Nurse Executives Impacting the Human Experience in Health Care.

The human experience in health care over the past 2 and one-half years has been unprecedented. Impacted by the COVID-19 pandemic, march on the United States Capitol, economic crisis, social injustice, and growing concerns regarding structural racism-our health system is under siege. Executive nurse leaders find themselves at the crossroads of motivating colleagues to provide excellence in nursing and patient care while confronting both a current and future shortage of registered nurses. Over 30 nurse leaders from around the globe meet and strategize on creating a preferred future for health care. The purpose of this article is to describe The Beryl Institute, the formation of the nurse executive council, the work of its members, and a collective call to action.

Cultivating a Culture:: Implementing Methods to Embrace Diversity and Inclusion.

Anne Arundel Medical Center, Annapolis, Maryland, was the recipient of this year's American Organization for Nursing Leadership's Prism Award, which recognizes an organization with advanced diversity efforts. The medical center, part of Luminis Health, added diversity as a core organizational value in 2009, making efforts to provide care to diverse populations and support an inclusive work environment for staff. Through strategic initiatives and community partnerships that target recruiting efforts, the organization has created a culture in which difficult conversations about differences are supported and encouraged. Additionally, targeted hiring initiatives have increased diversity among hospital leadership. Diversity and inclusion initiatives focus on creating a culture that welcomes and supports everyone, regardless race, ethnicity, or sexual orientation/identity.

Proteasome inhibitors in the clinical setting: benefits and strategies to overcome multiple myeloma resistance to proteasome inhibitors.

The majority of intracellular proteins undergo degradation through the ubiquitin-proteasome pathway. The proteasome pathway has a role in regulating cell proliferation, differentiation, survival and apoptosis. The naturally occurring proteasome inhibitor lactacystin was the first proteasome inhibitor noted to induce apoptosis in vitro. Compared with first-generation proteasome inhibitors, bortezomib (PS-341), a dipeptide boronic acid, has exhibited higher potency and specificity, and has been approved for the treatment of relapsed or refractory myeloma. However, there are some patients who do not respond to therapy or who respond briefly and then relapse. It is becoming increasingly clear that myeloma cells respond to the stress caused by proteasome inhibitors (bortezomib) via rapidly up-regulating pathways that suppress apoptosis, thus attenuating its antitumour activity. The delineation of these molecular pathways and mechanisms to circumvent them are needed to allow this important class of agents to remain vital in the armamentarium of the management of multiple myeloma and other malignancies.

Apo2L/TRAIL induction and nuclear translocation of inositol hexakisphosphate kinase 2 during IFN-beta-induced apoptosis in ovarian carcinoma.

Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-beta (interferon-beta) treatment and gamma-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-alpha2, IFN-beta is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-alpha2-resistant cells into cells that readily undergo apoptosis in response to IFN-alpha2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-beta, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-beta treatment. Cells expressing mutant NLS mutation were more resistant to IFN-beta. The IC50 value of IHPK2-expressing cells was 2-3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5Delta) inhibited the antiproliferative effects of IFN-beta. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-beta, and expression of the NLS mutant conferred resistance to IFN-beta. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-beta in ovarian carcinoma.

Effects of interferon beta on transcobalamin II-receptor expression and antitumor activity of nitrosylcobalamin.

BACKGROUND: The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl. METHODS: Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-beta, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively. RESULTS: Cancer cell lines were more sensitive to NO-Cbl (with ID(50)s [the dose that inhibits growth by 50%] as low as 2 microM) than normal cell lines (with ID(50)s of 85-135 microM). Single-agent NO-Cbl and IFN-beta treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-beta treatment increased TC II-R expression in vitro and uptake of [(57)Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-beta were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl. CONCLUSION: NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-beta resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.

IFN-alpha2b and thalidomide synergistically inhibit tumor-induced angiogenesis.

Angiogenesis is an absolute requirement for tumor growth and metastasis. The purpose of this study was to evaluate the antiangiogenic activity of interferon-alpha2b (IFN-alpha2b) and thalidomide, as single agents and in combination. The murine dermis model was used to assess tumor-induced angiogenesis in nude mice. Human ACHN (renal), NIH-OVCAR-3 (ovarian), LNCaP (prostate), and SK-Mel-1 (melanoma) tumor cells were inoculated intradermally into the flanks of nude mice. IFN-alpha2b and thalidomide, administered daily, were effective inhibitors of angiogenesis induced by all four tumor types. The combination of IFN-alpha2b and thalidomide caused a synergistic decrease in mean vessel count in tumors that were resistant to the antiproliferative effects of IFN-alpha2b and thalidomide in vitro. This enhanced suppression of angiogenesis translated into synergistic antitumor activity in a xenograft model. Pegylated IFN-alpha (PEG-IFN-alpha2b) (10(6) U) administered once in 10 days was as effective as daily IFN-alpha2b treatment (10(6) U x 10 days). IFN-alpha2b and thalidomide have potentiated antiangiogenic activity when used in combination. A single dose of PEG-IFN-alpha2b (10(6) U) was as effective at suppressing vessel growth as an equivalent dose of IFN-alpha2b given daily for 10 days.

Resistance to interferons in melanoma cells does not correlate with the expression or activation of signal transducer and activator of transcription 1 (Stat1).

Defects in expression or activation signal transducer and activator of transcription-1 (Stat1) in response to interferon-alpha2 (IFN-alpha2) have been implicated as a mechanism for IFN resistance in melanoma cells. To further determine the significance of this observation, 17 melanoma cell lines sensitive or resistant to the antiproliferative effects of IFN-alpha2 and IFN-beta, as well as 30 melanoma patient samples, were analyzed for Stat1 levels by either Western blot analysis or immunohistochemistry. Although the expression level varied between samples, all the cell lines except one and all melanoma biopsy specimens expressed Stat1. IFN-stimulated levels of Stat1 and Stat2, which constitute the transcriptional activation complexes, such as, gamma activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), for IFN-stimulated gene (ISG) induction were assessed in melanoma cell lines. Both IFN-alpha2 and INF-beta induced equivalent amounts of Stat1 and Stat2 proteins in cell lines, although compared with IFN-alpha2, IFN-beta had greater antiproliferative effects. No significant differences were observed in tyrosine or serine phosphorylation of Stat1 or the formation of GAF or ISGF3 complexes following IFN-alpha2 or IFN-beta treatment of IFN-resistant or IFN-sensitive cell lines. Comparable induction of two ISGs, ISG54 and IFN regulatory factor-1 (IRF-1), was observed in both sensitive WM9 and resistant A375 cells. Therefore, we report that defects in expression or activation of Stat1 or Stat2 were infrequent in melanoma cell lines and tumor samples and did not correlate with IFN resistance. Cellular resistance to IFNs likely results from defective quantitative or qualitative expression of specific ISGs.

Thrombospondin-1 expression in melanoma is blocked by methylation and targeted reversal by 5-Aza-deoxycytidine suppresses angiogenesis.

BACKGROUND: Reversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated. RESULTS: 5-Aza-dC reduced DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein, reversed promoter hypermethylation, and restored TSP1 expression in five melanoma cell lines, while having no effect on TSP1 protein levels in normal human melanocytes. In in vivo neovascularization studies, mice were implanted with melanoma cells (A375) either untreated or treated with 5Aza-dC. Vessels at tumor sites were counted by an observer blinded to treatments and the number of tumor vessels was significantly decreased at pretreated tumor sites. This difference occurred before a significant difference in tumor volumes was seen, yet in further studies the average tumor volume in mice treated in vivo with 5-Aza-dC was decreased by 55% compared to untreated controls. Knockdown of TSP1 expression with shRNA enhanced tumor-induced angiogenesis by 68%. Analyses of promoter methylation status of TSP1 in tumors derived from untreated and treated mice identified 67% of tumors from untreated and 17% of tumors from treated mice with partial methylation consistent with the methylation specific PCR analysis of A375 cells. Examination of methylation patterns in the promoter of TSP1 and comparison of aberrantly methylated TSP1 in melanoma with non-malignant nevi identified a significantly higher frequency of promoter methylation in tumor samples from melanoma patients. CONCLUSIONS: Pharmacological reversal of methylation silenced TSP1 had functional biological consequences in enhancing angiogenesis inhibition and inducing antitumor effects to decrease murine melanoma growth. Angiogenesis inhibition is an additional mechanism by which epigenetic modulators can have antitumor effects.

Proteomic identification of proteins conjugated to ISG15 in mouse and human cells.

Though the interferon-inducible protein ISG15 was one of the first ubiquitin-like modifiers to be discovered, much remains unknown about the identity of proteins conjugated to ISG15 or the biologic consequences of modification. To gain a better understanding of the cellular pathways affected by ISG15, we identified proteins targeted for ISGylation using a proteomic approach. Mass spectrometric analysis identified 76 candidate ISGylation targets in anti-ISG15 immunoprecipitates from interferon-treated mouse or human cells. Twenty-one proteins were found in both mouse and human samples, including STAT1, a known target of ISGylation. Candidates identified in both species were tested for ISGylation in a transfection system: 18 of 19 proteins tested were ISGylated in this system. Two candidates, EF-2 and VCP, were also shown to be ISGylated in an interferon-dependent manner in the absence of exogenous over-expression. Seven proteins identified from a single species, but functionally related to candidates found in both species, were also ISGylated in the over-expression system. Proteins that can be ISGylated play important roles in translation, glycolysis, stress responses, and cell motility. These data indicate that ISGylation targets proteins found in several fundamentally important cellular pathways and will contribute to understanding the physiologic role of interferon-induced ISG15 and ISG15 conjugation.

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis.

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.

High-throughput immunoblotting. Ubiquitiin-like protein ISG15 modifies key regulators of signal transduction.

ISG15 is a ubiquitin-like protein that conjugates to numerous proteins in cells treated with interferon or lipopolysaccharide. Dysregulation of protein ISG15 modification (ISGylation) in mice leads to decreased life expectancy, brain cell injury, and hypersensitivity to interferon. Although ISG15 was identified more than two decades ago, the exact biochemical and physiological functions of ISG15-modification remain unknown, and the proteins targeted by ISG15 have not been identified. The major purpose of this work was to identify ISG15 targets among well characterized proteins that could be used as models for biological studies. We purified ISGylated proteins from human thymus by immunoaffinity chromatography and analyzed ISG15 conjugates by a high-throughput Western blot screen (PowerBlot). We found that three key regulators of signal transduction, phospholipase Cgamma1, Jak1, and ERK1 are modified by ISG15. In addition to that, we demonstrate that transcription factor Stat1, an immediate substrate of Jak1 kinase, is also ISGylated. Using whole cell protein extracts and phospholipase Cgamma1 as an example we demonstrate that ISG15 conjugates are not accumulated in cells treated with specific inhibitors of proteasomes. Our work suggests a role for ISG15 in the regulation of multiple signal transduction pathways and offers attractive models to further elucidate the biochemical function of ISGylation.