Eur J Clin Chem Clin Biochem 1996; 34:23-30 © 1996 by Walter de Gruyter · Berlin · New York

Analysis of the In Vitro Secretory Activity of Human Pituitary Adenomas:Modification of Corticotropin Releasefrom Adenoma Tissue Explant Culturesby Addition of a Human Plasma Ultrafiltrate Bioactive Fraction

Neven Zarkovic1*2, Marianne Hayn2, Vesna Plavsic3, Kamelija Zarkovic4, Josko Paladino5, Nevenka HirsI1,Jasminka Golubic1, Simon Mikulandra6, Dunja Rogic3, Branka Salzer1, Biserka Pokric1, Rudolf J rg Schaur2,Franz Tatzber2, Heinz Faulhammer*, Bojan Benko9, Walter Dietrich10, Mislav Jurin1 and Mirko Korsic6

1 "Rudjer Boskovic" Institute, Zagreb, Croatia2 Institute of Biochemistry, Graz, Austria3 Clinical Hospital Centre "Rebro", Laboratory of Endocrinology, Zagreb, Croatia4 Clinical Hospital Centre "Rebro", Institute of Neuropathology, Zagreb, Croatia5 Clinical Hospital Centre "Rebro", Clinic for Neurosurgery, Zagreb, Croatia6 Clinical Hospital Centre "Rebro", Clinic for Internal Medicine, Zagreb, Croatia7 Clinical Institute of Laboratory Diagnosis, University Clinic "Rebro", Zagreb, Croatia8 Institute of Biochemistry, Bayreuth, Germany9 Institute of Immunology, Zagreb, Croatia

10 University of Oldenburg, Oldenburg, Germany

Summary: The lack of control of tumour behaviour is manifested in different ways, depending primarily on thetype of tumour. This results in numerous problems of tumour diagnosis and therapy. In the case of "benign"tumours, like pituitary adenomas, in vitro studies are often used for evaluation of the tumour. The use of tissueexplant cultures of human pituitary adenomas and the comparison of the feature of cultured tumours with theirbehaviour in vivo showed that corticotropin is released not only from the tumours associated with Gushing^ disease,but also from clinically non-functioning tumours. Hence, it was supposed that the release of corticotropin in vivofrom non-secreting tumours is probably under the influence of certain neuroendocrine and/or systemic humoralfactors. To test this possibility, samples of 22 tumours were cultured in plain culture medium or in the presenceof the "human plasma ultrafiltrate bioactive fraction" (tentatively termed as TBP) prepared by anion-exchangechromatography. In the presence of TBP the release of corticotropin was strongly inhibited in adenomas showingrelatively high spontaneous secreting activity in vitro (> 200 ng/1 in 24 hours), while immunohistochemistry ofthese tumours indicated accumulation of corticotropin inside the cells. In contrast, TBP stimulated corticotropinrelease from tumours that showed relatively low basic corticotropin release (< 200 ng/1 in 24 hours), with noobvious change in cellular corticotropin immunoreactivity. Such a dual activity of TBP was not observed for 8samples of adenomas cultured in the presence of surrounding pituitary tissue, probably because TBP did not affectcorticotropin secretion by the normal pituitary cells (as indicated by immunohistochemistry). From these results, itappears that TBP could be one of the humoral factors involved in the regulation of corticotropin release frompituitary adenoma tissue. Its possible involvement in the regulation of corticotropin release from normal pituitarytissue, however, is uncertain.

Introduction further variability of morphological features and hor-Ό ,. , , . .. .. .. ^ mone secreting activity (4-8). This in vitro analysis ofBy measuring blood hormone concentration, pituitary .fc, - . . , .,

, ι MT j . · « * - » / r *he characteristics of pituitary tumours also provides aadenomas are classified m vivo as "non-secreting" (elm- , . - . ,F . . ~ ~ i_ · L u. „ ο Α. . . ν , f - Λ basis for research on the activity of factors that might beically non-functioning tumours) or as hormone-secreting . J . *

• u· u · j r · ι Λ· «, κ involved m the regulation of tumour behaviour (9, 10).adenomas which cause recognised clinical disturbances 6 v

(galaetorrhoea and oligomenorrhoea, acromegaly, Cush- However, it is not known whether adenoma cells areing's disease, etc.) (1—3). The use of in vitro cultures of subject to control by the neuroendocrine system. Thepituitary adenomas and comparison of the features of the nature of factors that might be involved in the regulationcultured tumours with their behaviour in vivo revealed of hormone secreting activity of pituitary adenomas is

24 Zarkovic et al.: Release of corticotropin from pituitary tumours in vitro

also unknown. Thus, tumours associated with clinicalevidence of hormone excess show high hormone secret-ing activity if cultured in vitro, but clinically "non-func-tioning" tumours also display a low level of hormonesecreting activity in vitro (4, 6). Most of clinically "non-functioning" tumours, null cell adenomas and oncocyto-mas secret follitropin and lutropin in vitro, but occa-sional tumours (like normal pituitary gland) release so-matotropin, prolactin and corticotropin, as well (6).Similarly, we have noticed that in pituitary adenomascultured in vitro for 24 hours as tissue explant cultures(intact samples of the tumourous tissue), the release ofcorticotropin does not correspond to the hormonal activ-ity of the tumours in vivo (11).

Hence, it seems that the secretory activity of pituitary tu-mours may be influenced by systemic regulatory mecha-nisms, probably mediated by hypothalamic as well as bycertain humoral factors. To test this possibility and to fur-ther evaluate the hormone secreting activity of pituitarytumours, their sensitivity to the "bioactive fraction of thehuman plasma ultrafiltrate", tentatively termed as TBP1),(12) was analysed in vitro.

Materials and MethodsPatients

Pituitary adenomas of 22 patients were analysed (tab. 1). Elevenpatients (5 male and 6 female) 25-76 (52 ± 18) years old hadclinically non-functioning tumours. All of them had visual distur-bances and headaches, and preoperative CT imaging scans demon-strated a macroadenoma. Basal serum levels of hormones were inthe reference range, except prolactin which was elevated in 5 pa-tients (60.7 ± 20.9 μg/l) due to the stalk compression.

Five female patients, 29-40 (30 ± 5) years old, presented withthe syndrome of galactorrhoea and amenorrhoea associated withelevated serum prolactin levels (185.2 ± 59.1 μg/l). Three men,28-57 (42 ± 14) years of age, also had elevated serum prolactinlevels (274.1 ± 36.4 μg/l). In these two groups of patients the se-rum corticotropin concentrations were not elevated. However, thecorticotropin values were increased in the sera of 2 patients withmanifested Cushing's disease (one male and one female, 31 and66 years old respectively).

All the patients underwent pituitary operation, and subsequent mor-phological evaluation of the tumours revealed 10 null-cell adeno-mas, 8 sparsely granulated "prolactin cell" adenomas, two cortico-tropin cell adenomas, one oncocytoma and one mixed denselygranulated somatotropin- and sparsely granulated prolactin-cell ad-enoma. Samples of the tumours were cultured in vitro, and lateranalysis showed that 8 of them contained pituitary gland tissue,while the remaining 14 adenomas showed no indication of the pres-ence of pituitary gland.

Hormone assay

Corticotropin was determined in sera and culture media by IRMA,using the kit of CIS bio international (France). Coefficients of vari-ation were below 10% and the detection limit for corticotropin was2 ng/1 (reference plasma values were 10-75 ng/1). Background val-ues of corticotropin determined for the plain culture media used asa control were < 2 ng/1.

TBP = Tumour Basic Protein

Morphological studiesA portion of surgically removed pituitary adenoma was fixed in 40g/1 buffered paraformaldehyde, dehydrated in graded ethanol, andembedded in paraffin. An identical fixation procedure was appliedto the adenoma explants obtained after surgical removal of the tu-mour, which were also used for the in vitro studies. Paraffin sec-tions of 4 to 6 μηι thickness were stained with hematoxylineosin,Mallory 3-chrome and the periodic acid-Schijff technique. For theimmunohistochemical detection of the pituitary hormones, the im-munoperoxidase technique was used. Antisera for the followinghormones were used: thyrotropin (diluted 1 :500), prolactin(1 :300), corticotropin (1 :400), somatotropin (1:500), follitropin(1:150) and lutropin (1:700) (all by DAKO (Danmark)). Normalpituitary tissue obtained at the autopsy served as positive control.The specificity of each primary antiserum was previously verifiedby adsorption with its respective primary antigen. For the electronmicroscopic study, the portion of the adenoma tissue was fixedin buffered 40 g/i paraformaldehyde, processed in buffered 20 g/1osmium tetroxide, then embedded in Epon 812. Semithin sections,stained with toluidine blue, were examined to select the areas ap-propriate for the ultrastructural study. The ultrathin sections of 15nm stained with uranyl-acetate and lead-citrate, were studied in anOpton-Zeiss electron microscope EM 9S-2.

Preparation of the bioactive fraction of the humanplasma ultrafiltrate

For the preparation of the bioactive fraction of the human plasmaultrafiltrate (tentatively termed as TBP!)) the pooled EDTA-plasma of 49 healthy, male (20-35 years old) blood donors(hepatitis and HIV negative) was used. A sample of 1 1 wasrepeatedly filtered on an Amicon membrane ultrafiltration systemusing a 30000 MT cut-off membrane (Amicon, Ireland) undernitrogen pressure, in a stirred ultrafiltration cell. The eluant wasfurther repeatedly applied to the same ultrafiltration system usinga 3000 Λ/Γ cut-off membrane. The final retentate of 50 ml volumewas lyophilised and 5 mg of the lyophilisate was used for furtherpurification by anion exchange fast protein liquid chromatogra-phy (FPLC). Anion-exchange chromatography was performed onFPLC, Mod. LCC 500 (Pharmacia, Sweden) using a 1 ml Re-source-Q-column (Pharmacia, Sweden) equilibrated with 10mmol/1 Tris-buffer (pH 8.0).

Lyophilised 3000-30000 MT plasma ultrafiltrate was dissolved inwater (5 mg powder per 0.5 ml Tris H2O) and applied to the col-umn. Column elution was started with 5 ml of Tris-buffer, followedby a salt gradient of 0.5 ml NaCl in the same buffer within 30 min,increasing finally to 1 mol/l NaCl in 5 min (chromatogram in figure1). Two ml of fractions were collected and biological activity ofall the fractions was tested in vitro. According to its "immunomo-dulating" activity for cultured human lymphocytes (12), a particu-lar fraction (No. 9-TBP; eluted after 12 ml) was chosen for furtheranalysis. This was lyophilised and kept at + 4 °C. Further analysesof TBP composition (fig. 2) using SMART Mini-Q anion-ex-changer sepharose (Pharmacia, Sweden) revealed that TBP behavesas a "double-peak" compound with a dominating sharp peak elut-ing at approximately 35% of solvent B (10 mmol/1 Tris/HCl + 250mmol/1 NaCl). Due to lack of material, further purification andanalysis of TBP have not yet been possible.

For experimental purposes the sample of TBP was dissolved insaline, filtered through a 500 Mr cut-off membrane using saline aselution liquid. The retentate was passed through a 0.22 μηι filter(Millipore, USA) and aliquots were kept at -20 0C, until used invitro at a concentration of 100 g/I plasma equivalent concentration(approximately 5 μg/l).

Adenoma tissue explant culturesPituitary adenoma tissue was collected in iced saline with penicillinand streptomycin at the time of operation and washed twice withthe same solution. Afterwards, the weight of the wet tissue wasmeasured under sterile conditions, and the specimen was dissectedinto equal pieces of approximately 4-6 mm3 size. The weight ofthe tissue particles was measured again and the samples were

Zarkovic et al.: Release of corticotropin from pituitary tumours in vitro 25

Tab. 1 Characteristics of patients and pituitary adenoma tissue explants used for in vitro analysis.

Patient -sex/age

1. ?/652. <J/653. ?/654. ?/285. <J/286. ?/387. ?/288. ?/709. ?/37

10. ?/2511. c?/3312. ?/7013. <J/7614. ?/5115. ?/31

16. <J/57

17. cJ/41

18. <J/37

19. cJ/41

20. <?/66

21. ?/40

22. ?/28

Clinical findingsof adenomasecretory activity

non-functioningnon-functioningprolactinomaprolactinomaprolactinomanon-functioningprolactinomanon-functioningacromegaly

non-functioningnon-functioningnon-functioningnon-functioningnon-functioningCushing's disease

prolactinoma

prolactinoma

non-functioning

non-functioning

Cushing's disease

prolactinoma

prolactinoma

Pathological diagnosis of theoperated tumour specimen

null-cell adenomanull-cell adenomasparsely granulated prolactinomasparsely granulated prolactinomasparsely granulated prolactinomanull-cell adenomasparsely granulated prolactinomanull-cell adenomamixed densely granulated somatotropin

cell adenoma and sparsely granulatedprolactinoma

null-cell adenomanull-cell adenomanull-cell adenomaoncocytomanull-cell adenomacorticotropin-cell adenoma associated

with Gushing 's diseasesparsely granulated prolactinoma

sparsely granulated prolactinoma

null-cell adenoma

null-cell adenoma

corticotropin cell adenoma associatedwith Cushing's disease

sparsely granulated prolactinoma

sparsely granulated prolactinoma

Serumcorticotropin(ng/1)

12.28.5

12.322.519.99.9

12.916.31.5

16.524.828.1

1.50.6

228.6

27.6

7.0

11.3

22.1

221.0

17.4

31.7

Morphologyof tissue explant cultures*

chromophobic adenomachromophobic adenomachromophobic adenomachromophobic adenomachromophobic adenomachromophobic adenomachromophobic adenomachromophobic adenomachromophobic adenoma

chromophobic adenomachromophobic adenomachromophobic adenomaoncocytomachromophobic adenomachromophobic adenoma

mixed with pituitary glandchromophobic adenoma

mixed with pituitary glandchromophobic adenoma

mixed with pituitary glandchromophobic adenoma

mixed with pituitary glandchromophobic adenoma mixed

with pituitary glandchromophobic adenoma mixed

with pituitary glandchromophobic adenoma mixed

with pituitary glandchromophobic adenoma mixed

with pituitary gland

* — there were no major differences of the tissue morphology observed between the experimental andthe respective control adenoma tissue explant cultures

0.100 T -r 1.00

32 36 40

Elutiph volume [ml]

Fig. 1 Preparative anion-exchange liquid chromatography of thehuman plasma 3000-30000 MT membrane ultrafiltrate. Lyophili-sed 3000-30000 Mr. plasma ultrafiltrate was dissolved in water(5 mg powder/0.5 ml tris H2O-solvent A), applied to the columnand eluted with 5 ml of Tris-buffer followed 'by a salt gradient of0.5 ml NaCl in the same buffer (solvent B = solvent A + 1 mol/1NaCl) within 30 min, finally increasing to 1 mol/1 NaCl in 5 min.* — Bioactive fraction (No. 9) used for the in vitro experiments;tentatively termed as TBP.

placed in plastic Petri dishes (Greiner, Germany) containing Dul-becco's Modified Eagle's Medium Ham's F-12 supplemented withfetal calf serum (volume fraction 0.1) and 1 mU/l penicillin and1 mg/1 streptomycin. The culture medium of the tissue explant cul-tures treated with TBP contained the sample of bioactive plasmaultrafiltrate fraction at 100 g/1 plasma equivalent. The volume ofthe medium used for tissue explant cultures depended on theweight of adenoma tissue, thus the ratio was 2 ml medium per10 mg tissue. Tissue explants were first incubated at 37 °C in hu-midified air containing 5% COi for 1 hour without any treatment(for the comparison of the basic hormone secreting activity whichappeared to be equal for equivalent samples of the same tumour)and then for 24 hours in the plain medium or in the medium con-taining TBP.

Afterwards, the medium was removed and stored at -20 °C untilradioimmunoassay, while the tissue was fixed for morphologicalanalysis as described. Simultaneously, the same amount of mediumwas incubated in the same way without any pituitary tissue, fordetermination of the corticotropin background.

Statistics

Differences between the absolute values of corticotropin deter-mined in the culture media of the control and TBP incubatedadenoma tissue explants were evaluated by the Mann-Whitney-U test. Modulation by TBP of the corticotropin secretory activityof the pituitary adenomas in relation to their basic hormonesecretory activity was evaluated using the Spearman rank corre-lation.

26 2arkovic et al.: Release of corticotropin from pituitary tumours in vitro

Results

The values of corticotropin found in the culture mediaof adenomas that were surgically removed without pitu-itary gland tissue (as revealed by histology, tab. 1) thenincubated in the plain medium or in the medium con-taining TBP are presented in figure 3 and in table 2. Ifthe hormone values determined for the control mediawere relatively high (> 200 ng/1), addition of TBP todie culture medium resulted in a strong decrease of cor-

0.01 τ -r i.oo

GO

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Fig. 2 Analytical anion-exchange liquid chromatography of thebioactive fraction of human plasma 3000—30 000 MT membrane ul-trafiltrate.Lyophilised TBP (amount corresponding to approximately 50 mlof plasma) was dissolved in 25 μΐ of 10 mmol/1 Tris/HCl buffer(pH 8) - solvent A, diluted 1 : 10 by H2O and applied to SMARTMini-Q anion-exchanger Sepharose using a linear 250 mmol/1 NaClgradient in the same buffer (solvent Β = solvent A + 250mmol/1 NaCl).

οc-οωc

σ)c

10000η

1000- Γ ~ ~

ο.ο*-»οοtου

100- Γ > ! ' - -

8 9 10 11 12 13 14NF A NF NF NF NF NF

Patient

Fig. 3 The effects of TBP on corticotropin release in vitro fromthe pituitary adenomas incubated as tissue explants without pitu-itary gland tissue.Effects of TBP (shaded bars) on corticotropin release in the culturemedium (ng/1 · d) are presented in comparison with the respectiveadenoma incubated in the plain medium only (opened bars).* - patient number (according to the tab. 1) and clinical diagnosisof the respective adenoma secretory activity; NF - non-function-ing, P - prolactinoma, A - acromegaly.

ticotropin concentration (P = 0.0008). In contrast, if thetumours showed a lower capacity for corticotropinsecretion in vitro (< 200 ng/1), addition of TBPincreased their corticotropin secreting activity(P = 0.0283). Thus, for the in vitro high corticotropinsecreting adenomas the inhibition fof the hormone re-lease ranged from 47-99%, while the stimulated hor-mone release observed for the low corticotropin secret-ing tumours was 162—2370% of the respective basiccontrol hormone secreting activity. Furthermore, whileimmunohistochemistry of the control "high corticotropinsecreting" tumours revealed weak corticotropin positi-vity in only one adenoma tissue explant culture, cortico-tropin positivity was observed for almost all those ade-noma explants incubated with TBP, for which inhibitionof the corticotropin in the culture medium was noticed.

Such effects of TBP on the corticotropin secreting activ-ity of the pituitary tumours in vitro were observed onlyfor those tumours removed by surgery without pituitarygland tissue. If the samples of tissue explant culturescontained pituitary gland tissue in addition to adenoma-tous tissue (as revealed by histology, tab. 1) (fig. 4 andtab. 3), addition of TBP1) did not produce any regularand significant effect P > 0.1). Although high cortico-tropin concentrations (> 200 ng/1) were measured in themedium of all these cultures, a moderate decrease (ap-proximately 50% or less) of the corticotropin concentra-tion in the culture medium was noticed for three tissueexplant cultures only. Moreover, for two other samplesof this group TBP was even stimulatory (above 250%of the respective control value), in spite of the high cor-ticotropin concentrations in the control culture medium.Immunohistochemistry of the control tissue explant cul-tures showed moderate to strong corticotropin positivityin four tumours. For all of these, addition of TBP to theculture medium decreased corticotropin positivity in thetumour cells, although there was no obvious change ofthe hormone concentration in the culture medium. Wide-spread corticotropin staining of the normal pituitary tis-sue was noticed in all the tissue explants, in the controlsamples, and in the TBP treated tissue explants.

Furthermore, there was a strong negative correlation(r = -0.92, P < 0.01) between the dependence of thebiomodulating effects of TBP on the corticotropin secre-tory activity of the pituitary adenomas and their basichormone secretory activity (fig. 5); such a correlationwas not observed for the samples of pituitary adenomasincubated with the pituitary gland tissue (r = -0.14,P > 0.1). Thus, while TBP did not influence corticotro-pin release from the tissue explant cultures of thetumours mixed with the pituitary gland tissue, the dualeffect of TBP on corticotropin secretion in vitro, i. e.inhibition of the secreting activity of "high corticotro-pin secreting" tumours and stimulation of corticotropinsecretion of "low corticotropin secreting" tumours was

Zarkovic et al: Release of corticotropin from pituitary tumours in vitro 27

Tab. 2 Comparison of the corticotropin secretory activity and immunohistochemistry(corticotropin positivity) of the pituitary adenoma explants without normal pituitary gland tissue.

Patient -sex/age

1. 9/652. ί/653. 9/274. 9/285. <f/286. ?/387. ?/288. ?/709. 9/37

10. 9/2511. <ί/3312. ?/7013. c?/7614. ?/51

Clinical findingsof adenomasecretory activity

non- functioningnon-functioningprolactinomaprolactinomaprolactinomanon-functioningprolactinomanon-functioningacromegalynon- functioningnon-functioningnon:functioningnon-functioningnon-functioning

Controladenomatissue explantculture cortico-tropin secretoryactivity (ng/1)

9500.04619.03000.01698.0857.0761.0498.3308.0226.6127.762.142.822.816.5

Relative values ofadenoma tissueexplant culturecorticotropinsecretory activityin presence of TBP(% of the respectivecontrol value)

0.52.92.63.4

13.231.35.9

29.252.3

548.1162.2324.8409.7

2369.7

Immuno-histochemistry(corticotropinpositivity) of theadenoma cells in thecontrol adenomatissue explantcultures

negative*negative+negativenegativenegativenegativenegativenegativenegativenegativenegativenegativenegative

Immuno-histochemistry(corticotropinpositivity) of theadenoma cells in thetissue explantscultured in presenceof TBP

++ + ++ +++ ++ +negativenegativenot donenegativenegativenegativenetativenot done

negative no positive cellsweak positivity (< 5% of the cells)moderate staining (approximately 25% of thecells)strong staining (50-75% of the cells)

According to Katznelson et al. (13) tissue explants were culturedfor 24 hours in the plain medium (control) or in the medium con-taining bioactive fraction (100 g/1 plasma equivalent concentration)of the human plasma ultrafiltrate (TBP).

strong and regular, provided the adenoma tissue wasremoved without traces of the surrounding pituitarygland.

Discussion

The results obtained show that the human plasma ultra-filtrate bioactive fraction (TBP) contains potent factor(s)which regulate(s) secretion of corticotropin from the hu-man pituitary adenoma tissue explants in vitro. The na-

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Fig. 4 The effects of TBP on corticotropin release in vitro fromthe pituitary adenomas incubated as tissue explants in the presenceof pituitary gland tissue.Effects of TBP (shaded bars) on corticotropin release in the culturemedium (ng/1 · d) are presented in comparison with the respectiveadenoma incubated in the plain medium only (opened bars).* - patient number (according to the tab. 1) and clinical diagnosisof the respective adnoma secretory activity; NF — non-functioning,P - prolactinoma; C — Cushing's disease.

ture of the active component of TBP is not yet defined.However, it might be similar to the growth modifyingfactor found as a heat and acid resistant component indifferent tissues and culture media (12, 14, 15). Similarfactors modifying tumour cell behaviour have been de-scribed under various names (depending on the sourceof preparation, method of purification and the bioassayused for determination of the activity) (16—21). The ba-sic principle of the biological activity of TBP is stilluncertain, but it appears to be a ubiquitous factor thatmodifies cellular activity. Due to its ability to passthrough a Mr 30 000 cut-off membrane and inability topass through a MT 3000 cut-off membrane, the Mr ofTBP should be within the range of numerous bioactivepeptides and low Μτ proteins (20, 22). TBP cannot be anon-specific very low Mr component (like hydrocorti-sone, cAMP, etc.) since the MT of such factors is below3000. Thus it is not yet known, whether TBP is a newbioactive plasma (and/or tissue) factor. Preliminary dataindicate that TBP could also influence the reactivity ofthe human peripheral blood mononuclear cells to phyto-haemagglutinin, stimulating the reactivity of poorly re-active cells and inhibiting the reactivity of highly re-active cells (12).Similarly, the results show that the effects of TBP oncorticotropin release from the cultured pituitary adeno-mas depended on the basal corticotropin secreting activ-ity of the adenomas. Thus, TBP inhibited (on averagemore than 90%) corticotropin release from the adeno-mas that exerted relatively high basic hormone release

28 2arkovic et ah: Release of corticotropin from pituitary tumours in vitro

(200 ng/1 in 24 hours appeared to be the minimal re-leased control corticotropin value which was inhibited).In contrast, TBP stimulated corticotropin release (onaverage up to four-fold) by the adenomas that exertedrelatively low (< 200 ng/1 in 24 hours) basic hormonerelease. Furthermore, almost opposite results were ob-tained for corticotropin immunohistochemistry and cor-ticotropin concentration in the medium of the controland TBP treated "high corticotropin secreting" adeno-mas, indicating that TBP inhibition was actually pre-vented of the release of the hormone into the culture me-dium.

In tissue explant cultures of the "low corticotropinsecreting" tumours, increase of corticotropin concentra-tion in the culture medium was not associated with anyobvious change of the cellular corticotropin immunore-activity. Hence, either the TBP activity mechanism wasdifferent for the high and for the low corticotropin secre-^ting (or releasing) adenomas, or immunohistochemistrycould not reveal corticotropin in the "low corticotropinsecreting" tumours.

Corticotropin in TBP cultures,percent of control

Fig. 5 Dependence of the effects of TBP on the corticotropin se-cretory activity of the cultured pituitary adenomas in relation totheir basic cortieotropin secretion in vitro.A - samples of the pituitary adenomas cultured without pituitarygland; B - samples of the pituitary adenomas cultured with sur-rounding pituitary gland tissue.The relative effects of TBP (expressed .as percentage of the hor-,monal activity measured for the same'tumours incubated in theplain culture medium) were compared by using the Spearmanrank correlation.

Zarkovic et al.: Release of corticotropin from pituitary tumours in vitro 29

Such effects of TBP were not observed if the adenomasamples contained pituitary gland tissue. That was a sur-prising finding, since most of these samples showed avery high basic corticotropin secretion, so that a rela-tively strong inhibition by TBP could be expected. Thereare at least two possible explanations for this ineffec-tiveness of TBP. Thus, either TBP could not suppresscorticotropin release from the pituitary gland in thesetissue explant cultures, or it could not suppress cortico-tropin release from the adenoma tissue, due to interfer-ence by the tumour and pituitary gland corticotropinsecretion.

In favour of the first possibility is the presence of highcorticotropin in the culture media of the normal pituitarygland (23) and widespread immunohistochemical positi-vity to corticotropin in the normal pituitary surroundingadenoma tissue, which was not influenced by TBP treat-ment. The finding of decreased immunohistochemicalpositivity of the tumour cells after treatment with TBP,with no effect on corticotropin concentration in the cul-ture medium of the adenomas incubated with the sur-rounding pituitary gland, could indicate that TBP has adifferent effect on normal pituitary and adenoma cells,i. e. stimulating the release of corticotropin from the tu-mour cells, but not affecting corticotropin metabolismin the normal cells. However, this possibility should beverified by further experimental studies.

The second possibility cannot be excluded, since it wasnot possible to distinguish release of corticotropin fromthe adenoma and corticotropin release from the sur-rounding pituitary gland tissue. Moreover, cell to cellcommunication is of high importance in the regulationof corticotropin secretion (24), and the integrity of thetumourous and surrounding pituitary tissue was pre-served during incubation of these tissue explants.

On the other hand, it is possible that TBP influencedthe hormonal activity of "multipotential pituitary cells"

which mainly contain corticotropin and play an impor-tant role in the regulation of hormone secretion by theother pituitary cells (25).

Furthermore, post-surgical stress (removal of the pitu-itary tumour) as well as growth factor synthesis by thetumourous tissue in vitro modulate hormone secretion(26-28). It is possible that TBP (bioactive fraction ofthe human plasma ultrafiltrate) contains factor(s) in-volved in the stress reaction or paracrine regulation ofthe growth factor synthesis, thus indirectly affecting thehormone release, but these possibilities have to be fur-ther evaluated. Finally, it is not impossible that TBP, likesome other bioactive peptides (29-35), is involved inregulation of adenylate cyclase activity and/or calciummetabolism, thereby acting as a regulator of the hor-monal activity of the normal and tumourous pituitary tis-sue.

From the present results, however, it seems that TBPcould be the humoral factor involved in the regulationcorticotropin release from the pituitary adenoma tissue,but not in the regulation of corticotropin release fromnormal pituitary tissue. To verify this possibility, itwill be necessary not only to analyse the nature ofthis bioactive component of the human plasma ultrafil-trate but also to use the advantages of the tissueexplant culture method to evaluate the effects of TBPon the hormone release from the normal pituitarygland, as well as on the growth features of normaland tumour cells.

AcknowledgementsThis study was supported by Croatian Ministry of Science andTechnology, Austrian Science Foundation (FWF) and InternationalAssociation for Cancer Research (AICR). The authors would liketo express their sincere gratitude to Biomedica (Austria) and Mr.Herbert Zakarias for collaboration.

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Received January 23/May 29, 1995

Corresponding author: Dr. Neven Zarkovic, Rudjer BoskovicInstitute, Bijenicka 54, HR-10000 Zagreb, Croatia