SUMMARY A wide range of different kinds of malignant cell were fused with certain derivatives of the L cell line and the ability of the resulting hybrid cells to grow progressively in vivo was examined. In all cases the highly malignant character of the tumour cells was suppressed by fusion with the L cell derivatives, whether or not these had metabolic defects that facilitated selection of the hybrid cells. So long as the hybrid cells retained the complete chromosome complements of the two parent cells, their ability to grow progressively in vivo was very limited, for tumours composed of such unreduced hybrids were not found. However, when they lost certain specific, but as yet unidentified, chromosomes, the hybrid cells regained the ability to grow progressively in vivo and gave rise to a tumour. These findings thus indicated that the L cell derivatives contributed something to the hybrid that suppressed the malignancy of the tumour cell, and that this contribution was lost when certain specific chromosomes were eliminated.

I. hybrids between tumour cells and l cell derivatives

When the nucleus of a hen erythrocyte is introduced into the cytoplasm of a human or mouse cell in culture, it resumes the synthesis of RNA. The reactivated erythrocyte nucleus undergoes great enlargement, but it does not, for at least 2 or 3 days, develop nucleoli which can be discerned under the light microscope. During this period, the heterokaryon, although it may contain several active erythrocyte nuclei, does not synthesize any hen-specific surface antigens; and the hen-specific antigens introduced into the surface of the heterokaryon by the process of cell fusion are eliminated. But when, later, the erythrocyte nuclei do develop nucleoli, hen-specific antigens reappear on the surface of the heterokaryon and progressively accumulate. Before developing nucleoli, the erythrocyte nuclei synthesize little, if any, normal 28 S or 16 S RNA; but they do synthesize large amounts of the RNA which shows polydisperse sedimentation in conventional sucrose density gradients. Autoradiographic studies involving the use of a microbeam of ultraviolet light show, however, that this ‘polydisperse’ RNA is not transferred to the cytoplasm of the cell in detectable amounts so long as the erythrocyte nucleus lacks a definitive nucleolus. The inability of the erythrocyte nucleus at this stage to determine the synthesis of hen-specific surface antigens is thus attributable to the fact that it fails to transfer the RNA made on its chromosomes to the cytoplasm of the cell. When the erythrocyte nuclei develop nucleoli, however, the RNA which they make is transferred to the cytoplasm of the cell, and the synthesis of hen-specific surface antigens then begins. These experiments suggest that the nucleolus may play a decisive role in the transfer of information from nucleus to cytoplasm. The possible nature of this role is discussed.

The expression of genetic information: a study with hybrid animal cells.

Publisher Summary In analyzing the many and complex variables that affect the interaction between viruses and cells, present efforts can perhaps at best provide only model systems and working hypotheses that are not inconsistent with the available information. The study of virus-induced cell fusion is no exception. This chapter outlines the many areas in which further research is required. Many viruses are able to induce cell fusion. The increasing use in the past 5 years of virus-induced cell fusion as a research tool in cell biology, virology, and genetics is also a problem. Two distinct forms of virus-induced cell fusion are recognized. The first, so-called fusion from within is caused by viral products synthesized during intracellular replication of virus. The second type of fusion, so-called fusion from without, does not require intracellular virus replication and the synthesis of virus-coded products and is caused instead by components associated with the virus particle.

Mechanisms of virus-induced cell fusion.

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.

https://rupress.org/jcb/article-pdf/67/1/174/1266682/174.pdf

Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells.

I . Introduction . . . . . . . . . . . . . I1 . Synchrony in naturally occurring multinucleate cells . . . . . . . A . Protozoa . . . . . . . . . . . . . . . B . Algae . . . . . . . . . . . . . . . C . Fungi . . . . . . . . . . . . . . . D . Invertebrate somatic tissues . . . . . . . . . . . E . Germ cell formation . . . . . . . . . . . . F . Fertilization and co-ordination of pronuclei G . Early embryonic development . . . . . . . . . . . H . Cell multiplication in viwo and in witro

Nucleo-cytoplasmic interactions in the achievement of nuclear synchrony in dna synthesis and mitosis in multinucleate cells

When a chick erythrocyte nucleus is introduced into the cytoplasm of a mutant mouse cell lacking the enzyme inosinic acid pyrophosphorylase, synthesis of the enzyme is induced. However, this synthesis does not begin until the erythrocyte nucleus develops a nucleolus. The kinetics of synthesis of the enzyme are essentially similar to those previously described for the synthesis of surface antigens specified by the chick nucleus. The results of both sets of experiments indicate that the nucleolus plays a critical role in the transfer of information from the genes to the cytoplasm of the cell.

/pdf/synthesis-of-an-enzyme-determined-by-an-erythrocyte-nucleus-mktb3xv6t4.pdf

P. R. Harris

Papers

Synthesis of an enzyme determined by an erythrocyte nucleus in a hybrid cell.