Expression of recombinant platelet-derived growth factor A- and B-chain homodimers in rat-1 cells and human fibroblasts reveals differences in protein processing and autocrine effects.

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 receptor-competing 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.     

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.     

Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells

Cultured endothelial cells secrete a platelet-derived growth factor-like molecule (PDGFc). We examined the effects of purified human alpha-thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of alpha-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of alpha-thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha-thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell-mediated conversion of PDGFc from an inactive to an active form.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line

The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-beta), but no TGF-alpha production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-beta mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well as in (human) teratocarcinomas.     

Identification of a cell retention signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain.

The B-chain homodimer of platelet-derived growth factor (PDGF) is only very inefficiently secreted and remains largely associated with the producer cell; in contrast, the dimer of the short, and most common, splice variant of the A-chain is secreted. To identify the structural background to the differences in the secretory pattern between the different isoforms of PDGF, a set of chimeric PDGF A/B cDNAs was generated and expressed in COS cells. Analyses of the biosynthesis and processing of the corresponding products led to the identification of a determinant for cell association in the carboxy-terminal third of the PDGF B-chain precursor. Introduction of stop codons at various positions in the carboxy-terminal prosequence of the PDGF B-chain localized this determinant to an 11-amino-acid-long region (amino acids 219-229). This region contains an 8-amino-acid-long basic sequence that is homologous to a sequence present in an alternatively spliced longer version of the PDGF A-chain. In contrast to the short splice variant, the long splice A-chain version, like the B-chain, was found to remain predominantly cell associated. Thus, we have identified a conserved sequence that inhibits the secretion of some of the PDGF isoforms. Our data also suggest that switching of splicing patterns can be a mechanism to regulate the formation of secreted or cell-associated forms of PDGF-AA and possibly other growth factors.     

Growth of tumor cells with different sensitivities for murine natural killer cells in young and adult athymic nude mice

Of four tumor cell lines, the murine YAC lymphoma, the human K562 lymphoma, and the human prostatic carcinomas PC3 and PC93, the susceptibility to murine natural killer (NK) cells as well as the tumorigenicity in young (3.5-4 weeks old) and in adult (8-10 weeks old) nude mice were studied. In young nude mice, which exhibited a lower level of NK cell activity than adult nude mice, the formation of solid tumors after inoculation of tumor cell suspensions occurred more frequently and with a shorter time lag than in adult animals. These effects were observed not only with the NK-sensitive YAC cells, but also with the relatively NK-insensitive PC3 and PC93 cells, indicating that also factors other than NK cell susceptibility may influence the growth of tumor cells in nude mice. Therefore, the use of young nude mice may enhance the rate of success of heterotransplantation of human tumors, regardless of the NK cell susceptibility of the tumor cells.     

TGF-beta stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF-related peptides

Transforming growth factor-beta (TGF-beta) stimulates DNA synthesis in human foreskin fibroblasts after a prolonged lag period as compared with other growth factors. The mechanism of induction of DNA synthesis appears to be dependent on the synthesis and secretion of PDGF-related proteins as antibodies which are specific for PDGF can block the TGF-beta-induced DNA synthesis. Other growth factors such as PDGF, EGF, or FGF do not induce the synthesis of these PDGF-related proteins. Additionally, TGF-beta treatment of human foreskin fibroblasts induces the expression of the PDGF A-chain gene but not the B-chain gene. This phenomenon appears to function in vivo, as subcutaneous injection of TGF-beta in rat skin induces the expression of the PDGF A-chain gene. These data suggest that TGF-beta may stimulate the growth of fibroblastic cells via an autocrine production of PDGF-related proteins.     

Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins.

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.     

Platelet-derived growth factor receptor beta regulates migration and DNA synthesis in metanephric mesenchymal cells

Platelet-derived growth factor (PDGF) B-chain and PDGF receptor beta (PDGFR beta) are essential for glomerulogenesis. Mice deficient in PDGF B-chain or PDGFR beta exhibit an abnormal glomerular phenotype characterized by total lack of mesangial cells. In this study, we localized PDGFR beta in the developing rat kidney and explored the biological effects of PDGF in metanephric mesenchymal cells in an attempt to determine the mechanism by which PDGF regulates mesangial cell development. Immunohistochemical and in situ hybridization studies of rat embryonic kidneys reveal that PDGFR beta localizes to undifferentiated metanephric mesenchyme and is later expressed in the cleft of the comma-shaped and S-shaped bodies and in more mature glomeruli in a mesangial distribution. We also isolated and characterized cells from rat metanephric mesenchyme. Metanephric mesenchymal cells express vimentin and alpha-smooth muscle actin but not cytokeratin. These cells also express functional PDGFR beta, as demonstrated by autophosphorylation of the receptor as well as activation of phosphatidylinositol 3 kinase in response to PDGF B-chain homodimer. PDGF B-chain also induces migration and proliferation of metanephric mesenchymal cells. Taken together with the fact that PDGF B-chain is expressed in the glomerular epithelium and mesangial area, as demonstrated in the human embryonic kidney, we suggest that PDGF B-chain acts in a paracrine fashion to stimulate the migration and proliferation of mesangial cell precursors from undifferentiated metanephric mesenchyme to the mesangial area. PDGF B-chain also likely stimulates proliferation of mesangial cell precursors in an autocrine fashion once these cells migrate to the glomerular tuft.     

Malignant transformation of human cells by constitutive expression of platelet-derived growth factor-BB

Platelet-derived growth factors (PDGFs) comprise a family of growth factors strongly implicated in human oncogenesis. A number of human tumors overexpress PDGF family members or have translocations activating PDGF receptors. Whereas the epidemiologic evidence implicating PDGF in human tumors is strong, malignant transformation of human cells by overexpression of PDGF has not been demonstrated. We have previously developed a human cell line by the sequential introduction of large T cells and telomerase, and we have demonstrated that these cells express functionally active PDGF receptor (PDGFR) beta. In order to determine whether growth factor-mediated transformation of human cells could occur, these cells were transduced with a retrovirus encoding PDGF-BB. Constitutive expression of PDGF-BB led to malignant transformation in nude mice. This is the first demonstration of constitutive signaling causing malignant transformation of human cells. Some of the changes that occur because of constitutive growth factor expression can be reversed by the clinically approved tyrosine kinase inhibitor Glivec, whereas other changes are not reversible by tyrosine kinase inhibitors. Our model allows the assessment of epigenetic changes that occur during human carcinogenesis. In addition, these studies provide insight into the clinical failure of tyrosine kinase inhibitors as monotherapy for advanced malignancy.     

Platelet-derived growth factor gene expression in cultured human retinal pigment epithelial cells.

Gene expression of platelet-derived growth factor (PDGF) and its receptors in cultured human retinal pigment epithelial (RPE) cells was studied by using semiquantitative polymerase chain reaction. The RPE cells were found to express PDGF A- and B-chain genes as well as alpha- and beta-receptor genes with dominant expression of B-chain and beta-receptor isoforms. Phorbol myristate acetate (PMA) and thrombin increased the expression of PDGF B-chain gene to 19.8 +/- 1.75 and 15.9 +/- 1.84 fold (n = 3) of the control without affecting beta-receptor gene expression. PDGF produced by the RPE cells may play an important role in the pathogenesis of some ocular proliferative diseases.