BULGARICA ambvol. XXXIX - MEDICAL UNIVERSITY SOFIA - Central Medical Library
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MEDICAL UNIVERSITY − SOFIA
ACTA MEDICA
amb
BULGARICA
vol. XXXIX
2/2012
This journal is indexed in Global Health Database,
in Bulgarian Medical Literature Database and
in Scopus
Central Medical Library
Editor in chief
Prof. V. Mitev, MD, Ph. D. DSc
Editorial board
Prof. K. Tsachev, MD, Ph. D., DSc
Prof. M. Marinov, MD, Ph. D., DSc
Prof. D. Ziya, MD, Ph. D., DSc
Prof. N. Lambov, Mag. Ph., Ph. D.
L. Tacheva, MD
COMPARATIVE DETERMINATION OF GLYCATED HEMO-
GLOBIN WITH DIRECT PHOTOMETRIC IMMUNOASSAY
AND AFFINITY CHROMATOGRAPHY
E. Vucheva1, М. Genova2, Y. Niagolov3, N. Doncheva1, G. Peichinova1 and K. Tsatchev2
Department of Clinical Laboratory, Medical Institute − Ministry of Interior, Sofia
1
2
Department of Clinical Laboratory and Clinical Immunology, Medical University, Sofia
3
Department of Physiology, Medical University, Sofia
Summary. Glycated hemoglobin (HbA1c) is the gold standard in long-term
assessment of glycaemic control in patients with diabetes mellitus (DM). Liquid
Chromatography-Isotope Dilution-Mass Spectrometry is a reference method for
HbA1c, but other methods used in practice need standardization and compari-
son. This study was aimed to perform a comparative evaluation of the results from
HbA1c determination by direct immuno-photometric method (A) and affinity chro-
matography method (B). The study included 50 samples with values from all clini-
cally measurable areas. HbA1c in whole blood was studied using Olympus AU 400
analyzer and NycoCard READER II by, applying method A and method B, respec-
tiovely. The results are compared using the program Method Validator, as for the
reference method the chromatographic method was accepted. The imprecision in
series for method A has been traced in 10 parallel samples from two patients in
border and abnormal area of values. The inaccuracy was evaluated in normal and
pathological human control material for 25 consecutive days. The results from cor-
relation and regression analysis show linear regression coefficient r = 0.949, slope
= 0.919 and intercept = 0.59. Assessment of imprecision for method A shows CV
values of 1.21% and 1.91% for both cases, respectively. Accuracy monitoring of
method A with control materials shows d% 4.979 in the normal area, and 5.033 in
the pathological area. The results show high positive correlation and comparability
between the two methods. Direct immuno-photometric method has good accuracy
and precision and is applicable for analyzer Olympus AU 400.
Key words: Glycated haemoglobin, direct immuno-photometric method, affinity chro-
matography method, NycoCard READER
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 3
INTRODUCTION
G
lycated hemoglobin (HbA1c) is considered as a gold standard in long-
term assessment of glycaemic control in patients with diabetes mel-
litus (DM) [11,12,19]. Recently, standardization of hemoglobin HbA1c
measurement is of considerable interest, as long as it will provide quality assur-
ance in diabetes management [5,14]. It is recommended by the American Diabe-
tes Association for the diagnosis and screening of type 2 DM. There are over 20
methods of HbA1c determination, based on differences in structure, charge and
chemical reactivity, as the first clinically relevant method is described in 1978 [6,
7, 8, 14]. Most commonly used analytical methods are based on high performance
liquid chromatography (HPLC), electrophoresis, other chromatographic methods,
immune assays, etc. The reference method recommended and accepted in 2007
by the IFCC [9,10] is Liquid Chromatography-Isotope Dilution-Mass Spectrometry
(LC-ID-MS HP). Studies establish a difference in the results, which requires stan-
dardization and comparison of the methods used in practice. The transition to the
recently introduced to practice units (mmol/mol) is part of the standardization and
harmonization of the results.
This study was aimed to perform a comparative evaluation of the results from
HbA1c determination by direct immuno-photometric method (A) and affinity chro-
matography (B).
MATERIALS AND METHODS
The study included 50 patients (25 males and 25 females), aged between 28-
55 years. Thirty of them were randomly chosen and 20 were deliberately selected
according to values of HbA1c > 8.0%. The HbA1c values ranged from 5.0% to
12.70% and the measurement was performed in accordance with the requirements
of the local and international legislation. HbA1c in venous blood was obtained in 3
ml EDTA vacutainers and was kept at 4oC in order to preserve the stability of the
samples. The measurement was conducted within 2 days to avoid large differ-
ences in the results caused by on-going non-enzymatic glycation, especially at high
levels of HbA1c. The studied parameter was measured using Olympus AU 400
analyser, with the reagents, calibrators and controls for method A being produced
by PZ CORMAY S.A., Poland. The calibrators for method A are not certified to the
IFCC reference method and to the NGSP/DCCT recommended method, but are
evaluated using NCCLS protocols [21].Method A (candidate-method) is based on
a direct photometric immunoassay for HbA1c and requires manual preparation of
the hemolysate. The hemolysate is obtained by mixing 500 μl hemolysing agent
− NaN3 with 10 μl whole blood stored for 5 minutes or until complete hemolysis
occurres in accordance with the testing method procedures. The hemolysate is
mixed with latex reagent so that total Hb and HbA1c fraction competitively bound
to the latex particles. In the second stage, anti-human HbA1c (mouse) monoclo-
4 Comparative determination of glycated hemoglobin...
nal antibodies and anti-mouse IgG polyclonal antibodies (goat) are added. Goat
anti-mouse antibodies react with mouse anti-human HbA1c–latex complex causing
agglutination reaction which is measured photometrically. Linearity for this method,
according to the manufacturer, is up to 16%. Method B was performed using Nyco-
Card READER II device with reagents and controls produced by Axis-Shield PoC
AS, Norway, based on the principle of reflection refractometry. Method В requires
5 μL capillary blood or anticoagulated venous blood (EDTA, citrate or heparin) and
3 minutes assay time. It is a fast point of care test based on boronate affinity prin-
ciple. The calibrator of method B is compared to the primary reference standard,
certified September 2011 [22]. The linearity of method B is from 3 to 18%. Both
methods interfere with bilirubin up to 50 mg/dl, triglycerides up to 22 mmo/l, HbF up
to 30%, haemolysis > 3g/100 ml hemoglobin and ascorbic acid up to 50 mg/dl [16].
Correlation analysis is implemented in the verification of the results and coefficient
r is determined, as a criterion for the strength of the linear relationship between the
two groups of comparable variables. Statistical program for regression analysis
with Method Validator Software is applied to calculate the Slope, Intercept and r of
the results obtained by both methods [13,18,20]. Because of its standardization,
method B (affinity chromatography) was used as a reference method.Verification
for significantly deviating values („fugitives”) did not find results > 3.5 SD yx, indi-
cating that the random scatter follows a Gaussian distribution. Nevertheless, we
focused on Demming regression instead of simple linear regression. Simple linear
regression has many limitations and requires the independent variable to be deter-
mined without an error, i.e. determining with the reference method. Deming regres-
sion takes into account variation of both methods compared.
The imprecision in series for method A has been traced in 10 parallel samples
from two patients in the border and the abnormal values. The assessment is con-
ducted using standard deviation (SD) and coefficient of variation (CV).
Evaluation of the inaccuracy of method A is made through monitoring normal
and pathological human control material in 25 consecutive days [3]. Inaccuracy is ac-
cepted as a measure of total error (systematic + occasional error) and was calculated
by formula as d%. The absolute deviation (difference) from the target value (Bias) is
calculated as the difference between mean and target value of the controls.
Variation analysis and Student t-test were performed using GraphPad Prism
software and Interactive Statistical Calculation Pages.
RESULTS
The values of basic statistical parameters calculated by Method Validator Soft-
ware are presented in Table 1.The comparison of methods A and B (Fig. 1) showed
high positive correlation r = 0.949. Slope, as a criterion of the proportional error size
of the study method A, is 0.919 (0.822 to 1.016). Intercept, a criterion of the size of
the constant systematic error of the study method A, is 0.59 (-0.15 to 1.34) and ap-
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 5
proaches the ideal value. Figure 2 illustrates HbA1c values measured by methods
A and B for each of the patients studied. The application of variation analysis and
Student t-test shows no significant statistical differences, p > 0.05, between the
compared methods (p = 0.656). Mean ± SEM for method A was 7.85± 0.359 and for
method B was 7.89 ± 0.389 (Fig. 3), and presents the difference plot between the
two compared methods. Precision of method A, based on determination of HbA1c
in samples of two patients in border and pathological areas, are presented in Table
2 and Table 3. The mean values, standard deviations and coefficient of variance
were 6.42%, 0.079, 1.21% and 8.25%, 0.158, 1.91% for the patient in the border
area and the patient in the pathological area, respectively. Monitoring of inaccuracy
of method A with control material in normal and pathological area in 25 consecutive
days demonstrated the following results: for the low control – mean ± SEM = 5.144
± 0.066; SD = 0.327; Value for the control material (Хо) = 4.9 (3.9-5.9); d% = 4.979;
Bias = 0.244. For the high control – mean ± SEM = 11.396 ± 0.238; SD = 1.167; Хо
=12.0 (10.9-13.9); d% = 5.033; Bias = -0.604 (Table 4).
Table 1. Basic statistical parameters obtained by Method Validator
n = 50 “Reference” method B “Candidate – method” A
Mean 7.898 7.856
SD 2.095 1.935
SEM 0.296 0.274
Min 5.0 5.0
Max 12.7 12.1
Table 2. Results from HbA1c determination in samples of two patients in border and patho-
logical areas
1 2 3 4 5 6 7 8 9 10
Patient № 1 6.5 6.4 6.4 6.3 6.5 6.5 6.4 6.5 6.3 6.4
Patient № 2 8.3 8.4 8.3 7.9 8.2 8.4 8.3 8.2 8.1 8.4
Table 3. Statistial results for precision in series of method A
N Mean SD CV
Border area 10 6.42 0.079 1.21
Pathological area 10 8.25 0.158 1.91
Table 4. Monitoring of inaccuracy of method A with a control material in normal and patho-
logical area
N = 25 Mean х Х0 Bias d% SD SEM CV (%)
Control level 1 5.144 4.9 0.244 4.979 0.327 0.066 6.36
Control Level 2 11.396 12 -0.604 5.033 1.167 0.238 10.24
6 Comparative determination of glycated hemoglobin...
Fig. 1. Regression analysis − Deming regression (HbA1c) measured by method A − continours line
and method B − dashed line
Fig. 2. Parallel measurement of HbA1c in 50 patients by both methods
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 7
Fig. 3. Difference plot between the two methods
Fig. 4. Comparison of the mean values of HbA1c, assessed by both methods (variation analysis,
t-test)
CONCLUSIONS
Reproducibility (precision) is an assessment of coincidence between the re-
sults in identical conditions of repeatability. It is estimated by imprecision, which
reflects the dispersion of results around a mean value. Imprecision depends on the
random errors size in the analytical stage. The evaluation of imprecision data for
HbA1c by method A measured through a direct photometric immunoassay shows
CV < 2.0%. This result permits the method to be regarded as successfully appli-
cable in clinical practice. The maximal acceptable imprecision for HbA1c is pre-
8 Comparative determination of glycated hemoglobin...
sented by coefficient of variance less than 6%. According to the Medical Standard
of Clinical Laboratory the eligible d% for HbA1c is up to 12% [1, 2]. Method A has
d% 4.979 in the normal area and d% 5.033 in the pathological area. The used
control materials and the reagent are reliable and traceable in compliance with
the storage conditions, dissolving method, proper storage after reconstitution and
show good stability of the components. The high correlation coefficient for method
A and method B makes them comparable. Comparison between patient results
for both methods with Student t-test showed no significant differences (p > 0.05).
The results from the comparison between the two methods show high correlation
and comparability despite the different principles implicated. This perfect relation-
ship is due to standardization of calibrators in accordance with the IFCC reference
standard. It should be noted that the preliminary manual sample preparation in pre-
analytical phase could be a source of errors in the results.
The direct immune-photometric method has good accuracy and precision. It
is applicable for analyzer Olympus AU 400 and it is a good alternative to affinity
chromatography.
REFERENCES
1. Lambreva, L. et al. Ensuring quality in clinical laboratory. Pre-analytical stage.- a book, published
by “Diagnostics”, Sofia, 2010, 31-76 (in Bulgarian).
2. Medical Standard “Clinical laboratory” – Ordinance № 10 from May 2006 for the acceptance of
Medical Standard for Clinical Laboratory – State Gazette, Sofia, 44, 2006 (In Bulgarian).
3. Tzvetkova, T. et S. Danev. Analytical principles and procedures in clinical laboratory. Published by
“VAP”, Plovdiv, 2001.
4. C o n s e n s u s statement on the worldwide standardization of the hemoglobin A1C measurement:
the ADA, EASD, IFCC, and the IDF. An agreement by the ADA/EASD/IFCC/IDF as to how HbA1c
should be standardized and reported. – Diabetes Care, 30, 2007, № 9, 2399-2400.
5. E x e c u t i v e summary: Standards of medical care in diabetes. – Diabetes Care, 33, 2010,
(Suppl 1), S 4-10.
6. G a r c í a-A l c a l á, H., А. Ruiz-Argüelles et B. Cedillo-Carvallo. Effect of the method to measure
levels of glycated hemoglobin on individual clinical decisions: comparison of an immunoassay with
high – performance liquid chromatography. – Am. J. Clin. Pathol., 132, 2009, № 3, 332-335.
7. G e i s t a n g e r, A. et al. On the behalf of the IFCC Working Group on Standardization of He-
moglobin A1c. Statistical Methods for Monitoring the Relationship between the IFCC Reference
Measurement Procedure for Hemoglobin A1c and the Designated Comparison Methods in the
United States, Japan, and Sweden. – Clin. Chemistry, 54, 2008, №.8, 1379-1385.
8. G o o d a l l, I. HbA1c Standardisation Destination. Global IFCC Standardisation How, Why, Where and
When. A Tortuous Pathway from Kit Manufacturers, via Inter-laboratory Lyophilized and Whole Blood
Comparisons to Designated National Comparison Schemes. – Clin. Biochem., 26, 2005, № 1, 5-19.
9. J e p p s s o n , J.O. Approved IFCC reference method for the measurement of HbA1c in human
blood. – Clin. Chem. Lab. Med., 40, 2002, № 1, 78-89.
10. K a i s e r, P., T. Akerboom, R. Ohlendorf et H. Reinauer. Liquid chromatography-isotope dilution-
mass spectrometry as a new basis for the reference measurement procedure for hemoglobin A1c
determination. – Clin. Chem., 56, 2010, № 5, 750-754.
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11. L a r s e n, M. L., M. Hørder et E. F. Mogensen. Effect of long-term monitoring of glycosylated
hemoglobin levels in insulin-dependent diabetes mellitus. – N. Engl. J. Med. 323, 1990, № 15,
1021-1025.
12. L e n t e r s-W e s t r a, E. et R. J. Slingerland. Hemoglobin A1c determination in the A1C- Derived
Average Glucose (ADAG) study. – Clin. Chem. Lab. Med., 46, 2008, № 11, 1617-1623.
13. L i n n e t, K. Evaluation of regression procedures for method comparison studies. – Clin. Chem.,
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14. L i t t l e , R. R. et C. L. Rohlfing. HbA1c standardization: background, progress and current issues.
– Lab. Med., 40, 2009, № 2, 368-373.
15. Marquis, P. Method validator software. Centre hospitalier Metz – France Aug 6 1999 http://perso.
easynet.fr/~philimar.
16. N G S P. HbA1c assay interferences. Available from: http://www.ngsp.org/interf.asp (November,
2010).
17. Ö z ç e l i k, F. et al. Comparison of Three Methods for Measurement of HbA1c. – Turk.J. Biochem.,
35, 2010, № 4, 344-349.
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19. T h e I n t e r n a t i o n a l Expert Committee. International Expert Committee Report on the Role
of the A1C Assay in the Diagnosis of Diabetes. – Diabetes Care, 32, 2009, № 7, 1327-1334.
20. W e s t g a r d, J. O. Points of care in using statistics in method comparison studies. – Clin. Chem.,
44, 1998, № 11, 2240-2242.
21. http://www.pzcormay.pl
22. http://www.ngsp.org/docs/method.pdf
Address for correspondence:
Assoc. Prof. Nadezhda Doncheva, MD, PhD
Medical Institute − Ministry of Interior
Department of Clinical Laboratory
79 A, Skobelev str.
1606 Sofia
Bulgaria
9821-416
e-mail: ndon4eva@abv.bg
10 Comparative determination of glycated hemoglobin...RE-STIMULATION OF T-CELL ANTIGEN RECEPTOR
AND REGULATION OF THE TYROSINE
PHOSPHORYLATION IN PERIPHERAL T-CELLS
K. Hristov, J. Lindquist and B. Schraven
Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg,
GERMANY
Summary. TCR stimulation involves ZAP70 tyrosine phosphorylation and ac-
tivation that is essential for signal initiation and TCR degradation. Here we find that
re-stimulation of TCR fails to affect the tyrosine Y319 phosphorylation of ZAP70.
Vanadate is a reversible PTP inhibitor, sensitive to the presence of EDTA. By con-
trast, pervanadate inhibits irreversibly. We find that two successive washes for 10
min at 37oC remove the effects of vanadate on protein tyrosine phosphorylation in
T-cells. Hypothermia has been shown to affect the expression of different signalling
proteins within 1 hour. We find that incubation on ice does not affect the ability of
vanadate to increase the activatory phosphorylation of serine S473 at PKB/AKT,
and that the removal of the inhibitory effect of vanadate following two successive
washes with culture medium down-regulates to control levels the phosphorylation
of S473 at PKB/AKT.
Key words: cellular signalling, system biology, T-cell receptor, re-stimulation, tyrosine
phosphorylation, vanadate, pervanadate, PI3K – PKB/AKT signalling pathway
A
bbreviations: BAK, Bcl-2 homologous antagonist/killer; BCL-X, B-cell
lymphoma X protein; CBL, Casitas B-lineage lymphoma proto-onco-
gene; CD45, cluster of differentiation 45; CSK, C-Src kinase; CTLL2,
murine cytotoxic T-cell line; EDTA, ethylene glycol tetra-acetic acid; EGF, epithe-
lial growth factor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion
kinase; FOXP3, forkhead box protein 3; GAB1/2, GRB2-associated-binding pro-
tein 1/2; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HE-PTP, he-
matopoietic protein tyrosine phosphatase; HIF1α, Hypoxia-inducible factor 1 α; IL2,
interleukin 2; ITAM, immunoreceptor tyrosine-based activation motif; JAK, Janus
kinase; JNK1/2, c-Jun N-terminal kinase; LAT, Linker for activation of T-cells; LCK,
lymphocyte cell-specific protein-tyrosine kinase; LFA1, Leukocyte function-associ-
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 11ated molecule 1; LMW-PTP, low molecular weight protein tyrosine phosphatase;
MAPK, mitogen-activated protein kinase; MCF7 cell line, Michigan Cancer Foun-
dation – 7 cell line; MEK, MAPK/ERK kinase; MHC, major histocompatibility com-
plex; MKP1/3, mitogen-activated protein kinase phosphatase 1/3; mTOR, mam-
malian target of rapamycin; NFκB, nuclear factor κ B; PAC1, dual specificity protein
phosphatase 2; PDGF, platelet-derived growth factor; PI3K, phosphatidyl 3-kinase;
PKA, protein kinase A; PKB/AKT, protein kinase B / RAC-α serine/threonine-protein
kinase; PKC, protein kinase C; PTK, protein tyrosine kinase; PTP, protein tyrosine
phosphatase; SMAC, supramolecular adhesion cluster; SOCS, suppressor of cy-
tokie signalling; SRC, Raus sarcoma virus tyrosine kinase; TCR, T-cell antigen
receptor; TNFα, tumor necrosis factor α.
INTRODUCTION
TCR is a complex receptor that consists of two α-β, or δ-γ heterodimers, three
CD3 chains, and two additional ζ-ζ homodimers, or ζ-η heterodimers. The antigen
presented by the MHC molecule is recognized by α-β, or δ-γ heterodimers, and the
signal following the specific binding is initiated by the CD3-ζ-η complex. The phos-
phorylation of specific tyrosine residues involves SRC tyrosine kinases (LCK, and
FYN), ZAP70 (or SYK during the thymocyte development), CSK, and TEC kinases,
along with a number of adaptor molecules (GAB2, GRB2, SLP76, LAT, and PAG/
CBP) in a highly organized signalling cascades [1]. Several PTPs are involved in
the regulation of the phosphotyrosine status of these signalling molecules, and mi-
nor changes in the PTK/PTP activity have a major impact on TCR-triggered cellular
activation and proliferation.
A number of signalling molecules (GRB2, RAS-GAP, PI3K, SHC, and SRC-
PTKs) are shown to bind singly phosphorylated ITAMs. TCR-triggered ITAM phos-
phorylation is mediated mostly by LCK, and the double phosphorylated ITAMs bind
ZAP70 with high affinity, excluding other signalling molecules. Once recruited,
ZAP70 is activated by phosphorylation of tyrosine Y493 by LCK. Upon phospho-
rylation ZAP70 auto-phosphorylates, and phosphorylates LAT, creating docking
sites for other SH2-domain-containing molecules, comprising LCK-CD3/ζ-ZAP70-
LAT signal initiation cascade [2]. LCK is kept inactive by dephosphorylation of the
activatory tyrosine (Y394), and phosphorylation (by CSK through PAG/CBP, or
PKA) of the negative regulatory tyrosine (Y505). In T-cell lines, approximately half
of all LCK molecules are phosphorylated at Y505, and interestingly, changes in
this phosphorylation have not been observed during T-cell activation. Instead, the
phosphotyrosine content of LCK increases after T-cell activation, suggesting that
LCK activation does not involve acute dephosphorylation of Y505 [2]. CSK sets a
threshold for T-cell activation, shuttling between different anchor proteins in resting
and activated T-cells.
12 Re-stimulation of T-cell antigen receptor...Although there is necessity for sustained TCR stimulation, the continuous
stimulation reduces T-cell responsiveness. The reduction of TCR levels extinguish-
es the signalling and reduces the responsiveness to antigenic stimulation. In the
absence of cognate antigen, naive T-cells infrequently and transiently (2.6 ± 0.9
min) cluster, but do not internalize their TCR. TCR clustering is a rapid and brief
process (cSMACs persisting for up-to 10 min) that quickly (within 2 min) default to
internalization [3]. There are two TCR internalization pathways: PKC-dependent
that results in rapid internalization, and re-expression of TCR, and a SRC-PTKs-
dependent that leads to TCR degradation in the lysosomes. Clathrin heavy chain
phosphorylation is observed after 5 min of stimulation, persists up to 20 min, and
is mediated by LCK and ZAP70 [4]. ZAP70 is degraded rapidly, not in the lyso-
somes, but in a calpain-dependent mechanism, and its degradation is mandatory
for ζ-chain targeting to lysosomes. ZAP70 degradation reaches plateau after 120
min, and depends on SRC-PTKs activity (Y292) [5]. Tyrosine Y292 of ZAP70 is a
CBL-binding site, mediating TCR internalization and degradation [6]. Re-expres-
sion of ZAP70 is observed 48-72 hours after stimulation [5]. It is interesting that
stimulation of two specific for different antigens TCRs expressed on the same cell
results in independent biochemical and functional responses, including antigen-
induced TCR internalization [7].
TCR stimulation of peripheral T-cells results in their proliferation, and differen-
tiation into T-blasts, expressing signalling proteins (IL2R, PD1, CD95L, LCK, FYN,
RAC1/2, PKB/AKT, HPK1, STAT5, G1 cyclins D and E, c-MYC, BCL2, BCL-XL,
cIAP1, BIM) that alter their responsiveness [8, 9, 10, 11, 12, 13]. TCR re-stimulation
up-regulates the expression of SOCS proteins [14], and suppresses the cytokine se-
cretion [12]. Re-stimulation of these IL2-cultured T-blasts results in growth arrest and
apoptosis partially dependent on FAS (CD95L expression), and mitochondrial (HPK1
expression) cell death signalling pathways [15, 10, 11, 13]. Further, TCR stimulation
of peripheral T-cells results in dramatic changes in the lipid raft composition, increas-
ing surface monosialoganglioside GM1 expression, and the total amounts of raft-
associated lipids (sphingomyelin, cholesterol, and glycosphingolipids). While mRNA
levels of GlcCer, and LacCer synthetases and GM2 synthetase are increased, these
of GD3, and GM1 synthetases are decreased after 24 hr stimulation. Thus, the struc-
ture and function of lipid rafts in human activated T-cells is distinct from those in naïve
T-cells, which can explain the relative transient resistance of TCR signalling upon re-
stimulation [8]. Consistent with the altered protein content in the lipid rafts (increased
CSK, PAG/CBP, and FYN), TCR-mediated calcium response that depends entirely
on the raft integrity is inhibited in activated T-cells.
At least 30 PTPs, specific in their activity, are known to be expressed in T-
cells. There is strong pressure against tyrosine phosphorylation at the plasma
membrane, where PTP activity exceeds PTK activity by 1000-fold. CD45 is a posi-
tive regulator of TCR signalling, present initially in the central SMACs, activating
LCK [2]. Several PTPs have been described as positive (SHP2, LMPTP) and nega-
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 13tive (SHP1, PEP, PTPH1, VHX) regulators of TCR signalling. While HE-PTP and
MKP3 keep ERK inactive in the cytoplasm, PAC1 and MKP1 dephosphorylate ERK
in the nucleus [16]. LMW-PTP is a positive regulator, dephosphorylating (Y292)
ZAP70. This residue is important for c-CBL ubiquitin ligase complex binding, and
TCR internalization and degradation. Further, LMW-PTP negatively regulates FAK,
and TCR-induced LFA1-mediated cell adhesion during the formation of the immu-
nological synapse [17].
PTP, like all other proteins have a structure-determined temperature optimum
ranging from 75oC, for the low-molecular tyrosine phosphatase (Thermus ther-
mophilus HB27) [18], to −15oC for PPI (Shewanella) [19]. Hypothermia has been
shown to induce significant changes in the biological systems, altering the metabo-
lism and the cellular amounts of polyols, sugars, and amino acids [20, 21]; the ex-
pression of anti-freeze proteins and ice-nucleating proteins in the plasma; cellular
pH, water content (aquaporins and trehalose-6-phosphate synthase expression),
and the expression of signalling proteins (p38 MAPK, PKA, p70S6K, PKC, and Au-
rora family kinase I) [21]. In different cellular systems, hypothermia (33oC) induces
specifically the expression of MKP1 (after 1hr) that inhibits the activity of p38 MAPK
and JNK1/2, and prevents TNFα-triggered actin re-organization, caspase-3 pro-
cessing, and apoptosis [22]. Hypothermia (30oC) prevents the expression of p53
during ischemic injury, and increases the expression of the anti-apoptotic BCL-X,
but decreases the expression of the pro-apoptotic BAK [23]. Similarly, hypothermia
(30oC) prevents the ischemia-induced decrease in PKB/AKT and HIF1α levels [24].
Freezing (cryoablation) is a ubiquitous stress signal leading to cell death. It is in-
teresting that while the androgen-sensitive prostate cancer cell lines (LNCaP LP,
and PC3 AR) undergo cell death following freezing, androgen-insensitive prostate
cancer cell lines (LNCaP HP, and PC3) demonstrate decreased levels of cell death,
and caspase activity after freezing, which is associated with the increased expres-
sion of integrins α6 and β4 subunits [25].
Vanadate and pervanadate, the complexes of vanadate with hydrogen perox-
ide, used in this study, are two commonly used PTPs inhibitors. Vanadate is a phos-
phate analogue and a competitive, reversible inhibitor. Pervanadate, by contrast,
inhibits by irreversibly oxidizing the catalytic cyteine, which forms thiol-phosphate
intermediate during the catalysis. It is important to note that pervanadate is a mix-
ture of several different complexes of vanadate with hydrogen peroxide, and that
hydrogen peroxide, itself, is a much poorer inhibitor than pervanadate. HEPES is
one of the few buffers that does not react with vanadate. EDTA, commonly used
in assay buffers, forms a complex with vanadate, with a Keff 1.4 x 104 M-1 at pH
8.0. EDTA causes an immediate and complete reversal of the inhibition caused
by vanadate, and only partially reverses pervanadate inhibition. The presence of
excess DTT (or reducing agents like β-mercaptoethanol, and reduced glutathione)
converts pervanadate to vanadate [26].
14 Re-stimulation of T-cell antigen receptor...PI3K-PKB/AKT pathway is activated following TCR and CD28 co-stimulation,
regulating T-cell survival (BCL2, BCL-XL, SURVIVIN/AURORA-B) and prolifera-
tion (E2F, c-MYC, RB, p27-KIP1, CDK1/2, cyclins D2, D3, and E) [27, 28]. Further,
PI3K (p110δ and p110α) – PKB/AKT and mTOR signalling regulate the de novo
expression of FOXP3 in CD4 T-cells, their differentiation into regulatory T-cells,
and their migration (through CCR7 and CD62L) [29]. PI3K activity has been shown
to regulate PKCζ, necessary for the cytoskeletal reorganization during the cellular
proliferation, and the recruitment of PKCθ to the cSMAC, followed by activation of
NFκB and induction of IL2 transcription and secretion [30]. While mTOR remains
fully active 18 hr after TCR stimulation, PKB/AKT is not phosphorylated. When
T-cell activation is withdrawn, mTOR activity declines, and PKB/AKT become re-
phosphorylated [29]. Further, PI3K has been shown to mediate JNK activation pos-
sibly through HPK1 (PI3K-ITK-PLCγ-PKC-PKD1-HPK1 pathway) [1]. PI3K activity
is regulated by SRC-PTKs and JAK kinases. PI3K is phosphorylated on sever-
al tyrosine residues. Pervanadate-treated cells display increased basal tyrosine
phosphorylation, and interestingly, p85 tyrosine phosphorylation and PI3K activity
increase further following stimulation with IL2 [31].
GAB2-dependent activation of ERK depends on functional p85, and there is a
positive PI3K-MEK signalling mechanism, necessary for the optimal IL2-mediated
proliferative response. However, in some cell systems like CTLL2, PI3K inhibition
fails to affect ERK activity. Further, ERK-dependent phosphorylation of GAB1, fol-
lowing EGF treatment, inhibits the activity of PI3K. Another negative regulatory
mechanism is PKB/AKT-mediated GAB2 phosphorylation in MCF7 epithelial cells.
PKA activity has been shown to inhibit PI3K function in some cell systems, but not
in others like PDGF-mediating signalling in hep-G2 cells [27,28].
In this paper we describe the changes of the regulatory tyrosine Y319 phos-
phorylation of ZAP70 following re-stimulation of TCR in Jurkat T-cells, and the ef-
fects of vanadate and low temperature on cellular tyrosine phosphorylation.
MATERIALS AND METHODS
Cells: Murine, splenic, T-cell preparation: 119 ± 39 x 106 cells were isolated
on average from each C57/BL6 murine spleen (49 x 106 cells from OTII murine
spleen), with a yield of 8 ± 6 x 106 CD8+ T-cells (CD8+ T cell isolation kit II, mouse;
Miltenyi Biotec GmbH, Germany; Cat. number: 130-095-236), or 8 x 106 CD8+ T-
cells from each OTII mouse (Pan T cell isolation kit II, mouse; Miltenyi Biotec GmbH,
Germany; Cat. number: 130-095-130) . The murine, splenic CD8+ and the Jurkat T-
cells were cultured under routine cell culture conditions. Inhibitors: Vanadate (SOV,
sodium ortho-vanadate, 100 μM), and pervanadate (PV, 100 μl of 1mM vanadate
and 1 μl of 30% hydrogen peroxide, mixed with 900 μl culture medium, immediately
prior stimulation). Sample preparation: Whole cell lysates were prepared with 100
μl lysis buffer (100 μl 10% LM (lauryl maltoside, n-dodecyl-β-D-maltoside), 100 μl
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 1510% NP40, 10 μl 100 mM vanadate, 10 μl 100 mM PMSF, 50 μl 1 M Tris pH 7.5, 20
μl 500 mM NaF, 20 μl 500 mM EDTA pH 7.5, 33 μl 5 M NaCl, and ddH2O to 1000
μl), and after pelleting the nuclei, to the supernatants were added 30 μl sample
buffer (5 X sample buffer: 2 ml 0.5 M Tris pH 6.8, 5 ml glycerol, 0.250 ml 10% bro-
mphenolblue, 2.5 ml 20% SDS, and ddH2O to 10 ml; for the reducing buffer, 10 μl
β-mercapto-ethanol was added to 90 μl sample buffer). PAGE: Samples were load-
ed on 15% SDS-PAGE. The approximate molecular weight of the detected proteins
was determined with Fermentas PAGE Ruler (Fermentas, Inc., USA; Cat. number:
SM0671). Immunoblotting: The appropriate primary antibody was used for 1 hr in-
cubation at RT, or overnight at 4oC. Membranes were incubated with the secondary
antibodies for 1 hr at RT. Primary antibodies: Murine, anti-phosphotyrosine 4G10
antibody (4G10 hybridoma, IMKI, Otto-von-Guericke University; Magdeburg, Ger-
many); rabbit, anti-phosphotyrosine Y319 of ZAP70 antibody (Cell Signaling Tech-
nology, USA; Cat. number: 2701); and rabbit, anti-phosphoserine S473 of PKB/
AKT (Cell Signaling Technology, USA; Cat. number: 4058) were used. Secondary
antibodies: Donkey, anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc., USA;
Cat. number: sc-2317); and donkey, anti-mouse IgG-HRP (Santa Cruz Biotechnol-
ogy, Inc., USA; Cat. number: sc-2314) were used. Re-probing: Membranes were
incubated with 1% NaN3 for 1 hr at RT, and after washing, they were re-probed with
the appropriate primary and secondary antibodies. Detection: Kodak film was used
for the detection of the specific fluorescence (Amersham ECL™ Western Blotting
Detection Reagents; GE Healthcare; Cat. number: RPN2109).
RESULTS AND DISCUSSION
Stimulation of the TCR on Jurkat T-cells triggers rapid tyrosine phosphoryla-
tion of a number of proteins with important signalling functions [1, 2]. ZAP70 is a
key molecule for the function of TCR signalling. It is tyrosine phosphorylated and
activated immediately following TCR stimulation [2]. Similarly, we found ZAP70 ty-
rosine (Y319) phosphorylated after 2 min of stimulation with anti-CD3 (Figure 1,
lane 2). The murine, anti-human CD3 (C305) antibody (C305 hybridoma, IMKI,
Otto-von-Guericke University; Magdeburg, Germany) was used to stimulated TCR
of Jurkat cells (1 x 106 cells in 1 ml culture medium) for 2 min at 37oC (Figure 1,
lane 2), followed by a washing step (10 min: 2 x 3 min spin at 5000 rpm, and 2 min
for changing the culture medium or PBS) at RT (Figure 1, lane 3). A second stimu-
lation for 2 min followed the first wash (Figure 1, lane 4), and was followed by one,
or two washes (Figure 1, lane 5 and 6). The experiment was terminated with 500 μl
ice-cold PBS, and placing the tubes on ice. Washing (with culture medium) the cells
failed to extinguish the signal completely, and ZAP70 remained tyrosine (Y319)
phosphorylated, with phosphorylation levels about 50% of that observed after 2 min
of TCR stimulation. Re-stimulation of these cells failed to increase the phospho-
rylation of ZAP70. The level of ZAP70 tyrosine (Y319) phosphorylation was even
16 Re-stimulation of T-cell antigen receptor...decreased in the re-stimulated cells (Figure 1, lane 4). ZAP70 retained this level of
phosphorylation even after two successive washing steps (Figure 1, lanes 5 and 6).
We attribute the inability of anti-CD3 (C305) antibody re-stimulation to affect ZAP70
phosphorylation to its proteolysis [5, 6] during the TCR complex internalization and
degradation [3, 4, 6]. Determination of ZAP70 protein and phosphorylation levels
following re-stimulation with a different antigen would be interesting [7].
Fig. 1. Re-stimulation of Jurkat T-cell’s TCR. Jurkat cells were stimulated with anti-CD3 (C305) anti-
body for 2 min at 37oC (lane 2), followed by a washing step (10 min) at RT (lane 3), a second stimula-
tion for 2 min following the first wash (lane 4) was followed by one, or two washes (lane 5 and 6). The
immunoblot represents the phosphorylation of tyrosine Y319 at ZAP70
Further, to investigate the effect of vanadate on early TCR signalling, we
treated murine, splenic CD8+ T-cells (2 x 106 cells in 1 ml culture medium) with
100 μM vanadate for 30 min, which up-regulated the cellular tyrosine phosphory-
lation (Figure 2A, lane 2 and 2B, lane 2) of a group of protein with approximate
molecular weight between 55 and 100 kDa. The cells were incubated at 37oC,
and washed either ones or twice (10 min: 2 x 3 min spin at 5000 rpm and 2 min for
changing the culture medium or PBS). The experiment was terminated with 500
μl ice-cold PBS either immediately (0 hr), or after 10, 20, 30 min, or 1, 2, and 3 hr
incubation at 37oC (Figure 2), and placing the tubes on ice. While a single wash
(1 microfuge spin for 3 min at RT) failed to affect the vanadate-induced increase in
the tyrosine phosphorylation (Figure 2B, lanes 3-7), and tyrosine phosphorylation
level is retained, two successive washes at RT for total time of 10 min (Figure 2A,
lane 3) down-regulated vanadate-induced tyrosine phosphorylation. The tyrosine
phosphorylation level decreases progressively for up to 3 hr. This demonstrates
the reversibility of vanadate-induced PTP inhibition [26], and the increase in the
phospho-tyrosine content.
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 17Fig. 2. Vanadate, reversible protein tyrosine phosphatase inhibitor. Murine, splenic CD8+ T-cells are
incubated with or without (control) 100 μM vanadate for 30 min at 37oC, and washed either ones (A,
10 min) or twice (B, 2 x 10 min). The experiment was terminated either immediately (0 hr), or after 10,
20, 30 min, or 1, 2, and 3 hr incubation at 37oC. The immunoblot represents the total protein tyrosine
phosphorylation using the 4G10 antibody
To investigate the effect of low temperature (incubation on ice) on revers-
ibility of vanadate-induced inhibition, we treated murine, splenic CD8+ T-cells
(2 x 106 cells in 1 ml culture medium) with or without (control) 100 μM vanadate
(SOV), or pervanadate (PV). The cells were incubated for 30 min either on ice
(Figure 3, lane 2 and 3), or at 37oC (Figure 3, lane 1 and 4-11). After a wash-
ing step (10 min), the cells were incubated for additional 30 min either on ice
(Figure 3, lanes 2-5), or at 37oC (Figure 3, lane 1 and 6-11). The experiment
18 Re-stimulation of T-cell antigen receptor...was terminated with 500 μl ice-cold PBS, and placing the tubes on ice. The
pervanadate-treated cells were incubated at 37oC for 30 min before and after
the washes.
While it was impossible to detect anti-phosphotyrosine binding, but a band
with approximate molecular weight of 34 kDa in the vanadate-treated samples,
pervanadate induced significant increase in the tyrosine phosphorylation (Figure
3.A, lane 9). We found down-regulation of pervanadate-induced tyrosine phospho-
rylation after washing, and 30 min incubation in culture medium (Figure 3A, lane
11). We attributed the reversibility of pervanadate treatment on the presence of
vanadate that is removed during the washing step [26].
Fig. 3. The effect of low temperature (ice) on cellular tyrosine phosphorylation. Murine, splenic CD8+
T-cells are incubated with or without (control) 100 μM vanadate (SOV) or pervanadate (PV). The cells
were incubated for 30 min either on ice (lane 2 and 3), or at 37oC (lane 1 and 4-11). After a washing
step (10 min), the cells were incubated for additional 30 min either on ice (lanes 2-5), or at 37oC (lane
1 and 6-11). The immunoblots represents the total protein tyrosine phosphorylation using the 4G10
antibody (A), and the phosphorylation of serine S473 at PKB/AKT (B)
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 19Further to this, we probed the samples for PKB/AKT activatory, serine (S473)
phosphorylation (Figure 3B). Vanadate induced increase in PKB/AKT S473 phos-
phorylation (Figure 3B, lanes 2 and 4), in comparison with this in the control cells.
The phosphorylation levels were higher when the cells were treated at 37oC. Incu-
bation of the cells in culture medium for 30 min after washing removes the effect of
vanadate on S473 phosphorylation. The phosphorylation of PKB/AKT is similar to
this of control cells, regardless whether the cells are incubated on ice or at 37oC.
Similarly, the significant increase of PKB/AKT phosphorylation, observed in perva-
nadate-treated cells, is removed following washing and 30 min incubation in culture
medium (Figure 3.B, lanes 9 and 11). SRC-PTKs positively regulate PI3K-PKB/AKT
pathway [1, 27, 28, 31], and this is demonstrated by the increased activatory S473
phosphorylation of PKB/AKT in vanadate pre-treated cells. It is interesting that in-
crease in S473 phosphorylation is observed even in the samples pre-incubated on
ice, and that the washing step removes the increase of S473 phosphorylation even
in pervanadate-treated cells [26].
Acknowledgements: The authors would like to thank Tilo Beyer, Mandy Busse,
Slavyana Gurbiel, Michal Smida, and Clemens Cammann for the help during the
course of the study. This work was supported by grant from the German Ministry of
Education and Research (BMBF FOR-SYS program).
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Address for correspondence:
Kroum K. Hristov
Institute of Molecular and Clinical Immunology
Otto-von-Guericke University
Magdeburg, GERMANY
e-mail: kroum@dir.bg
22 Re-stimulation of T-cell antigen receptor...A RELATIONSHIP BETWEEN MICRORNA204
AND OCCLUDIN IN PROSTATE CANCER INFLAMMATION
SIGNALING
K. Todorova, N. Manolova, D. Zasheva and S. Hayrabedyan
Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences
Summary. For the first time the role of miR-20 in prostate cancerogenesis and
metastasis is implicated in the modulation of innate inflammatory signaling in pros-
tate cancer. The modulatory effect of miR-204 exogenous artificial upregulation
on human prostate cancer cell lines LNCaP (p53+/+, AR+) and PC3 (p53-/-, AR-),
challenged with synthetic analog of common for Gram-negative bacteria peptido-
glycan and minimal inducer of the NOD1 innate immunity receptor, was evaluated
using flow cytometry (FCS) and immunofluorescent detection of Occludin expres-
sion. The peptidoglycan minimal inducer challenge induced Occludin upregulation
in both cell lines - an effect modulated by miR-204 mimic towards preventing the
Occludin upregulation in LNCaP differentially from its null modulating effect in PC3
cells. Our data suggest that miR-204 modulation is at least p53-dependent and
impacts the already altered junctional signaling in prostate cancer cells.
Key words: prostate cancer, Occludin, microRNA, ie-DAP
INTRODUCTION
P
rostate cancer is a leading cancer in men and is the second leading
cause of death after lung cancer. Epithelial to mesenchymal transition
(EMT) is a developmental process characterized by the loss of adher-
ent junctions, desmosomes, tight junctions, as well as loss of epithelial markers,
accompanied by gain of mesenchymal markers and changes in the cellular mor-
phology and phenotype with increased ability for migration and invasion [8, 12, 4].
EMT is an important physiological process during embryogenesis and wound heal-
ing, but may also play a central role in cancer progression [8, 3]. The expression
of Occludin and Claudins, the two major transmembrane proteins that contribute
to formation of tight junctions, has been found to be altered in several types of
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 23cancer. Occludin, encoded by the OCLN gene in humans, is an integral plasma-
membrane protein located at the tight junctions, described for the first time in 1993
by Shoichiro Tsukita [7]. Recently, it became evident that the protein is a target of a
variety of pathogens, including viruses and bacteria, an interaction that sometimes
leads to its extra-junctional localization.
MicroRNAs are small non-coding RNAs that negatively regulate gene expres-
sion. Their role in prostate cancerogenesis is still unclear and only a limited number
of studies have investigated microRNA in prostate cancer [5]. Therefore, a better
understanding of the role of specific microRNAs for the prostate cancer develop-
ment and progression is needed.
The aim of our study was to investigate the potential role of microRNA-204
and its relation to Occludin after inflammatory challenge in prostate cancer cell
lines. Micro RNA miR-204 has a role in prostate cancerogenesis and metastasis.
For the first time it is implicated in the modulation of innate inflammatory signaling
in prostate cancer.
MATERIALS AND METHODS
Cell lines
LNCaP and PC3 prostate cancer cell lines were purchased from ATCC as fol-
lows: androgen receptor (AR) responsive (AR+), p53 enabled (p53+/+) for LNCaP
and bone marrow metastasis derived, p53 null (p53-/-) and AR-unresponsive (AR-)
for PC3, respectively.
Reagents
The minimal bioactive dipeptide present in the peptidoglycan (PGN) of all Gram-
negative and certain Gram-positive bacteria - γ-D-glutamyl-meso-diaminopimelic
acid (ie-DAP), was purchased from Invivogen and applied in concentrations of 4 μg/
mL in cell cultures of LNCaP and PC3. miR-204 mimic (Qiagen) is single stranded
synthetic microRNA resembling in vitro (and in vivo) the effects of mature miR-204
upon transfection in target cells.
Cell cultures induction with innate inflammatory ligands and miR-204 mimic
transfection
LNCaP and PC3 cell lines were maintained in RPMI and DMEM media (Sig-
ma), respectively, supplemented with 10% fetal bovine serum (FBS), and were
experimentally treated for 6h or 24h using 4 μg/ml ie-DAP or pre-transfected for
24h with miR-204 mimic and subsequently antibody treated, and consequently be-
came subject of immunofluorescent staining or flow cytometry analysis using goat
anti-Occludin polyclonal antibody or isotype IgG, and detected using an anti-goat
IgG FITC-conjugated secondary antibody (all antibodies are from SantaCruz Bio-
technologies).
24 A relationship between microrna204...Immunofluorescence (IFL)
Both cell lines were grown on glass coverslips in 12-well cell culture plates
and treated for 24h with ie-DAP. After incubation, cells were washed with PBS,
fixed with 4% paraformaldehyde for 10-15 min and subsequently permeabilized
with 0.2% Triton X-100 in PBS for 15 min at RT. After non-specific protein inter-
actions were blocked using 1% BSA-PBS incubation at RT for 1h, the cells on
coverslips were probed with primary antibody, diluted 1:50 in 1% BSA-PBS and
incubated overnight, at 4oC with anti-Occludin or isotype IgG. After 3 wash steps
with PBS, secondary mouse anti-goat IgG-FITC was used at 1:50 dilution in 1%
BSA-PBS, for 1h, RT (dark), and extensively PBS washed and Vector Shield - PI
mounting media included. Slides were visualized with an Olympus fluorescence
microscope.
Flow cytometry
Control and treated prostate cells were detached with 0.02% EDTA and
washed with cold 1% BSA-PBS. For detection of Occludin, the above mentioned
antibodies (Abs) were used. After 4% PFA fixation/Permeabilization solution wash,
and Staining solution block (eBiosciences), the specific primary Abs or the appro-
priate isotype control Abs were used at concentration of 0.5 μg/106 cells for 30 min
on ice, followed by BSA-PBS wash, secondary antibody incubation at 0.25 μg/106
cells for 30 min on ice (in the dark). Cells were gated using forward vs. side scat-
ter to exclude dead cells and debris. After washing, cells were analyzed with a BD
FACSCalibur flow cytometer (Becton Dickenson). Fluorescence of 104 cells per
sample was acquired in logarithmic mode for visual inspection of the distributions
and for quantifying the expression of the relevant molecules by calculating the me-
dian fluorescence intensity (referred to as MFI) in a histogram overlay graphics.
RESULTS
When miR-204 mimic was transfected for 24h and in some of the experi-
ments the prostate cancer cell lines subsequently challenged with ie-DAP for 6h,
a differential Occludin expression was observed in LNCaP vs. PC3 cell lines. MiR-
204 artificial exogenous over-expression (trough mimic) stratified the two cancer
phenotype model lines towards the ie-DAP affected Occludin expression levels.
The solely challenge with ie-DAP only of LNCaP and PC3 cells induced Occludin
expression in both cell lines, as seen on flow cytometry (Fig. 1a/b, red line), as well
as on immunofluorescence (Fig. 2). The transfection with miR-204 mimic only for
24h had already produced a second depression peak in the LNCaP histogram (Fig.
1a, blue line) suggestive of eventual down-regulation, although not very prominent.
The mimic transfection for 24h followed by 6h challenge with ie-DAP NOD1 minimal
inductor resulted in differential Occludin regulation on protein level, with Occludin
being down-regulated in LNCaP cells (Fig 1a, green line) and conversely – upregu-
lated in PC3 cells (Fig 1b, green line).
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 25Peak norm
O ccludin
Fig. 1. Histogram chart of flow cytometry detection of the Occludin expression in intact (pink shaded
histogram) LNCaP and PC3 cells, and accordingly after 6h ie-DAP treatment (red line), 24h miR-204
mimic transfection (blue line), and after 24h miR-204 transfection followed by a challenge for 6h with
ie-DAP (green line). In both cell lines ie-DAP induced positive change in MFI, while in LNCaP, miR-204
followed by ie-DAP resulted in decreased MFI in comparison to non-treated or miR-204 only treated
cells, while in PC3 cell line miR-204 alone or with ie-DAP challenge had no effect on occludin MFI and
only ie-DAP alone had induction effect on occludin
Occludin
non-treated ie-DAP neg ctrl.
LNCaP
PC3
Fig. 2. Immunofluorescent detection of occludin expression in non-treated and ie-DAP (4 μg/mL, 6h)
treated LNCaP and PC3 cells, using goat anti-human occludin polyclonal antibody and donkey anti-
goat FITC conjugated secondary antibody (SBCT). Negative controls of irrelevant goat IgG treated
specimens, probed and the same secondary antibody are also present. Ie-DAP treatment for 6h results
in an increased occludin green expression localized in the cell cytoplasm
26 A relationship between microrna204...DISCUSSION
The tight junction proteins claudins and occludins are abnormally regulated
in several human cancers. In particular, claudin-3 and -4 are frequently over-ex-
pressed in several neoplasias, including ovarian, breast, pancreatic, and prostate
cancers. Although the exact role of these proteins in tumorigenesis is still being
uncovered, it is clear that they represent promising targets for cancer detection,
diagnosis, and therapy [7, 11].
Previously regarded as tight junction “seal” protein, occludin has recently been im-
plicated to have a role in cell survival regulation. In epithelial cell monolayers the appli-
cation of occludin extracellular loop sequence as an exogenous ligand has invoked the
endogenous induction of occludin and its extrajunctional assembly. It has been reported
that this exogenous occludin treatment activates the extrinsic apoptotic pathway [2].
In our study we observed different expression of Occludin after ie-DAP chal-
lenge of two different prostate cancer cell lines - LNCaP (p53+/+, PTEN-/-, AR+)
and PC3 (p53-/-, PTEN-/-, AR-) which are differentially androgen responsive, and
both tumor suppressor gene PTEN (Phosphatase and Tensin Homolog) negative.
In non-malignant epithelial cells, the scaffold proteins loss is optimized by proteins
binding at the Occludin junctions, and preventing PTEN degradation. Normal PTEN
levels significantly decrease the cell proliferation activity of the Akt and prevent
junction disassembly, altering the levels of Occludins [9].
In human prostate cancer, PTEN deletion accounts for 63% of prostate meta-
static tumors, and its exogenous upregulation significantly suppresses metastasis
without affecting primary tumorigenesis [1]. Recent reports proposed a progressive
collaboration between early MYC amplifications, loss of PTEN, and finally, loss of
p53, leading to the development of invasive prostate cancer [10]. PTEN was also
found to physically interact with p53 and exert regulation of its DNA binding activity
[6]. In our study, both cell lines lack PTEN explaining the lack of occludin membrane
border expression, while in the p53+ context miR-204 was able to down-regulate
occludin expression in concomitant induction of NOD1 signaling pathway. The ob-
served differential occludin modulation by miR-204 is affected by two concomitant
phenomena: 1) when the NOD1 receptor is activated using the synthetic minimal
ligand this results in a NFkB pathway downstream activation, which is able to mod-
ulate the tight junction signaling and indirectly affects the p53 pathway as well; 2)
miR-204 down-regulates a transcription factor that modulates the p53 physical
complex with the c-MYC oncogene thus influencing the p53 downstream effects on
apoptosis and other signaling pathways, including tight junctions.
This study stresses the importance of regarding to the junctional molecules
affected by metastasizing not as end effectors, but as a part of a complex feed-back
mechanism, that recently become even more complicated with the understanding
of micro RNAs playing an additional modulating multilayered regulatory role.
Acknowledgements: This study was conducted under the DMU 03/27 project
Acta Medica Bulgarica, Vol. XXXIX, 2012, № 2 27You can also read
