Anna Alisi

Retinoic acid modulates the cell-cycle in fetal rat hepatocytes and HepG2 cells by regulating cyclin-cdk activities

Abstract: Retinoic acid (RA), the most biologically active metabolite of vitamin A, is known to modulate cell proliferation, apoptosis and differentiation, with different effects depending on the cellular context. Retinoic acid can exert its effects by directly or indirectly influencing the expression of genes involved in the control of cell proliferation. In the present report we investigate the possible correlation between the antiproliferative, differentiative and apoptotic effects previously observed on rat hepatocytes and HepG2 cells, with a possible modulation of cell‐cycle regulators. We demonstrate that RA induces growth arrest and differentiation in HepG2 cells by influencing the activities of cyclin‐cdk complexes involved in the regulation of G1/S transition and S‐phase progression, in particular by modifying the binding of these complexes to p21 and p27 inhibitors. In fetal cells, however, the induction of apoptosis and differentiation by RA was obtained via inhibition of cyclin D1‐cdk4 activity, as result of an increased binding to the p16 inhibitor. Retinoic acid also modulates c‐myc and Bcl‐2 expression. In conclusion, our data suggest that RA could be useful to regulate the reversion of transformed phenotype and could also be utilized as a chemiopreventive agent in cells of hepatic origin.

Thyroid hormones regulate DNA‐synthesis and cell‐cycle proteins by activation of PKCα and p42/44 MAPK in chick embryo hepatocytes

The molecular mechanism by which thyroid hormones exert their effects on cell growth is still unknown. In this study, we used chick embryo hepatocytes at different stages of development as a model to investigate the effect of the two thyroid hormones, T3 and T4, and of their metabolite T2, on the control of cell proliferation. We observed that T2 provokes increase of DNA‐synthesis as well as T3 and T4, independently of developmental stage. We found that this stimulatory effect on the S phase is reverted by specific inhibitors of protein kinase C (PKC) and p42/44 mitogen‐activated protein kinase (p42/44 MAPK), Ro 31‐8220 or PD 98059. Furthermore, the treatment with thyroid hormones induces the activation of PKCα and p42/44 MAPK, suggesting their role as possible downstream mediators of cell response mediated by thyroid hormones. The increase of DNA‐synthesis is well correlated with the increased levels of cyclin D1 and cdk4 that control the G1 phase, and also with the activities of cell‐cycle proteins involved in the G1 to S phase progression, such as cyclin E/A‐cdk2 complexes. Interestingly, the activity of cyclin‐cdk2 complexes is strongly repressed in the presence of PKC and p42/44 MAPK inhibitors. In conclusion, we demonstrated that the thyroid hormones could modulate different signaling pathways that are able to control cell‐cycle progression, mainly during G1/S transition.

PKR is a novel functional direct player that coordinates skeletal muscle differentiation via p38MAPK/AKT pathways

Myogenic differentiation is a highly orchestrated multistep process controlled by extracellular growth factors that modulate largely unknown signals into the cell affecting the muscle-transcription program. P38MAPK-dependent signalling, as well as PI3K/Akt pathway, has a key role in the control of muscle gene expression at different stages during the myogenic process. P38MAPK affects the activities of transcription factors, such as MyoD and myogenin, and contributes, together with PI3K/Akt pathway, to control the early and late steps of myogenic differentiation. The aim of our work was to better define the role of PKR, a dsRNA-activated protein kinase, as potential component in the differentiation program of C2C12 murine myogenic cells and to correlate its activity with p38MAPK and PI3K/Akt myogenic regulatory pathways. Here, we demonstrate that PKR is an essential component of the muscle development machinery and forms a functional complex with p38MAPK and/or Akt, contributing to muscle differentiation of committed myogenic cells in vitro. Inhibition of endogenous PKR activity by a specific (si)RNA and a PKR dominant-negative interferes with the myogenic program of C2C12 cells, causing a delay in activation of myogenic specific genes and inducing the formation of thinner myofibers. In addition, the construction of three PKR mutants allowed us to demonstrate that both N and C-terminal regions of PKR are critical for the interaction with p38MAPK and Akt. The novel discovered complex permits PKR to timely regulate the inhibition/activation of p38MAPK and Akt, controlling in this way the different steps characterizing skeletal muscle differentiation.

Thr 446 phosphorylation of PKR by HCV core protein deregulates G2/M phase in HCC cells

Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma (HCC). HCV core protein affects cell homeostasis, playing an important role in viral pathogenesis of HCC. We investigate the effects of HCV core protein expression on cell growth in HCC cell lines. Cell cycle distribution analysis of HepG2 polyclonal core positive cells reveals a peculiar accumulation of cells in G2/M phase. Different pathways mediate G2/M arrest: such as p53 and double strand RNA protein kinase (PKR). Flow cytometry in p53‐null cells demonstrates that p53 plays only a marginal role in inducing HCV core‐dependent G2/M phase accumulation that seems to be significantly affected by the functional inactivation of PKR. HCC core positive cells are characterized by a significant PKR phosphorylation in Thr 446 residue, which leads deregulation of mitosis. Moreover, we observe that the overexpression of the viral protein induces an upregulation of PKR activity, which does not correlate with an increased eIF‐2 phosphorylation. This uncommon behavior of PKR suggests that its activation by HCV core protein could involve alternative PKR‐dependent pathways, implicated in core‐dependent G2/M accumulation. The described biological effects of HCV core protein on cell cycle could be an additional viral mechanism for both HCV resistance to interferon (IFN) and HCC HCV‐related pathogenesis.

Involvement of PI3K in HCV-related lymphoproliferative disorders.

Hepatitis C virus (HCV) core protein has been shown to deregulate cell growth and programmed cell death in hepatoma cells, but only minimal informations are available about its possible role on B‐lymphoproliferative disorders (LPDs). The aim of our work was to analyze the biological activity of HCV core protein on B‐cell proliferation. We established Wil2‐ns and Ramos B‐cell lines that stably expressed the HCV core protein. Growth curve, thymidine incorporation analysis, as well as the expression of PCNA and activated‐ERKs demonstrated that HCV core protein induced an increased growth in both cell lines. Interestingly, the HCV core protein expression determined, in our model, a downregulation of DNp73 and an upregulation of DNp63, which was essential for the maintenance of viral‐dependent effects on cell growth. Finally, we have identified phosphoinositide 3‐kinase (PI3K) as mediator of HCV core‐dependent transcriptional increase of DNp63, which in turn correlated with the increasing of lymphocyte proliferation. In primary B‐lymphocytes, derived from HCV‐related low‐grade non‐Hodgkins lymphoma patients, consistent results were obtained. These findings provide evidence for a possible pathogenetic role played by HCV core protein in HCV‐related lymphomagenesis; it could occur through the deregulation of PI3K activity, consequent activation of Akt and overexpression of DNp63. J. Cell. Physiol. 214: 396–404, 2008.

Hepatitis C virus and alcohol: Same mitotic targets but different signaling pathways

BACKGROUND & AIMS Chromosomal aberrations are frequently observed in hepatitis C virus (HCV)- and alcohol-related hepatocellular carcinomas (HCCs). The mechanisms by which chromosomal aberrations occur during hepatocarcinogenesis are still unknown. However, these aberrations are considered to be the result of deregulation of some mitotic proteins, including the alteration of Cyclin B1 and Aurora kinase A expression, and the phosphorylation of gamma-tubulin. Our study aims at investigating changes in expression of the above mentioned proteins and related intracellular pathways, in in vitro and in vivo models of both HCV- and alcohol- dependent HCCs. METHODS In this study, the molecular defects and the mechanisms involved in deregulation of the mitotic machinery were analyzed in human hepatoma cells, expressing HCV proteins treated or not with ethanol, and in liver tissues from control subjects (n=10) and patients with HCV- (n=10) or alcohol-related (n=10) HCCs. RESULTS Expression of Cyclin B1, Aurora kinase A, and tyrosine-phosphorylated gamma-tubulin was analyzed in models reproducing HCV infection and ethanol treatment in HCC cells. Interestingly, HCV and alcohol increased the expression of Cyclin B, Aurora kinase A, and tyrosine-phosphorylated gamma-tubulin also in tissues from patients with HCV- or alcohol-related HCCs. In vitro models suggest that HCV requires the expression of PKR (RNA-activated protein kinase), as well as JNK (c-Jun N-terminal kinase) and p38MAPK (p38 mitogen-activated protein kinase) proteins; while, ethanol bypasses all these pathways. CONCLUSIONS Our results support the idea that HCV and alcohol may promote oncogenesis by acting through the same mitotic proteins, but via different signaling pathways.

Treatment with EGF increases the length of S-Phase after partial hepatectomy in rat, changing the activities of cdks.

Liver proliferation occurs in the presence of mitogenic stimuli such as partial hepatectomy or growth factors. In this work we investigate how partial hepatectomy and Epidermal Growth Factor (EGF) affect hepatocyte proliferation by modulating cell cycle regulators. EGF administered to non-operated rats increased PCNA (proliferating cell nuclear antigen) expression, whereas when EGF was administered to partially hepatectomized rats it was able to anticipate the increase in PCNA expression to 18h after PH and to prolong it to 34h. Cell cycle progression was examined by monitoring specific markers of late G1- and S-phases. Western blot analysis showed that both treatment with EGF alone and treatment with EGF after PH induce the expression of cyclins D1 and A and of p21cip1, but inhibites the expression of cyclin E and p27kip1. EGF administration after PH also significantly affected the activity of the cyclin D1-cdk4 and cyclin E-cdk2 complexes, mainly by changing their time progression: it accelerated the increase in activity to 18h and caused a subsequent drop in activity after 34h; it delayed the activity of the cyclin A-cdk2 complex to 34h. In conclusion we observed that EGF modulates the activity of cdk complexes and induces a different linkage with inhibitory proteins that demonstrates their dual role, depending on their association with different cyclin-cdk complexes.

Hepatitis C virus core protein enhances B lymphocyte proliferation.

HCV chronic infection leads to liver diseases and also to a wide range of extrahepatic disorders including benign, but pre-lymphomatous forms (mixed cryoglobulinemia) to frank hematological neoplasia (non-Hodgkins lymphoma). Recent data showed the involvement of p53 superfamily members in the pathogenesis of different lymphatic malignancies. In fact, tymomas and a subset of non-Hodgkins lymphomas (NHLs) express high levels of p63. Thus, we analyzed whether alterations in p53 superfamily gene expression are observable in B lymphocytes isolated from HCV-infected patients with and without lymphoproliferative disorders. We showed, by real-time PCR, a significant induction of DNp63 mRNAs in B lymphocytes obtained from HCV-positive low grade non-Hodgkins lymphoma patients. Since our current understanding of HCV proteins emphasizes the ability of the HCV core protein to deregulate the expression and activity of p53-related proteins, we established different B lymphocyte cell lines (Wil2-ns, Daudi and Ramos) stably expressing HCV core protein, in order to investigate the possible involvement of the viral protein in the upregulation of DNp63 in B lymphocytes. The analysis of p63 family transcripts showed no transcriptional changes for the p63 TA isoforms, whereas an increase (>5 times) of DNp63 mRNA occurred. In all cell lines, this abnormal expression was associated with a significant increase of cell proliferation that was specifically inhibited by silencing DNp63 mRNA. These findings suggest a pathogenetic role of the HCV core in HCV-related lymphomagenesis, through the induction of DNp63s pro-proliferative effects.