Transformation Of Primary Cells Research Articles

The role of centrosomal Nlp in the control of mitotic progression and tumourigenesis

The human centrosomal ninein-like protein (Nlp) is a new member of the γ-tubulin complexes binding proteins (GTBPs) that is essential for proper execution of various mitotic events. The primary function of Nlp is to promote microtubule nucleation that contributes to centrosome maturation, spindle formation and chromosome segregation. Its subcellular localisation and protein stability are regulated by several crucial mitotic kinases, such as Plk1, Nek2, Cdc2 and Aurora B. Several lines of evidence have linked Nlp to human cancer. Deregulation of Nlp in cell models results in aberrant spindle, chromosomal missegregation and multinulei, and induces chromosomal instability and renders cells tumourigenic. Overexpression of Nlp induces anchorage-independent growth and immortalised primary cell transformation. In addition, we first demonstrate that the expression of Nlp is elevated primarily due to NLP gene amplification in human breast cancer and lung carcinoma. Consistently, transgenic mice overexpressing Nlp display spontaneous tumours in breast, ovary and testicle, and show rapid onset of radiation-induced lymphoma, indicating that Nlp is involved in tumourigenesis. This review summarises our current knowledge of physiological roles of Nlp, with an emphasis on its potentials in tumourigenesis.

The Latent Origin of Replication of Epstein-Barr Virus Directs Viral Genomes to Active Regions of the Nucleus

The Epstein-Barr virus efficiently infects human B cells. The EBV genome is maintained extrachromosomally and replicates synchronously with the host's chromosomes. The latent origin of replication (oriP) guarantees plasmid stability by mediating two basic functions: replication and segregation of the viral genome. While the segregation process of EBV genomes is well understood, little is known about its chromatin association and nuclear distribution during interphase. Here, we analyzed the nuclear localization of EBV genomes and the role of functional oriP domains FR and DS for basic functions such as the transformation of primary cells, their role in targeting EBV genomes to distinct nuclear regions, and their association with epigenetic domains. Fluorescence in situ hybridization visualized the localization of extrachromosomal EBV genomes in the regions adjacent to chromatin-dense territories called the perichromatin. Further, immunofluorescence experiments demonstrated a preference of the viral genome for histone 3 lysine 4-trimethylated (H3K4me3) and histone 3 lysine 9-acetylated (H3K9ac) nuclear regions. To determine the role of FR and DS for establishment and subnuclear localization of EBV genomes, we transformed primary human B lymphocytes with recombinant mini-EBV genomes containing different oriP mutants. The loss of DS results in a slightly increased association in H3K27me3 domains. This study demonstrates that EBV genomes or oriP-based extrachromosomal vector systems are integrated into the higher order nuclear organization. We found that viral genomes are not randomly distributed in the nucleus. FR but not DS is crucial for the localization of EBV in perichromatic regions that are enriched for H3K4me3 and H3K9ac, which are hallmarks of transcriptionally active regions.

A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice

The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62DOK, and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62DOK and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant's ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.

Genetic and pharmacologic evidence implicating the p85α, but not p85β, regulatory subunit of PI3K and Rac2 GTPase in regulating oncogenic KIT-induced transformation in acute myeloid leukemia and systemic mastocytosis

AbstractOncogenic activation loop KIT mutations are observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants, the activation loop mutants are insensitive to imatinib mesylate. Furthermore, as prior studies primarily used heterologous cell lines, the molecular mechanism(s) underlying oncogenic KIT-induced transformation in primary cells is poorly understood. We demonstrate that expression of KITD814V in primary hematopoietic stem/progenitor cells (HSC/Ps) and mast cell progenitors (MCps) induces constitutive KIT autophosphorylation, supports ligand-independent hyperproliferation, and promotes promiscuous cooperation with multiple cytokines. Genetic disruption of p85α, the regulatory subunit of class IA lipid kinase phosphoinositol-3-kinase (PI3K), but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalizes KITD814V-induced ligand-independent hyperproliferation. Additionally, deficiency of p85α or Rac2 corrects the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V-expressing HSC/Ps and MCps. Treatment of KITD814V-expressing HSC/Ps with a Rac inhibitor (NC23766) or with rapamycin showed a dose-dependent suppression in ligand-independent growth. Taken together, our results identify p85α and Rac2 as potential novel therapeutic targets for the treatment of KITD814V-bearing AML and SM.

Rac1 GTPase Regulates Cell Genomic Stability and Senescence

The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.

The activation of TC10, a Rho small GTPase, contributes to v-Rel-mediated transformation

v-Rel is the oncogenic member of the Rel/NF-kappaB family of transcription factors and transforms hematopoietic cells and fibroblasts. Differential display was employed to identify target genes that exhibit altered expression in v-Rel transformed cells. One of the cDNAs identified encodes the chicken ortholog of TC10, a member of the Rho small GTPase family. The expression of TC10 was increased in v-Rel-transformed chicken embryonic fibroblasts (CEFs) 3 to 6-fold relative to control cells at both the RNA and protein levels. An elevated level of active, GTP-bound TC10 was also detected in v-Rel-transformed cells relative to control cells. Expression of a dominant-negative TC10 mutant (TC10T32N) decreased the colony formation potential of v-Rel-transformed cells. Furthermore, overexpression of wild-type TC10 or a gain-of-function mutant (TC10Q76L) greatly enhanced the ability of v-Rel transformed CEFs to form colonies in soft agar. In addition to enhance the transformation potential of v-Rel, the overexpression of wild-type TC10 or the gain-of-function mutant alone enhanced the saturation density of CEFs and was sufficient for their anchorage-independent growth in vitro. These results indicate that elevated TC10 activity contributes to v-Rel-mediated transformation of CEFs and demonstrate for the first time that a Rho factor alone is capable of inducing the in vitro transformation of primary cells.

YND1 Interacts with CDC55 and Is a Novel Mediator of E4orf4-induced Toxicity

Adenovirus E4orf4 (early region 4 open reading frame 4) protein induces protein phosphatase 2A-dependent non-classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Oncogenic transformation sensitizes cells to E4orf4-induced cell death. To uncover additional components of the E4orf4 network required for induction of its unique mode of apoptosis, we used yeast genetics to select gene deletions conferring resistance to E4orf4. Deletion of YND1, encoding a yeast Golgi apyrase, conferred partial resistance to E4orf4. However, Ynd1p apyrase activity was not required for E4orf4-induced toxicity. Ynd1p and Cdc55p, the yeast protein phosphatase 2A-B subunit, contributed additively to E4orf4-induced toxicity. Furthermore, concomitant overexpression of one and deletion of the other was detrimental to yeast growth, demonstrating a functional interaction between the two proteins. YND1 and CDC55 also interacted genetically with CDC20 and CDH1/HCT1, encoding activating subunits of the anaphase-promoting complex/cyclosome. In addition to their functional interaction, Ynd1p and Cdc55p interacted physically, and this interaction was disrupted by E4orf4, which remained associated with both proteins. The results suggested that Ynd1p and Cdc55p share a common downstream target whose balanced modulation by the two E4orf4 partners is crucial to viability. Disruption of this balance by E4orf4 may lead to cell death. NTPDase-4/Lalp70/UDPase, the closest mammalian homologue of Ynd1p, associated with E4orf4 in mammalian cells, suggesting that the results in yeast are relevant to the mammalian system.

P53 codon 72 polymorphism is associated with disease progression in adult T‐cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATL) is associated with the monoclonal integration of human T-lymphotropic virus type I (HTLV-I) proviral DNA into tumour cells, but the incidence of ATL is low (2–5%) among HTLV-I carriers and those who develop the disease usually have a 40- to 60-year latency period from the time of infection to progression to ATL. These phenomena suggest that additional mechanisms are involved in the progression of the disease, such as inactivation of tumour suppressor genes. P53 is one of the most important tumour suppressor genes that is involved in multiple pathways including apoptosis, cellular transcriptional control and cell cycle regulation. A common polymorphism of P53 at codon 72 results in either a variant protein with a proline (Pro) residue (CCC) or an arginine (Arg) amino acid (CGC) (Zhang et al, 2003). Recent studies have found that this polymorphism is associated with susceptibility or progression of several kinds of cancers (Zhang et al, 2003). However, data are not available as to whether this polymorphism plays a role in the pathogenesis of ATL among HTLV-I carriers. Human T-lymphotropic virus type I carriers (n = 81) were identified in the general population in an endemic region for HTLV-I, the Nagasaki district of Japan, after a mass medical and laboratory screening for individuals with anti-HTLV-I serum antibodies. ATL patients (n = 87) were diagnosed at the Department of Haematology, Nagasaki University School of Medicine. HTLV-I negative controls (n = 89) were included in the analysis. All subjects were Japanese. The genotype was performed as previously described (Zhang et al, 2003). Allele frequency of the P53 codon 72 polymorphism was not statistically different between 89 HTLV-I negative controls and 81 healthy HTLV-I carriers (P = 0·677) (Table I). These data suggest that genetic polymorphism in the P53 locus does not play a role in the infection of the HTLV-I. The allele frequency was not statistically different between 87 individuals with ATL and 81 healthy HTLV-I carriers (P = 0·627). These data suggest that this polymorphism does not play a role in the development of ATL among HTLV-I carriers. We previously found that a genetic polymorphism of the tumour necrosis factor gene enhanced susceptibility to ATL among HTLV-I carriers (Tsukasaki et al, 2001). In contrast, polymorphisms at the methylenetetrahydrofolate reductase locus did not alter susceptibility to ATL (Takeuchi et al, 2004). The frequency of the P53 codon 72 genotype in clinically defined subtypes of ATL is shown in Table I. Of note, the frequency of the arginine homozygous allele was higher in acute ATL than in chronic ATL (P = 0·0396). These results suggest that the P53 polymorphism plays an important role in the progression of ATL. Previous studies showed that mutation of the P53 gene in acute ATL was higher than that in chronic ATL (Hatta & Koeffler, 2002). We have previously reported that the frequency of adenomatous polyposis coli (APC) promoter methylation in acute type ATL (57%) was much higher than in chronic type ATL (13%) (P = 0·03) (Yang et al, 2005). Several differences occur among the P53 variants in their ability to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis and to repress the transformation of primary cells (Thomas et al, 1999). Polymorphism of the P53 gene is associated with increased susceptibility to the development of gastric, urinary tract and breast cancers (Zhang et al, 2003). Taken together, P53 may have functional importance in several types of malignancies including ATL, probably by modulating its efficiency as a transcription factor. This work was supported in part by grants from National Institutes of Health, Honara Shapiro Fund, Parker Hughes Trust, Sherly and David Weisberg Foundation, grant-in-aid from the Ministry of Education, Culture Sports, Science and Technology of Japan. H. P. K. is a member of the Jonsson Comprehensive Cancer Center and Molecular Biology Institute of UCLA, and holds the endowed Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center/UCLA School of Medicine.

Chromosome translocations in sarcomas and the emergence of oncogenic transcription factors

A subset of sarcomas is characterised by recurrent chromosome translocations that generate novel fusion oncoproteins. One or both of the genes involved in these translocations often encode transcription factors, and the resulting fusion proteins have aberrant transcriptional function compared to their wild-type counterparts. These fusion transcription factors disrupt multiple biological pathways by altering expression of target genes, and thereby result in a variety of altered cellular properties that contribute to the tumourigenic process. However, experimental data indicate that the fusion gene alone is not sufficient for transformation in primary cells (EWS–FLI1) or tumourigenesis in the mouse (PAX3–FKHR, FUS–CHOP), suggesting that additional collaborating genetic alterations are required. In addition to improving our understanding of the etiology of these tumours, this accumulating knowledge of the oncogenic properties of these fusion proteins, their downstream targets, and cooperating genetic alterations will permit the development of a variety of novel approaches to improve the therapy of these cancers.

Identification of novel mammalian growth regulatory factors by genome-scale quantitative image analysis

Functional profiling technologies using arrayed collections of genome-scale siRNA and cDNA arrayed libraries enable the comprehensive global analysis of gene function. However, the current repertoire of high-throughput detection methodologies has limited the scope of cellular phenotypes that can be studied. In this report, we describe the systematic identification of mammalian growth-regulatory factors achieved through the integration of automated microscopy, pattern recognition analysis, and cell-based functional genomics. The effects of 7364 human and mouse proteins, encoded by individually arrayed cDNAs, upon proliferation and viability in U2OS osteosarcoma cells were evaluated in a live-cell, kinetic assay using quantitative image analysis. Overexpression of more than 86 cDNAs (1.15%) conferred dramatic increases in the proliferation, as determined cell enumeration. These included several known growth regulators, as well as previously uncharacterized ones (LRRK1, Ankrd25). In addition, novel functional roles for two genes (5033414D02Rik, 2810429O05Rik), now termed Gatp1 and Gatp2, respectively, were identified. Further analysis demonstrated that these encoded proteins promoted cellular proliferation and transformation in primary cells. Conversely, cells depleted for Gatp1 underwent apoptosis upon serum reduction, suggesting that Gatp1 is essential for cell survival under growth-factor-restricted conditions. Taken together, our findings offer new insight into the regulation of cellular growth and proliferation, and demonstrate the value and feasibility of assessing cellular phenotypes through genome-level computational image analysis.

An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression.

Over the past decades, much has been learnt about the genes that contribute to oncogenic transformation of primary cells in vitro. However, much less is known about the genes that contribute to the later stages of tumor progression, in which cells of ever increasing malignancy arise through clonal selection in vivo. To search for genes that confer a tumor progression phenotype in vivo, we have used a functional genetic approach. We used adenovirus-transformed mouse embryo fibroblasts, which are tumorigenic in immunodeficient nude mice, but not in immunocompetent mice, due to strong cytotoxic T-cell-mediated immune rejection. We infected these cells in vitro with several high-complexity retroviral cDNA expression libraries and selected rare variants that formed tumors in immunocompetent mice. Using this approach, we identify here the TRK-T3 oncogene as a tumor progression gene. TRK-T3 does not inhibit T-cell reactivity towards the tumor cells. Instead, we find that cells expressing TRK-T3 enhances in vivo growth rate, most likely by stimulating anchorage-independent proliferation in growth factor-limiting conditions. Our data indicate that cDNA expression libraries can be used to identify tumor progression genes in vivo that cannot be readily identified using in vitro cell culture systems.

Involvement of Rho Family GTPases in p19Arf- and p53-Mediated Proliferation of Primary Mouse Embryonic Fibroblasts

The Rho family GTPases Rac1, RhoA, and Cdc42 function as molecular switches that transduce intracellular signals regulating gene expression and cell proliferation as well as cell migration. p19(Arf) and p53, on the other hand, are tumor suppressors that act both independently and sequentially to regulate cell proliferation. To investigate the functional interaction and cooperativeness of Rho GTPases with the p19(Arf)-p53 pathway, we examined the contribution of Rho GTPases to the gene transcription and cell proliferation unleashed by deletion of p19Arf or p53 in primary mouse embryo fibroblasts. We found that (i) p19(Arf) or p53 deficiency led to a significant increase in PI 3-kinase activity, which in turn upregulated RhoA and Rac1 activities; (ii) deletion of p19Arf or p53 led to an increase in cell growth rate that was in part dependent on RhoA, Rac1, and Cdc42 activities; (iii) p19(Arf) or p53 deficiency caused an enhancement of the growth-related transcription factor NF-kappa B and cyclin D1 activities that are partly dependent on RhoA or Cdc42 but not on Rac1; (iv) forced expression of the activating mutants of Rac1, RhoA, or Cdc42 caused a hyperproliferative phenotype of the p19Arf(-/-) and p53(-/-) cells and promoted transformation of both cells; (v) RhoA appeared to contribute to p53-regulated cell proliferation by modulating cell cycle machinery, while hyperactivation of RhoA further suppressed a p53-independent apoptotic signal; and (vi) multiple pathways regulated by RhoA, including that of Rho-kinase, were required for RhoA to fully promote the transformation of p53(-/-) cells. Taken together, these results provide strong evidence indicating that signals through the Rho family GTPases can both contribute to cell growth regulation by p19Arf and p53 and cooperate with p19Arf or p53 deficiency to promote primary cell transformation.

Suppression of Anchorage-Independent Growth of Human Cancer Cell Lines by the TRIF52/Periostin/OSF-2 Gene

In searching for genes that suppress the viral transformation of primary cells, we have isolated a number of TRIF (transcript reduced in F2408) genes that are expressed well in primary rat embryo fibroblasts (REFs) but poorly in spontaneously immortalized rat fibroblast cell lines derived from REFs. One of these genes, TRIF52, is a rat homologue of the mouse protein periostin, which is suspected of being involved in oncogenesis. We found here that periostin mRNA expression is markedly downregulated in a variety of human cancer cell lines and human lung cancer tissues. Human cancer cell lines with reduced endogenous periostin gene expression that were infected with a recombinant retrovirus containing the periostin gene had reduced anchorage-independent growth. Mutational analysis revealed that the C-terminal region of periostin is sufficient to convey the anchorage-independent growth-suppressive activity of the protein. These observations together suggest that periostin may serve to inhibit the development of human cancers by acting as a tumor suppressor.

Simian virus 40 large T antigen and two independent T-antigen segments sensitize cells to apoptosis following genotoxic damage.

The simian virus 40 (SV40) large tumor (T) antigen is sufficient to transform cells in cultures and induce tumors in experimental animals. Transformation of primary cells in cultures requires both overcoming growth arrest by stimulating the cell cycle and blocking cell death activities presumably activated by oncogene-mediated hyperproliferation signals. The study presented here examined the ability of specific regions and activities of T antigen to modulate apoptosis in cells treated with the genotoxic agent 5-fluorouracil (5-FU). The results showed that the expression of full-length T antigen rendered rat embryo fibroblasts (REF) sensitive to 5-FU-induced apoptosis. Thus, neither the p53-binding region nor the Bcl-2 homology region of T antigen was sufficient to prevent cell death induced by the DNA-damaging agent. T-antigen-mediated sensitization occurred independently of retinoblastoma protein or p53 and p300 binding. An N-terminal segment containing the first 127 T-antigen amino acids (T1-127) was sufficient to sensitize cells. A C-terminal segment consisting of T-antigen amino acids 251 to 708 (T251-708) also sensitized cells to 5-FU-induced apoptosis. This sensitization did not occur when T251-708 was targeted to the nucleus by inclusion of the SV40 nuclear localization signal. The introduction of mutations into the T-antigen J domain resulted in mutation-specific and variable inhibition of apoptosis. This result suggested that either the structural or the functional integrity of the J domain is required to sensitize cells to apoptosis. Treatment of REF or REF expressing full-length T antigen, an N-terminal segment, or T251-708 resulted in increased expression of the p53-responsive MDM2 gene; apoptosis occurred through a p53-dependent pathway, as p53-null cells expressing these T antigens were resistant to 5-FU-induced apoptosis. Possible mechanisms involved in sensitizing cells to a p53-dependent apoptosis pathway in spite of the ability of T antigen to bind and inactivate the transcriptional transactivating activity of p53 are discussed.

Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23.

We found that PPM1D, encoding a serine/threonine protein phosphatase, lies within an epicenter of the region at 17q23 that is amplified in breast cancer. We show that overexpression of this gene confers two oncogenic phenotypes on cells in culture: attenuation of apoptosis induced by serum starvation and transformation of primary cells in cooperation with RAS.

Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic ras-induced premature senescence.

In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.

Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1).

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21(Cip1/Waf1). Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-alpha for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2% vs. 4.3%, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58% of Ras-infected cells stained positive for senescence-associated beta-galactosidase activity as opposed to 2% in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21(Cip1/Waf1) in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-alpha induced-apoptosis. Surprisingly, senescence-associated beta-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21(Cip1/Waf1). Future studies have to investigate a potential role of Ras in human vascular biology.

The Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus Transactivates the Telomerase Reverse Transcriptase Promoter

Telomerase is a multi-subunit ribonucleoprotein holoenzyme that stabilizes telomere length through the addition of new repeat sequence to the ends of chromosomes. Telomerase reverse transcriptase is the subunit of this complex responsible for the enzymatic activity of telomerase. Expression of the reverse transcriptase is regulated at the level of transcription through the action of transcription factors that target its promoter. Most Kaposi's sarcoma tumor cells are latently infected with the Kaposi's sarcoma-associated herpesvirus, and the constitutive expression of a viral-encoded latency-associated nuclear antigen has been shown to be important for the maintenance of the viral episome. The proliferative nature of Kaposi's sarcoma suggests that this antigen may also play a critical role in viral-mediated oncogenesis. In this study telomerase reverse transcriptase promoter elements cloned into a luciferase reporter plasmid were analyzed to determine the ability of the latency-associated nuclear antigen to regulate transcription. The latency-associated nuclear antigen transactivated the full-length promoter in 293T, 293, and BJAB cell lines. Furthermore, truncation promoter studies implicated sequence from -130 to +5 in viral-mediated activation. This region contains five Sp1 transcription factor-binding sites. Electrophoretic mobility shift assays indicated that the latency-associated nuclear antigen targets and affects the Sp1-DNA complex in the context of BJAB nuclear extracts.

Induction of senescence by oncogenic ras

Publisher Summary This chapter discusses the induction of senescence by oncogenic Ras. Oncogenic Ras is able to transform efficiently most immortal rodent cell lines. In contrast, transformation of primary cells by Ras requires the cooperation of an immortalizing event. Transformation of primary cells through the combined action of immortalizing alterations and oncogenic Ras is the basis for several models of multistep carcinogenesis both in culture cells and in animal models. Normal, nonimmortal cells are therefore refractile to transformation by Ras, presumably because of the existence of built-in protection mechanisms against unlimited proliferation. A detailed study of the effects of Ras in primary cells has not been achieved until recently, mainly because of the lack of efficient gene transfer techniques. Improvements in retrovirus-based gene transfer methods have made it possible to analyze the effect of the sustained expression of oncogenic Ras in primary cells. Interestingly, it was observed that after an initial mitogenic response, prolonged action of Ras provokes a permanent cell cycle arrest.

Binding of bovine papillomavirus type 4 E8 to ductin (16K proteolipid), down-regulation of gap junction intercellular communication and full cell transformation are independent events.

The E8 open reading frame of bovine papillomavirus type 4 encodes a small hydrophobic polypeptide that contributes to primary cell transformation by conferring to cells the ability to form foci and to grow in low serum and in suspension. Wild-type E8 binds in vitro to ductin, a component of gap junctions, and this binding is accompanied by a loss of gap junction intercellular communication in transformed bovine fibroblasts. However, through the analysis of a panel of E8 mutants, we show here that binding of E8 to ductin is not sufficient for down-regulation of gap junction communication and that there is no absolute correlation between down-regulation of gap junction communication and the transformed phenotype.