Uptake and metabolism of mizoribine, an immunosuppressant, in L5178Y-R mouse lymphoma cells in vitro and peripheral blood mononuclear cells of rats and kidney transplant recipients in vivo.

The cellular uptake of mizoribine (MZR), an immunosuppressant, and metabolism of MZR to MZR-5'- monophosphate (MZRP), an active metabolite, were evaluated in L5178Y-R mouse lymphoma cells and peripheral blood mononuclear cells (PBMCs) of rats and kidney transplant recipients (KTRs, n = 22). Real-time PCR analysis revealed the expression of ENT1 and ENT2 mRNAs, but not of CNTs, in L5178Y-R cells and rat's PBMCs. In L5178Y-R cells, the uptake of MZR was suppressed by adenosine, a substrate for ENT1 and ENT2, but not by 5-(4-nitrobenzyl)-6-thioinosine (0.1 µM), an ENT1 inhibitor. Saturable metabolism of MZR to MZRP was observed. In rats, peak plasma concentrations of MZR and peak concentrations of MZR and MZRP in PBMCs were observed 3 h after oral administration. MZR disappeared from PBMCs in parallel with plasma MZR, but the disappearance of MZRP from PBMCs appeared to be slow. In KTRs, the mean plasma concentration of MZR 3-4 h after ingestion was 3.14 µg/ml and the mean MZRP concentration in PBMCs was 16.8% of MZR, reflecting the involvement of ENT in the uptake of MZR. A linear relationship was observed between plasma MZR concentrations ranging from 1 to 6 µg/ml and PBMC's MZRP concentrations ranging from 90 to 200 ng/ml.

Can the L5178Y Tk+/- mouse lymphoma assay detect epigenetic silencing?

The mouse lymphoma L5178Y Tk(+/-) assay is broadly used in toxicology to assess genotoxicity because of its known sensitivity to genotoxicants that act through a variety of mechanisms, which may include epigenetic DNA methylation. This brief article highlights the studies that have contributed to this conjecture and suggests an addition to the experimental design that could identify if the test substance is a potential epimutagen acting via hypermethylation.

Biological activity of hydantoin derivatives on P-glycoprotein (ABCB1) of mouse lymphoma cells.

BACKGROUND: Hydantoin derivatives possess a variety of biochemical and pharmacological properties. Although hydantoin compounds are studied extensively, there are not many studies that investigate their anticancer properties. MATERIALS AND METHODS: Thirty hydantoin compounds were evaluated for their efflux modulating effects in cancer cells using a rhodamine 123 accumulation assay and real-time fluorometry based on the intracellular accumulation of ethidium bromide. RESULTS: The 30 derivatives were screened by real-time fluorometry for rhodamine 123 accumulation. Among the selected derivatives, compounds SZ-7, LL-9, BS-1, MN-3, P3, RW-15b, AD-26, RW-13, AD-29 and KF-2 significantly increased the retention of rhodamine 123. Compounds AD-26, AD-29, RW-13, KF-2, BS-1, MN-3, RW-15b and JH-63 showed synergistic effect with doxorubicin on mouse lymphoma cells. Furthermore, compound SZ-7 had indifferent effect with doxorubicin. CONCLUSION: These results indicated the role of chemical modifications within the hydantoin ring for its potential inhibition of the ABCB1 transporter. The most active structures contained aromatic substituents as well as some tertiary amine fragments.

Constituents of Carpobrotus edulis inhibit P-glycoprotein of MDR1-transfected mouse lymphoma cells.

A bioassay-guided separation protocol, including the testing of the extracts, fractions and pure compounds for their ability to inhibit P-glycoprotein (the efflux pump responsible for the multidrug resistance of the used cell line) of mouse lymphoma cells containing the human efflux pump gene MDR1, led to the isolation of seven compounds from the chloroform and ethyl acetate soluble fractions of the methanolic extract of Carpobrotus edulis. The compounds were identified by 1D, 2D NMR and MS investigations as triterpens (beta-amyrin, uvaol and oleanolic acid), monogalactosyldiacylglycerol, catechin, epicatechin and procyanidin B5. Uvaol was the most effective and promising compound in the reversal of multidrug resistance in MDR mouse lymphoma cell line.

Differential effect of phenothiazines on MRP1 and P-glycoprotein activity.

BACKGROUND: Overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) or breast cancer resistance protein (BCRP) accounts for majority of cases of multidrug resistance (MDR) of cancer cells. MATERIALS AND METHODS: In the present work, the interactions of seven commercially available phenothiazine derivatives, known P-glycoprotein inhibitors, with this transporter and MRP1 were compared. By flow cytometry, it was shown that all the drugs increased the accumulation of rhodamine 123 in the P-gp-overexpressing lymphoma cell line L5178 MDR. On the other hand, phenothiazine derivatives stimulated MRP1-mediated efflux of fluorescent probe (BCPCF) out of human erythrocytes. RESULTS: In this way, these phenothiazine derivatives were identified as a group of atypical MDR modulators that differently interact with P-gp (as inhibitors) and MRP1 (as stimulators). CONCLUSION: This observation clearly shows that the activity of all new modulators should be tested for their effects towards different ABC transporters as a standard procedure.

Multidrug resistance modulation and apoptosis induction of cancer cells by terpenic compounds isolated from Euphorbia species.

BACKGROUND: One of the most promising strategies to overcome multidrug resistance (MDR) is to use compounds that can modulate P-glycoprotein and restore the cytotoxicity of anticancer drugs. Furthermore, the search for compounds that regulate and overcome apoptosis deficiency of cancer cells is also of great therapeutic importance. MATERIALS AND METHODS: Seven known pentacyclic triterpenes and one steroid were isolated from Euphorbia lagascae methanolic extracts and identified by physical and spectroscopic methods. These compounds, together with eleven terpenoids previously isolated from Euphorbia lagascae and E. tuckeyana were tested for their MDR-reversing and/or apoptosis induction activities by flow cytometry on L5178 human MDR1 gene-transfected mouse lymphoma cells. RESULTS: Four taraxastane-type triterpenes: 21alpha-hydroxytaraxasterol, 21alpha-hydroxytaraxasterol acetate, 3beta,30-dihydroxy-20(21)-taraxastene and 3beta-hydroxy-20-taraxasten-30-al, and two steroids: stigmastane-3,6-dione and ergosterol peroxide exhibited a significant MDR-Pgp modulation activity. Some aspects of structure-activity relationships are discussed. Regarding apoptosis induction, the most significant results were obtained for the polycyclic diterpenes ent-16alpha,17-dihydroxykauran-3-one and ent-16alpha,17-dihydroxyatisan-3-one.

Antitumor activity of alkaloids derived from Amaryllidaceae species.

The aim of the present study was to investigate the anticancer properties of five alkaloids isolated from Amaryllidaceae, including the inhibitory effect on P-glycoprotein (P-gp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for their multidrug resistance (MDR)-reversing activity on human MDR1-gene-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. Trisphaeridine and pretazettine increased the intracellular Rh-123 concentration 30- and 50-fold, respectively, as compared to the non-treated cells, and 2-O-acetyllycorine and trisphaeridine were demonstrated by means of the checkerboard method to enhance the antiproliferative activity of doxorubicin on L5178 MDR mouse lymphoma cells. The MTT assay revealed that pretazettine, trisphaeridine and 2-O-acetyllycorine displayed excellent antiproliferative effects on both the human and the mouse cell lines. The apoptosis-inducing activities of selected agents (2-O-acetyllycorine and trisphaeridine) were measured via acridine orange and ethidium bromide dual staining and flow cytometry of the subG1 population.

Antitumour properties of acridone alkaloids on a murine lymphoma cell line.

The aim of the present study was to investigate the anticancer properties of a set of furanoacridone alkaloids, arborinine and evoxanthine, including the inhibitory effect of P-glycoprotein (Pgp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for multidrug resistance (MDR)-reversing activity on human Pgp-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. The antiproliferative effects of natural compounds and their interactions with doxorubicin were determined in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Apoptosis-inducing activity was additionally measured by means of dual annexin V and propidium iodide staining. RT-PCR was used to test the expression of Pgp mRNA after acridone treatment. All of the acridones investigated increased the accumulation of Rh-123. Gravacridonetriol and gravacridonediol monomethyl ether increased the antiproliferative effect of doxorubicin on resistant L5178 cells. Treatment with these agents resulted in a decrease in Pgp mRNA levels. Naturally occurring acridone alkaloids exhibit a beneficial combination of anticancer effects and, accordingly, the acridone skeleton can be considered useful in the design of novel antiproliferative agents.

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk+/- mouse lymphoma cells.

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk+/- mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations>or=50 microM was reduced to <50%. In the dose range up to 25 microM, viability was >90%. Measures of comet-tail length and thymidine-kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 microM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at >or=100 microM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.

Annexin A2 regulates the levels of plasmin, S100A10 and Fascin in L5178Y cells.

Annexin A2 (ANXA2) was reported as the receptor, activator, expression enhancer, or cooperator for plasmin, S100A10, and others. To delineate the effect of ANXA2 on the proteins that are probably associated with tumor development and metastasis by a credible experimental method, we generated an ANXA2 gene knockout tumor cell line, ANXA2(-/-) L5178Y, and compared the expression levels of plasmin, S100A10 and fascin in the generated cell line with in wild type of L5178Y at mRNA and protein levels. The results showed that the mRNA level of plasminogen (PLG) was not substantially changed in cultured ANXA2(-/-) cells, but the protein level of plasmin was significantly lower in the cultured ANXA2(-/-) cells than in cultured ANXA2(+/+) cells. For S100A10 and fascin, their mRNA and protein levels were significantly lower in the cultured ANXA2(-/-) cells than in cultured ANXA2(+/+) cells. Results indicate that ANXA2 introduces the generation or expression of plasmin, S100A10, and fascin in tumor cells. ANXA2 affects PLG/plasmin level by a way post transcription and may be an inducer or enhancer to fascin expression at transcription level. By the regulations, ANXA2 enhances the development, invasion, and metastasis of tumor. The detailed mechanism for the regulations above remains to be further investigated, but our results show the potential of ANXA2 as a new target molecule for the strategies of tumor biotherapy or tumor gene therapy.

In vitro apoptotic activity of 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones in L5178Y cells.

Compounds containing B-N bonds have shown interesting biological activity. One class of such molecules is the 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones (3a-j), which contain a B-N bond, have an alpha-amino acid moiety in the heterocycle, and have an exocyclic moiety related to an amino acid. The purpose of this work was to determine the inhibitory effects of 3a-j on the proliferation of murine L5178Y lymphoma cells. A new five-membered heterocyclic nucleus with apoptotic activity was found. The target products showed potent cytotoxicity in the L5178Y cell line. Among them, 3a exhibited the highest antineoplastic activity in L5178Y cells with an IC(50) value of 22.5+/-0.2 microM.

Reversal of multidrug resistance of cancer cells in vitro: modification of drug resistance by selected carotenoids.

The development of multidrug resistance (MDR) causes difficulties in the chemotherapy of of human cancer. Investigation of the possibility of reversal of MDR has been greatly aided by the use of cell lines with acquired resitance to anticancer agents in vitro or transfected with the mdrl gene. The aim of this study was to examine new perspectives of chemotherapy focused on natural, carotenoid compounds, in connection with the modification of MDR. The function of the MDR protein was examined via the R123 drug accumulation of both cell lines in the presence of carotenoids. The fluorescence of the cell population was measured by flow cytometry. The most effective resistance modifiers Monoepoxy-beta-carotene, (SS, 8S)-capsochrome, (8'S) Luteoxanthin, (9Z)-Violaxanthin, (9Z)-Zeaxanthin, (13Z)-Zeaxanthin were assayed for their antiproliferative effects in combination with the anti-cancer drug epirubicin. (13Z)-Zeaxanthin was able to enhance the antiproliferative effect on human mdrl gene transfected mouse lymphoma and anthracycline resistant human breast cancer cell line MCF7. (8'S)-luteoxanthin, (5S, 8S)-capsochrome and (9Z)-zeaxanthin treatment revealed synergism with epirubicin on resistant mouse lymphoma. The enhanced antiproliferative activity of epirubicin combinated with (9Z)-Violaxanthin was more significant on MCF7 cells resistant to anthracycline.

Photomutagenicity of retinyl palmitate by ultraviolet a irradiation in mouse lymphoma cells.

Retinyl palmitate (RP), a storage form of vitamin A, is frequently used as a cosmetic ingredient, with more than 700 RP-containing cosmetic products on the U.S. market in 2004. There are concerns for the possible genotoxicity and carcinogenicity of RP when it is exposed to sunlight. To evaluate the photomutagenicity of RP in cells when exposed to ultraviolet A (UVA) light, L5178Y/Tk+/- mouse lymphoma cells were treated with different doses of RP alone/or in the presence of UVA light. Treatment of the cells with RP alone at the dose range of 25-100 microg/ml did not increase mutant frequencies (MFs) over the negative control, whereas treatment of cells with 1-25 microg/ml RP under UVA light (82.8 mJ/cm2/min for 30 min) produced a dose-dependent mutation induction. The mean induced MF (392 x 10(-6)) for treatment with 25 microg/ml RP under UVA exposure was about threefold higher than that for UVA alone (122 x 10(-6)), a synergistic effect. To elucidate the underlying mechanism of action, we examined the mutants for loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11, on which the Tk gene is located. The mutational spectrum for the RP + UVA treatment was significantly different from the negative control, but not significantly different from UVA exposure alone. Ninety four percent of the mutants from RP + UVA treatment lost the Tk+ allele, and 91% of the deleted sequences extended more than 6 cM in chromosome length, indicating clastogenic events affecting a large segment of the chromosome. These results suggest that RP is photomutagenic in combination with UVA exposure in mouse lymphoma cells, with a clastogenic mode-of-action.

Inhibition of P-glycoprotein transport activity in a resistant mouse lymphoma cell line by diterpenic lactones.

Multidrug resistance (MDR) is believed to be a major reason for the failure of cancer treatment. It is in most cases caused by the activity of the various ABC transporters, multidrug resistance (MDR) gene-encoded p-glycoproteins that pump anticancer drugs out of the cells. P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) are the most important and widely studied members of the ABC superfamily of transporters. The ability of four diterpenic lactones isolated from Euphorbia species to modulate the transport activity of P-gp in mouse lymphoma cells was evaluated by flow cytometry. The reversion of MDR was investigated by using a standard functional assay with rhodamine 123 as a fluorescent substrate analogue of doxorubicin. Verapamil was applied as a positive control. All the compounds were able to reverse the MDR of the tested human MDR1 gene-transfected mouse lymphoma cells, in a concentration-dependent manner from 4 to 40 microg/mL, in a short-term experiment below the cytotoxic doses.

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.

Cdc2 kinase-dependent disassembly of endoplasmic reticulum (ER) exit sites inhibits ER-to-Golgi vesicular transport during mitosis.

We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP-labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis.

Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines.

Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.

Genetic toxicology of remifentanil, an opiate analgesic.

Compounds that interact with opioid receptors are commonly used as analgesics. Opioid agonists vary in their potency and pharmacokinetic properties as well as in their affinity for distinct opioid receptors. The fentanyl opiate analogues are an important group of analgesics that interact with the mu opioid receptor. Remifentanil (GI87084) is a particularly interesting member of this group of opioids because its action is especially short in duration. This report examines the genetic toxicology of remifentanil. Remifentanil was not genotoxic in an Ames test, an in vitro chromosome aberration assay in Chinese hamster ovary cells, an in vivo micronucleus assay in rat erythrocytes, or an in vivo/in vitro unscheduled DNA synthesis assay in rat hepatocytes. In the in vitro L5178Y tk(+/-) mouse lymphoma assay, remifentanil produced a genotoxic response at dose levels >or=308 microg/mL only in the presence of rat liver S9 metabolic activation; primarily tiny and small mutant colonies were produced. This pattern of activity in a battery of genetic toxicology assays is not unique to remifentanil, but has also been observed for other pharmaceuticals, including the opioid fentanyl. A weight-of-evidence analysis, taking into consideration genotoxic mechanisms, in vivo results, and the conditions of clinical use, suggests remifentanil does not pose a genotoxic risk to patients.

Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.

TWEAK, a TNF family member, is produced by IFN-gamma-stimulated monocytes and induces multiple pathways of cell death, including caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-alpha-mediated cell death, in a cell type-specific manner. However, the TWEAK receptor(s) that mediates these multiple death pathways remains to be identified. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which was responsible for TWEAK-induced proliferation of endothelial cells and angiogenesis. Because Fn14 lacks the cytoplasmic death domain, it remains unclear whether Fn14 can also mediate the TWEAK-induced cell death. In this study, we demonstrated that TWEAK could induce apoptotic cell death in Fn14 transfectants. A pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive oxygen intermediates and cathepsin B-dependent pathway. By using newly generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is constitutively expressed on the cell surface of all TWEAK-sensitive tumor cell lines, and can transmit the multiple death signals. Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could totally abrogate TWEAK binding and TWEAK-induced cell death in all TWEAK-sensitive tumor cell lines. These results revealed that the multiple pathways of TWEAK-induced cell death are solely mediated by Fn14.

Membrane Fas ligand activates innate immunity and terminates ocular immune privilege.

It has been proposed that the constitutive expression of Fas ligand (FasL) in the eye maintains immune privilege, in part through inducing apoptosis of infiltrating Fas(+) T cells. However, the role of FasL in immune privilege remains controversial due to studies that indicate FasL is both pro- and anti-inflammatory. To elucidate the mechanism(s) by which FasL regulates immune privilege, we used an ocular tumor model and examined the individual roles of the membrane-bound and soluble form of FasL in regulating ocular inflammation. Following injection into the privileged eye, tumors expressing only soluble FasL failed to trigger inflammation and grew progressively. By contrast, tumors expressing only membrane FasL 1) initiated vigorous neutrophil-mediated inflammation, 2) terminated immune privilege, and 3) were completely rejected. Moreover, the rejection coincided with activation of both innate and adaptive immunity. Interestingly, a higher threshold level of membrane FasL on tumors is required to initiate inflammation within the immune privileged eye, as compared with nonprivileged sites. The higher threshold is due to the suppressive microenvironment found within aqueous humor that blocks membrane FasL activation of neutrophils. However, aqueous humor is unable to completely block the proinflammatory effects of tumor cells that express high levels of membrane FasL. In conclusion, our data indicate that the function of FasL on intraocular tumors is determined by the microenvironment in conjunction with the form and level of FasL expressed.