Murine Thymic Lymphoma Research Articles

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L‐dose dependency in HDAC1‐deficient thymic lymphoma

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL‐rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

Modification of radiation-induced murine thymic lymphoma incidence by curcumin

Introduction: Curcumin is a known antioxidant, preventing radiation damage including carcinogenesis. However, concentration and feeding schedule of curcumin in modification of radiation induced thymic lymphoma incidence in vivo model has not been studied. Materials and Methods: We report here modification of incidence of γ-radiation-induced thymic lymphoma in mice fed with different doses of curcumin (0.05 to 1*) in diet. Results: Female Swiss mice (6-8 weeks) fed with normal diet and exposed to 3 Gy whole body60Co γ-irradiation (WBI) showed 85 * incidence of thymic lymphoma (TL) at 120 days post-irradiation. A concentration of 1 * curcumin was found the most effective in TL incidence prevention than other fed concentrations. The TL incidence was remarkably reduced when curcumin was fed to the mice before than after the radiation exposure. The incidence of TL was reduced to 63* in mice fed with 1* curcumin in diet for 3 weeks after exposure to WBI. On the other hand, when animals were fed with same concentration of curcumin for 2 weeks and 3 weeks before WBI, the TL incidence was reduced to 55* and 35*, respectively. Curcumin feeding resulted in significant prevention in micronucleus formation in the bone marrow, which was corroborated with inhibition in DNA damage quantified by comet assay. Moreover, significant prevention in DNA damage was also observed in the peripheral blood cells in curcumin fed and irradiated mice, which however, was not prominent in thymus. Curcumin was able to prevent apoptotic death in thymus and bone marrow 4 h after irradiation, which however, got attenuated at longer post-irradiation period (24 h). Conclusion: These results suggest modification of TL incidence by curcumin in irradiated mice involving DNA damage and apoptotic death mechanisms.

The copper chelator ATN-224 induces caspase-independent cell death in diffuse large B cell lymphoma.

Bcl-2 and other anti-apoptotic proteins are associated with defective caspase-dependent apoptotic pathways, resulting in chemoresistance. We have previously shown that ATN-224, a copper chelator drug, induces cell death in murine thymic lymphoma cells transfected with Bcl-2. In the current study, we tested whether ATN-224 was effective in diffuse large B cell lymphoma (DLBCL) cells, which have increased anti-apoptotic proteins through translocation or amplification. We found that nanomolar concentrations of ATN-224 induced cell death in DLBCL cells independent of Bcl-2, Bcl-xL or Mcl-1 status. ATN-224 treatment resulted in mitochondrial dysfunction, release of apoptosis-inducing factor (AIF) and induction of caspase-independent cell death. In addition, ATN-224 degraded Mcl-1 and enhanced the effect of the BH3 mimetic ABT-263. These findings indicate that ATN-224 has potential as a therapeutic for the treatment of DLBCL. Induction of caspase-independent cell death in apoptosis-resistant DLBCL would provide a therapeutic alternative for the treatment of refractory disease.

Postchemotherapy and Tumor-Selective Targeting with the La-Specific DAB4 Monoclonal Antibody Relates to Apoptotic Cell Clearance

Early identification of tumor responses to treatment is crucial for devising more effective and safer cancer treatments. No widely applicable, noninvasive method currently exists for specifically detecting tumor cell death after cytotoxic treatment and thus for predicting treatment outcomes. We have further characterized the targeting of the murine monoclonal antibody DAB4 specifically to dead tumor cells in vitro, in vivo, and in clinical samples. We found that sustained DAB4 binding to treated cells was closely associated with markers of intrinsic apoptosis and DNA double-strand break formation. In a competition binding assay, DAB4 bound EL4 murine thymic lymphoma cells in preference to the normal counterpart of murine thymocytes. Defective in vivo clearance of apoptotic cells augmented in vivo accumulation of DAB4 in tumors particularly after chemotherapy but was unchanged in normal tissues. Tumor targeting of DAB4 was selective for syngeneic murine tumors and for human tumor xenografts of prostate cancer (PC-3) and pancreatic cancer (Panc-1) before and more so after chemotherapy. Furthermore, DAB4 was shown to bind to dead primary acute lymphoblastic leukemic blasts cultured with cytotoxic drugs and dead epithelial cancer cells isolated from peripheral blood of small cell lung carcinoma patients given chemotherapy. Collectively, these results further demonstrate the selectivity of DAB4 for chemotherapy-induced dead tumor cells. This postchemotherapy selectivity is related to a relative increase in the availability of DAB4-binding targets in tumor tissue rather than in normal tissues. The in vitro findings were translated in vivo to human xenograft models and to ex vivo analyses of clinical samples, providing further evidence of the potential of DAB4 as a marker of tumor cell death after DNA-damaging cytotoxic treatment that could be harnessed as a predictive marker of treatment responses.

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α.

Manganese Porphyrin, MnTE-2-PyP5 +, Acts as a Pro-Oxidant to Potentiate Glucocorticoid-Induced Apoptosis in Lymphoma Cells

Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP5+, augments glucocorticoid-induced apoptosis in WEHI7.2 murine thymic lymphoma cells, suggesting that it may have potential as a lymphoma therapeutic. However, the mechanism by which MnTE-2-PyP5+ potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H2O2]ss) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP5+ is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H2O2]ss levels. The combined treatment depletes GSH, oxidizes the 2GSH:GSSG ratio, and causes protein glutathionylation to a greater extent than glucocorticoid treatment alone. Removal of the glucocorticoid-generated H2O2 or depletion of glutathione by BSO prevents MnTE-2-PyP5+ from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP5+ glutathionylates p65 NF-κB and inhibits NF-κB activity. Inhibition of NF-κB with SN50, an NF- κB inhibitor, enhances glucocorticoid-induced apoptosis to the same extent as MnTE-2-PyP5+. Taken together, these findings indicate that: 1) H2O2 is important for MnTE-2-PyP5+ activity; 2) Mn-TE-2-PyP5+ cycles with GSH; and 3) MnTE-2-PyP5+ potentiates glucocorticoid-induced apoptosis by glutathionylating and inhibiting critical survival proteins, including NF-κB. In the clinic, over-expression of NF-κB is associated with a poor prognosis in lymphoma. MnTE-2-PyP5+ may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment.

Hydrogen peroxide signaling is required for glucocorticoid-induced apoptosis in lymphoma cells

Glucocorticoid-induced apoptosis is exploited clinically for the treatment of hematologic malignancies. Determining the required molecular events for glucocorticoid-induced apoptosis will identify resistance mechanisms and suggest strategies for overcoming resistance. In this study, we found that glucocorticoid treatment of WEHI7.2 murine thymic lymphoma cells increased the steady-state [H 2O 2] and oxidized the intracellular redox environment before cytochrome c release. Removal of glucocorticoids after the H 2O 2 increase resulted in a 30% clonogenicity; treatment with PEG–CAT increased clonogenicity to 65%. Human leukemia cell lines also showed increased H 2O 2 in response to glucocorticoids and attenuated apoptosis after PEG–CAT treatment. WEHI7.2 cells that overexpress catalase (CAT2, CAT38) or were selected for resistance to H 2O 2 (200R) removed enough of the H 2O 2 generated by glucocorticoids to prevent oxidation of the intracellular redox environment. CAT2, CAT38, and 200R cells showed a 90–100% clonogenicity. The resistant cells maintained pERK survival signaling in response to glucocorticoids, whereas the sensitive cells did not. Treating the resistant cells with a MEK inhibitor sensitized them to glucocorticoids. These data indicate that: (1) an increase in H 2O 2 is necessary for glucocorticoid-induced apoptosis in lymphoid cells, (2) increased H 2O 2 removal causes glucocorticoid resistance, and (3) MEK inhibition can sensitize oxidative stress-resistant cells to glucocorticoids.

Derivation of Thymic Lymphoma T-cell Lines from <em>Atm<sup>-/-</sup></em> and <em>p53<sup>-/-</sup></em> Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm(-/-) and p53(-/-) consistently develop thymic lymphoma early in life (1,2), and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols (1,3). Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain (1,4). We have successfully established several T-cell lines using this protocol and inbred strains ofAtm(-/-) [FVB/N-Atm(tm1Led)/J] (2) and p53(-/-) [129/S6-Trp53(tm1Tyj)/J] (5) mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study

BackgroundThe observation of cytokeratins (CK's) in mass spectrometry based studies raises the question of whether the identified CK is a true endogenous protein from the sample or simply represents a contaminant. This issue is especially important in proteomic studies of the corneal epithelium where several CK's have previously been reported to mark the stages of differentiation from corneal epithelial stem cell to the differentiated cell.MethodsHere we describe a method to distinguish very likely endogenous from uncertain endogenous CK's in a mass spectrometry based proteomic study. In this study the CK identifications from 102 human corneal samples were compared with the number of human CK identifications found in 102 murine thymic lymphoma samples.ResultsIt was anticipated that the CK's that were identified with a frequency of <5%, i.e. in less than one spot for every 20 spots analysed, are very likely to be endogenous and thereby represent a 'biologically significant' identification. CK's observed with a frequency >5% are uncertain endogenous since they may represent true endogenous CK's but the probability of contamination is high and therefore needs careful consideration. This was confirmed by comparison with a study of mouse samples where all identified human CK's are contaminants.ConclusionsCK's 3, 4, 7, 8, 11, 12, 13, 15, 17, 18, 19, 20 and 23 are very likely to be endogenous proteins if identified in a corneal study, whilst CK's 1, 2e, 5, 6A, 9, 10, 14 and 16 may be endogenous although some are likely to be contaminants in a proteomic study. Further immunohistochemical analysis and a search of the current literature largely supported the distinction.

Increased Manganese Superoxide Dismutase Expression or Treatment with Manganese Porphyrin Potentiates Dexamethasone-Induced Apoptosis in Lymphoma Cells

Glucocorticoid-induced apoptosis is exploited for the treatment of hematologic malignancies. Innate and acquired resistance limits treatment efficacy; however, resistance mechanisms are not well understood. Previously, using WEHI7.2 murine thymic lymphoma cells, we found that increasing the resistance to hydrogen peroxide (H(2)O(2)) by catalase transfection or selection for H(2)O(2) resistance caused glucocorticoid resistance. This suggests the possibility that increasing H(2)O(2) sensitivity could sensitize the cells to glucocorticoids. In other cell types, increasing manganese superoxide dismutase (MnSOD) can increase intracellular H(2)O(2). The current study showed that increased expression of MnSOD sensitized WEHI7.2 cells to glucocorticoid-induced apoptosis and H(2)O(2). Treatment of WEHI7.2 cells with the catalytic antioxidant Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)), a manganoporphyrin, mimicked the effects of increased MnSOD expression. MnTE-2-PyP(5+) also sensitized WEHI7.2 cells to cyclophosphamide and inhibited cell growth; it had no effect on the WEHI7.2 cell response to doxorubicin or vincristine. In primary follicular lymphoma cells, MnTE-2-PyP(5+) increased cell death due to dexamethasone. Treatment of H9c2 cardiomyocytes with MnTE-2-PyP(5+) inhibited doxorubicin cytotoxicity. The profile of MnTE-2-PyP(5+) effects suggests MnTE-2-PyP(5+) has potential for use in hematologic malignancies that are treated with glucocorticoids, cyclophosphamide, and doxorubicin.

A Novel Recurrent Chromosomal Inversion Implicates the Homeobox GeneDlx5in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

The oncogene v-akt was isolated from a retrovirus that induced murine thymic lymphomas. Transgenic mice expressing a constitutively activated form of the cellular homologue Akt2 specifically in immature T cells develop spontaneous thymic lymphomas. We hypothesized that tumors from these mice might exhibit oncogenic chromosomal rearrangements that cooperate with activated Akt2 in lymphomagenesis. Cytogenetic analysis revealed a recurrent clonal inversion of chromosome 6, inv(6), in thymic lymphomas from multiple transgenic founder lines, including one line in which 15 of 15 primary tumors exhibited this same rearrangement. Combined fluorescence in situ hybridization, PCR, and DNA sequence analyses showed that the distal inv(6) breakpoint resides at the T-cell receptor beta chain locus, Tcrb. The proximal breakpoint maps to a region near a locus comprising the linked homeobox/transcription factor genes Dlx5 and Dlx6. Expression analysis of genes translocated to the vicinity of the Tcrb enhancer revealed that Dlx5 and Dlx6 are overexpressed in tumors exhibiting the inv(6). Experimental overexpression of Dlx5 in mammalian cells resulted in enhanced cell proliferation and increased colony formation, and clonogenic assays revealed cooperativity when both Dlx5 and activated Akt2 were coexpressed. In addition, DLX5, but not DLX6, was found to be abundantly expressed in three of seven human T-cell lymphomas tested. These findings suggest that the Dlx5 can act as an oncogene by cooperating with Akt2 to promote lymphomagenesis.

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710 3. Kirstetter P, Thomas M, Dierich A, et al. Ikaros is critical for B cell differentiation and function. Eur J Immunol 2002 ; 32 : 720-3. 4. Koipally J, Renold A, Kim J, et al. Repression by Ikaros and Aiolos is mediated through histone deacetylase complexes. EMBO J 1999 ; 18 : 3090-100. 5. Lopez-Nieva P, Santos J, Fernandez-Piqueras J. Defective expression of Notch1 and Notch2 in connection to alterations of c-Myc and Ikaros in gamma-radiation-induced mouse thymic lymphomas. Carcinogenesis 2004 ; 25 : 1299-304. 6. Karlsson A, Soderkvist P, Zhuang SM. Point mutations and deletions in the znfn1a1/ikaros gene in chemically induced murine lymphomas. Cancer Res 2002 ; 62 : 2650-3. 7. Shimada Y, Nishimura M, Kakinuma S, et al. Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas. Radiat Res 2000 ; 154 : 293-300. 8. Winandy S, Wu P, Georgopoulos K. A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 1995 ; 83 : 289-99. 9. Papathanasiou P, Perkins AC, Cobb BS, et al. Widespread failure of hematolymphoid differentiation caused by a recessive niche-filling allele of the Ikaros transcription factor. Immunity 2003 ; 19 : 131-44. 10. Beverly L, Capobianco AJ. Perturbation of Ikaros isoform selection by MLV integration is a cooperative event in Notch(IC)-induced T cell leukemogenesis. Cancer Cell 2003 ; 3 : 551-64. Signal Notch

Glucose 6-Phosphate Dehydrogenase Overexpression Models Glucose Deprivation and Sensitizes Lymphoma Cells to Apoptosis

Glucocorticoids are one component of combined treatment regimens for many types of lymphoma due to their ability to induce apoptosis in lymphoid cells. In WEHI7.2 murine thymic lymphoma cells, altering catalase and glutathione peroxidase activity by transfection or the use of chemical agents modulates the ability of glucocorticoids to induce apoptosis. This suggests that the oxidative stress response is important in determining the glucocorticoid sensitivity of the cells. For glutathione peroxidase and catalase to detoxify reactive oxygen species (ROS), reducing equivalents in the form of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) are ultimately required. The major source of NADPH in the cell is glucose 6-phosphate dehydrogenase (G6PDH). Therefore, we created G6PDH-overexpressing WEHI7.2 variants to test whether G6PDH activity is a key determinant of glucocorticoid sensitivity in WEHI7.2 cells. G6PDH-overexpressing WEHI7.2 cells were more sensitive to oxidative stress and glucocorticoids. The G6PDH-overexpressing WEHI7.2 variants appeared similar to cells undergoing glucose deprivation with decreased adenosine triphosphate (ATP) synthesis by the mitochondria and increased basal levels of ROS. Overexpression of G6PDH also sensitized the cells to other standard lymphoma chemotherapeutics including cyclophosphamide, doxorubicin, and vincristine. The decreased ATP and elevated ROS due to G6PDH overexpression may be key factors in increasing the sensitivity of the WEHI7.2 cells to lymphoma chemotherapeutics.

Anticancer Effect of Dye-sensitized TiO2 Nanocrystals by Polychromatic Visible Light Irradiation

Abstract TiO2 nanocrystalline particles with monolayer adsorption of chlorin e6 trisodium salt (dye-TiO2 particles) were administered into a cancer cell, murine thymic lymphoma (EL-4). Irradiation of the polychromatic light (550–750 nm) that excites chlorin e6 significantly damaged the EL-4 cells. A higher cell-killing effect was found for the dye-TiO2 particle than for the system using chlorin e6 alone.

Regulation of the Expression of Caspase-9 by the Transcription Factor Activator Protein-4 in Glucocorticoid-induced Apoptosis

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at -1.55 and -1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases -1140 and -944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Photodynamic Therapy for Cancer Cells Using a Flash Wave Light Xenon Lamp

We determined photodynamic therapy (PDT) efficacy using a flash wave (FW) and a continuous wave (CW) light, of which the fluence rate was 70 μW/cm2, for murine thymic lymphoma cells (EL-4) cultivated in vitro. The irradiation frequency and the pulse width of the FW light were in the range of 1–32 Hz and less than one millisecond, respectively. 5-Aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) was used as a photosensitizer. When EL-4 with ALA administration was irradiated by the light for 4 h (irradiation fluence: 1.0J/cm2), the survival rate of EL-4 by the FW light was lower than that by the CW light, except for the FW light with irradiation frequency of 32 Hz, and decreased gradually with decreasing irradiation frequency. Moreover, the FW light, especially at lower irradiation frequency, was superior to the CW light for the generation of singlet oxygen in an aqueous PpIX solution. Therefore, thehigher PDT efficacy for EL-4 of the FW light would be caused by the greater generation of singlet oxygen in the cells.

Subversion of T lineage commitment by PU.1 in a clonal cell line system

Specification of mammalian T lymphocytes involves prolonged developmental plasticity even after lineage-specific gene expression begins. Expression of transcription factor PU.1 may maintain some myeloid-like developmental alternatives until commitment. Commitment could reflect PU.1 shutoff, resistance to PU.1 effects, and/or imposition of a suicide penalty for diversion. Here, we describe subclones from the SCID.adh murine thymic lymphoma, adh.2C2 and adh.6D4, that represent a new tool for probing these mechanisms. PU.1 can induce many adh.2C2 cells to undergo diversion to a myeloid-like phenotype, in an all-or-none fashion with multiple, coordinate gene expression changes; adh.6D4 cells resist diversion, and most die. Diversion depends on the PU.1 Ets domain but not on known interactions in the PEST or Q-rich domains, although the Q-rich domain enhances diversion frequency. Protein kinase C/MAP kinase stimulation can make adh.6D4 cells permissive for diversion without protecting from suicide. These results show distinct roles for regulated cell death and another stimulation-sensitive function that establishes a threshold for diversion competence. PU.1 also diverts normal T-cell precursors from wild type or Bcl2-transgenic mice to a myeloid-like phenotype, upon transduction in short-term culture. The adh.2C2 and adh.6D4 clones thus provide an accessible system for defining mechanisms controlling developmental plasticity in early T-cell development.

Ikaros mutation in murine thymic lymphoma induced by combined treatment of X-rays and N-ethyl-N-nitrosourea

Ikaros mutation in murine thymic lymphoma induced by combined treatment of X-rays and N-ethyl-N-nitrosourea

Size and Surface Charge Effect of 5-Aminolevulinic Acid-Containing Liposomes on Photodynamic Therapy for Cultivated Cancer Cells

5-Aminolevulinic acid (ALA)-containing liposomes having various average diameters and/or positive surface charges were prepared, and their photodynamic therapy (PDT) efficacy for murine thymic lymphoma cells, EL-4 cells, cultivated in vitro was investigated. The PDT efficacy for EL-4 cells and the accumulation of ALA-induced protoporphyrin IX (PpIX) in the cells increased with a decrease in the average diameter of liposomes. In particular, the ALA-containing liposomes smaller than 63.5 nm in diameter promoted the PDT efficacy in comparison with that of ALA alone. We also found no significant changes in PDT efficacy and PpIX accumulation with increasing positive surface charges of liposomes.

Involvement of protein kinase C-related anti-apoptosis signaling in radiation-induced apoptosis in murine thymic lymphoma(3SBH5) cells.

Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (alpha and betaI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (alpha, betaI and betaII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (alpha or beta) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.