American Association for Cancer Research

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Supplementary Figure S1 from Deoxycytidine Kinase Expression Underpins Response to Gemcitabine in Bladder Cancer

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journal contribution
posted on 2023-03-31, 19:10 authored by Martin Kerr, Helen E. Scott, Blaz Groselj, Michael R.L. Stratford, Katalin Karaszi, Naomi L. Sharma, Anne E. Kiltie

Supplementary Figure S1. Transfection efficiency of siRNA measured with fluorescence. 50 nM Block-iT fluorescent siRNA was transfected and cells fixed 6 h later.



Purpose: In a recent phase II clinical trial, low-dose (100 mg/m2) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines.Experimental Design: Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay.Results: Gemcitabine was cytotoxic in the low nanomolar range (10–40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma.Conclusions: Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors. Clin Cancer Res; 20(21); 5435–45. ©2014 AACR.