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Biomedical subjects

W Wharton

Publications and source records attributed to W Wharton.

At least 55 records · Page 3Linked to original sources
PubMed1984

Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements.

Cultures of human fibroblasts were prepared from chest skin obtained either from newborns (less than 3 months old) or adults (more than 35 years old) and maintained in vitro until they senesced. Adult cells grew logarithmically in medium supplemented with whole blood serum but not with platelet-poor plasma. Early passage cells obtained from newborns grew equally well in either plasma- or serum-supplemented medium. The difference in growth factor requirements between adult and newborn cells persisted through the lifespan of the cells; i.e., newborn cells did not develop adult hormonal requirements when maintained in culture. Thus, in vitro cellular aging can be distinguished from some types of differentiation.

Adult↗
PubMed1983

A method for staining 3T3 cell nuclei with propidium iodide in hypotonic solution.

A modification of the propidium-iodide hypotonic sodium citrate method has been developed specifically for high-resolution staining of mouse 3T3 cell nuclei for analysis by flow cytometry. The method employs a brief treatment of cells at 37 degrees C with Triton X-100 and RNAse in the presence of propidium iodide in hypotonic sodium citrate, followed by restoration to isotonicity with NaCl. The average CV obtained for the G1 peak was 3.5%, and the samples were stable for 1-2 weeks at 4 degrees C. Compared to this technique, previously described propidium iodide-staining methods gave poor resolution with 3T3 cells.

Animals↗
PubMed1983

Hormonal regulation of discrete portions of the cell cycle: commitment to DNA synthesis is commitment to cellular division.

Density-arrested human fibroblasts were stimulated to traverse G0/G1 and initiate DNA synthesis by the addition of medium containing either serum or a combination of platelet-derived growth factor and platelet-poor plasma. Medium containing a combination of epidermal growth factor and high concentrations of insulin also stimulated DNA synthesis in platelet factor-treated quiescent cells. Platelet factor was required only to initiate proliferation. Epidermal growth factor and insulin then allowed G1 traverse and commitment to DNA synthesis. Cells could complete S, G2, and M in unsupplemented medium lacking peptide growth factors.

Cell Division↗
PubMed1983

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts.

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.

Animals↗
PubMed1983

Human macrophage-like cell line U937-1 elaborates mitogenic activity for fibroblasts.

The human macrophage-like cell line U937-1 was shown to elaborate mitogenic activity for cultured fibroblasts. Activity was present in the culture medium when cells were maintained either in the presence or absence of serum components, although maximal levels were observed when the macrophage-like cells were incubated in medium supplemented with platelet-poor plasma. The mitogenic activity acted synergistically with platelet-poor plasma to allow density-arrested cells to initiate DNA synthesis and its action was potentiated by the addition of cholera toxin. The elaborated activity bound to DEAE Sephacel and was eluted in a single peak using an NaCl gradient.

Animals↗
PubMed1982

Macromolecular synthesis and stability in growth-arrested BALB/c-3T3 cells.

Concentrations of methylglyoxal bis-(guanylhydrazone) (mGBG) that inhibited serum-stimulated BALB/c-3T3 cells in late G1 caused a marked inhibition of 3H-leucine incorporation during a 20-min incubation. No decrease was observed in the incorporation of 3H-uridine during a 20-min incubation; however, the amount of acid-insoluble 3H-uridine in mGBG-treated cultures was decreased when the incubation period was longer than 20 min. The amount of the decrease in the accumulation of incorporated 3H-uridine was directly proportional to the length of the incorporation time. Between 10 and 12 h after quiescent BALB/c-3T3 cells were serum-stimulated in mGBG no additional 3H-uridine was accumulated. The stability of the incorporated 3H-uridine, as determined by acid-insoluble radioactivity remaining after the addition of actinomycin D, was less in cells cultured in mGBG. Exogenous spermine or spermidine reversed the inhibition of 3H-uridine accumulation in acid-insoluble material produced by mGBG as well as the decrease in stability of the incorporated 3H-uridine in acid-insoluble material. The effects of mGBG on both the incorporation of 3H-uridine and the stability of the incorporated 3H-uridine can apparently be accounted for by an effect on ribosomal RNA.

Animals↗
PubMed1982

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1.

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells.

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D1 (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-1 (lymphocyte activating factor).

Animals↗
PubMed1982

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells.

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of medium supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin., The addition of cholera toxin plus 30 microM isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Growth regulation by macrophages.

The evidence reviewed here indicates that macrophages, either acting alone or in concert with other cells, influence the proliferation of multiple types of cells. Most of the data indicate that these effects are mediated by soluble macrophage-elaborated products (probably proteins) although the role of direct cell-to-cell contacts cannot be ruled out in all cases. A degree of success has been achieved on the biochemical characterization of these factors, but such work has been hampered by the factors low specific activity in conditioned medium and the lack of rapid, specific assays. It is our belief that understanding the growth-regulating potential of macrophages is an important and needed area of research.

Animals↗
PubMed1982

Modulation of the epidermal growth factor receptor by platelet-derived growth factor and choleragen: effects on mitogenesis.

The addition of fresh medium supplemented with partially purified platelet-derived growth factor (PDGF) to quiescent density-arrested cultures of BALB/c-3T3 cells decreases the subsequent binding of radiolabeled epidermal growth factor (EGF). The decrease in EGF binding can be observed 1 hr after the addition of PDGF. This effect is maximal in 2-3 hr, and binding remains diminished for at least 6 hr. These effects can be accounted for by a decrease in the number of EGF receptors with no change in receptor affinity. The action of PDGF is concentration dependent, but even at very high concentrations of PDGF the reduction in EGF binding is never more than 50%. Similar decreases in EGF binding are produced by other treatments that render BALB/c-3T3 cells competent, such as the addition of fibroblast growth factor or medium previously exposed to the macrophage-like cell line P388D(1). Cholera toxin (choleragen), which alone had no effect on EGF binding, dramatically potentiated the ability of PDGF to down regulate EGF receptors. Two to three hours after the addition of PDGF and choleragen, EGF binding was reduced by 80-90% compared with control values. The ability of PDGF and choleragen together to decrease EGF binding was substantially inhibited by cycloheximide. Autoradiography of [(3)H]thymidine-labeled cells shows that choleragen potentiates the action of PDGF; lower concentrations of PDGF are required to make cells competent after choleragen treatment. Furthermore, cells treated with PDGF and choleragen no longer require EGF for traverse of G(1) phase and initiation of DNA synthesis in defined medium. The reduction in receptor number produced by choleragen and PDGF, which may be due to internalization of the EGF receptor, may mimic the action of EGF and thereby remove the EGF requirement for DNA synthesis.

Animals↗
PubMed1981

Inhibition of BALB/c-3T3 cells in late G1: commitment to DNA synthesis controlled by somatomedin C.

Methylglyoxal bis-(guanylhydrazone) (mGBG) blocked the stimulation of DNA synthesis in quiescent, density-inhibited BALB/c-3T3 cells treated with platelet-derived growth factor (PDGF) and platelet-poor plasma (PPP). Competence formation produced by a transient exposure to PDGF was not effected by mGBG. In contrast, mGBG effectively inhibited the PPP-stimulated progression of competent cells through the G1 phase of the cell cycle, although maximal inhibition was observed when mGBG was present during both the exposure to PDGF- and PPP-supplemented media. When quiescent cells were treated with PDGF and PPP-supplemented media in the presence of mGBG for 12--18 hours and the mGBG was then removed, cells entered the S phase after a 4 hour lag. The rate of entry into the S phase, but not the time necessary for the cells to progress from the mGBG block into the S phase, was dependent on the concentration of PPP present after removal of the mGBG. Either somatomedin C or insulin, but not epidermal growth factor, fibroblast growth factor, or PDGF were able to substitute for PPP in allowing cells to enter the S phase after the cells were released from the mGBG block. A marked inhibition of (3H)-leucine incorporation in serum-stimulated cultures was produced at mGBG concentrations which caused no decrease in the amount of (3H)-uridine incorporated during a short (15 minute) pulse. The ability of hormones to allow cells to progress to the late G1 phase and become committed to DNA synthesis after a mGBG inhibition was not related to their ability to restore the normal rate of protein synthesis as determined by (3H)-leucine incorporation.

Animals↗
PubMed1981

Regulation of uridine kinase activity in BALB/C-3T3 cells by serum components.

After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels or uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce and induction of thymidine kinase activity in late G1.

Animals↗
PubMed1980

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells.

Platelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine blood. Bovine platelet-poor plasma had properties similar to those previously described for human platelet-poor plasma; e.g., it would (a) support the growth of virally transformed but not normal BALB/c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.

Animals↗
PubMed1980

Inhibition by deoxycytidine, cytidine, and beta-cytosine arabinoside of the induction of alkaline phosphatase activity in HeLa cells.

The main objective of these experiments was the further examination of whether the induction of alkaline phosphatase activity in HeLa cells by 5-iodo-2'-deoxyuridine (IdUrd) depends on the incorporation of IdUrd into DNA. Thymidine (dThd), deoxycytidine (dCyd), cytidine, and beta-cytosine arabinoside (Ara-C) inhibited a dose-dependent manner the induction of alkaline phosphatase activity by IdUrd in HeLa cells, and 5-iodo-2'-deoxycytidine induced activity in a dose-dependent manner at concentrations similar to those of IdUrd. Three of these compounds, dThd, dCyd, and Ara-C, were studied with regard to degree of inhibition of induction and IdUrd incorporation into DNA. Although the various doses of these three compounds decreased the incorporation of IdUrd into DNA, there was no apparent linear correlation between the extent of inhibition of IdUrd incorporation and the degree of inhibition of the induction of alkaline phosphatase activity. dCyd also inhibited a dose-dependent manner the induction of alkaline phosphatase by hydrocortisone, sodium butyrate, and choline chloridee. These results, although not unequivocal, support the idea that IdUrd induction of alkaline phosphatase activity in HeLa cells does not require IdUrd incorporation into DNA. The dCyd altered the thermostability for alkaline phosphatase activity from control or IdUrd-treated cells, and for controls cells the change in thermostability occurred without a change in the enzyme specific activity.

Alkaline Phosphatase↗