- Количество слайдов: 1
1, 3 J. Cerman, 2 J. Čáp, 1 M. Mareková, 3 S. Němeček, 5 J. Marek, 4 M. Červinka 1 Dept. of Medical Biochemistry, 2 Second Dept. of Internal Medicine, 3 Center of Experimental Neurosurgery, and 4 Dept. of Medical Biology and Genetics: Faculty of Medicine in Hradec Kralové; 53 rd Dept. of Internal Medicine: 1 st Faculty of Medicine in Prague. Charles University in Prague; Czech Republic. • Aims • To investigate the role of apoptosis in the mechanism of action of conventional pituitary-adenoma treatment options: Materials and methods Results PATIENTS AND HISTOPATHOLOGY: Fourteen patients were treated with somatostatin analogue for three month until surgery. No medical therapy of acromegaly was introduced in 17 patients constituting the reference group. Formalin-fixed, paraffin-embedded (1) Histological examination of somatostatin analogue-treated adenomas: Classical histological examination revealed regressive changes (Fig. 6). Considerable fibrosis was more common than in untreated group (Table). The incidence of apoptosis was less frequent and the Ki-67 proliferative index significantly lower (Fig. 7, 8). (2) Cell lines: Somatostatin analogues were unable to induce apoptosis. Ionizing radiation induced apoptosis in the somatotroph GH 3 cell line 72 hours after irradiation by a dose of 20 and 50 Gy. Cells accumulate in G 2/M phase of cell cy 1515 cle 24 hours after irradiation and this cell-cycle arrest lasted for at least 10 days. In the corticotroph At. T 20 cell line apoptosis was detected after 6 hours using 10 Gy, without any cell-cycle block (Fig. 9, 10). Irradiation in the presence of somatostatin enhanced the effect on GH 3 cell line 15(Fig. 11). No radioprotective effect (suggested recently in a retrospective clinical study) was demonstrated. sections of pituitary adenomas were used for routine histopathology, immnocytochemical (ICC) detection for all pituitary hormones (Fig. 1 -3), markers pertinent to apoptosis: cytokeratin-18, apoptosis-specific fragment M 30, i. e. , caspase-cleaved formalin-resistant neo-epitope of cytokeratin-18, and cleaved 17 k. Da form of caspase-3. (Fig. 4, 5) Proliferation marker - the Ki-67 antigen was estimated and the Ki-67 labelling index calculated. CELL LINES Pituitary adenoma cell lines: GH 3 (rat somatotrophs) and At. T 20 (subclone D 16 v-F 2 of mouse corticotrophs). Flowcytometry: analysis of cell cycle and detection of apoptosis (subdiploid peek) after propidium iodide staining. Morphological changes typical of apoptosis were evaluated. IONISING RADIATION: 60 Co (dose rate 3 Gy/min, similar to that used during gamma-knife procedure) 2 5 4 3 6 CASPASE-3 1 • (1) ionizing radiation - and • (2) somatostatin analogues CK-18 Fig. 1, 2, 3: Illustration of hormonal status of some acromegalic adenomas in formalin paraffin sections. Fig. 1. Immunoperoxidase GH staining Fig. 2. The simultaneous double immunostaining revealed a mixed GH-prolactin adenoma (green and red fluorescent, resp. , ). Fig. 3. ICC detection of cytokeratin-18 (intensively reddish fluorochrom Alexa 546). Inset: double immunospecific fluorescence co-localizes CK-18 to GH-positive structures. M-30 Fig. 6. Photomicrograph of a somatostatin-analogue treated adenoma. (A) Loss of tumourous parenchyma with perivascular fibrosis. (B) Initial stage of interstitial fibrosis (Gömöri stain). Fig. 4. ICC detection of CK-18 and Fig. 5. apoptotic M 30 marker in acromegalic adenoma tissue. A case with high incidence of apoptosis 8 7 Fibrosis and apoptosis *p value of difference between treated and untreated group treated Table : Numbers of adenomas in the treated and untreated group with presence of significant fibrosis and apoptosis. Figure 7: Immunohistochemical staining for Ki-67. (A) A section from the treated adenoma; only isolated positive nucleus. (B) An untreated adenoma. High incidence of positive nuclei reflects high proliferative activity. At. T 20, 6 h 20 Gy Control G 1 1% G 2 APO 11% GH 3, 48 h 0% APO 55% Fig. 8. The Ki-67 labelling index in treated and untreated adenomas, respectively. The boxes express median (middle horizontal line) and 25 th to 75 th percentile. The bars show the range. The difference was statistical significant (p = 0. 049). 10 9 11 APO 12% 50 Gy Control 50 Gy untreated 200 Gy 0% Fig. 9. Flow-cytometric analysis of DNA content of At. T 20 and GH 3 cell lines after irradiation (6 and 48 h, resp. ). G 1 - cells in G 0/G 1 phase, G 2 - cells in G 2/M phase (duplex amount of DNA), APO - apoptotic cells (sub. G 1 peak). Note arrest in G 2 phase in GH-3 cells. Caspase-3 Fig. 10. ICC detection of caspase-3 in cell line At. T 20. Left: 6 hours after irradiation (50 Gy - 3 Gy/min), right: control. SA-B with DAB as chromogen. Conclusions Control 0. 2% 0 Gy 3. 0% 20 Gy 16. 4% 50 Gy 10. 7% 20 Gy 33. 2% 50 Gy Somatostatin 0% Fig. 11. Flow-cytometric analysis of DNA content of GH 3 cell line 10 days after irradiation (20 and 50 Gy). Red numbers = percentage of apoptotic cells (sub. G 1 peak). The presence of somatostatine 10 -8 mol/l enhances radiation induced apoptosis. • Somatostatin analogues (SSA) have an antiproliferative effect on GH-secreting adenomas that is mainly mediated by a drop in the number of replicating cells. • No induction of apoptosis by SSA has been demonstrated neither in humans nor in cell lines. Shrinkage in the size of some adenomas seems to be caused by the loss of functional parenchyma with compensatory fibrosis. • Ionising radiation in doses comparable to those used during gamma knife radiosurgery induced apoptosis in At. T 20 cell line after 6 hours, while the cell cycle arrest in G 2 phase dominated in the GH 3 line. SSA have no radioprotective effect.