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MOLECULAR BIOLOGY &
PATHOLOGY IN EPIDEMIOLOGY
JianYu Rao, M.D.
Associate professor of pathology and epidemiology
UCLA
Molecular Biology - Outline
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Introduction
Basic Principles of Molecular Biology
Core Techniques of Molecular Biology
High Throughput Technologies
Epigenetics – DNA Methylation
INTRODUCTION
• 1953 - Discovery of DNA double helix
(Crick & Watson)
• 1960s - DNA transcription mechanism
• 1970s - Recombinant DNA technology
• 1980s - PCR
• 1990s - Human genome project/DNA chips
• 2000 – Genome Wide Association (GWA)
Studies
Basic Principles of Molecular Biology
•
DNA structure
– 4 bases (nucleotide): 2
pyrimidines thymine (T) and
cytosine (C), and 2 purines
adenine (A) and guanine (G)
– Form double helix by baseparing through H-bond (A to T
and G to C) and a backbone
consists of sugars and
phosphate.
– The strands have polarity (3’ to
5’ or vice versa) and are
complementary to each other.
– Genetic information is organized
lineally:
• A codon is the basic unit with 3
consecutive nucleotides that
specifies a single aa.
5’
3’
• A gene is a segment of DNA (with
lineally linked multiple codeons)
that specifies a protein.
5' –CCT GGT CCT CTG ACT GCT - 3'
• A chromosome contains several
thousands genes and is the
smallest replicating unit (human
KHL…
has 46 chromosomes).
• The genome is the entire set of
information that an organism
contains.
Basic Principles of Molecular Biology
(cont.)
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Gene structure
– Gene is compose of a upstream 5’
regulatory region (TATA box or
CAAT box), several exons
(expressed gene sequences), and
intervening intrones
(nonexpressed sequence).
– There are a total of 100,000 genes
estimated in mammalian genome.
– Less than 30% of the genome is
ever transcribed into RNA, and
only a fraction of that is translated
into protein.
– More than 70% of entire genome is not
transcribed and is composed of many stretches
of repetitious sequences that can repeat on
scales of 5-10 bp, to 5000-6000 bp. Species
specific type of repeats, termed Alu sequences,
are useful as markers for identifying genes
transferred between species.
– A gene family are a number of closely linked
genes that code for structurally and functionally
related proteins.
Basic Principles of Molecular Biology
(Cont.)
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Gene transcription (DNA to
mRNA)
– mRNA (message RNA) is the
template for protein synthesis.
– Only the exon sequences of a
given gene is transcribed.
– Transcription begins by
binding of RNA polymerase II
on initiation site. This process
requires a transcription factor
which is a protein recognizing
the region of DNA to be
transcribed.
– A “primary transcript” which
ranges from the initiation site to
a termination site (including all
the exons and introns) is
produced initially, followed by
adding a cap (methylated G) at 5’
end and a Poly A tail at 3’end,
and finally by several steps of
splicing (cut off the introns).
– The produced mature mRNA is
then exported from nuclear to
cytoplasm by unknown
mechanisms for translation.
Basic Principles of Molecular Biology
(Cont.)
• Translation (mRNA to protein)
– The translation is taken place in
cytoplasm, in ribosomes.
– Proteins are further modified by
post-translational modification steps,
including proteolytic cleavage,
addition of carbohydrate or lipid
motifs, and modification of a.a..
• Gene expression in a cell is influenced
by both the micro (surrounding cell,
tissue, organ) and macro (endocrine and
paracrine) environments.
Core Techniques
• Restriction
Endonucleases
– Enzymes found in bacteria that
cleave DNA at precise sequences.
– Named by the organisms of origin
(eg. EcoRI is from E Coli R
strain).
– Size of fragments produced is a
function of the number of the
bases in the restriction site. (eg., 4
cutters produce DNA into smaller
fragments while 8 cutters produce
gene-sized DNA fragments).
Core Techniques (Cont.)
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Hybridization
– Based on the property of DNA
base paring (A to T and G to C).
– The principle is the recognition of
a complementary sequence (gene
to be detected) by a short sequence
(Probe) .
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The two strands of targeted DNA
needs to be separated into single
strands by a process of melting at
first, followed by annealing (reform
the double strand) after adding the
probe.
– The annealing depends on several
factors, including DNA concentration,
the time, the temperature, and the
concentration of salts. The stringency
of annealing is a function of temperature
and salt concentration.
– Examples:
• Dot or slot blot
• In situ hybridization (FISH, gene or
chromosome)
• Northern or Southern blot
– Needs to know the DNA sequence to be
fished.
Core Techniques (Cont.)
• Electrophoresis
– A technique to separate nucleic acids and proteins by size and
charge.
– All electrophoretic techniques are carried out using a supporting
gel of controlled pore size.
– Most separations are by size of moleculars (large one stay, the
small one migrate), while the charge governs the actual migration
of the moleculars.
• Polyacrylamide - for small noncharged moleculars (DNA)
• Agarose - for large noncharged moleculars (DNA/RNA)
• urea and SDS - for charged moleculars (protein)
– Procedure:
• Making a gel and buffers (loading and running
buffers)
• Apply sample into the well
• Apply voltage (100 to 1000s depends on the size of
gel)
• Visualize and detection (staining the gel, or transfer
the moleculars into membranes)
Core Techniques (Cont.)
• Sourthern blot - for DNA (RFLP)
• Northern blot - for RNA
• Western blot - for protein
Core Techniques (Cont.)
• Isolation of DNA and RNA
– It is crucial to have pure source of DNA or RNA for the accurate
analysis.
– The purity is indicated by the ratio of OD reading (OD 260 vs 280,
which measures nucleic acids vs protein, respectively)
– RNA is much less stable than DNA, due to the widely present
RNases.
– The major method for DNA isolation is the phenol-chloroform
extraction (phenol allows dissociation of DNA from protein,
whereas chloroform promotes the protein denaturation). Followed
by separation with centrifugation, the DNA is present at upper
phase.
– The major method for mRNA isolation is by
modified phenol-chloroform method that
requires a inhibition of RNase using
guanidinium and a purification step using either
oligo(dT) chromatography or beads.
– Source of DNA can be any fresh or archived
small amount materials (paraffin blocks, trace
amount of old blood, saliva, etc), while mRNA
usually requires large amounts of fresh or
immediately frozen samples.
Core Techniques (Cont.)
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PCR (Polymerase Chain Reaction)
– Revolutionize the detection
technique for nucleic acids (DNA
and RNA), also useful for cloning
and site-directed mutagenesis.
– The principle is by cycling the
temperature changes from
denaturation (95 C), annealing
(50C), and hybridization (70C), it
allows a molecular (single
stranded) to replicate itself
exponentially.
– Requires primers, DNA
polymerase, nucleoside
triphosphates, and magnesium ion.
– Limitations of PCR:
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Primer selectivity
Primer dimer formation
Contamination
Nonspecific priming
Temperature design for GC rich or AT rich genes (incomplete melting
or incomplete annealing, respectively).
– In epidemiological studies it is used for detecting the
presence/absence of genes (DNA or RNA), measures
the level of genes, or detect the specific forms of
mutations, etc.
Core Techniques (Cont.)
• Examples of Variant PCR
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LCR (for detection of point mutation)
Competitive PCR (for quantification of DNA copy #)
RT-PCR (for mRNA detection and quantification)
SSCP (for screening of gene mutation)
In situ PCR
TRAP (for telomerase activity detection)
Real-Time PCR
Core Techniques (Cont.)
• Monoclonal Antibodies
– Or so called immunoglobulins, are antibodies capable of recognizing only
one specific antigen (epitope).
– Developed by various techniques e.g., hybridoma, Phgae-display, etc.
– Used in molecular epidemiological studies to detect any protein products
(such as oncogene products, growth factors, receptors, etc) in a highly
specific and often quantitative manner by various methods such as ELISA,
EIA, immunohistochemistry, immunocytochemistry, etc.
– All these methods are basically use the same principle,
i.e.,antigen-antibody reaction. They can be either direct
(without amplification step) or indirect (with
amplification steps)and a detection step (with enzyme
colormatrix or fluorescence).
• 3 steps immunofluorescence to detect a tumor specific antigen
M344
– Step 1: Incubate cells with McAb (mouse anti human) against
M344
– Step 2: Incubate with biotinlated Goat (or rabbit) anti mouse IgG
(amplification)
– Step 3: Incubate with streptavidin-Texas Red
(amplification/detection)
QFIABiomarker Profile
G-actin: Texas-Red conjugated
DNase I
M344: FITC (or Rhodamin) 3Step Immunofluorescence
DNA: Hoechst or DAPI
Core Techniques (Cont.)
• RFLP - Microsattelite marker - SNP
– RFLP is the method to detect alterations (mutation) of one specific
gene.
– Microsattelite markers are simple tandem repeat polymorphisms of
several locus, which replaces RFLP as markers for disease
– SNP - are single nucleotide variants of entire genome - therefore
are much more powerful and may replace Microsattelite markers
or RFLP as markers of disease
• More prevalent in the genome than microsattelites in genome
• Some SNPs located in genes directly affect protein structure or
expression levels
• More stably inherited
• Better for high throughput analysis
SNPs - Definition
“Single base pair positions in genomic
DNA at which different sequence
alternatives (alleles) exist in normal
individuals in some population, wherein
the least frequent allele has an abundance
of 1% or greater” (Brookes, Gene, 1999).
How to Define SNPS?
Conventional way:
• develop sequence tagged sites (STS)
• identify DNA sequence variants
• estimate allele frequencies of the marker
• place the marker in human genome
• obtain DNA sequence
More powerful – Genome Wide Association
Studies (GWA)
Genome Wide Association (GWA) Study
• Help to identified genetic susceptibility markers for cancer
– Prostate: Chromosome 8q24 (Gudundsson, et al, Nature
genetics/Yeager, et al, Nature Genetics, 2007)
– Lung: Chromosome 15q25 (nicotinic acetylcholine receptor
subunits) (Huang, et al, Nature 2008/Amos, et al, Nature Genetics,
2008/Thorgerisson, et al, Nature genetic, 2008)
• Genes identified in these locus may also be the
targets for chemopreventive drug development
High Throughput Techniques
• Microarray technology
– DNA chips
• cDNA array format
• in situ synthesized oligonucleotide format (Affymetrix)
– Proteomics
– Tissue arrays
• These are powerful tools and high through put methods to
study gene expression, but they are not the answers
themselves
• Individual targets/patterns identified need to be validated
• In epidemiological studies, these methods can be used to
identify specific exposure induced molecular changes,
individual risk assessments, etc.
An example of our 9000 gene mouse-arrays using
differential expression analysis with Cy3 and Cy5
fluorescent dyes.
Proteomics
• Examine protein level expression in a high throughput manner
• Used to identify protein markers/patterns associated with
disease/function
• Different formats:
– SELDI-TOF (laser desorption ionization time-of-flight): the protein-chip
arrays, the mass analyzer, and the data-analysis software
– 2D Page coupled with MALDI-TOF (matrix-assisted laser desorption
ionization time-of-flight)
– Antibody based formats
A, GTE (20g/ml)
3.5 4.5 5.1 5.5
6.0
7.0
8.4
Fig 1
pI
9.5 3.5 4.5 5.1 5.5
6.0
7.0
9.53.5 4.5 5.1 5.5
8.4
6.0
7.0
8.4
9.5
MW (kDa)
217
116
98
8
55
2 10
7
17
6
30
13
7
16
12
5
11
13
2 10 1
9
8
1
5
11
37
9
6
17
16
18
12
14
14
3
20
15
B, GTE (40g/ml)
3.5 4.5 5.1 5.5
6.0
7.0
8.4
18
3
15
4
4
pI
9.5 3.5 4.5 5.1 5.5
6.0
7.0
8.4
9.53.5 4.5 5.1 5.5
6.0
7.0
8.4
9.5
MW (kDa)
217
116
98
55
5
1
10
11
20
19
13
37
10
5
11
17
12
14
30
17
18
16
12
14
15
20
48 hr
GTE:
-
16
15
4
Time:
1
24 hr
48 hr
+
+
13
18
Tissue Array
• Provide a new high-throughput tool for the study of gene dosage
and protein expression patterns in a large number of individual
tissues for rapid and comprehensive molecular profiling of cancer
and other diseases, without exhausting limited tissue resources.
• A typical example of a tissue array application is in searching for
oncogenes amplifications in vast tumor tissue panels. Large-scale
studies involving tumors encompassing differing stages and
grades of disease are necessary to more efficiently validate
putative markers and ultimately correlate genotypes with
phenotypes.
• Also applicable to any medical research discipline in which
paraffin-embedded tissues are utilized, including structural,
developmental, and metabolic studies.
Bladder Array
Gelsolin
HE
DNA Methylation
DNA methylation plays an important role in normal cellular
processes, including X chromosome inactivation, imprinting
control and transcriptional regulation of genes
It predominantly found on cytosine residues in CpG
dinucleotide, CpG island, to producing 5-Methylcytosine
CpG islands frequently located in or around the
transcription sites
DNA Methylation (Cont’d)
Aberrant DNA methylation are one of the most common
features of human neoplasia
Two major potential mechanisms for aberrant DNA
methylation in tumor carcinogenesis
Point mutation: C to T transition
(e.g. P53 gene)
Silencing tumor suppressor
genes (e.g. p16 gene)
Source:Royal Society of Chemistry
Promoter-Region Methylation
Promoter-region CpG islands methylation
• Is rare in normal cells
• Occur virtually in every type of human neoplasm
• Associate with inappropriate transcriptional silence
• Early event in tumor progression
In tumor suppressor genes
Most of the tumor suppressor genes are under-methylated
in normal cells but methylated in tumor cells. Methylation
is often correlated with an decreasing level of gene
expression and can be found in premalignant lesions
DNA methyltransferases
DNMTs catalyze the transfer of a methyl group (CH3) from Sadenosylmethionine (SAM) to the carbon-5 position of
cytosine producing the 5-methylcytosine
There are several DNA methyltransferases had been
discovered, including DNMT1, 3a, and 3b
Pathology - Objective
• To learn basic histopathological
terminology.
• To know different types of tumor.
What is the difference between
“tumor” vs “cancer”
Tumor – Either benign or malignant
Cancer – Usually malignant
Classification of Tumors
-Based on histological origin
(epithelial, mesenchyme, etc..)
-Based on biological behavior
(benign vs malignant)
PATHOLOGICAL REPORT
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Tumor histological type.
Tumor stage.
Tumor grade.
Other features (size, % necrosis,
lymphovascular invasion…)
CANCER HISTOLOGICAL TYPE
• Three Major Categories:
– Epithelial – “Carcinoma”
– Mesenchyme – “Sarcoma”
– Hematopoitic – “Leukemia/Lymphoma”
• Other Minor Categories:
– Nevocytic – “Melanoma”
– Germ cell – Teratoma, Seminoma, Yolk sac tumor,
Choriocarcinoma, etc…
– Endocrine/Neuro – Carcinoid/Insulinoma/small cell
carcinoma, etc…
CARCINOMA
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Squamous – Squamous Cell Carcinoma.
Glandular - Adenocarcinoma.
Transitional – Transitional Cell Carcinoma.
Small cell – Small cell carcinoma
SARCOMA
• Muscle
– Smooth muscle: Leiomyosarcoma
– Skeletal muscle: Rhabdomyosarcoma
• Fat – Liposarcoma
• Skeleton – Osteosarcoma
• Cartilage – Chondrosarcoma
Classification of tumor according to
their morphologic features (histology)
• Morphologic classification refers to the
histologic classification made by
pathologist based on microscopic
examination.
Benign vs Malignant Tumor
• The main distinction between benign and
malignant tumor is:
– Malignant tumor has invasion and metastatic
potential whereas benign tumor does not.
– Malignant tumor has features of abnormal
cellular differentiation whereas benign tumor
usually not.
Why histologic classification is
important in cancer epidemiology?
• Cancer is not ONE disease
• Different cancer types of same organ may
have different exposure etiology,
pathogenesis, as well as behavior, i.e.,
HETEROGENEITY
Carcinoma
• Carcinoma (Cancer of the epithelium) 85%
Epithelium is the term applied to the cells
that cover the external surface of the body
or that line the internal cavities, plus those
cells derived from the linings that form
glands.
Why most common cancers are
epithelial origin?
• These cells are the first point of contact of
the body with environmental substances,
either directly (squamous cells) or indirectly
(glandular cells).
• Epithelial cells usually have fast turn over
rate, i.e., fast cell division, and their DNA
can be damaged by carcinogens more often
than non-dividing cells.
Carcinoma: Squamous cell
• Originates from stratified squamous
epithelium of the skin, mouth, esophagus,
and vagina, as well as from areas of
squamous metaplasia, as in the bronchi or
squamocolumnar junction of the uterine
cervix. SCC is marked by the production of
keratin.
Skin Cancer
Squamous Cell Carcinoma
Carcinoma, Transitional Cell
• Transitional cell carcinoma - arise from the
transitional cell epithelium of the urinary
tract, such as bladder.
transitional cell carcinoma of the urothelium is shown here at low power
to reveal the frond-like papillary projections of the tumor above the
surface to the left. It is differentiated enough to resemble urothelium, but
is a mass. No invasion to the right is seen at this point.
TCC at high power
Carcinoma: Adenocarcinoma
• Adenocarcinoma - is carcinoma of
glandular epithelium and includes
malignant tumors of the gastrointestinal
mucosa, endometrium, and pancreas; and is
often associated with desmoplasia, tumorinduced proliferation of non-neoplastic
fibrous connective tissue, particularly in
adenocarcinoma of the breast, pancreas, and
prostate.
Prostate Ca
Ovarian Ca
Sarcoma
• Sarcoma is a malignant tumor of
mesenchymal origin
• Sarcoma is often used with a prefix that
denotes the tissue of origin of the tumor, as
in osteosarcoma (bone), leiomyosarcoma
(smooth muscle), rhabdomyosarcoma
(skeletal muscle), and liposarcoma (fatty
tissue).
Classification of tumor
according to stage
Stage
• -is clinical assessment of the degree of localization
or spread of the tumor.
• -generally correlated better with prognosis than
dose histopathologic grading.
• -is examplified by the generalized TNM system,
which evaluates size and extent of tumor (T),
lymph node involvement (N), and metastasis (M).
• -different staging systems (WHO, TNM, etc),
sometimes oriented toward specific tumors, e.g.,
Dukes system for colorectal carcinomas.
Classification of Tumor
according to its differentiation
(grade)
Grade of Disease
• Grading is histo-pathologic evaluation of the
lesion based on the degree of cellular
differentiation and nuclear features:
Well Differentiated (Grade I)
– more resemble to normal tissue/cell
Moderately differentiated (Grade II)
- less resemblance of normal tissue/cell
Undifferentiated (Grade III)
- lost resemblance to normal tissue/cell
Gleason's breakthrough was to
develop a reproducible description
of the glandular architecture, to
which one assigns a score from 1
to 5. The pathologist looks for a
major pattern and a minor pattern
to give a Gleason sum between 2
and 10. On the left is a picture
adapted from Gleason's 1977
article demonstrating the changes
in gland pattern as one goes from
grade 1 to grade 5 cancer. The
glands in grade 1 cancer are small
and round. Grade 5 cancer is
hardly forming glands at all.
Gleason Grade 1 Prostate Cancer
At right is Gleason 3 CaP.
The glands are irregularly
shaped. They are mixed in
with some normal glands.
This tumor is infiltrating
the prostate.
At higher
magnification, there
are nests of glands with
no intervening stroma.
This is characteristic of
higher grade CaP
Here is Gleason 5,
or poorly
differentiated
cancer. You can see
that it is invading
the seminal vesicle
(stage T4)
The cells are not
organized into glands,
but are infiltrating the
prostate as cords.
Precursors
from
intraepithelial neoplasia (IN)
to
carcinoma in situ (CIS)
NORMAL
CIN 1
NORMAL
LGSIL
CIN 2
HG SIL
CIN 3
HGSIL
Important for Epidemiologist
• Study nature history of disease progression
• Study genetic/environmental factors associate with
disease progression
• Develop tools for risk assessment/early detection
• Targets for chemoprevention
Exposure to Carcinogen
Birth
Precancerous
Intraepithelial
Lesions,
(PIN, CIN, PaIN..)
Additional
Molecular Event
Cancer
Surrogate End Point Markers
Genetic Suscep.
Marker
Markers for
Exposure
Markers of
Effect
CHEMOPREVENTION
Tumor
Markers
SUMMARY
• The key is to understand tumor hetergeneity:
– Human cancer is not one disease , but many different
types of diseases (Disease heterogeneity).
– The same type/stage/grade of tumor may behave
differently in different person (Behavior
heterogeneity).
– Even within the same tumor, there are may be different
histological appearances and molecular
expressions/changes (Expression heterogeneity).
• As an epidemiologist, we should know the basic
features of the disease, and design studies
accordingly
Thank You!
Thank You!!