An Inducible, Isogenic Cancer Cell Line System for Targeting the State of Mismatch Repair Deficiency

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells

MLH1-deficient NCI-H23 subclones exhibit increased resistance to chemotherapeutic drugs.

<p>NCI-H23 subclones that were uninduced or induced for MLH1 shRNA were treated with etoposide (A-C) or temozolomide (D-F). (A, D) Cells were treated with compound at the concentrations indicated, and cell viability was assessed at 4d using a Cell Titer-Glo assay. The graphs indicate the relative survival for duplicate samples from a single experiment. T tests to compare IC₅₀ values across multiple experiments determined p values of p=0.04 for cells treated with etoposide, and p=0.01 for cells treated with temozolomide. (B, E) Phase contrast images of cells at 0h and 96h time points during the cell viability experiment are shown. Cells were treated with 390 µM etoposide (B) or 500 µM temozolomide (E). (C, F) Cells were treated with compound for 24h at the concentrations indicated, and colony forming ability after compound washout was assessed. The graphs display percent survival at 10d for duplicate samples of cells treated with etoposide (C) or temozolomide (F). Comparison of the LD₅₀ values across multiple experiments by t-test determined p values of p=0.03 for cells treated with etoposide, and p=0.04 for cells treated with temozolomide.</p

MLH1 shRNA expression is inducible and reversible.

<p>NCI-H23 subclones 4-10 and 4-13 that were induced for MLH1 shRNA were split into two cultures, one maintained in inducing conditions (+ doxycycline) and the other grown in the absence of doxycycline (-) to allow re-expression of MLH1. Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting for expression of the MLH1 protein. Tubulin was used as a control for equal protein loading across samples. </p

MLH1-deficient NCI-H23 cells display increased cell sensitivity to rhodium metalloinsertor compounds.

<p>NCI-H23 subclones that were uninduced or induced for MLH1 shRNA were treated with the rhodium metalloinsertor compound [Rh(chrysi)(phen)(DPE)]³⁺. (A) Cells were treated at concentrations indicated, and cell viability was assessed after 4d using a Cell Titer-Glo assay. Percent viability of duplicate samples from a single experiment is shown. The p value was determined as p=0.01 by a t test. (B) Phase contrast images of cells treated with 5 µM [Rh(chrysi)(phen)(DPE)]³⁺ at 0h and 96h during the cell viability assay are shown. (C) Cells were treated with [Rh(chrysi)(phen)(DPE)]³⁺ for 24h, and then assayed for colony formation after 10d. The graphs show percent survival of duplicate samples of compound treated cells. The p value was determined as p=0.01 by a t test. </p

Induction of MLH1 shRNA decreases MLH1 protein levels.

<p>MLH1 protein levels in NCI-H23 subclones induced for the MLH1 928 shRNA were compared to MLH1 protein levels in the NCI-H23 parental cells (H23). Protein lysates were analyzed by SDS-PAGE and immunoblotting for the MLH1 and MSH2 protein levels. GAPDH was used as a control for protein loading. </p