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Aim Of This Experiment

Aim

The aim of this experiment was to determine the sensitivity of both human myeloid and lymphoid leukaemia cells to a cytotoxic drug used for leukaemia treatment in comparison to each other. Introduction

Leukaemia is a ‘cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream.’ (1)

It originates from malignant immature precursors and can severely affect many body functions including the immune system. Sufferers of Leukaemia undergo chemotherapy which involves administering cytotoxic drugs; a group of drugs containing chemicals that prevent the replication and growth of cells and therefore kill them. Healthy cells are significantly less sensitive to cytotoxic drugs than malignant cells.

In a healthy person, the body produces blood stem cells that mature over time. The stem cells can either become myeloid or lymphoid stem cells. When a myeloid blood stem cell matures, it develops into a red blood cell, white blood cell or platelets, all of which have different functions. Lymphoid stem cells develop into lymphocytes; B, T or natural killer cells. T-helper cells express the CD4 glycoproteins on their surfaces and assist other white blood cells in an immune response. Cytotoxic T cells express the CD8 glycoprotein and work to destroy tumour viral infected cells. However acute myeloid leukaemia is ‘a cancer in which the bone marrow makes abnormal myeloblasts, red blood cells or platelets’ (2). When immature white blood cells are produced, they haven’t developed the properties that allow white blood cells to fight infections and pathogens. When too many are in the blood, it can reduce the quantity of oxygen carrying red blood cells as well as blood platelets essential for blood clotting. It leaves the body open to an increased risk of infection. Lymphocytic leukaemia is when the bone marrow produces an excessive number of lymphocytes and affects red blood cells and platelets.

Principle of the Method

MTS viability colorimetric assay is a ‘method for sensitive quantification of viable cells in proliferation and cytotoxicity assay.’ (3) MTS tetrazolium is reduced by viable cells and results in a coloured formazan product, soluble in cell culture media. ‘The reduction reaction is carried out by NAD(P) H-dependent dehydrogenase enzymes in metabolically active cells.’ (3)

By measuring the absorbance at 495nm, the resulting dye can be quantified.

Protocol

The experiment was carried out using four samples:

Non-Treated THP-1 human acute myeloid leukaemia cells (CONTROL)

Non-Treated Jurkat T human T cell (lymphoid) leukaemia cells (CONTROL)

THP-1 cells treated for 24hrs with 2M Cytarabine

Jurkat T cells treated for 24hrs with 2M Cytarabine

Using a 1mL pipette, the cells were first re-suspended before 100L of each of the four samples was loaded on the 96 well plates provided. Each sample was loaded three times in order to later obtain an average reading. 20L of MTS was added to each sample and gently mixed before incubating at 37C for 60 minutes. Absorbance readings were taken at 495nm by a member of staff and the results were recorded as can be seen in table 1.0. A Fuchs-Rosenthal cell counting chamber was used to obtain information on how many cells were present in each of the four samples. 20L of each sample was injected into the sample area and placed under a microscope to count the number of cells in each large square which had a calculated volume of 0.2mm3. A mean value of the absorbance readings was calculated and normalised against the cell numbers obtained.

As we can see from table 1.1. the number of cells present was higher for both THP-1 human acute myeloid leukaemia cells and Jurkat T human T leukaemia cells controls where the cells had not been treated. The THP-1 cell sample that had been treated with cytarabine had 1.45×105 cells per 1mL fewer than the untreated THP-1 cell sample. We can see the same relationship between the Jurkat T cell samples, where the cells treated were 4.25×105 less frequent per 1mL than the untreated sample. From this we can see that the cytotoxic effect of the cytarabine is positive and killing the leukaemia cells.

Although the results from table 1.1 show what we expected to occur with the chemotherapy drug, some of the results from table 1.0 contradict this. When calculating the A495 average normalised against the cell numbers, we can see that the Jurkat T human T cells(lymphoid leukaemia cells) decrease when treated for 24hours with cytarabine from 6.9384×10-7 to 1.5507×10-6 showing that the lymphoid leukaemia cells are very sensitive to the drug. However the data for the THP-1 myeloid leukaemia cells shows an increase after treatment from 1.0481×10-6 to 2.2216×10-6; almost a 50% increase. This counters the data shown in table 1.1 and would suggest that cytarabine has an opposing effect on the cancerous cells. During the practical, there were many air bubbles present in the 96 wells plate which may have caused a difference in the absorbance reading. This would have been due to human error during the pipetting stage of the protocol. Conclusion

Both the lymphoid and myeloid leukaemia cells are sensitive to cytarabine however the drug does not directly kill the cancer cells, shown by the ratio between the MTS reduction and the cell numbers. Instead cytarabine prevents cell proliferation by inhibiting DNA replication as shown by the decrease in cell number. This can irrefutably be concluded for the lymphoid cells from all the data collected. Proliferation of the lymphoid cells (Jurkat T) seem to be more sensitive to the treatment of cytarabine as they show a larger reduction in cell numbers than the THP-1 myeloid cells. Although the results do not indicate that the treatment is effective on the myeloid leukaemia cells, it can still be seen than they are less sensitive to the cytarabine as the number of cells per 1mL dropped from 2.70×105 to 1.25×105.