Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Summary

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Abstract

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Introduction

Despite considerable advances in cancer treatment, resistance of malignant cells to chemotherapy, either intrinsic or acquired upon prolonged drug exposure, is the major reason for treatment failure in a wide range of leukemia and solid tumors¹.

In order to delineate the mechanisms underlying drug resistance, in vitro cell line models are developed by stepwise selection of cancer cells resistant to chemotherapeutic agents. This procedure mimics the regimes used in the clinical settings and therefore allows in depth investigation of relevant resistance mechanisms. Resistant cells which survive the treatment are then disti....

Protocol

1. Characterization of Drug Resistance Profiles through Cytotoxicity Assays

1. Leukemic Cell Line Culture
   1. Maintain the parental T-cell ALL cell line CCRF-CEM (CEM-WT) as well as its drug resistant sublines, including CEM/R30dm, CEM-R5 and CEM-C3, in 25 cm² in 10 ml RPMI-1640 medium containing 2.3 µM folic acid supplemented with 10% fetal calf serum and 100 units/ml penicillin G and 100 µg/ml streptomycin.
   2. Culture the cells in a humidified atmosphere at 37 °C in a 5% CO₂ incubator.
   3. Allow cell growth to concentrations between 2 - 3 x 10⁶ cell/ml.
   4. Split ....

Results

The cytotoxicity assays described in the protocol provide a reliable and robust method to assess the resistance of cancer cells to chemotherapeutic agents in vitro. By means of the MTT assay, sensitivity to Dex was determined in four T-ALL cell lines, including Dex-sensitive parental CEM-WT cells, and three Dex-resistant sublines: CEM/R30dm, CEM-R5 and CEM-C3. Two different concentration ranges had to be used due to the large difference in sensitivity between CEM-WT (2 µM - 0.97 .......

Discussion

Here we describe a novel approach that combines well-established cytotoxicity screening techniques and powerful NGS-based transcriptomic analyses to identify differential splicing events in relation to drug resistance. Spectrophotometric assays are convenient and robust high-throughput methods to assess drug sensitivity in in vitro cancer models and represent the first choice for many laboratories performing cytotoxicity screenings. Troubleshooting as well as possible variations for this method were extensively .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sulforhodamine B	Sigma-Aldrich	230162
Trichloroacetic acid	Sigma-Aldrich	251399
CCRF-CEM	ATCC, Manassas, VA, USA	ATCC CCL-119
Panc-1	ATCC, Manassas, VA, USA	ATCC CRL-1469
DMEM high glucose	Lonza, Basel, Switzerland	12-604F
RPMI-1640	Gibco, Carlsbad, CA, USA	11875093
Fetal bovine and calf serum	Greiner Bio-One, Frickenhausen, Germany	758093
penicillin G streptomycin sulphate	Gibco, Carlsbad, CA, USA	15140122
Tris(hydroxymethyl)-aminomethane	Sigma Aldrich	252859
MTT formazan	Sigma Aldrich	M2003
Anthos-Elisa-reader 2001	Labtec, Heerhugowaard, Netherlands		UV-Vis 96-well plate spectrophotometer
Greiner CELLSTAR 96 well plates	Greiner/Sigma	M0812-100EA
Trypsin/EDTA Solution 100 ml	Lonza, Basel, Switzerland	CC-5012
CELLSTAR Cell Culture Flasks 25 cm2	Greiner Bio-One, Frickenhausen, Germany	82051-074
CELLSTAR Cell Culture Flasks 75 cm3	Greiner Bio-One, Frickenhausen, Germany	82050-856
Phosphate Buffered Saline (NaCl 0.9%)	B.Braun Melsungen AG, Germany	362 3140