MOLECULAR BIOLOGY & PATHOLOGY IN EPIDEMIOLOGY

1. MOLECULAR BIOLOGY & PATHOLOGY IN EPIDEMIOLOGY JianYu Rao, M.D. Associate professor of pathology and epidemiology UCLA

2. Molecular Biology - Outline • Introduction • Basic Principles of Molecular Biology • Core Techniques of Molecular Biology • High Throughput Technologies • Epigenetics – DNA Methylation

3. 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

4. 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 base-paring 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.

5. Genetic information is organized lineally: • A codon is the basic unit with 3 consecutive nucleotides that specifies a single aa. • A gene is a segment of DNA (with lineally linked multiple codeons) that specifies a protein. • A chromosome contains several thousands genes and is the smallest replicating unit (human has 46 chromosomes). • The genome is the entire set of information that an organism contains. 5’ 3’ 5' –CCT GGT CCT CTG ACT GCT - 3' K H L …

6. Basic Principles of Molecular Biology (cont.) • 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.

7. 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.

8. Basic Principles of Molecular Biology (Cont.) • 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.

9. 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.

10. 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.

11. 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).

12. Core Techniques (Cont.) • 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) . • 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.

13. 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.

14. 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)

15. 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)

16. Core Techniques (Cont.) • Sourthern blot - for DNA (RFLP) • Northern blot - for RNA • Western blot - for protein

19. 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.

20. 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.

21. Core Techniques (Cont.) • 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.

22. Limitations of PCR: • 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.

23. Core Techniques (Cont.) • Examples of Variant PCR • 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

24. 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.

25. 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)

26. QFIABiomarker Profile • G-actin: Texas-Red conjugated DNase I • M344: FITC (or Rhodamin) 3- Step Immunofluorescence • DNA: Hoechst or DAPI

27. 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

28. 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).

29. 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)

30. 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

31. 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.

32. An example of our 9000 gene mouse-arrays using differential expression analysis with Cy3 and Cy5 fluorescent dyes.

33. 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

35. Fig 1 3.5 3.5 3.5 3.5 3.5 3.5 4.5 4.5 4.5 4.5 4.5 4.5 5.1 5.1 5.1 5.1 5.1 5.1 5.5 5.5 5.5 5.5 5.5 5.5 6.0 6.0 6.0 6.0 6.0 6.0 7.0 7.0 7.0 7.0 7.0 7.0 8.4 8.4 8.4 8.4 8.4 8.4 9.5 9.5 9.5 9.5 9.5 9.5 217 217 116 116 98 98 55 55 37 37 30 30 20 20 A, GTE (20g/ml) pI 9 MW (kDa) 8 10 9 8 2 2 10 1 1 5 5 11 11 13 13 17 7 7 6 6 17 18 18 16 16 12 12 14 14 3 3 15 15 4 4 B, GTE (40g/ml) pI 20 19 MW (kDa) 1 1 10 5 10 5 11 11 13 13 17 17 18 12 18 16 16 12 14 14 15 15 4 Time: 48 hr 24 hr 48 hr - + + GTE:

36. 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.

37. Bladder Array Gelsolin HE

38. 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

39. 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 Silencing tumor suppressor genes (e.g. p16 gene) Point mutation: C to T transition (e.g. P53 gene) Source:Royal Society of Chemistry

40. 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

41. DNA methyltransferases DNMTs catalyze the transfer of a methyl group (CH3) from S-adenosylmethionine (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

42. Pathology - Objective • To learn basic histopathological terminology. • To know different types of tumor.

43. What is the difference between “tumor” vs “cancer” Tumor– Either benign or malignant Cancer– Usually malignant

44. Classification of Tumors Based on histological origin (epithelial, mesenchyme, etc..) Based on biological behavior (benign vs malignant)

45. PATHOLOGICAL REPORT • Tumor histological type. • Tumor stage. • Tumor grade. • Other features (size, % necrosis, lymphovascular invasion…)

46. 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…

47. CARCINOMA • Squamous – Squamous Cell Carcinoma. • Glandular - Adenocarcinoma. • Transitional – Transitional Cell Carcinoma. • Small cell – Small cell carcinoma

48. SARCOMA • Muscle • Smooth muscle: Leiomyosarcoma • Skeletal muscle: Rhabdomyosarcoma • Fat – Liposarcoma • Skeleton – Osteosarcoma • Cartilage – Chondrosarcoma

49. Classification of tumor according to their morphologic features (histology) • Morphologic classification refers to the histologic classification made by pathologist based on microscopic examination.

50. 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.