Title

Determining expression of wild type PLK1 and drug-resistant mutant C67V PLK1 in cervical cancer cells

Author(s)

Ronit Kulkarni

School Name

Spring Valley High School

Grade Level

11th Grade

Presentation Topic

Biochemistry

Presentation Type

Mentored

Oral Presentation Award

1st Place

Written Paper Award

1st Place

Abstract

Polo-like kinase 1 has been identified as a potential oncology target due to its importance in regulating cell cycle progression; when highly expressed, wild-type PLK1 can cause cervical cancer, non-small cell lung cancer, gastric carcinoma, breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, and prostate cancer. A potential PLK1 inhibitor drug, volasertib, has been synthesized for treating cancers caused by aberrant expression of PLK1, but research in clinical trials with other kinase inhibitors has shown that patients with cancer develop resistance because mutations appear in PLK1, such as the drug-resistant mutant C67V PLK1. Therefore, it is crucial to study the expression of the C67V mutant in cancer cells so it can used as a model to determine treatments to those cancers that become resistant. Hence, it was hypothesized that drug-resistant mutant C67V PLK1 would exhibit higher expression in HeLa cells when compared to wild-type PLK1 expression in HeLa cells. The HeLa cells transfected with drug-resistant mutant C67V PLK1 would exhibit higher expression than HeLa cells transfected with wild-type PLK1 because resistance to a PLK1 inhibitor could only develop if the 67 amino acid cysteine is replaced by the invariant valine to the kinase active site, thus occluding a PLK1 inhibitor from attaching to the ATP-binding site. Three sets of HeLa cells were separately transfected with GFP plasmids, wild-type PLK1+GFP plasmids, and drug-resistant mutant C67V PLK1+GFP plasmids, and were subsequently mounted onto three microscopic slides; an additional three slides had the same sets of transfected HeLa cells, acting as a duplicate. Approximately three hundred cells from each of the six slides were counted using a fluorescence microscopic under green fluorescence and phase light to compare the number of cells expressing the specific plasmid to the number of total cells. Percentages for each slide were calculated and were then used to determine expression levels of each plasmid. After expressing the mutant C67V PLK1 in cancer cells, the next step in the process is to synthesize and test different drug inhibitors that may inactivate cancer cells expressing the mutant C67V PLK1.

Start Date

4-11-2015 10:45 AM

End Date

4-11-2015 11:00 AM

COinS
 
Apr 11th, 10:45 AM Apr 11th, 11:00 AM

Determining expression of wild type PLK1 and drug-resistant mutant C67V PLK1 in cervical cancer cells

Polo-like kinase 1 has been identified as a potential oncology target due to its importance in regulating cell cycle progression; when highly expressed, wild-type PLK1 can cause cervical cancer, non-small cell lung cancer, gastric carcinoma, breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, and prostate cancer. A potential PLK1 inhibitor drug, volasertib, has been synthesized for treating cancers caused by aberrant expression of PLK1, but research in clinical trials with other kinase inhibitors has shown that patients with cancer develop resistance because mutations appear in PLK1, such as the drug-resistant mutant C67V PLK1. Therefore, it is crucial to study the expression of the C67V mutant in cancer cells so it can used as a model to determine treatments to those cancers that become resistant. Hence, it was hypothesized that drug-resistant mutant C67V PLK1 would exhibit higher expression in HeLa cells when compared to wild-type PLK1 expression in HeLa cells. The HeLa cells transfected with drug-resistant mutant C67V PLK1 would exhibit higher expression than HeLa cells transfected with wild-type PLK1 because resistance to a PLK1 inhibitor could only develop if the 67 amino acid cysteine is replaced by the invariant valine to the kinase active site, thus occluding a PLK1 inhibitor from attaching to the ATP-binding site. Three sets of HeLa cells were separately transfected with GFP plasmids, wild-type PLK1+GFP plasmids, and drug-resistant mutant C67V PLK1+GFP plasmids, and were subsequently mounted onto three microscopic slides; an additional three slides had the same sets of transfected HeLa cells, acting as a duplicate. Approximately three hundred cells from each of the six slides were counted using a fluorescence microscopic under green fluorescence and phase light to compare the number of cells expressing the specific plasmid to the number of total cells. Percentages for each slide were calculated and were then used to determine expression levels of each plasmid. After expressing the mutant C67V PLK1 in cancer cells, the next step in the process is to synthesize and test different drug inhibitors that may inactivate cancer cells expressing the mutant C67V PLK1.