We have developed a culture system for guinea pig alveolar type II cells using an epithelium-denuded human amnion membrane as a substratum. The differentiated morphology was maintained for 3 wk by both air-interface feeding and immersion feeding when type II cells were cultured on the basement membrane side of the amnion with fibroblasts on the opposite side (coculture). Functionally, high levels of surfactant protein B (SP-B) and C (SP-C) messenger ribonucleic acids (mRNAs) were expressed even after the 3-wk cultivation and surfactant protein A mRNA was detected on day 10 of the culture. The differentiation was also maintained when fibroblasts were cultured on lower chambers of the culture plates (separate culture). In contrast, culture of type II cells without fibroblasts (monoculture) could not preserve the mature morphology. When the monoculture was supplemented with keratinocyte growth factor or hepatocyte growth factor, a monolayer of rather cuboidal type II cells with apical microvilli was maintained. However, the percent area of lamellar bodies in these cells was significantly less than that in freshly isolated type II cells, and mRNA expressions of SP-B and SP-C were also considerably suppressed. These findings suggest that other growth factors or combinations of these factors are necessary for the maintenance of the differentiated phenotype. As substratum, a permeable collagen membrane or a thin gel layer of Engelbreth–Holm–Swarm mouse sarcoma extracts did not preserve the mature characteristics. This culture system using an acellular human amnion membrane may provide novel models for research in type II cells.

We describe procedures for isolating and culturing airway epithelial cells from chronically infected human lungs. Experience in our laboratory demonstrated the need to balance pathogen eradication against antibiotic toxicity to epithelial cells. To provide a logical basis for antibiotic selection and dose, we systematically analyzed the cytotoxicity of antibiotics useful against typical pathogens. Alone, colistin, ciprofloxacin, doxycycline, and tobramycin were moderately toxic at concentrations close to those used in cell culture, whereas amphotericin, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, sulfamethoxazole/trimethoprim, and vancomycin were nontoxic even at concentrations many times the antimicrobial level. Epithelial cytotoxicity of combined antibiotics was additive, with no evidence of competition or synergism. Antibiotics had little effect on initial cell attachment and did not acutely lyse cells, but inhibited subsequent growth. Interestingly, cytotoxicity decreased markedly with increasing epithelial cell density. Cystic fibrosis (CF) and non-CF epithelial cells showed no differences in sensitivity to the antibiotics tested and initial exposure to antibiotics did not affect the electrophysiologic properties of resistance or short circuit current in well-differentiated cells. Tailored combinations of antibiotics at appropriate doses killed even multidrug-resistant bacteria. Thus, epithelial cells can usually be cultured from chronically infected CF airways.

Pancreatic islets, isolated from neonatal pigs, and Sertoli cells, isolated from prepubertal rats, were cocultured in simulated microgravity utilizing the NASA-developed highly accelerating, rotating vessel (HARV) biochamber. Following 5 d of incubation, three-dimensional Sertoli–islet cell aggregates (SICA) retained the ability to secrete insulin when exposed to elevated glucose. SICA contained FasL-positive Sertoli cells and insulin-positive β-cells randomly organized within the spherical construct. The addition of 1% Matrigel induced the reorganization of aggregates (SICAs formed in the presence of Matrigel [SICAmgs]) showing the peripherialization and epithelialization of Sertoli cells and the centralization of islets in association with lumen-like spaces. The Sertoli cells, but not Matrigel, aided in preserving the structural integrity of HARV-incubated islets. Neither Matrigel nor Sertoli cells appeared to interfere with the ability of SICA or SICAmg to secrete insulin and express FasL.

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) cyCD4. Phenotypically, LBC cells are CD5 , CD8 , CD16 , CD24 , CD25 , CD2^−/dim, CD3^−/dim, TCRβ^−/dim, TCRγδ⁻, CD154⁻, CD40⁻, and CD45R⁻. Coexpress cyTCRβ, cyCD3, cyCD4, and yet lack surface CD4 expression. Transplantation of LBC cells into mice resulted in an aggressive T-lymphoblastic lymphoma that infiltrated lymph nodes, thymus, spleen, liver, ovary, and uterus but not peripheral blood or bone marrow. LBC cells display a modal chromosome number of 39 and a near-diploid karyotype. Based on the characterization data, we demonstrated that the LBC cell line was derived from an early T-cell lymphocyte precursor. We propose that the malignant cell transformation of LBC cells could coincide with the transition stage from late double-negative, DN3 (CD4⁻, CD8⁻CD44^−/low, CD25 ) or DN4 (CD4^−/low, CD8^−/low, CD44⁻, CD25⁻) to double-positive (DP: CD4 CD8 ) stage of T-cell development. LBC cells provide a T-lymphoblastic lymphoma model derived from a malignant early T-lymphocyte that can be potentially useful as a model to study both cellular regulation and differentiation of T-cells. In addition, LBC tumor provides a short latency neoplasm to study cellular regulation and to perform preclinical trials of lymphoma-related disorders.

ECV304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1–2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy, Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement, transmembrane electrical resistance, and activity of γ-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as in T24 cells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.

The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human OSE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34° C), these cell lines underwent 52–71 population doublings (PD) compared to 15–20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34° C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin, varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24–43% of cells. At 39° C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d, the cells assumed more epithelial morphologies, keratin expression reached 85–100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collagen III at both temperatures. As indicated by β-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39° C by 6 h and were present in all cells after 24 h. However, the cells underwent 1–3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.

The insect steroid hormone 20-hydroxyecdysone initiates a cascade of regulatory events in a temporal and tissue-specific manner by first binding to a complex of an ecdysone receptor (EcR) protein and a ultraspiracle protein. Using an antisense (As) ribonucleic acid approach, we show that disruption of EcR expression in transfected C7-10 cells from the mosquito Aedes albopictus affects survival and growth. From stably transfected cells, we recovered a new isoform of A. albopictus AalEcRa, which is named AalEcRb. The deduced amino acid sequence of AalEcRb was almost identical to that of AalEcRa, with the exception of a seven amino acid sequence near the C-terminus. Using polymerase chain reaction followed by restriction enzyme analysis, we found that AalEcRa is the predominant species expressed by wild-type C7-10 cells, while cells transfected with As-EcR expressed both isoforms at approximately equal levels.

Squamous cell carcinomas (SCCs) are the most common malignancies in man. While clinical specimens are theoretically ideal to study tumor development and progression, practical difficulties such as normal cell contamination, the presence of different cell types, and limited material make preclinical studies of model systems involving a homogeneous population of normal or transformed cells preferable. Tumor-associated antigens (TAAs) found on the cell surface, including integrins, mucins, cadherins, growth factor receptors, membrane bound antigens, and glycoproteins are known to play an important role in squamous carcinogenesis. We hypothesized that (1) alterations in TAA expression in vitro predict in vivo alterations, (2) analysis of a group of TAAs would provide a better indication of SCC tumorigenesis than any single marker, and (3) SCCs with independence from exogenous growth factors in vitro would demonstrate the most aggressive growth in vivo. The cell line which grew best in vitro without serum or other supplements demonstrated the most rapid tumor growth, whereas cell lines which grew only with supplements rarely formed tumors. Normal keratinocytes, eight SCC and two immortal keratinocyte cell lines were evaluated by flow cytometry for the expression of 10 cell surface markers, including α and β integrins, minor blood group–related carbohydrate determinants, carcinoembryonic antigen–related proteins, E-cadherin, and GA733 (epithelial glycoprotein, epithelial cell adhesion molecule). None of the cell lines with abnormal expression of ≤2 markers formed tumors, whereas all lines with altered expression of ≥3 markers formed tumors. Using GA733 expression as an example, we found that altered TAA expression in vitro predicted the presence of TAA alterations in clinical specimens. In summary, in vitro independence from supplements for optimal growth and altered expression of ≥3 cell surface markers were good predictors of SCC tumorigenesis. These findings may be useful in decreasing the need for whole animal tumorigenicity experiments.

Keratins form the largest subfamily of intermediate filament proteins and show strict lineage- and differentiation-associated expression in epithelial cells. Little is known about the mechanisms that control keratin protein synthesis in these cells. We have examined the effect of the differentiation-modulating agent, 5′-bromo-2′-deoxyuridine (BrdU), on keratin 19 (K19) expression in two human lung carcinoma cell lines, DLKP and A549. Treatment of both cell lines with 10 μM BrdU for 7 d induced the expression of K19 protein in keratin-negative DLKP cells, and significantly increased K19 protein expression in A549 cells. K19 messenger ribonucleic acids (mRNAs) were detected by Northern blot and reverse transcriptase-polymerase chain reaction analyses in both cell lines, but no increase in K19 mRNA levels was detected in either cell line, even with treatment with BrdU for up to 21 d. This suggests that K19 protein synthesis is normally blocked at a posttranscriptional level in DLKP cells, and BrdU can somehow reverse this block, resulting in the induction of K19 protein synthesis. Treatment of HL60, a leukemic cell line, with BrdU, resulted in noninduction of K19 protein synthesis, and no K19 mRNA transcripts were detected before and after BrdU treatment, possibly suggesting that BrdU is acting in an epithelial-specific manner to reverse a block in K19 protein synthesis in DLKP keratin–negative lung cancer cells. Therefore, DLKP (and A549) may be useful cellular models to investigate if this represents a regulatory step in early lung development or a mechanism whereby tumor cells possess the ability to down-regulate the expression of a more-differentiated phenotype.