B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course. Recent studies have shown that expression of the protein tyrosine kinase ZAP-70 may serve ...as a prognostic marker in B-CLL. Employing a semiquantitative RT-PCR assay, we examined purified leukemia B cells of 39 CLL patients for the expression of ZAP-70 mRNA transcripts. Significant ZAP-70 mRNA levels exceeding those found in control samples with 5% T cells were detected in 36% of the CLL cases. Patients in the ZAP-70 positive cohort were characterized by an unfavorable clinical course with a significantly shorter progression-free survival as compared to the ZAP-70-negative patients (64%). These results were confirmed by flow-cytometric analysis of the ZAP-70 protein, and expanded to a larger patient cohort (n=67). A combined statistical analysis of 79 patients showed that the two patient subgroups also differed with regard to overall survival and a panel of known clinical prognostic factors including LDH, thymidine kinase serum levels and expression of the CD38 surface antigen by the leukemic cell clone. The level of ZAP-70 expression did not change over time in the majority of patients where sequential samples were available for analysis.
STAT factors act as signal transducers of cytokine receptors and transcriptionally activate specific target genes. The recently discovered protein PIAS3 binds directly to STAT3 and blocks ...transcriptional activation. Here, we present experimental evidence implementing the zinc finger protein Gfi‐1 as a new regulatory factor in STAT3‐mediated signal transduction. The interaction between the two proteins first became evident in a yeast two‐hybrid screen but was also seen in coprecipitation experiments from eukaryotic cells. Moreover, we found that both Gfi‐1 and PIAS3 colocalize in a characteristic nuclear dot structure. While PIAS3 exerts a profound inhibitory effect on STAT3‐mediated transcription of target promoters, Gfi‐1 can overcome the PIAS3 block and significantly enhances STAT3‐mediated transcriptional activation. In primary T cells, Gfi‐1 was able to amplify IL‐6‐dependent T‐cell activation. As Gfi‐1 is a known, dominant proto‐oncogene, our findings bear particular importance for the recently described ability of STAT3 to transform cells malignantly and offer an explanation of the oncogenic potential of Gfi‐1 in T lymphocytes.
Cells that lack PARP-1 activity are limited in their ability to repair DNA single strand breaks and respond to DNA damage with a strong accumulation of p53 and enhanced rates of apoptotic cell death. ...We have generated combinatorial mutant mice that both lack p53 and PARP-1 activity due to the expression of a dominant negative PARP-1 allele targeted to T-cells by the lck promoter. Here we report that these double mutant mice develop T-cell lymphoma at a significantly reduced latency period compared to single p53 null mice that are already cancer prone. We demonstrate that the absence of p53 does not only protect T-cells from lck-PARP-DBD transgenic mice from apoptosis but also abrogates the DNA damage induced cell cycle arrest in the G1 phase. T-cells from double mutant mice continue to proliferate after the induction of DNA strand breaks, are limited in their DNA repair capacity and cannot be eliminated by apoptosis. These results indicate that PARP-1 and p53 cooperate in the suppression of tumorigenesis by maintaining genomic integrity after DNA damage through the activation of a G1/S cell cycle checkpoint the initiation of DNA repair and the induction of cell death.
The c‐myc gene has been implicated in three distinct genetic programs regulating cell proliferation: control of cyclin E–cdk2 kinase activity, E2F‐dependent transcription and cell growth. We have now ...used p27−/− fibroblasts to dissect these downstream signalling pathways. In these cells, activation of Myc stimulates transcription of E2F target genes, S‐phase entry and cell growth without affecting cyclin E–cdk2 kinase activity. Both cyclin D2 and E2F2, potential direct target genes of Myc, are induced in p27−/− MycER cells. Ectopic expression of E2F2, but not of cyclin D2, induces S‐phase entry, but, in contrast to Myc, does not stimulate cell growth. Our results show that stimulation of cyclin E–cdk2 kinase, of E2F‐dependent transcription and of cell growth by Myc can be genetically separated from each other.
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies ...(ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
6.
Regulation of pre-T-cell development Möröy, T; Karsunky, H
Cellular and molecular life sciences : CMLS,
06/2000, Letnik:
57, Številka:
6
Journal Article
Recenzirano
One important pillar of cellular immune defense in mammals is the T-lymphoid compartment which produces cells that are able to specifically recognize foreign peptide antigens through a membrane-bound ...receptor. These T-cells can trigger a variety of defense mechanisms upon antigen stimulation ranging from the production of potent cytokines to the direct killing of virus-infected cells. The production of such highly specialized T-cells takes place in the thymus and requires a stringent process of differentiation and selection of precursor cells that are delivered from the bone marrow. In the thymus, several waves of proliferative expansion and selection ensure the production of a large repertoire of antigen-specific T-cells that each bear a unique T-cell receptor (TCR) which is able to recognize foreign antigens but can tolerate the own host-specific peptide structures. Education of precursors to mature T-cells in the thymus requires a dense network of regulatory processes acting at receptor-ligand interactions, signal transduction, genomic rearrangement of TCR gene loci, cell cycle progression, transcriptional control and programmed cell death.
The chromosomal translocation t(11:14) is associated with human lymphoid neoplasia affecting centrocytic B‐cells of intermediate differentiation. As a consequence the cyclin D1 (bcl‐1) gene is ...juxtaposed to the immunoglobulin heavy chain enhancer E mu. To show that transcriptional activation of cyclin D1 is causally involved in the generation of B‐cell neoplasia we have generated transgenic mice that carry a cyclin D1 gene under the transcriptional control of the E mu element. E mu cyclin D1 transgenic mice show only very subtle alterations in the cycling behaviour of B‐cell populations in the bone marrow compared with normal mice and do not develop lymphoid tumours. However, E mu‐directed coexpression of cyclin D1 and N‐MYC or L‐MYC in double transgenic mice reveals a strong cooperative effect between MYC and cyclin D1 provoking the rapid development of clonal pre‐B and B‐cell lymphomas. Interestingly, crossing of cyclin D1 transgenic mice with E mu L‐myc transgenics that express their transgene in both B‐ and T‐cells but predominantly develop T‐cell tumours leads in double transgenics exclusively to B‐cell neoplasia. The data presented here demonstrate that transcriptional activation of cyclin D1 can oncogenically transform B‐cells in concert with a myc gene. They establish cyclin D1 as a proto‐oncogene whose activity appears to depend on a specific cell type as well as on a specific cooperating partner and link disturbances in the regulation of cell cycle progression to the development of human malignancies.
The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 ...fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by dl-buthionine-(S, R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism.
Infecting mice with a mutant Moloney murine leukemia virus which contains the bacterial suppressor tRNA supF in its LTR allows rapid cloning of proviral integration sites from genomic tumour DNA. In ...a previous study Eµ pim-1/Eµ L-myc bitransgenic mice had been inoculated neonatally with MoMuLV supF virus. The retroviral infection led to acceleration of lymphomagenesis indicating the proviral activation of further oncogenes cooperating with myc and pim-1 in tumour development. Using a functional supF screen for analysis of genomic mouse tumour DNA libraries which had been constructed in the phage vector EMBL3A, a common proviral integration site on mouse chromosome 5 was cloned and found to be identical to the proviral integration site evi-5 which has recently been identified in an AKXD T-cell lymphoma and which is located 18 kb upstream of the gfi-1 gene. Tumours bearing evi-5 integrations showed an enhanced gfi-1 expression level suggesting that gfi-1 is the target gene for insertions at the evi-5 locus. Together with three other previously described Moloney integration clusters all responsible for enhanced gfi-1 expression the number of tumours from infected double transgenic Eµ L-myc/Eµ pim-1 transgenic mice with retrovirally activated gfi-1 added up to 53% underscoring the role of GFI-1 as an effective collaborator for MYC and PIM-1 in the process of lymphomagenesis.
We demonstrate in this paper that the G1 phase specific cell cycle regulator cyclin E is able to provoke focus formation when cotransfected with activated Ha-ras into primary rat embryo fibroblasts ...(REFs). Cyclin E/Ha-ras transformed cells are highly tumorigenic in synergeneic rats, are able to form colonies in soft agar and show protection towards apoptosis upon serum starvation or DNA damage compared to cells transformed by the combination of Myc, cyclin D1 or SV40 large T-antigen and Ha-ras. Lines that were established after cyclin E/Ha-ras or cyclin D1/Ha-ras transformation contain a large percentage of polyploid cells. This was not observed in cells transformed with other oncoproteins and Ha-ras pointing to an involvement of D- and E type cyclins in genomic instability. The cyclin dependent kinase inhibitors p21 and p27 but also p16 completely abrogate focus formation by cyclin E and Ha-ras suggesting that the oncogenic activity of cyclin E still requires functional G1 specific cyclin/CDK complexes. Moreover, inhibition of Myc function also blocks the oncogenic activity of cyclin E indicating a requirement of Myc for cyclin E function. The findings presented here demonstrate that cyclin E can act as an oncoprotein with a potential involvement in genomic instability and the prevention of cell death. Our data also present more evidence for a strict functional interdependency between G1 cyclin/CDK complexes and c-Myc.
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