38RW – Effects of Tyrosyl-DNA phosphodiesterase I modulation on ovarian cancer cell drug sensitivity

Status: Filled – Keana-Kelley Swanner
Intern: Keana-Kelley Swanner
Faculty Name: robert-van-waardenburg
Primary Faculty Appointment: UAB
UAB/HA Department: Pharmacology and Toxicology
Campus Address: VH 155
Telephone Number: (205) 934-4572
Email: rvanwaar@uab.edu or Click to Send E-Mail
For how many summers have you served as a preceptor: 0
CCC Research Area: Cancer Biology & Immunology
Number of hours per week that the preceptor will personally supervise or work with the intern: 25
Other faculty, staff, or graduate students who may help to supervise the intern:
1. Evan Brettrager
Title of Project: 38RW – Effects of Tyrosyl-DNA phosphodiesterase I modulation on ovarian cancer cell drug sensitivity
Project Description:

The majority of the new cases of ovarian cancer diagnosed in the US present with advanced disease. The consequence of this late detection is that ovarian cancer accounts for 5% of female cancer related deaths, with a 5 year survival of only 45%. An additional factor that contributes to this low survival rate is the frequent recurrence of this disease, despite the fact that ~80% of ovarian cancers initially respond to first-line therapy (cytoreductive surgery and chemotherapy with a platinum or taxanes). Second-line therapies may include other platinum combination regimens, liposomal doxorubicin, taxanes, topotecan, PARP inhibitors and anti-angiogenic agents.
Tyrosyl-DNA phosphodiesterase I (Tdp1) is a eukaryotic DNA repair enzyme that removes DNA-adducts from the ends of a DNA strand break by hydrolyzing the phosphodiester linkage. Tdp1 removes DNA-adducts via a “hand-off” of the adducted DNA-end to one of two active site histidines (the nucleophilic His) of Tdp1, to for a covalent Tdp1-DNA reaction intermediate. Tdp1 subsequently releases or “auto-hydrolyzes” itself from this covalent DNA-complex using the second active site histidine (the general acid/base His). These DNA-adducts range from small oxidative damaged nucleotides, nucleoside analogs, and DNA-embedded ribonucleotides, to larger, more complex adducts, such as the protein-DNA covalent complexes formed by DNA topoisomerase I or II and Tdp1 itself, or DNA-protein/peptide crosslinks formed by failed Schiff-base reactions such as PARP1-DNA peptide crosslinks.
Previously, we reported that DNA topoisomerase I (Top1) cleavage of DNA containing platinum adducts increases the stability of a covalent Top1-DNA reaction intermediate in ovarian cancer cell. As a consequence, ovarian cancer cells treated with cisplatin exhibit elevated levels of Top1-DNA complexes while increased expression of Top1 enhances cell sensitivity to platinum agents. Queries of the TCGA database indicate that Tdp1 mRNA levels are elevated in ~43% (316 samples) of HGSOC. We determined that ~43% of 68 HGSOC tumor samples stained for Tdp1 indeed exhibit elevated Tdp1 protein levels compared to surrounding stromal cells. Subsequent Kaplan-Meier and Log-Rank plot analyses revealed a negative association of ovarian tumors with elevated Tdp1 levels and progression-free, and overall survival. Furthermore, we detected a similar Tdp1 expression variation in high-grade serous ovarian carcinomas (HGSOC) cell lines.
Our aim is to understand the role of Tdp1 expression in ovarian cancer cell proliferation and sensitivity to standard of care and other chemotherapeutic combinations. We will us the CRISPRi system to knock-down Tdp1 levels, and ectopic overexpression to elevate Tdp1 levels, to study the effects of Tdp1 protein levels on cell drug sensitivity and proliferation. For further biochemical characterization of Tdp1 function in cells, we will us previous characterized toxic and catalytic inactive Tdp1 mutant proteins to study Tdp1 as a potential novel drug target for the development of novel small molecules and potential new treatment options.

Project Status: Already up and running
Location of Project: Birmingham, AL (UAB)
Proposed Start Date: May 17, 2021
Proposed End Date: August 6, 2021
Expected work schedule for intern: Flexible, intern can largely set his or her own schedule (as for students who are instructed how to proceed and are permitted to work independently with weekly guidance) and should contribute full-time effort.
Number of days that the student will be expected to come physically to UAB:
More than 1 day per week (prior to start of the internship, gain appropriate waivers and approvals for the student to be on site)
Category of Project: Laboratory Research
Cancer topic: Ovary
Does this project involve human subjects: No
Does this project involve animal subjects: No

Lab experiments including cell culture, drug sensitivity assays, transduction, plasmid isolation, protein isolation and analysis via immunoblotting.


Literature research


analysis of the obtained observaions/data

Preceptor will provide intern with access to the following: Office or desk space, Computer and printer, Laboratory work bench space, Supplies needed to complete project, Equipment needed to complete project
Likelihood that intern will be included as an author on one or more publications related to this summer research project: Very likely
Areas in which the ideal candidates will have experience: Biochemistry, Cell Biology, Laboratory Skills, basic knowledge, Molecular Biology