Cultures Of Human Mammary Epithelial Cells Research Articles

Development and Characterization of a Human Mammary Epithelial Cell Culture Model for the Blood–Milk Barrier—A Contribution from the ConcePTION Project

It is currently impossible to perform an evidence-based risk assessment for medication use during breastfeeding. The ConcePTION project aims to provide information about the use of medicines during lactation. The study aimed to develop and characterize an in vitro model of the blood–milk barrier to determine the extent of the milk transfer of xenobiotics, relying on either on human mammary epithelial cells (hMECs) or immortalized cell lines derived from breast tissue. The hMECs were cultured and characterized for epithelial markers; further, the ability to form an epithelial barrier was investigated. Drug transporter functionality in the cultured hMECs was analyzed with specific probe substrates. The hMECs showed an epithelial morphology and the expression of epithelial markers and tight junctions. They formed a reproducible tight barrier with a transepithelial electrical resistance greater than 400 Ωcm2, unlike immortalized cell lines. Different levels of mRNA expression were detected for 81 genes of membrane transporters. Functional assays showed no evidence for the transporter-mediated secretion of medicines across the hMECs. Nevertheless, the hMEC-based in vitro model covered a 50-fold range of permeability values, differentiating between passive transcellular and paracellular-mediated transport. The cultured hMECs proved to be a promising in vitro model for biorelevance; the wide characterization of hMECs makes them useful for studying medicine partitioning in milk.

An adaptive Epithelial-Mesenchymal Transition Program Enables Basal Epithelial Cells to Bypass Stress-Induced Stasis and Contributes to Metaplastic Breast Cancer Progenitor State.

Human mammary epithelial cell (HMEC) cultures encounter a stress-associated barrier termed stasis, during which most cells adopt a senescence-like phenotype. From these cultures, rare variants emerge from the basal epithelial population, re-initiating growth. Variants exhibit pre-malignant properties, including an aberrant epigenetic program that enables continued proliferation and acquisition of genetic changes. Following oncogenic transformation, variants produce tumors that recapitulate the histopathological characteristics of metaplastic breast cancer (MBC), a rare subtype characterized by squamous and mesenchymal differentiation. Using the conventional serum-free HMEC culture system, we probed the capacity for phenotypic plasticity inherent to basal epithelial cell populations from human breast tissue as they navigated stasis and emerged as variant populations. We observed robust activation of a TGF-β-dependent epithelial-mesenchymal transition (EMT) program in basal epithelial cells during stasis, followed by subsequent attenuation of this program in emerging variants. Inhibiting the TGF-β pathway or depleting the EMT regulators Snail or Slug allowed basal epithelial cells to collectively bypass stasis, demonstrating that cellular dysfunction and arrest resulting from TGF-β and EMT activation are central to this in vitro barrier. The spontaneous emergence of variants from stasis cultures was associated with a restricted EMT trajectory, which diverted cells away from a complete mesenchymal state characterized by irreversible growth arrest, and instead limited variants to epithelial and intermediate EMT states associated with greater proliferative capacity and stemness. Epigenetic mechanisms, which contributed to the dysregulated growth control characteristic of the variant phenotype, also contributed to the constrained EMT program in variants. By overcoming the cellular dysfunction and growth arrest resulting from TGF-β and EMT activation, variants exhibited increased oncogenic transformation efficiency compared to pre-stasis basal epithelial cells. Inhibiting the TGF-β pathway prior to stasis significantly reduced EMT in the basal epithelial population, alleviated selective pressure driving variant emergence, and enhanced oncogenic transformation efficiency, resulting in tumors with markedly diminished metaplastic differentiation. This study reveals how adaptive EMT reprogramming governs basal epithelial cell fate decisions and contributes to the development of MBC progenitors by restricting access to terminal mesenchymal states that induce growth arrest and, instead, favoring intermediate states with enhanced tumorigenic potential.

Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes

BackgroundA challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes.MethodsWe demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2–5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3–5 days to maintain subconfluent density.ResultsWhen compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components.ConclusionsPostconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.

Cellular context alters EGF-induced ERK dynamics and reveals potential crosstalk with GDF-15.

Cellular signaling dynamics are sensitive to differences in ligand identity, levels, and temporal patterns. These signaling patterns are also impacted by the larger context that the cell experiences (i.e., stimuli such as media formulation or substrate stiffness that are constant in an experiment exploring a particular variable but may differ between independent experiments which explore that variable) although the reason for different dynamics is not always obvious. Here, we compared extracellular-regulated kinase (ERK) signaling in response to epidermal growth factor treatment of human mammary epithelial cells cultures in either well culture or a microfluidic device. Using a single-cell ERK kinase translocation reporter, we observed extended ERK activation in well culture and only transient activity in microfluidic culture. The activity in microfluidic culture resembled that of the control condition, suggesting that shear stress led to the early activity and a loss of autocrine factors dampened extended signaling. Through experimental analysis we identified growth differentiation factor-15 as a candidate factor that led to extended ERK activation through a protein kinase C-α/β dependent pathway. Our results demonstrate that context impacts ERK dynamics and that comparison of distinct contexts can be used to elucidate new aspects of the cell signaling network.

Co-administering Melatonin With an Estradiol-Progesterone Menopausal Hormone Therapy Represses Mammary Cancer Development in a Mouse Model of HER2-Positive Breast Cancer.

Melatonin has numerous anti-cancer properties reported to influence cancer initiation, promotion, and metastasis. With the need for effective hormone therapies (HT) to treat menopausal symptoms without increasing breast cancer risk, co-administration of nocturnal melatonin with a natural, low-dose HT was evaluated in mice that develop primary and metastatic mammary cancer. Individually, melatonin (MEL) and estradiol-progesterone therapy (EPT) did not significantly affect mammary cancer development through age 14 months, but, when combined, the melatonin-estradiol-progesterone therapy (MEPT) significantly repressed tumor formation. This repression was due to effects on tumor incidence, but not latency. These results demonstrate that melatonin and the HT cooperate to decrease the mammary cancer risk. Melatonin and EPT also cooperate to alter the balance of the progesterone receptor (PR) isoforms by significantly increasing PRA protein expression only in MEPT mammary glands. Melatonin significantly suppressed amphiregulin transcripts in MEL and MEPT mammary glands, suggesting that amphiregulin together with the higher PRA:PRB balance and other factors may contribute to reducing cancer development in MEPT mice. Melatonin supplementation influenced mammary morphology by increasing tertiary branching in the mouse mammary glands and differentiation in human mammary epithelial cell cultures. Uterine weight in the luteal phase was elevated after long-term exposure to EPT, but not to MEPT, indicating that melatonin supplementation may reduce estrogen-induced uterine stimulation. Melatonin supplementation significantly decreased the incidence of grossly-detected lung metastases in MEL mice, suggesting that melatonin delays the formation of metastatic lesions and/or decreases aggressiveness in this model of HER2+ breast cancer. Mammary tumor development was similar in EPT and MEPT mice until age 8.6 months, but after 8.6 months, only MEPT continued to suppress cancer development. These data suggest that melatonin supplementation has a negligible effect in young MEPT mice, but is required in older mice to inhibit tumor formation. Since melatonin binding was significantly decreased in older mammary glands, irrespective of treatment, melatonin supplementation may overcome reduced melatonin responsiveness in the aged MEPT mice. Since melatonin levels are known to decline near menopause, nocturnal melatonin supplementation may also be needed in aging women to cooperate with HT to decrease breast cancer risk.

Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth

IntroductionThe Epithelial Cell Adhesion Molecule (EpCAM) has been shown to be strongly expressed in human breast cancer and cancer stem cells and its overexpression has been supposed to support tumor progression and metastasis. However, effects of EpCAM overexpression on normal breast epithelial cells have never been studied before. Therefore, we analyzed effects of transient adenoviral overexpression of EpCAM on proliferation, migration and differentiation of primary human mammary epithelial cells (HMECs).MethodsHMECs were transfected by an adenoviral system for transient overexpression of EpCAM. Thereafter, changes in cell proliferation and migration were studied using a real time measurement system. Target gene expression was evaluated by transcriptome analysis in proliferating and polarized HMEC cultures. A Chicken Chorioallantoic Membrane (CAM) xenograft model was used to study effects on in vivo growth of HMECs.ResultsEpCAM overexpression in HMECs did not significantly alter gene expression profile of proliferating or growth arrested cells. Proliferating HMECs displayed predominantly glycosylated EpCAM isoforms and were inhibited in cell proliferation and migration by upregulation of p27KIP1 and p53. HMECs with overexpression of EpCAM showed a down regulation of E-cadherin. Moreover, cells were more resistant to TGF-β1 induced growth arrest and maintained longer capacities to proliferate in vitro. EpCAM overexpressing HMECs xenografts in chicken embryos showed hyperplastic growth, lack of lumen formation and increased infiltrates of the chicken leukocytes.ConclusionsEpCAM revealed oncogenic features in normal human breast cells by inducing resistance to TGF-β1-mediated growth arrest and supporting a cell phenotype with longer proliferative capacities in vitro. EpCAM overexpression resulted in hyperplastic growth in vivo. Thus, we suggest that EpCAM acts as a prosurvival factor counteracting terminal differentiation processes in normal mammary glands.

Detection of Redundant Fusion Transcripts as Biomarkers or Disease-Specific Therapeutic Targets in Breast Cancer

Fusion genes and fusion gene products are widely employed as biomarkers and therapeutic targets in hematopoietic cancers, but their applications have yet to be appreciated in solid tumors. Here, we report the use of SnowShoes-FTD, a powerful new analytic pipeline that can identify fusion transcripts and assess their redundancy and tumor subtype-specific distribution in primary tumors. In a study of primary breast tumors, SnowShoes-FTD was used to analyze paired-end mRNA-Seq data from a panel of estrogen receptor (ER)(+), HER2(+), and triple-negative primary breast tumors, identifying tumor-specific fusion transcripts by comparison with mRNA-Seq data from nontransformed human mammary epithelial cell cultures plus the Illumina Body Map data from normal tissues. We found that every primary breast tumor that was analyzed expressed one or more fusion transcripts. Of the 131 tumor-specific fusion transcripts identified, 86 were "private" (restricted to a single tumor) and 45 were "redundant" (distributed among multiple tumors). Among the redundant fusion transcripts, 7 were unique to ER(+) tumors and 8 were unique to triple-negative tumors. In contrast, none of the redundant fusion transcripts were unique to HER2(+) tumors. Both private and redundant fusion transcripts were widely expressed in primary breast tumors, with many mapping to genomic loci implicated in breast carcinogenesis and/or risk. Our finding that some fusion transcripts are tumor subtype-specific suggests that these entities may be critical determinants in the etiology of breast cancer subtypes, useful as biomarkers for tumor stratification, or exploitable as cancer-specific therapeutic targets.

Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies

IntroductionMost human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated β-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth.MethodsPre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or γ-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated β-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses.ResultsCultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated β-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism.ConclusionsOur studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential.

An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here, we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying antiphosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that a majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.

Cellular senescence in human mammary epithelial cells (HMEC) is accompanied by an increase of elastin formation

Normal human mammary epithelial cells (HMEC) have a finite life span, regulated by a process termed cellular senescence. After an initial stress-associated senescence barrier, some cells are subjected to a self-selection process triggered by the loss of the G1 cell cycle regulator p16INK4a expression. This effect is associated with an escape from the growth plateau to resume cell cycle progression and to undergo further cell divisions. Thereafter, these so-called HMEC encounter a second senescence barrier, termed agonescence. Our recent work using an siRNA approach revealed an essential impact of the extracellular matrix metalloproteinase-7 (MMP-7) on the aging process of post-selection HMEC. Since MMP-7 is involved in a variety of cellular mechanisms, including processing of inactive precursor proteins, ectodomain shedding of extracellular signaling molecules and remodeling of the extracellular matrix (ECM), respectively, we could observe considerable alterations of the ECM during senescence of HMEC. Thus, the formation of characteristic structures similar to elastin was detectable, which markedly increased in agonescent HMEC cultures after passage 15. Moreover, Western blot analysis revealed an up-regulated expression of tropoelastin, the soluble precursor of the ECM protein elastin, during the aging process of HMEC. This was paralleled by an enhanced activity of the enzyme lysyl oxidase (LOX) which is responsible for cross-linking tropoelastin monomers to form insoluble elastic fibers. Thus, enhanced LOX activity was detectable in cellular homogenates as well as in the culture supernatant during HMEC aging between passage 12 up to passage 16. Together with the down-regulation of MMP-7, the increased formation of elastic fibers supported an essential effect of distinct ECM components and the microenvironment on HMEC aging [1].

A new paradigm for African American breast cancer involving stem cell differentiation.

Abstract Abstract #5051 Background: African American (AA) women have a higher incidence of breast cancer (BC) than white women before age 40, and are more likely to die from BC at every age. These differences are often attributed to socio-economic factors, however, there may be intrinsic biological differences in the breast tissue of AA and white women. For example, triple negative breast tumors are more prevalent in AA women than in non-Hispanic white women, although neither population is monolithic in its breast cancer phenotypes. We hypothesis that intrinsic biological differences between women of AA and European-white ancestry might be revealed by differences in growth and differentiation in vitro, reflecting underlying differences in the proportions and/or potency of breast epithelial stem cells.
 Methods: Our laboratory has developed a novel tissue engineering system for Human Mammary Epithelial Cells (HMEC), both normal and malignant. This system allows for long-term establishment of non-diseased primary cultures that begin as 3-dimensional attached “epispheres”, that are structures made up of 40-100 epithelial cells. These non-diseased epispheres subsequently differentiate into functional, organotypic branching ducts and lobules that demonstrate ESA and CK -18,-19 staining, lumen, polarized nuclei, desmosomes, microvilli on apical surfaces and casein secretion.
 Results: We have established primary HMEC cultures from 36/36 breast reduction mammoplasty tissues, including 9 AA donors matched in socioeconomic status. We have found: 1) The more children a woman had, the less likely her breast tissue was to form ductal structures in vitro. This finding is consistent with the idea that lactational differentiation decreases the number of pluripotent stem cells in the breast. 2) Pre-menopausal breast tissue was more likely to form ductal structures than post-menopausal tissue. 3) Race was a modifying factor in the ability to form ductal architecture in culture, significantly affecting the rate of differentiation. Putative mammary stem cells were analyzed by flow cytometry (CD44+, CD24-, MUC1-, α6 integrin+) and cloning. Stem cell proportions varied widely (range 0.5-15%), with no obvious association with race.
 Discussion: One interpretation of the hormonal risk factors for BC involves the susceptibility of undifferentiated breast tissue to transformation. In other systems, especially the gonads, interference with terminal differentiation is also associated with susceptibility to transformation. If AA breast tissue has more robust differentiation potential than that of white women, it may also contain stem cells more likely to transform into an aggressive tumor type. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5051.

Copper secretion from human breast epithelial cells is mediated by ATP7B and lactational hormones

Mammary epithelial cells that produce milk are unique in containing both of the Cu pumps, ATP7A and B, central to mammalian copper homeostasis but otherwise found individually. We have begun studies to clarify their roles in this gland using confocal microscopy. ATP7B was expressed in the luminal epithelial cells of human breast tissue, with a perinuclear location that shifted to a diffuse location with lactation. In the human mammary epithelial cell culture model (PMC42), ATP7B was also in the perinuclear region and redistributed to endosomes adjacent to, but not coincident with, the apical plasma membrane after treatment of the cultures with a lactational hormone regimen (3 days estrogen and progesterone then 3 days insulin, dexamethasone, prolactin). ATP7B was in a different subset of vesicles from those containing milk proteins and did not overlap with endosomes containing ATP7A. A physiological concentration of extracellular Cu was required for the redistribution effect, suggesting it was caused indirectly by increased intracellular Cu concentrations. Overexpression of ATP7B in the PMC42 cultures, grown as polarized monolayers, resulted in an enhanced efflux of Cu from the apical cell surface (determined with 64Cu). Our findings are consistent with the concept that ATP7B is important for the secretion of copper from the mammary gland into the milk. Supported by PHS Grant No. RO1 HD 46949.

Development of a human mammary epithelial cell culture model for evaluation of drug transfer into milk

In the present study, a human mammary epithelial cell (HMEC) culture model was developed to evaluate the potential involvement of carrier-mediated transport systems in drug transfer into milk. Trypsin-resistant HMECs were seeded on Matrigel-coated filters to develop monolayers of functionally differentiated HMEC. Expression of the specific function of HMEC monolayers was dependent of the number of trypsin treatments. Among the monolayers with different numbers of treatment (treated 1 to 3 times), the monolayer treated 3 times (3-t-HMEC monolayer) showed the highest maximal transepithelial resistance and expression of beta-casein mRNA as an index of differentiation. Transport of tetraethylammonium (TEA) across the 3-t-HMEC monolayer in the basolateral-to-apical direction was significantly higher than that in the apical-to-basolateral direction (p < 0.05), whereas such directionality was not observed for p-amino-hippurate, suggesting the existence of organic cation transporters, but not organic anion transporters. In fact, expression of mRNAs of human organic cation transporter (OCT) 1 and 3 were detected in the 3-t-HMEC monolayer. These results indicate that the 3-t-HMEC monolayer is potentially useful for the evaluation of carrier-mediated secretion of drugs including organic cations into human milk.

Human Mammary Epithelial Cells Express CYP27B1 and Are Growth Inhibited by 25-Hydroxyvitamin D-3, the Major Circulating Form of Vitamin D-3

1alpha,25-dihydroxycholecalciferol [1alpha,25(OH)2D3], the active form of cholecalciferol, is a negative growth regulator of breast cancer cells. CYP27B1 is a cytochrome P450-containing hydroxylase expressed in kidney and other tissues that generates 1alpha,25(OH)2D3 from an inactive vitamin D precursor 25-hydroxycholecalciferol [25(OH)D3]. In these studies, we tested the hypothesis that mammary cells express CYP27B1 and locally produce 1alpha,25(OH)2D3, which acts in an autocrine manner to regulate cell turnover. Using Western blot and quantitative real-time PCR, CYP27B1 mRNA and protein were detected in immortalized, nontumorigenic human mammary epithelial cell (HMEC) cultures. Furthermore, HMEC cultures were dose dependently growth inhibited by physiological concentrations of 25(OH)D3, suggesting that CYP27B1 converts this precursor cholecalciferol metabolite to 1alpha,25(OH)2D3, the ligand for the vitamin D receptor (VDR). In support of this suggestion, both 1alpha,25(OH)2D3 and 25(OH)D3 transactivated VDR in HMEC cultures, as measured by induction of a vitamin D responsive reporter gene and upregulation of CYP24, an endogenous VDR target gene. No induction of CYP24 by 25(OH)D3 was observed in mammary cells derived from CYP27B1 null mice. Similar results were observed in 2 independently derived immortalized HMEC lines as well as in primary cultures derived from human breast epithelium. These are the first studies to demonstrate that nontransformed human mammary cells express CYP27B1, that they are growth inhibited by physiologically relevant concentrations of 25(OH)D3, and that they provide a biological mechanism linking vitamin D status to breast cancer risk.

Mammary epithelial cell transformation: insights from cell culture and mouse models

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16INK4a, p21, p27 and p57), p14ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.

Estrogen-induced phosphorylation of P53 at Ser15 in aging HMEC in the absence of estrogen receptor

615 Background: A variety of studies have established primary human mammary epithelial cells (HMEC) as an ex vivo model. It was of interest therefore, to investigate aging-related functional alterations in response to estrogen treatment as part of a hormone replacement therapy (HRT). Method: Primary HMEC were cultured for 5d, 19d, and 50d, respectively. During this aging process, functional changes including cell cycle distribution and estrogen receptor expression were determined by flow cytometry, respectively. Expression of P53 and phosphorylated P53 were detected by Western blot analysis and quantified by a P53 phospho ELISA. Results: Primary HMEC cultures underwent significant morphological and functional changes during the process of aging. Cells in S phase significantly declined after 19d and accumulated in G0/G1 and G2/M phase after 50d. Simultaneously, the initial estrogen receptor expression of 32,3% dropped to less than 1% in 19d and 50d aged HMEC, respectively. Short term treatment of HMEC with 1μM β-estradiol demonstrated unaltered levels of P53 and phosphorylated P53 after 60min. Following 24h of estrogen exposure, however, Western blot analysis revealed a significantly enhanced phosphorylation of P53 at Ser15 in 5d and 19d HMEC populations, respectively. A phospho-P53 ELISA confirmed a more than 2-fold increased Ser15-phosphorylation of P53 in these two HMEC populations following 24h of estrogen stimulation, but no significant changes after 60min, respectively. Conclusions: According to the estrogen receptor depletion in aged HMEC, these data indicated a receptor-independent signaling and thus suggest a dual estrogen mechanism: 1) a receptor-dependent probably short-term transcriptional effect, and 2) a receptor-independent long-term effect associated with P53 phosphorylation and subsequent repair. Whereas these dual estrogen signaling pathways are accompanied by aging-related functional alterations of the breast tissue, this would also complicate HRT to balance protective and damaging effects of estrogen. No significant financial relationships to disclose.

In Vivo Assessment of the Corneal Limbal Microenvironment by Scanning Slit Confocal Microscopy

CXC chemokines bearing the glutamic acid–leucine–arginine (ELR) motif are crucial mediators in neutrophil-dependent acute inflammation. Interestingly, however, Interleukin (IL)-8/CXC ligand (CXCL) 8 is expressed in human milk in biologically significant concentrations, and may play a local maturational role in the developing human intestine. In this chemokine subfamily, there are six other known peptides beside IL-8/CXCL8, all sharing similar effects on neutrophil chemotaxis and angiogenesis. In this study, we measured the concentrations of these chemokines in human milk, sought their presence in human mammary tissue by immunohistochemistry, and confirmed chemokine expression in cultured human mammary epithelial cells (HMECs). Each of the seven ELR+ CXC chemokines was measurable in milk, and except for NAP-2/CXCL7, these concentrations were higher than serum. The concentrations were higher in colostrum (except for GRO-β/CXCL2 and NAP-2/CXCL7), and correlated negatively with time elapsed postpartum. IL-8/CXCL8, GRO-γ/CXCL3, and ENA-78/CXCL5 concentrations were higher in preterm milk. There was intense immunoreactivity in mammary epithelial cells for all ELR+ CXC chemokines, and the intensity of staining was higher in breast tissue with lactational changes. The supernatants from confluent HMEC cultures also contained measurable concentrations of all the seven ELR+ CXC chemokines. These results confirm that all ELR+ CXC chemokines are actively secreted by the mammary epithelial cells into human milk. Further studies are needed to determine if these chemokines share with IL-8/CXCL8 the protective effects on intestinal epithelial cells.

In situ analyses of genome instability in breast cancer.

Transition through telomere crisis is thought to be a crucial event in the development of most breast carcinomas. Our goal in this study was to determine where this occurs in the context of histologically defined breast cancer progression. To this end, we assessed genome instability (using fluorescence in situ hybridization) and other features associated with telomere crisis in normal ductal epithelium, usual ductal hyperplasia, ductal carcinoma in situ and invasive cancer. We modeled this process in vitro by measuring these same features in human mammary epithelial cell cultures during ZNF217-mediated transition through telomere crisis and immortalization. Taken together, the data suggest that transition through telomere crisis and immortalization in breast cancer occurs during progression from usual ductal hyperplasia to ductal carcinoma in situ.