Undergraduate Research in Cancer Biology

1. Undergraduate Research in Cancer Biology Mahima Venkatesh Mentor-Charmaine Ramlogan-Steel, MD (Postdoctoral Fellow)
Principal Investigator- George Atweh, MD
Department- Internal Medicine - Hematology/Oncology UCCOM

2. Research Focus: Stathmin • Stathmin is a 17 KDa regulatory protein that is important in mitosis and microtubule dynamics. It is necessary for the formation of the mitotic spindle which implies that its deficiency can lead to problems with cell division. • Stathmin knockout mice was developed in 1996 and found to be normal . This may imply that there are other proteins compensating for the loss of stathmin. • Stathmin is also found to be overexpressed in a number of cancers, including prostate, breast, ovarian and acute leukemias • Higher levels of stathmin have been shown to correlate with more aggressive disease, poorer prognosis and early recurrence in certain cancers

3. Objective • Our project’s goal is to identify the stathmin-like proteins or molecules which appear to compensate for the loss of stathmin.

4. Laboratory Techniques • Sterile Cell Culture • Western Blotting • PCR, RT-PCR, real time PCR (qPCR) • DNA isolation and RNA purification • Transfection • Wright-Giemsastains • Animal Handling

5. KO H WT KO KO WT H H Using PCR to determine stathmin genotype in mice • Ear punch collected for DNA • DNA isolated and purified from tissue • PCR performed • Electrophoresis of PCR product

6. Transfection of PC-3 and LNCaP cells with shRNA • PC-3 and LNCaP prostate cancer cell lines were grown in sterile cell culture conditions. • Cell lines were transfected with a plasmid expressing GFP +/- anti-stathmin shRNA using Lipofectamine • Cells were visualized 72 hrs after transfection using fluorescent microscopy. GFP without anti-stathmin GFP with anti-stathmin

7. Western Blotting showing stathmin knockdown. • 72 hrs after transfection, cells were lysed and protein collected. • Protein was run on a 12% polyacrylamide gel and then transferred to a • nitrocellulose membrane. • Membrane was stained with anti-stathmin and b-actin antibodies and then further stained with secondary antibodies for visualization. • Film was exposed to membrane and developed.

8. RT and qPCR to examine stathmin and stathmin like gene expression • 72 hrs after transfection, cells were collected and RNA isolated. • A reverse transcriptase reaction was performed to generated cDNA from RNA. • A qPCR using SYBR green was performed on cDNA to test primers for STAT1-STAT4

9. Thank you