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3.3 Cells and Radiation Cheat Sheet (DRAFT) by

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Linear­-Qu­adratic Model

For some cell lines, the survival curve appears to bend contin­uously so that the linear- quadratic relation is a better fit. In this case, n has no meaning.
This model assumes that there are two components to cell killing by radiation, one of which is propor­tional to dose and the other propor­tional to the square of the dose.
The idea is consistent with results from chromosome work in which many chromosome aberra­tions (for example, dicent­rics) are clearly the result of two separate breaks.

Hallmarks of Cancer

There are six classical hallmarks of malign­ancy:
Self suffic­iency in growth signals - malignant cells are able to grow without an external stimulus to do so.
Lack of response to growth inhibition - this is often due to loss of tumour suppressor genes, which would normally put the growth of the cell on hold.
Unlimited replic­ative capacity - normal cells may only multiply a set number of times before they become senescent (unable to divide further). Malignant cells circumvent this limit through activation of telome­rase.
Avoidance of apoptosis - normal cells trigger apoptotic pathways in response to uncont­rolled growth signal­ling. Apoptosis is often suppressed by malignant cells to avoid this fate.
Angiog­enesis - malignant tumours must form new blood vessels in order to expand locally. Angiog­enesis is also important for allowing malignant cells to metast­asise.
Invasion and Metastasis - malignant tumours invade surrou­nding normal tissues and may also spread throughout the body.
Two more were later added:
Deregu­lation of Cellular Energetics: Normal cells produce energy from glucose through glycolysis to pyruvate which then enters the citric acid cycle within the mitoch­ondria. Malignant cells upregulate glycolysis which forms an increased source of energy compared to normal aerobic cells. It can be utilised by FDG PET scanning to identify malignant cells.
Immune Avoidance: The immune system is hypoth­esised to provide protection again malignant transf­orm­ation of cells by detecting and destroying them. Malignant cells that survive to form a tumour mass must therefore have a means of immune avoidance, either by:
And two additional 'enabling charac­ter­ist­ics':
Genomic Instab­ility: The ability of malignant cells to develop a wide array of mutations in multiple oncogenes and tumour suppressor genes suggests a much higher rate of mutation than is seen in normal cells. Malignant cells have been shown to downre­gulate the normal cellular mechanisms that detect and prevent mutation, allowing them to accelerate the rate of mutation acquis­ition. The cells with the ability to mutate (or with mutations that have already been acquired) their genome to avoid destru­ction are those which survive and reprol­iferate the tumour.
Tumour Promoting Inflam­mation: Certain autoimmune condit­ions, such as ulcerative colitis or Sjogren's syndrome, promote the develo­pment of malignancy in the afflicted organ. This is due to the carcin­ogenic effects of inflam­mation on the target organ. Malignant tumours are frequently infilt­rated by cells of the immune system. It is thought that the inflam­mation caused by this infilt­ration, rather than helping to overcome the tumour, may in fact help to promote further mutation within the malign­ancy.

Cancer Cell Biology

Cancer cells are similar yet distinct to normal cells.
The features that make them different to normal cells allows them to be singled out for treatment; but these are limited by the simila­rities possessed between the cancer and normal cells.
Carcinomas (most common types of cancer), arise from the cells that cover external and internal body surfaces. Lung, breast, and colon are the most frequent cancers of this type.
Sarcomas are cancers arising from cells found in the supporting tissues of the body such as bone, cartilage, fat, connective tissue, and muscle.
Lymphomas are cancers that arise in the lymph nodes and tissues of the body's immune system.
Leukemias are cancers of the immature blood cells that grow in the bone marrow and tend to accumulate in large numbers in the bloods­tream.
Cancer arises from a loss of normal growth control. In normal tissues, the rates of new cell growth and old cell death are kept in balance. In cancer, this balance is disrupted.
This disruption can result from uncont­rolled cell growth or loss of a cell's ability to undergo cell suicide by a process called apoptosis.
This results in as gradual increase in the number of dividing cells, and creates a growing mass of tissue called a tumour or neoplasm.
If the rate of cell division is relatively rapid, and no "­sui­cid­e" signals are in place to trigger cell death, the tumour will grow quickly in size; if the cells divide more slowly, tumour growth will be slower. As more and more of these dividing cells accumu­late, the normal organi­sation of the tissue gradually becomes disrupted.
Cancers are capable of spreading throughout the body by two mechan­isms: invasion (the direct migration and penetr­ation by cancer cells into neighb­oring tissues) and metastasis (penetrate into lymphatic and blood vessels, circulate through the bloods­tream, and then invade normal tissues elsewhere in body).
Benign tumours cannot spread by invasion or metastasis (grow locally)
Malignant tumours ("ca­ncer“) are capable of spreading by invasion and metast­asis, making them a serious health problem.

Radios­ens­itivity in Radiation Therapy

When a single dose of radiation is delivered to a population of cells that are asynch­ronous, the effect will be different on cells occupying different phases of the cell cycle at the time of radiation exposure.
More cells will be killed in the sensitive portion of the cell cycle, such as those at or close to mitosis, while fewer of those in the DNA synthetic phase will be killed.
The overall effect is that a dose of radiation will, to some extent, tend to synchr­onise the cell population leaving the majority of cells in a resistant phase of the cycle.
In the clinical situation, the radiation is delivered in many separate dose fractions. In the time between these fractions, movement of cells through the cycle into more sensitive phases may be an important factor in 'sensi­tising' a cycling population of tumour cells to later doses in this treatment regime.
This process is termed sensit­isation due to reasso­rtment and it is the first of what are referred to as the five Rs of radiob­iology.

Variation of Radios­ens­itivity in the Cell Cycle

In the discussion on survival curves, we assume that the population of irradiated cells is asynch­ronous; that is, it consistes of cells distri­buted throughout all phases of the cell cycle
Techniques now make it possible to study the variation of radios­ens­itivity with the position or age of the cell in the cell cycle. These include: the mitotic harvest technique and the hydrox­yurea technique.
Mitotic Harvest Techniquecan be used for cultures that grow in monolayers attached to the surface of the growth dish.
Cells close to mitosis round up and become loosely attached. If the culture flasks are gently shaken, the mitotic cells will detach from the surface and float in the medium.
If these cells are removed and incubated in new dishes, the cells will move together synchr­onously in step through their mitotic cycle for a few cell division cycles. By delivering a dose of radiation at various times after the initial harves­tingwe can irradiate cells at various phases of the cell cycle.
Hydrox­yurea Technique involves the use of the drug hydrox­yurea.
Following the addition of the drug, cells that are in S phase (that is, synthe­sising DNA) are killedand cells that are in G2, M and G1 are halted at the end of the G1 period.
The drug is only added for a period equal to the combined G2, M and G1 time for that particular cell line. After this time, all the viable cells are poised at the end of G1 ready to enter S phase. If the drug is removed the synchr­onised cells proceed through the cell cycle. This technique can be used to produce synchr­onously dividing cell popula­tions in tissue as well as in culture.
Irradi­ation of Synchr­onously Dividing Cell Cultures
When Chinese hamster cells, harvested at mitosis, are irradiated with a single dose of x- rays at various times afterw­ards, the fraction of cells surviving varies with different phases of the cell cycle.
Following a dose of 6.6 Gy of x-rays, the surviving fraction is about 13% when the cells are in G1 and increases to more than 40% near the end of S phase.
Complete survival curves for mitotic cells (M), and for cells in G1 and G2 and for cells in early and late S can be obtained by repeating the procedure for various radiation doses. The most sensitive cells are those in M and G2 indicated by a steep curve with no shoulder. Cells in late S exhibit a survival curve that is less steep with a broad shoulder, and cells in G1 and early S are interm­ediate in sensit­ivity.
Applic­ation For Tissues
The variation in response with the phase of the cell cycle at which the radiation is given is very similar to that observed in many cells cultured in vitro. There is a radios­ens­itive period between G1 and S and maximum radior­esi­stance occurs in late S phase.
The reasons for the sensit­ivity changes through the cell cycle are not at all understood but a number of correl­ations have been observed:
1. minimum radios­ens­itivity coincides with DNA doubling in the S phase;
2. maximum radios­ens­itivity occurs just before mitosis when the chromo­somes condense; and
3. radios­ens­itivity varies with levels of naturally occurring sulfhydryl compounds (powerful radiop­rot­ectors) which are at their highest levels in S and at their lowest near mitosis.

The Cell Cycle

The Cell Cycle

Every biological species has its own sensit­ivity to ionising radiation, that is, its own radios­ens­iti­vity.
his is not the same in all phases of a cell's life; cell death requires a greater or lesser dose, depending on when in the cycle radiation is given.
The basic division of the cell cycle is into that of mitosis (M) and interphase (G1, S, G2).
Cells may also be in a special state known as G0 or ‘resting phase’, where the cell is not making any effort to divide.
We can divide the cell cycle into four recogn­isable stages:
G1 is the stage between reprod­uction episodes.
S is the stage when new DNA is synthe­sised.
G2 is the stage when certain protein and RNA molecules are synthe­sised.
M is the stage when cells, having replicated DNA and chromo­somes, divide to produce two cells from one.
Mitosis is subdivided into several events:
Prophase – The cell begins to assemble the mitotic spindle, a set of microt­ubules extending from the centro­meres which will later attach to the chromo­somes.
Promet­aphase – The nuclear envelope disint­egr­ates, and the microt­ubules of the mitotic spindle attach to the chromo­somes.
Metaphase – The chromo­somes are aligned on the mitotic spindle. There is a pause here to allow all chromo­somes to become attached.
Anaphase – The cohesion proteins which bind the sister chromatids together are cleaved and the chromo­somes are pulled apart by the mitotic spindle.
Telophase – The nuclear membrane recons­titutes around each set of chromo­somes.
The length of time required for the reprod­uction phases S, G2 and M does not vary very much among mammalian cells. It is the time between reprod­uction episodes (G1) that varies

Linear­-Qu­adratic Model cont.

A charac­ter­istic of the linear­-qu­adratic formul­ation is that the cell-s­urvival curve is contin­uously bending.
The extent of the curviness is a function of the relative values of α and β.
This does not coincide with what is observed experi­men­tally when survival curves are determined down to 7 or more decades (powers of 10) of cell killing.
Here, the curve closely approx­imates to an expone­ntial function of dose. However, in the first one or two decades of cell killing and up to any doses used as daily fractions in clinical radiot­herapy, the linear­-qu­adratic model is an adequate repres­ent­ation of the data.
Responses of tissues to radiot­herapy can be predicted from the ratio α/β

Linear­-Qu­adratic Model

Normal Cell Biology

There are two major compar­tments in the cell:
The cytoplasm contains the structures of the cell outside of the nucleus. The cell membrane forms the boundary of the cytoplasm and the cell.
The nucleus is the central, darker staining part of the cell which contains the chromo­somes.

Multi-­target Model

In this model, the survival curve is described in terms of:
1. D1, the dose required to reduce the fraction of surviving cells to 0.37 on the initial portion of the curve (singl­e-hit killing),
2. D0, the dose required to reduce survival from 0.1 to 0.037 or from 0.01 to 0.0037 on the final straight portion of the curve (multi-hit killing),
3. n, the extrap­olation number, or Dq, the quasi-­thr­eshold, which are a measure of the width of the shoulder.
If n is large (for example, 10 or 12), the survival curve has a broad shoulder. If n is small (for example, 1.5 to 2), the shoulder is narrow. A threshold dose is the dose below which radiation produces no effect, so there can be no true threshold; Dq, the quasi- threshold dose, is the closest thing.
For high-LET radiation D1 = D0 and Dq = 0, however for low-LET D1 > D0.
The value of D0 usually falls in the range of 1 to 2Gy.

High and Low LET survival curves

Cell Survival Curves

Survival curves are usually presented with dose plotted on a linear scale (x-axis) and surviving fraction on a logari­thmic scale (y-axis).
When cells are irradiated by single exposures of varying doses of high-LET radiation (for example, α-part­icles), an expone­ntial survival curve is obtained.
When cells are irradiated by single exposures of varying doses of ow-LET radiation (x-rays or γ-rays), the slope is not constant. Initially, the curve is relatively flat, but with a negative slope. This is followed by an inflection (called the shoulder) after which the curve also becomes expone­ntial.
It is difficult to explain the shape of the cell-s­urvival curve in terms of the biophy­sical events that have occurred and many theories have been proposed. Two models will be described here: Multi-­target Model and Linear­-Qu­adratic Model

Plating efficiency (PE)

Cell Culture Technique

A cell survival curve describes the relati­onship between the radiation dose and the proportion of cells that survive.
In the cell culture technique, a specimen of normal tissue is chopped into small pieces and treated with the enzyme trypsin.
This loosens the cells and separates them into a single cell suspen­sion. A known number of the single cells are plated in culture medium in a petri dish and here they attach themselves to the bottom of the dish, which is incubated at 37°C.
After about 10 days, small isolated colonies of cells are seen in the dish. These are the result of individual cells having undergone a series of cell divisions.
If the single cells are irradiated soon after they are plated, some of them will be killed and so will not produce colonies.
The ability to undergo five or more cell divisions following irradi­ation is an indication of cell survival, since these cells are capable of almost indefinite cell multip­lic­ation.
Conver­sely, cell death is indicated by a cell's inability to prolif­erate and give rise to a visible colony of some 32 - 64 cells (five or six successive doubli­ngs).
This is called reprod­uctive death (or mitotic death) to distin­guish it from death of cells that do not prolif­erate (for example, nerve, muscle, secretory cells) where cell death may be defined as loss of specific function.
The process is repeated for a range of doses and from this data, we can plot a cell-s­urvival curve. For higher radiation doses, more cells need to be plated in order to produce a statis­tically meaningful surviving fraction.

Eukaryotic Genes

A gene is a length of DNA, associated with some function, that may be inherited.
In eukaryotes (nucleated cells), a gene usually consists of:
A transc­ribed segment, which is translated into RNA for some effect:
The transc­ribed segment contains introns and exons; exons are expressed whereas introns are in between exons and are incised by splicing mechanics after transl­ation has occurred.
Each end of the transc­ribed segment contains a 5’ and 3’ untran­slated region. This may allow the cell to recognise sequences of genetic code after they have been transc­ribed into RNA.
Non-tr­ans­cribed parts, which include:
Regulatory segment(s), which are lengths of DNA allowing the cell to control which genes are expressed.
Start and stop segments which flank the transc­ribed segment, allowing transc­ription enzymes to bind.
The classical gene codes for a protein; the genetic code is transc­ribed into a strand of RNA by RNA polymerase.
This RNA, known as pre-me­ssenger RNA, undergoes splicing where the introns are removed. Once completed, the messenger RNA is transp­orted to the ribosomes in the cytoplasm for transl­ation into protein.
Other genes code for RNA products that do not require transl­ation into protein. This includes the RNA that forms the ribosome (ribosomal RNA), or micro-RNAs which are important in the post-t­ran­scr­iption control of gene expres­sion.
Gene regulatory segments need not be located immedi­ately adjacent to the transc­ribed segment. Regulatory segments can exert an influence over thousands of base pairs, and different regulatory segments can also interact to enhance (or further suppress) the transc­ription of a gene.

The Nucleus

Contains the DNA.
DNA is stored in 44 somatic and two sex chromo­somes.
may also contain a nucleolus, a site of ribosome manufa­ctu­ring. The double­-me­mbrane of the nucleus contains numerous pores which allow proteins and RNA to commun­icate with the cytoplasm.

The Cytoplasm

Contains numerous organelles which perform cellular functions:
Mitoch­ondria are energy producing organelles with their own mitoch­ondrial DNA.
Endopl­asmic reticulum is involved in the assembly of proteins. Rough endopl­asmic reticulum contains ribosomes which translate messenger RNA (Ribon­ucleic Acid) into protein.
The golgi apparatus is involved in the packaging of protein into membrane bound organe­lles, either for storage or for delivery to the cell membrane
Centrioles are small assemblies of microt­ubles arranged in a cylinder. They are important locali­sation of the chromo­somes during cell division.
The cytosk­eleton extends through the cytoplasm, attached to cell membrane proteins. The cytosk­eleton helps to cell to keep its shape and is also mobile in some cells (such as neutro­phils), altering the shape of the cell and allowing movement. Tumour cells frequently co-opt the cytosk­eleton to allow them to move through tissues.