Curtis L. Parker

Arachidonate metabolism during chondrogenesis in vitro

SummaryChick embryo limb bud mesenchyme cells undergoing chondrogenesisin vitro were labeled with [3H] arachidonic acid and [14C] palmitic acid, and stimulated by mechanical means to convert a portion of their incorporated [3H] to radiolabeled compounds which co-chromatographed with authentic prostaglandins in the appropriate thin layer chromatography system. Chondrogenesis was (1) inhibited by concentrations of indomethacin or eicosa-5,8,11,14-tetraynoic acid which inhibited conversion of [3H] to prostaglandinlike compounds; and (2) stimulated by prostaglandin E2. We interpret these data to mean that (1) cells undergoing chondrogenesisin vitro are able to metabolize endogenous arachidonic acid to prostaglandins, and (2) synthesis of prostaglandinlike compounds is requisite to chondrogenesisin vitro.

Development of the cyclic AMP response to parathyroid hormone and prostaglandin E2 in the embryonic chick limb.

SummaryThe developing chick limb was studied to determine the ability of parathyroid hormone (PTH) and prostaglandin E2 (PGE2) to increase intracellular cyclic AMP (cAMP) during various stages of development. All developmental stages examined (stages 20–21, 24–25, and 26–28) responded to PGE2 when the cells were assayed immediately following the removal of the limbs from the embryos. In contrast, only stage 26–28 limb cells responded to PTH when assayed in a similar manner. The response to PTH was temporally correlated with the appearance of cartilage matrix in vivo. Undifferentiated limb cells were also cultured and assayed at various times for hormone responsiveness. Stage 24–25 high-density cell cultures responded initially to PGE2 but not to PTH. However, by 36 h and in all subsequent itme intervals tested, the response to PTH was significantly greater than that to PGE2. The PTH receptor, in contrast to that of PGE2, was shown to be sensitive to trypsin treatment, but could be regenerated during subsequent cell culture. The majority of the hormoneresponsive cells were found in cartilaginous regions of the limb, and were shown to respond to both hormones in a dose-dependent manner. The PTH-induced cAMP response was affected by low cell density and mouse serum, both of which significantly inhibit the chondrogenic potential of cultured limb cells. These findings are consistent with a temporal correlation between the development of the PTH response and chondrogenesis in vivo.

The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture.

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20--21, 23--24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 microgram/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20--21, 23--24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20--21 or 23--24 embryos. It was determined that culture medium composed of Eagles MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Hams F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.

Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells.

Abstract Chick limb-bud mesoderm cells from embryonic stages 22–25 were cultured at high cell densities in media known to support chondrogenesis. Under these conditions the continuous presence of normal mouse serum, at a concentration of 10%, inhibits the ability of the cells to produce toluidine blue-stainable cartilage matrix materials. In contrast, mesodermal cells treated with comparable concentrations of other heterologous sera continue to differentiate much like the control cultures while growing in the presence of the test sera. The inhibitory effect of the serum was shown not to be the result of a general cytotoxic effect on protein synthesis or the inability of the cells to incorporate [ 3 H] d -glucosamine. There was a significant difference however, in the distribution of the incorporated glucosamine. Less label was associated with the cell layer of the treated series, while a greater amount of the incorporated material was found to be secreted into the medium when compared with the control cultures. Studies have shown also that the serum inhibitory response is dose dependent, while the factor(s) itself is non-dialysable, stable to heat and repeated freezing and is not a conventional serum lipoprotein. Following the addition of whole or delipidated mouse serum, a significant increase in lipid droplets appears in the cytoplasm of the cells. Biochemical analyses of mouse serum-treated cells indicate that there is a marked increase in their triglyceride content as compared to the control cells. While the nature of the serum inhibitory factor remains to be determined, the accumulation of triglyceride following mouse serum treatment suggests that this may play a role in modulating the expression of the chondrogenic phenotype.

The loss of Fc surface receptors by erythroleukemic cells following stimulation to differentiate.

Abstract Uninduced Friend virus-infected erythroleukemic cells (FLC) have been shown to carry the Fc receptor for immunoglobulin when assayed for their ability to form rosettes with antibody-coated sheep erythrocytes (EA-IgG). Cells induced to differentiate with dimethylsulfoxide (Me 2 SO), ultimately lose the ability to form rosettes with EA-IgG. Hence, it appears that the ability of FLC to form rosettes with EA-IgG is due to the precursor cell surface components which are either masked or lost following Me 2 SO-mediated induction as mature erythrocyte-specific membrane antigens appear. This suggestion is supported by a decrease in the number of Friend progenitor cells capable of binding fluorescein-conjugated antibodies, produced in rabbits against mouse gamma globulin, following Me 2 SO-induced differentiation.

Induction of hemoglobin synthesis in dimethylsulfoxide-treated friend erythroleukemic cells grown in the presence of cytochalasin B

Abstract The experiments reported here were designed to determine the necessity for nuclear and/or cell division for erythroid differentiation in the presence of the inducer, dimethylsulfoxide (Me 2 SO). Murine erythroleukemic cells are induced to synthesize hemoglobin only when the cells are cultured at a density which allows them to replicate in the presence of Me 2 SO. Hemoglobin is not detected in the cytoplasm of the cells prior to 48 h. Cells grown in the presence of inducer (Me 2 SO)+cytochalasin B (CB) do not divide, but do undergo karyokinesis thereby increasing in size as they become multinucleated. Under these experimental conditions, the CB-treated cells are also capable of synthesizing hemoglobin. Measurements of the rate of hemoglobin synthesis suggest that induced CB-treated cells accumulate more hemoglobin per induced cell than do cells induced by Me 2 SO alone. These results indicate that: (a) while cytokinesis is not required to induce hemoglobin synthesis, the ability of the cells to replicate their genomes is critical, and possibly allows Me 2 SO to interact with the genome to initiate the erythroid cell program; (b) CB does not alter the time course of hemoglobin synthesis; and (c) the increased levels of hemoglobin in the CB-treated cells is probably related to the increased cell-size mediated by the mold metabolite, and not to the induction of a higher percentage of cells.

Evidence for Fc surface receptors on friend murine erythroleukemic cells

Abstract Cloned Friend erythroleukemic cells (FLC) were assayed for their ability to form rosettes with antibody-coated sheep erythrocytes (EA). Erythroid progenitor cells were shown to consistently form a high percentage of EA-IgG rosettes at 4°C. The specificity of the rosetting phenomenon was determined by enzyme treatment of FLC with trypsin and trypsin-EDTA. Cells assayed immediately following the enzyme treatment showed a decrease in the percentage of cells forming rosettes. If, however, the cells were allowed to grow in culture for 24 h following the enzyme treatment, and then assayed for their ability to form rosettes, the rosetting percentages were again found to be at the control levels. This suggests that during the recovery period the altered cell surface receptors were regenerated. Rosettes were not produced when normal adult mouse erythrocytes were assayed in a similar manner.

The effect of cytochalasin B on chondrogenesis in chick limb-bud mesoderm cells grown in vitro

SummaryCytochalasin B (CB) has been shown to have many biological effects on cultured cells. We report that an initial 48-hr treatment of freshly plated chick embryo limb mesoderm cells with CB irreversibly inhibits chondrogenesis. A slight inhibition in the amount of matrix is seen when limb cells are allowed to grow in culture for 24 hr prior to treatment for the second 24 hr of culture. If the cells are allowed to plate-out and grow for 48 hr or longer prior to being treated with CB for 24 hr, the amount of matrix produced is essentially the same as that seen in the controls. However, if the initial 48-hr culture period is followed by a 48- or 72-hr treatment, chondrogenesis is reduced, but not to the same extent as that seen in cultures treated for the first 48 or 72 hr. The irreversible inhibition of chrondrogenesis does not appear to be due to irreversible inhibition of protein synthesis or hexose uptake because, although these are reduced during treatment, they return to control levels within 48 hr following the removal of the drug. We cannot mimic the effect of CB treatment using glucose-deficient medium, thereby eliminating the possiblity that a critical glucose level is necessary to permit chondrogenesis. Multinucleation of limb cells treated with CB is reversed within 4 to 7 days following the removal of the drug. Therefore multinucleation alone is probably not responsible for the CB effect on chondrogenesis. However, other subtle permanent changes may occur during the period of multinucleation which result in the irreversible inhibition of chondrogenesis.

Inhibition of erythroleukemic cell differentiation by normal mouse serum

Abstract The addition of normal mouse serum to otherwise normally cultured Friend erythroid progenitor cells has a significant inhibitory effect on the ability of the cells to differentiate during dimethylsulfoxide stimulation. In contrast, heterologous sera from five different species showed no inhibition of erythroid differentiation when compared to the non-mouse serum-treated control cells. Experiments designed to determine the nature of the serum inhibitory factor led to the following conclusions: (a) delipidating the serum prior to its use does not remove the inhibitory factor; (b) the differentiation-inhibiting factor (DIF) is nondialyzable and (c) the factor is stable to both heat (56°C for 60 min) and repeated freezing. While the nature of the serum inhibitory factor remains to be determined, the evidence obtained so far indicates that the inhibitory response is not due to a cytotoxic effect on the cells, but rather an interaction with the cells in such a manner so as to modulate the expression of the erythroid cell program.