Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?

Astrocytomas are among the most common brain tumors that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by astrocytoma-derived cytokines. Here, we propose that cell contact-mediated events also play a role, since we demonstrate the in vivo expression of Fas ligand (FasL/CD95L) by human astrocytoma and the efficient killing of Fas-bearing cells by astrocytoma lines in vitro and by tumor cells ex vivo. Functional FasL is expressed by human, mouse, and rat astrocytoma and hence may be a general feature of this nonlymphoid tumor. In the brain, astrocytoma cells can potentially deliver a death signal to Fas+ cells which include infiltrating leukocytes and, paradoxically, astrocytoma cells themselves. The expression of FasL by astrocytoma cells may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also show FasL expression by neurons. Overall, our findings suggest that FasL-induced apoptosis by astrocytoma cells may play a significant role in both immunosuppression and the regulation of tumor growth within the central nervous system.

Tumor expression of Fas ligand (CD95L) and the consequences.

Tumors of diverse origin have recently been shown to express CD95 ligand and to induce apoptosis in CD95-expressing targets in vitro; however, in vivo, enhanced tumor growth and rejection have both been observed as a consequence of either immunosuppressive or proinflammatory functions of CD95 ligand. The final in vivo outcome of CD95 ligand expression will depend upon a complex balance of interactions relevant for each tumor in its particular microenvironment.

Paper alert. Immunology.

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in immunology.

Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.

Identification of a multiprotein complex interacting with the c-fos serum response element.

The serum response element (SRE) is essential for serum and growth factor stimulation of the c-fos gene. We have examined the nuclear proteins, obtained from tissues with elevated expression of the c-fos gene (proliferating rat liver and hepatocarcinoma), that bind to the SRE sequence. A synthetic oligonucleotide containing the SRE sequence from the mouse c-fos gene promoter (-299 to -322) was radioactively labeled, used as a probe for the mobility shift assay and Southwestern (DNA-protein) blotting, and also used for sequence-specific affinity chromatography. We have identified a group of nuclear proteins of molecular sizes 36, 45, 62, 67, 72, and 112 kDa capable of interacting with the SRE sequence. The 36-, 67-, and 112-kDa proteins have DNA-binding properties, but the presence of the others in the SRE-protein complex could be the result of protein-protein interaction. All of these protein factors were present in nuclei obtained from intact and proliferating rat liver as well as from 5123tc Morris hepatoma. The DNA-binding activity (on Southwestern blots) of the 67- and 112-kDa proteins was not affected by alkaline phosphatase treatment, but the ability of the dephosphorylated nuclear proteins to form the complex with the SRE sequence under gel shift assay conditions was severely impaired. The same alkaline phosphatase treatment completely abolished the DNA-binding properties of the c-fos cyclic AMP-responsive element-specific proteins. Therefore, transcriptional activation of the c-fos gene at the SRE must require the presence of a multiprotein complex the formation of which is governed by phosphorylation. The binding of the 67- and 62-kDa proteins to the c-fos SRE has been previously reported; however, the 36-. 45-, 72-, and 112-kDa proteins are novel factors involved in the multifaceted regulation of c-fos gene expression in vivo.

Flecainide cardiotoxicity precipitated by electrolyte imbalance. Caution with thiazide diuretics.

A man presented with recurrent syncope, weakness and fatigue. His ECG showed marked QRS widening and he had gross hyponatraemia and hypokalaemia. His medications included bendroflumethiazide (long term) and flecainide (started 2 months previously). This presentation was consistent with flacainide cardiotoxicity exacerbated by electrolyte disturbance. The syncopal episodes probably represented life-threatening arrhythmias. The ECG and symptoms resolved completely once the electrolytes were corrected. Increased cardiotoxicity with hypokalaemia is documented, but not widely recognised. Hyponatraemia-induced flecainide cardiotoxicity has not been documented. The clinical effects of flecainide are due to use-dependent block of sodium channels. There are reports that support the use of hypertonic sodium salts to reverse flecainide toxicity via antagonism at the receptor. By this rationale, hyponatraemia would lead to Flecainide toxicity. Flecainide has been shown to reduce salt absorption in animal bowel. It is possible that in combination with bendroflumethiazide it acted synergistically to produce profound electrolyte disturbance. Flecainide cardiotoxicity has a significant mortality and can present non-specifically. Thus, early recognition is essential. This case demonstrates the importance of strict electrolyte control in patients who are on flecainide. We would discourage concomitant use of flecainide and bendroflumethiazide.

S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors.

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Changes in cyclic adenosine monophosphate-responsive element binding proteins in rat hepatomas.

We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) binding (CREB) proteins in rapidly growing, chemically induced 5123tc and 5123D Morris hepatomas. Using the CRE sequences from the c-fos, E2A, and somatostatin gene promoters, we identified in the nuclear proteins from normal unstimulated or proliferating rat liver cells six different protein factors of Mr 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of binding to the element. The Mr 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene expression. We could not find the Mr 47,000 CREB protein in the 5123tc and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering the protocol for extracting nuclear proteins, or (c) attempting to restore its DNA-binding property by phosphorylation [with endogenous protein kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and protein kinase C/dephosphorylation (with alkaline phosphatase)] were unsuccessful. The loss of tje Mr 47,000 CREB protein from solid tumors of the Morris hepatoma is likely to be related to the neoplastic properties of the tumor cell rather than to cell growth because the level of this protein remained unchanged during a 6-day period of liver regeneration. The nuclear extract from the Morris hepatoma that did not have the Mr 47,000 CRE-binding factor contained proteins immunologically related to the CREB, c-Jun, and c-Fos proteins. We conclude that the Mr 47,000 factor represents a distinct member of the CRE-binding protein family and that its absence from the hepatomas may lead to aberrant expression of cAMP-inducible genes.

Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes.

The effects of topoisomerase II-reactive epipodophyllotoxins etoposide and teniposide as well as amsacrine on the viability of thymocytes in primary culture has been examined. All three drugs were shown to produce DNA cleavage detectable by resolving isolated DNA by pulsed field agarose gel electrophoresis. The DNA cleavage was found to have two components. The first was due to the interaction of the drugs with topoisomerase II, whereas the second component was due to endonuclease cleavage caused by the drug-induced entry of the thymocytes into programmed cell death or apoptosis. This second component of the DNA cleavage was also detected in thymocytes undergoing apoptosis following exposure to the glucocorticoid analogue, dexamethasone. The effect of the drugs on programmed cell death is dependent upon new protein and RNA synthesis, indicating that topoisomerase II has a role in the very first stages of the process. These results are discussed in terms of the use of this class of topoisomerase II-reactive drugs in chemotherapy.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells.

Rats trained to the "8 + 16" controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from liver containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase a. Attempts of influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.

Degradation of chromatin in apoptotic cells.

We present here a model for the degradation of chromatin in cells undergoing apoptosis. This model rationalises all aspects of the fragmentation process that have been described to date, explaining not only the patterns of degradation seen within individual cells, but also the variability in extent of degradation seen in different cells. Although DNA fragmentation in apoptosis was initially considered to be solely internucleosomal, it is now apparent that the process is much more complex and most, if not all, cells also produce much larger DNA fragments. However, in the same way that internucleosomal DNA fragmentation is a reflection of chromatin structure, the generation of these larger fragments is a reflection of chromatin structure, too. By comparing the ionic requirements for the complete pattern of chromatin degradation in nuclei with those required for apoptosis, it is apparent that the whole process may be catalysed by two pools of Mg-activated\Ca-modulated DNase I-like enzyme activities.

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks.

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.

Induction of apoptosis in neoplastic cells by depletion of vitamin B12.

Methionine synthase, a critical enzyme in deoxyribonucleotide biosynthesis for DNA replication, requires vitamin B12 as a cofactor. We have tested the hypothesis that depletion of cells of vitamin B12 would block growth of neoplastic cells and divert them into apoptosis and could form the basis of a new therapeutic strategy for cancer treatment. Using nitrous oxide to inactivate vitamin B12 we show that, in a variety of cell lines in vitro, methionine synthase is rapidly inhibited, the cells cease proliferation and undergo apoptosis. The kinetics of cell death, once started, are similar to those observed following methotrexate treatment or serum withdrawal. This is the first observation of apoptosis being induced following depletion of an essential metabolite as opposed to the more conventional strategy of adding a toxic drug to damage cells thereby triggering apoptosis. Moreover, vitamin B12 depletion has no effect on the nonproliferating cell population.

Regulation of DNaseY activity by actinin-alpha4 during apoptosis.

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR.

Apoptotic DNA degradation could be initiated by the accumulation of single-strand (ss) breaks in vulnerable chromatin regions, such as base unpairing regions (BURs), which might be preferentially targeted for degradation by both proteases and nucleases. We tested this hypothesis in anti-Fas-treated apoptotic Jurkat cells. Several nuclear proteins known for their association with both MARs and the nuclear matrix, that is, PARP, NuMA, lamin B and SATB1, were degraded, but the morphological rearrangement of the BUR-binding SATB1 protein was one of the earliest detected changes. Subsequently, we have identified several genes containing sequences homologous to the 25 bp BUR element of the IgH gene, a known SATB1-binding site, and examined the integrity of genomic DNA in their vicinity. Multiple ss breaks were found in close proximity to these sites relative to adjacent regions of DNA. Consistent with our prediction, the results indicated that the initiation of DNA cleavage in anti-Fas-treated Jurkat cells occurred within the BUR sites, which likely became accessible to endonucleases due to the degradation of BUR-binding proteins.

A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution.

The development of T cell effector and memory responses against foreign antigens (Ags) involves the activation, differentiation and proliferation of naive T cells expressing distinct Ag-specific TCRs. Understanding the complexity of Ag-selected TCR repertoires in individual responders in terms of the sequences selected and their relative frequencies may provide indications about how a repertoire is established and suggest ways to influence the outcome of an immune response. Most methods of repertoire analysis are unsuitable for calculating the relative in vivo frequencies of Ag-specific clones (expressing distinct TCRs) selected during an immune response, whereas sequence data obtained by single-cell PCR analysis directly reflect cell frequencies if a sufficiently large number of cells is sampled. Using a CD8 T cell response in normal mice in which Ag-selected cells are identified by cell surface phenotype and rearranged TCRBV sequences are determined by PCR amplification of genomic DNA directly from single cells, we have analyzed a large number (>200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice. We found that each responder selects a unique Ag-specific TCR repertoire in which the various TCRBV sequences are present in a wide range of frequencies. However, the overall distribution of sequences is quite similar for different responder animals. Moreover, an individual's selected TCR repertoire is uniformly represented among Ag-specific CD8 cells circulating in the blood or localized in the spleen or liver. Relatively few sequences make up the bulk of the repertoire and account for the oligoclonality observed in earlier studies. We discuss various models that could account for this skewed distribution of an Ag-selected TCR repertoire.

Detection of the initial stages of DNA fragmentation in apoptosis.

DNA fragmentation during apoptosis proceeds through an ordered series of stages commencing with the production of DNA fragments of 300 kbp, which are then degraded to fragments of 50 kbp. The 50-kbp fragments are further degraded, in some but not all cells, to smaller fragments (10-40 kbp) and release the small oligonucleosome fragments that are recognized as the characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of the initial stages of DNA fragmentation using pulsed-field gel electrophoresis or a combination of pulsed-field gel electrophoresis and conventional agarose gel electrophoresis that allows detection of the DNA ladder in the same sample. A new method for the detection of high molecular weight DNA fragments on conventional agarose gels is presented, together with a rationale for the analysis of DNA fragmentation during apoptosis.

Variation in expression of mouse erythrocyte receptors on Epstein-Barr virus-induced B-cell lines.

To explain the variation in the percentage of mouse erythrocyte rosette-forming cells (MERFC) during culture of Epstein-Barr virus (EBV)-induced B-cell lines, we provide evidence that (i) there is an altered expression of mouse red blood cell (MRBC) receptors on cell line cells during the mitotic cycle, and (ii) putative receptor-negative cells are capable of de novo synthesis of the receptor, and passively adsorbing receptor shed from receptor-positive cells.

Effects of neoplastic transformation and teniposide (VM26) on protein kinase C isoform expression in rodent fibroblasts.

This study examined changes in protein kinase C (PKC) isoforms in rodent fibroblasts (rat F111 and mouse NIH3T3), transformed by the polyoma virus middle T antigen (mT) and undergoing apoptosis in response to teniposide (VM26). The mT-transformed cells up-regulated PKC delta and down-regulated both PKC epsilon and PKC lambda expression, and were more sensitive to the drug than their non-transformed counterparts. The drug treatment further lowered the expression of PKC epsilon, triggered nuclear translocation of PKC delta and its site-specific proteolysis, consistent with the notion that changes in specific PKC isoforms play a role not only in the neoplastic transformation of fibroblasts, but also in their apoptotic response.