Carcinogenesis/Mutagenesis

What is neoplasia?

New uncontrolled growth (tumours are referred to as neoplasm)

What are cancers?

Diseases in which abnormal cells divide without control and invade other tissues

How do cancers grow?

By clonal proliferation or expansion - as a result of a mutation a cell aquires characteristics that allow it to have a selective advantage over its neighbours (↑growth and ↓apoptosis)

What does it mean if a neoplasm is benign?

Cells are well differentiated, resemble normal cells but cannot control proliferation

What does it mean if a neoplasm is malignant?

Cells cannot control differentiation or proliferation

• Cells do not mature and function as it should

• Grow in absence of GF (+ignore growth inhibition)

• Evade apoptosis

What are the characteristics of benign tumours?

Contain cells that resemble normal tissue cells (well differentiated and may perfom function of tissue)

Grow slowly

Surrounded by fibourous capsule

DO NOT infiltrate, invade or metastasise

Can damage nearby organs by compression

What are the characteristics of malignant tumours?

Contain cells that do not resemble normal tissue cells

Autonomously proliferate and can mutate

Lack or have disrupted capsule

CAN infiltrate, invade and metastasise

Can damage or destroy organs

What are the two types of malignant tumours?

Solid tumours and Hematologic cancers

What are solid tumours?

Initially confined to a specific tissue or organ but eventually invade original tissue and may metastasise by entering blood and lymph

What are haemtologic cancers?

Involves cells normally found in the blood and immune system and so are desseminated from the begining (lukemia, lymphoma)

What are the characteristics of malignant neoplasms?

Genetic instability, abnormal growth properties and immortality

How are cancer cells genetically instable?

Cancer cells cannot correct errors in cell division (passes errors on)

May have multiple copies of chromosomes (incorrect mitotic spindle formation)

The mutations often affect growth regulation and cell cycle regulation (arrest)

What are the abnormal growth properties of cancer cells?

Malignant tumours can grow without GF (can either make their own or produce pre-activated growth receptors)

Lack cell density-dependent or contact inhibition (stop growing in crowded environment)

Lack anchorage dependence (cells don’t stop growing when unthethered)

/How are cancer cells immortal?

Cells turn on the enzyme telomerase which replaces lost telomeres (end of chromosome progressively lost through cell divisions) and therefore cells do not die

(favour proliferation and inhibit apoptosis)

Why can cancer cells be described as primitive?

As they only retain functions essential for survival and proliferation (e.g. can avoid immune system by producing foetal antigens)

What are the two categories of genes involved in cancer?

Proto-oncogenes and tumour supression genes

What are proto-oncogenes?

Genes which code for normal cell division proteins (GF, cell cycle proteins and inhibitors of apoptosis) which can mutate into oncogenes which have increased activity

What are tumour suppressor genes?

Genes which inhibit cell division and mutations either inhibit or decrease ability of cell to stop growth (e.g. p53 - prevents cell division if cell damaged but when mutated no stop to cell division)

What are the stages of carcinogenesis?

Initiation, promotion, progression

What is the initiation step in carcinogenesis?

The inital mutation and DNA damage

What is the promotion step in carcinogenesis?

Mutated cells are stimulated to divide and uncontrolled proliferation occurs

What is the progression step in carcinogenesis?

Tumour cells compete with one another and develop more mutations making them more aggressive

What impact does inheritance have on cancer?

Inheritance of genes involved with cell proliferation, DNA damage (BRCA1/2) and apoptosis

What impact do reproductive hormones have on cancer?

Hormones trigger cell growth and may promote cancerous cell division

What impact does obesity have on cancer?

Overproduction of insulin and IGF1 stimulate cell proliferation and apoptosis

What impact do immune survaillence and tumour antigens have on cancer?

When mutations or cell damage occurs the immune system removes it however this ability is reduced in immunodeficient diseases like AIDs

Cancer cells can also change their antigens to hide from the immune system (foetal antigens)

How can chemical carcinogens impact cancer?

Agents may damage DNA, RNA or proteins involved in cell proliferation (directly or indirectly via metabolite)

How can radiation impact cancer?

Causes direct changes to DNA resulting in gene or chromosome changes (X-ray etc)

How can virus/bacteria impact cancer?

Pathogens can stimulate cell changes resulting in tumour formation

What do stage 1 mutagenic tests look for?

Gene mutations, clastogenicity (disruption of chromosome structure), aneuploidy (change in no of chromosomes)

What are the stage 1 mutagenic tests?

Ames test and micronucleus assay

Describe how the Ames test works

Measures the affect of substances on the rate of reverse mutation (transforming mutant back to wt)

Uses salmonella typhimurium and mutate their histidine synthesis (histidine is needed for protein synthesis) therefore they cannot grow on histidine free medium

After culturing them with the possible carcinogens their ability to grow on histidine medium is assessed, if they grow the carcinogen mutated their DNA!

What does adding rat liver extract too the medium assess in the Ames test?

It tests if the metabolites are mutagenic

Describe how the micronucleus assay works

Determines if a chemical causes clastogenicity or aneugenicity by detecting DNA fragments or extra-chromosomes which maifest as micronuclei in the cytoplasm of daughter cells in interphase

Chinese hamster ovary cells are treated with chemical and their micronuclei frequency is assed with flow cytometry (inc = genotoxic)

What does stage 2 mutagenic tests test for?

Geneotoxicity in target tissues, germ cells and over long term

What are the tests used in stage 2 mutagenetic testing?

Bone marrow micronucleus assay, transgenic rodent assay, commet assay

Describe how the bone marrow micronucleus assay works

Detects calstogenicity by measuring no. of MN formed in young erythrocytes in BM or reticulocytes of peripheral blood

Usually test 2-3 doses each 24hrs apart and take measurements 24hrs after each dose

In mice MN in BM shows DNA damage in the last 72 hours

Describe how a transgenic rodent assay works

Malnipulated plasmid with reporter gene is added to rat who is then exposed to potential carcinogen for 28 days

The DNA is recovered 28 days post exposure and inserted into bacteria which are grown and examined for reporter gene compared to controls

(E.g. lac Z grows blue colonies on xgal)

Describe how the comet assay works

The alkaline assay measures different types of DNA damage in different cell types

Cells are collected from rodents exposed to potential genotoxic chemical, DNA is extracted and run on gel electrophoresis (pH>13)

Under alkaline conditions damaged DNA (strand break or adduct) is fragmented which increases the migration distance

DNA damage which results in cross links migrate less