Oncogene In Epithelial Cells Research Articles

Epstein-Barr Virus Gene BARF1 Expression is Regulated by the Epithelial Differentiation Factor ΔNp63α in Undifferentiated Nasopharyngeal Carcinoma.

Epstein–Barr Virus (EBV) BamHI-A rightward frame 1 (BARF1) protein is considered a viral oncogene in epithelial cells and has immune-modulating properties. During viral lytic replication BARF1 is expressed as an early gene, regulated by the immediate early EBV protein R. However, in viral latency BARF1 is exclusively expressed in epithelial tumors such as nasopharyngeal (NPC) and gastric carcinoma (GC) but not in lymphomas, indicating that activation of the BARF1 promoter is cell type specific. Undifferentiated NPC is characterized by high expression of ΔNp63 isoforms of the epithelial differentiation marker p63, a member of the p53 family of transcription factors. Transcription factor binding site analysis indicated potential p53 family binding sites within the BARF1 promoter region. This study investigated ability of various p53 family members to transactivate the BARF1 promoter. Using BARF1 promoter luciferase reporter constructs we demonstrate that only p63 isoform ΔNp63α is capable of transactivating the BARF1 promoter, but not the TAp63 isoforms, p53 or p73. Direct promoter binding of ΔNp63α was confirmed by Chromatin Immune Precipitation (ChIP) analysis. Deletion mutants of the BARF1 promoter revealed multiple ΔNp63 response elements to be responsible for BARF1 promoter transactivation. However, ΔNp63α alone was not sufficient to induce BARF1 in tumor cells harboring full EBV genomes, indicating that additional cofactors might be required for full BARF1 regulation. In conclusion, in EBV positive NPC and GC, BARF1 expression might be induced by the epithelial differentiation marker ΔNp63α, explaining BARF1 expression in the absence of lytic reactivation.

Scribble acts as an oncogene in Eμ-myc-driven lymphoma.

Scribble complex proteins maintain apicobasal polarity, regulate cell fate determination and function as tumour suppressors in epithelial tissue. Despite evidence that the function of Scribble is maintained in the lymphocyte lineage, we still understand little about its role as a tumour suppressor in haematological malignancies. Using the Eμ-myc model of Burkitt's lymphoma we investigated the role of Scribble in lymphomagenesis. We found that contrary to its well-documented tumour suppressor role in epithelial tissue, loss of Scribble expression delayed the expansion of peripheral B cells and delayed the onset of Eμ-myc-driven lymphoma. This was despite upregulated ERK phosphorylation levels in Scribble-deficient tumours, which are associated with loss of Scribble expression and the development of more aggressive Burkitt's lymphoma. Interestingly, the developmental stage of lymphoma was unaffected by Scribble expression challenging any role for Scribble in fate determination in the haematopoetic lineage. These data provide evidence for oncogenic properties of Scribble in Myc-driven B-cell lymphomagenesis, reinforcing recent findings that overexpression of a mutant form of Scribble can act as an oncogene in epithelial cells. Our results support the growing appreciation that the tumour regulatory functions of Scribble, and other polarity protein family members, are context dependent.

Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells.

Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation.

Metabotropic glutamate receptor 1 (Grm1) is an oncogene in epithelial cells

Non-neuronal expression of components of the glutamatergic system has been increasingly observed, and our laboratory previously had demonstrated the etiological role of ectopically expressed metabotropic glutamate receptor 1 (Grm1/mGluR1) in mouse models of melanoma. We hypothesize that inappropriate glutamatergic signaling in other cell types can dysregulate growth leading to transformation and tumorigenesis. As most cancers are carcinomas, we selected an immortalized primary baby mouse kidney (iBMK) cell model to assess whether Grm1 can transform epithelial cells. These iBMK cells, engineered to be immortal yet non-tumorigenic and retaining normal epithelial characteristics, were used as recipients for exogenous Grm1 cDNA. Several stable Grm1 expressing clones were isolated and the Grm1-receptors were shown to be functional, as evidenced by the accumulation of second messengers in response to Grm1 agonist. Additionally activated by agonist were MAPK and AKT signaling cascades, major intracellular pathways shown by many investigators to be critical in melanomagenesis and other neoplasms. These Grm1-iBMK cells exhibited enhanced cell proliferation in in vitro MTT assays and significant tumorigenicity in in vivo allografts. Persistent Grm1 expression was required for the maintenance of the in vivo tumorigenic phenotype as demonstrated by an inducible Grm1-silencing RNA. These are the first results that indicate Grm1 can be an oncogene in epithelial cells. Additionally, relevance to human disease in the corresponding tumor type of renal cell carcinoma (RCC) may be suggested by observed expression of GRM1/mGluR1 in a number of RCC tumor biopsy samples and cell lines, and the effects of GRM1 modulation on tumorigenicity therein. Moreover RCC cell lines exhibited elevated levels of extracellular glutamate, and some lines responded to drugs which modulate the glutamatergic system. These findings imply a possible role for glutamate signaling apparatus in RCC cell growth, and that the glutamatergic system may be a therapeutic target in renal cell carcinoma.

Epstein-Barr Virus Transcription Activator R Upregulates BARF1 Expression by Direct Binding to Its Promoter, Independent of Methylation

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides -554 and -327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.

The dawn of prophylactic vaccines against human papillomaviruses and cervical cancer

The dawn of prophylactic vaccines against human papillomaviruses and cervical cancer

E-Cadherin, β -Catenin and cadmium carcinogenesis

Cadmium (Cd2+) is an important industrial and environmental pollutant that has been classified as a human carcinogen. Studies reported in the literature indicate that cadmium may play a role in both the initiation of cancer, by activating oncogenes, and in the progression of cancer, by increasing the metastatic potential of existing cancer cells. However, the mechanisms underlying these effects have yet to be elucidated. Recent studies from our laboratory have shown that cadmium can disrupt the tight junctions between many types of epithelial cells by interfering with the normal function of E-cadherin, a Ca2+-dependent cell adhesion molecule that plays a key role in epithelial cell-cell adhesion. This finding may be especially significant because a large volume of evidence indicates that the disruption of E-cadherin-mediated cell adhesion can trigger the β-catenin-mediated activation of oncogenes in epithelial cells and increase the invasive potential of existing epithelial-derived cancers. The hypothesis that we are proposing is that the cadmium-induced disruption of E-cadherin dependent cell-cell junctions may represent a pivotal step in both the initiation of cancer by cadmium and in the tumor promoting actions of cadmium.

Absence of mutations in codon 61 of the Ha- ras oncogene in epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene

Epithelial cells of the respiratory tract of rats were transformed in vitro by 7,12-dimethylbenz(a)anthracene (DMBA) which has been reported to cause A→T transversion mutations of the second position of Ha- ras in codon 61 in several biological models. In this study Ha- ras exon 2 was amplified by the polymerase chain reaction (PCR) and then sequenced directly. In 10 transformed cell lines, of which 5 are known to be tumorigenic, no mutations in codon 61 were found. The results suggest that Ha- ras codon 61 mutations are not associated with cell transformation initiated with DMBA in this particular cell transformation system. These data imply that other genes (oncogenes) are responsible for transformation of these cells. The results are discussed in relation to observations in various transformation systems in vivo and in vitro.

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.