Down-regulation of T-STAR, a growth inhibitory protein, after SV40-mediated immortalization.

Normal human cells can undergo a limited number of divisions, whereas transformed cells may have an extended life span and can give rise to immortal cells. To isolate genes involved in the immortalization process, gene expression in SV40-transformed preimmortal human fibroblasts was compared with expression in SV40-transformed immortalized fibroblasts using an mRNA differential display. We found that the growth-inhibitory protein testis-signal transduction and activation of RNA (T-STAR) a homologue of cell-cycle regulator Sam68, is strongly down-regulated in immortalized cells. Overexpression of T-STAR in the SV40-transformed immortalized cells resulted in a strong reduction of colony formation, whereas deletion of the RNA-binding domain of T-STAR abrogated this effect. Down-regulation of testis-signal transduction and activation of RNA (T-STAR) expression is found only in immortal cells isolated after a proliferative crisis accompanied with massive cell death. The strict correlation of down-regulation of T-STAR expression only in those immortal cells that arose after a clear proliferative crisis suggests that the loss of T-STAR might be necessary to bypass crisis.

Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms.

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2-mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self-ubiquitylation.

WT1 proteins: functions in growth and differentiation.

The Wilms' tumor 1 gene (WT1) has been identified as a tumor suppressor gene involved in the etiology of Wilms' tumor. Approximately 10% of all Wilms' tumors carry mutations in the WT1 gene. Alterations in the WT1 gene have also been observed in other tumor types, such as leukemia, mesothelioma and desmoplastic small round cell tumor. Dependent on the tumor type, WT1 proteins might either function as tumor suppressor proteins or as survival factors. Mutations in the WT1 gene can also result in congenital abnormalities as observed in Denys-Drash and Frasier syndrome patients. Mouse models have proven the critical importance of WT1 expression for the development of several organs, including the kidneys, the gonads and the spleen. The WT1 proteins seem to perform two main functions. They regulate the transcription of a variety of target genes and may be involved in post-transcriptional processing of RNA. The WT1 gene encodes at least 24 protein forms. These isoforms have partially distinct biological functions and effects, which in many cases are also specific for the model system in which WT1 is studied. This review discusses the molecular mechanisms by which the various WT1 isoforms exert their functions in normal development and how alterations in WT1 may lead to developmental abnormalities and tumor growth.

Induction of the SAPK activator MIG-6 by the alkylating agent methyl methanesulfonate.

The alkylating agent methylmethanesulfonate (MMS) activates the c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and the p38 mitogen-activated protein kinase (p38MAPK) pathways via different mechanisms of action. Activation of p38MAPK by MMS involves the pp125 focal adhesion kinase-related tyrosine kinase RAFTK and the MAPK kinase 3. The way in which MMS can activate JNK/SAPK has not been elucidated. Here we describe the identification by differential display of human mitogen-activated gene-6 (MIG-6) as a novel MMS-inducible gene. Induction of MIG-6 by MMS was found in human diploid skin fibroblasts and in simian virus 40-transformed skin fibroblasts, indicating that the enhanced expression of MIG-6 after MMS-treatment did not require p53. The signal leading to activation of MIG-6 appeared to be independent of DNA damage. High MIG-6 expression was found in the liver, lung, and placenta. MIG-6 is an adapter protein that binds to the activated form of cdc42Hs and to 14-3-3 proteins, thereby activating JNK/SAPKs. Our results suggest that activation of JNK/SAPKs by MMS may involve the induction of MIG-6.

Aberrant expression of HDMX proteins in tumor cells correlates with wild-type p53.

It has been shown that the Hdmx gene is amplified in a subset of gliomas, but thus far, no data are available on HDMX protein expression in tumor cells. We now report that a significant fraction of tumor cell lines expresses increased HDMX levels compared with normal cells; in general, HDMX expression in these tumor cell lines correlates with the presence of wild-type p53. Analysis of tumor material showed that high HDMX expression is not a result of cell line establishment. Interestingly, several cell lines express alternative, shorter HDMX proteins. These results suggest that deregulated expression of HDMX plays a role in carcinogenesis as an alternative way to inactivate p53.

Mif1: a missing link between the unfolded protein response pathway and ER-associated protein degradation?

Eukaryotic cells have three different mechanisms to deal with the accumulation of unfolded proteins in the endoplasmic reticulum: (1) In cells in which unfolded polypeptides accumulate, translation initiation is inhibited to prevent further accumulation of unfolded proteins. (2) Expression of proteins involved in polypeptide folding is strongly enhanced by a process called the Unfolded Protein Response (UPR). (3) Proteins missing the proper tertiary structure are degraded by the ER-Associated protein Degradation (ERAD) mechanism. Recent studies in S. cerevisiae have shown that the processes of UPR and ERAD are functionally linked to each other. Cells lacking a functional ERAD show a constitutive activation of UPR. In addition, many of the components of ERAD are under the direct transcriptional control of UPR. Finally, while neither UPR nor ERAD are essential for cell viability, deletion of both pathways results in severe growth impairment. UPR and ERAD are conserved between yeast and mammalian cells. One of the components of mammalian UPR is the protease presenilin-1. Mutations in the gene for presenilin-1 cause early-onset familial Alzheimer disease. Interestingly, inhibition of proteolysis by the ubiquitin-26S proteasome system has also been described for Alzheimer s disease. This suggests a link between UPR and ERAD in mammalian cells. The recently identified gene Mif1 is a possible candidate to form a direct link between UPR and ERAD in mammalian cells. The Mif1 gene is under the direct control of UPR. Mif1 is a trans-ER-membrane protein, with both the N- and the C-termini facing the cytoplasmic side of the ER membrane. It contains an N-terminal ubiquitin-like domain. It is anticipated that Mif1 may associate through its ubiquitin-like domain with the 26S proteasome, in this way connecting the protein degradation machinery to the ER membrane and resulting in an efficient ERAD.

Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.

In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 +/- 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4 precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells.

The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e.g., many chemotherapeutic agents and radiation) require functional p53 and are inhibited by Bcl-2, Apoptin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptotic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis. Although Apoptin activity was not affected by CrmA, p35 did inhibit Apoptin-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. However, in most of these cells, cytochrome c is still released and the mitochondria are not stained by CMX-Ros, indicating a drop in mitochondrial membrane potential. These results imply that although the final apoptotic events are blocked by p35, parts of the upstream apoptotic pathway that affect mitochondria are already activated by Apoptin. Taken together, these data show that the viral protein Apoptin employs cellular apoptotic factors for induction of apoptosis. Although activation of upstream caspases is not required, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.

Hdmx stabilizes Mdm2 and p53.

The Mdm2 protein is a key regulator of p53 activity and stability. Upon binding, Mdm2 inhibits the transcription regulatory activity of p53 and promotes its rapid degradation. In this study we investigated the effect of the human Mdm2 homologue Hdmx on p53 stability. We found that Hdmx does not target p53 for degradation, although, like Mdm2, it inhibits p53-mediated transcription activation. On the contrary, Hdmx was found to counteract the degradation of p53 by Mdm2, and to stabilize both p53 and Mdm2. The RING finger of Hdmx was found to be necessary and sufficient for this stabilization, and it probably involves hetero-oligomerization with the RING finger of Mdm2, which may lead to inhibition of Mdm2's ubiquitin ligase activity. However, Hdmx does not relieve the inhibition by Mdm2 of transcription activation by p53, probably due to the formation of a trimeric complex consisting of Hdmx, Mdm2, and p53. We propose a model in which Hdmx secures a pool of largely inactive p53, which, upon the induction of stress, can be quickly activated.

Physical interaction between Wilms tumor 1 and p73 proteins modulates their functions.

The WT1 gene, which is heterozygously mutated or deleted in congenital anomaly syndromes and homozygously mutated in about 15% of all Wilms tumors, encodes tissue-specific developmental regulators. Through alternative mRNA splicing, four main WT1 protein isoforms are synthesized. All isoforms can bind to DNA via their zinc fingers, albeit with different affinities and specificities, and thereby modulate the transcriptional activity of their target genes. Several proteins bind to and alter the transcription regulatory properties of the WT1 proteins, including the product of the tumor suppressor gene p53. Interaction between WT1 and p53 was shown to modulate their ability to regulate the transcription of their respective target genes. Here, we report that all four isoforms of WT1 bind to p73, a recently cloned homologue of p53. p73 binds to the zinc finger region of WT1 and thereby inhibits DNA binding and transcription activation by WT1. Similarly, WT1 inhibits p73-induced transcription activation in reporter assays and counteracts p73-induced expression of endogenous Mdm2. This, taken together with our finding that WT1 also interacts with p63/KET, another p53 homologue, suggests that association between WT1 and the members of the p53 family of proteins may be an important determinant of their functions in cell growth and differentiation.

The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway.

In a search for genes induced by DNA-damaging agents, we identified two genes that are activated by methyl methanesulfonate (MMS). Expression of both genes is regulated after endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) pathway. The first gene of those identified is the molecular chaperone BiP/GRP78. The second gene, Mif1, is identical to the anonymous cDNA KIAA0025. Treatment with the glycosylation inhibitor tunicamycin both enhances the synthesis of Mif1 mRNA and protein. The Mif1 5' flanking region contains a functional ER stress-responsive element which is sufficient for induction by tunicamycin. MMS, on the other hand, activates Mif1 via an UPR-independent pathway. The gene encodes a 52 kDa protein with homology to the human DNA repair protein HHR23A and contains an ubiquitin-like domain. Overexpressed Mif1 protein is localized in the ER.

EGR-1 enhances tumor growth and modulates the effect of the Wilms' tumor 1 gene products on tumorigenicity.

The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family and is homozygously mutated or deleted in a subset of Wilms' tumors. Through alternative mRNA splicing, the gene is expressed as four main polypeptides that differ by a stretch of 17 amino acids just N-terminal of the four zinc-fingers and three amino acids between zinc fingers 3 and 4. We have previously shown that expression of the WT1(-/-) isoform, lacking both inserts, increases the tumor growth rate of the adenovirus-transformed baby rat kidney (AdBRK) cell line 7C3H2, whereas expression of the WT1(-/+) isoform, lacking the 17aa insert, strongly suppresses the tumorigenic phenotype. In the present study we show that expression of these splice variants does not affect the tumorigenic potential of the similar AdBRK cell line, 7C1T1. In contrast to the 7C3H2 cell line, this AdBRK cell line expresses high endogenous levels of EGR-1 (early growth response-1) protein, a transcription factor structurally related to WT1. Ectopic expression of EGR-1 in the 7C3H2 AdBRK cells significantly increases their in vivo growth rate and nullifies the tumor suppressor activity of the WT1(-/+) protein. Furthermore, we find that EGR-1 levels are elevated in some Wilms' tumors. These data are the first to show that EGR-1 overexpression causes enhanced tumor growth and that WT1 and EGR-1 exert antagonizing effects on growth regulation in baby rat kidney cells, which might reflect the situation in some Wilms' tumors.

cDNA micro array identification of a gene differentially expressed in adenovirus type 5- versus type 12-transformed cells.

Proteins encoded by non-oncogenic adenovirus type 5 and oncogenic adenovirus type 12 differentially affect expression of a number of cellular genes. We have used cDNA micro array analysis to identify a cellular gene that is expressed in Ad12- but not in Ad5-transformed cells. This cellular gene was found to be the gene encoding follistatin-related protein, a TGF-beta inducible gene. Consistently, a constitutive factor binding to Smad binding elements was found in adenovirus type 12-transformed cells.

Protein kinase C-alpha is an upstream activator of the IkappaB kinase complex in the TPA signal transduction pathway to NF-kappaB in U2OS cells.

Inactive nuclear factor kappaB (NF-kappaB) complexes are retained in the cytoplasm by binding to inhibitory proteins, such as IkappaBalpha. Various stimuli lead to phosphorylation and subsequent processing of IkappaBalpha in the 26S proteasome and import of the active NF-kappaB transcription factor into the nucleus. In agreement with our previous finding that p90(rsk1) is essential for TPA-induced activation of NF-kappaB in Adenovirus 5E1-transformed Baby Rat Kidney cells, we now report that the MEK/ERK/p90(rsk1) inhibitor U0126 efficiently blocks TPA-induced IkappaBalpha processing in these cells. However, in U2OS cells, the cytokine-inducible IkappaB kinase complex (IKK) is the essential component of the TPA signal transduction pathway. Activation of the IKK complex in response to TPA is mediated by PKC-alpha, since both the PKC inhibitor GF109203 and a catalytically inactive PKC-alpha mutant inhibit activation of endogenous IKK by TPA, but not by tumor necrosis factor-alpha (TNF-alpha). We conclude that IKK is an integrator of TNF-alpha and TPA signal transduction pathways in U2OS cells.

Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation.

Recombinant adenovirus vectors are popular tools for gene transfer and gene therapy. However biosafety constraints require that all handling of the vectors and vector-containing samples is restricted to dedicated containment laboratories, unless they had undergone a validated virus-inactivation procedure, which decontaminates the samples from any active virus. In this study we evaluated the feasibility of photodynamic treatment (PDT) with visible light to inactivate recombinant adenovirus vectors in biological samples, with minimum associated effects on other biological activities. Several photosensitizers were tested for their capacity to inactivate a model human adenovirus vector, AdCMVLuc, upon illumination. Four photosensitizers (methylene blue (MB), rose bengal (RB), uroporphyrin (UP) and aluminum phthalocynine tetrasulphonate (AIPcS4)) could inactivate the adenovirus, as measured by expression of the luciferase reporter gene and by plaque assay. Of these, MB demonstrated to be the most effective sensitizer in phosphate-buffered saline (PBS), giving > 7 log10 inactivation of the adenovirus. DNA isolated from MB- and light-treated virions was inefficient as a template for transcription. Furthermore, Southern blot analysis revealed fragmentation of the viral DNA. Based on its preference for DNA, MB is suited for adenovirus inactivation in blood plasma. Spiking experiments in which AdCMVLuc was added to plasma samples demonstrated a reduction (> 4 log10-fold) of reporter gene expression to almost background levels. In contrast to MB, photodynamic treatment with RB, UP or AIPcS4 did not lead to DNA damage. Although alterations of the viral capsid could not be detected, the binding pattern of the particles to target cells was significantly changed. Taken together, our data demonstrate that PDT is an efficient, convenient and useful method for the inactivation of adenovirus vectors in biological samples.

Specific tumor-cell killing with adenovirus vectors containing the apoptin gene.

Specificity is an essential prerequisite for cancer gene therapy. Recently we described that apoptin, a protein of 121 amino acids which is derived from the chicken anemia virus, induces programmed cell death or apoptosis in transformed and malignant cells, but not in normal, diploid cells (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843-5847). This protein has an intrinsic specificity that allows it to selectively kill tumor cells, irrespective of the p53 or Bcl-2 status of these cells. Hence, it is attractive to explore the use of the apoptin gene for therapeutic applications, viz cancer gene therapy. In this paper, we describe the generation and characterization of an adenovirus vector, AdMLPvp3, for the expression of apoptin. Despite the fact that apoptin ultimately induces apoptosis in the helper cells, which are transformed by the adenovirus type 5 early region 1 (E1), the propagation kinetics and yields of AdMLPvp3 are similar to those of control vectors. Infection with AdMLPvp3 of normal rat hepatocytes in cell culture did not increase the frequency of apoptosis. In contrast, in the hepatoma cell lines HepG2 and Hep3b, infection with AdMLPvp3, but not with control vectors, led to a rapid induction of programmed cell death. Experiments in rats demonstrated that AdMLPvp3 could be safely administered by intraperitoneal, subcutaneous or intravenous injection. Repeated intravenous doses of AdMLPvp3 were also well tolerated, indicating that the apoptin-expressing virus can be administered without severe adverse effects. In a preliminary experiment, a single intratumoral injection of AdMLPvp3 into a xenogeneic tumor (HepG2 cells in Balb/Cnu/nu mice) resulted in a significant reduction of tumor growth. Taken together, our data demonstrate that adenovirus vectors for the expression of the apoptin gene may constitute a powerful tool for the treatment of solid tumors.