Immortalization Of Epithelial Cells Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Murine alveolar epithelial cells and their lentivirus-mediated immortalisation.

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air-blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800Ω ·cm². Transport across the air-blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω·cm²) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.

Epigenetic silencing of lncRNA MORT in 16 TCGA cancer types

We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-AS1, Gene ID: 100128252), which is expressed in all normal cell types, but epigenetically silenced during cancer-associated immortalization of human mammary epithelial cells. Initial analysis of The Cancer Genome Atlas (TCGA) showed that 15 of 17 cancer types, which represent the 10 most common cancers in women and men, display DNA methylation associated MORT silencing in a large fraction of their tumors. In this study we analyzed MORT expression and DNA methylation state in the remaining 16 TCGA cancer types not previously reported. Seven of the 16 cancer types showed DNA methylation linked MORT silencing in a large fraction of their tumors. These are carcinomas (cervical cancer, and cancers of esophagus, stomach, and bile duct), and the non-epithelial tumors mesothelioma, sarcoma, and uterine carcinosarcoma. Together with the findings from our previous report, MORT expression is silenced by aberrant DNA methylation in 22 of 33 of TCGA cancer types. These 22 cancers include most carcinoma types, blood derived cancers and sarcomas. In conclusion, results suggest that the MORT gene is one of the most common epigenetic aberrations seen in human cancer. Coupled with the timing of MORT gene silencing during in vitro epithelial cell immortalization and its occurrence early in the temporal arc of human carcinogenesis, this provides strong circumstantial evidence for a tumor suppressor role for MORT.

Epigenetic silencing of lncRNA MORT in 16 TCGA cancer types.

We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-AS1, Gene ID: 100128252), which is expressed in all normal cell types, but epigenetically silenced during cancer-associated immortalization of human mammary epithelial cells. Initial analysis of The Cancer Genome Atlas (TCGA) showed that 15 of 17 cancer types, which represent the 10 most common cancers in women and men, display DNA methylation associated MORT silencing in a large fraction of their tumors. In this study we analyzed MORT expression and DNA methylation state in the remaining 16 TCGA cancer types not previously reported. Seven of the 16 cancer types showed DNA methylation linked MORT silencing in a large fraction of their tumors. These are carcinomas (cervical cancer, and cancers of esophagus, stomach, and bile duct), and the non-epithelial tumors mesothelioma, sarcoma, and uterine carcinosarcoma. Together with the findings from our previous report, MORT expression is silenced by aberrant DNA methylation in 22 of 33 of TCGA cancer types. These 22 cancers include most carcinoma types, blood derived cancers and sarcomas. In conclusion, results suggest that the MORT gene is one of the most common epigenetic aberrations seen in human cancer. Coupled with the timing of MORT gene silencing during in vitro epithelial cell immortalization and its occurrence early in the temporal arc of human carcinogenesis, this provides strong circumstantial evidence for a tumor suppressor role for MORT.

Excellent antitumor effects for gastrointestinal cancers using photodynamic therapy with a novel glucose conjugated chlorin e6

BackgroundPhotodynamic therapy (PDT) exploits the reaction between photosensitizer and irradiated light to generate potentially therapeutic reactive oxygen species such as singlet oxygen in cancer cells. We have reported several sugar-conjugated chlorins that express stronger antitumor effects in PDT than talaporfin sodium (TS), a second-generation photosensitizer clinically used in Japan. In this study, we developed a novel glucose-conjugated chlorin e6 (G-chlorin e6) and evaluated its antitumor effects. MethodsG-chlorin e6 was synthesized with a core photosensitizer chlorin e6 conjugated to glucose. We measured the half maximal inhibitory concentration (IC50) to compare the PDT effects of G-chlorin e6 and TS, and flow cytometry was performed to examine the accumulation of G-chlorin e6 in cancer cells. We also compared the accumulation of G-chlorin e6 between normal immortalized esophageal epithelial cells and esophageal cancer cells. Antitumor effects of G-chlorin e6 PDT were finally analyzed in allograft tumor mouse models. ResultsPDT in vitro using G-chlorin e6 elicited 9, 000–34,000 times stronger antitumor effects than TS, and there was 70–190 times more G-chlorin e6 accumulated than TS by flow cytometry. G-chlorin e6 accumulated more selectively in esophageal cancer cells than in esophageal immortalized epithelial cells, and in an allograft model, PDT with G-chlorin e6 showed very strong antitumor effects and a 40% complete response (CR) rate. ConclusionsG-chlorin e6 showed excellent tumor selectivity, and PDT using G-chlorin e6 revealed the strongest anti-tumor effects among all sugar-conjugated chlorins that we have studied. G-chlorin e6 is considered to be the best photosensitizer for next-generation PDT.

Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae.

Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.

Apoptosis induction of colorectal cancer cells HTL-9 in vitro by the transformed products of soybean isoflavones by Ganoderma lucidum.

Soybean isoflavones have been one of the potential preventive candidates for antitumor research in recent years. In this paper, we first studied the transformation of soybean isoflavones with the homogenized slurry of Ganoderma lucidum. The resultant transformed products (TSI) contained (703.21±4.35) mg/g of genistein, with transformed rates of 96.63% and 87.82% of daidzein and genistein, respectively, and TSI also could enrich the bioactive metabolites of G. lucidum. The antitumor effects of TSI on human colorectal cancer cell line HTL-9, human breast cancer cell line MCF-7, and human immortalized gastric epithelial cell line GES-1 were also studied. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay showed that TSI could dramatically reduce the viability rates of HTL-9 cells and MCF-7 cells without detectable cytotoxicity on GES-1 normal cells when the TSI concentration was lower than 100 μg/ml. With 100 μg/ml of TSI, HTL-9 cells were arrested in the G1 phase, and late-apoptosis was primarily induced, accompanied with partial early-apoptosis. TSI could induce primarily early-apoptosis by arresting cells in the G1 phase of MCF-7 cells. For HTL-9 cells, Western-blot and reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that TSI (100 μg/ml) can up-regulate the expression of Bax, Caspase-3, Caspase-8, and cytochrome c (Cyto-c), indicating that TSI could induce cell apoptosis mainly through the mitochondrial pathway. In addition, the expression of p53 was up-regulated, while the expression of Survivin and nuclear factor κB (NF-κB) was down-regulated. All these results showed that TSI could induce apoptosis of HTL-9 cells by the regulation of multiple apoptosis-related genes.

Hypermethylation of PGCP gene is associated with human bronchial epithelial cells immortalization

Cell immortalization is the initial step for cancer development. To identify the differentially expressed genes regulated by DNA methylation over the course of human primary bronchial epithelial cell (HPBECs) immortalization, an immortalized HBE cell line (HBETT) was generated via introduction of an SV40 LT and a catalytic subunit of human telomerase reverse transcriptase (hTERT) into the HPBECs. Microarrays of mRNA and DNA methylation were performed to compare the transcriptomes and DNA methylomes between these two types of cells. The results from the mRNA microarray revealed many genes whose expression changed upon cell immortalization. We identified signatures including global hypomethylation, perturbation of ECM-receptor interaction, focal adhesion, and PI3K-Akt pathways associated with cell immortalization. Moreover, we revealed 155 differentiated methylation regions (DMRs) within the CpG islands (CGIs) of 42 genes and the perturbation of several key pathways that might be involved in HBE cell immortalization. Among these genes, the hypermethylation of the plasma glutamate carboxypeptidase (PGCP) gene appeared specifically in lung cancer tissues. The inhibition of PGCP expression by promoter hypermethylation was observed in both immortal HBETT cells and benzo[a]pyrene (Bap)-transformed HBE cells. In conclusion, these findings provide new insight into the epigenetic modifications that are critical in the transition and maintenance of cell immortalization.

Identification of a Gene Encoding Slow Skeletal Muscle Troponin T as a Novel Marker for Immortalization of Retinal Pigment Epithelial Cells

Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeutic products (CTPs). In the development of hPSC-derived CTPs, it is imperative to ensure that they do not form tumors after transplantation for safety reasons. Because cellular immortalization is a landmark of malignant transformation and a common feature of cancer cells, we aimed to develop an in vitro assay for detecting immortalized cells in CTPs. We employed retinal pigment epithelial (RPE) cells as a model of hPSC-derived products and identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a novel marker of immortalized RPE cells by comprehensive microarray analysis. TNNT1 mRNA was commonly upregulated in immortalized RPE cells and human induced pluripotent stem cells (hiPSCs), which have self-renewal ability. Additionally, we demonstrated that TNNT1 mRNA expression is higher in several cancer tissues than in normal tissues. Furthermore, stable expression of TNNT1 in ARPE-19 cells affected actin filament organization and enhanced their migration ability. Finally, we established a simple and rapid qRT-PCR assay targeting TNNT1 transcripts that detected as low as 3% of ARPE-19 cells contained in normal primary RPE cells. Purified hiPSC-derived RPE cells showed TNNT1 expression levels below the detection limit determined with primary RPE cells. Our qRT-PCR method is expected to greatly contribute to process validation and quality control of CTPs.

Paeonol attenuates aging MRC-5 cells and inhibits epithelial-mesenchymal transition of premalignant HaCaT cells induced by aging MRC-5 cell-conditioned medium.

Senescence-associated secretory phenotype (SASP) factors, such as IL-6 and IL-8, are extremely critical in tissue microenvironment. Senescent human fibroblasts facilitate epithelial-mesenchymal transition (EMT) in premalignant epithelial cells mainly through the secretion of SASP factors. Meanwhile, premalignant human HaCaT Keratinocyte (HaCaT) cells as immortal epithelial cells are susceptible to malignant transformation. Paeonol, an herbal phenolic component found in peonies, exerts anti-aging and anti-tumor efficacies, while the molecular mechanisms of paeonol on EMT in premalignant HaCaT cells induced by SASP factors are unclear. In this study, we first established a senescent human fetal lung fibroblast MRC-5 cell model using hydrogen peroxide evaluated by senescence-associated β-galactosidase assay. Upon paeonol treatment, intracellular reactive oxygen species levels in aging MRC-5 cells were significantly decreased via regulation of nuclear translocation of Nrf2. Then we curiously studied whether the aging MRC-5 cell-conditioned medium could induce EMT in premalignant HaCaT cells, and the results showed that paeonol significantly reduced the clonogenic, migratory, and invasive capacities of premalignant HaCaT cells potentially induced by IL-6 and IL-8. Moreover, we found that paeonol notably altered pluripotency of EMT-associated markers via the modulation of ERK and TGF-β1/Smad pathway in premalignant HaCaT cells. These findings suggest that paeonol may be used as an adjuvant therapy for SASP factor-mediated EMT in premalignant lesion.

Abstract 2357: CDK4/6 inhibition in early stage triple negative breast cancer

Abstract Palbociclib (PD), a CDK4/6 inhibitor, has recently been approved for use in advanced ER positive breast cancer, demonstrating significant benefit in combination with endocrine therapies. Ductal carcinoma in situ (DCIS) is an inobligate precursor to invasive ductal carcinoma with anywhere from 14-53% of cases progressing. Progression is unpredictable and driven by poorly understood mechanisms. In this study, we assess the efficacy of PD in the context of this early stage disease using a triple negative DCIS model(MCF10DCIS), not only in inhibiting cell cycle but also in hindering the progression from in situ to invasive lesion. We demonstrate similar efficacy of PD in 2D on MCF10DCIS and immortal mammary epithelial cells (MCF10As) in preventing proliferation, inhibiting phosphorylation of RB and stopping cell cycle progression. Yet in 3D, MCF10DCIS cells form smaller, more organized spheres in the presence of drug while MCF10As appear unaffected, highlighting potential disease selectivity in vivo. Interestingly, when a cDNA array is performed on MCF10DCIS cells grown in 3D +/- PD, the most regulated genes are not well characterized cell cycle genes as one might predict based on previously published data and 2D response to treatment, suggesting alternative CDK4/6 targets may be responsible for the phenotype observed in 3D. We also demonstrate in a nude mouse xenograft model of MCF10DCIS that PD is able to significantly reduce overall tumor burden while simultaneously delaying the progression of lesions to invasive disease. Though the growth of PD treated MCF10DCIS lesions is delayed we demonstrate that proliferation is unaffected in vivo by the presence of the drug, based on equivalent Ki67 signal within the lesions. This reiterates the possibility that in 3D and in vivo contexts PD targets mechanisms outside of cell cycle regulation in order to slow tumor growth and delay invasive transition. We believe that investigation into alternative targets of CDK4/6 altered by PD in 3D and in vivo may provide insights on important mechanisms of DCIS progression in DCIS models and human patients. Citation Format: William B. Kietzman, Virginie Ory, Fransisco Saenz, Ghada Sharif, Anton Wellstein, Anna T. Riegel. CDK4/6 inhibition in early stage triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2357. doi:10.1158/1538-7445.AM2017-2357

Gene regulatory network underlying the immortalization of epithelial cells

BackgroundTumorigenic transformation of human epithelial cells in vitro has been described experimentally as the potential result of spontaneous immortalization. This process is characterized by a series of cell–state transitions, in which normal epithelial cells acquire first a senescent state which is later surpassed to attain a mesenchymal stem–like phenotype with a potentially tumorigenic behavior. In this paper we aim to provide a system–level mechanistic explanation to the emergence of these cell types, and to the time–ordered transition patterns that are common to neoplasias of epithelial origin. To this end, we first integrate published functional and well–curated molecular data of the components and interactions that have been found to be involved in such cell states and transitions into a network of 41 molecular components. We then reduce this initial network by removing simple mediators (i.e., linear pathways), and formalize the resulting regulatory core into logical rules that govern the dynamics of each of the network components as a function of the states of its regulators.ResultsComputational dynamic analysis shows that our proposed Gene Regulatory Network model recovers exactly three attractors, each of them defined by a specific gene expression profile that corresponds to the epithelial, senescent, and mesenchymal stem–like cellular phenotypes, respectively. We show that although a mesenchymal stem–like state can be attained even under unperturbed physiological conditions, the likelihood of converging to this state is increased when pro–inflammatory conditions are simulated, providing a systems–level mechanistic explanation for the carcinogenic role of chronic inflammatory conditions observed in the clinic. We also found that the regulatory core yields an epigenetic landscape that restricts temporal patterns of progression between the steady states, such that recovered patterns resemble the time–ordered transitions observed during the spontaneous immortalization of epithelial cells, both in vivo and in vitro.ConclusionOur study strongly suggests that the in vitro tumorigenic transformation of epithelial cells, which strongly correlates with the patterns observed during the pathological progression of epithelial carcinogenesis in vivo, emerges from underlying regulatory networks involved in epithelial trans–differentiation during development.

Next-Generation CDK2/9 Inhibitors and Anaphase Catastrophe in Lung Cancer

The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel). CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided. CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 μM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 μM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 μM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 μM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 μM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065. Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.

MicroRNA-9 inhibits the gastric cancer cell proliferation by targeting TNFAIP8.

MicroRNA-9 is frequently dysregulated in many human carcinoma types, including gastric cancer (GC). Previous studies demonstrated that the expression of TNFAIP8 in GC is correlated with tumour occurrence, development, invasion, metastasis and prognosis. However, till now, the relationship between MicroRNA-9 and TNFAIP8 in GC has not been reported. Levels of miR-9 and TNFAIP8 expression in GC tissues and in human GC cell lines were studied using qualitative real-time PCR (qRT-PCR) and Western blotting. Cell viability was detected using the CCK-8 and clone formation assays. A dual-luciferase reporter system was used to confirm the target gene of miR-9. We found that the expression level of MicroRNA-9 in GC tissues and cell lines was significantly lower than that in adjacent non-cancerous tissues and human immortalized gastric epithelial cell (GES) line, respectively. In addition, overexpression of MicroRNA-9 markedly inhibited GC cell proliferation in vitro and tumour growth in vivo. Further experiments revealed that TNFAIP8 was a direct and functional target of MicroRNA-9 in GC and overexpression of MicroRNA-9 obviously down-regulated the expression of TNFAIP8, which was involved in the gastric carcinogenesis and cancer progression. Our results suggested that MicroRNA-9-TNFAIP8 might represent a promising diagnostic biomarker for GC patients and could be a potential therapeutic target in the prevention and treatment of GC.

Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic Transformation.

The interior of cells is a highly complex medium, containing numerous organelles, a matrix of different fibers and a viscous, aqueous fluid of proteins and small molecules. The interior of cells is also a highly dynamic medium, in which many components move, either by active transport or passive diffusion. The mobility and localization of proteins inside cells can provide important insights into protein function and also general cellular properties, such as viscosity. Neoplastic transformation affects numerous cellular properties, and our goal was to investigate the diffusional and binding behavior of the important mismatch repair (MMR) protein MSH2 in live human cells at various stages of neoplastic transformation. Toward this end, noncancerous, immortal, tumorigenic, and metastatic mammary epithelial cells were transfected with EGFP and EGFP-tagged MSH2. MSH2 forms two MMR proteins (MutSα and MutSβ) and we assume MSH2 is in the complex MutSα, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSα-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14–24 μm2/s for EGFP and 3–7 μm2/s for MutSα-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSα produces an effective diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant K*. The MutSα nuclear K* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic K*for MutSα increased almost two orders of magnitude in going from noncancerous to immortal cells, suggesting that this quantity may be a sensitive metric for recognizing the onset of cancer.

Epigenetic Silencing of MORT Is an Early Event in Cancer and Is Associated with Luminal, Receptor Positive Breast Tumor Subtypes.

Immortality is an essential characteristic of cancer cells; a recent transcriptomic study of epithelial cell immortalization has linked epigenetic silencing of the long noncoding RNA Mortal Obligate RNA Transcript (MORT; alias ZNF667-AS1) to this process. This study evaluated the epigenetic and transcriptional state of MORT in two premalignant conditions—ductal carcinomas in situ and colon adenomas. Results show that MORT silencing is an early epigenetic event in human carcinogenesis, likely occurring near the point where premalignant cells gain immortality; this epigenetic silencing is maintained throughout malignant transformation and metastatic growth. Additional associations between MORT loss and clinical and molecular features of breast tumors showed that silencing of MORT occurs predominantly in luminal, receptor-positive breast cancer; is associated with overexpression of CCND1 and mutations of GATA3; and is negatively correlated with TP53 mutations. Taken in toto, MORT silencing occurs early in breast carcinogenesis, probably during cellular immortalization, and precedes the development of invasive luminal breast cancer.

Endometriotic cyst fluid induces reactive oxygen species (ROS) in human immortalized epithelial cells derived from ovarian endometrioma

ABSTRACTObjectives: Endometriotic cyst fluid (ECF) contains a large amount of reactive oxygen species (ROS), and endometriotic cysts are exposed to strong oxidative stress, which may cause malignant transformation. In this study, ROS production by ECF was clinically analysed.Methods: Human immortalized epithelial cells derived from ovarian endometrioma (EMosis-CC/TERT 1) were treated with ECF. In addition, ROS production in EMosis-CC/TERT 1 was measured, and its clinical significance was analysed.Results: A total of 38 ECF samples were obtained from patients diagnosed with endometriotic cysts. In EMosis-CC/TERT1, significantly higher levels of ROS were induced by ECF than by the vehicle control and ferric nitrilotriacetate. There were no significant differences in ROS production by laterality and preoperative serum CA125 values. There were several patients whose cyst sizes were approximately 5 cm and had relatively high ROS production. Production of ROS by ECF was relatively higher in patients older than 40 years of age than in those younger than 40.Discussion: Our study revealed that ROS are highly produced by ECF in EMosis-CC/TERT1 cells; therefore, exposure to ECF induced strong oxidative stress. Development of a therapeutic strategy to reduce ROS production might be useful for preventing malignant transformation of endometriotic cysts.

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue

Mammary gland regression at the cessation of lactation (involution) is an exquisitely orchestrated process of cell death and tissue remodelling in which Stat3 signalling has an essential role. The involution microenvironment of the mammary gland is considered to be pro-tumourigenic and a proportion of cases of pregnancy-associated breast cancer are suggested to originate in tandem with involution. However, the apparent paradox that STAT3 is required for cell death in normal mammary gland, but is associated with breast cancer cell survival, has not been resolved. Herein, we investigate Stat3-mediated regulation of expression of members of the calcium-activated chloride channel regulator (CLCA) family of proteins during involution and mammary carcinogenesis. Using the conditionally immortal mammary epithelial cell line KIM-2, together with mice exhibiting mammary epithelial cell-specific deletion of Stat3 during lactation, we demonstrate that expression of mCLCA1 and mCLCA2 is elevated in concert with activation of Stat3. By contrast, murine CLCA5 (mCLCA5), the murine orthologue of human CLCA2, is significantly upregulated at 24, 72 and 96 h of involution in Stat3 knockout mice, suggesting a reciprocal regulation of these proteins by Stat3 in vivo. Interestingly, orthotopic tumours arising from transplantation of 4T1 murine mammary tumour cells exhibit both phosphorylated Stat3 and mCLCA5 expression. However, we demonstrate that expression is highly compartmentalized to distinct subpopulations of cells, and that Stat3 retains a suppressive effect on mCLCA5 expression in 4T1 tumour cells. These findings enhance our understanding of the regulation of CLCA channel expression both in vitro and in vivo, and in particular, demonstrate that expression of mCLCA1 and mCLCA2 during involution is profoundly dependent upon Stat3, whereas the relationship between mCLCA5 and Stat3 activity is reciprocal and restricted to different subpopulations of cells.