Physiological induction and reversal of focus formation and tumorigenicity in NIH 3T3 cells.

NIH 3T3 cells undergo morphological transformation in response to conditions of constrained growth, such as occur in low serum concentrations or at confluence. Transformation is expressed in a small fraction of the cells by the appearance of discrete foci of multiplying cells on a confluent monolayer of quiescent cells. We isolated and expanded cell populations from three dense and three light foci. Cells from each of these populations efficiently reproduced foci of the same morphotype when grown on a background of nontransformed NIH 3T3 cells. Using cultures derived from one of the dense foci (subline D/2), we found that the number of focus-forming units was stable and the cells remained tumorigenic when they were subjected to repeated thrice-weekly passage in 2% calf serum. However, equivalent passage in 10% calf serum eventually rendered the cells incapable of both focus production and tumor formation. The results show that the capacity to produce tumors as well as morphological transformation are produced as a response to physiological constraints of growth and/or metabolism in the absence of carcinogens and that both properties can be reversed by lifting the constraints. This behavior is typical of an adaptational response and, taken together with other supporting evidence, shows that tumorigenesis does not require conventional genetic alteration.     

DNA-mediated alteration of the reversion frequency of transformed NIH/3T3 cells.

Cell selection immediately after DNA-mediated transfection of whole-cell DNA into mammalian cells has been used to select for specific DNA sequences that cause a phenotypic effect. Whole-cell mouse or human DNA was cleaved into a distribution of lengths (0.4-25 kilobase pairs) and transfected into anchorage-independent spontaneously transformed NIH/3T3 cells. Immediately after transaction, anchorage-dependent serum concentration-dependent reverents were selected. The Hirt supernatant, containing extrachromosomal DNA resulting from the transfection, was isolated from the revertants and transfected with high molecular weight carrier DNA into a second population of transformed cells; revertants were again selected. After five to seven cycles of transfection of Hirt supernatant DNA (obtained from revertants selected at the previous cycle) into new populations of transformed cells at each cycle, the reversion frequency had become 5-15 times greater than the spontaneous reversion frequency measured for several subclones of nontransfected or mocktransfected transformed NIH/3T3 cells. When nonmammalian genomic DNAs were used in transfecting a first population of cells, there was no effect on the reversion of frequency even after six cycles of selection. The reversion-enhancing activity of sixth-cycle Hirt supernatant DNA resulting after transfection at the first cycle with mouse or human sequences was destroyed by EcoRI but not by BamHI or Sal I. Sequences resembling human Alu I sequences were found in mouse whole-cell DNA isolated from sixth-cycle revertants generated after transfection of human sequences at the first cycle.     

Human papillomavirus 16 DNA in NIH3T3 cells transformed by colonic cancer cellular DNA.

Human papillomavirus (HPV) 16 DNA is closely associated with human cancers. It has been identified as an aetiological agent in cervical cancers and, recently, in colonic neoplasms. To further understand the role of HPV 16 DNA in colorectal carcinogenesis, NIH3T3 cells were transformed with high molecular weight DNA from colonic cancer cells and the expression of HPV 16 DNA detected. Both human Alu and HPV 16 DNA sequences were found in the type II foci of CC-M2T cells by Southern blot hybridisation. Additionally, 100% tumorigenicity in nude mice was seen. This study shows the transfection of HPV DNA from colonic cancers into NIH3T3 mouse cells and suggests that HPV type 16 might be associated with the malignant transformation of colonic cells.     

Antigenic properties of endogenous type-C viruses from spontaneously transformed clones of BALB-3T3

During long-term tissue culture of spontaneously transformed clones from BALB/c 3T3 mouse-embryo cells, some clones spontaneously begin to produce high titers of endogenous murine type-C viruses. The antigenic properties of these viruses have been analyzed by indirect immunoelectronmicroscopy and can be classified into two distinguishable populations: (a) BALB/c murine myeloma-associated extracellular viruses that carry a specific envelope antigen, xVEA, different from the typical murine leukemia viral envelope antigens; and (b) previously uncharacterized type-C viruses that have neither xVEA nor the murine leukemia viral envelope antigens. The former produces PC1 antigen and the latter might induce a new cell-surface antigen. Neither of these two populations of BALB/3T3 endogenous type-C viruses was able to infect BALB/c cells but both could infect NIH Swiss cells. A single BALB/3T3 clone, then, can release infectious endogenous type-C viruses with at least two different antigenic properties. We conclude that BALB/c somatic cells contain preexisting genetic information for production of at least closely related but, nevertheless, distinct type-C viruses.     

K+ efflux in NIH mouse 3T3 cells and transformed derivatives: dependence on extracellular Ca2+ and phorbol esters.

In culture medium deficient in Ca2+, NIH mouse 3T3 cells lose K+, gain Na+, and stop growing. A marked increase in the rate of K+ efflux accounts for this loss; Na+, K+-ATPase pump activity increases but does not fully compensate for enhanced K+ efflux. Phorbol esters and cycloheximide inhibit K+ loss in Ca2+-deficient medium. Phorbol esters inhibit K+ efflux from human fibroblasts as well, even at physiological levels of Ca2+. Two cell lines derived from NIH-3T3, one transformed by a simian virus 40 deletion mutant, the other by the polyoma virus oncogene encoding the middle-sized tumor antigen, retain K+ and can multiply in medium with low Ca2+. Efflux of K+ from these cells is relatively insensitive to reduced Ca2+ concentration, phorbol esters, and cycloheximide. The results suggest the following hypothesis: a channel, nonselective for K+ and Na+, opens when NIH-3T3 cells are in Ca2+-deficient medium; the channel is controlled by the receptor for phorbol ester (protein kinase C) and may also be regulated by a short-lived protein.     

Alteration of glycolipids in ras-transfected NIH 3T3 cells.

Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. We analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, we found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by 125I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1a as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, we found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. Interestingly, gangliotriaosylceramide appeared when the latter cells became confluent. These results indicated that ras oncogenes derived from human tumors are capable of inducing alterations in glycolipid composition.     

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.     

Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40.

Highly purified plasma membranes from hamster lymphocytes transformed by simian virus 40 (GD 248) were compared with the membranes of normal cells by crossed immune electrophoresis, crossed-line immune electrophoresis, and bidimensional isoelectric focusing-immune electrophoresis. Antiserum raised by inoculation of guinea pigs with GD 248 membranes was used as serologic reagent, either directly or after absorption with membranes from normal cells. Bidimensional immune electrophoresis reveals the presence in the plasma membranes of GD 248 cells of at least three antigens not detectable in the membranes from the normal cell population. At least two of these are also present in the mitochondrial membranes of GD 248 cells, but none could be detected in membranes of embryonic fibroblasts. Bidimensional isoelectric focusing-immune electrophoresis indicates that the distinctive antigens of the GD 248 membranes are glycoproteins.     

Pleiotrophin transforms NIH 3T3 cells and induces tumors in nude mice.

The pleiotrophin (PTN) gene (Ptn) encodes an 18-kDa protein that is highly conserved among mammalian species and that functions as a weak mitogen and promotes neurite-outgrowth activity in vitro. To further investigate the role PTN plays in regulating cell growth, we overexpressed the bovine PTN cDNA and now show that PTN phenotypically transforms NIH 3T3 cells, as evidenced by increased cell number at confluence, focus formation, anchorage-independent growth, and tumor formation in the nude mouse. The results demonstrate that the Ptn gene has the potential to regulate NIH 3T3 cell growth and suggest that PTN may influence abnormal cell growth in vivo.     

Self-normalization of highly transformed 3T3 cells through maximized contact interaction.

Nontransformed and moderately and highly transformed BALB/c 3T3 cells maintained on small coverslips in a large volume of medium multiplied to 2, 3, and 4 times higher population density, respectively, than they did in conventional cultures. Deprivation of Mg2+ caused highly transformed cells on coverslips to assume the appearance of nontransformed cells, decrease their rate of multiplication, and stop further growth at a much lower saturation density than the same cells in physiological Mg2+. The latter cells reached a saturation density of 10(6)/cm2 and their rate of DNA synthesis decreased progressively with increased crowding. At saturation density, cells in physiological Mg2+ took on an appearance and arrangement similar to normal fibroblasts. They developed a high requirement for serum to initiate DNA synthesis. When transferred at low density, they flattened out on a plastic surface and maintained the appearance of nontransformed cells for approximately 1 day. Onset of DNA synthesis and multiplication in the transferred cells was delayed for periods characteristic of quiescent nontransformed cells stimulated by fresh medium or transfer. Cells from crowded coverslips were approximately 1/10th as efficient at colony formation when suspended in agar as cells from uncrowded coverslips. They also had a significantly lower Mg2+ content. The crowded cells returned to their transformed morphological and growth behavior 2 to 3 days after transfer at low density. We conclude that a very high degree of crowding causes highly transformed cells to revert to the phenotype of nontransformed cells. Other treatments such as deprivation of Mg2+ or inorganic orthophosphate can achieve similar results. It appears that a balanced reduction in rates of metabolism and multiplication can restore the normal phenotype to transformed cells, implying that they differ only quantitatively from nontransformed cells. The putative role of Mg2+ in the regulation of multiplication and in transformation of animal cells is discussed.     

Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells.

Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into psi 2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected psi 2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylalanine hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normally synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.     

Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Ha-ras-1 gene derived from the human T24 bladder carcinoma cell line and compared them with untransfected NIH/3T3 cells. Whereas subcutaneous implantation of NIH/3T3 cells in the supraclavicular region produced palpable tumors that failed to metastasize, NIH/3T3 cells inoculated in the footpad gave rise to malignant tumors that metastasized to the lung. Under identical conditions and irrespective of the site of implantation, A51 cells formed rapidly growing primary tumors that produced pulmonary metastases. In an assay for experimental metastasis, intravenously injected NIH/3T3 cells gave rise to pulmonary nodules only at high cell inocula and in long-term survivors (90 days after injection). In contrast, A51 cells formed multiple lung tumor colonies detectable 14 days after injection. These results indicate that "normal" untransfected NIH/3T3 cultures contain subpopulations of cells that express malignant properties and that transfection of NIH/3T3 cells with activated c-Ha-ras-1 accelerates formation of metastases.     

Transfection of BLV containing DNA into NIH 3T3 cells

Cell DNA isolated from bovine leukosis virus (BLV) productive cell clones was transfected into the NIH3T3 cells. DNA from some cell clones was able to transform NIH3T3 cells. The transformed cells were cloned, and in 4 cell clones out of 33 bovine leukosis virus specific sequences were detected by hybridization with labeled BLV probe. According to the restriction analysis the BLV sequences were incomplete, they were rearranged, deleted, or both. The DNA from NIH3T3 transformants with BLV sequences was able to transform in the second round transfection experiments NIH3T3 cells again, but in these transformants BLV specific sequences were not detected. Cell DNA from sheep tumors induced by BLV was able to transform the NIH3T3 cells too, but BLV specific sequences were not present in the transformants. It appears that BLV specific sequences are not required for NIH3T3 cell transformation.     

S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C.

Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.     

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.     

Overexpression of the c-src protein does not induce transformation of NIH 3T3 cells.

NIH 3T3 mouse cells were transfected with plasmids that induce efficient expression of either (i) the Rous sarcoma virus v-src gene, (ii) the chicken c-src gene, or (iii) a recombinant gene combining the 5' portion of c-src with the 3' end of v-src. Focus formation in tissue culture and formation of large colonies in soft agar did not occur in cells transfected with c-src. Cells transfected with c-src expression plasmids did not form foci but were isolated using a coselectable biological marker. They display morphological and substrate-independent growth characteristics intermediate between those of normal and v-src-transformed mouse cells, and lysates from these cells have enhanced in vitro tyrosine kinase activity. Transfection with the c-src-v-src recombinant induced focus formation with an efficiency similar to that obtained with a v-src expression plasmid. These results imply that v-src-induced transformation does not result just from overexpression of an essentially normal cellular protein but, at least in part, depends on the mutations distinguishing the cellular and viral proteins.     

Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells.

Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.     

Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.     

Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts.

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.