Transcript uvb-induced effects on jurkat cells: a filter provided

1st ItalianWokshop on UltraViolet
Techniques and Applications
“UVB-INDUCED EFFECTS ON JURKAT
CELLS: A FILTER PROVIDED BY A
VEGETABLE MIXTURE”
L. Di Giambattista Lattanzi
Collaborators
P. Grimaldia, S.Gaudenzia, M. Grandid, S. Morronec, D. Pozzic,
I. Silvestric and A. Congiu Castellanoa
aDepartment
of Physics, Sapienza-Roma
cDepartment of Experimental Medicine, Sapienza-Roma
dPoliambulatory La Torre,Torino
What is the cellular response to UV radiation?
“UV radiation has a variety of physiological and biological effects
on the organism including the transformation to malignancy,
immune suppression, cellular aging, and the induction of DNA
repair and apoptosis……”
…………The cellular response is multifactorial!!
1/15
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2
3
2/15
We studied the induction of apoptosis in Jurkat cells by UVB
radiation (wavelength 290–320 nm) at a dose of 310 mJ/cm2.
We combined Fourier transform infrared (FTIR) spectroscopy with
flow cytometry to determine whether the combination of both
techniques could provide new and improved information about cell
modifications.
To do this, we looked for correspondences and correlations between
spectroscopy and flow cytometry data and found three highly
probable spectroscopic markers of apoptosis.
3/15
An example of result in the protein region (1800-1480 cm-1)…
1 0’
60’
20’
2
120’
3
210’
420’
Pozzi et al, Rad. Res. 168, 698-705 (2007)
4/15
Now……..
we have studied the induction of apoptosis in Jurkat cells by three
different treatments: UVB radiation, vegetable mixture and
vegetable mixture plus UVB radiation.
Following the same experimental methodology, we have analysed
and investigated the possible biological effects induced in tumoral
line cells.
At irradiation dose and drug concentrations utilized,we have
found that the apoptotic process of this cell line is modulated by
the drug which seems to filter the UVB radiation.
5/15
THE TREATMENT: vegetable mixture
Recent studies show how the products
of lichens are potential filters of UV
radiation, thanks to their absorption
in this region and to the antioxidant
power.
The mixture contains substances extracted from a lichen (Evernia
Prunastri) and other vegetable substances (Epilobium and Urtica dioica).
Evernia Prunastri contains a lichen metabolite, Usnic
Acid that has a good UV light filtering power.
Evernia Prunastri
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THE TREATMENTS: vegetable mixture plus UVB radiation
The use of this mixture inhibits synergically the apoptotic process
induced by UVB radiation in the Jurkat cell line.
We have analysed the following cellular samples:
 Cells treated with the drug in different concentrations (5,20,30 μl)
 Cells radiated with UVB-dose 310 mJ/cm2 at 30 cm from the UV-source
 UVB radiated cells for 30 minutes after treatment with the drug in
different concentrations (5, 20, 30 μl)
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WHAT IS THE APOPTOSIS ?
The apoptosis is an endogenous program of cellular suicide:
 it is a physiological process
 it is very different from necrosis
 its misfunctioning is at the basis of many diseases
The apoptotic process also plays a key role in various biological functions.
The difference between Apotosis and Necrosis
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THE APOPTOTIC PROCESS
The morphological changes that the cell undergoes during apoptosis are:
 cytoplasmic and nuclear condensation
 reduction of the cell volume
 maintenance of the membrane integrity
 formation of blebs or apoptotic bodies
The process causes no inflammation of
the surrounding tissue.
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THE OPTICAL MICROSCOPY:The morphology of Jurkat cells at 210 minutes
CTR
DRUG
UVB
DRUG
+
UVB
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THE RESULTS: Flow Cytometry
UVB
30 l + UVB
UVB
30 l+UVB
100
100
90
60
50
40
30
20
10
0
% Viable cells
80
70
80
70
60
50
Ti
210
m
60
e[
m
210
n]
]
60
[mi
0
in
Tim
e
40
30
20
10
0
360
360
2
90
% Apoptotic cells
1
0
UVB
30 l + UVB
30 l
UVB
30 l + UVB
100
10
5
80
60
40
% Viable cells
15
% Necrotic cells
20
20
0
0
3
360
24
Tim
e[h
210
60
Time[min]
0
ou
r]
0
4
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ANALYSE: Flow cytometry
To collection
Catcher Tube
Moves in and out
stream
To waste
Sample injection
Apoptosis was assessed using annexinV-FITC
apoptosis kit (Bender Med. Systems) or propidium
iodide PI (DNA probe) and flow cytometry.
In the first case , the percentage of apoptotic cells
was determined by the green fluorescence emitted
by AnnexinV-FITC bound to Phosphatidylserine
(PS) exposed to the outer leaflet of the apoptotic
cells membrane (flip-flop).
To distinguish the percentage of living, apoptotic
or necrotic cells, propidium iodide (PI) was added
to the cell.
In the second case, DNA content was assessed with
propidium iodide (PI); it enters into damaged cells,
intercalates into DNA staining them and emitting
a red fluorescence.
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THE RESULTS: FTIR spectroscopy
Fourier transform infrared (FTIR) spectroscopy, which is based on the
characteristic molecular vibrational spectra of cells, was used to investigate
spectral differences between untreated and treated Jurkat cells.
The Lipid (3000-2800 cm-1) and Protein – Nucleic Acids (1800 – 900 cm-1) regions
have been analysed.
Absorption peak (cm-1)
Assignments
2960 (L1)
–CH3 asymmetric stretching, lipids, proteins
2924 (L2)
–CH2 asymmetric stretching, lipids, proteins
2872 (L3)
–CH3 symmetric stretching, lipids, proteins
2852 (L4)
–CH2 symmetric stretching, lipids, proteins
1646 (A1)
amide I (–C=O stretching), proteins
1541 (A2)
amide II (–N–H bending, –C–N stretching), proteins
1454 (P1)
–CH2 scissoring/–CH3 asymmetric bending, proteins, lipids
1399 (P2)
–COO- symmetric stretching, proteins, lipids
1236 (P3)
-PO2- asymmetric stretching, nucleic acids, phospholipids
1085 (P4)
–PO2- symmetric stretching, nucleic acids, phospholipids
1050 (P5)
–C–O– stretching, carbohydrates
967 (P6)
–PO4- symmetric stretching, nucleic acids
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AN EXAMPLE OF THE RESULTS: FTIR spectroscopy
We have analysed the ratio between the region area (2800-2865) cm-1 of
symmetric CH2 groups (L4) and the region area (2865-2890) cm-1 of
symmetric CH3 groups (L3) : CH2 / CH3 (parameter R1) symmetric ratio
as function of time (between 0-360 minutes).
Correlation between the parameter R1 and the
% Apoptotic cells
A
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THE CONCLUSIONS
We have made a comparative study between the effects induced by UVB radiation on
Jurkat cells and the effects on the same cells treated with different concentrations of the
mixture.The mixture protects the cellular sample from the damage induced by the UVB
radiation at the level of some structure in the lipid (3000-2800 cm-1) and the proteinnucleic acids (1800 – 900 cm-1) regions.
Cytofluorometry measurements showed that the ability of UVB radiation to induce
apoptosis is attenuated by the treatment with Usnic Acid (lichen metabolite).
The mixture can be considered as a potential filter for the UVB radiation.
The FTIR spectroscopy measures have enabled to detect structural changes in the cells
related to the response to the stimulation of cells in the early stage of the apoptotic
process. We have found some highly probable spectroscopic markers of this process by
correlating spectroscopy and flow cytometry data.
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THE FUTURE OUTLOOK
Finally, the Fluorescence Microscopy is in progress in laboratory in
order:
to examine the autofluorescene of cellular sample treated with
the mixture plus UVB radiation
to observe and distinguish the different stages of the apoptotic
process
to establish a stastical model of the distribution of the light
peaks using the fluorescence image (software IMAGEJ)
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THANKS
TREATMENT: UVB radiation
The cell sample was irradiated for 30
minutes at a distance of 30 cm with a UV
lamp (Philips, TL20W/12 RS SLV) whose
emission maximum is at 310 nm.
310 nm
Wavelength [nm]
The measured radiation dose that hits the
sample is 310 mJ/cm2.
Irradiance on a surface I ( )  2 RP( ) l
with R( )  cos(r  )
Irradiance average I  21  I ( ) d
0
0 0
Final value
I
P sin( 0 )
2 0 r l
with
d 

 2r 
0  arctan 
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