Jan Paul Medema

An endogenous hybrid mRNA encodes TWE‐PRIL, a functional cell surface TWEAK–APRIL fusion protein

TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT–PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs. The encoded TWE‐PRIL protein is composed of TWEAK cytoplasmic and transmembrane domains fused to the APRIL C‐terminal domain. TWE‐PRIL mRNA is expressed and translated in human primary T cells and monocytes, and endogenous TWE‐PRIL protein was detected in primary human T lymphocytes and monocytic cell lines. TWE‐PRIL is membrane anchored and presents the APRIL receptor‐binding domain at the cell surface. It is a biologically active ligand, as it stimulates cycling in T‐ and B‐lymphoma cell lines. Much like membrane‐bound and secreted TNF‐α, the different cellular localizations of TWE‐PRIL and APRIL suggest that they exert distinct biological roles.

Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway

Hepatic natural killer (NK) cells are located in the liver sinusoids adherent to the endothelium. Human and rat hepatic NK cells induce cytolysis in tumor cells that are resistant to splenic or blood NK cells. To investigate the mechanism of cell death, we examined the capacity of isolated, pure (90%) rat hepatic NK cells to kill the splenic/blood NK‐resistant mastocytoma cell line P815. Cell death was observed and quantified by fluorescence and transmission electron microscopy, DNA fragmentation, and 51Cr release. RNA and protein expression were determined by real time reverse transcription‐polymerase chain reaction and Western blotting. Compared with splenic NK cells, hepatic NK cells expressed higher levels of perforin and granzyme B and readily induced apoptosis in P815 cells. Although P815 cells succumbed to recombinant Fas ligand (FasL) or isolated perforin/granzyme B, hepatic NK cells used only the granule pathway to kill this target. In addition, hepatic NK cells and sinusoidal endothelial cells strongly expressed the granzyme B inhibitor, protease inhibitor 9 (PI‐9)/serine PI‐6 (SPI‐6), and P815 cells and hepatocytes were negative. Transfection of target cells with this inhibitor resulted in complete resistance to hepatic NK cell‐induced apoptosis. In conclusion, hepatic NK cells kill splenic/blood NK‐resistant/FasL‐sensitive tumor cells exclusively by the perforin/granzyme pathway. Serine protease inhibitor PI‐9/SPI‐6 expression in liver sinusoidal endothelial cells may protect the liver microenvironment from this highly active perforin/granzyme pathway used to kill metastasizing cancer cells.

Cutting Edge: Cellular Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Inhibitory Protein Protects Germinal Center B Cells from Apoptosis During Germinal Center Reactions

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIPL), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIPL decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIPL degradation and blocks all signs of apoptosis, even in the presence of anti-Fas Abs. cFLIPL expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.

A serine protease is involved in the initiation of DNA damage-induced apoptosis.

AbstractCaspases are considered to be the key effector proteases of apoptosis. Initiator caspases cleave and activate downstream executioner caspases, which are responsible for the degradation of numerous cellular substrates. We studied the role of caspases in apoptotic cell death of a human melanoma cell line. Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did prevent DEVDase activity. It is highly unlikely that caspase-2, which is a relatively zVAD-fmk-resistant caspase, is mediating etoposide-induced PARP cleavage, as a preferred inhibitor of this caspase could not prevent cleavage. In contrast, caspase activation and PARP degradation were blocked by pretreatment of the cells with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). We therefore conclude that a serine protease regulates an alternative initiation mechanism that leads to caspase activation and PARP cleavage. More importantly, while zVAD-fmk could not rescue melanoma cells from etoposide-induced death, the combination with AEBSF resulted in substantial protection. This indicates that this novel pathway fulfills a critical role in the execution of etoposide-induced programmed cell death.

The uncertain glory of APRIL

AbstractThe tumour necrosis factor (TNF) family is intimately connected to the regulation of cellular pathways. A PRoliferation-Inducing Ligand (APRIL) is a rather new member of that family, named for its capacity to stimulate the proliferation of tumour cells in vitro. Subsequent publications also called this ligand TRDL-1 or TALL-2, respectively. APRIL and B-lymphocyte stimulator (BLyS; also termed BAFF, TALL-1, THANK, zTNF4) form a new subfamily of TNF-like ligands that are expressed in haematopoietic cells. Both ligands can bind the two members of the TNF receptor family, namely the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), as well as B-cell maturation antigen (BCMA). BLyS has recently been the subject of several reviews (for an extensive review, see Mackay et al.). The present review will thus focus on APRIL, and discuss BLyS only briefly for the sake of clarity.

Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles.

BackgroundThe molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis.ResultsAs microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma.ConclusionThese data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

TWE-PRIL; a fusion protein of TWEAK and APRIL.

TWEAK and APRIL are both members of the tumor necrosis factor family, which are involved in respectively angiogenesis and immune regulation. While TWEAK is processed at the cell surface, APRIL is processed inside the cell by a furin-convertase and is solely able to perform its function as a soluble factor. Recently, TWE-PRIL has been identified, which is an endogenous hybrid transcript between TWEAK and APRIL. TWE-PRIL is a transmembrane protein that consists of a TWEAK intracellular, transmembrane and stalk region combined with APRIL as its receptor-binding domain. As such TWE-PRIL is expressed at the cell surface. Although TWE-PRIL, like APRIL, can stimulate T and B cell lines, distinct biological functions that may result from its membrane anchoring cannot be excluded. Understanding the function of this newly identified protein will contribute to the elucidation of the complexity of the tumor necrosis factor family.

Expression and function of the apoptosis effector Apaf-1 in melanoma

Melanoma is characterised by its poor response to current therapeutic modalities. Soengas et al. showed that Apaf-1, a central component of the intrinsic apoptotic pathway, is often lost in metastatic melanoma, implying that Apaf-1 loss contributes to its remarkable resistance. In our attempts to corroborate their findings in a panel of melanoma lines, we have found no evidence for Apaf-1 downregulation as a major event. In the paper of Soengas et al., Apaf-1 loss correlates with the inability to execute an apoptotic program upon p53 activation, providing an explanation for the low frequency of p53 mutations in this tumour type. Recently, however, we found that DNA damage-induced cell death in melanoma cells can occur independently of caspase activity or cytochrome c release (not shown), and is dependent on an apical serine protease that can side-step the apoptosome to initiate cell death. Moreover, Apaf-1 and caspase-9 deficiency does not facilitate myc-induced lymphomagenesis in mice, arguing against a role for Apaf-1 loss in tumorigenesis. Together, these observations made us re-evaluate the high frequency and relevance of Apaf-1 inactivation in melanoma. We determined Apaf-1 protein levels in whole-cell lysates of 13 melanoma cell lines and three different melanocyte cultures and found that most lines, except for 607B, demonstrated higher Apaf-1 levels as compared to melanocyte cultures (Figure 1a and not shown). As Apaf-1 exerts its proapoptotic function in the cytoplasm, we also quantified Apaf-1 levels in cytosolic fractions of a selection of these melanoma cell lines. In line with the Apaf-1 levels in whole-cell extracts, cytosolic Apaf-1 levels were higher in most cell lines as compared to melanocytes (Figure 1b). This excludes the possibility that Apaf-1 is inactivated in these lines due to redistribution, similar to what has recently been reported for Burkitt’s lymphoma cell lines, and therefore indicates that loss of Apaf-1 expression is rarely found in our panel. Although our panel contains a relatively high incidence of p53 mutations (5/13), Apaf-1 loss is not correlated to p53 status. As a matter of fact, all melanoma lines that harbour wtp53 express relatively high levels of Apaf-1 (Figure 1a). The functionality of wt-p53 in these cell lines was confirmed by induction of the p53-inducible gene product p21 after triggering these cells with DNA damage (data not shown). Intriguingly, the only Apaf-1-low cell line in our panel, 607B, harbours mutant p53. These data therefore do not argue for a connection between loss of Apaf-1 and p53 mutation. We next set out to study the effect of Apaf-1 levels on caspase activation. As caspase-9 is activated by induced proximity and its cleavage is dispensable for its activity, it is not a good measure for enzymatic activity. We therefore measured cleavage of its substrate LEHD-afc in several etoposide-treated melanoma lines (Figure 1c, left panel). This analysis indicated that there is no correlation between caspase-9 activity and Apaf-1 expression (Figure 1a,c). For instance, cell line 607B expresses little Apaf-1 and displayed substantial LEHD-peptide cleavage, while 634 showed clear Apaf-1 expression and no LEHD-peptide cleavage. Besides caspase-9, which is directly regulated by Apaf-1, we also analysed effector caspase activation (DEVDase activity) that is likely to be more relevant for cell death. Also, these assays revealed no correlation between the levels of Apaf-1 and the caspase activity induced by etoposide treatment (Figure 1c, right panel). These results suggest that even though Apaf-1 may be downregulated in some melanoma lines, functional implications for such an event are not clear-cut. An attractive hypothesis that explains these observations is that an alternative, apoptosome-independent mechanism of initiating caspase activation plays an important role in melanoma. In conclusion, our data confirm that Apaf-1 levels vary in melanoma, but suggest that loss of expression may be less frequent. In this light, it is interesting to note that the Apaf-1 locus has recently been reassessed and shown to be located 40.3 Mbp centromerically on chromosome 12q. Several of the samples that were considered to be Apaf-1 LOH by Soengas et al. can therefore no longer be regarded as such when adhering to the rules set by Fujimoto. Although the discrepancy between Soengas’ and our data can possibly be explained by the type of melanoma or the treatment regimen given to the patients prior to isolation, we believe it is crucial to realise that loss of Apaf-1 does not necessarily lead to attenuation of caspase activation or of death. It is therefore of importance to nuance the concept that Apaf-1 downregulation is the key event that determines the resistance of melanoma to current treatment modalities.

The TNF family member APRIL promotes colorectal tumorigenesis

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in ApcMin mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.

GRANZYME A AND B-CLUSTER DEFICIENCY DELAYS ACUTE LUNG INJURY IN PNEUMOVIRUS-INFECTED MICE

Lower respiratory tract infection by the human pneumovirus respiratory syncytial virus is a frequent cause of acute lung injury in children. Severe pneumovirus disease in humans is associated with activation of the granzyme pathway by effector lymphocytes, which may promote pathology by exaggerating proapoptotic caspase activity and proinflammatory activity. The main goal of this study was to determine whether granzymes contribute to the development of acute lung injury in pneumovirus-infected mice. Granzyme-expressing mice and granzyme A- and B-cluster single- and double-knockout mice were inoculated with the rodent pneumovirus pneumonia virus of mice strain J3666, and were studied for markers of lung inflammation and injury. Expression of granzyme A and B is detected in effector lymphocytes in mouse lungs in response to pneumovirus infection. Mice deficient for granzyme A and the granzyme B cluster have unchanged virus titers in the lungs but show a significantly delayed clinical response to fatal pneumovirus infection, a feature that is associated with delayed neutrophil recruitment, diminished activation of caspase-3, and reduced lung permeability. We conclude that granzyme A- and B-cluster deficiency delays the acute progression of pneumovirus disease by reducing alveolar injury.