Cornelia Grimmel

PREDICTING CHEMORESISTANCE IN HUMAN MALIGNANT GLIOMA CELLS: THE ROLE OF MOLECULAR GENETIC ANALYSES

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we asked whether alterations in the p53 and RB pathways and the expression of six BCL‐2 family proteins predicted acute cytotoxicity and clonogenic cell death induced by BCNU, vincristine, cytarabine, teniposide, doxorubicin, camptothecin or β‐lapachone in 12 human malignant glioma cell lines. Neither wild‐type p53 status, nor p53 protein accumulation, nor p21 or MDM‐2 levels, nor differential expression of BCL‐2 family proteins predicted drug sensitivity, except for an association of BAX with higher β‐lapachone sensitivity in acute cytotoxicity assays. p16 protein expression was associated with high doubling time and chemoresistance. We conclude that some important molecular changes, which are involved in the development of gliomas and attributed a role in regulating vulnerability to apoptosis, may not determine the response to chemotherapy in these tumors. Int. J. Cancer (Pred. Oncol.) 79:640–644, 1998.

Secreted Frizzled-related proteins inhibit motility and promote growth of human malignant glioma cells

Cellular resistance to multiple proapoptotic stimuli and invasion of surrounding brain tissue by migrating tumor cells are main obstacles to an effective therapy for human malignant glioma. Here, we report that the Wnt family of embryonic differentiation genes modulate growth of malignant glioma cells in vitro and in vivo and inhibit cellular migration in vitro. sFRPs (soluble Frizzled-related proteins) are soluble proteins that bind to Wnt and interfere with Wnt signaling. We find that sFRP-1 and sFRP-2 are produced by the majority of longterm and ex vivo malignant glioma cell lines. Glioma cells that ectopically express sFRPs exhibit increased clonogenicity and enhanced resistance to serum starvation. In contrast, sFRPs do not modulate glioma cell susceptibility to apoptosis induced by the cytotoxic cytokines, CD95 (Fas/APO-1) ligand (CD95L) or Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL), or various cytotoxic drugs. sFRP-2 strongly promotes the growth of intracranial glioma xenografts in nude mice. In contrast, enhanced expression of sFRPs inhibits the motility of glioma cells in vitro. sFRP-mediated effects on glioma cells are accompanied by decreased expression and activity of matrix metalloproteinase-2 (MMP-2) and decreased tyrosine phosphorylation of β-catenin. Thus, sFRPs promote survival under non-supportive conditions and inhibit the migration of glioma cells. We suggest that the regulation of these cellular processes involves expression of MMP-2 and tyrosine phosphorylation of β-catenin. These data support a function for Wnt signaling and its modulation by sFRPs in the biology of human gliomas.

Taxol-mediated augmentation of CD95 ligand-induced apoptosis of human malignant glioma cells: association with bcl-2 phosphorylation but neither activation of p53 nor G2/M cell cycle arrest.

The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.

Hypericin-induced apoptosis of human malignant glioma cells is light-dependent, independent of bcl-2 expression, and does not require wild-type p53

Hypericin and tamoxifen are experimental agents for the adjuvant chemotherapy of malignant glioma. We report that hypericin and tamoxifen induce apoptosis of 7 human malignant glioma cell lines in a concentration- and time-dependent manner. Illumination is essential for the cytotoxicity of hypericin but not tamoxifen. Apoptosis is unaffected by inhibitors of RNA and protein synthesis or free radical scavengers, does not require wild-type p53 activity, and occurs in glioma cells expressing high levels of bcl-2. There is no correlation between hypericin and tamoxifen-induced cytotoxicity and inhibition of protein kinase C (PKC). Ectopic expression of a murine bcl-2 transgene provides modest protection from tamoxifen but does not affect hypericin toxicity. Hypericin and tamoxifen do not modulate glioma cell killing induced by tumor necrosis factor-alpha (TNF-alpha) or CD95 ligand. Both drugs augment the acute cytotoxicity of various cancer chemotherapy drugs but fail to shift their EC50 values in modified colony formation assays. These data do not provide further supportive evidence how to enhance the limited efficacy of tamoxifen treatment for human malignant glioma. However, hypericin is a promising agent for the treatment of malignant glioma if local photodynamic activation of hypericin in the glioma tissue can be achieved.

Bag-1 and Bcl-2 Gene Transfer in Malignant Glioma: Modulation of Cell Cycle Regulation and Apoptosis

Bag‐1 is a heat shock 70 kDa (Hsp70)‐binding protein that can collaborate with Bcl‐2 in suppressing apoptosis under some conditions. Here, we report that 11 of 12 human glioma cell lines express Bag‐1 protein in vitro. Moreover, 15 of 19 human glioblastomas expressed Bag‐1 as assessed by immunohistochemistry in primary tumor specimens. To examine the biological effects of Bag‐1 in glioma cells, we expressed Bag‐1 or Bcl‐2 transgenes in 2 human malignant glioma cell lines, LN‐18 and LN‐229. Bag‐1 significantly slowed glioma cell growth and reduced clonogenicity of both cell lines in vitro. Coexpressed Bcl‐2 abrogated these effects of Bag‐1. Intracranial LN‐229 glioma xenografts implanted into nude mice revealed a substantial growth advantage afforded by Bcl‐2. Bag‐1 had no such effect, either in the absence or presence of Bcl‐2. Upon serum starvation in vitro, Bcl‐2 prevented cell death whereas Bag‐1 did not. Both Bcl‐2 and Bag‐1 slowed proliferation of serum‐starved cells when expressed alone. Importantly, coexpression of Bcl‐2 and Bag‐1 provided a distinct growth advantage under conditions of serum starvation that is probably the result of (i) the death‐preventing activity of Bcl‐2 and (ii) the property of Bag‐1 to overcome a Bcl‐2‐mediated enhancement of exit from the cell cycle. In contrast to these Bcl‐2/Bag‐1 interactions observed under serum starvation conditions, Bag‐1 did not further enhance the strong protection from staurosporine‐, CD95 (Fas/Apo1) ligand‐, Apo2 ligand (TRAIL)‐ or chemotherapeutic drug‐induced apoptosis afforded by Bcl‐2. Taken together, these results indicate a role for Bag‐1/Bcl‐2 interactions in providing a survival advantage to cancer cells in a deprived microenvironment that may be characteristic of ischemic/hypoxic tumors such as human glioblastoma multiforme, and suggest that Bcl‐2/Bag‐1 interactions also modulate cell proliferation.

TGF‐β‐independent induction of immunogenicity by decorin gene transfer in human malignant glioma cells

Ectopic expression of the proteoglycan, decorin, abrogates the growth of experimental C6 gliomas in the rat. Since gliomas release large amounts of transforming growth factor‐β (TGF‐β) and since decorin is a TGF‐β antagonist, decorin gene transfer‐mediated abrogation of glioma growth in vivo may involve enhanced immunogenicity of the tumor cells. Here, we report that human glioma cells stimulate alloreactive immune responses when engineered to express decorin whereas parental glioma cells are non‐immunogenic in vitro. The alloreactive immune response is mediated by CD8+ and CD4+ T cells as well as by NK cells. The immunosuppression exerted by parental or mock‐transfected glioma cells is mediated by soluble factors and can in part be mimicked by exogenous TGF‐β. However, neutralizing anti‐TGF‐β antibodies do not reverse glioma‐mediated immunosuppression, suggesting that decorin abrogates glioma‐induced immune cell inhibition by interfering with the activity of other, so far unidentified glioma‐secreted mediators. We conclude that enhanced immunogenicity may mediate the antineoplastic effects of decorin gene therapy for malignant glioma but that factors other than TGF‐β may be responsible for glioma‐induced immunosuppression.

Para‐phenylenediamine‐specific lymphocyte activation test: a sensitive in vitro assay to detect para‐phenylenediamine sensitization in patients with severe allergic reactions

Please cite this paper as: Para‐phenylenediamine‐specific lymphocyte activation test: a sensitive in vitro assay to detect para‐phenylenediamine sensitization in patients with severe allergic reactions. Experimental Dermatology 2010; 19: 435–441.

Different Effects of Ranibizumab and Bevacizumab on Platelet Activation Profile

Purpose: The aim of the study was to evaluate the potential influence of ranibizumab and bevacizumab on platelet activation and aggregation, which are critical processes in the pathogenesis of arterial thromboembolic events (ATEs). Methods: For the assessment of platelet function, flow cytometry and aggregometry were employed. Platelets were isolated from healthy volunteers and exposed to ranibizumab (1 mg/ml and 150 ng/ml) and bevacizumab (2.5 mg/ml and 3 μg/ml) or their solvents for 10 and 30 min prior to the addition of TRAP (25 μM), PAR-4-AP (25 μM) or thrombin (0.02 U/ml). The surface expression of activated GP IIb/IIIa, P-selectin (CD62P) and platelet-bound stromal cell-derived factor-1 (SDF-1) was measured on resting (nonactivated) and activated platelets by flow cytometry. The platelet aggregation capacity was examined using light transmission aggregometry. Results: The expression of surface activation markers did not differ significantly between nonstimulated and TRAP-, PAR-4-AP- or thrombin-activated platelets after incubating with ranibizumab. However, GP IIb/IIIa, CD62P and SDF-1 were significantly downregulated in PAR-4-AP- and thrombin-activated platelets after exposure to bevacizumab 2.5 mg/ml. In addition, ranibizumab- and bevacizumab-FITC were significantly increased in all activated platelets. No significant differences were observed in the aggregation of activated platelets after incubation with ranibizumab or bevacizumab. Conclusion: All ranibizumab concentrations as well as the bevacizumab concentration of 3 μg/ml had no influence on platelet activation and aggregation. Therefore, this in vitro study did not show any relationship between the exposition of activated platelets to ranibizumab or bevacizumab and the development of ATEs. However, the highest level of bevacizumab interfered with platelet activation, leading to downregulation of platelet activation markers. This observation might explain why the systemic treatment with high-dose bevacizumab could be associated with an increased risk of bleeding. Regarding the use of lower intravitreal dosages, further research should focus on the complex interactions between platelets and other cells, such as endothelial cells, which might stronger relate to a potentially increased risk of ATEs and depend on systemic vascular endothelial growth factor levels. Facing the different activation profiles, the diverse effects of the drugs on the cellular level have to be critically scrutinized for their clinical relevance.

The clinical relevance of birch pollen profilin cross-reactivity in sensitized patients

Overlapping seasons and cross‐reactivity, especially to grass pollen profilin, can hamper the diagnosis of birch pollen allergy. To identify the primary sensitizing allergen and the clinical relevance of cross‐sensitization, we correlated sensitization profiles with in vitro and in vivo tests, symptom scores, and pollen counts.

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo. Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties. Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149–60. ©2018 AACR.