The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

A correlation between ovarian follicular maturity and anchorage-independent growth of bovine granulosa cells

Previous studies have shown that a subset of bovine ovarian granulosa cells can proliferate to form clones of functional cells in suspension in a semisolid agar matrix, without the requirement for attachment to the substratum. These clonogenic anchorage-independent granulosa cells are responsive to epidermal growth factor and exhibit properties of a primitive progenitor cell population. We have used this assay system to analyze the proliferation of granulosa cells during ovarian follicular maturation. As the follicle increases in size, there is a progressive decline in the ability of granulosa cells to clone in agar, and the proliferative potential of these cells as measured by colony size also decreases. The ratio of large colonies with high proliferative potential (greater than 250 micron in diameter) to small colonies with limited capacity for growth falls from 1.92 in follicles less than 7 mm in diameter, to 0.32 in follicles larger than 10 mm in diameter. This occurs as the follicular content of granulosa cells with more limited capacity for clonal growth in agar undergoes expansion. Analysis of colony-forming cells in follicles harvested at early and late estrus suggests that these cells are regulated by complex intraovarian factors rather than circulating gonadotropin levels. Our data indicate that the granulosa cell lineage is an age-structured continuum of maturing and differentiating cells with a progressively restricted proliferative capacity.     

On the autocrine growth regulation of Morris hepatoma cells

Chemically induced, transplantable Morris hepatoma 7777 cells (MH) were examined for their anchorage-dependent and anchorage-independent growth properties. MH cells were found to grow on solid surface in a density-dependent manner, with respect to serum factors and gelatinization. The correlation between anchorage independent growth of MH cells and agar or serum concentrations in the culture medium was described. The ability of MH cells to stimulation of soft agar colony formation of NRK-49F indicator cells in coculture assay was presented. Autocrine control of proliferation of MH cells by TGFs-like factor(s) was suggested.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

The detection of subclasses of granulosa cells with differing responsiveness to EGF, FGF, and gonadotropin preparations using an anchorage-independent clonal agar assay

Subpopulations of granulosa cells of differing responsiveness to epidermal growth factor (EGF), fibroblast growth factor (FGF), pituitary gonadotropin preparations, and rat erythrocyte suspensions (RBC) have been detected using an anchorage-independent clonal agar assay. All growth factor preparations were capable of enhancing colony formation when added alone, and elicited cloning efficiencies as high as 35% when added to the culture system at optimal concentrations in a variety of combinations. The FGF preparation was the single most effective stimulator of colony formation, augmenting both colony numbers and colony size at concentrations as low as 50 ng/ml. However, unlike the other growth factors in this assay system, a plateau in responsiveness could not be reached even at levels as high as 1 microgram/ml. NIH-FSH-P2 and NIAMMD-bLH-4 were considerably less potent than other growth factors. Both preparations were inactive at concentrations less than 1 microgram/ml and produced an optimal response at 10 micrograms/ml.     

Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23-transfected melanoma cells

Reduced expression of the nm23 gene in certain rodent model systems and human breast tumors has been correlated with high tumor metastatic potential. To investigate the functional effects of nm23 expression, we have transfected a constitutive murine nm23-1 expression construct into highly metastatic K-1735 TK murine melanoma cells. TK clones expressing the exogenous nm23-1 construct exhibited a reduced incidence of primary tumor formation, significant reductions in tumor metastatic potential independent of tumor cell growth, and altered responses to the cytokine transforming growth factor beta 1 in soft agar colonization assays, compared with control-transfected TK clones. In contrast, nm23-1-transfected TK clones exhibited no significant differences in intrinsic tumor cell growth, i.e., primary tumor size in vivo, anchorage-dependent growth rate in vitro, and anchorage-independent colony formation in soft agar in vitro. The data demonstrate a suppressive effect of nm23 on several aspects of the cancer process, including tumor metastasis.     

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).     

Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors

Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity. Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent conditions. D. W. Barnes     

Reciprocal effects of epidermal growth factor and transforming growth factor beta on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.     

Analysis of human ascites effect on clonogenic growth of human tumor cell lines and NRK-49F cells in soft agar

We studied the factors that control the capacity of tumor cells to grow in soft agar. And, we analyzed the effect of cell-free ascites (CFA) obtained from ovarian cancer patients in combination with various media on the cloning efficiency of H-134 and OVCAR-3nu ovarian carcinoma cell lines and the WiDr colon carcinoma cell line. Seven batches of CFA consistently enhanced the soft agar growth of tumor cells more efficiently than tested sera. The addition of charcoal-treated bovine serum albumin (BSA) lowered the amount of CFA required for optimal tumor cell growth. As little as 1.25 ng of epidermal growth factor (EGF)/ml further improved OVCAR-3nu soft agar growth in combination with all of the amounts of CFA tested. Thus, neither BSA nor EGF seems to account for the colony-stimulating effect of ascites on tumor cells. Four batches of CFA were tested for stimulating soft agar growth of normal rat kidney (NRK-49F) fibroblasts; all induced colonies of different morphologies. This effect was potentiated by EGF, which suggests the presence of several transforming growth factor-like activities in CFA. The results show that differences in cloning efficiency of tumor cells of one or two orders of magnitude can be found between standard (anchorage-dependent growth-supporting) media and media optimalized for soft agar growth, such as CFA in the presence of EGF. This paper will discuss the similarity in effects of CFA on various tumor cells and NRK cells, and possible implications of the stimulatory effects of CFA.     

The effect of oxygen tension on haemopoietic and fibroblast cell proliferation in vitro

The effects of providing low oxygen tension in the gas phase of two different types of cell culture systems were investigated. The clonal growth of granulocyte-macrophage progenitor cells in an agar culture system was improved markedly by incubation within a low oxygen tension gas phase (48 mmHg – 6.8%) instead of the conventional air (135 mmHg – 19%), the effects being measured by increases in numbers of colony forming cells detected and in the colony sizes. The increased efficiency of colony formation was observed both with mouse and human marrow cells. A similar effect was observed in a liquid adherence culture system with primary cultures of foetal mouse fibroblasts both at clonal and higher cell densities.     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Antigen-specific clones of proliferating T lymphocytes. I. Methodology, specificity, and MHC restriction

In this report we describe in detail a new method for cloning antigen-specific, proliferating T lymphocytes directly from primed murine lymph nodes after 3 days of activation in vitro. After expansion in liquid culture the cells from the colonies were shown to be antigen specific and to require I-A histocompatible, irradiated spleen cells for stimulation. For hapten-carrier-type antigens, the T cells were shown to be carrier specific in their recognition but they were also capable of distinguishing the presence of the hapten. Recloning of small numbers of these cells in soft agar under conditions of high plating efficiency yielded true clones (i.e., populations derived from a single cell) whose antigen specificity was identical to that of cells from the original colony. The fact that a clone of T cells was I-A restricted in its antigen recognition demonstrates that suppressor T cell function cannot account for the phenomenon of major histocompatibility complex restriction.     

Epidermal growth factor stimulates the anchorage-independent growth of human squamous cell carcinomas overexpressing its receptors

We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage-dependent growth, it stimulated anchorage-independent growth of all four cell lines tested. The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than -dependent growth, in cells overexpressing EGF receptors. Moreover, as EGF has been shown to stimulate the in vivo growth of squamous carcinoma cells overexpressing EGF receptors, it is also suggested that the in vitro EGF responsiveness of these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness.     

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha-transforming growth factors in ras-transformed mouse mammary epithelial cells

A mouse mammary epithelial cell line, NMuMG, exhibits a low capacity to grow in semisolid medium as colonies and it is not tumorigenic in nude mice. In contrast, NMuMG cells which have been transformed by an activated c-Harvey ras proto-oncogene, NMuMG/rasH, or by the polyoma middle T-transforming gene, NMuMG/pyt, are able to grow in soft agar and, when injected into nude mice, produce undifferentiated carcinomas. Human epidermal growth factor (EGF) or human alpha-transforming growth factor (alpha TGF) can stimulate, in a dose-dependent fashion, the anchorage-independent growth of NMuMG and NMuMG/pyt cells in soft agar but fail to enhance the anchorage-independent growth of the NMuMGrasH cells. Likewise, human EGF or human alpha TGF is also able to stimulate the anchorage-dependent growth of normal NMuMG cells and NMuMG/pyt cells in a serum-free medium supplemented with insulin, transferrin, fetuin, and laminin, or in medium containing low concentrations of serum, whereas these same growth factors under comparable culture conditions have little or no effect upon the anchorage-dependent growth of the ras-transformed NMuMG-rasH cells. The biological refractoriness of the NMuMG/rasH cells to human EGF or human alpha TGF is reflected by a reduction in the total number of cell surface receptors for EGF and by an absence of a high-affinity population of binding sites for mouse [125l]EGF on these cells as compared to the NMuMG or NMuMG/pyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMG/rasH and NMuMG/pyt cells contains a relatively higher amount of biologically active TGFs than CM obtained from comparably treated NMuMG cells as measured by the ability to induce the anchorage-independent growth of normal rat kidney cells in soft agar. The higher levels of biologically active TGFs found in the CM from the transformed cells relative to the NMuMG cells is paralleled by a corresponding increase in the CM from these cells in the amount of immunoreactive alpha TGF, by an increase in the amount of EGF receptor-competing activity, and by an increase in the levels of alpha TGF mRNA in the NMuMG/rasH cells. These results demonstrate that mammary epithelial cells which have been transformed by an activated ras proto-oncogene, but not by the polyoma middle T-transforming gene, become unresponsive to exogenous EGF or alpha TGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

The truncated form of the Epstein-Barr virus latent-infection membrane protein expressed in virus replication does not transform rodent fibroblasts.

The gene encoding the Epstein-Barr virus membrane protein LMP, expressed in latent infection, is known to induce morphologic changes and some loss of contact inhibition in NIH 3T3 cells as well as profound loss of contact inhibition and of anchorage dependence in Rat-1 cells. Another form of LMP (D1LMP), deleted for the amino terminus and first four putative transmembrane domains of LMP, was recently shown to be expressed late in Epstein-Barr virus replication. We now demonstrate that D1LMP has no transformation-associated phenotypic effect in Rat-1 cells and does not significantly affect LMP-induced Rat-1 cell transformation. LMP activity and D1LMP inactivity in inducing anchorage-independent growth are not restricted to Rat-1 cells, but are also evident in BALB/c 3T3 cells. In both cell types, loss of contact inhibition and anchorage independence are acutely evident after LMP expression. Although newly transfected polyclonal Rat-1 or BALB/c cells have a lower agar cloning efficiency than established LMP-expressing clones, this is attributable, at least in part, to their lower average LMP expression, since among clones of transfected cells, higher cloning efficiencies correlated with higher levels of LMP. LMP is bound to the vimentin cytoskeletal network in rodent fibroblasts as it is in transformed lymphocytes, whereas D1LMP showed no detectable cytoskeletal binding, suggesting that cytoskeletal association may be integral to LMP-mediated cell transformation. LMP association with the cytoskeleton in latently infected, growth-transformed lymphocytes and LMP-transformed rodent fibroblasts, correlated with the lack of both rodent cell-transforming activity and cytoskeletal association of D1LMP supports working hypothesis that cytoskeletal association is important in LMP transforming activity.     

Cloning of mouse myeloma cells and detection of rare variants

Cultured mouse myeloma cells have been cloned in soft agar using a modification of the method established by Pluznik and Sachs ('65, '66) and by Bradley and Metcalf ('66). A linear relationship existed between the number of cells plated and the number of colonies produced. Conditions for obtaining optimum cloning efficiency and colony size were determined for the MPC-11 cell line. Feeder cells of mouse, human and rabbit origin and conditioned growth medium obtained from mouse cultures and had an enhancing effect on colony formation. Immunoglobulin production by cloned cells was detected by overlaying the clones with anti-immunoglobulin antiserum. The antiserum had no adverse effect on cloning efficiency or colony size. A reconstruction experiment was performed to show that the plate assay could reliably detect rare variants of immunoglobulin producing cells. The plate assay was validated by studying immunoglobulin production following recovery of clones from dishes and their growth to mass suspension culture. Immunoglobulin formation in these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium, and by incubating the cells with radioactive amino acids and analyzing the intracellular and secreted immunoglobulin on polyacrylamide gels.     

Growth properties of RSV-transformed rat sarcoma (XC) cells

RSV-transformed rat sarcoma (XC) cells were characterized as to their ability to anchorage-dependent and anchorage-independent growth in various culture conditions. The proliferation of XC cells was dependent on serum concentration and initial cell density. The relationship between seeding density and the growth rate of XC cells indicated that the investigated cells produce factors with growth stimulating and growth inhibiting activity. The anchorage-independent growth inhibiting activity was found in ultrafiltrate (500 Mr 10,000) of XC cells conditioned medium. The obtained results suggests that anchorage-independent growth of XC cells may be regulated by two types of autocrine factors, which are antagonistic in their biological affects.