A practical EPR dosimetry system for routine use in radiotherapy: uncertainty analysis of lithium formate dosimeters at the therapeutic dose level.

In electron paramagnetic resonance (EPR) dosimetry, solid dosimeter materials such as alanine (AL) or, more recently, lithium formate monohydrate (LFM) are typically used. These materials offer high potential for applications in radiotherapy based on their favorable dosimetric properties. Nevertheless, EPR dosimetry is not widespread in the clinics. This work presents an uncertainty analysis of EPR dosimetry in the dose range from 1 to 70 Gy using a compact spectrometer and applying a practical procedure being suitable for routine use in radiotherapy. The performances of self-pressed LFM pellets and commercial AL pellets are compared side by side. All pellets had a diameter of 4 mm and a height of 2 mm (AL) or 4 mm (LFM). The mean pellet mass was 35.81 mg and 73.81 mg for AL and LFM, respectively. Before irradiation, the pellets were stored for at least 8 weeks at 34 ± 2% relative humidity. For irradiation, the pellets were put inside an airtight capsule. In total, 25 pellets per material were examined. The pellets were irradiated at a temperature of 25 ± 2.5 (2σ) °C to doses of either 1, 5, 20, 50 or 70 Gy (five pellets per dose value and material) by a clinical 6 MV photon beam. Measurement uncertainties were obtained from five independent readouts per pellet within five weeks following irradiation using a benchtop EPR spectrometer. The measurement time of a single readout was restricted to 10 min per pellet. Dose values were derived from EPR signal amplitudes using a specifically developed spectral fitting procedure. Signal fading characteristics were analyzed and taken into account during evaluation. The relative dose uncertainties (1σ) for a single readout at doses ≥ 5 Gy are below 2.8% (AL) and 1.1% (LFM) but increase to 12.3% (AL) and 2.6% (LFM) at 1 Gy. By averaging five independent readouts, the uncertainties at 1 Gy decrease to 2.6% (AL) and 0.8% (LFM). In terms of dose uncertainty, the LFM pellets are superior to the commercial AL pellets owing to their narrower EPR spectrum and approximately doubled mass resulting in higher EPR signal intensities. In case of the LFM pellets, the EPR dosimetry system shows a high level of precision (< 3%) down to 1 Gy being preferable for applications in radiotherapy. The uncertainties can be further decreased by averaging multiple dose values from independent readouts.

c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia.

BACKGROUND: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels. METHODS AND RESULTS: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter. CONCLUSIONS: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability.

The proteolytic activity of separase in BCR-ABL-positive cells is increased by imatinib.

Separase, an endopeptidase required for the separation of sister-chromatides in mitotic anaphase, triggers centriole disengagement during centrosome duplication. In cancer, separase is frequently overexpressed, pointing to a functional role as an aneuploidy promoter associated with centrosomal amplification and genomic instability. Recently, we have shown that centrosomal amplification and subsequent chromosomal aberrations are a hallmark of chronic myeloid leukemia (CML), increasing from chronic phase (CP) toward blast crisis (BC). Moreover, a functional linkage of p210BCR-ABL tyrosine kinase activity with centrosomal amplification and clonal evolution has been established in long-term cell culture experiments. Unexpectedly, therapeutic doses of imatinib (IM) did not counteract; instead induced similar centrosomal alterations in vitro. We investigated the influence of IM and p210BCR-ABL on Separase as a potential driver of centrosomal amplification in CML. Short-term cell cultures of p210BCR-ABL-negative (NHDF, UROtsa, HL-60, U937), positive (K562, LAMA-84) and inducible (U937p210BCR-ABL/c6 (Tet-ON)) human cell lines were treated with therapeutic doses of IM and analyzed by qRT-PCR, Western blot analysis and quantitative Separase activity assays. Decreased Separase protein levels were observed in all cells treated with IM in a dose dependent manner. Accordingly, in all p210BCR-ABL-negative cell lines, decreased proteolytic activity of Separase was found. In contrast, p210BCR-ABL-positive cells showed increased Separase proteolytic activity. This activation of Separase was consistent with changes in the expression levels of Separase regulators (Separase phosphorylation at serine residue 1126, Securin, CyclinB1 and PP2A). Our data suggest that regulation of Separase in IM-treated BCR-ABL-positive cells occurs on both the protein expression and the proteolytic activity levels. Activation of Separase proteolytic activity exclusively in p210BCR-ABL-positive cells during IM treatment may act as a driving force for centrosomal amplification, contributing to genomic instability, clonal evolution and resistance in CML.