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Aberrant expression of cell adhesion molecules correlates
with the malignancy of many tumors. Human MUC18 (huMUC18), a cell
adhesion molecule in the immunoglobulin gene super family, is expressed
on the surface of human malignant melanoma cells. MUC18 is expressed
in some healthy normal tissues. There is an association with the
increased expression of MUC18 and the malignant progression of human
prostate cancer and melanoma. Further evidence strongly suggests
that MUC18 expression is one of the key determinants, which cause
human prostate cancer and melanoma cells to metastasize. Identification
of the membrane-associated protein components differentially expressed
in the MUC18-expressing and non-MU18-epressing cancer cell lines
will greatly enhance our understanding of how MUC18 mediates cancer
metastasis. Before attempting two-dimensional electrophoresis, one-dimensional
SDS-gel electrophoresis was utilized to elucidate the best solubilization
method for MUC18 and other possible membrane proteins. Since conventional
solubilization protocols that employ the use of SDS could not be
used for the preparation of protein for isoelectric focusing (IEF),
different zwitterionic detergents were tested. Results from the
one-dimensional gels and western blots indicate that the detergent
ASB-14 was the most effective in producing the highest yield of
solubilized membrane proteins for IEF. Another parameter that affects
the resolution of membrane proteins in two-dimensional gels is the
lipid component of the membrane proteins. To alleviate the problem,
delipidation was attempted by extracting the solubilized proteins
with a solution consisting of tri-n-butyl phosphate, methanol, and
acetone. After resolving these potential problems, we began to use
the two-dimensional electrophoresis for the experiment. Results
of resolving membrane proteins from different cell lines by the
two-dimensional electrophoresis will be presented.
Over expression of MUC18, an integral membrane- protein,
has been shown to cause metastasis of certain cancers such as human
prostate cancer [7] and melanoma cells [8]. The way that MUC18 mediates
metastasis is unknown. Identification of the protein components
differentially expressed in these cancer cells will greatly enhance
our understanding of how specific proteins interact with each other
and possibly how these proteins accomplish metastasis. 2-D gels
[2] will give better resolution of important protein participants
in prostate cancer and melanoma cells that interact and cooperate
or modulate key players such as MUC18.
Tissue Culture
Mouse melanoma cell lines (K4, KM2, K3, and K9) were
maintained in EMEM-NaPyruvate plus 10% FBS and PSA medium [8]. The
cells were grown in 37 C in the presence of 7% CO2. Occasionally,
cells were trypsinized in order to disperse them or to transfer
them to new flasks to get more cells. Before the cells were trypsinized,
they were washed twice with PBS. After adding 0.25% trypsin, the
cells were put in a 37 C incubator for five minutes. Medium containing
FBS was added to stop trpsinization. Cells were dispersed and transferred
to new flasks or petri dishes.
Biotinylation of Membrane Proteins
Cells are washed twice with PBS before treatment with
EDTA and incubation at 37 C. The cells are dispersed via a pipet
and then transferred to a tube. The cells are centrifuged and subsequently
washed with a PBS solution containing calcium and magnesium. The
pellet is suspended in the same solution. An equal volume of 2mg/ml
of Sulfo-NHS-LC-LC-biotin is added to the pellet. Biotinylation
is allowed to occur at 4 C for an hour [1]. 5-10% FBS is added to
stop the biotinylation process. The pellet is washed two more times
with the PBS solution containing calcium and magnesium before suspending
the pellet in same solution and splitting them into portions that
will be later used in different solubilization methods.
Solubilization of Membrane Proteins
Membrane proteins were solubilized by a standard Western
Blot lysising buffer and by buffers consisting of 7M urea, 2M thiourea,
20mM DTT, 2% carrier ampholytes (pH 3, 5-10), and different zwitterionic
detergents [5]. The different detergents used were ASB-14, ASB-16,
Chaps, and Empigen BB. After biotinylation, the solubilization buffers
were added to the cells along with an anti-proteolytic cocktail.
SDS-Page (1-D)
In one-dimension gels, proteins are separated according
to their size in the presence of SDS. A glass plate sandwich is
assembled using 2 clean glass plates and two 0.75mm spacers. A separating
gel is prepared and pipetted to into the sandwich until the height
of the solution in the sandwich is approximately 11 cm. Isobutanol
is pipetted onto the top of the gel. The gel is allowed to polymerize
for 30 minutes at room temperature. The alcohol is then poured off.
A stacking gel solution is prepared and subsequently pipetted onto
the separating gel. A Teflon comb is inserted into the stacking
gel layer. The stacking gel solution is allowed to polymerize for
30 to 45 minutes at room temperature. The Teflon comb is removed.
Protein samples from the cell lysates are prepared. 1x SDS/electrophoresis
buffer is poured into the lower buffer chamber. The upper buffer
chamber is partially filled with 300 ml of 1x SDS electrophoresis
buffer so that the sample wells of the stacking gel are filled with
buffer. The prepared protein samples are loaded into the wells.
500 ml of 1x SDS/electrophoresis buffer is added to the upper buffer
chamber. The gel is allowed to run until the tracking dye has reached
the bottom of the separating gel. The gel is carefully removed and
stained for analysis.
Delipidation
Sometimes lipid components of membrane proteins interfere
with the resolution of a 2-D Gel [6]. To alleviate this problem,
organic solvents can be used to remove the interfering lipid portions
of the proteins. A delipidizing solution consisting of tri-n-butylphosphate,
acetone, and methanol was prepared according to a 1:12:1 ratio [4].
The solution was kept ice cold until it was added to the prepared
extracts. Incubation occurred at 4 C for 1 hr. The extract was centrifuged.
The supernatant was discarded. The pellets were washed with the
delipidation solution and resolubilized with the appropriate solubilization
buffers.
Western Blot
Western blot is a powerful assay that allows for the
identification of specific proteins by using the principle of antigen-antibody
recognition. The gels from the previous steps will be used. The
gel is placed in an electroblot apparatus with nitrocellulose filter
on anode side. 150V constant voltage is run through the apparatus
for 1 hour to overnight at 4 C. To visualize the transferred proteins,
Ponceau S solution is used. The membrane is then destained in water
for 2-3 minutes. The molecular weight markers on the membrane are
marked. The filter is blocked with blocking buffer for at least
2 hours in the room temperature. The primary antibody is diluted
in the blocking buffer and incubated with the filter for 2 hour
at 4 C (cold room). The filter is washed four times with 200ml TBST
solution. AP-conjugated anti-Ig secondary antibody is diluted in
the blocking buffer and incubated with the filter for 1 hour at
room temperature. The filter is washed four times with 200ml TBST.
NBT/BCIP substrate solution is used to stain the protein band on
the filter. The filter is washed with water and analyzed.
2-D Electrophoresis
Two-dimension gels give a much better resolution of
different proteins in a mixture by first separating proteins according
to their charges in an isoelectric focusing gel, and then separating
them according to their size in a second dimensional gel with SDS.
The first-dimension gel tube is assembled. A gel solution is prepared
and transferred to a glass tube via syringe. The gel solution is
allowed to polymerize for 1 hour. Any excess of acrylamide is removed.
Buffer for the upper (20mM NaOH) and lower (0.085% H3PO4) tank is
prepared. The tube gels are prefocused for 1 hr. at 200-400V. The
prepared protein samples are loaded onto the gel. The gel is allowed
to run for 16 hr. at 800V. The basic end of each gel is marked with
concentrated Bromphenol Blue (5mg/ml). The gels are extruded and
are put into equilibration buffer for a few minutes. The second-dimension
gradient gel is prepared. A glass plate sandwich is assembled using
2 clean glass plates and three 2.5mm spacers. The gel solutions
(7% acrylamide and 20% acrylamide) are prepared. The gel solutions
are poured into a gradient maker and then into the glass sandwich
to make a 7-20% gradient gel. A thin layer of isobutyl alcohol is
applied on top of the gels. The gels are allowed to polymerize for
20-30 minutes. A thin layer of agarose dissolved in equilibration
buffer is added over the slab gel.The first-dimension gel is put
on top of the second-dimension gel. A thin layer of agarose solution
is added over the first-dimension gel to make a continous contact
with the second-dimension gel. A well was made to accommodate the
MW marker. The gel is allowed to run for 1-1.5 hours at a constant
voltage of 250-300V . The gel is removed when the tracking dye reaches
the end of the gel. The gel is stained in silver stain solution
for further analysis.
The primary use of 1-D SDS gel electrophoresis for
this experiment was to try to find the best solubilization procedure
for membrane proteins, especially for MUC18, that will later be
used for running 2-D gels. Western blot membranes stained for total
protein and those stained for MUC18 indicated that the zwitterionic
detergent, ASB-14, was the most effective detergent. Proteins solubilized
in ASB-14 showed up much clearer on the membranes. Streaking on
2-D gels is a major problem that has to be considered if the primary
aim is resolution. Research that involves the use of 2-D gels has
used delipidation to eliminate some horizontal streaking. Delipidation
of the protein extracts was attempted in this study; however, due
to low protein concentrations, results obtained from delipidated
samples are inconclusive. Further experimentation employing delipidation
will have to be considered. 2-D gels of the different melanoma cell
lines differ in their staining pattern. More gels have to be run
before anything conclusive can be derived. Future experimentation
may rely on mass spectrometry to identify certain proteins.
The primary use of 1-D SDS gel electrophoresis for this experiment
was to try to find the best solubilization procedure for membrane
proteins, especially for MUC18, that will later be used for running
2-D gels. Western blot membranes stained for total protein and those
stained for MUC18 indicated that the zwitterionic detergent, ASB-14,
was the most effective detergent. Proteins solubilized in ASB-14
showed up much clearer on the membranes. Streaking on 2-D gels is
a major problem that has to be considered if the primary aim is
resolution. Research that involve the use of 2-D gels have used
delipidation to eliminate some horizontal streaking. Delipidation
of the protein extracts were attempted in this study; however, due
to low protein concentrations, results obtained from delipidated
samples are inconclusive. Further experimentation employing delipidation
will have to be considered. 2-D gels of the different melanoma cell
lines differ in their staining pattern. More gels have to be run
before anything conclusive can be derived. Future experimentation
may rely on mass spectrometry to identify certain proteins.
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No. 52003071 (Humphrey Lam) and by
the Department of Defense under Grant No. PC992041 (Dr. Guang-jer
Wu).
MUC18 is a membrane protein that is expressed in some
normal tissues in the body. Over expression of MUC18 correlates
with the malignant progression of a tumor. The specific aims of
this study was to analyze differently expressed proteins between
cancer cells that express MUC18 and those that do not express MUC18.
Having an understanding of the different proteins involved would
eventually allow for the elucidation of the pathway which MUC18
mediates metastasis.
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