Prospective isolation and characterization of committed and multipotent progenitors from immortalized mouse mammary epithelial cells with morphogenic potential.

INTRODUCTION: Utilizing single-cell cloning of the COMMA-D cell line engineered to express ß-galactosidase (CDß) cell line, which exhibits normal in vivo morphogenesis, distinct multipotent, ductal-limited, alveolar-limited and luminal-restricted progenitors, have been isolated and characterized. METHODS: A single-cell suspension of CDß cells was stained using Hoechst dye 33342, followed by analysis and sorting. Cells that effluxed the dye appeared on the left side of a FACS analysis panel and were referred to as side population (SP) cells. Cells that retained the dye appeared on the right side and were referred to as non-SP (NSP) cells. Cells from both SP and NSP regions were sorted and analyzed for outgrowth potential. Additionally, individual clones were derived from single cells sorted from each region. RESULTS: There was no difference in the outgrowth potential of the SP vs. NSP cells when 5,000 cells per fat pad were transplanted. However, individual clones derived from single cells sorted from either SP or NSP regions had varying growth potential. A total of nine clones were identified, four of which possessed in vivo mammary outgrowth potential and five of which lacked in vivo outgrowth potential. Two of the clones formed mammary lobuloalveolar structures that contained both ducts and alveoli and were termed multipotent. Two of the clones generated either ductal-only or alveolar-only structures and were referred to as ductal-limited or alveolar-limited progenitor clones, respectively. The ability to expand the clones in vitro allowed for the characterization of their unique molecular phenotypes. Among the mammary-specific markers tested, high cytokeratin 5 (CK5) expression was the only marker that correlated with the clones' outgrowth potential. Among the clones that did not show any in vivo outgrowth potential when transplanted alone, one clone showed in vivo growth and incorporated into the mammary lumen when mixed with normal mammary epithelial cells. This clone also showed the highest in vitro expression of CK8 and Elf5and may represent a luminal-restricted progenitor clone. In addition, six "biclones," each made from an SP cell plus an NSP cell, were analyzed. Of these six, three exhibited lobuloalveolar growth. CONCLUSIONS: Distinct immortalized mammary progenitors have been isolated and characterized. Importantly, the results of this study provide further evidence for the existence of distinct ductal and alveolar mammary progenitors.

MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells.

MicroRNAs (miRNAs) play important roles in many developmental processes, including cell differentiation and apoptosis. Transition of proliferative ovarian granulosa cells to terminally differentiated luteal cells in response to the ovulatory surge of luteinizing hormone (LH) involves rapid and pronounced changes in cellular morphology and function. MicroRNA 21 (miR-21, official symbol Mir21) is one of three highly LH-induced miRNAs in murine granulosa cells, and here we examine the function and temporal expression of Mir21 within granulosa cells as they transition to luteal cells. Granulosa cells were transfected with blocking (2'-O-methyl) and locked nucleic acid (LNA-21) oligonucleotides, and mature Mir21 expression decreased to one ninth and one twenty-seventh of its basal expression, respectively. LNA-21 depletion of Mir21 activity in cultured granulosa cells induced apoptosis. In vivo, follicular granulosa cells exhibit a decrease in cleaved caspase 3, a hallmark of apoptosis, 6 h after the LH/human chorionic gonadotropin surge, coincident with the highest expression of mature Mir21. To examine whether Mir21 is involved in regulation of apoptosis in vivo, mice were treated with a phospho thioate-modified LNA-21 oligonucleotide, and granulosa cell apoptosis was examined. Apoptosis increased in LNA-21-treated ovaries, and ovulation rate decreased in LNA-21-treated ovaries, compared with their contralateral controls. We have examined a number of Mir21 apoptotic target transcripts identified in other systems; currently, none of these appear to play a role in the induction of ovarian granulosa cell apoptosis. This study is the first to implicate the antiapoptotic Mir21 (an oncogenic miRNA) as playing a clear physiologic role in normal tissue function.

Rapid effects of LH on gene expression in the mural granulosa cells of mouse periovulatory follicles.

LH acts on periovulatory granulosa cells by activating the PKA pathway as well as other cell signaling cascades to increase the transcription of specific genes necessary for ovulation and luteinization. Collectively, these cell signaling responses occur rapidly (within minutes); however, presently no high throughput studies have reported changes before 4 h after the LH surge. To identify early response genes that are likely critical for initiation of ovulation and luteinization, mouse granulosa cells were collected before and 1 h after hCG. Fifty-seven gene transcripts were significantly (P<0.05) upregulated and three downregulated following hCG. Twenty-four of these transcripts were known to be expressed after the LH/hCG surge at later time points, while 36 were unknown to be expressed by periovulatory granulosa cells. Temporal expression of several transcripts, including the transcription factors Nr4a1, Nr4a2, Egr1, Egr2, Btg1, and Btg2, and the epidermal growth factor (EGF)-like ligands Areg and Ereg, were analyzed by quantitative RT-PCR, and their putative roles in granulosa cell function are discussed. Epigen (Epgn), another member of the family of EGF-like ligands was identified for the first time in granulosa cells as rapidly induced by LH/hCG. We demonstrate that Epgn initiates cumulus expansion, similar to the other EGF-receptor ligands Areg and Ereg. These studies illustrate that a number of changes in gene expression occur in vivo in response to LH, and that many of the differentially expressed genes are transcription factors that we would predict in turn modulate granulosa cell gene expression to ultimately impact the processes of ovulation and luteinization.

Quantitative RT-PCR methods for mature microRNA expression analysis.

This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a single RT reaction from one (as suggested by the manufacturer) to five. The second method uses a modified oligo(dT) technique to reverse transcribe all transcripts within an RNA sample; therefore, target miRNA and normalizing mRNA can be analyzed from the same RT reaction. We examined the level of miRNA-132, a miRNA known to be upregulated in granulosa cells following hCG treatment, using both of these methods. Data were normalized to GAPDH or snU6 and evaluated by DeltaDeltaCt and standard curve analysis. There was no significant difference (P > 0.05) in miRNA-132 expression between the stem-loop and modified oligo(dT) RT methods indicating that both are statistically equivalent. However, from a technical point of view, the modified oligo(dT) method was less time consuming and required only a single RT reaction to reverse transcribe both miRNA and mRNA.

Targeting the perpetrator: breast cancer stem cell therapeutics.

The hypothesis that tumors may originate from a rare population of cancer stem cells (CSCs) has gained tremendous popularity in recent years and is supported extensively by several pioneering works. Cancer therapies targeting CSCs have unlimited potential for relapse free survival of cancer patients. As a result, knowledge of biological pathways that govern CSCs is very important and this review is focused on the biology of CSCs with special emphasis on breast CSCs, and recent advances in therapeutic approaches targeting them.

A synonymous variant in scavenger receptor, class B, type I gene is associated with lower SR-BI protein expression and function.

OBJECTIVE: A synonymous variant within scavenger receptor class B type I gene (SCARB1), exon 8 rs5888, has been associated with altered lipid levels and cardiovascular risk in humans. The objective was to determine if rs5888 decreased SR-BI protein expression and function in vitro. METHODS: SR-BI RNA secondary structure, turnover, polysomal distribution and protein expression were examined in COS cells transfected with wild-type or rs5888-SR-BI plasmids by selective 2'-hydroxyl acylation and primer extension assays, actinomycin D inhibition, polysomal profiling, and western blotting. SR-BI function in murine macrophages stably expressing wild-type or rs5888-SR-BI was assessed by measuring the specific cell association of (125)I,(3)H-cholesteryl ester (CE) radiolabeled HDL. RESULTS: Rs5888 changed RNA secondary structure and led to marked differences in the polysomal profiles compared with wild-type transcript (p<0.02). As compared to wild-type cells, COS cells expressing rs5888 had significantly lower SR-BI protein expression (p<0.04), but no difference in total RNA transcript levels. There were no differences in SR-BI RNA turnover in murine macrophages, whereas specific cell association of (125)I (p<0.0001) or (3)H-CE (p<0.00001) was significantly lower in rs5888 cells. CONCLUSIONS: The rs5888 variant affected SR-BI RNA secondary structure, protein translation, and was significantly associated with reduced SR-BI protein expression and function in vitro.

RUNX2 transcription factor regulates gene expression in luteinizing granulosa cells of rat ovaries.

The LH surge promotes terminal differentiation of follicular cells to become luteal cells. RUNX2 has been shown to play an important role in cell differentiation, but the regulation of Runx2 expression and its function in the ovary remain to be determined. The present study examined 1) the expression profile of Runx2 and its partner CBFbeta during the periovulatory period, 2) regulatory mechanisms of Runx2 expression, and 3) its potential function in the ovary. Runx2 expression was induced in periovulatory granulosa cells of human and rodent ovaries. RUNX2 and core binding factor-beta (CBFbeta) proteins in nuclear extracts and RUNX2 binding to a consensus binding sequence increased after human chorionic gonadotropin (hCG) administration. This in vivo up-regulation of Runx2 expression was recapitulated in vitro in preovulatory granulosa cells by stimulation with hCG. The hCG-induced Runx2 expression was reduced by antiprogestin (RU486) and EGF-receptor tyrosine kinase inhibitor (AG1478), indicating the involvement of EGF-signaling and progesterone-mediated pathways. We also found that in the C/EBPbeta knockout mouse ovary, Runx2 expression was reduced, indicating C/EBPbeta-mediated expression. Next, the function of RUNX2 was investigated by suppressing Runx2 expression by small interfering RNA in vitro. Runx2 knockdown resulted in reduced levels of mRNA for Rgc32, Ptgds, Fabp6, Mmp13, and Abcb1a genes. Chromatin immunoprecipitation analysis demonstrated the binding of RUNX2 in the promoter region of these genes, suggesting that these genes are direct downstream targets of RUNX2. Collectively, the present data indicate that the LH surge-induced RUNX2 is involved in various aspects of luteal function by directly regulating the expression of diverse luteal genes.

Role of Dicer in female fertility.

Dicer is an RNAse III endonuclease that is essential for the biogenesis of microRNAs and small interfering RNAs. These small RNAs post-transcriptionally regulate mRNA gene expression through several mechanisms to affect key cellular events including proliferation, differentiation and apoptosis. Recently, the role of Dicer function in female reproductive tissues has begun to be elucidated through the use of knockout mouse models. Loss of Dicer within ovarian granulosa cells, luteal tissue, oocyte, oviduct and, potentially, the uterus renders females infertile. This review discusses these early studies and other data describing the current understanding of microRNAs and small interfering RNAs in female reproduction.

Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells.

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3' untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.