Lisa D. Sprague

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines.

BackgroundSquamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu.MethodsHIF-1α immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2 – 4 h) of hypoxic exposure (< 0.66 % O2), reoxygenation (24 h, 20 % O2), and radiation (0, 2, 5 and 10 Gy).ResultsHIF-1α expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells.ConclusionOur data suggest that both, short-term (~4 – 8 h) and long-term (~20 – 24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas reoxygenation of hypoxic tumour cells during fractionated radiotherapy could counteract the increased PAI-1 levels.

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei.

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin‐embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.

Effect of Reoxygenation on the Hypoxia-Induced Up-Regulation of Serine Protease Inhibitor PAI-1 in Head and Neck Cancer Cells

In squamous cell carcinoma of the head and neck (SCCHN), hypoxia is considered a crucial physiological modulator for malignant progression, wherebythe plasminogen activation system is involved in overlapping functions such as moulding of the extracellular matrix, cell proliferation and signal transduction. Little is known about the effects of reoxygenation on the plasminogen activation system in SCCHN cells. Three human SCCHN cell lines (BHY, CAL27, FaDu) and a non-transformed human fibroblast cell line (VH7) were exposed to hypoxic (<0.5% O2) conditions for up to 72 h and subsequently reoxygenated at normoxic conditions for 24 h. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein concentration and former protein activity were determined by ELISA and complex ELISA, respectively. Reoxygenation induced significant changes in cell-associated and secreted PAI-1 protein compared to the normoxic control. Significant increase in cell-associated and secreted uPA protein after reoxygenation was only observed for some of the cell lines. Determination of uPA-PAI-1 complex formation revealed the release of active protein into the cell supernatant. The beneficial role of reoxygenation during radiation therapy is widely accepted. However, reoxygenation does not seem to counteract the effects induced by hypoxia on the plasminogen activation system. Fatally irradiated reoxygenat- ed tumour cells might still produce sufficient amounts of ‘harmful’ protein and thus initiate a path for invasion and metastasis for surviving tumour cells.

Definition of Target Volume and Organs at Risk. Biological Target Volume

Target volume definition is an interactive process. Based on radiological (and biological) imaging, the radiation oncologist has to outline the GTV, CTV, ITV, and PTV and BTV. In this process, a lot of medical and technological aspects have to be considered. The criteria for GTV, CTV, etc. definition are often not exactly standardised, and this leads, in many cases to variability between clinicians; however, exactly defined imaging criteria, imaging with high sensitivity and specificity for tumour tissue and special training could lead to a higher consensus in target volume delineation and, consequently, to lower differences between clinicians. It must be emphasised, however, that further verification studies and cost-benefit analyses are needed before biological target definition can become a stably integrated part of target volume definition.