Tumor Biology Laboratory, School of Life Sciences, University of Nebraska, Lincoln, Nebraska, 68503, USA

ACTION OF GROWTH-PROMOTING HORMONES ON MACROMOLECULAR BIOSYNTHESIS DURING LOBULO-ALVEOLAR DEVELOPMENT OF THE ENTIRE MAMMARY GLAND IN ORGAN CULTURE

By

Rajendra

G. Mehta

and Mihir R.

Banerjee

ABSTRACT The entire 2nd thoracic mammary gland of the immature virgin BALB/c mouse was stimulated to full lobulo-alveolar (LA) growth after 120 h organ culture in hormone supplemented medium. The minimal hormonal combination required was insulin (I) + prolactin (Prl) + aldosterone (A). The corticosteroid was replaceable by oestradiol-17\g=b\ (E) + progesterone (P). The combination I alone or I + the steroid hormone(s) failed to induce the LA development and similar results were also evident in presence of Prl + the steroids. Incubation of the glands in medium with I + Prl + A activated a sequential rise of RNA, protein and DNA synthesis. A near maximal increase of RNA synthesis was present at 48 h in the medium with I + Prl, addition of the steroid hormones did not show further stimulatory effect. Supplementation of the medium with I + Prl and the adrenal or the ovarian steroids was needed for maximal activation of protein synthesis at 72 h and DNA replication at 96 h. The medium with I alone did not show a substantial rise of macromolecular biosynthesis in the mammary gland in organ culture. The highest level of DNA polymerase

Supported by USPHS Grant CA-11058 and a Contract NIH-N01-CP-33289 from the National Cancer Institute, USA. Portions of the results reported were presented at the Annual Meetings of the American Society of Cell Biology, 1972 (J. Cell Biol. 55 (1972)

174a).

0 The results presented in this report constitute a part of the thesis submitted for the degree of Doctor of Philosophy, University of Nebraska, Lincoln, Nebraska, USA.

activity

observed at 72 h in glands cultivated in medium with or E + P. Only a modest increase of DNA polymerase activity was present in glands cultivated with I alone or I + Prl. Prior treatment of the glands (cultivated with I + Prl + A) with actinomycin D or puromycin resulted into 44 and 40 % reduction of DNA polymerase activity suggesting hormone-induced synthesis of the enzyme before the rise of DNA synthesis in the mammary cells at 96 h in organ culture. Significance of these results with respect to the action of the "growth\x=req-\ promoting" hormones in the mammary gland in organ culture and in the animal has been discussed. I

+

was

Prl and A

The endocrine regulation of immature mammary gland development involves initial lobulo-alveolar growth, followed by functional differentiation of the alveolar structure into lactation. Evidence indicates that the lobulo-alveolar growth and the associated macromolecular events in the mammary gland in vivo is influenced by the ovarian steroids, oestradiol-17/5 and progesterone (Nandi 1958; Banerjee Sc Rogers 1970; Banerjee et al. 1971; Banerjee et al. 1913a)-). On the other hand, numerous studies using the conventional organ culture of mammary tissue fragments of mid-pregnant mice emphasized the role of I as the "mitogenic" (growth-promoting) hormone in mammary cell proliferation (Topper f968, 1970; Turkington 1972; Turkington et al. 1973). Problems pertaining to this variable nature of responsiveness of the mammary tissue to hormones in vivo and in the organ culture have been recently dis¬ cussed (Rivera 1971, 1974; Mayne Se Barry 1970; Mukherjee et al 1973). The conventional organ culture of the mammary gland involves incubation of tissue fragments in hormone supplemented, chemically defined medium (Rivera 1971; Elias 1957). Interest of most earlier studies using this organ culture system centered around the problems of hormonal regulation of func¬ tional differentiation of the mammary cell (Topper 1970; Mayne Se Barry 1970; Turkington et al 1973). Since little or no spatial proliferation of the epithelium is evident in the mammary fragment in organ culture, virtually no information is available regarding the role of the "growth-promoting" hor¬ mones on macromolecular events of lobulo-alveolar growth in organ culture. The development of the unique organ culture system of the entire mammary gland (Ichinose Se Nandi 1966; Singh et al. 1970; Banerjee et al. 1913b) now permits induction of full lobulo-alveolar structures in the immature gland in hormone supplemented organ culture. In this report we present the results of 2) Abbreviations and trivial

names

(steroids)

used for the hormones:

E, oestradiol-17/? (l,3,5(10)-oestratriene-3,17/3-dioI; A, aldosterone (ll/?,21-dihydroxy-3,20-dioxo-4-pregnen-18-al); P, progesterone (4-pregnene-3,20-dione); I, insulin; Prl, prolactin; GH, growth hormone.

on action of different protein and steroid hormones involved in of various macromolecular events associated with the lobuloregulation alveolar growth of the immature mouse mammary gland in organ culture. our

studies

MATERIALS AND METHODS

Animals and organ culture As a prerequisite for organ culture of the entire mammary gland 3-4 week old virgin BALB/c mice were injected subcutaneously daily with 1 µg E and 1 mg for 9 days as described (¡chinóse Se Nandi 1966; Singh et al. 1970). The animals were killed by cervical dislocation, 24 h after the last injection; the entire 2nd thoracic mammary gland was excised directly on a sterile dacron raft and transferred into a plastic organ culture dish (Bioquest, Falcon Plastics, California) containing 1 ml Waymouth MB 752/1 medium (Waymouth 1959) supplemented with 35 µg penicillin. 350 tig glutamine and the appropriate hormones. The glands were incubated at 37°C in presence of 95 % Oo and 5 % C02 atmosphere inside a humidified chamber. The detailed proce¬ dures for organ culture of the entire mammary gland have been previously described (Banerjee et al. 1973è). The cultures were terminated at different times by fixing the glands in a mixture of acetic acid and ethanol (1:3 v/v) for whole mount preparations or by freezing (in a mixture of dry ice and acetone) the glands for subsequent chemical analysis.

Nucleic acid and

protein synthesis

In order to measure RNA, DNA and protein synthesis, the macromolecules were pulse labelled with [5-3H] uridine (27.2 Ci/mM), methyl-[3H]thymidine (6.7 Ci/mM) or [4,5-3H]L-leucine (67.6 Ci/mM), 5 /¿Ci/ml each, for 1 h before termination of the culture. The frozen glands were thawed, weighed and homogenized in 2 ml of cold deionized water, and equal volume of cold 1 perchloric acid (PCA) was added to the homogenate and the mixture was allowed to stand for 30 min on ice. The acidinsoluble precipitate was sedimented by centrifugation at 5000 r. p. m. for 5 min and the supernatant was collected for determination of radioactivity in the acid-soluble fraction. The acid-insoluble material was washed twice in 0.5 PCA and then analysed for [3H] uridine, [3H] thymidine and [3H] leucine radioactivity. For deter¬ mining [3H] uridine radioactivity in RNA the precipitate was hydrolyzed in 2 ml of 0.3 µ at 37°C for 2 h and then equal volume of 0.8 M PCA was added to acidify the solution. The mixture was centrifuged and [3H] uridine radioactivity in the alkaline hydrolysate (supernatant) was used as a measure of RNA synthesis. For DNA syn¬ thesis the acid-insoluble material was hydrolyzed in 0.5 PCA at 90°C for 20 min and [3H] thymidine radioactivity in the hydrolysate was measured. For protein the PCA insoluble material was solubilized in 1 NaOH at 37°C for 1 h and [3H]leucine radioactivity in the hydrolysate was measured for protein synthesis. RNA and DNA were determined by UV-spectrometry (260 nm) of the PCA hydrolysate and protein was measured by the method of Lowry et al. (1951). Data represent typical values of 3-5 determination and 4-6 glands were pooled for each assay.

Sucrose

density gradient

of phenol extracted RNA was done according to the procedure previously (Luck Sc Hamilton 1972; Banerjee et al. 1973). Minced mammary tissue (200-300 mg) was homogenized in NaAc buffer [0.15 m Na Acetate, 0.1 m NaCI, 1 "la sodium laryl sucrosine (SLS), pH 5.1] containing equal volume of para-amino salicylate and 1 */o macaloid. The homogenate was mixed with 2 volumes of phenolcresol mixture and the aqueous phase was extracted by centrifugation. RNA in the aqueous phase was precipitated by chilled 95 "la ethanol and dissolved in NET (0.1 M Tris HCl, 0.01 m EDTA, 0.1 m NaCI, pH 7.4) buffer. RNA was layered (2-3, 260 nm absorbance units) on a linear 15-30% sucrose gradient and centrifuged at IS 000 r. p. m. in an SW 25.1 rotor in a Beckman L265B ultracentrifuge for 16 h at 4°C. The gradient was fractionated using a continuously recording density gradient fractionator (ISCO, Lincoln, Nebraska, USA) as described (Banerjee et al. 1973).

Analysis

described

·

DNA

polymerase assay The mammary tissue was weighed and homogenized in a DNA polymerase buffer (12 mM Tris HCl, 6 mMKCl, 0.25 M sucrose, pH 7.8) and the volume of the homo¬ genizing buffer was adjusted according to the weight of the tissue. The homogenate was centrifuged first at 2500 r. p. m. for 10 min and then the supernatant was cen¬ trifuged at 105 000 g for 1 h in a Spinco Model L ultracentrifuge. DNA polymerase (DNA nucleotidyl transferase) activity in the 105 000 g postmicrosomal supernatant was measured in the standard in vitro assay system using the deoxynucleotide tri[3H] thymidine 5'triphosphate phosphates including ([3H]dTTP) as substrates as de¬ scribed (Bollum Se Potter 1958; Bollum 1968; Banerjee el al. 1973a). The incubation was done at 37°C in a shaking water bath and the rate of incorporation of [3H]dTTP into trichloroacetic acid (TCA) insoluble material during incubation in vitro was used as a measure of DNA polymerase activity. [3H]dTTP radioactivity in the TCA in¬ soluble material was measured by the filter paper disc method (Bollum 1968) and the samples were counted in a Beckman LS350 liquid scintillation system. All procedures were carried out at 0-4°C unless otherwise indicated. Data represent typical value of 3-5 determinations and 8-10 glands were pooled for each assay. ·

Sources of hormones and chemicals The hormones: insulin (bovine), progesterone and oestradiol-17/? were from Calbio¬ chem, La Jolla, California; prolactin (ovine) and aldosterone were supplied by Sigma Chemical Company, St. Louis, Missouri; growth hormone (bovine) was obtained from Miles Laboratories, Kankakee, Illinois; deoxynucleotide triphosphates were obtained from Sigma Chemical Company, St. Louis, Missouri. The [3H]deoxythymidine 5'tri¬

phosphate, [5-3H]uridine, methyl-[3H] thymidine and [4,5-3H]L-lcucine by New England Nuclear, Boston, Massachusetts.

were

supplied

RESULTS

The morphology of the mammary parenchyma at the time of initiation of the culture was principally ductal with some terminal end-buds, but a dramatic development of lobula-alveolar (LA) structures was evident in the glands in-

cubated for 5 days in the medium containing the hormones I + Prl + A (Fig. la, lb). A similar LA development was also observed in the glands when GH + E + was added in the medium with I + Prl + A. The glands in medium with I only or I + A failed to show any LA development (Fig. 1 c). In Prl + GH + A containing medium the absence of LA development was accom¬ panied by a somewhat poor maintenance of the ductal structures (Fig. 1 d). The glands in I + Prl medium showed excellent maintenance of the parenchyma but little or no alveolar growth was detectable. Since LA development of the mammary gland cultivated with the hormones I + Prl + A or I + Prl + GH + E + + A was virtually identical after 120 h organ culture, supplementation by the hormone combination I + Prl + A was consid¬ ered as the complete "growth-promoting" medium. Results in Fig. 2 show that in complete medium DNA synthesis in the mammary tissue increased in 2 waves, rising to peaks at 48 h and 96 h during the 120 h culture period. RNA synthesis increased sharply to a near-maximal level at 48 h, reaching a peak at 72 h and then declined. Whereas, protein synthesis failed to show a sub¬ stantial rise at 48 h although it reached a peak at 72 h followed by a decline. The results on the pattern of incorporation of precursors of nucleic acids and protein into the tissue incubated in medium with different combination of protein and steroid hormones are shown in Fig. 3. As expected only a negligible level of synthesis of the macromolecules was observed when the glands were incubated without any hormone. Consistent with morphological characteristics only a modest rise of RNA and DNA synthesis was evident after incubation in I containing medium, protein synthesis, however, showed a somewhat greater increase in the same medium. In medium supplemented with I + Prl RNA syn¬ thesis increased to the maximal level and addition of the steroid hormones in the medium did not influence the RNA synthetic activity of the tissue. The presence of steroid hormones, however, was essential for maximal stimulation of DNA and protein synthesis. The adrenocorticoid or the ovarian steroids along with the protein hormones stimulated DNA synthesis to a similar extent and presence of A along with + did not show any additive effect (data

shown). Prolonged exposure to [3H] uridine was done to study possible differences in the biosynthetic pattern of the major molecular species (r-RNA, m-RNA and t-RNA) of RNA. No detectable difference in the type of RNA synthesized in

not

I, I + Prl or I + Prl + A was observed. However, the level of syn¬ thesis of all three molecular species of RNA was low in glands cultivated with insulin only (Fig. 4). Since both I + Prl + A or I + Prl + + hormone combinations were needed to stimulate DNA and protein synthesis, it was of interest to characterize the hormonal responsiveness of a protein(s) such as DNA polymerase involved in DNA synthesis. The in vitro assay of DNA polymerase (DNA nucleotidyl

presence of

those used in previous studies on mammary fragments in organ culture (Lockwood et al 1967; Turkington Se Ward 1969). Results of a typical assay are

o

o

DAYS IN CULTURE

Fig-

2.

The data on RNA, protein and DNA synthesis in the mammary tissue during in¬ cubation in organ culture medium containing I + Prl + A. Determinations at zero h were made on freshly excised glands from E + treated animals, the glands were pulse labelled for 1 h in organ culture medium without hormones. At zero h the cpm/100 µg RNA, protein and DNA were 8.58 IO"3, 4.15 IO3 and 51.3 IO"3, respectively. RNA, - ; Proteins, ·-·; DNA, o-O. Vertical lines indicate sem.

transferase) activity in the crude post-microsomal supernatant was similar to shown in Table 1. Little incorporation of [3H]dTTP was observed when the in vitro assay was done without cold dTTP, primer DNA or the enzyme and most [3H]dTTP radioactivity was rendered acid-soluble after DNase treat-

Morphology

Fig,

1.

of the entire 2nd thoracic mammary gland at zero h and after 120 h cultivation in organ culture medium supplemented with different combinations of hormones, a: Zero h, illustrates the freshly excised gland from a mouse after daily injections of E + for 9 days. Note the primarily ductal nature of the parenchyma. b: Gland cultivated in I + Prl + A containing medium. Note the dramatic LA develop¬ ment, c: Cultivated in medium with I only, d: Medium with Prl + GH + A. Glands cultivated in medium with I + Prl + GH + A + E + P or I + Prl + E + P produced LA development as in Fig. 1 b and I + A was as in Fig. 1 c. Replacement of Prl by GH showed poor LA developmtnt (not shown) 7. Concentrations of hormones in the medium: insulin, prolactin and growth hormone, 5 ,/íg/ml each; oestradiol-17/5, 0.001 //g/ml; progesterone and aldosterone, 1 /

\:

i

80-

-IO

70

.

60

-50 x--

60-

;-K>

40

20

rh•b

i Histogram showing

li

-4

-30

"20

I PRL

I PRL

I PR_

A

Fig.

3.

levels of RNA, DNA and protein synthesis in the mammary tissue after incubation of the entire mammary gland in organ culture medium with different hormone combination. The glands were cultivated in organ culture for 72 h for RNA and protein measurements and 96 h for DNA, corresponding to the period of maximal synthetic activity of the respective macromolecules. Hormone combination I + Prl + A + GH + + showed results as I + Prl + A. Replacement of Prl by GH showed reduced activity, I + A showed activity as NH (data not shown), NH, no hormone supplementation. G, RNA; , DNA:I///|, Protein. Vertical lines indicate sem.

ment. In medium

containing I + Prl + A measurable DNA polymerase activity the was present in gland throughout the culture period (Fig. 5) but the glands cultivated for 72 h showed a higher activity. The increased DNA polymerase activity in the mammary tissue at 72 h before the maximal rise of DNA syn¬ thesis at 96 h is in agreement with previous reports on high DNA polymerase activity before the rise of DNA synthesis (Baserga 1965). Fig. 6 shows DNA polymerase activity in the mammary tissue cultivated in organ culture with different combinations of hormones. DNA polymerase activity in glands cul¬ tivated without hormones was very low, addition of I in the medium led to a 2-fold increase of the enzyme activity and addition of Prl along with I did not change the level of enzyme activity. However, in medium containing

20

15

9 io r-

CJ