| 11. |
- Bülow, Elinor, et al.
(author)
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Sorting of neutrophil-specific granule protein human cathelicidin, hCAP-18, when constitutively expressed in myeloid cells.
- 2002
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In: Journal of Leukocyte Biology. - 1938-3673. ; 72:1, s. 147-153
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Journal article (peer-reviewed)abstract
- Neutrophil granulocytes carry storage organelles, e.g., azurophil and specific granules. Poorly understood are the mechanisms for retrieval from constitutive secretion followed by sorting for storage. Therefore, we asked whether the specific granule protein human cathelicidin (hCAP-18) could be sorted for storage in other granules when the biosynthetic window is widened to allow this. We observed that hCAP-18 was targeted for storage in lysosome-related organelles when expressed constitutively in the rat basophilic leukemia and the mouse promyelocytic (MPRO) cell lines. In addition, premature release of the antibiotic C-terminal peptide LL-37 was observed. Retention of hCAP-18 was diminished by induction of differentiation of MPRO cells. In conclusion, a specific granule protein with native conformation may be sorted for storage in lysosome-related organelles of myeloid cells and converted prematurely to a supposedly biologically active form.
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| 12. |
- Canny, G, et al.
(author)
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Functional and biochemical characterization of epithelial bactericidal/permeability-increasing protein
- 2006
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In: American Journal of Physiology: Gastrointestinal and Liver Physiology. - : American Physiological Society. - 1522-1547 .- 0193-1857. ; 290:3, s. 557-567
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Journal article (peer-reviewed)abstract
- Epithelial cells of many mucosal organs have adapted to coexist with microbes and microbial products. In general, most studies suggest that epithelial cells benefit from interactions with commensal microorganisms present at the lumenal surface. However, potentially injurious molecules found in this microenvironment also have the capacity to elicit local inflammatory responses and even systemic disease. We have recently demonstrated that epithelia cells express the anti-infective molecule bactericidal/permeability-increasing protein (BPI). Here, we extend these findings to examine molecular mechanisms of intestinal epithelial cell (IEC) BPI expression and function. Initial experiments revealed a variance of BPI mRNA and protein expression among various IEC lines. Studies of BPI promoter expression in IECs identified regulatory regions of the BPI promoter and revealed a prominent role for CCAAT/enhancer binding protein and especially Sp1/Sp3 in the basal regulation of BPI. To assess the functional significance of this protein, we generated an IEC line stably transfected with full-length BPI. We demonstrated that, whereas epithelia express markedly less BPI protein than neutrophils, epithelial BPI contributes significantly to bacterial killing and attenuating bacterial-elicted proinflammatory signals. Additional studies in murine tissue ex vivo revealed that BPI is diffusely expressed along the crypt-villous axis and that epithelial BPI levels decrease along the length of the intestine. Taken together, these data confirm the transcriptional regulation of BPI in intestinal epithelia and provide insight into the relevance of BPI as an anti-infective molecule at intestinal surfaces.
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| 13. |
- Ceballos, Eva, et al.
(author)
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c-Myc antagonizes the effect of p53 on apoptosis and p21WAF1 transactivation in K562 leukemia cells
- 2000
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In: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 19, s. 2194-2204
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Journal article (peer-reviewed)abstract
- c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two different methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53Vall35 mutant, which adopts a wild-type conformation at 32 degrees C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc significantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21WAF1, Bax and cytomegalovirus promoters. The impairment of p21WAF1 transactivation by c-Myc was confirmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21WAF1 mRNA protein were significantly reduced by c-Myc, while Bax levels were unaffected. Consistently, c-Myc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer.
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| 14. |
- Chylicki, Kristina, et al.
(author)
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Characterization of the molecular mechanisms for p53-mediated differentiation
- 2000
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In: Cell Growth and Differentiation. - 1044-9523. ; 11:11, s. 561-571
-
Journal article (peer-reviewed)abstract
- The p53 tumor suppressor protein can induce both apoptosis and cell cycle arrest. Moreover, we and others have shown previously that p53 is a potent mediator of differentiation. For example, expression of ptsp53, a temperature-inducible form of p53, induces differentiation of leukemic monoblastic U-937 cells. The functions of p53 have for long been believed to be dependent on the transactivating capacity of p53. However, recent data show that both p53-induced cell cycle arrest and apoptosis can be induced independently of p53-mediated transcriptional activation, indicating alternative pathways for p53-induced apoptosis and cell cycle arrest. The bcl-2 proto-oncogene contributes to the development of certain malignancies, probably by inhibition of apoptosis. Interestingly, Bcl-2 has been shown to inhibit p53-mediated apoptosis as well as p53-mediated transcriptional activation. Asking whether Bcl-2 would interfere with the p53-mediated differentiation of U-937 cells, we stably transfected bcl-2 to U-937 cells inducibly expressing p53. Although the established Bcl-2-expressing clones were resistant to p53-mediated apoptosis, we did not observe any interference of Bcl-2 with the p53-mediated differentiation, suggesting separable pathways for p53 in mediating apoptosis and differentiation of U-937 cells. Neither did expression of Bcl-2 interfere with p53-induced expression of endogenous p21, suggesting that p53-induced differentiation might be dependent on the transcriptional activity of p53. To further investigate whether the p53-mediated differentiation of U-937 cells depends on the transcriptional activity of p53, we overexpressed transactivation-deficient p53, a transcriptionally inactive p53 mutant in these cells. However, in contrast to the effects of wild-type p53, expression of trans-activation-deficient p53 did neither induce signs of apoptosis nor of differentiation in U-937 cells. Our results indicate that the transcriptional activity of p53 is essential both for p53-mediated apoptosis and differentiation of U-937 cells.
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| 15. |
- Dimberg, Lina Y., et al.
(author)
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Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma
- 2012
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In: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407 .- 1471-2407. ; 12, s. 318-
-
Journal article (peer-reviewed)abstract
- Background: Multiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM. Methods: To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS). Results: To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered. Conclusion: We conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro-apoptotic genes. However, this shift alone is not sufficient to alter apoptosis sensitivity in MM cells, suggesting that Stat1 independent pathways are operative in IFN mediated apoptosis sensitization.
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| 16. |
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| 17. |
- Egesten, Arne, et al.
(author)
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Phorbol ester-induced degranulation in adherent human eosinophil granulocytes is dependent on CD11/CD18 leukocyte integrins
- 1993
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In: Journal of Leukocyte Biology. - 1938-3673. ; 53:3, s. 93-287
-
Journal article (peer-reviewed)abstract
- Secretion of unique eosinophil granule constituents may play a role in allergic and parasitic reactions. Therefore we have investigated possible mechanisms for regulation of secretion in eosinophils. A hemolytic plaque assay and an enzyme-linked immunospot (ELISPOT) assay were developed for detection of secreted eosinophil cationic protein (ECP) from single adherent eosinophils. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) induced release of ECP in a dose-dependent fashion but 4-alpha-PMA, an analogue that does not activate protein kinase C, did not cause degranulation. Staurosporine and K252a, inhibitors of protein kinase C, decreased PMA-induced ECP secretion. Low concentrations of cytochalasin B enhanced PMA-induced secretion but high concentrations had an inhibitory effect. The calcium ionophores A23187 and ionomycin were weaker secretagogues than PMA. Tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, interleukin-5, N-formylmethionyl-leucyl-phenylalanine, and lipopolysaccharide caused little or no degranulation in adherent eosinophils. Preincubation of eosinophils with antibodies to CD18, the common beta chain of leukocyte adhesion proteins, resulted in inhibition of PMA-induced ECP release from adherent cells. 1,2-Bis(O-aminophenyl)-ethane-ethane-N,N,N',N'-tetraacetic acid (BAPTA), an agent that acts intracellularly by chelation of calcium, also inhibited PMA-mediated ECP release. In conclusion, PMA induces release of ECP from single adherent eosinophils and the effect appears to be mediated via protein kinase C and, in contrast to that in neutrophils, to be dependent on CD11/CD18 leukocyte integrins.
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| 18. |
- Egesten, Arne, et al.
(author)
-
The heterogeneity of azurophil granules in neutrophil promyelocytes: immunogold localization of myeloperoxidase, cathepsin G, elastase, proteinase 3, and bactericidal/permeability increasing protein
- 1994
-
In: Blood. - 1528-0020. ; 83:10, s. 94-2985
-
Journal article (peer-reviewed)abstract
- Azurophil granules of myeloid cells form in promyelocytes. They store cytotoxic and digestive agents which when released are involved in the defense against infection. In order to characterize the intragranular distribution of these agents, ultrastructural methods using immunogold were used on promyelocytes. Azurophil granules were divided into nucleated, large spherical (large azurophil) and small electron-dense (small azurophil) granules. Myeloperoxidase showed a peripheral distribution of large azurophils and a uniform distribution of small and nucleated azurophils, consistent with previous findings. Likewise, the major neutral proteases of azurophils, cathepsin G, granulocyte elastase, and proteinase 3, displayed a similar distribution, with a peripheral localization in large azurophils and a uniform distribution in small and nucleated azurophils, except for proteinase 3, which was associated with the crystalloid structure in nucleated azurophils. In contrast, the bactericidal/permeability increasing protein, which is bacteristatic and bactericidal for Gram-negative bacteria, was localized to the membrane area in all types of azurophil granules, consistent with a suggested association of this protein with the granule membrane. The observed differences in intragranular distribution of the proteins investigated may reflect variations in binding to matrix structures and granule membranes.
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| 19. |
- Ehinger, Mats, et al.
(author)
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Expression of the p53 tumor suppressor gene induces differentiation and promotes induction of differentiation by 1,25-dihydroxycholecalciferol in leukemic U-937 cells
- 1996
-
In: Blood. - 1528-0020. ; 87:3, s. 1064-1074
-
Journal article (peer-reviewed)abstract
- Leukemic U-937 cells, which lack normal p53, were stably transfected with a temperature-sensitive mutant of p53 to investigate the consequences for growth and differentiation. On induction of wild-type p53 activity at the permissive temperature, some of these cells underwent maturation as judged by the capacity for oxidative burst and the appearance of monocyte related cell surface molecules. Moreover, wild-type p53-expressing cells were more sensitive than p53-negative control cells to induction of differentiation by 1,25-dihydroxycholecalciferol; a twofold to fourfold increase of the fraction of cells showing signs of terminal maturation was observed when wild-type p53-expressing cells were incubated with 1,25-dihydroxycholecalciferol at concentrations that only slightly affected control cells. Whereas wild-type p53 activity per se induced maturation of certain cells, other underwent cell death judging from the reduced capability to exclude trypan blue and the appearance of fragmented DNA in flow cytometric analysis. The p53-induced cell death could be inhibited by incubation with 1,25-dihydroxy-cholecalciferol, but not all-trans retinoic acid. Thus, 1,25-dihydroxycholecalciferol, seemed to increase the survival of wild-type p53-expressing cells and to cooperate with wild-type p53 to induce differentiation. The data imply that p53-mediated maturation in U-937 cells depends on optimal regulation of signals for differentiation, survival and proliferation, and suggest a role for p53 in the differentiation induction of leukemic cells.
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| 20. |
- Ehinger, Mats, et al.
(author)
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Involvement of the tumor suppressor gene p53 in tumor necrosis factor-induced differentiation of the leukemic cell line K562
- 1995
-
In: Cell Growth and Differentiation. - 1044-9523. ; 6:1, s. 9-17
-
Journal article (peer-reviewed)abstract
- The cDNA of the human wild-type p53 tumor suppressor gene was constitutively overexpressed in the leukemic cell line K562 (which lacks detectable amounts of p53 protein) in order to investigate the consequences for growth and differentiation. Several stable clones were established by transfection of the expression vector pc53SN3. Expression of p53 protein was characterized by biosynthetic labeling and immunoprecipitation with the monoclonal antibodies pAb 1801 (reacting with wild-type and mutant human p53), pAb 240 (reacting with mutant human p53) and pAb 1620 (reacting with wild-type human p53). All clones which were 1801+, 240-, 1620- or 1801+, 240-, 1620+ were defined as "wild-type-like p53-expressing" clones. Our results show that expression of p53 protein is compatible with continuous proliferation of K562 cells. The growth characteristics of wild-type-like p53-expressing clones did not differ from that of control clones. However, the former were more sensitive than p53-negative control clones to growth inhibition by tumor necrosis factor (TNF), a cytokine with a potential role in growth and differentiation of myeloid leukemic cells. In addition, a 2- to 4-fold increase of the amount of hemoglobin, a marker of erythroid differentiation, was observed when wild-type-like p53 protein-expressing clones were incubated with TNF. This suggests that differentiation is the mechanism responsible for the increased TNF sensitivity of these clones. Our results support a role for p53 in mediating growth inhibitory and differentiation inducing signals by TNF.
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