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Biomedical subjects

C D Stiles

Publications and source records attributed to C D Stiles.

At least 91 records · Page 5Linked to original sources
PubMed1984

Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor.

Complementary DNA clones of genes induced by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells were isolated; one such clone contains a domain having nucleotide sequence homology with the third exon of c-fos. This nucleotide sequence homology is reflected in the predicted amino acid sequences of the gene products. Under low stringency conditions, the mouse v-fos gene cross-hybridizes with the PDGF-inducible complementary DNA clone. However, the messenger RNA transcripts of mouse c-fos and the new fos-related gene can be distinguished by gel electrophoresis and by S1 nuclease analysis. Expression of the authentic c-fos gene is induced by PDGF and superinduced by the combination of PDGF and cycloheximide.

Amino Acid Sequence↗
PubMed1984

Regulation of cytoskeletal architecture by platelet-derived growth factor, insulin and epidermal growth factor.

Addition of ng/ml quantities of the platelet-derived growth factor (PDGF) component of serum to the culture medium of quiescent monolayers of BALB/c-3T3 cells rapidly altered the actomyosin cytoskeletal system. PDGF triggered dispersal of microfilament bundles and induced phosphorylation of the 20 kD myosin light chain within 5 min of addition. Additional cytoskeletal responses followed the addition of insulin and of epidermal growth factor (EGF). Insulin alone induces a transient breakdown of well defined microtubule filaments. EGF and insulin together induced moderate perturbation of the cytoplasmic network of intermediate filaments which then reformed within a 90-180 min time frame. This response, however, was greatly enhanced in cells which were pretreated with PDGF and it appears to be a secondary event occurring as a consequence of cell growth. The PDGF-induced disruption of microfilament bundles could be blocked by phalloidin with no apparent inhibition of the mitogenic response. Insulin alone is only weakly mitogenic for 3T3 cells. The data thus indicate that the architecture of two discrete cytoskeletal elements (microtubules and microfilaments) is regulated directly by separate serum growth factors (insulin and PDGF). However the disruption of microfilament architecture by PDGF does not seem necessary for induction of cell growth and the disruption of microtubule filaments by insulin is not sufficient.

Animals↗
PubMed1983

The molecular biology of platelet-derived growth factor.

PDGF is a connective tissue mitogen that has been associated with clotted blood serum for at least 300 million years. It regulates the expression of cell cycle "early genes" in normal fibroblasts. Induction of early genes is preceded by stimulation of a tyrosine-specific kinase. The putative structural gene for PDGF has been acquired by an acutely transforming retrovirus and is expressed in many connective tissue tumors. Further work is needed to determine whether (i) production of PDGF by tumor cells confers a proliferative advantage on these cells, (ii) tyrosine-specific phosphorylations mediate the induction of cell cycle early genes by PDGF, and (iii) products of cell cycle early genes play any functional role in the 10-12 hr chain of events that culminates in replicative DNA synthesis and cell division. In the meantime, these very issues represent candidate functions for other viral oncogenes and their cellular homologs. Some of these genes could act at the onset of the mitogenic cascade by causing the production of automitogenic growth factors. Others may function in the interior of the cascade by promoting tyrosine-specific phosphorylations. Still others may be mutated or rearranged homologs of cell cycle early genes whose expression is normally modulated by extracellular growth factors.

Animals↗
PubMed1983

Molecular cloning of gene sequences regulated by platelet-derived growth factor.

We have screened a cDNA library for gene sequences that are regulated by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells. Of 8000 clones screened, less than 14 independent PDGF-inducible sequences were found. Two of these (KC and JE) were studied in detail. By hybrid-selection and translation the KC and JE mRNAs encode 10,000 and 19,000 dalton polypeptides, respectively. In the absence of PDGF, the JE and KC sequences correspond to low abundance mRNAs. One hour after addition of PDGF their abundance level can be increased 10- to 20-fold. Within 4 hr, a 60-fold induction of JE can be attained. Nanogram per ml quantities of pure PDGF regulate these sequences whereas microgram/ml quantities of chemically unrelated mitogens (EGF, insulin, or platelet-poor plasma) have either a weak or an undetectable effect. Inhibitors of protein synthesis block the progression of quiescent 3T3 cells through G1 into S phase; however these drugs do not block the induction of KC and JE by PDGF. This result indicates that these sequences correspond to "early genes" which are not induced as a consequence of cell growth, but rather are directly regulated by PDGF.

Animals↗
PubMed1983

Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor.

We show that c-myc is an inducible gene that is regulated by specific growth signals in a cell-cycle-dependent manner. Specifically, agents that initiate the first phase of a proliferative response in lymphocytes (lipopolysaccharide or Concanavalin A) and fibroblasts (platelet-derived growth factor) induce c-myc mRNA. Within one to three hr after the addition of these mitogens to the appropriate cells, c-myc mRNA concentration is increased between 10- and 40-fold. This induction of c-myc mRNA occurs in the presence of cycloheximide and, therefore, does not require the synthesis of new protein species. Consequently, the induction of c-myc mRNA is not secondary to growth. In addition, c-myc mRNA is "superinduced" by the combination of cycloheximide and mitogen, a finding consistent with a model that a labile protein may regulate c-myc levels in these cells. Further, this work suggests a regulatory linkage between the function of two oncogenes--c-myc and c-sis--the latter being the putative structural gene for PDGF.

Animals↗
PubMed1983

Persistence of the mitogenic response to platelet-derived growth factor (competence) does not reflect a long-term interaction between the growth factor and the target cell.

Quiescent BALB/c-3T3 cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA even if PDGF is removed from cell culture medium prior to the onset of DNA synthesis. We have suggested that persistence of the PDGF-induced competent state reflects a rapidly induced and relatively stable biochemical change within the target cells. Others suggest that the phenomenon reflects a long-term association between PDGF and its target cells or perhaps between PDGF and the cell culture dish. This controversy has been addressed (a) by examining the effect of anti-PDGF antibodies on PDGF-induced competence and (b) by studying the chemical fate of 125I-labeled PDGF. Anti-PDGF antibodies inactive both soluble and surface-bound PDGF. However, if quiescent 3T3 cells are exposed to PDGF for as little as 30 min, subsequent addition of these antibodies to the culture medium does not prevent the mitogenic response. Under conditions where the PDGF-induced competent state decays stochastically with a t1/2 of 18-20 h, cell-associated 125I-PDGF decays with a t1/2 of approximately 50 min. These data do not support the concept that persistence of the PDGF-induced competent state reflects a long-term association between PDGF and the target cells or between PDGF and the culture dish.

Animals↗
PubMed1982

Phylogenetic analysis of platelet-derived growth factor by radio-receptor assay.

Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo.

Animals↗
PubMed1981

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma.

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.

Animals↗
PubMed1981

Cytoplasmic transfer of the mitogenic response to platelet-derived growth factor.

BALB/c 3T3 mouse cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA and divide. When cells are treated with PDGF and then fused to untreated cells, the resulting heterokaryons become competent to replicate their DNA. Cytoplasts derived from PDGF-treated cells are also able to transfer the growth response to untreated cells. After cytoplasmic transfer to another cell, the strength of the PDGF-induced mitogenic signal is attenuated by a factor roughly proportional to the increase in total cytoplasmic volume. When RNA synthesis is blocked during PDGF treatment, cells do not acquire the capacity to transfer the PDGF growth signal to untreated cells. By contrast, exposure to cycloheximide during PDGF treatment has no effect. These observations suggest that cytoplasmic transfer of the growth response to PDGF (competence) is mediated by a PDGF-induced stable RNA rather than by PDGF itself or a PDGF--receptor complex. The onset of DNA synthesis in PDGF--control heterokaryons occurs a minimum of 11 hr after cell fusion. Thus the substance that is transferred in these cell fusions is not directly involved in DNA synthesis; rather, it seems to trigger a sequence of events culminating in DNA synthesis.

Animals↗
PubMed1980

Effect of ouabain on growth regulation by serum components in Balb/c-3T3 cells: inhibition of entry into S phase by decreased protein synthesis.

The effect of inhibition of the cell membrane Na+-K+ pump on the Balb/c-3T3 cell growth cycle was studied. Inhibition of the Na+-K+ pump resulted in a dose-dependent reduction of intracellular K+ concentration ((K+)i). However, inhibition of protein synthesis in Go/G1 and of subsequent entry into S phase occurred only after (K+)i fell below a critical threshold (50-60 mmoles/liter). Thus, when the (K+)i falls below a critical threshold, protein synthesis is inhibited, preventing cells from entering the S phase. The platelet-derived growth factor (PDGF) induces cells to become "competent" to traverse the cell cycle; the platelet-poor plasma component of serum allows competent cells to progress through G0/G1 and enter S phase. Inhibition of the Na+-K+ pump did not prevent the induction of competence by PDGF, but it did reversibly inhibit plasma-mediated events in early G0/G1. Similarly, cycloheximide inhibited plasma-mediated events but did not prevent PDGF-induced competence. Thus, protein synthesis may not be required for induction of competence; alternatively, the induction of the competent state may occur in these cells after removal of PDGF and protein synthesis inhibitor. Protein synthesis is required for subsequent plasma-mediated events in G0/G1.

Amino Acids↗
PubMed1980

Regulation of the Balb/c-3T3 cell cycle-effects of growth factors.

The platelet-derived growth factor (PDGF), which is found in serum but not in plasma, has been purified to homogeneity; it stimulates replication at a concentration of 10(-10) M. Brief treatment with PDGF causes density-inhibited Balb/c-3T3 cells to become competent to synthesize DNA; pituitary fibroblast growth factor (FGF) or precipitates of calcium phosphate also induce competence. Continuous treatment with plasma allows competent, but not incompetent, cells to synthesize DNA. A critical component of plasma is somatomedin, a group of hormones with insulin-like activity; multiplication-stimulating activity (MSA) or insulin replace plasma somatomedin in promoting DNA synthesis. We have studied the molecular correlates of competence and the role of SV40 gene A products in regulating DNA synthesis. Treatment of quiescent cells with pure PDGF or FGF causes the preferential synthesis of five cytoplasmic proteins (approximate molecular weight 29,000, 35,000, 45,000, 60,000, and 72,000 detected by SDS-PAGE under reducing conditions). Two of these competence-associated proteins (29,000 and 35,000 daltons) are insulin, or epidermal growth factor (EGF). PDGF, FGF, or calcium phosphate induce an ultrastructure change within the centriole of 3T3 cells; this ultrastructural modification of the centriole is detectable by immunofluorescence within 2 h or PDGF treatment. Plasma, EGF, or MSA do not modify the centriole. SV40 induces replicative DNA synthesis in growth-arrested 3T3 cells but does not cause this alteration in centriole structure. Gene A variants of SV40, including a mutant with temperature-sensitive (ts) T-antigen (ts A209), a deletion in t-antigen (dl 884), and several ts A209 strains containing t-antigen deletions were used to induce DNA synthesis in Balb/c-3T3 cells. Like wild type SV40, all strains induced DNA synthesis equally well under permissive or nonpermissive conditions. Addition of PDGF or plasma had little effect on SV40-induced DNA synthesis. Thus, the viral function that induces replicative DNA synthesis in Balb/c-3T3 cells. Like wild type SV40, all strains induced DNA synthesis equally well under permissive or nonpermissive conditions. Addition of PDGF or plasma had little effect on SV40-induced DNA synthesis. Thus, the viral function that induces replicative DNA synthesis in Balb/c-3T3 cells is not t and is not temperature sensitive. This SV40 gene function overrides the cellular requirement for hormonal growth factors. It does not induce transient centriole deciliation, a hormonally regulated event.

Animals↗