The ETS Factor Myeloid Elf-1-Like Factor (MEF)/Elf4 Is Transcriptionally and Functionally Activated by Hypoxia.

Hypoxia-inducible factor (HIF)-1α is a transcription factor belonging to the HIF family that is activated in mammalian cells during conditions of low oxygen tension or hypoxia to induce an adaptive response and promote cell survival. Some of the genes targeted by HIF-1α are important for angiogenesis and proliferation. Here, we found that the E26 transformation-specific (ETS) transcription factor myeloid elf-1-like factor (MEF)/Elf4 is activated by HIF-1α. MEF induces genes such as human beta-defensin 2 (HßD2) and perforin (PRF1), and is known to affect the cell cycle. Treatment with hypoxia mimetic CoCl2 or low O2 incubation up-regulated MEF mRNA and protein levels in various cell lines. HIF-1α overexpression in HEK293 cells also increased MEF mRNA and protein levels. In contrast, HIF-1α knockdown by small interfering RNA (siRNA) suppressed the induction of MEF in response to hypoxia. HIF-1α binds to the hypoxia response element in the MEF promoter region (-200 bp) and activates MEF promoter under hypoxia condition. The induction of MEF by hypoxia/HIF-1α correlated with the increase of MEF target genes HßD2 and PRF1. Intriguingly, the hypoxia-induced expression of HIF-1α target gene vascular endothelial growth factor (VEGF) was enhanced by the exogenous addition of MEF. Overall, these data indicate that hypoxia or HIF-1α positively regulates MEF expression and function.

The antitumor effects of methyl-ß-cyclodextrin against primary effusion lymphoma via the depletion of cholesterol from lipid rafts.

Primary effusion lymphoma (PEL) is a subtype of aggressive and chemotherapy-resistant non-Hodgkin lymphoma that occurs predominantly in patients with advanced AIDS. In this study, we examined the antitumor activity of methyl-ß-cyclodextrin (M-ß-CyD) in vitro and in vivo. M-ß-CyD quickly induced caspase-dependent apoptosis in PEL cells via cholesterol depletion from the plasma membrane. In a PEL xenograft mouse model, M-ß-CyD significantly inhibited the growth and invasion of PEL cells without apparent adverse effects. These results strongly suggest that M-ß-CyD has the potential to be an effective antitumor agent against PEL.

Essential immunoregulatory role for BCAP in B cell development and function.

BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell--independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca(2+) mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes.

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

Kiss1R Identification and Biodistribution Analysis Employing a Western Ligand Blot and Ligand-Derivative Stain with a FITC-Kisspeptin Derivative.

It is not always easy to establish specific antibodies against receptors. Most receptors are hydrophobic and have complicated three-dimensional structures, making them difficult to use as immunogens. Thus, we developed receptor detection methods with a fluorescein-labeled ligand as an antibody alternative, which we referred to as a western ligand blot (WLB) and ligand derivative stain (LDS). Kisspeptin receptor (Kiss1R) was detected by its ligand. Kiss1R expression was confirmed in eight human cell lines by the WLB and in four pathological tissues by the LDS. Next, Kiss1R was stained by LDS in organs, revealing Kiss1R expression by [67 Ga]Ga-DOTA-kisspeptin 10 accumulation. As a result, Kiss1R-expressing cells in each organ could be stained with fluorescein-labeled kisspeptin 14 instead of an antibody and observed by light microscopy. The combination of the WLB and LDS allows identification of receptors in tissues, which can be readily applied to target receptor detection by a synthetic ligand derivative.

Hybrid Light Imaging Using Cerenkov Luminescence and Liquid Scintillation for Preclinical Optical Imaging In Vivo.

PURPOSE: Cerenkov luminescence imaging (CLI) has recently emerged as a molecular imaging modality for radionuclides emitting ß-particles. The aim of this study was to develop a hybrid light imaging (HLI) technique using a liquid scintillator to assist CLI by increasing the optical signal intensity from both ß-particle and γ-ray emitting radionuclides located at deep regions in vivo. PROCEDURES: A commercial optical imaging system was employed to collect all images by HLI and CLI. To investigate the performance characteristics of HLI with a commercially available liquid scintillator (Emulsifier-safe), phantom experiments were conducted for two typical ß-particle and γ-ray emitters, sodium iodide (Na[(131)I]I) and 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG), respectively. To evaluate the feasibility of HLI for in vivo imaging, HLI was applied to a Na[(131)I]I injected nu/nu mouse and an [(18)F]FDG injected Balb-c mouse and compared with CLI alone. RESULTS: Measured HLI wavelength spectra with Emulsifier-safe showed higher signal intensities than for CLI at 500-600 nm. For material preventing light transmission of 12-mm thickness, CLI imaging provided quite low intensity and obscure signals of the source. However, despite degraded spatial resolution, HLI imaging provided sustained visualization of the source shape, with signal intensities 10-14 times higher than for CLI at 10-mm thickness. Furthermore, at 0, 4, and 8-mm material thicknesses, HLI showed a strong correlation between Na[(131)I]I or [(18)F]FDG radioactivity and signal intensity, as for CLI. In vivo studies also demonstrated that HLI could successfully visualize Na[(131)I]I uptake in the mouse thyroid gland in the prone position and [(18)F]FDG accumulation in the heart in the supine position, which were not observed with CLI. CONCLUSION: Our preliminary studies suggest that HLI can provide enhanced imaging of a ß-particle probe emitting together with γ-rays at deep tissue locations. HLI may be a promising imaging technique to assist with preclinical in vivo imaging using CLI.

Iodine-131 imaging using 284 keV photons with a small animal CZT-SPECT system dedicated to low-medium-energy photon detection.

OBJECTIVE: Iodine-131 is widely used for radionuclide therapy because of its ß-particle and for diagnostic imaging employing its principal gamma ray. Since that principal gamma ray has the relatively high energy of 364 keV, small animal single-photon emission computed tomography (SPECT) imaging systems may be required to possess the ability to image such higher energy photons. The aim of this study was to investigate the possibility of imaging I-131 using its 284 keV photons instead of its 364 keV photons in a small animal SPECT imaging system dedicated to the detection of low-medium-energy photons (below 300 keV). METHODS: The imaging system used was a commercially available preclinical SPECT instrument with CZT detectors that was equipped with multi-pinhole collimators and was accompanied by a CT imager. An energy window for I-131 imaging was set to a photopeak of 284 keV with a low abundance compared with 364 keV photons. Small line sources and two mice, one of each of two types, that were injected with NaI-131 were scanned. RESULTS: Although higher counts occurred at the peripheral region of the reconstructed images due to the collimator penetration by the 364 keV photons, the shape of the small line sources could be well visualized. The measured spatial resolution was relatively poor (~1.9 mm for full width at half maximum and ~3.9 mm for full width at tenth maximum). However, a good linear correlation between SPECT values and the level of I-131 radioactivity was observed. Furthermore, the uptake of NaI-131 to the thyroid gland for the two mice was clearly identified in the 3D-SPECT image fused with the X-ray CT image. CONCLUSION: We conclude that the use of an energy window set on the photopeak of 284 keV and the multi-pinhole collimator may permit I-131 imaging for a preclinical CZT-SPECT system that does not have the ability to acquire images using the 364 keV photons.

A novel EGFP-expressing nude mice with complete loss of lymphocytes and NK cells to study tumor-host interactions.

Enhanced green fluorescent protein (EGFP) expressing Balb/c nude mice strain with Rag-2 and Jak3 double mutants (Nude-R/J-EGFP mice) was established to improve the take rate of human tumors and to distinguish tumor and host cells. EGFP was ubiquitously expressed in all organs including the brain, lung, liver, heart, kidney, spleen, and gastrointestinal tract in Nude-R/J-EGFP mice. The mice showed complete loss of T lymphocytes, B lymphocytes, and NK cells, indicating a higher take rate of human tumor xenograft. M213-mCherry, an mCherry expressing the cholangiocarcinoma cell line, was successfully detected and tumor vessels derived from the host were clearly identified with fluorescence imager. Thus, dual-color fluorescence imaging visualizes the tumor-host interaction by non-invasive in vivo fluorescent imaging in Nude-R/J-EGFP mice. These finding suggests that Nude-R/J-EGFP mice are becoming a powerful tool to investigate human tumor-host interactions.

Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.

BACKGROUND: Nude mice are used in human xenograft research; however, only 25-35% of human tumors have been successfully transplanted into nude mice and their application is limited due to high natural killer (NK) cell activity. More severely immunodeficient mice with loss of NK activity are needed to overcome this limitation. MATERIALS AND METHODS: Balb/c nude Rag-2(-/-)Jak3(-/-) (Nude-RJ) mice were established by crossing Rag-2(-/-)Jak3(-/-) mice and nude mice. The K562 cell line was implanted subcutaneously to compare tumorigenicity between Nude-RJ mice and Nude mice. The cholangiocarcinoma mCherry expressing cell line (KKU-M213) was implanted subcutaneously, and fluorescence intensity and tumor weight were measured. RESULTS: Nude R/J mice showed complete loss of lymphocytes and NK cells. Xeno-transplantation of K562 cells showed higher proliferation in Nude R/J mice than nude mice. Subcutaneously-transplanted mCherry-transduced KKU-M213 cells were successfully detected with a fluorescence imager. CONCLUSION: Nude-R/J mice are valuable tools for in vivo imaging studies in biomedical research.

Therapeutic effects of γ-irradiation in a primary effusion lymphoma mouse model.

Primary effusion lymphoma (PEL) is a unique and recently identified non-Hodgkin's lymphoma in immunocompromised individuals. PEL is caused by the Kaposi sarcoma-associated herpes virus/human herpes virus 8 (KSHV/HHV-8) and has a peculiar presentation involving liquid growth in the serous body cavity, chemotherapy resistance and poor prognosis. In search of a new therapeutic modality for PEL, we examined the effect of γ-irradiation on PEL-derived cell lines (BCBL-1, BC-1, and BC-3) in vitro and in vivo. An MTT assay and trypan blue exclusion assay revealed that irradiation significantly suppressed cell proliferation in the PEL cell lines in a dose-dependent manner, and induced apoptosis. The PEL cell lines were relatively radiosensitive compared with other hematological tumor cell lines (Raji, Jurkat, and K562 cells). Inoculation of the BC-3 cell line into the peritoneal cavity of Rag2/Jak3 double-deficient mice led to massive ascites formation, and subcutaneous injection of BCBL-1 led to solid lymphoma formation. Total body irradiation (4 Gy × 2) with bone marrow transplantation resulted in the complete recovery of both types of PEL-inoculated mice. These results suggest that total body irradiation with bone marrow transplantation can be successfully applied for the treatment of chemotherapy-resistant PEL.

BANK negatively regulates Akt activation and subsequent B cell responses.

BANK is an adaptor protein that is highly expressed in B cells. To investigate its physiological role, we generated BANK-deficient mice. BANK-deficient mice displayed enhanced germinal center formation and IgM production in response to T-dependent antigens, whereas this phenotype was blocked in CD40-BANK double knockout mice. Involvement of BANK in CD40 signaling was further demonstrated by in vitro analysis. CD40-mediated proliferation and survival were significantly increased in BANK-deficient B cells, with enhanced Akt activation, whereas introduction of dominant-negative Akt into BANK-deficient B cells suppressed the augmented CD40-mediated responses. Together, our findings suggest that BANK attenuates CD40-mediated Akt activation, thereby preventing hyperactive B cell responses.