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1.
The JB6 mouse epidermal cell model system is being used to study the mechanism of promotion of transformation. Promotion of anchorage independence in JB6 cells occurs in response to second-stage but not first-stage promoters, and is inhibited by inhibitors of second-stage not first-stage promotion. A number of variants that are resistant to the phorbol diester TPA have been derived. Some are resistant to plateau density mitogenic stimulation by TPA; others are resistant to promotion of anchorage independence by TPA. Some of the mitogen-resistant variants were promotable by TPA, thus ruling out a requirement for TPA mitogenesis in promotion of transformation in JB6 cells. TPA promotable clones were also sensitive to mezerein and EGF while the TPA nonpromotable variants were also resistant to mezerein and EGF, suggesting that sensitivity to promoters in these JB6 cells is determined at a level distal to receptor binding. Promotion sensitivity did not require available EGF receptors since two TPA promotable variants were EGF receptorless. The mitogenic response of JB6 cells to TPA may however be mediated by EGF since four of four mitogen-resistant variants showed low to zero levels of EGF binding. Tumor promoting phorbol esters produce specific changes in cellular gangliosides. Certain of these changes occur in promotable but not nonpromotable variants of JB6 cells, suggesting that ganglioside changes may be involved in the process of promotion of transformation. 相似文献
2.
I. Eto 《Cell proliferation》1998,31(2):71-92
The molecular basis of tumour promotion is still largely unknown. In in vitro model of tumour promotion, the promotion-sensitive cells are induced to grow under anchorage-independent conditions in the presence of promoting agent. The customary way of providing such conditions is to immobilize these cells in soft agar, but such cells cannot be readily recovered to study the induced biochemical and molecular events. In the present report, we analysed these events using JB6 mouse epidermal cells maintained in suspension in liquid medium over agarose. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent synthesis of DNA in promotion-sensitive P+ (but not in promotion-resistant P?) JB6 cells and this TPA-induced synthesis of DNA positively correlated with TPA-induced formation of colonies in soft agar. The TPA-induced synthesis of DNA began on or shortly before 24 h after the introduction of TPA, peaked at about 48 h and then declined to the control levels over the next several days. All trans-retinoic acid and dexamethasone inhibited and calcitriol (1α,25-dihydroxyvitamin D3) synergistically stimulated this TPA-induced DNA synthesis. Western immunoblot analysis of cyclins (A, B1, D1 and E) and p27Kip1, a cyclin-dependent kinase inhibitor, indicated that TPA induced cyclin A and cyclin B1 expression in P+ (but not in P?) JB6 cells and this induction coincided in time with TPA-induced synthesis of DNA. TPA also strongly induced cyclin D1 expression in P+ (but not in P?) JB6 cells, but this induction started prior to the expression of cyclin A and cyclin B1. TPA did not affect the expression of either cyclin E or p27Kip1 to any significant extent. We also found that NMU38 rat mammary epithelial cells were operationally equivalent to the promotion-sensitive P+ JB6 cells, but in these cells 17β-oestradiol exerted a strong synergistic effect on TPA-induced synthesis of DNA. Based on these observations, we tentatively propose a sequence of molecular events which possibly lead to the anchorage-independent synthesis of DNA in these cells. 相似文献
3.
Effects of tert-butyl hydroperoxide on promotable and non-promotable JB6 mouse epidermal cells 总被引:1,自引:0,他引:1
Oxidants and agents that induce a cellular prooxidant state can act as carcinogens. We compared the effect of tert-butyl hydroperoxide (Bu-OOH) on DNA strand breakage, poly ADP-ribosylation of chromosomal proteins and the expression of the proto-oncogenes c-fos and c-myc between non-promotable clone 30 and promotable clone 41 of mouse epidermal cells JB6. These pathophysiological effects of oxidants are mechanistically related. Bu--OOH caused more DNA-strand breakage at high concentrations and more extensive poly ADP-ribose accumulation in clone 30 than in clone 41, in reactions which require intracellular free iron. Clone 41 exhibited constitutive c-myc expression while c-fos mRNA was very low in untreated cultures of both clones. Low concentrations of Bu-OOH induced c-myc and more strongly c-fos in clone 41. Both proto-oncogenes were strongly induced in clone 30. Our results allow insights into the mechanisms of action of a typical organic hydroperoxide in JB6 cells. However, they do not uncover the reasons for the differential promotability of the two JB6 clones by oxidants beyond the implication of the constitutive expression of c-myc in promotable clone 41. 相似文献
4.
Lenora R. Garrett C. E. Ackland-Berglund Clinton J. Jones Marvin A. Cuchens 《Experimental cell research》1993,205(2)
The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,6,10,14-tetramethylpentadecane (pristane) on gene expression and transformation were examined using two clones (P+, TPA transformation sensitive and P-, TPA resistant) of the mouse epidermal cell line JB6. Results from transformation studies indicated pristane was more efficient, i.e., lower concentrations were required to elicit an equivalent response, in transforming the P+, but not the P-, clone of JB6 compared to TPA. Furthermore, results from these studies demonstrated either TPA or pristane was effective in the transactivation of the chloramphenicol acetyltransferase gene under the regulatory control of most viral promoter/enhancer elements transfected into the P+, but not the P-, clone of JB6. However, if a consensus cAMP response element was linked to the simian virus 40 early promoter, pristane activation was observed in both P+ and P- cells. The differential effects of these two compounds suggest that while they have similar characteristics, they may utilize different pathways to elicit their effects. 相似文献
5.
Anne Tanner Stephanie A. Carlson Masatoshi Nukui Eain A. Murphy Bradford K. Berges 《Journal of virology》2013,87(22):12020-12028
Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2−/−γc−/− mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3− CD4+ CD8− and CD3+ CD4− cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4+ CD8+ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis. 相似文献
6.
Douwe Hoornstra Maria A. Andersson Vera V. Teplova Raimo Mikkola Liisa M. Uotila Leif C. Andersson Merja Roivainen Carl G. Gahmberg Mirja S. Salkinoja-Salonen 《Applied and environmental microbiology》2013,79(12):3534-3543
Bacillus cereus, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (∼1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)−1 (∼0.01 to 1 ng per 105 CFU). By DNA-fingerprint analysis, the isolates matched B. cereus F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (Picea abies). Exposure to cell extracts (1 to 10 μg of bacterial biomass ml−1) and to purified cereulide (0.4 to 7 ng ml−1) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml−1) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml−1 induced K+ translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K+ stores within 10 min. The ability of cereulide to transfer K+ ions across biological membranes may benefit the producer bacterium in K+-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells. 相似文献
7.
Wei SJ Trempus CS Ali RC Hansen LA Tennant RW 《The Journal of biological chemistry》2004,279(7):5993-6004
The molecular changes associated with early skin carcinogenesis are largely unknown. We have previously identified 11 genes whose expression was up- or down-regulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin keratinocyte progenitor cells (Wei, S.-J., Trempus, C. S., Cannon, R. E., Bortner, C. D., and Tennant, R. W. (2003) J. Biol. Chem. 278, 1758-1768). Here, we show an induction of a nucleoside diphosphate protein kinase B (NDPK-B) gene in response to TPA or UV radiation (UVR). TPA or UVR significantly induced the expression of NDPK-B both in vivo hyperplastic mouse skin and in vitro mouse JB6 Cl 41-5a epidermal cells. Indeed, this gene was also up-regulated in TPA or UVR-mediated skin tumors including papillomas, spindle cell tumors, and squamous cell carcinomas, relative to adjacent normal skins. Functional studies by constitutive expression of nm23-M2/NDPK-B in TPA susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b preneoplastic epidermal cell lines showed a remarkable gene dosage-dependent increase in foci-forming activity, as well as an enhancement in the efficiency of neoplastic transformation of these cells in soft agar but no effect on proliferation in monolayer cultures. Interestingly, stable transfection of the nm23-M2/NDPK-B del-RGD or G106A mutant gene in JB6 Cl 41-5a cells selectively abrogated NDPK-B-induced cellular transformation, implicating a possible Arg105-Gly106-Asp107 regulatory role in early skin carcinogenesis. 相似文献
8.
Rapid Colony Screening Method for Identifying Hydrogenase Activity in Rhizobium japonicum 总被引:10,自引:2,他引:8 下载免费PDF全文
Richard A. Haugland Frank J. Hanus Michael A. Cantrell Harold J. Evans 《Applied microbiology》1983,45(3):892-897
A method has been developed for the rapid screening of Rhizobium japonicum colonies for hydrogenase activity based on their ability to reduce methylene blue in the presence of respiratory inhibitors and hydrogen. Hydrogen uptake-positive (Hup+) colonies derepressed for hydrogenase activity were visualized by their localized decolorization of filter paper disks impregnated with the dye. Appropriate responses were seen with a number of Hup+ and Hup− wild-type strains of R. japonicum as well as Hup− mutants. Its specificity was further confirmed in selected strains on the basis of comparisons with chemolithotrophic growth and the presence of other genetic markers. Utilization of the method in identifying Hup+ colonies among 16,000 merodiploid derivatives of the Hup− mutant strain PJ17nal containing cloned DNA fragments of the Hup+ strain 122 DES has demonstrated its applicability as a screening procedure in the genetic analysis of the R. japonicum hydrogen uptake system. 相似文献
9.
Availability of Chloride Affects the Balance between Potassium Chloride and Potassium Malate in Guard Cells of Vicia faba L 总被引:3,自引:2,他引:1 下载免费PDF全文
Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0.1 equivalent of K+ per liter in the absence or presence of Cl−. In the absence of Cl−, iminodiacetate, a presumed impermeant zwitterion, served as anion. With no Cl− in the medium, 91% of the K+ imported into the guard cells during stomatal opening was neutralized by malate production; import of Cl− (presumably from the rest of the epidermal tissue) contributed 6%. In the presence of Cl−, 50% of the necessary negative charges were provided by malate synthesis, 45% by Cl− import. Stomatal opening was not obviously affected by the chloride concentration in the incubation medium, but malate production declined roughly linearly with the logarithm of [Cl−] between 10−5 and 10−1 equivalent per liter. 相似文献
10.
Un Yung Choi Ji Yeon Hur Myeong Sup Lee Quanri Zhang Won Young Choi Lark Kyun Kim Wook-Bin Lee Goo Taeg Oh Young-Joon Kim 《PloS one》2014,9(4)
To avoid excessive activation, immune signals are tightly controlled by diverse inhibitory proteins. TRIM30, a tripartite motif (TRIM)-containing protein is one of such inhibitors known to function in macrophages. To define the roles of TRIM30, we generated Trim30 knockout (Trim30
−/−) mice. Trim30 deletion caused no major developmental defects in any organs, nor showed any discernable defect in the activation of macrophages. But, Trim30
−/− mice showed increased CD4/CD8 ratio when aged and Trim30
−/− CD4+ T cells exhibited an abnormal response upon TCR activation, in particular in the absence of a costimulatory signal. Adoptive transfer of wild-type and Trim30
−/− CD4+ T cells together into lymphopenic hosts confirmed higher proliferation of the Trim30
−/− CD4+ T cells in vivo. Despite the enhanced proliferation, Trim30
−/− T cells showed decreased levels of NF-κB activation and IL-2 production compared to wild-type cells. These results indicate a distinct requirement for TRIM30 in modulation of NF-κB activation and cell proliferation induced by TCR stimulation. 相似文献
11.
12.
Sang-Mo Kwon Jun-Hee Lee Sang-Hun Lee Seok-Yun Jung Da-Yeon Kim Song-Hwa Kang So-Young Yoo Jong-Kyu Hong Ji-Hye Park Jung-Hee Kim Sung-Wook Kim Yeon-Ju Kim Sun-Jin Lee Hwi-Gon Kim Takayuki Asahara 《PloS one》2014,9(8)
Introduction
Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear.Methods
In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34+ and CD34− cells, and determined the optimal concentrations of CD34+ cells and CD34− cells for spindle-shaped EPC differentiation.Results
Functionally, the coculture of CD34+ and CD34− cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34+ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34+ and CD34− cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3+) markedly accelerated the early EPC status of CD34+ cells, while macrophages (CD11b+) or megakaryocytes (CD41+) specifically promoted large EPC colonies.Conclusion
Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34+ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy. 相似文献13.
Yasushi Mandai Daisuke Takahashi Koji Hase Yuuki Obata Yukihiro Furusawa Masashi Ebisawa Tomoo Nakagawa Toru Sato Tatsuro Katsuno Yasushi Saito Takeshi Shimaoka Osamu Yokosuka Kotaro Yokote Hiroshi Ohno 《PloS one》2013,8(6)
CD4+ T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4+ T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4+ T cells expressed CXCR6 in the CD45RBhigh T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn’s disease. Although surface marker analysis demonstrated that both CXCR6+ and CXCR6− CD4+ T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6+ subset produced IFN-γ and TNF-α compared to CXCR6− subset, and only the CXCR6+ subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6+ T cells into Rag1
−/− recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6− cells evoked colitis similar to that observed in CD4+CD45RBhigh T cell-transferred mice, and resulted in their conversion into CXCR6+ cells. Collectively, these observations suggest that the CXCR6+CD4+ T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6−CD4+ T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6+CD4+ T cells. 相似文献
14.
Yoshiko Fukuyama Daisuke Tokuhara Shinichi Sekine Kazuyoshi Aso Kosuke Kataoka Julia Davydova Masato Yamamoto Rebekah S. Gilbert Yuka Tokuhara Keiko Fujihashi Jun Kunisawa Yoshikazu Yuki Hiroshi Kiyono Jerry R. McGhee Kohtaro Fujihashi 《PloS one》2013,8(4)
We assessed the role of CCR5+/CCR6+/CD11b+/CD11c+ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5+/CCR6+/CD11b+/CD11c+ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b+/CD11c+ DCs were markedly decreased in both CCR5−/− and CCR6−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5−/− or CD11c-DTR and CCR6−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5+CCR6+ DCs play an important role in the induction of Ag-specific SIgA Ab responses. 相似文献
15.
16.
Kouji Hirota Eiichiro Sonoda Takuo Kawamoto Akira Motegi Chikahide Masutani Fumio Hanaoka Dávid Szüts Shigenori Iwai Julian E. Sale Alan Lehmann Shunichi Takeda 《PLoS genetics》2010,6(10)
Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη−/−/POLζ−/− cells from the chicken DT40 cell line. POLζ−/− cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη−/−/POLζ−/− cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ−/− cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells. 相似文献
17.
18.
Chang PL Tucker MA Hicks PH Prince CW 《The international journal of biochemistry & cell biology》2002,34(9):1142-1151
BACKGROUND AND AIMS: The expression of osteopontin (OPN), a protein postulated to play a role in tumorigenesis, is induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo and in the in vitro initiation-promotion skin carcinogenesis model (JB6 cells). Although TPA-induced OPN expression in JB6 cells has been suggested to involve protein kinase C (PKC), the PKC isoforms and the downstream pathway mediating OPN expression have not been extensively studied. METHODS: Using the JB6 cell model, we determined the involvement of PKC isoforms, mitogen-activated protein kinase kinase (MAPK kinase/MEK) and MAPK in TPA-induced OPN expression using inhibitors specific to PKC isoforms and MEK and performing Northern blot analyses. Western blot analyses of cells treated with specific inhibitors were also performed to determine whether PKC isoforms or MEK were involved in activation of MAPK. KEY RESULTS: TPA increased the steady-state level of OPN mRNA as early as 2-4h and this expression persisted for at least 4 days. TPA induction of OPN expression in JB6 cells is mediated through PKC epsilon and PKC delta, which also mediated the phosphorylation of MAPK. Additionally, inhibition of MEK activity, which activates MAPK, attenuated TPA-induced OPN expression. These findings suggest that activation of MAPK is important in mediating OPN expression. CONCLUSION: TPA-induced steady-state OPN mRNA expression in mouse JB6 cells involves the activation of MAPK mediated through PKC epsilon and/or PKC delta. 相似文献
19.
A plasmamembrane redox system and proton transport in isolated mesophyll cells 总被引:3,自引:2,他引:1 下载免费PDF全文
Potassium ferricyanide (K3Fe[CN]6) was added to aerated and stirred nonbuffered suspensions of mechanically isolated photosynthetically competent Asparagus sprengeri Regel mesophyll cells. Rates of Fe(CN)63− reduction and H+ efflux were measured with or without illumination. On the addition of 1 millimolar Fe(CN)63− to nonilluminated cell suspensions acidification of the medium indicated an H+ efflux of 1.54 nanomoles H+/106 cells per minute. Simultaneous Fe(CN)63− reduction occurred at a rate of 1.55 nanomoles Fe(CN)63−/106 cells per minute. Illumination stimulated these rates 14 to 17 times and corresponding values were 26.1 nanomoles H+/106 cells per minute and 22.9 nanomoles Fe(CN)63−/106 cells per minute. These two processes appeared to be tightly coupled and were rapidly inhibited when illuminated suspensions were transferred to darkness or treated with 1 micromolar 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Addition of 0.1 millimolar diethylstilbestrol eliminated ATP dependent H+ efflux in illuminated or nonilluminated cells but had no influence on Fe(CN)63− dependent H+ efflux. Recent reports indicate that a transmembrane redox system spans the plasma membrane of root cells and is coupled to the efflux of H+. The present report extends these observations to photosynthetically competent mesophyll cells. The results indicate a transport process independent of ATP driven H+ efflux which operates with a H+/e− stoichiometry of one. 相似文献
20.
Stéphane Hua Camille Lécuroux Asier Sáez-Cirión Gianfranco Pancino Isabelle Girault Pierre Versmisse Faroudy Boufassa Olivier Taulera Martine Sinet Olivier Lambotte Alain Venet 《PloS one》2014,9(7)