REYES, ANGELA P.

BSMT 1A-1
190122767
JUNE 6, 2020

CITATION
Zelig, U., Kapelushnik, J., Moreh, R., Mordechai, S., & Nathan, I. (2009). Diagnosis of cell
death by means of infrared spectroscopy. Biophysical journal, 97(7), 2107–2114.
https://doi.org/10.1016/j.bpj.2009.07.026

SUBJECT
Apoptosis, Cells, DNA, FTIR Spectometry, Lipid Protein, Necrotic

RESEARCH QUESTIONS AND HYPOTHESIS

What are the characteristic of the IR spectral patterns of necrotic cells?
H1: There is a significant characteristic of the IR spectral patterns of necrotic cells found out in
this research.
How does apoptosis and necrosis appear to affect protein structures?
H1: There is a significant relationship that apoptosis and necrosis appear to affect protein
structures.
How does FTIR spectroscopy can distinguish cell death mode?
H1: There is a significant relationship that FTIR spectroscopy can distinguish cell death mode.

EXPERIMENTAL STRATEGY, WITH MATERIALS AND METHODS

Fourier spectroscopy was developed as an accelerated spectroscopic tool for cell and
tissue biochemical analysis (FTIR). It appears as a successful clinical testing method, as it makes
it easy to track specific biological reactions in samples without reactants or complex sample
control. This method was used for the analysis of apoptosis as well as cell cycle, differentiation
and proliferation of various cell and tissue types, but there are still many concerns about the
interpretation of IR results. Leukaemia cell lines model system was used to address the FTIR
spectroscopic apoptosis analysis by the researchers and this approach was applied to the
treatment of necrotic death cells.

Some of the methods that the researchers introduced to the research are cell culture and
treatment, subcellular fractionation, western blot, fluorescent microscopy and FTIR microscopy.
In the cell culture and treatment, human leukaemia cell lines such as U937 and CCRF-CEM were
cultured and were incubated with AraC or known as the doxorubicin for 24 hours and 48 hours,
respectively, to induce apoptosis. While, to induce necrosis, U937 cells were subjected to a
freeze-thaw method or treated for 10 min with saponin or incubated with KCN in glucose free
RPMI for 7 hours. CCRF-CEM cells were induced to undergo apoptosis and necrosis by
treatment with H2O (Sigma) for 7 h. In the subcellular fractionation, the U937 cells were
processed and monitored and washed in cold saline buffered with phosphate two times. The
nuclei was cleaned and a 50 mL ice-cold buffer suspended the radioactive pellet. In dilutions, the
supernatant was frozen at 80 ° C. A colorimetric process of Lowry calculated the protein content
of nuclear and cytoplasmic extracts. In western blot, immunoblotting was carried out with
multiple different primary antibodies. In fluorescent microscopy, the cultured cells have been
centrifuged with a fixed concentration of 0.05 mg / mL at 650 grams for 10 minutes and treated
with AO and EB. A fluorescent microscope was used to determine the fatality mode for each cell
type. And lastly the FTIR microscopy, all cells of the same type are composed of biological
samples of common fundamental components that give relatively wide bands. It is also possible
to ignore shifts in FWHM bands.

As for the statistical analysis part, the researchers present a mean of 5 Se and a student’s
t-test was used were the p-value is less than 0.05 considered significant. The least-square method
was used for linear regressions and a data collection package was used to compare data sets.
RESULTS

FIGURE 1

FIGURE 2
FIGURE 3

FIGURE 4
 FIGURE 5
The process of cell death was traditionally determined by fluorescence microscopy of EB
and AO staining forms. See Figure 1. There are natural particles in the living cells and chromatin
tends to be green. See Figure 1a. Early apoptotic cells display shrink nuclei and green
chromatin. See Figure 1b. By comparison, compact, broken nucleus occur in late apoptotic cells,
and the chromatin in them is vividly stained with EB, thereby becoming orange. See Figure 1c.
The necrotic cells are regular in shape but EB stains the chromatin, which makes the nuclei look
orange. See Figure 1d.

At the same time, untreated U937 calls under IR-spectral review were handled and
analysed. See Fig 2. The domain of certain absorbing bonds expressed in the symmetric or ant
symmetric deformation of the CH3 and CH2 protein and lipid groups, 3000 to 2830 cm1
macromolecules. The amide 1 and amide 2, sometimes refer to the secondary structure of
proteins, in 1800–1500cm1, and the 1150–750cm1 region represents various protein,
carbohydrate, lipid and nucleic acid. See Figure 2a. The researchers analysed the second
derivative of a vector-normalized spectra and untreated control and apoptotic cell patterns to
improve accuracy when interpreting spectral data. Apart from these features, the increase in lipid
absorption and the decrease in DNA absorption are some features of apoptosis. See Fig 2b.

The proportion of apoptotic cells and the cell death stages have been assessed by blue
trypan and AO and EB stain. See Figure 3a&b. Lipids are 2852 cm1 in size. See Figure 3c&d.
At the same time the relative absorbance value of untreated U937 cells is derived from FTIR-
MSP analysis. See Figure 3c&h. Quantified DNA at 970 and 780 cm1. See Figure 3e&f. In
another area of interest, Amide I at ~1654 cm1, there are distinctive spectral variations, referring
to the secondary protein structure. See Figure 3g&h.

FTIR spectral analysis has been performed and the second derivative spectral pattern of
the treated and untreated cells has been compared. See Figure 4a&b.

AO and EB staining as well as TB staining had already generally characterized cell

viability and the type of death. See Figure 5a&b. The researchers also reveal LDH, the protein
activity for verification of the dying cell method and high mobility group nucleus release B1. See
Figure 5c. The second IR spectral patterns analysis reveals that KCN treatment leads to an
increasing lipid absorption and a 67 percent increase at high levels in untreated cells control. See
Figure 5d. Similarly, DNA absorption improves by 56%, but exceeds substantially after 10 mM
on a level. See Figure 5e. In addition, treatment with KCN tends to decrease the spontaneous
protein spindle size, as seen by a reduction in absorption by 1639 cm1, even though this effect is
not observed at 5 mm. See Fig 5f.

DISCUSSION AND CONCLUSION

The objective of this research is to analyse the utility of FTIR for the identification and
characterization of various types of cell death, using leukemic cell lines as a model method.
Arabinose and apoptosis inducing doxorubicin, as well as potassium cyanide, saponin, freezing
taws and H2O2 necroisinducers methods have been used for leukaemia cells. Both cell death
modes display major spectral changes during apoptosis and necrosis in lipid absorption, which
are similar and therefore cannot be used to differentiate between apoptosis and necrosis. The
researchers findings is that in contrast to the above confusing factor, the degree of DNA opacity
to infrared light can still be distinguished from apoptosis and necrosis. In addition, both modes of
cell death can also be differentiated from the cell's secondary structure of the total cellular
protein through its infrared absorbance. The researchers conclude that FTIR spectroscopy allows
clear and simple analysis of cell death modes and may be useful for biochemical and medical
applications, due to its ability to monitor multiple bio molecular parameters.

This research was easy to understand because the researchers discuss each method and
process to determine the cell death. The researchers really focused on the topic that they’re
looking for and found a solution on it. Research hypotheses proved that there’s a significant
relationship in all research questions. This research is really reliable and valid.

Researchers have found that increasing lipid-related methylene absorption is a feature of

the spectral necrotic cell IR patterns. Typical necrotic cell changes in the membrane include
cellular organelle swelling and plasma-like membrane integrity loss. As apoptosis and necrosis
do not require identical changes in the macro membrane, there is a chance that unexpected
membrane events that contribute to an increase in lipid absorption observed during each cycle.
Otherwise, the membrane shared by both cell death modes can be more subtle, underlying the
common changes in lipid absorption, perhaps signalling by ceramide, the FAS or other mortality
domain receptor.

This research also found out that FTIR spectroscopy's ability to monitor global changes
in secondary protein structure also reveals its capacity to identify the cell death mode, since
apoptosis and necrosis appear to affect the structures of proteins differently. Different models or
cell death induction systems could be used to provide the conflicting data, or the existence of
alternative apoptosis pathways. In contrast, the random coil structure of the total protein
decreases during necrosis. Reductions in the spontaneous bobbin structure are found in both
KCN and H2O2, but at different levels, given similar necrosis rates.

The research data shows that FTIR spectroscopy has shown that cell death mode can be
differentiated based on changes in DNA conformation and the secondary protein structure. In
addition, the researchers suggested a solution to the main problem of IR opacity of DNA, which
will affect other biomolecules in light of these results. As the FTIR spectroscopy provides
distinctive information on biochemistry and requires minimal treatment of samples and no
reagents, it can be a promising new tools for monitoring cell death in the clinic.