Robert Pollack

A nonselective analysis of SV40 transformation of mouse 3T3 cells

Abstract Mouse cells transformed by simian virus 40 show many alterations in their growth properties in vitro . In order to investigate the coordinate nature of these changes, we have analyzed the growth properties of 40 randomly selected colonies arising after SV40 infection of 3T3 cells. Clones of cells, established from these colonies, were characterized as to saturation density and doubling time in 10% and 1% calf serum, growth in methyl cellulose suspension, colony formation on monolayers of normal cells, and presence of viral antigens. This analysis revealed that only 5 of the clones were indistinguishable from 3T3 cells; the remaining 35 clones differed from 3T3 cells in that they grew as rapidly in 1% calf serum as standard SV40 transformed cells. Of these 35 clones, ten corresponded to standard transformants previously described. Another ten showed other growth properties intermediate between 3T3 cells and standard transformants. These intermediate clones had lower levels of viral T-antigen than standard transformants and showed considerable heterogeneity in staining from cell to cell. The remaining 15 clones were T-antigen negative and had saturation densities slightly higher than that of 3T3 cells. These changes in cellular behavior are stable on recloning.

Actin-containing cables within anchorage-dependent rat embryo cells are dissociated by plasmin and trypsin

Abstract The distribution of intracellular actin has been examined by specific immunofluorescence in a series of normal and SV40-transformed cell lines of rat origin. A consistent correlation was found between the presence of large thick sheathes of actin-containing material and anchorage-dependent growth control. Anchorage-independent growth by these cells has been shown to be associated with the production and secretion of a plasminogen activator, and dependent upon the presence of the active protease plasmin. We have found that these phenomena can be linked as follows. First, treatment with plasmin, but not urokinase or plasminogen, reversibly removes the actin-containing cables from normal rat embryo fibroblasts, and similar results are obtained with trypsin. Thrombin and chymotrypsin are relatively ineffective in causing the disappearance of the cables. Second, sera such as dog or monkey, which permit high levels of plasmin formation and activity, support cell growth in semi-solid media better than sera in which plasminogen is activated poorly or that are plasminogen-deficient; concomitantly, cables disappear in the former but not in the latter sera. The addition of a plasmin inhibitor prevents the disappearance of actin-containing cables.

Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton

We have examined the effect of the tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), on the actin-containing elements of the cytoskeleton of chick embryo fibroblasts (CEF). TPA at concentrations as low as 7.3 times 10-10M indices a reversible change in the cytoskeleton as visualized by indirect immunofluorescence using anti-actin antibodies. Cells incubated with TPA lose the ordered actin-containing structures found in normal cells and resemble Rous sarcoma virus-transformed cells in that the immunofluorescent actin pattern is diffuse. The TPA effects are both dose-and time-dependent. Analogs of TPA which are inactive as tumor promoters do not induce cytoskeletal changes at the concentrations tested, while a second tumor promoter, PDD, is also able to cause alterations in actin-containing structures. The action of TPA requires de novo synthesis of both RNA and protein. The direct cytoskeletal changes are neither plasmin-dependent nor subject to inhibition by incubating the cells with high levels of protease inhibitors during the exposure to TPA. However, plasminogen does increase the sensitivity of cells to TPA.

Isolation and characterization of T antigen-negative revertants from a line of transformed rat cells containing one copy of the SV40 genome

Negative selection with FUdR produced revertants from the transformed rat line 14B, which contains one insertion of the SV40 viral genome (Botchan, Topp and Sambrook, 1976). 14B contains nuclear T antigen, grows to a high density, grows in low serum and is anchorage-independent. The revertants fall into three classes with regard to viral DNA sequences: the SV40 DNA is retained; the SV40 DNA is retained but has undergone a deletion; and the SV40 DNA is lost, generating a cured cell. This heterogeneity is not a result of long-term passage. The revertants arise with a frequency of one in 8.4 X 10(5) cells after as few as 12 passages. All three classes of revertants are T antigen-negative, density-sensitive, more serum sensitive than 14B and anchorage-dependent. These data argue for a direct role of the functioning viral genome in the maintenance of the transformed state, and that with 14B, the phenotypes of transformation are not virus gene dosage-dependent.

Synthesis of infective poliovirus in BSC-1 monkey cells enucleated with cytochalasin B.

BSC-1 monkey kidney cells were enucleated by two cycles of centrifugation in the presence of cytochalasin B. The resulting populations contained 99.5 percent enucleated cells. After infection, newly synthesized poliovirus was recovered from the enucleated populations. Final virus yield per enucleated cell was about one-fifth the yield per infected untreated cell

Eugenics lurk in the shadow of CRISPR

![Figure][1] Eugenics on the horizon?PHOTO: LAWRENCE LAWRY / SCIENCE SOURCE IN CALLING THEIR Perspective “A prudent path forward for genomic engineering and germline gene modification” (3 April, p. [36][1]; published online 19 March), D. Baltimore et al. show at once the size of the

Anchorage independence: Analysis of factors affecting the growth and colony formation of wild-type and dl 5459 mutant SV40-transformed lines

Abstract We have studied in detail the parameters of anchorage-independent growth of normal rat cells and two clones of simian virus 40 (SV40)-transformed rat fibroblasts. Normal secondary rat embryo fibroblasts (REF) suspended in soft agar neither form large colonies (greater than 0.2-mm in diameter) nor show any appreciable increase in total cell volume. A clone of wild-type SV40-transformed REF grows in agar with a colony-forming efficiency of 54% and an increase in total cell volume greater than 10 3 . A clone of REF transformed by the SV40 early deletion mutant 884 has a colony-forming efficiency in agar of only 0.02%, but the total increase in cell volume is greater than 100-fold. In defining anchorage transformants, a distinction must be made between the ability to form large colonies and the ability to undergo a significant number of doublings. Transformation by the viable deletion mutant 884 apparently is impaired far more in the former aspect than in the latter.

Chapter 5 Methods for Obtaining Revertants of Transformed Cells1

Publisher Summary This chapter describes the three systems for the isolation of transformed cells based upon the ability of the transformed cells to grow in conditions where the normal cells cannot. These three assays measure (1) the maximum cell density attained by a line in excess serum, (2) the ability of a cell to establish an isolated colony suspended in agar or Methocel, and (3) the ability of a cell line to grow in limiting or depleted sera. In the case of assay (1), most normal cell lines grow in an oriented fashion and exhibit a density-dependent cessation of cell division. In the case of assay (2), a revertant cell line lacks at least one of the properties of a transformed cell line from which it is descended. A revertant may be selected by a modification of the protocol used to isolate the transformed parent. Three different assays for selecting transformants are in current use; therefore, three different selective assays for reversion are described.

Two integrated partial repeats of simian virus 40 together code for a super-T antigen.

We determined that the coding sequence for a 100-kilodalton super-T antigen found in Simian virus 40 mouse transformants spanned two separate partial repeats of the viral genome. The downstream repeat contained a complete Simian virus 40 large-T-antigen gene, whereas the upstream repeat was a truncated copy of the same gene. When the repeats were separated by subcloning, the capacity to code for the super-T antigen was lost. A small insertion or deletion in the origin-control region which preceded the second repeat could also destroy the ability to code for the 100-kilodalton protein. Our data suggest that differential splicing between parts of two gene copies was responsible for the additional molecular weight of this super-T antigen.

Integration, loss, and reacquisition of defective viral DNA in SV40-transformed mouse cell lines.

We have examined the state of viral DNA in a set of SV40-transformed mouse cell lines. Using restriction enzymes which cut SV40 DNA in one place, we demonstrate that anchorage-independent SV40-transformed mouse cells commonly contain one or more detectable defective monomers of integrated viral DNA. The defective viral DNA in one of these cell lines, SV101, was extensively mapped using single and double enzyme digests. The results of this analysis indicate that SV101 contains nondefective viral DNA as well as defective viral DNA of the following sizes: 5.0, 4.3, 3.7, 3.4, and 1.5 kb. Three of these defective monomers (4.3, 3.7, and 1.5 kb) preserve the amino terminal exon of large T antigen, and two monomers (4.3, and 3.7 kb) preserve the little t coding region. Anchorage-dependent subclones of SV101 preferentially lose the defective viral DNA, while retaining an intact SV40 early region and the ability to express lytic-sized large and small T antigens. Despite a considerable amount of viral DNA rearrangement which accompanies subcloning, anchorage-independent subclones of SV101 retain defective viral DNA, especially the 4.3- and 3.7-kb monomers. Also, when an anchorage-independent subclone is selected from an anchorage-dependent revertant of SV101, it reacquires defective viral DNA, although of a size not seen in SV101. We conclude that defective viral DNA plays a role in generating the anchorage-independent phenotype. In earlier studies, we have reported that anchorage-transformed mouse lines contain a variant (100kDa) T antigen. The possible role of defective viral DNA in generating this T antigen is discussed.