Background on mitosis movies

The project

In this project you will be provided with a culture of human cancer cells. You will treat these cells with anti-cancer drugs which interfere with cell division and then image them over a 24 hour period. You will then combine the images into a “mitosis movie” and analyse the movie to observe the effects of the drugs on the cells.

Getting started

Before you begin, make sure that you are familiar with the relevant theory behind the techniques we will be performing. There are several appendices which will provide you with this information :

• DNA
• DNA replication
• Using a micropipette
• Using a biological safety cabinet
• Glossary of terms

Make sure you read this information before proceeding. Additional information about cell and molecular biology can be found here.

Theoretical basis of the project

The cell cycle and cell division

The cell cycle represents the normal progression of cells through the division cycle to produce two identical daughter cells. It consists of four discrete stages (See Figure 1):

Figure 1 - The Cell Cycle

• G1 (Gap 1) phase occurs just after division. It is where the cell carries out normal metabolism and begins to grow in size and duplicate its organelles. Some cells arrest in G1 phase, staying in a stage called G0 phase and ceasing the cycle of cell division.

• S (Synthesis) phase represents the time where the genomic DNA is duplicated.

• G2 (Gap 2) phase is the time where the cell prepares for division.

Collectively, G1, S and G2 phases are known as Interphase.

• M (Mitosis) phase is the time when the replicated chromosomes are divided (Prophase, Prometaphase, Metaphase, Anaphase and Telophase (See Figure 2) and the cell splits into two identical daughter cells (Cytokinesis).

The aim of mitosis is to evenly divide the chromosomes replicated during S phase between the two daughter cells. By the end of S phase, each chromosome consists of a pair of chromatids, joined in the middle by a body called the centromere. During mitosis, it is these chromatids which separate.

The cell assembles several structures which allows the separation of chromatids to occur. The centrosomes are small bodies composed of protein microtubules. Centrosomes are replicated during S phase alongside the DNA. In prophase, the centrosomes migrate to opposite ends of the cell and form the anchor points of the mitotic spindle, a bundle of microtubules which runs between the two centrosomes. In metaphase, the chromosomes attach to the middle of the mitotic spindle at the centromere, via structures called the kinetochores. In anaphase, the kinetochores move towards the centrosomes, dragging the chromatids with them. In cytokinesis, the cell pinches off into two separate daughter cells, each one having a full complement of chromatids.

Normally, progression through the cell cycle is controlled by a series of checkpoints. If a cell cannot meet the conditions needed at each checkpoint, the cycle arrests at that point. This prevents cells being duplicated with significant errors. The major checkpoints occur at the G1/S interface (to ensure that the cell is ready to start DNA duplication), the G2/M interface (to ensure that DNA has been copied without major errors) and at the end of mitosis before the daughter cells divide (to ensure that chromosome separation has occurred correctly). If a cell is found to have problems at any one of these checkpoints (eg. damage to the DNA or an inappropriate number of chromosomes), the cell either repairs the mistake or, if this is not possible, the cell arrests at that point in the cell cycle or even undergoes a process of controlled cell death called apoptosis.

Cancer and the cell cycle

It is important to remember that most cells spend the majority of their time arrested in G0 phase. This is the time where the cells perform their normal functions. Entry into S phase (and ultimately G2 and M phases) only occurs if the cell needs to divide. The majority of body cells do not need to constantly divide – doing so would interfere with their normal function. Therefore most cells stop dividing after a certain number of generations. Replacing these cells is left to specific stem cells which are capable of dividing to produce body cells or more stem cells. Only in parts of the body where there is a high cell turnover (eg. on the body surfaces, in the parts of the bone marrow where blood cells are made, in the hair follicles and parts of the reproductive organs, or after damage has occurred) does one find high rates of cell division.

The collection of diseases called cancer result from a disturbance to this normal order. In cancer, the normal signals which limit cell proliferation are either absent or ignored, and cells spend a larger proportion of time progressing through the cell cycle and dividing. This can occur following mutations (changes in the genetic sequence) or damage to the DNA, particularly when this occurs in the genes which code for proteins which regulate the cell cycle. As a result, studying how the cell cycle is normally regulated is a major step in understanding why cancer develops and how it may be treated.

The cell cycle checkpoints are not physical barriers, but points in the cell cycle where the levels of regulatory proteins build up to a certain level. Each of these regulatory proteins can stimulate or inhibit the expression or function of other regulatory proteins, resulting in a complex feedback system. If something goes wrong with one of these regulatory proteins, it may affect the function of the others, and therefore the whole regulation process.

For example, in the lead-up to the G2/M checkpoint, the cell has just duplicated its DNA and certain cell structures such as the centrosome, which acts as the anchor point for the mitotic spindle. The process of DNA replication is a complex one and, on occasion, substitutions of nucleotide bases may occur. In addition, the cell’s DNA may be damaged by chemical or physical factors. For example, ultraviolet light may cause two adjacent thymidine nucleotides to chemically bond together, resulting in a double molecule. Since the polymerase enzymes that replicate DNA use each strand as a template for the formation of new DNA, this “thymine dimer” does not provide the necessary information to the polymerases for them to insert the correct complementary bases (in this case it would be two adenosine nucleotides) and a mutation is generated.

If a checking and correction mechanism was not present, any mistakes in the DNA would be passed on to the daughter cells. At the G2/M checkpoint, the cell checks the DNA to ensure that no such mistakes are present. If any are found, enzymes in the cell try to repair the damage. The cell pauses in the cell cycle at this point until the damage is repaired, whereupon the cell is released into mitosis. If the damage cannot be solved, the cell is not permitted to enter mitosis, thus limiting the problems associated with passing on these errors.

Problems arise when the errors occur in the genes involved in this regulatory process. If the proteins whose task it is to check the DNA, repair any damage or prevent the cell from progressing into mitosis until the damaged is repaired are themselves faulty, cells will be admitted into mitosis with the errors still in place and the cycle will continue. One of the hallmarks of cancer is hyperproliferation, where cells continually divide because the regulatory processes have been impaired. Cells which are constantly dividing do not carry out their normal function (eg. in leukaemia, a cancerous transformation of the stem cells which give rise to the white blood cells, a large number of white blood cells are produced, however these never mature to carry out their normal role of defending the body. Therefore people with leukaemia may be at a higher risk of developing infections).

Some external factors may interfere with the normal functioning of the regulatory process. The human papillomavirus has a pair of oncogenes (genes associated with the development of cancer). When the virus incorporates these into the genome of the host cell and they are expressed, the proteins produced inhibit the regulatory processes and the cells progress through the cell cycle. This suits the purposes of the virus, as it requires actively dividing cells in order to proliferate. However, the loss of cell cycle regulation means that more errors in DNA are allowed to pass and the cells accumulate enough of these mistakes to transform into cancerous cells.

Another characteristic of cancer is aneuploidy, where cells have an inappropriate distribution of chromosomes. This can occur in damaged strands of DNA which fragment during division. Under some circumstances the mechanisms which govern cell division may also be affected. In normal cells, the centrosome is replicated during S phase, and each centrosome migrates to opposite ends of the cell to create the mitotic spindle. When the daughter cells pinch off during cytokinesis, each cell should only have one centrosome. Sometimes an extra centrosome is retained or replicated in the cell, resulting in a mitotic spindle with three or even four poles. This means that when the chromatids are separated during anaphase, they may be pulled in 3 or even four different directions. The daughter cells which result from this sort of division are highly likely to be aneuploid and therefore lack important genes.

Cancer treatment – Chemotherapy and radiotherapy

Treating cancer is different to treating other diseases. If the body is being attacked by a foreign organism, we can target therapies which take advantage of how that organism is different to us. For example, some antibiotics like the penicillin family target bacterial cell walls, a structure lacking in our cells. With cancer, the disease agent is one that is derived from our own cells. To effectively treat cancer, we use therapies which target cellular functions that cancer cells do more than our normal body cells do – like constantly pass through the cell cycle.

Radiotherapy is the use of radiation to target cancer cells. It relies on the idea that certain types of ionizing radiation damages DNA. In normal cells this results in damage which either the cells can repair or a relatively low level of cell death, whereas in rapidly dividing cancer cells without functioning regulatory mechanisms and checkpoints, the damage is highly lethal. The principle is therefore to hit as many rapidly dividing cells as hard as possible, with the reasoning that the majority of these will be the cancer cells we want to kill. Unfortunately, the body also has rapidly dividing cells which are not cancerous, and so the side effects of radiotherapy may include symptoms which reflect this - anaemia and low white cell count as blood stem cells are affected, gastrointestinal upset as the cells lining the digestive tract are affected, loss of hair as the cells in the hair follicle are killed. Using radiotherapy therefore involves carefully targeting and control of the dose given to ensure that the number of normal cells lost is minimal.

Chemotherapy is the use of chemical drugs to target cancer cells. While some chemotherapeutic drugs use the same approach as radiotherapy (ie. targeting rapidly dividing cells), other drugs target systems more specific to the cancer cells. For example, a certain subset of breast cancers are stimulated to grow by the oestrogen hormones. Tamoxifen is a drug that blocks this stimulus by simulating the chemical structure of the oestrogens, thus slowing the progression of the cancer. However, not all oestrogen responsive cells are cancerous, and the drug still has some side effects. More recently, research has turned its attention to developing drugs which specifically target what has gone wrong in the normal regulatory processes of the cell cycle. These new agents may inhibit the expression of factors which promote uncontrolled cell proliferation, or they may stimulate or even mimic the regulatory proteins which limit progression through the cell cycle. The effects of these drugs can be seen by studying what happens to the cells as they divide.

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Investigating the effects of chemotherapeutic drugs using mitosis movies

Cancer cell lines are commonly used by cell biologists as models for how cells function. They have the benefit of being “immortal” (ie. they constantly pass through the cell cycle), so fresh cells do not need to be sourced from new tissue. The cancer cell line you will be using are called “HeLa”. These cells are derived from a tumour removed from a woman named Henrietta Lacks in the early 1950s, and were one of the first widely used cancer cell lines. HeLa cells are robust and serve as an effective model system for both cancer investigations and other cell biology studies. The HeLa cells you will be using have been genetically modified to contain a green fluorescent protein tag on one of its histones. In the nucleus, the DNA wraps around these proteins, so the green fluorescent signal can be used as an indication of where the DNA is in the cell. Under blue light, the nuclei of these cells emit a green fluorescence.

The drugs you will be using cover a range of modes of action, although each of them is effective in slowing cell proliferation :

• Taxol – derived from an extract of the Yew tree (Taxus brevifola), Taxol has been used as a chemotherapeutic agent for nearly 40 years. Taxol stabilizes the microtubules which form the mitotic spindle. The process of spindle formation is normally a dynamic process, involving the assembly of breakdown of the microtubules. In the presence of Taxol, this process is interrupted and the chromosomes cannot arrange appropriately on the spindle. Cells treated with Taxol tend to arrest at the M Phase checkpoint and often undergo apoptosis before cell division is complete.

• PLK1 Inhibitor – PLK1 (polo-like kinase 1) is an enzyme involved in the formation and maintenance of the mitotic spindle. Overexpression of the gene for PLK1 has been seen in cancerous cells, suggesting that it has a role in tumorigenesis. Conversely, removal of PLK1 from systems encourages cells to arrest in mitosis and enter apoptosis. This drug inhibits the action of PLK1, resulting in increased cell death.

• SBHA (Suberoyl bishydroxamic acid) – is a histone deacetylase inhibitor. These drugs work by promoting the expression of genes which inhibit the processes which lead to a loss of cell cycle regulation. Cells treated with histone deacetylase inhibitors undergo an aberrant mitosis and cytokinesis then  tend to undergo apoptosis.

• ICRF-193 - This drug inhibits the action of topoisomerase II, an enzyme involved in the repair and maintenance of the DNA strand. As errors are found in the DNA, damaged parts are cut out of the DNA strand and the two ends of the strands joined back together – both of these functions are carried out by topoisomerase enzymes. ICRF-193 prevents topoisomerase II from re-joining the DNA strands, resulting in fragmentation of the DNA. This causes the cells to arrest at the G2/M checkpoint and then enter mitosis where they may fail to divide their replicated genomes.

In addition to the four known drugs, you will also investigate the action of a new drug which has a similar mode of action to the others. By observing how this drug affects the cells, you should be able to classify it as being related to one of these drugs.