Malaysian Journal of Biochemistry and Molecular Biology (2007) 15(2), 80-83
80
Isolation of Perchloric-acid Soluble Protein from Normal
Human Monocytes
Mat Ludin Che Mat1, Hasenan Nordin2 and Wan Maziah Wan Mohamed3
1
Academic Laboratory Unit, 2Department of Chemical Pathology and 3Department of Pediatrics,
School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan
Abstract
Perchloric-acid-soluble protein (PSP), with molecular mass of approximately 14.5 kDa, was isolated from peripheral blood
monocytes of healthy donors by a combination of perchloric acid solubilization, trichloroacetic acid and ammonium sulfate
extraction, and CM-Sephadex chromatography techniques. However, this protein was not found in all cultured blast cells of acute
myeloblastic leukemia patients that were tested. The isolated protein showed strong cross-reactivity when tested with PSP
antibody, suggesting close similarity to the p14.5 PSP found in human mononuclear phagocytes isolated by earlier workers. PSP
did not exert any significant effect on the colony forming and proliferative activities of cultured blast cells from patients with acute
myeloblastic leukemia.
Keywords: Acute leukemia; perchloric-acid-soluble protein; blast cells; monocytes
Introduction
Perchloric-acid soluble protein (PSP) was first isolated
and characterized by Oka et al. [1] from the rat liver
(L-PSP1), followed by Ceciliani et al. [2] from the goat
liver, Samuel et al. [3] from the mouse liver, Asagi et al.
[4] from the rat kidney (K-PSP1) and Nordin et al. [5]
from the avian tissues (C-PSP). This protein exhibited
characteristics very similar to a group of proteins
belonging to the so-called YABJ and Y5GF isolated from
Bacillus subtilis and E. coli [2]. PSP is a homodimer
consisting of two identical subunits with a molecular
mass of 14 kDa. The cDNA of L-PSP1 contained 411 bp,
encoding a 137 amino-acid protein with a molecular
mass of 14,149 Da.
In 1996, Schmiedeknecht et al. [6] discovered the
presence of the translation inhibition protein (14.5 kDa)
designated as p14.5 from human mononuclear phagocytes.
The protein was found to show a remarkable similarity
with the PSP protein described by Oka et al. [1]. This
protein was also present in liver, kidney and vessel wall
sections when tested by immunohistochemical technique.
The expression of the mRNA of the translational inhibitor
p14.5 (human homologue of L-PSP1) was significantly
up-regulated with the induction of differentiation to
macrophages [6].
Another PSP-like protein which belongs to the same
family known as UK101 and UK 114 has been shown to
be involved in immune control of tumor growth [7]. The
therapeutic effect of UK101 and UK114 in experimental
mammalian tumors has been studied [8, 9], whereas their
role in the prevention of carcinogenesis has not been
investigated. Later work by Ghezzo et al. [10] showed
that the UK101 inhibits carcinogenesis of DMBA-induced
Syrian hamster cheek-pouch squamous cell carcinoma.
Blood cancer or leukemia is a purposeless, malignant,
neoplastic proliferation of abnormal leucocytes in
hematopoietic tissues. Based on the degree of
differentiation of the leukemic cell line, leukemias are
divided into acute and chronic forms. Acute myeloblastic
leukemia (AML) is characterized by progressive
accumulation of relatively immature, poorly functioning
myeloid blasts in the bone marrow (BM) and peripheral
blood (PB). AML is diagnosed morphologically, using
the criteria proposed by the French-American-British
(FAB) Co-operative group [11].
In the present study, we have isolated PSP from the
peripheral blood monocytes of normal healthy individuals.
The presence of PSP in the blast cells of AML patients
and the effect of PSP on colony forming and proliferative
activities of the patients’ blast cells were also investigated.
Materials and Methods
Blood samples
Ten healthy donors between the age of 20 to 45 years
old and five patients of confirmed cases of AML (Table
1) were used in this study. Fresh blood samples from
healthy donors were collected into containers with 1mM
EDTA and stored at 4˚C before used.
Extraction of human monocytic PSP
Human monocytes from healthy donors and blast cells
from AML patients were isolated by the OptiPrep density-
Author for correspondence: Associate Professor Dr. Hasenan
Nordin, Department of Chemical Pathology, School of Medical
Sciences, Universiti Sains Malaysia, Health Campus, 16150
Kubang Kerian. Tel: 609-7664751, E-mail: [email protected]
Isolation of PSP from normal human monocytes
81
Table 1: Characteristics of patients from whom the AML cell lines originated
Cell lines
AML-1
AML-2
AML-3
AML-4
AML-5
a
Age/Sex
a
12/M
9/F
10/M
12/M
8/F
Blast cell sources b
Phase of disease
PB
PB
PB
PB
PB
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
F, female; M, male; b PB, peripheral blood.
gradient medium technique as described by GrazianiBowering et al. [12]. The cells were then homogenized
in 50 mM Tris/HCL pH 7.4, 250 mM sucrose, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 1 µM leupeptin, 1 µM pepstatin A at 4°C
using a loose-fitting moto-driven glass/Teflon
homogenizer. Post-mitochondrial supernatant (PMS),
obtained after centrifugation at 10,000 g for 30 min, was
treated with 60% perchloric acid and recentrifuged at
10000 g for 15 min. The supernatant was made up to
25% with trichloroacetic acid. Precipitate was collected
by centrifugation at a similar speed, washed with cold
acetone and dried under vacuum. The dried material was
initially dialyzed against distilled water for 4 h, and then
extensively against 0.1 M sodium phosphate buffer (pH
7.5) overnight.
CM-Sephadex C-25 column chromatography and SDSPAGE
Proteins in the dialysate were fractionated with
saturated ammonium sulfate. The precipitate formed
between 0 and 40% saturation was collected by
centrifugation at 10 000 g for 10 min. The precipitate
was then dissolved in 0.1 M sodium phosphate buffer,
pH 7.5, and dialyzed against the same buffer. The dialyzed
product was applied through a 2 x 30 cm column of CMSephadex C-25. Absorbance readings for protein were
taken at 280 nm using the Ultraspec 3000
spectrophotometer (Pharmacia Biotech) and the flowthrough fractions were collected and electrophoresed by
SDS-PAGE according to the method described by
Laemmli [13]. Protein fractions that cross-reacted with
PSP antibody were pooled and again subjected to
electrophoresis. The corresponding protein fractions
obtained were used as the PSP-like protein in subsequent
studies.
Western blotting
The PSP-containing protein fractions in homogenate
from normal human monocytes and from blast cells were
analyzed by SDS-PAGE using 15% polyacrylamide gels
as described by Laemmli [13]. After electrophoresis, the
proteins on the gels were blotted onto nitrocellulose
membranes and immunoblotted with antisera against rat
PSP.
Immunohistochemistry
The cells were prepared for immunohistochemistry by
fixing in 10% neutral formalin for 2-3 days at room
temperature, after which the cells were pelleted by
centrifugation [14]. The cell pellet was suspended in
melted 2% agarose, and embedded in paraffin. Four µmthick sections were placed on slides and fixed with 70%
(v/v) alcohol for 30 min. The slides were then blocked
with PBS containing 20% normal goat serum and 1%
BSA for 1 h and incubated for 1 h with polyclonal
antibodies against PSP in PBS containing 0.1% BSA in a
moist chamber. The slides were washed extensively with
PBS and then incubated for 1 h with 8 µg/ml horseradish
peroxidase-conjugated goat anti-rabbit IgG. After washing
with PBS, the slides were stained by the addition of
0.05% diaminobentizine and 0.1% H2O2. After washing
with PBS, the smears were counterstained with methyl
green for 20 min, dehydrated and mounted. Negative
controls were prepared by using non-immunized rabbit
IgG as the primary antibody.
Establishment and characterization of AML cell lines
Mononuclear cells (including blast cells) were
separated by Opty-Prep density-gradient medium
technique as described by Graziani-Bowering et al. [12].
The cells were then cryopreserved at -70˚C in the
presence of 50% heat-inactivated FCS (Gibco, Grand
Island, NY, USA), 10% dimethyl sulfoxide (AldrichChemie, Steinhein, Germany) and α-minimal essential
medium (α-MEM, Gibco). Cells were quickly thawed,
washed twice with α-MEM and cultured at a high cell
density of 1-2 x 106/ ml in α-MEM and 10% FCS in the
presence of the following growth factors: 100 U/ml of
interleukin-3 and IL-6, 100 U/ml of granulocytesmacrophage colony-stimulating factor and 40 ng/ml of
mast cell growth factor. After culturing for 6 to 12
weeks, the cells were allowed to proliferate in 10%
FCS and α-MEM for the next three months. Thereafter
the cells were frozen at -70°C. For the experiments the
cells were thawed and continuously grown in the
presence of 10% FCS and α-MEM in a humid
atmosphere at 37°C with 5% CO2 at a cell density 3-5 x
105/ml. Fresh medium was generally changed every 34 days, and every other day for experiments.
Isolation of PSP from normal human monocytes
Colony forming assay
A modified semi-solid methylcellulose (mc) method
described by Buick and co-workers [15] was used to
investigate the effect of PSP on the formation of
colonogenic blast cell colonies. The cells were plated in
96-microwell plates in 0.1 ml of basic growth medium
[10% heat-inactivated FCS in α-MEM] and 0.9% mc
(Aldrich-Chemie, Steinheim, Germany) with or without
PSP and incubated in a humidified atmosphere at 37°C
with 5% CO2 at a cell density of 3 x 103 cell/well. All
cultures were performed in triplicate. Colony formation
was assessed and classified visually using an inverted
microscope. Colonies from three wells/sample containing
more than 20 cells were counted and the mean colony
number was calculated.
Suspension culture assay
In the suspension culture, 1 x 106 cells were incubated
in the presence or absence of PSP in 1 ml of basic growth
medium in 24 multi-well plates (Becton Dickinson &
Co., New Jersey, USA) as described previously by Nara
and McCulloch [16]. The cell number and viability were
determined by vital dye exclusion (0.4% trypan blue,
Sigma Chemical Co.Ltd., Irvin, UK) using a standard
hemocytometer.
Results
Isolation and purification of PSP from normal human
monocytes and blast cells of AML.
In the present study, a PSP-like protein was isolated
from the monocytes of normal healthy donors by
perchloric acid extraction and trichloroacetic acid
precipitation (Figure 1). However, this protein was not
detected in the blast cell extracts of AML patients. The
molecular mass of the PSP-like protein was found to be
14.5 kDa as assessed by SDS-PAGE. Results of the
1
2
82
Figure 2: Immunoblot analysis of PSP-like protein from
normal human monocytes. Protein samples were
electrophoresed in 15% SDS-PAGE and
immunoblotting was carried out as described in
the Methods and Materials section.
immunoblotting experiment showed a strong crossreaction with rat PSP antibody suggesting that the purified
protein is similar to the PSP purified from rat liver
(Figure 2).
Immunohistochemistry
To further support the data related to the detection of
PSP in monocytes and to prove that PSP was not present
in blast cells of AML, immunohistochemistry was
performed. In these preparations we showed that some
immunopositive deposits were seen in smears containing
monocytes obtained from the healthy donors but
immunoreaction was not observed in smears containing
blast cells from AML patients’ samples. No reactive
product was detected in any control smears prepared
using non-immunized rabbit IgG as the primary antibody
(Figure 3).
3
94000
67000
43000
30000
20000
14000
Figure 1: SDS-PAGE of the purified PSP protein. 1: Postmitochondrial supernatant of blast cells of AML
patients, 2. Post-mitochondrial supernatant of
normal human monocytes, 3: Molecular weight
markers.
Figure 3: Immunohistochemical staining of monocytes and
blast cells of AML for PSP-like protein. Left
panels (a and c) show negative controls, and right
panels (b and d) demonstrate cells immunostained
with anti C-PSP rabbit IgG. Immunoreactive
products are detected in the monocytes section
(b) but not in blast cells of AML patients (d).
Isolation of PSP from normal human monocytes
Effect of PSP on AML cell growth
When exposed to PSP, the in vitro AML patients’ blast
cell cultures failed to demonstrate significant difference
in the colony-forming and proliferative activities of the
cells.
Discussion
In this study, we described the isolation of PSP-like
protein found in the healthy donor monocytes for further
investigation. However, this protein was not found in the
blast cells of AML patients’ samples.
The protein, obtained by solubilization with perchloric
acid and precipitation with trichloroacetic acid and
ammonium sulfate of the post-mitochondrial supernatant
of cell extracts, was almost pure at this step. The PSPlike protein appeared in the flow through fractions of
CM-Sephadex chromatography and was shown to be
homogenous by SDS-PAGE. A single protein band with
molecular mass of 14.5 kDa was detected using the
method. Immunoblotting experiments showed a strong
cross-reaction with rat PSP antibody suggesting that the
purified protein is similar to the PSP purified from rat
liver.
83
Repeated extractions from ten healthy donor samples
were only able to generate minute amount of monocytic
PSP. This restricts further characterization of the protein.
Experiments performed on in vitro AML patients’ blast
cell cultures that were exposed to PSP failed to
demonstrate significant difference in the colony-forming
and proliferative activities of the cells although the rat
liver L-PSP1 has been previously shown to inhibit cell
growth in a cell-free lysate system [1]. Previous studies
have also shown that the p14.5 PSP from human
mononuclear cells plays an important role in protein
inhibition and regulation of cell proliferation [6].
Acknowledgements
We are very grateful to Associate Professor Mustapha
Musa, Head of Immunology Department for use of
laboratory and equipment. We are indebted to Ms Malisa
Yoong Abdullah, Ms Kamariah Abdul Rahim and Mr
Abdul Razak, from the Immunology laboratory, and Ms
Asiah from the cell culture section, PPSG, for their
technical assistance during the entire course of this study.
The research was financially supported by a short-term
grant (304/PPSP/6131243) from Universiti Sains
Malaysia.
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