FL Cells Research Articles

Molecular Ontogeny of Donor-Derived Lymphomas Occurring After Transplantation,

Abstract 3671Follicular lymphoma (FL) is characterized by the translocation t(14;18), which results in overexpression of the anti-apoptotic protein BCL2 through juxtaposition to the immunoglobulin heavy chain (IGH) locus. Additional genetic aberrations are required and recurrent mutations have been identified in FL, however their timing during lymphomagenesis remains unknown. We performed ultra-sensitive mutation detection to define in vivo clonal diversification in paired follicular lymphomas from a donor-recipient sibling pair that presented more than 9 years after hematopoietic cell transplantation. Briefly, a 41-year old woman with chronic myeloid leukemia (CML) underwent myeloablative bone marrow transplantation from her HLA-matched sister in 2000. She received three donor leukocyte infusions (DLI) for molecular relapse, with the last in June 2002. In November 2009, the donor was diagnosed with grade 2/3A FL. Six months later, the recipient was diagnosed with grade 2/3A FL. The FLs shared identical BCL2/IGH rearrangements, which was also recovered from the DLI at a frequency of 1-in-2000 cells. Both FLs also shared the same V(D)J rearrangement, with the exception of single base-pair mismatches and insertions/deletions (InDels) consistent with ongoing somatic hypermutation (SHM) during clonal divergence. Alignment with germline VH3-66 sequence indicated that the common ancestor had initiated SHM. Whole exome sequencing of both FLs identified 12 single nucleotide variants (SNVs) and 2 InDels in both lymphomas, 3 SNVs unique to the donor’s FL, and 4 unique to the recipient’s FL. All candidate mutations were validated and confirmed to be somatic by Sanger sequencing. Among the identical mutations identified in both FLs were two SNVs in BCL2, an in-frame deletion in EP300, and an in-frame insertion in KLHL6, which were recently found to be recurrently mutated in lymphoma. Among the SNVs unique to the recipient’s FL was an ARID1A (adenine-thymine (AT)-rich interactive domain-containing protein 1A) R1276 premature stop. Loss-of-function mutations in ARID1A have been reported in solid cancers, but not yet in hematologic malignancies. On immunohistochemical staining both lymphomas had decreased ARID1A/BAF250 protein expression, suggesting that loss of ARID1A occurred through separate mechanisms in each FL (i.e. convergent evolution). In fact, the donor’s lymphoma was found to have a copy number loss at this locus (1p35.3) by qPCR. To determine whether the somatic mutations that we identified were present at a low frequency within the DLI, we PCR amplified regions flanking each mutation site from the DLI and subjected the products to ultra-sensitive deep sequencing (average read coverage at mutation site, 361,723; range, 16,684–1,169,555). To correct for background frequencies of non-germline calls, we PCR amplified and deep sequenced the same positions from the donor’s buccal swab (average read coverage at the mutation site, 418,499; range, 20,711–1,070,734). Eleven of the 12 SNVs and the 2 InDels that were identified in both lymphomas were ‘enriched’ in the DLI, i.e., recovered at frequencies significantly above background, indicating that those mutations were present more than 7 years prior to presentation of either lymphoma. All 4 SNVs unique to the recipient’s FL and a RAFTLIN V254M mutation identified only in the donor’s FL were not enriched in the DLI, consistent with subsequent acquisition during clonal diversification. Of the final two mutations, one was detected only in the donor’s FL and was enriched in the DLI. The other was initially detected only in the donor’s FL, but deep sequencing recovered the mutation in 4.7% of reads from the recipient’s FL and demonstrated enrichment in the DLI. The presence of a mutation in the donor’s FL and DLI but not within the majority of recipient’s FL cells is consistent with at least two scenarios: (i) the recipient’s FL is derived from a clonally diversified population of ancestor cells transferred from the donor or (ii) the mutant allele was lost in a subset or in all cells of the recipient’s FL during clonal evolution. In conclusion, we utilized ultra-sensitive mutation detection to elucidate the molecular ontogeny of follicular lymphoma during clonal evolution in separate hosts. This approach has broad applicability for identifying genetic variants within tumor populations that confer phenotypes like therapeutic resistance or metastatic potential. Disclosures:No relevant conflicts of interest to declare.

GATA1s Exerts Fetal Stage-Specific Oncogenic Effects in Human Hematopoietic Stem and Progenitor Cells

Abstract 2355Approximately 10% of all neonates with Down syndrome (DS) have a transient leukemia (TL), which is characterized by the accumulation of immature megakaryoblasts in the peripheral blood, liver, and bone marrow. In the majority of cases, TL is asymptomatic and resolves spontaneously. However in 20 and 30% of DS children, TL can progress to acute megakaryoblastic leukemia (AMKL) within the first 4 years of life. Acquired mutations in the hematopoietic transcription factor GATA1 are consistently present in both entities. In mice Gata1s overexpression leads to hyperproliferation of a transient fetal population of megakaryocytic progenitors (MPs), while ostensibly similar adult MPs are largely unaffected. Thus, growing evidence supports that TL and DS-AMKL originate in utero from fetal hematopoietic cells, suggesting that intrinsic genetic programs in fetal hematopoietic stem and progenitor cells (HSPCs), which may differ from those in adult HSPCs, are essential for leukemogenesis. However, if human fetal HSPCs or MPs are equally sensitive to the effects of GATA1s as their murine counterparts, remained elusive.To address this issue, we first explored the intrinsic potential of adult (from mobilized peripheral blood; PB), neonatal (from cord blood; CB) and fetal (from livers of aborted fetuses, gestation weeks 13 to 18; FL) HSPCs to differentiate along the megakaryocytic lineage. In erythropoietin (EPO) and thrombopoietin (TPO) containing medium allowing erythroid and megakaryocytic differentiation, FL-HSPCs generated a higher percentage of CD41+ cells (29%) than PB- (16%), CB-HSPCs (24%). FL cells showed an 8-fold higher proliferation rate than PB cells and 1.4 higher proliferation rate than CB cells in stem cell expansion medium. The in vitro derived megakaryocytes from FL- and CB-HSPCs were much smaller in size than adult megakaryocytes from PB, consistent with previous reports.Next we explored the effect of GATA1s overexpression on human PB-, CB- and FL-HSPCs using a lentiviral system. Cells were sorted and differentiated along megakaryocytic lineage in liquid and semi-solid media. In liquid culture, GATA1s transduced FL-MPs expanded 10-fold by day 10 (p=0.016), whereas MPs from CB and PB expanded 8.4-fold (p=0.013) and 3.1-fold (p=0.035), respectively. GATA1 overexpression caused only a moderate proliferation of FL-MPs (6.3-fold, p=0.041), CB-MPs (6.8-fold, p=0.049) and PB-MPs (2.2-fold, p=0.11). In semi-solid media (MegacultC), GATA1s induced a remarkable increase of megakaryocytic colonies (CFU-MKs) in FL (383 ± 20) compared to CB (116 ± 7) and PB (6.5 ± 3.5). GATA1s-transduced FL-MPs gave rise to 4.9 times more CFU-MKs than empty vector (p=0.002) and 1.77 times more than GATA1-transduced cells (p=0.018). In CB-MPs, the increase was 4.37 times (p=0.003) and 1.69 times (p=0.04), respectively. In PB the difference between GATA1s, GATA1 and empty vector control was insignificant (p=0.18, p=0.51). The CFU-MKs of FL- and CB-MPs were not only more abundant but also larger in size. We obtained 90% CD41+CD42b+ cells in FL GATA1s megakaryocytic differentiation culture by day 11, 88% in FL GATA1 and only 19% in FL empty vector control culture.Thus, we demonstrated that FL-HSPCs have a higher intrinsic proliferative potential and are prone to differentiate along the megakaryocytic lineage. Furthermore, FL-MPs are more sensitive to GATA1s overexpression, leading to their hyperproliferation in liquid media and abundant formation of large CFU-MKs in semi-solid medium. However, without trisomy 21 in human cells we do not see oncogenic transformation and arisal of TL-like blasts, underscoring the requirement of the triad of factors (GATA1s mutation, fetal origin of the cells and trisomy 21) for DS-AMKL and DS-TL. Disclosures:No relevant conflicts of interest to declare.

A 1.8-GHz radiofrequency radiation induces EGF receptor clustering and phosphorylation in cultured human amniotic (FL) cells

Purpose: Many studies have shown that exposure to radiofrequency radiation (RFR) could activate cellular signal transduction pathways. In the present research, we investigated the effects of exposure to a 1.8-GHz RFR at different intensities on epidermal growth factor (EGF) receptor clustering and phosphorylation in human amniotic (FL) cells.Materials and methods: Receptor clustering on cellular membrane surface was analyzed using immunofluorescence assessed by confocal microscopy, and phosphorylation of EGF receptors was measured by western blot technology. EGF treatment served as a positive control.Results: The results showed that, compared with sham exposure, exposure to RFR at specific absorption rate (SAR) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min significantly induced EGF receptor clustering and enhanced phosphorylation on the tyrosine-1173 residue in FL cells, whereas exposure to a SAR 0.1 W/kg radiation for 15 min did not cause a significant effect.Conclusion: Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets that RFR interacts with cells, and the dose-rate threshold, in the case of EGF receptors, is between SAR of 0.1 and 0.5 W/kg. The results indicate a sigmoid dependence of RFR effects on intensity.

Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

Benzo[ a]pyrene induces complex H2AX phosphorylation patterns by multiple kinases including ATM, ATR, and DNA-PK

H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. While it has been reported that benzo[ a]pyrene (BaP) cannot induce γH2AX alone in several cell lines, we have shown that BaP alone could induce γH2AX in human amnion FL cells. Thus, we further examined the ability of BaP to induce γH2AX in different cell systems. It was shown that BaP-induced γH2AX in HeLa cells in a time- and dose-dependent manner. BaP also induced γH2AX in ATM −/− mouse fibroblasts, DNA-PKcs −/− mouse fibroblasts, and a genetically modified human osteosarcoma U2OS cell line. PI3K inhibitors caffeine and wortmannin were then used in an effort to identify the kinase(s) responsible for BaP-induced γH2AX. Unexpectedly, in BaP-treated HeLa cells, caffeine pretreatment did not inhibit but rather increased γH2AX level. On the other hand, caffeine or wortmannin can inhibit BaP-induced γH2AX in either U2OS, DNA-PKcs −/− or ATM −/− cells. Taken together, these data suggest that BaP alone can induce H2AX phosphorylation in certain cell systems, and that members of the PI3K family, including ATM, ATR, and DNA-PK can participate in the phosphorylation of H2AX in the various cell types.

Phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α) alleviates benzo[a]pyrene-7,8-diol-9,10-epoxide induced cell cycle arrest and apoptosis in human cells

Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage and inducing extensive cell responses regulating cell cycle, cell survival and apoptosis. However, the mechanism of cellular responses to BPDE exposure is not fully understood. In this study, we demonstrated the involvement of the phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α) in the cellular response to BPDE exposure and addressed the role of eIF2α phosphorylation in the regulation of the cellular stress. Phosphorylation of eIF2α was induced in a normal human FL amnion epithelial cell line, and the expression of ATF4, a conserved downstream transcriptional factor of eIF2α phosphorylation, was up-regulated after BPDE exposure; however, the four known primary kinases for eIF2α phosphorylation (GCN2, HRI, PKR, and PERK) were not found activated. While BPDE induced severe cell cycle arrest and apoptosis and decreased cell viability in FL cells, salubrinal, a selective inhibitor of eIF2α dephosphorylation, maintained the eIF2α phosphorylation and attenuated cell cycle arrest and apoptosis and promoted cell survival. The findings reveal that when BPDE causes cellular damages, it induces eIF2α phosphorylation as well, which produces a pro-survival and anti-apoptotic effect to alleviate the cellular damages. Thus, the present study proposes a new cellular defensive mechanism during the environmental mutagen and carcinogen attack.

The Effects of Ultraviolet Irradiation on FL Cells

The Effects of Ultraviolet Irradiation on FL Cells

Determination of in vitro „anti-aging„-effects of Ganoderma pfeifferi

Although the term „anti-aging“ is often used, it is not well defined. We used a perfusion cell culture model [1] for the sensitive detection of real „anti-aging“ effects and demonstrate here the results obtained with the new medicinal mushroom Ganoderma pfeifferi BRES. G. pfeifferi is a European relative of the medicinal mushroom Ganoderma lucidum (CURTIS: FR.) P. KARST (Reishi, LingZhi) used in Asian medicine for the prophylaxis and treatment of several disorders including aging processes [2]. A perfusion cell culture was characterised by the continuous addition of fresh nutrient medium and the withdrawal of an equal volume of used medium, allowing the realization of cell cultivation conditions that are approximated as closely as possible to the in vivo situation. We used a human amniotic epithelial cell line (FL cells, ATCC, CCL 62). Glucose consumption and lactate production were continuously measured as parameters of cell viability. Because the proliferation of the cells was inhibited by mitomycin the cell number was nearly the same over the whole time of the experiment. Application of extracts of Ganoderma pfeifferi or of Ganoderma Maresome® [3] caused a significant increase in the glucose consumption of the cells and prolonged their life span in comparison to the untreated controls. Whereas the control cells died about 210h after starting of the perfusion cell culture the treated cells live more than 270h. The effects occur also after prophylactic application of the mushroom preparation and those of Ganoderma pfeifferi are stronger than those of G. lucidum.

Response of human DNA polymerase ι promoter to N-methyl- N′-nitro- N-nitrosoguanidine

Human Pol ι is a highly distributed, low-fidelity DNA polymerase lacking intrinsic exonuclease proofreading activity, thus its effects are strictly regulated. We predicted and cloned the promoter region of the human POLI gene. Successively, by transfection of deletion constructs of the POLI promoter, we demonstrated that the regions −848/−408 and −30/+215 contained positive regulatory elements, and the region +215/+335 had proximal promoter activity. Overexpression of Sp1 significantly increased the transcriptional activity of the promoter, and mutation of the Sp1 site reversed Sp1-induced promoter transactivation. Quantitative RT-PCR showed that POLI mRNA expression was up-regulated in human amnion FL cells treated by the carcinogen N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Reporter gene assays demonstrated that MNNG also significantly increased the transcriptional activity of the predicted promoter (−848/+335) and the proximal promoter (+215/+335). However, the promoter with the Sp1 site mutation had no response to MNNG treatment, suggesting that Sp1 plays an important role in the transcriptional regulation of the POLI gene stimulated by MNNG. Our data suggest that abnormal regulation of Pol ι may be involved in the mutagenesis and carcinogenesis induced by environmental chemicals.

A preliminary study on role of acid sphingomyelinase in receptor clustering induced by 50-Hz magnetic fields

To investigate the relationship among a 50-Hz MF-induced epidermal growth factor receptor (EGFR) clustering, acid sphingomyelinase (A-SMase) and ceramide (CER), and to explore the possible mechanism of receptor clustering. Human amnion (FL) cells were exposed to a 50-Hz sinusoidal magnetic field at 0.4 mT for 15 min with or without imipramine, a specific inhibitor of A-SMase and ceramide pretreatment. EGF treatment served as the positive control and DMSO treatment served as the solvent control. The EGFR was labeled with polyclonal anti-EGFR antibody and the clustering of EGFR was analyzed using immunofluorescence and confocal microscopy. The percentage of cells with EGFR clustering was counted and compared. Both EGF treatment and 50-Hz MF exposure could induce EGFR clustering. However, the effect could be eliminated by imipramine pretreatment for 4 hours. When FL cells were incubated with ceramide following the imipramine pretreatment for 30 min, EGFR clustering induced by 50-Hz MF exposure could be recovered. EGFR clustering induced by 50-Hz MF depends on A-SMase activity, and ceramide, as the hydrolyzate from A-SMase might participate in the process of EGFR clustering.

Low concentration of anti‐7,8‐dihydroxy‐9,10‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene induces alterations of extracellular protein profile of exposed epithelial cells

7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) exposure induces adduct formation and oxidative damage on DNA, and consequently triggers complicated stress responses, including such responses as signaling pathway activation, cell cycle arrest, DNA repair, translesion DNA synthesis and mutagenesis. In the present study, 2-DE and MALDI-TOF MS were employed to analyze the differential extracellular protein patterns of human amniotic epithelial cells (FL cells) after exposure to 5 nM BPDE and control. As a result, one protein spot that appeared in the culture medium of BPDE treatment group was successfully identified as 14-3-3zeta, and three up-regulated protein spots were identified as annexin A3, annexin V and hydroxypyruvate isomerase homolog. Among them, 14-3-3zeta was further detected in some pleural fluid specimens also. These results demonstrate that BPDE exposure can induce alterations of extracellular protein profiles of exposed cells, which may be served as a starting point for searching candidate biomarkers for benzo[a]pyrene exposure.

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts.

The involvement of mitochondria and the caspase-9 activation pathway in rituximab-induced apoptosis in FL cells

Despite the wide use of anti-CD20 antibody rituximab in the cancer treatment of B cell malignancies, the signalling pathways of CD20-induced apoptosis are still not understood. By using dominant negative (DN)-caspase-9 overexpressing follicular lymphoma cells we demonstrated that the activation of caspase-9 was essential for rituximab-mediated apoptosis. The death receptor pathway mediated by caspase-8 activation was not involved in rituximab-mediated apoptosis since overexpression of FLIP(short) or FLIP(long) proteins, inhibitors of caspase-8 activation, could not inhibit rituximab-induced apoptosis. However, the death receptor pathway activation by anti-Fas antibodies showed an additive effect on rituximab-induced apoptosis. The stabilisation of the mitochondrial outer membrane by Bcl-x(L) overexpression inhibited cell death, showing the important role of mitochondria in rituximab-induced apoptosis. Interestingly, the rituximab-induced release of cytochrome c and collapse of mitochondrial membrane potential were regulated by caspase-9. We suggest that caspase-9 and downstream caspases may feed back to mitochondria to amplify mitochondrial disruption during intrinsic apoptosis.

Genomic and phenotypic analysis reveals a key role for CCN1 (CYR61) in BAG3—modulated adhesion and invasion

Chaperone protein quantity may regulate the balance of proteins involved in invasion and malignancy. BAG3 is a co-chaperone and pro-survival protein that has been implicated in adhesion, migration, and metastasis. We reported that BAG3 overexpression in MDA435 human breast cancer cells results in a significant decrease in migration and adhesion to matrix molecules that is reversed upon deletion of the BAG3 proline-rich domain (dPXXP). We now hypothesize that transcriptional analysis would identify proteins involved in matrix-related processes that are regulated by BAG3 and/or its PXXP domain mutant. Expression array analysis of MDA435 cells overexpressing either wild-type BAG3 (FL) or dPXXP identified CCN1 as a BAG3 target protein. CCN1 is a known AP-1 target. Increased AP-1 transcriptional activity and AP-1 DNA-binding was found in MDA435 dPXXP cells. Consistent with these findings, CCN1 quantity and secretion were increased in dPXXP mutants but suppressed in FL cells; both BAG3 forms resulted in up-regulated CCN1 in HeLa cells. CCN1 silencing in the BAG3 FL overexpressors reduced the already low phospho-integrin beta1 in response to attachment on collagen IV. Matrigel invasion of HeLa cells engineered with the BAG3 constructs was enhanced in FL cells and minimal in dPXXP cells. CCN1 silencing blocked a greater percentage of the serum-induced invasion in FL cells than in dPXXP cells. This implies a context-dependent function of BAG3 on CCN1 and thus mesenchymal behaviour. CCN1 may be necessary for adhesion and matrix-related signalling in FL cells, abrogating a negative signal of the PXXP domain when BAG3 is intact. We propose that BAG3 regulates CCN1 expression to regulate tumour cell adhesion and migration.

Differences in heating methods may account for variation in reported effects on γH2AX focus formation

Previously we have shown that heat shock cannot induce DNA damage in several mammalian cell lines, using γH2AX foci formation as an indicator for DNA damage. However, there have also been reports showing heat shock can induce DNA damage. In an effort to clarify why such differences exist among different studies, we examined the effects of five different heating methods, e.g., (1) heat block; (2) submerged in water bath; (3) half-submerged in water bath; (4) pre-heated medium; and (5) heating oven, on γH2AX foci formation in human amnion FL cells and Chinese hamster CHL cells. It was found that during a 24 h period, heating at 42 °C for 30 min showed no significant cytotoxicity, while 45 °C exhibited various degrees of cytotoxicity. The ability of these five methods to induce γH2AX foci was quite distinct, with only heat block inducing γH2AX foci at both 42 °C and 45 °C in both FL and CHL cells. The two cell types also responded differently to the different heating methods, with CHL being more sensitive to heat shock. Taken together, it is clear that certain heating methods can induce γH2AX foci formation; on the other hand, their effects differ in thermo-tolerant and non-tolerant cells. And the difference in γH2AX foci formation observed in different reports most likely is a consequence of the different heating methods used in the study. However, expression of γH2AX may not be used as a surrogate of cell killing.

Suppressive effects of a cyanine dye against herpes simplex virus (HSV)-1 infection

In this study, we demonstrate that a cyanine dye, lumin, significantly suppressed cytopathic effect by herpes simplex virus (HSV)-1 toward human amnionic FL cell and also it reduced replication of HSV-1 in a dose-dependent manner. In addition, lumin additively augmented the antiviral effect of interferon (IFN)-alpha. Furthermore, fluorescence microscopic study showed that lumin (not IFN-alpha) itself remarkably induced alkalinization of intracellular organelle, suggesting the inhibition of virus invasion into the cells. These results suggest that lumin exerts an antiviral action against HSV-1 with the independent pathways of IFN-alpha and also it would become a therapeutically effective drug in clinical practice.

Identification of Temporal Differentially Expressed Protein Responses to Microcystin in Human Amniotic Epithelial Cells

The increased distribution of microcystins (MCs) has become a global issue. However, the MC response machinery has not been completely studied and elucidated until now. Our previous dose-related analysis has revealed that different concentrations of mirocystin-RR (MC-RR) can trigger comprehensive and various alterations in the protein expression profile using a proteomic approach. To further dissect the kinetics of cellular responses associated with MC-RR cytotoxic effects, the present study has set out to investigate the temporal protein expression pattern at three time points (3, 12, and 24 h after exposure). The relative intensity has changed significantly in MC-RR-treated FL cells, as compared with the control, in a total of 127 protein spots. One hundred and two proteins have been further identified with high confidence by peptide mass fingerprinting. These proteins can be categorized into diverse functional classes such as signal transduction, apoptosis, protein degradation, cell cycle, cell differentiation, transporter, and so forth. The wide range of different proteins involved suggests that MC-RR has profound effects on the biological response and toxic consequences of the affected cells. The identification of p53 and its potential targets confirms a known role for p53 in the MC-RR response. Moreover, it is noteworthy that MC-RR can up-regulate the expression of the PP2A A subunit and down-regulate the expression of a number of proteins implicated in the ubiquitin-proteasome pathway. Therefore, the present expression data provide a global view of dynamic changes in cell responses to MC-RR and, more importantly, generate novel hypotheses regarding MC-RR-responsive mechanisms.

Analysis of Aurora B Kinase in Non-Hodgkin's Lymphoma.

Aurora B kinase is one of the key regulators of mitosis and has been reported to be over-expressed in a various malignancies including acute myelogenous leukemia; however, expression of Aurora B in non-Hodgkin's lymphoma (NHL) and its role in lymphoma genesis remain to be fully elucidated. This study explored the levels of Aurora B in seventy four lymph nodes and tumor specimens excised operatively from individuals with various types of NHLs including diffuse large B cell lymphoma (n=22, DLBCL), follicular lymphoma (n=12, FL), Burkitt's lymphoma (n=10, BL), mantle cell lymphoma (n=4, MCL), mucosa associated lymphoid tissue (n=4, MALT), adult T-cell leukemia/lymphoma (n=11, ATL), peripheral T-cell lymphoma (n=8, PTCL), and Hodgkin's lymphoma (n=3, HL). Immunohistochemical examination found that DLBCL (11/22, 50 %) and BL (8/10, 80 %) cells highly (percentage of positive cells, >20 %) expressed Aurora B in their nucleus. On the other hand, FL (11/12, 92 %), MCL (1/4, 25 %), and MALT (1/4, 25 %) cells slightly (percentage of positive cells, 5–20 %) expressed Aurora B and none of them, except for one case of FL, highly expressed this protein kinase, suggesting that expression of Aurora B correlated with histological grade in B cell NHLs (p=0.0004). We next examined whether Aurora B could be a molecular target for treatment of NHLs utilizing BALM-14, -18, and -27, Daudi, Ramos, and Raji Burkitt's lymphoma/leukemia cells. Exposure of these cells to AZD1152, a selective inhibitor of Aurora B kinase, potently induced apoptosis in association with activation of caspase 3, as measured by annexin V staining and Western blot analysis, respectively. Moreover, we found that AZD1152 synergistically enhanced the effects of vincristine (VCR), a tubulin depolymerizing agent, to induce growth arrest and apoptosis of these cells. Further experiments found that VCR increased levels of the phosphorylated forms of Aurora B (p-Aurora B) via activation of c-Jun N-terminal kinase (JNK), which was blocked in the presence of AZD1152 in BALM-27 cells. Taken together, Aurora B kinase is a promising molecular target for treatment of individuals with aggressive types of NHLs. The combined administration of AZD1152 and the conventional chemotherapeutic agents to patients with NHL warrants further investigation.

Morphological and biochemical changes associated with apoptosis induced by okadaic acid in human amniotic FL cells

The marine toxin okadaic acid (OA) is an apoptosis inducer and a tumor promoter. During recent years, extensive studies have demonstrated that OA can induce apoptosis in a wide variety of cell types. In contrast to the relatively longer incubation time or higher treatment concentrations of OA in apoptosis shown previously, relatively lower concentrations (<or=100 nM) and shorter time (4 h) were designed in the current study to observe the toxic effects of OA in human amniotic cells (FL cells). The present study was undertaken to determine the morphological and biochemical changes of FL cells induced by OA. Results indicated that externalization of phosphatidylserine, cytoskeletal disruption, DNA strand breaks and decrease of Bcl-2 protein expression levels as well as increase of PP2A-A subunit protein were all involved in the apoptosis of FL cells induced by OA. This work not only provided further evidence of apoptosis induced by OA but also suggested that PP2A might play a pivotal role in apoptosis induced by protein phosphatases inhibitors.

Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line

Synthetic pyrethroids (SPs) are used in preference to organochlorines and organophosphates due to their high efficiency, low toxicity to mammals, and ready biodegradability. Previous studies reported that enantioselective toxicity of SPs occurs in aquatic toxicity. Several studies have indicated that SPs could lead to oxidative damage in humans or animals which was associated with their toxic effects. Little is known about the differences in the effects of chronic toxicity induced by individual stereoisomers of chiral SPs. The present study was therefore undertaken to evaluate the enantioselectivity in cytotoxicity, genotoxicity caused by bifenthrin (BF) on human amnion epithelial (FL) cell lines and pesticidal activity on target organism. The cell proliferation and cytoflow analysis indicated that 1 S- cis-BF presented more toxic effects than 1 R- cis-BF above the concentration of 7.5 mg L −1 ( p > 0.05). FL cells incubated with 1 S- cis-BF exhibited a dose-dependent accumulation of intracellular reactive oxygen species (ROS). In the comet assay, the number of cells with damaged DNA incubated with 1 S- cis-BF was more than that with 1 R- cis-BF ( p < 0.01). While the LC 50 values of enantiomer to the target pest on Pieris rapae L. show that 1 R- cis-BF was 300 times more active than 1 S- cis-BF. These results indicate that the enantioselective toxicity and activity of BF between non-target organism and target organism was reversal. These implications together suggest that assessment of the environmental safety and new pesticides development with chiral centers should consider enantioselectivity.