Jürgen van der Bosch

Trichostatin A Modulates Expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in Human Lung Adenocarcinoma Cells

Abstract Lung adenocarcinoma cells treated for 16 h with trichostatin A (TSA), an inhibitor of histone deacetylases, and untreated cells were analyzed with respect to differential gene expression. Complex hybridization of cDNA arrays revealed repression of Bcl-xL, CRAB2 and TFIID/TAFII31 as well as induction of p21waf1/cip1, GATA-2, hsp86, ID1, ID2 and ID3 mRNA expression, which could be verified by Northern blotting. ID2 induction was further confirmed by Taqman realtime quantitative RT-PCR. The described alterations of gene expression due to TSA renders the lung adenocarcinoma cells susceptible to induction of apoptosis.

[46] The culture of human tumor cells in serum-free medium

Summary In this chapter we describe methods for growth in serum-free medium of several types of human tumors. Our laboratory and others have reported formulations of serum-free medium that will support the growth of other, additional types of human cells in culture that are not described in this chapter but are reviewed elsewhere.2,27 The serum-free medium HC described for the human colon tumors was originally devised to support the serum-free growth of a line of human colon tumor cells (HC84S) established in serum-containing medium and derived from a human colon tumor line (T84) transplantable in nude mice. Subsequently, it was found that this medium will support the serum-free growth of several (but not all) other transplantable human colon tumor lines in primary culture as well as primary culture of at least one tumor directly from a patient. The serum-free medium HA described for the human astrocytoma was devised to support the serum-free growth of a line of human astrocytoma cells (HA24) established in serum-containing medium and derived from a human astrocytoma line (T24) transplantable in nude mice. Both tumorigenic and nontumorigenic clones have been isolated from the HA24 cell line, and both will grow serum-free in the HA medium, although quantitative differences in responses of the different clones to omission of the individual components of the HA medium are seen. The serum-free medium LA described for the human lung adenocarcinoma was devised to support the serum-free growth of primary cultures of the T291 lung adenocarcinoma transplantable in nude mice. This medium will support the continuous serum-free growth of a line of lung adenocarcinoma cells originally established from cultures of the T291 tumor in serum-containing medium. The serum-free medium LE described for the human lung epidermoid carcinoma was devised to support the serum-free growth of primary cultures of the T222 lung epidermoid carcinoma transplantable in nude mice. Neither the LA nor the LE medium will support continuous, multipassage serum-free growth of cell cultures obtained from the T291 or T222 tumors. MCF-7 is an established cell line, derived in serum-containing medium from a metastatic pleural effusion of a human breast cancer.14 The medium developed for the continuous serum-free growth of the MCF-7 line will support growth in primary culture to some extent of some human breast tumors, but not all we have tested. In addition, we report in this section procedures for the partial purification of a factor from serum that stimulates the spreading of these cells, as well as others, in serum-free medium. We also describe in this chapter methods for the isolation of human tumor cells of epithelial origin for primary culture and methods for the quantitation of cells in such cultures. These methods, which are likely to be generally applicable in the isolation of most cell types for primary culture, allow us to obtain cultures with an acceptable plating efficiency while avoiding the ubiquitous problem of fibroblastic overgrowth of the epithelial population. Furthermore, the use of medium in which serum has been replaced by cell type-specific combinations of nutrients, hormones, binding proteins, and attachment factors allows us to reproduce more accurately in vitro the nutritional, hormonal and stromal influences to which the cells are subject in vivo.28 In this way we should minimize the genetic or epigenetic alteration and selection of an aberrant population of cells from those of the original culture. It is interesting to note that in several instances presented in this chapter both the growth and the differential properties of the cell types in their respective serum-free, hormone-supplemented media are closer to what we wish to attain in culture than what which we see in cultures of these cells in serum-supplemented medium. Human colon tumor cells, as well as human lung adenocarcinoma and epidermoid carcinoma, grow better in their serum-free media than in serum-containing medium. Furthermore, the keratinization of the epidermoid carcinoma cells is more marked in serum-free medium, as is the formation of villi-like, mucin-secreting structures in serum-free cultures of the colon tumor cells.3 It is our view that this is further evidence that, by the use of serum-free medium specifically designed for each cell type, we are more accurately reproducing the in vivo environment of the cells.

The chick oviduct in tissue culture: I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serums ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.

Density-dependent tumor cell death and reversible cell cycle arrest: Mutually exclusive modes of monocyte-mediated growth control

The population development of five human tumor cell lines is examined under the influence of elutriator-prepared human monocytes in a serum-free hormone- and growth factor-supplemented medium. Analysis was performed by electronic counting and sizing of tumor cell nuclei and flow cytometric detection of cell cycle phases. Tumor cell death is triggered at rather low monocyte:tumor cell ratios (1:2 to 1:4) whereas it is strongly reduced at high monocyte densities. Furthermore, it is shown that confluence of the target cell population is a necessary prerequisite for lysis. The data suggest that in monocyte/tumor cell cocultures the decision on target cell lysis is not made by the effector cell, but rather by the target cell and that the criterion for this decision is the target cells ability or inability to respond to a monocyte challenge by arresting the cell cycle in G1. Interactions between target cells play an important role in determining the result of this decision process. A common basis is suggested for this kind of density-dependent monocyte-triggered lysis and density-dependent cell death in 3T3 cell cultures as described previously.

A new in vitro model for studying human T cell differentiation: TH1/TH2 induction following activation by superantigens

A new T(H1)/T(H2) in vitro model for mechanistic studies and drug screening in human T cells was established working with ficoll-separated PBMCs or elutriated lymphocytes cultured in serum-free medium. Human T cells could be kept viable and reactive in this medium for several months. In this model, superantigens (SAs) were used to activate exclusively those T cell clones which were known to express specifically SA-binding Vbeta-chains of the T cell receptor. It was possible to identify the activated SA-specific T cells among the whole T cell population by using monoclonal antibodies against these Vbeta-chains and to follow responses involving receptor regulation and cytokine expression. The flow cytometric analysis revealed, that SA exposure caused an upregulation of the IL-2 receptor selectively in the SA-specific subpopulation. Only the T cells of this subpopulation could be shifted towards T(H1) or T(H2) differentiation, which was determined by the distribution of IFN-gamma and IL-4 positive cells. Regulation of IFN-gamma could be detected by flow cytometry after 18 h and that of IL-4 on the third day of cell culture. The differentiation status could be influenced by various measures: T(H1) shifts were achieved in the presence of IL-12 and anti-IL-4, whereas, T(H2) shifts were induced more slowly with monocyte-reduced elutriated lymphocytes in the presence of IL-4, IL-6, anti-IL-12, 1alpha,25-dihydroxy-vitamin-D3 or combinations thereof. It was found that sialidase stimulated whereas TGF-beta and pentoxifylline suppressed both kinds of T cell response. The T(H1)/T(H2) differentiation persisted for several weeks after primary activation if cells were expanded in IL-2 containing serum-free culture medium. Therefore, this human T(H1)/T(H2) in vitro model should be ideal for studying early and late events of infection, allergy, and autoimmunity as well as for investigating the cellular interactions involved. In addition, the early detection of the response pattern makes this model potentially useful for drug screening.

Complex population kinetics in 3T3 and SV40-3T3 cell cultures. Involvement of division, inhibition of division and death of cells.

Abstract Density dependent cell death is shown to be an important process during adjustment of stationary cell densities in 3T3 cell populations under certain conditions. The present data suggest a common mechanistic basis for density dependent cell death and density dependent inhibition of cell division. In SV40-3T3 cell populations density dependent growth-inhibiting processes may occur transiently.

Age-dependent decrease of growth responsiveness in density inhibited 3T3 cell populations and their interactions with SV 40-3T3 cells

An aging process has been detected in stationary 3T3 cell cultures, especially in the presence of plasma‐derived serum (PDS) from adult bulls. It leads to irreversible conversion of an increasing percentage of initially responsive cells of a stationary population into cells unresponsive to growth stimulation by newborn calf serum (NBCS) or reseeding at low cell density in the presence of NBCS. These unresponsive cells are viable in the sense that, following trypsinization, they reattach and spread on a new culture plate and can be maintained for many days. The conversion process is accelerated by increasing PDS concentration. It is antagonized by NBCS. It is accompanied by enhancement of growth‐inhibiting interactions exerted by stationary 3T3 cell populations on SV 40–3T3 cells.