Selective targeting of the mTORC1/2 protein kinase complexes leads to antileukemic effects in vitro and in vivo.

The BCR/ABL tyrosine kinase promotes leukemogenesis through activation of several targets that include the phosphoinositide 3-kinase (PI3K). Tyrosine kinase inhibitors (TKIs), which target BCR/ABL, induce striking clinical responses. However, therapy with TKIs is associated with limitations such as drug intolerance, inability to universally eradicate the disease and emergence of BCR/ABL drug-resistant mutants. To overcome these limitations, we tested whether inhibition of the PI3K/target of rapamycin (mTOR) signaling pathway has antileukemic effect in primary hematopoietic stem cells and BA/F3 cells expressing the BCR/ABL oncoprotein. We determined that dual inhibition of PI3K/mTOR causes growth arrest and apoptosis leading to profound antileukemic effects both in vitro and in vivo. We also established that pharmacologic inhibition of the mTORC1/mTORC2 complexes is sufficient to cause these antileukemic effects. Our results support the development of inhibitors of the mTORC1/2 complexes for the therapy of leukemias that either express BCR/ABL or display deregulation of the PI3K/mTOR signaling pathway.

CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor.

The PML tumor suppressor controls growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in hematopoietic and solid tumors. PML loss often correlates with tumor progression. Casein kinase 2 (CK2) is a stress-activated serine/threonine protein kinase that is oncogenic and frequently overexpressed in human tumor of multiple histological origins. In addition, CK2 overexpression due to gene amplification has been reported to be an adverse prognostic factor in non-small cell lung cancer. At the 5th International Conference on Protein Kinase CK2 in Padova, Italy, we reviewed our recent findings that PML undergoes ubiquitin/proteasome-mediated degradation in immortalized and tumor derived cell lines. PML degradation depends on direct CK2 phosphorylation of PML Ser517. PML mutants that are resistant to CK2 phosphorylation display increased tumor suppressive functions in assays measuring apoptosis, replicative senescence, and in xenograft models. More significantly, CK2 pharmacological inhibition enhances PML tumor suppressive property. These data identify a key post-translational mechanism that controls PML protein levels in cancer cells and suggest that CK2 inhibitors may be beneficial anti-cancer drugs.

The theory of APL revisited.

Acute promyelocytic leukemia (APL) is associated with reciprocal and balanced chromosomal translocations always involving the retinoic acid receptor alpha (RARa) gene on chromosome 17 and variable partner genes (X genes) on distinct chromosomes. RARalpha fuses to the PML gene in the majority of APL cases, and in a few cases to the PLZF, NPM, NuMA and STAT5b genes. As a consequence, X-RARalpha and RARalpha-X fusion genes are generated encoding aberrant chimeric proteins that exert critical oncogenic functions. Here we will integrate some of the most recent findings in APL research in a unified model and discuss some of the outstanding questions that remain to be addressed.

Cationic liposome-mediated gene delivery to the liver and to hepatocellular carcinomas in mice.

The potential of cationic liposomes as nonviral vectors for in vivo gene delivery to the liver and to intrahepatic hepatocellular carcinoma (HCC) was investigated. Mice were injected via the tail vein or portal vein with a cationic lipid complexed to plasmid DNA (pDNA) encoding the chloramphenicol acetyltransferase (CAT) reporter gene at various cationic lipid:pDNA molar ratios to analyze the efficiency of gene delivery after intravenous administration. Tail vein injection resulted in high CAT expression levels in lung and spleen and low levels in the liver. Portal vein injection, by comparison, significantly enhanced hepatic reporter gene expression but also resulted in pronounced hepatic toxicity. Gene delivery to intrahepatic tumors produced by intrahepatic injection of human HCC cells was analyzed in nude mice. Tail vein injection as well as portal vein injection resulted in low levels of gene expression in intrahepatic tumors. By comparison, high levels of gene expression were achieved by direct, intratumoral injection of liposome-pDNA complexes, with only minimal expression in the surrounding normal liver. Therefore, direct liposome-pDNA complex injection appears far superior to systemic or portal intravenous administration for gene therapy of localized intrahepatic tumors, and may be a useful adjunct in the treatment of human HCCs.

Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication.

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.

Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective.

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase "B domain" involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.

The small envelope protein is required for secretion of a naturally occurring hepatitis B virus mutant with pre-S1 deleted.

Naturally occurring deletions in the hepatitis B virus pre-S1 domain have been frequently found during persistent viral infection. In this study we have investigated the functional properties of a mutant viral genome that carries an in-frame deletion of 183 nucleotides in the pre-S1 region. This deletion removes the promoter of the small envelope gene. Transfection into human hepatocellular carcinoma cells of a replication-competent construct containing this deletion resulted in an increase of intermediate DNA replicative forms compared to those produced by wild-type hepatitis B virus. Northern blot analysis revealed that such cells lack the 2.1-kb transcripts encoding the small envelope protein and that hepatitis B surface antigen was absent as well. Furthermore, nucleocapsids containing the genome with pre-S1 deleted were not secreted, and the deleted large envelope protein was retained with the cytoplasm and exhibited a perinuclear pattern of distribution. However, coexpression with the small envelope protein was sufficient to restore virion secretion and to change the cellular distribution of the deleted large envelope protein. In addition, the creation of point mutations that prevent the synthesis of large or small envelope proteins also inhibited viral secretion and led to increased levels of hepatitis B virus intermediate replicative forms within the cell. These studies suggest that naturally occurring viral mutants with pre-S1 deletions involving the promoter region of the small envelope gene will generate a deleted large envelope protein that is retained in the endoplasmic reticulum, resulting in the accumulation of nucleocapsids containing viral DNA; transcomplementation with the wild-type small envelope protein will allow mutant virion secretion to occur.

Biologic properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame.

It is now well recognized that mutations in the hepatitis B virus (HBV) genome occur during the natural course of chronic viral infection. Regions of the viral genome that are frequently affected by such mutations, rearrangements, and/or deletions generally involve the precore promoter, precore, and core as well as the preS gene regions. However, little is known regarding the biologic consequences of these mutations on the functional properties of the variant viral strains with respect to effects on viral replication. In this study, we investigated the functional significance of precore promoter and precore gene mutations that reduce or abolish the synthesis of hepatitis B e antigen (HBeAg). We found that precore promoter mutations diminished the expression of HBeAg but did not affect the synthesis of pregenomic RNA. However, these precore mutations were associated with a modest increase in HBV replication. In contrast, a naturally occurring mutant that carries a termination codon in position 28 of the precore open reading frame demonstrated increased encapsidation of pregenomic mRNA into nucleocapsid particles. Consequently, this variant viral strain demonstrated a substantial increase in the level of viral replication compared to "wild-type" HBV and other precore promoter mutant viral strains. These studies suggest that substitutions in the precore promoter and precore gene not only alter the synthesis of HBeAg but also affect the level of viral replication.

Posttranscriptional regulation of hepatitis B virus replication by the precore protein.

Hepadnaviruses encode two core-related open reading frames. One directs the synthesis of the p21 core protein, which subsequently becomes a structural component of the viral nucleocapsid. The other produces a p25 precore protein that is targeted by a signal peptide to a cell secretory pathway where N-terminal processing will create a p22 species. This molecule will be further modified at the C-terminal region to generate p17, and the truncated protein is secreted from the cell as hepatitis B e antigen (HBeAg). The function of the precore gene in the biology of hepadnaviruses is unknown. We found that ablation of the precore gene resulted in the generation of a hepatitis B virus (HBV) species with a high-replication-level phenotype. More important, expression in trans of physiologic levels of p25 restored viral replication to wild-type levels. Moreover, transient or stable overexpression of the precore gene resulted in striking inhibition of HBV replication. The molecular species responsible for this viral inhibitory effect was identified as the p22 nonsecreted HBeAg precursor protein. By sucrose gradient sedimentation analysis, we determined that expression of p22 leads to the formation of nucleocapsids similar to those made with wild-type p21 core protein. Immunoprecipitation experiments revealed that the p21 and p22 physically interact and form hybrid nucleocapsid structures devoid of pregenomic viral RNA. These experiments suggest that expression of the precore gene may be important in the regulation of HBV replication and describe a possible molecular mechanism(s) for this effect.

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection.

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid-based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA-based immunization techniques to stimulate broad-based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the host's cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Recent advances in the molecular biology of hepatitis B virus.

Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus. Acute and chronic HBV infection causes significant liver diseases such as acute hepatis, fulminant hepatitis and chronic active hepatitis that may lead to liver cirrhosis and the development of hepatocellular carcinoma. The use of molecular biological techniques has substantially improved our understanding of the HBV life cycle. In this review, we discuss recent advances that have contributed to a better understanding of HBV biology. Recent studies in the understanding of the life cycle of HBV such as viral entry, replication, transcriptional regulation, viral regulatory proteins, viral assembly and secretion, and nucleic acid based approaches to antiviral therapy will be emphasized. These advances in molecular biology and relationship to clinical disease will be instrumental in developing effective therapeutic approaches for the estimated 300 million individuals worldwide chronically infected with HBV.

Characterization of hepatitis B virus core mutants that inhibit viral replication.

We have generated and functionally characterized dominant negative core protein variants of the hepadnaviruses to determine their effects on "wild type" viral replication. Plasmids expressing these constructs were introduced into hepatoma cell lines by transient transfection and effects on wild type woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) replication were evaluated by Southern blot analysis of purified viral core particles. WHV and HBV constructs expressing a truncated core protein fused in frame with the C-terminus of the small surface protein were found to inhibit viral replication by 90-95% due to disruption of the viral nucleocapsid assembly process and preventing encapsidation of pregenomic RNA. The antiviral effects were found to be specific for the targeted virus. These results demonstrate that mutants of hepadnaviral core protein may represent a novel class of antiviral agents.

Presence of HCV RNA in serum of asymptomatic blood donors involved in post-transfusion hepatitis (PTH).

To study the causes of residual posttransfusion hepatitis, serum from implicated donors was tested by PCR for the presence of HCV RNA. Of 20 anti HCV negative donors, 4 were HCV RNA positive and thus, infective. The results suggest that higher-level investigations are necessary for prospective donors who present blood enzyme abnormalities or other questionable characteristics.

Hepatitis B virus specific transcripts in peripheral blood mononuclear cells.

We report on the analysis of HBV transcription in peripheral blood mononuclear cells of chronically infected patients by polymerase chain reaction amplification. Our results suggest that in these cells gene expression occurs either as pregenomic or subgenomic transcripts.

Hepatocellular carcinoma: risk factors other than HBV.

The putative risk factors for hepatocellular carcinoma (HCC) are several, even in countries endemic for hepatitis B virus (HBV) infection. Cirrhosis characterizes more than 90% of HCC cases. The phases of inflammation, necrosis and regeneration, present for long periods in cirrhosis, might be most relevant in hepatocarcinogenesis. It is not clear what role is played by sex hormones while alcohol probably has a promoter role. Aflatoxins are known carcinogenins in the experimental animal: however it is difficult to evaluate the impact in human carcinogenesis due to the lack of reliable methods of measuring aflatoxin exposure in population studies. In conclusion, the aetiology of HCC is multifactorial and the main risk factor resides in the presence of underlying chronic liver disease.

PIP: Experimental and epidemiological studies of risk factors for hepatocellular carcinoma (HCC): cirrhosis, male sex, oral contraceptives, alcohol, smoking, and aflatoxins, are evaluated, with meta-analysis for oral contraceptives, alcohol, and smoking. It is likely that an initiating event and one or more promoting events interact, probably with prolonged inflammation, necrosis and regeneration, to cause cancer in several types of cirrhosis. Over 90% of HCC patients have cirrhosis, usually from hepatitis B virus. The viral post-necrotic liver is often chronically dysplastic, but other types of cirrhosis are associated with HCC if they endure long enough. The proportion of men with HCC increases as hepatitis progressors to cirrhosis and then to HCC. Meta-analyses of 3 oral contraceptive studies resulted in a risk of 2.8 for 8 years of use, but 9.9 for 8 years. Population studies do not show any concentration of HCC in countries with high pill use, so the rarity of this cancer may have biased the results. Large epidemiologic studies are needed to refine risk estimates for oral contraceptives and HCC. Alcohol abuse of 80 g/day gives a risk of about 1.65 in pooled studies, compared to a risk of 1.1 for 80 g/day. Smoking gives a risk of 1.9, but there is no evidence for a secular trend by country in proportion to dose, as is evident for lung cancer. There is good experimental evidence that aflatoxin acts as an initiator for liver cancer, but there is not practical way to judge exposure for clinical studies.

HCV RNA in serum of asymptomatic blood donors involved in post-transfusion hepatitis (PTH).

A group of blood donors involved in post-transfusion hepatitis was investigated for the presence of the anti-HCV antibody and of HCV RNA as a more direct infection marker. RNA was extracted from serum, reverse transcribed and amplified using primers which belonged to the non structural region. The amplified product of the PCR reaction was 582 base pairs. Seven (25.9%) of the 27 blood donors examined were found anti-HCV-positive by ELISA; five (71.4%) of these were HCV RNA positive. Among the 20 anti-HCV-negative blood donors, four (20.0%) were HCV RNA positive. ALT levels were below 45 UI/l in 18 donors, while the other nine had ALTs over the limit accepted for transfusion. The anti-HCV-negative HCV RNA-positive blood donors had normal ALTs. Our study offers a direct explanation for the substantial proportion of residual cases of anti-HCV-positive post-transfusion hepatitis and suggests the necessity of creating a register of blood donors who have at some time presented blood enzyme abnormalities and for whom second level investigations such as HCV RNA should be used.

Conserved core protein sequences in hepatitis B virus infected patients without anti-HBc.

The absence of detectable anti-HBc antibodies in some hepatitis B virus (HBV) infected patients may be due to altered core-protein (HBc) sequences. To investigate this possibility we sequenced the pre-C/C-region of HBV isolated from 12 juvenile cancer patients who incurred a nosocomial infection of HBV during chemotherapy but did not develop anti-HBc antibodies or acute cytolytic episodes. The sequences demonstrated the highest sequence homology to the pre-C/C region of a previously cloned HBV genome (subtype ayw) and no deletions or striking mutations were detected. Up to 7 years after infection almost all the survivors developed low titers of anti-HBc antibodies but no clinical signs of hepatic damage. These results suggest that chemotherapy may induce a tolerance status to HBcAg, the most immunogenic HBV protein.

Hepatitis B virus infection of peripheral blood mononuclear cells is common in acute and chronic hepatitis.

Hepatitis B virus (HBV) infection of peripheral blood mononuclear cells (PBMCs) has been observed in all stages of liver disease. Thus far all information about the physical state of HBV in mononuclear blood cells comes from Southern blot analysis and in situ hybridization. In this study we focused our attention on the presence of HBV DNA sequences in PBMCs of 30 patients with acute type B hepatitis and 6 patients with chronic active hepatitis by utilizing both Southern blot analysis and the polymerase chain reaction (PCR). Southern blot analysis showed no HBV DNA sequences in PBMCs of the acute hepatitis patients, although the sensitivity of our method enabled us to detect as little as 1 pg of cloned HBV insert. As far as the chronic hepatitis patients are concerned Southern blot analysis revealed the presence of HBV DNA sequences in 5 out of 6 patients but intermittently at successive follow-up times. On the other hand we were able to demonstrate the presence of HBV related sequences in 14 out of 30 acute hepatitis patients (5 HBeAg positive, 9 antiHBe positive) and in all 6 chronic hepatitis patients by PCR. Our results indicate that the involvement of PBMCs with HBV during acute HBV infection occurs at a very low level, often below the detection limit of the Southern blot technique.

[Estrogen receptors in the human liver]. / I recettori per gli estrogeni del fegato umano.

Human hepatic estrogen receptors (ER) were investigated in 17 healthy subjects (13 males and 4 females) and 70 patients with chronic liver disease (45 males and 25 females). Characterization of the estrogen binders in cytosol from human male liver showed two classes of binders, the first of them corresponding to estrogen receptor (Kd = 10(-10) M), and the second representing a low affinity binder (KD = 10(-8) M). Increased ER levels were found in males with chronic liver disease, patients with primary hepatic carcinoma (PHC) having about twice the levels of normal males. Normal females had basal values about three times higher than control males; during the progression of chronic liver disease, ER levels fell to arise again slowly so that, in PHC, values were about half of those in normal females. Prolonged alcohol abuse appeared to induce a marked increase in ER levels both in male and in female patients. The increase was maximal in patients who were still actively drinking and in those with histological signs of acute alcoholic hepatitis.

Detection of hepatitis B virus transcripts in patients with chronic liver disease.

Hepatitis B virus (HBV) transcription was studied by Northern blot analysis on total cellular RNA purified from liver biopsies in 70 patients with chronic liver disease (24 HBsAg positive, 15 antiHBs and/or antiHBc positive, 31 HBV negative). No transcripts were found in the HBV negative and in the antiHBs and/or antiHBc positive patients. In the others, three major RNA species were identified: i. a 3.5 kb transcript corresponding to the RNA pregenome; ii. 2.4-2.1 kb transcript corresponding to the s and preS1 gene RNA; iii. lower molecular weight species. All three forms were present simultaneously only in patients with active viral replication, with a strict relation between the presence of the 3.5 kb RNA in the liver and serum HBV-DNA. In conclusion, Northern blot analysis can easily be performed to study viral replication and it can contribute to a better understanding of the molecular processes underlying HBV infection and leading to liver disease in man.