Anchorage-independent Cells Research Articles

Shape-dependent regulation of cytoskeletal protein synthesis in anchorage-dependent and anchorage-independent cells.

We examine changes in protein synthesis that accompany suspension (i.e. shape alteration) of anchorage-dependent and anchorage-independent cells using a newly developed cell fractionation procedure based on detergent extraction. Using this procedure, a cell can be divided into four distinct and independent fractions: soluble, cytoskeleton, chromatin and nuclear matrix-intermediate filament. This fractionation procedure is used to investigate protein synthetic events associated with the release from anchorage-dependent growth, characteristic of transformed cells. Suspension results in several unexpected events in both anchorage-dependent (3T3) cells and anchorage-independent (SVPy 3T3) cells. Suspension of 3T3 cells results in a reduction of total protein synthesis; however, two proteins are enhanced in their amount of synthesis. Suspension of SVPy 3T3 results in about 20 proteins proceeding through short and long-term alterations in the rate of synthesis. The synthesis of some of these proteins is inhibited, others undergo a transient decrease in synthesis upon suspension and then increase above their rate in the anchored state, while the synthesis of others steadily increases after suspension. Suspension of anchorage-dependent cells results in a fraction specific shift in the rates of protein synthesis. Possible roles for these fraction-specific proteins are considered.

Differential regulation of polyamine synthesis and transmethylation reactions in methylthioadenosine phosphorylase deficient mammalian cells

The consumption of S-adenosylmethionine during polyamine synthesis and transmethylation reactions yields stoichiometric amounts of 5′-deoxy-5′-methylthioadenosine and S-adenosylhomocysteine, respectively. Information concerning the regulation of the two routes of S-adenosylmethionine metabolism in viable cells under changing growth conditions is limited. The present experiments have measured the time-dependent accumulation of 5′-deoxy-5′-methylthioadenosine and l-homocysteine in the medium of four malignant human and murine cell lines deficient in 5′-deoxy-5′-methylthioadenosine phosphorylase (5′-methylthioadenosine: orthophosphate methylthioribosyltransferase). Included in this group were anchorage-independent and anchorage-dependent cells. The enzyme-deficient cells did not detectably cleave 5′-deoxy-5′-methylthioadenosine, and did not appreciably metabolize homocysteine. A comparison of 5′-deoxy-5′-methylthioadenosine and homocysteine excretion therefore provided a noninvasive method for estimating the relative rates of polyamine synthesis and transmethylation. Early after the release of human CEM lymphoblasts from density dependent growth arrest, 5′-deoxy-5′-methylthioadenosine production increased, and exceeded homocysteine synthesis. 5′-Deoxy-5′-methylthioadenosine formation reached a maximum of 0.9 nmol/12 h per 10 6 cells prior to the onset of exponential growth. The kinetics of homocysteine synthesis were different. Homocysteine accumulation was proportional to the specific growth rate, and achieved a peak of 3.1 nmol/12 h per 10 6 cells during mid-exponential phase, at which time 5′-deoxy-5′-methylthioadenosine production was falling. Similar patterns of 5′-deoxy-5′-methylthioadenosine and homocysteine excretion were observed in other 5′-deoxy-5′-methylthioadenosine phosphorylase deficient cell lines. These data show that polyamine synthesis and transmethylation are differentially regulated during the growth cycle of mammalian cells.

Tyrosyl kinases acquired from anchorage-independent cells by a membrane-enveloped virus.

Tyrosyl kinase activity in vesicular stomatitis virus (VSV) acquired from host cells that differ in morphology was investigated. VSV grown in baby hamster kidney (BHK) cells with rounded morphology and a high efficiency of colony formation in soft agar (Rous sarcoma virus [RSV]-transformed and suspension BHK cells) was compared with VSV grown in BHK cells with a flattened morphology and lower efficiency of colony formation in soft agar (RSV-infected revertant and control BHK cells). Tyrosyl kinase activity measured with the substrates angiotensin II peptide or casein was found at 7-10-fold higher levels in virus released from the anchorage-independent BHK cells. Most of the VSV-associated tyrosyl kinases acquired from the RSV-transformed BHK cells reacted with antiserum to pp60src, whereas the activity acquired from the suspension BHK cells was unaffected by anti-src serum. The overall levels of tyrosyl kinase in subcellular fractions of the host BHK cells were also measured. Like the VSV released from them, the RSV-transformed cell extracts contained high levels. The suspension cells, however, contained the same low levels of tyrosyl kinase as was found in the control BHK cell extracts. Therefore, tyrosyl kinase was concentrated and acquired by VSV from the anchorage-independent suspension BHK cells. VSV-associated protein kinases acquired from other cell types followed a similar pattern. Tyrosyl kinase levels were high in VSV released from suspension cultures (Chinese hamster ovary and HeLa) and from virally transformed cells (Kirsten murine sarcoma virus-transformed rat kidney cells) and low in VSV released from an anchorage-dependent primary cell culture (chick embryo fibroblasts).

The mechanism of interferon effect on cell transformation by murine sarcoma virus.

Mouse interferon (IFN) was found to inhibit murine sarcoma virus (MSV)-induced neoplastic transformation of normal rat kidney (NRK) cells. This effect was observed upon examining the formation of foci of morphologically altered cells and colonies of anchorage-independent cells. IFN had no cytotoxic effect on MSV-transformed NRK cells, nor on their focus or colony-forming ability. It was therefore apparent that its inhibitory effect was directed against the viral role in cell transformation. In attempts to define the mechanism of this effect, we found that IFN delayed the initiation of the cytoplasmic viral DNA synthesis. However, the amount of this DNA eventually formed in IFN-treated cells was the same as in the control cells. Furthermore, the transport of this DNA to the nucleus was slower in IFN-treated cells, although all of it was finally transferred. However, while most of the viral DNA integrated into the genome of the control cells, very little integration occurred in IFN-treated cells. The unintegrated viral DNA of these cells was slowly degraded. Therefore, if the cells recovered from the antiviral effect of IFN when intact viral DNA molecules still existed in their nucleus, they could resume viral DNA integration and cell transformation. IFN was found to block viral DNA supercoiling. Since supercoiled viral DNA is considered to be a precursor to integrated provirus, it seems that the inhibition of both integration and cell transformation is due to this impaired coiling.

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.

Growth control and cell spreading: differential response in preneoplastic and in metastatic cell variants.

Growth control and sensitivity to changes in cell shape were studied in anchorage-dependent mouse fibroblasts (diploid fibroblasts, 3T3 and 3T6), in DNa tumor-virus-transformed mouse fibroblasts (SVPy 3T3), in four B16 melanoma and five uv-2237 fibrosarcoma cell variants that exhibit distinct metastatic properties. Differential adhesive conditions were established by precoating the plastic plates with poly (2-hydroxyethylmethacrylate) that allowed an accurate and reproducible control of cell shape, from flat to spherical. Mouse fibroblasts that form a continuum between rigorously controlled cells to fully anchorage-independent cells, display a direct correlation between degree of growth control and sensitivity to changes in cell spreading. In contrast, there is no apparent direct correlation between sensitivity of growth control to changes in cellular configuration and the metastatic potential of tumor cells.

Characteristics of benzo[ a]pyrene and A-ring reduced 7,12-dimethyl benz[ a]anthracene induced neoplastic transformation of human cells in vitro

The polynuclear aromatic hydrocarbons (PAH) benzo[ a]pyrene (BP) and the A-ring reduced analogue of 7,12-dimethylbenz[ a]anthracene (DMBA), 1,2,3,4-tetrahydro-7,12-dimethylbenz[ a]anthracene (TH-DMBA) are carcinogenic to human cells. The unsaturated PAH, DMBA exhibits no carcinogenic activity on human cells as measured by growth in soft agar. The TH-DMBA and BP treated cells exhibit a colony frequency in soft agar of 84 and 86, respectively. These anchorage independent cells, when seeded on the chick embryonic skin (CES) organ cultures, are invasive and form a fibrosarcoma. It is highly unlikely that TH-DMBA, which does not contain an aromatic A-ring, can undergo metabolism in human cells in culture to form a bay region 3,4-dihydrodiol-1,2-epoxide. These results suggest that an alternate mechanism for the induction of carcinogenesis is appropriate to explain the absence of bay region diol-epoxide metabolite as the ultimate form of the carcinogen in TH-DMBA induced carcinogenesis in human diploid cells.

Carcinogen-induced anchorage-independent growth and in vivo lethality of human MRC-5 cells.

Chemical carcinogens or u.v. irradiation induce in vitro anchorage-independent growth and in vivo lethal multicellular infiltrative growth of human MRC-5 cells. A single carcinogen treatment 8 h after release of MRC-5 cells from metabolic block induced by a deficiency of arginine and glutamine in the medium is followed within 4--5 weeks by formation of colonies of greater than 50 cells in 0.35% semi-solid agar medium. Anchorage-independent MRC-5 cells, moreover, when injected intracranially into immunologically deficient homozygous nude mice, 6--7 weeks post-carcinogen exposure, produce progressive neurological dysfunction 5--6 weeks later accompanied by lethal multifocal multicellular infiltrating lesions. The present investigation demonstrates for the first time, carcinogen-induced anchorage-independent growth and in vivo lethality of a well characterized human diploid fibroblast cell strain and indicates the potential value of MRC-5 cell transformation as a new model for the study of carcinogenesis in human cells.

Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors.

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorage-independent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas).

Multisequential Transformation of a Pig Cell Line (PFT): Correlations Between Tumorigenicity and Chromosome and Ultrastructural Markers<xref ref-type="fn" rid="FN2">2</xref>

Cells from representative subcultures of a continuous cell line of pig uterine tube (PFT) and carrying none, one, or two chromosome markers were inoculated in athymic nude mice (RCN) to determine the occurrence of the malignant transformation in this line. No tumors developed in mice after implantation of cells from either the 16th and 65th subcultures (no chromosome markers) or the 106th subculture (one chromosome marker, anchorage-dependent). Three adenomas (11.1%) were found in 27 mice that had been inoculated with anchorage-independent cells having one chromosome marker. Of 27 mice inoculated with cells showing two chromosome markers and gap junctions, 23 mice (85.1%) developed undifferentiated carcinomas. Ultrastructural traits and karyotypes of tumors were generally similar to those of the PFT cell inocula. However, annulate lamellae were found in 8 of 13 tumors examined by electron microscopy but were not seen in PFT cell inocula. The occurrence of a multisequential transformation was indicated by serial examination of the first 400 subcultures of the line and by comparison of the markers observed in three successive populations of cells differing in their genetic constitution.

Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability.

Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell-surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.

Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro.

Clonal isolates of mouse 3T3 cells and primary rat embryo cells, recovered nonselectively after infection by simian virus 40 (SV40), have been tested for tumorigenicity in the immune-deficient nude mice in order to determine the cellular growth properties in vitro specifically correlated with neoplastic growth in vivo. In addition, mouse 3T3 cells transformed by murine sarcoma virus (MuSV, Kirsten strain), and revertants isolated from cells fully transformed by either SV40 or MuSV were also studied. Results suggest that the single cellular property consistently associated with tumorigenicity in nude mice is the acquisition by virus-transformed cells of the ability to proliferate in vitro in the absence of anchorage. Other cellular parameters of virus-induced transformation, such as lack of sensitivity to high cell density and the capacity to grow in low serum concentration, are dissociable from cellular tumorigeneicity. This conclusion is supported further by the demonstration that specific selection in vivo for tumorigenic cells from anchorage-dependent cells results in the isolation of anchorage-independent cells. Conversely, a single-step selection in vitro for anchorage-independent cells from nontumorigenic cells results in a simultaneous selection of highly tumorigenic subclones.