cdkelly's blog

Chronic myelogenous leukemia is a cancer that affects the stem cells of white blood cells and causes them to become over-proliferative. These cancerous white blood cells look as if they properly differentiated, but they are not capable of fulfilling their proper function. Thus, the immune system suffers the consequences since white blood cells play such an important role. If a person with CML is left untreated, they will die in short order. Prior to the 1950s, patients would survive for an average of 2.5 years. Then, Busulfan was introduced to the market and the average life expectancy was increased to about 3.5 years. However, Busulfan was found to kill stem cells and was considered to be very detrimental to the health of the patient. Following the introduction of Busulfan, drug known as Hydroxyurea was created. It further increased the life expectancy to around 4.6 years, but also came with its own side effects. One more drug was introduced before oncologists discovered a drug that worked better than all of the others and had low side effects. This miracle drug was known as gleevec and as of 2011, it increased the survival rate of CML to 95%. Since the introduction of gleevec in 1998, the mortality rate of Chronic myelogenous leukemia has gone down dramatically, providing a ray of hope for those who suffer from the cancer.

Cells have control of where DNA replication is initiated as well how many times it is initiated. ORC (origin replication complex) initially binds to the origin of replication on the DNA and causes replication to go off in two directions. Once two replication complexes meet up in the middle, they finish their replication and dissociate. For our purposes we will assume that ORC is bound to DNA all the time. ORC binds, recruits Cdc6 (an ATPase), which then recruits other complexes, including helicase(MCM). Cdt1 brings helicase to the ORC. In S-phase, CDK phosphorylates the ORC, cdc6 and helicase. This effectively prevents the formation of the preRC (pre replication complex). This ensures that the DNA is only replicated once during one round of the cell cycle. PreRC assembly is known as origin licensing. When Rb lets go of E2F, the preRC is assembled. Phosphorylation of ORC stop cdc6 from binding, phosphorylation of cdc6 degrades it, phosphorylation of helicase cause exporitin to bind it and take it out of the nucleus. CDK is responsible for replication initiation as well as ensuring replication only happens once.

Cyclins provide binding interfaces for substrate targets, substrate specificity. Cyclins bind to CDKs and activate them. Cyclin levels rise and fall corresponding to the part of the cell cycle the cell is in! CAK (CDK activating kinase) adds phosphate to T-loop, enabling CDK to phosphorylate and activate its substrates. WEE1 kinase phosphorylates CDK at a different amino acid and holds it in an inactive state. Cdc25 removes that inactivating phosphate added to CDK by WEE1 and activates it (activating phosphatase). CDK phosphorylates Rb and causes its dissociation from E2F. Rb binds E2F, preventing it from causing over replication, when Rb is phosphorylated, it lets go of E2F. When E2F is active, the cell has committed to the cell cycle.  P53 can detect problems during replication and will act as a transcription factor for inhibitors of the cell cycle such as CDKI. It will wait until things are fixed or activate apoptosis if it goes on for too long. CDKs 1,2,4, and six are present in vertebrates. CDK1 is the most important.

The cell cycle consists of four main stages, G1 phase, S-phase, G2-phase, and M-phase. G1 is considered the start of the cell cycle and know as the growth or gap stage. In addition, G0 is the phase in which the cell stays in for the majority of its life, in a nondividing state. During G1, the cell prepares all of the various nutrients and materials needed to replicate its DNA. It can also encompass the synthesis of organelle copies and check for growth signals. At the end of G1, there is a checkpoint that checks for damaged DNA, enough nutrients, and correct growth factor signals. Following G1, the cell enters S-phase. During this phase, the cell copies all of its DNA and DNA replication can be halted to fix damaged DNA. If it cannot be repaired, then the cell undergoes apoptosis.  Next G2 phase begins and further growth proceeds as the cell prepares for chromosome segregation. The G2 checkpoint ensures that the copied DNA is not damaged or incomplete. Finally, the cell enters M-phase. Spindles, which are made up of microtubules and other proteins attach to the chromosomes and pull them apart, eventually resulting in cytokinesis and two copies of the same cell. The M-phase checkpoint ensures that chromosomes are all properly attached to the mitotic spindle. If not, CDKI prevent the function of CDKs and effectively stop the M-phase. Again, if the cell cannot fix the issue it undergoes programmed cell death.

When the chimeric gene is created as a result of a double stranded break in the DNA, it produces a chimeric protein. In the case of the Abl-Bcr chimeric gene, this lead to an overactive kinase and eventually cancer. Bcr replaces the N-terminal portion of the Abl protein, which in the wild type Abl is responsible for inactivating Abl at the right time. In addition, the CAP domain of the Abl and the myristate are not present on the chimeric Bcr-Abl gene, which normally keep Abl in the inactive state. Since Abl is normally involved in DNA damage response, regulation, and cell motility, when the chimeric form is present all of these processes are affected. Bcr-Abl does not have the ability to enter the nucleus, affecting its DNA damage response, and is hyperactive. Bcr-Abl is able to signal through additional pathways as well, including proliferative pathways. This eventually leads to CML.

When a large chromosomal rearrangement occurs, the chromosomes experience a break that leads to pieces of chromosome being swapped between one another. This causes the formation of a chimeric gene, because it incorporates two genes that are normally separate. In the case of chronic myelogenous leukemia, the chimeric gene produces a protein that actually functions. The resultant protein is a combination of the Abl kinase and Bcr kinase, creating Abl-Bcr kinase. Wild type Abl exists throughout our body, and is involved in many processes/pathways throughout the body. It is a non-receptor tyrosine kinase and is soluble in the cytosol. Abl has the ability to travel between the cytosol and the nucleus due to it having both a NLS signal and a NES signal. In addition, it also has both a SH2 domain and a SH3 domain which together work to prevent Abl from being overactive by binding to one another.

Chronic myelogenous leukemia is a cancer that affects the white blood cells and causes them to become over-proliferative. These cancerous white blood cells look as if they properly differentiated, but they are not capable of fulfilling their proper function. Thus, the immune system is negatively affected and if a person with CML is left untreated, they will die in short order. Prior to the 1950s, patients would survive for an average of 2.5 years. Then, Busulfan was introduced to the market and the average life expectancy was increased to about 4 years. However, Busulfan was found to kill stem cells and considered to be very detrimental to the health of the patient. Following the introduction of Busulfan, drug known as Hydroxyurea was created. It further increased the life expectancy to around 4.6 years, but also came with its own side effects. One more drug was introduced before oncologists discovered a drug that worked better than all of the others and had low side effects. This miracle drug was known as gleevec and as of 2011, it increased the survival rate of CML to 95%.

After allowing four days to elapse, all the data pertaining to the post-experiment web mass was collected. Values were compared to the original data collected for web mass. We observed that the condition with the lowest amount of web mass at the conclusion of the experiment was the cold condition. Furthermore, the warm and control conditions did not vary significantly in terms of the amount of web mass. The observation of the cold condition resulting in the lowest amount of web production had some confounding variables. Mainly, one of the spiders escaped its enclosure on the third day of the experiment. This would certainly lead to less web production since the spider was not present to produce web on the final day of the experiment. In addition, the ice replacement was relatively inconsistent due to time constraints. However, the other three spiders completed the entire experiment and the effect was still observed among those three.

The ice replacement was not as consistent throughout the experiment as we would have liked, and we believe this could have had an effect on the results. Due to the fluctuations in ice replacement and amount of ice added, the consistency of the temperature in the chambers was partially sacrificed. We were able to obtain a temperature reading from three of the four days of the experiment and consequently have an average temperature for the condition. But due to the methods of temperature reading acquisition and instrument used, we do not consider the values to be absolute. This takes away from the result of our experiment and for any future experiments of similar vein. To correct it, we would need to ensure a more consistent ice refresh schedule and implementation of better instruments to constantly observes the temperature as compared to periodically.

Other alterations to the design of the experiment that would be beneficial to the results include a more streamlined environment chamber design and a more consistent ice refreshing procedure. In particular, the cold condition chamber presented some challenges over the course of the experiment that were likely the reason one of the spiders escaped. Because ice was melting within the chamber at any given time, moisture was also accumulating within the environmental chamber. Although this was occuring at a slower rate compared to normal conditions due to the insulatory properties of the styrofoam box, it was still occuring. This in conjunction with ice being added periodically to the chamber lead to the deformation of the divider platform suspending the enclosures within the chamber. We believe that this created an opening and allowed one of the spiders to escape from its respective enclosure. To compensate for this, changes to the design and material for the cold condition environmental chamber would have to be made.