[Cystic diseases in urology : Recommendations for patients with systemic mast cell disease]. / Zystische Erkrankungen in der Urologie : Handlungsempfehlungen für Patienten mit systemischer Mastzellerkrankung.

The systemic mast cell disease (MCAD; prevalence 17%) may occur frequently in urological patients. MCAD-induced changes include cysts in all organs, also in the urogenital system. In the presence of MCAD, the surgical removal of such cysts must consider specific features of the MCAD in order to reduce surgical and complication risks. Vice versa, if in urological examinations multiple cysts are found, this could be an indication of a possibly existing, in some circumstances, unrecognized MCAD.

CYP3A4, CYP2C9 and CYP2B6 expression and ifosfamide turnover in breast cancer tissue microsomes.

Ifosfamide is a prodrug that requires bioactivation by cytochrome P450 for antitumour activity. Up to now, little is known, to what extent in addition to the liver the ifosfamide metabolism may occur intratumorally. For this purpose, we investigated the expression of CYP3A4, CYP2C9 and CYP2B6 in breast cancer tissue using Western Blotting. Ifosfamide turnover was determined by detection of metabolites of the ifosfamide 4-hydroxylation and N-dechloroethylation in tumour microsomal incubations using HPLC/UV and LC/MS. The results demonstrate that all mammary tumours (n=11) reveal CYP3A4 expression; contents varied from 0.5 to 63 pmol mg(protein)(-1). CYP2C9 (n=9) was present in all tested breast tumour samples, too, while CYP2B6 (n=10) protein could not be detected. All measured breast cancer microsomes (n=4) showed an ifosfamide N-dechloroethylation capacity in the range from 0.04 to 0.21 pmol min(-1) mg(protein)(-1), while metabolites of the 4-hydroxylation could not be determined. In conclusion, the detected presence of CYP3A4 and CYP2C9 in breast tumours offers the possibility of intratumoral turnover of ifosfamide. For the first time in the literature, we could demonstrate a turnover of ifosfamide by microsomal preparations from human breast cancer tissue. A calculated modulation of intratumoral ifosfamide turnover could considerably influence its therapeutic efficiency.

CYP2C and IL-6 expression in breast cancer.

Besides hepatic P450 (cytochrome P450) metabolism, there is increasing interest in the possibility of intratumoral activation of oxazaphosphorines by P450. Therefore, we investigated the expression of P450 (CYP2C8, CYP2C9, CYP2C18, and CYP2C19) by RT (reverse transcriptase)-polymerase chain reaction (PCR) and of CYP2C9 by Western blotting in 10 different breast tumor samples. Since P450 may be down regulated by interleukin (IL) IL-6, the receptor (R) for IL-6 was analyzed by RT-PCR and IL-6 in supernatants was calculated from ELISA data. None of the breast tumors was positive for CYP2C18 and CYP2C19 mRNA, whereas CYP2C8 and CYP2C9 were detected in all 10 breast tumors. Correspondingly, all breast tumors tested (9 of 10) revealed low, but nevertheless positive, staining of the CYP2C9 protein. All 10 samples were positive for the IL-6 receptor mRNA. ELISA measurement of IL-6 cytokine in supernatants revealed that all measured samples (8 of 10) were producing IL-6, the amounts ranging from 0.004 to 3.1 ng/g(tumor tissue). In summary, we have demonstrated that tumors of the breast express two out of four members of the CYP2C family, indicating that activation of such prodrugs as oxazaphosphorines may take place intratumorally. The presence of the IL-6 receptor and of IL-6 cytokine, which is produced in an autocrine manner, opens up the possibility that the well-known down regulating effect of IL-6 also takes place in breast tumors and might explain the weak or even absent expression of different CYP2C members.

NGF increases cell survival rates under serum deprived conditions.

The known effects of nerve growth factor (NGF) are induction of differentiation and promotion of survival. We analysed the effects of exogenously added NGF on rat C6 and 9L glioma cells and the rat pheochromocytoma cell line PC12. Cells were seeded into 96-well plates and exposed to different concentrations of FCS (10%, 5%, 1%, 0.5% and no FCS) supplemented with or without 50 ng/ml NGF for up to 120 hours. Cell survival was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-assay. In this study we could clearly show two different effects: (1) proliferation was not influenced by NGF under high, medium or low serum and (2) survival rates increased under dramatic or complete serum deprivation, indicating that NGF acts as survival factor against cell death or cell cytostasis.

Time-dependent inhibition of glioblastoma cell proliferation by dexamethasone.

Because of the outstanding importance of the glucocorticoid Dexamethasone (DEX) as supportive therapy in the management of brain tumours, the direct effect of DEX on tumour cell proliferation is of particular interest. Previous in vitro studies led to contradictory results. To characterise more precisely the influence of DEX, we investigated the glioblastoma multiforme (GM) cell lines A172, T98G and 86HG39. Cells were treated with DEX concentrations ranging from 5 x 10(-9) to 5 x 10(-5) M from 24 to 240h under different treatment conditions. Influence of DEX on glioma cell viability was assessed daily for 5 days by MTT-assay: (I) with continuous DEX incubation (acute treatment), (II) in a recultivation period without DEX after 5 days of DEX pre-incubation (pre-treatment), (III) with continuous DEX incubation after 5 days of DEX pre-incubation (combination treatment). DEX acute treatment led to strongly decreased proliferation of A172 cells, whereas T98G and 86HG39 cells remained uninfluenced. In opposite, a time-delayed inhibition of cell proliferation was observed in all three cell lines after DEX pre-treatment. Combination treatment induced a significant increase of the inhibitory effect in A172 and T98G cells. These data show a variable, partial time-dependent inhibitory effect of DEX on the proliferation of GM cells and may open new treatment strategies for malignant brain tumours.

Different effects of valproic acid on proliferation and migration of malignant glioma cells in vitro.

Valproic acid (VPA) has been considered as a possible treatment agent for malignant gliomas. In order to characterise the possibilities of VPA, we investigated the effects on cell migration and proliferation. Human cell lines T98G, A172, 85HG66 and 86HG39 were treated with VPA or left untreated, afterwards Boyden chamber assay was used for measuring vertical migration. In a second assay cells were stimulated to create spheroids and spheroid migration was measured. Proliferation was assessed using a cell counter. VPA decreased proliferation of 86HG39 > A172 > 85HG66 cells, whereas T98G remained uninfluenced. The influence of VPA on migration was different; whereas VPA dose-dependently stimulated migration of 86HG39 cells, migration of T98G and 85HG66 decreased, whereas A172 cells remained uninfluenced. Only 86HG39 and A172 cells created spheroids. In both cell lines Boyden-chamber-findings were confirmed by analysing the influence of VPA on spheroid migration. These non-uniform data demonstrate that the benefit of VPA in glioma treatment is not clear and needs further investigation.

Interferon-gamma inhibits growth and migration of A172 human glioblastoma cells.

Malignant gliomas are highly proliferative and invasive tumors with poor prognosis. We investigated the influence of Interferon-gamma (IFN-gamma) on the human malignant glioma cell line A172, measuring cell viability (MTT-test), proliferation (3H-thymidine-uptake), cell death (FACS) adhesion to hyaluronic acid (HA, adhesion-assay) and migration (Boyden-chamber). IFN-gamma significantly decreased cell viability and proliferation. Measured by FACS, an up-regulation of CD95 expression has been shown in combination with an increased rate of cell death, first seen after 96 hours IFN-gamma treatment. Adhesion to HA was decreased after pre-treatment with IFN-gamma. This was not mediated by down-regulation of the main HA-receptor CD44, since IFN-gamma did not change CD44 expression. IFN-gamma-treated cells showed a significantly diminished migration rate through a native or HA-coated 8-microm polycarbonate membrane. To summarise, IFN-gamma influences both the main characteristics of malignancy: it decreases cell proliferation and induces cell death, further it diminishes migration of A172 human glioblastoma cells.

IL-1 receptor type I expression in breast cancer.

The cytokine interleukin (IL)-1 is known to be involved in angiogenesis and metastasis. A prerequisite for IL-1 signalling is the presence of its receptor. Previously we have shown that glioblastoma cells express the IL-1 receptor type I (IL-1RI). In this study we analysed 11 breast tumour specimens for IL-1RI expression using the reverse transcriptase (RT) polymerase chain reaction (PCR). We found all the 11 breast tumours were positive for IL-1RI. This suggests that paracrine or autocrine produced IL-1 mediated signalling via IL-IRI might take place in breast tumours to control the production of pro-tumourigenic factors such as angiogenic factors and support further progression of tumour growth and metastasis.

Interferon gamma inhibits proliferation and hyaluronic acid adhesion of human malignant glioma cells in vitro.

Malignant gliomas are frequent and the prognosis is poor. The cytokine interferon gamma (IFN-gamma) enhances several immune phenomena and may be used in immunotherapy of tumours. Therefore we investigated the influence of IFN-gamma on human cell lines T98G, U87MG, 86HG39 and 85HG66, measuring cell viability (MTT-test) and proliferation (3H-thymidine uptake). IFN-gamma markedly decreased viability and proliferation of all investigated cell lines. Expression of CD44 and adhesion to hyaluronic acid (HA) are involved in glioma invasion. Influence of IFN-gamma on these two features has also been investigated. IFN-gamma markedly decreased HA-adhesion in all three investigated cell lines, whereas CD44 expression remained uninfluenced. To summarise, IFN-gamma strongly decreased cell growth and HA-adhesion of malignant glioma cell lines in vitro. We suggest further investigations to characterise better the role of IFN-gamma as a treatment opportunity for malignant gliomas.

Interferon-gamma increases IL-6 production in human glioblastoma cell lines.

BACKGROUND: Various immunotherapeutical approaches are presently evaluated for their efficacy to eradicate glioma cells. Complicating the concepts, these tumors secrete cytokines which modulate the immune response. MATERIALS & METHODS: We analyzed the influence of interferon gamma (IFN-gamma) on the cytokine production and IFN-gamma receptor expression in T98G and U87-MG human glioma cells. RESULTS: The IFN-gamma receptors were own-regulated after IFN-gamma treatment. Secretion of interleukin-6 (IL-6) protein was elevated by factors of 6.4 in T98G cells and 5.2 in U87-MG. Other cytokines were increased as well, but less constantly: IL-8, and VEGF were elevated significantly only in T98G, but not in U87-MG. Increases of IL-1 beta, IL-1 alpha and TGF beta-2 were only detectable at the mRNA level. TNF was not detectable in any of the cell lines, and TGF-beta, alpha FGF and HG were not influenced by IFN-gamma. CONCLUSION: IL-6 produced by glioma cells in response to IFN-gamma might support immune eradication of glioma cells.

P450-expression in brain tumors.

Oxazaphosphorines are inactive anticancer prodrugs that are bioactivated by hepatic cytochrome P450. Besides hepatic metabolism, there is increasing interest in the possibility of intratumoral activation of oxazaphosphorines by P450. Therefore, we investigated the expression of P450 (CYP3A4, CYP3A5, CYP2C9) by RT-PCR in 10 different brain tumor samples. Because P450 may be downregulated by interleukin-1 (IL-1) and IL-6, the receptors for IL-1 and IL-6 were analyzed. None of the brain tumors was positive for CYP3A4 whereas CYP3A5 was detected in 3 out of 10 tumors (two meningeomas, one medulloblastoma grade IV). All five gliomas, an ependymoma, and a lymphoma-metastase gave no signal. CYP2C9 mRNA was present in every sample studied. All samples were positive for IL-1 and IL-6 receptors. In summary, we have demonstrated that tumors of the CNS express P450, indicating that activation of prodrugs like oxazaphosphorines may take place intratumorally. However, the most abundantly hepatically expressed CYP3A4 enzyme is absent in the brain tumor samples. The presence of the IL-1 and IL-6 receptors opens the possibility that the wellknown downregulating influence of these cytokines also takes place in brain tumors.