Murine Embryonic Fibroblast Cell Line Research Articles

Drug-eluting biodegradable metals and metal-ceramic composites

Biodegradable metals emerged as promising temporary bone implants. The integration of local drug delivery (LDD) can prevent biomaterial-centered infections that are difficult to treat. LDD is achieved by drug-eluting coatings or porous implants where the drug is impregnated after implant fabrication because the high temperatures used during conventional production processes would result in their thermal decomposition. This work reports on the production and characterization of vancomycin hydrochloride (VH)-loaded Fe and Fe-iron oxide (Fe2O3) composites (Fe-Fe2O3) by a cold sintering technique. We use focused ion beam milling for the first time to investigate the drug-metal interface. We investigate the mechanical and degradation properties of VH-free and VH-loaded Fe and Fe-Fe2O3. Results show very high mechanical strength of drug-eluting Fe and Fe-Fe2O3 composites (up to than 780 MPa under compression, exceeding the maximum strength of cancellous bone more than three times) accompanied by a delayed drug release. Then, we confirm the good antimicrobial activity against Staphylococcus aureus and cell compatibility with the murine embryonic fibroblast cell line NIH/3T3 in vitro. Overall results confirm the promise of drug-eluting metals and metal-ceramic composites for LDD in bone.

New amides derived from sclareolide as anticholinesterase agents

A series of 25 amides (15 new) derived from (3aR)-(+)-sclareolide were prepared and subjected to Ellman’s assay to determine their efficacies as inhibitors for AChE or BuChE. Five amides (9, 13, 14, 15 and 17) caused inhibition of one of the enzymes greater than 60%; thereby those that inhibited BuChE were more active than positive control galantamine, and they showed better Ki values (1.07 to 8.49). In general, it was found that molecules holding a meta-substituted phenyl group showed a higher percentage of enzymatic inhibition. Molecular modelling calculations indicated the putative interactions of compounds with the amino acids residues of both enzymes AChE and BuChE. The cytotoxicity of compounds 9, 13, 14, 15 and 17 was evaluated against a non-malignant murine embryonic fibroblast cell line (NIH 3T3). Of special note is compound 15, as it presented the second-best Ki value for BuChE (1.71), was not cytotoxic (EC50 > 30 μM). Compound 15 also does not violate Lipinski rules, and showed permeability in the blood brain barrier, indicating that it can be considered a lead for the development of new drugs to treat Alzheimer’s disease.

Control of doxorubicin-induced, reactive oxygen-related apoptosis by glutathione peroxidase 1 in cardiac fibroblasts

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 μM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 μM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1–2 μM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.

Anticancer Activity of a Highly Potent Small Molecule Tubulin Polymerization Inhibitor, AB8939

We have identified the small chemical molecule AB8939 as being a structurally novel, synthesized tubulin inhibitor that can circumvent resistance mechanisms known to limit the effectiveness of existing tubulin inhibitors; e.g., P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance. A series of in vitro preclinical studies provide proof-of-concept that AB8939 has broad applicability as a potent anticancer drug, particularly in tumors of hematopoietic and lymphoid tissues, including acute myeloid leukemia (AML). Regarding mechanism of action, x-ray crystallography demonstrated that AB8939 binds to the colchicine-binding site on the beta-subunit of tubulin. Cell cycle arrest in the G2/M phase was evaluated using HCT116 cells (a human colorectal tumor), treated at various concentrations of AB8939 for 24 hours. It was seen that AB8939 produced a strong mitotic arrest at the sub-micromolar concentration range (90% of cells in G2/M phase at 10 nM), which was of comparable strength to that of established microtubule targeting agents, each at a concentration of 100 nM. Additional assays using cytarabine (Ara-C) resistant MOLM14 AML cells confirmed this activity, also demonstrating dose dependent (2 to 20 nM) G2/M phase cell cycle arrest in patient-derived AML blasts and that G2/M cell cycle arrest lead to cellular death by apoptosis at nanomolar concentrations. The effect of AB8939 (100 nM) on the integrity of the microtubule and actin networks was tested in 3T3NIH cells (murine embryonic fibroblast cell line). AB8939 induced a rapid (within 1 hour) and radical destabilization of the microtubule network but did not affect the actin network. Similarly, destabilization of the microtubule network was observed in human primary cardiomyocytes and primary human lung fibroblast cells treated for 24 hours at 10 to 1000 nM AB8939. Further in vitro analysis showed that AB8939 produces a direct and potent, dose-dependent depolymerization effect (50% inhibition of in vitro microtubule polymerization at around 1 µM, with 100% inhibition at &gt;5 µM). The potential of AB8939 to overcome resistance to chemotherapeutic agents in Pgp-dependent multidrug-resistant cell lines was assessed using the drug-sensitive human sarcoma cell line MES-SA (parental) and its multidrug-resistant counterparts MES-SA/MX2 and MES-SA/Dx5 in a 6-day proliferation/survival assay. AB8939 efficiently inhibited each of these cells with an IC50 ≤10 nM. By comparison, the MES-SA/MX2 and MES-SA/Dx5 cell lines were highly resistant to the chemotherapeutic agents of doxorubicin and vincristine, as compared with the effect on parental cells (IC50 &lt;1.5 - 2.0 µM versus 20 nM, respectively). Additional tests showed that AB8939 is a very poor substrate of Pgp efflux pump, comparable with combretastatin-4, and therefore has the potential to overcome multidrug resistance in cancer patients. The anti-proliferative activity of AB8939 in various hematopoietic tumors and solid tumors was evaluated using a colorimetric cell proliferation and viability assay. AB8939 produced good anti-tumor activity after 72 hours (IC50 of ≤50 nM) in 19 hematopoietic tumor cell lines tested, including AML (3 cell lines), B cell lymphoma (8 cell lines), T cell lymphoma (6 cell lines), and multiple myeloma (2 cell lines). AB8939 also showed good anti-tumor activity after 6 days (IC50 of ≤10 nM) in several solid tumor cell lines, including breast, colon, glioblastoma, head and neck, lung, kidney, melanoma neuroblastoma, ovary, pancreas and prostate cell lines. The therapeutic potential of AB8939 in refractory/resistant AML was investigated further on doxorubicin-resistant AML cell lines (HL60 and U937), doxorubicin being a commonly used AML induction drug and Pgp substrate. AB8939 produced a strong anti-proliferative effect in both cell lines whereas both were resistant to doxorubicin, thus demonstrating AB8939's potential to overcome refractory/resistant AML. Notably, HL60 and U937 are respectively MPO-positive and MPO-negative, indicating that unlike vinca alkaloids (e.g. vincristine or vinblastine) AB8939 it is not deactivated by this myeloid enzyme. These data show that AB8939 is a prolific and highly potent (nanomolar concentrations) Pgp-independent, next-generation microtubule-destabilizer drug for cancer therapy; in particular, difficult to treat hematopoietic tumors such as relapsed/refractory AML. Disclosures Humbert: AB Science: Employment. Goubard:AB Science: Employment. Mansfield:AB Science: Employment, Patents &amp; Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Dubreuil:AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. AB8939 Study Group:AB Science: Consultancy, Employment.

Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines.

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”.

Search for Chemical Compounds for Pharmacotherapy of Neurofibromatosis Type 2

A murine embryonic fibroblast cell line (MEF NF2-/-) was generated to model neurofibromatosis type 2 (NF2), a serious inherited disease for which there is no pharmacotherapeutic cure. Screening of a library of low-molecular-weight bioactive compounds using the experimental cell line MEF NF2-/- and MEF NF2f/f as a control cell line identified two agents, lovastatin and cerulenin, as potential candidates for NF2 pharmacotherapy.

Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress‑mediated cell death in multiple myeloma cells.

The inhibitory effects of macrolide antibiotics including clarithromycin (CAM) on autophagy flux have been reported. Although a macrolide antibiotic exhibits no cytotoxicity, its combination with bortezomib (BZ), a proteasome inhibitor, for the simultaneous blocking of the ubiquitin (Ub)-proteasome and autophagy-lysosome pathways leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (ER). As misfolded protein cargo is recruited by histone deacetylase 6 (HDAC6) to dynein motors for aggresome transport, serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways and aggresome formation concomitantly in MM cells. Pronounced apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SAHA); potently inhibits HDAC6] with CAM and BZ compared with each reagent or a 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome formation along with the accumulation of autolysosomes in the perinuclear region, whereas they were inhibited in the presence of SAHA. The SAHA/CAM/BZ combination treatment maximally upregulated genes related to ER stress including C/EBP homologous protein (CHOP). Similarly to MM cell lines, enhanced cytotoxicity with CHOP upregulation following SAHA/CAM/BZ treatment was shown by a wild-type murine embryonic fibroblast (MEF) cell line; however, a CHOP-deficient MEF cell line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced cytotoxicity and CHOP induction along with the cancellation of aggresome formation. Targeting the integrated networks of aggresome, proteasome, and autophagy is suggested to induce efficient ER stress-mediated apoptosis in MM cells.

Murine Embryonic Fibroblast Cell Lines Differentiate into Three Mesenchymal Lineages to Different Extents: New Models to Investigate Differentiation Processes

Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation.

Concomitant Targeting Aggresome Formation and Intracellar Proteolytic Pathways Enhances ER-Stress Mediated Cell Death In Myeloma Cells

ER-stress is caused by an imbalance between the amount of unfolded or misfolded protein in the ER lumen and the capacity of the ER machinery to refold these proteins. This stress induces a coordinated cellular response known as unfolded protein response (UPR). The main functions of UPR are to reduce the amount of protein that enters the ER by suppressing the translational rate and to increase the folding capacity of the ER via translational activation of chaperon proteins. Additionally, if proteins cannot be folded correctly in the ER, they are retrotranslocated to the cytoplasm for degradation via the ubiquitin-proteasome pathway for adaptation. However, if these adaptation strategies fail, apoptosis is triggered with the induction of the pro-apoptotic transcription factor CHOP/GADD153 and with the IRE1 involved in signaling via caspase-12. Autophagy is a highly conserved cellular process in eukaryotes. Intracellular proteins and organelles, including ER, are engulfed in a double-membrane vesicle known as an autophagosome, and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, evidence indicates a selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1. Thus, the two major intracellular protein degradation systems are directly linked. We previously reported that macrolide antibiotics including clarithromycin (CAM) block autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in myeloma and breast cancer cells, whereas treatment with CAM alone results in almost no cytotoxicity (Moriya S, et al. Int J Oncol. 2013, Komatsu S, et al. Biochem Biophys Res Commun. 2013). HDAC6, a microtubule-associated deacetylase, is a component of the aggresome and has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors. HDAC6 recruits misfolded protein cargo to dynein motors for transport to aggresomes, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy. Therefore, evidence suggests the existence of an elaborate intracellular network system for processing unfolded proteins. In the present study, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6 (IC50 37 nM), with CAM and BZ in myeloma cell lines. SAHA exhibited some cytotoxicity along with an increased acetylation level of α-tubulin, a substrate of HDAC6. Combined treatment of SAHA, CAM, and BZ potently enhanced the apoptosis-inducing effect compared with treatment using each reagent alone or a combination of two of the three. Concomitant treatment with CAM and BZ for blocking autophagy flux and proteasome activity dramatically enhanced aggresome formation in perinuclear region, whereas this aggresome formation was almost completely eliminated in the presence of SAHA. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP, were maximally induced by the simultaneous combination of three reagents. Like myeloma cell lines, a wild-type murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and maximally up-regulated Chop after combined treatment with SAHA, CAM, and BZ; however, a Chop KO MEF cell line almost completely canceled this enhanced effect. The specific HDAC6 inhibitor tubacin also exhibited a pronounced cytocidal effect with a combination of CAM plus BZ. These data suggest that simultaneous targeting of intracellular proteolytic pathways and HDAC6 enhances ER-stress-mediated apoptosis in myeloma cells, and also provide novel insight into “ER-stress loading therapy” for myeloma patients. Disclosures:Miyazawa:Taisho Toyama Phamaceutical Co. Ltd.: Research Funding.

Combined treatment with SAHA, bortezomib, and clarithromycin for concomitant targeting of aggresome formation and intracellular proteolytic pathways enhances ER stress-mediated cell death in breast cancer cells

The ubiquitin–proteasome pathway and the autophagy–lysosome pathway are two major intracellular protein degradation systems. We previously reported that clarithromycin (CAM) blocks autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in breast cancer cells, whereas treatment with CAM alone results in almost no cytotoxicity. Since HDAC6 is involved in aggresome formation, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6, with CAM and BZ in breast cancer cell lines. SAHA exhibited some cytotoxicity along with an increased acetylation level of α-tubulin, a substrate of HDAC6. Combined treatment of SAHA, CAM, and BZ potently enhanced the apoptosis-inducing effect compared with treatment using each reagent alone or a combination of two of the three. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP (GADD153), were maximally induced by the simultaneous combination of three reagents. Like breast cancer cell lines, a wild-type murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and maximally up-regulated Chop after combined treatment with SAHA, CAM, and BZ; however, a Chop knockout MEF cell line almost completely canceled this enhanced effect. The specific HDAC6 inhibitor tubacin also exhibited a pronounced cytocidal effect with a combination of CAM plus BZ. These data suggest that simultaneous targeting of intracellular proteolytic pathways and HDAC6 enhances ER-stress-mediated apoptosis in breast cancer cells.

Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells

The specific 26S proteasome inhibitor, bortezomib (BZ) potently induces apoptosis as well as autophagy in metastatic breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of clarithromycin (CAM) and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 μg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 μg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP (CADD153) and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wild-type murine embryonic fibroblast (MEF) cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that endoplasmic reticulum (ER)-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis.

Abstract 2513: Molecular dissection of cell death mechanisms following ionizing radiation in vitro and in vivo

Abstract Purpose: A majority of human cancers have the ability to evade cell death with dysregulated cell death and survival mechanisms. This may have a direct impact on treatment resistance and tumor recurrence. This study aims to elucidate the roles of apoptosis, autophagy and necrosis induced by ionizing radiation (IR). Materials/Methods: Immortalized murine embryonic fibroblast (MEF) cell lines with various genetic backgrounds were used in the study. Xenograft tumors were generated by injecting tumor cells (engineered by retrovirally transfecting the above MEF cell lines with oncoprotein E1A and KRAS) to bilateral flanks of nude mice. Mice bearing xenograft tumors were treated with radiation by using an orthovoltage X-ray machine. Tissue cultures were irradiated by using a Gammacell 40 irradiator with a Cs137 source. Cell survival was analyzed with several commonly accepted methods. Morphology changes before and after treatments were analyzed by phase contrast microscopy as well as transmission electron microscopy (TEM). Western blotting and Immunohistochemistry (IHC) methods were carried out as described. Results: There was no significant difference in the level of tumor regression between xenografted tumors lacking Bax and Bak (Bax−/−Bak−/−) and WT tumors after radiation. While apoptotic cell death was observed in immortalized WT MEF cell line and xenograft tumors after irradiation, the intrinsic apoptosis pathway was not required Autophagy was found to be activated after radiation, and pharmacologic inhibition of autophagy by 3-methyladenine (3MA) resulted in higher survival fraction after radiation, suggesting a cytotoxic role of autophagy following radiation. However, there was no difference in radiation response when an essential autophagy gene ATG5 was stably knocked down, suggesting that autophagy activation may not be required for radiation induced cell death. We then investigated the role of poly(ADP)-ribose polymerase (PARP) in radiation response by using PARP-1−/− immortalized MEFs. These cells were more resistant to MAF (an active metabolite of cyclophosphamide) induced necrosis as was shown previously, but were more sensitive to radiation both in vitro and in vivo (as in xenograft). We also found that a specific PARP inhibitor ABT-888 can significantly sensitize xenograft tumors to radiation treatment. Conclusion: While apoptosis and autophagy occur after radiation, the intrinsic apoptosis and autophagic pathways is not required for tumor regression following IR. Conversely, PARP protein plays an important pro-survival role after IR, and deficiency of PARP renders cells more sensitive to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2513. doi:10.1158/1538-7445.AM2011-2513

Transmembrane Delivery and Biological Effect of Human Growth Hormone Via a Phage Displayed Peptide In Vivo and In Vitro

For a long time, people have been looking forward to being able to clinically deliver bio-drugs systemically by a noninvasive method. Here, we show that a synthetic peptide, TD (ACSSSPSKHCG) was efficient in transferring human growth hormone (GH) across various kinds of membranes and the blood-brain barrier (BBB) in vivo via rectal administration, resulting in elevation of GH level in serum, acetylcholine and O-choline acetyltransferase activities and GH /IGF-1 contents in brain tissues, manifesting great therapeutic effects on chronic age-related dementia in mice and ameliorating neuronal damage in the brain. Furthermore, the effects of Aβ and TD/GH on LDH release, apoptosis and its relevant gene expression, involving bcl-2 and bax/caspase-3, were observed in a human neuroblastoma cell line (SH-SY5Y). Results indicated that GH decreased LDH release, apoptosis, and bax/caspase-3 activity, and increased bcl-2 expression compared with Aβ treatment, moreover, TD/GH may enhance the effects due to existence of TD, which might be dependent on TD assisted cross-membrane delivery of GH. The transdermal/transmembrane-enhancing activity of the TD peptide was also manifested on porcine abdominal skin in vivo and the murine embryonic fibroblast cell line (3T3 cell) in vitro, which was further shown through interaction between TD and lecithin (one constituent of the cell membrane) by ESI-MS. In conclusion, TD/GH counteracted brain defects in aged mice in vivo and cell apoptosis induced by Aβ in vitro might explain several underlying mechanisms by which GH could ameliorate learning and memory deficits in aged mice. Mixed TD/GH transmembrane delivery might be a promising therapy of Alzheimer's disease.

Role of Topoisomerase IIβ in DNA Damage Response following IR and Etoposide

The role of topoisomerase IIβ was investigated in cell lines exposed to two DNA damaging agents, ionising radiation (IR) or etoposide, a drug which acts on topoisomerase II. The appearance and resolution of γH2AX foci in murine embryonic fibroblast cell lines, wild type and null for DNA topoisomerase IIβ, was measured after exposure to ionising radiation (IR) or etoposide. Topoisomerase II-DNA adduct levels were also measured. IR rapidly triggered phosphorylation of histone H2AX, less phosphorylation was seen in TOP2β−/− cells, but the difference was not statistically significant. IR did not produce topoisomerase II-DNA adducts above control levels. Etoposide triggered the formation of topoisomerase II-DNA adducts and the phosphorylation of histone H2AX, the γH2AX foci appeared more slowly with etoposide than with IR. Topoisomerase II-DNA complexes in WT cells but not TOP2β−/− cells increased significantly at 24 hours with the proteasome inhibitor MG132, suggesting topoisomerase IIβ adducts are removed by the proteasome.

Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

Mechanisms of Plk4 haploinsufficiency in hepatocellular carcinogenesis

11100 Background: The pathogenesis of hepatocellular carcinoma (HCC) is multifactorial and includes genetic predisposition, though the precise nature of the latter is as yet poorly understood. Mice heterozygous for the polo like kinase Plk4 develop spontaneous HCC and human HCC cases show a high rate of LOH (loss of heterozygosity) at the Plk4 locus. We hypothesize that Plk4 is a haploinsufficient tumor suppressor in man. The purpose of this study was to investigate the mechanisms by which a 50% reduction of Plk4 leads to carcinogenesis. Methods: We established Plk4± and Plk4+/+ murine embryonic fibroblast cell lines (MEFs) in vitro. 5 of 5 Plk4+/+ MEF lines senesced at passage 7–8 as expected, while 9 of 9 Plk4±MEF lines immortalized in culture under NIH 3T3 protocol. The latter were used at passage 15–20 for in vivo tumorigenesis studies. Results: Plk4± MEFs demonstrated increased ploidy with increased passage number. By passage 4, Plk4± MEFs showed a near tetraploid karyotype as well as multiple chromosomal rearrangements and deletions, as assessed by Spectral Karyotype (SKY) Analysis. 5 out of 9 late passage Plk4± MEF lines injected into NOD-SCID mice grew tumors, with a latency of 3 to 12 weeks. By contrast, injection of Plk4+/+ MEFs yielded no tumors. Tumors generated from injection of Plk4± MEFs were harvested, cultured and submitted to SKY analysis; this showed increased clonal rearrangements and deletions compared to the parent cell lines. Conclusion: A 50% reduction in Plk4 expression results in immortalization, aneuploidy and chromosomal instability in vitro, and tumorigenicity in vivo. These results demonstrate that insufficient levels of Plk4, whether due to mutation or LOH, can contribute to the pathogenesis of HCC. No significant financial relationships to disclose.

Lidocaine Inhibits NIH-3T3 Cell Multiplication by Increasing the Expression of Cyclin-Dependent Kinase Inhibitor 1A (p21)

We explored molecular mechanisms by which lidocaine inhibits growth in the murine embryonic fibroblast cell line NIH-3T3. Local anesthetics can adversely affect cell growth in vitro. Their effects on wound healing are controversial. We examined the effects and novel mechanisms by which lidocaine affects in vitro multiplication of the murine fibroblast cell line NIH-3T3. NIH-3T3 cells were grown in culture with lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM]. Cell multiplication was assessed by determining cell counts on subsequent days, while mechanisms by which inhibition occurred were evaluated by bromodeoxyuridine uptake, gene expression using polymerase chain reaction array, and Western blot analysis to verify increased levels of affected proteins. Lidocaine caused dose-dependent inhibition of multiplication of NIH-3T3 cells. Effects ranged from no inhibition [0.05 and 0.5 mM] and mild inhibition [1 mM], to severe inhibition [2 and 5 mM] [P = 0.006]. Lidocaine 2 mM inhibited bromodeoxyuridine uptake at day 3.5 [P = 0.02 versus control, and P = 0.0495 vs 1 mM lidocaine]. On day 1.5, lidocaine upregulated expression of cyclin-D1 and cyclin-dependent kinase inhibitor 1A [p21]. On day 2.5, lidocaine increased the levels of p21 protein. Low concentrations of lidocaine, as would be seen in plasma after spinal, epidural, or plexus anesthesia, do not significantly affect multiplication of fibroblasts. Higher doses of lidocaine arrest cell multiplication at the S-phase of the growth cycle by upregulation of p21, an extremely potent inhibitor of cell multiplication. Higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing.

Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.

Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

Lack of Mcl-1 Confers Resistance to Bortezomid in Multiple Myeloma.

Bortezomib is a proteasome inhibitor recently FDA approved for treatment of relapsed refractory MM. To date, however, mechanisms of MM cell sensitivity versus resistance to Bortezomib are undefined. Mcl-1 (42kd) is an anti-apoptotic protein which protects MM cells against spontaneous and chemotherapy-induced apoptosis. Here we examine the role of Mcl-1 in protection against Bortezomib-induced apoptosis. We studied modulation of Mcl-1 expression triggered by Bortezomib in MM1S cells and two murine embryonic fibroblast cell lines (MEFs): Mcl-1 deleted (Mcl-1 Δ/null) and its wild type (Mcl-1wt/wt). We similarly evaluated conventional therapies (doxorubicin and melphalan) for MM. Treatment with doxorubicin, melphalan and Bortezomib for 48h inhibits MM1S cell growth, measured by MTT assay, in a dose dependent-manner with IC50 of 50 nM, 9 M and 5 nM, respectively. At 6h and 24h Bortezomib, but not melphalan (up to 40 μM) or doxorubicin (up to 200nM), induces appearance of cleaved-form (28kd) of Mcl-1 associated with Caspases-8, -9, and -3, as well as PARP cleavage.Down-regulation and cleavage of Mcl-1 is observed after 24h at Bortezomib concentrations as low as 2.5nM. The pan-caspase inhibitor Z-VAD-FMK inhibits induction of Mcl-1 cleavage, suggesting that it is caspase-dependent. We therefore hypothesized that Mcl-1 may be an early target in Bortezomib-induced-apoptosis. To address this issue, we compared the percentage of apoptotic Mcl-1Δ/null vs Mcl-1wt/wt MEFs, defined as the percentage of cells in sub-G1 phase by flow cytometry (PI staining). Both cell lines were sensitive to doxorubicin: 50% vs 45% apoptosis at 50nM and 85% vs 80% apoptosis at 100nM in Mcl-1wt/wt and Mcl-1Δ/null MEFs, respectively. In contrast, melphalan (10 μM) failed to induce apoptosis in Mcl-1Δ/null MEFs: 60% vs 10% apoptotic cells in Mcl-1wt/wt and Mcl-1Δ/null MEFs, respectively. Interestingly, in melphalan treated-cells PARP is cleaved, but no morphological apoptosis is observed. Furthermore, cell cycle analysis in melphalan treated-cells demonstrates accumulation of Mcl-1Δ/null MEFs in G2/M phase. Bortezomib (up to 20nM) similarly failed to induce apoptosis in Mcl-1Δ/null MEFs: 80% vs 17% apoptotic cells in Mcl-1wt/wt and Mcl-1Δ/null MEFs, respectively. Lack of induction of sub-G1 Mcl-1Δ/null cells by Bortezomib correlated with absence of both PARP cleavage and morphological signs of apoptosis. Interestingly and as in MM1S cells, only Bortezomib induces Mcl-1 cleavage in Mcl-1wt/wt MEFs. These results suggest that Bortezomib and melphalan, but not doxorubicin, trigger distinct Mcl-1-dependent induced-apoptosis pathways: Bortezomib triggers Mcl-1 cleavage prior to PARP cleavage; and melphalan triggers PARP cleavage, with downstream Mcl-1 required for progression from G2/M to sub-G1 phase. In conclusion, our study demonstrates that Mcl-1 is not only an anti-apoptotic protein but also an important mediator of Bortezomib and melphalan, but not doxorubicin, induced-apoptosis.