Tm experimental investigation of hormone sensitivity in breast cancer has been handicapped by lack of a good experimental model. It seems likely that this deficiency may be remedied since mammary gland tumours can now be readily induced in rats by intragastric instillation of certain chemical carcinogens (Shay et ul., 1949-50; Huggins, Briz,iareK and Sutton, 1959; Huggins, Grand and Brillantes, 1959, 1961); the induction period is short and many of the tumours have been hormone-sensitive and have a structure resembling human breast cancer. The purpose of this paper is to present our results in inducing such tumours in rats commercially available in the United Kingdom, and the effects of oiiphorectomy and subsequent substitution of ovarian steroids on tumour growth and histology.MATERIAL AND METHODS Non-inbred female rats from stock imported from SpragueDawley of Madison, Wisconsin, and bred Commercially in Great Britain were used throughout. At Sokl days they were given a single dose of 9,1Odimethyl-1,2-benzanthracene (DMBA) in corn oil by gastric tube. In early experiments 20 mg. DMBA in 2 ml. corn oil was given, but in later experiments the dose was raised to 50 mg. in 2 ml. No further dose of carcinogen was given. In all, 222 rats were Used.From 4 wk onwards the animals were'examined twice weekly for tumours. Two diameters of the tumour were measured with calipers, one the long axis, the other at right-angles to it; the mean of the measurements was used as the measure of tumour size. Oiiphorectomy was performed through a single middorsal incision and was carried out only on rats whose mean tumour diameter was 15 mm. or more and was still increasing. Rats were maintained on diet G.R. 25 with water ad libitum.The dose of oestradiol-178 was 2pg. and of progesterone 8 mg. Whether they were used singly or together, they were given by subcutaneous injection dissolved in 0-4 ml. of corn oil, daily for 6 days per wk, usually for a period of 4 wk.This period was sometimes reduced to 3 wk in the case of a rapidly growing tumOW.Portions of tumour were fixed primarily in 4 per cent. neutral buffered formaldehyde and secondarily in formol-sublimate. Sections were cut at 4 p from par& blocks and stained with haematoxylin and eosin. Frozen sections of some tumours were stained for fat.

SUMMARY The uptake and intracellular localization of [6,7-3H]oestradiol has been studied in liver, kidney and mammary adenocarcinoma from female rats treated with dimethylbenzanthracene (DMBA). The tumour is similar to other oestrogen-responsive tissues in that the major oestrogen* within the tissue is oestradiol which is mainly associated with cell nuclei. The tumour has the ability to retain the oestrogen for at least 4 hr. Virtually no oestrogen is associated with the nuclei from liver and this result is discussed in relation to the intranuclear localization of oestrogen and the mechanism whereby oestrogen can enter the nuclei of some cells. A significant amount of oestradiol is associated with nuclei from kidney.

The intranuclear localization of [6,7-3H]oestradiol-17\g=b\(oestradiol) has been studied in nuclei from dimethylbenzanthracene (DMBA)-induced rat mammary adenocarcinoma, anterior pituitary, uterine epithelium and kidney. The evidence suggests that, with the possible exception of uterine epithelium, most of the oestrogen is associated with an acid-labile nonhistone component of the chromatin. No evidence has been found to suggest that oestradiol is associated with any particular type of chromatin. These results are compatible with the view that oestradiol affects cellular function in some tissues by altering the priming ability of the chromatin DNA.

MAMMARY gland tumours can be readily induced in rats by intragastric instillation of a chemical carcinogen (Shay, Aegerter, Gruenstein and Komarov, 1949; Dao and Sutherland, 1959; Huggins, Briziarelli and Sutton, 1959). It has been shown that a proportion of these tumours stop growing and some regress spontaneously (Young, Cowan and Sutherland, 1963). The purpose of this paper is to describe in greater detail the " natural history ", histology and hormone responsiveness of tumours which behave in this way. It is based on a study of 181 tumours which have been induced in 86 rats. MATERIAL AND METHODSNon-inbred female rats, descended from stock imported from Sprague-Dawley of Madison, Wisconsin, and bred commercially in Great Britain, were used throughout. They were maintained on diet G.R.25 with water ad libitum. At 50 ± 1 days of age they were given intragastrically 50 mg. 9,10-dimethyl-1,2-benzanthracene (DMBA) dissolved in 2 ml. of corn oil. This was the only dose of carcinogen to be given.Starting 4 weeks after the carcinogen, each rat was examined twice weekly for tumours. These were measured with calipers in two diameters at right angles to one another, one of which was the long axis of the tumour. The arithmetic mean of these two measurements was used as the measure of tumour size.Oophorectomy was carried out through a single mid-dorsal incision. Oestradiol-17,/ and progesterone were given together, subcutaneously, dissolved in corn oil. The doses were: oestradiol-17,/ 2 ,ag. and progesterone 8 mg., in 0-4 ml. oil, or oestradiol-17,/ 1 ,tg. and progesterone 4 mg., in 02 ml. oil. Bovine growth hormone 0.5 mg. in 1 ml. saline, and cortisone acetate 10 mg., were given subcutaneously. All hormones were given daily, 6 days a week for 3 or 4 weeks.Intercurrent infection or the development of progressively growing tumours caused us to kill most of the animals before they were a year old. Survivors were killed 1 year after giving the carcinogen when the experiments were discontinued.Portions of tumour were fixed primarily in 4 per cent neutral buffered formaldehyde for frozen sections, and fixed secondarily in formol-sublimate, or primarily in Bouin for paraffin sections, followed by haematoxylin and eosin staining.

SUMMARY.-Circulating antibody response to flagella antigen has been measured in three groups of Sprague-Dawley rats after feeding with 7.12.DMBA in an attempt to differentiate carcinogen and tumour growth as causative agents in the depression of immune response seen in these animals. DMBA fed female rats developing tumours had progressive depression of both primary and secondary response as compared to control animals, and 7S and 19S antibody fractions were equally affected. Removal of tumours did not result in recovery of response. Attempts to prevent tumour development by mammectomy after DMBA feeding were unsuccessful, but the similar number of tumours found in this group was associated with an equal degree of antibody depression to that seen in the first experiment. Male animals fed DMBA did not develop malignant tumours, and showed no depression of immune response. Results suggest that tumour development plays a part in the depression of circulating antibody response seen in these animals, but that it is not directly related to the number of tumours, and is not reversible by tumour excision.