Tumor Fas (APO-1/CD95) up-regulation results in increased apoptosis and survival times for rats with intracranial malignant gliomas.

OBJECTIVE: The cellular "death" receptor Fas has been proposed to be a potential specific target for anti-glioma therapy. However, little is known regarding the effects of Fas expression on glioma viability in vivo. The goal of this study was to clarify the relationships among Fas expression, apoptosis, and survival rates for high-grade astrocytomas. METHODS: Fas expression was measured in several human glioblastoma multiforme cell lines and a malignant rat glioma cell line (36B10), before and after Fas up-regulation by gene transfer. Expression was correlated with the degree of Fas-mediated cytotoxicity and apoptosis induced after Fas activation. Subsequently, rats underwent intracranial implantation of either wild-type or genetically altered 36B10 cell lines, for study of the effects of Fas up-regulation on survival rates. RESULTS: Low levels of cell surface Fas expression in glioblastomas multiforme were correlated with their limited susceptibility to Fas-mediated cytotoxicity. Through Fas receptor up-regulation, relationships among increased Fas expression, Fas-mediated cytotoxicity, and apoptosis were demonstrated. The percentage of cells undergoing apoptosis after exposure to a Fas ligand-producing cell line increased from 4% in the sham-transfected line (36B10-) to 27% in the Fas-transfected line (36B10-Fas). After intracranial implantation of these tumors into rats, the median survival time increased significantly from 14 days (36B10 and 36B10-) to 24.5 days (36B10-Fas), which represents a 75% increase in the survival time for the greater Fas-expressing group (P = 0.0005). CONCLUSION: It seems that the overall low rate of apoptosis in high-grade astrocytomas is related to low levels of cell surface Fas expression. With increases in cellular Fas expression, rates of Fas-mediated apoptosis and survival rates were increased.

Soluble Fas-ligand (sFasL) in human astrocytoma cyst fluid is cytotoxic to T-cells: another potential means of immune evasion.

Gliomas of all grades have been shown to express FasL, an apoptosis-inducing protein. Because of the ability of FasL to be cleaved from cell surfaces by metalloproteinases, soluble FasL can be released by FasL bearing cells into surrounding tissues. In the present study, we demonstrate the presence of sFasL in the cyst fluids of astrocytomas. Additionally, a human T-cell line, Jurkat, exposed to astrocytoma cyst fluid resulted in significantly increased cytotoxicity as compared to controls, an effect blocked by FasL neutralizing antibodies. This suggests that sFasL, may be utilized as a means of escaping immune surveillance by these tumors.

The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme.

OBJECTIVE: We investigated whether the hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor (EGFR) genes would lead to a loss of tumorigenicity of a glioblastoma multiforme cell line. METHODS: Glioblastoma multiforme cells were treated in vitro with 0 (HU0) or 100 micromol/L (HU100) hydroxyurea and then injected into the flanks of nude mice. Survival and tumor volumes were evaluated. Pulsed-field gel electrophoresis, Southern blot hybridization, and slot-blot analysis were used to determine EGFR amplification levels. Flow cytometry and immunofluorescent staining were used for cell-cycle analysis and EGFR protein expression. RESULTS: Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HU0 cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresis analysis showed that HU100 tumors had reamplified the EGFR gene as double-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the absence of hydroxyurea, the EGFR gene also reamplified. HU100 cells grew at less than half the rate of untreated HU0 control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 micromol/L) cells showed decreased EGFR protein expression. CONCLUSION: The EGFR gene is important for tumorigenicity in mice and growth in culture. Hydroxyurea induces the loss of double-minute chromosome-amplified EGFR genes against a selection gradient and significantly delays the onset of tumors. These results support the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.

Hydroxyurea accelerates the loss of epidermal growth factor receptor genes amplified as double-minute chromosomes in human glioblastoma multiforme.

OBJECTIVE: We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions. METHODS: Pulsed-field gel electrophoresis and Southern blot hybridization were used to demonstrate EGFR genes amplified as dmin. Giemsa-stained metaphase spreads were prepared in an attempt to visualize dmin. A GBM cell line containing amplified EGFR genes was treated continuously in vitro with 0 to 150 mumol/L hydroxyurea, and slot blot analysis was used to show the loss of amplified EGFR genes. RESULTS: Amplified EGFR genes were found on dmin in 4 of 11 (36%) fresh human GBM biopsy specimens. None of the GBMs contained EGFR genes amplified as homogeneously staining regions. Amplified dmin were not microscopically visible when stained with Giemsa because of their small size. Slot blot analysis showed that these low doses of hydroxyurea accelerated the loss of amplified EGFR genes in a dose- and time-dependent fashion. Pulsed-field gel electrophoresis and Southern blot analysis confirmed that EGFR gene loss was accompanied by amplified dmin loss in a dose-dependent fashion. CONCLUSION: These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.

Synthesis and antitumor properties of activated cyclophosphamide analogues.

A series of 5- and 6-substituted cyclophosphamide analogues has been prepared, and their 31P NMR kinetics of phosphoramide mustard (PDA) release and in vitro and in vivo cytotoxicity have been evaluated. cis-4-Hydroxy-5-methoxycyclophosphamide equilibrated very slowly and to a minor extent with the ring-opened aldophosphamide analogues in aqueous buffer; release of PDA was observed to a minor extent and only at high (1 M) buffer concentrations. This analogue was essentially inactive in vitro against L1210 and P388 leukemia cells. 6-Phenylcyclophosphamide and its 4-hydroperoxy derivative were potent inhibitors of blood acetylcholinesterase and were lethal at therapeutic doses in mice. In contrast, 4-hydroperoxy-6-(4-pyridyl)cyclophosphamide did not inhibit acetylcholinesterase and showed significant antitumor activity in vitro and in vivo against both wild-type and cyclophosphamide-resistant L1210 leukemia. The 4-hydroperoxy-6-arylcyclophosphamides were generally active in vitro against both wild-type and cyclophosphamide-resistant L1210 and P388 cells, and several analogues showed significant activity in vivo. Surprisingly, there was no correlation between antitumor activity in vitro and the rate of PDA release in aqueous buffer. Several compounds that showed essentially no release of PDA in aqueous buffer over several hours were highly cytotoxic to leukemia cells following a 1-h exposure in vitro. These results show that activated cyclophosphamide analogues substituted at the 6-position are not cross-resistant in these leukemia cell lines, and that a specific intracellular activation mechanism may be catalyzing PDA release in these analogues.