Chromosome, Microdissection and Microcloning
49
assigns color intensity to ratios of gene
expression: for example, shades of red
represent genes that are upregulated in
prostate cancer (1–4 fold increase); shades
of green are downregulated (1–4 fold de-
crease); black, genes that are unchanged
between tumor and benign tissue; gray,
missing elements (not present). Selected
genes were then validated at the transcript
level using Northern blot or RT-PCR or at
the protein level with immunoblots.
Similarly, the feasibility of combining
LCM and cDNA microarray hybridiza-
tion has been demonstrated in several
reports showing reproducible differences
in gene expression between normal and
pathologic tissue samples. Furthermore,
a combination of LCM, cDNA arrays,
and real-time quantitative PCR was used
to
show
altered
gene expression pat-
terns at various stages of breast cancer
progression. Several expression libraries
from puri±ed cell populations derived
from microdissected normal, preneoplas-
tic, and neoplastic breast, prostate, ovary,
lung, and liver tissues with large num-
bers of partially sequenced clones are
already available at the CGAP website.
Knowledge of the gene sequence or the
quantity of gene expression using DNA
chips is not suf±cient to predict the bio-
logical nature and function of a protein.
The identi±cation and quantitation of ex-
pressed proteins is more challenging. Pro-
teomics is the global analysis of expressed
proteins (including posttranslational mod-
i±cations)
and
seeks
to
establish
the
relationship between genome sequence,
expressed proteins, protein–protein inter-
actions, and cell and tissue phenotype.
The bene±t of global protein analysis is
immense because identi±cation of distinc-
tive protein signatures associated with cell
function may provide novel therapeutic
targets, molecular markers of disease, and
increased understanding of determinants
of cell phenotype.
Proteomics-based pro±ling uniquely al-
lows delineation of global changes in
protein
expression
patterns
resulting
from transcriptional and posttranscrip-
tional control, posttranslational modi±-
cations, and shifts in proteins between
different cellular compartments. Some of
the current technologies for proteome pro-
±ling and the application of proteomics
to the analysis of tumor tissues are be-
ing used concurrently with microdissec-
tion. Given that comprehensive expression
pro±les obtained using genomics and
proteomics are highly complementary, a
combined approach to pro±ling may well
uncover expression patterns that could
not be predicted using a single approach.
Hanash used LCM and high-throughput
microarrays/proteomics to study in paral-
lel with Alzheimer’s disease and showed
that consistent with cDNA microarray
evidence, the expression of proteins in-
volved in synaptic activities was also al-
tered in the brains of early Alzheimer’s
disease cases. The study demonstrated
t
h
a
tp
r
o
t
e
i
nc
h
i
pa
r
r
a
y
sc
a
nb
eu
s
e
d
to examine sequential changes in dis-
tinctive gene expression patterns in the
brain as a function of the progression of
Alzheimer’s disease.
Currently, the most widely used pro-
teomic tool for analysis of disease is two-
dimensional protein gel electrophoresis
(2-DE), which can display protein expres-
sion patterns to a high degree of resolution.
2D-PAGE is being complemented by new,
exciting approaches to multiparametric
protein characterization. As an alterna-
tive, an equally powerful technology is
being employed to generate protein expres-
sion patterns from whole tissue extracts.
The technology uses surface-enhanced
previous page 1369 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1371 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off