Cholecystokinin-2 receptor modulates cell adhesion through beta 1-integrin in human pancreatic cancer cells.

Several lines of evidence suggest that gastrin and the CCK-2 receptor (CCK2R) could contribute to pancreatic carcinogenesis by modulating processes such as proliferation, cell adhesion or migration. In the current study, we used a 'cancer gene array' and identified beta1-integrin subunit as a new gastrin-regulated gene in human pancreatic cancer cells. We also demonstrated that Src family kinases and the phosphatidylinositol-3-kinase (PI-3-kinase) pathway play a crucial role in the expression of beta1-integrin induced by gastrin. Our results also showed that gastrin modulates cell-substrate adhesion via beta1-integrin. Indeed, using blocking anti-beta1-integrin monoclonal antibodies, we completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we observed that in response to gastrin, beta1-integrin is tyrosine phosphorylated by Src family kinases and associates with paxillin, a scaffold protein involved in focal adhesion and integrin signalling. This mechanism might be involved in gastrin-induced cell adhesion. Moreover, we showed in vivo that targeted CCK2R expression in the pancreas of Elas-CCK2 mice leads to the overexpression of beta1-integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals.

Role of c-myc regulation in Zta-mediated induction of the cyclin-dependent kinase inhibitors p21 and p27 and cell growth arrest.

Latency-associated Epstein-Barr virus (EBV) gene expression induces cell proliferation. Unlike the latency associated genes, lytic gene expression in EBV, as well as other herpesviruses, elicits cell cycle arrest. Previous studies have shown that the EBV immediate early lytic transactivator, Zta, induces a G(0)/G(1) cell cycle arrest through induction of the cyclin-dependent kinase inhibitors, p21 and p27. Here we show that while EBV latency is intimately linked to activation of the protooncogene, c-myc, Zta represses c-myc expression. We also show that inhibition of c-myc expression is required for Zta-mediated growth arrest and for maximal induction of p21 and p27. Nevertheless, induction of p21 and p27 is also influenced by a c-myc-independent mechanism. A detailed genetic analysis of Zta's basic/DNA binding region identified two distinct subregions that contribute to full induction of p21 and p27. One subdomain influences p21 and p27 expression through the c-myc-dependent mechanism and the other subdomain influences p21 and p27 induction through the c-myc-independent pathway. Together, these studies further our understanding of the complex nature of Zta-induced growth arrest.

A mineral sunscreen affords genomic protection against ultraviolet (UV) B and UVA radiation: in vitro and in situ assays.

Ultraviolet (UV) radiation has been shown to be responsible for different biological effects on human skin, including the initiation of photocarcinogenesis. Both UVB and UVA have been described as mutagenic, but the processes by which they alter the DNA are different. Although cells can repair DNA damage, some deleterious mutations nevertheless appear and can promote cancer. The risk of photocarcinogenesis is acknowledged and the frequency of photogenodermatosis is increasing. In order to evaluate the protection efficacy of a high sun protection factor (SPF) mineral sunscreen against UVB- and UVA-induced genomic alterations, we have followed two approaches. First, we have tested the sunscreen for its ability to decrease the unscheduled DNA synthesis response in vitro in human fibroblasts, as an indirect measure of UVB-induced lesions (0.005 and 0.01 J/cm2), and second, we have verified its ability to reduce the in situ end-labelling intensity in human skin as a direct measure of UVA-induced single-strand breaks (10 J/cm2). Microscopic analysis clearly demonstrated the protective effect of the sunscreen against UVB and UVA. A dose-dependent effect of mineral sunscreens was observed. There was also a relationship between the SPF and genomic protection. By limiting the accumulation of UV-induced lesions on DNA, this mineral sunscreen could limit the mutation frequency.

Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21.

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1/Waf1) plays an essential role in the control of cell proliferation by modulating the activity of cyclin/CDK complexes in response to various intracellular or extracellular signals. Small variations in p21 expression levels may determine whether it acts as an inhibitor or an assembly factor for cyclin/CDK complexes. It is therefore critical to better characterize the mechanisms regulating p21 abundance. Here, we show, using a tetracycline-regulated system in p53-deficient DLD-1 human colon cancer cells, that p21 protein levels and stability are regulated by the proteasome-dependent degradation pathway and by association with its partners, CDKs and PCNA. A p21 mutant deficient for interaction with CDKs, p21CDK-, displayed an enhanced stability and greatly reduced sensitivity to proteasome-mediated proteolysis, indicating that association with cyclin/CDK complexes may trigger p21 degradation. In contrast, a p21 mutant impaired in the interaction with PCNA, p21PCNA-, exhibited a decreased stability, suggesting that association with PCNA protects p21 from proteasome-dependent degradation. Furthermore, the abundance of p21 itself, in addition to protein-protein interactions, may also modulate p21 stability since we found that high levels of p21 expression overcome proteasome-dependent regulation of p21 accumulation.

A novel Xenopus mix-like gene milk involved in the control of the endomesodermal fates.

Here we describe a novel Xenopus homeobox gene, milk, related by sequence homology and expression pattern to the vegetally expressed Mix.1. As is the case with Mix.1, milk is an immediate early response gene to the mesoderm inducer activin. milk is expressed at the early gastrula stage in the vegetal cells, fated to form endoderm, and in the marginal zone fated to form mesoderm. During gastrulation, expression of milk becomes progressively reduced in the involuting mesodermal cells but is retained in the endoderm, suggesting that it may play a key role in the definition of the endo-mesodermal boundary in the embryo. Overexpression of milk in the marginal zone blocks mesodermal cell involution, represses the expression of several mesodermal genes such as Xbra, goosecoid, Xvent-1 or Xpo and increases the expression of the endodermal gene, endodermin. In the dorsal marginal zone, overexpression of milk leads to a severe late phenotype including the absence of axial structures. Ectopic expression of milk in the animal hemisphere or in ectodermal explants induces a strong expression of endodermin. Taken together, we propose that milk plays a role in the correct patterning of the embryo by repressing mesoderm formation and promoting endoderm identity.

UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.

DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion.

p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells.

A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases delta and epsilon. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK-, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA-, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest.

[Prevention of venous thrombosis: role of heparins]. / Prophylaxie de la thrombose veineuse: place des héparines.

Prevention of venous thromboembolism is of major importance because deep vein thrombosis is an economic burden. To prevent pulmonary embolism, whether fatal or not, and the postphlebitic syndrome, virtually all patients' level of risk should be assessed in order to provide adequate prophylactic measures against venous thromboembolism. Non-pharmacological, pharmacological or combined modalities can reduce the frequency of venous thrombosis. Evidence-based guidelines are available for most situations in surgical patients. However, in medical patients there are fewer data and there are wide variations of opinion. Systematic reviews should be performed and updated to obtain practice guidelines. Cost and effectiveness as well as patients' preferences should be taken into account. Randomized control trials are ongoing: low-molecular-weight heparins are being evaluated in general medical patients; other forms of prophylaxis or combined methods are also being investigated.

Use of the two-hybrid system to identify protein-protein interaction temperature-sensitive mutants: application to the CDK2/p21Cip1 interaction.

We describe the application of the two-hybrid system to the identification of protein-protein interaction temperature-sensitive mutants. We applied this strategy to the interaction between the human CDK2 cell cycle regulator and the p21Cip1 regulatory subunit. A library of randomly generated CDK2 mutant proteins was screened for interaction with p21Cip1 at different temperatures. This approach resulted in the isolation of single point mutations in CDK2 causing temperature-sensitive interaction with p21Cip1. Our results demonstrate that the two-temperature two-hybrid screen is an efficient approach for the rational design and screening of protein-protein interaction conditional mutations.

G0/G1 growth arrest mediated by a region encompassing the basic leucine zipper (bZIP) domain of the Epstein-Barr virus transactivator Zta.

The Epstein-Barr virus (EBV) immediate early transactivator Zta is a basic leucine zipper (bZIP) transcription factor that causes G0/G1 cell cycle arrest through induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase inhibitors, p21 and p27 (Cayrol, C., and Flemington, E. K. (1996) EMBO J. 15, 2748-2759). Here, we report a genetic analysis of Zta-mediated G0/G1 growth arrest and p21 induction. The majority of the Zta transactivation domain can be deleted (ZDelta1-128) without significantly affecting the ability of Zta to elicit growth arrest. A larger amino-terminal deletion (ZDelta1-167) abrogates the ability of Zta to inhibit proliferation, mapping the growth-inhibitory domain to a carboxyl-terminal region encompassing the bZIP domain (amino acids 128-245). The integrity of the bZIP domain is required for growth suppression since a two-amino acid mutant which is defective for homodimerization, fails to induce cell cycle arrest. Western blot analysis of p21 expression in cells expressing Zta mutants reveals that the ability of Zta mutants to cause G0/G1 growth arrest is intimately related to their capacity to induce p21 expression. Together, these data demonstrate that a carboxyl-terminal region of Zta that includes the bZIP domain is sufficient to mediate G0/G1 growth arrest and p21 induction.

The Epstein-Barr virus bZIP transcription factor Zta causes G0/G1 cell cycle arrest through induction of cyclin-dependent kinase inhibitors.

While oncoproteins encoded by small DNA tumor viruses and Epstein-Barr virus (EBV) latent antigens facilitate G1/S progression, the EBV lytic switch transactivator Zta was found to inhibit growth by causing cell cycle arrest in G0/G1 in several epithelial tumor cell lines. Expression of Zta results in induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase inhibitors, p21 and p27, as well as accumulation of hypophosphorylated pRb. Up-regulation of p53 and p27 occurs by post-transcriptional mechanisms while expression of p21 is induced at the RNA level in a p53-dependent manner. Inactivation of pRb by transient overexpression of the human papillomavirus E7 oncoprotein indicates that pRb or pRb-related proteins are key mediators of the growth-inhibitory function of Zta. These findings suggest that EBV plays an active role in redirecting epithelial cell physiology to facilitate the viral replicative program through a Zta-mediated growth arrest function.

A novel TGF-beta-like gene, fugacin, specifically expressed in the Spemann organizer of Xenopus.

Using a differential screening strategy, we have cloned a novel Xenopus gene, fugacin, related to the transforming growth factor beta superfamily. Transcripts were detected primarily in the dorsal marginal zone of late blastula. Thereafter, they became highly localized to the blastopore lip of early gastrula and were not observed at later stages. This gene, which is most homologous to the mouse gene nodal, displays a new pattern of cysteine residues. These findings highlight the potential role of these growth factors during early vertebrate development.

Recovery of respiration following the SOS response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase.

Agents that damage DNA in Escherichia coli or interfere with its replication induce DNA repair and mutagenesis via the SOS response. This well-known activity is regulated by the RecA protein and the LexA repressor. Following repair or bypass of the DNA lesion, the cell returns to its resting state by a largely unknown process. We found that 2-keto-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate aldolase; EC 4.1.3.16) is necessary for the recovery of respiration and that it is regulated by the SOS response. This protein was induced by DNA-damaging agents. Induction required RecA activation. When the LexA regulon was repressed, activation of RecA was not sufficient for induction, indicating the requirement for an additional protein under LexA control. Finally, a mutant in the corresponding hga gene was UV sensitive. 2-Keto-4-hydroxyglutarate aldolase also plays a role in respiratory metabolic pathways, which suggests a mechanism for respiration resumption during the termination of the SOS response.

A subset of HLA-DR9 molecules is detected by a polymorphic monoclonal antibody on lymphoblastoid cell lines but not on peripheral blood lymphocytes.

Some mAbs recognizing polymorphic epitopes of HLA-DR molecules exhibit striking differences of reactivity with the same HLA-DR molecules expressed by different cell types. In this study, we investigated the basis for the differential reactivity of the polymorphic anti-DR mAb OHA TM901 with HLA-DR9 molecules expressed by human PBLs or LCLs. By immunoprecipitation experiments we showed that OHA TM901 recognizes a subset of HLA-DR9 molecules from LCLs. This subset corresponds to HLA-DR9 molecules containing immature-type oligosaccharides. The absence of OHA TM901 reactivity with HLA-DR9 PBLs, as revealed by cytofluorometry analysis, suggests that this subset is either not expressed or expressed at a very low level on PBLs. These results indicate that overexpression of HLA-DR molecules in immortalized LCLs could lead to cell-surface expression of underglycosylated forms which are generally not found on the cell surface of PBLs.

Identification of cellular target genes of the Epstein-Barr virus transactivator Zta: activation of transforming growth factor beta igh3 (TGF-beta igh3) and TGF-beta 1.

The lytic switch transactivator Zta initiates the ordered cascade of Epstein-Barr virus gene expression that culminates in virus production. Zta is a sequence-specific DNA-binding protein that transactivates early viral promotes via cis-acting sequences. Activation of some of these genes is mediated through binding to consensus AP-1 promoter elements. This observation suggests that Zta may also regulate the expression of cellular genes. While many targets of Zta have been identified in the Epstein-Barr virus genome, putative host cell targets remain largely unknown. To address this issue, a tetracycline-regulated Zta expression system was generated, and differential hybridization screening was used to isolate Zta-responsive cellular genes. The major target identified by this analysis is a gene encoding a fasciclin-like secreted factor, transforming growth factor beta igh3 (TGF-beta igh3), that was originally identified as a gene that is responsive to the potent immunosuppressor TGF-beta 1. Northern (RNA) blot analysis demonstrated that induction of Zta expression results in a 10-fold increase in TGF-beta igh3 mRNA levels. Zta was also found to increase TGF-beta 1 mRNA levels as well as the amount of active TGF-beta 1 secreted into the medium. Interestingly, alpha 1-collagen IV, which has been shown to potentiate the effects of TGF-beta 1, is also a cellular target of Zta. These results suggest that Zta could play a role in modulating the host cell environment through activating the expression of secreted factors.

DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.

Characterization of dinY, a new Escherichia coli DNA repair gene whose products are damage inducible even in a lexA(Def) background.

Bacteriophage Mu dX(Ap lac) was used to isolate a mutation in an Escherichia coli lexA(Def) strain representing a previously undescribed gene (dinY) which does not seem to be under the direct control of LexA. The insertion created a dinY::lacZ fusion in which beta-galactosidase expression required a DNA-damaging treatment (UV irradiation or mitomycin) and activable RecA protein. This strain showed a decreased Weigle reactivation of bacteriophage lambda. However, it was fully inducible for UV mutagenesis. Two-dimensional gel electrophoresis analysis identified two spots absent in the mutant which were both UV inducible only in the presence of activated RecA protein (RecA*). This finding suggests that the dinY::lacZ fusion lies in a gene either that is under the direct control of activated RecA or whose product undergoes RecA*-dependent posttranscriptional/posttranslational modification(s). The dinY gene may also control the expression of some other gene(s) and/or lie in an operon. The fusion was mapped at a position between 41 and 41.5 min on the E. coli chromosome, in the vicinity of the ruv operon.

New polymorphic HLA-DR epitopes recognized by three monoclonal antibodies produced against DR103 transfected L cells.

Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non-MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103-transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA-DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B-lymphoblastoid cell lines (B-LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA-DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B-LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA-DR products and react in western blots with DR alpha/beta-dimer but not with free alpha- or beta-chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.