Mutations + Cancer 1

Mutations in somatic vs germline cells

Mutations in somatic cells are not passed on to the next generation. Mutations in somatic cells can cause disease (aging, cancer)

Mutations in germ-line cells are passed on to the next generation, thus are heritable. Most germ-line mutations in humans have NO phenotypic effects because most human DNA is junk. Mutations that do have a phenotypic effect are mostly negative, but can be positive

How can mutations arise

1. Errors during DNA replication

2. Damage to DNA from various sources

3. Integration of retroviral genomes and transposable elements

4. Chromosomal rearrangements

Errors in DNA replication and how it fixes itself

DNA polymerase is an enzyme with high fidelity (gets it right 999,999/1 million bases). Misincorporation rate: 1 in 10⁵. Wrong base is incorporated in newly synthesized strand

1. DNA polymerase has “proofreading” activity. DNA polymerase can cleave off the newly synthesized, incorrect base (if no additional bases have been synthesized) and resynthesize the base

2. Post-replication mismatch repair. When a mismatch occurs during DNA synthesis, the correct base is on the parental strand and the incorrect base is on the newly synthesized strand. DNA poly I excises nucleotides on the new strand and fills in the gap from 5’ to 3’. DNA ligase links the new and old nucleotides together

Errors in replication occur during S phase and can be repaired in G2, hence the G2 checkpoint for DNA damage

how does DNA damage happen

First step in generating a mutation is DNA damage

it is any modification to DNA bases

1. damage from byproducts of cellular metabolism, ESPECIALLY oxidative phosphorylation (oxygen radicals, hydrogen peroxide, etc.)

2. damage from UV radiation

3. damage from exposure to mutagens

Damage rate (excluding replication) > 50,000 lesions/cell*day MOST FROM NORMAL METABOLISM

how to prevent DNA damage

1. Separate DNA from sites of metabolism (nucleus and mitochondria)

2. Use enzymes and compounds to neutralize potentially damaging compounds

repairing DNA damage

Excision Repair

1. Enzymes detect an irregularity in DNA structure and cut out the damaged strand.

2. An enzyme excises nucleotides on the damaged strand.

3. DNA polymerase fills in the gap in the 5 3 direction.

4. DNA ligase seals the phosphodiester backbone.

how to know if damage will turn into mutation

The critical concept for DNA damage and mutation: As long as the original sequence is available on one of the strands of DNA, the mutation can be repaired! That means that DNA damage is NOT the same as mutation.

Second step in generating a mutation is DNA synthesis. Any incorrect or damaged DNA base not repaired prior to DNA synthesis can lead to the incorporation of the incorrect base in the newly synthesized strand. the error/damage is now permanent!

The G1/S and G2/M checkpoints for DNA damage help reduce the number of DNA damaging events that get converted into mutations by preventing DNA synthesis/mitosis until DNA damage is very low.

Silent and missense mutations

Silent: change one nucleotide so amino acid stays the same

Missense: Change one nucleotide to change one amino acid

Genetic code has evolved so that many mutations are silent or missense mutations that substitute a chemically similar amino acid (e.g., non-polar for non-polar)

Nonsense and frameshift mutations

Nonsense: a nucleotide substitution that creates a stop codon

Frameshift: Insertion or deletion of 1 or 2 (or any number not divisible by 3) bases results in disruption/shifting of the reading frame

somatic mutations

Mutations in somatic cells are not passed on to the next generation. Mutations in somatic cells can cause disease. Lead to cancerous cells, contribute to aging. Cancer is the accumulation of specific cellular defects as the result of genetic changes in individual cells

germline mutations

• Germline mutations generate variation in DNA sequences among individuals in a population

• Cause heritable defects – mutations that lead to less active or defective proteins are much more common than those that provide a benefit

• Mutations can create new, beneficial enzymatic activities or protein functions

• Mutations can create new expression patterns

• Generate phenotypic variation between individuals that can be the basis for selection

cancer phenotypes

Increased proliferation, Increased cell growth, Upregulated metabolism, Decreased apoptosis (cell death), Escape immune response, Ability to migrate, Ability to colonize new locations, Upregulated telomerase activity

apoptosis

Cells in certain development settings undergo programmed cell death (e.g., the cells between each finger during early development). Apoptosis is also a key mechanism to remove cells with abnormal proliferation and/or metabolism (pre-cancerous cells). The apoptotic pathway is a way to prevent cancer development. Many factors within a cell either promote or inhibit apoptosis

immune system in clearing precancerous cells

The immune system also screens for cells with abnormalities. It can trigger apoptosis within the abnormal cell or kill it in other ways

Changes within cells that block this immune cell function are another cancer phenotype

Cancer metastasis

When cancer cells gain the ability to migrate and colonize new locations, that results in metastasis: new tumors growing in many locations (e.g., a metastatic brain tumor can colonize the lungs, colon, etc.)

how do cancer phenotypes arise

Cancer is the result of uncontrolled proliferation and other cellular changes in individual cells

Cancer phenotypes occur as a result of genetic changes within individual cells

Cancer is not a bimodal state. In other words, the idea that cells are either ‘normal’ or ‘cancer’ is false. Cancer encompasses a broad range of cellular phenotypes that vary dramatically. Cells can develop various subsets of cancer phenotypes

mutations in RB

Retinoblastoma = RB is a Tumor Suppressor

Mutations in RB are a causal factor in many individual cancers. RB normally locks the transcription factors E2F and DP in an inactive state, preventing them from activating S-phase transcription.

A tumor-suppressor is a gene that normally blocks cancer phenotypes. Loss-of-function mutations in tumor suppressor genes lead to cancer phenotypes. Loss-of-function mutations are relatively common, but you need to inactivate both alleles – in the same cell!

protoonco genes

A proto-oncogene is a gene that normally promotes cancer phenotypes, but in a regulated manner. When hyper-activated by mutation (gain-of-function), proto-oncogenes become oncogenes and promote cancer phenotypes. Oncogenes drive the unregulated cellular behaviors found in cancers.

Your cells need ALL of the cancer phenotypes in some cells/in a regulated fashion. For example, you need cells that do not undergo apoptosis, you need cells with telomerase, you don’t want your immune system to kill off all of your cells, you need cells that proliferate

Ras GTP hydrolysis

GTP unable to hydrolyze back to GDP in Ras pathway, which means Ras gets stuck on the GTP-bound “on” state.

K-Ras oncogenic mutations are always missense mutations in amino acids 12 or 13 (called K-Ras- GOF, for “Gain Of Function”) These substitutions lock K-Ras into a permanently active state by blocking GTP hydrolysis This leads to constitutive signaling and cancer phenotypes

Gain-of-function mutations are exceedingly rare, but you only need to hyper-activate one allele.

standard chemotherapy drugs

Standard chemotherapy drugs typically target proliferating cells

These drugs do not distinguish between cancerous cells and normal cells. Thus, they have serious side-effects that can make patients very sick. However, in many cases cancerous cells are particularly affected by the drugs because they lack cell cycle checkpoints

targeted chemotherapy drugs

Targeted chemotherapy drugs are tailored to an individual tumor in a specific patient.

The first step is to determine WHY a particular tumor has cancer phenotypes. What genetic changes have occurred in this specific tumor? Can be done with protein stains, cDNA analysis and/or genomic sequencing. Some chemotherapy drugs, for example, will work very well on one lung cancer tumor, but will be completely infective on a different lung cancer tumor. Because these target a specific protein or pathway, targeted chemotherapy drugs typically have far fewer/less severe side effects.