New Therapeutic Options for Advanced Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), one of the most common lethal diseases in the world, has a 5-year survival rate of only 7%. Hepatocellular carcinoma has no symptoms in the early stage but obvious symptoms in the late stage, leading to delayed diagnosis and reduced treatment efficacy. In recent years, as the scope of HCC research has increased in depth, the clinical development and application of molecular targeted drugs and immunotherapy drugs have brought new breakthroughs in HCC treatment. Targeted therapy drugs for HCC have high specificity, allowing them to selectively kill tumor cells and minimize damage to normal tissues. At present, these targeted drugs are mainly classified into 3 categories: small molecule targeted drugs, HCC antigen-specific targeted drugs, and immune checkpoint targeted drugs. This article reviews the latest research progress on the targeted drugs for HCC.

Ramucirumab: A Review in Hepatocellular Carcinoma.

Ramucirumab (Cyramza®), a fully human anti-VEGFR-2 monoclonal antibody, has been approved as monotherapy for the treatment of patients with hepatocellular carcinoma (HCC) and α-fetoprotein levels ≥ 400 ng/mL who have been treated with sorafenib. Ramucirumab significantly prolonged overall survival (OS) and progression-free survival (PFS) relative to placebo in this population in the randomized, double-blind phase 3 REACH 2 trial. These benefits were seen in key prespecified subgroups based on demographic and disease characteristics. Ramucirumab had an acceptable tolerability profile and manageable safety profile in these patients, with the majority of treatment-related adverse events being mild or moderate in severity. The safety profile of ramucirumab was consistent with that expected for agents targeting the VEGF/VEGFR axis. Currently, ramucirumab is the only therapy specifically tested in patients with α-fetoprotein levels ≥ 400 ng/mL, which is associated with an aggressive disease and poor prognosis. Therefore, ramucirumab is an important treatment option for patients with HCC and α-fetoprotein levels ≥ 400 ng/mL who have been treated with sorafenib.

Serum Alpha-fetoprotein Levels and Response to Direct Antiviral Therapy in Patients with Chronic Hepatitis C: Real-world Results from 1716 Patients in Egypt.

BACKGROUND & AIMS: Direct Antiretroviral Agents (DAAs), sofosbuvir-based therapies, have opened a new era in the treatment of chronic HCV infection. The aim of the study was to investigate the potential use of baseline and in serial serum, AFP levels as a predictor for response to DAAs in patients with Chronic Hepatitis C. METHODS: This multicenter observational study was carried out on 1716 chronic hepatitis C virusinfected patients who received direct anti-viral drugs for 12 weeks. The primary end point was sustained virological response at 12 weeks after the end of treatment determined by quantitative PCR for HCV RNA. Serum AFP was quantitatively assessed at baseline then after 12week after stoppage of treatment (SVR12). RESULTS: SVR12 rate was 97.8%. Elevated serum AFP was significantly higher in non -SVR group p value (<0.001). There was a significantly marked decrease in AFP after treatment in comparison to pretreatment values. The multivariate logistic regression analysis on the resulting significant variable from the univariate analysis revealed that only AFP was significantly related to the response to direct antiviral therapy in patients with chronic hepatitis C with p <0.001, OR 1.10 (95% CI 1.07:1.12). Other sociodemographic (e.g. Age, gender, BMI, ..) or laboratory factors (Hb, ANC, WBCs, …) did not show any significant association with the patients' response to treatment. CONCLUSIONS: Serum AFP levels were a predictor for response in patients with chronic HCV with the administration of direct antiviral drugs.

Knockdown of alpha-fetoprotein expression inhibits HepG2 cell growth and induces apoptosis.

AIMS: To explore the biological roles of alpha-fetoprotein (AFP), a tumor-associated antigen in human hepatocellular carcinoma (HCC). MATERIALS AND METHODS: After knockdown of AFP in HepG2 cells by transfection of specific Stealth™ RNAi, the expression of AFP were detected by reverse transcription polymerase chain reaction at mRNA level and by enzyme-linked immunosorbent assay at the protein level. Then, the effect of silenced AFP on cell proliferation was assessed by dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide assay, and apoptosis assessment with Hoechst33258 and flow cytometry (double stain with fluorescein isothiocyanate/propidium iodide), the roles of AFP in the cell cycle regulation were assessed by flow cytometry. We also detected the expression of some key proteins related to apoptosis pathway by Western immunoblot analysis. RESULTS: After the transfection for 48 h, the expression of AFP gene was almost abolished, the cell proliferation was inhibited by 47.61%, the number of cells undergoing early apoptosis was significantly increased to 59.47%; cell cycle was arrested with the increase of G0/G1 phase cells from 45.3% to 58.4%. Inhibition of AFP expression also results in decreasing of transforming growth factor-ß (TGF-ß), mutant P53 expression, and increasing of Bax/Bcl-2 ratio, activation of caspase-3. CONCLUSIONS: The results suggest that AFP may positively regulate cell proliferation by enhancing the apoptosis resistance via effect on TGF-ß and p53/Bax/caspase-3 signaling pathway in HepG2 cells. As such, the knockdown of AFP gene should be further investigated in vivo as a novel approach to HCC treatment.

Antibody to human α-fetoprotein inhibits cell growth of human hepatocellular carcinoma cells by resuscitating the PTEN molecule: in vitro experiments.

It has been proposed that α-fetoprotein (AFP) is a new member of the intracellular signaling molecule family of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway via interaction with the phosphatase and tensin homolog (PTEN). In this study, the effects of anti-human AFP antibody on the functions of PTEN were examined using an AFP-producing human hepatoma cell line. The antibody caused significant inhibition of cell growth, compared to a normal IgG control, with the accumulation of intracellular immune complexes followed by significant reduction of cytosolic functional AFP. Decrease in the amount of AKT phosphorylated on serine (S) 473 indicated that PI3K/AKT signaling was suppressed in the cells. S380-phosphorylated PTEN increased markedly by the second day after antibody treatment, with slight but significant increase in the PTEN protein level. Since phosphorylation at S380 is critical for PTEN stability, the increase in S380-phosphorylated PTEN indicated maintenance of the number of PTEN molecules and the related potential to control PI3K/AKT signaling. p53 protein (P53) significantly, but slightly increased during antibody treatment, because PTEN expression increased the stability and function of P53 via both molecular interactions. P53 phosphorylated at S20 or at S392 dramatically increased, suggesting an increase in the stability, accumulation and activation of P53. Glucose transporter 1 (GLUT1) increased immediately after antibody treatment, pointing to a deficiency of glucose in the cells. Immunofluorescence cytology revealed that antibody-treatment re-distributed GLUT1 molecules throughout the cytoplasm with a reduction of their patchy localization on the cell surface. This suggested that translocation of GLUT1 depends on the PI3K/AKT pathway, in particular on PTEN expression. Antibody therapy targeted at AFP-producing tumor cells showed an inhibitory effect on the PI3K/AKT pathway via the liberation, restoration and functional stabilization of PTEN. PTEN simultaneously induced both P53 activation and intracellular translocation of GLUT1, since these are closely associated with PTEN.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PURPOSE: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. EXPERIMENTAL DESIGN: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. RESULTS: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). CONCLUSIONS: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

Resveratrol prevents liver fibrosis via two possible pathways: Modulation of alpha fetoprotein transcriptional levels and normalization of protein kinase C responses.

OBJECTIVE: Liver fibrosis is a global health problem that causes approximately 1.4 million deaths per year. It is associated with inflammation, oxidative stress, necrosis and ends with cirrhosis, liver cancer, or liver failure. Therefore, the present study was constructed to investigate the protective effect of resveratrol (RVT) on liver fibrosis, focusing on the possible involvement of alpha 1-fetoprotein and protein kinase C signaling. MATERIALS AND METHODS: Rats received thioacetamide (TAA) (200 mg/kg, intraperitoneal) twice weekly, for 4 successive weeks to induce liver fibrosis. RVT (30 mg/kg, per os) and vehicle were administered orally for 1 month before and another month during TAA intoxication. Body weights and mortality rate were assessed during the experiment. Liver functions and protein concentration were determined in serum, while liver tissues were analyzed for oxidative and fibrotic biomarkers. Moreover, histological examinations were performed to liver biopsies. RESULTS: RVT prevented the debility of TAA; liver functions including alanine aminotransferase, aspartate aminotransferase, bilirubin, and albumin were also protected. RVT prevented TAA oxidative stress, and normal liver contents of malondialdehyde and reduced glutathione were markedly preserved. In addition, RVT abolished the stimulant effect of TAA to fibrosis markers and conserved normal liver contents of nuclear factor kappa B, hydroxyproline, and alpha fetoprotein. Histological examinations indicated normal liver architecture in RVT-administered rats as compared to their TAA-administered peers. CONCLUSION: RVT was able to enhance liver functions, prevent oxidative stress, and eliminate liver fibrosis. Hence, the present data highlight the therapeutic potential of RVT as a protective agent against liver fibrosis.

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX.

Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed.

Ionizable amphiphilic dendrimer-based nanomaterials with alkyl-chain-substituted amines for tunable siRNA delivery to the liver endothelium in vivo.

A library of dendrimers was synthesized and optimized for targeted small interfering RNA (siRNA) delivery to different cell subpopulations within the liver. Using a combinatorial approach, a library of these nanoparticle-forming materials was produced wherein the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length, and evaluated for their ability to deliver siRNA to liver cell subpopulations. Interestingly, two lead delivery materials could be formulated in a manner to alter their tissue tropism within the liver-with formulations from the same material capable of preferentially delivering siRNA to 1) endothelial cells, 2) endothelial cells and hepatocytes, or 3) endothelial cells, hepatocytes, and tumor cells in vivo. The ability to broaden or narrow the cellular destination of siRNA within the liver may provide a useful tool to address a range of liver diseases.

Effects of AFP gene silencing on apoptosis and proliferation of a hepatocellular carcinoma cell line.

Alpha fetoprotein (AFP) is an oncoembryonal protein that is highly expressed in the majority of hepatocellular carcinomas. Previous studies have shown that AFP may be involved in multiple cell growth regulating, differentiating, and immunosuppressive activities. We investigated the effects of AFP gene silencing by siRNA on apoptosis and proliferation of hepatocellular carcinoma cell line EGHC-9901, which highly expresses AFP and may serve as an ideal model for investigation of AFP functions. siRNA expressing plasmid targeting the AFP gene was first established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; cells were then divided into three groups: siRNA-afp, transfected with AFP-siRNA; siRNA-beta-actin, transfected with siRNA-beta-actin as the positive group; and vector control, transfected with empty vector as the blank control group. After G418 positive clone selection for a couple of weeks, Western blot and RT (reverse transcription)-PCR assay demonstrated that AFP expression was almost completely inhibited by siRNA-afp, which indicates that siRNA expressing plasmid targeting the AFP gene has been successfully established. Furthermore, MTT (methyl thiazolyl tetrazelium) assay showed that cells transfected with siRNA-afp proliferated at a significantly lower speed than the other two groups and flat plate clone formation assay also witnessed less clones with diameters of more than 75 µm in siRNA-afp immunofluorescence indicating that the apoptosis rate of cells transfected with siRNA-afp was significantly higher than the other two groups. Furthermore, flow cytometry manifested approximately 20% more cells of siRNA-afp within G1 phase than those of the negative group, indicating that inhibition of AFP expression may cause G1 phase arrest. Finally, Western blot and RT-PCR assay demonstrated that siRNA-afp induced a higher expression of caspase-3 than the other two groups whereas there was no difference in expression of caspase-8, caspase-9, and Bcl-2 between the three groups.

AFP-specific immunotherapy impairs growth of autochthonous hepatocellular carcinoma in mice.

BACKGROUND AND AIMS: In this study, we have assessed the potential of antigen-specific immunotherapy against hepatocellular carcinoma (HCC) in conditions of low tumour burden, in an autochthonous HCC model. METHODS: Diethylnitrosamine (DEN) injected into infant mice results in the development of multi-nodular HCC in which alpha-fetoprotein (AFP) is re-expressed. DEN-injected animals received an antigen-specific immunization with a synthetic vector consisting of a low dose of AFP-encoding plasmid formulated with the amphiphilic block copolymer 704 (DNAmAFP/704). Animals were treated at 4 and 5 months, before macroscopic nodules were detected, and were sacrificed at 8 months. The tumour burden, as well as liver histology, was assessed. AFP and MHC class I molecule expression in the nodules were monitored by qRT-PCR. RESULTS: The AFP-specific immunotherapy led to a significant (65%) reduction in tumour size. The reduced expression of AFP and MHC class I molecules was measured in the remaining nodules taken from the DNAmAFP/704-treated group. CONCLUSIONS: This is the first study demonstrating the relevance of antigen-specific immunotherapy in an autochthonous HCC model. In this context, we validated the use of an anti-tumour immunotherapy based on vaccination with nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer. Our results demonstrate the potential of this strategy as adjuvant immunotherapy to reduce the recurrence risk after local treatment of HCC patients.

Galectin-1-asialofetuin interaction is inhibited by peptides containing the tyr-xxx-tyr motif acting on the glycoprotein.

Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.

Alpha fetoprotein is a novel protein-binding partner for caspase-3 and blocks the apoptotic signaling pathway in human hepatoma cells.

Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase-3-mediated signaling of apoptosis. Our results showed that low doses of TNF-related apoptosis-inducing ligand (TRAIL) elevated the activity of caspase-8, but not caspase-3. Caspase-3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all-trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase-3 and block onward transmission of signaling from caspase-8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase-3 signaling. No intermolecule interaction occurred between AFP and caspase-8, nor was caspase-8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase-3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors.

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells.

AIM: To study the function of alpha-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescence activated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.

N-substituted 4-aminobenzamides (procainamide analogs): an assessment of multiple cellular effects concerning ion trapping.

Procainamide and related triethylamine-substituted 4-aminobenzamides, such as metoclopramide and declopramide, exert cellular effects potentially exploitable in oncology at millimolar concentrations (DNA demethylation, nuclear factor-kappaB inhibition, apoptosis) and display anti-inflammatory properties. However, these drugs induce massive cell vacuolization at similar concentrations, a response initiated by vacuolar (V-) ATPase-dependent ion trapping into and osmotic swelling of acidic organelles. We have examined whether this overlooked response might be related to the effects on cell proliferation and viability using cultured vascular smooth muscle cells and tumor-derived cell lines (Morris 7777 hepatoma, HT-1080 fibrosarcoma). Giant vacuole formation, of confirmed trans-Golgi origin (labeled with C5-ceramide, p230, golgin-97), is a cellular response to all tested amines in the series (> or = 2.5 mM), including triethylamine. These drugs and the V-ATPase inhibitor bafilomycin A1 inhibited smooth muscle cell proliferation, suggesting that acidification of a cellular compartment is essential to cell division. The cytotoxicity was maximal with metoclopramide, and this effect was minimally influenced by bafilomycin A1; furthermore, metoclopramide (2.5 mM) induced apoptosis in tumor cells as judged by poly(ADP-ribose)polymerase (PARP) cleavage. Triethylamine and procainamide exhibit a low level of cytotoxicity variably reduced by bafilomycin co-treatment. In Morris cells, the secretion of alpha-fetoprotein is inhibited by amines, consistent with the impairment of the secretory pathway. The most highly substituted 4-aminobenzamides are significant NF-kappaB inhibitors in smooth muscle cells. Although some effects of 4-aminobenzamides are independent of V-ATPase-driven ion trapping (inhibition of NF-kappaB nuclear translocation, agent-specific cytotoxicity, PARP cleavage), other effects are dependent on this phenomenon (vacuolization, a component of the cytotoxicity, inhibition of secretion).

Angiogenesis of AFP producing gastric carcinoma: correlation with frequent liver metastasis and its inhibition by anti-AFP antibody.

We studied roles of angiogenesis in patients with alpha-fetoprotein (AFP)-producing gastric carcinoma (APGC), which is well known to have a poor prognosis and frequent liver metastases. Immunohistochemical analyses were conducted using antibodies against alpha-fetoprotein, factor VIII (endothelial cells) and vascular endothelial growth factor (VEGF). Archival specimens of APGC (n=25) and non-APGC (n=68) were studied. Expressions of vessel density and VEGF were significantly higher in APGC than those in non-APGC (p<0.001). There is a correlation among the AFP expression, the vessel density and the VEGF expression in APGC (p<0.001). Next, we studied the effects of anti-AFP antibody on APGC xenotransplanted in nude mice. There is significant inhibition of tumor growth in the treatment groups compared to the control groups in 2 APGC lines (p<0.01). There were also significant differences in serum AFP and VEGF levels between treatment groups and non-treatment groups in 2 APGC lines, but not in a non-APGC line. Moreover, vessel densities of the treatment groups were significantly lower than those of the control groups in the two lines. These findings thus suggest that the biological behavior of APGC is angiogenesis-dependent. Down-regulation of angiogenesis by anti-AFP antibody suggest that AFP itself may up-regulate angiogenesis, and the treatment by antibody could have anti-angiogenic effects, inhibiting metastasis, especially liver metastasis in APGC.

ING1 represses transcription by direct DNA binding and through effects on p53.

The ING family of proteins is involved in the regulation of diverse processes ranging from cell cycle and cellular senescence to apoptosis. These effects are most likely through activation of acetylation-dependent pathways that ultimately alter gene expression. Despite reports linking ING to p53 activation, the molecular basis of how ING activates p53 function has not been elucidated. In this study, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) up-regulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein.

Do overexpressed oncoproteins cause malignant growth of cancer cells?--Studied by antisense oligonucleotides.

Antisense oligonucleotides were used to demonstrate the importance of insulin-like growth factor II and alpha-fetoprotein for the growth of hepatoma cell lines. The level of insulin-like growth factor II was found to correlate positively with cell proliferative activity, whereas alpha-fetoprotein was not. We have developed an in vitro system for the screening of antisense oligonucleotides effective for inhibiting target protein production. Using this system, the effectiveness of antisense oligonucleotides can be determined even when a specific antibody or activity assay method is not available. These approaches will be useful for verifying the physiological role of other oncoproteins or proteins in living cells, and antisense oligonucleotides may be developed as new therapeutic agents.