branches of complex N-glycans and reduced bisecting branches in vitro and in vivo, which leads to the increase in migration and metastatic capability of hepatocarcinoma cells. Conversely, down-regulated expression of GnT-IVa in Hca-F cells showed reduced tetra-antennary branches of N-Glycans, and significantly decreased the migration and metastatic capability. Furthermore, we found that the regulated GnT-IVa converts the heterogeneous N-glycosylated forms of CD147 in Hepa1-6 and Hca-F cells, and significantly changed the antennary oligosaccharide structures on CD147. These results suggest that GnT-IVa could be acting as a key role in migration and metastasis of mouse hepatocarcinoma cells through altering the glycosylation of CD147. These findings should be valuable in delineating the important function of GnT-IVa during the process of hepatocarcinoma growth and metastasis.

Growth Inhibition and Apoptosis Induced by Osthole, A Natural Coumarin, in Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most commonly diagnosed tumors worldwide and is known to be resistant to conventional chemotherapy. New therapeutic strategies are urgently needed for treating HCC. Osthole, a natural coumarin derivative, has been shown to have anti-tumor activity. However, the effects of osthole on HCC have not yet been reported.Methods and FindingsHCC cell lines were treated with osthole at various concentrations for 24, 48 and 72 hours. The proliferations of the HCC cells were measured by MTT assays. Cell cycle distribution and apoptosis were determined by flow cytometry. HCC tumor models were established in mice by subcutaneously injection of SMMC-7721 or Hepa1-6 cells and the effect of osthole on tumor growths in vivo and the drug toxicity were studied. NF-κB activity after osthole treatment was determined by electrophoretic mobility shift assays and the expression of caspase-3 was measured by western blotting. The expression levels of other apoptosis-related genes were also determined by real-time PCR (PCR array) assays. Osthole displayed a dose- and time-dependent inhibition of the HCC cell proliferations in vitro. It also induced apoptosis and caused cell accumulation in G2 phase. Osthole could significantly suppress HCC tumor growth in vivo with no toxicity at the dose we used. NF-κB activity was significantly suppressed by osthole at the dose- and time-dependent manner. The cleaved caspase-3 was also increased by osthole treatment. The expression levels of some apoptosis-related genes that belong to TNF ligand family, TNF receptor family, Bcl-2 family, caspase family, TRAF family, death domain family, CIDE domain and death effector domain family and CARD family were all increased with osthole treatment.ConclusionOsthole could significantly inhibit HCC growth in vitro and in vivo through cell cycle arrest and inducing apoptosis by suppressing NF-κB activity and promoting the expressions of apoptosis-related genes.

Abstract 4427: Role of cytochrome P450 (CYP)1A2 in the sustained induction of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1)

Abstract Humans are constantly exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (PAHs) through cigarette smoke, diesel exhausts, charcoal-broiled meats, etc. Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC ithdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in Cyp1a2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 nduction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this henomenon. Hepa-1 cells were treated ith the MC (2.5 µM), or dimethylsulfoxide (DMSO) as control, and at selected ime points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. We fund that MC markedly and persistently induced CYP1A1 promoter activity, transcription, apoprotein expression, and the CYP1A1 associated ethoxyresorufin O-deethylase (EROD) activities for up to 5 days. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter ctivity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly ttenuated by Cyp1a2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans. (Supported in part by 2R01 ES009132.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4427. doi:1538-7445.AM2012-4427

Efficient siRNA Delivery Using a Polyamidoamine Dendrimer with a Modified Pentaerythritol Core

Delivery of siRNA into cells remains a critical challenge. Our lab has shown a novel polyamidoamine (PAMAM) dendrimer with modified pentaerythritol derivative core (PD dendrimer) to exhibit high plasmid DNA transfection efficiency and low cytotoxicity. Here, we evaluate PD dendrimer as a siRNA carrier. Agarose gel electrophoresis and AFM were used to confirm formation of generation 5 (G5)-PD dendrimer/siRNA nanoparticles (NPs). G5 PD dendrimer/anti-luciferase siRNA NPs were used to transfect SK Hep-1 cells with stable luciferase expression. Effects of various endocytic pathway inhibitors on uptake of G5 PD dendrimer/siRNA NPs in SK Hep-1 cells were also investigated. Agarose gel electrophoresis indicated that G5 PD dendrimer and siRNA formed NPs at weight ratios >0.5:1. G5 PD dendrimer showed effective luciferase gene silencing when weight ratio was 3.0:1 and above. Treatment with endocytosis inhibitors showed that clathrin-mediated endocytosis was the main endocytic pathway by which G5-PD dendrimer/siRNA NPs enter the cell. These results show that the novel G5 PD dendrimer has high siRNA delivery activity and is promising as a delivery agent for its therapeutic application.

Hepatitis B vaccine induces apoptotic death in Hepa1–6 cells

Vaccines can have adverse side-effects, and these are predominantly associated with the inclusion of chemical additives such as aluminum hydroxide adjuvant. The objective of this study was to establish an in vitro model system amenable to mechanistic investigations of cytotoxicity induced by hepatitis B vaccine, and to investigate the mechanisms of vaccine-induced cell death. The mouse liver hepatoma cell line Hepa1-6 was treated with two doses of adjuvanted (aluminium hydroxide) hepatitis B vaccine (0.5 and 1μg protein per ml) and cell integrity was measured after 24, 48 and 72h. Hepatitis B vaccine exposure increased cell apoptosis as detected by flow cytometry and TUNEL assay. Vaccine exposure was accompanied by significant increases in the levels of activated caspase 3, a key effector caspase in the apoptosis cascade. Early transcriptional events were detected by qRT-PCR. We report that hepatitis B vaccine exposure resulted in significant upregulation of the key genes encoding caspase 7, caspase 9, Inhibitor caspase-activated DNase (ICAD), Rho-associated coiled-coil containing protein kinase 1 (ROCK-1), and Apoptotic protease activating factor 1 (Apaf-1). Upregulation of cleaved caspase 3,7 were detected by western blot in addition to Apaf-1 and caspase 9 expressions argues that cell death takes place via the intrinsic apoptotic pathway in which release of cytochrome c from the mitochondria triggers the assembly of a caspase activation complex. We conclude that exposure of Hepa1-6 cells to a low dose of adjuvanted hepatitis B vaccine leads to loss of mitochondrial integrity, apoptosis induction, and cell death, apoptosis effect was observed also in C2C12 mouse myoblast cell line after treated with low dose of vaccine (0.3, 0.1, 0.05μg/ml). In addition In vivo apoptotic effect of hepatitis B vaccine was observed in mouse liver.

Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs) is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm) are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

SiRNA-optimized Modifications for Enhanced In Vivo Activity

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

Mouse hepatoma cell lines differing in aryl hydrocarbon receptor-mediated signaling have different activities for glucuronidation

For studies on the aryl hydrocarbon receptor (AhR)-dependent toxicity of the mycotoxins alternariol (AOH) and alternariol methyl ether (AME), three mouse hepatoma (Hepa-1) cell lines with intact and with compromised AhR signaling were compared with respect to their activities for hydroxylation, methylation, and glucuronidation. Whereas the activities of cytochrome P450-mediated monooxygenase and catechol-O-methyl transferase were very low and did not differ between the three cell lines, a pronounced difference was observed for UDP-glucuronosyl transferase activity, which was much higher in Hepa-1c1c4 than in c1c7 and c1c12 cells. In all three cell types, the rate of glucuronidation of AOH was about four times higher than that of AME. Whereas AME caused a concentration-dependent G2/M arrest in each cell line, AOH arrested Hepa-1c1c7 and c1c12 cells but not c1c4 cells. However, Hepa-1c1c4 cells were arrested by AOH when β-glucuronidase was added to the incubation medium in order to reverse the formation of AOH glucuronides. We conclude that the failure of AOH to cause cell cycle inhibition in Hepa-1c1c4 cells is due to its efficient glucuronidation. The considerable UDP-glucuronosyl transferase activity of Hepa-1c1c4 cells should be taken into account when other compounds are studied in this cell line. Moreover, we demonstrate that differences in glucuronide formation between cell types can be overcome by the addition of β-glucuronidase to the cell culture medium.

The heterotrimeric G protein subunits Gαq and Gβ1 have lysophospholipase D activity

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gαq and Gβ1 respectively. When FLAG-tagged Gαq or Gβ1 was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg2+ dependency and substrate specificity of Gαq were similar to those of lysoPLD purified from the rat brain. Mutation of Gαq at amino acids Lys52, Thr186 or Asp205, residues that are predicted to interact with nucleotide phosphates or catalytic Mg2+, dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gαq overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated Km and Vmax values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gαq and Gβ1 as an enzyme with lysoPLD activity. Tag-purified Gα11 also exhibited a high lysoPLD activity, but Gαi and Gαs did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gαq and Gα11 siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.

Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors

Hepatoma cells, which are derived from liver carcinoma, are able to proliferate infinitely under culture conditions. However, the liver functions of hepatoma cells are generally low compared with those of hepatocytes in a liver. Here, we attempted to create genetically engineered hepatoma cells with enhanced liver functions by overexpression of liver-enriched transcription factors (LETFs), which are associated with the transcription of liver-specific genes and hepatic differentiation. For this purpose, genes for eight LETFs, hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β, HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ, were obtained from the mouse liver. Mouse hepatoma Hepa1-6 cells were transduced with retroviral vectors, in which inducible expression cassettes for the LETF genes were introduced. Cell clones with inducible expression of high liver functions were established. Upon overexpression of the LETF genes, cell proliferation ceased and the cells exhibited an epithelial morphology, indicating hepatic maturation of hepatoma cells. This approach for genetic modification of hepatoma cells may be promising for the construction of cells for use in bioartificial liver support systems.

Response of peripheral blood mononuclear cells to conditioned medium from cultured oral squamous cell carcinomas

The current study investigated the capacity for tumor factors secreted by head and neck squamous cell carcinoma (HNSCC) cell lines, KB, KB16, and HEP, to induce the secretion of various cytokines from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples collected from six healthy volunteers and these cells were incubated for 6, 24, 48, or 72 hours in the presence of 50% conditioned medium collected from cultured cell lines pretreated with, or without, stimulants such as phytohemagglutinin (PHA) or lipopolysaccharides (LPS). Aliquots of each supernatant were then assayed for levels of IFN-Γ, vascular endothelial growth factor (VEGF), TNF-α, and IL-4 using enzyme linked immunosorbent assays (ELISAs). Data collected were analyzed using Student's t-test, an ANOVA test followed by Tukey's test, and tests of Pearson's Correlation. PBMCs cultured with KB16-conditioned medium produced the highest levels of IFN-Γ. VEGF was also detected in conditioned media collected from all of the squamous cell carcinoma (SCC) cell lines used, and a significant difference in VEGF levels between control and KB- or KB16-conditioned media was observed. TNF-α was secreted by all PBMC groups within 6 hours of receiving conditioned media, and these levels increased up to the 24 hour timepoint, after which levels of TNF-α stabilized. In contrast, none of the supernatant samples contained detectable levels of IL-4. In combination, these data suggest that direct contact between fresh human PBMCs and conditioned media from tumor cells induces the secretion of TNF-α and VEGF by PBMCs, and this represents an initial angiogenic response.