Transformation Of Cells In Culture Research Articles

Cytokine associated neopterin response of peripheral blood mononuclear cells to in vitro Epstein–Barr virus transformation process of B lymphocytes

The Epstein-Barr Virus (EBV) is a human herpes virus that infects B lymphocytes latently and immortalises the cells due to transformation. EBV infection is asymptomatic in childhood, while it may cause a self-limiting lymphoproliferative disorder in adolescence. This study aimed to show the association between in vitro transformation of B lymphocytes via EBV and the levels of neopterin with different accompanying cytokine responses. Materials and methods: Mononuclear cells were isolated from peripheral blood samples of healthy donors and transformed with an EBV secreting cell-line, B95-8. Neopterin, interferon (IFN)-\gamma, tumour necrosis factor (TNF)-\alpha, and interleukin (IL)-6 levels were measured in culture supernatants using enzyme immunoassay. Results: Neopterin levels increased dependent on time and independent of EBV transformation. In transformed cell cultures, TNF-\alpha and IFN-\gamma levels reached their peak in the first week and decreased in the third week; however there were no significant differences between the 3 weeks. IL-6 reached its maximum level in the third week. Conclusion: According to these results, neopterin levels, which increased with time and independently of EBV transformation, may be a helpful marker for evaluating the acute response to viral infection, but not for B lymphocyte transformation

Akt-Mediated Regulation of Autophagy and Tumorigenesis Through Beclin 1 Phosphorylation

Aberrant signaling through the class I phosphatidylinositol 3-kinase (PI3K)-Akt axis is frequent in human cancer. Here, we show that Beclin 1, an essential autophagy and tumor suppressor protein, is a target of the protein kinase Akt. Expression of a Beclin 1 mutant resistant to Akt-mediated phosphorylation increased autophagy, reduced anchorage-independent growth, and inhibited Akt-driven tumorigenesis. Akt-mediated phosphorylation of Beclin 1 enhanced its interactions with 14-3-3 and vimentin intermediate filament proteins, and vimentin depletion increased autophagy and inhibited Akt-driven transformation. Thus, Akt-mediated phosphorylation of Beclin 1 functions in autophagy inhibition, oncogenesis, and the formation of an autophagy-inhibitory Beclin 1/14-3-3/vimentin intermediate filament complex. These findings have broad implications for understanding the role of Akt signaling and intermediate filament proteins in autophagy and cancer.

RhoA triggers a specific signaling pathway that generates transforming microvesicles in cancer cells

Vesicular structures called microvesicles (MV) that are shed from the surfaces of cancer cells are capable of transferring oncogenic cargo to recipient cancer cells, as well as to normal cells, sending mitogenic signals that greatly enhance tumor growth. Because MVs are stable in the circulation, they also may play a key role in secondary colonization and metastasis. Thus, understanding how MVs are generated could have important consequences for interfering with cancer progression. Here we report that the small GTPase RhoA triggers a specific signaling pathway essential for MV biogenesis in various human cancer cells. Inhibiting the activity of different proteins comprising this pathway blocks MV biogenesis in the donor cancer cells and prevents oncogenic transformation in cell culture as well as tumor growth in mice. While RhoA has often been implicated in human cancer, these findings now highlight a previously unappreciated role for this GTPase in malignant transformation, and demonstrate that blocking MV biogenesis may offer novel approaches for interfering with malignant transformation.

Abstract A177: Phase 1, dose-escalation, safety, pharmacokinetic and pharmacodynamic study of single agent PF-03758309, an oral PAK inhibitor, in patients with advanced solid tumors.

Abstract Background: P21 activated kinase (PAK), a family of serine-threonine kinases, is a downstream effector of the Rho family and functions as a signaling node for various oncogenic pathways. The capability to transform cell cultures and the consistent anti tumor response associated with PAK inhibition in preclinical studies has raised the interest of PAK as a potential target for oncology. PAK consists in 6 isoforms divided into 2 subgroups. PAK isoforms 1 and 4 have been more frequently related to cancer biology. PF309 is a potent ATP-competitive inh of PAK1, 4, 5 and 6. Methods: Adult pts with advanced solid tumors were enrolled in a Ph 1 single agent dose escalation study with a standard 3 + 3 design. Oral PF309 was given on a continuous daily dosing. Tumor response was assessed at baseline and every 8 wks thereafter. AEs were recorded according NCI CTC v3.0. PK assessment was a study objective and PD endpoints (downstream substrates of the PAK pathway, eg: GEFH1, p-PAK4, p-cofilin) were to be explored in pre and post-treatment tumor biopsies as well as in surrogate tissue (hair follicles). Results: 33 pts (17 M and 16 F) were treated with PF309 among 8 dose levels (1 mg QD to 60 mg BID). Median age was 62.0 years (range 24–80) and median ECOG PS was 1. The most frequent tumors were CRC (16 pts), NSCLC (4 pts), breast cancer (2 pts), osteosarcoma (2 pts) and pancreatic cancer (2 pts). Median number of cycles was 3 (range 1 to 10). All subjects experienced treatment emergent AEs; 9 subjects experienced G3–4 AEs, and 2 SAEs were reported from 2 subjects: abdominal pain and hemoptysis (both at 60 mg BID). One patient died while on study due to PD. At the time of this report 2 patients experienced DLTs at 60 mg BID: G3 diarrhea (1 pt), and G2 nausea, vomiting and diarrhea, and G1 abdominal cramping leading to the administration of less than 80% of the planned dose during C1 (1pt). As a result 60 mg BID is the maximum administered dose (MAD). Treatment-related AEs were mainly from the GI tract: G3 diarrhea (1 pt), G1–2 diarrhea (10 pts), G1–2 vomiting (8 pts), G1–2 nausea (8 pts each), abdominal pain (5 pts) and fatigue (3 pts). G1–2 QTcF prolongation was observed in 11 pts (G1 in 6 pts, G2 in 5 pts), 6 of whom had an abnormal pre-treatment QTcF. No QTcF increase > 60 ms above baseline was observed in any patient. G3 lab abnormalities were rarely reported and included hyponatremia, thrombocytopenia, neutropenia and increased bilirubin (1 pt each). The PK data demonstrated that after attainment of Cmax within 5 hours after dosing, PF309 plasma concentrations declined in a multi-exponential manner with an average terminal half-life of 12.4 to 17.8 hs across doses. There was a lack of dose proportionality in the dose range of 10 to 60 mg, likely due to the high inter-patient variability. The inter-patient variability of AUCtau and Cmax at steady-state ranged from 21 to 177% and 45 to 184%, respectively, across doses. So far no tumor responses have been observed. A total of 10 pts have been on study treatment for ≥ 12 weeks. One heavily pretreated pt with CRC and progressive increase of the CEA serum level experienced a decrease in the CEA value from 1343.3 to 625.2 ng/ml, and remained on study for 4 months. Conclusions: GI toxicity is the most relevant toxicity associated with PF309 and 60 mg BID is the MAD. MTD confirmation is ongoing. Clinical development of PF309 is on hold due to PK findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A177.

Viruses and microRNAs: RISCy interactions with serious consequences

Analyses of small RNA expression profiles have revealed that several DNA viruses-including particularly, herpesviruses-express high levels of multiple viral microRNAs (miRNAs) in infected cells. Here, I review our current understanding of how viral miRNAs influence viral replication and pathogenesis and discuss how viruses reshape the pattern of cellular miRNA expression. Indeed, viruses are now known to both activate and repress the expression of specific cellular miRNAs, and disrupting this process can perturb the ability of viruses to replicate normally. In addition, it is now clear that virally encoded miRNAs play a key role in inhibiting antiviral innate immune responses and can also promote cell transformation in culture. While our understanding of how viruses interact with miRNAs remains somewhat rudimentary, it is nevertheless already clear that these interactions can play a critical role in mediating viral pathogenesis and therefore may represent novel and highly specific targets for therapeutic intervention.

Abstract 2359: Regulation of cytoskeleton organization by sphingosine in a mouse cell model of ovarian cancer progression

Abstract Ovarian cancer is the fifth leading cause of cancer deaths among women mainly because of late detection of the disease due the lack of symptoms and reliable biomarkers. Approximately 90% of ovarian cancers are derived from the surface epithelium of the ovary. To further investigate the events that lead to progression of epithelial ovarian cancer, we previously developed a cell model of ovarian cancer. Mouse ovarian surface epithelial (MOSE) cells that have undergone spontaneous transformation in cell culture were categorized into distinct morphological and phenotypical stages based on passage history: early passage cells (MOSE-E) represent a pre-neoplastic, non-tumorigenic phenotype whereas late passage cells (MOSE-L) exhibit a highly aggressive malignant phenotype both in vitro and in vivo. In depth studies of genetic changes during MOSE cell progression in our labs have identified the cytoskeleton and its regulatory genes as critical early progression events. These changes in gene expression led to an increasingly dysregulated cytoskeleton which may affect the intracellular localization of signaling intermediates, and, thereby, important downstream signaling events involved in cancer progression. Sphingosine (SO), a bioactive metabolite of complex sphingolipids, has been shown to have chemopreventive activity against cancer, including cancer of the colon, breast and ovarian. In the present study, we have investigated the hypothesis that SO prevents or reverses the observed aberrant cytoskeleton organization, and thereby affects the cells metastatic potential. In MOSE-L cells, SO treatment did not significantly influence the gene expression or protein levels of several cytoskeleton components or regulatory proteins. However, immunostaining of SO-treated MOSE-L cells revealed a reversal of the aberrant cytoskeleton organization and mislocalization of regulatory proteins, many of which are associated with cell migration. This corresponds to observations that SO reduces cell motility in MOSE cells. Our data indicates that cytoskeleton re-organization induced by exogenous SO treatment may be involved in the regulation of signaling pathways that reduce proliferation, migration and invasion of the MOSE cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2011-2359

Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression Model

BackgroundOvarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment.Methodology/Principal FindingsMouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII.Conclusions/SignificanceOur studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression.

Influence of fungal elicitation on glycyrrhizin production in transformed cell cultures of Abrus precatorius Linn.

Background:Glycyrrhizin, obtained from Abrus precatorius (Indian liquorice), is a phytoconstituent of importance for pharmaceutical and food industries.Materials and Methods:High producing and fast growing cell lines of A. precatorius were developed by transformation with Agrobacterium tumefaciens for glycyrrhizin production. Its maximum transformation efficiency of 85% was obtained by infecting leaves with A. tumefaciens MTCC-431 supplemented with 50 μM acetosyringone. Thorough culture growth kinetics with sugar consumption profiles was established.Results:A twofold increase in glycyrrhizin productivity was obtained in transformed A. precatorius cell suspension cultures over the untransformed cultures. The fungal elicitors prepared from Aspergillus niger and Rhizopus stolonifer were tested at different concentrations to enhance glycyrrhizin production in transformed cell suspension cultures of A. precatorius. Maximum enhancement of 4.9- and 3.8-fold in glycyrrhizin contents, were obtained with A. niger (7.5% v/v) and R. stolonifer (5.0% v/v), respectively, on the 5th day after elicitor treatment.Conclusion:This study indicates the prospective of the amalgamation of elicitation methodology with transformed cell cultures for the large-scale production of glycyrrhizin.

Evidence and controversies on the role of XMRV in prostate cancer and chronic fatigue syndrome

The recent discovery of xenotropic murine leukaemia virus-related virus (XMRV) in prostate cancer tissues and in the blood of individuals suffering from chronic fatigue syndrome has attracted considerable interest. However, the relevance and significance of XMRV to human disease remain unclear, since the association has not been confirmed in other studies. XMRV is the first gammaretrovirus to be found in humans. XMRV and murine leukaemia viruses share similar structures and genomic organisation. Human restriction factors such as APOBEC3 or tetherin inhibit XMRV replication. Although XMRV induces low rates of transformation in cell culture, it might be able to induce cancer by low-frequency insertional activation of oncogenes or through the generation of highly active transforming viruses. A preference for regulatory regions of transcriptional active genes has been observed after a genomic-wide analysis of XMRV integration sites. Genes related to carcinogenesis and androgen signalling have been identified in the vicinity of integration sites. The XMRV genome contains a glucocorticoid responsive element, and androgens could modulate viral replication in the prostate. Evidence supporting the involvement of XMRV in chronic fatigue syndrome is still very weak, and needs further confirmation and validation. Currently approved anti-retroviral drugs such as zidovudine, tenofovir and raltegravir are efficient inhibitors of XMRV replication in vitro. These drugs might be useful to treat XMRV infection in humans. The identification of XMRV has potentially serious health implications for the implementation of novel techniques including gene therapy or xenotransplantation, while raising concerns on the need for screening donated blood to prevent transmission through transfusion.

CDPK gene expression in salt tolerant rolB and rolC transformed cell cultures of Panax ginseng

CDPKs (calcium-depended protein kinases) are of great importance for the activation of defense reactions in plants. In this study, we aimed to find a connection between CDPK expression and increased salt tolerance in Panax ginseng. Treatment of P. ginseng cell cultures with W7 (CDPK protein inhibitor) showed that CDPK proteins were necessary for salt tolerance. Expression of PgCDPK1c, PgCDPK2c and PgCDPK4a was significantly increased in the cells treated with 60 mM NaCl compared to control cells, whereas expression of PgCDPK1b and PgCDPK3a was decreased. In the NaCl-treated cells, new CDPK transcripts also appeared (PgCDPK3c, PgCDPK4as). We also used rolC and rolB transformed cultures and the effects of the rol genes on CDPK expression were similar to the effects of salt stress: they caused a significant increase in the expression of PgCDPK1c, PgCDPK2c, and PgCDPK4a and decreased expression of PgCDPK3a, in addition to the appearance of the "short" CDPK transcripts.

Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate

Dedifferentiated Taxus media cell cultures presenting the same genetic characteristics as the parent culture were established from transformed roots. Two transformed cell lines were studied: Rol C, carrying the T-DNA of A. rhizogenes 9,402 and TXS, carrying both the T-DNA of A. rhizogenes and the txs transgene of T. baccata under the control of the 35S CaMV promoter. In the second part of a previously optimized two-stage system, the transformed cell lines were cultured in a production medium supplemented with the elicitor methyl jasmonate. Taxane production in the transformed cultures was compared with an untransformed T. media cell line cultured in the same conditions. The highest taxane production was observed in the TXS cell line when cultured in the optimized production medium with methyl jasmonate, being 265% greater than in the untransformed control and 170% greater than in the Rol C cell line. However, txs expression and the activity of the enzyme taxadiene synthase in the TXS cells were lower than in the line carrying only the rol genes (Rol C). It is also noteworthy that the taxane production as well as the txs gene expression and TXS activity in all the cell lines, both transformed and untransformed, were clearly dependent on the elicitor action.

SCCRO Promotes Glioma Formation and Malignant Progression in Mice

Originally identified as an oncogene activated by amplification in squamous cell carcinomas, several lines of evidence now suggest that squamous cell carcinoma-related oncogene (SCCRO; aka DCUN1D1) may play a role in the pathogenesis of a wide range of human cancers including gliomas. SCCRO's oncogenic function is substantiated by its ectopic expression, resulting in transformation of cells in culture and xenograft formation in nude mice. The aim of this study was to assess the in vivo oncogenicity of SCCRO in a murine model. Ubiquitous expression of SCCRO resulted in early embryonic lethality. Because SCCRO overexpression was detected in human gliomas, its in vivo oncogenic activity was assessed in an established murine glioma model. Conditional expression of SCCRO using a replication-competent ASLV long terminal repeat with splice acceptor/nestin-(tumor virus-A) tv-a model system was not sufficient to induce tumor formation in a wild-type genetic background, but tumors formed with increasing frequency and decreasing latency in facilitated background containing Ink4a deletion alone or in combination with PTEN loss. Ectopic expression of SCCRO in glial progenitor cells resulted in lower-grade gliomas in Ink4a-/- mice, whereas its expression in Ink4a-/-/PTEN-/- background produced high-grade glioblastoma-like lesions that were indistinguishable from human tumors. Expression of SCCRO with platelet-derived growth factor-beta (PDGF-β) resulted in an increased proportion of mice forming glioblastoma-like tumors compared with those induced by PDGF-β alone. This work substantiates SCCRO's function as an oncogene by showing its ability to facilitate malignant transformation and carcinogenic progression in vivo and supports a role for SCCRO in the pathogenesis of gliomas and other human cancers.

The Cancer/Testis Antigen CAGE with Oncogenic Potential Stimulates Cell Proliferation by Up-regulating Cyclins D1 and E in an AP-1- and E2F-dependent Manner

A cancer/testis antigen, CAGE, is widely expressed in various cancer tissues and cancer cell lines but not in normal tissues except the testis. In the present study, ectopic expression of CAGE in fibroblast cells resulted in foci formation, suggesting its cell-transforming ability. Using stable HeLa transfectant clones with the tetracycline-inducible CAGE gene, we found that CAGE overexpression stimulated both anchorage-dependent and -independent cell growth in vitro and promoted tumor growth in a xenograft mouse model. Cell cycle analysis showed that CAGE augments the levels of cyclin D1 and E, thereby activating cyclin-associated cyclin-dependent kinases and subsequently accelerating the G(1) to S progression. Moreover, increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even in a growth arrested state, indicating a direct effect of CAGE on G(1) cyclin expression. CAGE-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced retinoblastoma phosphorylation and subsequent E2F-1 nuclear translocation. In contrast, small interfering RNA-mediated knockdown of CAGE suppressed the expression of G(1) cyclins, activation of AP-1 and E2F-1, and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclins D1 and E.

Abstract 2875: Regulation of gene expression by sphingosine in a mouse cell model of ovarian cancer progression

Abstract Approximately 90% of ovarian cancers are derived from the surface epithelium of the ovary. To study and further understand the early events that lead to epithelial ovarian cancer, we previously developed the syngeneic mouse ovarian surface epithelial (MOSE) model; MOSE cells have undergone spontaneous transformation in cell culture and were categorized into distinct stages by their phenotype, with early passages (MOSE-E) representing a pre-neoplastic, non-tumorigenic phenotype to late passages (MOSE-L) that demonstrate a highly aggressive malignant phenotype both in vitro and in vivo. Sphingosine, a bioactive metabolite of complex sphingolipids, has been shown to have chemopreventive properties in cancer, including colon and ovarian. In these studies we have investigated the hypothesis that sphingosine can prevent or reverse the aberrant gene expression in MOSE-L cells. Using a complete mouse genome microarray, we have identified 1412 genes that were significantly changed in the late malignant MOSE-L versus non-tumorigenic MOSE-E cells. Upon treatment of MOSE-L cells with non-toxic concentrations of sphingosine, microarray analysis revealed the down-regulation of 172 of 701 upregulated genes and up-regulation of 176 of 711 down-regulated genes in MOSE-L cells. Gene ontology analyses showed that these genes were overrepresented in specific categories with up-regulated genes involved in lipid and steroid metabolism, ER, and innate immune response while down regulated genes are involved in cell cycle, cytoskeleton and its regulation, and chromosome stability. A comparison of upregulated genes in MOSE-L cells treated with 5’-azaDC and sphingosine identified genes that are potentially epigenetically silenced by promoter methylation as cells transition from pre-neoplastic to malignant. Also, in addition to changes in gene expression, sphingosine reversed aberrant cytoskeleton organization. Taken together, this indicates that sphingosine acts as a chemopreventive agent by regulating multiple pathways including gene expression. 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 2875.

Rethinking “Cancer as a Dynamic Developmental Disorder” a Quarter Century Later

In a Perspectives article of 1985 in Cancer Research, I discussed evidence for a nonmutational origin of teratocarcinoma, and cited related claims in liver carcinogenesis, which I later broadened to include spontaneous transformation in cell culture. Further research on spontaneous transformation cast doubt on that interpretation, and motivated re-examination of the cases for teratocarcinomas and liver cancer. This re-examination led to the conclusion that mutation and selection underlie the development of neoplasia in all three cases. At the same time, it affirmed the primary role of hierarchical tissue structure and intercellular relations in maintaining homeostasis, which was the major theme of the 1985 article. Tumor promotion disrupts intercellular relations to allow progressively selective proliferation of initiated cells and accompanying clonal accumulation of genetic change.

Human T-cell leukemia virus type-1 antisense-encoded gene, Hbz, promotes T-lymphocyte proliferation

Human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ) is dispensable for HTLV-1-mediated cellular transformation in cell culture, but is required for efficient viral infectivity and persistence in rabbits. In most adult T-cell leukemia (ATL) cells, Tax oncoprotein expression is typically low or undetectable, whereas Hbz gene expression is maintained, suggesting that Hbz expression may support infected cell survival and, ultimately, leukemogenesis. Emerging data indicate that HBZ protein can interact with cAMP response element binding protein (CREB) and Jun family members, altering transcription factor binding and transactivation of both viral and cellular promoters. Herein, lentiviral vectors that express Hbz-specific short hairpin (sh)-RNA effectively decreased both Hbz mRNA and HBZ protein expression in transduced HTLV-1-transformed SLB-1 T cells. Hbz knockdown correlated with a significant decrease in T-cell proliferation in culture. Both SLB-1 and SLB-1-Hbz knockdown cells engrafted into inoculated NOD/SCID(gammachain-/-) mice to form solid tumors that also infiltrated multiple tissues. However, tumor formation and organ infiltration were significantly decreased in animals challenged with SLB-1-Hbz knockdown cells. Our data indicate that Hbz expression enhances the proliferative capacity of HTLV-1-infected T cells, playing a critical role in cell survival and ultimately HTLV-1 tumorigenesis in the infected host.

Improved podophyllotoxin production by transformed cultures of Linum album

Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.

DIGE Analysis of Ras-Transformed Fibroblast Cell-Derived Exosomes

Ras is a small GTP binding protein. Specific point mutations can lead to a constant activation of Ras molecule. Over expression of mutated Ras molecule induced morphological transformation of NIH3T3 cells in culture and these transformed cells had the ability to induce tumors in nude mice. The activation mutations in Ras molecule play a significant role in tumor formation and development. The critical role of Ras signaling in human cancer is proved by the fact that it is one of the most highly mutated genes in human cancer, with approximately 90% incidence of activating mutation in pancreatic cancer, 50% in colon and 30% in many other types.

Constitutively active Rheb induces oncogenic transformation

Rheb (Ras-homolog enriched in brain) is a component of the phosphatidylinositol 3-kinase (PI3K) target of rapamycin (TOR) signaling pathway, functioning as a positive regulator of TOR. Constitutively active mutants of Rheb induce oncogenic transformation in cell culture. The transformed cells are larger and contain more protein than their normal counterparts. They show constitutive phosphorylation of the ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1, two downstream targets of TOR. The TOR-specific inhibitor rapamycin strongly interferes with transformation induced by constitutively active Rheb, suggesting that TOR activity is essential for the oncogenic effects of mutant Rheb. Rheb-induced transformation is also dependent on a C-terminal farnesylation signal that mediates localization to a cellular membrane. An engineered N-terminal myristylation signal can substitute for the farnesylation. Immunofluorescence localizes wild-type and mutant Rheb to vesicular structures in the cytoplasm, overlapping with the endoplasmic reticulum.

Developmental Study of Fragile X Syndrome Using Human Embryonic Stem Cells Derived from Preimplantation Genetically Diagnosed Embryos

We report on the establishment of a human embryonic stem cell (HESC) line from a preimplantation fragile X-affected embryo and demonstrate its value as an appropriate model to study developmentally regulated events that are involved in the pathogenesis of this disorder. Fragile X syndrome results from FMR1 gene inactivation due to a CGG expansion at the 5'UTR region of the gene. Early events in FMR1 silencing have not been fully characterized due to the lack of appropriate animal or cellular models. Here we show that, despite the presence of a full mutation, affected undifferentiated HESCs express FMR1 and are DNA unmethylated. However, epigenetic silencing by DNA methylation and histone modification occurs upon differentiation. Our unique cell system allows the dissection of the sequence by which these epigenetic changes are acquired and illustrates the importance of HESCs in unraveling developmentally regulated mechanisms associated with human genetic disorders.