Ulrike Naumann

APO2 ligand: a novel lethal weapon against malignant glioma?

APO2L (TRAIL) is a novel CD95L (Fas/APO‐1‐L) homologous cytotoxic cytokine that interacts with various receptors which transmit (DR4, DR5) or inhibit (DcR1, DcR2) an apoptotic signal. Here, we report that human glioma cell lines preferentially express mRNAs for agonistic death receptors DR4 (8/12) and DR5 (11/12) rather than the death‐inhibitory decoy receptors DcR1 (4/12) and DcR2 (2/12). Ten of 12 cell lines are susceptible to APO2L‐induced apoptosis. The resistant cell lines, U138MG and U373MG, are cross‐resistant to CD95L‐induced apoptosis. Similar to CD95L‐induced apoptosis, APO2L‐induced apoptosis is inhibited by ectopic expression of the caspase inhibitor, crm‐A, or of bcl‐2, or by coexposure to the corticosteroid, dexamethasone, or the lipoxygenase inhibitor, nordihydroguaretic acid. There is no correlation between p53 genetic status of the cell lines and their susceptibility to APO2L‐induced apoptosis, but the latter is moderately enhanced by ectopic expression of wild‐type p53. APO2L targeting may be a promising approach for selectively targeting apoptosis to human malignant glioma cells.

Monocyte chemoattractant protein–1 increases microglial infiltration and aggressiveness of gliomas

Macrophages are thought to represent a first line of defense in anti‐tumor immunity. Despite infiltration by microglial cells, however, malignant gliomas are still highly aggressive tumors. We here identify monocyte chemoattractant protein‐1 (MCP‐1) as a critical chemoattractant for glioma‐infiltrating microglial cells. MCP‐1–transfected rat CNS‐1 gliomas were massively infiltrated by microglial cells. Whereas MCP‐1 did not promote the growth of CNS‐1 cells in vitro, intracerebral CNS‐1–transfected tumors grew more aggressively than control‐transfected tumors. This provides the first functional evidence that MCP‐1 recruits microglial cells to gliomas and promotes their growth in vivo. Microglial cells may support rather than suppress glioma growth.

Transforming Growth Factor-β: A Molecular Target for the Future Therapy of Glioblastoma

The median survival of patients with glioblastoma treated by surgery, radiotherapy and chemotherapy is in the range of 12 months. These limits in the efficacy of current treatment modalities call for the development of novel therapeutic approaches targeting the specific biological features of this type of cancer. Glioblastomas are a rich source of immunosuppressive molecules which may interfere with immune recognition and rejection as well as clinical strategies of active immunotherapy. The most prominent glioblastoma-associated immunosuppressant is the cytokine, transforming growth factor (TGF)-beta, a multifunctional cytokine which not only interferes with multiple steps of afferent and efferent immune responses, but also stimulates migration, invasion and angiogenesis. The complex regulation of TGF-beta bioavailability includes its synthesis as a proprotein, proteolytic processing by furin-like proteases, assembly in a latent complex, and finally liberation from latency by multiple effector mechanisms, a process collectively referred to as activation. Several in vitro paradigms and rodent glioma models have been used to demonstrate that the antagonism of TGF-beta holds promise for the treatment of glioblastoma, employing antisense strategies, inhibition of pro-TGF-beta processing, scavenging TGF-beta by decorin, or blocking TGF-beta activity by specific TGF-beta receptor (TGF-betaR) I kinase antagonists. Moreover, the local application of TGF-beta(2) antisense oligonucleotides is currently evaluated in a randomized clinical trial for recurrent malignant glioma. In summary, we propose that TGF-beta-antagonistic treatment strategies are among the most promising of the current innovative approaches for glioblastoma, particularly in conjunction with novel approaches of cellular immunotherapy and vaccination.

Transforming growth factor-β and p-21: multiple molecular targets of decorin-mediated suppression of neoplastic growth

Decorin is a member of the small leucine-rich proteoglycan (SLRP) gene family that has recently become a focus in various areas of cancer research. The decorin protein consists of a core protein and a covalently linked glycosaminoglycan chain. Decorin binds to collagens type I, II and IV in vivo and promotes the formation of fibers with increased stability and changes in solubility. Further, the decorin core protein binds to growth factors, including transforming growth factor-β (TGF-β), to other intercellular matrix molecules such as fibronectin and thrombospondin, and to the decorin endocytosis receptor. Decorin may directly interfere with the cell cycle via the induction of p21WAF1/CIP1 (p21), a potent inhibitor of cyclin-dependent kinases (CDKs). Here, we discuss interactions of decorin with TGF-β and with p21, both of which are relevant to carcinogenesis and tumor progression. TGF-β is released by tumors of various histogenetic origins and promotes immunosuppression in the host and tumor immune escape by induction of growth arrest and apoptosis in immune cells, by downregulation of MHC II antigen expression and by changes in the cytokine release profiles of immune and tumor cells. Moreover, TGF-β may modulate tumor growth in an autocrine and paracrine fashion, may mediate drug resistance, and may facilitate tumor angiogenesis. Decorin binds to TGF-β, thus inhibiting its bioactivity, and is a direct or indirect negative modulator of TGF-β synthesis. Ectopic expression of decorin results in the regression of rat C6 gliomas, an antineoplastic effect attributed to the reversal of TGF-β-induced immunosuppression. On the other hand, de novo expression of decorin in colon cancer cells and some other tumor cells, even though not in glioma cells, results in an upregulation of p21 expression and a cell cycle arrest, presumably in a TGF-β-independent manner. Decorin expression is downregulated in many tumors but upregulated in the peritumoral stroma. By virtue of its growth regulatory and immunomodulatory properties, decorin promises to become a novel target for the experimental therapy of human cancers.

PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis.

The tumor suppressor gene PTEN (MMAC1, TEP1) encodes a dual-specificity phosphatase and is considered a progression-associated target of genetic alterations in human gliomas. Recently, it has been reported that the introduction of wild type PTEN into glioma cells containing endogenous mutant PTEN alleles (U87MG, LN-308), but not in those which retain wild-type PTEN (LN-18, LN-229), causes growth suppression and inhibits cellular migration, spreading and focal adhesion. Here, we show that PTEN gene transfer has no effect on the chemosensitivity of the four cell lines. Further, a correlational analysis of the endogenous PTEN status of 12 human glioma cell lines with their sensitivity to seven different cancer chemotherapy drugs reveals no link between PTEN and chemosensitivity. In contrast, ectopic expression of wild type PTEN, but not the PTENG129R mutant, in PTEN-mutant gliomas markedly sensitizes these cells to irradiation and to CD95-ligand (CD95L)-induced apoptosis. PTEN-mediated facilitation of CD95L-induced apoptosis is associated with enhanced CD95L-evoked caspase 3 activity. Protein kinase B (PKB/Akt), previously shown to inhibit CD95L-induced apoptosis in nonglial COS7 cells, is inactivated by dephosphorylation. Interestingly, both PTEN-mutant U87MG and PTEN-wild-type LN-229 cells contain phosphorylated PKB constitutively. Wild-type PTEN gene transfer promotes dephosphorylation of PKB specifically in U87MG cells but not in LN-229 cells. Sensitization of U87MG cells to CD95L-apoptosis by wild-type PTEN is blocked by insulin-like growth factor-1 (IGF-1). The protection by IGF-1 is inhibited by the phosphoinositide 3-OH (PI 3) kinase inhibitor, wortmannin. Although PKB is a down-stream target of PI 3 kinase, the protection by IGF-1 was not associated with the reconstitution of PKB phosphorylation. Thus, PTEN may sensitize human malignant glioma cells to CD95L-induced apoptosis in a PI 3 kinase-dependent manner that may not require PKB phosphorylation.

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.

CP-31398, a novel p53-stabilizing agent, induces p53-dependent and p53-independent glioma cell death

CP-31398 is a prototype small molecule that stabilizes the active conformation of p53 and promotes p53 activity in cancer cell lines with mutant or wild-type p53. Here, we report that CP-31398 induces p53 reporter gene activity and p21 expression in all of 11 glioma cell lines harboring wild-type or mutant p53, but not in p53-null LN-308 cells. Upon prolonged exposure to CP-31398, all glioma cell lines undergo caspase-independent and bcl-xL-insensitive cell death with EC50 concentrations of 10–36 μM. By comparing p53 wild-type U87MG and p53-null LN-308 cells expressing the temperature-sensitive p53V135A mutant, we delineate two pathways of CP-31398-induced cell death: an early, p53-dependent pathway that requires (new p53) protein synthesis and a late, p53-independent pathway characterized by aurintricarboxylic acid -sensitive calcium release and epiphenomenal free radical formation. Post-transcriptional repression of p53 synthesis by an intracellularly transcribed short interfering RNA confirmed the presence of these two pathways of cell death. These observations point out some of the liabilities of CP-31398 as a prototype p53-based therapeutic and define a rationale for further refinement of small molecules that specifically target the p53 pathway, but lack the p53-independent effects.

Decorin gene transfer-mediated suppression of TGF-β synthesis abrogates experimental malignant glioma growth in vivo

Cytokines such as transforming growth factor-β (TGF-β) are thought to mediate escape from immune surveillance in human malignant glioma. Here, we report that ectopic expression of the small TGF-β-binding proteoglycan, decorin, inhibits not only TGF-β bioactivity but also TGF-β1 and TGF-β2 mRNA transcription and TGF-β protein synthesis by human LN-18, LN-229, T98G and rat C6 glioma cells in vitro. Ectopic expression of decorin in C6 rat glioma cells results in strong inhibition of tumor formation in vivo. Decorin-expressing C6 gliomas grow initially but regress to very small residual tumors at 12 weeks after implantation whereas all control animals die or have to be killed within 4 weeks. Decorin-expressing tumors show a four-fold increase of infiltration by activated T cells and a 1.6-fold increase in total B and T cells. Chronic steroid-mediated immunosuppression abrogates the inhibitory effects of decorin gene transfer. We conclude that decorin-induced inhibition of TGF-β release by glioma cells significantly enhances antiglioma immune responses in vivo. Clinical evaluation of decorin gene therapy for human malignant gliomas may be warranted.

Processing of Immunosuppressive Pro-TGF-β1,2 by Human Glioblastoma Cells Involves Cytoplasmic and Secreted Furin-Like Proteases

TGF-β is a putative mediator of immunosuppression associated with malignant glioma and other types of cancer. Subtilisin-like proprotein convertases such as furin are thought to mediate TGF-β processing. Here we report that human malignant glioma cell lines express furin mRNA and protein, exhibit furin-like protease (FLP) activity, and release active furin into the cell culture supernatant. FLP activity is not modulated by exogenous TGF-β or neutralizing TGF-β Abs. Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the TGF-β1 and TGF-β2 precursor molecules and, consequently, the release of mature bioactive TGF-β molecules. Ectopic expression of PDX, a synthetic antitrypsin analog with antifurin activity, in the glioma cells inhibits FLP activity, TGF-β processing, and TGF-β release. Thus, subtilisin-like proprotein convertases may represent a novel target for the immunotherapy of malignant glioma and other cancers or pathological conditions characterized by enhanced TGF-β bioactivity.

The histone acetyltransferase p300 promotes intrinsic axonal regeneration

Axonal regeneration and related functional recovery following axonal injury in the adult central nervous system are extremely limited, due to a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. As opposed to what occurs during nervous system development, a weak proregenerative gene expression programme contributes to the limited intrinsic capacity of adult injured central nervous system axons to regenerate. Here we show, in an optic nerve crush model of axonal injury, that adenoviral (cytomegalovirus promoter) overexpression of the acetyltransferase p300, which is regulated during retinal ganglion cell maturation and repressed in the adult, can promote axonal regeneration of the optic nerve beyond 0.5 mm. p300 acetylates histone H3 and the proregenerative transcription factors p53 and CCAAT-enhancer binding proteins in retinal ganglia cells. In addition, it directly occupies and acetylates the promoters of the growth-associated protein-43, coronin 1 b and Sprr1a and drives the gene expression programme of several regeneration-associated genes. On the contrary, overall increase in cellular acetylation using the histone deacetylase inhibitor trichostatin A, enhances retinal ganglion cell survival but not axonal regeneration after optic nerve crush. Therefore, p300 targets both the epigenome and transcription to unlock a post-injury silent gene expression programme that would support axonal regeneration.