Oncogene database

957

ENTPD5

ectonucleoside triphosphate diphosphohydrolase 5;ENTPD5;ectonucleoside triphosphate diphosphohydrolase 5

CD39 antigen-like 4|CD39-like 4|ER-UDPase|GDPase ENTPD5|NTPDase 5|Pcph proto-oncogene protein|UDPase ENTPD5|guanosine-diphosphatase ENTPD5|nucleoside diphosphatase|proto-oncogene CPH|uridine-diphosphatase ENTPD5

14q24

protein-coding

We report here the isolation and characterization of a cDNA from mouse thymus encoding the murine homolog of the protein product of the Syrian hamster Pcph proto-oncogene. The single open reading frame identified in the cDNA sequence encoded a protein predicted to have 428 amino acids, which shared 93.7% amino acid identity with the Syrian hamster Pcph within the first 412 residues but had a shorter, highly dissimilar C-terminus. Northern and western analyses revealed that Pcph mRNA and protein were widely distributed in mouse embryo and adult tissues, with the highest expression in adults detected in kidney and liver. The mouse Pcph proto-oncogene was mapped by linkage analysis to within 3.3+/-2.3 cM of Pkch-rs1 on chromosome 12. These data should prove valuable in designing studies to define the cellular function of the Pcph proto-oncogene.

We identified a human cDNA encoding a 47-kDa protein that shares 78% and 87% identity with the products of the Syrian hamster and mouse PCPH proto-oncogenes respectively. The human homolog was localized by radiation-hybrid mapping to chromosome band 14q24.3, a region syntenic to the Pcph location on mouse chromosome 12. Northern analyses revealed that PCPH mRNA was widely distributed in normal human adult tissues, but its expression varied significantly among human tumor cells and cell lines of several tissue types, regardless of the level of expression in the corresponding normal tissues. The highest levels of PCPH mRNA and protein were detected in kidney and liver. However, PCPH was not expressed in the majority of human neoplasms tested, including kidney tumors. These data provide suggestive evidence for a possible association of the lack of PCPH expression to the neoplastic phenotype of human tumor cells. Our results should prove instrumental in designing studies to define the cellular function of the human PCPH proto-oncogene.

expression of the Protein Product of the PCPH proto-oncogene in Human tumor Cell Lines. Exposure of Syrian hamster embryo fibroblasts to chemical carcinogens resulted in the oncogenic activation of the PCPH proto-oncogene by induction of a single base-pair deletion that generated a truncated PCPH oncoprotein (mutated PCPH). Recently, we isolated and characterized the cDNA for the human PCPH proto-oncogene and determined that in humans PCPH is a single-copy gene located in chromosome 14 (14q24.3). Pilot mRNA expression studies indicated that PCPH was expressed in the majority of normal organs tested, particularly in liver and kidney, but it appeared to be expressed either at low levels or not at ALL in tumor cells or cell lines derived from the high-expressing tissues. We have generated an antiserum against bacterial recombinant Syrian hamster PCPH. This antiserum recognizes both the normal and truncated, oncogenic Syrian hamster PCPH proteins and cross-reacts with the yeast, mouse, rat and human homologue proteins. Using this antibody, we have performed a study of PCPH expression in a larger sample of human neoplastic cell lines, including some derived from breast, nervous system, colon, lung and pancreas tumors. Results confirmed the frequent lack of PCPH expression in malignant cells and identified several immunoreactive forms of PCPH being differentially expressed in cells of diverse tissue origins.

PCPH was initially defined as a proto-oncogene on the basis of its frequent detection as an activated oncogene in tumorigenic Syrian hamster embryo fibroblast cell lines converted to the neoplastic state by a single treatment with the carcinogen 3-methylcholanthrene (MC). Further studies identified the translation product of the PCPH gene as a ribonucleotide-binding protein with special affinity for ribonucleoside diphosphates. Later, we showed that the PCPH protein was homologous to the product of the yeast GDA1 gene and demonstrated that it had intrinsic guanosine diphosphatase activity, although it did not complement the disrupted phenotype when expressed in gda1 null Saccharomyces cerevisiae strains. These results indicated that the primary function of PCPH was unlikely to be related to the ribonucleotide recycling function that its yeast counterpart performs in the Golgi during the process of protein glycosylation. However, taken together, our data strongly suggested that the normal cellular function of PCPH was related to ribonucleotide metabolism. We now report that PCPH is structurally and functionally identical to the mammalian ectonucleoside triphosphate diphosphohydrolase CD39L4 (ENTPD5), recently described as a member of the lymphoid activation antigen () CD39 protein family. These results may help to establish the normal cellular function of the PCPH proto-oncogene product and its role in neoplastic development during carcinogenesis.

The PCPH proto-oncogene was identified by its frequent activation in Syrian hamster fetal cells exposed to 3-methylcholanthrene. We previously isolated human PCPH cDNA and studied its expression in normal human tissues. We report herein the pattern of PCPH expression in normal rat tissues. Each tissue expressed one major PCPH polypeptide that varied in molecular mass in different tissues. Normal mammary gland expressed a single PCPH polypeptide of 27 kDa. This PCPH form also was expressed in lactating mammary glands but at significantly greater levels. These results suggest the existence of tissue-specific regulatory mechanisms for PCPH expression that may be influenced by the differentiation stage. Our previous studies on the involvement of PCPH in human cancer showed that human breast tumor cell lines have frequent alterations in PCPH, including multiple PCPH polypeptide forms that are not expressed in normal cells. These cell lines also have frequent loss of a 27-kDa form identified as the only PCPH polypeptide expressed by normal human breast epithelial cells. In this study, we found that these same alterations occurred in vivo during mammary carcinogenesis in Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene, in both benign and malignant tumors, indicating that stable changes in PCPH expression took place early in the neoplastic process. Results showed that this experimental system is relevant to human breast carcinogenesis and provides an excellent model to study the molecular basis of the regulation of PCPH expression during normal differentiation and pathologic stages of neoplasia of the mammary gland and to analyze the role of PCPH in the carcinogenic process. Furthermore, the detection of atypical PCPH polypeptides in tumors suggests that PCPH immunodetection may be applied as a diagnostic tool for the early identification of neoplastic breast epithelial cells.

Promotion of cellular resistance to stressful stimuli, including ionizing radiation and chemotherapeutic drugs, contributes to the transforming activity of the PCPH oncogene. The mechanism of this action, however, has remained unknown. Consistent with its intrinsic ATP diphosphohydrolase activity, expression of the PCPH oncoprotein in cultured cells has now been shown to result in partial depletion of intracellular ATP and consequent inhibition of the c-JUN NH2-terminal kinase-mediated stress signaling pathway. Supplementation of cells expressing the PCPH oncoprotein with exogenous ATP restored both stress-response signaling and sensitivity to cisplatin-induced apoptosis. In contrast, overexpression of the wild-type PCPH protein had a minimal effect on stress-induced signaling and on the cellular ATP content and did not protect cells from apoptosis. These results suggest that the PCPH oncoprotein confers resistance to stressors by reducing the cellular ATP concentration to levels below those required for optimal stress-induced signaling and apoptosis. Treatment with adenosine or nucleoside analogues may thus enhance the response to radiation or chemotherapy of tumors that express the PCPH oncogene.

We performed a study on the expression of the PCPH protein in samples corresponding to normal, pre-malignant and malignant stages of the human mammary gland by using protocols of immunohistochemistry and Western blot analysis with anti-PCPH specific antibodies. Results obtained from the immunohistochemical study showed that PCPH was undetectable in samples of normal breast and of benign diseases, with the exception of glands presenting apocrine metaplasia, in which an intense PCPH stain was observed both in the basal cytoplasm of the secretory cells and in the apocrine secretion. On the contrary, an intense labeling was observed in the cytoplasm of neoplastic cells in samples of both ductal and lobular carcinoma in situ, with this immunostaining increasing even further in samples of infiltrating carcinoma, both ductal and lobular. Western blot analyses of the same set of samples detected a 47 kDa form as the main PCPH polypeptide present in ALL cases studied. However, whereas this 47 kDa polypeptide was the only PCPH form detected in normal and pre-malignant samples, multiple forms could be detected in carcinoma samples, indicating the presence of altered PCPH polypeptides at these disease stages. These results were in agreement with those from the immunohistochemical study and together indicated that PCPH protein expression represents a good molecular marker to follow the process of human breast carcinogenesis. Furthermore, these results suggested that characterization of the pattern and level of PCPH expression may be a useful tool for early identification of breast cancers.

expression of the ENTPD5/mt-PCPH onco-protein and overexpression of the normal ENTPD5/PCPH protein contribute to the malignant transformation of diverse mammalian cell types, and PCPH is mutated and/or deregulated in various human tumor types. expression of PCPH or mt-PCPH caused similar phenotypes, yet the effects promoted by mt-PCPH expression were consistently and substantially greater. ATP depletion and increased stressresistance are phenotypes commonly associated with PCPH and mt-PCPH expression. It was suggested that the intrinsic nucleoside triphosphate diphosphohydrolase (NTPDase) activity of PCPH and mt-PCPH may be responsible for these phenotypes, but direct supporting evidence remains to be established. Results from experiments designed to test such hypothesis demonstrate that, as expected, mt-PCPH expression in human colorectal carcinoma (CRC) cells decreased their ATP levels and conferred resistance to oxaliplatin, a colorectal cancer-relevant chemotherapeutic agent. Using a combination of site-directed mutagenesis, immunoprecipitation methods, in vitro enzyme activity assays and in situ enzyme activity determinations in live cells, this report also demonstrates that the mt-PCPH oncoprotein lacks detectable NTPDase activity, indicating that direct ATP cleavage by mt-PCPH did not cause the ATP depletion observed in mt-PCPH-expressing CRC cells. These results strongly suggest that the mt-PCPH oncoprotein may regulate the cellular energy levels and subsequent chemoresistance by an NTPDase-independent mechanism. Understanding possible alternative mechanisms will be essential to devise strategies for the successful treatment of predictably therapeutically resistant tumors expressing either increased PCPH levels or, particularly, the mt-PCPH oncoprotein.