Introduction: AML, causes, prevalence, symptoms, prognosis and treatment Cytarabine: mechanism of action, dosage for AML, side effects and cytotoxicity Issues on Drug resistance in AML and why research into new treatments is important.Isothiocyantes: what they are, where the found, why they are of interest in cancer but there is lack of information on blood cancers.MAPK1 is target gene in PEITC, what the pathway is and involvement in cancerRationale- determine the synergistic potential of phenyl isothiocyanates with cytarabine, as there are limitations in current method. And determine what MAPK1 expression means in AML patients Aims of experiment- evaluate synergistic potential of phenyl isothiocyanate using AML cell lines as a model system as well as investigating MAPK1 expression in AML patients.Methods:Kasumi-1 and HL60 were donated by supervisorMaking of RPMI complete- 174ml RPMI-1640, 2ml 2mM L-glut, 4ml 10IU Pen-Strep and 20ml 10%v/v foetal bovine serum.Cell passage-
RPMI-complete and cell culture were incubated for 30 mins in 5% CO2 incubator. 5ml of cell culture was transferred to falcon tube, centrifuged at 300G for 5 mins. The supernatant was discarded and the cell pellet was resuspended in 1ml of RPMI-complete.A 2 minute interval was given to allow the cells to settle. 2ul of the resuspension was added to a 1.5ml eppendorf tube as well 38ul of trypan blue. A interval time of 5 minutes was given to allow the cells to be adequately stained and 10ul of the stained suspension was loaded onto a hemocytometer.
Total cell count (cells/ml) = number of cells x 20 x 10^4/ number of squares counted on haemocytometer (cells in 1ml)
Doubling times:
Both cells were counted very 24hrs for 5 days to calculate to produce growth curve which was plotted on excel and used to work out doubling times of both cell lines
Cell viability was calculated using MTS assay, cells were treated with cytarabie at 0,0.125,0,25,0,5,1,2,4,8uM and phenyl-isothiocyanates at 0,10,25,50,75,100. Cells were incubated for 48hrs before 20ul MTs was added, plate was incubated at 37C for 2hrs and absorbance read at 490nm. The cell viability was then calculated and plotted using excel.
MAPK1 expression was recorded using Bloodspot which measured expression at 229847, 212271 and 224621 probes for AML t(15;17) and AML t(18;21). The expression was plotted on excel.
Results-
Data has been generated just needs to be discussed
Discussion
Discuss results further using articles, why knowing doubling times is important, MTS assay didn’t work for both cells and drug talk about what could have caused this, what expected results would have been, what MAPK1 expression means for AML patients and prognosis.
Talk about the importance of synergy and novel treatments in AML, why knowledge of MAPK1 activity in cancer is important for treatment.
Concluding statement summarising the discussion and need for further research in the field.