Molecular Basis of Cancer: Chromosomes

Molecular Basis of Cancer

• Alleles are different versions of genes
• An organism is heterozygous if it has two different alleles
• The cell cycle consists of a long interphase, where genes are expressed and chromosomes are replicated

Chromosomes

• Chromosomes remain together as sister chromatids during interphase
• Individual chromosomes are only distinguishable during mitosis
• Sister chromatids condense into mitotic chromosomes during mitosis, which are then separated into two daughter cells
• Each chromosome is an individual unit that replicates, separates, and partitions into daughter cells
• Three types of specialized nucleotide sequences in DNA control these functions
• Centromere DNA sequences allows each copy to be pulled into daughter cells during division
• The kinetochore is protein complex, attaches chromosomes to the mitotic spindle
• Telomeres are specialized DNA sequences forming the ends of chromosomes.
• Writer proteins mark nucleosomes, while reader proteins recognize these marks and bind tightly to nucleosomes

Chromatin

• Is a complex histones and nonhistone chromosomal proteins
• Binds DNA to form chromosomes
• Nucleosomes use ATP to move DNA relative to the histone core
• The histone code involves writers adding modifications, erasers removing modifications, and readers recognizing and recruiting other proteins
• Euchromatin is less condensed, heterochromatin is more condensed
• Chromatin is folded in loops and coils
• Acetylation loosens chromatin to activate transcription
• Methylation activate or repress transcription, depending on its location
• Phosphorylation and ubiquitination activates transcription
• Methylated CpG islands cause gene silencing, while unmethylated CpG islands act as active promoters
• DNA methyltransferase mostly performs this process

Barr Lichaam

• Is an inactive, compacted X chromosome
• Mutations can be inherited through the germ line
• Somatic mutations aren't heritable
• Each centromere pulls one copy of a chromosome to a daughter cell
• A promoter's activity can be studied using whole-genome sequencing or second-generation sequencing

Polymerase II

• Transcribes genes encoding proteins
• Transcription factor TTD binds to the TATA box close to the promoter, which then allows the DNA helicase to unwind the DNA, giving access to the template strand
• After transcription, mRNA undergoes splicing, capping, and 3' polyadenylation
• mRNA exits the nucleus through pores, and then translated into a protein

Regulation of Gene Expression

• Regulation of gene expression dictates when and how often a gene transcribed
• RNA processing, transport, and localization control
• Translational and mRNA degradation control
• Protein activity control
• Transcription regulators regulate transcription of genes
• Coactivators and repressors bind to transcription regulators on DNA to regulate gene transcription
• Barrier sequences prevent the spread of heterochromatin, and insulator DNA regions prevent cis-regulatory regions from inappropriately activating genes
• RNA interference machinery can selectively shut off the synthesis of target RNAs
• The RITS protein complex modifies histones, directing heterochromatin formation, so that transcription is prevented
• piRNAs transcriptionally silence transposon genes and destroy any RNA produced
• Quantitative RT-PCR used to measure individual RNA amounts in cells
• A DNA microarray can be used to analyse which mRNAs are produced in a cell
• mRNA is turned into cDNA and labelled fluorescently, then hybridize to fragments, then a microscope reveals which mRNA was present

Transcription

• Controlled by gene control regions
• Silencers bind repressors
• Insulators form circles around genes and promoters, preventing enhancers from reaching them

Analysis of Genetic Defects

• DNA Cloning: accomplished by inserting a DNA fragment into a purified genome of a self-replicating genetic element
• Purified plasmid DNA circles = cut with restriction nucleases to create linear DNA molecules
• DNA ligase adds foreign DNA to the plasmid
• Recombinant DNA circles are added to bacteria, which grow and replicate the recombinant plasmids with the foreign DNA
• Cells are lysed, and the plasmid DNA is extracted

Gel Electrophoresis

• Separates DNA by size
• DNA is stained for band visualization

Hybridization

• Detects specific nucleotide sequences
• Labeled probes are added and visualized where they bind, indicating the presence of specific gene sequence, FISH

Southern Blot

• Electrophoresis with probe hybridization detects specific DNA sequences
• DNA is separated by size, the membrane is blocked, and a probe is added that binds to specific sequences

Northern Blot

• Gen expression is studied using RNA
• Separates with gel electrophoresis, adds a probe, then bands are analyzed

PCR

• In vitro gene cloning, ssDNA, primers, hybridization, translation with TAQ pol, and DNA amplification, resulting in a band on the blot due to same length DNA fragments located on the membrane since they span the primers
• qPCR quantifies a given sequence in a sample based on labeled color intensity, reflecting gene experience

Sanger Sequencing

• Determines the seq of DNA or RNA
• Occurs by hybridizing a primer to DNA, adding excess dNTPs and DNA polymerase, then adding one type of base into four different tubes, then observing length of fragments which stop due to ddNTPs, then band patterns reveals the sequence

CRISPR/Cas9

• Modifies genome
• Transfects cells with Cas9 protein and guide RNA plus plasmids containing DNA for these proteins, this also replicates the plasmids in which it is contained
• The proteins then cuts the genome in the HR region which causes specific mutations to occur to these genes it contains

Mass Spectometry

• Identifies which amino acids are sumoylated via analyzing ions from a sample which are separated the ratio of mass to charge to view how frequently they all appear
• SUMO means "small ubiquitin-like modification."

SNP Array

• Detects polymorphism within a population as well as deletion and duplications
• SNPs are small polymorphisms that differ by one nucleotide, also identifies loss of heterozygosity

Fusion Analysis

• Analyzes fusions between two separate genes
• Uses fluorescence in situ hybridization for fusions with an area already documented, NGS to sequence for fusions that are new

The Nature of Cancer

• A neoplasm is a tumor, but a tumor is not necessarily neoplastic
• Cancer is a malignant tumour
• Behavioral classification is either benign or malignant
• Histogenetic classification is a cell of origin; morphology like different tissue types. Everything with the suffix -oma is benign. Adenoma follows glands, papilloma finger like projections, a polyp is not a true neoplasm. Benign mesenchymal neoplasms; lipoma, chondroma.
• Everything with -sarcoma is malignant
• Germ Layers: teratoma, hamartoma, malignant or benign
• Disorganized mass of tissue whose cell types are native to the site of the lesion are known are hamartomas
• Misnomers that have -omas but are actually malignant; melanoma, seminoma, lymphoma
• Morphology immunohistochemistry and molecular analysis are how benign vs malignant cells are typed

Grading Tumors

• Low grade tumors have a better outcomes
• The grade of tumor is degree to which it is different or mature as viewed through a microscope, this involves growth patterns, the extent of differentiation, and nuclear aspects and cell cycle information
• Ki-67 gives cell cycle information, as it is present in all proliferating cells
• G1 = well-differentiated (low grade), G2 = moderately differentiated, G3 = poorly differentiated (high grade), and G4 = undifferentiated
• Objective grading; DNA aneuploidy , TP53 mutations (helps preserve genome), and gene expression profile

Tissue Structure

• Normal tissue contains an epithelial cell layer - basement membrane - stroma (supportive connective tissue cells)
• Squamous cell carcinomas arise from epithelial cells that serve as barriers
• Adenocarcinomas arise from epithelial cells that secrete products into vessels they contain
• Mesoderm produces the connective tissues forming and immune cells 
• Mesoderm is derived from cells in the system, CNS and PNS
• Melanomas come from neural crest melanocytes

Hyperplastic growths

• Deviate from normal cells, excessive numbers of them

Metaplasia

• Normal cell layer displaced by one of its neighboring cells

Dysplastic

• Appearance of cells no longer standard
• Hyperplasia and metaplasia are flexible and dysplasia causes mutations and invasion
• Genetic mutations and invasion are not reversible
• Key irreversible events: Loss of tumor suppressor genes, activation of oncogenes, chromosomal instability/aneuploidy

Types of Tumors

• Monoclonal tumour is derived from one single ancestor, while polyclonal derives from genetically distinct subpopulations
• Origin is from monoclonal cells

Procarcinogens

• Dramatically different rates of cancer across the world and the world
• Environmental factors, physical factors, and lifestyle all increase the risk of it occurring
• Some of the cancer is also caused by screening, early detection; factors, include chemical factors.

Rous Sarcoma

• Discovered to lead to cancer
• Hen experiment, after cells were infected a petri dish to isolate the species causing the disease, led to the understanding of the nature of cancer and viruses
• Foci formed; clusters of cells in a petri dish
• Viruses cause it to kill their host cells (virulent), while or allowing the cells to not die, but not long-term with limited viability for an intermediate rate of time.
• Normal cells in a petri dish stop because of their inability to connect with other cells
• Genome of RNA cells can be manipulated to do this, and can stop at a time when there is much layering occurring

Viral Transformation

• Transformation phenotype was transferred from initially infected transformed chicken cell to its descendants
• 41degrees reverts cell to the shape and growth patterns from its past experience
• The cell reverts if it doesn't want the disease in the first place

HPV

• Protein coat and caries a DNA genome
• Closely related viruses SV40 and the mouse polyomavirus and Shope´s Papillomavirus: groups of DNA tumor viruses

Cell Culture

• Normal cells need certain factors to live
• Cancer cells can live independently without factors
• They can also live for an unusual amount of extended period of time
• Normal cells need a substrate, called “anchorage dependence” to live

Viral Integration

• DNA virus in a host cell becomes transcribed in genomes for cells that divide
• HPV integrates an oncogene withing the genome to replicate cancer cells.

Retroviruses

• Convert their RNA back to DNA

• Three genes are in viral rep

  • 2 for the virus to assemble and function
  • 1 to transform the cell into a cancer cell

• Viral V - src is closely related to the c - src  in the chicken and it has a drastic function shift

Retroviruses

• Evolved pre-existing cellular DNA for their own use
• MC29 was acquired to have a cellular genome, termed V - myc oncogene
• These have a high tumorigenic potential in infected chickens Myc had its genes remodelled to gain oncogenic characteristics

The Classes of Tumors

• Retroviruses grouped
  • 1 that induces cancer rapidly and carry oncogenes SRC for an example -2 carry oncogenes slowly for tumors proto-oncogenes (ALV → myc, MLV),
  • 3 T - lymphotropic leukemia virus which gives cancer encoded in the viral genome with no known oncogenes

In order for bacterial cells to cause cancer, specific criteria has to be meet.

• H pyloni, the cell has to allow a protein, CAG a bacteria product to inhibit the gut, therefore reducing cell to cell signaling, to the effect of enhanced proliferation, apoptosis inhibition.
• Testing tumorigenicity with mice via impairing its immune system

Cellular oncogenes

• To Determine if nonviral oncogenes exist chemically transferred, if so they are then transplanted into mice.
• Foci Were created on the body; meaning its potential to cause a cancerous outcome.
• DNA was extracted from those which were 3 - MH, transformed mouse cell lines, into NIH, 3T3 recipient which also formed foci, and were anchorage and tumorigenic

Human Tunor

• Oncogenes which were retro virus associated were present at high number, by amplifying cell genomes. increased amount of potein to to cancer cell
• erbB2: amplified form often increased amount of potein which drives a breast cancer
• Development: the growth of Tunor can cause passengers with causal issues with development
• Genes responsible for the loss of growths
• The normal regulation might enhance

Point mutation for cancer

• By analyzing site structure and looking molecular restriction nucleuses and their site analysis. In the H-ras
• Where one G was swapped out for the proto-oncogene with a T in oncogene

K-ras

• Protooncogene regulatory sequences, controlling for expressions
• Which causes substitution of amino acid
• An normal gene can be turned into a oncogene by the number of copies it expresses, due to the translocation of the proto- oncogene.
• Mhyc gene occurs through translocations, and its also activated
• Translocations is done by deleting miRNA which prevents the cell destruction. can also cause a gene to be ocogenic by deletting sites for mirna
• Translocation create hibrbrid poteins and change their functions
• Growth stem signals can be sent even if EGF receptors are taken away

Growth factor & the pathway

• Gleevec is a selective tyrosine kinase inhibitor which drives proliferation
• Growth factors are proteins sent to give messages to other cells
• These GF’s are also know as mitogens, and this allows an ability to have the cells able to proliferate
• Which leads to dramatic changes in cell expression Src: functions to remove phosphate from 5 ATP and transfers it to a portein substrate, to then be covalently bound with more acid chains The EGF & the pathway

EGF-R

• Protrudes into the extracellular space
• Transmembrane is in the hydrophobic volume which turns the membrane into the plasma
• The ligand stabilizes at some point they are monomers
• Then phospyrolates the tyrosine, leading to the signals

The Jaks polypeptide Cytokine

• Responds through ligand bining
• TGF-B function as Herter Dimmers
• Notch 1 and notch 2 receptors
• Patched, which are primary for the cilium’s
• wnt Factors Ras: can go for both an anchored cytoplasmic membrane and go into the cytoplasm and then to its core.
• Ras activation is triggered by exchange GEF as inactivation is set forth 
• Lack of GTPase Activity, a mutation will make it  continue even the event its purpose is already fulfilled by it
• Immediate early genes MyC: protein rises after stimulation but decrease as stimluation is gone, due to its short half life, to be replaced by src, the proto is created
• This makes the proto more vulnerable

Protein Activation

• Shc: for ras activations then becomes a bridge between others factors & proteins which then allows a tyrosines to create a phosphate compound of the tails

Grb-2: then activates receptors, which then the SOS allows the RAS to activate downstream Raf: to the serine

Akt molecule

• Become tethered to the PIP3 to reach PH Domain
• Activation creates a stimulus of prolifiting cells with higher chance of metastasis

Cancer Cell proliferation

• Driven by cancer ce dimerizaiton Jaks separate poly peptides
• Jak's phosphorylation activates c receptors

Rora Responds to winths

• B - catenin is the most important , it is what has the most protien loss in the event of cancer.

Tumors

• If cell is absent in the absence of proliferation they will undergo APO - kins
• In a petri dish, cells tether which the body creates and forms the stability for the cells in it
• Ras is  able to mimic signals

Cytooplasmic Signaling

• A GEF for Ga, leads stimulates of cell proliferation
• Duals pathways operate then dispatchs protiens to normal cells

Tumor Suppressor Genes

• Cells were in fused whewre alleles were forced to co exisit, synctiyumn of two cells from various origins
• Tumor cells fused with normal cells create a lost to form
• Malignant phenotype was recessive

Point

The cell is to lose its genes. So in that sense they are more prone to the spread than that to start tumor, The tumor can not cause enough if the body can not have its defenses. If first allele is inactivated by chance mutation, the second can be lost during recombinations or the chromosomes themselves.

How the allele is caused the cause

• Through DNA being elongated during cell division

• This can cause the disease to come out

• 13q14, for what part in the body of the receptor is responsible

• Restriction fragment lengths, help show the lOH SNPs with PCR helps complement CpG methylation cause gene silence in these type of cancer mutations They are related to gtp-ase activating

Familial Adenomatour polposis

• Has APC as well Enterocyte encounter
• What they must have for there to be loss

Capable of Induce

• DNA damage regulated signals
• P53 activation on a TF after sythesizing the TF and then destruction
• Survival signals activaie the alkt leading to tumor cell surivival and or growth PI3: gets activated

E2F Activity

• Leading to AF is an expression

• P3 leads inhibits the function

• It is very hard to study because it spreads so commonly and so easily

• Attrition : a tumor that comes and goes depending on the number of factors at play and to what effect that it is being triggered is more than the cause.

Hemotiximality

• By the flow if you understand you’ll undersetand there importance. to each individual.

Explore the molecular basis of cancer with a focus on chromosomes. The cell cycle includes a long interphase where genes are expressed and chromosomes are replicated. Discover how sister chromatids condense into mitotic chromosomes during mitosis and are separated into daughter cells. Learn about centromeres, kinetochores and telomeres.