Methionine-restricted diet inhibits growth of MCF10AT1-derived mammary tumors by increasing cell cycle inhibitors in athymic nude mice.

BACKGROUND: Dietary methionine restriction (MR) improves healthspan in part by reducing adiposity and by increasing insulin sensitivity in rodent models. The purpose of this study was to determine whether MR inhibits tumor progression in breast cancer xenograft model and breast cancer cell lines. METHODS: Athymic nude mice were injected with MCF10AT1 cells in Matrigel® and fed a diet containing either 0.86 % methionine (control fed, CF), or 0.12 % methionine (MR) for 12 weeks. Plasma amino acid concentrations were measured by UPLC, and proliferation and apoptosis were examined using RT-PCR, immunohistochemistry, and Cell Titer 96® Aqueous One Solution Cell Proliferation assay. RESULTS: Mice on the MR diet had reduced body weight and decreased adiposity. They also had smaller tumors when compared to the mice bearing tumors on the CF diet. Plasma concentrations of the sulfur amino acids (methionine, cysteine, and taurine) were reduced, whereas ornithine, serine, and glutamate acid were increased in mice on the MR diet. MR mice exhibited decreased proliferation and increased apoptosis in cells that comprise the mammary glands and tumors of mice. Elevated expression of P21 occurred in both MCF10AT1-derived tumor tissue and endogenously in mammary gland tissue of MR mice. Breast cancer cell lines MCF10A and MDA-MB-231 grown in methionine-restricted cysteine-depleted media for 24 h also up-regulated P21 and P27 gene expression, and MDA-MB-231 cells had decreased proliferation. CONCLUSION: MR hinders cancer progression by increasing cell cycle inhibitors that halt cell cycle progression. The application of MR in a clinical setting may provide a delay in the progression of cancer, which would provide more time for conventional cancer therapies to be effective.

Introduction of the human growth hormone gene into the guinea pig mammary gland by in vivo transfection promotes sustained expression of human growth hormone in the milk throughout lactation.

We tested the feasibility of transfecting mammary tissue in vivo with an expression plasmid encoding the human growth hormone (hGH) gene, under the control of the cytomegalovirus promoter. Guinea pig mammary glands were transfected with plasmid DNA infused through the nipple canal and expression was monitored in control and transfected glands by radioimmunoassay of milk samples for hGH. Sustained expression of hGH throughout lactation was attained with a polyion transfection complex shown to be optimal for the transfection of bovine mammary cells, in vitro. However, contrary to expectations, hGH expression was consistently 5- to 10-fold higher when DEAE-dextran was used alone for transfection. Thus polyion complexes which are optimal for the transfection of cells in vitro may not be optimal in vivo. The highest concentrations of hGH in milk were obtained when glands were transfected within 3 days before parturition. This method may have application for studying the biological role or physical properties of recombinant proteins expressed in low quantities, or for investigating the regulation of gene promoters without the need to construct viral vectors or produce transgenic animals.

Associations of the epithelial mucin, PAS-1, with yield, health, and reproductive traits in Holstein dairy cows.

Five polymorphic forms of the epithelial mucin, PAS-1, were identified in milk by SDS-PAGE. The sizes of PAS-1 ranged from 194.5 to 163 kDa. Mature equivalent milk, fat, and protein yields and protein and fat percentages from 139 cows in The Pennsylvania State University herd were analyzed with two models. Both models contained a year and season combination of calving and parent average. In Model [1], yields were regressed on the mean size of PAS-1 either linearly or linearly and quadratically. Model [2] included PAS-2 phenotype as a discrete variable. Lactation mean of somatic cell scores (119 cows), number of times bred (136 cows), and days open (136 cows) were analyzed with the same two models excluding parent average (data not available). Mean size of PAS-1 was not linearly related to the traits evaluated. The effects of PAS-1 phenotypes as a discrete variable were significant for mature equivalent fat and protein yields, fat percentage, number of times bred, number of times bred restricted to 3, and days open restricted to 150 d. Overall, there was little indication of an important linear or quadratic relationship between PAS-1 size and traits measured.

Polymorphic forms of the epithelial mucin, PAS-I (MUC1), in milk of Holstein cows (Bos taurus).

The polymorphic epithelial mucin, PAS-I (also known as MUC1), in individual milk samples from 119 Holstein cows was resolved into bands on SDS-gels. Mobility indices established for these bands provided evidence of four and possibly five polymorphic forms. Sialic acid, a major component of the oligosaccharide portion of PAS-I, was removed from the mucin by treatment of milk samples with neurominidase. This reduced the mobility of the mucin bands but did not alter their mobility relationships within a sample or among samples. Consideration of evidence from this and other studies indicates that the four or five polymorphic forms correspond to alleles, which are inherited, one each from sire and dam, and co-dominantly expressed. It appears that the Holstein population may carry several more alleles for PAS-I than do Ayrshire, Jersey or Brown Swiss cattle. In addition to these breed differences, some remarkable molecular differences have been noted between MUC1 (PAS-I) of human and mouse suggesting that research regarding molecular evolution of this mucin could provide another approach to understanding relationships among species.