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.

Expression of the fgr protooncogene product as a function of myelomonocytic cell maturation.

The fgr protooncogene is a member of the src family of protein tyrosine kinases. Recent studies have shown that normal myelomonocytic cells and tissue macrophages are the major sites of fgr mRNA expression. In the present study, we have identified the fgr protooncogene protein product in HL60 cells and have examined its expression as a function of HL60 cell maturation. Whether induced toward monocytic or granulocytic lineages, p55c-fgr accumulated in HL60 cells during maturation. In differentiated cells, the protein was active as a protein tyrosine kinase and was localized to peripheral cell membranes. Demonstration that a myristyl group was covalently bound to the protein probably accounted for its subcellular distribution. These findings establish developmental regulation of p55c-fgr in a lineage that represents its natural site of expression.

Primary structure of the human fgr proto-oncogene product p55c-fgr.

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.

Mutations in the p53 gene in radiation-sensitive and -resistant human squamous carcinoma cells.

Five of six human squamous cell carcinoma (SCC) cell lines characterized as radiation sensitive (SQ-38, SCC-9, SQ-9G) or radiation resistant (SQ-20B, SCC-35, JSQ-3) exhibited alterations of the p53 gene. The point mutations and a deletion were detected by using single-stranded conformational polymorphism analysis and polymerase chain reaction-direct sequencing. Interestingly, three of three radiation-sensitive and two of three radiation-resistant cell lines revealed mutations in the p53 gene. Point mutations were located in exons 4, 6, and 8 (at codons 72 and 298 in JSQ-3; 273 in SCC-35; 196 in SQ-38), and deletions consisted of 32 base pairs between codons 274 and 285 in SCC-9 and 1 base pair at codon 271 in SQ-9G. Three mutations resulted in substitutions for an arginine residue. Immunocytochemical analysis confirmed p53 protein overexpression in SCC-35 cells which contained a missense mutation at codon 273. In contrast to previous studies which linked alterations in ras, myc, and raf expression with radiation resistance, this study indicates that mutations in the tumor suppressor gene, p53, do not directly correlate with such resistance.

Enhanced expression of calreticulin in the nucleus of radioresistant squamous carcinoma cells in response to ionizing radiation.

Ionizing radiation has been shown to modulate gene and protein expression as well as cellular signal transduction pathways. However, the biochemical and molecular mechanisms that underlie the cellular response to radiation are not fully understood. The effects of ionizing radiation on the expression of nuclear proteins have now been investigated in radiorestistant human head and neck squamous carcinoma cells (SQ-20B cells) using the techniques of two-dimensional PAGE, silver staining, and computer-assisted quantitative analysis. Radiation (600 cGy) induced the expression of 10 proteins and suppressed the expression of 5 proteins in the nuclei of SQ-20B cells as detected 4 h after treatment. Electroelution and NH2-terminal amino acid sequence analysis revealed that one of the radiation-induced proteins was the Ca(2+)-binding protein calreticulin. The expression of calreticulin was increased approximately 6-fold in the nuclei of irradiated SQ-20B cells. Calreticulin and the other proteins whose expression was affected by radiation may contribute to the radioresistance phenotype of SQ-20B cells.

Radiation-induced apoptosis of Ewing's sarcoma cells: DNA fragmentation and proteolysis of poly(ADP-ribose) polymerase.

Ewing's sarcoma (ES) cells express high levels of poly(ADP-ribose) polymerase (PADPRP) and are responsive to killing by ionizing radiation. We have determined that ionizing radiation induced a pronounced but reversible G2-M phase cell cycle arrest that was maximum by 24 h after exposure. Following the release from this block, floating cells began to appear. These floating cells were shown to be apoptotic by flow cytometric and DNA fragmentation analyses. We found that apoptosis is a significant component of radiation-induced death in ES cells and that this is accomplished in conjunction with proteolytic cleavage of PADPRP. Two fragments of M(r) 25,000 and M(r) 29,000 containing the PADPRP DNA-binding domain were identified in floating (apoptotic) cells, whereas only the full-length M(r) 116,000 native protein was detected in adherent cells that retained DNA intact. These data are consistent with PADPRP cleavage being an early step in the apoptotic cascade of biochemical events in ES cells after ionizing radiation exposure.

3-Methylcholanthrene inactivates the p53 gene in Syrian hamster embryo fibroblasts by inducing a specific intronic point mutation.

The expression of the tumor suppressor gene p53 was studied in Syrian hamster embryo cells neoplastically initiated with a single dose of 3-methylcholanthrene. Ten randomly selected individual 3-methylcholanthrene-transformed colonies were established in culture independently. Eight of these cell lines contained levels of p53 mRNA similar to those in primary embryo cells (p53+ cell lines), as measured by Northern blot analysis of total RNA, whereas two of them (81C43 and 81C47) showed no detectable levels of p53 mRNA (p53- cell lines). However, Southern blot and karyotype analyses did not reveal any significant changes in copy number or gross rearrangements of the p53 gene in any of the p53- cell lines. A 3-kilobase genomic fragment cloned from p53- cells (81C47) containing both upstream and downstream promoters of the p53 gene was able to drive the expression of a CAT reporter gene when transfected into either p53+ or p53- cells. Furthermore, run-on assays performed on nuclei of p53- cells showed that the p53 gene was transcriptionally active, demonstrating that the genetic defect leading to the lack of p53 expression was not due to alterations in the promoter region. Detection of mRNA species corresponding to p53 mRNA precursors in Northern blot analysis of polyadenylated RNA from both p53+ and p53- cells indicated that the lack of p53 expression was not caused by mutations in the 3' regulatory region of the p53 gene affecting transcription termination and/or polyadenylation of p53 precursor mRNA. PCR amplification and nucleotide sequence analysis of extensive internal regions of the gene revealed that both p53- cell lines were homozygous for the same unique point mutation on the splice acceptor site of the fifth intron, a G to C transversion in the last nucleotide of the intron. The presence of this mutation in both p53- cell lines strongly suggests that it was induced specifically by 3-methyl-cholanthrene treatment and indicates that the resulting splicing malfunction may account for the lack of p53 gene expression.

Quercetin mediates the down-regulation of mutant p53 in the human breast cancer cell line MDA-MB468.

The effects of the bioflavonoid quercetin (3,3',4',5,7-pentahydroxyflavone) on the growth and cell cycle progression of the human breast cancer cell line MDA-MB468 have been studied. Quercetin inhibited cell proliferation with an IC50 (a drug concentration which inhibited growth by 50% following a 3-day exposure) value of 7 micrograms/ml. In actively growing cultures, the addition of quercetin resulted in the accumulation of cells at the G2-M phase. We have correlated these effects on cell proliferation with the observation that quercetin strongly inhibited, in a time- and dose-dependent fashion, the expression of the mutated p53 protein, which is the only form present at high levels in this cell line. This inhibition takes place at the translational level. Quercetin did not affect the steady-state mRNA levels of p53, but prevented the accumulation of newly synthesized p53 protein. This quercetin action appeared to be somewhat specific for p53 because the drug did not alter the amount of other proteins present in MDA-MB468 cells such as P-glycoprotein and did not prevent the induction of the synthesis of epidermal growth factor receptor in response to epidermal growth factor.

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.

Activation of phospholipase D participates in signal transduction pathways responsive to gamma-radiation.

Early responses of mammalian cells to ionizing radiation include the activation of a protein kinase C implicated in the regulation of gene expression, the stimulation of tyrosine kinase activities, and the enhancement of phosphatidylinositol turnover. In the present report we show that clinically relevant doses of gamma-radiation (2 Gy) stimulate phosphatidylcholine hydrolysis in human squamous carcinoma cells. Radiation induced the accumulation of intracellular [3H]choline and the simultaneous increase in [3H]myristoyl-phosphatidic acid, followed by a small increase in the levels of [3H]myristoyl-diacylglycerol. Furthermore, in the presence of ethanol, gamma-radiation stimulated the appearance of [32P]phosphatidylethanol, an indicator of phospholipase D transphosphatidylation activity. These data demonstrate for the first time that phospholipase D activation participates in signaling pathways in response to gamma-radiation.

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.

Frequent activation of non-ras transforming sequences in neoplastic Syrian hamster cells initiated with chemical carcinogens.

Carcinogen-caused transformation of Syrian hamster embryo cells has been widely used as a model for experimental carcinogenesis. However, analysis of the molecular mechanisms of hamster cell transformation has been limited. To expand the understanding of the molecular basis of this system, 22 independently derived Syrian hamster neoplastic cell lines initiated with chemical carcinogens were screened for the presence of dominant transforming sequences by DNA transfection into mouse NIH3T3 cells. High molecular weight DNAs from 12 (55%) of these cell lines transformed NIH3T3 cells through serial transfection cycles. NIH3T3 transformants contained hamster-specific repetitive sequences, which co-segregated with the transformed phenotype in successive transfection rounds. Results from Southern hybridization analyses and p21ras mobility assays indicated the presence of N-ras oncogenes, presumably activated by point mutations at codon 61, in 3 of the 12 (25%) transfection positive lines, all initiated with sodium bisulfite; non-ras transforming sequences were apparently activated in the remaining 9 (75%) lines. DNA prepared from NIH3T3 transformants derived from cell line 81C39 was analysed by Southern hybridization with a battery of 38 probes including non-ras oncogenes known to score as positive in the NIH3T3 assay as well as other retroviral and mammalian oncogenes. Each probe hybridized to DNA fragments showing the mobility characteristic of NIH3T3 protooncogenes, but failed to detect homolog sequences of hamster origin, even under hybridization conditions which allowed their detection in hamster DNA. Results show that ras activation occurs at a low frequency in hamster neoplastic transformation and strongly suggest that novel transforming sequences are activated, thus validating the use of this system for investigating the role of non-ras transforming sequences in neoplasia.

Oncogenic activation of the tyrosine kinase domain of the human trk proto-oncogene by fusion to a cell adhesion molecule.

To investigate the mechanisms of radiation-induced neoplastic conversion, DNA from X-ray transformed human epidermal keratinocytes (RHEK-1) was used in sequential cycles of NIH3T3 transfection followed by nude mice tumorigenicity assays. NIH3T3-derived transformants retained discrete DNA fragments hybridizing to human alu probes. Four clones were isolated from a cosmid library prepared from one of these transformants (49-7G) using human DNA as the probe. Analyses of DNAs from 49-7G cells and the four cosmid clones with probes for a number of human oncogenes demonstrated that the cloned sequences were related to the trk oncogene. Transfection of NIH3T3 cells with the cosmid DNAs did not result in the appearance of transformed foci when the murine fibroblasts were cultured on plastic. However, foci developed when transfected cells were cultured on plates coated with various extracellular matrix (ECM) components. Neomycin-resistant cosmid-transfected NIH3T3 cells did induce tumors in nude mice, and their tumorigenicity correlated with their level of trk expression. Nucleotide sequence analyses of cDNA clones isolated from a 49-7G library with a human trk probe revealed that the cloned sequences resulted from the fusion between 5' sequences from the human beta-1,4-galactosyltransferase gene, which encodes a membrane protein involved in cell-cell and cell-matrix interactions, and 3' sequences from the human trk proto-oncogene. The 76 kDa protein product of the chimeric gene, designated bgt-trk, has been identified in NIH3T3 cells transfected with cosmid 19/2 or with bgt-trk cDNA expression constructs, and its phosphorylation in tyrosine has been found to increase when the transfected cells were seeded on plates coated with ECM components which also elicited foci formation in NIH3T3 transformation assays. The fusion of the trk tyrosine kinase domain to a cell adhesion molecule may explain the ECM dependence for the expression of the full transforming potential of the resulting oncogene product.

Induction of human cervical squamous cell carcinoma by sequential transfection with human papillomavirus 16 DNA and viral Harvey ras.

Clinical and epidemiological data are consistent with the hypothesis that human papillomaviruses (HPVs) are a factor in genital, particularly cervical cancer. Although HPV16 and 18 are found primarily in cervical malignancy, the transfection of HPV16 or 18 DNA into cervical cells results in immortalization but not tumorigenicity. The addition of activated Ha-ras, an oncogene found in some cervical cancers expressing HPV16 or 18, to HPV16-immortalized human cervical cells results in malignancy as proven by the formation of cystic squamous cell carcinomas by HPV16-Ha-ras cells in nude mice. This two-stage model utilizing relevant human cells demonstrates that HPVs play a critical role in cervical malignancy and provides a system for elucidating critical cellular changes associated with progression to malignancy.

cph, a novel oncogene which cooperates with H-ras in the transformation of NIH3T3 fibroblasts.

We have performed the molecular cloning of the non-ras transforming sequences previously detected in neoplastic Syrian hamster embryo fibroblasts initiated in vitro with 3-methylcholanthrene (MCA) (Notario et al., 1990). These sequences were isolated using cosmid-rescue techniques from a third-cycle NIH3T3 transformant obtained by sequential transfections of genomic DNA from MCA-initiated hamster fetal cells. Rescued (C-5) clones encompassed about 42.5 kbp of Syrian hamster genomic DNA containing hamster-specific repetitive elements (HRS). An internal 19 kbp BamHI fragment (B-1) was the only C-5 fragment which recognized specific transcripts in poly(A)+ RNA from hamster embryo cells. The same mRNA species were present in both normal and MCA-initiated neoplastic cells: a major transcript of about 2.5 kb, and other less abundant ones, ranging from approximately 2.0 kb to 5.0 kb. These mRNA species were detected consistently by each of several B-1 DNA subfragments located at positions spanning almost the entire B-1 length. The nucleotide sequence of some transcript-positive (S5P2 and S6) genomic B-1 fragments was determined. No significant homology exists between the nucleotide sequences of these B-1 subfragments and established DNA databases. Therefore, the C-5 cosmid clone contains novel genomic sequences. Transfection of C-5 DNA into mouse NIH3T3 cells resulted in the appearance of transformed foci (about five foci per microgram of DNA) within 25 days post-transfection, thus demonstrating the transforming activity of the C-5 clone, which was consequently renamed as the cph oncogene. Co-transfection of the cph oncogene with the human H-ras oncogene (T24), demonstrated a synergistic action between the two oncogenes in the transformation of murine fibroblasts.

Hyperactive autocrine loop mediated by a NDF-related factor in neoplastic hamster embryo fibroblasts expressing an activated cph oncogene.

We described previously the characterization of a novel oncogene, cph, activated in primary Syrian hamster embryo fibroblasts by exposure to 3-methylcholanthrene (Velasco et al., Oncogene 9:2065-2069, 1994). The present report describes the participation in the neoplastic conversion of cph-expressing (81C39) hamster fibroblasts of a hyperactive autocrine loop involving a neu differentiation factor [NDF]-like protein. The tyrosine phosphorylation of the p185erbB-2 receptor in the human breast carcinoma MDA-MB-453 cells was stimulated by conditioned medium from neoplastic 81C39 cells. The extent of this stimulatory effect was much greater than that induced by conditioned medium from normal 84-3 hamster cells. The p185erbB-2 tyrosine phosphorylation-stimulating activity was partially blocked by the heparin analogue pentosan polysulfate [PPS], a known antagonist of p185erbB-2 ligands, and was partially purified from 81C39 conditioned medium by heparin-Sepharose chromatography. The level of p185erbB-2 tyrosine phosphorylation-stimulating activity in the heparin-Sepharose fractions correlated directly with their content in NDF-like protein as immunodetected with an anti-rat NDF antibody. Consistently, the steady-state level of NDF-related mRNA was found to be four times greater in neoplastic 81C39 cells than in normal 84-3 cells. However, the levels of erbB-2 mRNA were similar in both cell types, while the expression of erbB-4 mRNA was upregulated in the neoplastic fibroblasts. The ability of 81C39 conditioned medium to stimulate protein tyrosine phosphorylation and to induce other PPS-sensitive growth responses on 81C39 cells themselves suggested the involvement of an autocrine loop in their neoplastic conversion. The participation of a NDF-related factor in this autocrine loop was confirmed by the ability of an anti-NDF antibody to block the mitogenic activity present in their own conditioned medium. The involvement of the cph oncogene in the upregulation of NDF-related expression was evidenced when cph-transformed NIH3T3 fibroblasts showed elevated levels of NDF-related mRNA, and their conditioned medium induced tyrosine phosphorylation on MDA-MB-453 cells, reproducing the effect of the medium from 81C39 hamster cells.

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.

Co-regulated expression of dbl and poly(ADP-ribose) polymerase in Ewing's sarcoma cells and dbl-transformed NIH3T3 fibroblasts.

Poly(ADP-ribose) polymerase (PADPRP) is a ubiquitous enzyme constitutively expressed at low levels in the majority of eukaryotic cells, including most mammalian tumors and tumor-derived cell lines. Overexpression of PADPRP following the introduction of cDNA recombinant constructs into various cell types results in cell death. An exception to this effect are Ewing's sarcoma (ES) cells, which have been shown to contain elevated steady-state levels of PADPRP mRNA and high constitutive levels of protein and polymerase activity. In fact, this excess of PADPRP has been suggested to participate in the intrinsic radiosensitivity of Ewing's sarcomas, a highly malignant childhood bone tumor frequently curable with radiotherapy. It appears that ES cells might possess a hitherto unknown mechanism(s) by which PADPRP overexpression is controlled and made compatible with cell survival and proliferation. In order to investigate the contribution of other genetic alterations to PADPRP regulation in ES cells, we analysed the expression levels of PADPRP and of other genes, such as oncogenes and tumor suppressor genes, which may enhance the proliferative potential of ES cells. We have detected a positive correlation between the expression levels of the DNA-repair enzyme poly(ADP-ribose) polymerase and the dbl proto-oncogene in Ewing's sarcoma cells. The co-regulated expression of these genes has been established in NIH3T3 cells transformed by the human dbl oncogene or by overexpression of the dbl proto-oncogene. In both instances, the increase in dbl expression resulted in elevated levels of PADPRP mRNA and polymerase activity. The dbl oncogene was more efficient than the proto-oncogene in upregulating PADPRP expression. The inability of other oncogenes to upregulate PADPRP upon transformation of NIH3T3 cells demonstrated the specificity of the dbl in the process. Transfection of dbl-transformed NIH3T3 cells with retroviral PADPRP vectors resulted in the establishment of clones with PADPRP levels higher than those detectable in untransformed NIH3T3 cells transfected with the same retroviral constructs. These results suggest that dbl plays a role in the mechanism by which mammalian cells autoregulate their endogenous levels of PADPRP. Post-translational modification of the dbl or proto-dbl proteins by cytoplasmic PADPRP does not participate in the mechanism(s) underlying the observed PADPRP/dbl co-regulation.

Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X.

Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994). We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes. The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes. The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle. Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe. Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution. Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome. FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7. Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts. The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

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.