C. Sparwasser

Intercomparison of apoptosis morphology with active DNA cleavage on single cells in vitro and on testis tumours

Apoptosis morphology (DNA condensation) and endonucleolytical DNA cleavage (TdT assay) were measured simultaneously on double fluorescence labelled cells employing confocal laser scanning and conventional immunofluorescence microscopy. In vitro experiments on irradiated HL‐60 cells revealed a high correspondence of non‐apoptotic (normal) cells without detectable DNA cleavage, versus apoptotic cells and apoptotic bodies showing DNA cleavage. Experiments performed on histological slides of testis tumours reflected a heterogeneous picture: non‐apoptotic (normal) cells, apoptotic cells, and apoptotic bodies appeared either with or without detectable DNA cleavage. These data allowed the characterization and quantitation of the grade of disturbance/heterogeneity of the apoptosis programme in vivo. Furthermore, the measured apoptotic index (AI) based on apoptosis morphology was lower than the AI assessed by DNA cleavage, in contrast to published work. Taken together, these methods represent a new approach and might be suitable for improved correlation with clinical parameters. In addition, the data presented confirm frequently published doubts regarding the ability of the TdT assay to detect apoptosis as defined by morphological criteria in tumours.

APOPTOSIS IN HUMAN EMBRYONAL CELL CARCINOMA: PRELIMINARY RESULTS

Abstract Disorders in the regulation of apoptotic cell death may contribute to cancer. Furthermore, lymphocytes are supposed to play a role in counteracting tumorigenesis by inducing apoptosis in different human tumors. In this study, for the first time, tumor cell and lymphocyte apoptosis were investigated systematically in human embryonal cell carcinoma. DNA fragmentation and DNA condensation were measured simultaneously on double-fluorescence-labeled testis tumor sections using immunofluorescence microscopy. Different apoptotic indices (AIs), based either on biochemical (DNA fragmentation) or morphological criteria (DNA condensation) alone or on a combination of both, were determined in different histological regions in and around the tumor. Using morphological criteria alone, 40–75% of all apoptotic cells were not detected. Based on previous observations this finding might be related to subsets of apoptotic cells which induce the process of DNA condensation without activation of processes responsible for DNA fragmentation. Moreover, the AIs of tumor cells and lymphocytes were highest in the tumor region, compared with regions around the tumor and distant from it; these findings are discussed in the context of the Fas/FasL system.

Comparative analysis of different apoptosis detection methods in human testicular cancer

In situ end-labeling (ISEL) of internucleosomal 3′ DNA strand breaks and the morphological proof of nuclear chromatin condensation are two widely used methods to investigate and quantify apoptosis. However, it is still unclear whether both processes are linked with each other and if quantifying apoptosis by both methods leads to comparable results. Therefore, internucleosomal DNA fragmentation and chromatin condensation were measured simultaneously on double-fluorescence-labeled sections of 62 testicular tumors (47 nonseminomatous tumors and 15 seminomas) using immunofluorescence microscopy. Different apoptotic indices (AI), based on DNA fragmentation and/or morphological criteria were determined. The AI were quantified. Morphologically obtained AI ranged between 1.99% for non-seminomatous tumors and 0.88% for seminomas. The detection of DNA fragmentation values ranged between 8.15% for non-seminomatous tumors and 2.70% for seminomas. Only about 30% of all apoptotic cells could be detected with the morphological method compared to 80% using ISEL in both tumor entities. Therefore, the equivalence of investigations using different apoptosis detection methods in human testicular cancer seems questionable.

Significance of apoptosis in metastasizing testis tumors

Testis tumors of embryonal origin (ten metastasized, six non-metastasized) and 17 mixed testis cell carcinomas (eight metastasized, nine non-metastasized) were examined. A triple immunofluorescence microscopic labeling procedure allowed the simultaneous detection of two features of apoptosis, namely morphological changes in the nucleus (DNA condensation visualized by DAPI staining) and the process of DNA fragmentation (TdT-assay) in tumor cells as well as T-cells (recognized by their CD45RO epitope). Both methods for apoptosis detection showed similar apoptotic indices (AI) only in 2.6% of all tumors. Most tumors (81.6%) showed more cells with DNA fragments than condensed chromatin, but in a number of cases (10.5%) the opposite pattern was found. These data add to the few published in vivo examinations of apoptosis using different methods and help to explain differences in the judgment of apoptosis significance for tumor prognosis. With regard to tumorigenesis, non-metastasized testis tumors were characterized by higher AIs of tumor cells and T-cells compared with metastasized tumors, which could be interpreted as a characteristic of tumors in an earlier stage of their development into an apoptosis-resistant phenotype. For the first time, in metastasized tumors a 5 to 25-fold increase of the T-cell’s AIs over the corresponding AIs of tumor cells was shown. This suggests a successful counterattack of tumor cells, thus supporting the process of metastasis. However, only ten out of 33 tumors revealed these AI changes, which again highlights that tumor biology cannot be predicted by a single parametric approach. It remains to be seen whether these characteristics might be suitable for a reliable prediction of metastasis.

Apoptosis in Non-Tumorous Adult Human Testis Tissue

Introduction: Apoptosis seems to play an important role in tumorigenesis, prognosis and therapy of testicular tumors. To understand its biological significance, it is importantto quantify the amount of apoptosis and to compare the rate of apoptosis to that of a normal, unaffected reference tissue. Usually tissue from the unaffected site of the testis in patients with testicular cancer or testis tissue from patients who underwent surgical castration due to prostate cancer is used as the reference tissue. However it is not known, if both tissues are equivocal with respect to their apoptotic index. The purpose of the study was to compare the two most often used reference tissues for the quantification of apoptosis in testicular tissues with regard to their apoptotic index. Materials and Methods: The apoptotic indices of both tissues were compared, using two standard apoptosis detection methods, i.e. in situ end labeling and a morphological approach. Results: The apoptotic index in testis tissue from patients who were surgically castrated for antihormonal treatment of prostate cancer was shown to be significantly higher than the apoptotic index of tumor free but tumor-associated testicular tissue of testis cancer patients. There was a strong relationship between the apoptotic index and the age of the patients. Conclusion: Although there might be genetic changes in the tumor-associated testicular tissue influencing the apoptotic index, it seems advisable to use tumor-associated tissue rather than testis tissue of patients with prostate cancer as the reference tissue, due to the significant age dependence of the apoptotic index.

Imaging Studies in the Diagnosis of Urogenital Trauma

Polytraumatized patients often present with urological injuries. After hemodynamic stability is maintained urologists are consulted to evaluate diagnostic and therapeutic interventions. The following article describes how to handle the work-up of patients with injuries to specific urogenital organs: the importance of clinical examination, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), angiography as well as organ-specific radiologic studies such as intravenous pyelography or cystography are discussed. Conclusion: Even though injuries to the urogenital tract are rarely initially life – threatening, a fast, reliable and adequate diagnostic algorithm has to be established to avoid any delay of specific treatment. Urologists should be familiar with the indications, range and accuracy of these procedures in the diagnosis of urogenital trauma.

Role of intracellular Ca2+ stores in smooth muscle of human penile erectile tissue

Objective: In human erectile tissue smooth muscle contraction and detumescence are highly dependent on an increase in cytosolic [Ca2+]. The Ca2+ influx can be derived from the extracellular space or from intracellular sarcoplasmic stores. The role of both pathways was evaluated in an organ bath study on human cavernosal strips. Patients and methods: The tissue was obtained from 12 patients with chronic erectile dysfunction. The effects of Ca2+-free solution, ryanodine, caffeine and of nifedipine on electrically and adrenergically induced contractions were evaluated. Results: Following an incubation period of 10 min in Ca2+-free solution the electrically induced contraction was reduced to 20%, whereas the contraction induced by phenylephrine (PE) was only reduced to 64 ± 6% (mean ± SEM). Ryanodine inhibited the PE-contraction to 30 ± 6% and the additional application of caffeine or nifedipine further reduced the contraction to 11% and 8%. Conclusion: The results give evidence for a role of intracellular Ca2+-stores in human cavernosal tissue. Whether the more marked effect of ryanodine in tissue from patients with erectile failure in comparison with similar experiments in rabbit cavernosal tissue might be a sign of an increased cavernosal contractility in these patients remains to be shown in future experiments with normal erectile tissue.

Testis cancer cells have a genetic determination for a high sensitivity to apoptosis inducing stimuli

OBJECTIVE The effect of cytotoxic therapy in testicular tumors (TGCT) has been shown to be mediated mainly by the induction of apoptosis. So far, it is not known which genes play a role for this inherent sensitivity to apoptosis inducing drugs. The aim of this study was to investigate the differential gene expression of apoptosis regulating genes in testicular tumors and in normal testis tissue using a quantitative method. As a premature S-phase entry was shown to induce apoptosis, genes controlling the G1/S-phase checkpoint were also investigated. MATERIAL AND METHODS Gene expression levels of a representative subset of 19 genes involved in apoptosis and cell cycle control were investigated in vivo in 19 TGCTs using real-time quantitative PCR. Measurements were performed in tumor tissues of both tumor entities, seminomatous and non-seminomatous tumors (SGCT and NSGCT), and in corresponding biopsies from the unaffected site of the resected testis. RESULTS There was an up-regulation of genes that play a role in facilitating apoptosis, such as FasL, TRAIL, and Bax in both tumor entities. Genes inhibiting apoptosis, such as Bcl-2 were predominantly down-regulated. Regarding cell cycle regulators, a gene expression profile was found that corresponds to a premature S phase entry and subsequent apoptosis induction. CONCLUSION This study for the first time identified in vivo a panel of genes that give TGCT an inherent sensitivity to apoptotic stimuli after exposure to DNA damaging agents. Studies on these genes in therapy-refractory cancers should provide further insight into the mechanisms of chemotherapy resistance. Furthermore, these genes are promising targets for a future targeted therapy of testis cancer.

Apoptosis: a key effector mechanism of lymphocyte action in human nonseminomatous testicular carcinoma?

To correlate the number of tumour‐infiltrating T lymphocytes (TILs) with the extent of apoptosis in testicular germ cell tumours, as TILs are considered to be a favourable prognostic factor of human testicular tumours, especially of seminomas, but the mechanism by which TIL contribute to an improved outcome is unclear.

Modified approach for apoptosis detection reveals changes in apoptotic processes in the seminoma-associated tissue

Apoptosis morphology (DNA condensation) and internucleosomal DNA cleavage (TdT assay) were measured simultaneously on double fluorescence labeled testis tumor sections, employing conventional immunofluores cence microscopy. Six different apoptosis indices (AI) were determined based either solely on morphological or biochemical criteria, or on a combination of both processes. Measurements were performed in metastasized and non-metastasized seminoma, and in histological regions located distantly and associated with the tumor. Preliminary results on 19 histologies revealed that up to 66% of apoptotic cells were not detected, depending on the method used for apoptosis detection. Besides, no changes of solely morphologically defined AI was found in the different histological regions. By contrast, significant changes (p < 0.0004) in the different histological regions were detected when measuring AIs, e.g., defined by DNA fragmentation occuring without DNA condensation in apoptotic cells. Those changes were not detected in metastasized seminoma. These data, for the first time allow a comparison of two widely used approaches for apoptosis detection. Furthermore, the results reveal differences in apoptotic processes in tissue associated with non-metastasized seminoma detectable by a modified evaluated TdT assay but not by morphological changes, although this TdT method fails to show the total amount of apoptotic cells.