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1.
Abstract Three mouse mammary tumour lines (66, 67, and 68H) derived from a single mouse mammary tumour were investigated for their growth kinetics and development of quiescent cells in unfed monolayer cultures. All three lines develop pure quiescent populations when grown in unfed plateau cultures. A dramatic cell-cycle redistribution accompanied the proliferating (P) to quiescent (Q) transition, with the percentage of cells having a G1 DNA content increasing from 50% in the P state to <97% in the Q state. As the cultures progressed from exponential to plateau growth, a decrease of 50% in cellular RNA was observed in all three lines. This property enables the clear identification of P v. Q cells by flow cytometry using the two-step acridine orange assay. Autoradiographic data verified that these plateau cells were quiescent since >2.5% of the cells incorporated [3H]TdR when labelled for approximately two doubling times. Further comparison of the P and Q cells showed that: (a) the Coulter volume of Q cells was approximately half that of P cells in all three lines; (b) viability, as measured by dye exclusion was >95% in all cultures regardless of their proliferative state; and (c) colony-forming ability decreased as the cells entered the quiescent state. In each of these cell lines the development of Q-cell populations was marked by similar changes in all measured parameters. These quiescent tumour cells provide a relatively simple model to evaluate what, if any, important differences exist between the response of P v. Q cells to various therapeutic agents. 相似文献
2.
Landa Zeenelabdin Ali Salim Rozana Othman Mahmood Ameen Abdulla Karim Al-Jashamy Hapipah Mohd Ali Pouya Hassandarvish Firouzeh Dehghan Mohamed Yousif Ibrahim Fatima Abd Elmutaal Ahmed Omer Syam Mohan 《PloS one》2014,9(12)
BackgroundThymoquinone is an active ingredient isolated from Nigella sativa (Black Seed). This study aimed to evaluate the in vitro and in vivo anti-leukemic effects of thymoquinone on WEHI-3 cells.ConclusionThymoquinone promoted natural killer cell activities. This compound showed high toxicity against WEHI-3 cell line which was confirmed by an increase of the early apoptosis, followed by up-regulation of the anti-apoptotic protein, Bcl2, and down-regulation of the apoptotic protein, Bax. On the other hand, high reduction of the spleen and liver weight, and significant histopathology study of spleen and liver confirmed that thymoquinone inhibited WEHI-3 growth in the BALB/c mice. Results from this study highlight the potential of thymoquinone to be developed as an anti-leukemic agent. 相似文献
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Brigitte Mack Carola Eggert Katharina Eder Sannia Imrich Philipp Baumeister Ulrich Harréus Olivier Gires 《PloS one》2013,8(1)
The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm3 cubes termed explants, which are cultured 1–3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines. 相似文献
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Mariam B. Gonzalez-Hernandez Thomas Liu Luz P. Blanco Heather Auble Hilary C. Payne Christiane E. Wobus 《Journal of virology》2013,87(23):12685-12693
Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells'' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host. 相似文献
7.
The role of a stimulating factor in cell recruitment and the kinetics of its secretion were investigated by in vivo and in vitro techniques. the association of these two methods made it possible to demonstrate that a non-cycling population liberates a factor which in turn stimulates quiescent bone marrow stem cells into DNA synthesis. Moreover, it seems that undamaged cells are capable of secreting this factor. A stimulating factor responsible for cell recruitment was also demonstrated in an experimental EMT6 tumour and the kinetics of its secretion reported. 相似文献
8.
N. M. Barfod 《Cell proliferation》1981,14(4):389-394
A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G1 DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period. 相似文献
9.
Kinetics of Cell Volume Changes of Murine Lymphoma Cells Subjected to Different Agents In Vitro 总被引:1,自引:0,他引:1
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Cell volume distributions obtained with an electronic particle analyzer were used to study the changes in volume of individual cells in the absence of cell division. Cultures of murine lymphoma (strain L5178-Y) cells in suspension were used in these studies. During a division delay following ionizing radiation, individual cells increased exponentially in volume with equal rate constants; these rate constants were indistinguishable from that describing the increase in cell number of an unirradiated population. When an originally log phase population of cells was prevented from increasing in number by inhibitors of DNA synthesis, individual cells increased exponentially in volume for about one generation time with the same rate constant as observed after exposure to ionizing radiation; thereafter, only the cells defining the upper half of the volume distribution continued to increase in volume, and they apparently did so with a first order rate constant proportional to their amount of DNA exceeding that present in one diploid complement of chromosomes in G(1). Cells arrested in mitosis with colchicine increased in volume for approximately 4 hr after which they remained constant in volume for almost one generation time; eventually these cells again increased in size. Inhibitors of protein and RNA synthesis inhibited the cell volume growth of irradiated cells. 相似文献
10.
THE possibility of an oncogenic virus in human breast cancer has been increased by recent findings. Virus-like particles resembling the mammary tumour virus (MTV) were found in electron micrographs of human breast cancer tissue1 and particles physically and morphologically similar to MTV particles have been seen in human milk2. These particles were found more frequently in the milk of American women with a history of breast cancer in their immediate families and in the milk of Parsi women in Bombay than in the milk of nonselected American women3. Parsi women are three times more likely to have breast cancer than other women in Bombay3. The detection of RNA-dependent DNA-polymer-ase activity in such particles isolated from human milk emphasized the possibility that these particles represent an oncogenic RNA virus4. In addition to the physical and morphological resemblance of human milk particles and MTV an immunological relationship between these two kinds of particles seems probable; sera from breast cancer patients neutralize the biological activity of MTV whereas normal human sera did not do so5. We report data supporting the hypothesis of an immunological cross relationship between MTV and human breast cancer. 相似文献
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Júlia Kurkó András Vida Tímea Ocskó Beata Tryniszewska Tibor A. Rauch Tibor T. Glant Zoltán Szekanecz Katalin Mikecz 《PloS one》2014,9(11)
Background
Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.Methods
Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.Results
BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.Conclusions
Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA. 相似文献12.
S. NANDI 《Nature: New biology》1971,230(13):146-147
THE classical mammary tumour virus (MTV), also referred to as the Bittner virus, is transmitted in milk from mother to offspring. Studies1–4 of the fate and biological behaviour of MTV in BALB/cfC3H (C+) mice suggested to us that MTV goes through a cycle in the infected host. Milk-borne MTV (M-MTV), assumed to be B particles, is abundantly present in milk and mammary tissues of infected mice. It seems likely that, on entering the host, M-MTV first infects the erythropoietic cells; subsequently the virus is carried into the general circulation chiefly inside the reticulocyte fraction2 of red blood cells (RBC) in a form which we have called R-MTV4. We have also suggested4 that, in the MTV cycle, R-MTV functions in the infection of mammary tissues, which are the primary sites of production of M-MTV (or B particles). The latter form of the virus, in turn, is involved in the transport of the viral genome to the young through milk. 相似文献
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From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro. 相似文献
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The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti-Ia serum on the response were studied. The incorporation of [3H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti-Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti-Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia-negative bone marrow cells generate Ia-positive cells during the culture period, and the Ia-positive cells are responsive cells to LPS. The proliferative response of 1- or 2-week-old spleen cells was easily suppressed by anti-Ia serum when compared with that of 4-week-old spleen cells. Furthermore, the responses of spleen cells obtained from γ-irradiated and syngeneic bone marrow cell-reconstituted mice were prominently suppressed by anti-Ia serum in comparison with that of normal adult spleen cells. These findings suggest that LPS-responsive lymphocytes in the developmental stage are quite sensitive to anti-Ia serum. The effect of anti-Ia serum on the maturation of bone marrow-derived lymphocytes was discussed. 相似文献
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D. L. Fine L. O. Arthur J. K. Plowman E. A. Hillman F. Klein 《Applied microbiology》1974,28(6):1040-1046
An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C(3)H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 10(10) particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus. 相似文献
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Migration of Fibroblastoid Stromal Cells In Murine Blood 总被引:1,自引:0,他引:1
A. H. Piersma R. E. Ploemacher K. G. M. Brockbank P. G. J. Nikkels C. P. E. Ottenheim 《Cell proliferation》1985,18(6):589-595
Abstract. This paper describes the kinetics of fibroblastic colony forming units (CFU-f) in murine blood after phenylhydrazine-induced haemolytic anaemia and their subsequent migration into haemopoietic organs. Murine blood contained 5.3 φ 0.8 CFU-f per 106 nucleated cells. Absence of particle ingestion and factor VIII-related antigen in addition to the enzyme pattern in CFU-f-derived cells confirmed that these cells did not have a macrophage-like or endothelial nature. Phenylhydrazine treatment of mice resulted in a 3-fold increase in blood CFU-f numbers which was accompanied by increases in blood cellularity and granulocyte-macrophage progenitor numbers. When both partners of CBA/N and CBA/T6T6 mice in parabiosis had been treated with phenylhydrazine, spleens and femoral bone marrow of both mice were shown to contain partner-derived CFU-f. These data suggest that circulating CFU-f represent a stromal cell population which can migrate into haemopoietic organs. 相似文献
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Kathleen Church 《Genetics》1965,52(4):843-849
19.
Relationship Between Organization of Mammary Tumors and the Ability of Tumor Cells to Replicate Mammary Tumor Virus and to Recognize Growth-Inhibitory Contact Signals In Vitro 总被引:2,自引:3,他引:2
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Mammary tumor virus (MTV) replication was confined primarily to cells organized as acini in intact mouse mammary glands. Primary mammary tumors maintained a high degree of acinar organization and cells therein continued to replicate MTV vegetatively. Nonacinar mammary cells, derived by serial transplantation of acinar tumor cells, no longer actively replicated MTV. This suggests that phenotypic differences exist among mammary epithelial cells in their ability to support virus replication, that a fundamental relationship exists between the organization of epithelium for secretion and active virus replication, and that this relationship is not altered as a primary consequence of neoplastic transformation. Mammary epithelial cells from pregnant, non-tumor-bearing, MTV-infected BALB/cfC3H mice or from acinar mammary tumors from a number of mouse strains were grown in primary monolayer cultures. Such cell cultures under the influence of insulin and cortisol exhibited the ability to organize into discrete three-dimensional structures called "domes." MTV replication in such cultures took place primarily in cells within the organized domes. Cells cultured from nonacinar tumors did not exhibit any propensity to organize into domes, nor did they replicate MTV in primary culture. This suggests that the cell organizational requirement for MTV replication observed in vivo is conserved in primary culture. Dome formation is not an effect of virus replication, as cells from uninfected BALB/c animals organized into domes in culture without concomitant MTV replication. Growth-regulating signals, exerted between contiguous cells in cultures of non-MTV-infected mammary epithelium, were not modified by the occurrence of active virus replication nor as a direct consequence of neoplastic transformation. Cells derived from nontumor BALB/cfC3H glands and from spontaneous tumors exhibited cell growth kinetics, saturation densities, and deoxyribonucleic acid synthesis kinetics nearly identical to those of noninfected normal mammary epithelium in primary culture. Cell to cell growth regulatory signals were modified in cultures of nonalveolar tumor cells wherein evidence of overgrowth is documented. 相似文献
20.
An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent, i.e., may be generated from a single mammary epithelial stem cell. Early studies showed that any portion of an intact murine mammary gland containing epithelium could recapitulate an entire mammary epithelial tree on transplantation into an epithelium-free mammary fat pad. More recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium and that their behavior and maintenance is dependent on signals generated both locally and systemically. In this review, we have attempted to develop the scientific saga surrounding the discovery and characterization of the murine mammary stem/progenitor cell hierarchy and to suggest further approaches that will enhance our knowledge and understanding of these cells and their role in both normal development and neoplasia.Before the 1980s there was little if any thought that the epithelium in murine mammary glands might be engendered by or supported by a mammary epithelial specific stem cell. In 1980, Rudland et al. wrote a review entitled “Stem cells in rat mammary development and cancer: A review” and noted that dimethylbenz [α] anthracene (DMBA)-induced rat carcinomas contained all three main types of epithelium found in the normal rat gland, those lining the ductal lumina, those lining the alveolar lumina, and myoepithelial cells (Rudland et al. 1980). They suggested, based on the two types of morphologically distinct epithelial (luminal and myoepithelial) cancer cells in the clonally derived Rama 25 cell line, that a single cell might give rise to both types and this also held true when these cells were inoculated into hosts and produced tumors. Williams and Daniel (Williams and Daniel 1983) suggested that the cap cells at the tip of the growing ducts in the mouse could give rise to both luminal and myoepithelial cells during ductal morphogenesis. However, no direct evidence that a single cell could produce both epithelial cell types in vivo was available. Nevertheless, in retrospect there was evidence that full regenerative activity for mammary epithelial existed in every part of the adult mammary epithelial tree.The experiments that originally showed the potential existence of stem cells in the mouse mammary gland were the pioneering studies of DeOme and his students, Les Faulkin and Charles Daniel. The approach they developed and optimized was serial transplantation of normal mammary gland into the cleared mammary fat pad of syngeneic mice (Deome et al. 1959; Faulkin and Deome 1960). The cleared mammary fat pad allowed the transplantation and growth of normal mammary cells into their normal anatomical site and under the influence of a normal physiological environment. Using this method, DeOme and coworkers showed that all portions of the normal mammary gland contains cells that will grow and fill the fat pad with a normal ductal mammary tree and respond to hormones with a normal differentiation program (Daniel 1975; Daniel et al. 1975). The progeny of the transplanted cells could be serially transplanted into the appropriate recipients for multiple times; however, unlike preneoplastic or neoplastic cells, the normal cells always senesced after multiple serial transplants, generally five to eight transplant generations (Daniel 1975). This was interpreted as indicating mammary stem cells possessed a finite proliferative activity (i.e., life span). This finite life span was a fundamental difference between normal and preneoplastic/neoplastic mammary cells. Cells with an indefinite in vivo life span (i.e., immortalized) have been identified in numerous mammary model systems, including MMTV-induced alveolar hyperplasia''s (Callahan and Smith 2000), chemical carcinogen-induced ductal and alveolar hyperplasia''s (Smith et al. 1978, 1980), hormonally induced alveolar hyperplasia, spontaneously immortalized ductal hyperplasia''s (Medina 2000, 2002), and cells containing specific genetic alterations (i.e., p53 deletion, Polyoma mT antigen) (Maglione et al. 2001; Medina et al. 2002).Subsequent studies showed that stem cells were located along the entire mammary tree and represented in all the different developmental states of the mammary gland. These stages included primary and tertiary ducts from 6- and 16-wk virgin glands, uniparous and multiparous regressed gland, 15-d pregnant and 10-d lactating glands (Smith and Medina 1988). Host age and reproductive history had little influence on the frequency of stem cells as measured by percent successful takes and life span assay (Young et al. 1971; Smith and Medina 1988). Mammary cells taken from 26-mo-old virgin mice had the same transplant potential as cells taken from 3-wk-old mice. Cell populations, from both, senesced after five transplant generations. Similarly, mammary cells in 12-mo-old multiparous mice had the same serial transplant potential as cells from 3-wk-old virgin mice (Young et al. 1971). Finally, continuous hormone stimulation did not induce additional loss of ductal growth potential. These results have important implications for understanding the role of mammary stem cells in normal mammary development because they emphasize that the mammary stem cell is a relatively quiescent cell that is only activated under conditions of gland repopulation (i.e., fetal growth stage and pubertal growth phase). 相似文献