The impact of simulated and real microgravity on bone cells and mesenchymal stem cells.

How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering.

Mechanisms of apoptosis in irradiated and sunitinib-treated follicular thyroid cancer cells.

The multikinase inhibitor sunitinib (S) seems to have promising potential in the treatment of thyroid cancer. We focused on the impact of S and/or irradiation (R) on mechanisms of apoptosis in follicular thyroid cancer cells. The effects of R, S and their combination were evaluated 2 and 4 days after treatment, using the human thyroid cancer cell line CGTH W-1. The transcription of genes involved in the regulation of apoptosis was investigated using quantitative real-time PCR. Western blot analyses of caspases and survivin were also performed. S elevated BAX (day 4), CASP9, CASP3, BIRC5 (day 4) and PRKACA (day 4) gene expression, whereas the mRNAs of BCL2, CASP8, PRKCA, ERK1, and ERK2 were not significantly changed. S, R and R+S clearly induced caspase-9 protein and elevated caspase-3 activity. Survivin was down-regulated at day 4 in control cells and the expression was blunted by S treatment. R+S induced survivin expression at day 2 followed by a reduction at day 4 of treatment. Sunitinib and the combined application with radiation induced apoptosis in follicular thyroid cancer cells via the intrinsic pathway of apoptosis. In addition, sunitinib might induce apoptosis via decreased expression of the anti-apoptotic protein survivin. These findings suggest the potential use of sunitinib for the treatment of poorly differentiated follicular thyroid carcinomas.

Impact of sunitinib on human thyroid cancer cells.

BACKGROUND/AIMS: Thyroid cancer accounts for about 1% of all cancer cases. Multikinase inhibitors like sunitinib (S) have a promising potential in thyroid cancer therapy. Therefore, the principal aim of this study was to investigate the impact of sunitinib on the secretion of cytokines of follicular thyroid cancer cells. METHOD: The effects of irradiation (R), S, and their combination (R+S) on cytokine secretion by the human thyroid cancer cell lines ML-1 and CGTH W-1 were evaluated after two (d2) and four days (d4) of treatment. RESULTS: Multi-Analyte Profiling of cytokine release showed a decrease after S treatment (CGTH W-1: IFN-γ, IL-4, IL-8 d2, MIP-1a, MMP-2, TNF-α and TNF-ß; ML-1: IFN-γ, IL-4, IL-6, IL-7, IL-8; MIP-1α, MMP-2, MCP-1, TNF-α and TNF-ß). R elevated significantly the release of cytokines (exception ML-1: MCP-1, MMP-2; CGTH W-1: IL-4, TNF-ß). In contrast, R+S treatment resulted in a reduction of IFN-γ, IL-4, and MMP-2 in both cell lines. IL-6, IL-8 and MCP-1 proteins in the supernatant correlated with the data obtained by quantitative RT-PCR. VEGFD mRNAs were significantly elevated by R+S. CONCLUSION: A target-based therapy with R+S changed VEGFD, IL-6 and IL-8 in follicular thyroid cancer cells. These in vitro-experiments suggest IL-6, IL-8, VEGFD and TNF-α as interesting biomarkers to be investigated in vivo. Different reactions of the cell lines under equal treatment might be due to their different origin and characteristics.

Interaction of proteins identified in human thyroid cells.

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains.

Gravity-sensitive signaling drives 3-dimensional formation of multicellular thyroid cancer spheroids.

This study focused on the effects induced by a random positioning machine (RPM) on FTC-133 thyroid cancer cells and evaluated signaling elements involved in 3-dimensional multicellular tumor spheroid (MCTS) formation. The cells were cultured on the RPM, a device developed to simulate microgravity, and under static 1-g conditions. After 24 h on the RPM, MCTSs swimming in culture supernatants were found, in addition to growth of adherent (AD) cells. Cells grown on the RPM showed higher levels of NF-κB p65 protein and apoptosis than 1-g controls, a result also found earlier in endothelial cells. Employing microarray analysis, we found 487 significantly regulated transcripts belonging not only to the apoptosis pathway but also to other biological processes. Selected transcripts were analyzed with quantitative real-time PCR using the same samples. Compared with 1-g IL-6, IL-8, CD44, and OPN were significantly up-regulated in AD cells but not in MCTSs, while ERK1/2, CAV2, TLN1, and CTGF were significantly down-regulated in AD cells. Simultaneously, the expression of ERK2, IL-6, CAV2, TLN1, and CTGF was reduced in MCTSs. IL-6 protein expression and secretion mirrored its gene expression. Thus, we concluded that the signaling elements IL-6, IL-8, OPN, TLN1, and CTGF are involved with NF-κB p65 in RPM-dependent thyroid carcinoma cell spheroid formation.

Cell separation by countercurrent centrifugal elutriation: recent developments.

Countercurrent centrifugal elutriation (CCE) is a cell separation technique that separates particles predominantly according to their size, and to some degree according to their specific density, without a need for antibodies or ligands tagging cell surfaces. The principles of this technique have been known for half a century. Still, numerous recent publications confirmed that CCE is a valuable supplement to current cell separation technology. It is mainly applied when homogeneous populations of cells, which mirror an in vivo situation, are required for answering scientific questions or for clinical transplantation, while antibodies or ligands suitable for cell isolation are not available. Currently, new technical developments are expanding its application toward fractionation of healthy and malignant tissue cells and the preparation of dendritic cells for immunotherapy.

Short-term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells.

This study focused on the effects of short-term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements. ECs were investigated during four German Space Agency (Deutsches Zentrum für Luft- und Raumfahrt) parabolic flight campaigns. Hoechst 33342 and acridine orange/ethidium bromide staining showed no signs of cell death in ECs after 31 parabolas (P31). Gene array analysis revealed 320 significantly regulated genes after the first parabola (P1) and P31. COL4A5, COL8A1, ITGA6, ITGA10, and ITGB3 mRNAs were down-regulated after P1. EDN1 and TNFRSF12A mRNAs were up-regulated. ADAM19, CARD8, CD40, GSN, PRKCA (all down-regulated after P1), and PRKAA1 (AMPKα1) mRNAs (up-regulated) provide a very early protective mechanism of cell survival induced by 22 s microgravity. The ABL2 gene was significantly up-regulated after P1 and P31, TUBB was slightly induced, but ACTA2 and VIM mRNAs were not changed. ß-Tubulin immunofluorescence revealed a cytoplasmic rearrangement. Vibration had no effect. Hypergravity reduced CARD8, NOS3, VASH1, SERPINH1 (all P1), CAV2, ADAM19, TNFRSF12A, CD40, and ITGA6 (P31) mRNAs. These data suggest that microgravity alters the gene expression patterns and the cytoskeleton of ECs very early. Several gravisensitive signaling elements, such as AMPKα1 and integrins, are involved in the reaction of ECs to altered gravity.

18F-FET-PET-based dose painting by numbers with protons.

PURPOSE: To investigate the potential of (18)F-fluoroethyltyrosine-positron emission tomography-((18)F-FET-PET-)based dose painting by numbers with protons. MATERIAL AND METHODS: Due to its high specificity to brain tumor cells, FET has a high potential to serve as a target for dose painting by numbers. Biological image-based dose painting might lead to an inhomogeneous dose prescription. For precise treatment planning of such a prescribed dose, an intensity-modulated radiotherapy (IMRT) algorithm including a Monte Carlo dose-calculation algorithm for spot-scanning protons was used. A linear tracer uptake to dose model was used to derive a dose prescription from the (18)F-FET-PET. As a first investigation, a modified modulation transfer function (MTF) of protons was evaluated and compared to the MTF of photons. In a clinically adapted planning study, the feasibility of (18)F-FET-PET-based dose painting with protons was demonstrated using three patients with glioblastome multiforme. The resulting dose distributions were evaluated by means of dose-difference and dose-volume histograms and compared to IMRT data. RESULTS: The MTF for protons was constantly above that for photons. The standard deviations of the dose differences between the prescribed and the optimized dose were smaller in case of protons compared to photons. Furthermore, the escalation study showed that the doses within the subvolumes identified by biological imaging techniques could be escalated remarkably while the dose within the organs at risk was kept at a constant level. CONCLUSION: The presented investigation fortifies the feasibility of (18)F-FET-PET-based dose painting with protons.

Changes of apoptosis, p53, and bcl-2 by irradiation in poorly differentiated thyroid carcinoma cell lines: a prognostic marker for the prospect of therapeutic success?

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) has an unfavorable prognosis. Surgical management is the principal treatment approach. In addition, radioiodine treatment and external beam radiotherapy (EBRT) are given to reduce the risk of local relapse. Despite aggressive therapy, the response to treatment tends to become increasingly poorer over time. The objective of this study was to investigate the induction of apoptosis by EBRT as a function of p53 and bcl-2 protein levels in PDTC. The predictive value of these molecules with respect to treatment efficacy was evaluated. MATERIALS AND METHODS: Two different cell lines of PDTC (FTC-133 and ML-1) were irradiated with a dose of 30 Gy. Apoptotic cells were quantified using terminal deoxynucleotidyltransferase-dUTP nick-end labeling staining without irradiation, 48 and 96 hours after irradiation. The protein levels of p53 and bcl-2 were measured simultaneously using flow cytometry and western blotting. The cell cycle distribution was determined. RESULTS: Untreated FTC-133 cells showed a high rate of apoptosis, a high protein level of p53, and a low bcl-2 protein level. Forty-eight hours after irradiation, a slight reduction in apoptotic cells was observed in conjunction with an increase in bcl-2 and p53 protein levels. The slightly reduced fraction of apoptotic cells remained at the same level up to 96 hours after irradiation, whereas the p53 protein level was further downregulated. The cell cycle distribution showed a significant G2/M arrest after 48 hours and recovery 96 hours after irradiation. ML-1 cells did not show any detectable p53 levels and revealed a low rate of apoptosis which significantly increased 48 hours after irradiation. Ninety-six hours after irradiation, a decrease in apoptosis was detectable. The protein level of bcl-2 increased significantly within 48 hours and decreased 96 hours after irradiation. The cell cycle distribution showed a G2/M arrest after 48 hours without a recovery 96 hours after irradiation. CONCLUSIONS: The p53 and bcl-2 expression profiles and the observed apoptotic rates of FTC-133 and ML-1 under irradiation are consistent with a more aggressive FTC-133 phenotype. Alterations in p53- and bcl-2 protein levels yield predictive information for EBRT efficacy.

Staged surgery with neoadjuvant 90Y-DOTATOC therapy for down-sizing synchronous bilobular hepatic metastases from a neuroendocrine pancreatic tumor.

PURPOSE: Treatment with DOTA-d-Phe(1)-Tyr(3)-octreotide (DOTATOC), labeled with beta-emitting radioisotope yttrium-90 ((90)Y-DOTATOC), has successfully been used for the palliative treatment of patients with advanced somatostatin receptor-expressing neuroendocrine tumors (NETs). However, controversy persists as to whether patients with metastatic NETs of the pancreas should undergo radical (salvage) surgery or receive palliative therapy. We proposed that (90)Y-DOTATOC could be used in a neoadjuvant intention for improving therapy of hepatic NET metastases. MATERIALS AND METHODS: We investigated a novel therapy concept in a 49-year-old patient presenting with a neuroendocrine tumor of the pancreatic tail and synchronous multiple bilobular hepatic metastases. After surgical removal of the large primary tumor by extended left en bloc resection of the pancreatic tail, the patient received neoadjuvant (90)Y-DOTATOC for therapy of primarily non-resectable bilobular hepatic metastases. RESULTS: The (90)Y-DOTATOC therapy resulted in an impressive regression of hepatic lesions, thus facilitating surgical removal of all remaining liver metastases in a second operation (staged surgery). In addition, one lesion was ablated using radiofrequency ablation (RFA). At 1-year of follow-up after hepatic R0 resection/RFA, there was no evidence of tumor recurrence or extrahepatic metastasis. CONCLUSIONS: The neoadjuvant use of (90)Y-DOTATOC therapy could prove valuable for treatment of advanced pancreatic NETs metastatic to the liver in terms of facilitating R0 resection by applying staged surgery concepts.

Radiolabeled annexin V for imaging apoptosis in radiated human follicular thyroid carcinomas--is an individualized protocol necessary?

INTRODUCTION: Induction of apoptosis is a widely used strategy for cancer therapy, but evaluating the degree and success of this therapy still poses a problem. Radiolabeled annexin V has been proposed to be a promising candidate for detecting apoptotic cells in tumors following chemotherapy in vivo. In order to see whether radiolabeled annexin V could be a suitable substance for the noninvasive in vivo detection of apoptosis in thyroid tissue and to establish an optimized study protocol, we investigated two poorly differentiated thyroid carcinoma cell lines: ML-1 and FTC-133. METHODS: Apoptosis was evaluated before as well as 2 and 4 days after in vitro irradiation with 30 Gy X-rays. In this study, binding of FITC- and of (125)I-labeled annexin V was measured in comparison to other apoptosis markers such as Bax, caspase-3 and Fas, which were determined by flow cytometry and Western blot analysis with densitometric evaluation. RESULTS: ML-1 and FTC-133 cells showed a significant increase in annexin V binding 48 h after irradiation. Ninety-six hours after irradiation, the annexin V absorption capability of ML-1 cells was still maximal, while the living fraction of FTC-133 increased significantly. The amount of caspase-3 and Bax was clearly increased 48 h after irradiation and had normalized after 96 h in both cell lines. Fas protein concentrations remained unchanged in ML-1 cells but were significantly enhanced in FTC-133 cells. CONCLUSION: The binding of FITC- and (125)I-labeled annexin V showed a significant accordance. A reliable evaluation of apoptosis induced by radiotherapy in thyroid tumors was possible 48 h after irradiation, when binding of radiolabeled annexin V is most significantly enhanced. Using two poorly differentiated cell lines of thyroid carcinoma, one may expect to find a nearly similar response to external irradiation. In contrast, the cell lines showed a completely contrary response. However, an individualized study protocol for each type of tumor and probably within each type is necessary.

A biologically adapted dose-escalation approach, demonstrated for 18F-FET-PET in brain tumors.

PURPOSE: To demonstrate the feasibility of a biologically adapted dose-escalation approach to brain tumors. MATERIAL AND METHODS: Due to the specific accumulation of fluoroethyltyrosine (FET) in brain tumors, (18)F-FET-PET imaging is used to derive a voxel-by-voxel dose distribution. Although the kinetics of (18)F-FET are not completely understood, the authors regard regions with high tracer uptake as vital and aggressive tumor and use a linear dose-escalation function between SUV (standard uptake value) 3 and SUV 5. The resulting dose distribution is then planned using the inverse Monte Carlo treatment- planning system IKO. In a theoretical study, the dose range is clinically adapted from 1.8 Gy to 2.68 Gy per fraction (with a total of 30 fractions). In a second study, the maximum dose of the model is increased step by step from 2.5 Gy to 3.4 Gy to investigate whether a significant dose escalation to tracer-accumulating subvolumes is possible without affecting the shell-shaped organ at risk (OAR). For all dose-escalation levels the dose difference Delta D of each voxel inside the target volume is calculated and the mean dose difference Delta D and their standard deviation sigma Delta D are determined. The dose to the OAR is evaluated by the dose values D OAR 50% and D OAR 5%, which are the dose values not exceeded by 50% and 5% of the volume, respectively. RESULTS: The inhomogeneous dose prescription is achieved with high accuracy (Delta D < 0.03 +/- 0.3 Gy/fraction). The maximum dose can be increased remarkably, without increasing the dose to the OAR (standard deviation of D OAR 50% < 0.02 Gy/fraction and of D OAR 5% < 0.05 Gy/fraction). CONCLUSION: Assuming that regions with high tracer uptake can be interpreted as target for radiotherapy, (18)F-FET-PET-based "dose painting by numbers" applied to brain tumors is a feasible approach. The dose, and therefore potentially the chance of tumor control, can be enhanced. The proposed model can easily be transferred to other tracers and tumor entities.

Significance of scintigraphy for the localisation of obscure gastrointestinal bleedings.

AIM: To determine the role of scintigraphy in patients with gastrointestinal (GI) bleeding of unknown localisation. METHODS: We performed retrospective analyses on 92 patients receiving scintigraphies from 1993 to 2000 in the University of Regensburg hospital, which were done for localisation of GI bleeding as a diagnostic step after an unsuccessful endoscopy. In addition to the scintigraphies, further diagnostic steps such as endoscopy, angiography or operations were performed. In some of the scintigraphies with negative results, a provocation test for bleeding with heparinisation was carried out. RESULTS: 73% of all scintigraphies showed a positive result. In 4.5% of the positive results, the source was located in the stomach, in 37% the source was the small bowel, in 25% the source was the right colon, in 4.5% the source was the left colon, and in 20% no clear localisation was possible. Only 4% of all scintigraphies were false positive. A reliable positive scintigraphy was independent of the age of the examined patient. A provocation test for bleeding with heparin resulted in an additional 46% of positive scintigraphies with a reliable localisation in primary negative scintigraphies. CONCLUSION: Our results show that scintigraphy and scintigraphy with heparin provocation tests are reliable procedures. They enable a reliable localisation in about half of the obscure GI-bleeding cases. Scintigraphy is superior to angiography in locating a bleeding. It is shown that even in the age of video capsule endoscopy and double-balloon enteroscopy, scintigraphy provides a reliable and directed localization of GI bleeding and offers carefully targeted guidance for other procedures.

Innovative strategies in in vivo apoptosis imaging.

Apoptosis (programmed cell death) plays a key role in the pathogenesis of many disorders including cerebral and myocardial ischemia, autoimmune and neurodegenerative diseases, infections, organ and bone marrow transplant rejection, and tumor response to chemotherapy and/or radiotherapy. Apoptosis in itself represents a complex mechanism where numerous (pro-apoptotic and anti-apoptotic) molecules interact in an elaborate manner. Since the original description by Kerr et al. in 1972, clinical assessment of apoptosis has always required biopsies or aspirated material for in vitro investigations. Several well-established methods are available for in vitro tests using tissue specimens. However, a non-invasive detection of apoptosis would be of great benefit for many patients in various situations. Today, non-invasive techniques for direct in vivo detection of apoptotic cells are rare and urgently need improvement. The early in vivo detection of apoptotic cells can provide the physician with important information to develop further therapeutic strategies in chemotherapy or radiotherapy of tumors, in transplantation of organs, or in healing of infarct areas. In some preliminary publications, several authors reported on the in vivo use of caspase-inhibitors and annexin V, labeled with indium-111, technetium-99m, iodine-123, iodine-124 or fluoride-18. In the present paper, we review the current applicability of both techniques for in vivo apoptosis imaging, and discuss the methodical problems.

Clinical value of planar and tomographic dual-isotope scintigraphy using 99mTc-methylene diphosphonate and 131I in patients with thyroid cancer.

PURPOSE: (131)I whole-body scintigraphy is a highly sensitive method for the detection of differentiated thyroid tumours and metastases. However, a lack of anatomical landmarks and the physiological excretion of the tracer complicates the evaluation of the images. Therefore, we determined whether additional bone scintigraphy in combination with (131)I scintigraphy, simultaneously acquired via planar and tomographic techniques, positively contributes to the treatment plan in patients with non-conclusive (131)I images. METHODS: Twenty-one patients with differentiated thyroid cancer and known metastases or unclear findings in the (131)I whole-body scan underwent dual-isotope scintigraphy (DIS) within 2-7 days after application of 5000-8000 MBq (131)I. Dual-energy planar and tomographic data were acquired simultaneously and the results compared with other imaging modalities. RESULTS: In 48% of the cases (10 of 21), DIS supplied important additional information that either altered the treatment plan or staging of the patients. In 28% (six of 21), DIS provided new information that was not known before, but did not change the staging of the patients. In five cases (24%), DIS did not add any new data regarding the extent of the disease. CONCLUSIONS: The simultaneous acquisition of (131)I and (99m)Tc-methylene diphosphonate provides clear landmarks and facilitates the localization of functioning metastases from differentiated thyroid cancer as well as improves the fusion with morphological images. It can be performed easily and also transferred to other isotope combinations.

Retinoid- and sodium-butyrate-induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology.

Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.

Evaluation of the clinical value of bone metabolic parameters for the screening of osseous metastases compared to bone scintigraphy.

BACKGROUND: Bone metastases are common in many types of cancer. As screening methods different imaging modalities are available. A new approach for the screening of osseous metastases represents the measurement of bone metabolic markers. Therefore aim of this study was to evaluate the usefulness of the determination of bone metabolic markers aminoterminal propeptide of type I procollagen (PINP, osteoblastic activity) and the carboxyterminal pyridinoline cross-linked telopeptide of type I collagen (ICTP, osteoclastic activity) for the detection of bone metastases associated with other malignancies. METHODS: 88 patients aged 21 - 82 years with malignant tumors were prospectively studied. The serum concentrations of PINP and ICTP were measured and compared to the results of bone scintigraphy, radiological bone series, CT, MRI and clinical follow-up. RESULTS: Osseous metastases were found in 21 patients. 19 of them were correctly identified by bone scintigraphy (sensitivity: 90%). For bone metabolic markers results were as follows: ICTP sensitivity: 71%, specificity: 42%; PINP sensitivity: 24%, specificity: 96%. CONCLUSIONS: As markers of bone metabolism PINP and ICTP showed low sensitivity and/or specificity for the detection of osseous metastases. The presented markers did not seem to be sufficient enough to identify patients with bone metastases or to replace established screening methods.

Effects of PTK787/ZK222584, a tyrosine kinase inhibitor, on the growth of a poorly differentiated thyroid carcinoma: an animal study.

The process of neoangiogenesis is induced by several mediators. Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis including thyroid carcinomas. The principal aim of this study was to test the hypothesis that inhibition of VEGF activity by PTK787/ZK222584 (PTK/ZK), a specific blocker of both VEGF-receptor tyrosine kinases, could inhibit the growth of a poorly differentiated thyroid cancer. Human follicular thyroid tumor xenografts were implanted sc into nude mice. Eight days following implantation, the animals were randomized into two groups (n = 10 each group). One group received PTK/ZK daily, and the other was treated with sodium chloride (control). Treatment was orally administered using a gastric tube. All animals were killed after 4 wk. Tumors, blood, and samples of other organs were taken for further examinations. Treatment with PTK/ZK induced a 41.4% reduction in tumor volumes. Necrosis of the tumors was detectable earlier in PTK/ZK-treated mice compared with controls. Immunohistochemistry revealed a significant decrease in neoangiogenesis in tumors of PTK/ZK-treated animals. Moreover, no compensatory overexpression of VEGF protein was detectable in the treated group. The compound was well tolerated by the animals without significant side effects on body weight or in general. These results showed that VEGF receptor blockade is a rational approach to the therapy of thyroid cancer. The combination of radioiodine or external radiation with VEGF receptor tyrosine kinase inhibitors might be a new option, especially for poorly differentiated thyroid cancers with limited or no response to conventional therapy.

Heat shock protein 70 (Hsp70) membrane expression on head-and-neck cancer biopsy-a target for natural killer (NK) cells.

PURPOSE: Heat shock protein 70 (Hsp70) was detected on the cell membrane of human tumor cell lines, but not on normal cells. Here we studied Hsp70 membrane expression as a target for natural killer (NK) cells on tumor material and control tissues of head-and-neck cancer patients. METHODS AND MATERIALS: Membrane-bound Hsp70 was determined by flow cytometry on single-cell suspensions of tumors and the corresponding normal tissues of head-and-neck cancer patients. The cytolytic activity of NK cells against Hsp70-positive tumor cells was measured in a standard cytotoxicity assay. RESULTS: In total, 54 of 74 primary tumors were found to be Hsp70 membrane-positive (73%); tongue/mouth, 21 of 24 (88%); oropharynx, 13 of 20 (65%); hypopharynx, 3 of 6 (50%); larynx, 8 of 11 (73%); trachea 1 of 2 (50%); esophagus, 4 of 5 (80%); lymph node metastases, 4 of 6 (67%). The corresponding control tissue was negative for membrane-bound Hsp70. Biopsies (6 of 6) of patients after in vivo gamma-irradiation (fractionated 5 x 2 Gy) were strongly Hsp70 membrane-positive. Irradiated, Hsp70-positive tumor cells are targets for Hsp70-peptide stimulated NK cells. CONCLUSION: An irradiation-inducible, tumor-selective Hsp70 membrane localization provides a target structure for Hsp70-peptide stimulated human NK cells.