Loss of the tumor suppressor Pten promotes proliferation of Drosophila melanogaster cells in vitro and gives rise to continuous cell lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.     

Muscle cell culture as a tool in animal growth research

Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.     

The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [3H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [3H]thymidine and [3H]ouabain. Cycling cells which had [3H]thymidine-labeled nuclei did not have notably higher labeling with [3H]ouabain. However, [3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.     

Properties of the H-4-II-E tumor cell system. II. In vitro characteristics of an experimental tumor cell line.

The growth and cell proliferation characteristics of the H-4-II-E cell line, giving rise to hepatoma H-4-II-E when inoculated into male ACI rats, were studied in vitro. Following seedling of 2 x 10(5) cells into culture dishes, exponential cell growth occurs in cultures fed both at 24 hr and 48 hr intervals with a population doubling time of 18-4 hr. Plateau phase growth conditions are established on day 7 and day 5 for cultures fed at 24 hr and 48 hr intervals respectively. Both the plateau phase cell density and the maintenance of plateau phase appear dependent on the frequency of feeding. For cultures fed daily, the transition from exponetial growth to plateau phase results from both a reduction in the number of proliferating cells (99% v. 35%) as well as an elongation of the cell cycle (17-7 hr v. 128-4 hr). The cell proliferation characteristics of the culture are further discussed in reference to both cell growth and feeding schedules of other cell lines.     

Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation.

We have previously used human Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines for the identification and purification of a novel cytokine, natural killer cell stimulatory factor (NKSF/IL-12), that has pleiotropic effects on human lymphocytes. B cell lines are also routinely employed as feeder cells for the culture of T and natural killer (NK) cells. In this report we describe the ability of two NKSF/IL-12 producing B cell lines (RPMI-8866 and Cess) and two nonproducing lines (Raji and Daudi) to stimulate the proliferation of T and NK cells in 8-day PBL cultures. We demonstrate, using an anti-NKSF/IL-12 neutralizing monoclonal antibody, that the endogenous production of NKSF/IL-12 in these cultures can significantly enhance the proliferation and cytotoxic activity of T and NK cells. We also report that the addition of exogenous rNKSF/IL-12 can greatly increase the number of T and NK cells obtained from the cultures following stimulation by the B cell lines. Aside from the possible practical applications, the enhanced proliferation of T and NK cells consistently observed in the presence of endogenously produced NKSF/IL-12 or exogenously added rNKSF/IL-12 in this system may further our understanding of the role of this cytokine during an in vivo immune response.     

Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet

Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed “bipotential murine oval liver” (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic.

These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.     

IL-2-dependent proliferation of thymic accessory cells

Phagocytic cells of the thymic reticulum are Ia-positive accessory cells continuously produced in long term thymic stroma cultures. We show in the present paper that phagocytic cells of the thymic reticulum have R for IL-2. They share this property with splenic accessory cells produced in vitro by the same technique but have the unique property of proliferating in the presence of rIL-2. This proliferation is not enhanced by Con-A, is not linked to a lymphoid contaminant, and is specifically inhibited by an anti-IL-2R mAb.     

Sponge-Cell Culture? A Molecular Identification Method for Sponge Cells

Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea avara specimen was sequenced and compared to eukaryotic 18S rDNA sequences picked up from a proliferating cell culture that originated from a dissociated Dysidea avara specimen. Our method proved unambiguously that this was not a sponge-cell culture. Therefore, it provides a valuable tool for further research on sponge-cell cultures.     

Long-term culture of muscle explants from Sparus aurata

Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.     

Characterization and optimization of a simple, repeatable system for the long term in vitro culture of aligned myotubes in 3D

Increased recent research activity in exercise physiology has dramatically improved our understanding of skeletal muscle development and physiology in both health and disease. Advances in bioengineering have enabled the development of biomimetic 3D in vitro models of skeletal muscle which have the potential to further advance our understanding of the fundamental processes that underpin muscle physiology. As the principle structural protein of the extracellular matrix, collagen-based matrices are popular tools for the creation of such 3D models but the custom nature of many reported systems has precluded their more widespread adoption. Here we present a simple, reproducible iteration of an established 3D in vitro model of skeletal muscle, demonstrating both the high levels of reproducibility possible in this system and the improved cellular architecture of such constructs over standard 2D cell culture techniques. We have used primary rat muscle cells to validate this simple model and generate comparable data to conventional established cell culture techniques. We have optimized culture parameters for these cells which should provide a template in this 3D system for using muscle cells derived from other donor species and cell lines.     

Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.     

Conditionally Immortalized Neural Cell Lines: Potential Models for the Study of Neural Cell Function

Studies on primary cell cultures have contributed significantly to our understanding of neural cell function. Nevertheless, for many studies the value of these primary cell cultures has been limited by the time the cultures survivein vitro,the quantity of cellular material available for analysis, and the need to prepare the cells on a regular basis from fresh tissue. Techniques for immortalizing cells have existed for some time, but the repertoire of immortalizing genes has grown significantly. This has expanded our ability to generate useful cell lines of specific neural types that are better models of thein vivophenotype than previously. The constitutive expression of oncogenes keeps cells in a proliferative state that could lead to the loss of differentiated gene expression and function. An appealing improvement of immortalization methodology is the use of temperature-sensitive oncogenes that generate cell lines that can proliferate at a permissive temperature and “differentiate” at a nonpermissive temperature. The proliferation of such conditionally immortalized cell lines can be suppressed simply by increasing the temperature. Cell lines maintained at the nonpermissive temperature can enter into a stage in which they express differentiated properties of the cell. The potential ability of conditionally immortalized neural cell lines to accurately reflect theirin vivofunction has now been demonstrated on several occasions through transplantation experiments. In this report, the generation of these cell lines is described along with a discussion of their potential applications in neurobiology.     

The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures

It is well established that cell proliferation in batch (unfed) hematopoietic cell cultures is greatly inhibited relative to that in cultures with feeding. What is not known, however, is the nature of this inhibition. On the basis of our observations in hematopoietic cultures that cell proliferation ceases when the lactate concentration ([lactate]) exceeds 20 mM (accompanied by a decrease in culture pH), we investigated the effect of lactate accumulation on cell proliferation, metabolism, and differentiation. We differ in our approach from previous efforts in that we have tried to more accurately recreate the manner in which lactate accumulates in culture by employing a daily feeding protocol in which [lactate] and/or pH in the fresh medium was adjusted to match the conditions prior to feeding. We conclude that the decrease in pH associated with lactate accumulation significantly inhibits both cell proliferation and metabolism. Although inhibition in cultures with high [lactate] and low pH is similar to that in unfed cultures, pH control in unfed cultures does not alleviate the inhibition, indicating that other inhibitory factors are also present. Thus, pH control is necessary, but not sufficient, to eliminate inhibition of cell growth and metabolism in unfed hematopoietic cell cultures. We also conclude that high [lactate] and low pH have little effect on cell differentiation in fed cultures, although there is evidence to suggest that low pH may play a role in monocyte differentiation in unfed cultures.     

Systematic classification of melanoma cells by phenotype-specific gene expression mapping

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.     

Long Term Culture of Cells Derived from Mouse Blastocysts

The development of mouse blastocysts in primary culture has been followed for up to two months. The trophectoderm layer of the blastocyst gives rise to a monolayer of trophoblast cells; cells resembling both ectoplacental cone cells and primary giant cells are observed. The former can transform to giant cells, presumably secondary trophoblast, after several days in culture. Giant trophoblast cells are evident in the culture for much longer than the normal gestation period. Under the culture conditions described, the proportion of blastocysts showing substantial inner cell mass (ICM) proliferation in vitro is higher than that noted in previous studies. The ICM clumps develop into either egg cylinder-like structures, or, more commonly, into spherical, fluid-filled vesicles. The vesicles, which resemble yolk sac morphologically and biochemically [10, 11], continue to enlarge in size during several weeks of culture. The vesicles are attached to the underlying trophoblast monolayers by a stalk. Cells appear to migrate from this stalk out along the culture dish. The result after two to four weeks of culture is the appearance of a mixed monolayer containing a variety of different cell types. Secondary cultures of blastocyst cells have been continuously maintained in vitro for more than one year. Four lines of cells, all developing from the same pool of blastocysts, have been monitored for morphological, growth and biochemical properties, as well as chromosome number. Each line contained two or more morphologically distinct cell types, clearly indicated by cloning studies after eight months of culture. Doubling times and saturation densities among the four lines differed, as did biochemical properties. Although none of the cell lines resembled trophoblast biochemically after 7.5 months in culture, one line, MB4, possessed a number of biochemical properties in common with midgestation yolk sac. After a further five months of culture, some enzymes in the four lines were relatively unchanged; in other cases, notably with alkaline phosphatase, a sharp drop in enzyme activity was observed. One cell line, MB2, and specifically one of the cell types in this line, produced a yellow-orange pigment with a spectrum resembling that of a heme protein. After 7.5 months of culture, two of the four lines, MB21 and MB31, contained large numbers of cells with a diploid number of chromosomes. However, by 12.5 months in culture, the large majority of metaphases in all four cell lines possessed a hypotetraploid chromosome number. In a number of studies carried out to date, none of the cell lines generated tumors when injected into syngeneic hosts.     

Improved in vitro model systems for gastrointestinal infection by choice of cell line, pH, microaerobic conditions, and optimization of culture conditions

BACKGROUND: Commonly used in vitro infection cultures do not mimic the human gastrointestinal tract with regard to pH and microaerobic conditions. Furthermore, despite the importance of mucin-Helicobacter interactions, the cell lines used have not been selected for appropriate mucin expression. To make in vitro studies more applicable to human disease, we have developed coculture methods taking these factors into account. MATERIALS AND METHODS: Nine human gastrointestinal epithelial cell lines (MKN1, MKN7, MKN28, MKN45, KATO3, HFE145, PCAA/C11 Caco-2, and LS513) were investigated. Expression and glycosylation of mucins (MUC1, 2, 3, 4, 5AC, 5B, 6, 12, 13, and 16) were determined by immunohistochemistry. We analyzed the effect of microaerobic conditions and acidic pH on cell proliferation, viability, and apoptosis. RESULTS: Microaerobic culture, which is more physiological for the bacteria, did not adversely affect mammalian cell viability, proliferation, or induce apoptosis The cell lines varied in mucin expression, with MKN7 and MKN45 being most similar to gastric mucosa and Caco-2 and LS513 to intestinal mucosa, although none exactly matched normal mucosa. However, changes in culture conditions did not cause major changes in the mucin expression within cell lines. CONCLUSIONS: Culture conditions mimicking the natural environment and allowing the bacterial cells to thrive had no effect on cell viability or apoptosis, and very little influence on mucin expression of human gastrointestinal cells. Thus, it is feasible, using the simple methods we present here, to substantially improve bacterial-mammalian cell in vitro coculture studies to make them more reflective of human infection.     

Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions

Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand management of proliferating cells, AK1 and AK1beta were expressed in mouse neuroblastoma N2a cells and in human colon carcinoma SW480 cells. Glucose deprivation, galactose feeding, and metabolic inhibitor tests revealed a differential energy dependency for these two cell lines. N2a cells showed a faster proliferation rate and strongest coupling to mitochondrial activity, SW480 proliferation was more dependent on glycolysis. Despite these differences, ectopic expression of AK1 or AK1beta did not affect their growth characteristics under normal conditions. Also, no differential effects were seen under metabolic stress upon treatment with mitochondrial and glycolytic inhibitors in in vitro culture or in solid tumors grown in vivo. Although many intimate connections have been revealed between cell death and metabolism, our results suggest that AK1- or AK1beta-mediated high-energy phosphoryl transfer is not a modulating factor in the survival of tumor cells during episodes of metabolic crisis.     

Relation between sister chromatid exchange,cell proliferation and proportion of B and T cells in human lymphocyte cultures

Summary Human B and T lymphocytes differ in the rate of cell proliferation and frequency of sister chromatid exchange (SCE) when cultured separately in short-term cultures. This difference could theoretically be responsible for part of the variation in the SCE-frequency previously observed among healthy subjects since there is individual variation in the proportion of B and T cells in the peripheral blood. We have therefore studied cell proliferation and SCE-frequency in conventional short-term cultures of lymphocytes from 28 healthy subjects with different proportions of B and T cells. The percentage, of B or T lymphocytes did not correlate with the SCE-frequency, nor with the rate of cell proliferation in culture. However, a significantly higher SCE-frequency was found in slowly proliferating cultures than in cultures with a high rate of turn over. Thus, the rate of cell proliferation appears to be an important determinant of the SCE-frequency in conventional lymphocyte cultures. Although the data do not exclude attribution of the difference in SCE-frequency between rapidly and slowly growing cultures to differences in subpopulations of lymphocytes, it appears less likely that B and T cells constitute these tentative subpopulations.