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By utilizing the selection system, a population of hybrid cells with the gene coding HGPRT and sensitive to 8-azaguanine (8-AG) was isolated from a mixed population of hybrid cells derived from malignant diploid HGPRT+ mouse lymphosarcoma cells (LS/BL) and L cells resistant to 8-AG (HGPRT-, R-AG/20 strain). During back-selection of this hybrid population , cultivated in vitro, HGPRT- negative fractions of the cell population, resistant to 8-AG, appeared in a high frequency. As a rule, the telocentric chromosomes (or chromosome), originated from the HGPRT+ parental cells (LS/BL), were lost in these cells. The X chromosome of the LS/BL cells coding the HGPRT enzyme was not present in all cells of the hybrid HGPRT+ population. Hence it is assumed that the fusion of HGPRT+ (LS/BL) and HGPRT- (R-AG/20) cells resulted in a reexpression of the HGPRT activity from the R-AG/20 parental cells.

Mouse lymphosarcoma LS/BL cells growing in tissue culture were irradiated with 100, 316 and 1000 R respectively and fixed 5, 15 or 60 minutes later. Semithin sections were studied under a light and ultrathin under an electron microscopes. Under the light microscope, differences between the morphology of individual nuclei were found in all groups, including controls; the rate of their occurrence was without any relation to the irradiation. They may reflect the differences in the state of LS/BL cells which were not completely adapted to cultivation in small glass tubes. On the other hand, the frequency of some ultrastructural changes seen under the electron microscope showed a relation to the irradiation. They were: undulation of the outer nuclear membrane and dilatation of the space between the outer and inner nuclear membranes, slots in karyosomes adjacent to the surface of the nuclei, enlargement of cisternae and vesicles of endoplasmic reticulum and Golgi apparatus. The importance of a careful examination of control groups in such experiments is stressed.

The growth-promoting alpha-globulin necessary for long-term cultivation of mammalian cells in serum-free medium is a complex of several proteins which also stimulates the incorporation of exogenous DNA into recipient L cells. Biological activity responsible for the stimulation of DNA incorporation into L cells can be concentrated because one of two major protein components, compared to the whole GPAG complex, increases incorporation into cell nuclei by 100%.

Colchicine and its derivatives (demecolcine, 2-demethyldemecolcine and acetylated 2-demethyldemecolcine) blocked the mitotic activity of L cells and inhibited proliferation and DNA synthesis. This effect depended on the length of the exposure and the concentration of the substances tested (from 0.05 micrograms/ml up to 2.6 micrograms/ml). The greatest values of plating efficiency, cell proliferation, mitotic index and inhibition of DNA synthesis, respectively, were found after the application of colchiciline (0.2 micrograms/ml). These effects were reversible, since L cells underwent regeneration of the mitotic spindle and renewal of their function after the removal of colchiciline. Reversible effects were much less with other colchicine derivatives tested. The different effects of colchicine and its derivatives were due to their chemical structure, which determines the interactions with respective L cell receptors and thus modifies the mechanism of action on the mitotic apparatus of L cells.

GPAG stimulates the uptake of exogenous 3H-DNA by L cells and facilitates its retention in host cell nuclei during 48-h postcultivation; the nuclei contain 1.5 times more radioactivity than GPAG-free controls. Owing to the well-known properties of GPAG, the formation of the 3H-DNA-GPAG complex may affect the incorporation of exogenous 3-H-DNA and its intracellular transport and retention in the host cell nuclei.

Hybrid cells (HY SS2 and HY SS6) arising from the fusion of diploid cells of the mouse lymphosarcoma LS/BL and L cells resistant to 8-azaguanine (HGPRT-) showed slower growth and a longer generation time than the parent lines. The inter- and intrachromosomal timing and patterns of early chromosome DNA replication of parent cells was preserved in the hybrid genome and was not influenced by loss of telocentric chromosomes from LS/BL or L (HGPRT-) cells. Thus DNA chromosome replication sequences are not dependent on the presence of a complete set of chromosomes of the parent cells and do not therefore seem to be a result of interaction between chromosomes not segregated in the hybrid genome.

Isologous and heterologous 3H-DNA (optimum concentration 25 microgram/ml of medium) are incorporated into L-cells from the medium during short-term incubation (up to 60 min). The incorporation of DNA is stimulated by a protein complex from calf serum--the growth-promoting alpha-globulin (GPAG) in the concentration 0.8 mg/ml of medium), which is rapidly taken into cells by pinocytosis. GPAG increases the frequency of incorporation of exogenous 3H-DNA into L-cells and the quantity of DNA incorporated. The quantity of 3H-DNA incorporated is further increased on the incubation of L-cells in a medium containing the complex 3H-DNA + GPAG, which is formed by the joint preincubation of the two components at 37 degrees C (20 hr); under these conditions the quantity of DNA incorporated is 150% greater than when 3H-DNA is used alone. GPAG acts as an activator of exogenous DNA transfer, stimulates its pinocytosis by the cells, and at the same time accelerates its intracellular transfer to the cell nuclei.

Incorporation of isologous 3H-DNA of exogenous origin into chromosomes of the L cells was investigated by means of autoradiographical technique. It was shown that during short time intervals of incubation of 3H-DNA with the host cells (1--3 hr), nearly double quantity of the mitoses was labelled, when compared with 3H-TdR used as a control. During longer time intervals, no difference between number of the mitoses labelled with 3H-TdR and exogenous isologous 3H-DNA was observed.

Ninety-six clonal populations were derived from a wild mouse neuroblastoma cell population C 1300 in a serum-free medium containing commercially available serum growth-promoting proteins (GPP). From among these 96 lines the clonal population E 7 was chosen for further work because it displayed maximum spontaneous morphological differentiation. The neuroblastoma clonal population differs morphologically from the original population; it was defined both cytogenetically and by means of growth parameters. The cells of the neuroblastoma clone E 7 are hypertetraploid with two chromosome number modals - 88 and approximately 180-200. The majority of telocentric chromosomes in metaphases with a modal number of 88 chromosomes are identical with the chromosomes of mouse diploid cells. The cell generation time is 22 hours. The cells of the clonal population E 7 are highly sensitive to the action of ethanolamine, which induces morphological differentiation, so that the processes of 30% of the cells in the population are over 40 micron long. Electrophysiological studies showed that the cells of the neuroblastoma clonal population E 7 retain the character of excitable cells and they are thus suitable for studying some of the properties of nervous tissue cells.

The initial and terminal patterns of DNA replication in the chromosomes of the hybrids arising from a fusion of mouse lymphosarcoma LS/BL cells and L cells (HGPRT-) were studied. The replication of chromosomes in the hybrids compared to the parent cells was not modified by the alterations in generation time of the hybrids not by segregation of telocentric chromosomes. It can be assumed that there is a gross control system in LS/BL X L hybrids that is responsible for the patterns and timing of chromosome DNA replication and does not depend on interactions between genomes of the parent cells.

Hybrid cells were obtained by fusion of irradiated and non-irradiated mouse cells of two different lines; they differed from the parent lines and from the hybrid cells of non-irradiated parents in their morphological, growth and karyological properties. The frequency of their occurrence was lower than in hybrids from non-irradiated cells, and unlike the irradiated cells of the parent line, these hybrid cells were capable of permanent proliferation in vitro. Chromosomes of the irradiated parent line were preferentially eliminated from the karyotype of the hybrids.

This paper describes the proliferative and morphological properties of cybrid cells derived from fusion of the whole cells of a mouse lymphosarcoma LS/BL and cytoplasts prepared from L cells or their biochemically marked mutants (HGPRT-). In cybrids, decondensation of parent LS/BL nuclei and their cooperation with heterologous cytoplasm was observed. Cybrids incorporated the precursors of nucleic acid and protein synthesis up to 70 h after fusion.

The karyotype of HY 5/3--4 hybrid cells formed by fusion of diploid and heteroploid murine cells (LS/BLxR-AG/20) consisted of 60--75 chromosomes (modal number 72). The G-banding technique made it possible to identify the origin of the chromosomes; it is concluded that due to chromosomal segregation the hybrid genome lost preferentially the chromosomes derived from the diploid parental line LS/BL. Even after long-term culture in vitro, the chromosomal complement of hybrid cells retained all bi-armed chromosomes and most of the telocentrics, derived from the heteroploid parental cells.

For the biosynthesis of macromolecules in amounts sufficient for indefinite growth or survival in dividing as well as in nondividing metazoan cells, a specific serum protein is required. As the addition of this factor to the medium triggers off a chain of events which leads to RNA, DNA, and protein synthesis, we have called it the pleiotropin. Pleiotropin is taken into the cells by pinocytosis. Pinocytic uptake of pleiotropin is strongly inhibited by a mutual cell-to-cell contact; diploid cells in the stage of contact inhibition of pleiotropin pinocytosis are nutritionally limited, and some changes in life processes may be expected. In continouous culture and in tissues in vivo, these changes may accumulate and ultimately result in an irreversible damage of cells so that old cells are characterized by a low pinocytic activity and by a reduced formation of macromolecules. As pleiotropin increases the saturation density and prolongs the life span of diploid cells in vitro, it is suggested that the loss of division potential in metazoan cells is not a programmed event at the cellular level and that the ageing of static cell populations may be caused by the same nutritional deficiency as that of proliferating cells.

Chinese hamster cell line (line K 431, HGPRT+) and its mutant (line K II, HGPRT-) are similar in the numberical composition of the karyotype (modal number 22 chromosomes), but differ in its structureal constitution (occurrence of trisomy, breaks in the centromeric region, chromosome losses and others). Deletion and rearrangement of some heterochromatic segments of the X chromosome appeared with a higher frequency in HGPRT- line and can constitute one of the conceivable causes of the HGPRT activity loss in the mutant line.