No abstract

The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

Communicated by J.PontenAs human fibroblasts in culture senesce their response to platelet-derived growth factor (PDGF) becomes attenuated. To clarify at which level such cells are blocked in the prereplicative part of the cell cycle, we have analysed PDGFinduced pre-replicative events in senescent (phase III) cultures. We found that phase III cells retain a normal number of PDGF receptors and that these are functional with regard to PDGF-induced receptor autophosphorylation. Phase III cells also respond to PDGF by rapid actin reorganization and increased levels of c-fos and c-myc mRNA, similar to growth-arrested phase H fibroblasts. However, the expression of the nuclear antigen K-67, which in phase II cell is induced in S-phase and continues to be expressed throughout the cell cycle, is not induced in phase III cells in response to PDGF. We conclude that phase III human fibroblasts, although blocked with regard to proliferation, still retain a functional growth factor receptor system, and display early responses when exposed to growth factors, such as changes in the cytoskeleton and the expression of proto-oncogenes.

U-1810, a human large-cell lung cancer line, was found to express a PDGF-like growth factor. 35S-cysteine labelling and immunoprecipitation revealed the synthesis and secretion of a 31-kDa PDGF-like protein. Serum-free conditioned medium contained PDGF-receptor-competing and mitogenic activity when tested on human fibroblasts. Whereas the receptor-competing activity was fully neutralized by anti-PDGF antibodies, the mitogenic activity was only partially affected. We therefore probed U-1810 mRNA with a panel of growth-factor DNA clones. We found expression of the genes for PDGF A- and B-chains, TGF-alpha, TGF-beta and IGF-II but not EGF or IGF-I. U-1810 cells lacked specific binding sites for PDGF but showed specific binding of EGF and expressed EGF-receptor transcripts. Thus, U-1810 is an example of a human tumor cell line that expresses multiple growth factor genes; in the intact tumor the corresponding growth factors may operate in autocrine stimulation of the tumor cells as well as in paracrine growth reactions (i.e. stroma recruitment).

The autocrine effects of platelet-derived growth factor (PDGF) A-and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A-and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A-and B-chain transfectants released PDGF receptorcompeting activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms.The discovery of a close structural homology between the platelet-derived growth factor (PDGF) B-chain and the predicted protein product of the simian sarcoma virus (SSV) oncogene, v-sis (11, 13, 43), provided the first direct evidence that the uncontrolled expression of a growth factor may alter cell growth patterns in a way that might contribute to malignant transformation. Additional examples of growth factor genes that under transcriptional control of viral promoters provide a transforming signal to relevant cells include the colony-stimulating factors GM-CSF (27) and CSF-l (M-CSF) (37), transforming growth factor-Cx (TGF-oL) (15. 36, 42), epidermal growth factor (EGF) (38), and basic fibroblast growth factor (bFGF) (34). Two potential oncogene products, the hst (41) and int-2 (12) . Thus, the functions specified above which are specific for B-chain containing PDGF dimers appear to be triggered through the type B receptor.The different biological properties of PDGF-AA and PDGF-BB are of interest in relation to the differential expression of the PDGF-A and -B genes in human tumor cell lines (4). The autocrine action of a growth factor is dependent not only on its biological activity in the extracellular space, but also on its synthesis rate, secretion, and processing. In order ...