Electron paramagnetic resonance oximetry as a novel approach to monitor the effectiveness and quality of red blood cell transfusions.

BACKGROUND: The goal of red blood cell transfusion is to improve tissue oxygenation. Assessment of red blood cell quality and individualised therapeutic needs can be optimised using direct oxygen (O2) measurements to guide treatment. Electron paramagnetic resonance oximetry is capable of accurate, repeatable and minimally invasive measurements of tissue pO2. Here we present preclinical proof-of-concept of the utility of electron paramagnetic resonance oximetry in an experimental setting of acute blood loss, transfusion, and post-transfusion monitoring. MATERIALS AND METHODS: Donor rat blood was collected, leucocyte-reduced, and stored at 4 °C in AS-3 for 1, 7 and 14 days. Red blood cell morphology, O2 equilibrium, p50 and Hill numbers from O2 binding and dissociation curves were evaluated in vitro. Recipient rats were bled and maintained at a mean arterial pressure of 30-40 mmHg and hind limb muscle (biceps femoris) pO2 at 25-50% of baseline. Muscle pO2 was monitored continuously over the course of experiments to assess the effectiveness of red blood cell preparations at different stages of blood loss and restoration. RESULTS: Red blood cell morphology, O2 equilibrium and p50 values of intra-erythrocyte haemoglobin were significantly altered by refrigerated storage for both 7 and 14 days. Transfusion of red blood cells stored for 7 or 14 days demonstrated an equivalently impaired ability to restore hind limb muscle pO2, consistent with in vitro observations and transfusion with albumin. Red blood cells refrigerated for 1 day demonstrated normal morphology, in vitro oxygenation and in vivo restoration of tissue pO2. DISCUSSION: Electron paramagnetic resonance oximetry represents a useful approach to assessing the quality of red blood cells and subsequent transfusion effectiveness.

Adjuvant chemotherapy for elderly patients (aged 70 or older) with gastric cancer after a gastrectomy with D2 dissection: A single center experience in Korea.

AIMS: Adjuvant chemotherapy is recommended for gastric cancer after a gastrectomy with D2 dissection. However, its survival benefit in elderly patients is unclear. Here we investigated the use of adjuvant chemotherapy in patients ≥70 years old with stage II or III gastric cancer. METHODS: Patients ≥70 years old diagnosed with stage II or III gastric cancer at Ulsan University Hospital were identified. A retrospective analysis of electronic and paper patient records was performed. RESULTS: From 2008 to 2012, 277 patients ≥70 years old underwent gastrectomy with D2 dissection. Of these patients, 94 were pathologically diagnosed with stage II or III; 55 of these patients (58.5%) received adjuvant chemotherapy and 39 received regular checkups without chemotherapy. Fluoropyrimidine-alone regimens, including TS-1 composed of tegafur, gimestat and otastat potassium (n = 26) and doxifluridine (n = 22), were more commonly used than fluoropyrimidine-platinum combination regimens (n = 7). With a median follow-up of 30.9 (range 0.8-65.5) months, the median relapse-free survival of patients with adjuvant chemotherapy or regular follow-up only was 35.5 and 20.4 months, respectively (P = 0.030). Multivariate analysis revealed that adjuvant chemotherapy is associated with longer relapse-free survival (hazard ratio 0.50; 95% confidence interval 0.27-0.96). There was a trend toward an improved overall survival in the adjuvant chemotherapy group compared with the follow-up only group (P = 0.242). CONCLUSIONS: Although well-designed prospective studies are required, adjuvant chemotherapy may confer a potential survival benefit in elderly patients (aged 70 or older) with stage II or III gastric cancer after a gastrectomy with D2 dissection.

Four-and-a-half LIM domain proteins inhibit transactivation by hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that promotes angiogenesis, metabolic reprogramming, and other critical aspects of cancer biology. The four-and-a-half LIM domain (FHL) proteins are a family of LIM domain-only proteins implicated in transcriptional regulation and suppression of tumor growth. Here we describe functional interactions between the FHL proteins and HIF-1. FHL1-3 inhibit HIF-1 transcriptional activity and HIF-1α transactivation domain function by oxygen-independent mechanisms. FHL2 directly interacts with HIF-1α to repress transcriptional activity. FHL1 binds to the p300/CBP co-activators and disrupts binding with HIF-1α. FHL3 does not bind to HIF-1α or p300, indicating that it regulates transactivation by a novel molecular mechanism. Expression of the FHL proteins increased upon HIF-1α induction, suggesting the existence of a feedback loop. These results identify FHL proteins as negative regulators of HIF-1 activity, which may provide a mechanism by which they suppress tumor growth.

MCM proteins are negative regulators of hypoxia-inducible factor 1.

MCM proteins are components of a DNA helicase that plays an essential role in DNA replication and cell proliferation. However, MCM proteins are present in excess relative to origins of replication, suggesting they may serve other functions. Decreased proliferation is a fundamental physiological response to hypoxia in many cell types, and hypoxia-inducible factor 1 (HIF-1) has been implicated in this process. Here, we demonstrate that multiple MCM proteins bind directly to the HIF-1α subunit and synergistically inhibit HIF-1 transcriptional activity via distinct O(2)-dependent mechanisms. MCM3 inhibits transactivation domain function, whereas MCM7 enhances HIF-1α ubiquitination and proteasomal degradation. HIF-1 activity decreases when quiescent cells re-enter the cell cycle, and this effect is MCM dependent. Exposure to hypoxia leads to MCM2-7 downregulation in diverse cell types. These studies reveal a function of MCM proteins apart from their DNA helicase activity and establish a direct link between HIF-1 and the cell-cycle machinery.

Spermidine/spermine N(1)-acetyltransferase-1 binds to hypoxia-inducible factor-1alpha (HIF-1alpha) and RACK1 and promotes ubiquitination and degradation of HIF-1alpha.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis that controls the expression of genes encoding proteins that play key roles in angiogenesis, erythropoiesis, and glucose/energy metabolism. The stability of the HIF-1alpha subunit is regulated by ubiquitination and proteasomal degradation. In aerobic cells, O(2)-dependent prolyl hydroxylation of HIF-1alpha is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, which then recruits the Elongin C ubiquitin-ligase complex. SSAT2 (spermidine/spermine N-acetyltransferase-2) binds to HIF-1alpha and promotes its ubiquitination/degradation by stabilizing the interaction of VHL and Elongin C. Treatment of cells with heat shock protein HSP90 inhibitors induces the degradation of HIF-1alpha even under hypoxic conditions. HSP90 competes with RACK1 for binding to HIF-1alpha, and HSP90 inhibition leads to increased binding of RACK1, which recruits the Elongin C ubiquitin-ligase complex to HIF-1alpha in an O(2)-independent manner. In this work, we demonstrate that SSAT1, which shares 46% amino acid identity with SSAT2, also binds to HIF-1alpha and promotes its ubiquitination/degradation. However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1alpha with RACK1. Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O(2)-independent and O(2)-dependent degradation of HIF-1alpha.

RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha.

Hypoxia-inducible factor 1 (HIF-1) regulates transcription in response to changes in O(2) concentration. O(2)-dependent degradation of the HIF-1alpha subunit is mediated by prolyl hydroxylase (PHD), the von Hippel-Lindau (VHL)/Elongin-C/Elongin-B E3 ubiquitin ligase complex, and the proteasome. Inhibition of heat-shock protein 90 (HSP90) leads to O(2)/PHD/VHL-independent degradation of HIF-1alpha. We have identified the receptor of activated protein kinase C (RACK1) as a HIF-1alpha-interacting protein that promotes PHD/VHL-independent proteasomal degradation of HIF-1alpha. RACK1 competes with HSP90 for binding to the PAS-A domain of HIF-1alpha in vitro and in human cells. HIF-1alpha degradation induced by the HSP90 inhibitor 17-allylaminogeldanamycin is abolished by RACK1 loss of function. RACK1 binds to Elongin-C and promotes ubiquitination of HIF-1alpha. Elongin-C-binding sites in RACK1 and VHL show significant sequence similarity. Thus, RACK1 is an essential component of an O(2)/PHD/VHL-independent mechanism for regulating HIF-1alpha stability through competition with HSP90 and recruitment of the Elongin-C/B ubiquitin ligase complex.

Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B.

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.