Daniel Goldenberg

Differential mRNA stability of the cspA gene in the cold-shock response of Escherichia coli

Exposure of bacterial cells to temperature changes induces the synthesis of a set proteins. We investigated the control of expression of the cspA gene, coding for the major cold‐shock protein of Escherichia coli. This protein was shown to be transiently induced upon shift to low temperature. We demonstrated that the cspA mRNA is extremely unstable at 37°C with a half‐life of approx. 10 s. Upon shift to 15°C cspA mRNA becomes highly stable. This mRNA stability is transient and is lost once the cells are adapted to the low temperature. Transcription fusions of lacZ containing part or most of the cspA gene do not show the rapid degradation at high temperature. Our results suggest that mRNA stability plays a major role in the control of the cspA gene. The expression of cspA is also regulated, to a smaller extent, by the relative increase in transcription after transfer to low temperature. A model by which cspA mRNA is regulated in response to temperature shift is discussed.

HCV Causes Chronic Endoplasmic Reticulum Stress Leading to Adaptation and Interference with the Unfolded Protein Response

Background The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance. Methods and Findings The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells. Conclusions Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.

Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is considered to be the outcome of chronic liver inflammation. Currently, the main treatment for HCC is surgical resection. However, survival rates are suboptimal partially because of tumor recurrence in the remaining liver. Our aim was to understand the molecular mechanisms linking liver regeneration under chronic inflammation to hepatic tumorigenesis. Mdr2-KO mice, a model of inflammation-associated cancer, underwent partial hepatectomy (PHx), which led to enhanced hepatocarcinogenesis. Moreover, liver regeneration in these mice was severely attenuated. We demonstrate the activation of the DNA damage-response machinery and increased genomic instability during early liver inflammatory stages resulting in hepatocyte apoptosis, cell-cycle arrest, and senescence and suggest their involvement in tumor growth acceleration subsequent to PHx. We propose that under the regenerative proliferative stress induced by liver resection, the genomic unstable hepatocytes generated during chronic inflammation escape senescence and apoptosis and reenter the cell cycle, triggering the enhanced tumorigenesis. Thus, we clarify the immediate and long-term contributions of the DNA damage response to HCC development and recurrence.

Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the Mdr2-knockout mice.

Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model.

Molecular Mechanisms of Liver Carcinogenesis in the Mdr2-Knockout Mice

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of β-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for β-catenin–negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes. (Mol Cancer Res 2007;5(11):1159–70)

Analysis of differentially expressed genes in hepatocellular carcinoma using cDNA arrays

Hepatocellular carcinoma (HCC) is characterized by multiple somatic mutations, including DNA rearrangements, that affect many cell‐growth regulatory pathways. Many genes differentially expressed in HCC have been reported previously, but the patterns of expression varied significantly between patients who bore different risk factors for HCC. To identify genes whose differential expression could serve as a “signature” for diagnosis and prognosis of HCC, we performed analyses of differentially expressed genes in three cases of HCC with different risk factors using the Atlas Human Cancer cDNA Expression Arrays. Among all 597 genes present on the array, only three were found to be coordinately differentially expressed in all three HCC cases, in agreement with published data. These three genes, Cu/Zn superoxide dismutase, osteonectin/secreted protein acidic and rich in cysteine, and matrix metalloproteinase 14, could serve as candidates for the HCC “signature.” Ten genes were found to be coordinately differentially expressed in only two of three tested HCC cases. On the other hand, many genes that had been reported previously as differentially expressed in HCC failed to show the described pattern of expression in this group. The results of this study confirm the great variability in gene‐expression patterns in HCC and establish the utility of the array technology for identifying both the HCC signature genes and individual gene‐expression patterns for purposes of patient‐oriented therapy.

Inflammation‐induced hepatocellular carcinoma is dependent on CCR5 in mice

Human hepatocellular carcinoma (HCC) is an inflammation‐induced cancer, which is the third‐leading cause of cancer mortality worldwide. We investigated the role of the chemokine receptors, CCR5 and CCR1, in regulating inflammation and tumorigenesis in an inflammation‐induced HCC model in mice. Multidrug resistance 2 gene (Mdr2)‐knockout (Mdr2‐KO) mice spontaneously develop chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC. We generated two new strains from the Mdr2‐KO mouse, the Mdr2:CCR5 and the Mdr2:CCR1 double knockouts (DKOs), and set out to compare inflammation and tumorigenesis among these strains. We found that in Mdr2‐KO mice lacking the chemokine receptor, CCR5 (Mdr2:CCR5 DKO mice), but not CCR1 (Mdr2:CCR1 DKO), macrophage recruitment and trafficking to the liver was significantly reduced. Furthermore, in the absence of CCR5, reduced inflammation was also associated with reduced periductal accumulation of CD24+ oval cells and abrogation of fibrosis. DKO mice for Mdr2 and CCR5 exhibited a significant decrease in tumor incidence and size. Conclusions: Our results indicate that CCR5 has a critical role in both the development and progression of liver cancer. Therefore, we propose that a CCR5 antagonist can serve for HCC cancer prevention and treatment. (Hepatology 2013;53:1021–1030)

Molecular mechanisms of the chemopreventive effect on hepatocellular carcinoma development in Mdr2 knockout mice

Dietary antioxidants and selenium compounds were shown to have a therapeutic effect against hepatocellular carcinoma in several mouse models. We tested the effects of tannic acid and selenomethionine on hepatocellular carcinoma development in Mdr2 knockout (Mdr2-KO) mice. Mdr2-KO and age-matched Mdr2 heterozygous control mice were fed with tannic acid or selenomethionine during the first 3 months of life. Then, several mice from each group were sacrificed, and liver tissue samples were removed for analysis. The remaining mice were fed a regular diet until the age of 16 months, at which time the number and size of liver tumors were determined. Liver tissue samples of 3-month-old mice were subjected to gene expression profiling analysis using cDNA macroarrays containing probes for 240 genes that regulate responses to oxidative stress and inflammation or lipid metabolism. Both tannic acid and selenomethionine had partial chemopreventive effect on development of hepatocellular carcinoma in Mdr2-KO mice: they reduced the incidence of large tumor nodules (diameter >1 cm) at age 16 months. Both agents inhibited gene expression and reversed up-regulation of many genes that control inflammation or response to oxidative stress in Mdr2-KO livers at age 3 months. This inhibitory effect on gene expression correlated with the ability of agents to reduce incidence of large tumors: selenomethionine was more active than tannic acid in both aspects. Understanding the molecular mechanism of chemoprevention effect could improve our therapeutic modalities while using these agents. [Mol Cancer Ther 2007;6(4):1283–91]

Interstrain differences in chronic hepatitis and tumor development in a murine model of inflammation‐mediated hepatocarcinogenesis

Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)–knockout (KO) mouse (adenosine triphosphate–binding cassette b4−/−), a model of inflammation‐mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2‐KO strains: Friend virus B‐type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2‐KO/B6 mice versus the Mdr2‐KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2‐KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age‐matched Mdr2‐KO/FVB males. A comparative genome‐scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain‐specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin‐1 (Gal‐1), possesses both anti‐inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal‐1–KO mutation (lectin galactoside‐binding soluble 1−/−) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal‐1 protected the liver against concanavalin A–induced hepatitis with the B6 genetic background but not the FVB genetic background. Conclusion: Decreased chronic hepatitis in Mdr2‐KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2‐KO/FVB strain. We found candidate factors that may determine strain‐specific differences in the course of chronic hepatitis and HCC development in the Mdr2‐KO model, including inefficient anti‐inflammatory activity of the endogenous lectin Gal‐1 in the FVB strain. (HEPATOLOGY 2013 )

HCV Tumor Promoting Effect Is Dependent on Host Genetic Background

Background The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. Methodology/Principal Findings We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. Conclusion These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner.