J. F. Nagelkerke

Caspase-3 Activity Predicts Local Recurrence in Rectal Cancer

Purpose: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy. Experimental Design: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay. Results: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001). Conclusion: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.

PHOTODYNAMIC TREATMENT OF YEAST WITH CHLOROALUMINUM-PHTHALOCYANINE: ROLE OF THE MONOMERIC FORM OF THE DYE

The effect of photodynamic treatment on the yeast Kluyveromyces marxianus with aluminum‐phthalocyanines has been studied. It was found that the nonsulfonated sensitizer caused light‐dependent loss of colony‐forming capacity, whereas the mono‐ and tetrasulfonated forms did not induce loss of clonogenicity. The effect of the nonsulfonated sensitizer increased with longer preincubation periods of cells with the dye. Formation of cellattached, mostly intracellularly localized monomelic sensitizer also increased with time. The amount of cell‐bound multimeric nonsulfonated phthalocyanine did not vary with time. Experiments designed to specifically increase the amount of cell‐attached monomers led also to an increased photoinactivation of the cells. It is therefore concluded that the photodynamic effect of the nonsulfonated Al‐phthalocyanine is mediated by the monomeric form of the dye.

Allyl Alcohol and Acrolein Toxicity in Isolated Rat Hepatocytes is Independent of Lipid Peroxidation

For many years now the relation between lipid peroxidation and cell death, induced by xenobiotics, has been debated, the question being whether lipid peroxidation is the cause or the consequence of toxic events leading to cell death (Smith et al. 1983, Younes and Siegers 1981, Kappus and Muliawan 1982). A major defence system against lipid peroxidation is based on the reducing activity of glutathione (GSH) in which it is converted to the oxidized form (GSSG). Compounds that are conjugated with GSH can induce lipid peroxidation because they deplete GSH and therefore eliminate the defences against lipid peroxidation (Smith et al. 1983; Reiter and Wendel 1982). Acrolein is formed by alcohol dehydrogenase from the hepatotoxin allyl alcohol. It can be conjugated with GSH or it can be further oxidized to the nontoxic agent acrylic acid by aldehyde dehydrogenase. Recently it was demonstrated (Ohno et al. 1985) that acrolein is responsible for the toxic effects of allyl alcohol. The present study was undertaken to obtain a better insight into the relation between lipid peroxidation and cell death, induced by allyl alcohol and acrolein.

Video Microscopy and confocal Laser Scan Microscopy to study the mechanisms of cytotoxicity in individual living cells. Application and new developments

SummaryIn the past decade numerous studies have been performed using Video Microscopy and Confocal Laser Scan Microscopy to investigate in real time the effect of compounds on cells. An overview of the techniques themselves and the necessary hardware, the major types of calculations and how to handle cells and probes is presented in this paper.Although VM and CLSM are very powerful techniques they have some drawbacks. Therefore, recently, technological innovations have emerged to overcome these drawbacks. The new techniques are Multi-Photon Excitation Microscopy, Fluorescence Life Time Microscopy and determination of Fluorescence Resonance Energy Transfer. Also the discovery of the Green Fluorescent Protein and subsequent development of derivatives thereof which all emit light upon excitation, without the need of a prosthetic group, has created new opportunities. Especially, transfection with vectors including GFP-DNA is very powerful to study protein localisation and, in addition, is very suitable to perform FRET studies. Recently, also transgenic mice expressing GFP-tagged proteins at specific sites of the body have become available to performin vivo studies.

Use of video microscopy to study the mechanisms of cytotoxicity in vitro in individual living cells

Recent technological innovations have made it possible to determine simultaneously multiple biochemical parameters in individual living cells. This new technique, commonly designated as video microscopy, makes use of fluorescent probes, specific for a certain cellular parameter. Cells are loaded with the probes and placed under a fluorescence microscope. After excitation at a specific wavelength the probes emit light of a longer wavelength. The intensity of the emitted light is proportional to the magnitude of the specific parameter. Emission is recorded with a video camera and the signal transferred to an image processor for extraction of quantitative data from the image, or to improve image quality. An overview of the technique itself and the necessary hardware, the major types of calculations and how to handle cells and probes is presented in this paper.