Engineered Cre and the mutated ligand-binding domain of estrogen receptor fusion recombinase (CreERT) allow temporal control of Cre activity.Cre/T, which show 4-hydroxytamoxifen (4-OHT)-inducible and spontaneous (4-OHT-independent) Cre activities, referred to Tg(BK5-CreERT)I and Tg(BK5-CreERT)S, respectively. The transgenic construct is driven by the bovine Keratin 5 promoter, which is active in the basal epithelial lineage of stratified and pseudo-stratified epithelium across multiple organs. Despite the difference in 4-OHT dependency, the Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mouse lines shared similar Cre-mediated recombination among various organs, except for unique mammary epithelial Cre activity in Tg(BK5-CreERT)S females.In this study, we have generated two distinct transgenic mouse lines expressing CreERThese two new transgenic mouse lines for the analysis of basal epithelial function and for the genetic modification have been created allowing the identification of these cell lineages and analysis of their differentiation during embryogenesis, during perinatal development and in adult mice. Howeveiewed in ,2) offerstradiol ,4. Thus,loxP recombination activity through female germ-line only, which potentially confines the breeding strategy available for the analysis of tissue-specific gene ablation, that is in generalized germ-line deleted strains [loxP-mediated recombination in utero or at adult stage; these have proved to be powerful genetic resources and have mostly concentrated on the analysis of epidermal development and disease [T driven by the bovine K5 promoter on an inbred (C57BL/6J) background in this report.Keratin 5 (K5) is a member of type II keratins and expresses with its type I keratin partner keratin 14 (K14) in the basal layer of stratified squamous epithelium (SSC) . Utilizi strains ,14. As a disease -19. To sBK5-CreERT transgenic plasmid , a 1.8-kb of EcoRI-digested/Klenow filled-in of Cre-ERT fusion gene derived from pCre-ER(T) plasmid (kindly provided by Dr. Richard R. Behringer with an agreement of Dr. Pierre Chambon), a 0.5-kb SV40 polyadenylation signal (pA) and 2 copies of the ~1.2-kb HS4 insulator sequence from the 5' region of the chicken β-globin locus . This 5'-HS4 served as a barrier element that protects genes from any chromosomal position effect, which has been thought to result in histone modifications via USF proteins [NotI/SalI-digested transgene was eluted and separated from the pBluscript vector backbone for pronuclear microinjection [The d Figure  was creaproteins ,21. The njection .Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mice bearing the same transgene, BK5-CreERT 26Sortm1Sor; the Jackson Laboratory) on a C57BL/6;129 mix background are referred to as R26R mice in this report. The Tg(BK5-CreERT)I/+;R26R/+ and Tg(BK5-CreERT)S/+;R26R/+ bigenic mice were generated by crossing R26R/R26R females with Tg(BK5-CreERT)I/+ and Tg(BK5-CreERT)S/+ males, respectively. Occasionally, the offspring of R26R/R26R males and Tg(BK5-CreERT)S/+ females were generated and are referred to as R26R;Tg(BK5-CreERT)S, as they are the progeny of Tg(BK5-CreERT)S/+ females; these were used for the analysis of spontaneous Cre activity through the female germ-line. The genotyping protocol for the R26R allele was according to the methodology for PCR genotyping presented on the web site of the Jackson Laboratory. Genotyping of the Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mice was determined by a 300-bp PCR product amplified by a forward primer (5'-GGACATGTTCAGGGATCGCCAGGCG-3') and a reverse primer (5'-CGACGATGAAGCATGTTTAGCTG-3'). The PCR conditions were 5 min at 94°C, 28 cycles for 1 min at 94°C, 1 min at 64°C, 1 min at 72°C and a final 2 min extension at 72°C. All animals were housed in microisolator cages (up to 5 mice per cage) using specific pathogen free husbandry. All experiments with mice were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at National Yang-Ming University.The T Figure  were gen4-hydroxytamoxifen  was dissolved in dimethyl sulfoxide  at a concentration of 25 mg/ml. The 4-OHT solution was emulsified in sunflower seed oil (Sigma) by vortex followed by mixing on a rotator for 4~6 hours. The mice were injected intraperitoneally with three doses of 4-OHT (4~5 mg/kg body weight) every other day.The detailed experimental procedures had been described previously . After XBK5-CreERT transgenic founders were obtained. Each transgenic founder was backcrossed to C57BL/6J for expansion of the individual transgenic line. All transgenic founders were fertile and transmitted BK5-CreERT transgenes to their progeny. To examine the Cre recombinase activity, BK5-CreERT mice were bred with ROSA26 Cre Reporter (R26R) mice [Tg(BK5-CreERT);R26R bigenic mice; this was followed by intraperitoneal 4-OHT injection. Among these four transgenic mouse lines, three lines exhibited 4-OHT induced Cre recombinase activity. We selected an efficient and tightly controlled 4-OHT inducible BK5-CreERT transgenic mouse line, referred to here as Tg(BK5-CreERT)I, to be the representative line for further study.Through pronuclear microinjection, four 6R) mice  to generTg(BK5-CreERT)I;R26R bigenic mice was assessed in terms of reporter (LacZ) gene expression S. The detailed mechanism that triggers this spontaneous Cre activity remained unclear. X-gal stained tissues of Tg(BK5-CreERT)S;R26R bigenic mice revealed that Cre activity initiated reporter LacZ gene expression started at around embryonic day (E) 14.5 S;R26R mice was undetectable at birth S mice might not exactly match that of endogenous K5 expression during organogenesis. Furthermore, in the urogenital system of the Tg(BK5-CreERT)S;R26R females at P7, the transitional epithelia lining of the bladder and the stratified epithelia lining of sinus vagina (SV) markedly exhibited β-galactosidase activity, whereas very few X-gal positively stained epithelial cells in the Müllerian vagina (MV) could be detected S transgenic mice exhibited female germ-line Cre activity, we obtained offspring S) from a cross of a R26R/R26R male and a Tg(BK5-CreERT)S/+ female and analyzed spontaneous β-galactiosidase expression compared to of control R26R mice. Our results showed that the X-gal positively stained patterns remained specific to the K5-expressing cell types as shown by examples of skin and trachea S skin, the β-galactosidase activity showed tissue/lineage-specific expression in the epidermal, hair follicles and sebaceous cells S trachea, intense X-gal-stained cells were found in the basal cells of the pseudo-stratified epithelium S mouse line does not behave as a female germ-line deleter as described in earlier reports [Tg(BK5-CreERT)S mice enabled us to bypass the female germ-line deletion effect and to use this line for Cre-mediate recombination in a tissue-specific manner during embryonic and early postnatal development.Previously Hafner et al and Ramirez et al. independently demonstrated that K5-Cre and K14-Cre express in female oocytes and have suggested their applicability as female germ-line deleter strains ,14. To da Figure  &3g. In s Figure , suggest reports ,14. The Tg(BK5-CreERT)S transgenic mice, but not in our Tg(BK5-CreERT)I transgenic mice, Cre-mediated recombination in the mammary gland was detected and X-gal-stained cells were observed in both the luminal epithelium and the basal epithelial cells of the mammary glands at P7 (data not shown), in virgins S;R26R anterior prostate (AP) lobes S and Tg(BK5-CreERT)I, which exhibit spontaneous and 4-OHT-inducible Cre activity driven by a bovine K5 promoter, respectively. For analysis of Cre/loxP-mediated recombination at the embryonic and early postnatal stages using Tg(BK5-CreERT)S mouse line, a breeding scheme that considers whether the male or the female is carrying the BK5-CreERT transgene is not necessary because there was no female germ-line Cre-mediated recombination in our Tg(BK5-CreERT)S females. The 4OHT-induced Tg(BK5-CreERT)I mouse line will enable us to perform conditional genetics in temporally and spatially controlled manner.Taken together, we reported two new transgenic mouse lines, These genetic resources generated in this study will not only help us to understand the biology of the skin, but will also support studies of the basal epithelial lineages of pseudo-stratified, stratified and transitional epithelia as they appear in the thymic medulla, the esophagus, the forestomach, the trachea, the bronchus, the bladder, the cervicovagina, etc. Moreover, glandular organs such as prostate glands, which are composed of K5-expressing basal epithelium, can also be a target organ using these new transgenic mouse lines; this will allow the genetic fine-dissection of basal epithelial function in terms of epithelial identity and differentiation.The authors declare that they have no competing interests.CCL performed the experiments and analyzed the data. LRY and JLC participated in the data interpretation. TFT contributed the materials for the initial design of transgenic construct. CMC performed the experiments, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.


M) dose-dependently decreased basal IL-6 secretion into medium, and also suppressed tumour necrosis factor (TNF-α)-induced IL-6 secretion. Both basal and TNF-α-induced IL-6 mRNA levels were dose-dependently lowered by MPA. Moreover, intramuscular injections of MPA (100 mg kg−1 twice a week) into nude mice bearing KPL-4 transplanted tumours significantly decreased serum IL-6 levels without affecting tumour growth and preserved the bodyweight of recipient mice. These findings suggest that suppression of IL-6 secretion from tumour cells, at least in part, causes the anticachectic effect of MPA. © 1999 Cancer Research CampaignInterleukin 6 (IL-6) is a multifunctional cytokine. Recent reports suggest that circulating IL-6 secreted from tumour cells plays an important role in cancer-induced cachexia. Medroxyprogesterone acetate (MPA) has been used as an endocrine therapeutic agent for patients with breast cancer. It has been suggested that MPA decreases serum IL-6 levels and preserves the bodyweight of patients with advanced breast cancer. However, the mechanisms of action responsible for the anticachectic effect of MPA have not been elucidated. Therefore, the effects of MPA on IL-6 secretion were studied both in vitro and in vivo using a human breast cancer cell line, KPL-4, which secretes IL-6 into medium and induces cachexia when injected into female nude mice. MPA (10–1000 n


Therefore, 99mTc-MIBI was evaluated in the GLC4 cell line and its doxorubicin-resistant MRP-, but not P-gp-, overexpressing GLC4/ADR sublines as well as in the S1 cell line and its MRP-transfected subline S1-MRP. 99mTc-MIBI concentration decreased in the GLC4/ADR sublines with increasing MRP overexpression and was lower in S1-MRP than in S1. 99mTc-MIBI plus vincristine increased 99mTc-MIBI concentration in GLC4 lines compared with 99mTc-MIBI alone. 99mTc-MIBI efflux raised with increasing MRP expression in the GLC4 lines. Glutathione depletion elevated 99mTc-MIBI concentration in GLC4/ADR150x. Cross resistance for 99Tc-MIBI, used to test cytotoxicity of the Tc compound, was observed in GLC4/ADR150x vs GLC4. 99Tc-MIBI induced a synergistic effect on vincristine cytotoxicity in GLC4/ADR150x. These results show that 99mTc-MIBI is involved in MRP-mediated efflux. The fact that 99mTc-MIBI efflux is influenced by MDR1 and MRP expression must be taken into account when this gamma-rays-emitting complex is tested for tumour efflux measurements.


Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different sets of target genes at various times and in diverse cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. We have used the Nuclear Spot Assay and GFP reporters to examine PHA-4 interactions with target promoters in living embryos and with single cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen of candidate nuclear factors identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx, but emr-1 did not modulate PHA-4 binding in the intestine. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, may facilitate promoter access and productive transcription. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells, preventing target gene expression in that organ. PHA-4 binding within the pharynx is limited by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.Central regulators of cell fate, or selector genes, establish the identity of cells by direct regulation of large cohorts of genes. In  Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different target genes in different cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. Here we examine PHA-4 interactions with target promoters in living embryos and with single-cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, facilitates promoter access. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells and is limited in the pharynx by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.Central regulators of cell fate establish the identity of cells by direct regulation of large cohorts of genes. In  Selector genes govern the fates of groups of cells related to each other by virtue of their cell type, position or affiliation to an organ pha-4/FoxA plays a broad role in the development and physiology of the C. elegans digestive tract. PHA-4 establishes the diverse cell types of the C. elegans pharynx during early embryogenesis, and drives differentiation and morphogenesis at later stages The selector gene Studies have implicated the nuclear periphery for modulation of gene transcription. Active and inducible genes are recruited to nuclear pores C. elegans is composed of a single B-family lamin . These constructs revealed that pax-1 was expressed in 14 pharyngeal cells, which included nine marginal cells, the e2 epithelial cells and the pm8 muscle, based on morphology, position and co-staining for marginal cell filaments  upstream of the transcriptional start site  lies within Delta14 and Delta16, and an inverted repeat (AAGCTCT) lies within Delta14 and Delta18, suggesting one or both of these may be recognition sites for transcription factors  to examine association of PHA-4 with pharyngeal promoters in vivo. The NSA allowed us to track PHA-4 binding to promoters in living embryos, with precise spatial and temporal resolution. For this assay, we constructed a transgene array or “pseudo-chromosome” that carried multiple copies of a target promoter and the Lac operator C. elegans' sequences C. elegans arrays are relatively stable through mitosis and meiosis, and are incorporated into chromatin C. elegans chromosomes, and they likely differ from endogenous chromosomes in some regards.The mutational analysis suggested that both myo-2 and pax-1 are direct PHA-4 targets (C44H4.1: pax-1) or three-fold (myo-2) stages. The robust association of PHA-4 to its target promoters required a consensus PHA-4 binding site since pseudo-chromosome arrays that carried a promoter with mutated PHA-4 binding sites We observed multiple pharyngeal cells with PHA-4::YFP enriched on pseudo-chromosome arrays, supporting the notion that C44H4.1: . The assmyo-2What are the repercussions of PHA-4 association to target genes? Previous genetic studies suggested that PHA-4 and its orthologues influence the chromatin environment myo-2 or pax-1 promoters, detectable as a large, diffuse dot  and the bean stage for pax-1 (p = 0.007). For myo-2, many pseudo-chromosomes became decompacted prior to transcription at the 2-fold stage and remained decompacted. For pax-1, decompaction began at the comma stage and was maximal at the 1.5 and 2-fold stages, when pax-1 is transcribed ), whereas pax-1 mutP arrays bearing a mutated PHA-4 binding site did not (−14). This result suggests that productive transcription is not essential for decompaction, and that PHA-4 association is sufficient. To test this idea more stringently, we created arrays bearing three repeats (3X) of a PHA-4 binding site derived from pax-1, but lacking additional promoter sequences. The 3X repeats were sufficient for PHA-4::YFP recruitment to the pseudo-chromosome and caused large-scale decompaction  in one mid-section focal plane. Pharyngeal binding was detected in ∼67% of embryos at the bean stage (10/15), ∼58% at the comma stage (7/12), and ∼76% at the 1.5-fold stage . By contrast, binding was almost never detected in the intestine at any stage . An optical section through a 1.5-fold embryo sampled approximately 10 pharyngeal nuclei and 10 intestinal nuclei, indicating that the differential association of PHA-4 did not reflect different numbers of nuclei in each organ.PHA-4 is expressed broadly, including the pharynx, intestine, rectum, somatic gonad and some neurons ntestine . We wondpax-1::GFP. HS::PHA-4 induced widespread expression of pax-1::GFP in many cells. However, we did not observe pax-1::GFP in the developing intestine (0/50)  . This abe (0/50) . We detee (0/50) , nor didpax-1 promoter because pax-1-containing arrays typically bound PHA-4::YFP in fewer pharyngeal cells than myo-2-containing arrays (data not shown). We surveyed genes involved in chromatin modification such as histone demethylation, methylation, acetylation and RNA interference. Given the proximity of the pseudo-chromosomes to the nuclear lamina, we also tested genes involved in nuclear envelope structure and function. We counted the number of nuclei with bound PHA-4::YFP in a section that passed through the pharynx and intestine of comma to 1.5 fold embryos.To begin to understand the selective binding of PHA-4 in different cell types, we conducted a small RNAi screen for nuclear factors that modulate PHA-4 binding to target promoters. We used SM1634 carrying a mutant emr-1/Emerin had the most dramatic effect on PHA-4 binding  embryos (1 of 88 embryos (1.25%) in three experiments). These results reveal that the nuclear lamina interferes with binding of PHA-4::YFP to its targets within pharyngeal cells, but that additional processes function in the intestine.Of 28 genes surveyed,  binding . In the n embryo . Inactivn embryo . Althougemr-1(RNAi) strongly lowered expression of EMR-1 protein (emr-1(RNAi) embryos could be a consequence of increased accessibility. 32% (28/88) of emr-1(RNAi) embryos had decondensed arrays compared to 17% (9/52) for wild-type  embryos was not surprising given that set-1 has been implicated in transgene silencing set-1(RNAi) embryos did not lead to increased PHA-4::YFP association  nuclei, and a greater proportion of arrays were decondensed compared to those in wild-type embryos  embryos had PHA-4::YFP localized to pseudo-chromosomes , and affected embryos are healthy and viable . HS::pha-4 cannot activate pax-1::GFP within intestinal cells (this study) or convert nascent intestinal cells to a pharyngeal fate C. elegans MyoD homolog hlh-1 induces the body-wall muscle program throughout the embryo, including developing intestinal cells The association of PHA-4::YFP with pseudo-chromosomes was not constitutive, but responded to the cellular milieu in two ways. First, within the pharynx, PHA-4::YFP binding was restricted by EMR-1/emerin. EMR-1/emerin resides at the nuclear lamina, which suggests that tethering of pharyngeal genes or trans-acting factors at the nuclear periphery may modulate PHA-4 binding. We note that the nuclear lamina appears normal after mutA promoter or 3X PHA-4 binding site repeats recruit PHA-4::YFP and undergo decompaction, in the absence of GFP production. In Drosophila, nucleosomes are lost rapidly at heat-shock loci prior to transcription, and this loss extends across several kilobases upstream and downstream of the activated gene What is the nature of PHA-4-induced chromatin restructuring? The global decompaction we observe is consistent with a disordered structure, such as decondensation by loss of nucleosomes and/or reconfiguring of chromatin into loops or coils pax-1 expressing cells is that they share a lineage relationship. We identified 11 of the 14 pax-1::GFP+ cells unambiguously, and found that each of these cells derived from the posterior daughter of the penultimate cell division  cell divisions in which pairs of A-P siblings are distinguished by high (anterior) or low (posterior) levels of nuclear POP-1, a TCF transcription factor (A/T)CAAAG pax-1 and A-P specification remains a mystery.An interesting feature of n factor . Loss ofpax-1 expression in fourteen pharyngeal cells. The first was an enhancer element likely recognized by PHA-4 and defined by D6. PHA-4 can bind this sequence in vitroin vivo (this study). Moreover, this site is required for pharyngeal expression (this study), and multimers of this sequence respond to PHA-4 in vivopax-1 expression within the pharynx, it also led to ectopic expression in non-pharyngeal cells such as epidermis. M05B5.2 and T05E11.3 are two additional PHA-4 target genes C. elegans Fox genes did not result in ectopic expression in lines carrying the wildtype M05B5.2 reporter . This result suggests that multiple Fox proteins function redundantly to repress epidermal expression, or alternatively, that an unrelated protein acts through the predicted PHA-4 binding site.Our promoter analysis identified four regulatory elements that establish pax-1 activation. The Delta16 region contains a match to a GATA-2,3 binding site . We note that the sequence TTGAGA lost in Delta16 is half of a direct repeat, with a second copy located within Delta14  lies within these sequences, with one copy entirely within Delta20 and a second spanning Delta22 and Delta24. An appealing model is that PHA-4 promotes expression within the pharynx in combination with Delta16 sequences. The broad activation is refined by the repression elements embedded in Delta14/Delta18 and Delta20/Delta22/Delta24. The combination of four cis-regulatory sites explains why pan-pharyngeal PHA-4::YFP can bind its target promoters, yet those targets become transcriptionally active in only a subset of pharyngeal cells and after PHA-4 is first expressed.A second enhancer element defined by Delta16 contributes to  Delta14 . Abuttin Delta18 . Two addin vivo results expand our understanding of PHA-4/FoxA function in driving pharyngeal transcriptional programs. Moving beyond the Nuclear Spot Assay, it will be interesting to investigate the binding and down-stream consequences of PHA-4 in its native environment, at endogenous loci.We have demonstrated that the master regulator PHA-4 binds to its pharyngeal targets hours before the onset of gene expression. PHA-4 binding and activity is restricted in the intestine and negatively regulated by EMR-1/emerin in the pharynx. The association of PHA-4 with target promoters led to large-scale chromatin decompaction, which may facilitate chromatin-associated processes such as transcription. These Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR), unless stated otherwise. Bristol N2 was used as the wild-type strain. The following mutation was used LGIV: cha-1(p1182). For pax-1::GFP analysis the following transgenic strains were used: SM202 pxls2(pax-1::GFP + pRF4), SM699 N2(pax-1::GFP + pRF4), SM707 N2(pax-1 mutP-pro::GFP + pRF4), SM658 N2(pax-1 mutA-pro::GFP + pRF4), SM660 N2(pax-1 Delta14-pro::GFP), SM700 N2(pax-1Delta18-pro::GFP). For Heat Shock: SM259 pxEx(HS::PHA-4 + pax-1::GFP + UL8::lacZ + pRF4 + 1 KB ladder + Herring Sperm DNA)cha-1(p1182); pxEx(cha-1 + his-24pro::CFP::LacI + pha-4::yfp+ lacO + Herring Sperm DNA), SM1476 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP +myo-2proWT + lacO + Herring Sperm DNA), SM1429 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP +myo-2proWT + lacO + Herring Sperm DNA), SM1443 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP +myo-2 mutP + lacO + Herring Sperm DNA), SM1444 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP +myo-2 mutP + lacO + Herring Sperm DNA), SM1432 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP + pax-1proWT + lacO + Herring Sperm DNA), SM1434 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP + pax-1proWT + lacO + Herring Sperm DNA), SM1463 cha-1(p1182); pxEx(cha-1 + htz-1pro::CFP::LacI + PHA-4::YFP + pax-1 mutP + lacO + Herring Sperm DNA), SM1628 cha-1(p1182); pxEx(cha-1 + his-24pro::CFP::LacI + PHA-4::citrineYFP + pax-1Delta6proMut + lacO + Herring Sperm DNA), SM1564 cha-1(p1182); pxEx(cha-1 + his-24pro::CFP::LacI + PHA-4::citrineYFP + pax-1 mutA + lacO + Herring Sperm DNA), SM1634 cha-1(p1182); pxEx(cha-1 + his-24pro::CFP::LacI + PHA-4::citrineYFP + pax-1 mutA + lacO + Herring Sperm DNA), SM1523 cha-1(p1182); pxEx(cha-1 + his-24pro::CFP::LacI + PHA-4::citrineYFP + 3X low affinity pha-4 site + lacO + Herring Sperm DNA). SM1876 cha-1(p1182); stIs10389 (pha-4::gfp::3xFLAG); pxEx. Transgenic worms used for the Nuclear Spot Assay (NSA) were grown at 24°C on an E. coli OP50 lawn or on RNAi plates (see below).Strains were maintained as described in pax-1::GFP cytoplasmic translational fusion construct (BSEM74): a 4.6 kb genomic SacI DNA fragment was cloned from K07C11.1 into pBluescriptIISK+. The resulting plasmid was digested with NsiI, which is located within the pax-1 locus, and XbaI from the polylinker, to generate a 3 kb pax-1 fragment that was inserted into PstI/XbaI-digested pPD95.77 (A gift from Dr. Andrew Fire). The resulting pax-1::GFP reporter contained approximately 2.4 KB upstream sequences, with GFP fused to pax-1 within the second predicted exon of pax-1. The transcriptional pax-1 nuclear construct (BSEM274) was made starting with the cytoplasmically-expressed pax-1::GFP translational construct, we created a transcriptional fusion by removing all coding sequence. We performed inverse PCR using primers containing BglII tails that flanked the region to be deleted. The linear PCR product was then digested with BglII and re-ligated, placing the GFP translational start site at the same position as the one removed for pax-1. PCR products for injection were generated using pax-1 5′ as the forward primer and pax-1 3′ as the reverse primer.For the unc-54 poly A addition site was removed. This generates a pax-1::GFP transcriptional fusion with the coding sequence for histone H2B fused to the 3′ end of GFP. 276, 277,279,280.The transcriptional fusion construct was modified for use in the scanning mutagenesis. A 1.2 kB Bst1071I fragment was removed from BSEM274, and replaced with a 1.9 kb Bst1107I/ApaI fragment from pAP.10 that extends from within the GFP coding region through the pha-4::citrineYFP for the nuclear spot assay was created using QuickChange site directed mutagenesis . Two mutations, V68L and Q69M (5′-GTT-CAA-3′ mutated to 5′-CTT-ATG-3′), were introduced into the YFP sequence of the pha-4::YFP (SEM962) YFP into citrineYFPYFP FW CitrineCTTATGTGCTTCGCCAGATACCCAGATC-3′5′-GTCAC TACTTTCGGTTATGGT and YFP RV CitrineCATAAGACCATAACCGAAAGT AGTGAC-3′5′GATCTGGGTATCTGGCGAAGCA.TATTTGTCCCTACTATTTGTCCCTACTATTTGTCC-3′5′-CTAC Bottom ACAAATAGTAGGGACAAATAGTAGGGACAAATAGTA-3′5′ GGG (underlined are the PHA-4 binding sites). Oligos were diluted to a concentration of 2 mg/ml and heated to 95°C for 3 minutes. The temperature was dropped 0.10/sec until 20°C was reached to hybridize the oligos.3X low affinity PHA-4 binding site oligos were designed as in rol-6(su1006)) as a co-injection marker Reporter constructs were injected into the germ line of hermaphrodites and stable F2 transgenic roller lines examined. All reporter constructs were injected at 0.5 ng/ml as PCR fragments. Low concentrations of PCR products circumvented artificial expression in pharyngeal cells that has been observed with plasmid constructs Antibody staining was performed as described previously All constructs for the scanning mutagenesis were constructed using an inverse PCR strategy. For each mutant, we used a specific pair of primers that flank the 10 bp region to be altered. These primers each carry 5′ tails that contain a restriction site (PstI or ClaI). Following inverse PCR (BSEM279 as template), the linear PCR product was digested with the appropriate restriction enzyme (PstI or ClaI) and re-ligated. Each resulting reporter plasmid contains the restriction site plus a variable number of base pairs in place of the 10 bp of wild-type sequence. For injection, PCR products were generated from each mutant plasmid. All constructs were sequenced to confirm the predicted sequence.pha-4 binding sites (SM1523), myo-2 wild-type promoter  bearing two high affinity PHA-4 binding sites myo-2 promoter bearing mutagenized FoxA sites  pax-1 wild-type promoter , pax-1 promoter with a mutagenized FoxA site  and pax-1 promoter with a mutagenized positive regulator site  (see below). SM1560 was created by injecting cha-1(p1182) worms with Xho1-linearized pha-4::citrineyfp plasmid (bSEM1045) (1 ng/µl), his24promoter::CFP::LacI PCR product cha-1 plasmid  linearized with Apa1 (2 ng/µl) for rescue, and sheared herring sperm DNA to make 100 ng/µl total DNA. For SM1476 and SM1429, 499 bp of the endogenous myo-2 promoter upstream of the start codon was used in addition to the components listed for SM1560 with one difference, CFP::lacI expression was driven by the htz-1 promoter (BSEM995) myo-2 promoter bearing two mutated PHA-4 binding sites pax-1 promoter upstream of the start codon was used (the fragment contains one PHA-4 binding site (TGTTTGC)). SM1463 carried an altered version of the 240 (bp) pax-1 promoter in which the PHA-4 binding site was mutated from TGTTTGC to ATCGATT (MutP). Both SM1463 and SM1434 were injected with htz-1pro::CFP::LacI. For SM1564 and SM1634 a positive regulator site −40 to −50 upstream of the TSS was mutated from TTGAGATTAA to CAATCGATTG. SM1876 was created by injecting SM1754 cha-1(p1182); stIs10389 (pha-4::gfp::3xFLAG) worms with cha-1, a 440 (bp) M05B5.2 promoter fragment htz-1pro::mCherry::LacI that has a premature stop codon at the end of the mCherry sequence, thus failing to make any mCherry::LacI. SM1876 was used to examine whether decompaction reflected an artificial interaction between LacI and PHA-4. Nuclear spot assays were performed as described previously pax-1 mutA) or a Leica DM RXE confocal (for everything else).Transgenic lines for the Nuclear Spot Assay were as follows: no target control (SM1560), 3X low affinity cha-1(p1182) (25°), and treated with bleach to synchronize embryos. Four 10 cm OP50 plates of moving, nonCha-1 L3 animals were harvested for DNA isolation by phenol chloroform extraction and ethanol precipitation. qPCR was performed for promoter regions and normalized to act-1 using a LightCycler PCR machine with LightCycler FastStart DNA MasterPlus SYBR Green 1 kit (Roche) for quantitation. qPCR indicated a copy number of ≤200 for each promoter.To determine copy number, worms were grown at restrictive temperature for Gravid mothers were dissected and embryos collected in a PCR tube. Heat-shock was administered in a PCR machine. Embryos were initially incubated at 20°C for 75 min. After the initial incubation, the temperature was raised gradually to 33°C at a rate of 0.1°C/second. Embryos were then incubated at 33°C for 30 minutes. Following heat shock, the temperature was gradually lowered to 20°C at a rate of 0.1°C/second, and embryos incubated at 20°C for 5 hours.+ cells using an intensity threshold. The CFP::LacI classifier included separation of touching objects, removal of noise and exclusion of objects smaller than 0.25 micron2. The PHA-4::YFP classifier was modified to remove noise. CFP::LacI areas within PHA-4::YFP+ cells were considered “inside the pharynx” and the remainder as “outside the pharynx.” Proofreading of selections was performed blind by comparing measurements with images. Area measurements (INT Area (micron2)*10) were analyzed using Cox regression models to evaluate differences in chromosome area with location (inside the pharynx versus outside), developmental stage, or transgenic line. While Cox regression models were originally developed for analyzing survival data, their semi-parametric nature made them suitable for analyzing data following a non-standard distribution that was difficult to capture parametrically.Perkin Elmer Volocity was used to calibrate images for true X and Y pixel dimensions to ensure accurate spatial measurements. Classifiers were designed to select CFP::LacI areas and PHA-4::YFPgfp, spr-5, rbr-2, npp-11, emr-1, nhr-60, ima-3, lem-3, zyg-12, lmn-1, lin-49, lin-59, set-17, set-16, set-2, set-1, met-2, hda-3, hda-4, tsn-1, prg-1, sago-2, prg-2, csr-1, top-1, ergo-1, chd-3, tam-1, lin-35, or hil-7 were grown in liquid cultures for 8 hours and seeded onto plates containing 8 mM IPTG (Sigma) and 50 g/ml Carbenicillin (Sigma). All RNAi clones were derived from the Ahringer library 5′-GAGTGAGCTGATACCGCTCG-3′ and pPD129_rev 5′-CACGACGGTGTATTTCGACGGC-3′ primers at the Dana-Farber/Harvard Cancer Center DNA Resource Core. Adult SM1634 worms were bleached and ∼50 embryos were placed on RNAi plates (Po). For every experiment, F1 progeny embryos from 5 6 cm plates were collected by bleaching and analyzed, and each experiment was repeated at least twice. Images were acquired from live embryos using an Olympus FluoView FV1000 confocal microscope and DeltaVision RT Deconvolution System and SoftWoRx software (Applied Precision). A multitrack setting was used to acquire separate CFP and YFP images from slices through the pharynges of comma to 1.5fold-stage embryos. Embryos were scored for general non-tissue specific extrachromosomal array decompaction and for the number of nuclei containing PHA-4-::YFP colocalized with CFP::LacI.RNAi by bacterial feeding was performed similarly to Figure S1pax-1::GFP+ pharyngeal cells. (A) Cell nuclei positions in the pharynx (Adapted from pax-1::GFP. (B) the Lineage of pax-1::GFP+ pharyngeal cells. Eleven of these express pax-1::GFP, and the 12th (e1VR) may as well, although this has not been confirmed unambiguously.Lineage of (10.21 MB TIF)Click here for additional data file.Figure S2pax-1 promoter. The PAX-1::GFP cytoplasmic expression construct  was used for this analysis. Expression is pharyngeal, but identification of individual cells was difficult. Magenta box indicates the PHA-4 binding site (TGTTTGC). Progressively larger deletions from the original 2.5 kb upstream sequence resulted in a gradual loss of GFP intensity, with eventually a complete loss of expression when the predicted PHA-4 site was removed. Images representative of strong, weak and no GFP expression are shown below the schematic.Deletion analysis of the (5.10 MB TIF)Click here for additional data file.Figure S3pax-1MutP::GFP reporter in epidermal cells (A) and seam cells (B).Ectopic expression of (2.30 MB TIF)Click here for additional data file.Figure S4pax-1 promoter. (A) D18 resulted in an increased number of pharyngeal cells expressing the GFP reporter (20 cells), but with no significant non-pharyngeal expression. (B) D20 displayed increased numbers of GFP-expressing cell in the pharynx, as well as non-pharyngeal expression, epidermal expression is shown here.Characterization of negative regulatory elements in the (1.78 MB TIF)Click here for additional data file.Figure S5PHA-4 binding to an additional pharyngeal target. (A) Quantitation of embryos with co-localized CFP::LacI and PHA-4::YFP in two transgenic lines bearing a WT C44H4.1 (1 kb) promoter. (B) Numbers of embryos scored for binding for all transgenic lines in this study. (C) PHA-4::GFP binding to a Nuclear Spot Assay array bearing the promoter of M05B5.2 in a transgenic line that lacks mCherry::LacI. Binding to the array and decompaction is observed as an intense PHA-4::GFP signal  compared to a transgenic line expressing PHA-4::GFP without any target promoter (Right image). (D) PHA-4 binding is maintained on mitotic chromosomes (Arrows) (E) The diameter of pharyngeal nuclei at different developmental stages.(8.08 MB TIF)Click here for additional data file.Figure S6emr-1 RNAi reduces the expression of EMR-1 in all cells. (A) EMR-1 antibody stain reveals its position at the nuclear periphery in a nuclear spot assay transgenic line (B) EMR-1 signal is lost after RNAi. A secondary antibody against LacI was used as a positive control for antibody staining .(5.57 MB TIF)Click here for additional data file.Figure S7pax-1 promoter or a MutP pax-1 promoter. Three embryos per stage were analyzed. Each dot on the plot represents a pseudo-chromosome.Comparison of area measurements versus volume measurements for array size. (A) Area or (B) Volume of pseudo-chromosomes in the pharynx were measured at the comma and 1.5Fold stage in transgenic lines carrying either a WT (7.06 MB TIF)Click here for additional data file.Table S1pax-1 reporters are activated at the bean stage. (A) GFP expression assayed in two transgenic lines. (A) is a line carrying a transcriptional fusion of pax-1WTpro::GFP. Onset of expression was detected at the bean stage. This expression pattern was recapitulated using an integrated PAX-1::GFP translational fusion. (B) Mutations in a predicted PHA-4 binding site (mutP) or a second activation site (mutA) interfere with activation at any stage. n =  number of embryos.(2.64 MB TIF)Click here for additional data file.Table S2Number of nuclei assayed for pseudo-chromosome size. The numbers are broken down per promoter, developmental stage and for the location of pseudo-chromosomes inside the pharynx versus outside the pharynx.(6.97 MB TIF)Click here for additional data file.Table S3Number of embryos assayed for de-compaction for each RNAi treatment. (A) Colors indicate different categories. (B) the number of embryos assayed for the proportion of bound arrays.(9.28 MB TIF)Click here for additional data file.


Hs578T human breast cancer cells are an oestrogen receptor (ER)-negative cell line. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in formation of a 6.9 S nuclear aryl hydrocarbon (Ah) receptor complex, which bound to a [32P]dioxin-responsive element in a gel electrophoretic mobility shift assay. However, TCDD does not induce CYP1A1 gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNH11c or pMCAT5.12, which are Ah-responsive plasmids derived from the 5'-flanking region of the human and murine CYP1A1 genes respectively. Restoration of Ah responsiveness was investigated by co-transfecting Hs578T cells with pRNH11c or pMCAT5.12 and plasmids that express the ER (hER), Ah receptor (AhR) and AhR nuclear translocator (Arnt) proteins. ER expression resulted in significantly increased basal CAT activity; however, TCDD did not induce CAT activity in the transiently transfected cells. Expression of the AhR or Arnt proteins did not alter basal or inducible CAT activity. Expression of N- or C-terminal truncated ER in Hs578T resulted in differential regulation of Ah responsiveness. In Hs578T cells transiently expressing the ER, which contains C-terminal deletions (amino acids 282-595), basal CAT activity was also increased; however, Ah responsiveness was not restored. In contrast, transient expression of N-terminal-deleted (amino acids 1-178) ER resulted in a marked decrease in basal CAT activity but a restoration of Ah responsiveness. These results suggest that basal and inducible CAT activity in Hs578T cells transiently transfected with pRNH11c is modulated differentially by ER domains that are present in the N- and C-terminal regions of the ER.


We have investigated the effects of 50 min whole-body hyperthermia  on the toxicity and pharmacokinetics of the radiosensitizer Ro 03-8799 in mice. WBH markedly reduced Ro 03-8799 LD50/7d from 779 to 259 micrograms g-1 (P less than 0.001). Pharmacokinetics were studied at 175 micrograms g-1 (approximately 0.6 WBH LD50/7d) with and without heat and 437 micrograms g-1 (approximately 0.6 control LD50/7d) without heat. WBH increased Ro 03-8799 plasma concentrations and prolonged its elimination t1/2 by 26% (P less than 0.01). Total plasma area under the curve (AUC0-infinity) was increased by 22%, but was still less than 50% of the unheated high-dose value. Ro 03-8799 concentrated 300-400% in tumour and brain relative to plasma. Absolute tumour and brain levels were unaltered by WBH, giving reduced tissue/plasma ratios. WBH greatly inhibited glomerular filtration (51Cr EDTA clearance) during heating, contributing to the increased plasma Ro 03-8799 concentrations. WBH increased peak plasma concentrations of the Ro 03-8799 N-oxide metabolite Ro 31-0313 by 61% and the beta-phase AUC of i.v. administered Ro 31-0313 by 36%. Since Ro 31-0313 levels were increased to a greater extent after Ro 03-8799 and WBH than Ro 31-0313 and WBH, WBH must both increase metabolite production and decrease its plasma clearance. WBH had no effect on Ro 31-0313 tumour concentrations or its exclusion from brain. These complex effects of WBH on Ro 03-8799 pharmacokinetics may contribute to the enhanced toxicity, possibly through hyperthermia-stimulated bioreductive drug activation, but do not wholly explain it.


The possibility that cultured keratinocytes produceendothelins were investigated. The results showed thatcultured keratinocytes derived from normal humanskin produce endothelin-1. Moreover, keratinocyteendothelin-1 production was completely inhibited by thepresence of actinomycin D in the medium. As in the caseof endothelial cells, recombinant interleukin-1β wascapable of promoting endothelin-1 production inkeratinocytes, whereas herapin inhibited it. Thrombinalso inhibited endothelin-1 production. These results indicatethat the mechanism of endothelin-1 production inkeratinocytes is slightly different from the mechanism invascular endothelial cells.


NK- and T-cells are closely related lymphocytes, originating from the same early progenitor cells during hematopoiesis. In these differentiation processes deregulation of developmental genes may contribute to leukemogenesis. Here, we compared expression profiles of NK- and T-cell lines for identification of aberrantly expressed genes in T-cell acute lymphoblastic leukemia (T-ALL) which physiologically regulate the differentiation program of the NK-cell lineage.This analysis showed high expression levels of HOXA9, HOXA10 and ID2 in NK-cell lines in addition to T-cell line LOUCY, suggesting leukemic deregulation therein. Overexpression experiments, chromatin immuno-precipitation and promoter analysis demonstrated that HOXA9 and HOXA10 directly activated expression of ID2. Concomitantly elevated expression levels of HOXA9 and HOXA10 together with ID2 in cell lines containing MLL translocations confirmed this form of regulation in both ALL and acute myeloid leukemia. Overexpression of HOXA9, HOXA10 or ID2 resulted in repressed expression of apoptosis factor BIM. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including components of polycomb repressor complex 2 (PRC2), indicated lacking expression of EZH2 in LOUCY and exclusive expression of HOP in NK-cell lines. Subsequent treatment of T-cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively. Moreover, siRNA-mediated knockdown of EZH2 in JURKAT enhanced HOXA10 expression, confirming HOXA10-repression by EZH2. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression.Our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 which therefore contributes to T-cell leukemogenesis. Adult lymphopoiesis starts with progenitor cells which originate from CD34+ hematopoietic stem cells (HSC) in the bone marrow. While the development of natural killer (NK)- cells completes primarily in the bone marrow, T-cells finalize their differentiation in the thymus -3. NeverChromosomal juxtaposition of the HOXA gene cluster with T-cell receptor (TCR)-beta via inv(7)p15q34) or t results in ectopic expression of several HOXA genes ,15. Tranq34 or t, PRC1 and PRC2, have been identified, comprising, firstly, BMI1 together with CBX4 and, secondly, EED together with EPC1, EZH2 and SUZ12 -24. EZH2The aim of our study was to identify developmental oncogenes and their deregulating mechanisms in T-ALL cells. Therefore, we compared gene expression profiles of NK- and T-cell lines and identified the conspicuous expression of HOXA9, HOXA10 and ID2 which may represent the physiological situation in the differentiation process of NK-cells but aberrant activity in one T-ALL cell line. Analysis of genes, coding for chromatin activators/repressors, revealed the (de)regulatory impact of two PRC2 components in lymphoid HOXA gene expression.Cell lines were supplied by the DSMZ . Cultivation was performed as described by Drexler . For stiTotal RNA was extracted from cells using TRIzol reagent . cDNA was subsequently synthesized from 5 μg RNA by random priming, using Superscript II (Invitrogen).http://rana.lbl.gov/EisenSoftware.htm.For quantification of gene expression via profiling we used DNA chips U133A Plus 2.0 obtained from Affimetrix, . Chip-data analysis was performed as described recently . AnalysiQuantitative expression analysis was performed using the 7500 Real-time System, commercial buffer and primer sets for BIM, EZH2, HOP, HOXA9, HOXA10, KCNQ1 and RUNX2 . Expression analysis of pri-miR-17-92 was performed as described recently . For norThe following oligonucleotides were used for reverse transcription (RT)-PCR were generated by MWG Eurofins : miR26-for: 5'-CCGGTTGAAATCGATGGAAC-3'; miR26-rev: 5'-TCAGGTCCTTCACGTAGTTC-3'; miR101-for: 5'-TCCTCATGCAAATAGCGGGAAG-3'; miR101-rev: 5'-AAGCCATGGCATTGCAGTCCTC-3'; TEL-for: 5'-AGGCCAATTGACAGCAACAC-3'; TEL-rev: 5'-TGCACATTATCCACGGATGG-3'; TFDP1-for: 5'-TCTTCTCTGGGAAGGTGAAC-3'; TFDP1-rev: 5'-TCCTACCACCAGGGTGTTTG-3'. Obtained PCR products were analyzed by agarose gel electrophoresis.EcoRI, the promoter via HindIII/BamHI into the backbone of the pcDNA3 vector [To quantify the impact of HOXA10 on the ID2 promoter we cloned a corresponding genomic fragment containing the HOXA10 binding site in front of a reporter gene. PCR using oligonucleotides ID2-for (5'-GCAAGCTTATCTAGCCCTCCCTCTAGCTG-3') and ID2-rev (5'-AAGGATCCTGACAGATAGGTGGCCCTAGC-3') generated a promoter fragment of 1550 bp in length. The validity of the construct was confirmed by sequence analysis (MWG Eurofins). As described previously, the reporter gene consists of a genomic fragment of the HOXA9 gene, comprising exon1-intron-exon2, allowing the quantification of the transcribed and spliced reporter mRNA via RQ-PCR . The rep3 vector . A cotraFISH analysis was performed as described recently . The folExpression constructs for HOXA9, HOXA10, ID2 and TFDP1 (in pCMV-XL5/4 vectors) were obtained from Origene . Transfection of expression constructs into cervix carcinoma cell line HELA was performed using transfection reagent SuperFect . Transfection experiments were performed at an efficiency of about 30% as determined by fluorescence microscopy. Expression construct for ID2 and siRNAs directed against EZH2 and HOP (Qiagen) were electroporated into cell lines using the EPI-2500 impulse generator . Transfection and electroporation experiments have been performed twice.VSV.G-pseudotyped lentiviral particles were generated by calcium phosphate co-transfection of embryonal kidney 293T cells and viral supernatants were concentrated as previously described . LentiviChIP analysis was performed with the ChIP Assay Kit . Antibody anti-HOXA10 was obtained from Santa Cruz Biotechnology . The subsequent PCR analysis was carried out, using nested ID2-oligonucleotides.For the HOXA10-binding site: ID2-for: 5'-TATTGGGCGTGCTGAAACAG-3', ID2-rev: 5'-GATTAAGGCAGTGCCTTCTC-3', ID2-for-nested: 5'-CACTAGTAACTTAGGCCTCG-3', ID2-rev-nested: 5'-CCCTGATGTTAGTAAAATGGC-3'.For control: ID2-for: 5'-CCAGGGTGTTCTCTTACTTG-3', ID2-for-nested: 5'-GCCCTTTCTGCAGTTGGA-3', ID2-rev: 5'-CAGCATTCAGTAGGCTTGTG-3', ID2-rev-nested: 5'-GGATCCTTCTGGTATTCA-3'.For protein analysis Western blotting was performed as described previously . BrieflyCell lines were treated for 16 h with 100 μM etoposide (Sigma), which has been dissolved in dimethylsulfoxide, and subsequently prepared for standardized MTT -2,5-diphenyltetrazolium bromide; obtained from Sigma) assays. The absorbance was determined at 570 nm by an ELISA reader .Two NK-cell lines, NK-92 and YT, together with eight T-cell lines and one sample of primary CD34+ HSC were examined by expression profiling and subsequently by cluster analysis Fig. . Three mTo identify specifically expressed genes within the NK-cell lineage we subtracted averaged values of NK- and T-cell data sets, respectively, and ranked these differences to generate a list of differentially expressed genes. From this list we analyzed the top-200 over- and under-expressed genes as heat maps Fig. . These cWe subsequently focused on potential mechanisms which might conceivably deregulate these candidate oncogenes in LOUCY. FISH analysis using flanking probes excluded chromosomal translocations at 7p15, 2p25 and 6p12 which might have been responsible for high expression levels of HOXA9/HOXA10, ID2 and RUNX2, respectively (data not shown). Sequence analyses of promoter regions, according to genome browser data of the University of California Santa Cruz (UCSC), demonstrated binding sites for HOXA9/HOXA10 within the promoter regions of ID2 (at -2167 bp) and of RUNX2 (at -5249 bp) which may indicate regulative interactions. Accordingly, in osteoblasts HOXA10 has been shown to activate expression of RUNX2 . TherefoFirst, we transfected HELA cells with CMV-driven constructs for overexpression of transcription factors HOXA9 or HOXA10. Subsequent RQ-PCR analysis demonstrated elevated expression levels of ID2 in HOXA9 and HOXA10 transfected cells. However, the expression levels of RUNX2 showed no significant alteration, failing to support a regulatory connection Fig. . Thus, tTo analyze this mechanism in T-ALL we lentivirally transduced JURKAT cells with constructs of HOXA9 or HOXA10 for overexpression. Subsequent expression analysis by RQ-PCR demonstrated elevated and unaltered levels of ID2 and RUNX2, respectively Fig. . GreaterBoth, HOXA9 and HOXA10 are targets of MLL-fusion proteins in AML and ALL malignancies . RQ-PCR TCF3 controls many cellular processes during thymocyte development, including differentiation and survival. Accordingly, gene expression analysis of TCF3 target genes has identified activation of the pro-apoptosis gene BIM . Since IMLL and SET1 proteins together with polycomb-related proteins are chromatin components which regulate transcription of developmental genes, including those of the HOXA cluster ,20,39-42Therefore, we treated EZH2-negative LOUCY and EZH2-positive JURKAT cells with DZNep, an inhibitor of EZH2/PRC2 . WesternAs shown recently, EZH2 activity is reduced upon phosphorylation via AKT-signaling . To analHOP has been shown to interact with PRC2 component EPC1 . To analTo examine possible causes underlying the lack of EZH2 expression in LOUCY cells we looked for chromosomal deletions by consulting genomic array data published by the Sanger centre . However, the EZH2 locus at 7q36 showed no copy number alterations, thus excluding genomic deletion. Moreover, treatment of LOUCY cells with AZA to reactivate DNA-methylated genes showed no effect on EZH2 when analyzed by RQ-PCR (not shown), thus excluding aberrant methylation at this locus. Although, EZH2 is the target of at least two micro-RNAs, namely miR-26 and miR-101 ,49, RT-PAs shown previously, expression of EZH2 is regulated by the E2F1 transcription factor which in turn is deregulated by overexpressed micro-RNAs of the miR-17-92 gene cluster, reducing E2F1 protein levels in T-ALL cells ,50. But T-cell leukemogenesis involves ectopic activation of proto-oncogenes which often regulate physiological development. In this study, comparison of gene expression profiles of NK/T-cell lines identified the apparently aberrant expression of developmental homeobox genes, HOXA9 and HOXA10, a bHLH gene, ID2, and RUNX2 in T-ALL cell line LOUCY. These T-ALL oncogenes are physiologically expressed in hematopoietic progenitor cells and regulate the differentiation lineage of the closely related NK-cells ,9,33. DuWe showed that HOXA9 and HOXA10 activated ID2 in T-cells (and HELA) while RUNX2 was not regulated by these homeodomain proteins. Coexpression of HOXA9/HOXA10 and ID2 was detected in cell lines expressing MLL-fusion proteins which have been shown to activate particular HOXA genes, including HOXA9 and HOXA10 -18. ThusID2 belongs to the basic helix-loop-helix family of transcription factors. Physiologically, ID2 is expressed in progenitors of NK-cells and contributes essentially to the differentiation of this lymphocytic lineage -10. TogeHOXA genes are physiologically expressed in lymphocytic progenitor cells and become downregulated during T-cell development ,56. DuriDrosophila mutations in each of ten tested different PRC components, including the EZH2-homologue, results in ectopic HOX-gene expression, supporting the necessity of a complete complex for its gene repression activity [Inhibition of EZH2 by DZNep or siRNA consistently increased expression of HOXA10 while its activation by rapamycin decreased HOXA10 levels. The latter result may influence the treatment of HOXA-positive T-ALL patients because of frequent aberrant activity of the AKT-pathway in this disease . T-ALL cactivity .By examining the cause of EZH2 silencing in LOUCY we identified reduced TFDP1 expression. Increased expression of TFDP1 in LOUCY via lentiviral transduction of an expression construct restored EZH2 expression. TFDP1 is a cofactor for E2F transcription factors, acting as heterodimers, which have been shown to regulate EZH2 transcriptional expression . TherefoHOP has also been shown to interact with PRC2 component EPC1 . Forced Activating chromatin complexes contain HMT activity mediated by closely related MLL/SET1 proteins ,20. ChroOur data indicate that PRC2 contributes to the differentiation of NK/T-cells via regulation of HOXA9/HOXA10 gene expression. Deregulated function of PRC2 is involved in T-cell leukemogenesis, resulting in ectopic activation of NK-cell lineage-associated HOXA9/HOXA10 genes and their target ID2.The authors declare that they have no competing interests.SN designed the research and wrote the paper, LV and MS performed lentiviral cloning and transduction, VM supplied DZNep together with a protocol, CM and MK performed cell culture and labwork, RM performed cytogenetic analysis and HD wrote the paper. All authors read and approved the final manuscript.


Total cath B activity (cath BAT) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath BA7.5) were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The immunostaining pattern of cath B was determined in 239 lung tumour tissue sections, showing the presence of the enzyme in tumour cells (cath BT-I) and in tumour-associated histiocytes (cath BH-I). The median levels of cath BAT, cath BA7.5 and cath BC were 5.6-, 3.2- and 9.1-fold higher (P < 0.001), respectively, in tumour tissue than in non-tumourous lung parenchyma. Out of 131 tissue sections from patients with squamous cell carcinoma (SCC), 59.5% immunostained positively for cath B, while among the 108 adenocarcinoma (AC) patients 48.2% of tumours showed a positive reaction. There was a strong relationship between the levels of cath BAT, cath BA7.5, cath BC and cath BT-I in the primary tumours and the presence of lymph node metastases. Significant correlation with overall survival was observed for cath BT-I and cath BA7.5  in patients suffering from SCC. In these patients positive cath B in tumour cells (cath BT-I) and negative cath B in histiocytes (cath BH-I) indicated significantly shorter survival rate compared with patients with negative cath BT-I and positive cath BH-I (P < 0.0001). In contrast, in AC patients, both, positive cath BT-I and positive cath BH-I, indicated poor survival probability (P < 0.014). From these results we conclude that the proteolytic enzyme cath B is an independent prognostic factor for overall survival of patients suffering from SCC of the lung. © 1999 Cancer Research CampaignIn order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human non-small cell lung cancer (NSCLC) we examined cath B concentrations (cath B


Viruses lack self-propulsion. To move in multi-cellular hosts they must therefore manipulate infected cells. Herpesviruses provide an archetype for many aspects of host manipulation, but only for alpha-herpesviruses in is there much information about they move. Other herpesviruses are not necessarily the same. Here we show that Murine gamma-herpesvirus-68 (MHV-68) induces the outgrowth of long, branched plasma membrane fronds to create an intercellular network for virion traffic. The fronds were actin-based and RhoA-dependent. Time-lapse imaging showed that the infected cell surface became highly motile and that virions moved on the fronds. This plasma membrane remodelling was driven by the cytoplasmic tail of gp48, a MHV-68 glycoprotein previously implicated in intercellular viral spread. The MHV-68 ORF58 was also required, but its role was simply transporting gp48 to the plasma membrane, since a gp48 mutant exported without ORF58 did not require ORF58 to form membrane fronds either. Together, gp48/ORF58 were sufficient to induce fronds in transfected cells, as were the homologous BDLF2/BMRF2 of Epstein-Barr virus. Gp48/ORF58 therefore represents a conserved module by which gamma-herpesviruses rearrange cellular actin to increase intercellular contacts and thereby promote their spread. Viruses are inherently non-motile. They must therefore rely for their movement on manipulating infected cells. In lytic infection, this means maximizing the number of uninfected neighbours each infected cell contacts and the distance each round of replication travels. Both parameters depend on cell shape. Vaccinia virus promotes its spread by inducing actin tail formation with A36R in vitro. However, their lytic genes are generally conserved in other family members, which can therefore tell us how EBV and KSHV are likely to work. Murine gamma-herpesvirus-68 (MHV-68) in vivo without an obvious cell-free viraemia in vivo replication than those involved in cell-free virion binding Most information on herpesvirus spread comes from the neurotropic alpha-herpesviruses. For example, the Herpes simplex virus gE/gI complex promotes viral spread across tight junctions We have previously identified ORFs 27 and 58 as important for intercellular MHV-68 spread A cell's shape is determined largely by its cytoskeleton. Thus, different patterns of actin polymerization generate structures such as filopodia, lamellopodia or membrane ruffles under the control of Rho GTPases The MHV-68 gp48 accumulates on fine protrusions that reach out from infected cells + dots changed noticeably in distribution over 10 second intervals. The infected cell plasma membrane was also highly dynamic , RhoA (RhoN19) and Cdc42 (Cdc42N17) were kindly provided by Dr. P. Digard . pGEX-KG-TAT-C3 was kindly provided by Dr. Michael Way , and expressed in E.coli by standard methods Retrovirus-expressed human CD8α BHK-21 cells, 293T cells, NIH-3T3 cells, the CD8α-transduced derivative 3T3-CD8 and the cre-transduced derivative 3T3-CRE AgeI-restricted and AseI-restricted primers and cloned into the AgeI and AseI restriction sites of pEGFP-C2-ORF58. This added a second genomic flank to the eGFP coding sequence. The flanked eGFP was then subcloned into the pST76-SR shuttle vector as a BamHI/KpnI fragment, using a BamHI site in pEGFP-C2 and a KpnI site in the AseI-restricted primer. The eGFP coding sequence was then recombined into the MHV-68 BAC ORF58 locus by standard protocols ScaI site as described −) was reconstituted into infectious virus by transfection into BHK-21 cells using Fugene-6 . The loxP-flanked BAC cassette was removed by passaging the virus through 3T3-CRE cells. ORF27-deficient and ORF58-deficient mutants on a wild-type MHV-68 background g, 3 min) and discarded. Virions were then recovered from supernatants by high-speed centrifugation , and stored at −70°C. Viruses were titered by plaque assay on BHK-21 cells MHV-68 genomic co-ordinates 78254-79298 32P-dCTP-labeled probes  were generated by random primer extension . Membranes were hybridized with the probe , washed in 30 mM sodium chloride/3 mM sodium citrate/0.1% sodium dodecyl sulfate at 65°C, and exposed to X-ray film.DNA was extracted from virus stocks by alkaline lysis − MHV-68. The cells were then trypsinized and stained  with mAb 6D10 (gp48), mAb 3F7 (gN) or T1A1 (gp150), plus phycoerythrin-conjugated goat anti-mouse IgG pAb . The cells were washed ×2 in PBS after each antibody incubation and analyzed on a FACSort .BHK-21 cells were left uninfected or infected  with eGFP-ORF58 or eGFP-ORF58/27BHK-21 cells were infected with wild-type MHV-68  then fixed, stained and embedded for transmission electron microscopy as described C.difficile Toxin B (5 ng/ml) or recombinant TAT-C3 protein (1 µg/ml) for 4 h at 37°C. All the cells were then fixed , permeabilized  and blocked . MHV-68 proteins were stained using mAbs 3F7 (gN) 2. All images were captured with a Leica SP2 confocal microscope and analysed using ImageJ.NIH-3T3, 3T3-CD8, BHK-21 or 293T cells on glass cover slides were infected (1 p.f.u./cell) or not with MHV-68, or transfected with plasmids. Where indicated, the cells were treated with Brefeldin A (18 µM) or Nocodazole (20 µM) for 1 h at 37°C, or with Figure S1MHV-68 induces ORF27-dependent membrane fronds on NMuMG epithelial cells. A. NMuMG cells were infected  with ORF27+ or ORF27- MHV-68, each with eGFP-tagged ORF58. The cells were then fixed, permeabilized and stained for the ORF65 (capsid) with mAb MG-12B8 plus Alexa568-conjugated goat anti-mouse IgG pAb. Nuclei were counter- stained with DAPI. EGFP fluorescence was visualized directly. EGFP+ membrane fronds were seen only when ORF27 was intact. B. NMuMG cells were infected with ORF27+ eGFP-ORF58-tagged MHV-68 , then fixed, permeabilized and stained for gN with mAb 3F7 plus Alexa568-conjugated goat anti-mouse IgG pAb. EGFP fluorescence was visualized directly. The punctate gN staining in more distal membrane fronds presumably corresponds to virions. C. NMuMG cells were infected as in B, then fixed, permeabilized and stained for actin with Alexa-568-conjugated phalloidin. EGFP fluorescence was visualized directly.(5.79 MB TIF)Click here for additional data file.Figure S2Gp150 is accessible on membrane fronds without permeabilization. NIH-3T3 cells were infected with gM-eGFP expressing MHV-68 , then either stained intact or first fixed with paraformaldehyde and permeabilized with Triton-X100. ORF65 was visualized with mAb MG-12B8 and gN with mAb 3F7. The intact cells were fixed and permeabilized after staining. EGFP-ORF58 was viewed directly and nuclei were counter-stained with DAPI. The zoomed images correspond to the boxed regions. The lack of difference between permeabilized and non-permeabilized gN staining, particularly in the punctate staining of the distal membrane fronds, argued against virions being contained within the fronds.(7.50 MB TIF)Click here for additional data file.Movie S1BHK-21 cells were infected with gM-eGFP-tagged MHV-68 , then imaged every 7 sec over 2.5 min. The top panel shows gM-eGFP, the bottom panel phase contrast images.(3.14 MB MOV)Click here for additional data file.Movie S2This is a higher magnification view of (10.58 MB AVI)Click here for additional data file.Movie S3A single long process of an infected  BHK-21 cell is shown, with images captured every 10 sec over 10 min. Note the motility of the plasma membrane over this time. The fine fronds branch off from this process, as can be seen from its “hairy” phase contrast appearance. The eGFP+ dots are probably individual virions.(6.19 MB MOV)Click here for additional data file.Movie S4A higher magnification view of (3.49 MB MOV)Click here for additional data file.Movie S5NIH-3T3 cells were infected  with eGFP-ORF58-tagged MHV-68. Images were captured every 10 sec over 5 min. ORF58 is expressed more on the plasma membrane than in virions, and therefore lights up finer membrane fronds. These are seen to reach out to a neighbouring, uninfected cell. Note also the highly mobile, even pulsatile membrane of the infected cell. This was a consistent feature of MHV-68 infection.(3.93 MB MOV)Click here for additional data file.Movie S6NIH-3T3 cells were infected  with gp48-deficient eGFP-ORF58-tagged MHV-68, then imaged every 20 sec over 10 min. Note the lack of eGFP+ connections between infected and uninfected cells.(6.23 MB MOV)Click here for additional data file.Movie S7NIH-3T3 cells were infected  with gp48+ eGFP-ORF58-tagged MHV-68, then imaged every 20 sec over 10 min. Membrane fronds are seen to extend out from the central infected cell, mostly towards an uninfected cell on its lower left. These fronds grow noticeably in length over 10 min.(5.16 MB MOV)Click here for additional data file.


Using a murine model of herpes simplex virus (HSV)-1 encephalitis, our laboratory has determined that induction of proinflammatory mediators in response to viral infection is largely mediated through a Toll-like receptor-2 (TLR2)-dependent mechanism. Published studies have shown that, like other inflammatory mediators, reactive oxygen species (ROS) are generated during viral brain infection. It is increasingly clear that ROS are responsible for facilitating secondary tissue damage during central nervous system infection and may contribute to neurotoxicity associated with herpes encephalitis.-/- mice. Intracellular ROS production in cultured murine microglia was measured via 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) oxidation. An assay for 8-isoprostane, a marker of lipid peroxidation, was utilized to measure free radical-associated cellular damage. Mixed neural cultures obtained from β-actin promoter-luciferase transgenic mice were used to detect neurotoxicity induced by HSV-infected microglia.Purified microglial cell and mixed neural cell cultures were prepared from C57B/6 and TLR2-/- microglia displayed delayed and attenuated ROS production following viral infection. HSV-infected TLR2-/- microglia produced less neuronal oxidative damage to mixed neural cell cultures in comparison to HSV-infected wild-type microglia. Further, HSV-infected TLR2-/- microglia were found to be less cytotoxic to cultured neurons compared to HSV-infected wild-type microglia. These effects were associated with decreased activation of p38 MAPK and p42/p44 ERK in TLR2-/- mice.Stimulation with HSV-1 elevated intracellular ROS in wild-type microglial cell cultures, while TLR2These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection. ROS production, while beneficial in clearing invading pathogens, can also cause irreparable harm through bystander damage to crucial host cells.Exposure to an invading pathogen initially triggers robust innate immune responses. These responses involve recruitment and activation of phagocytic macrophages, neutrophils and, during central nervous system infection, the activation of resident microglia. One effective method that immune cells utilize to eliminate invading pathogens is through the rapid and robust production of reactive oxygen species (ROS), termed the respiratory burst, which facilitates damage to the invading pathogen. Some of the reactive species generated include superoxide (O2Herpes simplex virus (HSV)-1 infection of the brain results in devastating necrotizing encephalitis. Using a murine model of HSV-1 encephalitis our laboratory has shown that intranasal delivery of HSV induces a robust activation of brain microglial cells, production of proinflammatory mediators, and focal tissue damage which, if left untreated, can result in prolonged brain inflammation, and compromised brain function or death . It is bin vitro studies in our laboratory implicate microglial cells as the primary mediator of proinflammatory cytokines and chemokines during HSV-1 infection, little is known about ROS production and its impact on the brain during viral encephalitis. While reactive species themselves are short lived and difficult to measure, detection of secondary products such as lipid peroxidation (F2-isoprotanes and F4-neuroprostanes), tyrosine nitrosylation and DNA-oxidation (8-hydroxydeoxyguanosine) are useful indicators of oxidative damage in vivo. Indeed, our laboratory has detected increases in 8-isoprostane, 8-hydroxydeoxyguanosine and 3-nitrotyrosine during active experimental herpes encephalitis [Microglial cell production of ROS is implicated in neurototoxicity associated with HIV-associated dementia, Alzheimer's disease, Parkinson's disease and Amyotrophic lateral sclerosis ,8. Whilephalitis . These s-/- mice are less susceptible to HSV infection, highlighting the importance of this receptor in the outcome of HSV encephalitis [Microglial cells express a myriad of receptors from the Toll-like receptor family (TLR) of pathogen recognition receptors, including TLR2. TLR2phalitis ,10. Usinphalitis ,11-13. Iphalitis -16. Data-/- mice. Additionally, compared to wild-type microglia, TLR2-/- microglia displayed attenuated HSV-induced lipid peroxidation and neurotoxicity. These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.In this study, we tested the hypothesis that HSV-induced neural cell oxidative tissue damage and cytotoxicity are mediated by microglial cell ROS through a TLR2-dependent mechanism. We detected elevated intracellular ROS in HSV-infected microglia obtained from wild-type mice. In contrast, the virus failed to induce ROS in microglia obtained from TLR2-/- and transgenic β-actin promoter-luciferase Balb/c mice. Following dispersion of fetal (d 15 of gestation) cerebral cortices with trypsin, cells (5 × 105 or 2 × 105/500 ml) were plated into collagen coated wells of 24-well plates with DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. On day 5, the culture medium was replaced with DMEM containing 10% heat-inactivated FBS, uridine (33.6 mg/ml), and fluorodeoxyuridine (13.6 mg/ml) to suppress glial cell growth. After 24 h, cells were replaced with DMEM containing 10% heat-inactivated FBS. Culture medium was changed every 4 days thereafter. On day 7 the neural cell cultures consisted of approximately 85-90% neurons  and containing characteristic processes and birefringent cell bodies), 10-15% astrocytes , and < 2% microglia .Neural cell cultures were established from C57B/6, TLR2-/-, mice were dissociated after a 30- min trypsinization (0.25%) and were plated in 75-cm2 Falcon culture flasks in DMEM containing 10% heat-inactivated FBS and antibiotics. The medium was replenished 1 and 4 days after plating. On day 12 of culture harvested cells were plated in a 60-mm petri dish and incubated for 15 min at 37°C. After extensive washing with culture medium, adherent cells (microglia) were collected with a rubber policeman and centrifuged at 1000 rpm for 10 min. Purified microglial cell cultures were comprised of a cell population in which > 98% stained positively with MAC-1 antibodies and < 2% stained positively with antibodies specific to glial fibrillary acid protein, an astrocyte marker.Murine cerebral cortical cells from 1-day-old C57B/6 and TLR2HSV-1 strain 17 syn + was propagated and titrated using plaque assay on rabbit skin fibroblasts .4/well) in 96-well plates or chamber slides were infected with HSV-1 (MOI = 2). At designated time points, cells were washed and incubated with HBSS containing DCFH-DA (20 μM) for 30 min. After incubation, cells were excited at 485 nm and DCFH-DA fluorescence read at 530 nm emission on a fluorescence plate reader or viewed and photographed under a fluorescence microscope. Each sample was run in triplicate and sample means were normalized to their respective controls (% of control).The production of intracellular ROS was measured by 2',7'-Dichlorofluorescin diacetate  oxidation. Murine microglial cultures seeded (4 × 10-/- mice were infected with HSV-1 (2 MOI). Cells were harvested at 90 min p.i. and 5 × 104 or 1 × 105 microglia were transferred onto mixed neural cultures (5 × 105/well) from C57B/6 or TLR2-/- mice creating microglia to neuron ratios of 1:10 and 1:5, respectively. To free the transferred microglia of residual HSV-1, the microglia were treated prior to harvest with 0.05% trypsin for 10 minutes, followed by 3 washes with 10% FBS containing DMEM. Transfer of 5 × 104 or 1 × 105 uninfected microglia served as a control. Oxidative damage to lipids was quantified by using an 8-isoprostane EIA kit according to the manufacturer's instructions .Purified murine microglia from wild-type and TLR2-/- mice were infected with HSV-1 (2 MOI). Cells were harvested at 90 min p.i. and transferred, at the designated microglia to neuron ratios , onto cultured neurons obtained from β-actin promoter-luciferase transgenic mice. D-Luciferin potassium salt  was added to each well and luciferase activity measured on a plate reader 48 h after microglial cell transfer.Purified murine microglia from wild-type and TLR26/well) were infected with HSV-1 (MOI = 2). Thirty minutes following HSV-1 treatment cell lysates were collected, electrophorezed in 12% acrylamide/bis-acrylamide electrotransfered onto nitrocellulose membrane and probed with rabbit anti-phospho-p38 MAP Kinase (Thr180/Tyr182) and rabbit anti-phospho-p44/42 MAP Kinase (Thr202/Tyr204) antibodies . Rabbit anti-p38 MAP Kinase and rabbit anti-p42/p44 MAP kinase antibodies were used to detect total p38 and p42/p44 levels . Alkaline phosphatase-conjugated secondary antibodies with chemiluminescence detection was used with a Kodak Image Station to capture protein band images.Microglia  was performed followed by either Fisher's protected least significant difference (PLSD)-test or Scheffe-test. For comparison of means of pairs of data a two-tailed Student's T-test for paired samples was applied.in vitro. Purified microglial cell cultures from wild-type mice were inoculated with HSV-1 (MOI = 2), and at designated times following infection, cells were loaded with the ROS fluorescence indicator DCFH-DA. A kinetic analysis of microglial ROS production following HSV-1 challenge revealed increased intracellular ROS production as early as 3 h.p.i. ROS continued to increase at 8 h and 24 h, reaching peak ROS accumulation by 48 h.p.i. , 8 h (p = 0.026), 24 h (p = 0.022), and 48 h.p.i. (p = 0.042). These data clearly demonstrate the importance of TLR2 in HSV-induced microglial ROS production.To determine the role of microglia and TLR2 on HSV-1 induced oxidative damage, we first examined ROS production in wild-type microglia following HSV-1 infection in vitro. HSV-1 (MOI = 1) was added to C57B/6 or TLR2-/- mixed neural cell cultures  and the infected cultures were assessed for 8-isoprostane levels, a marker of lipid peroxidation. Based on the peak HSV-1-induced ROS production in microglial cells demonstrated above, we chose to assess oxidative damage at 48 h.p.i. HSV-1 infection was found to increase 8-isoprostane levels in wild-type mixed neural cultures (p = 0.002). In contrast, 8-isoprostane levels in TLR2-/- neural cultures did not differ from untreated controls (p = 0.40), demonstrating the importance of TLR2 in HSV-1 induced oxidative damage , 1:20 (p = 0.067), and 1:5 (p = 0.052) microglia:neuron ratios and, hence, induced less cytotoxicity. Interestingly, the addition of C57B/6 background microglia to the non-syngeneic Balb/c background mixed neuronal cultures was not problematic in these experiments as evidenced by negligible neurotoxicity upon addition of uninfected C57B/6 microglia to Balb/c luciferase transgenic mixed neural cultures.Using neural cultures derived from β-actin promoter-luciferase transgenic Balb/c mice, we were able to assess neural cytotoxicity resulting from the addition of exogenously added HSV-infected C57B/6 microglia. In this assay, HSV-infected or control purified microglial cells were added at specific ratios to mixed neural cultures  and luciferin emission measurements from each experimental group was normalized to luciferin readings from untouched mixed neural cultures (expressed as % of control). Because β-actin promoter-luciferase transgenic mice ubiquitously express the luciferase enzyme, in these studies, a decrease in luciferin intensity indicated a decrease in neural cell number. Addition of HSV-infected wild-type C57B/6 microglia resulted in a significant decrease in neural culture luciferin emission at ratios of 1:20 (p < 0.001) and 1:5 (p < 0.001) indicating increased neurocytotoxicity , may be potential targets in regulating unwanted bystander damage to healthy neurons during viral brain infection.The authors declare that they have no competing interests.SJS designed and performed experiments, and drafted the manuscript. SH performed and designed experiments, and co-conceived of the study. MRL performed experiments. JRL co-conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors have read and approved the final version of this manuscript.