Density-dependent Inhibition Of Cell Growth Research Articles

The Extracellular Domain of p185c-neuInduces Density-Dependent Inhibition of Cell Growth in Malignant Mesothelioma Cells and Reduces Growth of MesotheliomaIn Vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.

Evaluating function of transmembrane protein tyrosine phosphatase CD148 in lymphocyte biology.

The transmembrane protein tyrosine phosphatase CD148 is expressed on numerous cell types, including most cells of the hematopoietic lineage. CD148 has been shown to regulate density-dependent inhibition of cell growth as well as cellular differentiation in nonhematopoietic cells and has been shown to regulate signal transduction processes in several nonlymphoid hematopoietic cell types. Analysis of CD148 expression on lymphoid cells has demonstrated that CD148 is expressed at low levels on T cells and that it is upregulated in response to activation. Several groups have observed that CD148 negatively regulates T cell activation in response to crosslinking of the T cell antigen receptor, suggesting that it may play a role in feedback inhibition of the T cell immune response. In the B cell compartment, CD 148 expression appears to be restricted to the memory subpopulation, raising the possibility that it serves a unique function in these cells, which has yet to be determined. Recent studies have shown that CD148 interacts with the PDZ domain-containing protein syntenin, raising the possibility that its function or its localization with substrates in T and B cells may be controlled through this or a related interaction with another PDZ domain protein.

Reduced by Contact

Reactive oxygen species (ROS) and the redox state of a cell have been implicated in several signal transduction cascades including those that regulate cell proliferation. Specifically, signaling by several growth factors, including epidermal growth factor (EGF), produces a transient increase in ROS. One effect of increases in ROS is the inactivation of tyrosine phosphatases. Pani et al. found that the redox status of cells in culture was different in sparse and dense cultures (sparse cultures have more ROS than dense cultures) and that this difference correlated with whether the cells exhibited contact inhibition. Dense cultures had lower levels of the active [guanosine triphosphate (GTP)-bound] form of the small GTPase Rac-1. Treatment of sparse cultures with drugs to inhibit leukotriene biosynthesis decreased the levels of ROS to those measured in dense cultures, suggesting that Rac-1 may stimulate leukotriene biosynthesis as a mechanism to increase ROS in sparsely cultured cells. The authors also used several other drugs to regulate tyrosine phosphatase activity (sodium orthovanadate) and redox status (dithiothreitol) of the cells; however, none of the agents used were very specific. With that caveat, the results were consistent with the hypothesis that dense cells have lower levels of ROS, which allow increased activity of tyrosine phosphatases, thereby attenuating the ability of growth factors to stimulate proliferation. Thus, the regulation of the levels of ROS through regulation of the activity of proteins such as Rac-1 may be one mechanism by which the inhibition of cell proliferation in response to cell-cell contact occurs. Pani, G., Colavitti, R., Bedogni, B., Anzevino, R., Borrello, S., and Galeotti, T. (2000) A redox signaling mechanism for density-dependent inhibition of cell growth. J. Biol. Chem. 275 : 38891-38899. [Abstract] [Full Text]

Surface area and anchorage-dependent growth of Chinese hamster ovary cells.

Cell growth can be affected by many factors. For study of the cell responses under various environmental changes, the traditional method using batch cultures may not provide precise answers. It sometimes even leads to misapprehension. A flow system was therefore designed to eliminate the variations in chemical environment during the cell growth. An anchorage Chinese hamster ovary (CHO) cell line was chosen for study under this system. With the help of time-lapse video recording and image analysis, it was found that the exposed surface area (Ae) of Chinese hamster ovary (CHO) cells was significantly affected by the serum concentration (S) in the medium. Cells appeared to be larger when cultivated in medium of low serum content. Comparing the growth rates of cells on different substrata, we have found that, although they were different at a given serum concentration, the growth curves could be correlated to a Monod type equation by replacing the substrate term with AeS. Furthermore, when we studied the effect of density-dependent inhibition of cell growth, we found that there exists a minimum value of AeS required for stimulating cell proliferation. Once AeS falls below this value, cell proliferation stops. These observations seemed to indicate that the interaction between serum factors and cell surface, i.e., AeS, is an important parameter of the growth of CHO cells.

Detection of a phosphatidylinositol-specific phospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth.

The metabolism and intracellular distribution of a fluorescent analog of phosphatidylinositol (PI), 1,2-[oleoyl,N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl) aminocaproyl)]-PI (C6-NBD-PI), was examined in monolayer cultures of Swiss 3T3 cells following its insertion into the plasma membrane. Evidence is presented that the exogenously supplied C6-NBD-PI was hydrolyzed by a calcium-dependent PI-specific phospholipase C (PI-PLC) at the external cell surface and that this PI-specific phospholipase C may play a role in the density-dependent inhibition of cell growth: (i) When confluent monolayer cultures were incubated with C6-NBD-PI for 60 min at 7 degrees C, the lipid spontaneously transferred to the cells, and prominent labeling of intracellular membranes was observed. Lipid extraction and analysis demonstrated that more than 60% of the fluorescent lipid in these cells was fluorescent diacylglycerol (DAG). However, when the corresponding fluorescent analogs of phosphatidylcholine or phosphatidylethanolamine were used, the fluorescent lipids readily transferred to cells, but no hydrolysis to fluorescent DAG occurred. (ii) Both intracellular labeling and hydrolysis of C6-NBD-PI to -DAG were inhibited in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. (iii) When myo-[2-3H]inositol-labeled C6-NBD-PI was incubated with cells, [3H]inositol phosphate was released into the incubation medium, but no water-soluble 3H-labeled products were found associated with the cells. (iv) The level of C6-NBD-PI hydrolysis increased dramatically with increasing density of 3T3 cells in monolayer culture.

Density-dependent inhibition of cell growth by transforming growth factor-beta 1 in normal human fibroblasts.

We report here that transforming growth factor-beta 1 (TGF-beta 1) inhibits platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human fibroblasts in a cell density-dependent manner; no inhibition was seen in sparse cultures, approximately 50% inhibition in confluent cell cultures, and an almost total inhibition in dense cultures. The PDGF-inducible genes c-myc and c-fos were induced also by TGF-beta 1. Simultaneous addition of TGF-beta 1 and PDGF resulted in sustained, rather than transient, expression of c-fos mRNA; c-fos mRNA was detected as long as 24 hr after addition of PDGF and TFG-beta 1. TGF-beta 1 also induced mRNA for the A chain, but not the B chain, of PDGF. Conversely, PDGF induced TGF-beta 1 mRNA in sparse but not in dense cultures. These data indicate the existence of a complex interdependent regulation of PDGF and TGF-beta mRNA expression which is influenced by the cell density.

Specific inhibition of endothelial cell proliferation by isolated endothelial plasma membranes

Cultures of human vascular endothelial cells were used to study the phenomenon of density-dependent inhibition of cell growth. Endothelial cells were disrupted by nitrogen cavitation, and a plasma membrane-enriched fraction was prepared by differential centrifugation followed in some cases by sucrose density gradient fractionation. Membrane suspension was added to low-density early-passage endothelial cultures grown in microwells. Hemocytometer cell counts and 6 hr 3H-thymidine pulses were performed in triplicate wells at varying intervals. Plasma membranes suppressed cell proliferation in a reversible, dose-dependent fashion. Increasing the ambient concentration of endothelial cell growth factor did not alter the inhibitory effect. The antiproliferative effect was sensitive to heat and trypsin and to incubation with 0.1 M sodium carbonate, pH 11.5. Membrane vesicles selectively derived from the apical cell surface also suppressed proliferation. This phenomenon showed at least some specificity for cell type and species in both human and bovine models. Therefore, cell-cell contact is capable of regulating endothelial cell proliferation in vitro despite the presence of available growth surfaces and of optimally supportive culture medium.

Optimization of environment for high density Vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites.

This study was initiated for optimization of the environment of a technologically useful mammalian cell line for high density production. Cultures of Vero cells on microcarriers were perfused with 100%, 50%, 25% and 12.5% modified L15 media (galactose was replaced with 10 mM-fructose, with 4 mM-glutamine and 5% foetal bovine serum) in phosphate-buffered saline at either 4 or 8 vol. day-1. Cell growth, pH, dissolved oxygen, and changes in the metabolites, lactate to pyruvate and lactate to ammonia indices, demonstrated that under the conditions used in the present study, perfusion of cultures with 50% L15 medium in PBS at 8 vol. day-1 provided the optimum microenvironment for Vero cell growth. The highest cell density in the perfused cultures was 3 X 10(7) cells ml-1, which at these conditions was ten times higher than the maximum cell density (3 X 10(6) cells ml-1) obtained in a batch culture. Nutrient supply and conditioning factors were the most probable growth-limiting factors in cultures that were perfused with 12.5% and 25% L15 media, while multilayering, limitation of available oxygen, and accumulation of metabolic end products in the cellular microenvironment were the most probable causes of a density-dependent inhibition of cell growth observed under the optimized and overfed (supply of 100% L15 medium at the rate of 8 vol. day-1) culture conditions. Under the optimized environmental condition, the major source of energy was probably glutamine during the first week. However, significant utilization of fructose became evident at higher cell densities during the second week, when lactate production dramatically declined and reached an almost undetectable level, while respiration progressively assumed the predominant role in energy production. It is postulated that 'available' oxygen in the multicell-layered microenvironment of the optimized cultures was higher than in the overfed culture due to the greater utilization rate of oxygen for oxidation of excess nutrients in the overfed culture.

Inhibition by quercetin of the release of density dependent-inhibition of cell growth in RSV-transformed chicken cells

The expression of src gene in dense cultures of chick embryo fibroblasts (CEF) infected by a thermosensitive mutant (NY68) of RSV released densitydependent inhibition of growth and induced in these cells a large increase in DNA, RNA and protein synthesis. This stimulation of cellular metabolism was abolished in the presence of quercetin. Furthermore, quercetin added to the culture medium also inhibited the stimulation of pp60src kinase due to the expression of transformation.

Inability of Serum to Completely Reverse Density-Dependent Inhibition of Cell Growth in Culture2

Secondary cultures of mouse embryo cells showed density-dependent inhibition of growth when the medium contained 10% serum. After change of medium or addition of fresh serum, denslty-lnhlblted cells started synthesizing DNA and dividing. Synthesis of DNA began after a lag of about 12 hours, reached a maximum between 18 and 24 hours, and then declined gradually. The capacity of fresh serum to stimulate growth depended on the nutritional status of the medium to which it was added. Increase in the amount of serum in the culture medium increased the rate of DNA synthesis. However, DNA synth esis declined drastically as the medium became depleted, though it contained higher concentrations of serum. This indicated that nutrients and regulating micromolecules in the chemically defined medium are important in controlling the growth stimulated by serum and that serum does not have a nutritional function in monolayer cell culture.

Effect of adenosine 3'-5'-cyclic monophosphate on cell proliferation.

Secondary cultures of human diploid fibroblasts, which demonstrate density-dependent inhibition of cell growth, were used to study the effect of adenosine 3'-5'-cyclic monophosphate (cAMP) on cell proliferation. DNA synthesis in nonconfluent cultures and in contact-inhibited cultures stimulated to grow by refeeding with fresh medium was found to be inhibited by exogenous cAMP. The properties of this inhibition of DNA synthesis, together with the alterations in cAMP metabolism observed in confluent cultures of cells stimulated with fresh medium to resume growth, strongly suggest that cAMP is involved in contact-inhibition of cell proliferation.