The adhesion forces of cervical carcinoma cells in tissue culture were measured by using the manipulation force microscope, a novel atomic force microscope. The forces were studied as a function of time and temperature for cells cultured on hydrophilic and hydrophobic polystyrene substrates with preadsorbed proteins. The cells attached faster and stronger at 37°C than at 23°C and better on hydrophilic than on hydrophobic substrates, even though proteins adsorb much better to the hydrophobic substrates. Because cell adhesion serves to control several stages in the cell cycle, we anticipate that the manipulation force microscope can help clarify some cell-adhesion related issues.Many of the activities of mammalian cells in vivo, such as embryogenesis, mitosis, morphogenesis, cell orientation, cell motility, and survival depend on attachment to neighboring cells and the extracellular matrix (1-5). Attachment is often mediated by integrins, transmembrane glycoproteins that bind to ligands such as collagens, laminin, (LAM) or fibronectin (FN) (1, 6). Cell-substrate adhesion involves a cascade of events leading to integrin activation and strong adhesion (6), whereas a force applied to an integrin receptor induces a local strengthening response of the integrin-cytoskeleton linkages (7). Cell attachment is an active process and therefore is temperature-dependent. Umbreit and Roseman (8) concluded from studies of cell-aggregation kinetics that cell-cell attachment involved two steps, an initial recognition event and an active attachment process expending metabolic energy.In vitro, most mammalian cells are anchorage-dependent and attach firmly to the substrate. Several attempts have been made to quantify cell adhesion. The simplest attachment assay is based on rinsing the surface to remove weakly attached cells from the substratum and counting the remaining cells (9-13). Cells adhering with a force less than the rinsing force are removed, but this force is difficult to control and therefore ill-defined.Quantitative force measurements have been made using centrifugal assays (14, 15). Here, the discrimination force is better defined, and a probability distribution is obtained by counting the number of remaining cells for a given series of applied forces. This method was recently improved (16,17), solving the technical problems in applying large forces in the earlier centrifugal assays.Significant progress has been made in measuring the traction forces of cells. By cultivating cells on an elastic siliconrubber membrane (18-20) or on protein films formed at fluorocarbon oil-water interfaces (21), it is possible to estimate the forces applied by the cell to the substrate by observing the deformation of the elastic films. Recently, a related technique employing a micromachined array of small pads supported by springs was used to resolve the traction force exerted by the different parts of a locomoting cell (22).The atomic force microscope (23) has found many uses as a tool in biology. As a microscope scanning biological...

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Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/I neg at Telelab (Skien, Norway) and group I neg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp5؉-Gp6؉ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp؉, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp؉, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6-mm deep, stagnant, medium layer covering a cell culture. The measurement set-up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.

Summary.-The photosensitizing effect of haematoporphyrin (HP) on human cells of the established line NHIK 3025 has been studied. Fluorescence measurements show that HP is bound to these cells. Serum proteins also bind HP, and the presence of 10% human serum during incubation with HP (3 x 10-4M) reduces the cellular uptake of HP by 75%o or more. The photosensitized inactivation is enhanced when the cells are suspended in D20-buffer during irradiation. This indicates that singlet oxygen is involved in the inactivation.Two findings indicate that the photoinduced damage is repairable: firstly, the fraction of cells surviving a given light dose decreases with decreasing irradiation temperature, and secondly, the survival curves have a shoulder at low exposures of light.

The method of synchronizing cells by means of mitotic selection has been adapted to the human line NHIK 3025. Increase in cell number as a function of time in asynchronous and synchronous populations was studied as well as mitotic index as a function of time after selection of synchronized populations. Phase durations of the cell cycle of synchronous populations were determined by 3 H‐thymidine incorporation and scintillation counting. The relative phase durations of exponentially growing asynchronous populations were determined by mathematical analysis of DNA‐histograms recorded by flow cytofluorimetry. Both the generation time and the various phase durations of the cell cycle were found to be the same in asynchronous and synchronous populations. It was found that NHIK 3025 cells are damaged by cooling to 4 and 0°C so that cooling of selected cells in order to increase the yield would reduce the quality of the synchronized populations.

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