Saddleback Journal of Biology - Saddleback College

Fall 2009 Biology 3B Paper
hepatolenticular degeneration (Scheinberg and Gitlin,
1952). There is a disruption of normal copper
metabolism and copper deposition in tissues (Gitlin,
1975).
To study the copper components in milk we
used a mammary epithelial cell model, PMC-42 cells,
grown on a Transwell® membrane as a monolayer with
tight junctions. PMC-42 cells are very similar to
normal breast epithelial tissue in cell composition and
physiological responses (Krishnan and Cleary 1990).
They are polarized and mimic the basal and apical
sides of mammary epithelial cells. The basal (blood)
side is where copper is delivered on different plasma
proteins, such as albumin and transcuprein (Git et al.,
2008). Medium is collected on the apical, or milk
secretion side and we analyze the secretions. Mammary
explants obtained from virgin female mice were also
used as a model system to test for the effect of
lactational hormone on CTR1 and ceruloplasmin.
Based on findings from Whitehead et al. (1984),
PMC42 cells have been shown to respond to hormonal
stimuli as would normal breast epithelial tissue upon
treatment with prolactin or stimulation of growth.
Culture media were also analyzed after 48 and 96 hours
post-incubation. We expect lactational hormones to
increase the expression of CTR1 and ceruloplasmin at
the mRNA level as well as at the protein level.
Methods
PMC 42 cell culture / Copper transport study
PMC 42 cells were trypsinized and 100 ul of siCTR
and non-targeting siRNA (as negative control) were
added onto Transwells treated with Matrigel for cell
polarization. The cells were incubated with RPMI
containing 10% Fetal Bovine Serum at 37 C.
Radioactive 64 Cu (5uM)–His (50uM) was delivered
on the basal side 48-90h post transfection with
siRNAs,. Apical and cellular secretions were counted
with gamma counter.
Quantifying expression of CTR1 mRNA
CTR1 mRNA was isolated using RNABee solution and
performing phenol chloroform extraction. The
supernatant aqueous layer was collected, and
isopropanol was added to it and allowed to sit
overnight. cDNA was synthesized by mixing isolated
RNA with reverse transcriptase, deoxynucleoside
triphosphates, and random primer. The expression of
CTR1 was quantified by performing Real Time PCR
(50 cycles in triplicates with a fluorescent reporter
probe), relating the expression to 18S ribosomal RNA
as an internal control.
Determining protein expression of CTR1knockdown
siCTR (100nM) cells on the apical membrane were cut
out of the Transwells. Five hundred ul of lysis buffer
(10mM tris, 2% SDS, pH 7.2) was added, and the
samples were incubated with shaking for 5 minutes.
Five ul 4x sample treatment buffer was mixed with 20
ul of sample and then loaded into 12.5% acrylamide
gel. Gel electrophoresis was performed with 25 ul
loaded in each well, and the gel was transferred onto a
membrane with transfer buffer (192 mM glycine, 25
mM Tris base, 150ml MeOH, 0.375g SDS, ddH2O).
The membrane was blocked overnight with 5% dry
milk (1g). Two ul of CTR1 primary antibody (rabbit
anti-CTR1 from Jack Kaplan) was added afterwards
and left to shake over night. It was then washed with
TTBS 3 times (100ml 1x TBS + 100µL Tween 20) and
2 ul of CTR1 secondary antibody (Sigma monoclonol
antirabbit antibody produced in mouse) was added and
allowed to shake overnight. The membrane was
washed twice with 1x TTBS. The membrane was then
placed in developing solution [50 ml development
buffer (pH 9.4), 500 ul BCIP + 500 ul NBT] in the dark
and allowed to develop. After bands appeared, the
membrane was placed in ddH2O. We expected
Mammary Explants
Mammary tissue from virgin female mice (6-8 weeks
old) was harvested and sliced. They were then grown
on wells plated with collagen and immersed in DMEM
medium with and without lactational hormones
(insulin, prolactin, dexamethazone). The wells were
then incubated at 37 C for 96 hours. Secretions were
collected from the apical side at 48 hours and 96 hours
after incubation. Tissues that had been grown were
weighed, and a 40% solution was made for each
sample with SDS tank buffer (18g Tris base, 86.4g
glycine, 60 ml 10% SDS, 30 ml 2% NaN3, H2O). The
samples were then homogenized to prepare for gel
electrophoresis. The concentrations of secretions were
measured based on the Bradford protein assay. Two
hundred ul of Bradford reagent was combined with
water and BSA in multiples of 10 ul beginning at 0 ul
and ending at 70 ul. The absorbance values at 595 nm
were obtained with a spectrophotometer, and a
standard curve was generated based on the known
concentrations of BSA. Fifty ul of each sample was
combined with 750 ul of water and 200 ul of Bradford
reagent, where the absorbance at 595 nm was taken.
Based on the absorbance values plotted against the
standard curve, the concentrations were determined.
Determining protein expression of CTR1 and CP from
mammary explants
Five ul 4x sample treatment buffer was mixed with 20
ul of sample (either from mammary tissue or
secretions) and then loaded into 12.5% acrylamide gel
(7% for ceruloplasmin). Gel electrophoresis was
performed with 25 ul loaded in each well, and the gel
was transferred onto a membrane with transfer buffer
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Saddleback Journal of Biology
Spring 2010