Addition Of Doxycycline Research Articles

A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages

SummaryPrecise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants—along with engineered mutants—in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.

Combination of amikacin and doxycycline against multidrug-resistant and extensively drug-resistant tuberculosis

The objective of this study was to assess the activity of amikacin in combination with doxycycline against clinical strains of Mycobacterium tuberculosis in the search for new strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. The study included 28 clinical M. tuberculosis strains, comprising 5 fully susceptible, 1 isoniazid-resistant, 17 MDR, 1 poly-resistant (streptomycin/isoniazid), 1 rifampicin-resistant and 3 XDR isolates, as well as the laboratory strain M. tuberculosis H37Rv. Minimum inhibitory concentrations (MICs) were determined using a modified chequerboard methodology in a BACTEC™ MGIT™ 960 System. Fractional inhibitory concentration indices (FICIs) were calculated, and synergy, indifference or antagonism was assessed. Whole-genome sequencing was performed to investigate the genetic basis of synergy, indifference or antagonism. The MIC50 and MIC90 values (MICs that inhibit 50% and 90% of the isolates, respectively) were, respectively, 0.5mg/L and 1.0mg/L for amikacin and 8mg/L and 16mg/L for doxycycline. The combination of amikacin and doxycycline showed a synergistic effect in 18 of the 29 strains tested and indifference in 11 strains. Antagonism was not observed. A streptomycin resistance mutation (K43R) was associated with indifference. In conclusion, the benefit of addition of doxycycline to an amikacin-containing regimen should be explored since in vitro results in this study indicate either synergy or indifference. Moreover, doxycycline also has immunomodulatory effects.

Establishment of cell lines derived from the genus Macaca through controlled expression of cell cycle regulators.

Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines.

Activation of Rac1 through FARP1 Induces Dexamethasone Resistance in Multiple Myeloma Cells

Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients’ survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Infrared Microspectroscopy Allows Direct Identification of Leukemic Cells Expressing T315I-Mutated BCR-ABL Via a Unique Spectral Signature

The use of tyrosine kinase inhibitor (TKI) therapies has dramatically changed the prognosis of chronic myeloid leukemia (CML) and modified the natural history of the disease. However, the generation of ABL-kinase domain mutations due to the genetic instability of leukemic cells continues to be very significant challenge, especially in advanced phases of the disease. Amongst these mutations, T315I is the most problematic as it reduces the binding of Imatinib, Dasatinib or Nilotinib to their target, leading to a total resistance to all three TKIs. Although the mechanism of the appearance of this mutation is not completely known, it is possible that it induces a signalling pathway not entirely similar to that induced by classical wild-type BCR-ABL. From the mechanistic point of view, the mechanism of resistance of T315I-mutated BCR-ABL to Imatinib is related to the substitution of Isoleucine to Threonine, which normally forms a hydrogen bond with Imatinib, this bond being absolutely required for binding of the drugs to the catalytic site. T315I mutation can be identified by direct sequencing, D-HPLC, allele specific PCR or double gradient gel electrophoresis of the PCR products amplified from the BCR-ABL kinase domain. However these techniques cannot identify directly the leukemic cells expressing T315I-mutated BCR-ABL which are known to co-exist with native BCR-ABL-expressing cells in bone marrow. Infrared microspectroscopy allows the identification of metabolic features of mammalian cells, based on their protein, lipid, amid and nucleic acid contents, generating a spectral signature. In this work we asked whether infrared microspectroscopy can be used to identify metabolic changes occurring in single cells bearing BCR-ABL T315I mutation. For this purpose we have used human (UT7) and murine embryonic stem cells (GS2) expressing either native (N) or T315I-mutated BCR-ABL. In the human UT7 cells expressing N-BCR-ABL, we were able to clearly distinguish at the single cell level, cells expressing BCR-ABL from wild type cells using principal component analysis. However, the presence of T315I mutation induced a clearly different signature, allowing a highly significant separation by the use of spectral signature. To confirm the specificity of this signature, we have used UT7 cells engineered to express either native or T315I-mutated BCR-ABL under the control of Doxycycline (DOX) -sensitive promoters. In this TET-OFF system, the addition of doxycycline to the culture medium inhibits BCR-ABL expression as monitored by Western blots, in approximately 6 days. Using this system, at day 0, UT7 cells expressing N-BCR-ABL and T315I mutated BCR-ABL were clearly distinguishable from parental UT7 cells. Upon inhibition of BCR-ABL expression by addition of Doxycycline, the spectral signature of N-BCR-ABL and T315I-mutated BCR-ABL cells became similar at day 2 and indistinguishable at day 4, but still distinguishable from wild type UT7 cells expressing DOX-inducible GFP. To confirm these results in a different cell context we have used a murine embryonic stem (ES) cell line (GS2) which was transduced with either N-BCR-ABL or T315I-mutated BCR-ABL-expressing lentiviral vectors. BCR-ABL-expressing individual clones grown were analyzed to analyze the spectral signature. In this cell line also, infrared microspectroscopy was able to distinguish ES cells expressing BCR-ABL T315I as compared to N-BCR-ABL. Thus, these results suggest that T315I mutation clearly induces metabolic changes different from N-BCR-ABL in leukemic cells, rendering them identifiable by the analysis of nucleic acid, lipid, amid and sugar contents. This new methodology can now be applied to the identification of primary CML leukemic cells harboring T315I at the single cell level and could be of interest for rapid identification of leukemic cells in a single step. This technique can also be used for rapid screening of novel compounds active against T315I mutation using microfluidic technologies leading to novel drug discoveries. DisclosuresTurhan:Bristol Myers Squibb, Novartis: Honoraria, Research Funding.

A Matched Case Control Study of Doxycycline Added to Chemotherapy for Reducing Early Mortality in Patients with Advanced Cardiac AL Amyloidosis from the Alchemy Study Cohort

Background: Survival of patients with AL amyloidosis has improved over the last decade due to availability of novel agent based therapy. Bortezomib combination regimes, such as CyBorD, give unprecedented response rates in early stage disease but cannot overcome poor prognosis in patients with advanced AL amyloidosis (Venner et al and Palladini et al Leukemia 2014); which has remained poor and essentially unchanged over the last 25 years. Heart failure occurs in AL amyloidosis not only due to mechanical impairment of cardiac function from physical amyloid deposition but also due to direct cardiomyocyte light chain toxicity as shown in animal models by groups from Pavia and Boston. Doxycycline appeard to reduce light chains muscle toxicity in a C.Elegans model and hence may have a potential role to improve outcomes in cardiac AL.Methods: We report here the outcomes of a cohort of patients with advanced cardiac amyloidosis treated with addition of doxycycline (as an off-label indication) to chemotherapy compared to matched group of similarly treated control patients without doxycycline, identified from the ALChemy study database of the UK National Amyloidosis centre. ALChemy is a prospective observational study of patients with AL amyloidosis undergoing treatment and has currently recruited just over 800 patients. All patients who received doxycycline with their front line chemotherapy regime were identified These patients were matched with controls based on updated Mayo disease stage, age, SBP >100 mm of Hg, dFLC and NT-proBNP levels. Since all but one patient in the doxycycline cohort received a velcade triplet regime, only patients treated with a velcade triplet regime were included in the matching cohort.Patients and results: A total of 16 patients and 22 controls were identified. The median age was 60 yrs (range 40-81 yrs), all patients had cardiac involvement, 35 patients had Mayo stage 4 disease, 3 had Mayo stage 3 disease. The median NT-proBNP was 5621 pMol/L (range 1211-17365), high sensitivity troponin 0.1 mcg/L (range 0.022-0.58), eGFR 60 ml/min (range 15-100) and dFLC 490 mg/L (range 54-5825). Two patients from each group had NT-proBNP >8500 ng/L. There was no significant difference at baseline between the treated and control groups in terms of length of follow up, body mass index, supine systolic BP, LV wall thickness, ejection fraction, NT-proBNP, Hs-TNT, proteinuria or dFLC. 34 (89%) started with CyBorD, 1 each with Bor-MelDex, Vel-Rev-Dex and IV intermediate dose melphalan. 4 patient (3 in the doxycycline and one in the control group) switched due to lack of clonal response at cycle 2 or 3. 14 patients received doxycycline 100 mg daily and two patients received 200 mg daily. Three patients had to discontinue doxycycline early (2 months each) due to photosensitive rash in 2 and nausea in one patient. Overall 60% achieved a complete haematological response (CR), 8 % VGPR and 13% PR. In the doxycycline group 56% achieved a CR (89% CR+VGPR) compared to 63% CR (no VGPR’s) in the control group. The median follow up of the whole cohort is 13 months and there were a total of 10 deaths – 9 deaths in the control group (median 3.5 months) and one in the doxycycline treated group (at 2.3 months). The 3, 6 and 12 month survival in the doxycycline treated group was 94% and in the control group was 86%, 68% and 55% respectively; median not reached in either group (log rank p =0.04; Figure 1). On an intent to treat basis, 57% of the doxycycline group and 36% of the control group achieved cardiac responses by NT-proBNP criteria.Conclusions: Treatment with doxycycline in combination with a bortezomib triplet regime significantly reduces early mortality (with only one death in the doxycycline treated group over one year median follow up) in patients with advanced cardiac AL amyloidosis which translated into a higher CR/VGPR rate compared to controls and greater cardiac responses. This is one of the first interventions to have a significant impact on early mortality in AL. Larger studies are urgently needed to confirm the findings of this simple intervention to substantially improve outcomes in cardiac AL amyloidosis. [Display omitted] DisclosuresOff Label Use: Doxycycline for reducing early cardiac mortality.

A Novel DNA Repair Chip Assay Allows Rapid Identification of DNA Repair Abnormalities Induced By BCR-ABL

BCR-ABL expression in leukemic cells is associated with several types of DNA repair abnormalities, involving homologous recombination (HR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), mismatch repair (MMR) and base-excision repair (BER) pathways. These defects, combined with the genetic instability inherent to chronic myeloid leukemia (CML), are thought lead to ABL-kinase domain mutations which remain poorly repaired and selected by tyrosine kinase inhibitor (TKI) therapies, leading to drug resistance. Currently, there is no functional, practical, single step assay to determine if BCR-ABL expression in a given leukemic cell is predominantly associated with a specific DNA repair pathway. For this purpose, we have used a novel DNA repair test based on biopchips evaluating the functional capacity of cells to achieve DNA repair through the use of a functional excision/synthesis repair assay that establish DNA Repair Enzyme Signatures. This assay allows monitoring the repair of a set of DNA lesions handled by BER (8oxoG, alkylated bases, glycols and Abasic sites) and NER (photoproducts, psoralen and cisplatin adducts). For modelling CML, we have used the UT7 cell line expressing BCR-ABL and T315I-mutated BCR-ABL. Parental (P)-UT7 cells were used as controls. We have tested the effects of BCR-ABL tyrosine kinase activity on the DNA repair assays using two strategies: The first was the evaluation of the effects of the nuclear extracts of UT7 cells expressing either native (N) BCR-ABL or T315I-mutated BCR-ABL before and after incubation of the cells in the presence of Imatinib (IM) or Nilotinib whereas the second tested the effects of BCR-ABL in an inducible system without the use of TKI. As expected, UT7 cells with N-BCR-ABL were sensitive to the inhibitory effects of these drugs whereas UT7 cells with T315I-mutated BCR-ABL were totally resistant. Using this system, we have found that the inhibition of BCR-ABL by IM stimulated the DNA repair capacity of the cells and specially BER (8oxoG, alkylated bases and glycols) and NER (photoproducts and psoralen adducts) after 4 or 8 hours of treatment, as compared to baseline repair capacities ( p< 0.05). No effect was detected for the repair of Abasic sites and Cisplatin adducts. When the cells were treated with Nilotinib in the same conditions, we have observed also the stimulation of DNA repair capacities for all lesions except for the Abasic sites ( p< 0.125). The fact of treating P-UT-7 cells or UT7-T315I cells with IM or Nilotinib had no effect and led conversely, to the inhibition of DNA repair activity via Abasic and Cisplat adducts pathways (p< 0.05) or Pso (p < 0.125). To confirm directly the effect of the BCR-ABL activity in the repair modifications observed, we have used UT7 cell lines expressing N-BCR-ABL or T315I-mutated BCR-ABL under the control of a Doxycycline-inducible promoter. In this TET-OFF system, BCR-ABL expression is inhibited upon Doxycycline addition to the culture and BCR-ABL protein becomes undetectable by Western blots at day 8. In this system, the analysis of DNA repair capacity demonstrated a statistically significant stimulation of repair of Alkylated bases, Glycols, Psoralen adducts and photoproducts ( p < 0.05) upon inhibition of N-BCR-ABL or that of T315I-mutated BCR-ABL. Interestingly, the fact of reducing BCR-ABL T315I in the DOX-inducible system stimulated DNA repair capacity whereas in the non-inducible UT7 cells expressing T315I, IM did not have a stimulatory effect, whereas Nilotinib induced a slight stimulation of both NER and BER pathways after 8 hours of treatment. Overall, these results demonstrate that the novel microarrays that we describe can be efficiently detect the DNA repair ability of the leukemic cells and the reversal of these abnormalities on TKI therapies. The preliminay results suggest clearly a differential effect of IM as compared to Nilotinib in terms of the stimulation of DNA repair capacity. This new methodology can now be used to quantify the abnormality of the DNA repair pathways in CML cells at diagnosis, allowing the monitoring of the tyrosine kinase inhibitor therapies and potentially predicting the occurrence of ABL-kinase mutations according to the preferential involvement of NER or BER pathways. DisclosuresTurhan:Bristol Myers Squibb, Novartis: Honoraria, Research Funding. Sauvaigo:LXRepair: Employment, Equity Ownership.

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Cell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promoters.

The GTPases RhoA and Rac1 are key regulators of cell spreading, adhesion, and migration, and they exert distinct effects on the actin cytoskeleton. While RhoA classically stimulates stress fiber assembly and contraction, Rac1 promotes branched actin polymerization and membrane protrusion. These competing influences are reinforced by antagonistic crosstalk between RhoA and Rac1, which has complicated efforts to identify the specific mechanisms by which each GTPase regulates cell behavior. We therefore wondered whether RhoA and Rac1 are intrinsically coupled or whether they can be manipulated independently. To address this question, we placed constitutively active (CA) RhoA under a doxycycline-inducible promoter and CA Rac1 under an orthogonal cumate-inducible promoter, and we stably introduced both constructs into glioblastoma cells. We found that doxycycline addition increased RhoA activity without altering Rac1, and similarly cumate addition increased Rac1 activity without altering RhoA. Furthermore, co-expression of both mutants enabled high activation of RhoA and Rac1 simultaneously. When cells were cultured on collagen hydrogels, RhoA activation prevented cell spreading and motility, whereas Rac1 activation stimulated migration and dynamic cell protrusions. Interestingly, high activation of both GTPases induced a third phenotype, in which cells migrated at intermediate speeds similar to control cells but also aggregated into large, contractile clusters. In addition, we demonstrate dynamic and reversible switching between high RhoA and high Rac1 phenotypes. Overall, this approach represents a unique way to access different combinations of RhoA and Rac1 activity levels in a single cell and may serve as a valuable tool for multiplexed dissection and control of mechanobiological signals.

Abstract 137: Pharmacologically Controlled IGF-1 Release For Paracrine Support Of Engineered Heart Muscle

Tissue engineered heart repair is developing rapidly, but needs refinement before clinical translation. We tested the hypothesis that force generating human engineered heart muscle (EHM) can be enhanced by integration of insulin-like growth factor-1 (IGF-1) secreting fibroblasts. Methods: TetOn lentiviral particles encoding for IGF-1 and the Tet-transactivator (tTA) were cloned and used to stably transduce human foreskin fibroblasts (HFF). Baseline and doxycycline induced IGF-1 release from HFFTetOn+IGF1 was quantified by ELISA. HFFwt and HFFTetOn+IGF1 conditioned medium was layered over human embryonic stem cell (HES2) derived cardiomyocytes followed by an analysis of AKT-phosphorylation. EHMs were assembled from HES2-derived cardiomyocytes and HFF (HFFwt or HFFTetOn+IGF1) at 70:30 ratio. Transgene activation was induced by addition of doxycycline (10 ng/ml) for 7 days. Twitch forces and response to pharmacological stimuli were measured to assess the functional consequences of IGF-1 release. EHMs were subsequently subjected to morphological analysis or dissociated into single cells to assess cellular composition of EHMs. Results: HFFTetOn+IGF1 released IGF-1 upon doxycycline stimulation (3.3x10-6 vs 8.3x10-8 [HFFwt] ng/ml/cell/day. Secreted IGF-1 from HFFTetOn+IGF1 induced Akt phosphorylation in HES2-derived cardiomyocytes (2.4±0.6 fold increase of [HFFwt]; n=3). EHMs with HFFTetOn+IGF1 developed significantly higher twitch forces than EHMs with HFFwt (0.24±0.03 vs 0.15±0.02 mN; n=10) under baseline conditions (IGF-1 leak). Doxycycline induced IGF-1 release further enhanced (P&lt;0.05) EHM twitch force (0.26±0.03 mN; n=10). Single cell analysis from EHMs demonstrated cardiomyocyte hypertrophy in response to paracrine IGF-1 release (154±11% of [HFFwt] control; n=4). Histological analyses demonstrated that HFFTetOn+IGF1 supplemented EHMs contained thicker muscle bundles and enlarged cardiomyocytes. Conclusion: EHM can be functionally enhanced by integration of drug-controllable IGF-1 release. Drug controllable, cell based paracrine release of protective factors may not only be exploited to enhance tissue engineered myocardium in vitro but also to achieve better survival and integration of EHM grafts in vivo.

Answer to June 2014 Photo Quiz

Answer: Dysgonomonas capnocytophagoides. This isolate, which was nonmotile and catalase and oxidase negative, was identified by the Vitek 2 system (bioMerieux, Inc., Hazelwood, MO) as Rhizobium radiobacter, while RapID NF (Remel, Lenexa, KS) gave no identification. The isolate was submitted for identification by partial 16S rRNA gene sequencing as well as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Sequencing identified the isolate as Dygonomonas capnocytophagoides. Blood cultures remained positive for 13 days despite treatment with meropenem alone followed by dual therapy with piperacillin-tazobactam and levofloxacin. The MICs of ceftriaxone, erythromycin, ampicillin, clindamycin, and tetracycline for this organism were ≥256 μg/ml, 8 μg/ml, 32 μg/ml, 0.25 μg/ml, and 0.5 μg/ml, respectively. The addition of doxycycline based on these susceptibility results quickly sterilized the patient's bloodstream. Ultimately, the hepatic abscesses were excised en bloc on hospital day 48 with the explantation and reimplantation of the patient's second orthotopic liver. His postoperative course was uneventful, and he has since made a full recovery. The genus Dysgonomonas is subdivided into three major species, Dysgonomonas mossii, D. gadei, and D. capnocytophagoides (formerly designated CDC group DF-3), with a fourth species “Dysgonomonas hofstradii,” proposed (1,–3). Colonies measure 1 to 2 mm in diameter after 24 h of growth, have a distinct strawberry-like odor, and do not spread or adhere. Microscopically, Dysgonomonas spp. appear as small, Gram-negative rods or cocci. All members are oxidase negative, do not reduce nitrate or produce H2S, ferment glucose, lactose, and xylose, and produce alkaline phosphatase and β-galactosidase but not arginine dehydrolase. Although the species show few biochemical differences, the distinguishing features of D. capnocytophagoides include negative indole and catalase reactions (2, 3). The pathogenic potential of this uncommon bacterium seems to be limited to immunocompromised individuals. In 1988, D. capnocytophagoides was reported to have caused diarrhea in an immunocompromised host and later bacteremia in two leukemic patients, one of whom had the strain isolated from stool (1). When D. capnocytophagoides causes clinical disease, a gastrointestinal source should be strongly considered because most strains have been isolated from stool, although the organism has also been isolated from urine and skin abscesses (1, 3). Another alarming feature of Dysgonomonas spp. is their well-established resistance to beta-lactams, macrolides, aminoglycosides, and quinolones (4), a susceptibility profile similar to that of our patient's organism. Identification of this organism with conventional identification techniques can be difficult. MALDI-TOF MS misidentified the organism as D. gadei, an error previously documented in the medical literature because of gaps in the MALDI-TOF MS database. A negative catalase reaction in this situation would indicate that the correct identification is indeed D. capnocytophagoides (5). Though 16S rRNA gene sequencing correctly identified the pathogen as D. capnocytophagoides, this procedure is time-consuming and is also considerably more expensive to perform than MALDI-TOF MS. MALDI-TOF MS may replace such molecular techniques in identifying such rare pathogens as D. capnocytophagoides once the databases become more accurate and reliable (6). (See page 1811 in this issue [doi:10.1128/JCM.00349-13] for photo quiz case presentation.)

Heparanase enhances myeloma progression via CXCL10 downregulation.

In order to explore the mechanism(s) underlying the pro-tumorigenic capacity of heparanase, we established an inducible Tet-on system. Heparanase expression was markedly increased following addition of doxycycline (Dox) to the culture medium of CAG human myeloma cells infected with the inducible heparanase gene construct, resulting in increased colony number and size in soft agar. Moreover, tumor xenografts produced by CAG-heparanase cells were markedly increased in mice supplemented with Dox in their drinking water compared with control mice maintained without Dox. Consistently, we found that heparanase induction is associated with decreased levels of CXCL10, suggesting that this chemokine exerts tumor-suppressor properties in myeloma. Indeed, recombinant CXCL10 attenuated the proliferation of CAG, U266 and RPMI-8266 myeloma cells. Similarly, CXCL10 attenuated the proliferation of human umbilical vein endothelial cells, implying that CXCL10 exhibits anti-angiogenic capacity. Strikingly, development of tumor xenografts produced by CAG-heparanase cells overexpressing CXCL10 was markedly reduced compared with control cells. Moreover, tumor growth was significantly attenuated in mice inoculated with human or mouse myeloma cells and treated with CXCL10-Ig fusion protein, indicating that CXCL10 functions as a potent anti-myeloma cytokine.

A Comprehensive System for Generation and Evaluation of Induced Pluripotent Stem Cells Using piggyBac Transposition

The most stringent criterion for evaluating pluripotency is generation of chimeric animals with germline transmission ability. Because the quality of induced pluripotent stem cell (iPSC) lines is heterogeneous, an easy and accurate system to evaluate these abilities would be useful. In this study, we describe a simple but comprehensive system for generating and evaluating iPSCs by single transfection of multiple piggyBac (PB) plasmid vectors encoding Tet-inducible polycistronic reprogramming factors, a pluripotent-cell–specific reporter, a constitutively active reporter, and a sperm-specific reporter. Using this system, we reprogrammed 129 and NOD mouse embryonic fibroblasts into iPSCs, and then evaluated the molecular and functional properties of the resultant iPSCs by quantitative RT-PCR analysis and chimera formation assays. The iPSCs contributed extensively to chimeras, as indicated by the constitutively active TagRFP reporter, and also differentiated into sperm, as indicated by the late-spermatogenesis–specific Acr (acrosin)-EGFP reporter. Next, we established secondary MEFs from E13.5 chimeric embryos and efficiently generated secondary iPSCs by simple addition of doxycycline. Finally, we applied this system to establishment and evaluation of rat iPSCs and production of rat sperm in mouse–rat interspecific chimeras. By monitoring the fluorescence of Acr-EGFP reporter, we could easily detect seminiferous tubules containing rat iPSC–derived spermatids and sperm. And, we succeeded to obtain viable offspring by intracytoplasmic sperm injection (ICSI) using these haploid male germ cells. We propose that this system will enable robust strategies for induction and evaluation of iPSCs, not only in rodents but also in other mammals. Such strategies will be especially valuable in non-rodent species, in which verification of germline transmission by mating is inefficient and time-consuming.

MTOR, autophagy, and reprogramming.

mTOR, autophagy, and reprogramming.

Three-dimensional patterning of multiple cell populations through orthogonal genetic control of cell motility

The ability to independently assemble multiple cell types within a three-dimensional matrix would be a powerful enabling tool for modeling and engineering complex tissues. Here we introduce a strategy to dynamically pattern distinct subpopulations of cells through genetic regulation of cell motility. We first describe glioma cell lines that were genetically engineered to stably express constitutively active or dominant negative Rac1 GTPase mutants under the control of either a doxycycline-inducible or cumate-inducible promoter. We culture each population as multicellular spheroids and show that by adding or withdrawing the appropriate inducer at specific times, we can control the timing and extent of Rac1-dependent cell migration into three-dimensional collagen matrices. We then report results with mixed spheroids in which one subpopulation of cells expresses dominant negative Rac1 under a doxycycline-inducible promoter and the other expresses dominant negative Rac1 under a cumate-inducible promoter. Using this system, we demonstrate that doxycycline and cumate addition suppress Rac1-dependent motility in a subpopulation-specific and temporally-controlled manner. This allows us to orthogonally control the motility of each subpopulation and spatially assemble the cells into radially symmetric three-dimensional patterns through the synchronized addition and removal of doxycycline and cumate. This synthetic biology-inspired strategy offers a novel means of spatially organizing multiple cell populations in conventional matrix scaffolds and complements the emerging suite of technologies that seek to pattern cells by engineering extracellular matrix properties.

Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.

Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP

Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1μg/mL and removing doxycycline from the media 60min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation.

A Robust Screening Assay For Diamond Blackfan Anemia Candidate Drugs

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome associated with ribosomal protein (RP) deficiency caused by mutations in RP genes. 25% of patients carry mutations in the RPS19 gene. A salient feature of DBA is proliferative arrest of erythroid progenitors. It is still not clear how defects in RPs, which are essential to all cell types, affect the erythroid compartment more severely than the others. Today, DBA patients are treated with glucocorticoids and/or blood transfusions, often for long periods, causing severe adverse effects. We hypothesize that novel drugs with more disease-specific therapeutic mechanisms can be developed and that such drugs will be superior to current therapies. Towards this aim we have developed a method for screening chemical libraries to identify novel drug candidates.Use of DBA patient cells is not feasible as their limited availability severely limits the size of the applied screening library. Immortalized cell lines are also less suitable since the disease mechanism involves TP53 activation and most cell lines have perturbations in the TP53 pathway. Hence, primary c-Kit+ E14.5-15.5 fetal liver erythroid progenitor cells from a mouse model of DBA with doxycycline inducible expression of rps19-shRNA were used (Jaako et. al. Blood, 2011). The DBA phenotype is induced by adding doxycycline to the culture medium which reduces erythroid proliferation >80%. Rescue of this proliferation defect is a simple and relevant readout for large-scale screening.Assay development in 96 well microtiter plates allowed rational liquid handling. Firstly, the proliferation readout method was optimized. Use of CellTiter-Glö Luminescent Cell Viability Assay generated data with superior linear correlation with cell numbers compared to Prestobluë and high content screening microscopy approaches. To enable large-scale screening, the assay was further optimized for cell numbers per well, media composition, culture duration, doxycycline concentration and timing of induction and of addition of test chemicals. We also controlled for evaporation during incubation to significantly reduce plate edge effects. During these optimizations, luminescence readout from uninduced cells was set to represent 100% rescue and readout from doxycycline-induced controls to represent 0% recue. This allows reliable normalization between plates and gives toxic chemicals a negative value and chemicals rescuing proliferation a positive value with 100% meaning complete rescue. To objectively quantify how changes of different parameters improved variability in both induced and uninduced cells, we calculated the Z factor as described by Zhang et. al. (JBS, 1999). The Z factor takes into consideration both the range and variability of data to calculate the suitability of an assay for high throughput screening. It is represented as:▪(SD: Standard Deviation). Where Z value is meaningful in the range of -1 < Z ² 1. The larger the value of Z, the higher is the data quality and a Z-factor above 0.5 is considered very robust.After assay development we arrived at the conditions shown in the table, resulting in the Z factor of 0.7:Culture medium100 ng/ml mSCF, 2 U/ml Epo, 100 nM dexamethasone in serum-free medium. (doxycycline: 0.5 μg/ml).Culture days4 (addition of test chemicals 24 hours after doxycycline induction)Cells per well2000 (murine cKit+ fetal liver cells with inducible rps19-shRNA)ReadoutLuciferase based ATP detection (CellTiter-Glo¨).For screening involving small molecule based libraries we added the compounds 24 hours after cell seeding and doxycycline addition, which allows for the induction of the proliferation perturbed phenotype. We have pharmacologically validated the assay by testing and quantifying the impact of IL-3, a cytokine known to have a positive effect on erythroid progenitors. IL-3 had a 25% rescue value in our assay. Ongoing experiments (>11,000 compounds screened to date) show that more than 50% of compounds with >20% rescue score in the screen could be confirmed to rescue proliferation in dose-dependent experiments.In conclusion, we have established a robust scalable assay for screening molecules that rescue proliferation arrest caused by Rps19-deficiency in mouse erythroid progenitors. We are currently using this assay to screen small molecule libraries in our search for novel tool compounds for DBA research and drug candidates for DBA treatment. Disclosures:No relevant conflicts of interest to declare.

Interferon-Inducible Protein 16 (IFI-16) Is Upregulated In Chronic Myeloid Leukemia (CML) and Is Involved In Oncogene-Induced Senescence

IFI-16 is a transcriptional factor induced by IFN-γ and α in human cells and is involved in several cellular processes including cell cycle regulation, telomere length control and cellular senescence. Recently it has been shown that, similar to other oncogenes such as RAS and BRAF in solid tumors, acute expression of BCR-ABL in hematopoietic cells can induce senescence (Wajapayee et al, Blood 2010). This oncogene-induced senescence (OIS) which is a barrier for tumor development, is obviously bypassed by secondary events leading to malignant transformation. The molecular basis of the BCR-ABL-induced senescence in CML cells is not determined. In the UT7 cell line model expressing native (unmutated) BCR-ABL and its T315I-mutated counterpart, we identified by a gene profiling, a major increase of the expression IFI-16 (42-fold and 18-fold increase, respectively) as compared to parental UT7 cells. This major increase of IFI-16 expression in UT7 cells expressing native BCR-ABL was confirmed in western blots, whereas there was no detectable IFI-16 protein in parental UT7 cells. To determine if IFI-16 protein levels were dependent on BCR-ABL expression, we have used doxycycline-inducible BCR-ABL UT7 model. In this TET-Off system, the addition of doxycycline to cultures inhibits the expression of BCR-ABL by day7 and this was accompanied by a concomittant inhibition of IFI-16 expression. As expected, IFN-α (1000 U/ml) induced IFI-16 expression in parental UT7 cells and to a much higher degree in UT-7 BCR-ABL cells. In the UT7-T315I cells, IFN- α showed also a synergistic effect for IFI-16 expression which was less prominent. As IFI-16 expression can be modulated by oxidative stress, we have performed experiments in the presence of N-acetyl-cysteine which did not modify IFI-16 protein levels. To further study the role of IFI-16 in this model, we have transfected UT7 cells and their BCR-ABL expressing counterparts using shRNA-IFI-16 lentiviral vectors allowing silencing of IFI-16 expression. UT7 clones resistant to puromycin were amplified and the clones with reduced or absent expression of IFI-16 were selected for further analyses. In senescence-induction experiments, we have found that native BCR-ABL expressing UT7 silenced for IFI-16 expression, had reduced beta-galactosidase activity suggesting an inhibition of senescence phenotype. We have then checked the localization of IFI-16 in BCR-ABL and BCR-ABL T315I-expressing UT7 cells. In BCR-ABL-UT7 cells. IFI-16 was mainly in the cytoplasm and was relocated to nucleus upon IFN-α treatment. On the other hand, in UT7-T135I cells, IFI-16 was distributed between cytoplasm and nucleus and IFN-α had no effect on IFI-16 localisation. To determine the potential involvement of IFI-16 in human CML, we have analyzed IFI-16 expression by Q-RT-PCR. In a cohort of 40 Chronic Phase (CP) CML patients at diagnosis, the expression of IFI-16 was found to be significantly increased (2-Fold) as compared to controls (n=30). Thus, our results suggest strongly that IFI-16 induction could be one of the mechanisms of OIS in in BCR-ABL expressing cells. We also demonstrate for the first time that this mechanism is operational in primary CML cells. Further exploitation of this senescence pathway could be of therapeutic interest with the goal of enhancing this phenomenon especially at the level of the most primitive, TKI-resistant CML stem cells. Current experiments are underway to study the role of IFI-16 in the most primitive leukemic stem cell compartments. Disclosures:Guilhot:Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.