Transformation Of Human Epithelial Cells Research Articles

Polymorphism of E6 and E7 Genes in Human Papillomavirus Types 31 and 33 in Northeast China.

Persistent infection with human papillomavirus (HPV) types 31 and 33 is an important causative factor for cervical cancer. The E6/E7 genes are key oncogenes involved in the immortalization and transformation of human epithelial cells. Genetic polymorphism may lead to differences in the virus' carcinogenic potential, the immune reaction of the host, and the potencies of vaccines. Few studies on HPV31/33 E6/E7 genetic polymorphism have been carried out. To study the genetic polymorphism of HPV31 and HPV33 E6/E7 genes in northeast China, these genes (HPV31 E6/E7, n = 151; HPV33 E6/E7, n = 136) were sequenced and compared to reference sequences (J04353.1, M12732.1) using BioEdit. Phylogenetic trees were constructed by the neighbor-joining method using MegaX. The diversity of the secondary structure was estimated using the PSIPred server. The positively selected sites were analyzed using PAML4.9. The major histocompatibility complex (MHC) class I and MHCII epitopes were predicted using the ProPred-I server and ProPredserver. B-cell epitopes were predicted using the ABCpred server. In the 151 HPV31E6 sequences, 25 (25/450) single-nucleotide mutations were found, 14 of which were synonymous mutations and 11 were nonsynonymous. In the 151 HPV31E7 sequences, 8 (8/297) nucleotide mutations were found, 3 of which were synonymous mutations and 5 were nonsynonymous. In the 136 HPV33E6 sequences, 17 (17/450) nucleotide mutations were observed, 7 of which were synonymous mutations and 10 were nonsynonymous. C14T/G (T5I/S) was a triallelic mutation. Finally, in the 136 HPV33E7 sequences, 9 (9/294) nucleotide mutations were observed, 3 of which were synonymous mutations and 6 were nonsynonymous. C134T/A (A45V/E) and C278G/A (T93S/N) were triallelic mutations. Lineage A was the most common lineage in both HPV31 and HPV33. In all of the sequences, we only identified one positively selected site, HPV33 E6 (K93N). Most nonsynonymous mutations were localized at sites belonging to MHC and/or B-cell predicted epitopes. Data obtained in this study should contribute to the development and application of detection probes, targeted drugs, and vaccines.

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.

The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells

Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes.

Effects of Mercury Chloride and Nitrosamine on Neoplastic Transformation of Human Epithelial Cells: an in vitro Study

Effects of Mercury Chloride and Nitrosamine on Neoplastic Transformation of Human Epithelial Cells: an in vitro Study

Anti-oncogene of opioid binding protein/cell adhesion molecule-like methylation status in malignant transformation of human bronchial epithelial cells induced by glycidyl methacrylate

To analyze the opioid binding protein/cell adhesion molecule-like (OPCML) methylation status at different stages of malignant transformation of human bronchial epithelial (16HBE) cells induced by Glycidyl Methacrylate (GMA) and to explore the effect of OPCML methylation in the process of malignant transformation. Cells were harvested at different stages (the 10th generation, the 20th generation and the 30th generation). To verify the Methylation chip result of OPCML methylation status in the process of malignant transformation, we detected it by methylation-specific-PCR (MSP); Real-time fluorescence Quantitative PCR (qPCR) were applied to measure the gene expression levels of OPCML at different transformed stage, and compared with the control groups (treated with DMSO). Based on the result of methylation chip, the gene of OPCML methylation occurred in all stages, which was consistent to the result of MSP; qPCR showed that the levels of gene expression decreased in the 20th generation and 30th generation. Methylation status of OPCML gene promoter could be considered as a stable and specific biomarker in the transformation process.

Epidermal growth factor receptor protein overexpression and gene amplification are associated with aggressive biological behaviors of esophageal squamous cell carcinoma.

Alterations of the epidermal growth factor receptor (EGFR), including overexpression or gene mutations, contribute to the malignant transformation of human epithelial cells. The aim of this study was to assess EGFR overexpression or gene amplification in esophageal squamous cell carcinoma (ESCC) tissue samples and investigate their correlations with biological behaviors. Tissue specimens from 56 patients with surgically resected ESCC were obtained for immunohistochemical analysis of EGFR expression and fluorescence in situ hybridization analysis of EGFR amplification. The data were statistically analyzed to determine the associations with patient clinicopathological and survival data. EGFR was overexpressed in 30 of the 56 (53.6%) ESCC samples and was associated with poor tumor differentiation (P=0.047). EGFR amplification was detected in 13 cases (23.2%) and was associated with advanced pathological stage (P=0.042) and tumor lymph node metastasis (P=0.002). The univariate analysis identified no association between EGFR overexpression and the overall survival (OS) of the patients. By contrast, EGFR amplification predicted ESCC prognosis (P=0.031), while the multivariate analysis revealed a marginal statistical significance for the association between EGFR amplification and OS (P=0.056). EGFR overexpression and increased EGFR copy number were common events in ESCC and contributed to malignant biological behaviors, including tumor dedifferentiation and lymph node metastasis. EGFR amplification may therefore be useful in predicting OS in patients with ESCC.

Hijacking Dlg1 for oncogenic phosphatidylinositol 3-kinase activation in human epithelial cells is a conserved mechanism of human adenovirus E4-ORF1 proteins.

The E4-ORF1 gene of human adenoviruses encodes a 14-kDa protein that promotes viral replication as well as cellular metabolic reprogramming, survival, and transformation by constitutively activating cellular phosphatidylinositol 3-kinase (PI3K). We recently reported that the E4-ORF1 protein from subgroup D human adenovirus type 9 upregulates and oncogenically activates PI3K by a novel mechanism involving separate interactions of E4-ORF1 with cellular discs large 1 (Dlg1) and PI3K to form a ternary complex that translocates to the plasma membrane (K. Kong, M. Kumar, M. Taruishi, and R. T. Javier, PLoS Pathog. 10:e1004102, 2014, doi:10.1371/journal.ppat.1004102). The current study was carried out to investigate whether other human adenovirus E4-ORF1 proteins share this mechanism of action. The results showed that in human MCF10A epithelial cells, stable expression of E4-ORF1 proteins encoded by representative human adenovirus serotypes from subgroups A to D induce ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein elevation, Dlg1 and PI3K membrane recruitment, and PI3K-dependent cellular transformation. The first three of these E4-ORF1 activities were also observed in MCF10A cells infected with each wild-type human adenovirus from subgroups A to D. Our findings indicate that most, if not all, human adenovirus E4-ORF1 proteins share a conserved molecular mechanism of PI3K activation, which confers a common capacity to promote oncogenic transformation in human epithelial cells. PI3K activation by the adenovirus E4-ORF1 protein mediates oncogenic cellular transformation by human adenovirus type 9, augments viral protein expression and replication by human adenovirus type 5, and dysregulates cellular glucose and lipid metabolism by human adenovirus type 36. For the first time, we report that E4-ORF1 proteins from human adenoviruses in subgroups A to D evolved a conserved molecular mechanism to mediate constitutive PI3K activation that can provoke oncogenic transformation in human epithelial cells. The results raise potential safety concerns about the use of vectors encoding the E4-ORF1 gene in human gene therapy and vaccination. Our findings further suggest that the conserved mechanism revealed here may be targeted for development of therapeutic drugs to treat and prevent adenovirus-associated human diseases.

Tumor necrosis factor-α and NF-κB play a role in macrophage-like THP-1 cells promoting coal tar pitch extract-induced tumorigenic transformation of human bronchial epithelial cells

To characterize the role of tumor necrosis factor-α (TNF-α) and NF-κB play a role in macrophage-like THP-1 cells promoting coal tar pitch extract (CTPE)-induced tumorigenic transformation of human bronchial epithelial cells (BEAS-2B). From passage 10, CTPE-induced BEAS-2B cells cocultured with THP-1 cells were treated with NF-κB inhibitor-Pyrrolidine dithiocarbamate (PDTC) every 3 passages and TNF-α antibody every passage. Alterations of cell cycle, karyotype and colony formation in soft agar of BEAS-2B cells at passages 20, indicative of tumorigenicity, were determined, respectively. In addition, mRNA and protein levels of TNF receptor associated factor2 (TRAF2) and Cyclin D1 in BEAS-2B cells were measured with Real Time-PCR and Western blot, respectively. The percentages of S-phase BEAS-2B cells at passage 20 in PDTC group and TNF-α antibody group were (33.97±2.16)% and (34.29±2.04)% respectively, which were less than that in Co-culture+CTPE group of 20th passage [(44.46±0.83)%], P < 0.05; The number of cells with aneuploidy in 100 cells in 20th passage PDTC group and TNF-α antibody group were 40 and 37, and there were significantly different when comparing to that of 20th passage Co-culture+CTPE group (75); The number of colony formation and the rate of colony formation of BEAS-2B cells in soft agar at passage 20 in PDTC group were (15.17±2.48) and (1.51‰±0.25‰), (13.33±2.58)and (1.33‰±0.26‰) in TNF-α antibody group, which were less that those in 20th passage Co-culture+CTPE group [(172.33±12.09) and (17.23‰±1.20‰)], P < 0.05; at the same time, the mRNA and protein levels of TRAF2 and Cyclin D1 in BEAS-2B cells were decreased after PDTC and TNF-α antibody treatment. TNF-α and NF-κB could play an important role in THP-1 cells promoting coal tar pitch extract-induced tumorigenic transformation of BEAS-2B cells by influencing the expression of TRAF2 and Cyclin D1.

Changes of centrosome and related protein in malignant transformation of BEAS-2B cell induced by coal tar pitch smoke extracts

To analyze the centrosome abnormalities in the malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by coal tar pitch smoke extracts and to investigate the role and action mechanism of centrosome in the lung cancer induced by coal tar pitch. Medium-temperature coal tar pitch smoke extracts were used to treat immortalized human bronchial epithelial cells (BEAS-2B) and establish a malignant transformation model. The treated BEAS-2B cells were used as exposure group, and solvent control group and normal control group were also set for passage culture. The changes of centrosome in BEAS-2B cells seeded on coverslips were evaluated by indirect immunofluorescence assay. The mRNA expression of p53, p21, and cyclin E in BEAS-2B cells was measured by real-time quantitative RT-PCR, and their protein levels in BEAS-2B cells seeded on coverslips were measured by semiquantitative immunohistochemical analysis. The overall rate of centrosome abnormalities in BEAS-2B cells at passage 20 was 6.56±1.01% in the exposure group, significantly higher than those in the normal control group (3.40±0.86%) and solvent control group (3.14±0.59%) (P < 0.05). In addition, the exposure group had a significantly higher overall rate of centrosome abnormalities in BEAS-2B cells at passage 30 compared with the normal control group and solvent control group (22.39±9.5% vs 4.34±1.04%, P < 0.05; 22.39±9.5% vs 4.33±1.20%, P < 0.05). Compared with the normal control group and solvent control group, the exposure group had significantly decreased mRNA and protein expression of p53 and significantly increased mRNA and protein expression of cyclin E in BEAS-2B cells at passages 20 and 30 (P < 0.05). Centrosome abnormalities occur before the malignant transformation in BEAS-2B cells treated with coal tar pitch smoke extracts, and they may be mediated by the p53/p21/cyclin E signaling pathway.

Methylation status of P16 gene during malignant transformation of human bronchial epithelial cells induced by glycidyl methacrylate

To analyze the methylation status of P16 gene at the different stages of malignant transformation of human bronchial epithelial cells (16HBE) induced by glycidyl methacrylate (GMA) and to explore the DNA methylation mechanisms. The cells exposed to GMA were harvested at the end of exposure (early stage), the 10th generation (protophase) and the 30th generation (anaphase), respectively. The methylation status of P16 promotor was detected by Methylation-specific PCR (MSP). The transformed 16HBE cells were compared with the normal 16HBE cells and the cells exposed to DMSO for methylation status. At the early stage and protophase stage, the non-methylation status in P16 gene promotor of the normal 16HBE cells and the cells exposed to DMSO appeared, the methylation status in P16 gene promotor of the 16HBE cells exposed to GMA was detected to some extension. At the anaphase stage, the methylation status in P16 gene promotor of the 16HBE cells exposed to GMA or DMSO was detected to some extension. Methylation status of P16 gene promoter was specific at the early stage and protophase stage of malignant transforming in 16HBE cells induced by GMA, which can serve as an early sensitive biological indicator for malignant transforming in 16HBE cells induced by GMA.

Abstract 4813: EMT inducers catalyze malignant transformation of mammary epithelial cells and drive tumorigenesis towards claudin-low tumors

Abstract The malignant transformation of human epithelial cells is generally described as a multistep process resulting from the accumulation of five to seven rate-limiting changes. Here, we challenge this dogma and demonstrate that this number is radically reduced when cells undergo an epithelial-mesenchymal transition (EMT). EMT is an embryonic transdifferentiation process that consists of the conversion of polarized epithelial cells into motile mesenchymal ones. EMT-inducing transcription factors, including Twist and Zeb proteins, are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. We demonstrate that, beyond their prometastatic potential, EMT inducers also act as potent drivers of tumorigenesis. We indeed show that the Twist1 EMT-inducing transcription factor promotes breast and skin cancer development in vivo in cooperation with the K-RasG12D oncoprotein. Importantly, in the model of breast tumorigenesis, transgene expression in differentiated mammary epithelial cells leads to the development of undifferentiated tumors exhibiting all the characteristic features of the claudin-low subtype. These observations challenge the concept that this tumor subtype specifically arises from transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Consistently, oncogenic cooperation assays performed in human mammary epithelial cells with Twist or Zeb EMT-inducers in combination with H-RasG12V generate transformed cell lines displaying all characteristics of claudin-low tumors including mesenchymal features, undifferentiated traits, and stem-cell-like properties. EMT might thus drive the development of claudin-low tumors by exhibiting a dual role in cell transformation and dedifferentiation. In other terms, the claudin-low tumor subtype of breast cancers might thus constitute a first example of human adult malignancies driven by aberrant reactivation of an embryonic transdifferentiation program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4813. doi:1538-7445.AM2012-4813

The effects of monocyte-macrophages on malignant transformation of human bronchial epithelial cells induced by extracts from coal tar pitch

To study the effects of monocyte-macrophages (THP-1) in malignant transformation of human bronchial epithelial cells (BEAS-2B) cells induced by coal tar pitch (CTP) and the expression of TNF-α in the process of the cell malignant transformation. BEAS-2B cells and THP-1 Cells were divided into four groups: coal tar pitch (CTP) group, benzo(a)pyrene [B(a)P] group, dimethyl sulfoxide (DMSO) group, BEAS-2B and THP-1 co-culture (co-culture group) group. Carcinogenesis model was established. The soft agar colony formation, chromosome aberrations and cell cycle tests were used to detect the cellular malignant transformation. The ELISA assay was utilized to measure the levels of TNF-α in the supernatant of CTP group and co-culture group. The chromosome number abnormalities could be observed in early stage of the experiment (the 10th generation cells), which showed the increased ratio of aneuploid to polyploid, and the decreased number of diploid. The colony formation rate of co-culture group (the 20th generation cells) was 17.63‰ ± 0.97‰, which was significantly higher than that (13.94‰ ± 0.84‰) of CTP group and that (12.96‰ ± 1.62‰) of B(a)P group (P < 0.05). The proportion of S phase cells in the co-culture group was 44.49% ± 0.68%, which was significantly higher than that (38.19% ± 1.26%) of CTP group and that (36.41% ± 1.19%) of B(a)P group (P < 0.05). The TNF-α level in the co-culture group was significantly higher than that in CTP group (P = 0.001). Monocyte-Macrophages can accelerate the malignant transformation of BEAS-2B cells induced by CTP and increase the expression level of TNF-α.

Purification and direct transformation of epithelial progenitor cells from primary human prostate

Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without previous immortalization via genetic influences such as SV40 T-antigen, thus limiting our knowledge of the events that can transform naive human epithelium. Here we describe a system developed in our laboratory to directly transform freshly isolated primary human prostate epithelial cells without previous culture or immortalization. Prostate tissue is obtained from patients and benign tissue is separated from malignant tissue. Benign and malignant tissues are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated on the basis of differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. At 8-16 weeks after transplantation into immune-deficient mice, the development of lesions, histologically classified as benign prostate, prostatic intraepithelial neoplasia and adenocarcinoma, can be evaluated.

Smoking-induced Human Breast Cancer Formation through Induction of α9-nicotinic Acetylcholine Receptor

Tobacco is a substance that contains human carcinogens and may contribute to the risk for tumor formation in the lung and colon via nicotinic acetylcholine receptors (nAChRs). Nicotine receptors are a family of ligand-gated ion channels that mediate the effects of the neurotransmitter acetylcholine and are among the most well-understood receptors. The α4β2 nAChR is known to exist in the central nervous system (CNS) and functions as a transmitter, whereas the α7-nAChR is a well-established molecule that is involved in lung cancer tumorigenesis. Previously, however, there was no direct evidence to provide a link between tobacco carcinogens and cellular molecules involved in breast tumorigenesis. Recently, two large cohort epidemiological studies undertaken in the USA and Japan have indicated that breast cancer risk is associated with both active and passive smoking. Analysis of α9-nAChR expression in clinical tumor tissues indicated that theα9-nAChR is important for tumor carcinogenesis in vivo and nicotine-induced transformation of normal human breast epithelial cells in vitro. Long-term exposure to estrogen and nicotine significantly increases α9-nAChR expression and forms a positively regulated loop and that can be blocked by several chemicals and natural compounds. In this paper, the structure and functional relationship of the nAChR will be discussed. The role of nAChR in breast cancer formation and its implication for prevention of cancer formation will also be discussed.

Analysis of expression and structure of the TSG101 gene in cervical cancer cells

Human papillomavirus (HPV)-mediated transformation of human epithelial cells has been recognized as a multi-step process in which additional unknown factors and (epi)genetic events are required. The tumor susceptibility gene 101 (TSG101) was discovered in mouse NIH3T3 fibroblast cells as a gene whose functional knockout leads to transformation. TSG101 protein is involved in a variety of important biological functions, such as ubiquitination, transcriptional regulation, endosomal trafficking, virus budding, proliferation and cell survival. It is suggested that TSG101 is an important factor for maintaining cellular homeostasis and that perturbation of TSG101 functions leads to cell transformation. Interestingly, a recent report showed up- or down-regulation of TSG101 in several human malignancies. At present, the role of TSG101 in cervical tumorigenesis is unexplained. TSG101 expression in tumors, where carcinogenesis is connected with viral infection, and a mechanism of TSG101 expression regulation in cancer cells are also unknown. The aim of our study was to estimate the TSG101 mRNA and protein level in cervical cancer and non-tumor epithelial cells. We also analyzed the TSG101 coding and promoter sequence using the PCR-SSCP technique and methylation pattern of the TSG101 promoter. Our real-time PCR and Western blot analysis showed decreased TSG101 mRNA and protein level in cervical cancer tissue in comparison to normal (non-tumor) HPV(-) and HPV16(+) epithelial cells. Our results suggest that TSG101 down-regulation in cervical cancer cells is not regulated by genetic or epigenetic events. However, we detected novel single nucleotide polymorphisms in the promoter of this gene.

Mouse mammary tumor virus-related sequences in Mus musculus genome and human breast cancer tissues.

Abstract Abstract #3087 Background: Mouse mammary tumor virus is the causative agent for breast cancer (BC) in susceptible mouse lines. MMTV –homologous sequences were detected in breast carcinoma tissue DNA samples from 14-75% BC patients in Mexico, China, USA, Australia and Tunis. However, the sequences weren't found in Japanese, Austrian, English and some other human populations. Earlier, we have found the env MMTV –homologous sequences in BC tissues and peripheral blood lymphocyte DNA samples in 39-42% sporadic and 52% familial BC as well as in 64% of BC during pregnancy and/or lactation. These population differences in MMTV-related retrovirus frequencies might associated with geographic distribution of Mus musculus sp.domesticus – native MMTV reservoir. Material and methods: To demonstrate if wild commensal mice species are MMTV carriers we investigate 11 mice ( 3 males, 8 females) related to Mus musculus sp.domesticus from Moscow region (Russia) using specific PCR and RT-PCR followed by PCR products subcloning in pGEM-T vector and sequencing. A gp52 and ITAM-coding areas of the env MMTV gene, significant for MMTV infection and epithelial cells transformation , as well as a Sag-coding area of 3'LTR were analyzed. MMTV superantigen (Sag) – mediated amplification of MMTV in lymphoid cells is required for efficient viral transformation and dissemination in the mammary gland. Results: The sequences homologous to these functionally significant MMTV genome areas were detected in the DNA samples from mouse liver, timus and peripheral blood. In 63% (7/11) animals analyzed the sequences were expressed. The sequences homology with MMTV genome (C3H strain) according BLAST was 93-96%; homology with human MMTV- related provirus detected in BC patients was 86-95%. Recently MMTV-induced human epithelial cell transformation and important role of ITAM-coding area of the env MMTV gene was demonstrated in vitro. Conclusion: MMTV-related sequences were found in wild commensal mice, Mus musculus sp.domesticus, from Moscow region. In 60% cases these sequences, homologous to functionally significant env- and Sag-coding virus genome regions were expressed. Preliminary data support the possibility of the horizontal MMTV transmission from mice to humans, except of vertical one from mother to children. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3087.

Expression Profiling of Galectin-3-Depleted Melanoma Cells Reveals its Major Role in Melanoma Cell Plasticity and Vasculogenic Mimicry

Galectin-3 (Gal-3) is a beta-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells. In vitro, Gal-3 silencing resulted in loss of tumor cell invasiveness and capacity to form tube-like structures on collagen ("vasculogenic mimicry"). cDNA microarray analysis after Gal-3 silencing revealed that Gal-3 regulates the expression of multiple genes, including endothelial cell markers that appear to be aberrantly expressed in highly aggressive melanoma cells, causing melanoma cell plasticity. These genes included vascular endothelial-cadherin, which plays a pivotal role in vasculogenic mimicry, as well as interleukin-8, fibronectin-1, endothelial differentiation sphingolipid G-protein receptor-1, and matrix metalloproteinase-2. Chromatin immunoprecipitation assays and promoter analyses revealed that Gal-3 silencing resulted in a decrease of vascular endothelial-cadherin and interleukin-8 promoter activities due to enhanced recruitment of transcription factor early growth response-1. Moreover, transient overexpression of early growth response-1 in C8161-c9 cells resulted in a loss of vascular endothelial-cadherin and interleukin-8 promoter activities and protein expression. Thus, Gal-3 plays an essential role during the acquisition of vasculogenic mimicry and angiogenic properties associated with melanoma progression.

Establishment and application of oncogene over expressed human epithelial cell transformation model

To establish human bronchial epithelial cell lines over expressing oncogene and to investigate its application in detection of carcinogen-induced cell transformation. Mediated by retrovirus infection, human telomerase catalytic subunit, hTERT was introduced into immortal human bronchial epithelial cells (16HBE) and followed by introduction of the oncogenic allele H-Ras(V12), or c-Myc or empty vector, creating cell lines 16HBETR, 16HBETM and 16HBETV, respectively. Biological characteristics of these cell lines including morphology, proliferation, and chromosomal aberration were examined to access whether they were transformed. Soft agar experiment and nude mice subcutaneous injection were performed using pre-transformed 16HBE cells induced by known carcinogens, nickel sulfate (NiSO4) and 7, 8, -dihydrodiol-9, 10-epoxide benzo[a] pyrene (BPDE). With detection of telomerase activity and Western blotting, the expression of target proteins was verified. Thus, the transgenic 16HBE cell lines were successfully established. Cells expressing oncogene H-Ras or c-Myc grew 30.3% or 10.4% faster than control cells. However, these cells failed to form colonies in soft agar or form tumor in nude mice. 16HBETR, 16HBETM cells obtained transformed phenotype at 5 wks, 11 wks, respectively after treatment with BPDE, which are 15 wks and 9 wks earlier than control cells 16HBETV (20 wks). Meanwhile, 16HBETR, 16HBETM cells obtained transformed phenotype at 11 wks, 14 wks, respectively after treatment with nickel sulfate, which are 21 wks and 18 wks earlier than control cells (32 wks). With the advantage of shorter latency, transgenic human cell transformation models could be used in potent carcinogen screening and applied to chemical-carcinogenesis mechanism study.

Serum and tissue levels of insulin-like growth factor-I in women with dysplasia and HPV-positive cervical cancer

The ectocervical epithelium is the target of the oncogenic human papillomavirus (HPV), which acts as an etiological agent in the development of cervical carcinoma. However, the HPV-mediated transformation of human epithelial cells is a multi-step process dependant on unknown factors additional to the virus, which is a necessary but, in and of itself, insufficient catalyst. In the present study, we characterized the role of insulin-like growth factor-I (IGF-I) in this process. IGF-I is an endocrine hormone with an autocrine and paracrine role in many tissues. Our data demonstrated that autocrine secretion of IGF-I can contribute to HPV-induced carcinogenesis of the epithelium. An immunohistochemical study showed that IGF-I was present in the nuclei of the reproductive layer of the paraepidermal epithelium in 89% of cases of intraepithelial neoplasia (CIN I-III) and 60% of cases of invasive cervical cancer. The presence of IGF-I at a nuclear localization in the cells studied suggests that it may also have intranuclear actions.

Id-1 modulates senescence and TGF-β1 sensitivity in prostate epithelial cells

Loss of sensitivity to TGF-beta1 (transforming growth factor beta1)-induced growth arrest is an important step towards malignant transformation in human epithelial cells, and Id-1 (inhibitor of differentiation or DNA binding-1) has been associated with cell proliferation and cell-cycle progression. Here, we investigated the role of Id-1 in cellular sensitivity to TGF-beta1. Using an immortalized prostate epithelial cell line, NPTX cells, we suppressed Id-1 expression through antisense strategy. We found that inhibition of Id-1 expression suppressed cell proliferation and at the same time induced cellular senescence and G2/M cell-cycle arrest. In addition, inactivation of Id-1 made cells more vulnerable to TGF-beta1-induced growth arrest. The sensitization effect on TGF-beta1 was associated with up-regulation of two downstream effectors of the TGF-beta1 pathway, p21WAF1/Cip1 and p27KIP1. Our results indicate that endogenous Id-1 levels might be a crucial factor in the development of resistance to TGF-beta1-induced growth suppression in human prostate epithelial cells.