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Aspects of interferon alpha signalling in hematopoetic cells
Umeå University, Faculty of Medicine, Molecular Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment.

We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002.

In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes.

The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.

Place, publisher, year, edition, pages
2004. , 51 p.
Series
Umeå University medical dissertations, ISSN 0346-6612
Keyword [en]
Molecular biology, interferon, human, B-lymphocyte, survival, proliferation, myeloma
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-318ISBN: 91-7305-713-4 (print)OAI: oai:DiVA.org:umu-318DiVA: diva2:143082
Public defence
2004-10-08, Major Groove, 6L, NUS, Umeå Universitet, Umeå, 10:00
Opponent
Supervisors
Available from: 2004-09-20 Created: 2004-09-20Bibliographically approved
List of papers
1. Interferon-alpha promotes survival of human primary B-lymphocytes via phoshatidylinositol 3-kinase
Open this publication in new window or tab >>Interferon-alpha promotes survival of human primary B-lymphocytes via phoshatidylinositol 3-kinase
2001 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, ISSN --, Vol. 284, no 3, 583-586 p.Article in journal (Refereed) Published
Abstract [en]

Signaling pathways for the antiviral and antiproliferative biological effects of type I interferons (IFN) are well established. In this report we demonstrate a novel signaling pathway for IFN-α, as it induced rapid phosphorylation of both PKB/Akt and its substrate forkhead. The PI3-kinase inhibitor LY294002 abolished these phosphorylations. PI3-kinase has been implicated in cell survival mediating its effect through the second messenger PIP3 and the subsequent activation of PKB/Akt. We could show that IFN-α inhibited spontaneous apoptosis of primary B-lymphocytes, in the absence of a mitogenic stimulus. This effect was inhibited by LY294002. Thus, our data suggests that IFN-α promotes survival of peripheral B-lymphocytes via the PI3-kinase-PKB/Akt pathway. In addition, IFN-α stimulation of anti-IgM activated cells resulted in downregulated expression of the cell cycle inhibitor p27/Kip1.

Keyword
apoptosis, B-lymphocytes, interferon, phosphatidylinositol 3-kinase, PKB/Akt; forkhead, p27/Kip1
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4109 (URN)10.1006/bbrc.2001.5025 (DOI)
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2017-12-14Bibliographically approved
2. IFN-alpha induced proliferation of human primary B-lymphocytes
Open this publication in new window or tab >>IFN-alpha induced proliferation of human primary B-lymphocytes
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-4110 (URN)
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2012-08-30Bibliographically approved
3. Characterisation of IFN resistance in a myeloma cell line with an unstable genome
Open this publication in new window or tab >>Characterisation of IFN resistance in a myeloma cell line with an unstable genome
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-4111 (URN)
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2012-08-30Bibliographically approved

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