Stem cells are enabling an improved understanding of the peripheral arterial disease, and patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic ...modality. However, applications of patient-specific induced pluripotent stem cell (iPSC)-derived ECs are limited by rapid loss of mature cellular function in culture. We hypothesized that changes in autophagy impact the phenotype and cellular proliferation of iPSC-ECs. Endothelial cells were differentiated from distinct induced pluripotent stem cell lines in 2D culture and purified for CD144 positive cells. Autophagy, mitochondrial morphology, and proliferation were characterized during differentiation and over serial passages in culture. We found that autophagy activity was stimulated during differentiation but stagnated in mature iPSC-ECs. Mitochondria remodeled through mitophagy during differentiation and demonstrated increasing membrane potential and mass through serial passages; however, these plateaued, coinciding with decreased proliferation. To evaluate for oxidative damage, iPSC-ECs were alternatively grown under hypoxic culture conditions; however, hypoxia only transiently improved the proliferation. Stimulating mTOR-independent ULK1-mediated autophagy with a plant derivative AMP kinase activator Rg2 significantly improved proliferative capacity of iPSC-ECs over multiple passages. Therefore, autophagy, a known mediator of longevity, played an active role in remodeling mitochondria during maturation from pluripotency to a terminally differentiated state. Autophagy failed to compensate for increasing mitochondrial mass over serial passages, which correlated with loss of proliferation in iPSC-ECs. Stimulating ULK1-kinase-driven autophagy conferred improved proliferation and longevity over multiple passages in culture. This represents a novel approach to overcoming a major barrier limiting the use of iPSC-ECs for clinical and research applications.
Cancer Letters: Highlights for Review • Blockade of the progestogen-PR is a untested strategy for breast cancer prevention • TPA opposed the pro-hyperplastic effects of MPA in mouse mammary glands • ...TPA decreased tumor incidence, pHH3, angiogenesis, and ER/PR expression in rats • Physiological estradiol plus progesterone increased the growth of T47D spheroids • TPA, and UPA are superior to mifepristone in growth suppression of T47D spheroids
Soy isoflavone consumption may protect against breast cancer development. We conducted a phase IIB trial of soy isoflavone supplementation to examine its effect on breast epithelial proliferation and ...other biomarkers in the healthy high-risk breast. One hundred and twenty-six consented women underwent a random fine-needle aspiration (rFNA); those with 4,000 or more epithelial cells were randomized to a double-blind 6-month intervention of mixed soy isoflavones (PTIG-2535) or placebo, followed by repeat rFNA. Cells were examined for Ki-67 labeling index and atypia. Expression of 28 genes related to proliferation, apoptosis, and estrogenic effect was measured using quantitative reverse transcriptase PCR. Hormone and protein levels were measured in nipple aspirate fluid (NAF). All statistical tests were two-sided. Ninety-eight women were evaluable for Ki-67 labeling index. In 49 treated women, the median Ki-67 labeling index was 1.18 at entry and 1.12 post intervention, whereas in 49 placebo subjects, it was 0.97 and 0.92 (P for between-group change: 0.32). Menopausal stratification yielded similar results between groups, but within premenopausal soy-treated women, Ki-67 labeling index increased from 1.71 to 2.18 (P = 0.04). We saw no treatment effect on cytologic atypia or NAF parameters. There were significant increases in the expression of 14 of 28 genes within the soy, but not the control group, without significant between-group differences. Plasma genistein values showed excellent compliance. A 6-month intervention of mixed soy isoflavones in healthy, high-risk adult Western women did not reduce breast epithelial proliferation, suggesting a lack of efficacy for breast cancer prevention and a possible adverse effect in premenopausal women.
The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from ...these agents will be a critical factor for their clinical success.
We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines.
TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites.
By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mouse mammary organ culture (MMOC) is used to evaluate the efficacy of chemopreventive agents against the development of carcinogen-induced preneoplastic lesions and is highly correlative to
...carcinogenesis models. Here, we developed a new
MMOC model, by introducing human breast cancer cells into the mouse mammary gland. This novel model, termed human breast cancer in MMOC (BCa-MMOC), mimics
orthotopic breast cancer mouse models. To develop this model, estradiol- and progesterone-sensitized female mice were injected with letrozole-sensitive and -resistant T47D breast cancer cells in the mammary glands and then euthanized. The glands were cultured
with hormone-supplemented media. On day 25, the glands were fixed and processed by histopathology and immunohistochemistry to evaluate for the presence of T47D cells, growth pattern, cancer markers and estradiol responsiveness. Histopathological analyses demonstrated an identical pattern of growth between the breast cancer cells injected
and
Interestingly, clusters of cancer cells in the mammary gland stroma appeared similar to those observed in human breast tumors. The injected T47D cells survived and proliferated for 15 days maintaining expression of estrogen receptor alpha (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and aromatase. The aromatase-overexpressing T47D grown in the BCa-MMOC sufficiently metabolized estrogen, resulting in enhanced cell proliferation, induction of estrogen target genes (i.e. ER and PR-B), and showed typical changes to estrogenic milieu. In summary, here we show a novel, inexpensive
model, to potentially study the effects of therapeutic agents on cancer cells grown in an orthotopic micromilieu.This article has an associated First Person interview with the first author of the paper.
The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: ...nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including
tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.
Previous studies have shown that progesterone concentrations in serum and nipple aspirate fluid (NAF) are significantly correlated
in premenopausal women, but estradiol concentrations are not. We ...therefore sought to ascertain the patterns of both steroids
in NAF throughout the menstrual cycle and in postmenopausal women. Simultaneous samples of blood and NAF were obtained from
40 premenopausal and 16 postmenopausal women. Premenopausal samples were backdated from the following menstrual period. Steroids
were purified by high-performance liquid chromatography before quantification by immunoassays. Serum steroids and NAF progesterone
followed the expected pattern across the menstrual cycle, with a midcycle peak of estradiol and a midluteal peak of progesterone.
However, the estradiol peak in NAF occurred about a week after the serum peak in the midluteal phase, when serum estradiol
had declined to less than half the value at midcycle. NAF estrone was also elevated at the midluteal phase. Potential estrogen
precursors androstenedione, estrone sulfate, and dehydroepiandrosterone sulfate declined in NAF from midcycle to the midluteal
phase as NAF estradiol was increasing. Progesterone concentrations were significantly lower in NAF in postmenopausal women
than in premenopausal women, but estrogen concentrations were not. This is the first description of the temporal relationships
of sex steroids in NAF and serum relative to the menstrual cycle. These results provide insights into the lack of correlation
of NAF and breast tissue estrogens with serum estrogens, and generate new hypotheses. Cancer Epidemiol Biomarkers Prev; 19(1);
275–9
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