Retinoic acid and hydrocortisone strengthen the barrier function of human RPMI 2650 cells, a model for nasal epithelial permeability

The nasal pathway represents an alternative route for non-invasive systemic administration of drugs. The main advantages of nasal drug delivery are the rapid onset of action, the avoidance of the first-pass metabolism in the liver and the easy applicability. In vitro cell culture systems offer an opportunity to model biological barriers. Our aim was to develop and characterize an in vitro model based on confluent layers of the human RPMI 2650 cell line. Retinoic acid, hydrocortisone and cyclic adenosine monophosphate, which influence cell attachment, growth and differentiation have been investigated on the barrier formation and function of the nasal epithelial cell layers. Real-time cell microelectronic sensing, a novel label-free technique was used for dynamic monitoring of cell growth and barrier properties of RPMI 2650 cells. Treatments enhanced the formation of adherens and tight intercellular junctions visualized by electron microscopy, the presence and localization of junctional proteins ZO-1 and β-catenin demonstrated by fluorescent immunohistochemistry, and the barrier function of nasal epithelial cell layers. The transepithelial resistance of the RPMI 2650 cell model reached 50 to 200 Ω × cm², the permeability coefficient for 4.4 kDa FITC-dextran was 9.3 to 17 × 10⁻⁶ cm/s, in agreement with values measured on nasal mucosa from in vivo and ex vivo experiments. Based on these results human RPMI 2650 cells seem to be a suitable nasal epithelial model to test different pharmaceutical excipients and various novel formulations, such as nanoparticles for toxicity and permeability.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9493-7) contains supplementary material, which is available to authorized users.     

Coexpression of tumor-associated alpha 2-macroglobulin and growth factors in human melanoma cell lines.

alpha 2-Macroglobulin (alpha 2M) is known as an inhibitor of various proteinases and to bind several of the growth factors. We previously demonstrated that clonal variation exists in the production of alpha 2M in a human melanoma and that this variation may be associated with growth stimulation. We have now analyzed six human melanoma cell lines for the simultaneous expression of TGF-alpha, TGF-beta, PDGF-A chain, PDGF-B chain, and tumor-associated alpha 2M. In Northern blot analysis TGF-alpha was detected in four of the cell lines, TGF-beta in all, PDGF-A chain in three, and PDGF-B chain in none of the cell lines. alpha 2M, detected by immunoblotting, varied significantly between the different melanoma cell lines and only one cell line was found to be negative. Evaluation of growth-promoting activity in conditioned media suggested that alpha 2-macroglobulin, secreted by these tumor cell lines, is a significant modulator of melanoma cell growth.     

Monoclonal antibody 425 inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived EGF/TGF-alpha

Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha. Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors. Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha. Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media. All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha. EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels. EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media. In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha. Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media. Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion.     

Production of an auto-stimulatory growth factor by human hepatoma cells abrogates requirement for a brain-derived factor

Summary Human hepatoma cells grow at high cell density in the absence of exogenous growth factors. At low cell density, two different hepatoma cell lines required a novel growth factor from brain tissue. A factor with similar physico-chemical properties in the concentrated medium from high density cultures completely substituted for the brain extract. The autogenous secretion of a novel liver cell growth factor that is concentrated in brain tissue may underlie in part the unregulated growth of hepatomas. EDITOR'S STATEMENT Collective properties of growth factor activities for hepatoma cells in bovine brain extract and the medium of hepatoma cells suggest a neural tissue-derived liver cell growth factor that may be autogenously produced by liver tumor cells. Purification and characterization of such a factor may lead to selective inhibition of hepatoma, cell proliferation. David W. Barnes     

Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth

Summary A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [¹²⁵I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 μM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 μg/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells.     

Melanoma growth stimulatory activity: isolation from human melanoma tumors and characterization of tissue distribution

Melanoma growth stimulatory activity (MGSA) is an acid and heat stable, auto-stimulatory growth factor which was first isolated from culture medium conditioned by the Hs294T human melanoma cell line. In this report, we describe the purification of MGSA from acid ethanol extracts of Hs294T tumors grown in nude mice using a series of Bio-Gel P30, reverse phase-high performance liquid chromatography and heparin-sepharose steps. This modified procedure provides a 10-fold improved yield of MGSA over previously published procedures. Purified MGSA-stimulated melanoma cell growth in both 3H-thymidine and cell number assays over a concentration range of 0.06 to 6 ng/ml. The MGSA bioactivity was primarily associated with fractions which exhibited molecular weights of 16 and 13-14 Kd based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor-beta (TGF beta), and epidermal growth factor (EGF) in combination with TGF beta did not stimulate 3H-thymidine incorporation in Hs294T cells under the conditions used for MGSA bioassay. Monoclonal antibody to MGSA was used to screen melanoma and benign nevus cultures as well as fixed sectioned tissue for MGSA. The majority of the melanoma cultures were MGSA positive, while most nevus cultures were MGSA negative. However, when fixed sectioned tissue was screened for MGSA immunoreactivity, melanoma tissue was MGSA positive and three-fourths of the benign nevi were MGSA positive. In addition, epidermal keratinocytes and several tissues exhibiting proliferative disorders contained immunoreactive MGSA. These data suggest that MGSA may be a normal regulator of growth and that the microenvironment of the cell may regulate both production of MGSA and response to MGSA.     

A cell line of human malignant astrocytoma producing autocrine growth factor

Summary A cell line was established from an anaplastic astrocytoma from a 69-yr-old female. The cells have been serially subcultured over 300 times for 6 yr without showing any sign of cell senescence. Their doubling time is about 36 h. The cells are fusiform and often hexagonal in sparse culture, but become spindle-shaped and formed mosaic structure in confluent culture. Under electron microscopy, intermediate filaments were randomly distributed in the cytoplasma, especially in the perinuclear space. The chromosome number was near tetraploid and varied from 86 to 94 chromosomes with a modal number of 91. The alpha and beta subunits of S-100 protein, vimentin, and glial fibrillary acidic protein (GFAP), which are reliable markers of astrocytic cells, were demonstrated in a large number of cells by immunoperoxidase staining. The results of immunoblotting showed that the expression of vimentin was much higher than that of GFAP. The tumorigenicity of the cells was revealed by xenografting into nude mice, which were X-irradiated before inoculation. Culture medium conditioned by the cells promoted growth of these cells in serum-free conditions and of normal rat glial cells in serum-depleted culture. The growth-promoting effect of conditioned medium was lost by trypsinization and reduced by boiling. These findings suggest that these cells are derived from neoplastic astrocytic cells and secrete a self-acting polypeptide growth-promoting factor into the culture medium. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.     

N-terminal amino acid sequence of leukemia derived growth factor (LGF) from human erythroleukemia cell culture

Summary A human erythroleukemia cell line, K-562 T1, was adapted to a protein-free chemically defined medium (1); that is, the medium does not contain any proteins such as exogenous hormones, growth factors, serum and serum albumin. The K-562 T1 cells which can proliferate in a protein-free medium are one of the model systems suitably supporting the autocrine hypothesis (2), which claims that cancer cells produce and respond to their own growth factors (3). The K-562 T1 cells were cultured in a protein-free medium at large scale and the growth factors were purified from the conditioned medium. It was found that K-562 T1 cells produce at least two growth factors; one is LGF-I (leukemia-derived growth factor-I) which can stimulate the proliferation of a wide range of human leukemia cell lines and the other is LGF-II (leukemia-derived growth factor-II), which can contribute to the growth of fibroblasts. LGF-I was purified using QAE-Sephadex, Bio Gel P-60 and Mono S FPLC. The purified protein was found to be homogenous by SDS-polyacrylamide gel electrophoresis and NH₂-terminal sequence analysis. The molecular weight of LGF-I was 20,000 by SDS-polyacrylamide gel electrophoresis. The 30 NH₂-terminal residues of LGF-I are the same as that of ubiquitin. Ubiquitin is a protein found in eukaryotic cells with molecular weight of 8,600. In the nucleus ubiquitin is conjugated to histone 2A to form the nuclear protein A24 which may play a role in regulation of chromatin structure (3), and in the cytoplasm is part of an ATP-dependent non-lysosomal proteolytic pathway (4). However, its physiological significance has not yet been fully resolved. Ubiquitin purified from bovine thymus did not show cell proliferating activity for any cells tested. The results suggest that LGF-I is a new autocrine growth factor with a molecular weight of 20,000 daltons, containing ubiquitin at the NH₂-terminal end. This work was supported by funds obtained under the Research and Development Project of Basic Technologies for Future Industries from the Ministry of International Trade and Industry of Japan. Editor’s Statement Identification of sequences identical to ubiquitin associated with the leukemia-derived growth factor described in this report is particularly intriguing considering recent reports of association of ubiquitin with surface membrane receptors of lymphocytes and fibroblasts. References: SCIENCE vol. 231, pgs, 823–829; NATURE vol. 323, pgs 226–232, 1986. David W. Barnes     

Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

Summary To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Cells were studied in Passages 2 to 8. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue. Supported in part by grant DK 31063 from the National Institutes of Health, Bethesda, MD.     

Heparin-binding (fibroblast) growth factors are potential autocrine regulators of esophageal epithelial cell proliferation

Summary A serum-free culture system supplemented with neural tissue extract for normal and tumor human esophagi was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life span but retained morphology and other characteristics of normal epithelial cells. The cells formed a small cyst consisting of keratined squamous epithelium in syngenic hosts. A screen for growth factors that stimulated growth of the nonmalignant MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed large invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 and proliferating nonmalignant MEE cells had properties similar to HBGF-1 (acidic fibroblast growth factor). These results constitute a cultured mouse esophageal epithelial cell model for study of conversion of immortalized premalignant cells to malignant cells, and suggest that conversion from a state of cell cycle-dependent autocrine expression of one or more members of the HBGF family to a state of constitutive expression correlates with and may contribute to malignancy. The work was supported in part by grants CA37589 and DK35310 to Dr. McKeehan, from the National Cancer Institute, Bethesda, MD.     

Production and reception of growth factors in placenta during physiological pregnancy and the pregnancy, complicated with gestosis

30 women with physiological pregnancy and 28 women with gestosis were examined. The content of epidermal growth factor (EGF), vascular-endothelial growth factor (VEGF) and their receptors were studied in the early chorion obtained after abortion and in the full-term placenta using the ELISA method. The process of normal gestation was characterized by the increase of the placental production both of the EGF and VEGF. During the pregnancy complicated with gestosis and miscarriage in the first trimester the content of EGF and its receptor was lower compared to the physiological values. For VEGF and its receptor opposite changes were found: the increase of quantity of the growth factor and the decrease of its receptor. In the case of gestosis and term of pregnancy the content of the both growth factors and their receptors was lower than in corresponding controls. The changes in production of the angiogenic growth factors and their receptors in the placenta may have the pathogenic importance in the development of gestosis.     

Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.     

Amino acid transport in a human neuroblastoma cell line is regulated by the type I insulin-like growth factor receptor

Insulin-like growth factor I (IGF-I) and IGF-II stimulate cancer cell proliferation via interaction with the type I IGF receptor (IGF-IR). We put forward the hypothesis that IGF-IR mediates cancer cell growth by regulating amino acid transport, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply. We examined the effects of alphaIR3, the monoclonal antibody recognizing IGF-IR, on cell growth and amino acid transport across the cell membrane in a human neuroblastoma cell line, SK-N-SH. In the presence of alphaIR3 (2 micro/ml), cell proliferation was significantly attenuated in both control (2 mM glutamine) and glutamine-deprived (0 mM glutamine) groups. Glutamine deprivation resulted in significantly increased glutamate (system X(AG)(-)), MeAIB (system A), and leucine (system L) transport, which was blocked by alphaIR3. Glutamine (system ASC) and MeAIB transport was significantly decreased by alphaIR3 in the control group. Addition of alphaIR3 significantly decreased DNA and protein biosynthesis in both groups. Glutamine deprivation increased the IGF-IR protein on the cell surface. Our results suggest that activation of IGF-IR promotes neuroblastoma cell proliferation by regulating trans-membrane amino acid transport.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Autocrine stimulation by insulin-like growth factor I is involved in the growth, tumorigenicity and chemoresistance of human esophageal carcinoma cells

Insulin-like growth factor I (IGF-I) receptor (IGF-IR)-mediated signals are known to be involved in cell growth and transformation and prevention of apoptosis. In this study, we demonstrated the coexpression of IGF-I and IGF-IR in human esophageal carcinoma tissues. We also demonstrated the IGF-I autocrine system in esophageal carcinoma cell lines. Both the CE48T/VGH and CE81T/VGH cell lines showed proliferative responses to IGF-I stimulation. Autokinase activity of IGF-IR in these cells can be triggered by the exogenous addition of IGF-I. In addition, an IGF-I peptide antagonist, JB1, specifically inhibited ligand-induced receptor autophosphorylation in a dose-dependent manner. Under serum-free conditions, JB1 also reduced the degree of IGF-IR phosphorylation and cell numbers. Furthermore, the addition of JB1 decreased the number of CE81T/VGH colonies formed in methyl cellulose agar and the size and the incidence of tumors which grew in mice with severe combined immunodeficiency. These results imply that an IGF-I autocrine system in human esophageal carcinoma cells could stimulate tumor growth. Finally, we found that IGF-I prevented the apoptosis of CE81T/VGH cells induced by chemotherapeutic drugs, such as cisplatin, 5-fluorouracil and camptothecin. Thus, interruption of IGF-IR function may provide a way to retard tumor growth and increase the sensitivity of esophageal carcinoma to chemotherapy.     

Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

INTRODUCTIONAsaderivativeofvitaminA,RAcaninhibittheproliferati0nofmanymalignantcel1sallde1icitdifferentiationinsometumorce1lsI1-5].RecentstudieshaveshownthatRAmodu1atessynthesisofover4Oproteinsthroughitsnucleicrecept0r[6].Forinstance,RAinducesthesynthesis0ffibronectin(FN)incertaintumorcellsandactivatesthegenecodingfOrB1sllbunitoflaminin(LN)causingitsexpressi0n[7,8].FNandLN'areknownt0bethemostimportantcomponentsofnon-c0llageng1ycoproteinsintheextracellu1armatrixandareclose1yrelatedt…