Early gastrula-stage Drosophila embryo cells will differentiate in vitro to form several cell types, including neurons. We report here the morphological appearance of cultured embryo cells, the pattern of DNA synthesis, and the expression of neurotransmitter-metabolizing macromolecules. The cells initially exhibit no overt morphological differentiation, and all cells incorporate 3H-thymidine following a 1 hr pulse-labeling period. As cells undergo morphological differentiation, fewer total cells as well as qualitatively different cell types incorporate label. By the time cells are 8 or 9 hr old, no myocytes or myotubes are labeled. In contrast, some neurons are labeled with a thymidine pulse as late as 18 hr. We have also stained cultured cells of various developmental ages with the insect neuron-specific antibody: anti-HRP. Some positive cells can be detected as early as 5 hr, when no overt morphological differentiation is apparent. As the cells differentiate, the staining is limited to the small, round neuronal type and its processes. These findings suggest that this neuron-specific cell marker is expressed very early in cultured gastrula-stage cells and may be used to identify neuronal precursor cells. We have studied the patterns of expression of several macromolecules involved in acetylcholine metabolism using these cultures. The appearance of choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh production, is first detected in 5-hr-old cells. There is an initial phase of low-level expression, followed by a rapid rise in activity shortly after the differentiating neuron clusters make contact with one another. ChAT activity reaches a plateau in 36-48-hr-old cells. Acetylcholinesterase activity can be detected several hours before ChAT and also shows a period of low-level expression followed by a rapidly increasing phase, reaching a plateau at around 36-48 hr. 125I-alpha-bungarotoxin binding appears in cells about 4 hr old and rapidly approaches maximum levels by about 36 hr. The in vitro expression pattern for ChAT and AChE is similar to that seen in vivo. AChE activity has been localized histochemically to the neurons and their processes in vitro. The normal in vitro expression pattern for ChAT and AChE can be altered by adding various cholinergic drugs to the culture medium during cell differentiation. Medium conditioned by older cultures can also result in lower levels of ChAT and AChE expression.(ABSTRACT TRUNCATED AT 250 WORDS)

https://www.jneurosci.org/content/jneuro/7/1/10.full.pdf

In vitro neuronal differentiation of Drosophila embryo cells

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NPl cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrbsinc specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW ∼ 11,000 daltons and 7,000 daltons, respectively) is ∼ 0.4 μM. Protamine II (MW ∼ 4,800 daltons) was equally active (half maximum inhibition concentration ∼ 0.4 μM); protamine IV (MW ∼ 3,300 daltons) was substantially less active (half maximum inhibition concentration ∼ 2.8 μM).

These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

A fibrotic focus (FF) is a scar-like area within invasive ductal carcinoma (IDC) of the breast, and has been shown to be a marker of high aggressiveness of IDC. In order to investigate the mechanism of FF formation in IDC, expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor (FGFR) was studied. One hundred and forty-nine IDCs were divided into solid tumors and scirrhous tumors. Immunohistochemistry was used to determine the expression of bFGF and FGFR proteins in both tumor cells and fibroblasts forming FF. Scirrhous tumors with FF showed a significantly higher frequency of bFGF protein expression than those without (P = 0.017), whereas, in solid tumors, the presence of FF was not significantly associated with the frequency of bFGF protein expression (P = 0.143). In addition, scirrhous tumors showed a significantly higher frequency of FGFR protein expression than solid tumors (P = 0.001). Among IDCs having FF and expressing bFGF protein, a significantly larger number of fibroblasts expressing FGFR protein within FF was observed in scirrhous tumors than in solid tumors (P = 0.016). The results of this study suggest that in scirrhous tumors the interaction between tumor cells and stromal fibroblasts plays an important role in the formation of FF, and that there is a paracrine mechanism between bFGF protein from tumor cells and FGFR protein in fibroblasts.

Significance of Basic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor Protein Expression in the Formation of Fibrotic Focus in Invasive Ductal Carcinoma of the Breast

Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. Implications for existence of cryptic EGF receptors.

Loss of responsiveness in senescent human TIG-1 cells to the DNA synthesis-inducing effect of various growth factors

Histone fractions prepared from various sources stimulated DNA synthesis in quiescent BALB/ 3T3 cells in sparse culture. Of the various sources investigated, the stimulatory activity was highest in the histone fraction prepared from rat Rhodamine fibrosarcoma. The maximal activity at optimal concentration of the histones from the tumor was about half that in the presence of an excess concentration of calf serum. The histone fraction was fractionated by column chromatography on Bio-Gel P-60. All histone species thus obtained had stimulatory activity for DNA synthesis.Although protamine, poly-L-lysine and poly-L-arginine also stimulated DNA synthesis in BALB/ 3T3 cells, the former two polypeptides were less effective than any of the histones.In addition, we found that the nonhistone protein fraction from tumor chromatin stimulated DNA synthesis in BALB/ 3T3 cells. The maximal activity at optimal concentration of nonhistone protein corresponded to that achieved by excess calf serum. From these findings, we postulate the role of a growth factor located in cell nuclei in controlling cell proliferation.

DNA Synthesis-Stimulating Activities for BALB/3T3 Cells Present in Histone and Nonhistone Protein Fractions from Rat Rhodamine Fibrosarcoma

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DNA Synthesis-Stimulating Activities for BALB/3T3 Cells Present in Histone and Nonhistone Protein Fractions from Rat Rhodamine Fibrosarcoma