T Antigen Research Articles

Patients With Sporadic Colorectal Cancer or Advanced Adenomatous Polyp Have Elevated Anti-JC Virus Antibody Titer in Comparison With Healthy Controls

JC virus (JCV) is thought to infect approximately 80% of the human population. Antibodies against JCV can be found in the sera of many people with and without colorectal carcinoma (CRC). We hypothesized that JCV antibody titer will be higher in CRC patients than in healthy controls. To evaluate this hypothesis in a cohort of patients undergoing colonoscopy. We compared JCV antibody titers in patients with simple adenoma, advanced adenomatous polyp (AAP), CRC, and healthy controls, and evaluated JCV DNA in the tissue. Ninety-seven patients undergoing colonoscopy offered to participate in the study. Normal colonoscopy, simple adenoma, AAP, and CRC were found in 41, 19, 12, and 25 cases, respectively. A blood sample was taken for JCV DNA isolation and serology. In 18 patients with CRC or AAP tissue samples were taken for JCV DNA isolation and T-antigen (T-Ag) detection. A positive correlation was found between a JCV antibody titer and advanced colonic pathology. The average titer for normal controls, simple polyp, AAP, and CRC was 2.61+/-0.72, 2.95+/-0.77, 3.33+/-0.76, and 3.30+/-0.50 log, respectively (P<0.001). Viral DNA could not be shown in the serum. The presence of neoplastic tissue T-Ag (in 33.3% of the patients) was not associated with a difference in the log titer of serum antibody. In this study we showed that patients with advanced neoplasia, compared with patients with normal colonoscopy, harbor a higher JCV antibody titer in the serum. If confirmed, our finding may serve as a marker for CRC or for an earlier stage of AAP.

Mouse CSB protein is important for gene expression in the presence of a single-strand break in the non-transcribed DNA strand

CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3′-OH and 5′-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibited no effect on the expression of the nicked DNA in human fibroblasts immortalised by SV40 large T-antigen but caused a very strong decrease of gene expression in spontaneously transformed mouse embryonic fibroblasts (MEFs). Compared to the corresponding CSB-proficient MEFs, the effect was on average 6.7-fold stronger for a defined SSB located in the non-transcribed DNA strand, but only 2.4-fold for a SSB in the transcribed strand and 1.7-fold for a SSB located in the non-genic region. At the same time, CSB deficiency did not compromise the overall efficiency of repair of SSBs generated by treatment of the cells with hydrogen peroxide. The gene expression data thus indicate that CSB prevents irreversible transcription failures at the sites of DNA damage, acting preferentially at SSBs located in the non-transcribed DNA strand of the transcribed genes. We further conclude that SSBs in the non-transcribed DNA strand are commonly more harmful for transcription than those situated in the transcribed strand.

Brain stem cell transplantation daring mild hypothermia treatment of patients with traumatic brain injuries

Objective To study the feasibility of stem cell transplantation under mild hypothermia so as to provide a prerequisite for stem cell transplantation in patients with traumatic brain injuries (TBI) during mild hypothermia treatment. Methods After transfecting plasmid containing temperature-sensitive simian virus 40 large T-antigen (tsSV40LT) into temperature-sensitive umbilical cord mes-enchymal stem cells (tsUCMSCs) , the changes of cell morphology, nuclear proliferation index (PIx) and telomerase activity were detested when the tsUCMSCs were cultured at 33℃ and 37℃. After the mouse model with tTBI treated with mild hypothermia was established, tsUCMSCs were transplanted into semi-injury area to detect survival rate, proliferation and apoptosis indices and perform neurological deficit scoring. Results When cultured at 33℃, the tsUCMSCs displayed long spindle-shaped and highly refractive, with higher proliferation index and telomerase activity than those cultured at 37℃. Compared with control group (non-temperature-sensitive UCMSCs transplantation), tsUCMSCs in semi-injury area showed much higher cell survival and proliferating cell nuclear antigen expression ( P < 0.05 ) , with fewer apoptotic cells and better neurological function (P < 0.05). Conclusion The establishment of temperature-sensitive stem cell line enables stem cell transplantation during treatment of TBI with mild hypothermia, as provides us a new direction for treatment of TBI. Key words: Brain injuries; Hypothermia, artificial; Stem cells; Transplantation

CPLA2 Regulates the Expression of Type I Interferons and Intracellular Immunity to Chlamydia trachomatis

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

Transplantation of Immortalized Human Fetal Hepatocytes Prevents Acute Liver Failure in 90% Hepatectomized Mice

Aim The aim of this study was to investigate whether human fetal hepatocytes are amenable to simian virus 40 large T-antigen (SV40Tag) mediated immortalization and whether the immortalized cells rescue mice with acute liver failure induced by 90% hepatectomy. Methods We constructed a retroviral vector expressing a thermolabile mutant SV40Tag for transfer into primary human fetal hepatocytes. We quantitatively detected the synthetic ability for albumin and urea by the immortalized cells, which were subcutaneously inoculated into mice with severe combined immunodeficiency (SCID) to evaluate tumorigenzcity. The immortalized cells were also transplanted into the spleens of mice with acute liver failure. Results One clone resulting after selection, referred to as HepCL, was highly differentiated, growing steadily in a chemically defined serum-free medium. HepCL cells were positive for albumin, cytokeratin 18, and cytokeratin 19 immunocytochemical staining. The average synthetic efficacies of HepCL cells for albumin and urea were comparable to that of unmodified primary human fetal hepatocytes. The population doubling time of HepCL cells in the logarithmic growth phase was approximately 17 hours. HepCL cells showed no oncogenicity in immunodeficient mice at 16 months. Mice receiving HepCL cells (G1) and primary human fetal hepatocytes (G2) showed significantly lower blood ammonia levels after 90% hepatectomy. Pairwise comparisons between the 4 groups showed that xenotransplantation of HepCL (G1) or primary fetal hepatocytes (G2) significantly improved survivals of recipient mice. Conclusions HepCL may be useful as a source of hepatic function for cell-based therapeutics in acute liver failure.

A Conditional Immortalized Mouse Müller Glial Cell Line Expressing Glial and Retinal Stem Cell Genes

Müller glia have multiple functions in the retina, including synthesis of neurotrophic factors, uptake and metabolism of neurotransmitters, spatial buffering of ions, maintenance of the blood-retinal barrier, and response to injury. A population of Müller glia has some stem cell-like characteristics both in vivo and in vitro. The purpose of this study was to generate and characterize novel Müller glial cell lines from the postnatal mouse retina. Cells were cultured from postnatal day (P) 10 double heterozygous transgenic (H-2K(b)-tsA58/+; HRhoGFP/+) or C57BL/6 mice after papain dissociation. Interferon gamma (IFNγ) induction of the SV40 T-antigen (TAg) was assayed by immunohistochemistry and Western blot analysis. Proliferation was assayed by BrdU uptake and cell counts of calcein AM/ethidium bromide-stained cells. Gene expression was analyzed by RT-PCR and immunohistochemistry. Conditionally immortalized (ImM10 [Immortmouse Müller P10]) and spontaneously immortalized (C57M10 [C57BL/6 Müller P10]) Müller glial cell lines were selected by differential adherence to laminin; both consisted of adherent flat cells with large, diffusely staining nuclei and an epithelial morphology. TAg induction stimulated BrdU uptake by Müller glia in mixed retinal cultures from H-2K(b)-tsA58/+; HRhoGFP/+ mice and increased the proliferation of ImM10 cells. ImM10 and C57M10 cells expressed genes characteristic of Müller glia but not genes characteristic of differentiated retinal neurons. ImM10 cells also expressed retinal stem cell genes. The ImM10 cell line is a novel, conditionally immortalized Müller glial cell line isolated from the P10 mouse retina that expresses genes characteristic of Müller glial and retinal stem cells.

JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity

BackgroundThe human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg) and three T′ proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses.Methodology and FindingsWe investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A), and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector.ConclusionsJCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions.

Merkel Cell Polyomavirus-Infected Merkel Cell Carcinoma Cells Require Expression of Viral T Antigens

Merkel cell carcinoma (MCC) is the most aggressive skin cancer. Recently, it was demonstrated that human Merkel cell polyomavirus (MCV) is clonally integrated in approximately 80% of MCC tumors. However, direct evidence for whether oncogenic viral proteins are needed for the maintenance of MCC cells is still missing. To address this question, we knocked down MCV T-antigen (TA) expression in MCV-positive MCC cell lines using three different short hairpin RNA (shRNA)-expressing vectors targeting exon 1 of the TAs. The MCC cell lines used include three newly generated MCV-infected cell lines and one MCV-negative cell line from MCC tumors. Notably, all MCV-positive MCC cell lines underwent growth arrest and/or cell death upon TA knockdown, whereas the proliferation of MCV-negative cell lines remained unaffected. Despite an increase in the number of annexin V-positive, 7-amino-actinomycin D (7-AAD)-negative cells upon TA knockdown, activation of caspases or changes in the expression and phosphorylation of Bcl-2 family members were not consistently detected after TA suppression. Our study provides the first direct experimental evidence that TA expression is necessary for the maintenance of MCV-positive MCC and that MCV is the infectious cause of MCV-positive MCC.

Structure of a DNA Polymerase α-Primase Domain That Docks on the SV40 Helicase and Activates the Viral Primosome

DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

Abstract 2294: IGF-independent regulation of EMT by insulin-like growth factor binding protein-3 in transformed human esophageal cells

Abstract Introduction: Insulin-like growth factor (IGF) binding protein-3 (IGFBP3), a key regulator of IGF bioactivity, exerts either proapoptotic or growth stimulatory effects in a cellular context dependent manner. IGFBP3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP3 in esophageal tumor biology remains elusive. Methods: Human esophageal epithelial cells transformed by combinations of either Ha-RasV12 and SV40 T-antigen (T-Ag) or epidermal growth factor receptor (EGFR) and p53R175H were stably transduced with wild-type (WT) or I56G/L80G/L81G (GGG) mutant IGFBP3, the latter incapable of binding IGFs. Short hairpin RNAs (shRNAs) were used to knockdown IGFBP3. Gene expression was determined by real-time RT-PCR, Western blotting and immunofluorescence. Organotypic 3-D culture, a form of tissue engineering was done. Xenograft transplantation was also performed. Results: TGF-beta induced IGFBP3 robustly upon epithelial to mesenchymal transition (EMT) in EGFR-p53R175H transformed cells where a cadherin-class switch, induction of other mesenchymal markers and a cellular morphological change were documented. Interestingly, IGFBP3 knockdown prevented TGF-beta from inducing EMT while ectopically expressed WT or GGG-mutant IGFBP3 promoted EMT. In organotypic 3-D culture, IGFBP3 knockdown greatly impaired the invasive growth of the transformed cells. By contrast, WT or GGG-mutant IGFBP3 stimulated invasion. Upon xenograft transplantation, WT-IGFBP3 induced massive apoptosis and completely abrogated tumor formation by the Ras-transformed cells. By contrast, both empty vector control and GGG-IGFBP3 allowed tumor formation at a comparable rate. Interestingly, however, immunofluorescence revealed two major cell populations within the GGG-mutant IGFBP3 expressing tumors; SV40 T-Ag positive, pancytokeratin bright, and fibroblast specific protein 1 (FSP1) negative epithelioid tumor cells and T-Ag positive, pancytokeratin dim, and FSP1 positive spindle shaped tumor cells. The latter, representing a predominant subset of tumor cells, implied EMT and grew more aggressively. Such EMT-compatible tumor cells were present minimally in the empty vector control transduced tumors, suggesting that IGFBP3 may also facilitate EMT in vivo through an IGF-independent mechanism. Conclusions: Our innovative approach reveals that xenografted tumors may require IGF signaling for adaptation to the microenvironment in the host tissue where WT-IGFBP3 may antagonize a prosurvival effect of IGFs in vivo. However, once tumors are adapted to the local microenvironment, IGFBP3 may promote EMT in an IGF-independent fashion, a novel mechanistic finding. This has implications upon EMT in other cancers and may offer platforms for cancer therapy. Grants: NCI P01-CA098101 and NIH R01-DK077005 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2294.

Abstract 3276: Characterization of tumors in mouse oviduct-specific glycoprotein 1(Ogp1) promoter-driven SV40 large T antigen

Abstract It was well accepted for many years that ovarian surface epithelium is the origin of epithelial ovarian carcinomas. However, recent molecular and pathologic evidence indicate that high-grade serous carcinomas of the ovary, which are the most common type of epithelial carcinomas, may also arise from the distal part of fallopian tubes or tubal carcinoma implants. Gene expression profiles of tubal epithelium from BRCA mutation carriers resemble those of high-grade serous carcinomas of the ovary, and ovarian carcinomas and tubal intraepithelial carcinomas have identical p53 mutations. Oviductal glycoprotein 1 (Ogp1) is a glycosylated protein found in the epithelial cells in the oviduct. So far, Ogp1 is the only specific tubal marker for the oviduct. Ogp1 promoter-driven SV40 large T-antigen transgenic mice develop tumors in the female reproductive organs. To characterize these tumors, we did immunohistochemical analysis in frozen and paraffin-embedded sections. Mice were sacrificed at 8 weeks old. Macroscopically the cervico-vaginal region and the fallopian tubes were markedly enlarged. Histologically, three distinct abnormal proliferations were observed; (i) epithelial hyperplasia with cytologic atypia in the oviduct (with endometrioid architectural and cytologic features), (ii) leiomyosarcomatous proliferation in the myometrium, and (iii) squamous carcinoma in situ in the cervix with extensive papillary architecture. SV40 was expressed in these tumor sites. The oviduct demonstrated weak and spotty p53 expression. The expression of Ogp1 and Pax8 (a marker of the salpingeal secretory cells) in the oviduct epithelium showed patchy pattern. Ogp1 expression was not associated with cellular atypia, while Pax8 was expressed in normal cells. Expression of DNA replication licensing factor, Mini-chromosome maintenance protein 7(MCM7), was also mixed. MCM7 expression was higher in neoplastic cells, but atypical cells had less MCM7 expression. A serous marker, WT1, and a Müllerian-specific epithelial marker, Pax2, showed lower expressions in Ogp1-SV40 transgenic mouse compared to wild type control mouse. WT1 and Pax2 expression were uniform. In ovarian surface epithelium, there was no malignant transformation and no different expression of these markers in control and Ogp1-SV40 mice. In conclusion, this study demonstrated that Ogp1-SV40 transgenic mouse develops abnormal oviductal, myometrial, and cervical proliferations with no involvement of ovaries. Weak and spotty p53 expression in the oviduct indicates that p53 pre-dysfunction status in non-malignant precursor lesions. These results indicate that Ogp1-SV40 transgenic mouse is a useful model for studies involving fallopian and low genital tract carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3276.

Abstract 5774: Identification of novel targeted therapies for human triple-negative breast cancer

Abstract Triple Negative (TN) breast cancers are characterized by the lack of estrogen receptor, progesterone receptor, and Her2 in the tumors. Currently, unlike breast cancers that are hormone responsive or Her2+, there are no targeted therapies for this subtype of breast cancer. Additionally, TN breast cancer is frequently diagnosed in younger women, often prior to the age of routine mammography with young African-American women having a disproportionately higher incidence and mortality from TN breast cancers than Caucasians. Despite advances in the treatment of breast cancer, molecular characteristics of this subtype remain poorly understood and improved therapies are critically needed. Gene expression profiling has identified genetically engineered mouse (GEM) models that share important correlations with human breast tumor subtypes. In particular, the C3(1)/TAg GEM model appears to best represent basal-like/triple negative human breast cancers in several ways. The C3(1)TAg GEM model develops primary and metastatic mammary tumors following a progressive course characteristic of human breast tumor development, loss of p53 and Rb functions via SV40 T-antigen expression and amplifications of syntenic regions (hu. Chr. 12p12; mus. Chr. 6, a region containing K-RAS.) We have previously identified a critical proliferation/DNA repair/cell cycle gene signature (TAg signature), in primary mammary tumors from SV40 TAg mice that is also characteristic of human basal-like breast cancers. The TAg signature was also found to be highly predictive of human breast cancer prognosis. By targeting the up-regulated genes identified in the TAg signature, we have identified novel targets for the treatment of TN breast cancer. We have also performed synthetic lethality studies in which gene knockdown has been combined with common chemotherapy agents (cisplatin and paclitaxel) and agents currently in clinical trials (gemcitabine, Chk1 and Notch signaling inhibitors) to identify novel combination therapies. Several combinations are being tested in in vivo tumor transplant and human xenograft experiments. These studies have shown that the TAg signature can be used for the identification of new targets through siRNA screens of up-regulated genes in the signature and help determine new targets for preclinical testing of candidate drugs in novel combinations for treatment of TN breast cancer. The results of these studies may be translated into potentially effective therapies for women with aggressive forms of TN breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5774.

Abstract 944: Preventive effects of UV radiation and dietary vitamin D on autochthonous murine mammary tumors

Abstract Epidemiologic studies support an inverse correlation between solar UV radiation and breast cancer incidence and mortality, a matter of some concern in a society being urged to avoid sun exposure. We assessed directly whether UV radiation of skin has any preventive effect on mammary tumor carcinogenesis in the C3(1)SV40 Tag transgenic mouse. Mice fed diets depleted of vitamin D (+ rescue minerals) were treated with 0 [group A] or 350mJ/cm2 UV 3 times a week without [Group B] or with [Group C] topical 7-dehydrocholesterol, the precursor of cutaneous vitamin D3. Control mice were fed an otherwise identical diet but containing normal levels of vitamin D (1,000IU/kg + rescue minerals) [Group D]. We also fed mice a separate diet containing high (20,000IU/kg) [Group E] or normal (1,000IU/kg) [Group F] vitamin D. We examined mice and excised all palpable tumors (“lumpectomies”) every two weeks at ages 16-28 weeks. Blood was collected at the end of the study. Analyses were performed using Krusskal Wallis test, Mann Whitney test, Spearman's test, life table survival analysis, and log rank test. Circulating 25-hydroxyvitamin D3 (25D3) levels in 59 mice, measured in ng/mL [median (interquartile range)] in groups A-F were 3.0(1.0) 34.0(26.3), 26.0(18.0), 28.0(13.0), 121.0(82.5), and 42.0(15.5), respectively (p&amp;lt;0.001). We found no significant correlation among groups between 25D3 levels and tumor number, mean size, or total tumor volume/mouse. Mice fed the vitamin D depleted diet had a tumor burden that was reduced by UV radiation or by addition of dietary vitamin D, and UV-treated mice had a longer tumor free survival than did the D-depleted mice not so treated. By contrast, the high vitamin D diet, despite tripling the circulating 25D3 level, by comparison to the control diet did not influence mammary tumor number or size or tumor free survival. We interpret these data to suggest that although UV can inhibit development of autochthonous murine mammary tumors, the failure of tumor development to correlate strictly with circulating 25OH D3 levels suggests that its effects may be mediated in part by non-obvious pathways and/or that the anti-mammary cancer effects of vitamin D may plateau. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 944.

Regulation of FGF1 Gene Promoter through Transcription Factor RFX1

Fibroblast growth factor 1 (FGF1) has been suggested to have an important role in cell growth, proliferation, and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven green fluorescence (F1BGFP) has been shown to monitor endogenous FGF1 expression. F1BGFP could also be used to isolate neural stem/progenitor cells from embryonic, neonatal, and adult mouse brains or to isolate glioblastoma stem cells (GBM-SCs) from human glioblastoma tissues. Here, we present evidence that transcription factor RFX1 could bind the 18-bp cis-elements (−484 to −467) of the F1B promoter, modulate F1BGFP expression and endogenous FGF1 expression, and further regulate the maintenance of GBM-SCs. These observations were substantiated by using yeast one-hybrid assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, gain- and loss-of-function assays, and neurosphere assays. Overexpression of RFX1 was shown to down-regulate FGF-1B mRNA expression and neurosphere formation in human glioblastoma cells, whereas RNA interference knockdown of RFX1 demonstrated the opposite effects. Our findings provide insight into FGF1 gene regulation and suggest that the roles of FGF1 and RFX1 in the maintenance of GBM-SCs. RFX1 may negatively regulate the self-renewal of GBM-SCs through modulating FGF-1B and FGF1 expression levels by binding the 18-bp cis-elements of the F1B promoter.

Cross‐talk between T‐Ag presence and pRb family and p53/p73 signaling in mouse and human medulloblastoma

The formation and progression of mudulloblastoma (MB) is poorly understood. However, somatic inactivation of pRb/p105, in combination with a somatic or a germ-line TP53 inactivation, leads to MB in a mouse model. Presently, there is no specific evidence of pathway/s alterations for the other two members of the retinoblastoma family, pRb2/p130 and/or p107 in MB. JC virus (JCV) is a human polyomavirus. Although there is no firm evidence that this virus plays a causal role in human neoplasia, it has been clearly proven that JCV is highly oncogenic when injected into the brain of experimental animals. The mechanism of JCV-induced tumorigenesis is not entirely clear. However, several studies relate the oncogenic properties of JCV mainly to its early protein large T-antigen (T-Ag), which is able to bind and inactivate both TP53 and Rb family proteins. Here, we compared the protein expression profiles of p53, p73, pRb family proteins, and PCNA, as main regulators of cell proliferation and death, in different cell lines of mouse primitive neuroectodermal tumors (PNET), either T-Ag-positive or -negative, and in human MB cell lines. Our goal was to determine if changes in the relative expression of these regulators could trigger molecular perturbations underlying MB pathogenesis in mouse and human cells. Our results support that the presence of JCV T-Ag may interfere with the expression of pRb family proteins, specific p73 isoforms, and p53. In turn, this "perturbation" may trigger a network of signals strictly connected with survival and apoptosis.

Modulation of tumorigenesis and oestrogen receptor-α expression by cell culture conditions in a stem cell-derived breast epithelial cell line

The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ER alpha (oestrogen receptor alpha) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T-antigen transfection and X-ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone-deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and alpha 6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44+/high/CD24-/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial-to-mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX-2 (cyclo-oxygenase-2) through a CD44-mediated mechanism. An oestrogen-responsive cell line with ER alpha and CD44+/CD24-/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ER alpha-positive breast cancers, (2) CD44+/CD24-/low breast tumour stem cells and (3) the metastatic ability of breast cancer.

Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells

Early Growth Response-1 (Egr-1) is overexpressed in human prostate tumors and contributes to cancer progression. On the other hand, mutation of p53 is associated with advanced prostate cancer, as well as with metastasis and hormone independence.This study shows that in prostate cell lines in culture, Egr-1 overexpression correlated with an alteration of p53 activity due to the expression of SV40-TAg or to a mutation in the TP53 gene. In cells containing altered p53 activity, Egr-1 expression was abolished upon pharmacological inhibition or RNAi silencing of p53. Although forced expression of wild-type p53 was not sufficient to trigger Egr-1 transcription, four different mutants of p53 were shown to induce Egr-1. Direct binding of p53 to the EGR1 promoter could not be detected. Instead, Egr-1 transcription was driven by the ERK1/2 pathway, since it was abrogated by specific inhibitors of MEK. Egr-1 increased the transcription of HB-EGF, amphiregulin and epiregulin, resulting in autocrine activation of the EGFR (epidermal growth factor receptor) and downstream MEK/ERK cascade. Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway. Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen

Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.

Precise conditional immortalization of mouse cells using tetracycline‐regulated SV40 large T‐antigen

Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline-regulated system for the expression of SV40 large T-antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand-binding domain (LBD) of the androgen receptor (irtTA-ABD) or of a mutated glucocorticoid receptor (irtTA-GBD*). Induction of T-antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA-ABD or dexamethasone for irtTA-GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T-antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T-antigen.

Prevention of Senescence Progression in Reversibly Immortalized Human Ensheathing Glia Permits Their Survival After Deimmortalization

Reversible immortalization holds great potential for primary tissue expansion to develop cell-based therapies as well as for basic research. Human olfactory ensheathing glia (hOEG) are promising candidates for treating spinal cord injury and for studying extrinsic neuroregenerative mechanisms. We used lentivectors with Cre/loxP technology to achieve reversible gene transfer of BMI1, SV40 large T antigen (TAg), a short hairpin RNA against p53 (shp53), and the catalytic subunit of telomerase (TERT) in primary cultures of hOEG from human donor cadaver olfactory bulbs. Several combinations of these genes were able to immortalize hOEG, conserving their antigenic markers and neuroregenerative properties but only those transduced by BMI1/TERT did not accumulate karyotypic alterations or increase senescence marker levels. Strikingly, these were also the only cells which continued to proliferate after transgene removal by Cre recombinase delivery, whereas hOEG immortalized by shp53 or TAg in combination with TERT entered into growth arrest and died. These data support the idea that immortalization and halting senescent changes are separate processes; hOEG immortalized by BMI1/TERT can revert back to their former primary cell replicative state when deimmortalized, whereas those transduced by the other combinations depend on the presence of these transgenes to maintain their aberrant proliferative state.