Growth Of Human Epithelial Cells Research Articles

TGFβ activates insulin‐like growth factor 1‐mediated signaling pathway in human liver cancer cells (144.9)

Transforming growth factor β (TGFβ) potently inhibits the growth of human epithelial cells. However, neoplastic epithelial cells become resistant to TGFβ‐mediated mitoinhibition, and the mechanisms for this alteration during tumorigenesis are not fully understood. In this study, we demonstrate that TGFβ sensitises PLC/PRF/5 cells (Human hepatocellular carcinoma cell line) to insulin‐like growth factor 1 (IGF1), and enhances cell survival in the presence of sorafenib, a therapeutic agent used to treat advanced stage HCC. Firstly, we observed that TGFβ significantly increased mRNA and protein expression of insulin like growth factor 1 receptor (IGF1R) in PLC cells. To test how this result would affect IGF1 signaling, PLC cells were treated with TGFβ for up to 72 hrs, then treated with IGF1 for 30 min. PLC cells pretreated with TGFβ had a much greater response to IGF1 treatment, exhibiting significantly increased levels of pIGF1R and pAkt compared with IGF1 treatment only. Since IGF1 is a pro‐survival mitogen, we further tested whether an increase in IGF1 sensitivity would reduce apoptosis in the presence of sorafenib. The cells were treated with TGFβ for 48 hrs before the incubation with sorafenib for up to 24 hrs. We found that TGFβ pretreatment led to a significant reduction in both cleaved caspase 7 and cleaved PARP, and a significant increase in cell viability compared with sorafenib treatment only. Taken together, our data suggest that TGFβ can regulate the survival of HCC cells via the activation of IGF1 pro‐survival pathway and prevent Sorfafenib‐induced apoptosis.Grant Funding Source: NIH/NCI

Abstract 4280: Differential influence of naïve & tumour-conditioned mesenchymal stem cells on the growth of human epithelial cells in in vitro monolayer co-cultures & 3D hollow fibre systems

Abstract Background: Tumour cell behaviour is altered by the non-neoplastic portion of the stroma composed of fibroblasts, mesenchymal stem cells (MSC), pericytes, endothelial and inflammatory cells actively intertwined with the tumour parenchyma. Epithelial to mesenchymal transition (EMT) is a key phenotypic change involved in carcinoma progression, resulting in profound phenotypic changes to malignant cells e.g. loss of cell-cell adhesion, loss of cell polarity and acquisition of migratory and invasive properties. Human MSC can give rise to endothelial-like or pericyte-like cells, and are also capable of differentiating into carcinoma associated fibroblasts (CAFs) when exposed to soluble factors produced by tumour cells, which support the malignant epithelial cells resulting in overall enhanced tumour growth and survival in vitro and in vivo. EMT is common within primary tumours and may be further promoted by associated CAFs. Objectives: The objectives of our current research are to recapitulate the human tumour micro-environment within the hollow fibre model (HF) and apply a real-time bio-imaging component to enable the non-invasive analysis of biological parameters associated with tumour progression. Results: Using tumour cells stably expressing a bioluminescent reporter gene, we have investigated tumour growth in the presence and absence of naive (MSC) and tumour-conditioned (tcMSC) human mesenchymal stem cells both within in vitro cell cultures and the HF model. Real-time imaging of bioluminescent tumour cells demonstrated that these cells can actively proliferate within the HFs; and the presence of MSC or tcMSC significantly enhances tumour growth compared with tumour cells alone. Immuno-staining analysis revealed that a stromal network is established around the periphery of the fibre and that with prolonged culture, MSCs acquire a ‘myofibroblastic’ phenotype (elevated expression of αSMA and FSP1) resulting in growth promoting effects similar to CAFs. Here, we also demonstrate downregulation of E-Cadherin – a hallmark of EMT- and upregulation of genes of the EMT trascriptome, in both direct and indirect co-cultures.Conclusions: Here we provide experimental evidence that naive MSC or tcMSC may provide a tumour cell-protective setting and alter tumour behaviour both in vitro and in the hollow fibre model. Real-time PCR data obtained from direct and indirect co-cultures also provide an insight in the co-evolution of the carcinoma and surrounding stroma with the overall microenvironment. Finally, this recapitulation of the human tumour micro-environment provides a relevant model by which to identify and characterise novel anti-cancer compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4280. doi:10.1158/1538-7445.AM2011-4280

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.

Differential Influence of Normal and Cancer-Associated Fibroblasts on the Growth of Human Epithelial Cells in an In vitro Cocultivation Model of Prostate Cancer

The prostate is composed of a number of different cell populations. The interaction between them is crucial for the development and proper function of the prostate. However, the effect of the molecular cross talk between these cells in the course of carcinogenesis is still unclear. Employing an approach wherein immortalized epithelial cells and immortalized human fibroblasts were cocultured, we show that normal associated fibroblasts (NAF) and cancer-associated fibroblasts (CAF) differentially influenced the growth and proliferation of immortalized human prostate epithelial cells. Whereas NAFs inhibited the growth of immortalized epithelial cells but promoted the growth of metastatic PC-3 cells, CAFs promoted the growth of immortalized epithelial cells but not of PC-3. Cytokine arrays revealed that NAFs secreted higher levels of tumor necrosis factor-alpha compared with CAFs whereas CAFs secreted higher levels of interleukin-6 (IL-6) compared with NAFs. The growth-inhibiting effects of NAFs were counteracted by the addition of IL-6, and the growth-promoting effects exerted by the CAFs were counteracted by tumor necrosis factor-alpha. Furthermore, CAFs induced the migration of endothelial cells in an IL-6-dependent manner. Here, we show that normal fibroblast cells have a protective function at very early stages of carcinogenesis by preventing immortalized epithelial cells from proliferating and forming new blood vessels whereas CAFs aid immortalized epithelial cells to further develop.

Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells

Recently, TWIST, a basic helix-loop-helix transcription factor, is suggested to be an oncogene because of its over-expression in many types of human cancer and its positive role in promoting cell survival. The aim of this study was to investigate the role of TWIST on the growth of human epithelial cells. Using two immortalized human prostate epithelial cell lines, we demonstrated that inactivation of TWIST by small RNA technology led to the promotion of cellular senescence and growth arrest, suggesting that TWIST plays a key role in the continuous proliferation of immortalized cells. Over-expression of TWIST, in contrast, resulted in suppression of cellular senescence in response to genotoxic damage and promotion of cell proliferation with DNA damage accumulation, indicating that TWIST promotes genomic instability. In addition, we also found that the TWIST-mediated cellular senescence was regulated through its negative effect on p14(ARF) and subsequent suppression of MDM2/p53 and Chk1/2 DNA damage response pathways. Our results suggest that over-expression of TWIST results in down-regulation of p14(ARF), which leads to the impairment of DNA damage checkpoint in response to genotoxic stress. This negative effect of TWIST on DNA damage response facilitates uncontrolled cell proliferation with genomic instability and tumorigenesis in non-malignant cells.

Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-β

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

Transforming Growth Factor-β (TGF-β) Activates Cytosolic Phospholipase A2α (cPLA2α)-mediated Prostaglandin E2 (PGE)2/EP1 and Peroxisome Proliferator-activated Receptor-γ (PPAR-γ)/Smad Signaling Pathways in Human Liver Cancer Cells: A NOVEL MECHANISM FOR SUBVERSION OF TGF-β-INDUCED MITOINHIBITION

Transforming growth factor-beta (TGF-beta) potently inhibits the growth of human epithelial cells. However, neoplastic epithelial cells become resistant to TGF-beta-mediated mitoinhibition, and the mechanisms for this alteration during tumorigenesis are not fully understood. This study was designed to determine whether there is an association between the cytosolic phospholipase A2alpha (cPLA2alpha)-controlled eicosanoid metabolism and the growth response to TGF-beta in human liver cancer cells. TGF-beta treatment induced simultaneous Smad-mediated gene transcription and phosphorylation of cPLA2alpha. Whereas Smad activation inhibited tumor cell growth, phosphorylation of cPLA2 alpha promoted growth and counteracted Smad-mediated mitoinhibition. TGF-beta1 failed to prevent the growth of cells with high basal expression of cPLA2alpha, but inhibition of cPLA2 alpha, cyclooxygenase-2 (COX-2), or EP1 receptor restored mitoinhibition by TGF-beta1 in these cells. These results suggest that resistance of tumor cells to TGF-beta-mediated mitoinhibition involves activation of cPLA2alpha/COX-2/EP1 signaling. Furthermore, the TGF-beta1-induced Smad transcriptional activity and mitoinhibition were blocked by overexpression of cPLA2alpha or peroxisome proliferator-activated receptor-gamma (PPAR-gamma) but enhanced by depletion of cPLA2alpha or PPAR-gamma. These findings, along with the observations that cPLA2alpha activates PPAR-gamma and that PPAR-gamma binds Smad3, illustrate novel cPLA2alpha/COX-2/EP1 and cPLA2alpha/PPAR-gamma/Smad signaling pathways that counteract the mitoinhibition by TGF-beta in human cancer cells.

Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

Modulation of enzymatic activity and biological function of Listeria monocytogenes broad-range phospholipase C by amino acid substitutions and by replacement with the Bacillus cereus ortholog.

The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.

Immune control of Chlamydial growth in the human epithelial cell line RT4 involves multiple mechanisms that include nitric oxide induction, tryptophan catabolism and iron deprivation.

The antimicrobial activity of T cell-derived cytokines, especially interferon (IFN)-gamma, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia-susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine-induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine-mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for approximately 20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.

Epstein–Barr virus latent membrane protein inhibits human epithelial cell differentiation

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.

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.

An inhibitory effect of tumour promoters on human epithelial cell growth can be dissociated from an effect on junctional communication

Studies with rodent cells have indicated that the abilities of various tumour promoters to inhibit metabolic cooperation correlate with their potencies as mitogens. Here we have examined the effects of the most potent phorbol ester tumour promoter 12- O-tetradecanoyl phorbol-13-acetate (TPA), on metabolic cooperation and growth of human epidermal cells transformed by SV40 (SVK14 cells). In this system, TPA inhibits Junctional communication and at the same concentration also inhibits growth in a reversible fashion. These effects appear to be mediated by binding of phorbol ester to a single class of high affinity binding site with a Kd similar to that reported for rodent cells ( K d = 20.9 nM at 4 ° C). Further studies on the effects of phorbol esters on other human epithelial cell lines reveal that the inhibitory effects of TPA on growth and metabolic cooperation may be completely dissociated. Alternative mechanisms by which TPA may exert its growth-inhibitory effects are discussed.

Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells.

The growth of human epithelial cells is stimulated by cholera toxin and analogues of cyclic AMP, while the growth of breast derived fibroblasts is inhibited. These compounds have little effect on DNA synthesis in the absence of other mitogens but show a synergistic effect with serum and/or EGF. The results suggest that high intracellular levels of cyclic AMP in human mammary epithelial cells increase the growth response of the cell to mitogens.

EGF growth promoting activity is neutralized by phorbol esters

The tumor-promoter, TPA, stimulates growth of human epithelial cells but not that of normal fibroblasts. In the presence of either EGF or FGF, TPA antagonizes the growth stimulatory activity of both factors in both cell lines.