The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

Nucleotide sequence analysis identifies the human c-sis proto-oncogene as a structural gene for platelet-derived growth factor

The simian sarcoma virus transforming gene, v-sis, encodes a protein, p28sis , that is closely related to human platelet-derived growth factor (PDGF). The human locus related to v-sis was cloned and shown to contain at least five exons corresponding to the v-sis coding region. Nucleotide sequence analysis of these exons revealed that the predicted amino acid sequence of human c-sis differed by 6% from that of the woolly monkey-derived v-sis. These findings imply that the sis proto-oncogene has been well conserved during primate evolution. By comparison of the known amino acid sequences of PDGF peptides with the predicted human c-sis protein, it was possible to demonstrate that this human proto-oncogene is the structural gene encoding one of the two major polypeptides of this potent mitogen for connective tissue cells.     

The Parodi-Irgens feline sarcoma virus and simian sarcoma virus have homologous oncogenes, but in different contexts of the viral genomes

We have identified the oncogene and the putative transforming protein of the Parodi-Irgens feline sarcoma virus (PI-FeSV). The PI-FeSV is defective and needs a helper virus for its replication. The v-onc sequences in the PI-FeSV were found to be related to the v-sis sequences of the simian sarcoma virus (SSV). PI-FeSV nonproducer cells express two viral RNAs, a 6.8-and a 3.3-kilobase RNA. The 6.8-kilobase RNA contains gag, sis, and env sequences but lacks the pol gene. The 3.3-kilobase RNA, on the other hand, contains only env sequences. We have detected one feline leukemia virus-related protein product in these cells, namely, a 76-kilodalton protein which contains determinants of the feline leukemia virus gag proteins p15 and p30. The v-sis sequences in the PI-FeSV have been located near the 5' end of the viral genome. Taken together, these results imply that the p76 protein contains both feline leukemia virus gag and sis sequences and probably is the transforming protein of this virus. In contrast, in SSV the sis sequences are located towards the 3' end of the viral genome, and the sis protein is thought to be expressed via a subgenomic RNA. PI-FeSV and SSV therefore use different schemes to express their onc-related sequences. The v-sis sequences in the PI-FeSV contain restriction sites which reflect the different origin of the v-sis sequences in the PI-FeSV and SSV. The homologous oncogenes of the PI-FeSV and SSV thus were transduced by two different retroviruses, feline leukemia virus and the simian sarcoma-associated virus, apparently from the genomes of different species.     

Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.     

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.     

A highly efficient retroviral vector allows detection of the transforming activity of the human c-fps/fes proto-oncogene.

We have constructed an efficient new retroviral vector containing strong promoting elements derived from the Friend murine leukemia virus (F-MuLV) long terminal repeat (LTR) and have used the vector to demonstrate that overexpression of human c-fps/fes can transform established mouse cells. When a c-fps/fes cDNA was cloned into the vector, this viral DNA and the recovered virus induced very high levels of the c-fps/fes product NCP92 and tumorigenic transformation of NIH 3T3 cells. Compared with an isogenic vector under control of a Moloney MuLV-derived LTR, the vector driven by the F-MuLV LTR induced 3- to 10-times-higher levels of expression of c-fps/fes, a higher level of phosphotyrosine in cellular proteins, and a virus whose transforming activity was 2 orders of magnitude greater. We conclude (i) that normal c-fps/fes can induce morphologic transformation and that its transforming activity is a function of the level of expression of NCP92 and (ii) that the vector based on the F-MuLV LTR is more efficient than the vector driven by a Moloney MuLV LTR in inducing high levels of expression and measurable biological activity.     

Isolation and expression of cDNA for different forms of hepatocyte growth factor from human leukocyte

Human leukocyte cDNA library was screened to isolate cDNA clones coding for hepatocyte growth factor using cDNA from human liver as a probe. Nucleotide and deduced amino acid sequences were analyzed for two of four clones obtained. One of them contained an open reading frame coding for a polypeptide chain of 728 amino acid residues like that of cDNA clone derived from human liver. In another clone a spontaneous deletion of 15 base pairs was found within the coding sequence. When expressed transiently using COS-1 cells both clones produced protein with similar biological activity against rat hepatocyte in vitro.     

Structure of viral DNA in a rat cell line transformed by the cloned EcoRI-C fragment of adenovirus 12.

A DNA segment carrying viral DNA was cloned from a rat cell line transformed by the cloned EcoRI-C fragment (0 to 16.4 map units) of human adenovirus type 12(Ad12), and the viral sequence in the clone was analysed. The cloned segment contained the region from nucleotide positions 118 to 3520 of the Ad12 genome in the middle. No unique structure was found at the viral and non-viral DNA junctions. When examined the transforming activity, the conserved viral sequence was able to transform rat 3Y1 cells efficiently. Southern blotting analysis of the viral sequence in five re-transformed cell lines showed that the viral sequence was inserted at different sites of cellular DNA. These results indicate that (I) the Ad12 DNA moiety from the enhancer-promoter region of the E1A gene to the end of the E1B gene contains enough information for efficient transformation of the rat cell, and (II) integration of the viral sequence at unique cellular sites is not prerequisite for transformation.     

Identification and characterization of novel human endogenous retroviral sequences prefentially expressed in undifferentiated embryonal carcinoma cells.

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.     

Construction and Characterization of a Replication-Competent Retroviral Shuttle Vector Plasmid

We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.     

Platelet-derived growth factor in human malignancy.

Platelet-derived growth factor (PDGF) was first implicated in the process of transformation when one of its peptide chains was found to be homologous to the viral sis oncogene (v-sis). Since that time, there have been multiple demonstrations of the transforming activity of v-sis in fibroblasts. Because of the near identity of the v-sis protein with the PDGF B chain, v-sis is thought to transform through an autocrine stimulatory mechanism of cell growth. Consistent with this view are studies which demonstrate inhibition of v-sis-mediated transformation by anti-PDGF antibodies. Expression of the cellular sis gene (c-sis) and its receptors, and secretion of PDGF-like factors have been demonstrated in many types of human malignant cells. Nevertheless, a causative role for c-sis in inducing or maintaining the transformed phenotype in human malignancies remains to be established. There are significant differences in structure between v-sis and c-sis. Studies of transforming ability have yielded conflicting results in transfection models, depending on the transfected vector and target cell type utilized. While there is compelling evidence for the involvement of PDGF in an autocrine growth mechanism in transformed fibroblasts, the evidence in human epithelial tumor types is less convincing because PDGF receptors are usually not detectable on the cell surface. The recent demonstration of intracellular co-localization of active PDGF precursors and PDGF receptors, however, supports the existence of an internal autocrine pathway independent of PDGF secretion. Further investigation of such a mechanism in de novo human malignancies is warranted to establish the role of PDGF in the development of these neoplasms.     

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.