Mutation of p53 gene in hepatocellular carcinoma cell lines with HBX DNA

It has been known that the incidence of p53 mutation is very rare in HBX-positive primary human hepatocellular carcinoma (HCC) tissues. The frequency of p53 mutation, however, in established cell lines with integrated HBV DNA and/or HBX has not been well studied. To know p53 mutational frequency, and to investigate whether the presence of HBX DNA sequence correlates with the absence of p53 mutation in the established HCC cell lines, we studied the p53 mutation and the presence of HBX sequence in 12 recently characterized HCC cell lines. As a result, all 12 (100%) lines showed mutation in the p53 gene. Three (25%) cell lines had transversion of codon 215 while no mutation of codon 249 was found. In contrast with previous reports, although HBX DNA was present in 11 cell lines, p53 mutation had occurred, indicating that the presence of HBX viral DNA does not correlate with a lack of p53 mutation in established HCC cell lines. Our results suggest that the frequency of p53 mutation is extremely high even in HBX DNA positive HCC cell lines. © 1996 Wiley-Liss, Inc.     

Effect of promoter methylation of the p16 gene on phosphorylation of retinoblastoma gene product and growth of hepatocellular carcinoma cells.

The biological significance of hypermethylation of p16 gene promoter in human hepatocellular carcinoma (HCC) cells remains to resolved. In order to clarify the significance of methylation of p16 gene promoter, we examined the methylation status of p16 gene in association with phosphorylation of retinoblastoma gene product (pRb) and cell growth in human HCC cell lines. The presence of methylation was examined by methylation-specific PCR. Expression and phosphorylation of p16 and pRb were examined by Western blot analysis. Genetic changes were analyzed by multiplex PCR and DNA sequencing. The effect of demethylation of p16 was assessed by cell growth. p16 gene promoter was methylated in HuH7 and HLF cells. The demethylating agent, 5-aza-2-deoxycytidine (5-Aza-CdR), upregulated p16 mRNA in HuH6 and HuH7 cells. 5-Aza-CdR increased p16 protein expression in HuH6, HuH7, and HLF cells, and it clearly decreased the phosphorylation level of pRb in HuH6, HuH7 and PLC/PRF/5 cells. Treatment with 5-Aza-CdR inhibited the growth of HuH7 cells. Homozygous deletion and significant mutations were absent. Methylation in the p16 promoter region is biologically significant, being associated with phosphorylation of pRb and cell growth in human HCC cells.     

Association between HBX status,aflatoxin-induced R249S TP53 mutation and risk of hepatocellular carcinoma in a case-control study from Thailand

Hepatocellular carcinoma (HCC) is associated with hepatitis B virus (HBV) chronicity and dietary exposure to aflatoxin, a mutagen targeting codon 249 of tumor suppressor TP53 (R249S mutation). Based on a case-control in Thailand, we have measured R249S and the status of HBX gene in plasma DNA of 176 cases and 133 referents. Detection of HBX complete sequences was associated with R249S in HCC with no documented prior cirrhosis but not in HCC developing in a context of cirrhosis or in non-cancer chronic liver diseases. Thus, R249S may specifically cooperate with HBX in a pathway to HCC that bypasses cirrhosis.     

Activation of AMP‐activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib

To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all‐trans retinoic acid (ATRA), NIK‐333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK‐333 and Am80. We found that ATRA induced AMP‐activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT‐1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase‐3, was observed. Suppression of AMP‐activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells.     

TP53 R249S mutation, genetic variations in HBX and risk of hepatocellular carcinoma in The Gambia

In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B(1) (AFB(1)) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3'-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case-control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3'-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ(2) = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762(T)/1764(A) was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50-60.11]; HCC: OR: 11.29 [95% CI 2.07-61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.     

Role of caveolin‐1 in hepatocellular carcinoma arising from non‐alcoholic fatty liver disease

The molecular features of hepatocellular carcinoma arising from non‐alcoholic fatty liver disease (NAFLD‐HCC) are not well known. In this study, we investigated the mechanism by which NAFLD‐HCC survives in a fat‐rich environment. We found that caveolin (CAV)‐1 was overexpressed in clinical specimens from NAFLD‐HCC patients. HepG2, HLE, and HuH‐7 HCC cell lines showed decreased proliferation in the presence of the saturated fatty acids palmitic acid and stearic acid, although only HLE cells expressed high levels of CAV‐1. HLE cells treated with oleic acid (OA) showed robust proliferation, whereas CAV‐null HepG2 cells showed reduced proliferation and increased apoptosis. CAV‐1 knockdown in HLE cells attenuated the OA‐induced increase in proliferation and enhanced apoptosis. Liquid chromatography–tandem mass spectrometry analysis revealed that the levels of OA‐containing ceramide, a pro‐apoptotic factor, were higher in HepG2 and CAV‐1‐deficient HLE cells than in HLE cells, suggesting that CAV‐1 inhibits apoptosis by decreasing the level of OA‐containing ceramide. These results indicate that CAV‐1 is important for NAFLD‐HCC survival in fatty acid‐rich environments and is a potential therapeutic target.     

Functional characterization of p53 molecules expressed in human squamous cell carcinomas of the head and neck

Mutation of the p53 tumor suppressor gene has been demonstrated in a large proportion of human head and neck squamous cell carcinomas (HNSCCs) and has been assumed to play a role in the pathogenesis of these tumors, although no formal evidence of functional aberration has been demonstrated. In this study, we isolated cDNA clones encoding the entire p53 coding region from six human HNSCC cell lines that showed aberrant patterns of p53 expression in the parental cells, analyzed their nucleotide sequences, and characterized their function in vivo. cDNAs cloned from four cell lines harbored alterations within the p53 coding sequence (one missense mutation, one missense mutation plus in-frame deletion, one splice donor mutation, and a 1-nt insertion). HN30 cells, which contained wild-type p53 nucleotide sequences, showed a high constitutive level of protein expression. HN26 cells contained wild-type coding sequences but did not express the 53-kDa protein, although the mRNA was transcribed and a molecule of increased molecular mass (70 kDa) was observed by western blotting. Functional studies revealed that none of the four proteins encoded by mutant cDNAs were able to transactivate expression of a reporter plasmid containing a wild-type p53 consensus binding site when cotransfected into p53-null cells, whereas molecules encoded by wild-type p53 cDNAs increased reporter gene expression about a hundredfold over uninduced levels. Co-expression of each mutant cDNA with wild-type p53 cDNA and a wild-type p53-responsive reporter gene demonstrated that each of the proteins encoded by mutant cDNAs harbored some degree of inhibitory activity that varied depending on the mutation present. Thus, aberrant p53 function as a result of mutation or altered expression characterizes oral squamous cell carcinomas. The inhibitory activity of these molecules may be a mechanism for deregulation of the function of co-expressed wild-type p53 that may be of importance during the early stages of tumor development. Mol. Carcinog. 18:89–96, 1997. © 1997 Wiley-Liss, Inc.     

Screening the p53 status of human cell lines using a yeast functional assay

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promoter in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30°C but not at 37°C. Four temperature-sensitive p53 mutations were isolated: CAT→CGT at codon 214 (H214R), TAC→TGC at codon 234 (Y234C), GTG→ATG at codon 272 (V272M), and GAG→AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization. Mol. Carcinog. 19:243–253, 1997. © 1997 Wiley-Liss, Inc.     

Augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells.

Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1.     

Adenovirus-mediated tBid overexpression results in therapeutic effects on p53-resistant hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53-resistant while PLC/PRF/5 a p53-sensitive. A recombinant adenovirus-Ad/AFPtBid, which contained a tBid gene driven by an alpha-fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL-positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP-producing cells but not those non-AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status.     

Downregulation of microRNA‐15b by hepatitis B virus X enhances hepatocellular carcinoma proliferation via fucosyltransferase 2‐induced Globo H expression

Globo H, a cancer‐associated carbohydrate antigen, is highly expressed in various types of cancers. However, the role of Globo H in hepatocellular carcinoma (HCC) remains elusive. In our study, we performed glycan microarray analysis of 134 human serum samples to explore anti‐Globo H antibody changes and found that Globo H is upregulated in hepatitis B virus (HBV)‐positive HCC. Similarly, immunohistochemistry showed that Globo H expression was higher in tumors compared to normal tissues. In addition, fucosyltransferase 2 (FUT2), the main synthetic enzyme of Globo H, was also increased in HCC cells overexpressing HBV X protein (HBX). HBX plays an important role in promoting cell proliferation and may be related to increased levels of FUT2 and Globo H. Furthermore, using microRNA profiling, we observed that microRNA‐15b (miR‐15b) was downregulated in patients with HCC and confirmed association of FUT2 expression with expression of its product, Globo H. Therefore, our results suggest that HBX suppressed the expression of miR‐15b, which directly targeted FUT2 and then increased levels of Globo H to enhance HCC cell proliferation. Additionally, proliferation of HBX‐overexpressing HCC cells was significantly inhibited by treatment with Globo H antibody in vitro. In xenograft animal experiments, we found that overexpression of miR‐15b effectively suppressed tumor growth. The newly identified HBX/miR‐15b/FUT2/Globo H axis suggests one possible molecular mechanism of HCC cell proliferation and represents a new potential therapeutic target for HCC treatment.     

Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo

Alterations in the p53 tumor-suppressor gene occur in 35–60% of human glioblastomas, and re-introduction of p53 can suppress neoplastic growth. To evaluate the potential for p53 gene therapy of glioblastoma, we have analyzed the response of human glioblastoma cell lines in vitro and in vivo to experimental therapy with replication-deficient recombinant adenoviruses encoding wild-type p53 (rAd-p53). Western blot analyses showed high-level expression of p53 protein after treatment with rAd-p53, and transgene expression was dependent on promoter strength. A p53-specific dose-dependent inhibition of in vitro cellular proliferation was observed in 5 of 6 cell lines, and growth inhibition corresponded to adenovirus-mediated gene transfer and expression. p53-specific cell death was quantitated by release of the lactate dehydrogenase enzyme. Fragmentation of DNA into nucleosomal oligomers and the occurrence of a hypodiploid cell population detected by flow cytometry provided evidence for apoptosis. Studies in nude mice demonstrated that ex vivo infection with rAd-p53 suppressed the tumorigenic potential of human glioblastoma cells. Furthermore, direct injection of rAd-p53 into established s.c. xenografts inhibited tumor growth. Our observations suggest that re-introduction of wild-type p53 may have potential clinical utility for gene therapy of glioblastoma. © 1996 Wiley-Liss, Inc.     

miR-1228 promotes the proliferation and metastasis of hepatoma cells through a p53 forward feedback loop

Background:

The effective mechanisms of microRNAs (miRNAs) functions as oncogenes or tumour suppressors in human hepatocellular carcinoma (HCC) are still obscure. Here, we investigated the function and expression of miR-1228 in HCC.

Methods:

The role of miR-1228 in HCC was determined by colony formation, transwell, and nude mice xenograft experiments. miR-1228 target gene were identified by EGFP reporter assays, real-time PCR, and western blot analysis. Dual-luciferase reporter assay and real-time PCR analysis are used to examine the regulation of p53.

Results:

miR-1228 promoted proliferation and metastasis, and facilitated the transition of cell cycle in hepatoma cells. miR-1228 downregulated p53 expression by binding to its 3′UTR. The ectopic expression of p53 abrogated the phenotypes induced by miR-1228. An inverse correlation existed between miR-1228 and p53 expression in hepatoma tissues compared with the adjacent tissues and three hepatoma cell lines. Moreover, we found that p53 suppressed the expression and promoter activity of miR-1228.

Conclusions:

miR-1228 functions as an oncogene by promoting cell cycle progression and cell mobility and negatively regulates the expression of p53. p53 downregulation in turn leads to an increase in miR-1228 expression, thereby forming a positive feedback loop that contributes to cancerogenesis in HCC.