Studies on the surface antigens and surface properties of animal cell heterokaryons

A study has been carried out on the surface antigens and surface properties of heterokaryons formed by fusing animal cells of different species with UV-inactivated Sendai virus. HeLa cells of human origin were fused with mouse Ehrlich ascites cells, mouse L strain fibroblasts, hen and chick erythrocytes, rat lymphocytes, chick fibroblasts, and mouse and rabbit macrophages. L cells were fused with Ehrlich cells and with hamster embryo fibroblasts.
In the first section of the thesis experiments using inactivated virus to produce artificial heterokaryons were reviewed. The structure and function of the cell membrane and cell surface were considered as well as some of the methods, problems and results of antigen identification in tissue culture.
In experimental work the mixed antiglobulin technique of mixed haem-adsorption was found to combine great sensitivity with effective labelling and was applicable to living monolayer cells. It was the main method used for studying the surface antigens of heterokaryons.
HeLa-Ehrlich heterokaryons and mononuclear hybrid cells carried both human and mouse surface antigens and these appeared to be intimately mixed. A double labelling technique using erythrocytes and killed bacteria as marker particles was developed and permitted each species antigen to be identified on the same cell* Small clones of fibroblastic cells bearing each surface antigen appeared some days after fusion, but the division rate v/as slow and hybrid cells did not live more than a few weeks.
HeLa-Ehrlich heterokaryons could be lysed with antiserum against each species antigen but were slightly more resistant to the cytolytic effect of anti-HeLa serum than single HeLa cells and considerably more resistant to anti-Ehrlich serum than single Ehrlich cells. Combinations of dilute antisera were more effective in producing cytolysis than single antisera at the same concentrations.
Heterokaryons were also produced by the fusion of HeLa cells with UV-irradiated Ehrlich cells. A. combination of autoradiography and mixed haemadsorption showed that Ehrlich antigen could persist on heterokaryons in the absence of RNA. synthesis by Ehrlich nuclei. HeLa-L cell heterokaryons were also found to carry the antigens of each species but only occasional pairs of mononuclear hybrid cells were seen and no"hybrid clones.
Hen species antigen was shown on HeLa-hen erythrocyte and HeLa-chick erythrocyte heterokaryons, but the percentage of heterokaryons carrying this antigen decreased daily after cell fusion in spite of RNA. synthesis by enlarging hen or chick nuclei. The presence of hen antigen on some single HeLa cells soon after fusion was thought to represent incomplete fusion of erythrocytes. The disappearance of this antigen within 24 or 48 hours could indicate turnover and synthesis of HeLa cell surface. The theoretical and experimental evidence for cell membrane turnover was reviewed and the lack of definite data noted.
HeLa-chick fibroblast heterokaryons could be identified for three days and the proportion labelled for chick antigen did not decrease.
Rat species antigen was demonstrated on HeLa-rat lymphocyte heterokaryons but disappeared daily following fusion in a pattern similar to the loss of chick antigen in the HeLa-erythrocyte system. Gamma-irradiation of HeLa cells prior to cell fusion was found to delay nuclear fusion and prolong the life-span of HeLa-lymphocyte and HeLa- erythrocyte heterokaryons.
Ehrlich-mouse macrophage heterokaryons adhered to glass. The macrophage surface property of adsorbing erythrocytes sensitised with antibody was expressed on these heterokaryons initially but lost within two days of fusion. HeLa-rabbit macrophage heterokaryons lost the ability to adsorb sensitised erythrocytes within 24 hours of fusion and were not affected by concentrations of leucocidin which killed all single macrophages.
A series of experiments was performed in which HeLa cells were labelled with dilute antibody which did not affect cell multiplication. A. method of preventing antibody transfer was developed and antibody labelled cells were detected daily by mixed haemadsorption. The possible use of such a method in studying antigen turnover was considered.
Heterokaryon surface characteristics such as persistence of parental antigens on some heterokaryons and disappearance of antigens from others, loss of specialised surface properties, and unusual morphology and surface activity were discussed. A correct explanation may not be possible until the structure, synthesis and metabolism of the cell surface are better understood.