Neointimal Hyperplasia after Carotid Transection and Anastomosis Surgery is Associated with Degradation of Decorin and Platelet Derived Growth Factor Signaling.

OBJECTIVE: Intimal hyperplasia (IH) is the expansion of the vascular intimal region after intervention, which can lead to stenosis and eventual failure of vascular grafts or interventional procedures such as angioplasty or stent placement. Our goals were to investigate the development of IH in a rabbit open surgical model and to evaluate the associated pathophysiological processes involving decorin and the platelet derived growth factor-BB / platelet derived growth factor receptor-ß / mitogen activated protein kinase (PDGF/PDGFR-ß/MAPK) pathway. METHODS: We conducted carotid transection and primary anastomosis on five New Zealand White rabbits to induce IH and examined the associated pathophysiological changes. Tissue was obtained for histological and protein analysis on post-operative day 21 using the contralateral vessel as a control. Intimal medial thickness (IMT) was calculated to measure IH and compared with the unoperated side. Western blot analysis was performed on tissue lysates to determine the expression of decorin core protein, PDGF-BB, PDGFR-ß, and phosphorylated-MAPK (ph-MAPK). Immunofluorescence microscopy was used to assess tissue distribution of matrix metalloproteinase-2 (MMP-2) and phosphorylated-PDGFR-ß (ph-PDGFR-ß). RESULTS: Bilateral carotid arteries were harvested on postoperative day 21. We compared the IMT in operated with unoperated specimens. IMT was significantly elevated in operated arteries vs. unoperated arteries in all 5 animals (148.6 µm +/- 9.09 vs. 103.40 µm +/- 7.08; 135.2 µm +/- 8.30 vs. 92.40 µm +/- 2.35; 203.1 µm +/- 30.23 vs.104.00 µm +/- 4.52; 236.2 µm +/- 27.22 vs. 141.50 µm +/- 9.95; 226.9 µm +/- 11.12 vs. 98.8 µm +/- 3.78). Western blot analysis revealed degradation of decorin protein in the operated tissue, including loss of a 50 kDa band and the appearance of a cleaved fragment at 10 kDa. Decorin and MMP-2 were observed, via immunofluorescence microscopy, in the neointima of the operated vessels. Western blot analysis also revealed increased PDGF-BB, PDGFR-ß, and ph-MAPK levels in operated tissue. Immunofluorescent staining for ph-PDGFR-ß primarily localized to the neointima, indicating increased signaling through PDGF in this region. CONCLUSION: Carotid transection and primary reanastomosis in rabbits induced IH that was associated with MMP-2 activation, degradation of decorin, and activation of the PDGF/PDGFR-ß /MAPK pathway. The findings in this study should lead to further mechanistic evaluation of these pathways to better understand the potential to modify the intimal hyperplastic response to surgery.

Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2).

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

p27/Kip1 functions as a tumor suppressor and oncoprotein in osteosarcoma.

The p27/kip1 (p27) tumor suppressor inhibits cyclin/cyclin-dependent kinase (CDK) complexes and halts cell cycle progression. p27 further regulates invasion and migration in cancer cells, suggesting p27 also functions as an oncoprotein. Using a human osteosarcoma tissue microarray we identified high expression of cytoplasmic p27 in metastatic tumors. We demonstrated a positive correlation between mRNA and protein expression of p27 and expression of key metastatic markers, vimentin, snail-2, ß-catenin and stathmin-1 (STMN1) in patient tumors. Our results show that T198 phosphorylation of p27 controls the interaction between p27 and STMN1 that regulates microtubule stabilization and the invasion and migration of osteosarcoma cells. We found that anti-tumoral activity of gemcitabine and the Wee1 kinase inhibitor AZD1775 in osteosarcoma cells, was dependent on drug sequencing that relied on p27 stabilization. Gemcitabine activated caspase-3 and synergized with AZD1775 through caspase-mediated cleavage of p27, that dissociated from STMN1 and effectively induced apoptosis. Further, blockage of nuclear export of p27 by inhibition of Exportin-1 (XPO1) promoted growth arrest, demonstrating that the biological effects of agents relied on the expression and localization of p27. Together, these data provide a rationale for combining chemotherapy with agents that promote p27 tumor suppressor activity for the treatment of osteosarcoma.

Development of Chemotherapy with Cell-Cycle Inhibitors for Adult and Pediatric Cancer Therapy.

Preclinical and clinical development of agents that inhibit cell-cycle progression have brought an understanding of the feasibility of targeting various cell-cycle regulators in patients with cancer. Small molecule inhibitors targeting key proteins that participate in cell-cycle progression including the cyclin-dependent kinases and checkpoint kinases induce cell-cycle arrest and apoptosis in neoplastic cells. Early phase I studies demonstrate targeted inhibitors can be administered safely in adult and pediatric cancer patients, but these agents generally show limited clinical benefits as single agents. In this review, we discuss biological mechanisms that support dual combination strategies of cell-cycle inhibition with chemotherapeutic agents that are anticipated to achieve rationally targeted therapies for cancer patients. The rationale for evaluating these combination strategies is that DNA damage renders tumors highly responsive to irreversible cell-cycle arrest therapy. This approach is predicted to generate less intensive therapies and to maximize the efficacy of individual agents against solid tumors and hematologic malignancies. Cancer Res; 78(2); 320-5. ©2018 AACR.

Recent Advances of Cell-Cycle Inhibitor Therapies for Pediatric Cancer.

This review describes the pivotal roles of cell-cycle and checkpoint regulators and discusses development of specific cell-cycle inhibitors for therapeutic use for pediatric cancer. The mechanism of action as well as the safety and tolerability of drugs in pediatric patients, including compounds that target CDK4/CDK6 (palbociclib, ribociclib, and abemaciclib), aurora kinases (AT9283 and MLN8237), Wee1 kinase (MK-1775), KSP (ispinesib), and tubulin (taxanes, vinca alkaloids), are presented. The design of mechanism-based combinations that exploit the cross-talk of signals activated by cell-cycle arrest, as well as pediatric-focused drug development, are critical for the advancement of drugs for rare childhood diseases. Cancer Res; 77(23); 6489-98. ©2017 AACR.

The Microtubule Network and Cell Death Are Regulated by an miR-34a/Stathmin 1/ßIII-Tubulin Axis.

MicroRNA-34a (miR-34a) is a master regulator of signaling networks that maintains normal physiology and disease and is currently in development as a miRNA-based therapy for cancer. Prior studies have reported low miR-34a expression in osteosarcoma; however, the molecular mechanisms underlying miR-34a activity in osteosarcoma are not well-defined. Therefore, this study evaluated the role of miR-34a in regulating signal transduction pathways that influence cell death in osteosarcoma. Levels of miR-34a were attenuated in human osteosarcoma cells and xenografts of the Pediatric Preclinical Testing Consortium (PPTC). Bioinformatics predictions identified stathmin 1 (STMN1) as a potential miR-34a target. Biotin pull-down assay and luciferase reporter analysis confirmed miR-34a target interactions within the STMN1 mRNA 3'-untranslated region. Overexpression of miR-34a in osteosarcoma cells suppressed STMN1 expression and reduced cell growth in vitro Restoration of miR-34a led to microtubule destabilization and increased ßIII-tubulin expression, with corresponding G1-G2 phase cell-cycle arrest and apoptosis. Knockdown of the Sp1 transcription factor, by siRNA silencing, also upregulated ßIII-tubulin expression in osteosarcoma cells, suggesting that miR-34a indirectly affects Sp1. Validating the coordinating role of miR-34a in microtubule destabilization, when miR-34a was combined with either microtubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed and there was greater cytotoxicity in osteosarcoma cells. These results demonstrate that miR-34a directly represses STMN1 gene and protein expression and upregulates ßIII-tubulin, leading to disruption of the microtubule network and cell death.Implications: The miR-34a/STMN1/ßIII-tubulin axis maintains the microtubule cytoskeleton in osteosarcoma, and combining miR-34a with microtubule inhibitors can be investigated as a novel therapeutic strategy. Mol Cancer Res; 15(7); 953-64. ©2017 AACR.

Simulating the slow to fast switch in cytochrome c oxidase catalysis by introducing a loop flip near the enzyme's cytochrome c (substrate) binding site.

The mitochondrial enzyme cytochrome c oxidase catalyzes the reduction of molecular oxygen in the critical step of oxidative phosphorylation that links the oxidation of food consumed to ATP production in cells. The enzyme catalyzes the reduction of oxygen at two vastly different rates that are thought to be linked to two different conformations but the conformation of the "fast enzyme" remains obscure. In this study, we demonstrated how oxygen binding at haem a3 could trigger long-distance conformational changes and then simulated a conformational change in an eight-residue loop near the enzyme's substrate (cytochrome c) binding site. We then used this modified cytochrome c oxidase (COX) to simulate a stable COX-cytochrome c enzyme-substrate (ES) complex. Compared to ES complexes formed in the absence of the conformation change, the distance between the redox centers of the two proteins was reduced by half and instead of nine, only four COX amino acid residues were found along the axis linking the electron entry point and the CuA redox center of COX: We proposed that intramolecular electron transfer in COX occurs via a charge/hydrogen relay system involving these four residues. We suggest that the conformational change and resulting shortened electron pathway are features of fast-acting COX.

Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Osteosarcoma is the most frequently occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common. Eribulin is a synthetic microtubule inhibitor that has demonstrated activity in preclinical osteosarcoma models. The effects of eribulin were evaluated in two human osteosarcoma cell lines as well as in eribulin-sensitive and -resistant osteosarcoma xenograft tumors of the Pediatric Preclinical Testing Program (PPTP) by characterizing cell viability, microtubule destabilization, mitotic arrest and mechanism of cell death. Eribulin demonstrated cytotoxic activity in vitro, through promotion of microtubule dynamic instability, arrest of cells in the G2/M phase, mitotic catastrophe and cell death. The microtubule-destabilizing protein stathmin-1 (STMN1) was coimmunoprecipitated with the cyclin-dependent kinase inhibitor p27 indicating that these cytoplasmic complexes can protect cells from the microtubule destabilizing effect of eribulin. Increased tumoral expression of P-glycoprotein (P-gp) and TUBB3 were also associated with lower drug sensitivity. In summary, eribulin successfully blocked cells in G2/M phase but interfered with mitochondria activity to inhibit proteins involved in apoptosis. Understanding the complex and inter-related mechanisms involved in the overall drug response to eribulin may help in the design of therapeutic strategies that enhance drug activity and improve benefits of eribulin in pediatric patients with osteosarcoma.

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways.

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS.

MicroRNAs and Potential Targets in Osteosarcoma: Review.

Osteosarcoma is the most common bone cancer in children and young adults. Surgery and multi-agent chemotherapy are the standard treatment regimens for this disease. New therapies are being investigated to improve overall survival in patients. Molecular targets that actively modulate cell processes, such as cell-cycle control, cell proliferation, metabolism, and apoptosis, have been studied, but it remains a challenge to develop novel, effective-targeted therapies to treat this heterogeneous and complex disease. MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating cell processes including growth, development, and disease. miRNAs function as oncogenes or tumor suppressors to regulate gene and protein expression. Several studies have demonstrated the involvement of miRNAs in the pathogenesis of osteosarcoma with the potential for development in disease diagnostics and therapeutics. In this review, we discuss the current knowledge on the role of miRNAs and their target genes and evaluate their potential use as therapeutic agents in osteosarcoma. We also summarize the efficacy of inhibition of oncogenic miRNAs or expression of tumor suppressor miRNAs in preclinical models of osteosarcoma. Recent progress on systemic delivery as well as current applications for miRNAs as therapeutic agents has seen the advancement of miR-34a in clinical trials for adult patients with non-resectable primary liver cancer or metastatic cancer with liver involvement. We suggest a global approach to the understanding of the pathogenesis of osteosarcoma may identify candidate miRNAs as promising biomarkers for this rare disease.

A phase I trial and viral clearance study of reovirus (Reolysin) in children with relapsed or refractory extra-cranial solid tumors: a Children's Oncology Group Phase I Consortium report.

BACKGROUND: Reovirus is a naturally occurring human virus that is cytopathic to malignant cells possessing an activated Ras signaling pathway. We conducted a phase I trial of Reolysin, a manufactured, proprietary isolate of purified reovirus, in children with relapsed/refractory extracranial solid tumors to define the recommended phase 2 dose (RP2D), toxicities, and pharmacokinetic properties when administered as a single agent or in combination with cyclophosphamide. PROCEDURES: Reolysin was administered intravenously for 5 consecutive days, every 28 days. Using a 3 + 3 design, the following dose levels were evaluated: 3 × 10(8) Tissue Culture Inhibitory Dose 50% (TCID50 )/kg; 5 × 10(8) TCID50 /kg (maximum dose was 3 × 10(10) TCID50 ); and 5 × 10(8) TCID50 /kg plus oral cyclophosphamide (50 mg/m(2) /day × 21 days). RESULTS: Twenty-nine patients were enrolled; 28 were eligible and 24 were evaluable for toxicity and response. There were no hematologic dose-limiting toxicities. Grade 5 respiratory failure and a Grade 5 thromboembolic event were reported, both in the setting of progressive disease. The median time to clear the reovirus viremia was 6.5 days. Eight of 24 patients were viremic beyond the 5 days of therapy, all were negative by day 17. No patient had detectable viral RNA in saliva or stool. There were no objective responses. CONCLUSIONS: Reolysin at a dose of 5 × 10(8) TCID50 /kg daily for 5 days was well tolerated in children alone and in combination with oral cyclophosphamide. Virus was cleared rapidly from the serum and shedding in stool and saliva was not detectable.

Wilms' tumor protein induces an epithelial-mesenchymal hybrid differentiation state in clear cell renal cell carcinoma.

The Wilms' tumor transcription factor (WT1) was originally classified as a tumor suppressor, but it is now known to also be associated with cancer progression and poor prognosis in several malignancies. WT1 plays an essential role in orchestrating a developmental process known as mesenchymal-to-epithelial transition (MET) during kidney development, but also induces the reverse process, epithelial-to-mesenchymal transition (EMT) during heart development. WT1 is not expressed in the adult kidney, but shows elevated expression in clear cell renal cell carcinoma (ccRCC). However, the role of WT1 in this disease has not been characterized. In this study, we demonstrate that WT1 is upregulated in ccRCC cells that are deficient in the expression of the von Hippel-Lindau tumor suppressor protein (VHL). We found that WT1 transcriptionally activated Snail, a master transcriptional repressor that is known to induce EMT. Although Snail represses E-cadherin and induces mesenchymal characteristics, we found partial maintenance of E-cadherin and associated epithelial characteristics in kidney cells and ccRCC cells that express WT1, since WT1 upregulates E-cadherin expression and competes with Snail repression. These findings support a novel paradigm in which WT1 induces an epithelial-mesenchymal hybrid transition (EMHT), characterized by Snail up-regulation with E-cadherin maintenance, a tumor cell differentiation state in which cancer cells keep both EMT and MET characteristics which may promote tumor cell plasticity and tumor progression.

Oncolytic viruses for potential osteosarcoma therapy.

Since the first anecdotal reports of dramatic tumor responses following a viral infection in early 1900s, the field of oncolytic virotherapy has evolved at a rapid pace finally making its way into clinical trials. A large number of both wild-type and genetically altered viruses with a preferential replication-competency for tumor cells have been studied in tissue cultures, animal models and in humans, with an ever increasing repertoire of new viruses being added to this pool. Although oncolytic viruses have caused dramatic antitumor responses in cell cultures and mouse models, their clinical effects in humans have been modest. Therefore, the current research is focused on understanding the mechanisms by which viruses kill tumor cells, the barriers to successful viral delivery and penetration into tumor cells, the role of the immune system in viral oncolysis and generating stronger target specific and replication competent viruses. Osteosarcoma is a challenging malignancy to identify novel targets for therapy due to its complex genetic make-up. Oncolytic virotherapy may be a promising approach as a novel therapeutic, not dependent on consistent expression of a single target. In this review we summarize the supportive evidence and rationale for use of viral oncolysis in osteosarcoma along with the specific challenges it may face.

Xenograft and genetically engineered mouse model systems of osteosarcoma and Ewing's sarcoma: tumor models for cancer drug discovery.

INTRODUCTION: There are > 75 histological types of solid tumors that are classified into two major groups: bone and soft-tissue sarcomas. These diseases are more prevalent in children, and pediatric sarcomas tend to be highly aggressive and rapidly progressive. Sarcomas in adults may follow a more indolent course, but aggressive tumors are also common. Sarcomas that are metastatic at diagnosis, or recurrent following therapy, remain refractory to current treatment options with dismal overall survival rates. A major focus of clinical trials, for patients with sarcoma, is to identify novel and more effective therapeutic strategies targeted to genomic or proteomic aberrations specific to the malignant cells. Critical to the understanding of the potential for targeted therapies are models of disease that are representative of clinical disease and predictive of relevant clinical responses. AREAS COVERED: In this article, the authors discuss the use of mouse xenograft models and genetically engineered mice in cancer drug discovery. The authors provide a special focus on models for the two most common bone sarcomas: osteosarcoma (OS) and Ewing's sarcoma (ES). EXPERT OPINION: Predicting whether a new anticancer agent will have a positive therapeutic index in patients with OS and ES remains a challenge. The use of mouse sarcoma models for understanding the mechanisms involved in the response of tumors to new treatments is an important step in the process of drug discovery and the development of clinically relevant therapeutic strategies for these diseases.

A review of targeted therapies evaluated by the pediatric preclinical testing program for osteosarcoma.

Osteosarcoma, the most common malignant bone tumor of childhood, is a high-grade primary bone sarcoma that occurs mostly in adolescence. Standard treatment consists of surgery in combination with multi-agent chemotherapy regimens. The development and approval of imatinib for Philadelphia chromosome-positive acute lymphoblastic leukemia in children and the fully human monoclonal antibody, anti-GD2, as part of an immune therapy for high-risk neuroblastoma patients have established the precedent for use of targeted inhibitors along with standard chemotherapy backbones. However, few targeted agents tested have achieved traditional clinical endpoints for osteosarcoma. Many biological agents demonstrating anti-tumor responses in preclinical and early-phase clinical testing have failed to reach response thresholds to justify randomized trials with large numbers of patients. The development of targeted therapies for pediatric cancer remains a significant challenge. To aid in the prioritization of new agents for clinical testing, the Pediatric Preclinical Testing Program (PPTP) has developed reliable and robust preclinical pediatric cancer models to rapidly screen agents for activity in multiple childhood cancers and establish pharmacological parameters and effective drug concentrations for clinical trials. In this article, we examine a range of standard and novel agents that have been evaluated by the PPTP, and we discuss the preclinical and clinical development of these for the treatment of osteosarcoma. We further demonstrate that committed resources for hypothesis-driven drug discovery and development are needed to yield clinical successes in the search for new therapies for this pediatric disease.

Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer.

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.

Substrate binding-dissociation and intermolecular electron transfer in cytochrome c oxidase are driven by energy-dependent conformational changes in the enzyme and substrate.

Reduction of O2 by cytochrome c oxidase (COX) is critical to the cellular production of adenosine-5'-triphosphate; COX obtains the four electrons required for this process from ferrocytochrome c. The COX-cytochrome c enzyme-substrate complex is stabilized by electrostatic interactions via carboxylates on COX and lysines on cytochrome c. Conformational changes are believed to play a role in ferrocytochrome c oxidation and release and in rapid intramolecular transfer of electrons within COX, but the details are unclear. To gather specific information about the extent and relevance of conformational changes, we performed bioinformatics studies using the published structures of both proteins. For both proteins, we studied the surface accessibility and energy, as a function of the proteins' oxidation state. The residues of reduced cytochrome c showed greater surface accessibility and were at a higher energy than those of the oxidized cytochrome c. Also, most residues of the core subunits (I, II, and III) of COX showed low accessibility, ∼35%, and compared to the oxidized subunits, the reduced subunits had higher energies. We concluded that substrate binding and dissociation is modulated by specific redox-dependent conformational changes. We further conclude that high energy and structural relaxation of reduced cytochrome c and core COX subunits drive their rapid electron transfer.

Expression of Na,K-ATPase-beta(1) subunit increases uptake and sensitizes carcinoma cells to oxaliplatin.

PURPOSE: The ovarian carcinoma subline A2780/C10B (C10B) is an oxaliplatin resistant clone derived from the human ovarian carcinoma cell line A2780. The C10B cells are characterized by mesenchymal phenotype, decreased platinum uptake and increased glutathione levels (Hector et al. in Cancer Lett 245:195-204, 2007; Varma et al. in Oncol Rep 14:925-932, 2005). Na,K-ATPase-beta subunit (Na,K-beta(1)) functions as a cell-cell adhesion molecule in epithelial cells and is reduced in a variety of carcinoma cells that show mesenchymal phenotype. The purpose of this study is to evaluate the relationship between Na,K-beta expression and sensitivity to oxaliplatin. METHODS: Cell lines used include A2780, C10B, C10B transfected with Na,K-beta(1) (C10B-Na,K-beta) and a canine kidney carcinoma cell line MSV-MDCK also transfected with Na,K-beta(1) (MSV-MDCK-beta subunit). Cytotoxicity studies were performed by sulforhodamine-blue assay. The Na,K-alpha(1) and Na,K-beta(1) subunit localization and expression were by immunofluorescence microscopy and Western blot analysis. Platinum accumulation measurements were by atomic absorption spectrophotometry. RESULTS: C10B cells express highly reduced levels of Na,K-beta(1) subunit. Exogenous expression of Na,K-beta(1) increased platinum accumulation and sensitized C10B cells to oxaliplatin. The pharmacological inhibitor of Na,K-ATPase ouabain did not alter the oxaliplatin accumulation indicating that Na,K-beta(1) sensitizes cells in a Na,K-ATPase enzyme activity independent manner. These findings were also confirmed in MSV-MDCK-beta subunit cells. CONCLUSIONS: This study for the first time reveals that reduced expression of the Na,K-beta(1) protein is associated with oxaliplatin resistance in cancer cells and demonstrates a novel role for this protein in sensitizing the cells to oxaliplatin. This study suggests a potentially important role for Na,K-beta(1) in both prognosis and therapy of oxaliplatin resistant malignancies.

Failure of immunosuppressive drug levels to predict T-cell reactivity in pediatric transplant patients.

PURPOSE: In children, therapeutic management of immunosuppression relies on allograft function, drug levels, and clinical insight. Using a Food and Drug Administration-approved test for T-cell response, T-cell activation in vitro can be measured to monitor the immune response. METHODS: In a retrospective study, 92 posttransplant children who received either a liver and/or kidney transplant and were followed by routine screening had their T-cell response tested by the Cylex ImmuKnow assay (Columbia, MD). After phytohemagglutinin-L stimulation of T-cells, adenosine triphosphate (ATP) concentrations were measured. In this assay, light emission at lambda = 562 nm is proportional to the ATP concentration (ng/mL). Immunosuppressive drug trough levels were also measured. Quantitative real-time polymerase chain reaction Epstein-Barr virus (EBV) viral titers were determined for 2 patients. RESULTS: Separating the results into younger than 12 years and 12-year or older populations, we found that for the younger than 12 years, 28% of patients were in the low immune function category, 47% in the moderate, and 25% in the high category. For the 12 years or older, 25% of patients were in the low immune function category, 47% in the moderate, and 28% in the high category. The immune function distribution was not different (P = not significant) between the younger than 12 years and 12-year or older groups. Tacrolimus trough levels were 6.3 +/- 2.4 ng/mL for younger than 12 years and 5.6 +/- 3.3 ng/mL for 12 years or older (P = not significant), and rapamycin was similar, but both showed no correlation to immune function. We observed increased ATP values with decreased EBV viral loads. CONCLUSIONS: These results suggest that tacrolimus and/or rapamycin levels do not adequately determine the biologic effect of immunosuppression. We expect that future T-cell activation monitoring will allow us to diminish rejection and infection events posttransplantation and lead to a healthier pediatric transplant population.

MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells.

Regulation of the MYC oncogene remains unclear. Using 10058-F4, a compound that inhibits MYC-MAX transcription factor, MYC protein and gene expression were down-regulated in Namalwa cells, a Burkitt lymphoma. Compound 10058-F4 decreased MYC mRNA (45%), MYC protein (50%), and cell growth (32%). MYC-MAX transcription factor was disrupted 24 h after treatment, resulting in transcriptional inhibition of target genes. Because microRNAs (miRNA) disrupt mRNA translation, let-7a, let-7b, and mir-98 were selected using bioinformatics for targeting MYC. Inhibition of MYC-MAX transcription factor with 10058-F4 increased levels of members of the let-7 family. In inhibited cells at 24 h, let-7a, let-7b, and mir-98 were induced 4.9-, 1.3-, and 2.4-fold, respectively, whereas mir-17-5p decreased 0.23-fold. These results were duplicated using microRNA multianalyte suspension array technology. Regulation of MYC mRNA by let-7a was confirmed by transfections with pre-let-7a. Overexpression of let-7a (190%) decreased Myc mRNA (70%) and protein (75%). Down-regulation of Myc protein and mRNA using siRNA MYC also elevated let-7a miRNA and decreased Myc gene expression. Inverse coordinate regulation of let-7a and mir-17-5p versus Myc mRNA by 10058-F4, pre-let-7a, or siRNA MYC suggested that both miRNAs are Myc-regulated. This supports previous results in lung and colon cancer where decreased levels of the let-7 family resulted in increased tumorigenicity. Here, pre-let-7a transfections led to down-regulation of expression of MYC and its target genes and antiproliferation in lymphoma cells. These findings with let-7a add to the complexity of MYC regulation and suggest that dysregulation of these miRNAs participates in the genesis and maintenance of the lymphoma phenotype in Burkitt lymphoma cells and other MYC-dysregulated cancers.