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The Biology of Retinoblastoma

The Biology of Retinoblastoma

Abstract :
Retinoblastoma (RB) is one of the most common intraocular malignancy tumors that occur during childhood and infancy. There are two forms: heritable and non-heritable. The RB gene is located on chromosome 13 and it produces a protein (pRb) responsible for the regulation of the cell cycle. The theory of “the two hits” explains the occurrence of this disease. Several types of mutation can inactivate both alleles of the retinoblastoma gene and therefore cause a tumor. Point mutations are the most commons type of mutations associated with retinoblastoma. According to its size and localization, tumors are classified by groups. The diagnosis of the disease is essentially clinical. Multiple treatments exist such as chemotherapy, laser, radiotherapy, external beam and as a last resource enucleation. Nowadays, clinicians are trying to optimize its treatment by reinforcing the efficiency of directed chemotherapy with minimal side effects. In the future, a better understanding of the molecular biology of retinoblastoma will allow us to improved therapeutic treatments.

Introduction :
Retinoblastoma (RB) is a disease of early childhood, most of the cases are discovered by the age of 5.
It affects the retina of the child in one eye (unilateral) or in both eyes (multilateral). Studies have shown that disease incidence is around 1 in 20000 live births.
Overall, 40% of the retinoblastoma patients have hereditary retinoblastoma, the other 60% have non-hereditary retinoblastoma. (In the hereditary form, the two eyes are usually affected whereas in the non-hereditary form only one eye is touched.)
In both cases, the most commons symptoms are leukocoria (a white reflex of the pupil) and strabismus (squint eyes). Leukocoria is only visible under certain light conditions. In some cases, parents find out their child have leukocoria by taking pictures of them. Indeed, in case of leukocoria the fight of the flash colours the eye in white instead of red. As for strabismus, it is easy to diagnose and it reflects the weakness of the vision.
The people affected by retinoblastoma have an abnormal karyotype, with a mutation of the Rb1 gene in the locus 14.2 of chromosome 13. The earlier the diagnostic of the disease, the more the patient maintains his vision and lives a safe life.
The RB1 gene and its protein action on the cell cycle:
As previously mentioned, the Rb1 gene is located at the locus 14.2 of the chromosome 13. It is a tumor suppressor gene composed of 27 exons. Tumor suppressor genes encode molecules involved in the regulation of the cell cycle. There are two tumor suppressor genes that regulate directly the cell cycle: the retinoblastoma gene and the p53 gene.
The Rb gene produces the Rb protein (pRb) responsible of the regulation of the cell cycle. The cell cycle consists of 4 phases: G1, S, G2, and the mitosis. At the end of a cell cycle the DNA of the cell will be replicated. This process is true for each of nucleated cells of the body. When the cells are not cycling they are at phase G0 so to re-enter the cycle the cells need to received intra and extracellular signals. When the cell reaches the checkpoint G1 to enter the S phase it needs to build the proteins or enzymes necessary for the DNA replication. To do so, the protein E2F, binds to the DNA sequence to allow its transcription. This protein E2F, also called a transcription factor, binds itself to the pRB protein to form a complex (1) The function of the pRB protein is to inactivate the E2F protein in order to stop the replication of the cell DNA, as a consequence, the cell cycle doesn’t reach the G1 checking point. For the cells to divide further, the pRB protein needs to be inactivated. This is achieved through a process called phosphorylation, during which a phosphate group is added to the protein. The transcription factor E2F can then allow the cells to go to the phase S, and controls the rest of the cell division.
However, if the gene Rb is mutated, then the pRB protein formed might not be recognized. Therefore, it won’t combined with the E2F protein, and this transcription factor will be allowed to move the cells directly to phase S with an uncontrolled cell division.
Mutation
In the 70’s Alfred Knudson made a theory called « the two hit » where he proposed that in the inherited form of Rb, one mutation is inherited via one of the parents: this is called the « first hit », and the second occurs in somatic cells. In the non-hereditary form, both mutations occur in somatic cells randomly. The « two hits » means that the tumor formation occurs after two mutations. They are two alleles in the Rb gene in every nucleated cell of the body (2).
In the hereditary form, one of the parents carries a mutated non-functioning Rb gene that is transmitted to the child. Every cell of the child’s body will therefore be carrying one faulty copy of the Rb gene. The cells of the child will only develop the tumor after the remaining other normal copy is mutated, which corresponds to the « second hit ». (figure 1) (3)

Figure 1: The genetic mechanisms that cause retinoblastoma
We can see here that in the hereditary RB, one of the parents has a mutated non-functional RB gene that is transmitted to the child. The remaining correct, or non-altered, copy is then inactivated by a random event (a mutation or an epigenetic event). The cell proliferation will then lead to the development of retinoblastoma.
In the non-hereditary form, the person has two normal copies of the gene. Two mutations are required for retinoblastoma to happen, which is rare.
In a normal individual, a cell can inactivate one of the two goods copies but this is not enough to lead to retinoblastoma.
Source: Molecular Biology of the Cell (6th edition) (3)
In the non-hereditary form, one cell inactivates one of its two RB genes. Later on, second hit will inactivate the second good copy of the RB gene, leading to retinoblastoma.
In the non-hereditary form, the person has two normal copies of the gene. Two mutations are required for retinoblastoma to happen, which is rare.
In a normal healthy individual, a cell can inactivate one of the two copies of its RB gene but as we described before, two mutations need to happen for someone to get retinoblastoma.
The occurrence of retinoblastoma both in the hereditary and non-hereditary RB is the result of multiple categories of mutations such as nonsense, frameshift, splice, missense or epigenetic mutations or even to a mutation in the promoter sequence of the gene.
The possible mutations are regrouped in the following table: (figure 2)

Figure 2 : Genes Mutations in the RB gene
The most frequent mutation is a substitution that leads to premature termination or splicing or missense. We can also have deletions mutations or less frequently, insertions.
Source : RB1 Gene Mutations in Retinoblastoma (4)
The majority of mutations identified in the hereditary form of RB are point nonsense mutations. They are distributed over the 27 exons or in the promoter sequence of the Rb gene. Most of the time there is a point mutation from a C nucleotide to a T nucleotide. A point mutation induces a premature stop codon. In this case, a CGA (Arginine) becomes a TGA (a stop codon). This premature arrest of the making pRB protein results in a shorter protein that may not function. (4).
Diagnostic/classification of the disease
The diagnostic of a child with suspected retinoblastoma is accomplished by some ocular examination, a test that allows the ophthalmologist to see inside the eye with an ophthalmoscope. An ultrasonography can also help to detect the mass of the tumor. The international system of classification of the retinoblastoma is based on the size of the tumor as well as on its localization. The type of retinoblastoma determines the treatment choice. There are several groups of disease: group A corresponds to a retinoblastoma tumor size of up to 3 mm, group B is for tumor bigger than 3 mm, group C: retinoblastoma with localized seeds, group D: retinoblastoma with diffuse seeds and group E: retinoblastoma that needs enucleation, the removal of the eye (5).
Treatments
Retinoblastoma has evolved throughout the years. Before it was a deadly childhood cancer, now currents treatments are evolving to provide the best vision possible to the patient.
There are several kinds of treatments for retinoblastoma (6).
Chemotherapy aims to reduce to a smaller size the tumor so that a laser treatment can then be effective. Laser treatment is generally for tumors that have been shrunk by chemotherapy. The laser is pointed on the dilated pupil using an indirect ophthalmoscope. There are two types of lasers ; one is a 532nM green light and the other is an 810nM infrared light. The second one is preferred to heat the tumor slowly. Cryotherapy is the use of a probe in the sclera of the eye. It produces low freezing temperature to kill the tumor. This is usually used for large peripheral or closed retina tumors. There is also radiotherapy, preferably, used when the tumor is not responsive to any treatments. It uses an external beam therapy or a radioactive plaque and can have important consequences such as other malignancies within the radiation field or dry eyes. Finally, enucleation, the oldest treatment of retinoblastoma, is curative for uniocular retinoblastoma or for the worse bilateral cases. It is reserved for extensive retinoblastoma where there is no hope to keep vision. The eye is removed and sends to histology to study the DNA sequence of the tumor (6). A prosthesis is then inserted to replace the shape of the eye.

Future treatment
Over the last few years, researchers tried to focus on developing new delivery chemotherapy methods with fewer side effects. For example, they develop techniques based on periocular injections of carboplatin and other agents in order to increase the efficacy of the drug used. One of the most promoting advances is the interventional radiological technique of intra-arterial chemotherapy (IAC). This technique allows the delivery of a chemotherapeutic agent directly into the ophthalmic artery, with few side effects. Current researches are directed towards gene therapy, stem cells and microarray technology to prevent tumor apparition.
Conclusion
Retinoblastoma is a child tumor that can be genetic or non-genetic: it can affect one or both eyes. The most common symptoms are leukocoria and squint. Nowadays, multiple treatments exist as the choice of therapy depends on the tumor type whether the diagnosis is done early or not.
References
Yannek I. Leiderman, Szilárd Kiss,Shizuo Mukai, Molecular Genetics of RB1—-The Retinoblastoma Gene, Seminars in Ophthalmology, 2007, 22:4, 247-254, Available from DOI: 10.1080/08820530701745165
Milam A. Brantley, Jr.

Effect of Sugar Concentration on Respiration in Yeast

APPLIED SCIENCE-Biology
RATE OF RESPIRATION
Respiration is the set of which processes and metabolic reactions take place in cells of organisms; these metabolic reactions and processes can also be the core of producing adenosine triphosphate (ATP) rather than breaking it down.
The simpler word equation can be written as:
Glucose Oxygen  Carbon Dioxide water ATP
The balanced equation for cellular respiration can be written as:
C6H12O6 6O2  6CO2 6H2O ATP
This ATP synthesised during respiration can be used in a plethora of ways within organisms such as; growth and repair in cells, nerve impulses, active transport of molecules or ions and even muscle contraction.
The two types of respiration are aerobicandanaerobic.
Aerobic respiration is the process of which 36 ATP is produced due to multistep processes (Glycolysis, Citric Acid Cycle and the Electron Transfer Chain).This type of respiration is key as it takes place in the presence of Oxygen, produces large amounts of useful energy but Carbon dioxide and water are produced as waste products. A prime example of aerobic respiration is a long distance race, this is because a large amount of energy will be produced during key stages of a race rather than one quick burst, which would leave the athlete tired. The long distance race in summary is a cardio exercise so the heart must maintain a steady rate of roughly 60%to 80% of it maximum to sustain oxygen for shared muscle power. Glucose Oxygen  Carbon Dioxide water ATP
Anaerobicrespiration is the process by which only 2 ATP is produced due to the process of only glycolysis due to the absence of Oxygen. It also is the process of which glucose is broken down without oxygen, this results in the production of lactic acid rather than Carbon Dioxide and water. A Prime example is an athlete completing a short distance sprint, this means they’ll needed to contract and release muscles really quickly, as a result, due to the lack of oxygen, anaerobic respiration takes place. In most cases the heart rate would increase to a significantly high number but only for a short period of time. Glucose Enzymes  Carbon Dioxide Ethanol
Aerobic
Anaerobic
Long term
Organism and body cells
No lactic acid
38 glucose molecules
Short term
Muscle and red blood cells
Lactic Acid
2 Glucose molecules
As you may have noticed in the anaerobic word equation, ethanol is produced. This can also take place in microorganisms such as yeast cells. This can also be referred to as fermentation.
Ways of measuring the rate of respiration
A respiratory meter can be used to measure the rate of respiration of an organism by keeping track of its exchange of Carbon dioxide and Oxygen. It also considers factors such as age, and the effect of light.
Figure 1 is an example of the use of a respiratory meter to measure the rate of respiration. In summary, as the organism’s intake oxygen from the air available, they give out Carbon Dioxide, this results in the manometer fluid rising towards the organisms as the pressure and volume is decreased. The manometer with no organisms would be expected to not move but it could due to temperature changes. By measuring the distance moved by the liquid in the manometer in a period of time, it can give you the rate of respiration of an organism, repeating this multiple times can result in a more precise and accurate verdict.
An investigation of the effect of sugar concentration on respiration in yeast.
Introduction
Yeast is a single-celled fungus. It can respire aerobically and anaerobically. During aerobic respiration, CO2is produced. In bread-making, the yeast starts off respiring aerobically, producing water and also carbon dioxide to make the dough rise. When the air runs out, the yeast begins to respire anaerobically producing ethanol and continuing to produce CO2.
My group carried out the experiment with the given sugar concentrations three times in total.
Sugar Carbon Dioxide Alcohol Energy to make ATP
Materials Needed
• Glucose to dissolve to the correct concentrations of solutions (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100/ litre)
• Dry yeast (7g per repetition).
• Measuring Cylinder.
• Conical flask 100ml.
• Gas syringes to measure CO2 production.
• Marker pen.
• Timer.
Safety precautions.
Contains small parts. Do not allow children under the age of seven to have access to any kit components.
Never taste or ingest any materials provided.
Do not eat, drink, and apply make-up or contact lenses while performing experiments.
Wash your hands before and after performing experiments.
Goggles and gloves should be worn.
Variables
Dependent variable: Total vol of gas produced
Independent variable: Glucose concentration
Fixed (or control) variables: Mass of Yeast
Hypothesis
As the concentration of glucose increases within a conical flask, the volume of gas produced will increase gradually.
Method
During this experiment we worked collectively, each individual repeated a maximum of two glucose concentrations 3 times and shared the results with the class – others relied heavily on our results Method.
1. Dissolve the correct amount of glucose into 100ml of water. Label your conical flask your glucose concentration (= ……..g/l).
2. Set up your equipment as shown below in the diagram but do not connect up the rubber bung.

3. Start your timer as you add the 7g of dried yeast directly to the sugar solution in the conical flask (yeast 7%) after 3 minutes seal with the bung and continue timing for 10 minutes. Remember to check the temp of the water bath ~35oC.
4. Measure the gas produced in 10 minutes recording the volume every minute. If the syringe fills quickly, carefully remove the bung push the syringe back and replace the bung. Remembering the volume produced previously.
5. Record the total volume of gas produced in your table.
6. Repeat steps 2-5 for the 2nd and 3rd repeats
7. Collect data from the other class members to complete the table of results.

Individual results
Sugar concentration ……….. (g/l)
Vol of gas produced (cm3) each minute
Total vol of gas produced (cm3)
0
1
2
3
4
5
6
7
8
9
10
rep 1
0
0
0
0
0
0
0
0
0
0
0
0
0
rep 2
0
2
2
2
2
2
2
2
2
2
2
2
0
rep 3
0
3
3
3
3
3
3
3
3
3
3
3
0
Average vol of gas produced
0
Sugar concentration ……….. (g/l)
Vol of gas produced (cm3) each minute
Total vol of gas produced (cm3)
0
1
2
3
4
5
6
7
8
9
10
rep 1
50
1
2
2
10
19
19
32
35
43
48
55
54.00
rep 2
50
4
9
14
22
30
36
54
69
76
89
98
94.00
rep 3
50
2
5
9
14
14
24
30
36
47
55
64
62.00
Average vol of gas produced
70.00
Class pool Results Conclusion

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