Mehmet Ozturk

P53 FUNCTIONS AS A CELL CYCLE CONTROL PROTEIN IN OSTEOSARCOMAS

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

p53 mutation in hepatocellular carcinoma after aflatoxin exposure

Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.

Smad2 and Smad4 gene mutations in hepatocellular carcinoma

TGF-β is a negative regulator of liver growth. Smad family of genes, as mediators of TGF-β pathway, are candidate tumor suppressor genes in hepatocellular carcinoma (HCC). We studied 35 HCC and non-tumour liver tissues for possible mutations in Smad2 and Smad4 genes. Three tumours displayed somatic mutations; two in Smad4 (Asp332Gly and Cys401Arg) and one in Smad2 (Gln407Arg) genes. All three mutations were A:T → G:C transitions suspected to result from oxidative stress as observed in mitochondrial DNA. These observation demonstrate that TGF-β pathway is altered in hepatocellular carcinoma.

Retinoblastoma and p53 tumor suppressor genes in human hepatoma cell lines.

We analyzed the status of retinoblastoma and p53 genes in 10 human hepatoma cell lines. Polyclonal anti‐peptide antibodies generated against peptides homologous to COOH‐terminal and leucine‐zipper domains of the retinoblastoma protein allowed us to identify two cell lines (Hep 3B and FOCUS) with abnormal expression. The same cell lines have both lacked p53 expression. In contrast to the retinoblastoma gene, the expression of the p53 gene was abnormal in six additional cell lines. Indeed, only the Hep G2 hepatoblastoma cell line (and its derivative Hep G2/2215) appeared to have normal p53 and retinoblastoma gene expression. Our studies indicate that p53 abnormalities are common but retinoblastoma gene aberrations are rare in human hepatoma cell lines.—Puisieux, A., Galvin, K., Troalen, F., Bressac, B., Marcais, C., Galun, E., Ponchel, F., Yakicier, C., Ji, J., Ozturk, M. Retinoblastoma and p53 tumor suppressor genes in human hepatoma cell lines. FASEB J. 7: 1407‐1413; 1993.

Transforming growth factor‐beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth

Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16Ink4a) are frequently inactivated in these cancers. We tested whether transforming growth factor‐β (TGF‐β) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF‐β signaling that leads to small mothers against decapentaplegic (Smad)‐targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF‐β1 (1‐5 ng/mL) treatment. Upon treatment, c‐myc was down‐regulated, p21Cip1 and p15Ink4b were up‐regulated, and cells were arrested at G1. The expression of p16Ink4a was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21Cip1 and p15Ink4b recapitulated TGF‐β1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N‐acetyl‐L‐cysteine, or silencing of the NOX4 gene, rescued p21Cip1 and p15Ink4b accumulation as well as the growth arrest in response to TGF‐β. Human HCC tumors raised in immunodeficient mice also displayed TGF‐β1–induced senescence. More importantly, peritumoral injection of TGF‐β1 (2 ng) at 4‐day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF‐β receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. Conclusion: TGF‐β induces p53‐independent and p16Ink4a‐independent, but Nox4‐dependent, p21Cip1‐dependent, p15Ink4b‐dependent, and ROS‐dependent senescence arrest in well‐differentiated HCC cells. Moreover, TGF‐β–induced senescence in vivo is associated with a strong antitumor response against HCC. HEPATOLOGY 2010

p53 mutation as a source of aberrant β-catenin accumulation in cancer cells

β-catenin is involved in both cell–cell interactions and wnt pathway–dependent cell fate determination through its interactions with E-cadherin and TCF/LEF transcription factors, respectively. Cytoplasmic/nuclear levels of β-catenin are important in regulated transcriptional activation of TCF/LEF target genes. Normally, these levels are kept low by proteosomal degradation of β-catenin through Axin1- and APC-dependent phosphorylation by CKI and GSK-3β. Deregulation of β-catenin degradation results in its aberrant accumulation, often leading to cancer. Accordingly, aberrant accumulation of β-catenin is observed at high frequency in many cancers. This accumulation correlates with either mutational activation of CTNNB1 (β-catenin) or mutational inactivation of APC and Axin1 genes in some tumors. However, there are many tumors that display β-catenin accumulation in the absence of a mutation in these genes. Thus, there must be additional sources for aberrant β-catenin accumulation in cancer cells. Here, we provide experimental evidence that wild-type β-catenin accumulates in hepatocellular carcinoma (HCC) cells in association with mutational inactivation of p53 gene. We also show that worldwide p53 and β-catenin mutation rates are inversely correlated in HCC. These data suggest that inactivation of p53 is an important cause of aberrant accumulation of β-catenin in cancer cells.

Enhanced Expression of an Exocrine Pancreatic Protein in Alzheimer's Disease and the Developing Human Brain

Pancreatic thread protein (PTP) is a major exocrine secretory protein that in vitro forms filamentous bundles reminiscent of the paired helical filaments of Alzheimers disease (AD). We previously described increased PIP immunoreactivity in AD brains and now report high levels in the developing human brain. Using a full-length cloned bovine PTP cDNA and synthetic oligonucleotides corresponding to human PTP cDNA, which is identical to human islet cell regeneration factor, we analyzed the expression ofPTP in pancreas and brain. A major 0.9-kb as well as several minor transcripts were identified in human pancreas. In AD brain, the same size transcripts were detected by Northern analysis, primer extension assay, or polymerase chain reaction amplification of cDNAs generated by reverse transcriptase assay. There were significantly higher levels of PIP mRNA in brains with AD compared with aged controls, with increased amounts of 1.2-, 0.6-, and 0.4-kb transcripts by Northern analysis. In situ hybridization localized expression to pyramidal neurons in the cerebral cortex, the same population that contains neurofibrillary tangles and high levels ofimmunoreactive PIP. These findings suggest thatAD is associated with enhanced expression of PTP-related transcripts with intraneuronal accumulation of PTIP-like proteins. (J. Clin. Invest. 1990. 86:1004-1013.) Key words: pancreatic thread protein* Alzheimers disease * Downs syndrome * central nervous system

Enhanced expression of the protein kinase substrate p36 in human hepatocellular carcinoma.

A basic phosphoprotein defined by a monoclonal antibody named AF5 was found to be highly abundant in human hepatocellular carcinoma by Western immunoblotting. Under the same conditions, the levels of this phosphoprotein were low or undetectable in normal liver extracts. The AF5 antibody was used to screen a cDNA expression library of a human hepatoma cell line named FOCUS. A 960-base-pair cDNA was isolated and found to be a partial cDNA encoding the human protein-tyrosine kinase substrate p36, also known as lipocortin II. p36 expression was highly abundant in hepatocellular carcinomas at both the transcript and protein levels. Its expression was not induced significantly during rat liver regeneration following a partial hepatectomy. These results suggest that the induction of p36 expression is associated with malignant transformation of hepatocytes. p36 was previously shown to be phosphorylated upon transformation of normal fibroblasts by retroviral oncogenes without significant modulation of expression. We report here the initial description of the association of increased p36 expression with malignant transformation.

Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells.

p53 and p73 proteins activate similar target genes and induce apoptosis and cell cycle arrest. However, p53, but not p73 is considered a tumour-suppressor gene. Unlike p53, p73 deficiency in mice does not lead to a cancer-prone phenotype, and p73 gene is not mutated in human cancers, including hepatocellular carcinoma. Here we report that normal liver cells express only ΔN-p73 transcript forms giving rise to the synthesis of N-terminally truncated, transcriptionally inactive and dominant negative p73 proteins. In contrast, most hepatocellular carcinoma cells express TA-p73 transcript forms encoding full-length and transcriptionally active p73 proteins, in addition to ΔN-p73. We also show that together with the acquired expression of TA-p73, the ‘retinoblastoma pathway’ is inactivated, and E2F1-target genes including cyclin E and p14ARF are activated in hepatocellular carcinoma. However, there was no full correlation between ‘retinoblastoma pathway’ inactivation and TA-p73 expression. Most TA-p73-expressing hepatocellular carcinoma cells have also lost p53 function either by lack of expression or missense mutations. The p73 gene, encoding only ΔN-p73 protein, may function as a tumour promoter rather than a tumour suppressor in liver tissue. This may be one reason why p73 is not a mutation target in hepatocellular carcinoma.

Senescence and immortality in hepatocellular carcinoma

Cellular senescence is a process leading to terminal growth arrest with characteristic morphological features. This process is mediated by telomere-dependent, oncogene-induced and ROS-induced pathways, but persistent DNA damage is the most common cause. Senescence arrest is mediated by p16(INK4a)- and p21(Cip1)-dependent pathways both leading to retinoblastoma protein (pRb) activation. p53 plays a relay role between DNA damage sensing and p21(Cip1) activation. pRb arrests the cell cycle by recruiting proliferation genes to facultative heterochromatin for permanent silencing. Replicative senescence that occurs in hepatocytes in culture and in liver cirrhosis is associated with lack of telomerase activity and results in telomere shortening. Hepatocellular carcinoma (HCC) cells display inactivating mutations of p53 and epigenetic silencing of p16(INK4a). Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment.