1. Conclusions THE ROLE OF APOPTOSIS IN PITUITARY
treated untreated
THE ROLE OF APOPTOSIS IN PITUITARY
ADENOMAS IN THE FIELD OF CONVENTIONALLY
USED THERAPEUTIC APPROACHES
1,3J. Cerman, 2J. Čáp, 1M. Mareková, 3S. Němeček, 5J. Marek, 4M. Červinka
To investigate the role of apoptosis in the mechanism
of action of conventional pituitary-adenoma treatment options:
1Dept. of Medical Biochemistry, 2Second Dept. of Internal Medicine, 3Center of Experimental Neurosurgery, and 4Dept. of Medical Biology and Genetics: Faculty of Medicine in Hradec Kralové; 53rd Dept. of Internal Medicine: 1st Faculty of Medicine in Prague. Charles University in Prague; Czech Republic.
Results
(1) Histological examination of somatostatin analogue-treated adenomas: Classical histological examination revealed regressive changes (Fig. 6). Considerable fibrosis was 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 GH3 cell line 72 hours after irradiation by a dose of 20 and 50 Gy. Cells accumulate in G2/M phase of cell cy1515cle 24 hours after irradiation and this cell-cycle arrest lasted for at least 10 days. In the corticotroph AtT20 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 GH3 cell line 15(Fig. 11). No radioprotective effect (suggested recently in a retrospective clinical study) was demonstrated.
Materials and methods 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 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 M30, i.e., caspase-cleaved formalin-resistant neo-epitope of cytokeratin-18, and cleaved 17 kDa 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: GH3 (rat somatotrophs) and AtT20 (subclone D16v-F2 of mouse corticotrophs). Flow-cytometry: analysis of cell cycle and detection of apoptosis (subdiploid peek) after propidium iodide staining.
Morphological changes typical of apoptosis were evaluated.
IONISING RADIATION: 60Co (dose rate 3 Gy/min, similar to that used during gamma-knife procedure)
Fig. 4. ICC detection of CK-18 and
Fig. 5. apoptotic M30 marker in acromegalic adenoma tissue. A case with high incidence of apoptosis
CASPASE-3 5 4
CK-18
M-30
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. 3 2 1
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. 9. Flow-cytometric analysis of DNA content of AtT20 and GH3 cell lines after irradiation (6 and 48 h, resp.). G1 - cells in G0/G1 phase, G2 - cells in G2/M phase (duplex amount of DNA), APO - apoptotic cells (subG1 peak). Note arrest in G2 phase in GH-3 cells.
Control
50 Gy
200 Gy
APO
55% 0%
20 Gy 1%
11%
12%
AtT20, 6h
GH3, 48h G1 G2 9
Fig. 8. The Ki-67 labelling index in treated and untreated adenomas, respectively. The boxes express median (middle horizontal line) and 25th to 75th percentile. The bars show the range. The difference was statistical significant (p = 0.049).
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 AtT20 cell line after 6 hours, while the cell cycle arrest in G2 phase dominated in the GH3 line. SSA have no radio-protective effect. 11
Fig. 11. Flow-cytometric analysis of DNA content of GH3 cell line 10 days after irradiation (20 and 50 Gy). Red numbers = percentage of apoptotic cells (subG1 peak). The presence of somatostatine 10-8 mol/l enhances radiation induced apoptosis.
Fig. 10. ICC detection of caspase-3 in cell line AtT20. Left: 6 hours after irradiation (50 Gy - 3Gy/min), right: control. SA-B with DAB as chromogen.
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. 7 8
16.4%
33.2%
3.0%
10.7%
0.2%
Somatostatin
0 Gy 6
Conclusions
Table : Numbers of adenomas in the treated and untreated group with
presence of significant fibrosis and apoptosis.
Fibrosis and apoptosis
*p value of difference between treated and untreated group
• (1) ionizing radiation - and • (2) somatostatin analogues
• A i m s • 10
Caspase-3