The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to ...binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr(843) and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr(843) or Tyr(863) is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of alpha-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab')(2) blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.
MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have ...been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.
Abstract
Cell surface molecules expressed on cancer cells can be used as diagnostic and therapeutic tools. In breast cancer, CD44 and CD24 were identified as cell surface markers characterizing ...cancer cell stemness that may correlate with prognosis. Breast cancer cells with high CD44 and low CD24 expression have been shown to exhibit proliferative, invasive and metastatic properties that may relate to drug sensitivity and metastatic risk in patients. In addition to stemness, immune evasion is another hallmark of cancer with CD47 “don’t eat me” and CD274 “don’t find me” molecules playing important roles in antitumor immunity. Although each of these molecules are of interest as therapeutic targets, current therapeutic paradigms involve multimodal approach with radiation therapy often having a central role. Hence, the effect of irradiation, and related radio-sensitizers, on the expression of cancer cells surface markers to be targeted with an adjuvant therapeutic mode must be considered when designing such therapies. The goal of this study was to determine the dynamics of CD44, CD24, CD47 and CD274 expression in radio-sensitized and irradiated MDA-MB 231 breast cancer cells. We have recently reported that gold nanoparticles (AuNPs) sensitized breast cancer cells to irradiation at kV and MV energies. Here, we hypothesize that AuNP may also modulate the irradiation altered expression of cell surface markers. We explored this by using 10 MV energies and 6 Gy radiation dose. MDA-MB-231 cells were incubated for 3 hours with 14nm AuNPs, irradiated and allowed to grow for 24h and 72h after which percenteges of positive cells were determined by flow cytometry. Results are expressed as a percent of control, non-irradiated cells that was set at 100%. At 24h non-irradiated cells pre-incubated with AuNP exhibited a decrease in the percentage of CD24+ and CD44+ cells, while no significant change in the percentage of CD47+ and CD274+ cells was observed. Irradiation with 6Gy at 10 MV induced a decrease in CD24+ and an increase in CD44+ cells and AuNP potentiated this effect. However, 72h post-irradiation, percentages of CD24+ and CD44+ cells significantly increased, compared to non-irradiated controls and this effect was of lesser magnitude in AuNP pretreated cells. Percentage of CD47+ and CD274+ cells also significantly increased 24h post-irradiation, but with less magnitude in AuNP treated cells. At 72h in AuNP treated irradiated cells no change was observed in the percentage of CD274+ cells, while the percentage of CD47+ cells significantly increased in comparison to the irradiated cells not exposed to AuNPs. Preliminary data shown here indicate that expression of molecules important for cancer progression, metastasis and immune evasion undergo changes in response to irradiation, and that these changes are affected by AuNPs. Further studies will shed more light on the mechanisms behind these observed effects.
Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin Chetty, Benjamin Movsas, Ning Wen. Effect of irradiation and gold nanoparticle on expression dynamics of cell surface markers in MDA-MB 231 breast cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4862.
The HIV Nef protein is thought to promote HIV immune evasion by downmodulating MHC-I and protecting infected cells from CTL killing. In addition, we demonstrated that Rev, an HIV regulatory protein ...needed for expression of the HIV late genes, can influence CTL killing. When Rev activity level was reduced by virtue of amino acid alterations in the Rev protein sequence, infected cells were more resistant to anti-Gag and anti-Env CTL killing. A screen of primary viral isolates revealed that viruses derived from asymptomatic, infected people had lower Rev activity, lower Gag levels, and greater resistance to anti-Gag CTL killing. Thus,
rev alleles with low activity may have a selective advantage in infected people with effective immune responses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag ...presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that regulates B cell function. In ex vivo B cells primed by Ag receptor (BCR) cross-linking and incubation with IL-4, or B cell lines such as K46-17 micromlambda, class II ligation leads to the activation of protein tyrosine kinases, including Lyn and Syk and subsequent phospholipase Cgamma-dependent mobilization of Ca(2+). In this study, experiments demonstrated reciprocal desensitization of class II and BCR signaling upon cross-linking of either receptor, suggesting that the two receptors transduce signals via common processes and/or effector proteins. Because class II and BCR signal transduction pathways exhibit functional similarities, additional studies were conducted to evaluate whether class II signaling is regulated by BCR coreceptors. Upon cross-linking of class II, the BCR coreceptors CD19 and CD22 were inducibly phosphorylated on tyrosine residues. Phosphorylation of CD22 was associated with increased recruitment and binding of the protein tyrosine phosphatase SHP-1. Similarly, tyrosine phosphorylation of CD19 resulted in recruitment and binding of Vav and phosphatidylinositol 3-kinase. Finally, co-cross-linking studies demonstrated that signaling via class II was either attenuated (CD22/SHP-1) or enhanced (CD19/Vav and phosphatidylinositol 3-kinase), depending on the coreceptor that was brought into close proximity. Collectively, these results suggest that CD19 and CD22 modulate class II signaling in a manner similar to that for the BCR.
Abstract
Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double ...strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect.
Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin J. Chetty, Benjamin Movsas, Ning Winston Wen. Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1376.
Abstract
GDF15 (growth/differentiation factor 15), also known as MIC-1 (Macrophage inhibitory cytokine 1), is a divergent member of the TGFβ superfamily of cytokines and is highly expressed in ...prostate tumors, but its role in prostate carcinogenesis and utility as a prognostic biomarker is unclear. We studied 91 prostate cancer cases that underwent surgery as their primary treatment and were then subsequently followed for biochemical recurrence (BCR). These cases also had a benign prostate biopsy at least one year before their prostate cancer diagnosis. In both the benign biopsy and tumor specimens, we quantified the intensity of GDF15 expression and characterized the presence of tumor associated macrophages by measuring the density of CD68-positive stained cells, and the M2 macrophage marker CD204 by immunohistochemical analysis. Marker expression was measured in prostate a) benign biopsy (BS), b) tumor-adjacent benign (BGS) and c) tumor tissue (MGS) using an automated multi-image processing macro developed in the ImageJ software. A Cox proportional hazards model was used to test the association of time to BCR of the low expression group compared with medium or high biomarker expression. The risk of BCR differed with the extent of GDF15 expression in the prostatic epithelial tissue of benign and tumor regions of the prostate. For instance, in BGS, high expression of GDF15 was associated with increased risk of BCR (hazard ratio (HR)=2.58, p=0.07). After controlling for PSA at diagnosis, Gleason grade and pathologic stage, high expression of GDF15 in BGS had a stronger association with BCR (HR=3.30, p=0.04). The risk of BCR increased with increasing level of infiltration of CD204/CD68 positive macrophages in BGS (HR=4.19, p=0.02) and MGS (HR=4.16, p=0.01) even after controlling for PSA at diagnosis, Gleason grade and pathologic stage. The combined expression levels of GDF15 and CD204/CD68 showed that prostate cancer cases with small GDF15 expression changes between BGS and MGS and high CD204/CD68 expression in BGS were at 3-fold higher risk of BCR (HR=3.67, p=0.05). In summary, expression levels of GDF15 and CD204 M2 macrophages in different regions of the prostate can change as the disease progresses from benign to a tumorigenic state and serve as markers for aggressive disease. Further evaluation of these dynamic differences in the prostate immune cellular profile in the pre-malignant and malignant state may offer additional insight into inflammatory-mediated prostate carcinogenesis.
Citation Format: Sudha M. Sadasivan, Yalei Chen, Nilesh Gupta, Sean Williamson, Dhananjay Chitale, Xiaoxia Han, Kevin Bobbitt, Andrew Rundle, Deliang Tang, Benjamin A. Rybicki. Changes in GDF15 (growth/differentiation factor 15) expression and M2 macrophages during prostate carcinogenesis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2337.
Primary brain tumors, or Glioblastomas (GBMs), are devastating to each patient diagnosed due to the inability of current therapy to prolong survival. Thus, GBMs are prime candidates for trials of ...novel therapeutic modalities. One such novel therapy is oncolytic herpes simplex viruses (OHV). Oncolytic viral therapy (OV) has been explored as a potential therapy for many tumor types for many years. However, significant gains in ability to treat GBMs with OVs have not occurred. A significant problem with OV for GBM therapy is the inability of the virus to overcome the migratory infiltrative nature of tumor growth, a unique problem with GBMs. Thus, we determined the ability of neural stem cells (NSCs) to deliver OHV to sites of infiltrative tumor growth following intratumoral delivery of the stem cells. For this end, we utilized an infiltrative, HSV-sensitive GBM tumor model in syngenic, immunocompetent C57/Bl6 mice. We see greater distribution of the virus through the main injected tumor mass following NSC delivery than compared with that seen following OHV alone delivery. Furthermore, NSCs and virally infected cells are found in distal infiltrative tumor sites following NSC/OHV delivery. Delivery of the OHV via NSCs also leads to an alteration in the activation of the immune response to both viral and tumor antigens. Thus, delivery of infectious virus through the use of neural stem cells may be a feasible way to gain greater distribution of therapeutic agents such as OHV.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Rising prostate‐specific antigen (PSA) levels are associated with both increased risk of prostate cancer and prostatic inflammation. The confounding effects of inflammation on the utility ...of PSA kinetics to predict prostate cancer may be partially mitigated by anti‐inflammatory drug use. We investigated the influence of anti‐inflammatory drug use on the association of PSA kinetics with prostate cancer risk.
Methods
We studied 488 prostate cancer case‐control pairs (290 white, 198 African American (AA)) nested in a retrospective cohort of men with a benign prostate biopsy. A series of multivariable models estimated prostate cancer risk associated with PSA velocity (PSAV) at different levels of anti‐inflammatory drug use while adjusting for the presence of both clinical and histologic prostatitis.
Results
In men with one, two, or three or more courses of anti‐inflammatory drug use, for each ng/mL/year increase in PSAV, prostate cancer risk increased 1.21‐fold, 1.83‐fold, and 1.97‐fold, respectively (
P < 0.0001). In controls with histologic prostatitis, anti‐inflammatory drug use was associated with a significantly lower PSAV (
P < 0.0001). This association was not observed in men with histologic prostatitis who were subsequently diagnosed with prostate cancer. A positive interaction between anti‐inflammatory drug use and PSAV‐associated prostate cancer risk was only observed in AA men, as well as a strong positive association between any anti‐inflammatory drug use and clinical prostatitis (
P = 0.004).
Conclusions
In men with benign prostate biopsy, accounting for the presence of histologic prostatitis and anti‐inflammatory drug use, particularly in AA men, may help distinguish between men with rising PSA because of prostatitis vs undiagnosed cancer.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Background Mitochondria support critical cellular functions, such as energy production through oxidative phosphorylation, regulation of reactive oxygen species, apoptosis, and calcium homeostasis. ...Objective Given the heightened level of cellular activity in patients with asthma, we sought to determine whether mitochondrial DNA (mtDNA) copy number measured in peripheral blood differed between individuals with and without asthma. Methods Whole genome sequence data was generated as part of the Trans-Omics for Precision Medicine (TOPMed) Program on participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity (SAPPHIRE) and the Study of African Americans, Asthma, Genes, & Environment II (SAGE II). We restricted our analysis to individuals who self-identified as African American (3,651 asthma cases and 1,344 controls). Mitochondrial copy number was estimated using the sequencing read depth ratio for the mitochondrial and nuclear genomes. Respiratory complex expression was assessed using RNA-sequencing. Results Average mitochondrial copy number was significantly higher among individuals with asthma when compared with controls (SAPPHIRE: 218.60 vs. 200.47, P<0.001; SAGE II: 235.99 vs. 223.07, P<0.001). Asthma status was significantly associated with mitochondrial copy number after accounting for potential explanatory variables, such as participant age, sex, leukocyte counts, and mitochondrial haplogroup. Despite the consistent relationship between asthma status and mitochondrial copy number, the latter was not associated with time-to-exacerbation or patient-reported asthma control. Mitochondrial respiratory complex gene expression was disproportionately lower in individuals with asthma when compared with individuals without asthma and other protein-encoding genes. Conclusions We observed a robust association between asthma and higher mitochondrial copy number. Asthma having an effect on mitochondria function was also supported by lower respiratory complex gene expression in this group.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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