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1st Italian Wokshop on UltraViolet Techniques and
Applications
UVB-INDUCED EFFECTS ON JURKAT CELLS A FILTER
PROVIDED BY A VEGETABLE MIXTURE
L. Di Giambattista Lattanzi
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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 dPoliambulato
ry La Torre, Torino
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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!!
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We studied the induction of apoptosis in Jurkat
cells by UVB radiation (wavelength 290320 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.
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An example of result in the protein region
(1800-1480 cm-1)
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Pozzi et al, Rad. Res. 168, 698-705 (2007)
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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.
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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
UVB
DRUG UVB
DRUG
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THE RESULTS Flow Cytometry
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ANALYSE Flow cytometry
Apoptosis was assessed using annexin V-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 Annexin V-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.
To collection
Catcher Tube Moves in and out stream
To waste
Sample injection
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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 (CO stretching), proteins
1541 (A2) amide II (NH bending, CN 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) CO 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 protein-nucleic 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
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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. The measured radiation dose that hits the
sample is 310 mJ/cm2. Irradiance on a
surface with Irradiance average Final value with
310 nm
Wavelength nm
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