Growth Hormone Mrna in Mammary Gland Tumors of Dogs and Cats
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Growth hormone mRNA in mammary gland tumors of dogs and cats. J A Mol, … , A Rijinberk, G R Rutteman J Clin Invest. 1995;95(5):2028-2034. https://doi.org/10.1172/JCI117888. Research Article We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin- stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals. Find the latest version: https://jci.me/117888/pdf Growth Hormone mRNA in Mammary Gland Tumors of Dogs and Cats Jan A. Mol, Evert van Garderen,* Paulus J. Selman, Jeannette Wolfswinkel, Ad Rijnberk, and Gerard R. Rutteman Department of Clinical Sciences of Companion Animals, and *Department of Pathology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands Abstract higher risk of developing breast cancer at a young age (4-6). The question of whether this increased risk is attributable to We have shown recently that in the dog progestin adminis- the estrogen or the progestin content of the contraceptives has tration results in mammary production of immunoreactive not been answered satisfactorily, but there is good reason to growth hormone (GH). At present we demonstrate the ex- think that this increased risk may be due to the progestin compo- pression of the gene encoding GH in the mammary gland nent (7-9). This hypothesis is supported by the observations of dogs and cats using reverse-transcriptase PCR. that the highest proliferation rates of mammary epithelium are GH mRNA was found in the great majority of normal found in the progesterone-dominated luteal phase of the men- mammary tissues as well as benign and malignant mam- strual cycle and in women receiving progestin-only formula- mary tumors of the dog and was associated with the pres- tions of contraceptives (3, 10), indicating a strong mitogenic ence of immunoreactive GH in cryostat sections. The mam- action of progestins upon mammary epithelium. mary PCR product proved to be identical to that of the In dogs and cats, as in humans, spontaneous forms of benign pituitary. The highest expression levels were found after and malignant mammary gland tumors are found. These animals prolonged treatment with progestins. In carcinomas GH may therefore provide a model, in addition to the carcinogen- mRNA was also found in progesterone receptor-negative induced mammary tumors in rodents, for the study of compara- tissue samples, indicating that after malignant transforma- tive aspects of mammary tumor formation. In the dog many tion GH gene expression may become progestin indepen- toxicity studies have been carried out with estrogens and proges- dent. GH mRNA was also present in mammary tissues of tins. Prolonged administration of estrogens does not increase cats with progestin-induced fibroadenomatous changes. the incidence of mammary tumors in the dog ( 11), but treatment It is concluded that GH gene expression occurs in nor- of female dogs with progestins causes mammary tumor develop- mal, hyperplastic, and neoplastic mammary tissue of the ment in a dose-dependent manner ( 11-15 ). Many of the neo- dog. The expression in normal tissue is stimulated by proges- plastic changes are benign, but malignant tumors have also been tins and might mediate the progestin-stimulated develop- encountered (14, 15). ment of canine mammary tumors. The demonstration of Some 14 yr ago it was shown that exogenous (16-18) and progestin-stimulated GH expression in mammary tissue of endogenous (19) progestins may induce a syndrome of growth cats indicates that the phenomenon is more generalized hormone (GH)' excess in the dog, leading to acromegalic fea- among mammals. (J. Clin. Invest. 1995.95:2028-2034.) Key tures and glucose intolerance. The GH excess is reversible upon words: cats * dogs * mammary tumor * progestins * growth progestin withdrawal. Very recently we reported that this pro- hormone gestin-induced growth hormone excess originates from foci of hyperplastic ductular epithelium of the mammary gland (20). Introduction In cats progestins also induce mammary hyperplasia, some- times resulting in extensive fibroadenomatous changes (21). The role of progestins in the pathogenesis of human breast This proliferation of mammary duct epithelium and stroma oc- cancer has been highly debated ( 1-4). Although there is con- curs in the luteal phase of the estrous cycle, in the early stages vincing evidence that ovarian hormones play a prominent role of pregnancy, and after administration of progestins (22). In at all stages of mammary development, epidemiologic studies cats, however, administration of progestins does not result in in general do not support an overall increased risk of breast increased circulating concentrations of GH (23). cancer after progestin treatment. However, oral contraceptives In this study we investigated the sequence of the cDNA or depot medroxyprogesterone acetate does not decrease the encoding mammary GH in the dog and compared this sequence risk of female breast cancer, in contrast to inhibitory effect on with the cDNA encoding pituitary GH. The presence of the the risk of developing endometrial cancer. Furthermore, several GH mRNA was investigated in normal mammary tissue and in epidemiologic studies have linked the use of contraceptive benign and malignant mammary tumors of the dog. The pres- agents during adolescence or before a full-term pregnancy to a ence of GH mRNA was also investigated in cats with fibroade- nomatous changes and other proliferative lesions in the mam- mary gland. Address correspondence to Dr. Ir. J. A. Mol, Department of Clinical Sciences of Companion Animals, Utrecht University, P. 0. Box 80.154, Methods 3508 TD Utrecht, The Netherlands. Phone: 313-053-1709; FAX: 313- 051-8126. Origin of mammary tissue. Eight mammary tissue samples were ob- Received for publication 31 August 1994 and in revised form 30 tained from female experimental beagle dogs that had been treated with December 1994. long-acting progestins after ovariohysterectomy. Full details of treat- J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: ER, estrogen receptor; GH, growth 0021-9738/95/05/2028/07 $2.00 hormone; MPA, medroxyprogesterone acetate; PR, progestin receptor; Volume 95, May 1995, 2028-2034 RT, reverse transcriptase. 2028 Mol et al. A 1095 >. 495 > Figure 1. Size separation by elec- trophoresis on 1.5% agarose of the lI ll I l RT-PCR products obtained from Tumour 1 tig total RNA of canine mam- Normal Tissue MPA Prol Benign mary tissues using porcine specific GH primers. The RT-PCR prod- ucts were hybridized after a Southern blot using a 32P-labeled canine GH cDNA probe. The codes for the products correspond to the data for the tissue samples in Table I. (A) Samples 1-8 were obtained from female dogs at vari- ous stages of the reproductive cy- 495 > cle. Samples 9-16 were obtained after pretreatment with the syn- ..m1: 'I Asid <igf :: 4 l .... thetic progestins MPA or proliges- A B A BA A B A B 1 2 3 4 5 6 7 8 9 10 ton (Prol). Samples 17-21 were classified as benign mammary tu- mors. (B) Samples derived from Malignant Mammary Carcinom( malignant tumors. ment and hormonal changes have been published previously (20). primers derived from the sequence of the porcine GH gene from the Briefly, four dogs were treated with medroxyprogesterone acetate second to the fifth exon (upstream primer: 5 '-TTCCCAGCCATGCCC- (MPA; Upjohn, Ede, The Netherlands; 10 mg/kg body wt), and four TTGTCC; downstream primer: 5 '-CTTGAAGCAGGAGAGCAG- dogs were treated with proligestone (Mycofarm, De Bilt, The Nether- CCC). The upstream primer coincides with the start codon of the GH lands; 50 mg/kg body wt). The progestins were administered at 3-wk sequence, which is 60 bp downstream from the start codon of the prepro- intervals for a total of 8 injections, stopped for 6 mo, and then resumed hormone starting with a signal peptide. The downstream primer ends at the same doses and intervals for a total of 5 additional injections. within the coding sequence of the fifth exon. To the RT mixture was Mammary glands were collected immediately after excision, frozen in added 69.5 Id sterile bidistilled water, 0.5 ul Ampli Taq DNA polymer- liquid nitrogen, and stored at -700C until analysis. The other mammary ase (5 U/IA; Promega Corp.), 8 pi 10 x Taq buffer, and 1 p0l of each specimens were obtained in the same manner from privately owned primer (10 pmol). After an initial denaturation for 5 min at 950C the dogs and cats that were referred to the Utrecht University Clinic for cDNA was amplified by 35 cycles of 1 min at 950C, 1 min at 550C, Companion Animals. Mammary tumors and unaffected mammary tissue and 1 min at 720C. Samples were kept for 10 min at 720C after the last were well separated from each other during collection at surgery or, in amplification cycle.