III RESULTS
1. TLS is an Co-activator of the RANC
Firstly, we analyzed the effect of a transitory surexpression
of TLS on the expression of a gene rapporteur (luciférase) under the
control of the RARß2 promoter (Figure 4A). We used as cellular model
hematopoietic cells HL-60. These cells are électroporées in the
presence of Tkßgal, to evaluate the effectiveness of transfection and to
standardize the results, of ßRARE-Luc, used like gene rapporteur to
evaluate the transcriptionnelle activity and of various quantities of vector of
expression of TLS, as indicated. pCS3, a plasmide control is used in such way
that all the transfections have the same total quantity of plasmide of
expression (Figure 4). The cells are collected 24 H after and the
luciférase activity is measured. The values represent the averages of
the doublets carried out. Similar results were obtained in several independent
experiments.
Expressed results corresponding to the increase in the
activity of the luciférase compared to a negative witness (cells
transfectées with pCS3 only, in absence of ligand).
At the time of the transfection in the presence of AR alone (1
'), an increase in the luciférase activity is observed. Co-transfection
by pcs3-MT-TLS alone and Rare-Luc (2 and 3) does not have an effect on
induction of the luciférase activity, except in the presence of AR (ATRA
10-6 M) (2 ' and 3 ', multiplied by 25, in the presence of 5 ug and 10 uG of
TLS (Figure 4B). These results suggest that TLS increases the
transcriptionnelle activity induced by the endogenous receivers in cells
HL-60.
In order to determine that Co-activation by TLS acts by and
thanks to the RANC, we used Cos-6 cells having few receivers to the endogenous
rétinoïdes which we transfectées with the plasmides
expression of RAR and RXR. The Cos-6 cells are thus Co-transfectées with
calcium chloride with the plasmides TKgal and RARE, as previously described.
Figure 4 . TLS increases the transcriptionnelle
activity of the nuclear receivers to the rétinoïque acid in cells
HL-60. With, descriptive diagram of the construction used in the test
of transactivation. B, the transcriptionnelle activation of RARE A place with
AR alone or TLS in a way dependant on AR in cells HL-60.
In Figure 5, controls (1 and 4) indicate the basal
transactivation of the gene rapporteur. At the time of the transfection in the
presence of AR alone (1 ' and 4 ', multiplied by 40) or with TLS (2 ', 3 ', 5
', 6 ' and 7 '), an induction is observed. Induction is optimal with 2 ug of
TLS (3 ' and 5 ', multiplied by 85). TLS is thus an Co-activator of the complex
transcriptionnel receivers RAR and RXR, depend on AR. With larger quantities, 5
ug and 10 ug of TLS (6 ' and 7 '), a reduction in induction in the presence of
AR, lower at the level reached in absence of TLS, are observed. This reduction
could be due for a purpose of « quenching » or with the
effect TLS which, in great quantities, would prevent RXR from binding to the
ADN.
The whole of these results indicate that protein TLS is an
Co-activator of the transcription by the receivers with AR. TLS thus seems
implied in the way of indication of AR.
Figure 5 A and B. TLS act like Co-activator of the
RANC, way proportions dependant. The two experiments are carried out
independently.
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