Cell Proliferation In Primary Cultures Research Articles

Stimulatory effects of pleural fluids from mesothelioma patients on CD44 expression, hyaluronan production and cell proliferation in primary cultures of normal mesothelial and transformed cells

Cell lines established from human malignant mesotheliomas, but not from normal mesothelial cells, have been shown to possess hyaluronan receptors, and to secrete factors that stimulate hyaluronan production by fibroblasts and normal mesothelial cells. In the present study we investigated the generality of this observation, namely the presence of hyaluronan receptors and factors which induce stimulation of hyaluronan synthesis in primary mesothelioma and mesothelial cell cultures. Functionally active hyaluronan-binding sites on the surface of malignant mesothelioma cells in primary cultures, established from pleural effusions of 3 different patients, were demonstrated using 3H-hyaluronan. Primary cultures of normal mesothelial cells from non-mesothelioma effusions did not exhibit any binding ability. Pleural fluids from mesothelioma patients both stimulated hyaluronan synthesis and promoted proliferation of normal mesothelial cells to a larger extent than non-mesothelioma fluids. The hyaluronan-stimulatory activity was only slightly neutralized by antibodies against PDGF-BB or TGF-beta; antibodies against bFGF had no effect. Although the concentration of hyaluronan was much higher in pleural fluids from mesothelioma than from non-mesothelioma patients, its molecular weight was almost the same. The hyaluronan-binding capacity of early-passage mesothelioma cells derived from pleural effusions can be an additional marker, in combination with other diagnostic tools, to distinguish between mesothelioma and mesothelial cells.

Effect of medroxyprogesterone acetate (MPA) and serum factors on cell proliferation in primary cultures of an MPA-induced mammary adenocarcinoma

The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on the in vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblast-enriched cultures using 3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10(-11) to 10(-7) M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10(-11) M. At nM concentrations, neither DEXA nor DHT stimulated 3H-thymidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10(-7)-10(-9) M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 +/- 115 fmoles/10(5) cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10(5) cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of 3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals. It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.

Toxicity and metabolism of ochratoxin A.

The frequent occurrence of ochratoxin A (OA) in food and feed commodities and the high incidence of human exposure, as confirmed by different surveillance studies initiated several investigations devoted to elucidating the molecular mechanisms underlying OA toxicity. Previous studies indicated that the primary effects of OA are the inhibition of tRNA synthetase, the inhibition of mitochondrial respiration, and a disturbance of intracellular calcium homeostasis inherent to lipid peroxidation processes. We here report the effect of OA on a number of toxicological endpoints including cytotoxicity in different cell lines and effects on macromolecular synthesis and cell proliferation in primary cultures of hepatocytes at concentrations corresponding to overall exposure levels. These studies provide evidence that prominent toxicological effects might be linked to biotransformation processes. Analysis of hepatic biotransformation resulted in the detection of a number of distinct stable OA-metabolites. As metabolic activation has also been identified as an essential step in OA mutagenicity, the biological relevance on this mechanistic data is discussed.

Pulmonary epithelial cell proliferation in primary culture of alveolar type II cells.

A small subpopulation of pulmonary epithelial cells (PE) proliferates in low-density primary culture of alveolar type II cells and forms colonies of cells that could be passaged for several generations and that in some respects maintain a differentiated phenotype of the alveolar type II cells. At this time it is not known if these cells are some form of progenitor epithelial cells or type II cells that are not fully differentiated in vitro. The proliferation of the PE cells was dependent on serum, alveolar macrophage-conditioned medium, and insulin being included in the culture medium. Under these conditions, approximately 0.5-1.0% of the seeded cells that adhered to the culture dishes were capable of forming colonies. Efficiency of colony formation increased to 5-10% in subsequent passages. PE cells maintained a high level (> 40%) of saturated phosphatidylcholine (PC) as a percentage of total PC throughout the culture period (> 28 days). However, the saturated PC content was not constant throughout the long-term culture period and the subsequent passages (41.3% at 29 days and 37.3% in the 3rd passage). These cells also contained numerous lamellar bodies and were able to bind the Maclura pomifera lectin. PE cells also expressed cytokeratin No. 19, as well as alkaline phosphatase activity, both possible markers for differentiated type II cells. However, PE cell synthesized low levels of Pg (approximately 2%), were squamous, and tended to form multiple strata, unlike the cuboidal type II cells in vivo. The cells did not exhibit immunocytochemically demonstrable surfactant-associated protein A (SP-A). Additional factors and culture requirements may be necessary for complete maturation of cultured PE cells. This was demonstrated by culturing PE cells on EHS matrix. Aggregates of cells surrounding a central lumen were formed after a few hours in culture and were maintained for 20 days. The cells contained lamellar bodies and some intercellular junctions. PE cells can be regarded as a highly selected subpopulation of pulmonary epithelial cells that concomitantly maintain proliferation and aspects of differentiated alveolar type II cells in long-term culture.

Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells

The purpose of our experiments was to examine variables affecting early events in the establishment of rat tracheal epithelial (RTE) cultures as well as factors regulating long-term RTE cell growth. The experiments showed that when RTE cells were seeded into complete serum-free medium between 13 and 30% of the seeded cells attached. Of the seeded cells, only approximately 2% entered into DNA synthesis and underwent repeated cell divisions to form colonies containing greater than 20 cells. Coating the dishes with extracellular matrix components had little effect on cell attachment or colony forming efficiency (CFE). However, coating the dishes with fetal bovine serum markedly increased CFE. The media components bovine serum albumin and bovine pituitary extract were shown to be important in promoting cell attachment as well as CFE. Cholera toxin on the other hand had no effect on cell attachment but significantly increased CFE. These and other studies showed that cell attachment and cell proliferation are independently regulated. Studies on long-term culture growth indicated that the number of progeny produced per colony forming unit (CFU) is inversely proportional to the number of CFUs seeded. Inasmuch as the cultures did not become confluent under any of the culture conditions tested and media obtained from high density cultures were shown to be growth inhibitory, these findings suggest that a diffusible growth restraining factor is being produced by the cultures limiting clonal expansion. Experiments showing growth inhibitory effects of media conditioned by high cell density cultures support this interpretation. The putative factor reaches critical concentrations earlier in cultures seeded with high numbers of CFU than in cultures seeded with low numbers of CFU. Because the cultures are known to produce transforming growth factor-beta, this growth regulator probably plays a role in controlling RTE cell proliferation. However, it is likely than other events, such as depletion of growth factors from the media, also are significant in regulating the growth of the cultures.

Transferrin Stimulates Proteoglycan Accumulation by Fetal Lung Cells in Culture

The role of transferrin in growth and the formation of extracellular matrix was investigated by comparing its effects on proteoglycan metabolism and cell proliferation in primary cultures of fetal rat lung fibroblasts and Type II epithelial cells. Transferrin specifically stimulated the accumulation of dermatan/chondroitin sulfate proteoglycans associated with the cells and matrix in a dose-dependent manner (0-200 micrograms/ml, r = .850 in fibroblasts and r = .810 in Type II cells). This effect was not due to increased synthesis since there was a corresponding decrease in proteoglycans and their degradation products released into the medium. The effect is probably mediated via an action on the proteoglycan core protein, since there was no effect of transferrin on enzyme activity promoting glycosaminoglycan synthesis on the synthetic initiator beta-D-xyloside. The effect of transferrin on proteoglycan distribution was not a secondary effect caused by changes in collagen synthesis and was not linked to cell proliferation or the concentration of Fe3+ ions in the culture medium.

Activation of excitatory amino acid receptors reduces thymidine incorporation and cell proliferation rate in primary cultures of astrocytes

Addition of quisqualate (a heterocyclic analogue of glutamate) reduced [methyl-3H]thymidine incorporation and cell proliferation in primary cultures of rat cortical astrocytes. The inhibitory action of quisqualate was mimicked by glutamate and ibotenate, whereas kainate, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were inactive. These results suggest that activation of a specific class of excitatory amino acid receptors contributes to the regulation of growth and proliferation of glial cells in primary culture.

Growth of non-pigmented ciliary epithelial cells in serum-free hormone-supplemented media

Bovine non-pigmented ciliary epithelial (NPE) cells are cultured following a dissecting procedure that preserves the original cellular polarity. Cell growth of primary cultures of NPE cells is supported by hormonally defined serum-free culture media and an artificial substrate with high binding affinity to attach cells in culture. Cell proliferation of primary cultures of bovine NPE cells was limited to 3-5 weeks. Nerve growth factor (NGF) was found to have a profound effect on the cellular junctions of growing NPE cells when added to the culture media. Nerve growth factor induced the formation abundant microfilament bundles associated with junctional complexes of undetermined nature between adjacent cells. Electron microscopy of NPE in culture shows a high intracellular transport activity with abundant coated pits and clathrin-coated vesicles, a complex network of microfilaments, microtubules and intermediate filaments associated with desmosomes.

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture.

A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferring (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium.

Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen.

Primary cultures of rabbit endometrial cells grown on collagen substrates exhibit cyclic changes in DNA content throughout extended periods of culture. These cycles are characterized by periods of significant increases and decreases in the DNA content of the cultures or number of cells present, yet through the entire duration of culture there is no net change in the total DNA. The rates of cell proliferation and cell death change through time in culture with the same periodicity as the changes in DNA. Neither changes in the rate of cell proliferation nor the rate of cell death alone are sufficient to account for the changes in DNA. Rather, there appears to be a feedback mechanism operating between cell proliferation and cell death such that when one increases, the other increases concomitantly in order to maintain a homeostasis in total culture mass. This homeostasis appears to be mediated by a soluble cell proliferation factor (CPF) and a cell death factor (CDF) produced by the cells. CPF and CDF may be obtained from either conditioned media or cultured cell extracts. These biological activities are heat and trypsin sensitive. The major mode of cell death in these cultures appears to be apoptosis or programmed cell death, characteristic of renewing epithelia. The data suggest that this tissue culture model system represents a renewing cell population containing stem cells and their progeny, whose total growth is strictly regulated by CPF and CDF. As such, it provides a model system in which to study homeostasis and how it may be altered in hyperplasia and neoplasia, as well as its regulation by hormones.

Collagen required for proliferation of cultured connective tissue cells but not their transformed counterparts.

THE proline analogue, cis-hydroxyproline, when incorporated into collagen, prevents collagen secretion1 and also inhibits the growth of fibroblasts in vitro2. We describe here studies on the mechanism of action of cis-hydroxyproline on cell proliferation of primary cultures of murine connective tissue cells and of their spontaneously transformed, tumorigenic counterparts. We find that the analogue inhibits the attachment, spreading and proliferation of the connective tissue cells and that these effects are completely reversed when the cells are grown on collagen-coated dishes. The tumorigenic cells are less affected by cis-hydroxyproline even though their collagen synthesis is inhibited to a similar extent to the connective tissue cells. These results suggest that the presence of a collagen matrix is required for the growth of some connective tissue cells in vitro but not for the growth of their tumorigenic counterpart.

Microdensitometric and autoradiographic analysis of cell proliferation in primary culture of Nicotiana glauca pith tissue

Microdensitometric measurements of nuclear DNA content in Nicotiana glauca pith cells during the first days of culture indicate the presence of two concomitant phenomena: an amplification process, evidentiated by the non geometric increase of DNA content, and a general DNA reduplication which changes DNA values from 2 and 4 C to about 16 C. At the onset of proliferation, large nuclei show fragmentation to pieces with DNA content of about 2 C values. Tritiated thymidine pulse treatment and cell cycle length measurements are in agreement with the hypothesis of the coexistence of two cellular populations, one deriving from nuclear fragmentation, the other from cells which follow a normal synthesis mitosis synthesis cycle.