Changes in ErbB2 (her-2/neu), ErbB3, and ErbB4 during growth, differentiation, and apoptosis of normal rat mammary epithelial cells.

Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs.     

Induction of HL-60 monocytic cell differentiation promoted by a perturbation of DNA synthesis: hydroxyurea promotes action of TPA

Control of terminal cell differentiation was studied using the human promyelocytic leukemia cell line, HL-60. HL-60 cells are known to undergo terminal monocytic differentiation when continuously exposed to 1.6 nM tetradecanoylphorbol acetate (TPA). The dose-response relationship between TPA concentration and induced differentiation is relatively steep. TPA (1.1 nM) induces little G1/0 specific growth inhibition or phenotypic differentiation. In contrast, pretreating the cells with a pulse exposure to hydroxyurea promotes their capability to terminally differentiate in response to TPA. Initially exponentially proliferating cells exposed for 20 h, approximately one doubling time, to 0.3 mM hydroxyurea, a subcytotoxic dose, underwent rapid G1/0 specific growth arrest and cell differentiation in response to subsequent exposure to 1.1 nM TPA. The extent of terminal differentiation was comparable to that induced by 1.6 nM TPA. The results support the hypothesis that early events in induction of terminal HL-60 cell differentiation depend on an S phase-specific process which may involve gene amplification.     

TGF-β Inhibition Restores Terminal Osteoblast Differentiation to Suppress Myeloma Growth

Background

Multiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-β, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-β and its inhibition in bone formation and tumor growth in MM.

Methodology/Principal Findings

TGF-β suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-β. Inhibitors for a TGF-β type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-β inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-β inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-β inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models.

Conclusions/Significance

The present study demonstrates that TGF-β inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-β appears to be an important therapeutic target in MM bone lesions.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.     

Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine

Tight regulation of self-renewal and differentiation of adult stem cells ensures that tissues are properly maintained. In the Drosophila intestine, both commitment, i.e. exit from self-renewal, and terminal differentiation are controlled by Notch signaling. Here, we show that distinct requirements for Notch activity exist: commitment requires high Notch activity, whereas terminal differentiation can occur with lower Notch activity. We identified the gene GDP-mannose 4,6-dehydratase (Gmd), a modulator of Notch signaling, as being required for commitment but dispensable for terminal differentiation. Gmd loss resulted in aberrant, self-renewing stem cell divisions that generated extra ISC-like cells defective in Notch reporter activation, as well as wild-type-like cell divisions that produced properly terminally differentiated cells. Lowering Notch signaling using additional genetic means, we provided further evidence that commitment has a higher Notch signaling requirement than terminal differentiation. Our work suggests that a commitment requirement for high-level Notch activity safeguards the stem cells from loss through differentiation, revealing a novel role for the importance of Notch signaling levels in this system.     

Primary culture in chemically defined medium of human fetal mammary epithelial cells within reconstituted matrix]

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal human fetal mammary epithelial cells within an extracellular matrix preparation. Human fetal mammary epithelial cells were isolated from the mammary glands of human female fetuses, 17 to 39 weeks-old. The "organoids" were embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm (EHS) sarcoma according to the method of Hahm and Ip. "Organoids" were grown in either serum-free medium or in medium with fetal calf serum (FCS). The "organoid" proliferated over a 2 to 3 weeks culture period and remained viable for 1 or 2 months within the basement membrane matrix in serum free medium. Several types of colonies were observed; including alveolar-like budding clusters obtained from cultures of mammary gland from fetuses of over 20 weeks age, units with ductule-like projections and stellate-type colonies. Cell proliferation was dependent on the culture medium (with FCS no proliferation was obtained) and on the substratum (without matrix, significantly less growth and development occurred). These types of colonies are obtained when a glandular differentiation of cells budding from the malpighian epithelium is observed. Light microscopic and transmission electron microscopic studies were undertaken at the time of culture. This unique system using normal fetal mammary epithelial cells thus provides a model in which the regulation of human mammary development can be investigated.     

Dynamic histology of the antral epithelium in the mouse stomach: III. Ultrastructure and renewal of pit cells

The pit (foveola) of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice, using light and electron microscopy, sometimes combined with 3H-thymidine radioautography. Reconstruction of units from serial sections revealed that, on the average, the pit measured 151 microns in length and was lined by 184 mucus-containing pit cells. Of these, 164 were located along the wall of the pit, whereas 20 surrounded its opening on the free surface. For ultrastructural examination the pit was divided into equal thirds. The proximal third, located next to the isthmus and referred to as pit base, was composed of cells showing electron-dense mucous granules greater in number but similar in density and diameter to those of isthmal dense granule cells. Nucleoli were rather large, irregular, and reticulated; these and other features were indicative of partial differentiation. The appearance of the cells gradually changed with the distance from the isthmus. In the middle third or mid pit, cells had small, fairly rounded nucleoli, while mucous granules were more numerous than in the pit base but similar in appearance and size; these cells were considered to be mature. In the distal third or pit top-surface, granules became elongated and nucleoli shrank, and lysosomes and vacuoles greatly increased in number, indicating that cells were at a terminal stage. Indeed, some of the cells were extruded into the stomach lumen while others were phagocytosed by adjacent cells. Following a single injection of 3H-thymidine, labeling was found only in a small cohort of cells in the pit base. At the end of 1 day of continuous infusion, the cohort of labeled cells had reached the mid pit; by 2 days, the pit top; and by 3 days, the free surface, where cells were eventually lost. The renewal time of pit cells was assessed at 2.98 days (t1/2 = 1.8 days), giving a turnover rate of 33.5% per day. It is estimated that the divisions of pit base cells provide two-thirds of the cells needed daily for pit-cell renewal, while the other third is supplied by an influx of dense granule cells from the isthmus. These cells enter the pit and continuously migrate toward the gastric lumen, while differentiating in the pit base, maturing in the mid pit, and reaching a terminal stage at the pit top-surface. The progressive and orderly migration of pit cells is described as a "pipeline pattern" of renewal. It is completed in about 3 days when terminal cells are lost at the pit top-surface.     

A simple mathematical model based on the cancer stem cell hypothesis suggests kinetic commonalities in solid tumor growth

Background

The Cancer Stem Cell (CSC) hypothesis has gained credibility within the cancer research community. According to this hypothesis, a small subpopulation of cells within cancerous tissues exhibits stem-cell-like characteristics and is responsible for the maintenance and proliferation of cancer.

Methodologies/Principal Findings

We present a simple compartmental pseudo-chemical mathematical model for tumor growth, based on the CSC hypothesis, and derived using a “chemical reaction” approach. We defined three cell subpopulations: CSCs, transit progenitor cells, and differentiated cells. Each event related to cell division, differentiation, or death is then modeled as a chemical reaction. The resulting set of ordinary differential equations was numerically integrated to describe the time evolution of each cell subpopulation and the overall tumor growth. The parameter space was explored to identify combinations of parameter values that produce biologically feasible and consistent scenarios.

Conclusions/Significance

Certain kinetic relationships apparently must be satisfied to sustain solid tumor growth and to maintain an approximate constant fraction of CSCs in the tumor lower than 0.01 (as experimentally observed): (a) the rate of symmetrical and asymmetrical CSC renewal must be in the same order of magnitude; (b) the intrinsic rate of renewal and differentiation of progenitor cells must be half an order of magnitude higher than the corresponding intrinsic rates for cancer stem cells; (c) the rates of apoptosis of the CSC, transit amplifying progenitor (P) cells, and terminally differentiated (D) cells must be progressively higher by approximately one order of magnitude. Simulation results were consistent with reports that have suggested that encouraging CSC differentiation could be an effective therapeutic strategy for fighting cancer in addition to selective killing or inhibition of symmetric division of CSCs.     

3T3-L1 adipocytes promote the growth of mammary epithelium

Murine mammary epithelium grows in association with predominantly adipocyte stroma in vivo. To investigate potential growth-promoting effects of adipocytes on mammary epithelium, we developed a co-culture system of mammary epithelium and adipocytes by taking advantage of the 3T3-L1 cell line. These cells undergo adipocyte differentiation when the culture reaches confluence and growth ceases. Mid-pregnant murine mammary epithelium was plated on lethally irradiated feeder layers of 3T3-L1 adipocytes, undifferentiated 3T3-L1 cells, 3T3-C2 fibroblasts (a subclone of 3T3 cells that does not undergo adipocyte differentiation), or tissue culture plastic. Mammary epithelial colony size on adipocyte feeder layers was 2-fold larger than colonies on 3T3-C2 cells and 4-fold larger than colonies on tissue culture plastic. Measurement of tritiated thymidine [3H]TdR incorporation and labelling index in mammary cells was significantly higher on adipocytes than on other feeder layers or plastic. There was a 6-fold increase in mammary cell number after 5 days in culture when mammary epithelium was plated on substrate-attached material ('extracellular matrix') derived from 3T3-L1 cells and a 4-fold increase in cell number when plated on plastic in conditioned medium derived from 3T3-L1 adipocytes compared with growth on plastic in unconditioned medium. We conclude that interaction of mammary epithelium with adipocytes results in a marked increase in proliferation of mammary epithelium and that extracellular components may mediate this effect.     

Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium

A serum-free medium (LEP-1) has been developed for mouse epidermal keratinocytes. LEP-1 consists of "Ca2+-free" Eagle's MEM with non-essential amino acids and seven added supplements (transferrin, 5 micrograms/ml; epidermal growth factor (EGF), 5 ng/ml; hydrocortisone, 0.5 microM; insulin, 5 micrograms/ml; phosphoethanolamine and ethanolamine, each 50 microM; bovine pituitary extract, 180 micrograms of protein/ml). Although serum-free the culture system was dependent for growth on bovine pituitary extract as the only still undefined supplement. LEP-1 supports sustained multiplication of mouse keratinocytes for 25 or more population doublings. A clonal growth assay was developed to investigate the action of growth factors, hormones and other supplements on keratinocytes. Cells grown in LEP-1 (calcium concentration was 0.03 mM) maintained a high proliferative rate and presented the typical morphology of basal epidermal cells. When the calcium concentration of the medium was raised to 1.0 mM, the cells were triggered to differentiate terminally. The epithelial nature of the cells was demonstrated both by electron microscopy and by immunostaining with anti-keratin antibody. The maturation stage of the keratinocytes was defined by several morphological features during the proliferative phase and in terminally differentiating cultures. This serum-free system supported a useful number of cell divisions while keratinocytes retained the capacity to undergo terminal differentiation when given the appropriate stimulus. It provides, therefore, provides a useful model for investigations on growth, differentiation and malignant transformation of epidermal cells in culture.     

Data-driven modeling of cellular stimulation, signaling and output response in RAW 264.7 cells

Background

Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny.

Results

In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabe^puro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the β-casein gene in response to lactogenic hormones. We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3β hyperphosphorylation and β-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed.

Conclusion

This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to β-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.     

Morphogenesis during asexual reproduction in Pygospio elegans Claparede (Annelida, Polychaeta)

The spionid Pygospio elegans reproduces both asexually and sexually. Using scanning electron and bright field microscopy, we examined morphogenesis following asexual reproduction to determine how "lost" body regions were regenerated after a worm spontaneously divided. Asexual reproduction occurred through transverse fission and divided the parent worm into 2 to 6 fragments (architomy). All fragments retained their original anterior-posterior polarity. Regeneration in all fragments followed a specific series of events: wound healing (day 1); extension of the blastema to generate lost body regions-specifically, the head and thorax for posterior fragments and the tail and pygidium for anterior fragments (days 2-3); segmentation (days 3-6); and differentiation of segment- or region-specific structures (days 4-8). This pattern occurred regardless of where the original division took place. Subsequent growth occurred through addition of terminal setigers anterior to the pygidium followed by differentiation of tail setigers into abdominal setigers, leaving the tail region about 6 to 10 setigers in size. Division rates were compared in worms from three populations in Nova Scotia, Canada. Worms from two populations (Conrad's Beach, Starr's Point) divided more frequently (about 1.2 and 1.3 weeks between divisions, respectively) than worms from Bon Portage Island (3.5 weeks between divisions). Fragments containing the original head (original mouth intact, generally much larger fragment) had a higher survivorship than fragments containing the original tail.     

A simplified method for passage and long-term growth of human mammary epithelial cells

Summary A method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca⁺⁺ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca⁺⁺ to overcome “renewal inhibition” and to stimulate growth. In low Ca⁺⁺ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca⁺⁺, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca⁺⁺, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 10⁵ cells/cm² of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: (a) they formed domes and exhibited renewal inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments, and (d) they were diploid.     

TOPOGRAPHY OF DNA SYNTHESIS IN THE MAMMARY GLAND OF THE C3H MOUSE AND ITS CONTROL BY OVARIAN HORMONES: AN AUTORADIOGRAPHIC STUDY

Tritium-labelled thymidine was injected 45 min before sacrifice into virgin female C3H/HeJ mice 7–23 weeks of age, as well as into 10-week-old mice which had been ovariectomized and treated daily with 1 μg of oestradiol-17β and/or 1 mg of progesterone. Autoradiographs were made of squash preparations of the mammary glands, stained by Feulgen's method. The following results were obtained: (1) During normal development of the gland, cells synthesizing DNA are abundant in terminal buds and virtually absent in duct epithelium. Hence ductal growth takes place by the addition of cells produced in the terminal end structures. (2) At 5–6 months, when mammary growth has ceased, a considerable number of cells synthesizing DNA can still be found in alveoli, though not in duct epithelium. Hence the alveolar cells constitute a renewal population in the adult virgin. Because they maintain the potentiality to divide, duct cells are a G₀ population. (3) Ovariectomy results in arrest of DNA synthesis within 3–5 days. Both oestradiol and progesterone restore DNA synthesis in alveoli but only progesterone is able to induce DNA synthesis in duct epithelium, and the differentiation of terminal buds into alveoli. This finding provides an explanation for the resumed proliferation of duct cells in pregnancy. (4) The number of cells engaged in DNA synthesis varies considerably among identical structures within the same gland. This may be due either to synchrony of cell replication and/or to fluctuations of proliferative activity in the gland.     

The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line

We have previously postulated an in vivo pathway of thymic epithelial (TE) cell maturation in pre- and postnatal thymus, whereby endocrine medullary TE cells terminally differentiate to form Hassall's bodies. Epithelial-cell differentiation has been well documented in vitro using epidermal keratinocytes. Therefore, to characterize TE-cell differentiation in vitro, we observed clones of the rat TE cell line, IT26R21, after 4 and 14 days in culture. We found alterations in cell morphology, the cessation of cell proliferation, and the acquisition of a differentiation antigen defined by monoclonal antibody TE-19 (a marker of terminally differentiated epithelial cells). At light and electron microscopy, we detected progressive TE-cell stratification and squamous-cell formation between 4 and 14 days of culture. Autoradiography on day 14 showed that squamous TE cells in stratified layers did not incorporate tritiated thymidine, while surrounding smaller cells adhering to the substratum continued to synthesize DNA. At indirect immunofluorescence, only 3% of cells reacted with monoclonal antibody TE-19 at day 4, while on day 14, 22% of the TE cells were TE-19 positive (P less than 0.02). Antibody-TE-19 reactivity was limited to stratified, squamous TE cells. Additionally, we isolated a clone of the IT26R21 cell line that did not undergo these changes characteristic of TE cell differentiation. We conclude that IT26R21 TE cells are capable of undergoing programs of both terminal differentiation and cell renewal in vitro.     

Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.

Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.     

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres ¹. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo ². The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.Download video file.^{(57M, mp4)}