Differentiation Of Human Epithelial Cells Research Articles

Vav1 Sustains the In Vitro Differentiation of Normal and Tumor Precursors to Insulin Producing Cells Induced by all-Trans Retinoic Acid (ATRA)

All-trans retinoic acid (ATRA) promotes the development and the function of insulin producing cells and induces partial differentiation of pancreatic tumor cells. A number of evidences clearly indicate that the ATRA mediated signaling may have a substantial role in therapeutic approaches based on restoration of functional β-cells. Among the proteins up-regulated by ATRA, Vav1 is involved in maturation and function of haematopoietic cells and is essential for retinoids induced differentiation of tumor promyelocytes. The presence of Vav1 in solid tissues, including pancreas, is considered ectopic and no role in the differentiation of human epithelial cells has so far been described. We demonstrated here that Vav1 sustains the maturation to β-cells of the normal precursors human Biliary Tree Stem/progenitor Cells (hBTSCs) induced by a differentiation medium containing ATRA and that, in the mature normal pancreas, insulin-producing cells express variable levels of Vav1. Using pancreatic ductal adenocarcinoma (PDAC)-derived cells, we also revealed that the ATRA induced up-modulation of Vav1 is essential for the retinoid-induced trans-differentiation of neoplastic cells into insulin producing cells. The results of this study identify Vav1 as crucial molecule in ATRA induced maturation of insulin producing cells and suggest this protein as a marker for new strategies ended to restore functional β-cells.Graphical abstract

Epithelial–mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro

Epithelial-mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase-20 (MMP-20), and kallikrein-4 (KLK-4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum-free medium. Both the epithelial cells and fibroblasts were co-cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in-situ hybridization and RT-PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP-20 and KLK-4 in epithelial cells of Malassez co-cultured with periodontal ligament fibroblasts. in-situ hybridization and RT-PCR confirmed significant mRNA expression of these factors in co-cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial-mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.

Profilin1 facilitates staurosporine‐triggered apoptosis by stabilizing the integrin β1–actin complex in breast cancer cells

Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5β1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5β1. These results suggest that the insufficient fibronectin caused by the integrin α5β1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin β1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5β1, and suggested a new target for breast cancer therapy.

A combined in vitro and in vivo system for culture of epithelial cells from distal human airways

A system for combined in vitro and in vivo culture of epithelial cells from distal human airways was established. Lung tissues that appeared to be generally normal were obtained from lungs removed surgically from patients with lung cancer. Small pieces of peripheral lung tissue were placed on culture dishes and cultured in F-12 complete medium containing serum and various growth factors, to obtain outgrown cells. For in vivo culture, rat tracheal grafts were de-epithelialized by freezing and thawing and were then used as culture vessels. Outgrown cells were harvested after 4 weeks of in vitro culture, inoculated into the denuded tracheal grafts, and then implanted into nude mice. For comparative purposes, bronchial fragments were also cultured in vitro and in vivo, by the same method. In vitro efficiency of colony formation was about the same for cells derived from peripheral lung tissue and from bronchial tissue (14.8 +/- 8.9% and 16.0 +/- 4.7%, respectively). Four weeks after implantation, the grafts were retrieved and processed for morphologic evaluation. By that time, grafts in both groups had totally re-epithelialized. Therefore, the growth potential of the cells derived from peripheral lung tissue and from bronchi in vivo appeared to be almost the same. Newly formed epithelial cells in grafts showed the same well-developed pseudostratified columnar form in both groups at 4 weeks. The time course of epithelial cell differentiation was also studied, with outgrown cells from lungs. Two days after implantation, undifferentiated cells were attached to the inner surface of the grafts as a single cell layer, and at 4 days, small cell nests containing mitotic cells were observed. At 1 week, the grafts were totally covered with undifferentiated cells. Over 2 to 3 weeks, differentiated cells (ciliated, secretory, and basal cells) appeared, and the epithelia had become fully developed by 4 weeks. As reported previously, cells that outgrew from lung explants were considered to be derived from bronchioles. Therefore, this system may be useful for studies of growth and differentiation of human bronchiolar epithelial cells under various conditions.

Monetary incentives in research and participation in HIV/STD risk reduction programmes.

<h3>Abstract</h3> Organoids development relies on the self-organizing properties of adult stem cells to create structures which recapitulate the architecture, functionality, and genetic signature observed in original tissues. Little is known...

Effects of retinoic acid combined with interferon-gamma on the expression of a tumor rejection antigen (heat shock protein gp96) in human cervical cancer

Santin AD, Hermonat PL, Ravaggi A, Chiriva-Internati M, Pecorelli S, Parham GP. Effects of retinoic acid combined with interferon-γ on the expression of a tumor rejection antigen (heat shock protein gp96) in human cervical cancer. Int J Gynecol Cancer 1998; 8: 158–163. Retinoids and interferons are important regulators of human epithelial cell differentiation and have been used successfully in the clinical treatment of HPV-involved cervical cancer. Hsp-gp96 molecules recently have been implicated in the presentation pathways of tumor and viral antigens and have resulted in an effective protein vaccine in several tumor animal models. In this study, we analyzed at the transcriptional (northern blot) and post-transcriptional levels (western blot) the effects of retinoic acid and interferon-γ alone or in combination on the expression of the tumor rejection antigen heat shock protein-gp96 molecules (Hsp-gp96) in two human cervical carcinoma cell lines (SiHa and HT-3). Exposure to retinoic acid or IFN-γ was able to upregulate the expression of Hsp-gp96 molecules in both cervical cell lines studied. Exposure to the combination of IFN-γ plus retinoic acid significantly upregulated (in an additive manner) Hsp-gp96 molecules when compared to the levels obtained after exposure to IFN-γ or retinoic acid alone. Northern blot analysis of mRNA for Hsp-gp96 molecule confirmed that the activity of both of these biologic response modifiers was at the transcriptional level. Taken together, these data indicate that the combination of these modalities could induce a previously unrecognized additive effect on the expression of another immunologically important antigen, the Hsp-gp96 tumor rejection antigen, in human cervical cancer cells. These findings extend previous reports on the effects of retinoids and IFN-γ on tumor cells and provide further support for the combined use of these powerful biologic response modifiers in the adjuvant treatment of cervical cancer.

Effects of retinoic acid combined with interferon-gamma on the expression of major-histocompatibility-complex molecules and intercellular adhesion molecule-1 in human cervical cancer.

Retinoids and interferons are important regulators of human epithelial cell differentiation and have been successfully used in the clinical treatment of HPV-involved cervical cancer. In this study, 2 HPV-positive human cervical-carcinoma cell lines were analyzed for their surface expression of MHC-Class-I, MHC-Class-II and ICAM-1 surface antigens before and after exposure to all-trans retinoic acid, interferon-gamma and the combination of the 2 compounds. In addition, the effects on HLA-Class-I-mRNA expression were evaluated after such treatments. Both cell lines expressed MHC-Class-I molecules, and their levels were markedly up-regulated after exposure to IFN-gamma. Similarly, MHC-Class-II and ICAM-1 antigens were either induced or significantly up-regulated by IFN-gamma. Exposure to all-trans retinoic acid was also able to significantly increase the expression of MHC-Class-I and ICAM-I antigens as compared with untreated tumor cells. However, unlike IFN-gamma, retinoids were not able to induce the expression of HLA-Class-II surface antigens. Exposure to the combination of IFN-gamma plus retinoic acid significantly up-regulated (in an additive manner) HLA-Class-I and ICAM-1 molecules as compared with the levels obtainable after exposure to IFN-gamma alone. Finally, Northern-blot analysis of HLA-Class-I-mRNA expression confirmed that the activity of both of these biologic response modifiers was at transcriptional level. These data indicate that the combination of these modalities could induce an additive effect on the expression of immunologically important surface antigens on human cervical-cancer cells. These findings, together with the known anti-proliferative effects mediated by retinoids and IFN-gamma on tumor cells, further support the combination of these agents in the treatment of pre-invasive and invasive human cervical cancer.

Effect of beta-lymphocyte- and NPC-derived EBV-LMP1 gene expression on in vitro growth and differentiation of human epithelial cells.

The effect of expression of the Epstein-Barr-virus (EBV) latent membrane protein (LMP1) derived from B-lymphocytes (B) and nasopharyngeal carcinoma (NPC) (C) on the in vitro growth and differentiation of a human keratinocyte line, Rhek-1, was analyzed in clonal growth and in in vitro differentiation assays. In contrast to the polygonal parental cells, the B-LMP1-expressing sublines were spindle-shaped while the C-LMP1-expressing cells were pleomorphic. Both B- and C-LMP1-expressing sublines showed increased proliferation as evidenced by: (1) higher colony-forming efficiency (CFE) and larger colony size at reduced serum levels; (2) an increased number of epithelial cell layers formed in the air-liquid-interface culture system and (3) increased expression of proliferative cell nuclear antigen (PCNA). At low serum concentration, the C-LMP1-expressing sublines formed larger colonies than those expressing B-LMP1. In the air-liquid-interface culture system, both B- and C-LMP1-expressing lines showed reduced epithelial differentiation resulting in reduced stratification and reduced involucrin expression similar to those of the cancer cell line, Siha. The results of the present study indicate that the expression of LMP1 in human keratinocytes is associated with morphological transformation and predisposes these cells to a more neoplastic phenotype. The structural difference between the 2 genes responsible for the functional differences and transforming ability will be pinpointed in further experiments.

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: Differentiation response distinguishes KGF from EGF family

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.

Growth and differentiation of human nasal epithelial cells in culture. Serum-free, hormone-supplemented medium and proteoglycan synthesis.

Ham's F12 medium supplemented with insulin (Ins), transferrin (Tf), epidermal growth factor (EGF), hydrocortisone (HC), T3, cholera toxin (CT), and bovine hypothalamus extract (BHE) was developed for in vitro growth of human nasal epithelial (HNE) cells. The HNE cells were dissociated from freshly excised nasal polyps or turbinates with protease. Colony-forming efficiency of primary HNE cells was approximately 5%. Growth studies showed Ins, BHE, and CT were essential for growth; HC, EGF, Tf, and T3 were also stimulatory for growth. The growth rate in this serum-free, hormone-supplemented medium was 24 h per population doubling. Up to 20 population doublings and 3 passages of dissociated HNE cells could be achieved. Addition of serum to this culture medium inhibited epithelial cell growth. Vitamin A had no apparent effect on cell growth but induced an alteration in the morphologic characteristics of the cell. The epithelial nature of cultured cells was confirmed by positive staining with antihuman keratin antibody, ultrastructural studies, and by formation of a columnar, ciliated epithelium in denuded tracheal grafts repopulated by these cultured HNE cells. Biochemical analyses of glycoproteins (labeled with 3H-glucosamine and/or 35S-sulfate) secreted by cultured HNE cells were unable to demonstrate the secretion of mucinlike glycoproteins in culture. Instead, major secretory products of cultured cells were hyaluronate and heparan sulfate. These results were in agreement with morphologic observations that showed no mucus-secreting granules in cultured cells. Dome formation was observed in high cell density cultures. We conclude that HNE cells can be cultured in well-defined culture media. As indicated by formation of domes, these cells may be useful for in vitro ion transport studies. Further differentiation, however, may be required for studies of mucin synthesis.