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What is cancer?
Cancer is cell growth out of control, causing the development of tumours which disrupt normal body function. Cancer may
occur in any part of the body and tumours may be solid masses of cells such as a breast cancer tumour, or they may be liquid
tumours consisting of blood cells that have become cancerous such as in lymphoma and leukaemia.
The cell cycle (Fig 1) controls growth and reproduction in cells.
Healthy cells have a limited life span and respond to signals
which regulate cell growth and division. Cells will replicate a
certain number of times and then go through programmed
cell death (apoptosis), to be replaced by new cells. Unlike
normal cells, cancer cells are immortal. They are not
responding correctly the regulatory signals and will go on
reproducing themselves over and over again. This fast rate of
reproduction creates excess cells, forming tumours.
Mitosis
The cell divides to
produce two
identical daughter
cells.
Gap 2
Gap 1
The cell grows, produces
RNA, & synthesises
proteins. A check system
ensures that the cell is
ready for DNA synthesis.
If the check finds that
there is a problem, the
cell will be destroyed.
Synthesis
The DNA is copied in
readiness for cell replication.
Identical copies of all
chromosomes are
synthesised.
Uncontrolled
Growth
Evading
Death
Promoting
Mutations
1. Uncontrolled Growth
Normal cells grow or stop growing in response to
signalsstart or stop. Cancer cells do not
respond to these signals they will grow when
Angiogenesis
there are no growth signals and will continue to
grow when they are getting stop signals. So no
matter what colour the traffic lights are they will Fig 2. The Hallmarks of
Cancer (After Hanahan &
go!
Weinberg, 2000)
2. Evading Death
Invading Tissues
& Avoiding
Detection
Becoming
Immortal
4. Becoming Immortal
Normal cells have a limited number of times that they can divide before they stop growth. Sequences of DNA on the ends
of chromosomes called telomeres are responsible for making sure that chromosomes do not fuse end-to-end. Each time a
cell divides, the telomeric DNA get slightly shorter. When it gets so short that it cannot protect the chromosome the cell
dies. In cancer cells the temoreric DNA does not get shorter because an enzyme called telomerase is released which
lengthens the telomeres so the cell can keep on replicating endlessly.
6. Promoting Mutations
The development of cancer requires an accumulation of mutations in a number of genes over a period of time. Some of
these changes accelerate the rate at which mutations occurmeaning that mutations are acquired at a faster rate.
Uncontrolled
Growth
Cancer Cells
Normal Cell
Invasion
Angiogenesis
Capillary
Poor
Prognosis
Metastasis
Technology
Gene Profiling
About 5-10% of cancer patients inherit a genetic defect that gives them a susceptibility to cancer
over their lifetimes. Many years of research using a number of biotechnologies went into achieving
this understanding for example: PCR, DNA sequencing and gene mapping. Gene profiling allows
scientists to identify individuals that carry specific alleles that increase their risk of developing cancer
in their lifetime.
Scientists have been able to study gene expression one gene at a time for many years using PCR
technology. Primers specific to the gene of interest were used in the PCR mix to find out whether
that gene was being expressed in the tissue.
Microarrays are used to compare the expression levels of thousands of genes all at the same time,
enabling scientists to study a genetic profile. The microarray technology can identify which specific
genes a cell is using at a particular point in time. This means that we can compare which genes are
turned on or off in different conditions (e.g. when cancer is present compared to when cancer is
absent).
Genome Analysis
The information from the microarray gives an overview of which genes are turned on or off, over or
under expressed. To confirm how much more or less a gene is being expressed in a cancer cell line
Real Time PCR technology is used. This is using PCR with primers specific to the gene as usual but a
real time PCR allows scientists to further quantify the change in gene expression.
Often the microarray analysis will identify genes that are already known to have a role in cancer.
However, sometimes an experiment will identify a known gene which has not previously been
associated with cancer. This is useful information as it identifies new targets for cancer therapy.
Cell Culture:
Making & culturing
transgenic cell lines
Human cells, including cancer cells, can be cultured outside the human body in plastic flasks kept in
incubators. There are many genes that scientists have noted are expressed differently in cancer cells.
Once a gene has been identified it can be introduced to a cell line and the subsequent effects on the
cells studied by growing these cells in cell culture.
The gene must first be cloned and inserted into a plasmid vector and then amplified in bacteria. The
vector is then transferred into mammalian cells using cell transfection technologies such as
liposomes. These cells are incubated at human body temperatures and supplied with nutrients. By
comparing the gene expression in these cells with control cells scientists can determine the changes
in the cells that have been caused by the inserted gene.
Fig 5: PCR and Gel Electrophoresis are used to study the expression of genes
in individual tumour samples. These gel photos show Beta-Actin, a house
keeping gene that is used as a loading control and two genes of interest hGH
and the cell surface receptor for hGH, hGHR.
Microarray technology is used to scan 19,000 genes to look for differences in gene expression
What is on the microarray chip?
A microarray is a small glass slide
that contains tiny fragments of
known DNA sequences in different
spots on a slide. A Human Genome
microarray will contain small
fragments of each of the genes in
the genome. They are made
robotically, with DNA probes being
attached on vertical stacks onto the
glass slide.
Each spot on the slide contains
multiple copies of the same probe.
When the cDNA solution is washed
over the slide, the fluorescently
labeled cDNA pieces that match the
complimentary base pairs on the
slide will bond. When the slide is washed, the bonded
cDNA fragments will remain in place and the other
fragments will wash away. The fluorescent spots on
the slide are read using computer technology.
Reading a Microarray
Red Spots
Green Spots
Fig 11: The microarray image produced from the Liggins Institute
Human Growth Hormone Study
http://LENS.auckland.ac.nz