Effect of anchorage dependency on growth rate and monoclonal antibody production of hybridoma cells

Hybridoma cells (S3H5/2bA2) are found to grow either in suspension or as attached to the surface of cell culture T flask. Cell growth rates and monoclonal antibody (MAB) production rates of both suspended and attached cells were examined. Although the percentage of viable cells was higher for the attached cells, cells growing in suspension showed almost the same charateristics as cells attached to the flasks with respect to cell growth and MAB production rate. Cell attachment increased with increasing serum concentrations up to 5% and remained essentially constant at cell densities of about 2·10⁵/cm².No differences in cell growth rate and MAB production could be attributed to anchorage dependent growth.     

Rac, membrane heterogeneity, caveolin and regulation of growth by integrins

Anchorage dependence of growth blocks cell proliferation in inappropriate environments, thereby inhibiting cancer cell invasion and metastasis. Inhibition of growth regulatory pathways, including Rac, Erk and PtdIns 3-kinase in non-adherent cells mediates this effect. Here, we review recent work showing that integrin-mediated adhesion controls Rac binding to membranes. Rac binding sites can be found within cholesterol-enriched membrane domains, which are internalized when cells are deprived of adhesion. Endocytosis of these domains is mediated by caveolae and regulated by caveolin-1 phosphorylated on Tyr 14. This mechanism can account for the control of multiple pathways by integrins, thus providing an important mechanism for anchorage dependence of growth.     

Competitive nuclear export of cyclin D1 and Hic-5 regulates anchorage dependence of cell growth and survival

Anchorage dependence of cell growth and survival is a critical trait that distinguishes nontransformed cells from transformed cells. We demonstrate that anchorage dependence is determined by anchorage-dependent nuclear retention of cyclin D1, which is regulated by the focal adhesion protein, Hic-5, whose CRM1-dependent nuclear export counteracts that of cyclin D1. An adaptor protein, PINCH, interacts with cyclin D1 and Hic-5 and potentially serves as an interface for the competition between cyclin D1 and Hic-5 for CRM1. In nonadherent cells, the nuclear export of Hic-5, which is redox-sensitive, was interrupted due to elevated production of reactive oxygen species, and cyclin D1 was exported from the nucleus. When an Hic-5 mutant that was continuously exported in a reactive oxygen species-insensitive manner was introduced into the cells, cyclin D1 was retained in the nucleus under nonadherent conditions, and a significant population of cells escaped from growth arrest or apoptosis. Interestingly, activated ras achieved predominant cyclin D1 nuclear localization and thus, growth in nonadherent cells. We report a failsafe system for anchorage dependence of cell growth and survival.     

Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.

The mitogen-activated protein (MAP) kinase pathway is a critical regulator of cell growth, migration, and differentiation. Growth factor activation of MAP kinase in NIH 3T3 cells is strongly dependent upon integrin-mediated adhesion, an effect that contributes to the anchorage dependence of normal cell growth. We now show that expression of constructs that constitutively activate focal adhesion kinase (FAK) rescued the defect in serum activation of MAP kinase in suspended cells without directly activating MAP kinase. Dominant negative FAK blocked both the rescue of suspended cells by the activated construct and the serum activation of MAP kinase in adherent cells. MAP kinase in FAK(-/)- mouse embryo fibroblasts was adhesion-insensitive, and reexpression of FAK restored its adhesion dependence. MAP kinase activity in ras-transformed cells is still decreased in suspension, but expression of constructs that constitutively activate FAK enhanced their anchorage-independent growth without increasing adherent growth. V-src, which activates both Ras and FAK, induced MAP kinase activation that was insensitive to loss of adhesion, and that was blocked by a dominant negative FAK. These results demonstrate that FAK mediates the integrin requirement for serum activation of MAP kinase in normal cells, and that bypassing this mechanism contributes to anchorage-independent growth in transformed cells.     

Evidence for two distinct mechanisms of anchorage stimulation in freshly explanted and 3T3 Swiss mouse fibroblasts

When single cells were allowed to attach to circular islands of adhesive substratum, their proliferation was strongly dependent on island area over the range 500 micron2 to 5000 micron2. The number of freshly explanted whole mouse embryo fibroblasts that performed DNA synthesis corresponded closely with a simple geometrical measure of area of cell surface exposed to the medium: freely suspended cells were only slightly less stimulated than attached cells exposing an equal surface area; hemispherical cells were less stimulated than cells of any other shape. In contrast, 3T3 cells were stimulated sixfold by islands too small to allow any increase in area. These experiments show that anchorage can stimulate by two different mechanisms. They offer a general method of measuring substrate contact stimulation.     

Chemically and virally transformed cells able to grow without anchorage in serum-free medium: evidence for an autocrine growth factor

BA10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serum-free (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin. BA10-IR cells and FR3T3-Ki cells also release beta-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable beta-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors. Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980). The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

The surface growth and activity ofNitrobacter

The effect of surface attachment on oxidation of nitrite to nitrate byNitrobacter was studied in batch culture, on glass coverslips, and in continuous culture on glass beads and anion exchange resin beads in an air-lift column fermenter. In batch culture, the surfaces stimulated specific growth rate, while in continuous culture, activity of attached cells was less than that of freely suspended cells. Nitrate productivity, free cell productivity, and attached cell concentration increased exponentially at the same specific rate, termed the colonization rate, and nitrate productivity was found to be a convenient estimate of biomass concentration. Permanent attachment was mediated by production of slime material. Surface growth resulted in multiple steady states and the ability to respond quickly to changes in dilution rate. The air-lift column fermenter system provided a convenient system for the study of growth and activity of attached cells and was most suitable when using ion exchange resins as a substratum for attachment.     

Emergence of hamster fibroblast tumors in nude mice—evidence for in vivo selection leading to loss of growth factor requirement

The Chinese hamster lung fibroblast cell line (CC139) has high anchorage dependence for growth and has retained the high serum dependence of secondary cultures of adult fibroblasts. This cell line is tumorigenic in nude mice; however, the resulting tumor cells have different properties than those of the cell line injected. The tumor-derived cells had strongly reduced or even lost both the high anchorage and the high serum dependence of CC139 cells. This finding suggests that an in vivo selection is necessary for CC139 cells to acquire the malignant phenotype. After mutagenesis, which increases the frequency of CC139 colony formation in agarose up to 8-fold, we selected and analyzed 15 anchorage-independent colonies. No correlation between the colony-forming ability in agarose and serum-growth factor requirement for DNA synthesis was observed. Each of these clones were injected into nude mice and the growth factor dependence of the ensuing tumor cells was compared to that of corresponding injected cells. All of the anchorage-independent colonies with the exception of one (A71), had acquired in vivo a stable phenotype allowing for partial or total escape of growth factor requirement. A71, the only clone which maintained the same growth factor requirement after two passages in vivo (A71 T1 and A71 T2) had already gained, in vitro, the minimal growth factor “relaxation” compatible with in vivo growth. A71 and A71 T1 tumor cells arrested in G₀/G₁ can reinitiate DNA synthesis in the presence of mouse plasma, low concentrations of serum, or thrombin. The fact that none of the tumors analyzed (more than 20) were found to have retained the high serum dependence of CC139 cells strongly suggests that the partial loss of serum growth factor requirement acquired in vivo is an essential malignant character for bypassing the hormonal growth restraints imposed by the host upon CC139 cells.     

The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abl's effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia.     

Regulation of mammalian cell growth by autocrine growth factors: analysis of consequences for inoculum cell density effects

Effects of inoculum cell density on mammalian cell growth in culture have been observed in a variety of experimental systems. Although these effects have been attributed generally to medium conditioning by the cells, there has previously been no quantitative theory proposed for this phenomenon based on developments in molecular and cell biology. In this article, we offer such a theory founded on the regulatory action of autocrine growth factors. A particularly relevant example of these is platelet- derived growth factor (PDGF), which is produced by fibroblastic cells in response to stimulation by transforming growth factor beta (TGFbeta), a common serum constituent, and provides a mitogenic signal for the same cells. A simple mathematical model for the production, diffusive transport, and binding of autocrine growth factors to cell surface receptors, coupled to a model for the dependence of cell proliferation on growth factor receptor binding allows prediction of initial cell population growth rate as a function of inoculum cell density. We focus on situations involving anchorage-dependent cell growth, in which the cells are attached to a surface. A number of clear results are obtained, most notably the following: 1) for cells cultured on spherical microcarrier bead surfaces, the inoculum cell density needed to produce a given growth rate is linearly proportional to the bead radius; and 2) all other factors being equal, the inoculum cell density on a unit surface area basis needed to produce a given growth rate is greater for spherical microcarrier surfaces than for flat culture dish surfaces. These two results are consistent with the experimental observations of Hu and coworkers(1,2) for fibroblast growth in minimal medium plus serum. The model also allows elucidation of the influence of other system parameters, both biological and physical, on initial cell proliferation rate and the inoculum cell density dependence.     

Reduced root anchorage of freshwater plants in sandy sediments enriched with fine organic matter

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The anchorage mechanics of deep rooted larch, Larix europea L. japonica

The anchorage of deep rooted 16-year-old larch trees, Larixeuropea japonica, has been studied by combining winching testswith analyses of strain around the base of the trunk and rootsystem and mechanical tests on individual roots. These showedthat anchorage is provided by the laterals which emerge fromaround the stem base, sinker roots which emerge along theirlength, and tap roots positioned directly underneath the bole.During anchorage failure the leeward laterals are bent and eventuallybreak close to their base, whilst the windward laterals arepulled out of the ground, with their sinker roots intact. Afterinitially being confined by the soil and bending, the tap rootrotates in the soil. Anchorage failure is similar when the soilis dry as when it is wet, but failure occurs closer to the trunk.Strain measurements along the lateral roots revealed that thestresses were highest close to the trunk and that these regionsof the roots contribute most to tree stability. The two major components of anchorage were found to be the resistanceof leeward laterals to bending and the resistance of tap rootsand windward sinkers to uprooting. Bending tests on leewardlaterals revealed that they provide around 25% of tree anchorage.Almost 75% of the anchorage strength must, therefore, be providedby the windward sinkers and tap roots. Anchorage strength ofroots was positively correlated to their cross-sectional area.The vertical orientation of the sinkers makes the anchoragesystem of larch more efficient than the plate system formedby Sitka spruce on waterlogged soils and means that no root-soilplate is formed. Key words: Anchorage, root architecture, sinker roots, root bending strength, windthrow     

A comparison of the growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis in the presence of suspended and attached Escherichia coli K12

The growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis were investigated in the presence and absence of Escherichia coli on an agar surface or within shaken suspensions. The amoebae perceived all the suspended systems to be unfavourable for growth, despite being challenged with high levels of prey, and as a consequence they exhibited a starvation response. However, the response differed between species, with A. castellanii producing characteristic cysts and H. vermiformis producing round bodies. These amoebic forms were reactivated into feeding trophozoites in the presence of bacterial aggregates, which formed in the suspended systems after 68 h of incubation. In contrast, both species of amoebae grew well in the presence of attached E. coli at a concentration of 1 x 10(6) cells cm(-2) of agar and yielded specific growth rates of c. 0.04 h(-1). Starvation responses were induced at the end of the growth phase, and these were equivalent to those recorded in the suspended systems. We conclude that, when suspended, amoebae in the 'floating form' cannot feed effectively on suspended prey, and hence the starvation response is initiated. Thus the majority of amoebic feeding is via trophozoite grazing of attached bacterial prey.     

Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes

Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4-8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine.     

The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells.

This study is concerned with the use of freshly harvested bovine endothelial cells attached to microcarrier beads in the production of the endothelium-derived relaxing factor (EDRF). The results are compared to production of EDRF by endothelial cells grown in tissue cultures. We found that freshly harvested cells attach themselves to microcarrier beads within minutes. This results in large surface/area volume ratio and permits superfusion of cells suspension on a filter (pore size of 25-30 microns), resulting in cell free filtrate. When superfusing an endothelium-deprived pulmonary artery strip, the effluent causes relaxation; the response depends on the number of superfused endothelial cells. The number of viable freshly harvested cells attached to microcarrier beads in 5 ml Krebs-Henseleit solution is small (30%), as compared to almost 100% for cultured cells. Despite this difference, percent relaxation induced for the same number of viable cells is identical for both groups. Scanning electromicrographs confirm anchorage of endothelial cells to microcarrier beads. While cultured cells cover the entire surface and are individually attached, freshly harvested cells are anchored as cell aggregates leaving some of the surface free. Attachment of freshly harvested endothelial cells to microcarrier beads offers an alternative for the study of the role of endothelial cells in the production of vasoactive substances.     

Anchorage dependence and Ca2+ requirement are independently modulated by hydrocortisone hormone in rat C6 glioma cells

We had previously shown that hydrocortisone (Hy), a glucocorticoid hormone, regulates the expression of the transformed phenotype of rat C6 glioma cells. The hormone reversibly renders C6 cells dependent on anchorage and high Ca2+ concentration for growth. We had also isolated Hy-resistant C6 variants in agarose suspension cultures. Here we report that Hy-resistant variants selected in high (1.8mM) Ca2+ medium become growth-arrested in low (30 microM) Ca2+ medium upon hormone treatment. We conclude that Hy-induced anchorage dependence and Hy-induced high Ca2+ requirement for growth of C6 glioma cells, are two independent phenotypes.     

Growth behaviors of bacteria in biofouling cake layer in a dead-end microfiltration system

The growth behaviors of three bacterial species, i.e. Escherichia coli, Pseudomonas putida and Aquabaculum hongkongensis, in biofouling cake layer (attached form) were investigated using an unstirred dead-end continuous microfiltration system, and were compared with those in suspended form. Results showed that all the three bacteria had larger average growth rates in suspended form than in attached form under high substrates levels. Under oligotrophic conditions, the average growth rates in the attached form were faster than those in the suspended form, especially for A. hongkongensis. The growth behaviors analysis presented the same results due to all the tested bacteria had higher maximum growth rate and saturation constant in suspended form than attached form, indicating the dominant growth mode would be shifted from attached form to suspended form with substrate concentration increase. Finally, total filtration resistance determined in the experiments increased significantly with the bacterial growth in filtration system.     

Identification of negative regulator of interleukin-3 (NIL-3) in bone marrow

We have reported that an inhibitor of interleukin-3 (NIL-3) is produced from murine bone marrow cells in response to excess stimulation of interleukin-3. In this report, we attempted the purification of the NIL-3 activity from bone marrow culture supernatant in the presence of interleukin-3. The purified NIL-3 activity was a protein with relative molecular weight of 54.5 kDa (SDS-PAGE), which inhibited the growth of IL-3 dependent DA-1 cell growth in a dose dependent manner. The N-terminal amino acid sequence of purified NIL-3 activity was determined to be homologous to beta-2 glycoprotein I (apolipoprotein H: APO-H). The gene expression of APO-H was detected by nested-PCR in STIL-3 C5-CM stimulated total bone marrow cells and STIL-3 C5-CM stimulated bone marrow fraction 2 (Fr. 2) which has been reported as a hematopoietic stem cell rich fraction. These observations indicate the possibility that the APO-H is the NIL-3 which was produced from bone marrow cells in response to excess IL-3 stimuli.     

Increased mass transfer to microorganisms with fluid motion

The effect of fluid flow and laminar shear on bacterial uptake was examined under conditions representative of the fluid environment of unattached and attached cells in wastewater treatment bioreactors. Laminar shear rates below 50 s(-1) did not increase leucine uptake by suspended cultures of Zoogloea ramigera. However, leucine uptake by cells fixed in a flow field of approximately 1 mm s(-1) was 55-65% greater than uptake by suspended cells. Enhanced microbial uptake with advective motion is consistent with mass transfer rates calculated using Sherwood number correlations. Advective flow increases microbial uptake by increasing collisions between substrate molecules and cells through compression of the concentration boundary layer surrounding a cell. The rate of leucine uptake suggests that binding proteins used to transport leucine into the cell can occupy approximately 1% of the cell surface area.     

Microfilament bundles in cultured cells : Correlation with anchorage independence and tumorigenicity in nude mice

The distribution of actin microfilament bundles in cell lines 3T3B, CHO, HeLa and CLID extracted with 0.1% Triton X-100 was examined by indirect immunofluorescence using human actin antibodies and by electron microscopy of whole cells grown directly on support grids. Anchorage dependence as determined by growth in soft agar and tumorigenicity in nude mice was also investigated. Immunofluorescent staining showed that CHO and HeLa cells have normal numbers and distributions of actin microfilament bundles as compared with similarly spread control 3T3B cells. A significant fraction of the mouse CLID cells showed comparable numbers of microfilament bundles as 3T3B cells but their distribution was markedly different. In many cases the bundles radiated from a region close to the cell's centre or near its projections and usually penetrated the projections. The presence of diffuse staining in 4% of the cell population also indicated the existence in these cells of disorganized actin. Electron microscope studies of well spread regions of negatively-stained, Triton-extracted cells corroborated the observations made with the immunofluorescence technique. In 3T3B, CHO and CLID cells abundant microtubules were found, colinearly arranged with actin filaments in the thin cytoplasmic extensions. While CLID, CHO and HeLa cells showed the capacity to grow in soft agar, only CLID and HeLa cells produced tumours in athymic nude mice. The observations suggest that a reduction or disorganisation of the actin microfilament bundles may not in itself be essential at least for the non-virally transformed cells studied to show anchorage independence or to produce tumours in nude mice.