Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.

p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein.

A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53-/- mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53-/- mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared with p53-/- basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor alpha also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor alpha.

The antiapoptotic decoy receptor TRID/TRAIL-R3 is a p53-regulated DNA damage-inducible gene that is overexpressed in primary tumors of the gastrointestinal tract.

Both DR4 and DR5 have recently been identified as membrane death receptors that are activated by their ligand TRAIL to engage the intracellular apoptotic machinery. TRID (also named as TRAIL-R3) is an antagonist decoy receptor and lacks the cytoplasmic death domain. TRID protects from TRAIL-induced apoptosis by competing with DR4 and DR5 for binding to TRAIL. TRID has been shown to be overexpressed in normal human tissues but not in malignantly transformed cell lines. DR5 is a p53-regulated gene and we have recently reported that DR5 expression is induced in response to genotoxic stress in both a p53-dependent and independent manner (Sheikh et al., 1998). In the current study, we demonstrate that TRID gene expression is also induced by the genotoxic agents ionizing radiation and methyl methanesulfonate (MMS) in predominantly p53 wild-type cells, whereas UV-irradiation does not induce TRID gene expression. Consistent with these results, exogenous wild-type p53 also upregulates the expression of endogenous TRID in p53-null cells. Thus, TRID appears to be a p53 target gene that is regulated by genotoxic stress in a p53-dependent manner. Using primary gastrointestinal tract (GIT) tumors and their matching normal tissue, we also demonstrate for the first time that TRID expression is enhanced in primary tumors of the GIT. It is, therefore, possible that TRID overexpressing GIT tumors may gain a selective growth advantage by escaping from TRAIL-induced apoptosis.

Cloning and characterization of a human genotoxic and endoplasmic reticulum stress-inducible cDNA that encodes translation initiation factor 1(eIF1(A121/SUI1)).

We report the cloning and characterization of a DNA damage-inducible (DDI) transcript DDI A121. The full-length human DDI A121 cDNA contains an open reading frame of 113 amino acids, corresponding to a protein of 12.7 kDa. The deduced amino acid sequence of A121 shows high homology to the yeast translation initiation factor (eIF) sui1 and also exhibits perfect identity to the partial sequence of recently purified human eIF1. Expression of human A121 corrected the mutant sui1 phenotype in yeast, demonstrating that human A121 encodes a bona fide translation initiation factor that is equivalent to yeast sui1p. The mammalian A121/SUI1 gene exhibits two transcripts (1.35 kilobases and 0.65 kilobases) containing a common coding region but differing in their 3'-untranslated region. The long and short A121/SUI1 mRNAs are differentially regulated by genotoxic and endoplasmic reticulum stress. The genotoxic stress induction of A121/SUI1 mRNA is conserved in both humans and rodents and occurs in a p53-independent manner. Our identification of a stress-inducible cDNA that encodes eIF1 suggests that modulation of translation initiation appears to occur during cellular stress and may represent an important adaptive response to genotoxic as well as endoplasmic reticulum stress.

Genetic deletion of p21WAF1 enhances papilloma formation but not malignant conversion in experimental mouse skin carcinogenesis.

Tumor suppression by p53 is believed to reside in its ability to regulate gene transcription, including up-regulation of p21WAF1. In p53(-/-) mice, chemical- or oncogene-induced skin tumors undergo accelerated malignant conversion. To determine the contribution of the p21WAF1 gene product to epidermal carcinogenesis, animals +/+, +/-, and -/- for a null mutation in the p21WAF1 gene were treated once with 25 nmol 7,12-dimethylbenz[a]anthracene, followed by 5 microg of TPA two times/week for 20 weeks. Papilloma frequency was higher in the p21WAF1-deficient mice. However, the frequency of malignant conversion was similar among all three genotypes. After TPA treatment, all genotypes developed epidermal hyperplasia, although the labeling index was lower in p21WAF1 (-/-) epidermis compared with p21WAF1 (+/+). Furthermore, the expression of differentiation markers was the same across genotypes in untreated or TPA-treated epidermis. Similar frequencies of malignant conversion were also observed in an in vitro assay. Thus, p21WAF1 suppresses early stages of papilloma formation but not malignant progression in mouse skin carcinogenesis, and decreased levels of p21WAF1 do not account for the enhanced malignant conversion of p53 null epidermal tumors.

Potato carboxypeptidase inhibitor, a T-knot protein, is an epidermal growth factor antagonist that inhibits tumor cell growth.

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in many aspects of the development of carcinomas, including tumor cell growth, vascularization, invasiveness, and metastasis. Because EGFR has been found to be overexpressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. Here we report that potato carboxypeptidase inhibitor (PCI), a 39-amino acid protease inhibitor with three disulfide bridges, is an antagonist of human EGF. It competed with EGF for binding to EGFR and inhibited EGFR activation and cell proliferation induced by this growth factor. PCI suppressed the growth of several human pancreatic adenocarcinoma cell lines, both in vitro and in nude mice. PCI has a special disulfide scaffold called a T-knot that is also present in several growth factors including EGF and transforming growth factor alpha. PCI shows structural similarities with these factors, a fact that can explain the antagonistic effect of the former. This is the first reported example of an antagonistic analogue of human EGF.