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  Director : Gordon Langsley (langsley@pasteur.fr)


  abstract

 

Theileria-induced transformation of bovine leukocytes is unique in that it is entirely reversible as upon drug-induced parasite death the leukocyte reverts to a quiescent cell that will die from apoptosis unless further stimulated. The reversible nature of the transformed state provides a powerful model to study host-parasite relationships in the context of leukocyte activation and we use this model to address three basic questions: 1) Leukocyte survival i.e. how does the parasite prevent the activated leukocyte from dying of apoptosis? 2) Leukocyte proliferation i.e. how does the parasite induce the uncontrolled proliferation of its host cell? 3) Metastasis i.e. how does the parasite induce its host cell to metastasise?



  report

cale

Specific subjects

1) Leukocyte survival: the role of the serine/threonine phosphatase PP1 & PP2A.

Recent results suggest a new pivotal role for the serine/threonine protein phosphatases PP1/PP2A in the regulation of apoptosis. Specifically, we examine the contribution of PP1/PP2A in the control of PI3-K- and PKA-mediated survival pathways that regulate the Bcl-2 family of proteins. As Theileria parasites protect their host lymphocytes from apoptosis transfection of parasite-specific cDNA into infected lymphocytes (see ETS project) followed by drug-induced parasite death could lead to the isolation of the Theileria gene(s) responsible for the parasite-mediated anti-apoptotic mechanism. Surviving lymphocytes should be those expressing the parasite anti-apoptosis inducing gene(s).

Go to the PP1 Signature search web site.

2) Leukocyte proliferation: induction of the transcription factor E2F.

The reversible nature of Theileria-induced transformation implies that uncontrolled lymphocyte proliferation is not due to mutations in for example, p53 or retinoblastoma protein (RB) which is the case in many tumour cells. RB is one of the major regulators of the G1/S transition via its interaction with the transcription factor E2F. The growth-suppressing activity of RB and its related

p107 and p130 proteins, collectively referred to as the pocket proteins, is mainly exerted by binding to and inhibiting members of E2F family thus, leading to a G1 growth arrest. It would appear that Theileria intervenes in the PI3-K pathway of E2F activation downstream of cyclin D3. Whether this is done by a putative RB (and p130) binding protein, or by some other mechanism that induces their phosphorylation is the focus of our current studies. We plan to use genetic selection to identify putative Theileria RB- and p130-binding proteins via transfection of the Theileria-specific cDNA library combined with drug-selection for E2F activation.

3) Metastasis of Theileria-infected leukocytes.

We have previously described the parasite-dependent induction of the transcription factor AP-1 and currently we are examining the contribution of AP-1 induction to the metastatic phenotype of Theileria-transformed lymphocytes. Via the induced expression of a dominant negative c-Jun mutant we have observed that AP-1 inhibition leads to a significant reduction in the ability of the transformed B cells to degrade extra cellular matrix. Since Theileria-transformed lymphocytes give rise to tumours in immuno-compromised mice, we are currently testing whether this reduced capacity to degrade synthetic extra cellular matrix is reflected in altered tumorigenesis in vivo. We plan to isolate the parasite gene(s) responsible for AP-1 induction by transducing the

Theileria-specific retroviral cDNA library into non-parasitised B cells (BL3). In BL3 cells AP-1 is not induced and the resistant phenotype will only occur if BL3 receives a retrovirus expressing a parasite cDNA encoding an AP-1 activator.

4) A Theileria annulata ETS project.

In order to gain access to transcripts of the transforming stage of Theileria, we have developed an improved protocol for purifying parasites from infected lymphocytes. Using T. annulata macroschizonts purified from infected macrophages we have made parasite specific cDNA libraries. In collaboration with Genopole Pasteur we have initiated a 10, 000 tag ETS project and to date have generated 1000 sequence tags. The parasite-specific cDNA is being cloned in retroviral transfection vectors and we have already established retroviral transfection of Theileria-infected leukocytes. Retroviral transduction of the cDNA libraries will be used in the different projects outlined above.

Photo: Hoechst DNA stain of infected B cells (left) and B cells, where the parasite has been killed by buparvaquone treatment that results in lymphocyte apoptosis and nuclear condensation.

Keywords: Theileria, transformation, apoptosis, proliferation, metastase



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  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Houssin, Wendy (whoussin@pasteur.fr) Gordon LANGSLEY (langsley@pasteur.fr) Directeur de Recherche, CNRS

Madeleine COCHET (mcochet@pasteur.fr) Directeur de Recherche, CNRS

Alphonse GARCIA (agarcia@pasteur.fr) Chef de Laboratoire, I.P.
Veronique Hospital Post-doc

Frédéric Dessauge Etudiant en thèse d'Immunologie

Julien Guergnon Etudiant en thèse d'Immunologie

Regina Luzundia VetMed Etudiante en DEA d'Immunologie

Marie-Françoise MOREAU Assistant ingénieur au CNRS (50%)

Brigitte Blumen TCS CNRS


Activity Reports 2002 - Institut Pasteur
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