[Metabolit-induced bioluminescence for tumor prediction: detection of metabolites in tumors of the head and neck region]. / Metabolitinduzierte Biolumineszenz zur Tumorprädiktion: Detektion von Metaboliten in Tumoren des Kopf-Hals-Bereiches.

Solid tumors show an altered metabolism with respect to glycolysis in comparison to normal tissue. Recently, the determination of different glycolytic metabolites for tumor diagnosis and therapeutic decision-making became the focus of interest for various research groups. In particular an increased lactate concentration in tumor tissue appears to be a predictor of an adverse prognosis. Imaging of induced bioluminescence in rapidly frozen tumor biopsies is an established technique for the detection of selected substances. In this method the metabolites of interest are biochemically linked to luciferases. A microscopic photon counting system registers the light intensity and after calibration reflects the concentration distribution of metabolites. In contrast to other methods it is possible to detect different metabolites from one specific area of tissue. Preliminary results of a pilot study on oral cancer patients suggest a prognostic impact in terms of high lactate concentrations being associated with poor survival. This technique could increase the validity and significance of tumor grading and might be supportive decision guidance for tumor therapy in the future.

Circulatory and metabolic responses of malignant tumors during localized hyperthermia.

The effect of localized hyperthermia on the circulatory responses and on the oxygen and glucose supply has been evaluated in tissue-isolated rat tumors utilizing an in situ perfusion system. On the average, localized hyperthermia caused a significant increase in total tumor blood flow after raising of the mean tumor temperature from 37 degrees C to 39.5 degrees C. At higher temperatures (42 degrees C) total tumor blood flow decreased to a level somewhat below the flow during normothermia. However, there were great interindividual differences in the response of blood flow to temperature. The changes in blood flow were paralleled by variations of the O2-consumption and of the glucose uptake of the tumor tissue. The alteration of the oxygen and glucose supply seem to be predominantly mediated through changes of tumor blood flow with temperature. Tumor microcirculation appears to be improved at moderate hyperthermic temperatures (39 degrees C -40 degrees C) and deteriorated at higher temperatures. Whereas a vasodilation of tumor vessels seems to be a paramount factor for flow improvements, a reduction of red blood cell flexibility due to severe tissue acidosis, multiple microthromboses as well as occlusions of microvessels should be taken into consideration as factors for flow impairments at higher tumor temperatures.

Tumor blood flow and O2 availability during hemodilution.

Experiments were performed to study the influence of localized and systemic hemodilution on tumor blood flow and on O2 availability in the tumor tissue. The results obtained clearly show that both localized and systemic hemodilution can distinctly improve nutritive blood flow through solid tumors. This can be utilized to enhance pharmacokinetics of antitumor drugs. Due to the improved metabolic status, the pharmacodynamics of some antitumor drugs should also be enhanced. To achieve a maximum improvement of the oxygen supply to the tumor, hematocrit values should not be decreased below 0.20 in localized hemodilution, and 0.30 in systemic hemodilution.

Impact of various thermal doses on the oxygenation and blood flow in malignant tumors upon localized hyperthermia.

Upon localized hyperthermia at modest thermal doses an increase in tumor blood flow can be observed in many tumors which is paralleled by an improvement of the oxygenation status of the tissue. At intermediate or high thermal doses a pronounced restriction of the tumor circulation becomes obvious leading to a deterioration of the tumor oxygenation. As a consequence, a further enhancement of the thermal response of tumors relative to normal tissues has to be expected at intermediate or high thermal doses.

Hemodilution in isolated tumor perfusion.

Experiments are performed to study the influence of local hemodilution on tumor blood flow, oxygen availability in tumor tissue and O2 consumption of cancer cells. The results obtained clearly show that hemodilution in isolated tumor perfusion can distinctly improve nutritive blood flow through solid tumors. This can be utilized to enhance pharmacokinetics of antitumor drugs. Due to the improved metabolic status, the pharmacodynamics of some antitumor drugs should also be enhanced. To achieve a maximum improvement of the O2 supply to the tumor, hematocrit values should not be decreased below 0.20.

Effect of hyperthermia on tumor blood flow.

Differences in blood perfusion rates between tumors and normal tissue can be utilized to selectively heat many solid tumors. Blood flow in normal tissues is considerably increased at temperatures commonly applied during localized hyperthermia. In contrast, tumor blood flow may respond to localized heat typically in two different blood flow patterns: Flow may either decrease continuously with increasing exposure time and/or temperature or flow may exhibit a transient increase followed by a decline. A decrease in blood flow at high thermal doses can be observed in most of the tumors, whereas an increase in flow at low thermal doses seems to occur less frequently. The inhibition of blood flow at high thermal doses may lead to physiological changes in the microenvironment of the cancer cells that increase the cell killing effect of hyperthermia. Flow increases at low thermal doses can enhance the efficiency of other treatment modalities, such as irradiation or the administration of antiproliferate drugs.

Cytostatic activity of aeroplysinin-1 against lymphoma and epithelioma cells.

(+/-)-Aeroplysinin-1, an optically active 1,2-dihydroarene-1,2-diol, was isolated from the marine sponges Verongia aerophoba (+-isomer) and Ianthella ardis (- -isomer). For the experiments presented we used the +-isomer from Verongia aerophoba. Here we describe the hitherto unknown biological and pharmacological property of this compound to display pronounced anticancer activity against L5178y mouse lymphoma cells (ED50: 0.5 microM). Friend erythroleukemia cells (ED50: 0.7 microM), human mamma carcinoma cells (ED50: 0.3 microM) and human colon carcinoma cells (ED50: 3.0 microM) in vitro. Furthermore, aeroplysinin caused a preferential inhibition of [3H]thymidine (dThd) incorporation rates in L5178y mouse lymphoma cells if compared with murine spleen lymphocytes in vitro. At concentrations between 1.1 and 28.5 microM, the [3H]dThd incorporation rates in L5178y cells were suppressed to 28%-0% but only to 78%-18% in murine spleen lymphocytes. The same differential effect in vitro was found with the following epithelial cells: 14.70 microM of the compound were required to inhibit normal human fibroblasts to 50%, but only 2.9 microM in the assays with human malign keratinocytes or malignant melanoma cells to observe the same inhibitory effect. Moreover, aeroplysinin-1 displayed antileukemic activity in vivo using the L5178y cell/NMRI mouse system; administered at a dose of 50 mg/kg for five consecutive days, the T/C (%) value was determined to be 338. Preliminary toxicology studies revealed an acute LD50 of 202 mg/kg and a subacute LD50 of 150 mg/kg. Aeroplysinin-1 is neither a direct mutagen nor a premutagen in the umu/Salmonella typhimurium test system.

On the cross-section structure of the mitochondrial cristae-membrane as revealed by x-ray diffraction.

Mitochondria were isolated using sorbitol and high buffer concentration in the medium. X-ray diffraction patterns arising from the mitochondrial cristae-membrane were recorded in the fully dried state and in two different states in humidity. The Q-function evaluation of these X-ray diffraction diagrams resulted in electron density cross-section profiles, which consist of two main peaks of opposite sign and one, respectively two, smaller peaks. The total thickness of the membrane amounts to 120 A in the dry and 140 A to 160 A in the wet state. An interpretation of the cross-section profile is tentatively proposed.

A pressure chamber for removing cryobiopsies from small laboratory animals.

A pressure chamber for small laboratory animals was designed that can be operated up to 4 bar. It enables the removal of tissue cryobiopsies at liquid nitrogen temperatures during pressurisation. Relevant systemic parameters of the animals investigated, such as mean arterial blood pressure or rectal temperature, can be monitored throughout the experiments. In addition, intravenous infusions of blood, blood substitutes or drugs can be administered. The main goal of constructing the chamber presented is the investigation of tumour oxygenation under hyperbaric conditions. This is performed by measuring the oxyhaemoglobin saturation (HbO2) of single red blood cells in tumour microvessels using a cryophotometric micromethod. In a preliminary series of experiments with rats bearing DS-carcinosarcoma it could be demonstrated that the present experimental set-up leads to HbO2 values in solid tumours which are in accordance with results obtained from previous investigations during exposure to normal air using a different sampling technique.

Respiratory gas exchange in the rat spleen in situ and intrasplenic oxyhemoglobin saturation.

Measurements of splenic respiratory gas exchange and of HbO2 saturations in the red pulp of the rat spleen have shown that there are no indications of a reduced intrasplenic O2 availability during normoxia. The present studies provide evidence that, in the normal spleen, the intrasplenic sequestration of red blood cells cannot be explained by an O2 deficiency in the red pulp since the commonly accepted notion of an intrasplenic hypoxia is not true.

Effect of CO2 and lactic acid on intracellular pH of ascites tumor cells.

The effect of CO2 and of lactic acid (L.A.) on the extracellular (pHe) and intracellular pH (pHi) of ascites tumor cells (DS-carcinosarcoma) in rats was studied by in vitro equilibration of ascites with CO2 and alteration of lactic acid concentration. pHi was determined by the distribution of DMO. The effects of lactic acid and CO2 on pH were additive and could be expressed as pHe = 8.872 - 0.745 logPCO2 - 0.0355 (L.A.) (R = 0.867, n = 201) pHi = 8.218 - 0.436 logPCO2 - 0.0275 (L.A.) (R = 0.812, n = 143) delta pHi/delta pHe was dependent on the way pHe was changed: If the change in pHe was due to lactic acid, delta pHi/delta pHe was 0.91; if it was due to CO2 delta pHi/delta pHe was 0.625. pHi exceeded pHe if either PCO2 and/or the concentration of lactic acid was raised above a critical level. The results render it questionable to predict intracellular pH values within solid tumor from pH measurements within the extracellular fluid.

Low light level in vitro monitoring of cellular and antigen-antibody reactions using a photon detection camera system--new perspectives for clinical diagnosis and research.

This article briefly describes the use of a photon counting system (ARGUS-100) in the detection of low levels of light. The ARGUS-100 was used in determining ATP in cell sections from tumor tissues and in measuring a luminescence-enhanced immunoluminometric assay, using ferritin as the analyte, based on the luminol-peroxide-4-iodophenol reaction with peroxidase as the enzyme. The aim is not so much the presentation of data, but rather to show the potentials of the photon counting camera in increasing our knowledge of the cellular and subcellular levels, as well as lowering the detection limits in already sensitive systems, such as immunoassays.

Terminally differentiated postmitotic tumor cells in a rat rhabdomyosarcoma cell line.

A permanent rat rhabdomyosarcoma cell line (BA-HAN-1C) has been established, the phenotype of which is characterized by the coexistence of undifferentiated mononuclear cells and differentiated multinuclear myotube-like giant cells. The failure of attempts to separate these two cell types by repeated recloning procedures indicates their close histogenetic relationship and suggests that differentiation in this tumor proceeds in a similar manner to that in normal striated muscle where postmitotic myotubes arise from mononuclear myoblasts by fusion. The morphologically undifferentiated mononuclear tumor cells were shown to be actively proliferating and to incorporate thymidine methyl-3H(3H-TdR). The myotube-like giant cells neither incorporated 3H-TdR nor underwent mitosis or exhibited any clonogenic potential. After retransplantation into syngenic rats, tumor growth was markedly retarded when the tumor cell inoculum contained a high percentage of myotube-like giant cells. These data show that proliferative activity in this rhabdomyosarcoma cell line is confined to the mononuclear tumor cell compartment, the multinuclear myotube-like giant cells having withdrawn from the cell cycle and represent terminally differentiated postmitotic cells. This cell line should provide a valuable tool for further investigation of coherent aspects of proliferation and differentiation using various differentiation inducers.

The establishment of two rat colonic carcinomas in tissue culture. A basic prerequisite for standardized experiments on the biology of colonic carcinomas in vivo.

Two rat colonic carcinomas (DMH-Co-1 and DMH-Co-2) derived from dimethyl-hydrazine-induced metastasizing adenocarcinomas were established as permanent cell lines. By means of electron microscopy, immunofluorescence microscopy and biochemical analysis of cytoskeletal components, it has been shown that both tumor cell lines retain in vitro the phenotypic characteristics of the primary tumors. The in vitro growth properties revealed only minor differences between the two cell lines. After retransplantation in vivo, DMH-Co-2 gave rise to moderately differentiated adenocarcinomas, whereas the tumors arising from DMH-Co-1 exhibited a continuum of differentiation encompassing adenocarcinomas, undifferentiated carcinomas and squamous cell carcinomas. These permanent cell lines offer the opportunity for isolating divergent subpopulations by in vitro cloning and facilitate standardized experiments on their biological behaviour in vivo.

Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions.

Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb+ is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca2+ or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in cellular ATP levels evoked by low toxin doses correlated temporally with a fall in oxygen consumption, which was interpreted to reflect breakdown of mitochondrial respiration. The lethal event could not be thwarted by manipulating the extracellular K+ or Ca2+ concentrations. Realization that alpha-toxin may form very small pores in nucleated cells is important for future research on cellular toxin effects and membrane repair processes.

Inhibition of intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex from human breast cancer cells by the marine sponge metabolite (+)-aeroplysinin-1.

1. (+)-Aeroplysinin-1, a naturally occurring tyrosine metabolite from the marine sponge Verongia aerophoba, was found to inhibit the phosphorylation of lipocortin-like proteins by a highly purified preparation of the epidermal growth factor (EGF) receptor-tyrosine protein kinase complex from MCF-7 breast carcinoma cells. 2. (+)-Aeroplysinin-1 blocked the EGF-dependent proliferation of both MCF-7 and ZR-75-1 human breast cancer cells and inhibited the ligand-induced endocytosis of the EGF receptor in vitro. 3. Treatment with aeroplysinin-1 in the concentration range at 0.25-0.5 microM resulted in a time- and dose-dependent total tumor cell death in vitro. 4. At a 10-fold higher concentration the compound did not reveal any cytostatic activity in normal human fibroblasts. 5. From these data we conclude that (+)-aeroplysinin-1 represents a compound which displays a strong anti-tumor effect on EGF-dependent tumor cell lines.