The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma.

BACKGROUND: Prolyl hydroxylation is a post-translational modification that affects the structure, stability and function of proteins including collagen by catalysing hydroxylation of proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4 (C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC symbols and names at http://www.genenames.org/ and Entrez database at http://www.ncbi.nlm.nih.gov/gene) leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1), leprecan-like 2 (Leprel2) and two paralogs Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are found in humans. The C-P4Hs are tetrameric proteins comprising a variable α subunit, encoded by the P4HA1, P4HA2 and P4HA3 genes and a constant ß subunit encoded by P4HB. METHODS: We used RT-PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting and immunohistochemistry to investigate expression and regulation of the C-P3H and C-P4H genes in B lymphomas and normal bone marrow. RESULTS: C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with methylation in the CpG islands and is detected in almost all common types of B-cell lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple C-P3H and C-P4H genes is present in some lymphomas, particularly Burkitt's lymphoma. CONCLUSIONS: Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.

The prioritisation of invasive alien plant control projects using a multi-criteria decision model informed by stakeholder input and spatial data.

Invasions by alien plants are a significant threat to the biodiversity and functioning of ecosystems and the services they provide. The South African Working for Water program was established to address this problem. It needs to formulate objective and transparent priorities for clearing in the face of multiple and sometimes conflicting demands. This study used the analytic hierarchy process (a multi-criteria decision support technique) to develop and rank criteria for prioritising alien plant control operations in the Western Cape, South Africa. Stakeholder workshops were held to identify a goal and criteria and to conduct pair-wise comparisons to weight the criteria with respect to invasive alien plant control. The combination of stakeholder input (to develop decision models) with data-driven model solutions enabled us to include many alternatives (water catchments), that would otherwise not have been feasible. The most important criteria included the capacity to maintain gains made through control operations, the potential to enhance water resources and conserve biodiversity, and threats from priority invasive alien plant species. We selected spatial datasets and used them to generate weights that could be used to objectively compare alternatives with respect to agreed criteria. The analysis showed that there are many high priority catchments which are not receiving any funding and low priority catchments which are receiving substantial allocations. Clearly, there is a need for realigning priorities, including directing sufficient funds to the highest priority catchments to provide effective control. This approach provided a tractable, consensus-based solution that can be used to direct clearing operations.

Epstein-Barr virus and Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive B-cell malignancy with endemic, sporadic and immunodeficiency-associated variants. It has been known for many years that the fundamental transforming event in BL is the translocation of the MYC gene, and the events that bring about this translocation and those that allow cells to survive with the constitutive expression of MYC have been the subject of intense investigation. Epstein-Barr virus (EBV) infection, malaria, immunodeficiency and spontaneous, somatic mutation can all contribute to the origin and maintenance of this cancer and their mechanisms are the subject of this review.

Preferential retention of codon 72 arginine p53 in squamous cell carcinomas of the vulva occurs in cancers positive and negative for human papillomavirus.

We have sought to determine the basis for preferential loss of the codon 72 proline (72P) rather than the arginine (72R) allele in squamous cell carcinoma of the vulva with loss of heterozygosity (LOH) in p53. The proportion of cases containing human papillomavirus (HPV) 16 was not statistically different among individuals with either 72RR or 72RP in the germ line (P > 0.99), but p53 LOH was significantly more common in individuals heterozygous 72RP than in 72RR individuals (P = 0.04). LOH more commonly involved the 72P allele in both HPV-positive and HPV-negative cancers. Our results imply that preferential loss of the 72P allele in vulval squamous cell carcinoma occurs by HPV-dependent and -independent mechanisms.

The BZLF1 protein of EBV has a coiled coil dimerisation domain without a heptad leucine repeat but with homology to the C/EBP leucine zipper.

The EBV transactivator protein BZLF1 can bind many sites in the EBV genome, most of which have homology to a consensus AP-1 site, the binding site for the fos/jun family of transcription factors. Here we present evidence that BZLF1 can also recognise the binding site for the CCAAT/enhancer binding protein C/EBP and that a BZLF1 binding site within the BZLF1 promoter is recognised by the C/EBP protein. Analysis of the BZLF1 DNA binding domain suggests that the BZLF1 protein binds to DNA as a dimer using sequences adjacent to a basic DNA binding motif. The BZLF1 dimerisation domain does not have a heptad repeat of leucine residues common to leucine zipper proteins but does have characteristics of a coiled coil structure, as judged by site directed mutagenesis. We therefore propose that the dimerisation domain of BZLF1 is structurally related to the coiled coil structure of leucine zippers but lacks the highly conserved leucine repeat. We show that the PZLF1 dimerisation domain has residues in common with the C/EBP leucine zipper and discuss the possible implications of this relationship.

Epstein-Barr virus exploits the normal cell pathway to regulate Rb activity during the immortalisation of primary B-cells.

Infection of human primary B-lymphocytes with Epstein-Barr virus (EBV) drives quiescent cells into continual proliferation and results in the outgrowth of immortal cell lines. This requires re-programming of the mechanisms that, in the absence of appropriate antigenic stimulation, normally prevent the proliferation of B-lymphocytes. Since the Retinoblastoma protein (pRb) and its relatives, p107 and p130, play critical roles in controlling the mammalian cell division cycle, we have investigated the expression and phosphorylation status of these proteins following EBV immortalisation of primary B-lymphocytes. In this report, we show that EBV drives the hyperphosphorylation of pRb. This is achieved by a strategy involving the altered expression of several components of the signal transduction pathway that normally regulates the phosphorylation status of pRb, including the up regulation of a number of cyclins and cyclin-dependent kinases and the down regulation of a subset of cyclin-dependent kinase inhibitors. The net result is the formation of active cyclin-dependent kinase complexes that are capable of phosphorylating and inactivating pRb. The results presented here identify the activation of a normal signal transduction pathway as an important component of the strategy used by EBV to drive cell proliferation.

Epstein-Barr virus nuclear antigen (EBNA)3C is an immortalizing oncoprotein with similar properties to adenovirus E1A and papillomavirus E7.

Epstein-Barr virus (EBV) requires six genes to efficiently immortalize human B cells. We have shown that one of these, EBNA3C, can cooperate with activated (Ha-)ras in co-transfection assays to immortalize and transform rat embryo fibroblasts (REFs). EBNA3C also augmented transformation by (Ha-)ras and a mutant p53 to a similar extent as human papilloma virus E7. As with E7 this effect was not inhibited by cotransfection with the cyclin-dependent kinase inhibitor (CDKI), a p16INK4A, which can normally activate the retinoblastoma protein (pRb) and induce growth arrest. Also like E7/ras and E1A/ras transformed cells the EBNA3C/ras transformants are very susceptible to apoptotic cell death. In vitro EBNA3C binds to pRb in a manner which is dependent on the integrity of the pocket domain; this suggests that EBNA3C, even though it lacks the LXCXE pRb binding motif found in E7 and E1A, may interact with pRb in vivo. We conclude that EBNA3C functions as an oncoprotein which directs cell cycle progression through the G1 phase restriction point when conditions might signal arrest. For the first time this demonstrates that EBV encodes a protein, functionally but not necessarily mechanistically, similar to the pRb-neutralizing nuclear antigens encoded by the 'small' DNA tumor viruses.

DNA binding of USF is required for specific E-box dependent gene activation in vivo.

Although USF-1 and -2 are the major proteins that bind to Myc-regulated E-box (CACGTG) elements in many cells, there is no clear role for USF during Myc-dependent gene regulation. Using dominant negative alleles of USF-1 we now show that DNA binding by USF at a Myc-regulated E-box limits the ability of another E-box binding factor, TFE-3, to activate a target gene of Myc in vivo and to stimulate S phase entry in resting fibroblasts. Similarly, dominant negative alleles of USF-1 relieve the restriction that prevents activation of the IgH enhancer by TFE-3 in non B-cells. DNA binding activity of USF complexes is abundant in primary human B-cells and is significantly downregulated during B-cell immortalization. Re-expression of USF-1 in immortalized B-cells retards proliferation. Our data establish an essential role for USF in restricting E-box dependent gene activation in vivo and suggest that this control is relaxed during cellular immortalization.

Epstein-Barr virus immortalizing genes.

Epstein-Barr virus (EBV) is linked to several types of human cancer and immortalizes human B cells very efficiently; at least six viral genes are required for this. The mechanism by which EBV immortalizes cells is particularly interesting because its immortalization genes are not obvious homologues of those of other DNA tumour viruses.

Cell cycle arrest following exposure of EBV-immortalised B-cells to gamma irradiation correlates with inhibition of cdk2 activity.

The exposure of Epstein-Barr virus immortalised B cells (LCLs) to the genotoxic effects of gamma irradiation causes a decreased proliferation of the cells. The early events in this process have been investigated here. The induction of p53 expression correlates with a cell cycle arrest in the G1 and G2/M phases of the cell cycle within 24 h of exposure. The molecular mechanism governing the decreased proliferation appears to involve the induction of the cyclin dependent kinase (cdk) inhibitor p21CIP1 and its functional association with cdk2.

Regulation of cell growth and death by Epstein-Barr virus.

Epstein-Barr virus (EBV) efficiently induces growth of human B cells and prevents cell death. Considerable progress has been made in understanding these processes, the role of EBV in human cancer cells and the relationship of viral gene expression to virus persistence and cancer.

Use of flow cytometry to rapidly optimize the transfection of animal cells.

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.

A new and sensitive method of screening for human papillomavirus infection.

Human papillomaviruses (HPVs) have been implicated in the development of cervical cancer, and HPV screening may one day supplement cytologic analysis of cervical samples. Detection of the small amounts of HPV DNA in cervical scrapes has been very difficult. The polymerase chain reaction is a new technique that can specifically amplify target DNA to facilitate its detection. We have amplified a region of HPV type 16 DNA using this technique and have shown the method to be both specific and sensitive (a tenfold improvement when compared with Southern blotting). Among 21 cervical scrapes, five from women with normal smears and 16 from women with dyskaryotic smears, HPV 16 DNA was present in scrapes from two women (40%) with normal smears and in scrapes from 12 women (75%) with dyskaryotic smears. The polymerase chain reaction technique is rapid, requires no radioactive probes, and should be the method of choice in future studies to determine the prevalence of HPV infection of the cervix.

Screening of transformed insect cell lines for recombinant protein production.

Nine insect cell lines were evaluated for their potential as host systems for recombinant protein production using a new expression vector permitting the continuous high-level expression of secreted glycoproteins by transformed insect cells (Farrell et al., 1998). As a means of preliminary screening, all nine insect cell lines were transfected with the green fluorescence protein. Growth in static and suspension culture was then examined as a further method of screening. On the basis of their transfection efficiencies and cell growth characteristics, five insect cell lines, Bm5, High Five, IPLB-LdFB, IZD-MB-0503, and Sf-21, were selected for stable transformation to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). These five cell lines were stably transformed using an antibiotic resistance scheme and evaluated as a polyclonal population. Increasing the antibiotic concentration was found to cause not only a decrease in the specific growth rate but also an increase in the specific protein production rate and final GM-CSF concentration. The transformed High Five cells exhibited by far the greatest specific protein production rate of 5.1 x 10(-)(6) microgram/(cell.h), resulting in the highest final GM-CSF concentration of 22.8 mg/L when grown in static culture. One cloned High Five cell line produced a GM-CSF concentration of 46 mg/L in static culture and 27 mg/L in suspension culture.

Epstein-Barr virus genes and cancer cells.

Human B lymphocytes infected with Epstein-Barr virus (EBV) express 11 viral genes, of which six are essential for efficient transformation. The protein products of these genes appear to cause cell growth by modifying cell signal transduction pathways. For example, EBNA-2 mimics the Notch 1 pathway and LMP-1 interacts with the signalling from CD40/CD40-L, which promotes growth in normal B cells. In the human cancers linked to EBV, most of the viral transforming genes are not expressed. It is likely that growth of these cells is controlled by a combination of the EBV genes whose expression continues with altered cell proto-oncogenes and tumour suppressor genes, but other explanations of the role of EBV in cancer cells are also possible. The presence of the virus in the tumour cells of EBV-associated cancers constitutes a potentially useful tumour specific marker that might be used to direct therapy to the tumour cells.

Association of innate immune activation with latent Epstein-Barr virus in active MS lesions.

OBJECTIVE: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. METHODS: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. RESULTS: We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFNα in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFNα was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFNα production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFNα production in vitro. CONCLUSION: These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFNα production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.