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author:("Kim, kyunghee")
1.  Homotypic NK cell-to-cell communication controls cytokine responsiveness of innate immune NK cells 
Scientific Reports  2014;4:7157.
While stationary organ cells are in continuous contact with neighboring cells, immune cells circulate throughout the body without an apparent requirement for cell-cell contact to persist in vivo. This study challenges current convention by demonstrating, both in vitro and in vivo, that innate immune NK cells can engage in homotypic NK-to-NK cell interactions for optimal survival, activation, and proliferation. Using a specialized cell-laden microwell approach, we discover that NK cells experiencing constant NK-to-NK contact exhibit a synergistic increase in activation status, cell proliferation, and anti-tumor function in response to IL-2 or IL-15. This effect is dependent on 2B4/CD48 ligation and an active cytoskeleton, resulting in amplification of IL-2 receptor signaling, enhanced CD122/CD132 colocalization, CD25 upregulation, and Stat3 activation. Conversely, ‘orphan' NK cells demonstrate no such synergy and fail to persist. Therefore, our data uncover the existence of homotypic cell-to-cell communication among mobile innate lymphocytes, which promotes functional synergy within the cytokine-rich microenvironment.
PMCID: PMC4256668  PMID: 25475707
2.  The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A 
Nature medicine  2008;14(9):931-938.
The immunosuppressive action of the calcineurin inhibitor cyclosporine A (CsA) stems from the inhibition of nuclear factor of activated T cells (NFAT) signaling in T cells. CsA is also used for the treatment of proteinuric kidney diseases. As it stands, the antiproteinuric effect of CsA is attributed to its immunosuppressive action. Here we show that the beneficial effect of CsA on proteinuria is not dependent on NFAT inhibition in T cells, but rather results from the stabilization of the actin cytoskeleton in kidney podocytes. CsA blocks the calcineurin-mediated dephosphorylation of synaptopodin, a regulator of Rho GTPases in podocytes, thereby preserving the phosphorylation-dependent synaptopodin–14-3-3β interaction. Preservation of this interaction, in turn, protects synaptopodin from cathepsin L–mediated degradation. These results represent a new view of calcineurin signaling and shed further light on the treatment of proteinuric kidney diseases. Novel calcineurin substrates such as synaptopodin may provide promising starting points for antiproteinuric drugs that avoid the serious side effects of long-term CsA treatment.
PMCID: PMC4109287  PMID: 18724379
3.  A Comparison of Podocyturia, Albuminuria and Nephrinuria in Predicting the Development of Preeclampsia: A Prospective Study 
PLoS ONE  2014;9(7):e101445.
Preeclampsia, a hypertensive multisystem disease that complicates 5–8% of all pregnancy, is a major cause for maternal and fetal mortality and morbidity. The disease is associated with increased spontaneous and evoked preterm birth and remote cardio-renal disorders in the mother and offspring. Thus the ability to predict the disease should lead to earlier care and decreased morbidity. This has led to fervent attempts to identify early predictive biomarkers and research endeavors that have expanded as we learn more regarding possible causes of the disease. As preeclampsia is associated with specific renal pathology including podocyte injury, early urinary podocyte (podocyturia), or the podocyte specific proteinuria nephrin in the urine (nephrinuria), as well as the more easily measured urinary albumin (albuminuria), have all been suggested as predictive markers. We performed a prospective study recruiting 91 pregnant women (78 of whom were high risk) and studied the predictive ability of these three urinary biomarkers. The subjects were recruited between 15–38 weeks of gestation. Fourteen patients, all in the high-risk obstetric group, developed preeclampsia. The levels of podocyturia, nephrinuria, and albuminuria were variably higher in the high-risk pregnant patients who developed preeclampsia. The sensitivities and specificities for podocyturia were 70% and 43%, for albuminuria were 36% and 96%, and for nephrinuria were 57% and 58%, respectively. Also, abnormal nephrinuria (69%) and podocyturia (38%) were detected in low risk women who had uncomplicated gestations; none of these women exhibited albuminuria. In our study, none of the three urinary markers achieved the minimum predictive values required for clinical testing. The lack of excessive albuminuria, however, may indicate a preeclampsia-free gestation. Given a discrepant literature, further studies with larger sample size should be considered.
PMCID: PMC4092019  PMID: 25010746
4.  Augmentation of natural cytotoxicity by chronic low-dose ionizing radiation in murine natural killer cells primed by IL-2 
Journal of Radiation Research  2012;53(6):823-829.
The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR.
PMCID: PMC3483842  PMID: 22915781
Low-dose radiation; natural killer cells; natural cytotoxicity; innate immunity
5.  High Throughput Screening of Small Molecule Libraries for Modifiers of Radiation Responses 
An unbiased approach of drug discovery through high-throughput screening (HTS) of libraries of chemically defined and bioactive small molecule compounds was used to identify modulators of radiation injury with an emphasis on radioprotectors and mitigators rather than radiosensitizers. Assay system endpoints included radiation-induced genotoxicity and DNA damage in yeast and apoptosis in murine lymphocytes. Large-scale data mining of chemically diverse libraries identified agents that were effective with all endpoints. HTS of bioactive compound libraries against murine lymphocytes profiled tetracycline and fluoroquinolone antibiotics and cyclopiazonic acid as having activity, and structure-activity analysis showed a common pharmacophore. Purine nucleosides, the interferon inducer tilorone, and linoleic acid were also identified as potential mitigators of radiation damage that often were also radioprotective. Many of these compounds enhance DNA repair, have anti-inflammatory activity, and stimulate hematopoiesis. Selected compounds within these initial verified hits from both types of libraries identified potent mitigators of lethal whole body irradiation (WBI) in mice.
In spite of the fact that in vitro HTS has limitations and is unable to fully recapitulate all aspects of the complex in vivo acute radiation response, it identified several classes of molecules that had activity as radioprotectors and radiomitigators of the hematopoietic system in vivo. In the future, addition of 3 dimensional (3D) or stem cell cultures or pathway analysis, may improve the power of HTS, but our findings indicate that common, evolutionary conserved, canonical pathways can be identified that could be exploited to mitigate radiation-induced defects.
PMCID: PMC3203687  PMID: 21401317
High throughput screening; radioprotector; mitigator; antibiotics
7.  Ionizing radiation activates the Nrf2 antioxidant response 
Cancer research  2010;70(21):8886-8895.
The transcription factor Nrf2 binds the antioxidant DNA response element (ARE) to activate important cellular cytoprotective defense systems. Recently several types of cancers have been shown to overexpress Nrf2, but its role in the cellular response to radiation therapy has yet to be fully determined. In this study, we report that single doses of ionizing radiation from 2-8Gy activate ARE-dependent transcription in breast cancer cells in a dose-dependent manner, but only after a delay of 5 days. Clinically relevant daily dose fractions of radiation also increased ARE-dependent transcription, but again only after 5 days. Downstream activation occurred of Nrf2-ARE-dependent gene and protein markers, such as heme oxygenase-1, whereas Nrf2-deficient fibroblasts were incapable of these responses. Compared to wild-type fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of reactive oxygen species that increased greatly five days after radiation exposure. Further, in vitro clonogenic survival assays and in vivo sublethal whole body irradiation tests demonstrated that Nrf2 deletion increased radiation sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our results indicate that the Nrf2-ARE pathway is important to maintain resistance to irradiation, but that it operates as a second-tier antioxidant adaptive response system activated by radiation only under specific circumstances, including those that may be highly relevant to tumor response during standard clinical dose-fractionated radiation therapy.
PMCID: PMC2970706  PMID: 20940400
Ionizing Radiation; Nrf2; Antioxidant Response Element; Delayed Gene Expression; Heme Oxygenase-1
8.  Modifying Radiation Damage 
Current drug targets  2010;11(11):1352-1365.
Radiation leaves a fairly characteristic footprint in biological materials, but this is rapidly all but obliterated by the canonical biological responses to the radiation damage. The innate immune recognition systems that sense “danger” through direct radiation damage and through associated collateral damage set in motion a chain of events that, in a tissue compromised by radiation, often unwittingly result in oscillating waves of molecular and cellular responses as tissues attempt to heal. Understanding “nature’s whispers” that inform on these processes will lead to novel forms of intervention targeted more precisely towards modifying them in an appropriate and timely fashion so as to improve the healing process and prevent or mitigate the development of acute and late effects of normal tissue radiation damage, whether it be accidental, as a result of a terrorist incident, or of therapeutic treatment of cancer. Here we attempt to discuss some of the non-free radical scavenging mechanisms that modify radiation responses and comment on where we see them within a conceptual framework of an evolving radiation-induced lesion.
PMCID: PMC3079263  PMID: 20583981
TBI; cytokines; RDS; inflammation; NF-κB
9.  Anti-inflammatory function of arctiin by inhibiting COX-2 expression via NF-κB pathways 
Arctiin, isolated from Forsythia suspensa has been reported to have anti-inflammatory, anti-oxidant, antibacterial, and antiviral effects in vitro. However, there has been a lack of studies regarding its effects on immunological activity. The aim of this study is to investigate the anti-inflammatory potential and possible mechanisms of arctiin in LPS-induced macrophages.
We investigated the mRNA and protein levels of proinflammatory cytokines through RT-PCR and western blot analysis, followed by a FACS analysis for surface molecule changes.
Arctiin dose dependently decreased the production of NO and proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and PGE2, and it reduced the gene and protein levels as determined by RT-PCR and western blot analysis, respectively. The expression of co-stimulatory molecules such as B7-1 and B7-2 were also inhibited by arctiin. Furthermore, the activation of the nuclear transcription factor, NF-κB in macrophages was inhibited by arctiin.
Taken together these results provide evidence of the bioactivity of arctiin in inflammatory diseases and suggest that arctiin may exert anti-inflammatory effect by inhibiting the pro-inflammatory mediators through the inactivation of NF-kB.
PMCID: PMC3146809  PMID: 21733191
10.  High Throughput Screening Identifies Two Classes of Antibiotics as Radioprotectors: Tetracyclines and Fluoroquinolones 
Discovery of agents that protect or mitigate normal tissue from radiation injury during radiotherapy, accidents, or terrorist attacks, is of importance. Specifically, bone marrow insufficiency, with possible infection due to immune suppression, can occur after total body irradiation (TBI) or regional irradiation and is a major component of the acute radiation syndrome. The purpose of this study was to identify novel radioprotectors and mitigators of the hematopoietic system.
Experimental Design
High throughput screening of small molecule libraries was performed using viability of a murine lymphocyte line as a read out with further validation in human lymphoblastoid cells. The selected compounds were then tested for their ability to counter TBI lethality in mice.
All of two major classes of antibiotics, tetracyclines and fluoroquinolones, which share a common planar ring moiety, were radioprotective. Furthermore, tetracycline protected murine hematopoietic stem/progenitor cell populations from radiation damage and allowed 87.5% of mice to survive when given before and 35% when given 24 h after lethal TBI. Interestingly, tetracycline did not alter the radiosensitivity of Lewis lung cancer cells. Tetracycline and ciprofloxacine also protected human lymphoblastoid cells, reducing radiation-induced DNA double strand breaks by 33% and 21%, respectively. The effects of these agents on radiation lethality are not due to the classical mechanism of free radical scavenging but potentially through activation of the histone acetyl transferase Tip60 and altered chromatin structure.
Tetracyclines and fluoroquinolones can be robust radioprotectors and mitigators of the hematopoietic system with potential utility in anti-cancer radiotherapy and radiation emergencies.
PMCID: PMC2787903  PMID: 19920105
11.  Epidermal Growth Factor Receptor vIII Expression in U87 Glioblastoma Cells Alters Their Proteasome Composition, Function, and Response to Irradiation 
Molecular cancer research : MCR  2008;6(3):426-434.
Little is known about the factors that influence the proteasome structures in cells and their activity, although this could be highly relevant to cancer therapy. We have previously shown that, within minutes, irradiation inhibits substrate degradation by the 26S proteasome in most cell types. Here, we report an exception in U87 glioblastoma cells transduced to express the epidermal growth factor receptor vIII (EGFRvIII) mutant (U87EGFRvIII), which does not respond to irradiation with 26S proteasome inhibition. This was assessed using either a fluorogenic substrate or a reporter gene, the ornithine decarboxylase degron fused to ZsGreen (cODCZsGreen), which targets the protein to the 26S proteasome. To elucidate whether this was due to alterations in proteasome composition, we used quantitative reverse transcription-PCR to quantify the constitutive (X, Y, Z) and inducible 20S subunits (Lmp7, Lmp2, Mecl1), and 11S (PA28α and β) and 19S components (PSMC1 and PSMD4). U87 and U87EGFRvIII significantly differed in expression of proteasome subunits, and in particular immunosubunits. Interestingly, 2 Gy irradiation of U87 increased subunit expression levels by 16% to 324% at 6 hours, with a coincident 30% decrease in levels of the proteasome substrate c-myc, whereas they changed little in U87EGFRvIII. Responses similar to 2 Gy were seen in U87 treated with a proteasome inhibitor, NPI0052, suggesting that proteasome inhibition induced replacement of subunits independent of the means of inhibition. Our data clearly indicate that the composition and function of the 26S proteasome can be changed by expression of the EGFRvIII. How this relates to the increased radioresistance associated with this cell line remains to be established.
PMCID: PMC2877368  PMID: 18337449
12.  In Vivo Imaging, Tracking, and Targeting of Cancer Stem Cells 
There is increasing evidence that solid cancers contain cancer-initiating cells (CICs) that are capable of regenerating a tumor that has been surgically removed and/or treated with chemotherapy and/or radiation therapy. Currently, cell surface markers, like CD133 or CD44, are used to identify CICs in vitro; however, these markers cannot be used to identify and track CICs in vivo. The 26S proteasome is the main regulator of many processes within a proliferating cell, and its activity may be altered depending on the phenotype of a cell.
Human glioma and breast cancer cells were engineered to stably express ZsGreen fused to the carboxyl-terminal degron of ornithine decarboxylase, resulting in a fluorescent fusion protein that accumulates in cells in the absence of 26S proteasome activity; activities of individual proteases were monitored in a plate reader by detecting the cleavage of fluorogenic peptide substrates. Proteasome subunit expression in cells expressing the fusion protein was assessed by quantitative reverse transcription—polymerase chain reaction, and the stem cell phenotype of CICs was assessed by a sphere formation assay, by immunohistochemical staining for known stem cell markers in vitro, and by analyzing their tumorigenicity in vivo. CICs were tracked by in vivo fluorescence imaging after radiation treatment of tumor-bearing mice and targeted specifically via a thymidine kinase–degron fusion construct. All P values were derived from two-sided tests.
Cancer cells grown as sphere cultures in conditions, which enrich for cancer stem cells (CSCs), had decreased proteasome activity relative to the respective monolayers (percent decrease in chymotryptic-like activity of sphere cultures relative to monolayers—U87MG: 26.64%, 95% confidence interval [CI] = 10.19 to 43.10, GL261, 52.91%, 95% CI = 28.38 to 77.43). The cancer cells with low proteasome activity can thus be monitored in vitro and in vivo by the accumulation of a fluorescent protein (ZsGreen) fused to a degron that targets it for 26S proteasome degradation. In vitro, ZsGreen-positive cells had increased sphere-forming capacity, expressed CSC markers, and lacked differentiation markers compared with ZsGreen-negative cells. In vivo, ZsGreen-positive cells were approximately 100-fold more tumorigenic than ZsGreen-negative cells when injected into nude mice (ZsGreen positive, 30 mice per group; ZsGreen negative, 31 mice per group), and the number of CICs in tumors increased after 72 hours post radiation treatment. CICs were selectively targeted via a proteasome-dependent suicide gene, and their elimination in vivo led to tumor regression.
Our results demonstrate that reduced 26S proteasome activity is a general feature of CICs that can easily be exploited to identify, track, and target them in vitro and in vivo.
PMCID: PMC2727141  PMID: 19244169
13.  Survival and self-renewing capacity of breast cancer initiating cells during fractionated radiation treatment 
Recent data indicate a hierarchical organization of many solid cancers, including breast cancer, with a small number of cancer initiating cells (CICs) that have the ability to self-renew and exhibit multi-lineage potency. We, and others, have demonstrated that CICs in breast cancer and glioma are relatively resistant to ionizing radiation if compared to their non-tumorigenic counterparts. However, the extent of the remaining self-renewing capacity of CICs after fractions of radiation is currently unknown. We hypothesized that CICs, in contrast to their non-tumorigenic counterparts, not only survive fractions of ionizing radiation but also retain the CIC phenotype as defined by operational means.
We used two marker systems to identify breast CICs (CD24-/low/CD44high, or lack of proteasome activity) and performed sphere-forming assays after multiple clinical fractions of radiation. Lineage tracking was performed by membrane staining. Cell cycle distribution and RNA content were assessed by flow cytometry and senescence was assessed via β-galactosidase staining.
We demonstrated that irradiated CICs survived and retained their self-renewal capacity for at least four generations. We show that fractionated radiation not only spared CICs but also mobilized them from a quiescent/G0 phase of the cell cycle into actively cycling cells, while the surviving non-tumorigenic cells were driven into senescence.
The breast CIC population retains increased self-renewal capacity over several generations and therefore, we conclude that increases in the number of CICs after sublethal doses of radiation have potential clinical importance. Prevention of this process may lead to improved clinical outcome.
PMCID: PMC2880434  PMID: 20158881
14.  Imaging of radiation effects on cellular 26S proteasome function in situ 
The classical radiobiological paradigm is that DNA is the target for cell damage caused by ionising radiation. However, evidence is accumulating that other constituents, such as the membrane, organelles, and proteins, are also important targets. We have shown that the isolated 26S proteasome is one such target and here we wish to substantiate it within the cell, in situ.
Materials and methods
We used confocal microscopy to quantitatively detect and subcellularly localise radiation-induced 26S proteasome inhibition in cells expressing an ornithine decarboxylase degron that targets a fused Zoanthus species green (ZsGreen) fluorescent protein reporter specifically to the 26S proteasome.
Exposure of cells to a range of radiation doses, even as low as 0.05 Gy inhibited 26S activity within minutes. Initially, punctate nuclear ZsGreen fluorescence was observed that became cytoplasmic after seven hours – a pattern distinct from the diffuse homogeneous fluorescence of cells incubated in the conventional proteasome inhibitor MG-132.
Our study clearly indicates that the 26S proteasome is a radiation target with physiological consequences and introduces a new perspective in mechanistic investigations of cellular responses to stresses.
PMCID: PMC2775098  PMID: 19401903
Proteasome; fluorescence confocal microscopy; ionising radiation; ornithine decarboxylase degron-Zs-Green
15.  Effects of Recombinant Erythropoietin on Breast Cancer-Initiating Cells1 
Neoplasia (New York, N.Y.)  2007;9(12):1122-1129.
Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs). In breast cancer, CICs can be identified by phenotypic markers and their fate is controlled by the Notch pathway.
In this study, we investigated the effect of erythropoietin on CICs in breast cancer cell lines. Levels of erythropoietin receptor (EpoR), CD24, CD44, Jagged-1 expression, and activation of Notch-1 were assessed by flow cytometry. Self-renewing capacity of CICs was investigated in sphere formation assays.
EpoR expression was found on the surface of CICs. Recombinant human Epo (rhEpo) increased the numbers of CICs and self-renewing capacity in a Notch-dependent fashion by induction of Jagged-1. Inhibitors of the Notch pathway and PI3-kinase blocked both effects.
Erythropoietin functionally affects CICs directly. Our observation may explain the negative impact of recombinant Epo on local control and survival of cancer patients with EpoR-positive tumors.
PMCID: PMC2134908  PMID: 18084619
CD24-/low/CD44+ breast cancer cells; breast cancer-initiating cells; rhEpo; Notch; Epor
16.  Synaptopodin regulates the actin-bundling activity of α-actinin in an isoform-specific manner 
Journal of Clinical Investigation  2005;115(5):1188-1198.
Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. Synaptopodin-deficient (synpo–/–) mice lack the dendritic spine apparatus and display impaired activity-dependent long-term synaptic plasticity. In contrast, the ultrastructure of podocytes in synpo–/– mice is normal. Here we show that synpo–/– mice display impaired recovery from protamine sulfate–induced podocyte foot process (FP) effacement and LPS-induced nephrotic syndrome. Similarly, synpo–/– podocytes show impaired actin filament reformation in vitro. We further demonstrate that synaptopodin exists in 3 isoforms, neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). The C terminus of Synpo-long is identical to that of Synpo-T. All 3 isoforms specifically interact with α-actinin and elongate α-actinin–induced actin filaments. synpo–/– mice lack Synpo-short and Synpo-long expression but show an upregulation of Synpo-T protein expression in podocytes, though not in the brain. Gene silencing of Synpo-T abrogates stress-fiber formation in synpo–/– podocytes, demonstrating that Synpo-T serves as a backup for Synpo-long in synpo–/– podocytes. In concert, synaptopodin regulates the actin-bundling activity of α-actinin in highly dynamic cell compartments, such as podocyte FPs and the dendritic spine apparatus.
PMCID: PMC1070637  PMID: 15841212

Results 1-16 (16)