No abstract

Alphafetoprotein (AFP), a major serum protein synthesized during the embryo-fetal and postnatal period (in the yolk sac, then in the liver), is also an oncoprotein. The intracellular presence of AFP and of serum albumin (SA) in normal and neoplastic neural crest and neural tube derivatives was previously demonstrated. In this work we have studied the comparative expression of AFP and SA in primitive neuroectoblastic structures of mouse embryos (6 and 7 days "post coitum") and mouse teratocarcinomas (derived from the PCC4 cell line). Using immunofluorescence technique, antibodies to SA gave a positive reaction in embryos of 7 days, while AFP was not detected during this period. By mRNA in situ hybridization, SA mRNA gave a strong signal in both 6 and 7 day embryos, whereas AFP mRNA gave a weak signal only in 7-day embryos. The distribution of SA and AFP and their mRNAs was investigated in primitive neuroectoblastic structures of the teratocarcinomas by in situ hybridization and immunostaining. Only SA protein was detectable by immunostaining. SA mRNA gave a strong signal in differentiating structures as well as in undifferentiated cell clusters. AFP mRNA was observed only in differentiating structure. Dot-blot hybridization indicated that the level of SA transcripts was at least 6-fold higher than that of AFP transcripts in the teratocarcinomas investigated. In teratocarcinoma-bearing mice injected intraperitoneally with 125I-radiolabeled SA and AFP, significant accumulations of both SA and AFP were demonstrated in the tumors, SA being about 3-fold higher than that of AFP after normalization to quantity of uptake in liver. External in vivo photoscanning confirmed this relationship of accumulated radiolabeled proteins. The last observation could be useful in vivo for diagnosis of teratocarcinoma. We conclude that the expression of SA relative to AFP and the external cellular uptake of SA relative to AFP are similar in normal embryonic developing tissues and in the corresponding morphologically neoplastic tissues of the teratocarcinomas. The same SA:AFP relationship constitutes an oncofetal marker of primitive neuroectoblastic structures.

Exogenous chicken α-Fetoprotein (AFP) was added to embryonic chick dorsal root ganglia plated on gelatin-coated tissue culture dishes at different stages during the differentiation process and its intracellular uptake demonstrated by immunocytochemical methods. Other embryonic serum proteins were added as a control. Morphologically well-differentiated neurons (ganglion cells) appeared positively labeled for AFP, contrasting with weakly stained other cell types: these included spindle-shaped cells identified as Schwann cells and larger, often closely packed cells, which could be less mature neurons. Fibroblasts were found negative or faintly stained. No AFP was noticeable in cultures grown in the absence of the protein. These results suggest that the presence of AFP in the developing CNS is for the most part, if not entirely, due to protein uptake as opposed to in situ synthesis.

Α-Fetoprotein (AFP) and AFP-gene transcripts were demonstrated in vitro in Schwann (S) cell and fibroblast (F) cell cultures of neonatal mouse origin. All S and F cells of primary cultures and of established cell lines expressed the AFP gene. AFP mRNA was detected by an in situ hybridization technique using a ³⁵S-AFP-cDNA probe. AFP was localized by immunocytoperoxidase labelling using purified anti-AFP antibodies. The amounts of stained endogenous AFP, estimated semiquantitatively, were about 3-fold higher in S cells than in F cells. After incubating the cultures with exogenous mouse AFP, both S and F cells showed significant ability to take up the protein; the amount of internalized protein was found to be higher in F cells than in S cells. Moreover, the uptake of AFP fluorescein conjugates (FITC-AFP), estimated quantitatively by fluorometry, also gave higher values for F cells. The cytoplasm of F cells exhibited a characteristic fluorescence pattern, strongly illuminated and dispersed grains; the cytoplasm of S cells was regularly labelled. If exogenous FITC-AFP uptake could be used to distinguish labelled F cells from S cells (with application for identification and selection of F cells), the immunocytochemically stained endogenous AFP could allow S cells to be distinguished from F cells (using the dilutions of antibodies still staining the S cells but which lead to the absence of F-cell labelling). The two procedures, which can be used independently or together, may constitute differential markers for S cell and F cultures in, i.e., nerve regeneration of neurofibroma studies using the model of mixed S and F culture also containing other types of cells.

Internalization of exogenous fluoresceinated alpha-fetoprotein (FITC-AFP) was studied at different stages of development on embryonic chick neural retinas maintained, for short periods, in organ cultures. Cellular localization of endogenous, native AFP was carried out by immunohistoperoxidase methods. Cells which specifically internalized exogenous FITC-AFP (neurons and their processes) were precisely those showing positive immunolabeling for the endogenous, native protein. Such a result supports the hypothesis of a predominantly exogenous origin of intracellular neuroretinal AFP. A precise topography and temporal sequence of AFP labeling after internalization in retinal structures is given. AFP uptake was not displayed by undifferentiated cell precursors or germinal cell layers but was apparent in cells with phenotypic characteristics of maturing neurons. Nerve fibers and synaptic layers actively internalized FITC-AFP at specific stages of development. Fully differentiated neurons and processes did not internalize AFP. The possible role of AFP, a carrier of biologically active substances such as fatty acids, in neural retina differentiation is discussed.