Howard Green

Ribosomal RNA synthesis in human-mouse hybrid cells

Abstract Human and mouse 28 s ribosomal RNA when extracted at room temperature have identical mobility in polyacrylamide gel electrophoresis, but when heated, the mobility of the human type changes, whereas that of the mouse does not. It was therefore possible to distinguish the 28 s RNA of the two species in mixtures and to examine the nature of the ribosomal RNA synthesized by human-mouse somatic cell hybrids. Only the mouse type of 28 s could be detected, even in hybrids with up to 35 human chromosomes per cell. Several possible explanations are considered; perhaps the most likely is that in these hybrids the transcription of human ribosomal genes is repressed.

The production of hyaluronate by spontaneously established cell lines and viral transformed lines of fibroblastic origin

Human diploid fibroblast strains and established mouse fibroblast lines continue to synthesize hyaluronate over many cell generations in culture. However, this property may be lost in the course of many years of serial culture of established lines as it is no longer detectable in three well-known mouse lines of fibroblastic origin, including L-cells. Transformation of the established mouse fibroblast line 3T3 by SV40 or polyoma virus and of human diploid fibroblast strains by SV40 leads in each case to a marked diminution in the rate of hyaluronate synthesis.

Somatic Cell Hybrid between the Established Human Line D98 (Presumptive HeLa) and 3T3

Somatic cell hybrids have been made between an established human cell line with a long culture history and established mouse fibroblast line. When first analyzed, the hybrid cells contained nearly twice as many mouse chromosomes as the mouse parent line and a human chromosome complemnent of about half that of the human parent. There was further loss of human chromosomes on continued cultivation. This behavior resembles that of other human mouse hybrids and appears to be characteristic of the human-mouse combination. However, the number of human chromosomes is greater than in hybrids made from human diploid fibroblasts. Some clones contain more than a haptoid quantity of human DNA per cell and should synthesize a much greater number of human gene products.

Collagen synthesis on polyribosomes of cultured mammalian fibroblasts

Abstract Ribosomes attached to the endoplasmic reticulum have been separated from those unattached to cytoplasmic membranes of cultured, stationary mammalian fibroblasts. In linear sucrose gradients, approximately one-half of the unattached ribosomes were recovered as 80 s particles, the rest as polyribosomes. The ribosomes freed by deoxycholate from the endoplasmic reticulum were recovered predominantly as polyribosomes. Free and attached polyribosomes had about the same size distributions in the gradients, were equally active in protein synthesis, and nascent collagen and non-collagen proteins were synthesized over all size distributions of polyribosomes. Polyribosomes sedimenting at 180 s were most abundant, and fractions sedimenting at 210 to 220 s were most active in collagen synthesis. There was no evidence in this system for appreciable synthesis of collagen or non-collagen protein on polyribosomes sedimenting faster than 330 s. Hydroxylation of prolyl residues in nascent collagen occurred while the peptide chains were attached to polyribosomes of the endoplasmic reticulum. Pulse-chase experiments indicated that as these nascent collagen chains neared completion, as judged by their hydroxyproline content, they moved with the fastest sedimenting polyribosomes. Release of completed collagen molecules presumably occurred from such units. Nascent collagen appeared to help stabilize polyribosomes of the endoplasmic reticulum.

Antigenic and cultural properties of cells doubly transformed by polyoma virus and SV40

Abstract Cells of the established mouse line 3T3 were transformed in vitro by polyoma virus. Two of the resulting polyoma transformed lines were then transformed by SV40. Several doubly transformed clones were isolated and their properties were studied. Although most of them did not release either virus, all produced both virus-specific complement-fixing (CF) cellular antigens. The titers of each of these CF antigens in the double transformants were as high as those of the single antigens in cells transformed by only one of the viruses. Polyoma transformants and SV40 transformants of 3T3 may be distinguished by their colonial morphology. Colonies of the doubly transformed cells had, in general, characteristics of each of the single transformants. However, certain doubly transformed lines showed a preponderance of either the polyoma or SV40 character. The phenotypes of the doubly transformed lines demonstrate that each of the viruses is capable of acting independently of the other and each may have its distinctive effect on the same host cell.

THE ACTION OF ANTIBODY AND COMPLEMENT ON MAMMALIAN CELLS

The studies discussed in this paper are concerned principally with the action of heterologous antibodies on mammalian cells. Cell-antibody interactions are most conveniently studied in such systems, for it is a simple matter to obtain powerful heterologous antisera. However, the action of homologous antibodies now has been studied 1-4 (see also, C . A. Stetson and E. Jensen, this monograph) sufficiently to make it clear that they produce the same kind of changes in cells as do heterologous antibodies. There is therefore no reason to doubt that the mechanism of action on cells is substantially the same for the two kinds of antibodies. The effects of antibodies will be described as they are observed in vitro under conditions in which it is possible to distinguish between the effects of antibody alone and those requiring the presence of complement. How these effects may be modified in vivo when the cells are in the form of a solid tissue transplant will not be considered in this paper. Most studies of the in vitro effects of antibodies on animdl cells have utilized erythrocytes because they represent a homogeneous population of cells uniformly exposed to the action of substances in the medium. Methods of obtaining tissue cells in free suspension or in monolayers have provided similar advantages in the use of structurally more complex cells than the anucleate erythrocyte. Although the events occurring in such cells when they are exposed to antibody and complement differ in detail from those in the erythrocyte, the mechanism of immune lysis in the two cases appears to be the same.

Serum Albumin Supplemented Medium for Long Term Cultivation of Mammalian Fibroblast Strains.

Summary Serially transferred hamster or human fibroblasts maintained on synthetic medium containing 10% calf serum could be carried through about 10 and about 50-70 cell generations respectively, before growth ceased. Addition of crystallized serum albumin to the medium to a concentration of 20 mg/ ml affected the growth potential so that in both cases over 100 cell generations could be obtained. The human fibroblasts after 95 cell generations showed no chromosomal abnormalities while in the hamster fibroblasts some deviation from the diploid number did occur. In neither case was there evidence of the evolution of improved growth properties that characterize established cell lines.

ENHANCEMENT BY THYMIDINE ANALOGS OF SUSCEPTIBILITY OF CELLS TO TRANSFORMATION BY SV40.

Abstract The established mouse cell line, 3T3, is readily transformed by SV40. Infection of exponentially growing cultures results in a greater transformation frequency (3.6–6.8%) than does infection of stationary phase, non-DNA-synthesizing cells (1.8–2.6%). The thymidine analogs IUDR and BUDR are without effect on cell viability or transformation frequency when added to nongrowing cultures. Dividing cultures lose viability in the presence of these drugs to a degree dependent on the concentration and duration of exposure. Under conditions that lead to loss of viability of a portion of the population, surviving the cells are transformed by SV40 with a considerably higher frequency. Since this effect occurs whether the cells are exposed to the analogs before or after infection, they do not appear to act through modification of viral growth. At analog concentrations where cell killing is not too expensive, the absolute number of transformed colonies is increased, indicating a direct effect of the analogs on the susceptibility of the cells to transformation.

SUCCESSIVE TRANSFORMATIONS OF AN ESTABLISHED CELL LINE BY POLYOMA VIRUS AND SV40.

Two different oncogenic viruses, polyoma and SV40, are capable of transforming mouse cell line 3T3. The properties of the transformed cells produced by the two viruses are in some ways similar, but in other ways they are specific for the infecting virus. This fact permits testing whether a cell line transformed by the one oncogenic virus is still susceptible to the transforming action of the second virus. Two different clonally isolated polyoma-transformed lines when infected with SV40 give rise to cells with properties characteristic of SV40-transformed cells. The frequency of transformation, however, is considerably reduced compared to that of the parent cell line, 3T3.

Collagen Synthesis by Human Fibroblast Strains.

Summary Human diploid fibroblast strains derived from fetal lung, adult skin and keloids, produce collagen in vitro when allowed to grow to confluence and remain without transfer. This property may be retained throughout most of their culture life. Keloid fibroblasts produced the smallest quantities of collagen and had the lowest growth potential. All strains produced more collagen when ascorbic acid was added to the medium. Up to 3% of the total protein being made in the presence of ascorbic acid consisted of collagen. An appreciable fraction of the collagen failed to precipitate in the cell layer and escaped to the medium both in the presence and in the absence of added ascorbate.