[4] Isolation and use of liver cells

Moldéus, Peter; Högberg, Johan; Orrenius, Sten

doi:10.1016/s0076-6879(78)52006-5

Cited by 1,099 publications

(389 citation statements)

References 20 publications

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“…3D). Therefore, we prepared primary mouse hepatocytes 14 to transfect with either 1.3xHBV or 1.3xHBV- X KO , an HBV point mutant which does not express the HBx protein. Remarkably, the wild-type, but not the mutant HBV that is defective in HBx expression, induced R2 transcription (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Hepatitis B Virus Activates Deoxynucleotide Synthesis in Nondividing Hepatocytes by Targeting the R2 Gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2010

Hepatology

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Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 10 7 to 10 11 virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA-transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene. Conclusion: Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV-host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs. (HEPATOLOGY 2010;51:1538-1546 H epatitis B virus (HBV) is a widespread pathogen responsible for acute and chronic hepatitis and is a causative factor of hepatocellular carcinoma (HCC). 1 HBV is a small-enveloped noncytopathic virus containing a circular partially doublestranded DNA genome, and exhibits strong tropism for human liver cells. 2 During replication, the viral polymerase reverse-transcribes the pregenomic RNA to DNA using deoxyribonucleotide triphosphates (dNTPs). HBV preferentially replicates in nondividing cells, 3 in which the concentration of dNTP is low, which raised the question whether dNTP concentration is adequate to support viral yield. The level of dNTPs in a nondividing adult liver cell is <0.4 lM. 4 The Michaelis constant (K m ) of the viral polymerase at a dNTP concentration of 0.4 lM is only 62%, whereas a 4 lM concentration gives 93.3% activity. 5 Furthermore, one hepatocyte produces 50-300 hepatitis B virions per day, 6 and because the HBV genome is approximately 3.2 kilobases, between 3 Â 10 5 to 2 Â 10 6 dNTPs are consumed per day in this process. Considering cell volume as 500 fL, 7 a resting cell contains approximately 1.2 Â 10 5 dNTP molecules. Thus, the total amount of dNTPs used for HBV production per day exceeds the amount found in a nondividing hepatocyte. Because HBV does not activate the cell cycl...

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Section: Resultsmentioning

confidence: 99%

“…Isolation of primary mouse hepatocytes was done according to the method of Moldeus et al 14 The method is based on collagenase digestion and separation of liver parenchymal cells.…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus Activates Deoxynucleotide Synthesis in Nondividing Hepatocytes by Targeting the R2 Gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2010

Hepatology

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“…Method Hepatocytes were isolated from male Sprague Dawley rats (180-260 g), which had ad libitum access to food and water, by collagenase (from Gibco BRL Life Technologies, Paisley, Scotland) perfusion (Moldeus et al, 1978). Viability of the hepatocyte suspensions was typically 80-95%, as determined by Trypan Blue exclusion.…”

Section: 2mentioning

confidence: 99%

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

Stevenson

Grant

2005

Bioreactors for Tissue Engineering

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“…The LDH activity was measured 26 by mixing 10 ml of the medium with 10 ml of 50 mM sodium pyruvate (Merck, Amsterdam, The Netherlands) solution in 1 ml TEA buffer (containing 500 mM triethanolamine, 100 mM MgCl 2 , 50 mM EDTA and 0.2 mM NADH, (Sigma Aldrich Chemie, Zwijndrecht, The Netherlands)).…”

Section: Mtt Assay and Ldh Releasementioning

confidence: 99%

The protective effect of cardiac gene transfer of CuZn–sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells

et al. 2003

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Doxorubicin-induced cardiotoxicity is related to its production of free radicals that specifically affect heart tissue because of its low antioxidant status. Monohydroxyethylrutoside (monoHER), a potent antioxidant flavonoid, is under development as a protector against doxorubicin-induced cardiotoxicity. The overexpression of high levels of superoxide dismutase (sod) protects against free radical damage in transgenic mice. Seeking alternatives besides the few cardioprotectors that are presently under investigation, the aim of the present study was to investigate the protective effect of cardiac gene transfer of CuZn-sod compared with that of the presently most promising cardioprotector monoHER against doxorubicin-induced cardiotoxic effects on neonatal rat cardiac myocytes (NeRCaMs) in vitro. NeRCaMs were infected with different multiplicity of infections (MOIs) of adenovirus encoding CuZn-sod (AdCuZn-sod). A control infection with an adenovirus vector encoding a nonrelated protein was included. The overexpression of CuZn-sod was characterized within 3 days postinfection.For doxorubicin treatment, NeRCaMs were divided into three groups. The first group was infected with AdCuZn-sod before treatment with doxorubicin (0-50 mM). The second and third groups were treated with doxorubicin (0-50 mM) alone and with 1 mM monoHER, respectively. The LDH release and survival of treated cells were measured 24 and 48 hours after doxorubicin treatment. The beating rate was followed during the 3 days after doxorubicin (0-100 mM) treatment.At the third day after infection with an MOI of 25 plaque-forming unit (PFU) of AdCuZn-sod/cell, the activity of CuZn-sod significantly increased (five-fold, P ¼ .029). Higher MOI produced cytopathic effects (CPEs).Doxorubicin alone produced significant concentration-and time-dependent reduction in NeRCaMs beating rate and survival (Po.0005). Doxorubicin (X50 mM)-treated cells ceased to beat after 24 hours. This cytotoxicity was associated with an increase in the LDH release from the treated cells (Po.0005).The five-fold increase in the activity of CuZn-sod did not protect against any of the cytotoxic effects of doxorubicin on NeRCaMs. In contrast, monoHER (1 mM) protected against the lethal effects of doxorubicin on the survival, LDH release and the beating rate of NeRCaMs (Po.004) during 48 hours after doxorubicin treatment. Doxorubicin-treated (r100 mM) cells continued beating for 472 hours in the presence of monoHER.The present study showed the lack of adenoviral CuZn-sod gene-transfer to protect myocardiocytes against doxorubicin-induced toxicity and confirms the efficacy of monoHER cardioprotection. Thus, a gene-therapy strategy involving overexpression of CuZnsod to protect against doxorubicin-induced cardiotoxicity is not feasible with the currently available adenovirus vectors.

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[4] Isolation and use of liver cells

Cited by 1,099 publications

References 20 publications

Hepatitis B Virus Activates Deoxynucleotide Synthesis in Nondividing Hepatocytes by Targeting the R2 Gene

Hepatitis B Virus Activates Deoxynucleotide Synthesis in Nondividing Hepatocytes by Targeting the R2 Gene

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

The protective effect of cardiac gene transfer of CuZn–sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells

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