Impact of erythropoietin on the effects of irradiation under hypoxia

Purpose

Head and neck squamous cell carcinoma (HNSCC) represents an ideal model for assessing the impact of anemia and tumor hypoxia on the response to radiotherapy (RT). Various treatment strategies aimed at increasing tumor oxygenation in HNSCC patients have been studied and these studies have been fueled by evidence that hypoxia and, unexpectedly, erythropoietin (EPO), adversely affect the radiosensitivity of cells. The purpose of the present study was to experimentally examine the relationship between hypoxia, EPO, its receptor EPOR, EGFR and their effects on the survival and radiosensitivity of HNSCC cells underwent hypoxia.

Methods

We used Cal-166 head and neck cell line to investigate different cellular responses after RT given in oxic and hypoxic conditions, focusing on the role of EPO administration in cell proliferation and in regulating response to RT.

Results

Our results show that EPO do not evoke a physiologic response on EPOR-bearing tumor cells as assessed by cellular growth and proliferation. In addition, we present some indications that EPO could activate opposite signals related to proliferation, DNA repair and apoptosis. Among them, EGFR and AKT phosphorylation may play a role in radioresistance.

Conclusions

We concluded that the expression of the EPOR in Cal-166 cells does not seem essential for their growth and that administration of EPO does not affect RT efficacy.     

Quantitative determination of guanidinoacetate and creatine in dried blood spot by flow injection analysis-electrospray tandem mass spectrometry

BACKGROUND: Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of primary creatine disorders. The aim of this study was to develop and validate a method for the determination of GAA and Cr in dried blood spot through the use of stable isotope dilution and flow injection analysis (FIA)-ESI-MS/MS. METHODS: Dried blood spots were extracted using methanol-water solution containing D3-Cr. After evaporation and formation of butyl esters, samples were analyzed using multiple reaction monitoring mode (m/z 174.2-->101.1 for GAA, 188.3-->90.1 for Cr and 191.3-->93.1 for D3-Cr). RESULTS: The analysis was very fast (1 min). The detection limits were 0.34 micromol/l of blood and 0.30 micromol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25-12.5 micromol/l of blood for GAA and 3.57-624.7 micromol/l of blood for Cr. Recovery range was 93-101% for Cr and 94-105% for GAA and between-run CVs were 5.3% for GAA and 4.5% for Cr. Ion suppression effect was also studied. The method was applied to spots obtained from two patients affected by GAMT deficiency, four patients affected by AGAT deficiency (including a newborn) as well as 282 healthy subjects. CONCLUSIONS: The detection of GAA in dried blood spot by FIA-ESI-MS/MS is a highly reliable and high throughput method for the diagnosis of GAMT and AGAT deficiencies and a possible tool for newborn screening of both these tractable disorders.     

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34(+) cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34(+) cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34(+) cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34(+) cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.     

A mutation on exon 6 of guanidinoacetate methyltransferase (GAMT) gene supports a different function for isoform a and b of GAMT enzyme

A new patient affected by Guanidinoacetate methyltransferase (GAMT) deficiency was reported. This 13-year-old girl presented with mental retardation, as main symptom, associated with a typical pattern of biochemical and neurochemical (brain magnetic resonance spectroscopy) alterations. Molecular study detected a L197P transition on exon 6 of the GAMT gene. Since this mutation leaves the isoform B of the GAMT enzyme unaffected, the occurrence of biochemical alterations and disease in this subject testifies against the possibility that isoform b had GAMT activity.     

The fusiform face area is not sufficient for face recognition: evidence from a patient with dense prosopagnosia and no occipital face area

We tested functional activation for faces in patient D.F., who following acquired brain damage has a profound deficit in object recognition based on form (visual form agnosia) and also prosopagnosia that is undocumented to date. Functional imaging demonstrated that like our control observers, D.F. shows significantly more activation when passively viewing face compared to scene images in an area that is consistent with the fusiform face area (FFA) (p < 0.01). Control observers also show occipital face area (OFA) activation; however, whereas D.F.'s lesions appear to overlap the OFA bilaterally. We asked, given that D.F. shows FFA activation for faces, to what extent is she able to recognize faces? D.F. demonstrated a severe impairment in higher level face processing--she could not recognize face identity, gender or emotional expression. In contrast, she performed relatively normally on many face categorization tasks. D.F. can differentiate faces from non-faces given sufficient texture information and processing time, and she can do this is independent of color and illumination information. D.F. can use configural information for categorizing faces when they are presented in an upright but not a sideways orientation and given that she also cannot discriminate half-faces she may rely on a spatially symmetric feature arrangement. Faces appear to be a unique category, which she can classify even when she has no advance knowledge that she will be shown face images. Together, these imaging and behavioral data support the importance of the integrity of a complex network of regions for face identification, including more than just the FFA--in particular the OFA, a region believed to be associated with low-level processing.     

The role of parietal cortex in visuomotor control: what have we learned from neuroimaging?

Research from macaque neurophysiology and human neuropsychology has implicated the parietal cortex in the sensory control of action. Functional neuroimaging has been very valuable in localizing and characterizing specific regions of the human brain involved in visuomotor actions involving different effectors, such as the eyes, head, arms and hands. Here, we review the areas discovered by human neuroimaging, including the putative functional equivalents of the following macaque regions: parietal eye fields (PEF), ventral intraparietal (VIP) area, parietal reach region (PRR) and the anterior intraparietal (AIP) area. We discuss the challenges of studying realistic movements in the imaging environment, the lateralization of visuomotor function, caveats involved in proposing interspecies homologies and the limitations and future directions for neuroimaging studies of visuomotor control.