Morphology and growth characteristics of epithelial cells from classic Wilms' tumors

The ability to establish cell cultures representing the epithelial component of Wilms' tumor was determined for 18 cases of classic Wilms' tumors. From these 18 cases only two resulted in the culture of epithelial cells. Although the tumors from both cases were composed of a prominent epithelial component, other classic tumors not producing epithelial cell cultures also possessed appreciable epithelial components. Likewise, heterotransplants of these two primary tumors failed to give rise to epithelial cell cultures, although cultures of the blastemal element were produced. This suggests that Wilms' tumors may be prone to differentiate in different directions at varying times during tumor growth, possibly dependent on local tumor environment. Epithelial cells from these two classic cases were grown in culture in basal medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with selenium, insulin, transferrin, hydrocortisone, tri-iodothyronine, and epidermal growth factor, on a collagen type I matrix with absorbed fetal calf serum proteins. One of the two cases also required the addition of bovine pituitary extract, ethanolamine, prostaglandin E1, and putrescine for optimum growth. Morphological analysis disclosed that the cultured cells were very similar to normal renal tubular cells in culture, except that the cells displayed little evidence for differentiated active ion transport and tended to grow in a multilayered arrangement. The culture of the epithelial cells from classic Wilms' tumors provides a model system for the study of tumor differentiation and progression.     

Cell culture for the study of epithelial cells

Keratinocytes depend on the support of fibroblasts or fibroblast products to grow from single cells into colonies. The essentials of a human stratified squamous epithelium can be constructed from single human epidermal keratinocytes and lethally irradiated fibroblasts. Established lines of mouse keratinocytes obtained from teratomas have many of the same properties. In this way it is possible to study many aspects of this epithelial tissue or organ under essentially cell culture conditions.     

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40--50% while effecting only a 10-15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.     

Receptor-mediated gene transfer to airway epithelial cells in primary culture

The reproducibility of tooth tapping frequencies was measured in young and elderly dentate subjects. Six rates of tapping, i.e. 40, 60, 90, 120, 160 and 200 times per min, were practised to the accompaniment of a metronome for 15 s before recording. After a 15-s break, subjects were asked to reproduce the same rate of tapping without metronome accompaniment, and these movements were recorded. It was determined that the young subjects regulated tooth tapping frequencies by controlling velocity of mandibular movement. On the other hand, the elderly subjects regulated tooth tapping frequency by controlling opening width.     

Primary culture and transfection of epithelial cells of human small intestine

OBJECTIVE: To evaluate perioperative and long-term morbidity in patients undergoing selective evaluation of coronary artery disease prior to abdominal aortic aneurysm (AAA) repair. DESIGN: Case series. SETTING: University and Veterans' Administration medical centers. PATIENTS: One hundred eighty-nine consecutive patients undergoing AAA repair between January 1989 and September 1996 were selectively evaluated for coronary artery disease and assigned to 1 of 3 groups: group 1, no abnormal cardiac history, normal electrocardiogram; group 2, minimal symptoms, history of myocardial infarction (MI), older than 70 years, diabetes mellitus, or congestive heart failure; or group 3, severe or unstable angina, ventricular dysfunction. INTERVENTIONS: Group 1 patients proceeded to AAA repair without further workup. Group 2 patients underwent pharmacologic or exercise stress testing followed by coronary angiography and intervention as required. Group 3 patients went directly to coronary angiography and intervention as needed. MAIN OUTCOME MEASURES: Perioperative MI, arrhythmias, or death. Long-term follow-up measures included MI and death. RESULTS: Adequate documentation was available on 171 patients. Twenty-four patients (14%) were in group 1. Of 136 patients (79.5%) in group 2, coronary angiography was performed in 36 (26%), followed by percutaneous transluminal coronary angioplasty (PTCA) in 9 (7%) and coronary artery bypass (CAB) in 5 (4%). Of 11 patients in group 3, 3 (27%) each received PTCA and CAB. Remote CAB or PTCA had been performed in 32 (19%) and 12 (7%) patients, respectively. Two perioperative deaths (1.1%) occurred in the 189 patients, one due to MI in a group 2 patient. There were 2 (1%) nonfatal MIs, both in group 2 patients who had no preoperative intervention. Arrhythmias and/or congestive heart failure occurred in 17 (9%) cases, 7 (39%) having had recent coronary revascularization (P = .001). By univariate analysis, only preoperative renal dysfunction predicted perioperative complications (P = .03) Overall survival by lifetable analysis was 87.9% and 69.7% at 3 and 5 years, respectively. CONCLUSION: Coronary artery disease is common in patients undergoing AAA repair, with 35.7% having preoperative coronary revascularization at some point. Selective preoperative coronary artery disease screening achieves excellent perioperative and late results in this population.     

Unique phosphorylation of vimentin filaments in glomerular epithelial cells in culture and in disease

Three cDNA clones for the Machado-Joseph disease gene (MJD1) were isolated, two of which have a new exon sequence and a distinct 3' terminal nucleotide sequence resulting in a new carboxyl terminal domain in the translated product. The nucleotide sequence of the other one is similar to the previously published one except for five polymorphisms, one of which is a single nucleotide substitution resulting in a change from the stop codon (TAA; allele A) to a tyrosine residue (TAC; allele C). Genetic analysis results suggest that Japanese MJD mutations are associated with allele A.     

Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells

Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.     

Effects of oxygen on pulmonary macrophages and alveolar epithelial type II cells in culture

Cultures of Chinese hamster lung fibroblasts and explants of 20-day-old rat lungs were exposed to 95% oxygen with 5% CO2 in vitro. The Chinese hamster cells had stopped dividing after 17 hours exposure and cell death occurred at a mean time of 67 hours (s.d. 15 hours). The rat lung explants showed macrophages moving over a monolayer of alveolar type II epithelial cells. Both cell types appeared to function normally for 24 hours but cell division in the type II cells was about 50% of control between 12 and 24 hours of exposure and virtually ceased after 26 hours. Cell death commenced after 4 days and was complete in 9 days. Macrophages divided freely in the control cultures but only one division was seen during exposure to oxygen and that occurred during the first 24 hours. Motility was reduced by 50% during the second day of exposure and stopped during the 3rd day. No live macrophages were seen after 4 days exposure. These culture systems appear very suitable for screening drugs for their protective effect against oxygen toxicity.     

Salivary epithelial cells in primary culture: characterization of their growth and functional properties

Mouse submandibular salivary gland cells were grown in primary explant culture. After an initial period of degeneration within the explant, surviving epithelial cells proliferated rapidly and duct-like structures recolonized the explant. Autoradiographic studies showed that a peak of DNA synthesis occurred after 4 days in vitro and that proliferation was enhanced by insulin and hydrocortisone. These cells retained specialized secretory function (protease activity) for at least 2 weeks in vitro. This enzyme is a differentiated product of granular tubule cells in vivo. Between 6 and 10 days, explants attached to the substrate. An outgrowth developed, consisting largely of ultrastructurally identifiable epithelial cells which formed pseudoglandular structures in the monolayer. Epithelium survived for over 6 months in primary culture but could not be serially transferred. Secondary cultures were rapidly overgrown by mesenchymal cells.     

A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.     

Effect of 5-hydroxytryptamine on bioelectric properties of airway epithelial cells in culture

Waardenburg syndrome type 1 is caused by mutations in PAX3. Over 50 human PAX3 mutations that lead to hearing, craniofacial, limb, and pigmentation anomalies have been identified. A PAX3 mutant allele, segregating in a family, can show reduced penetrance and variable expressivity that cannot be explained by the nature of the mutation alone. The Mus musculus Pax3 mutation Spd (Splotch-delayed, Pax3Spd), coisogenic on the C57BL/6J (B6) genetic background, produces in heterozygotes a white belly spot with 100% penetrance and very few other anomalies. By contrast, many Spd/+ BC1 progeny [F1 female Spd/+ (female Spd/+ B6 x male +/+ Mus spretus) x male +/+ B6] exhibit highly variable craniofacial and pigmentary anomalies. Of the BC1 Spd/+ progeny, 23.9% are estimated to be nonviable, and 32.1% are nonpenetrant for the white belly spot. The penetrance and expressivity of the Spd/+ genotype are controlled in part by the genetic background and the sex of the individual. A minimum of two genes interact with Spd to influence the craniofacial features of these mice. One of these genes may be either X-linked or sex-influenced, while the other is autosomal. The A-locus (Agouti) or a gene closely linked to A also plays a role in determining craniofacial features. At least one additional gene, possibly the A-locus or a gene linked to A, interacts with Spd and determines the presence and size of the white belly spot. The viability of BC1 mice is influenced by at least three factors: Spd, A-locus alleles or a gene closely linked to the A-locus, and the sex of the mouse. These BC1 mice provide an opportunity to identify genes that interact with and modify the expression of Pax3 and serve as a model to identify the genes that modify the expression of human PAX3 mutations.     

Substratum-dependent and region-specific control of attachment and proliferation of gastrointestinal epithelial cells in primary serum-free culture

A system for the primary serum-free culture of fetal rat gastrointestinal epithelial cells was used to examine the role of the extracellular matrix (ECM) in the attachment and proliferation of these epithelial cells. Forestomach epithelial cells (FSEC) were able to attach to and proliferate on plastic dishes without a substratum, while glandular stomach epithelial cells (GSEC) and duodenal epithelial cells (DEC) were unable to do so. The presence of a substratum promoted the attachment and proliferation of these epithelial cells. The effects of various components of the ECM differed depending on the type of cell. FSEC attached most efficiently to a substratum of fibronectin, while GSEC did so to laminin. DEC attached more efficiently to type I collagen and fibronectin than to any other substratum. FSEC proliferated most rapidly on laminin, while GSEC and DEC did so on collagen gels. These substrata induced the most efficient attachment and proliferation of FSEC, and they were effective in promoting the attachment and proliferation of GSEC and DEC in decreasing order of efficiency, indicating the existence of a head-to-tail gradient in the response of epithelial cells to substrata. The expression of c-myc mRNA in these cells differed depending upon the substratum on which they were cultured and the mRNA level was well correlated with the extent of the cell proliferation, indicating that the cell proliferation is mediated by c-myc gene expression, which is regulated by cell-ECM interactions. The results of the present study demonstrate that proliferation of gastrointestinal epithelial cells is regulated region-specifically not only by soluble factors but also by insoluble components of the ECM.     

Isolation, cell culture, and characterization of oviduct epithelial cells of the cow

This report describes an easy method of isolation and cell culture of the epithelial cells of cow oviduct. Incubation of cow oviduct with 0.1 mg/ml collagenase in the lumen for 90 minutes helped to dislodge large numbers of ciliated and secretory epithelial cells. The isolated cells, when seeded on plastic, proliferated very quickly and became confluent in 8-10 days in 35 mm Petri dishes. The isolated ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The cultured epithelial cells were keretin positive. The isolated bovine oviduct epithelial cells, when cultured on plastic precoated with 10 mg/ml matrigel, organized themselves into hollow tubes or spheres with microvilli directed towards the lumen. The epithelial cells seeded on 2 mg/ml matrigel became subconfluent in 15-20 days after seeding. The histoarchitecture of the secretory cells growing in vitro on matrigel resembled that of intact oviduct secretory epithelial cells. Occasional ciliated cells containing large number of mitochondria were observed in the monolayer cultured on 2 mg/ml matrigel substratum but possessed few cilia. The oviduct epithelial cells cultured on 2 mg/ml matrigel incorporated 35S-methionine linearly into protein up to 8 hours in the presence of estradiol or progesterone. The fluorograph of the newly synthesized proteins indicated the presence of an additional 60 kd protein in the cell extract of epithelial cells incubated with estradiol.     

Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid

Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mM aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 10(6) cells cm(-2) (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mM and 2.72 mM respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 microM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml(-1)). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 microg ml(-1)) or actinomycin D (10 microg ml(-1)) even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 10(6) cells cm(-2)), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h(-1) at a cell density of 0.02 x 10(6) cells cm(-2). Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurring in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.     

Differential effects of gamma interferon on Chlamydia trachomatis growth in polarized and nonpolarized human epithelial cells in culture

The effects of gamma interferon (IFN-gamma) on Chlamydia trachomatis growth in polarized epithelial cells were examined. The range of IFN-gamma concentrations causing aberrant chlamydial growth was wider in polarized than in nonpolarized cultures. Results indicate that chlamydial growth modulation in polarized cells readily leads to persistence and better reflects in vivo conditions.     

A simple technique for the long-term non-polarised and polarised culture of human fallopian tube epithelial cells

Fallopian tubes were obtained from 25 women undergoing abdominal hysterectomy. Pieces of fallopian tube mucosa were placed in culture flasks containing minimum essential medium in Earle's salts supplemented with fetal bovine serum. First passage was carried out after 7-10 days and subcultures in 4-5 days. For polarised cell culture, epithelial cells were seeded onto an extracellular matrix system. New epithelial cells were seen on day 2-3 of the primary culture and epithelial patches on day 7-10. Cells reached confluence in 4-5 days in subcultures. The cells could be subcultured for 7-11 passages with a life span of 42-60 days. Epithelial origins of the cells were confirmed by immunofluorescence staining with anti-cytokeratin antibody. Polarised cells showed a columnar pattern, microvilli on their apical surface and basally located nucleus whereas non-polarised cells were flat. It was concluded that the human fallopian tube epithelial cells can be cultured in vitro to create non-polarised and polarised cell layers by using a simple and reproducible technique and this system can be a potential model to study function of the fallopian tube.     

Changes in DNA distributions and ploidy of CHO cells as a function of time in culture

Chinese hamster ovary (CHO) cells maintained in continuous culture for 3 to 5 months may undergo subtle changes in drug sensitivity response, growth kinetics, plating efficiencies, et cetera. Our studies done independently in two different laboratories, using flow cytometry, indicate that the DNA histogram patterns change at about 11 wk, from populations with an approximate diploid DNA content to populations also composed of triploid and tetraploid cells. Chromosome counts also change from distributions of 21 to 22 to populations of cells having 21 to 22, 34 to 35 and 44 to 46 chromosomes. These alterations occur earlier (at 8 to 9 wk) in cell populations previously treated with anticancer drugs.