Insulin receptors in normal and transformed fibroblasts: relationship to growth and transformation

The insulin receptors in normal and transformed lines of mouse Balb/3T3 fibroblasts have been studied. In the normal fibroblasts, the binding of insulin was low in growing cells and increased 2–9 fold in confluent stationary cells. Insulin binding was increased whether growth arrest was due to contact inhibition of growth or serum starvation. When serum-starved cells were stimulated to grow by the addition of fresh serum, insulin binding declined. In cells transformed by simian virus 40, Kirsten, Moloney, and Harvey sarcoma viruses, methylcholanthrene, X rays, or spontaneously, the binding was low, in the same range as growing normal cells. In simian virus 40-transformed cells, insulin binding increased 4 fold as the cells reached higher densities in culture. No relationship to changes in cell size was found. The differences in binding were due to changes in the concentration of the receptors, without changes in their affinity for the hormone.     

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.     

2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines.

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2-amino-isobutyric acid and 3-O-methyl-D-glucose were measured in sparse, confluent, serum-starved, and serum-stimulated cultures. As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101. The density revertant F1SV101, which has regained density regulation of growth similar to 3T3 cells, has also regained density regulation of transport. Neither growth nor transport were serum dependent. The serum revertants AgammaSV7 and LsSV6 have regained both density and serum regulation of growth, but not according to the original mechanism of 3T3 cells of entry into a Go state. Transport was high under conditions of confluence or serum deprivation. Thus for these cells rates of transport were not reduced simply as a consequences of slower cell growth nor were low transport rates responsible for growth arrest. The data are consistent with the possibility that growth arrest specifically in the G0 state could shut off a number of cellular activities, including transport.     

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.     

Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate.     

Cyclic nucleotides and cell growth

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G₀ by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction. Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G₀ arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2-5-fold in resting as compared to growing cultures, and increased 10-20-fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum-activated cultures of transformed cells. Measurement of guanylcyclase under unphysiological conditions (2 mM Mn⁺⁺) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G₁ phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP-pleiotypic response mechanism at the restriction point of their cell cycle.     

Alpha-lipoic acid induces p27Kip-dependent cell cycle arrest in non-transformed cell lines and apoptosis in tumor cell lines

alpha-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regulating metabolism. We report here that alpha-lipoic acid induces hyperacetylation of histones in vivo and has differential effects on the growth and viability of normal versus transformed cell lines. The human tumor cell lines FaDu and Jurkat, as well as a Ki-v-Ras-transformed Balb/c-3T3 murine mesenchymal cell line, all initiated apoptosis following exposure to alpha-lipoic acid. In contrast, treatment of non-transformed cell lines with alpha-lipoic acid resulted only in reversible cell cycle arrest in G0/G1. Treatment with butyrate, another short-chain fatty acid, induced a G0/G1 arrest in both transformed and non-transformed cell lines. alpha-Lipoic acid caused a post-translational elevation in the levels of the cyclin-dependent kinase inhibitor p27Kip1. Studies using p27Kip1-deficient MEF cells demonstrated that p27Kip1 was required for the alpha-lipoic acid-mediated cell cycle arrest. The mechanism of apoptosis was independent of Fas-mediated signaling, as alpha-lipoic acid-treated Jurkat cell mutants deficient in Fas or FADD retained sensitivity to apoptosis. The differential selectivity of the pro-apoptotic effects of alpha-lipoic acid for transformed cells supports its potential use in the treatment of neoplastic disorders.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Density dependent inhibition of both growth and T-antigen expression in revertants isolated from simian virus 40-transformed mouse SVT2 cells.

Phenotypic revertants were isolated from simian virus 40-transformed cells in order to examine the relationship between simian virus 40 T-antigen expression and G1 arrest of growth. Revertant clones with increased adherence were selected from cultures of SVT2, a simian virus 40-transformed BALB/c mouse cell line, and screened to find arrestable revertant clones which inhibited DNA synthesis when crowded. The clones selected from untreated SVT2 were unstable and showed little or no inhibition of DNA synthesis when crowded. Stable revertants were found after treatment of SVT2 with Colcemid to increase ploidy. The stable revertants all lost most transformed growth properties tested, including tumorigenicity, but only a few showed the same degree of inhibition of DNA synthesis at high cell density as BALB/3T3. All revertant clones expressed T antigen at low cell density. Three revertants showed coordinate inhibition of DNA synthesis and apparent loss of T antigen at high cell density. We suggest that changes in gene dosage rather than mutations caused the altered properties of the new revertants and that continued DNA synthesis in confluent cultures may be the transformed phenotype that requires the least simian virus 40 T antigen.     

Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation.

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.     

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.     

Elevated level of gas3 gene expression is correlated with G0 growth arrest in human fibroblasts

The gas3/PMP22 gene product is a dual function protein, involved in both peripheral nerve myelination and cell proliferation. gas 3/PMP22 is highly expressed in myelinating Schwann cells and is required for normal PNS development. In addition, a more general function for gas3 is suggested by its expression in non-neural tissues and upregulation by growth arrest in cultured rodent fibroblasts. In the present work, the expression of the gas3 gene has been studied in human fibroblasts. We have confirmed that gas3 mRNA is upregulated when cells are serum starved or grown to high cell density (G0 arrest). When quiescent cells were stimulated by serum or platelet-derived growth factor-BB (PDGF-BB), gas3 mRNA was down regulated. In contrast, we found that the expression of gas3 mRNA was neither upregulated in senescent cells nor in cells arrested in G1 using Lovastatin. Thus, high expression of gas3 is not related to growth inhibition in general, but more probably to the G0 growth arrest state. Furthermore, we found that in two malignant fibrous histiocytoma cell lines, gas3 expression was lower than in normal fibroblasts, suggesting an altered regulation of the gas3 gene in transformed cells.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Cell cycle and cyclic AMP-dependent phosphorylation of plasma membrane proteins p14 and p24: defects in smooth surface transformed cells

Two proteins which are localized to the cytoplasmic surface of the plasma membrane, p14 and p24, undergo cyclic AMP-dependent phosphorylation in rapidly growing nontransformed murine embryo cells. In this cell system, growth arrest in the G1 phase of the cell cycle induced by growth factor deprivation is associated with the reversible loss in ability to phosphorylate these substrates. By contrast, Simian virus 40 and methylcholanthrene transformed cells show both defective G1 growth control and defects in their ability to phosphorylate p14 and p24 under all tested growth conditions. These data suggested a correlation between defects in the physophorylation of p14 and p24 and defects in the ability of transformed cells to G1 growth arrest. The results of the current studies by contrast show that 3T3 T proadipocytes which have been transformed by the smooth surface tumorigenesis method show different characteristics. They retain the ability to G1 growth arrest in serum-deficient medium. They show cyclic AMP-dependent phosphorylation of p14 and p24 during exponential growth. They do not, however, down regulate p14 and p24 phosphorylation in association with G1 growth arrest. These observations suggest that neoplastic transformation is not necessarily associated with absolute defects in the ability to phosphorylate p14 and p24. Rather, the results of the current study suggest that the inability to modulate the cyclic AMP-dependent phosphorylation of plasma membrane p14 and p24 proteins during the G1 phase of the cell cycle may be more tightly associated with neoplastic transformation.     

Lack of evidence of chalone activity in used medium and extract of JB-1 tumor cells in vitro. A flow cytometry study.

In cell-free mouse ascites fluid from the JB-1 ascites tumor in the plateau phase of growth low-molecular chalone substances have been found which reversibly and specifically arrest JB-1 cells in the G1 and G2 phase of the cell cycle. The aim of this study was to investigate whether chalones were involved in the regulation of in vitro growth of JB-1 tumor cells. Used medium and cell extract from confluent, stationary JB-1 cell cultures were investigated for proliferation-inhibitory properties. JB-1 cells from stationary cultures were explanted in test cultures and the traverse of cells through the S phase was investigated by means of flow cytometry (FCM). Inhibition--expressed as a delay of the traverse of cells through the S phase--was not observed when a surplus of used medium, concentrated and fractionated used medium or concentrated and fractionated cell extract from JB-1 cells in vitro was added to test cultures. On the contrary, used medium and concentrated and fractionated used medium stimulated growth. Thus, no involvement of chalones in the growth regulation of JB-1 tumor cells in vitro was detected.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Human chromosome 3 mediates growth arrest and suppression of apoptosis in microcell hybrids.

Chemotherapeutic treatment of tumor cells leads either to tumor cell death (usually by apoptosis) or to the formation of drug-resistant subpopulations. Known mechanisms of cancer cell drug resistance include gene amplification and increased expression of drug transporters. On the other hand, normal cells survive many forms of chemotherapy with minimal damage probably because of their capacity for growth arrest and stringent control of apoptosis. Microcell hybrids between B78 (murine melanoma) and HSF5 (normal human fibroblasts) were analyzed to identify a new human chromosomal region involved in the promotion of drug-induced growth arrest and suppression of apoptosis. In these hybrids, the presence of human chromosome 3 was strongly associated with suppression of apoptosis via G1 and G2 growth arrest during exposure to the antimetabolite N-phosphonoacetyl-L-aspartate (PALA), suggesting that a gene(s) on chromosome 3 serves an antiproliferative role in a drug-responsive growth arrest pathway.     

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.     

p21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-beta-mediated G1 arrest through modulation of IL-2 responsiveness

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8(+) T cells from DKO mice, TGF-beta is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15(Ink4b) in T cells stimulated in the presence of TGF-beta is not essential, as TGF-beta also efficiently suppressed proliferation of T cells from p15(Ink4b-/-) mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-beta, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3(-/-) T cells to the induction of growth arrest by TGF-beta. In summary, the growth suppressive effects of TGF-beta in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-beta by lowering thresholds for a maximal mitogenic response.