The Cell Biologist.........!
I was involved in my master thesis with the topic Evaluation of Cyclin D1 and H-Ras gene expression patterns in leukemic blood samples using Real-Time PCR at the Regional Cancer Center, Trivandrum, India. This thesis mainly focuses on the signaling pathway, via c-myc/MAP kinase, resulting in the over expression of cyclin D1 and H-Ras. While doing my research thesis, I found that in most of the Chronic Myeloid Leukemia (CML) cases, there is an overexpression of cyclin D1, the reason for the unregulated proliferation of cells from G1 to S phase. Similarly, H-Ras, too, is found to be over expressed in most of the CML cases indicating that the cyclin D1 over expression is as a result due to the H-Ras, c-myc/MAP kinase pathway. Meanwhile, after reading research articles/journals on CML, I learnt that there are other pathways and may be interacting as well, in a complex web that influence the cyclin D1 expression pattern. Samples (5ml of blood or bone marrow) were collected in heparinized vials, mononuclear cells were isolated, using standard procedure, and stored in RNase later solution at -20 degree Celsius until isolation of RNA.RNA from the samples were disjuncted using procedure supplied in the kit. After disjunction (of RNA), analysis of integrity of RNA was performed on 1% agarose gel and observed under the gel dock The quality of RNA was analyzed by comparing the intensity of 28s and 18s rRNA. Concentration and purity of RNA was also determined using spectrophotometry. After analysis of RNA, construction of cDNA was performed using polymerase chain reaction (PCR).After this step, Cyclin D1 and H-Ras expression were analyzed using Real-Time Polymerase Chain Recation (RT-PCR). I also studied blood samples of treated CML patients. In this result, I found that the cyclin D1 is still over expressed but H-Ras expression is found to be normal in most of the samples. This result raises questions like has cyclin D1 acquired any resistance against targeted therapy? Or is cyclin D1 independently over expressed irrespective of signaling pathways? Or is there any other oncogenic pathway still responsible that is yet to be understood? All these questions were revealed in my master thesis, and my education background in the field of cell and molecular biology and immunology invigorated me to look in another direction in this research area. I have carefully read several research papers related to signaling pathways in CML. From these papers, I noticed that there are several blanks in this research field waiting to be filled in.
More research to follow soon!