Identification of pigment epithelium-derived factor protein forms with distinct activities on tumor cell lines.

PURPOSE: Pigment epithelium-derived factor (PEDF) is a multifunctional serpin. The purpose of this study is to identify PEDF protein forms and investigate their biological activities on tumor cell lines. METHODS: Recombinant human PEDF proteins were purified by cation- and anion-exchange column chromatography. They were subjected to SDS-PAGE, IEF, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, rat retinal R28, and human ARPE-19 cells. RESULTS: Two PEDF protein peaks were identified after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 had higher pI value and lower apparent molecular weight than PEDF-2. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. CONCLUSION: Two distinct biochemical forms of PEDF varying in overall charge have distinct biological effects on tumor cell viability and migration. The existence of PEDF forms may explain the multifunctional modality of PEDF.

Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth.

Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-Myc(T58A)+H-RasG12V). The transformed cells, WT(t) and DKO(t) respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKO(t) cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/10(7) cells from 9 to 11 nmol/10(7) WT(t) or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKO(t) cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.

Identity between the PCPH proto-oncogene and the CD39L4 (ENTPD5) ectonucleoside triphosphate diphosphohydrolase gene.

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.

Alteraciones frecuentes en la expresión del oncogén PCPH en sistemas experimentales de cáncer de mama / Frequent alterations in the expression of the PCPH oncogene in experimental models of breast cancer

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Expression of the protein product of the PCPH proto-oncogene in human tumor cell lines.

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.

Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1.

Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH.

The human PCPH proto-oncogene: cDNA identification, primary structure, chromosomal mapping, and expression in normal and tumor cells.

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.

cDNA isolation, expression, and chromosomal localization of the mouse pcph proto-oncogene.

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.

Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor.

Ewing's sarcoma (EWS) cells accumulate elevated steady-state levels of poly (ADP-ribose) polymerase (PARP) mRNA and protein. To understand the molecular mechanisms underlying PARP upregulation, we cloned and analysed the 5'-flanking region of the PARP gene from EWS cells. Nucleotide sequence analysis demonstrated no variations in the PARP promoter region in EWS cells. The PARP promoter encompasses multiple binding motifs for the ETS transcription factor. We have also observed that there is a coordinated up-regulation of the expression of both PARP and ETS1, relative to cells of other human tumor types expressing lower levels of PARP. Transient co-expression of ETS1 in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of ETS in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with Ets1 antisense cDNA constructs. Antisense-mediated down-regulation of endogenous ETS1 resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for ETS regulation of PARP expression levels, and implicate ETS transcription factors in the radiation response of EWS cells.

Detection of heterogeneity of apoptotic fragments of poly (ADP-ribose) polymerase in MDA-MB-468 breast cancer cells: two-dimensional gel analysis.

Caspace-mediated proteolysis of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) (EC 2.4, 2.30) is a biochemical marker of cell death in response to various apoptotic stimuli. Anti-PARP antibodies identifying the 89 kDa polypeptide from the C-terminus as well as the 113 kDa native enzyme are often used to demonstrate evidence of apoptosis-associated, interleukin converting enzyme (ICE)-mediated limited cleavage. Recent evidence points to redundancy of caspases, heterogeneity of their cleavage sites, and a possibility of generating distinct context-specific, and cell-specific PARP fragments. In the present study, we employed antibodies directed to multiple sites in PARP and probed two-dimensionally resolved proteins of the estrogen receptor negative MDA-MB-468 breast tumor cells, induced to undergo apoptosis by ionizing radiation (IR). Our results revealed that the 24 kDa apoptotic fragment of PARP, from the N-terminus, consists of at least three isoforms, located at a p/more basic than the full length enzyme. We also report a hitherto unrecognized feature of an anti-PARP antiserum, VIC-5, detecting both the 89 kDa and the 24 kDa caspase-generated fragments of PARP. Thus, application of two-dimensional electrophoresis combined with antisera directed to multiple sites would be valuable in distinguishing PARP cleavage site- and inhibitor specificities of proteases during apoptosis.

The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress.

Cph was isolated from neoplastic Syrian hamster embryo fibroblasts initiated by 3-methylcholanthrene (MCA), and was shown to be a single copy gene in the hamster genome, conserved from yeast to human cells, expressed in fetal cells and most adult tissues, and acting synergistically with H-ras in the transformation of murine NIH3T3 fibroblasts. We have now isolated Syrian hamster full-length cDNAs for the cph oncogene and proto-oncogene. Nucleotide sequence analysis revealed that cph was activated in MCA-treated cells by a point-mutational deletion at codon 214, which caused a shift in the normal open reading frame (ORF) and brought a translation termination codon 33 amino acids downstream. While proto-cph encodes a protein (pcph) of 469 amino acids, cph encodes a truncated protein (cph) of 246 amino acids with a new, hydrophobic C-terminus. Similar mechanisms activated cph in other MCA-treated Syrian hamster cells. The cph and proto-cph proteins have partial sequence homology with two protein families: GDP/GTP exchange factors and nucleotide phosphohydrolases. In vitro translated, gel-purified cph proteins did not catalyze nucleotide exchange for H-ras, but were able to bind nucleotide phosphates, in particular ribonucleotide diphosphates such as UDP and GDP. Steady-state levels of cph mRNA increased 6.7-fold in hamster neoplastic cells, relative to a 2.2-fold increase in normal cells, when they were subjected to a nutritional stress such as serum deprivation. Moreover, cph-transformed NIH3T3 cells showed increased survival to various forms of stress (serum starvation, hyperthermia, ionizing radiation), strongly suggesting that cph participates in cellular mechanisms of response to stress.

Molecular aspects of neoplasia of Syrian hamster cells transformed in vitro by chemical carcinogens.

The addition of environmental agents (carcinogens) induces transformation that can be quantitated. Its frequency follows a linear relationship with dose and is consistent with a 'one hit' phenomenon. Transformed colonies produce transformed lines with attributes of neoplastic cells including production of tumors. The results parallel in vivo activity. Although, molecular analysis of most animal assay indicate the presence of activated oncogenes belonging to the ras family, ras activation is a low frequency event in the neoplastic conversion of Syrian hamster cells just as is found with human malignancies. In our analysis of 22 independently derived lines N-ras activation was found only with sodium bisulfite transformed lines. A novel oncogene named carcinogenesis promotion hamster (cph) because its association with the carcinogenic process has been identified. This resulted from focusing on Syrian hamster cells transformed with a single dose of 3-methylcholanthrene (MCA) and cosmid-rescue-techniques from a third-cycle NIH3T3 transformant obtained by sequential transfections of genomic DNA from MCA-initiated hamster fetal cells. cph transforms NIH3T3 cells and acts synergistically with Ha-ras to transform murine fibroblasts. Gene expression analysis using cph genomic fragments from normal and neoplastic cells identifies a number of transcripts including a major mRNA of 2.5 kb as well as several larger transcripts. cph is actively transcribed in different tissues and different species. In the hamster it is a single copy gene localized by FISH to the euchromatic short arm of the X chromosome, at region Xpa7. cph does not have any significant global homology to sequences deposited in date banks, confirming that it is a novel gene. The transforming gene codes for a truncated 246 amino acids whereas the normal cph has a residue of 469 amino acids. In conclusion cph oncogene is activated by a single point-mutation; its activation appears an important mechanism for the conversion of carcinogen treated hamster cells to malignancy and because homologous sequences occur in human DNA it may also be important to the neoplastic conversion of human cells.

Sequence and expression analysis of bovine pigment epithelium-derived factor.

PEDF, a member of the serpin superfamily of proteins related through their highly conserved folded conformation, has neurotrophic properties, including promotion of neurite-outgrowth and neuronal survival. Previously, we have purified and characterized PEDF protein from extracellular matrixes of bovine eyes. Here, we show the cDNA sequence and expression analysis of bovine PEDF. Northern analysis of RNA from bovine retinal pigment epithelium (RPE) and neural retina using a human PEDF cDNA fragment reveals expression of the PEDF gene only for RPE. Sequence analysis of a cDNA clone isolated from bovine RPE predicts a polypeptide of 416 amino acid residues that shares 88.6% and 85% amino acid identity with human and mouse PEDF, respectively. It has an N-terminal signal peptide, a consensus glycosylation site and homology with serpins including the conserved residues required for maintaining the serpin tertiary structure. Cell-free expression of the bovine PEDF cDNA by in vitro transcription and translation yields a precursor polypeptide of 45,000-Mr that immunoprecipitates with an antibody to human PEDF. Expression analysis in stably transfected baby hamster kidney cells shows that the recombinant bovine protein is secreted to the culture media as a mature 50,000-Mr protein, which induces neurite-outgrowth on retinoblastoma cells, like the naturally-occurring PEDF protein. Thus, the bovine PEDF cDNA isolated here codes for a functional soluble secreted PEDF glycoprotein.

Secretion of neu differentiation factor-like polypeptides by cph-transformed fibroblasts: cloning and characterization of Syrian hamster neuregulin cDNAs.

We reported previously that expression of the cph oncogene in Syrian hamster and mouse cells leads to the secretion of a polypeptide factor or factors structurally and functionally related to neu differentiation factor (NDF) and the establishment of an autocrine loop mediated through the simultaneous overexpression of the erbB4 receptor. To identify the nature of this factor and to characterize its role in the neoplastic conversion of Syrian hamster embryo cells, we cloned cDNAs hybridizing to rat NDF-derived probes by screening a library prepared from neoplastic 81C39 cells, which harbor an activated cph oncogene and secrete active NDF-like polypeptides. Sequence analysis of the isolated clones revealed a high level of homology between the hamster neuregulin and its rat and human counterparts and the existence of various neuregulin cDNA variants in Syrian hamster cells, presumably originating from alternatively spliced RNA species. Interestingly, some of these neuregulin cDNAs were longer (up to 5.5 kb) than those isolated before from any other system, suggesting that the Syrian hamster neuregulin precursor mRNA accumulates at greater concentrations than in other species. We also detected different hamster neuregulin protein isoforms by using in vitro transcription-translation analyses. Peptide sequence analysis identified the major NDF-like polypeptide secreted by 81C39 cells as an alpha2b-neuregulin. Northern blot analyses with the cloned cDNA inserts showed that neuregulin overexpression was commonly associated with the neoplastic conversion of chemically initiated hamster embryo fibroblasts. This, along with the detection of elevated erbB2- or erbB4-specific transcripts in most (six of eight) neoplastic cell lines tested, supports the notion that autocrine neuregulin signaling plays an important role in maintaining the transformed phenotype by providing a growth advantage to cph-transformed cells.

Altered processing of precursor transcripts and increased levels of the subunit I of mitochondrial cytochrome c oxidase in Syrian hamster fetal cells initiated with ionizing radiation.

Treatment of Syrian hamster fetal cells (SHFC) with ionizing radiation resulted in the establishment of 21 transformed cell lines. Relative to unirradiated controls, cells from early post-irradiation passages (p.3) showed marked morphologic alterations, increased growth rate and extended life span, and they were contact-inhibited and not tumorigenic in nude mice, although they became tumorigenic after extended passaging in culture (p. > 30). Differential mRNA display analyses of normal cells (84-3) and radiation-initiated cell lines at early passage showed that the latter contained increased steady-state levels of the precursor (4-fold) and mature (1.7-fold) transcripts of the mitochondrial (mt) gene encoding the subunit I of cytochrome c oxidase (CO I). These molecular alterations were consistently observed in 57% of the irradiated (HDR) cell lines, and were stably maintained during continuous passaging (p. > 50). Further analyses of one of these cell lines (HDR-3) demonstrated that the accumulation of CO I precursor transcripts was the result of mRNA stabilization and increased replication and/or amplification of the mt DNA. Radiation-initiated cells contained elevated levels of the CO I protein, showed a 75% reduction in cytochrome c oxidase (CO) activity, and a 5-fold increase in the concentration of hydrogen peroxide secreted into their culture medium compared with cells with no alterations in CO I mRNA processing. Our findings suggest that alterations in mt CO I processing may play a role in the neoplastic conversion of mammalian cells by ionizing radiation.

Structure and expression of the c-Myc/Pvt 1 megagene locus.

A chromosomal translocation (Tx) that interrupts the transcription of either c-Myc or Pvt 1 is the principal lesion in many B cell malignancies including Burkitt's Lymphoma (BL), AIDs-NHL, mouse plasmacytoma (Pct) and possibly multiple myeloma (MM). There is a restriction associated with this Tx such that only the immunoglobulin (Ig) heavy chain gene is found juxtaposed to c-Myc and only the Ig light chain gene is found juxtaposed to Pvt 1. Over the past several years, our laboratory has been instrumental in the elucidation of the structure of the mouse Pvt 1 locus as a means of understanding the relationship between these two divergent Txs which, nevertheless, produce indistinguishable disease phenotypes. In the mouse, we have identified a uniform Pvt1/Ig Ck fusion product which is consistently found in all tumors harboring Pvt 1 associated Txs. We have recently constructed transgenic mice harboring a translocated Pvt 1/Ck segment in order to determine whether 1). these mice produce the Pvt 1/Ck fusion product 2). these mice are immunocompromised and 3). these mice develop tumors of a B cell origin.

Radiation-initiated, immortal Syrian hamster embryo fibroblasts release elevated levels of H2O2 and show divergent MnSOD and catalase activities.

To characterize molecular events associated with the neoplastic conversion of primary cells by ionizing radiation, we studied the activities and mRNA expression of manganese superoxide dismutase (MnSOD) and catalase (CAT) in Syrian hamster embryo fibroblasts during the early stages of immortalization after treatment with gamma-rays. The irradiated cells showed divergent MnSOD and CAT responses relative to unirradiated controls. At passage 6, MnSOD activity was increased about 50-fold, although the concentration of MnSOD mRNA increased only 1.6-fold. By contrast, CAT activity diminished 2-fold despite an increase of 1.6-fold in the concentration of CAT mRNA. This divergence between the MnSOD and CAT activities was maintained upon culturing and, at passage 12, MnSOD was 35-fold increased and CAT 3.7-fold decreased, relative to unirradiated cells. The amount of H2O2 released into the culture medium by the radiation-initiated cells was 6-fold greater than in control media. Because H2O2 is a causative agent in the induction of malignant transformation in vitro, our results suggest that the elevated production of H2O2 caused by the imbalance between the activities of MnSOD and CAT may participate in the immortalization and subsequent malignant conversion of Syrian hamster embryo fibroblasts by ionizing radiation.

Increased manganese superoxide dismutase activity, protein, and mRNA levels and concurrent induction of tumor necrosis factor alpha in radiation-initiated Syrian hamster cells.

The levels of the antioxidant enzyme manganese superoxide dismutase (Mn-SOD) are frequently decreased in tumor cells and increased in normal cells upon treatment with ionizing radiation. We studied Mn-SOD at different stages during the neoplastic conversion of radiation-initiated Syrian hamster embryo HDR-3 cells. Mn-SOD activity and the concentration of Mn-SOD protein and mRNA increased gradually during the malignant transformation of HDR-3 cells after radiation exposure; fully neoplastic cells showed greater Mn-SOD levels than preneoplastic and normal 84-3 cells. Inhibitors of superoxide (SO) anion production (thenoyltrifluoroacetone and rotenone) decreased the concentration of Mn-SOD mRNA, raising the possibility that the generation of SO radicals participated in the upregulation of Mn-SOD in cells transformed by exposure to radiation. We observed an increase in the concentration of tumor necrosis factor alpha (TNF alpha) in HDR-3 cells relative to mock-irradiated cells. Together with the observation that TNF alpha stimulates the production of SO by mitochondria and increases the level of Mn-SOD mRNA in other experimental systems, our results suggest that as normal 84-3 cells undergo malignant transformation induced by ionizing radiation they produce TNF alpha, to which the cells respond by increasing the concentration of Mn-SOD mRNA and protein and the activity of the enzyme.