Integration of radiation oncology teaching in medical studies by German medical faculties due to the new licensing regulations : An overview and recommendations of the consortium academic radiation oncology of the German Society for Radiation Oncology (DEGRO).

The new Medical Licensing Regulations 2025 (Ärztliche Approbationsordnung, ÄApprO) will soon be passed by the Federal Council (Bundesrat) and will be implemented step by step by the individual faculties in the coming months. The further development of medical studies essentially involves an orientation from fact-based to competence-based learning and focuses on practical, longitudinal and interdisciplinary training. Radiation oncology and radiation therapy are important components of therapeutic oncology and are of great importance for public health, both clinically and epidemiologically, and therefore should be given appropriate attention in medical education. This report is based on a recent survey on the current state of radiation therapy teaching at university hospitals in Germany as well as the contents of the National Competence Based Learning Objectives Catalogue for Medicine 2.0 (Nationaler Kompetenzbasierter Lernzielkatalog Medizin 2.0, NKLM) and the closely related Subject Catalogue (Gegenstandskatalog, GK) of the Institute for Medical and Pharmaceutical Examination Questions (Institut für Medizinische und Pharmazeutische Prüfungsfragen, IMPP). The current recommendations of the German Society for Radiation Oncology (Deutsche Gesellschaft für Radioonkologie, DEGRO) regarding topics, scope and rationale for the establishment of radiation oncology teaching at the respective faculties are also included.

Proliferation and micromilieu during fractionated irradiation of human FaDu squamous cell carcinoma in nude mice.

PURPOSE: Previous functional radiobiological experiments demonstrated a significant acceleration of repopulation after 3 weeks and reoxygenation after 12 days of radiotherapy of FaDu tumours. Owing to the temporal coincidence between repopulation and reoxygenation, it was hypothesized that the improved oxygenation status during fractionated irradiation might be the preceding stimulus for increased proliferation. The study investigated whether these changes in repopulation and re-oxygenation are reflected by histological parameters of proliferation and the tumour micromilieu. MATERIALS AND METHODS: Human FaDu squamous cell carcinomas in nude mice were irradiated with three to 18 fractions of 3 Gy daily or every second day under normal blood flow and clamp hypoxia. At different time points, tumours were excised and stained for Ki67, BrdUrd, epidermal growth factor receptor (EGFR) and markers of the micromilieu (HOECHST 33342, pimonidazole, ER-MP12). RESULTS: On average, Ki67 and BrdUrd labelling indices decreased initially and increased again at later times during the course of fractionated radiotherapy. A similar kinetic pattern was found for the staining intensity of the EGFR. The vascular density in the viable tumour area remained constant during the whole course of irradiation, while the perfused fraction of vessels decreased within the first week of irradiation and returned to baseline values after 2 weeks. There was a corresponding increase in perfusion and a decrease in cellular hypoxia. CONCLUSIONS: The histological results were in surprisingly good agreement with the kinetics of clonogen repopulation and re-oxygenation determined previously using functional assays. The results support that the kinetics of repopulation of FaDu squamous cell carcinoma in response to fractionated irradiation are determined not only by intracellular processes, but also by a complex interaction of proliferation parameters with a changing microenvironment.

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay, but not local control, after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse.

PURPOSE: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments. MATERIALS AND METHODS: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5 microM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50 mg kg(-1) body weight orally) or carrier. Tumour growth delay and dose-response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks. RESULTS: BIBX1382BS blocked radiation-induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6 Gy (95% confidence interval 55; 73) for irradiation alone and 67.8 Gy (60; 77) for the combined treatment (p=0.5). CONCLUSIONS: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.

Impact of the tumour bed effect on microenvironment, radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse.

PURPOSE: To investigate the impact of the tumour bed effect (TBE) on histological parameters of the micromilieu, radiobiological hypoxic fraction and local control after fractionated irradiation in FaDu squamous-cell carcinoma in the nude mouse. This tumour has previously shown a clear-cut TBE caused by increased necrotic cell loss at a constant cell production rate in the viable tumour compartment. MATERIALS AND METHODS: Human FaDu tumours were studied in the NMRI nude mouse. Tumours were transplanted either into unirradiated subcutaneous (s.c.) tissues (controls) or s.c. tissues pre-irradiated with 12.5 Gy (TBE group). In both groups we measured the volume doubling time (VDT), potential doubling time (T(pot)), relative necrotic area, and in the viable tumour compartment the relative vascular area (9F1 mAb), relative hypoxic area (NITP or pimonidazole), relative perfused area (Hoechst 33342), and the perfused fraction of vasculature. The tumour control dose 50% (TCD 50), radiobiological hypoxic fraction (rHF) and dose-modifying factors (DMF) for the comparison of tumours in the TBE and control groups were determined from local tumour control data after treatment with single doses under ambient conditions or under clamp hypoxia, and after irradiation with 30 fractions under ambient conditions within 6 weeks using maximum-likelihood analysis. RESULTS: A clear-cut TBE (VDT = 4.0 days (95%CI 2.9;4.4) for the control group versus 7.2 days (6.4;8.9) for the TBE group; p <0.0001) caused by increased necrosis (mean relative necrotic area of 12% (5;20)) versus 33% (10;41); p = 0.07) at a constant cell production rate (T(pot) = 2.2 days (1.4;2.3) versus 2.2 days (1.7;2.6); p = 0.30) was confirmed. Histological analysis of the micromilieu within the vital subarea revealed no systematic differences between the TBE and control groups. The rHF of 2% (0.1;27) for control tumours was lower than the 15% (95% CI 2;91) for the TBE group, but this difference was nonsignificant (p = 0.12). Compared with control tumours, the TCD50 for irradiation under clamped hypoxia was in a statistical trend lower for tumours in the TBE group (DMF 1.11 (0.98;1.28), p = 0.09). After fractionated irradiation, tumours of the TBE group were significantly more radiosensitive (TCD50 56.6 Gy (46;70) versus 78.7 Gy (63;100); p = 0.003). CONCLUSIONS: The results on FaDu tumours growing in pre-irradiated tissues indicate that increased necrosis caused by impairment of the vascular supply may increase the radiosensitivity of tumours treated by fractioned irradiation.

Repopulation of FaDu human squamous cell carcinoma during fractionated radiotherapy correlates with reoxygenation.

PURPOSE: FaDu human squamous cell carcinoma (FaDu-hSCC) showed a clear-cut time factor during fractionated radiotherapy (RT) under ambient blood flow. It remained unclear whether this is caused solely by proliferation or if radioresistance resulting from increasing hypoxia contributed to this phenomenon. To address this question, repopulation of clonogenic FaDu cells during fractionated RT under clamp hypoxia was determined by local tumor control assays, and compared to the results after irradiation with the same regimen under ambient blood flow. METHODS AND MATERIALS: FaDu-hSCC was transplanted into the right hind leg of NMRI nu/nu mice. In the first set of experiments, irradiation was performed under clamp hypoxia. After increasing numbers of 3 Gy fractions (time intervals 24 h or 48 h), graded top-up doses were given to determine the TCD(50) (dose required to control 50% of the tumors). In the second set of experiments, all 3 Gy fractions were applied under ambient conditions, but as in the previous experiments the graded top-up doses were given under clamp hypoxia. A total of 26 TCD(50) assays were performed and analyzed using maximum likelihood techniques. RESULTS: With increasing numbers of daily fractions, the top-up TCD(50) under clamp hypoxia decreased from 39.4 Gy [95% CI 36, 42] after single dose to 19.8 Gy [15, 24] after 18 fractions in 18 days and to 37.8 Gy [31, 44] after 18 fractions in 36 days. The results were consistent with biphasic repopulation, with a switch to rapid repopulation after about 22 days [13, 30]. The clonogen doubling time (T(clon)) decreased from 9.8 days [0, 21] in the beginning of RT to 3.4 days after 22 days. Under ambient blood flow the top-up TCD(50) decreased from 37.6 Gy [34, 40] after single dose irradiation to 0 Gy [0, 1] after 18 fractions in 18 days and 22.4 Gy [18, 27] after 18 fractions in 36 days. Similar to results from irradiations under clamp hypoxia, the ambient data were consistent with a biphasic course of clonogen inactivation. Comparison of both data sets revealed significant reoxygenation after 12 fractions. CONCLUSIONS: Our data are most consistent with a biphasic course of clonogen repopulation during fractionated RT of FaDu-hSCC under clamp hypoxia with a switch in T(clon) after about 22 days of treatment ("dog-leg"). A similar biphasic course of cell repopulation was observed under ambient conditions. The temporal coincidence between repopulation and reoxygenation suggests that the latter might be the stimulus for proliferation in FaDu tumors.

Radiobiological hypoxia, oxygen tension, interstitial fluid pressure and relative viable tumour area in two human squamous cell carcinomas in nude mice during fractionated radiotherapy.

Very little is known about the correlation between the radiobiological hypoxic fraction (rHF) and other measures of tumour oxygenation during fractionated irradiation. In the present study the rHF is determined in untreated human FaDu and GL squamous cell carcinoma in nude mice and in tumours irradiated with 10 fractions in 2 weeks and 20 fractions in 4 weeks, using tumour control as the experimental endpoint. The results were compared with measurements of the pO2, the interstitial fluid pressure (IFP) and the relative viable tumour area. In FaDu tumours the radiobiological hypoxic fractions (rHFs) before and during irradiation were not statistically different from 100%. Depending on the assumptions made for D0, the rHFs of GL tumours were between 0.2 and 4% or 30 and 53%. The median pO2 values were 2.8 mmHg for untreated FaDu tumours and 0.2 mmHg for GL tumours (p < 0.001). The median IFP values were 2.6 mmHg in FaDu and 5.3 mmHg in GL tumours (p = 0.01). No important changes in the pO2 and IFP values were observed during fractionated irradiation. The relative viable tumour area during irradiation decreased by 83% in FaDu tumours (p = 0.002) and by 54% in GL tumours (p = 0.003). It is concluded that differences in rHF exist between FaDu and GL tumours before and during fractionated irradiation and that these differences are not reflected by pO2 and IFP values and the relative viable tumour area.

Impact of preirradiation of the tumour bed on cell production rate and cell loss of human FaDu squamous cell carcinoma growing in nude mice.

PURPOSE: To explore whether the tumour bed effect (TBE) in FaDu squamous cell carcinoma growing in nude mice is caused by a reduced tumour cell production rate and/or by increased tumour cell loss. MATERIALS AND METHODS: Human FaDu tumours were studied in NMRI nude mice. The volume doubling time (VDT) between 100 and 400 mm3 was determined for tumours in unirradiated subcutaneous (sc) tissues (group 1), tumours in sc tissues preirradiated with 12.5 Gy (group 2), tumours irradiated in situ with 12.5 Gy (group 3), and tumours from group 3 re-transplanted into unirradiated sc tissues (group 4). Labelling index (LI), potential doubling time (Tpot), relative necrotic area and apoptotic index (AI) were evaluated in tumours from groups 1 and 2. RESULTS: The median VDT were 2.6 days (95% CI 2-4) in group 1 and 7.0 days (4-15) in group 2 (p<0.001). The VDT were not significantly different between groups 2 and 3, and group 1 and 4. In groups 1 and 2, the Tpot values (3.1 +/- 0.6 days (SD) versus 2.9 +/- 0.5 days) and the LI were identical (10 +/- 1.5%). The median relative necrotic area was significantly larger in group 2 (37% [23-42]) compared with group (6% [0.3-27]). The apoptotic index was low (0.2%) and did not differ between groups 1 and 2. CONCLUSIONS: The results indicate that the TBE in FaDu squamous cell carcinoma is not caused by a reduced cell production rate in the viable tumour compartment. Rather, the TBE reflects a decreased viable tumour cell compartment due to increased cell loss. Necrosis appears to be the major component of the tumour bed induced cell loss in FaDu tumours, whereas apoptosis has no impact on the TBE in this model.

5 alpha-reductase activity in the human hair follicle concentrates in the dermal papilla.

Hair growth depends on a close interaction of different cell populations of the hair follicle. In certain regions of the body, androgens interfere with this highly regulated cooperation in a yet poorly understood manner. The response of hair follicles to androgens can be categorized as androgen-dependent, e.g. in the beard, androgen-sensitive, e.g. in the frontal scalp of affected individuals, or androgen-independent, e.g. in the occipital scalp. At the target cell level, the balance between 5 alpha-reductase, 17 beta-hydroxysteroid-dehydrogenase (17 beta-HSD) and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) yields metabolites with different androgenic potential. We examined this target cell-specific androgen metabolism in microdissected intact sub-units of dermal papillae, connective tissue sheaths (CTS) and root sheaths. In dermal papillae, 5 alpha-reductase predominated with an accumulation of the strong androgen 5 alpha-dihydrotestosterone. The specific activity of 5 alpha-reductase in the papillae exceeded those in the other hair follicle compartments by a factor of at least 14 in the scalp (5.4, 0.4 and 0.1 pmol/h per mm3 in the papilla, CTS and root respectively and at least 80 in the beard (16.0, 0.2 and 0.4 pmol/h per mm3 in the papilla, CTS and root respectively). The root sheath keratinocytes expressed low 5 alpha-reductase levels, but high 17 beta-HSD levels, with androstenedione as the major metabolite. The CTS expressed both 5 alpha-reductase and 17 beta-HSD, resulting in androstenedione, 5 alpha-androsterone and 5 alpha-androstanedione. In the CTS and the root sheath, only minor amounts of 5 alpha-DHT were found. In beard papillae, the 5 alpha-reductase activity was three times that in the occipital scalp papillae. These results indicate that the androgen response of hair follicles depends on a differentiated intrafollicular androgen metabolism and that the dermal papilla might be a primary target in this process.

Phorbol-myristate-acetate, but not interleukin-1 beta or insulin-like growth factor-I, regulates protein kinase C isoenzymes in human dermal papilla cells.

The in vitro growth of human hair follicles is inhibited by interleukin (IL)-1 beta and phorbol esters, such as phorbol-myristate-acetate (PMA), but enhanced by insulin-like growth factor (IGF)-I. Although this process is only incompletely understood, the dermal papilla as a pivotal part of the hair follicle is almost certainly involved. Since protein kinase C (PKC) isoenzymes are activated by phorbol esters and are key enzymes in signalling pathways of several hormones, neurotransmitters, and growth factors, we addressed the question whether the action of the above-mentioned hair growth-modulating substances may affect PKC isoenzymes in cultured dermal papilla cells (DPC). By Western blot analysis, protein kinase C alpha, -epsilon, -gamma, -iota, -lambda, and the RACK1 receptor protein were detected in dermal papilla cell cultures, whereas the isoenzymes delta and mu were expressed only at low levels and protein kinase C-beta, -theta, and -zeta, were not present. After PMA stimulation, the PKC alpha, -epsilon, and -gamma were translocated from the cytosol to the membrane fraction and subsequently down-regulated. PKC iota was down-regulated but not translocated, and PKC lambda and RACK1 were not affected by PMA. Neither, IL-1 beta nor IGF had an effect on PKC or RACK1 expression. We conclude that cultured DPC express a distinct PKC isoenzyme pattern and that the PMA-induced growth arrest in cultivated hair follicles may be transmitted via protein kinases, whereas the effects of IL-1 beta or IGF may be transduced via other signal transduction pathways or other cell types.

Interleukin-1beta-induced inhibition of hair growth in vitro is mediated by cyclic AMP.

Interleukin (IL)-1 has been shown to be a potent inhibitor of hair growth in vitro. We hypothesized that this cytokine might be a decisive factor causing hair loss during the lymphocytic attack in alopecia areata. Neither the intracellular pathways involved in hair growth inhibition mediated by IL-1beta nor the signal transduction processes within hair follicles in general are known. We therefore investigated the intracellular signals involved in human hair growth in vitro. Hair follicles were isolated from scalp biopsies by microdissection, and hair growth was measured daily by image analysis. We assessed intracellular signal transducing elements using specific inhibitors or activators either alone or in combination with IL-1beta. The calcium ionophore A 23187 induced a rapid and complete arrest of hair growth, and phorbol-12-myristate-13-acetate (PMA), genistein, or IL-1beta decreased hair growth by approximately 60%-80%. IL-1beta-elicited hair growth arrest was not antagonized by calphostin C, a specific inhibitor of protein kinase C. In contrast, coincubation of IL-1beta with pertussis toxin or H 1004 neutralized the effect of IL-1beta, and dibutyryl-cAMP and cholera toxin, an activator of adenylate cyclase, inhibited hair growth. These data suggest that cAMP acts as a second messenger for IL-1beta-induced inhibition of hair growth. Moreover, our data indicate that in vitro hair growth is dependent on intracellular Ca2+ levels and activation of tyrosine kinase as well as protein kinase C. We were unable to detect a signal transducing element responsible for enhanced hair growth in vitro.

Cytokines and growth factors influence hair growth in vitro. Possible implications for the pathogenesis and treatment of alopecia areata.

Factors that influence the growth of the anagen hair follicle or initiate the switch to a catagen growth pattern have so far not been definitely determined, but there is increasing evidence that cytokines and growth factors play an important role during these processes. Recently we detected an aberrant in situ expression pattern of cytokines of the Th1 type (IFN gamma, IL-2) plus IL-1 beta expression in untreated alopecia areata (AA), and a switch to high levels of IL-10 TGF-beta 1 expression after successful treatment with the contact allergen diphenylcyclopropenone (DCP). Hence the question arose as to whether cytokines are able to arrest hair growth and whether IL-10 or TGF beta 1 have the capacity to antagonize this process. Using whole-organ cultures of microdissected human hair follicles we studied the effect of a panel of cytokines and growth factors on hair growth and on the gross morphology of the hair follicles in vitro. IL-2, IL-10 and IFN-gamma had no effect in this regard, whereas TGF beta 1 partially inhibited hair growth and EGF, TNF alpha and IL-1 beta completely abrogated it. EGF and TNF alpha induced the formation of a club-like hair follicle, similar to catagen morphology of the hair bulb, whereas hair follicles grown in the presence of IL-1 beta or TGF beta 1 showed no particular morphological changes. We conclude that cytokines and growth factors are pivotal regulators of hair growth at least in vitro. IL-1 is suggested as playing an important role during the pathogenesis of AA. Possible mediators of therapeutic contact dermatitis (IL-10, TGF beta 1, TNF alpha, PGE2) are, at least in vitro, not able to antagonize the IL-1 beta-triggered hair growth inhibition. Therefore, we infer that these mediators rather "modulate' the immune response in AA.

Immunocytochemical evidence for differential subcellular localization of 5 alpha-reductase isoenzymes in human tissues.

To contrast the previously described distribution of 5 alpha-reductase isoenzyme 2 in human organs we have raised synthetic C-terminal peptides of human 5 alpha-reductase isoenzyme 1 (h 5 alpha r1) and isoenzyme 2 (h 5 alpha r2) respectively, and studied the cellular and subcellular distribution of both isoforms of this key enzyme of testosterone metabolism using the human prostate gland as a reference. h 5 alpha r1, which in Western blots of human prostatic proteins had an apparent molecular weight of 23 kDa, was localized immunohistochemically in the nucleus of prostatic epithelial and stromal cells. Ultrastructurally, it was closely associated with the nuclear matrix. The apparent molecular weight of h 5 alpha r2 was 26 kDa in Western blotting of human prostatic proteins. In immunohistochemically processed sections, it was seen in the cytoplasm of prostatic epithelial as well as stromal cells. An organ screening with genital and extragenital tissues of males and females was performed and to elucidate the distribution of the isoenzymes. Presence of both isoenzymes was demonstrated for a number of tissues, including the central nervous system, the urogenital tract, the respiratory tract, the gastrointestinal tract, skin, and the endocrine system. The divergent localization of 5 alpha-reductase isoenzymes points to potentially different functions in various organs. In view of the nuclear localization of isoenzyme 1, its close spatial relationship with the androgen receptor is presumed to indicate a close association with the receptor mechanism.

Immunohistochemical evidence for differential distribution of 5 alpha-reductase isoenzymes in human skin.

Antibodies raised against fragments of synthetic peptides of human 5 alpha-reductase isoenzymes 1 (h5 alpha r1) and 2 (h5 alpha r2) were applied to paraffin sections of human skin (scalp, eyelid, lip, breast, scrotum). Immunoreactive sites were differentially distributed, in that h5 alpha r1 immunoreactivity was present in the nuclei of cells in the stratum germinativum (basal and lower portion of the spinous layer) of the epidermis, subepithelial fibroblasts, adipocytes, smooth muscle cells of the scrotal tunica dartos, basal cells of sebaceous glands, excretory duct cells of sweat glands, cells of the dermal papilla and fibrous and outer epithelial sheath of hair roots, as well as endothelial cells of small vessels and Schwann cells of cutaneous myelinated nerves. In contrast, immunoreactivity for h5 alpha r2 was found in the cytoplasm of the cells of the spinous layer (and far less intensely in the basal layer) of the epidermis, subepidermal fibrocytes, and especially in subcutaneous adipocytes. Immunoreactivity was strongest in the non-keratinized portion of the inner epithelial sheath and the cuticle of hair follicles, whereas other portions of the hair root were negative. Sweat glands were stained, whereas sebaceous glands showed only weak diffuse immunoreactivity. In mucocutaneous zones, salivary glands and conjunctival epithelium showed immunoreactive cells. Vascular endothelium displayed immunoreactivity only in the genital region. We present experimental evidence for a differential distribution of 5 alpha-reductase isoenzymes in human skin. This might reflect a diversity in the response of different areas of the skin to androgenic challenge.

Distribution of rat hepatic steroid 5 alpha-reductase 1 as shown by immunohistochemistry.

Peptide fragments of rat liver 5 alpha-reductase were synthesized according to the published primary sequence data. The peptide were used (i) in free form and (ii) linked to keyhole limpet hemocyanin (KLH), respectively, for immunization of rabbits. Resulting antisera showed positive reaction with the respective peptide-antigen in an ELISA. In Western blot experiments the antisera specifically recognized a 26,000 mol wt protein in extracts from female and male rat liver. All antisera, as well as isolated immunoglobulins, showed inhibition of 5 alpha-reductase activity in microsomes prepared from rat liver. Positive immunohistochemical reactions were observed in the perinuclear cytoplasm of hepatocytes. No reaction was seen in Kupffer- and connective tissue cells. Acinar heterogeneity of the immunoreaction was seen in the male rat liver with a gradient from the portal canal to the central vein. After androgen-deprivation a considerable increase in immunoreactivity was seen in male rat liver cells, indicating androgen-dependent suppression of the enzyme in male liver.

Hormonally induced changes in apocrine secretion of transglutaminase in the rat dorsal prostate and coagulating gland.

Coagulating gland and dorsal prostate of the rat are peculiar in secreting transglutaminase, a protein-cross linking enzyme that is released in an apocrine fashion. To elucidate whether or not the intracellular pathway and the unusual extrusion mechanism proceed constitutively or were differentially regulated, transglutaminase immunoreactivity was studied both at the light and electron microscopic levels. In addition, ultrastructural morphometry and scanning densitometry were applied to quantitate hormone-dependent distribution of transglutaminase. Coagulating glands and dorsal prostate, respectively, from sexually active rats were compared to those from sexually inactive, castrated, estradiol-treated or testosterone-substituted castrated animals. In intact, sexually active animals, no labeling of the cisternae of rough endoplasmic reticulum was seen, but instead the hyaloplasm was labeled. In the supranuclear portions of the cells an increase in labeling density of the hyaloplasm subjacent to the plasma membrane was found, whereas no labeling of either Golgi stacks or vesicles was observed. Apical blebs projecting into the acinar lumen were densely labeled. In castrated animals, epithelium showed a reduction of rough endoplasmic reticulum, loss of secretory blebs, and a decrease in cell size. Morphometric analysis of immunolabeling of coagulating gland epithelium from experimental animals resulted in a highly significant reduction of labeling of the hyaloplasm and apical blebs which was reversed by testosterone supplementation of castrated animals. After estrogen treatment, the reduction in immunolabeling was less pronounced, but morphology of apical blebs was obviously changed. Results from scanning densitometry of Western blots correlated with quantitative immunoelectron microscopical findings. Northern blot analysis using a secretory transglutaminase cDNA probe showed characteristic changes at the RNA levels. Our results indicate that apocrine secretion of transglutaminase in rat coagulating gland and dorsal prostate is a hormonally controlled process, where androgen deprivation results in impaired biosynthesis and release of transglutaminase, whereas estradiol treatment only partially inhibits secretion, but changes morphological features of the glandular epithelium, especially apocrine bleb formation.

Immunocytochemical localization of human 5 alpha-reductase 2 with polyclonal antibodies in androgen target and non-target human tissues.

We studied the tissue distribution and cellular localization of 5 alpha-reductase 2, the human prostatic isoenzyme, in different human tissues, both cryostat-sectioned and paraffin-embedded. Polyclonal antibodies raised in rabbits against a native peptide (C-terminal amino acids 229-254) or synthetic peptides (amino acids 234-245), either as carrier-conjugated linear peptides or multiple antigen peptides (MAP), were assayed for specificity and sensitivity with Western blotting and an ELISA system. One antibody showing monospecificity on Western blots and in ELISA was used for immunohistochemical detection of the respective antigen in tissues from male and female subjects. Positive cells were found (with decreasing intensity) in inner epithelial sheath of hair follicles, pyramidal cells of the cerebral cortex, hepatocytes and bile duct cells, prostate epithelial cells, seminal vesicle epithelial cells, endothelial cells of small vessels, fat cells, fibrocytes of genital and extragenital organs, and smooth muscle cells of prostate and seminal vesicles. Some variation in the immunoreactivity of testis and ovary tissue was seen with different antibodies. 5 alpha-Reductase 2 is obviously not restricted to androgen target organs in the male, but is present in a large number of cells and tissues in both males and females.

[Current aspects on morphology and functions of the prostate]. / Aktuelle morphologische und funktionelle Aspekte der Prostata.

The human prostate has a dual function in that it produces a number of secretory compounds conditioning the urethral surface for sperm passage and acting on spermatozoa as well as on vesicular coagulation proteins (semen liquefaction). In addition to differentially distributed and regulated steroid hormone receptors in epithelium and stroma, the prostate contains a large number of growth factors and their receptors. An incompletely understood paracrine regulation of growth and differentiation exists between epithelial cells, such as secretory, basal and neuroendocrine cells, as well as the underlying stroma (smooth muscle cells, fibrocytes). The presently available (malignant and non-malignant) prostatic cell lines have a number of disadvantages that render them of limited value in prostate cancer research.