Identification of a PDGF-like mitoattractant produced by NIH/3T3 cells after transformation with SV40

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Expression of growth-regulated genes in normal and SV40 transformed hamster fibroblasts.

Transformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth-regulated genes in SV40-transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1-specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk-ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk-ts13/1%), was also included in these studies. The low serum adapted SV40-transformed cells and the spontaneous tk-ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental.(ABSTRACT TRUNCATED AT 250 WORDS)     

SV40 transformation of Swiss 3T3 cells can cause a stable reduction in the calcium requirement for growth

A well-characterized SV40-transformed Swiss 3T3 line, SV101, and its revertants were tested for the ability to grow in reduced Ca++ (0.01 mM). Transformants and revertants did not differ from the parent 3T3 line in their Ca++ requirements. All three classes of cells grew less well in low Ca++ than in regular Ca++ (2.0 mM). SV40 transformants were then selected for the ability to grow in reduced Ca++. This new class of transformants was found to grow in 1% serum, grow in soft agarose, have a reorganized actin cytoskeleton, and express viral T antigens, as well as grow well in low Ca++. One of the selected clones was found to be T antigen-negative, yet was transformed in the serum, anchorage, actin, and Ca++ assays. It is possible that this clone was a spontaneous transformant. However, Southern blot analysis revealed the presence of integrated SV40 DNA. In addition, this analysis revealed the absence of an intact early region fragment, which codes for the viral T antigens. One explanation of this result may be that the mechanism of viral transformation for growth in low Ca++ involves viral-host DNA interactions that may not require a fully functional T antigen. In this case SV40 integration may be acting as a nonspecific cellular mutagen.     

Replicon sizes in non-transformed and SV40-transformed cells, as estimated by a bromodeoxyuridine photolysis method

Replicon sizes were measured in Simian Virus 40 (SV40)-transformed and untransformed normal human, xeroderma pigmentosum (XP), and mouse 3T3 cells with an X-ray plus bromodeoxyuridine (BUdR) photolysis method. Replicon sizes in SV40-transformed cells were at least twice those in untransformed counterparts, but DNA fork displacement rates were only slightly increased.     

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.     

Transport of 6-deoxy-D-glucose and D-xylose by untransformed and SV40-transformed 3T3 cells

Transport rates of the nonphosphorylated D-glucose analogs 6-deoxy-D-glucose and D-xylose were measured in quiescent and serum-stimulated cultures of mouse 3T3 cells, in SV40-transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (Fl-SV101). Initial rates of both entry and exit of 6-deoxy-D-glucose and D-xylose were more than threefold higher in serum-stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum-stimulated 3T3. Confluent cultures of Fl-SV101 showed lower rates of transport than serum-stimulated Fl-SV101, but not as low as quiescent 3T3 cells. These data confirm previous findings of others with other analogs that glucose transport is one of the cell functions that is depressed when 3T3 cells enter the quiescent G₀ state, but emphasize that SV40-transformed 3T3 cells do not show higher activity of the D-glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.     

Cell density-dependent increase in chromatin-associated ADP-ribosyltransferase activity in simian virus 40-transformed cells.

ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.     

Increased ouabain-sensitive 86Rb+ uptake and sodium and potassium ion-activated adenosine triphosphatase activity in transformed cell lines.

During the log phase of growth both the active, ouabain-sensitive K+ uptake, measured as 86Rb+, and the sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase) activity of SV40-transformed 3T3 cells were 2.5-and 5,5-fold higher, respectively, than in untransformed 3T3 cells. A similar higher active K+ uptake was found for Rous sarcoma virus and SV40-transformed baby hamster kidney cells compared with untransformed BHK cells. The active K+ uptake in SV403T3 and normal 3T3 cells decreased when the growth rate of both cell types diminished. Reduction in ouabain-sensitive ATP hydrolysis only occurred later, however, when appreciable decreases in cell viability were seen. Arrhenius plots of the (Na+ + K+)-ATPase activity of SV403T3 cells indicated a discontinuity at 24 degrees, whereas no similar discontinuity was indicated for 3T3 cells. The consequences of elevated K+ transport and (Na+ + K+)-ATPase activity in transformed cells and the possibility that the increased activity might be related to differences inphospholipid fatty acyl chain fluidity are discussed.     

Alterations in SVT2 cell transfer RNAs in response to cell density and serum type.

The chromatographic elution profiles of tRNA-A sn, tRNA-A sp, tRNA-H is and tRNA-T y r from SV40-transformed BALB-3T3 cells grown in fetal calf serum or cald serum-supplemented media have been examined. The relative proportions of certain of the isoaccepting species of these four tRNAs are altered in a similar fashion depending on the serum type. It is suggested that the elution profile alterations reflect the extent of modifications of a specific G residue to the minor nucleoside Q, and that this process differs between untransformed and transformed cells. In addition, cell density appears to influence the Q content of these tRNAs, though other density-dependent tRNA modifications also appear to occur.     

The growth requirements of SV40 virus transformed Balb/c-3T3 cells in serum-free monolayer culture

The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.     

Density-dependent changes in the amount of sulfated glycosaminoglycans associated with mouse 3T3 cells.

The relative amount of sulfated glycosaminoglycans associated with the cell layer of parent and SV40-transformed Swiss mouse 3T3 cells was determined from the incorporation of labeled sulfate (35SO4) into macromolecular material. In cultures of SV40-transformed cells, the glycosaminoglycan content per cell was constant over a wide range of densities. In cultures of parent 3T3 cells, the glycosaminoglycan content per cell increased directly with density, the highest values being found in contact-inhibited cultures. At high cell densities, the glycosaminoglycan content of 3T3 cells was several-fold higher than that for SV40-transformed cells. Most of the density-dependent increase in glycosaminoglycans of 3T3 cells was accounted for by chondroitin sulfate (dermatan sulfate) which was over 6-fold higher in confluent cultures than in low density cultures.     

"Transformation" of human endothelial cells by SV40 virions

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages. The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.     

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.     

The growth arrest-specific gene, gas1, is involved in growth suppression.

This report describes the structure of the mRNA, the protein product, and the growth-regulating activity of one of the growth arrest-specific genes, gas1. From the predicted amino acid sequence, in vitro translation of gas1 mRNA, and immunofluorescence of cells in culture, it appears that the gas1 protein is an integral plasma membrane protein whose expression is linked to growth arrest. When gas1 is overexpressed from a constitutive promoter in quiescent cells, the serum-induced transition from the G0 to the S phase of the cell cycle is inhibited without affecting the normal early serum response. Ectopic expression of the gas1 gene by microinjection in normal and transformed NIH 3T3 cell lines with the notable exception of SV40-transformed 3T3 cells leads to inhibition of DNA synthesis. Thus, gas1 appears to be one component of a negative circuit that governs growth suppression. Its effect is, however, abolished in SV40-transformed cells.     

Isolation and Characterization of Revertant Cell Lines VI. Susceptibility of Revertants to Retransformation by Simian Virus 40 and Murine Sarcoma Virus

The susceptibility of two classes of revertants of Simian virus 40 (SV40)-transformed 3T3 cells to retransformation by SV40 or murine sarcoma virus (MSV) was studied. Both serum-sensitive and density-sensitive revertants are not retransformable by SV40. MSV can transform both types of revertants. The MSV-transformed revertants grow to high cell densities and form colonies when suspended in semi-solid methylcellulose medium, but are unable to grow in 1% calf serum. The MSV-transformed revertants produce infectious MSV and murine leukemia virus and possess the same number of chromosomes as the untransformed revertants.     

Neutral glycolipids and gangliosides of concanavalin A-selected SV3T3 revertant cells and of normal and SV40-transformed Balb/c 3T3 cells.

The structural analysis of neutral glycolipids and gangliosides of the SV40 transformed Balb/c3T3 cells (SV3T3 cells) and concanavalin A-selected SV3T3 revertant cells, both compared with untransformed Balb/c3T3 cells, has shown: (i) a content of neutral glycolipids in revertant cells near to that found in the untransformed parental cells; (ii) a similar decrease of the higher gangliosides in transformed and revertant cells; (iii) a content of ganglioside GM3 in revertant cells much higher than that found in both SV3T3 and untransformed Balb/3T3 cells. The possible role of ganglioside GM3 in growth control is discussed.     

Isolation and characterization of revertant cell lines. 3. Isolation of density-revertants of SV40-transformed 3T3 cells using colchicine

Treatment of the SV40 transformed 3T3 cell line SV101 with colchicine permits the isolation of polyploid revertant sublines Which have lower saturation densities than SV101. These low saturation density lines have also reverted to a high serum requirement for growth, and are unable to form colonies in methocel. Normal SV40 has been recovered from these revertants. 3T3 cells are more resistant to colchicine than SV3T3 cells at all cell densities. Colchicine revertants do not display a 3T3-like resistance to colchicine at low density, but do survive colchicine at confluent cell densities, presumably due to their increased contact inhibition.     

Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice.

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.