Proteomics Analysis of the Polyomavirus DNA Replication Initiation Complex Reveals Novel Functional Phosphorylated Residues and Associated Proteins.

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract and originate from the interstitial cells of Cajal (ICC), which is the pacemaker for peristaltic movement in the gastrointestinal tract. Existing GIST cell lines are widely used as cell models for in vitro experimental studies because the mutation sites are known. However, the immortalization methods of these cell lines are unknown, and no Chinese patient-derived GIST cell lines have been documented. Here, we transfected simian virus 40 large T antigen (SV40LT) into primary GIST cells to establish an immortalized human GIST cell line (ImGIST) for the first time. The ImGIST cells had neuronal cell-like irregular radioactive growth and retained the fusion growth characteristics of GIST cells. They stably expressed signature proteins, maintained the biological and genomic characteristics of normal primary GIST cells, and responded well to imatinib, suggesting that ImGIST could be a potential in vitro model for research in GIST to explore the molecular pathogenesis, drug resistance mechanisms, and the development of new adjuvant therapeutic options.

Establishment and characterization of an immortalized epicardial cell line.

Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40-LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.

Reversible expansion of pancreatic islet progenitors derived from human induced pluripotent stem cells.

Transplantation of pancreatic islets is an effective therapy for severe type 1 diabetes. As donor shortage is a major problem for this therapy, attempts have been made to produce a large number of pancreatic islets from human pluripotent stem cells (hPSCs). However, as the differentiation of hPSCs to pancreatic islets requires multiple and lengthy processes using various expensive cytokines, the process is variable, low efficiency and costly. Therefore, it would be beneficial if islet progenitors could be expanded. Neurogenin3 (NGN3)-expressing pancreatic endocrine progenitor (EP) cells derived from hPSCs exhibited the ability to differentiate into pancreatic islets while their cell cycle was arrested. By using a lentivirus vector, we introduced several growth-promoting genes into NGN3-expressing EP cells. We found that SV40LT expression induced proliferation of the EP cells but reduced the expression of endocrine lineage-commitment factors, NGN3, NEUROD1 and NKX2.2, resulting in the suppression of islet differentiation. By using the Cre-loxP system, we removed SV40LT after the expansion, leading to re-expression of endocrine-lineage commitment genes and differentiation into functional pancreatic islets. Thus, our findings will pave a way to generate a large quantity of functional pancreatic islets through the expansion of EP cells from hPSCs.

Establishing a papillary craniopharyngioma cell line by SV40LT-mediated immortalization.

BACKGROUND: Craniopharyngioma represents a troublesome tumor of the intracranial sellar region. There are currently no available well-characterized craniopharyngioma cell lines. This lack of reliable, immortal cell lines is a major reason for the slow progress in fundamental research related to craniopharyngioma. METHODS: We describe the development of an immortal papillary craniopharyngioma (PCP) cell line by transfecting primary PCP cells with the pLenti-simian virus 40 large T antigen(SV40LT). RESULTS: Three clones have been cultured for more than 14 months so far, while non-transfected cells ceased proliferation within three months of isolation. The established immortal PCP cell lines were identified to have BRAFV600E mutations, while no mutations in tumor suppressor genes were found in primary cells or immortal cells. Immortal cells had higher proliferation rates and formed tumors when implanted in the bran of nude mice. BRAF inhibition in immortal PCP cells altered cell morphology, inhibited cell proliferation and promoted apoptosis. CONCLUSION: We successfully developed PCP cell lines by SV40LT-mediated immortalization. These cell lines represent a powerful tool for fundamental and therapeutical studies on craniopharyngioma.

The RB gene family controls the maturation state of the EndoC-ßH2 human pancreatic ß-cells.

The functional maturation of human pancreatic ß-cells remains poorly understood. EndoC-ßH2 is a human ß-cell line with a reversible immortalized phenotype. Removal of the two oncogenes, SV40LT and hTERT introduced for its propagation, stops proliferation, triggers cell size increase and senescence, promotes mitochondrial activity and amplifies several ß-cell traits and functions. Overall, these events recapitulate several aspects of functional ß-cell maturation. We report here that selective depletion of SV40LT, but not of hTERT, is sufficient to revert EndoC-ßH2 immortalization. SV40LT inhibits the activity of the RB family members and of P53. In EndoC-ßH2 cells, the knock-down of RB itself, and, to a lesser extent, of its relative P130, precludes most events triggered by SV40LT depletion. In contrast, the knock-down of P53 does not prevent reversion of immortalization. Thus, an increase in RB and P130 activity, but not in P53 activity, is required for functional maturation of EndoC-ßH2 cells upon SV40LT-depletion. In addition, RB and/or P130 depletion in SV40LT-expressing EndoC-ßH2 cells decreases cell size, stimulates proliferation, and decreases the expression of key ß-cell genes. Thus, despite SV40LT expression, EndoC-ßH2 cells have a residual RB activity, which when suppressed reverts them to a more immature phenotype. These results show that the expression and activity levels of RB family members, especially RB itself, regulate the maturation state of EndoC-ßH2 cells.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen.

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I.

Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer.

Construction and Identification of a Human Colorectal Adenoma Epithelial Cell Line by SV40 Large T-Antigen Transfection.

BACKGROUND: Colorectal adenoma represents the critical step in the development of colorectal cancer. The establishment of an immortalized epithelial cell line of colorectal adenoma of human origin would provide a tool for studying the mechanism of precancerous lesions, screening the efficacy of novel drugs, and constructing in vivo disease models. Currently, there is no commercially available stable supply of epithelial cells from precancerous lesions. AIMS: This study aimed to establish a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection. METHODS: Simian vacuolating virus 40(SV40), SV40-LT overexpressed lentivirus vector, was transfected into primary human colorectal adenomatous polyp epithelial cells. The transfected cells were screened, and the screened cells were amplified to obtain the epithelial cell line: IHCRA- CELL. The cells were identified by morphological observation, cell proliferation, Quantitative real-time PCR (qPCR), and Short Tandem Repeats (STR) experiments. Morphologically, the cells showed epithelial-like characteristics, such as polygon shape, desmosomes mitochondria, and strong positive keratin staining. There was no significant difference between the transfected cells and the primary cells. Through the STR identification experiment, no matching cell lines were found in the cell lines retrieval. CONCLUSION: We successfully established a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection, which has been patented and is now preserved in the Chinese Typical Culture Preservation Center. It was verified that the transformed cells maintained the phenotype and biological characteristics of epithelial cells. This cell line can be used to study the mechanism of precancerous lesions, screen the efficacy of novel drugs, and construct in vivo disease models.

Construction and identification of an immortalized rat intestinal smooth muscle cell line.

BACKGROUND: Although intestinal smooth muscle cells (ISMCs) play an important role in the remodeling of the intestinal structure, considerably less is known about the molecular mechanisms that mediate the development and growth of ISMCs. A possible reason for this lag is the lack of cell lines that recapitulate ISMCs in vivo. METHODS: In this study, we separated the primary ISMCs from the rat intestinal tract and integrated the Simian Vacuolating virus 40 Large T antigen (SV40 LT) gene into the genome of the primary ISMC to construct an immortalized cell line named ISMC-Hc. KEY RESULTS: ISMC-Hc proliferated persistently without any signs of senescence up to 50 passages and without neoplasticity. Analysis of the genome isolated from ISMC-Hc confirmed that the SV40LT gene recombined in the genome, and mRNA reverse transcription PCR suggested that SV40LT could be expressed normally. In addition, ISMC-Hc had few morphological differences compared with the primary ISMC. Furthermore, ISMC-Hc showed the expression of the specific protein markers (alpha-smooth muscle actin and desmin) through immunofluorescence analysis. Further studies showed that ISMC-Hc had enhanced contractility and expressed the glial-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor after co-stimulation with IL-1 beta and TNF-alpha. CONCLUSIONS AND INFERENCES: ISMC-Hc showed characteristics similar to that of primary ISMC and can be used as an in vitro model to explore the cellular and molecular mechanisms of ISMC. Additionally, the immortalized ISMCs could help investigate the basic functional mechanisms of intestinal diseases.

Stem cells immortalized by hTERT perform differently from those immortalized by SV40LT in proliferation, differentiation, and reconstruction of matrix microenvironment.

Although matrix microenvironment has the potential to improve expanded stem cell proliferation and differentiation capacity, decellularized extracellular matrix (dECM) deposited by senescent cells does not contribute to the rejuvenation of adult stem cells, which has become a barrier to personalized stem cell therapy. Genetic modification is an effective strategy to protect cells from senescence but it carries the increased risk of malignant transformation and genetic instability. In this study, lentivirus carrying either human telomerase reverse transcriptase (hTERT) or simian virus 40 large T antigen (SV40LT) was used to transduce human infrapatellar fat pad-derived stem cells (IPFSCs). We found that virus transduction modified the proliferative, chondrogenic, and adipogenic abilities of IPFSCs. Interestingly, dECM deposited by immortalized cells significantly influenced replicative senescent IPFSCs in proliferation and differentiation preference, the effect of which is hinged on the approach of immortalization using either SV40LT or hTERT. Our findings indicate both dECM expansion and immortalization strategies can be used for replicative senescent adult stem cells' proliferation and lineage-specific differentiation, which benefits future stem cell-based tissue regeneration. This approach may also work for adult stem cells with premature senescence in elderly/aged patients, which needs further investigation. STATEMENT OF SIGNIFICANCE: Adult stem cells are a promising solution for autologous cell-based therapy. Unfortunately, cell senescence due to donor age and/or ex vivo expansion prevents clinical application. Recent progress with decellularized extracellular matrix provides a potential for the rejuvenation of senescent stem cells by improving their proliferation and differentiation capacities. Given the fact that the young matrix can provide a healthy and energetic microenvironment, in this study, two approaches using lentivirus transduction of hTERT and SV40LT were compared. The goal was to immortalize donor cells for deposition of decellularized extracellular matrix. The matrix was demonstrated to contribute diverging effects on the chondrogenic and adipogenic differentiation of expanded stem cells and exhibited proliferation benefits as well. These findings provide an invaluable asset for stem cell-based tissue regeneration.

CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity.

BACKGROUND: Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in multiple myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding. METHODS: Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three serine residues (Ser316, Ser320 and Ser321) in 308-325 aa region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser316, Ser320 and Ser321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis. RESULTS: Here, we confirmed that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser316, Ser320 and Ser321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation. CONCLUSIONS: Che-1 phosphorylation at Ser316, Ser320 and Ser321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.

Simian Virus 40 Large T Antigen as a Model to Test the Efficacy of Flouroquinolones against Viral Helicases.

Simian virus 40 large T-antigen (SV40 LT-Ag) is a 708 amino acid nuclear phosphoprotein. Among many functions of LT-Ag is its ability to perform as an ATPase-helicase, catalyzing the unwinding of viral genome during replication. The LT-Ag has been employed in the studies of helicase structure and function, and has served as a model helicase for the screening of antiviral drugs that target viral helicase. In this study, using in vitro enzyme assays and in silico computer modeling, we screened a batch of 18 fluoroquinolones to assess their potential as antivirals by virtue of their inhibition of the LT-Ag helicase. We found all fluoroquinolones to be inhibitory to the helicase activity of LT-Ag. In our docking analysis, most of these tested drugs showed similarity in their interactions with LT-Ag. Our study shows the potential of fluoroquinolones as antiviral drugs and of SV40 LT-Ag as a model protein for screening drugs against viral helicases.

A porcine model system of BRCA1 driven breast cancer.

BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1 expression is down-regulated in ~30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer. In contrast to mice, pig biological systems, and cancer genetics appear to more closely resemble their human counterparts. To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.

Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.