Jocelyn H. Wright

RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability.

The protein encoded by the RAP1 gene of S. cerevisiae binds in vitro to a consensus sequence occurring at a number of sites in the yeast genome, including the repeated sequence C2-3A(CA)1-6 found at yeast telomeres. We present two lines of evidence for the in vivo binding of RAP1 protein at telomeres: first, RAP1 is present in telomeric chromatin and second, alterations in the level of RAP1 protein affect telomere length. The length changes seen with under- and overexpression of RAP1 are consistent with the interpretation that RAP1 binding to telomeres protects them from degradation. Unexpectedly, overproduction of the RAP1 protein was also shown to decrease greatly chromosome stability, suggesting that RAP1 mediates interactions that have a more global effect on chromosome behavior than simply protecting telomeres from degradation. Such interactions may involve telomere associations both with other telomeres and/or with structural elements of the nucleus.

Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice

Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end‐joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ‐deficient (Ku70−/−), heterozygote (Ku70 +/−), and wild‐type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70−/− mice injected with DEN displayed accelerated HCC development. Ku70−/− HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70−/− HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. Conclusion: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC. (HEPATOLOGY 2008;47:2078–2088.)

Expression of the Casein Kinase 2 Subunits in Chinese Hamster Ovary and 3T3 L1 Cells Provides Information on the Role of the Enzyme in Cell Proliferation and the Cell Cycle

In order to investigate the in vivofunctions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2α and Myc-CK2β, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2β subunit exhibited a severe growth defect, as shown by a much lower value of [3H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2α showed a slightly but consistently higher value of [3H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2β overexpression were partially rescued by coexpression of Myc-tagged CK2α. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2α and Myc-CK2β subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2β, but not Myc-CK2α, caused a growth defect. By measuring [3H]thymidine incorporation, it was found that expression of Myc-CK2β prolonged the G1phase and inhibited up-regulation of cyclin D1 expression during G1. In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2β-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2β inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Role of Smad3 in Platelet-Derived Growth Factor-C induced liver fibrosis

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic (PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters

The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines results in serious, potentially life-threatening, adverse effects. By contrast, nucleic acid transfer via electroporation (EP) is a safe and effective method of delivering plasmid-encoded cytokines to tumors. Intratumoral delivery of IL-12 plasmid DNA by electroporation (IT-pIL12-EP) produced objective response rates in Phase 2 clinical trials in metastatic melanoma. However, only 17.9% of patients receiving IT-pIL12-EP show a complete therapeutic response. Here, we sought to improve the antitumor efficacy of our clinical IT-pIL12-EP plasmid electroporation platform. We evaluated multiple plasmid designs for IL-12 expression. IL-12 expression from a plasmid incorporating a picornavirus-derived co-translational P2A site was the most effective in expressing IL-12p70. In addition, modifying the electroporation parameters improved transfection efficiency and expression of plasmid-derived IL-12p70, as well as its downstream effector IFN-γ in vivo. Finally, using a murine melanoma model that is representative of the intended target patient population, we show that combining modified electroporation conditions with the pIL12-P2A plasmid expression enhances the systemic antitumor response. These improvements to the IT-pIL12-EP platform may improve patient clinical response rates and survival when translated to clinical trials.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1hi CD8+ effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated “armoring” of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: Evidence for stromal induction of hepatocellular carcinoma: Stromal activation in PDGF-C transgenic mice

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.