SV40 immortalization of adult human mesenchymal cells from neuroretina. Biological, functional and molecular characterization

Abstract

Human adult mesenchymal cells from neuroretina (human choroid cells, HC) have acquired an infinite lifespan, following phenotypic transformation with a wild-type SV40. Immortalized cells (HC/SV40) contain high numbers of free circular viral DNA, and integrated molecules in a head-to-tail array in the cellular DNA. HC/SV40 cells express both the virus-coded "T" antigens and the cell-coded p53 transformation-associated protein. The transformed phenotype was further characterized by loss of contact inhibition of cell division, inability to induce the retraction of a fibrin clot and to spread within fibrin, and the existence of an altered distribution of actin cables. For the first time we also describe a coupling of the immunofluorescence and the quantitative cytofluorometric analyses, a new transformation parameter, since we show that SV40 transformation causes reorganization of the cell membrane by inducing the unmasking of the antigen recognized by the 4F2 monoclonal antibody, which is present in a "cryptic" form in the untransformed cells. Though the HC/SV40 cells have been continuously passaged over a 3-year period, they have not yet achieved a fully malignant phenotype, since they retain serum-dependency and the presence of a well developed fibronectin pericellular network, and they are not tumorigenic in nude mice. Thus this human immortal cell line constitutes a very useful tool for studying the progression toward full malignancy and the relationships between evolution of transformation parameters and changes in the viral and cellular genome interplay.

Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2.

“…Incubation of lymphocytes with the 4F2 monoclonal antibody decreases the level of activation (1,10) and inhibits the growth ofthe tumor cell line HT-1080 in vitro (7). That molecule may be involved in Na'-dependent, non-ATP-dependent calcium uptake by cells, functioning either as the exchange molecule itself or in a regulatory capacity in association with the actual transporter (11).…”

“…Several groups have reported the expression of the molecule on actively proliferating cells of various origins, including embryonic skin and lung, basal-layer keratinocytes, fibrosarcomas, osteosarcomas, and rhabdomyosarcomas as well as cells transformed by the DNA tumor virus simian virus 40 (6-9). In fibroblasts, the 4F2 antigen is detectable at low levels on the surface of nondividing cells; however, its expression increases dramatically upon transformation by simian virus 40 and the onset of active cell division (7,9).…”

“…Several groups have reported the expression of the molecule on actively proliferating cells of various origins, including embryonic skin and lung, basal-layer keratinocytes, fibrosarcomas, osteosarcomas, and rhabdomyosarcomas as well as cells transformed by the DNA tumor virus simian virus 40 (6-9). In fibroblasts, the 4F2 antigen is detectable at low levels on the surface of nondividing cells; however, its expression increases dramatically upon transformation by simian virus 40 and the onset of active cell division (7,9).Although the function of the 4F2 molecule is unknown, its pattern of expression and distribution on cells has indicated a possible role in cell division. Incubation of lymphocytes with the 4F2 monoclonal antibody decreases the level of activation (1, 10) and inhibits the growth ofthe tumor cell line HT-1080 in vitro (7).…”

Evolution in the structure and distribution of 4F2‐antigen from the oncofetal to the adult phenotype of human fibroblasts

“…The same cells transformed by SV40 were 100% positive both in suspension and when attached to a substratum. Normal and SV4O-transformed cells exhibited a similar mean surface density (MSD) of the 4F2 antigen (Daya-Grosjean et al, 1984). These data suggest (i) that the difference between transformed embryonic and normal adult cells consists in a different cellular distribution of the 4F2 antigen, and (ii) that in normal adult cells, either it moves to a membrane domain which is inaccessible to antibodies or it is internalized.…”

“…Moreover, 4F2 antigen was also detected on 100% of adherent sarcoma cells and SV4O-transformed adult cells (Azzarone et al, 1984;Daya-Grosjean et al, 1984). In a quantitative FACS I1 analysis of the 4F2 antigen it was shown that normal adult fibroblasts in suspension were 100 % positive; however, when spread on a coverslip, they were negative.…”