Transformation Of Rodent Cells Research Articles

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

Abstract 2052: The transcriptional co-regulator and emerging cancer drug target C-terminal binding protein (CtBP) is a transforming oncogene

Abstract The paralogous C-terminal binding proteins (collectively CtBP) 1 and 2 are evolutionarily conserved transcriptional co-regulators which demonstrate oncogene-like properties such as allowing for the progression of EMT and cellular invasion/migration, and supporting cellular survival through suppression of tumor suppressors (PTEN, p16, E-cadherin, BRCA-1) and pro-apoptotic genes (Bik, Puma, Noxa). Overexpression of CtBP is found in colon, ovarian, breast, and prostate cancers, and often correlates with loss of the tumor suppressors ARF and APC and with poorer prognosis for the patient. CtBP is also an emerging cancer drug target, encoding a dehydrogenase domain that can be targeted with small molecule inhibitors to kill cancer cells. CtBP appears to fit the criteria for a driver oncogene, though has never been fully characterized as such. To investigate the hypothesis that CtBP is a transforming oncogene, we subjected CtBP2 to multiple assays for cellular oncogenic activity including loss of contact inhibition, invasion, and anchorage independent growth. Previously immortalized NIH 3T3 mouse fibroblasts expressing CtBP2 or mutant H-Ras as a positive control were analyzed for focus formation, Matrigel-invasion, and soft agar colony formation. Consistent with the hypothesis that CtBP exhibits properties of an oncogene, CtBP2 potently induced focus formation, Matrigel-invasion, and soft agar colony formation with similar potency to oncogenic mutant H-Ras. Moreover, CtBP2 cooperated with the adenovirus E1A oncoprotein but not H-Ras to transform primary mouse embryonic fibroblasts (MEFs). Taken together, the 3T3 and MEF transformation data suggest CtBP2 acts as a “Ras-like” oncogene in rodent cell transformation assays. Previous research from our lab has shown that CtBP's ability to induce migration in HCT116 cells depends in part on its activation of T-cell lymphoma invasion and metastasis 1 (Tiam1). Work is ongoing to characterize critical downstream targets for CtBP's transforming activity. Targeting CtBP and its dehydrogenase domain in cancers where the protein is found to be overexpressed, or where its regulators, ARF and APC are found to be inactive, could lead to a therapeutic approach specific for an emerging driver oncogene. Citation Format: Evan T. Sumner, Steven R. Grossman. The transcriptional co-regulator and emerging cancer drug target C-terminal binding protein (CtBP) is a transforming oncogene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2052. doi:10.1158/1538-7445.AM2015-2052

Rapamycin induces pluripotent genes associated with avoidance of replicative senescence

Primary rodent cells undergo replicative senescence, independent from telomere shortening. We have recently shown that treatment with rapamycin during passages 3–7 suppressed replicative senescence in rat embryonic fibroblasts (REFs), which otherwise occurred by 10–14 passages. Here, we further investigated rapamycin-primed cells for an extended number of passages. Rapamycin-primed cells continued to proliferate without accumulation of senescent markers. Importantly, these cells retained the ability to undergo serum starvation- and etoposide-induced cell cycle arrest. The p53/p21 pathway was functional. This indicates that rapamycin did not cause either transformation or loss of cell cycle checkpoints. We found that rapamycin activated transcription of pluripotent genes, oct-4, sox-2, nanog, as well as further upregulated telomerase (tert) gene. The rapamycin-derived cells have mostly non-rearranged, near-normal karyotype. Still, when cultivated for a higher number of passages, these cells acquired a chromosomal marker within the chromosome 3. We conclude that suppression mTORC1 activity may prevent replicative senescence without transformation of rodent cells.

Homologous SV40 RNA trans-splicing

Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5′ donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5′ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5′ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes.

P53 suppresses expression of the 14-3-3gamma oncogene

Background14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro.MethodsqRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC). Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter.ResultsQuantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3gamma expression.ConclusionIncreased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

Sodium butyrate induces cell senescence in transformed rodent cells resistant to apoptosis

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts and transformed by E1A + E1B19 kDa oncogene has been studied. These transformants are resistant to apoptosis in response to γ-irradiation and the deprivation of growth factors. The process of cell senescence was investigated by analyzing cell growth curves, G1/S and G2/M cell cycle arrest and senescent associated β-galactosidase expression. The irreversibility of the antiproliferative activity of sodium butyrate was analyzed by clonogenic assay.

Abstract 4315: Transgenic expression of human Thymidylate Synthase cooperates with an Ink4a/Arf mutation to enhance tumor progression

Abstract While thymidylate synthase (TS) is an established tumor prognostic biomarker and therapeutic target, it was only recently shown to exhibit an unexpected direct role in tumorgenesis. Overexpression of catalytically active human TS (hTS) transformed rodent cells in vitro and in vivo, while TS missence mutants with loss of enzymatic function were non-transforming (Cancer Cell v5, p341, 2004). In addition, transgenic hTS mice developed hyperplasia and tumors of the endocrine pancreas (Oncogene v26, p4817, 2007). These mice showed high levels of hTS expression in pancreatic tissue and lower levels in most other organs. Since hTS transgenic animals developed islet cell adenomas after long latency, we asked whether deregulated hTS would cooperate with other genetic lesions in the initiation and progression of tumor development. To address this, we have crossed hTS transgenic animals with Ink4a/Arf null mice and evaluated tumor spectra, incidence, latency and tumor burden. The Ink4a/Arf null control mice developed predominantly lymphomas and fibrosarcomas while the hTS/Ink4a/Arf -/- mice develop liposarcoma, rabdomyosarcoma, teratoma, hystiocytic neoplasms as well as fibrosarcoma and lymphoma. Soft tissue sarcomas (excluding fibrosarcoma) were large and developed only in hTS/Ink4a/Arf -/- mice ranging from 2.8 to 10 grams. In addition, tumors isolated from hTS/Ink4a/Arf -/- mice contained blood filled cyst-like structures in the lymph nodes, liver, lung, spleen and pancreas as well as on the surface of teratoma and liposarcoma suggesting increased or dysplastic angiogenesis. The frequency of histiocytic neoplasms in hTS/Ink4a/Arf -/- mice was significantly enhanced (42%) as compared to control mice (8%) and these tumors spread to multiple sites (as many as 9 different organs) as compared to the control mice where tumors were localized predominantly in lymph nodes and spleen. Similarly, lymphomas spread to many extranodal organs, while the control tumors were localized predominantly to lymph nodes or spleen. In summary, we observed that transgenic expression of hTS in Ink4a/Arf null mice accelerated tumor growth leading to more aggressive disease with higher tumor burden. Understanding the mechanism of TS-mediated tumor progression may lead to development of new agents that may act synergistically with known TS inhibitors. The observation that TS promotes tumor growth and it is not just a passive biomarker for cell proliferation will have an important impact for the prevention and treatment of human cancers given the widespread over-expression of TS in the common adult tumors including lymphomas, colorectal, breast, pancreatic, sarcomas and lung cancers. 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 4315. doi:10.1158/1538-7445.AM2011-4315

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr(161) by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr(14) and Tyr(15) phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G(2)/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr(15). In addition, Tyr(15)-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G(2) and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr(161) phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G(2)/M is tightly coupled and regulated by Cdc25 phosphatases.

Adenovirus type 5 early encoded proteins of the E1 and E4 regions induce oncogenic transformation of primary rabbit cells

Analysis of the molecular mechanisms of viral-mediated oncogenesis has contributed enormously to the understanding of the basic principles of normal/malignant cell growth. Transformation by human adenoviruses is a multi-step process involving the modulation of numerous cellular pathways, leading to inhibition of apoptosis and growth arrest. However, the molecular mechanism of how the adenovirus oncogenes facilitate transformation of rodent cells, while concurrently failing to do so for human cells, remains elusive. In this report, we demonstrate for the first time that the transformation capabilities of adenovirus type 5 oncogenes are not restricted to rodent cells, but include cells of the related mammalian order Lagomorpha, inducing considerable morphological alterations, enhanced cell growth and tumour induction in vivo. Furthermore, the established cell lines may represent a suitable tool for further development to generate E4-mutated adenoviruses, which has so far been difficult as mutations within the E4 region often prove to be lethal without a helper-cell system.

Papillomavirus transforming functions.

The bovine papillomavirus type 1 (BPV-1) has served as a model for unravelling the molecular genetics of the papillomaviruses. BPV-1 transformation of rodent cells in tissue culture has provided a means to study the viral functions involved in latent infection of cells and in the induction of cellular proliferation functions. BPV-1 has been shown to encode two independent transforming genes, each of which can induce cellular transformation in susceptible rodent cells. These two genes apparently act synergistically in transforming mouse C127 cells. Deletion mutagenesis studies have shown that the expression of one of these genes (E6) is required for efficient tumorigenesis and anchorage independence. BPV-1 also encodes functions which may act indirectly to affect transformation. BPV-1 contains transcriptional enhancers which can act in a position-independent and orientation-independent manner to increase the transcriptional activity of a heterologous gene. One of these elements, which is located in a non-coding region of the genome, can be trans-activated by a specific viral gene product encoded by the E2 open reading frame. Mutations which eliminate this trans-activation function also have a dramatic effect on transformation and on stable plasmid maintenance.

Anticancer activity of an adenoviral vector expressing short hairpin RNA against BK virus T-ag

The human polyomavirus BK (BKV) is oncogenic in rodents and induces malignant transformation of rodent cells in vitro. Although its role in human tumorigenesis is still debated, BKV represents an excellent model to evaluate molecularly targeted antineoplastic approaches. Here, we have tested whether stable suppression of the T antigen (T-ag) oncogene expression could inhibit the in vitro and in vivo malignant phenotype of BKV-transformed mouse cells. An adenovirus vector system that expresses small hairpin RNAs (shRNAs), which are converted into active small interfering RNAs (siRNA) molecules against the BKV T-ag, was developed. This vector was able to inhibit the expression of BKV T-ag through a highly efficient in vitro and in vivo delivery of the siRNA molecule. In addition, it allowed a stable expression of siRNA for a period of time sufficient to elicit a biological effect. Inhibition of T-ag expression results in reduction of the in vitro growth rate of BKV-transformed cells, which is, at least in part, caused by restoration of p53 activity and induction of apoptosis. In vivo studies proved that adenovirus vectors expressing anti-T-ag siRNA were able to suppress tumorigenicity of BKV-transformed cells. Moreover, adenovirus vector direct treatment of growing tumors resulted in a significant reduction of tumor growth. This study indicates that siRNAs delivery via a viral vector have a potential usefulness as in vivo anticancer tool against viral and cellular oncogenes.

E6AP-Dependent Degradation of DLG4/PSD95 by High-Risk Human Papillomavirus Type 18 E6 Protein

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.

Adenovirus: from foe to friend

Human adenoviruses (HAdVs) can cause mild respiratory, gastrointestinal, urogenital and ocular disease. Knowledge about HAdVs has been expanding for more than five decades putting them amongst the most-studied viruses. This continued interest stems, to a great extent, from the fact that these double-stranded DNA viruses have proven to be a versatile tool to probe the basic phenomena of eukaryotic cells. HAdV research has led to the discovery of, for instance, RNA splicing and greatly contributed to our knowledge of processes as fundamental as replication, transcription and translation. Moreover, the transformation of rodent cells by HAdVs has provided a system to unravel the molecular pathways that control cell proliferation. As a result, the genetic organisation of these agents is known in great detail allowing the straightforward manipulation of their genomes. In addition, the virus itself became renowned for its ability to produce large amounts of progeny and to efficiently infect mammalian cells regardless of their cell cycle status. These features contributed to the broad use of recombinant HAdVs as gene carriers particularly in in vivo settings where the vast majority of target cells are post-mitotic. The most advanced type of HAdV vectors can accommodate up to 37 kb of foreign DNA and are devoid of viral genes. With the aid of these high-capacity HAdV vectors large physiologically responsive transcriptional elements and/or genes can be efficiently introduced into target cells while minimising adaptive immune responses against the transduced cells. This article provides information on HAdV especially on the aspects pertinent to the design, production and performance of its recombinant forms. The development and characteristics of the main HAdV-based vector types are also briefly reviewed.

Progression elevated gene-3 (PEG-3) induces pleiotropic effects on tumor progression: modulation of genomic stability and invasion.

Progression elevated gene-3 (PEG-3) is a novel rodent gene, identified and cloned by subtraction hybridization, that associates with transformation progression in virus- and oncogene-transformed rat embryo (RE) cells. Previous reports document that ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed/tumorigenic phenotype. Moreover, PEG-3 expression in rodent tumor cells correlates directly with genomic instability, as indicated by chromosomal alterations and gene amplification, and it promotes angiogenesis. The present studies were designed to further elucidate the functional significance and role of PEG-3 in cancer progression with a specific focus on genomic instability and cancer invasion. Genomic instability was assessed by micronucleus assays and staining of centrosomes to define centrosomal amplification. Immunocytochemical observations revealed that overexpression of PEG-3 in transformed rodent cells induced a loss of chromosomes as established by the appearance of micronuclei and staining of the centrosomes with gamma-tubulin antibody, thereby confirming centrosome amplification. Overexpression of PEG-3 modulated the expression of several genes involved in centrosomal duplication, such as p21CIP1/WAF1/MDA-6, nucleophosmin (NPM), and aurora-A kinase. In vitro invasion of transformed rodent cells was augmented by PEG-3, which correlated with an increase in the transcription and activity of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), which play important roles in local invasion during cancer progression. These findings demonstrate that PEG-3 plays a central role in augmenting tumor progression by modulating several critical parameters of the carcinogenic process, such as genomic stability and local tumor cell invasion.

E1A-based determinants of oncogenicity in human adenovirus groups A and C.

A broad spectrum of genetic and molecular investigations carried out with group C, Ad2 and Ad5, and with group A, Ad12, have shown that early region1 (E1) gene products are sufficient for complete transformation of rodent cells in vitro by these viruses. During the past quarter century, the processes by which E1A proteins, in cooperation with E1B proteins, perturb the cell cycle and induce the transformed phenotype, have become well defined. Somewhat less understood is the basis for the differential oncogenicity of these two groups of viruses, and the processes by which the E1A proteins of Ad12 induce a tumorigenic phenotype in transformants resulting from infection of cells in vivo and in vitro. In this chapter we review previous findings and present new evidence which demonstrates that Ad12 E1A possesses two or more independent functions enabling it to induce tumors. One of these functions lies in its capacity to repress transcription of MHC class I genes, allowing the tumor cells to avoid lysis by cytotoxic T lymphocytes. We have shown that class I repression is mediated through increased binding of repressor COUP-TF and decreased binding of NF-kB to the class I enhancer. In addition to mediating immune escape, E1A also determines the susceptibility of transformants to Natural Killer (NK) cell lysis, and in this case, also, Ad12 transformants are not susceptible. By using Ad12 mutants containing chimeric E1A Ad12-Ad5 genes, point mutations, or a specific deletion, we have shown that the unique spacer region of Ad12 E1A is an oncogenic determinant, but is not required for transformation in vitro. Given that the E1A regions responsible for class I repression are first exon encoded, we have examined a set of cell lines transformed by these altered viruses, and have found that while they display greatly reduced tumorigenicity, they maintain a wildtype capacity to repress class I transcription. Whether the spacer contributes to NK evasion remains unresolved. Lastly, we discuss the properties of the Ad2/Ad5 E1A C-terminal negative modulator of tumorigenicity, and examine the effects on transformation, tumor induction and transformant tumorigenicity, when the Ad5 negative modulator is placed by chimeric construction in Ad12 E1A.

Putative phosphatidylinositol 3-kinase (PI3K) binding motifs in ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway.

Proteasome-mediated regulation of the hDlg tumour suppressor protein.

The Dlg tumour suppressor protein is intimately involved in the control of cell contact and polarity. Previous studies have shown that hDlg is a target for a number of viral transforming proteins. In particular, the high risk human papillomavirus (HPV) E6 proteins target hDlg for proteasome-mediated degradation, an activity that appears to contribute to HPV-induced malignancy. However, little information is available concerning the normal regulation of hDlg. In this study we have investigated the role of the proteasome in the regulation of endogenous hDlg protein levels in epithelial cell lines. We demonstrate that hDlg is, indeed, degraded via the proteasome both in the presence and absence of HPV, in a fashion that is dependent on the ability of the cells to form cell junctions. By western blot and immunofluorescence analysis we show that hDlg is efficiently degraded in isolated cells; however, upon cell-cell contact, hDlg is both hyper-phosphorylated and stabilised. Strikingly, in both transformed rodent cells and undifferentiated cervical cancer cells, this ability to stabilise Dlg upon increased cell density is lost. These results demonstrate a complex pattern of hDlg regulation by phosphorylation and proteasome degradation in response to cell contact. Loss of this regulation probably represents a significant step in the development of malignancy.

The cytoplasmic amino-terminus of the Latent Membrane Protein-1 of Epstein-Barr Virus: relationship between transmembrane orientation and effector functions of the carboxy-terminus and transmembrane domain.

The Latent Membrane Protein 1 (LMP-1) protein of Epstein-Barr virus (EBV) is localized in the plasma membrane of the infected cell. LMP-1 possesses a hydrophobic membrane spanning domain, and charged, intracellular amino- and carboxy-termini. Two models have been proposed for the contribution of the amino-terminus to LMP-1's function: (i) as an effector domain, interacting with cellular proteins, or (ii) as a structural domain dictating the correct orientation of transmembrane domains and thereby positioning LMP-1's critical effector domains (i.e. the carboxy-terminus). However, no studies to date have addressed directly the structural contributions of LMP-1's cytoplasmic amino-terminus to function. This study was designed to determine if LMP-1's cytoplasmic amino-terminus (N-terminus) encodes information required solely for maintenance of proper topological orientation. We have constructed LMP-1 chimeras in which the cytoplasmic N-terminus of LMP-1 is replaced with an unrelated domain of similar size and charge, but of different primary sequence. Retention of the charged amino-terminal (N-terminal) cytoplasmic domain and first predicted transmembrane domain was required for correct transmembrane topology. The absolute primary sequence of the cytoplasmic N-terminus was not critical for LMP-1's cytoskeletal association, turnover, plasma membrane patching, oligomerization, Tumor Necrosis Factor Receptor-associated factor (TRAF) binding, NF-kappaB activation, rodent cell transformation and cytostatic activity. Furthermore, our results point to the hydrophobic transmembrane domain, independent of the cytoplasmic domains, as the primary LMP-1 domain mediating oligomerization, patching and cytoskeletal association. The cytoplasmic amino-terminus provides the structural information whereby proper transmembrane orientation is achieved.

Aneuploidy and cancer.

Numeric aberrations in chromosomes, referred to as aneuploidy, is commonly observed in human cancer. Whether aneuploidy is a cause or consequence of cancer has long been debated. Three lines of evidence now make a compelling case for aneuploidy being a discrete chromosome mutation event that contributes to malignant transformation and progression process. First, precise assay of chromosome aneuploidy in several primary tumors with in situ hybridization and comparative genomic hybridization techniques have revealed that specific chromosome aneusomies correlate with distinct tumor phenotypes. Second, aneuploid tumor cell lines and in vitro transformed rodent cells have been reported to display an elevated rate of chromosome instability, thereby indicating that aneuploidy is a dynamic chromosome mutation event associated with transformation of cells. Third, and most important, a number of mitotic genes regulating chromosome segregation have been found mutated in human cancer cells, implicating such mutations in induction of aneuploidy in tumors. Some of these gene mutations, possibly allowing unequal segregations of chromosomes, also cause tumorigenic transformation of cells in vitro. In this review, the recent publications investigating aneuploidy in human cancers, rate of chromosome instability in aneuploidy tumor cells, and genes implicated in regulating chromosome segregation found mutated in cancer cells are discussed.

PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis.

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.