Circadian clock gene expression in human peripheral blood mononuclear cells

Circadian clock genes have been identified in humans but information regarding their expression has remained very limited. However from a basic point as well as in a diagnostic and therapeutic perspective, it is important to evaluate molecular clock gene expression. Peripheral blood mononuclear cells represent an ideal material to investigate non-invasively the human clock at the molecular level. Several studies including ours reported rhythmic expression of clock genes in these cells, with significant intersubject variability of expression. In addition, our results reveal the existence of different chronotypes of clock gene expression patterns and suggest specific regulatory mechanisms in these human cells as compared to other peripheral tissues.     

Circadian clock and cell cycle: Cancer and chronotherapy

The circadian clock is an endogenous timing system that ensures that various physiological processes have nearly 24 h circadian rhythms, including cell metabolism, division, apoptosis, and tumor production. In addition, results from animal models and molecular studies underscore emerging links between the cell cycle and the circadian clock. Mutations in the core genes of the circadian clock’ can disrupt the cell cycle, which in turn increases the possibility of tumors. At present, tumor chronotherapy, which relies on a circadian clock mechanism, is developing rapidly for optimizing the time of drug administration in tumor treatment to improve drug efficacy and safety. However, the relationship between the circadian clock and the cell cycle is extremely complicated. This review summarizes the possible connection between the circadian clock and the cell cycle. In addition, the review provides evidence of the influence of the circadian clock on senescence and cancer.     

Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland

Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland was examined under light microscopic histochemistry and electron microscopy. To avoid loss of water-soluble glycogen during histological tissue preparation, fixation with ethanol and embedding in hydrophilic glycol methacrylate resin was used for light microscopy, and high-pressure freezing/freeze substitution for electron microscopy. Glycogen was detected on periodic acid-Schiff stain, periodic acid-thiosemicarbazide-silver proteinate reaction, and the digestion test with alpha-amylase. On embryonic day 14, glycogen began to accumulate in the proximal portions of the developing epithelial cords. On embryonic day 17, marked glycogen particles were seen at the basal portion of the ductal epithelial cells and an abrupt increase of glycogen accumulation occurred in the secretory cells in the terminal bulbs. Ultrastructural observation indicated large clumps of glycogen particles localized in the basal portion of the terminal bulb cells. The initiation of glycogen accumulation preceded the formation of lumens in the ducts and terminal bulbs. Furthermore, proliferation analysis by bromodeoxyuridine labeling showed that this glycogen accumulation followed the cessation of the epithelial cell proliferation. Postnatally, glycogen accumulation in the terminal bulbs became gradually inconspicuous and completely disappeared by postnatal day 3, but that in the ducts was retained until around postnatal day 12. Temporary glycogen accumulation after the cell proliferation and before/during the lumen formation and secretory granule formation suggests significant involvement of the carbohydrate metabolism in the organogenesis of the submandibular gland.     

Histamine synthesis by mouse mammary gland epithelial cells in primary culture. The effects of mammary gland differentiation stage (pregnancy and lactation)

Inflammation Research -     

Histamine concentration and metabolism in mouse mammary gland during the estrous cycle

Inflammation Research -     

s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP⁺ cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP⁺ cells can regenerate the mammary epithelial network. GFP⁺ mammary epithelial cells are p63⁺, CD24^mod, CD49f^high, and CD29^high; are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP⁺ basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells.     

Mapping oxytocin receptor gene expression in the mouse brain and mammary gland using an oxytocin receptor-LacZ reporter mouse

The hypothalamic nonapeptide oxytocin (OT) has an established role as a circulating hormone but can also act as a neurotransmitter and as a neuromodulator by interacting with its central OT receptor (OTR). To understand the role of the OTR in the mouse brain we investigated the expression of the OTR gene at the cellular level. We targeted the lacZ reporter gene to the OTR gene locus downstream of the endogenous OTR regulatory elements. Using lactating mouse mammary gland as a control for OTR promoter directed specificity of lacZ gene expression, X-gal histochemistry on tissue sections confirmed that gene expression was restricted to the myoepithelial cells. We also identified for the first time in mice the expression of the OTR gene in neighbouring adipocytes. Further, investigation in the mouse brain identified numerous nuclei containing neurons expressing the OTR gene. Whilst some of these regions had been described for rat or sheep, the OTR-LacZ reporter mouse enabled the identification of novel sites of central OTR gene expression. These regions include the accessory olfactory bulb, the medial septal nucleus, the posterolateral cortical amygdala nucleus, the posterior aspect of the basomedial amygdala nucleus, the medial part of the supramammillary nucleus, the dorsotuberomammillary nucleus, the medial and lateral entorhinal cortices, as well as specific dorsal tegmental, vestibular, spinal trigeminal, and solitary tract subnuclei. By mapping the distribution of OTR gene expression, depicted through histochemical detection of beta-galactosidase, we were able to identify single OTR gene expressing neurons and small neuron clusters that would have remained undetected by conventional approaches.These novel sites of OTR gene expression suggest additional functions of the oxytocinergic system in the mouse. These results lay the foundation for future investigation into the neural role of the OTR and provide a useful model for further study of oxytocin functions in the mouse.     

Circadian rhythms of kinetics of mouse esophageal epithelial cells

The circadian rhythm of cell division and DNA synthesis and parameters of the mitotic cell cycle during the maximum and minimum of proliferation in the course of the 24-h period were studied in the epithelium of the mouse esophagus. The number of mitoses and DNA-synthesizing cells increased regularly at 1–7 a.m. and 10 p.m. –4 a.m. respectively. When [³H]-thymidine was injected into the mice at 2 a.m. the duration of the periods of the mitotic cycle was as follows: G_{2 min}=1 h, G₂+1/2M=2 h, S=7.1 h, and G₁+1/2M=15.9 h. When [³H]-thymidine was injected at 2 p.m. the duration of the S-period was increased to 8.2 h, and that of G₁+1/2M to 14.8 h. The total duration of the mitotic cycle in both series of experiments was 25 h. The duration of the individual phases of the mitotic cycle thus depends on the time of day and correlates with rhythmic changes in the number of dividing and DNA-synthesizing cells. The duration of the mitotic cycle is the same for cells passing through it at different times of day and is approximately the same as the period of the circadian rhythm of mitosis and DNA synthesis in the esophageal epithelium.Department of Biology and Genetics, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 341–343, September, 1979.     

The mouse mammary microenvironment redirects mesoderm-derived bone marrow cells to a mammary epithelial progenitor cell fate

Mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue leads to a reduced capacity of dispersed epithelial cells to recapitulate complete functional mammary structures. Previous studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the mammary stroma, nonmammary cells of ectodermal germ origin may be sequestered and reprogrammed to perform mammary epithelial cell (MEC) functions, including those ascribed to mammary stem/progenitor cells. To test whether tissue cells from organs derived from different germ layers could respond to mammary epithelial-specific signals, we utilized fluorescence-activated cell sorting-purified Lin(-) and Lin(-)/cKit+adult male bone marrow cells to mix with MECs. Our evidence shows that the signals provided by the mammary microenvironment are capable of redirecting mesoderm-derived adult progenitor cells to produce functional MEC progeny.     

Cysteine string protein expression in mammary epithelial cells

Cysteine string protein (Csp) is a secretory vesicle protein previously demonstrated to be required for Ca2+-regulated exocytosis in neurons and endocrine cells. It has been suggested to function by regulating voltage-gated Ca2+ channels or, alternatively, to have a more direct effect on the regulated exocytotic machinery. Here we demonstrate the expression of Csp in mammary epithelial cells and in the KIM-2 mammary cell line. In KIM-2 cells, Csp was found to be associated with a population of small vesicles and showed partial co-distribution with the vesicle protein cellubrevin. KIM-2 cells do not express detectable levels of voltage-gated Ca2+ channels, ruling these out as a site of action. Using the release of transfected growth hormone (GH) as an assay of secretion, we found that GH is secreted in an exclusively constitutive manner from KIM-2 cells. Overexpression of Csp1 inhibits regulated exocytosis in other cell types but has no effect on constitutive GH release by KIM-2 cells. These results suggest that Csp does not have a major function in constitutive exocytosis.     

EMT tumorigenesis in the mouse mammary gland

The term EMT (epithelial-mesenchymal transition) is used in many settings. This term is used to describe the mechanisms facilitating cellular repositioning and redeployment during embryonic development and tissue reconstruction after injury. Recently, EMT has also been applied to potential mechanisms for malignant progression and has appeared as a specific diagnostic category of tumors. In mice, most 'EMT' tumors have a spindle cell phenotype. The definition of EMT is controversial because spindle cell tumors are not common in humans, especially in human breast cancers. Spindle cell tumors of the mouse mammary gland have been observed for many years where they are usually classified as sarcomas or carcinosarcomas. Genetically engineered mice develop mammary spindle cell tumors that appear to arise in the epithelium and undergo EMT. To better understand the origin and evolution of these spindle cell tumors in progression and metastases, seven cohorts of spindle cell tumors from the archives of the University of California, Davis Mutant Mouse Pathology Laboratory were studied. This study provides experimental and immunohistochemical evidence of EMT showing that dual epithelial and mesenchymal staining of tumor spindle cells identifies some, but not all, EMT-type tumors in the mouse. This suggests that potential EMT tumors are best designated EMT-phenotype tumors.