1. The study examines the long-term effects of PMLIV overexpression on gene expression in breast cancer cells. Cells were treated with doxycycline to induce PMLIV expression, then grown without doxycycline. 2. Proliferation rates recovered after removal of doxycycline. Expression of cell cycle genes TOPOIIA, CyclinB1, MCM6 increased in cells previously exposed to PMLIV, suggesting a memory effect. 3. Further experiments are needed to fully characterize changes in protein levels, proliferation rates after repeat treatment, and other genes involved in the response. Understanding this pathway could provide insights into breast cancer therapy.

1. ΠΡΑΚΤΙΚΗ ΑΣΚΗΣΗ
ΚΑΡΑΚΟΥΣΗ ΤΡΙΝΤΑΦΥΛΛΙΑ 1977
Επιστημονικός υπεύθυνος:ΙΩΣΗΦ ΠΑΠΑΜΑΤΘΑΙΑΚΗΣ
Επιβλέπων καθηγητής:ΓΕΩΡΓΙΟΣ ΓΑΡΙΝΗΣ
“PML MEMMORY EFFECT”
INTRODUCTION
PML protein is encoded by the PML gene and it exists in a both nuclear and
cytoplasmic form. It was characterized for the first time in the case of Acute
Promyelocytic Leukemia (APL) as a fusion protein with the retinoic acid receptor α
(RARα) (de The H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. The
PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute
promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991; 66:675–84).
PML belongs to the tripartite motif (TRIM)-containing protein superfamily, which is
structurally characterized by a RING domain (R), two B-box domains (B) and a
coiled-coil domain (CC),many members of which are ubiquitin ligases that generate
subcellular structures through autoassembly (Reymond et al. 2001;Meroni and Diez-
Roux 2005). The human PML protein can be found in 7 isoforms occurring from
alternative slpicing. In our experiment we used the PMLIV which is nuclear. PML is
the main organizer of discrete nuclear structures referred as PML nuclear bodies
(PML-NBs). These structures are spheres of 0.1–1.0 µm in diameter found in most
cell types. PML NBs are proteinaceous, tightly bound to the nuclear matrix,
proposed to anchor and regulate many nuclear functions, including DNA replication,
transcription, or epigenetic silencing (Stuurman et al 1990). Additionally PML
influences or regulates other key processes such as apoptosis, senescence,
response to DNA-damage or resistance to micro-organisms too.(Salomoni and
Pandolfi 2012;Everett and Pandolfi 2007;Bernardi et al. 2008PML can regulate both
negatively or positively the transcription process according to its interacting partners
(Zhou et al, 2013). PML-NBs might control transcription either directly by modulating
the availability or activity of transcription factors, or indirectly by participating in
chromatin-remodelling processes and establishing chromatin architecture that
facilitates transcription.In this report, we use a tetracycline-inducible transgenic
system expressing PMLIV and GFP through a bi-directional promoter integrated in
MDA-MB-231 cells (mesenchymal aggressive breast adenocarcinoma), to examine
the long term effect of PMLIV on gene expression.

2. culture cells 5-6 days
+dox to the
medium
remove dox
from the
medium
culture cells 7 days
+dox to the
medium
collect
Results
The proliferation rate of MDA MB 231 cells recovers after PMLIV
overexpression .
In our lab we have generated a mammary cell line (MDA-MB-231) in which
doxycycline (dox) induces transient PML IV isoform expression. Using this inducible
system recent, unpublished work showed that the proliferation of breast cancer cells
is repressed after Dox treatment. In fact, Real Time PCR analysis demonstrated that
the expression levels of important cell cycle or DNA replication/repair markers such
as Cyclin B1 and TOP2A, MCM6, BRAC1 respectively, were reduced. To address
whether PML has a long term effect on gene expression we treated the transgenic
cell line according to the following set up (figure 1). We cultured the cells with dox for
5-6 days and after we removed the dox from the cells. We cultured the cells again for
5-6 days with out dox and then we added dox again in the medium and cultured the
cells for some days before we collect them.
FIG 1. A time point scheme in which we show the way we treated the cells with dox.
The proliferation rate of the cells was assessed when cells were cultured in the
presence of doxycycline for 5 days and when cells were cultured after the removal of
doxycycline. (figure 2) In the first growth curve the blue line demonstrates the control
cells and the red one the cells which were treated with Dox. As we would expect the
growth rate of the treated cells was decreased in contrast with the control ones. In
the second growth curve the blue line demonstrates the control cells and the red one
the cells that have been treated with dox once tand they have been left to grow in the
absence of dox. Surprisingly these cells grew normally and their proliferation rate
was the same as the control cells.

3. D0 D1 D2 D3 D4
0
50000
100000
150000
200000
250000
300000
control
+dox
D0 D2 D4 D6
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
never treated
treated once
Figure 2: In this figure it is shown the result of the PML overexpression in contrast with the
control cells. The ψ axis shows the number of the cells and the χ axis the time in days.
The expression of TOPOIIA,CyclinB1 and MDM6 is following a PML dependent
profile.
Next we examined the transcriptional profile of transgenic MDA MB 231 cells after
the treatment with doxycycline for the second time. For this reason we used real time
PCR to check PML mRNA levels as well as for the mRNA levels of the genes
(TOPOIIA, Cyclin B1,MCM6) which have been shown to be downregulated after
PML IV orverexpression. The data from the real time PCRs are shown above (figure
3).

4. MCM6/18S
TOPOIIA/18S
control control +dox treated treated + dox
0
5
10
15
20
25
30 0.02
2.17
0
24.11
PML
control control +dox treated treated + dox
0.00E+00
2.00E-05
4.00E-05
6.00E-05
8.00E-05
1.00E-04
control control + dox treated treated + dox
0.00E+00
5.00E-07
1.00E-06
1.50E-06
2.00E-06
2.50E-06
7.91E-07
1.16E-07
2.16E-06
3.04E-07

5. CyclinB1/18S
FIG3. The real time PCR results normalized with 18S for PML, MCM6, TOPOIIA, CyclinB1.
DISCUSSION
The overexpression of PML leads to decreased proliferation and growth problems to
the cells as it was already suggested from past publications. Here we see that after the
overexpression of the PMLIV the cells recover and start to proliferate normally.
Moreover when we checked for the RNA levels of the TOPOIIA,CyclinB1 and
MCM6 genes we came out with the result that the RNA level of each of these genes
was increased a lot in the case of the cells which had been exposed once to high levels
of PMLIV in contrast with the control cells. Does the cells in order to survive
overproduce these significant proteins? To see if this increased level of the RNA
levels reflect an increased protein level too a western blot must be done. A question
that has to be answered in this point is whether the protein or RNA levels of these
genes are decreased after the recovery to the normal levels or they are remaining high
and for how long. Does the cell storage them in order to be ready for a next PML
overexpression? Additionally the dox treatment process must be done more times and
growth curves as long as western blots and real time PCRs must be demonstrated for
each time point. Which is the proliferation rate of the cells after the second time of
treatment with dox? Based on our PCR results the RNA levels after the second
treatment are increased in contrast with the levels after the first time in the case of
CyclinB1 and TOPOIIA. Do the cells recover sooner? Could there exist a memory
mechanism by which the cells gain a form of resistance to the PML overexpression
control control +dox treated treated + dox
0.00E+00
2.00E-08
4.00E-08
6.00E-08
8.00E-08
1.00E-07
1.20E-07
5.95E-08
1.35E-08
1.09E-07
3.11E-08

6. effects? Taking advantage of the si RNA or the auxin-based degron system technology
we could investigate whether the knock down of these proteins has the same effect on
the proliferation rate and how the proliferation rate would be in the loss of each of
them alone. Moreover it would be interesting to investigate which other genes are
implicated in the process. For this reason micro arrays analyses could be
demonstrated in order to have a complete view of the expression profile of the cells
before the PML overexpression, after the first recovery and after the second recovery.
Finally which is the exact role of the PMLIV isoform? Does the overexpression of
other PML isoforms has the same result? Are the PML nuclear bodies have a role in
this process? Understanding all these insights of the PML mediated recovery pathway
would be very important and it could lead to the better understanding and therapy of
breast cancer.
MATERIAL AND METHODS
Cell culture: ΜDΑ ΜΒ 231 cells with a tetracycline-inducible transgenic system
expressing PMLIV and GFP through a bi-directional promoter were cultured as normal
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal
bovine serum, ++. For the growth curve experiments 100.000 cells were plated per well
in 12wells plate. Doxycycline (10mg/ml) was used in 1:1000 concentration.
RNA extraction:
1)PBS wash
2)add trizol (cell lysis) 1ml / 3.5 cm diameter well (6-well)
3)homogenise by pipetting several times (mechanic lysis)
4)5 min at RT for complete dissociation of nucleoprotein complexes
5)add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)
6)shake for 15 sec (Eccles protocol: do not vortex)
7)incubate 2-5 min at RT
8)spin max. 12000g, 5-15 min, 2-8°C
9)select the upper phase
10)add isopropanol (70% of aqueous phase or 1/2 trizol volume)
11)incubate 10min at RT)

7. 12)spin max g, 10-15 min, 4ºC
13)remove supernatant
14)wash pellet 70% EtOH (add & vortex briefly) ,70% ethanol prepared with RNase-free
water
15)desolve in ddH2O 20μl
Reverse Transcription :
Reaction:
5x buffer 8μl
10x DTT 4μl
6mers 2μl
dNTPS 2μl
RNAasin 0.25μl
RT O.2μl
ddH2O 3.55μl
and fort the control samples noRT.
Real time PCR:
Template 2μl
10X Taq buffer 2μl
MgCL2 (25mM) 2 μl
F (100pmol) 0.1μl
R (100pmol) 0.1μl
10mM dNTPs 0.1μl
Taq(5u) 0.1μl
SYBER GREEN I 1μl
The program of Real time PCR in the Opticon Monitor System
1. 5 min at 94 degrees
2. 20s at 94 degrees
3. 20s at 60 degrees
4. 20s at 72 degrees
5. 1 sec at 80-89 depending on the Tm of each PCR product
6. measuring optical absorption
7. step 2 again for 35 cycles
8. Melting at 72 degrees

8. 9. melting curve (0.5 degree from 72 to 94 degrees)
The real time PCR data were analyzed with the Opticon Monitor Software. All the
results were normalized with the 18sRNA.