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V Defendi

Publications and source records attributed to V Defendi.

At least 37 records · Page 2Linked to original sources
PubMed1983

Transformation and immortalization of human keratinocytes by SV40.

We have studied the appearance of transformed properties following infection of human epidermal keratinocytes by the oncogenic virus SV40. Shortly after infection, only a small fraction of the cells are positive for SV40 T antigen by immunofluorescence; this fraction progressively increases upon serial subcultivation concomitant with an increase in plating/colony-forming efficiency and growth rate. The capacity of the cells to differentiate progressively decreases, as indicated by cytochemical staining and cornified cell-envelope formation induced by suspension in methyl cellulose. The infected cells enter a period of growth crisis characterized by cytopathology and cell death as the level of T antigen synthesis reaches about 90 percent positive cells at about the tenth serial passage. Viable cells emerging from the crisis period are found to exhibit anchorage-independent growth, as indicated by the formation of viable colonies in semisolid media, but there is considerable variability in colony formation among clones isolated from anchorage-independent populations. The emergent population also manifests phenotypic instability in terms of the appearance of variants, which, in contrast to uninfected cells, expresses a well-defined actin cytoskeleton. The infected cells eventually become "immortalized," as evidenced by an indefinite lifespan, i.e., replication capability maintained well beyond the ordinary time of senescence for uninfected cells. We present these findings in the context of a stage-specific model of epithelial transformation in vitro.

Actins↗
PubMed1983

A scanning electron microscope study of normal and SV40-infected human keratinocytes.

We have studied the changes in the surface ultrastructure of human epidermal keratinocytes after infection by the oncogenic virus, SV40. The surfaces of uninfected cells show patterns of dense microvilli and microridges in varying proportion with microridges predominating in the center of the developing keratinocyte colony and on the fully mature keratinocytes (squames) and the microvilli being more prevalent on the immature proliferative cells at the colony periphery. As the transformation process progressed over time highly microridged surfaces became less numerous. After many months in culture cell surfaces were found to exhibit more sparse, predominantly villous ultrastructure. The surface characteristics of the infected cells in long term culture were similar to those observed in a cell line derived from a squamous cell carcinoma.

Carcinoma, Squamous Cell↗
PubMed1982

Effects of 12-O-tetradecanoylphorbol-13-acetate on the differentiation of simian virus 40-infected human keratinocytes.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on the differentiation of human epidermal keratinocytes infected by the oncogenic virus Simian Virus 40. The cells in monolayers exposed to a concentration of 12-O-tetradecanoylphorbol-13-acetate as low as 5 X 10(-10) M redistributed into dense patches within 48 hr. This morphological change was accompanied by a 10- to 30-fold increase in the production of cells cytochemically stained by orange G:acid fuchsin indicating keratinization and a 2- to 3-fold increase in the exfoliation of cornified envelope-bearing cells. The induced cellular differentiation occurred in parallel with an inhibition of cell growth. The biologically inactive congener 4 alpha-phorbol-12,13-didecanoate at equimolar concentrations did not affect the growth or differentiation of these cells. Binding studies using [3H]12-O-tetradecanoylphorbol-13-acetate revealed a binding site with characteristics similar to those found for other cell types (Kd = 17 nM; 1.25 X 10(5) available sites/cell).

Cell Differentiation↗
PubMed1981

Receptor activities for low-density lipoprotein and acetylated low-density lipoprotein in a mouse macrophage cell line (IC21) and in human monocyte-derived macrophages.

IC21 macrophages, a permanent culture of a line of cells derived from a single colony of mouse peritoneal macrophages transformed with simian virus 40, demonstrate most of the characteristics of lipoprotein metabolism that have been described for primary cultures of rodent or canine peritoneal macrophages. IC21 macrophages have low but demonstrable low-density lipoprotein (LDL) receptor activity. They actively degrade acetylated LDL (AcLDL), which has a negative charge and is not recognized by the LDL receptor. Incubation of IC21 macrophages with human lipoprotein-depleted serum leads to a marked increase in cholesterol synthesis, as measured by incorporation of labeled acetate into sterols. Sterol synthesis is inhibited by further incubation with AcLDL; incubation with LDL also decreases cholesterol synthesis with an accumulation of radioactivity from acetate in sterol intermediates, which indicates that some uptake of LDL occurs. Incubation with AcLDL but not LDL leads to a marked stimulation of cholesterol esterification, as measured by labeled oleic acid incorporation into cholesteryl esters, and a concomitant increase in cellular cholesteryl ester content. IC21 macrophages as compared with human monocyte-derived macrophages are shown to have marked difference in their abilities to degrade native LDL and AcLDL. Human monocyte-derived macrophages degrade LDL at low concentrations at a rate sevenfold greater than do IC21 macrophages. The rate of cholesteryl ester synthesis after LDL receptor induction and incubation with LDL increases linearly with LDL concentration in HMD macrophages, but no increase was found in similarly incubated IC21 macrophages. IC21 macrophages degrade AcLDL at a rate two- to fourfold greater than do human monocyte-derived macrophages.

Acetates↗
PubMed1981

Patterns of cell communication and differentiation in SV40 transformed human keratinocytes.

Fluorescein dye microinjection was used to demonstrate changes in communication between human epidermal keratinocytes grown in vitro after infection by the oncogenic virus, SV40. Whereas keratinocytes are normally fully coupled to each other, dye spread becomes progressively restricted to small cell subpopulations after infection, although dye coupling is increased when the infected cells attain high densities. Reduction or enhancement of dye coupling is correlated with similar changes in the extent of cytochemical differentiation and cornified envelope formation.

Cell Communication↗
PubMed1981

Centrifugal assessment of cell adhesion.

The design of a chamber for determining the centrifugal force necessary to detach cells from various substrates is presented. Cells from an SV40-transformed murine peritoneal macrophage line and human erythrocytes were used to assess the feasibility of using the chamber for studies of cell adhesion. This work was confirmed the usefulness of the chamber and provided data concerning the force necessary to detach the cells. These data indicated that the percentage of cells detached from a glass substrate was not a function of force alone. The number of cells detaching increased with the impulse applied to the cells when they were exposed to a constant force. Similarly, when the impulse applied to the IC21 cells was maintained at a constant level, the percentage of cells detached by a centrifugal force increased with the magnitude of the force.

Animals↗
PubMed1981

Instability of integrated viral DNA in mouse cells transformed by simian virus 40.

The state and organization of simian virus 40 (SV40) DNA in tsA mutant-transformed mouse clones were examined early after agar selection in an attempt to elucidate the mechanisms that actively generate the diverse integration patterns found in transformed cells. Although recently selected as a cloned population from agar, A21 cells displayed extremely heterogeneous SV40 DNA patterns when analyzed by agarose gel electrophoresis and Southern blot hybridization. Reselection of clones in agar from A21 at 33 degrees C or 39.5 degrees C and DNA analysis by hybridization demonstrated (i) simplification of the number of integration sites in the new clones; (ii) new sites of integrated SV40 DNA in high molecular weight cell DNA fragments generated by digestion with restriction endonuclease Bgl II; (iii) relatedness between clones with respect to integrated viral sequence arrangement; and (iv) persistence of free viral DNA forms. The majority of free viral DNA appeared to be full length, nondefective SV40 DNA, although a subpopulation of defective viral molecules was also detected. No detectable free SV40 DNA could be observed in A21 clonal derivatives isolated by growth in agar at 39.5 degrees C, indicating that the persistence of free viral forms was regulated by the A gene. These results suggest that the heterogeneity in viral sequences in the A21 cells was generated within a cloned population from which new clones can be derived with different transformed phenotypes and integration patterns.

Animals↗
PubMed1981

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.

Animals↗
PubMed1980

Amplification and rearrangement of integrated SV40 DNA sequences accompany the selection of anchorage-independent transformed mouse cells.

Clones of SV40 tsA mutant-transformed mouse embryo cells with a temperature-independent transformed growth phenotype were derived from a parental line with a temperature-dependent growth phenotype, using the selective pressure of anchorage independent growth in agar at 40 degrees C. The parental J78 clone contained a complex tandem structure of integrated SV40 DNA within two cellular DNA fragments of 10 and 14 kb, generated by Bgl II digestion. The new clonal derivatives, in addition to an altered growth phenotype, displayed additional high molecular weight sites of SV40 DNA integration. A marked structural similarity among integration sites suggested that the new sites may have arisen by duplication and translocation of an original integration structure, or via an unequal crossover event. The rearrangement of viral DNA sequences appears to be specifically associated with the emergence of temperature-independent clones, since the isolation of new clones under nonselective conditions, that is, growth in semi-solid medium at 33 degrees C, was not accompanied by modification in cellular sequences containing SV40 DNA.

Animals↗
PubMed1979

Increased mutation rates in doubly viral transformed Chinese hamster cells.

An untransformed parental Chinese hamster cell line, one polyoma transformed clone, and two clones containing both SV40 and polyoma DNA were tested for the appearance of variants resistant to 6-thioguanine or oubain. The frequency of such variants was found to be highest in doubly transformed cells. The mutation rate at these loci was correlated with the level of transformation. The mutants observed were stable and had low reversion frequencies with no gross cytogenetic changes. These results establish a quantitative basis for the evaluation of phenotypic variability observed in transformed cell populations.

Animals↗
PubMed1979

Altered pattern of growth and differentiation in human keratinocytes infected by simian virus 40.

Human epidermal keratinocytes were infected by simian virus 40 in vitro. The structure of the developing keratinocyte colony reflects the spatial separation of cell division and keratinization in intact skin; thymidine-incorporating cells were primarily localized at the colony periphery whereas nondividing, histologically differentiated cells accumulated in the interior. Viral infection produced a dramatic increase in the size of the proliferative population as, simultaneously, differentiation was reduced in the colony interior. These changes were manifest when simian virus 40 T-antigen synthesis was detectable in only a small percentage of the cells; differentiation became increasingly density dependent as the percentage of T-antigen-positive cells rose over serial passage. The disruption of the normal pattern of growth/differentiation localization coincided with a loss of dependence on serum for growth, but preceded the appearance of other virus-induced properties associated with transformation; i.e., the ability to form colonies in soft agar and independence of growth from fibroblasts.

Antigens, Neoplasm↗
PubMed1979

Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.

Cell Cycle↗