Clifford W. Welsch

Dietary fish oil inhibits human breast carcinoma growth: A function of increased lipid peroxidation

Female athymic nude mice were implanted subcutaneously with human breast carcinoma MDA-MB231. Seven to ten days later, the mice were divided into groups and fed a purified diet containing the following types of fat (% of diet):(i) 20% corn oil (CO); (ii) 15% CO:5% fish (menhaden) oil (FO); (iii) 10% CO:10% FO; (iv) 5% CO:15% FO; (v) 1% CO:19% FO; and (vi) 1% CO:19% FO plus antioxidants (α-tocopherol acetate, 2000 IU/kg diet and tertiary butyl-hydroquinone, 2% of the total fat). The linoleic acid levels (% of diet) of the groups were 12.0, 9.1, 6.2, 3.3, 0.9 and 0.9%, respectively. After 6–8 wk, the carcinomas were assessed for tumor volume (cm3) and assayed for thiobarbituric acid reactive substances (TBARS). Human breast carcinoma growth was suppressed in mice consuming FO diets without antioxidants as compared to mice fed CO; the greater the amount of dietary FO fed, the greater the carcinoma growth suppression (P<0.05). The addition of antioxidants to the FO diet significantly (P<0.05) reversed the FO-induced carcinoma growth suppression. Concentrations of TBARS in the human breast carcinomas were increased in all the FO (without antioxidants) fed mice, compared to mice fed CO; the level of increase in TBARS was directly related to the increase in the level of FO fed (P<0.05). The addition of antioxidants to the FO diet significantly (P<0.05) reduced the concentration of TBARS in the breast carcinomas. Thus, these results provide evidence that dietary FO can significantly suppress growth of human breast carcinoma MDA-MB231, even in the presence of substantial amounts of linoleic acid (3.3–9.1%). The inhibitory effect of FO on growth of these carcinomas was associated with an increased concentration of TBARS in the tumor tissue. In conclusion, dietary FO induced suppression of human breast carcinoma growth is a function, at least in part, of an accumulation of lipid peroxidation products in the tumor tissues.

The effect of estrogen, progesterone, thyroxine, and human placental lactogen on DNA synthesis of human breast ductal epithelium maintained in athymic nude mice.

Specimens of human breast tissues from the periphery of excised benign tumors, obtained from 17 pre‐menopausal patients, were processed into slices and transplanted subcutaneously to 122 female Balb/c athymic nude mice. Subsequently, the host mice were treated with estrogens, progesterone, thyroxine, and human placental lactogen (HPL) alone or in combination. Growth of the ductal epithelium within the human breast transplants, as a function of the hormone treatment, was assessed by 3H‐thymidine autoradiographic analysis, i.e., the number of 3H‐thymidine radiolabeled ductal cells per unit area of ductal epithelium (labeling index [LI]). The administration of estrogen or thyroxine alone significantly increased the LI. Progesterone or HPL treatments alone did not substantially influence LI. HPL treatment, but not progesterone or thyroxine treatments significantly enhanced the stimulatory effect of estrogen on LI. The athymic nude mouse as host to human breast tissues in these in vivo/ex vivo studies, has been instrumental in producing information that effectively increases understanding of the hormonal factors influencing DNA synthesis in human breast ductal epithelium.

Review of the effects of dietary fat on experimental mammary gland tumorigenesis: role of lipid peroxidation.

The purpose of this communication is threefold, that is, (1) to review and critique the studies designed to examine the interrelationship between dietary fat and experimental rodent mammary gland tumorigenesis, (2) to assess the influence of dietary fat on growth of human breast carcinoma transplants in immunodeficient mice, and (3) to examine and discuss the role of products of lipid peroxidation in these tumorigenic processes. It is concluded from this review and critique that the amount and type of dietary fat can significantly influence the development and/or growth of rodent mammary gland tumors and growth of human breast carcinomas in immune deficient mice. Dietary fat can be either stimulatory or inhibitory to these tumorigenic processes, phenomena that could be a function, at least in part, of the generation of products of lipid peroxidation.

Dna synthesis of human, mouse, and rat mammary carcinomas in vitro. Influence of insulin and prolactin

Carcinomatous mammary tissues, derived from six spontaneously arising mouse mammary tumors, six DMBA‐induced rat mammary tumors, and 26 biopsy specimens of human breast tumors, were processed into slices and each tumor was individually cultured for two days in Medium 199. The influence of bovine insulin (5.0 μg/ml) and ovine prolactin (10.0 μg/ml) on H3‐thymidine incorporation into DNA was determined on the cultured tumor slices. Insulin consistently (p<0.05–0.01) increased the incorporation of H3‐thymidine into DNA of the organ cultures of mouse, rat, and human mammary carcinoma slices. The stimulatory effect of insulin was quantitatively more prominent in the mouse tumor slices than in the rat or human slices. The addition of prolactin to the insulin‐containing culture medium further increased significantly (p<0.001) the incorporation of H3‐thymidine into DNA of rat mammary carcinoma slices but had no significant effect on cultures of either mouse or human mammary carcinomas. The addition of prolactin to insulin and hydro‐cortisone‐enriched medium containing slices of 20 individually cultured human breast carcinomas did not significantly influence the mean incorporation of H3‐thymidine into DNA. However, a very small fraction (≅ 15%) of these human breast carcinomas responded to prolactin by increasing the incorporation of H3‐thymidine into DNA to a degree quantitatively comparable to the prolactin‐sensitive, DMBA‐induced rat mammary carcinoma. These results suggest that a very small fraction of human breast malignancies may respond to the growth‐stimulatory effects of prolactin, but that the vast majority mimic more closely the prolactin‐independent mouse mammary carcinoma.

GROWTH REQUIREMENTS AND NEOPLASTIC TRANSFORMATION OF TWO TYPES OF NORMAL HUMAN BREAST EPITHELIAL CELLS DERIVED FROM REDUCTION MAMMOPLASTY

SummaryA chemically defined culture medium was developed to support the growth of two distinctly different types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty. Type I cells expressed luminal epithelial cell markers and were deficient in gap junctional intercellular communication (GJIC), whereas Type II cells expressed basal epithelial cell markers and were efficient in GJIC. In this study, we examined and compared the growth factor and hormone requirements of these two types of cells and a series of cell lines that were obtained by sequential transfection with SV40 DNA (extended lifespan, nontumorigenic), treatment with 5-bromodeoxyuridine (BrdU)/black light (immortal and weakly tumorigenic), and infection of a virus carrying the neu oncogene (highly tumorigenic). Growth of Type I cells was inhibited by withdrawing epidermal growth factor (EGF), hydrocortisone (HC), or insulin (INS) from the culture media, but was enhanced by fetal bovine serum (FBS) supplementation. Growth of Type II cells was inhibited by withdrawal of EGF, HC, or INS from the media, and was inhibited by FBS supplementation. Withdrawal of human transferrin (HT) or 17β-estradiol (E2) from the media did not alter the growth of Type I or Type II cells. SV40 transfected Type I cell lines still required EGF, HC, or INS for optimal growth. However, the highly tumorigenic cell line did not show a growth dependence on EGF, HC, or INS but did appear to require HT and 3,3′,5-triiodo-D.L. thyronine (T3) for optimal growth. In addition, FBS stimulated the growth of these cell lines. Thus, this study shows that Type I HBEC are distinctly different from Type II HBEC in growth response to FBS and that neoplastically transformed Type I cells could become growth factor and hormone independent.

Suppression of growth by dietary fish oil of human breast carcinomas maintained in three different strains of immune-deficient mice

It has been reported that high levels of dietary fish (menhaden) oil, compared with corn oil, suppress the growth of MDA-MB231 and MCF-7 human breast carcinomas maintained in female athymic nude (T lymphocyte-deficient) mice. The purpose of this study was to determine whether dietary fish (menhaden) oil, compared with corn oil, can also suppress the growth of these carcinomas when maintained in female beige-XID-athymic nude (T lymphocyte- and NK/LAK cell-deficient) mice and in female severe combined immune-deficient (SCID) mice (total lack of functional T and B lymphocytes). Results clearly show that dietary fish (menhaden) oil can significantly (p < 0.05) suppress the growth of these carcinomas in the beige-XID-athymic nude mouse and the SCID mouse. Such results provide evidence that the growth suppression of MDA-MB231 and MCF-7 human breast carcinomas, induced by dietary fish oil, is not mediated by immune system mechanisms involving T lymphocytes, B lymphocytes, and/or NK/LAK cells.

Interrelationship between dietary lipids and calories and experimental mammary gland tumorigenesis

The purpose of this communication was to review and critique the studies designed to examine the interrelationship between dietary fat and calories in experimental rodent mammary gland tumorigenesis. The results of these studies clearly show that hyperalimentation of fat, either saturated or unsaturated, significantly stimulates this tumorigenic process. This has been demonstrated in an impressive array of carcinogen‐induced, transplantable, spontaneous, and metastatic experimental rodent mammary gland tumor systems. The stimulatory effect of high levels of dietary fat appears to act primarily at the promotional stage of this tumorigenic process. Whether the mammary tumor stimulatory effect of high levels of dietary fat is a result of the metabolic activity of the fat per se or is due to an excessive energy (caloric) intake has been examined. Data obtained from the experimental studies that address this issue support the viewpoint that the mammary tumorigenic‐enhancing activities of a high fat diet is, at least in part, through a caloric mechanism.

ESTABLISHMENT AND CHARACTERIZATION OF BOVINE MAMMARY EPITHELIAL CELL LINES

SummaryOne bovine mammary epithelial cell clone, designated PS-BME-C1, and two bovine mammary epithelial cell lines, designated PS-BME-L6 and PS-BME-L7, were derived from mammary tissue of a pregnant (270 day) Holstein cow. The cells exhibit the distinctive morphologic characteristics of mammary epithelial cells and express the milk fat globule membrane protein, PAS-III. They form domes when cultured on plastic substrata and acinilike aggregates when cultured on a collagen matrix. These cells are capable of synthesizing and secretingα-lactalbumin andα-s1-casein when cultured on a collagen matrix in the presence of insulin, cortisol, and prolactin. The cells have a near-normal diploid number and do not grow in suspension culture. When transplanted to the cleared mammary fat pads of female athymic nude mice, the cells readily proliferate forming noninvasive palpable spherical cellular masses within 8 wk after inoculation. The cells may become a useful tool to study the regulation of ruminant mammary epithelial cell growth and differentation.

Estrogen induced growth of human breast cancer cells (MCF-7) in athymic nude mice is enhanced by secretions from a transplantable pituitary tumor

MCF-7, a human breast carcinoma cell line, was maintained s.c. in female athymic nude mice for a period of 5-6 weeks. Administration of estrogen (s.c. pellet of 17 beta-estradiol and estrone in drinking water, 0.5 mg/l) to these mice resulted in sustained (P less than 0.001) growth of MCF-7 tumors. Grafting of a prolactin and growth hormone secreting rat pituitary tumor to the estrogen-treated mice resulted in an increased (P less than 0.05) rate of MCF-7 tumor growth. MCF-7 did not grow in athymic nude mice grafted with rat pituitary tumor alone or in mice without hormone treatment (controls). Thus, secretions of pituitary hormones alone are not capable of promoting in vivo growth of MCF-7 although such secretions significantly enhance estrogen-induced growth of this cell line. A synergism between pituitary hormones and estrogen for in vivo growth of a human breast carcinoma has been demonstrated in this study.

Differential effects of estrogen and prolactin on DNA synthesis in organ cultures of DMBA-induced rat mammary carcinoma.

Summary Explants from mammary carcinoma of DMBA-treated Sprague-Dawley rats were cultured for 5 days in medium 199 containing insulin (5 μg/ml) and corticosterone (1 μg/ml). Estradiol-17β (0.0001–10.0 μg/ml) and/or ovine prolactin (5μg/ml) in various combinations were added to the media. Media were replaced at 2 and 4 days. 3H-thymidine (0.5 μCi/ml) was added to each group of cultures 4 hr prior to termination and its rate of incorporation into DNA was used in the assessment of DNA synthesis. A highly significant increase in incorporation of 3H-thymidine into DNA was obtained in the prolactin-treated cultures. Estrogen, at the lower levels (0.0001–1.0 μg/ml) or at the higher levels (5.0–10.0 μg/ml), was without significant effect or inhibitory, respectively, to DNA synthesis. These data demonstrate an important role for prolactin in stimulating DNA synthesis in vitro, in contrast to estrogen, which was consistently found to be without stimulatory effect.