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The interaction of steroid hormones with methylcholanthrene in the rat prostate gland grown in organ culture
Europ. 07. CatwerVol. 1, pp. 289-293. Pergamon Press 1965. Printed in Great Britain
The Interaction of Steroid Hormones with Methylcholanthrene in the Rat Prostate Gland Grown in Organ Culture ILSE LASNITZKI
Strangeways Research Laboratory Cambridge, England
treatment relative to the exposure to the carcinogens, to variations in general systemic factors between different groups of animals or, most important of all, to the interference of other hormones present in the organism. In culture these complications are largely eliminated, the variables can be isolated and the effect of individual hormones and their interaction with carcinogens studied under controlled experimental conditions. In earlier work [7, 8] the effect of methylcholanthrene alone on the rat prostate gland in organ culture has been studied. The carcinogen induced a rapid and extensive hyperplasia of the prostatic epithelium which persisted after transfer to control medium. Recently the influence ofoestrone, oestradiol, testosterone and hydrocortisone on the effect of methylcholanthrene has been examined in organ cultures of the ventral prostate gland of the adolescent rat. These hormones play an important role in the maintenance of this organ, its growth and function depend on the presence of androgenic hormones and are impaired by exposure to oestrogens.
INTRODUCTION IT Is well known that the development and growth of certain h u m a n tumours such as the carcinoma of the cervix, of the m a m m a r y gland and of the prostatic gland are connected with changes in the hormonal status of the host, but the exact nature of" the hormonal action is not known. The role of hormones in the experimental induction of animal tumours by chemical carcinogens has been widely investigated but has not produced clear cut results. Dunning et al. [1] found that methylcholanthreneinduced tumours of the rat prostate gland grew equally well in female and male hosts, Mirand and Staubitz [2], on the other hand, reported that such tumours could be transplanted more successfully into female rats and Allen [3] obtained a higher incidence of prostatic tumours in castrates than in intact rats or in castrates treated with testosterone. The influence of corticosteroids on the induction of tumours has also been studied. Asboe-Hansen [4] and Baserga and Shubik [5] showed that cortisone delayed tumour production in mouse epidermis painted with DMBA or methylcholantlhrene while Green and Savigear [6] claimed that cortisone did not reduce mitosis in mouse epidermis made hyperplastic with DMBA. These contradicl;ory results may have been due to differences in the timing of the hormonal
MATERIALS AND M E T H O D S Ventral prostate glands obtained from 6 to 10 week old rats were grown in natural and semidefined medium by techniques described in detail elsewhere [7]. One group of explants was treated with oestrone, testosterone or 289
290
Ilse Lasnitzki Table 1. The influence of oestrogens, testosterone and hydrocortisone on the effect of methyleholanthrene ( M C ) in organ cultures of the rat prostate grown in natural medium. Treatment
MC MC MC MC
Total number of cultures
only for 11 days and oestrone for 11 days and testosterone for 11 days and hydrocortisone for 11 days
M C only for 11 days followed by control medium for 6 days M C only for 11 days followed by oestrone or oestradiol for 6 days M C for 11 days followed by testosterone for 6 days M C for 11 days followed by hydrocortisone for 6 days *
Number of treated cultures showing various degrees of hyperplasia* 0
I
37 16 11 18
4 2 7 16
13 0 3 2
II 17 6 1 0
III 3 8 0 0
21
1
2
9
9
15
0
0
7
8
12
0
12
0
0
10
0
4
3
3
0---no hyperplasia I--less than half the number of alveoli show mild hyperplasia I I - - h a l f to two-thirds of the alveoli show medium to marked hyperplasia I I I - - m o s t of the alveoli show marked hyperplasia.
hydrocortisone alone and fixed after 11 days' cultivation;
another
group
was
exposed
the carcinogen for 11 days and then transferred to medium containing hormone for an additional 6 days. These were compared with explants pretreated with MC and maintained in control medium for 6 days. (also see Tables 1 and 2). The explants were fixed with 3% acetic Zenker's solution, embedded in paraffin, serially sectioned at 6 ~t and stained by the periodic acid Schiff reagent after diastase digestion.
to
methylcholanthrene alone. This latter group was subdivided into two sets, one of which was fixed after 11 days' growth in the presence of the carcinogen, while the other was treated with methylcholanthrene (MC) for 11 days and transferred to natural control medium for an additional 6 days. Another series was exposed to three different carcinogen-hormone combinations. MC (4 Ixg/ml) and oestrone (4 ~tg/ml) or oestradiol (1.5 lxg/ml). MC (4 ~g/ml) and testosterone (30 lag/ml). MC (4 ~tg/ml) and hydrocortisone (7 I~g/ml). One set of this series was treated with the carcinogen and hormone simultaneously, fixed after 11 days' cultivation and compared with explants grown with the carcinogen alone. In another set the cultures were first treated with
RESULTS Controls The prostate gland consists of alveoli lined with one row of slightly folded cuboidal or columnar secretory epithelium (Fig. 1). After explantafion into medium without additional hormones the epithelium undergoes regressive
Table 2. The influence of oestrone, testosterone and hydrocortisone on the effect of methylcholanthrene ( M C ) in organ cultures of the rat prostate gland grown in semi-defined medium. Treatment
MC MC MC MC *
only for 11 days and oestrone for 11 days and testosterone for 11 days and hydrocortisone for 11 days
Total number of cultures 14 10 12 10
Number of treated cultures showing various degrees of hyperplasia* 0 5 0 12 10
I 9 4 0 0
O---no hyperplasia I--less than half the number of alveoli show mild hyperplasia I I - - h a l f to two-thirds of the alveoli show medium to marked hyperplasia I I I - - m o s t of the alveoli show marked hyperplasia.
II 0 5 0 0
III 0 I 0 0
All sections have been stained by the periodic
Fig.
Fig.
1.
2.
acid Schzj’reagent
after diastase disgestion (PAS)
Section through a ventral prostate gland of an 8 week rat before explantation. x 130. folded epithelium.
Note high, slightly
Se.ction through a similar gland grown for 11 days in natural control medium. .Note low e/&helium x 130. and increase of connective tissue.
(to face p. 290)
Fig. 3.
Section through a gland grown for 11 days in natural medium in the Presence of methylcholanthrene, x 130. showing marked hyperplasia of the alveolar epithelium.
Section through a gland grown for 11 days in natural medium in the Presence of me,thylchol, rnthren Fig. 4. and transferred to natural control medium for 6 days. Note persistence of hyperplasia. x 130.
Fig.
5.
Fig. 6.
Section through a gland grown in natural medium for oestrone showing extensive hy$erplasia.
11 days with methylcholanthrene and x 130.
Set:tion through a gland grown for 11 days in natural medium with methybcholanthrene and transferred to medium containing oestradiol. Note hyberplasia.
x 130.
Fig.
7.
Section through a gland treated in natural medium with methylcholanthrene 11 days. .Note absence of hy@@sia. x 130.
and testosterone for
Fig. 8. Section through a gland grownjirst with methylcholanthrene in natural medium and transferred to medium containing testosterone. Or+ two alveoli at the perz$hery show very mild hyperplasia. x 130.
Fig. 9.
Fig.
10.
Sect.ion through a gland treated in natural medium with methylcholanthrene and hydrocorti ‘sane for x 130. I 1 d+ys. Note absence of hyperplasia and high epithelium.
Section through a gland treatedjirst with methylcholanthrene and transferred to medium containing hydrocortisone. Note both normal alveoli and hyperplastic foci. x 130.
Interaction of Hormones with Methylcholanthrene
291
changes. The alveolar lumen becomes narrow and straight and the cells become lower and cease to secrete. In the natural medium this regression is accompanied by an increase in the connective tissue (Fig. 2).
or oestradiol for 6 days, the hormones also enhanced the effect of methylcholanthrene; a greater number of cultures showed hyperplasia which was more extensive than in controls grown without hormones (Table 1, Fig. 6).
Effect of Hormones
Effect of methylcholanthrene and testosterone
Addition of oestrone or oestradiol to the medium accelerated the atrophy of the epithelium in some alveoli, in others it produced a slight epithelial hyperplasia. In contrast, treatment with either testosterone or hydrocortisone prevented the regression of the epithelium observed[ in the control medium, and the alveoli were lined with tall actively secreting elements.
Simultaneous application of the two compounds drastically reduced the incidence and the extent of hyperplasia induced by the carcinogen alone. In the natural medium hyperplasia was present in only four out of eleven explants; in three of these it was very mild and increased cell proliferation was seen in only one explant. In the remaining seven cultures all alveoli were lined with one row of secretory ceils (Fig. 7, Table I). In the semi-defined medium hyperplasia was 'completely absent in all explants (Table 2). Transfer of carcinogen-treated tissue to medium containing testosterone for a further 6 days also diminished the hyperplastic changes. Thus, in most cultures only a few peripheral alveoli showed mild hyperplasia (Fig. 8, Table
Effect of methylcholanthrene These experiments confirmed the results of earlier work [8]. In the natural medium the carcinogen induced[ a rapid multiplication of the alveolar cells in most treated cultures (Table I); layers of crowded cells were formed which partially or completely filled the alveolar lumen (Fig. 3). The superficial cells continued to secrete but at a later stage they were shed and replaced by flat non-secretory elements. After the transfe, r of the tissue to control medium the hyperplasia became more marked (Fig. 4). Thus, all except one explant showed hyperplastic loci and in a greater number the hyperplasia was more extensive than in explants exposed to methylcholanthrene for 11 days only (Table 1). In the semi-defined medium the carcinogen was less effective, the epithelial cells in a few alveoli had multiplied to form not more than 2-3 layers and a smaller proportion of explants were affected (Table 2).
Effect of methylcholanthrene and oestrogens During simultaneous treatment in the natural medium, the hormone increased the effect of the carcinogen to a moderate degree. A similar proportion of treated cultures as those grown with MC alone showed hyperplasia but in most of these the changes were more extensive (Table 1, Fig. 5). In the semi-defined medium, however, the action of the hormone was much more pronounced; more treated cultures showed hyperplasia and in most of them cell multiplication was active (Table 2). In both media the secretory ceils at the lumen were shed and replaced by flat cells at an earlier stage than in the MC controls. In explants pretreated with the carcinogen and transferred to medium containing oestrone
1). Effect of methycholanthrene and hydrocortisone Simultaneous treatment was followed by an almost complete suppression of epithelial hyperplasia (Table 1). In the natural medium hyperplasia was absent in sixteen out of eighteen treated explants and in the remaining two only a very few alveoli at the periphery showed an increase to 2-3 rows of cells; elsewhere the alveoli were lined with a single layer of tall columnar epithelium (Fig. 9). In the semi-defined medium hyperplasia was absent in all explants (Table 2). Transfer of pretreated cultures to medium containing hydrocortisone for 6 days was also followed by a reduction 6f cell proliferation, but the inhibition of growth was less complete than after simultaneous application (Table 1). In three out of ten explants hyperplasia was still extensive, three showed medium and four mild hyperplasia (Fig. 10).
DISCUSSION The results show that methylcholanthrene induces a rapid and extensive hyperplasia of the prostatic epithelium in the natural medium and a mild hyperplasia in the semi-defined medium. The lower activity of the carcinogen is probably due to its reduced solubility and hence, its lower concentration in the tissue in the protein poor semi-defined medium.
292
Ilse Lasnitzki
The oestrogens enhance the hyperplasia to a moderate degree in the natural medium and considerably so in the semi-defined medium where the MC induced hyperplasia is mild. The hormones also accelerate the dedifferentiation of the epithelium. Testosterone or hydrocortisone, on the other hand, suppresses or inhibits the hyperplasia in both media and prevents the regression of the epithelium. Although the promotion or suppression of hyperplasia varies in degree in the natural and the semi-defined medium, the direction of hormonal action is identical under both conditions. This suggests that it is independent of the traces of other hormones or substances present only in the natural medium. Possible mechanisms for these effects were considered. The promotion or inhibition of epithelial hyperplasia may be related to the degree of differentiation of the cells, in the sense that only cells which have lost their functional activity, i.e. in explants grown in control medium or in medium containing oestrogens are susceptible to the carcinogen, whereas epithelium which is maintained in an actively secreting state by either testosterone or hydrocortisone is unable to respond. A direct interaction of the carcinogen with the hormones cannot be ruled out however.
Heidelberger and his group [9, 10] showed that carcinogenic hydrocarbons are taken up by cytoplasmic key proteins and testosterone or hydrocortisone may either prevent the binding by competing for sites on the protein molecule during simultaneous application, or break the protein-carcinogen bond when given to cultures pretreated with methylcholanthrene. Another possibility involves the lysosomes. Allison and Mallucci [11] found that in cells treated in vitro with carcinogenic hydrocarbons these substances were localised within the lysosomes. The authors contend that the compounds may initiate carcinogenesis by altering the pattern of lysosomal enzymes. Measurements of lysosomal acid phosphatase in the rat prostate in culture [12] indicate that testosterone or hydrocortisone protects the lysosomal membranes while oestrogens increase their fragility. On this basis it could be argued that testosterone and hydrocortisone may impede the entry of the carcinogen into the lysosomes and that the oestrogens facilitate it. Experiments to test this view have begun.
Acknowledgements--I would like to thank Professor Dame Honor Fell, F.R.S. for constructive criticism in the preparation of this manuscript and Mr. G. Lenney, A.I.T.S. for the microphotographs.
RESUME L'influence des oestrog~nes, de la testost#one et de l'hydrocortisone sur l'action du rngthylcholanthr~ne sur la prostate du rat a gtg gtudige en culture organotypique dans des milieux naturels et semi-synthgtiques. Dans le milieu naturel, le mgthylcholanthr~ne provoque une tr~s forte hyperplasie de lYpitMlioma prostatique. Dans le milieu semi-syntMtique, l'hyperplasie est plus mod~rge. L'oestrone et l°oestradiol augmentent l'hyperplasie gpitMliale, tandis que la testosterone et l'hydrocortisone la suppriment dans les deux milieux.
SUMMARY The influence of oestrogens, testosterone and hydrocortisone on the effect of methylcholanthrene on the rat prostate gland grown as organ cultures in natural and semi-defined medium has been examined. The hormones were either administered simultaneously with methylcholanthrene or applied to cultures pretreated with the carcinogen. In natural medium methylcholanthrene alone causes a rapid and extensive hyperplasia of the prostatic epithelium. In the semi-defined medium it causes a mild hyperplasia. In natural medium oestrone and oestradiol increase the hyperplasia to a moderate degree, in the semi-defined medium they enhance it considerably. Testosterone and hydrocortisone inhibit or suppress the hyperplasia in both media.
Interaction of Hormones with Methyleholanthrene ZUSAMMENFASSUNG Der Einfluss yon Oestron, Oestradiol, Testosteron und Hydrocortison auf die Wirkung yon Methylcholanthren wurde in Organkulturen der Rattenvorsteherdriise (Prostata), die in natiMichen und semi-synthetischen Medien geziichtet wurden, untersucht. Methylcholanthren aUein verursacht eine starke Hyperplasie des Prostataepithels. Oestron und Oestradiol steigern diese Wirkung, wiihrend Testosteron und Hydrocortison sie in beiden Medien hemmen. REFERENCES 1. W. F. DUNm_~O, R. R. CURTISand A. SEOALOFF,Methylcholanthrene squamous cell carcinomas of the rat prostate with skeletal metastases and failure of the rat liver to respond to the same carcinogen. CancerRes. 6, 256-62 (1946). 2. E.A. MIRANDand W.J. STAUBITZ,Prostatic neoplasms of the Wistar rat induced with methylcholanthrene. 07. Natl. Cancer Proc. Soc. Exp. Biol. Med. 93, 457-62 (1956). 3. J. M. ALLEN,Responses of the rat prostate gland to methylcholanthrene, 07. Exp. Zool. 123, 289-13 (1953). 4. G. A~BoE-Ha~sEN, Hormonal effects on connective tissues. Connective Tissues Reaction Trans. 5th Conf., New York. J. Macy Junior Foundation, 123-82 (1954). 5. A. BASEROAand P. SHtmIK,The action of cortisone on transplanted and induced tumours in mice. Cancer Res. 14, 12-16 (1954). 6. H. N. GREENand M. SAVIG~.AR,Shock and Neoplasia. Brit. Med. 07. 1, 498-500 (1951'1. 7. I. LASmTZKI,The action and interaction of hormones and methylcholanthrene on the ventral prostate gland of the rat in vitro. 07. Natl. Cancer Inst. In press (196511. 8. I. LAmITZKI,The effect of methylcholanthrene on rat prostate glands grown in natural and semi-defined medium. Cancer Res. 24, 973-76 (1964). 9. C. W. ABETL and C. HEm~.LBERG~R,Interaction of carcinogenic hydrocarbons with tissue. VIII. Binding of tritium labelled hydrocarbons to the soluble proteins of mouse skin. CancerRes. 22, 931-46 (1962) 10. B. C. GIOVANELLA,L. E. MCKINN~.Yand C. HEIDELBERGER,On the reported solubilisation of carcinogenic hydrocarbons in aqueous solutions of DNA. 07. Mol. Biol. 8, 20-27 (1964). 11. A.C. ALLISONand L. MALLUCCI,Uptake of hydrocarbon carcinogens by lysosomes. Nature 203, 1024-27 (1964). 12. I. LASNITZKI,S. WAITEand J. T. DING~-~,The effect of hormones on the lysosomal acid phosphatase in the rat prostate in culture. Brit. Emp. Cane. Camp. Report, 375 (1964).
293
The Interaction of Steroid Hormones with Methylcholanthrene in the Rat Prostate Gland Grown in Organ Culture ILSE LASNITZKI
Strangeways Research Laboratory Cambridge, England
treatment relative to the exposure to the carcinogens, to variations in general systemic factors between different groups of animals or, most important of all, to the interference of other hormones present in the organism. In culture these complications are largely eliminated, the variables can be isolated and the effect of individual hormones and their interaction with carcinogens studied under controlled experimental conditions. In earlier work [7, 8] the effect of methylcholanthrene alone on the rat prostate gland in organ culture has been studied. The carcinogen induced a rapid and extensive hyperplasia of the prostatic epithelium which persisted after transfer to control medium. Recently the influence ofoestrone, oestradiol, testosterone and hydrocortisone on the effect of methylcholanthrene has been examined in organ cultures of the ventral prostate gland of the adolescent rat. These hormones play an important role in the maintenance of this organ, its growth and function depend on the presence of androgenic hormones and are impaired by exposure to oestrogens.
INTRODUCTION IT Is well known that the development and growth of certain h u m a n tumours such as the carcinoma of the cervix, of the m a m m a r y gland and of the prostatic gland are connected with changes in the hormonal status of the host, but the exact nature of" the hormonal action is not known. The role of hormones in the experimental induction of animal tumours by chemical carcinogens has been widely investigated but has not produced clear cut results. Dunning et al. [1] found that methylcholanthreneinduced tumours of the rat prostate gland grew equally well in female and male hosts, Mirand and Staubitz [2], on the other hand, reported that such tumours could be transplanted more successfully into female rats and Allen [3] obtained a higher incidence of prostatic tumours in castrates than in intact rats or in castrates treated with testosterone. The influence of corticosteroids on the induction of tumours has also been studied. Asboe-Hansen [4] and Baserga and Shubik [5] showed that cortisone delayed tumour production in mouse epidermis painted with DMBA or methylcholantlhrene while Green and Savigear [6] claimed that cortisone did not reduce mitosis in mouse epidermis made hyperplastic with DMBA. These contradicl;ory results may have been due to differences in the timing of the hormonal
MATERIALS AND M E T H O D S Ventral prostate glands obtained from 6 to 10 week old rats were grown in natural and semidefined medium by techniques described in detail elsewhere [7]. One group of explants was treated with oestrone, testosterone or 289
290
Ilse Lasnitzki Table 1. The influence of oestrogens, testosterone and hydrocortisone on the effect of methyleholanthrene ( M C ) in organ cultures of the rat prostate grown in natural medium. Treatment
MC MC MC MC
Total number of cultures
only for 11 days and oestrone for 11 days and testosterone for 11 days and hydrocortisone for 11 days
M C only for 11 days followed by control medium for 6 days M C only for 11 days followed by oestrone or oestradiol for 6 days M C for 11 days followed by testosterone for 6 days M C for 11 days followed by hydrocortisone for 6 days *
Number of treated cultures showing various degrees of hyperplasia* 0
I
37 16 11 18
4 2 7 16
13 0 3 2
II 17 6 1 0
III 3 8 0 0
21
1
2
9
9
15
0
0
7
8
12
0
12
0
0
10
0
4
3
3
0---no hyperplasia I--less than half the number of alveoli show mild hyperplasia I I - - h a l f to two-thirds of the alveoli show medium to marked hyperplasia I I I - - m o s t of the alveoli show marked hyperplasia.
hydrocortisone alone and fixed after 11 days' cultivation;
another
group
was
exposed
the carcinogen for 11 days and then transferred to medium containing hormone for an additional 6 days. These were compared with explants pretreated with MC and maintained in control medium for 6 days. (also see Tables 1 and 2). The explants were fixed with 3% acetic Zenker's solution, embedded in paraffin, serially sectioned at 6 ~t and stained by the periodic acid Schiff reagent after diastase digestion.
to
methylcholanthrene alone. This latter group was subdivided into two sets, one of which was fixed after 11 days' growth in the presence of the carcinogen, while the other was treated with methylcholanthrene (MC) for 11 days and transferred to natural control medium for an additional 6 days. Another series was exposed to three different carcinogen-hormone combinations. MC (4 Ixg/ml) and oestrone (4 ~tg/ml) or oestradiol (1.5 lxg/ml). MC (4 ~g/ml) and testosterone (30 lag/ml). MC (4 ~tg/ml) and hydrocortisone (7 I~g/ml). One set of this series was treated with the carcinogen and hormone simultaneously, fixed after 11 days' cultivation and compared with explants grown with the carcinogen alone. In another set the cultures were first treated with
RESULTS Controls The prostate gland consists of alveoli lined with one row of slightly folded cuboidal or columnar secretory epithelium (Fig. 1). After explantafion into medium without additional hormones the epithelium undergoes regressive
Table 2. The influence of oestrone, testosterone and hydrocortisone on the effect of methylcholanthrene ( M C ) in organ cultures of the rat prostate gland grown in semi-defined medium. Treatment
MC MC MC MC *
only for 11 days and oestrone for 11 days and testosterone for 11 days and hydrocortisone for 11 days
Total number of cultures 14 10 12 10
Number of treated cultures showing various degrees of hyperplasia* 0 5 0 12 10
I 9 4 0 0
O---no hyperplasia I--less than half the number of alveoli show mild hyperplasia I I - - h a l f to two-thirds of the alveoli show medium to marked hyperplasia I I I - - m o s t of the alveoli show marked hyperplasia.
II 0 5 0 0
III 0 I 0 0
All sections have been stained by the periodic
Fig.
Fig.
1.
2.
acid Schzj’reagent
after diastase disgestion (PAS)
Section through a ventral prostate gland of an 8 week rat before explantation. x 130. folded epithelium.
Note high, slightly
Se.ction through a similar gland grown for 11 days in natural control medium. .Note low e/&helium x 130. and increase of connective tissue.
(to face p. 290)
Fig. 3.
Section through a gland grown for 11 days in natural medium in the Presence of methylcholanthrene, x 130. showing marked hyperplasia of the alveolar epithelium.
Section through a gland grown for 11 days in natural medium in the Presence of me,thylchol, rnthren Fig. 4. and transferred to natural control medium for 6 days. Note persistence of hyperplasia. x 130.
Fig.
5.
Fig. 6.
Section through a gland grown in natural medium for oestrone showing extensive hy$erplasia.
11 days with methylcholanthrene and x 130.
Set:tion through a gland grown for 11 days in natural medium with methybcholanthrene and transferred to medium containing oestradiol. Note hyberplasia.
x 130.
Fig.
7.
Section through a gland treated in natural medium with methylcholanthrene 11 days. .Note absence of hy@@sia. x 130.
and testosterone for
Fig. 8. Section through a gland grownjirst with methylcholanthrene in natural medium and transferred to medium containing testosterone. Or+ two alveoli at the perz$hery show very mild hyperplasia. x 130.
Fig. 9.
Fig.
10.
Sect.ion through a gland treated in natural medium with methylcholanthrene and hydrocorti ‘sane for x 130. I 1 d+ys. Note absence of hyperplasia and high epithelium.
Section through a gland treatedjirst with methylcholanthrene and transferred to medium containing hydrocortisone. Note both normal alveoli and hyperplastic foci. x 130.
Interaction of Hormones with Methylcholanthrene
291
changes. The alveolar lumen becomes narrow and straight and the cells become lower and cease to secrete. In the natural medium this regression is accompanied by an increase in the connective tissue (Fig. 2).
or oestradiol for 6 days, the hormones also enhanced the effect of methylcholanthrene; a greater number of cultures showed hyperplasia which was more extensive than in controls grown without hormones (Table 1, Fig. 6).
Effect of Hormones
Effect of methylcholanthrene and testosterone
Addition of oestrone or oestradiol to the medium accelerated the atrophy of the epithelium in some alveoli, in others it produced a slight epithelial hyperplasia. In contrast, treatment with either testosterone or hydrocortisone prevented the regression of the epithelium observed[ in the control medium, and the alveoli were lined with tall actively secreting elements.
Simultaneous application of the two compounds drastically reduced the incidence and the extent of hyperplasia induced by the carcinogen alone. In the natural medium hyperplasia was present in only four out of eleven explants; in three of these it was very mild and increased cell proliferation was seen in only one explant. In the remaining seven cultures all alveoli were lined with one row of secretory ceils (Fig. 7, Table I). In the semi-defined medium hyperplasia was 'completely absent in all explants (Table 2). Transfer of carcinogen-treated tissue to medium containing testosterone for a further 6 days also diminished the hyperplastic changes. Thus, in most cultures only a few peripheral alveoli showed mild hyperplasia (Fig. 8, Table
Effect of methylcholanthrene These experiments confirmed the results of earlier work [8]. In the natural medium the carcinogen induced[ a rapid multiplication of the alveolar cells in most treated cultures (Table I); layers of crowded cells were formed which partially or completely filled the alveolar lumen (Fig. 3). The superficial cells continued to secrete but at a later stage they were shed and replaced by flat non-secretory elements. After the transfe, r of the tissue to control medium the hyperplasia became more marked (Fig. 4). Thus, all except one explant showed hyperplastic loci and in a greater number the hyperplasia was more extensive than in explants exposed to methylcholanthrene for 11 days only (Table 1). In the semi-defined medium the carcinogen was less effective, the epithelial cells in a few alveoli had multiplied to form not more than 2-3 layers and a smaller proportion of explants were affected (Table 2).
Effect of methylcholanthrene and oestrogens During simultaneous treatment in the natural medium, the hormone increased the effect of the carcinogen to a moderate degree. A similar proportion of treated cultures as those grown with MC alone showed hyperplasia but in most of these the changes were more extensive (Table 1, Fig. 5). In the semi-defined medium, however, the action of the hormone was much more pronounced; more treated cultures showed hyperplasia and in most of them cell multiplication was active (Table 2). In both media the secretory ceils at the lumen were shed and replaced by flat cells at an earlier stage than in the MC controls. In explants pretreated with the carcinogen and transferred to medium containing oestrone
1). Effect of methycholanthrene and hydrocortisone Simultaneous treatment was followed by an almost complete suppression of epithelial hyperplasia (Table 1). In the natural medium hyperplasia was absent in sixteen out of eighteen treated explants and in the remaining two only a very few alveoli at the periphery showed an increase to 2-3 rows of cells; elsewhere the alveoli were lined with a single layer of tall columnar epithelium (Fig. 9). In the semi-defined medium hyperplasia was absent in all explants (Table 2). Transfer of pretreated cultures to medium containing hydrocortisone for 6 days was also followed by a reduction 6f cell proliferation, but the inhibition of growth was less complete than after simultaneous application (Table 1). In three out of ten explants hyperplasia was still extensive, three showed medium and four mild hyperplasia (Fig. 10).
DISCUSSION The results show that methylcholanthrene induces a rapid and extensive hyperplasia of the prostatic epithelium in the natural medium and a mild hyperplasia in the semi-defined medium. The lower activity of the carcinogen is probably due to its reduced solubility and hence, its lower concentration in the tissue in the protein poor semi-defined medium.
292
Ilse Lasnitzki
The oestrogens enhance the hyperplasia to a moderate degree in the natural medium and considerably so in the semi-defined medium where the MC induced hyperplasia is mild. The hormones also accelerate the dedifferentiation of the epithelium. Testosterone or hydrocortisone, on the other hand, suppresses or inhibits the hyperplasia in both media and prevents the regression of the epithelium. Although the promotion or suppression of hyperplasia varies in degree in the natural and the semi-defined medium, the direction of hormonal action is identical under both conditions. This suggests that it is independent of the traces of other hormones or substances present only in the natural medium. Possible mechanisms for these effects were considered. The promotion or inhibition of epithelial hyperplasia may be related to the degree of differentiation of the cells, in the sense that only cells which have lost their functional activity, i.e. in explants grown in control medium or in medium containing oestrogens are susceptible to the carcinogen, whereas epithelium which is maintained in an actively secreting state by either testosterone or hydrocortisone is unable to respond. A direct interaction of the carcinogen with the hormones cannot be ruled out however.
Heidelberger and his group [9, 10] showed that carcinogenic hydrocarbons are taken up by cytoplasmic key proteins and testosterone or hydrocortisone may either prevent the binding by competing for sites on the protein molecule during simultaneous application, or break the protein-carcinogen bond when given to cultures pretreated with methylcholanthrene. Another possibility involves the lysosomes. Allison and Mallucci [11] found that in cells treated in vitro with carcinogenic hydrocarbons these substances were localised within the lysosomes. The authors contend that the compounds may initiate carcinogenesis by altering the pattern of lysosomal enzymes. Measurements of lysosomal acid phosphatase in the rat prostate in culture [12] indicate that testosterone or hydrocortisone protects the lysosomal membranes while oestrogens increase their fragility. On this basis it could be argued that testosterone and hydrocortisone may impede the entry of the carcinogen into the lysosomes and that the oestrogens facilitate it. Experiments to test this view have begun.
Acknowledgements--I would like to thank Professor Dame Honor Fell, F.R.S. for constructive criticism in the preparation of this manuscript and Mr. G. Lenney, A.I.T.S. for the microphotographs.
RESUME L'influence des oestrog~nes, de la testost#one et de l'hydrocortisone sur l'action du rngthylcholanthr~ne sur la prostate du rat a gtg gtudige en culture organotypique dans des milieux naturels et semi-synthgtiques. Dans le milieu naturel, le mgthylcholanthr~ne provoque une tr~s forte hyperplasie de lYpitMlioma prostatique. Dans le milieu semi-syntMtique, l'hyperplasie est plus mod~rge. L'oestrone et l°oestradiol augmentent l'hyperplasie gpitMliale, tandis que la testosterone et l'hydrocortisone la suppriment dans les deux milieux.
SUMMARY The influence of oestrogens, testosterone and hydrocortisone on the effect of methylcholanthrene on the rat prostate gland grown as organ cultures in natural and semi-defined medium has been examined. The hormones were either administered simultaneously with methylcholanthrene or applied to cultures pretreated with the carcinogen. In natural medium methylcholanthrene alone causes a rapid and extensive hyperplasia of the prostatic epithelium. In the semi-defined medium it causes a mild hyperplasia. In natural medium oestrone and oestradiol increase the hyperplasia to a moderate degree, in the semi-defined medium they enhance it considerably. Testosterone and hydrocortisone inhibit or suppress the hyperplasia in both media.
Interaction of Hormones with Methyleholanthrene ZUSAMMENFASSUNG Der Einfluss yon Oestron, Oestradiol, Testosteron und Hydrocortison auf die Wirkung yon Methylcholanthren wurde in Organkulturen der Rattenvorsteherdriise (Prostata), die in natiMichen und semi-synthetischen Medien geziichtet wurden, untersucht. Methylcholanthren aUein verursacht eine starke Hyperplasie des Prostataepithels. Oestron und Oestradiol steigern diese Wirkung, wiihrend Testosteron und Hydrocortison sie in beiden Medien hemmen. REFERENCES 1. W. F. DUNm_~O, R. R. CURTISand A. SEOALOFF,Methylcholanthrene squamous cell carcinomas of the rat prostate with skeletal metastases and failure of the rat liver to respond to the same carcinogen. CancerRes. 6, 256-62 (1946). 2. E.A. MIRANDand W.J. STAUBITZ,Prostatic neoplasms of the Wistar rat induced with methylcholanthrene. 07. Natl. Cancer Proc. Soc. Exp. Biol. Med. 93, 457-62 (1956). 3. J. M. ALLEN,Responses of the rat prostate gland to methylcholanthrene, 07. Exp. Zool. 123, 289-13 (1953). 4. G. A~BoE-Ha~sEN, Hormonal effects on connective tissues. Connective Tissues Reaction Trans. 5th Conf., New York. J. Macy Junior Foundation, 123-82 (1954). 5. A. BASEROAand P. SHtmIK,The action of cortisone on transplanted and induced tumours in mice. Cancer Res. 14, 12-16 (1954). 6. H. N. GREENand M. SAVIG~.AR,Shock and Neoplasia. Brit. Med. 07. 1, 498-500 (1951'1. 7. I. LASmTZKI,The action and interaction of hormones and methylcholanthrene on the ventral prostate gland of the rat in vitro. 07. Natl. Cancer Inst. In press (196511. 8. I. LAmITZKI,The effect of methylcholanthrene on rat prostate glands grown in natural and semi-defined medium. Cancer Res. 24, 973-76 (1964). 9. C. W. ABETL and C. HEm~.LBERG~R,Interaction of carcinogenic hydrocarbons with tissue. VIII. Binding of tritium labelled hydrocarbons to the soluble proteins of mouse skin. CancerRes. 22, 931-46 (1962) 10. B. C. GIOVANELLA,L. E. MCKINN~.Yand C. HEIDELBERGER,On the reported solubilisation of carcinogenic hydrocarbons in aqueous solutions of DNA. 07. Mol. Biol. 8, 20-27 (1964). 11. A.C. ALLISONand L. MALLUCCI,Uptake of hydrocarbon carcinogens by lysosomes. Nature 203, 1024-27 (1964). 12. I. LASNITZKI,S. WAITEand J. T. DING~-~,The effect of hormones on the lysosomal acid phosphatase in the rat prostate in culture. Brit. Emp. Cane. Camp. Report, 375 (1964).
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