Density-dependent inhibitory effect of transforming growth factor-β on human fibroblasts involves the down-regulation of platelet-derived growth factor α-receptors

We have previously found that transforming growth factor-β1 (TGF-β1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-β1 on the PDGF α-receptor, which binds all PDGF isoforms, as well as on the β-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-β1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-β1, there was an complete inhibition of ³H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-β1 was seen in PDGF-BB treated cells, although less pronounced. The binding of ¹²⁵I-labeled PDGF-AA and PDGF-BB to the α-receptor was significantly reduced after treatment with TGF-β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α-receptor mRNA was also seen. The levels of β-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-β1 is cell density-dependent and involves down-regulation of PDGF α-receptors. © 1993 Wiley-Liss, Inc.     

The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein.

The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions.     

Growth factors induce early pre-replicative changes in senescent human fibroblasts.

As 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 pre-replicative part of the cell cycle, we have analysed PDGF-induced 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 II 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.     

Bovine amyloid protein AA: isolation and amino acid sequence analysis

Amyloid-laden renal glomeruli were selectively isolated from a cow with a history of multiple organ inflammatory diseases which terminated in amyloid-induced glomerulopathy and severe proteinuria. Lyophilized amyloid fibrils obtained by water extraction procedures were dissolved in 6M guanidine hydrochloride and gel filtered on Sepharose CL6B and Sephacryl S-300 Superfine columns for slab gel electrophoresis, analytic isoelectric focusing, and amino acid sequence analyses. Electrophoresis of material from the major retarded peak of the elution profile revealed that bovine protein AA moves as one band with an apparent molecular mass of about 14,000 Daltons. Several distinct bands between approximately pH 4.0 and 5.0 were observed when this material was evaluated by analytic isoelectric focusing, thus having a pattern resembling that of human and dog protein AA. A blocked N-terminus was demonstrated when protein from the major retarded peak was subjected to amino acid sequencing, but cyanogen bromide cleavage followed by gel filtration produced 3 peptide fragments for amino acid sequence analysis. These peptides had a high degree of homology with positions 4-14, 18-24 and 25-49 of human protein AA. An apparent complete homology between bovine protein AA and protein AA from other species was apparent at positions 35-45, providing further evidence that this is a functionally significant part of the serum protein AA (SAA) molecule.     

Demonstration of an ATPase at the cell surface of intact normal and neoplastic human cells in culture

An ATPase reaction has been studied at the surface of intact normal and neoplastic human cells in culture. For this purpose a sensitive method has been developed which permits repeated monitoring of enzyme activity without interference with cellular viability. Experiments could be performed with the cells cultured in a single dish. The cells were firmly attached to the supporting medium throughout the experiment. The incubation medium could easily be separated from the cells at the end of the reaction. There was no diffusion of the surface-located ATPase into the incubation medium. Another advantage was the possibility of microscopic control of the appearance of the cells during the reaction. High ATPase activity was found in glia-like cells derived from normal adult brain cells while lines from gliomas had very low activity. One SV40 transformed glia line had an extremely low ATPase activity in contrast to the uninfected cells. Normal fibroblasts and sarcoma cells had about the same low activity as the glioma cells.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF β-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transtormed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists. © 1994 Wiley-Liss, Inc.