RRM1 is a determinant of gemcitabine efficacy in cancer patients. However, the precision of predicting tumor response based on RRM1 levels is not optimal. We used gene-specific overexpression and RNA interference to assess RRM1's impact on different classes of cytotoxic agents, on drug-drug interactions, and the modulating impact of other molecular and cellular parameters. RRM1 was the dominant determinant of gemcitabine efficacy in various cancer cell lines. RRM1 also impacted the efficacy of other antimetabolite agents. It did not disrupt the interaction of two cytotoxic agents when combined. Cell lines with truncation, deletion, and null status of p53 were resistant to gemcitabine without apparent relationship to RRM1 levels. Pemetrexed and carboplatin sensitivity did not appear to be related to p53 mutation status. The impact of p53 mutations in patients treated with gemcitabine should be studied in prospective clinical trials to develop a model with improved precision of predicting drug efficacy.
The regulatory subunit of ribonucleotide reductase (RRM1) has been identified as the key molecular determinant of gemcitabine efficacy both
An association between intratumoral RRM1 levels and efficacy of systemic therapy that includes gemcitabine as a single-agent or in combination with a platinum-agent or pemetrexed has also been reported [
Here we studied associations between RRM1 expression levels and sensitivities to frequently used chemotherapeutic single agents and combinations as well as cell lines characteristics in an effort to determine the impact of RRM1 on relevant classes of agents and to identify parameters that might modify the RRM1-gemcitabine efficacy interaction.
The cell lines used in this study were obtained from the American Type Culture Collection (ATCC) or the originators. MCF7 human mammary adenocarcinoma cells were maintained in MEM-
We have generated three human cell line models derived from lung (H23), breast (MCF7), and colon (HCT8) cancers, with increased and decreased RRM1 expression by stable transfection as previously described [
Dharmacon on-TARGETplus Smartpool siRNA to TP53, ERCC1, and RRM1 (Dharmacon RNAi Technologies) were delivered to H23, A549, H292, and H460 NSCLC cell lines using Lipofectamine RNAiMAX (Invitrogen) following manufacturer’s instructions. Nontarget Pool siRNA was used as control.
Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen), and cDNA was synthesized with the Superscript amplification kit (Invitrogen). Quantitative real-time PCR was employed to measure the expression of RRM1 using 18s-rRNA as internal reference standard. The RRM1 primers were forward AAGAG CAGCG TGCCA GAGAT, reverse ACACA TCAAA GACCA GTCCT GATTA G, and probe
The following anticancer drugs were tested: gemcitabine and pemetrexed (Eli Lilly), methotrexate, carboplatin, hydroxyurea, and 5-fluorouracil (Sigma Aldrich); docetaxel (Sanofi-Aventis); cisplatin (Ben Venue Laboratory), vinorelbine (Sicor), and etoposide (Bedford Laboratory). At the time of use, the drugs were freshly prepared and diluted stepwise to the desired concentration in the proper solvent or culture medium.
Cell viability in response to various drugs was assessed with a cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in 96-well plates (Corning). Briefly, 1,000–4,000 viable cells were seeded in triplicate in 100
Alternately, drug activity was assessed using the CellTiter-Blue viability assay in 384-well plates (Promega). In this format, 800 or 1,200 cells (for 5-day or 3-day experiments, respectively) were plated in each well by using a Precision XS automated pipetting system (Bio-Tek Instruments) and allowed to attach overnight at
For both, the 96-well and 384-well experiments, fluorescence data were transferred to a spreadsheet program to calculate the percent viability relative to the replicate control cell wells that did not receive drug. Data analysis for
For drug combination experiments, the IC50 values obtained from single drug assays were used to design the experiments, and the cell viability assays were performed as described above. The results were analyzed for synergistic, additive, or antagonistic effects using the combination index (CI) method developed by Chou [
Tumor cells were cultured as described above. Crude cell extract proteins were suspended in RIPA buffer in the presence of a protease inhibitor cocktail. After determination of the protein concentration, extracts were separated on 8%–10% SDS-PAGE gels, transferred to membranes, and the expression profiles analyzed by immunoblotting. Monoclonal antibodies or antisera to RRM1 (T-16, cat # sc-11733, lot # H0608), RRM2a (I-15, cat # sc-10848, lot # G1806), RRM2b (N-16, cat # sc-10840, lot # E2107), P38 (H-147, cat # sc-7149, lot # I149), ERCC1 (FL-297, cat # sc-10785, lot # G1103), and MCM2 (N-19, cat # sc-9839, lot # I1907) were purchased from Santa Cruz Biotechnology, and monoclonal antibody to TS (TS-106, cat # MS-471-p1, lot # 471P708B) was from Anatomical Pathology and to TP53 (p53, cat # 554293, lot # 0000045190) from BD Bioscience. The bound antibody was detected using the ECL detection system according to manufacturer’s instructions (Amersham Pharmacia Biotech). The intensity values of specific bands were quantified with a Personal Densitometer SI (Molecular Dynamics). To compare expression values among the different cell lines, the target protein values were normalized by comparison with the house keep gene
DNA sequencing of the p53 and K-ras genes was done using the Applied Biosystems 3130XL genetic analysis system. Genomic DNA from tumor cell lines was obtained with PureLink Genomic DNA kits (Invitrogen). Exons of the p53 gene were amplified using previously reported primers with minor modifications [
We had previously described the stably transfected RRM1 up- and down-regulated clones of NSCLC cell line H23 [
Modification of RRM1 expression by stable transfection with RRM1 and shRRM1 expression vectors in cell lines H23, MCF7, and HCT. Wt, wild-type cell lines, R1, clones of cell lines transfected with RRM1; Ct, clones transfected with an out-of-frame RRM1 vector; shR1, clones transfected with a small hair-pin RRM1 vector; shCt, clones transfected with a random control small hair-pin vector. (a) RRM1 protein (red) and mRNA (green) expression in stably transfected clones of H23, MCF7, and HCT8. (b) Western blots of H23, MCF7, and HCT8 clones. (c) Cytotoxicity of MCF7 clones following gemcitabine treatment for 6 days. Each point is the mean of at least three independent experiments. The dashed line indicates the 50% survival fraction.
To evaluate the impact of RRM1 on different classes of chemotherapeutic agents, clones with high and low RRM1 levels and their respective controls were treated with each agent over a broad range of concentrations. Dose response blots were generated and mean
H23 | MCF7 | HCT8 | |||||||||||||
Control | RRM1 up | RRM1 down | Control | RRM1 up | RRM1 down | Control | RRM1 up | RRM1 down | |||||||
IC50 | IC50 Ratio | IC50 Ratio | IC50 | IC50 Ratio | IC50 Ratio | IC50 | IC50 Ratio | IC50 Ratio | |||||||
Mean | Mean | Mean | Mean | Mean | Mean | ||||||||||
Antimetabolites | |||||||||||||||
Gemcitabine | 0.010 | 0.22 | 0.06 | 0.008 | 0.92 | 0.12 | 0.013 | 0.16 | 0.08 | ||||||
Hydroxyurea | 686.400 | 0.87 | 0.01 | 1.18 | 0.05 | 0.960 | 1.11 | 0.04 | 0.05 | 1547.900 | 1.20 | 0.10 | 0.02 | ||
Methotrexate | 0.025 | 0.21 | 0.87 | 0.26 | 0.022 | 1.01 | 0.07 | 1.14 | 0.06 | 0.026 | 1.03 | 0.12 | 1.03 | 0.17 | |
Pemetrexed | 0.105 | 0.25 | 0.80 | 0.02 | 0.036 | 0.03 | 1.20 | 0.09 | 0.109 | 0.07 | 0.38 | ||||
5-Fluorouracil | 0.911 | 0.97 | 0.05 | 3.183 | 1.27 | 0.26 | 39.223 | 0.50 | |||||||
Platinum Compounds | |||||||||||||||
Cisplatin | 2.692 | 0.06 | 0.02 | 25.161 | 1.13 | 0.02 | 0.80 | 0.04 | 5.653 | 0.04 | 1.04 | 0.11 | |||
Carboplatin | 66.328 | 0.61 | 0.16 | 120.000 | 0.97 | 0.08 | 1.15 | 0.09 | 21.558 | no data | no data | ||||
Mitosis Inhibitors | |||||||||||||||
Docetaxel | 0.003 | 1.21 | 0.44 | 0.90 | 0.17 | 0.001 | 0.89 | 0.12 | 0.79 | 0.12 | 0.013 | 0.94 | 0.16 | 1.18 | 0.04 |
Vinorelbine | 0.034 | 0.96 | 0.16 | 1.00 | 0.21 | 0.010 | 1.02 | 0.10 | 0.98 | 0.14 | 0.072 | 0.98 | 0.10 | 1.14 | 0.14 |
Topoisomerase Inhibitors | |||||||||||||||
Etoposide | 0.227 | 1.15 | 0.06 | 1.14 | 0.06 | 0.291 | 0.89 | 0.10 | 1.06 | 0.11 | 0.183 | 1.18 | 0.14 | 1.03 | 0.17 |
For the platinum agents cisplatin and carboplatin, high or low RRM1 induced minimal resistance or sensitivity in H23 and had no consistent impact in MCF7 and HCT8. There was no observable relationship between RRM1 levels and efficacy of docetaxel, vinorelbine, and etoposide in all three model systems.
We next assessed if RRM1 modulation would impact on the cytotoxicity of combinations of two agents. For this, we chose four commonly used chemotherapy doublets focused on antimetabolites in NSCLC; that is, gemcitabine
Impact of RRM1 expression on drug combinations.
Drug combination | Clones of H23 | |||
H23-Ct | H23-R1 | H23-shCt | H23-shR1 | |
control | R1 increased | control | R1 decreased | |
Gemcitabine & Carboplatin | 0.93 ( | 0.83 ( | 0.56 ( | 0.72 ( |
Gemcitabine & Docetaxel | 2.8 ( | 2.1 ( | 3.1 ( | 1.4 ( |
Gemcitabine & Pemetrexed | 1.5 ( | 1.4 ( | 1.7 ( | 1.7 ( |
Pemetrexed & Carboplatin | 0.79 ( | 0.94 ( | 0.73 ( | 0.64 ( |
To confirm if RRM1 downregulation would increase gemcitabine efficacy in other NSCLC cell lines, we transfected 20 nM of target-specific short interfering RNA (siRNA) and nonspecific random siRNA for control purposes into cell lines A549, H292, and H460. Since ERCC1 (excision repair cross complementing group 1) expression levels in lung cancers are positively correlated with those of RRM1, we also used ERCC1-specific siRNA as a control. Immunoblot analysis demonstrated efficient knock-down of the specific target proteins RRM1 and ERCC1 in all three cell lines (Figure
Knock-down of RRM1 and ERCC1 expression in three NSCLC cell lines by RNA interference and impact on gemcitabine efficacy. (a) Western blots showing that RRM1-specific siRNA reduced RRM1 protein expression to undetectable levels, while random control and ERCC1-specific siRNAs did not affect RRM1 expression. Likewise, ERCC1-specific siRNA reduced ERCC1 protein expression to undetectable levels, while random control and RRM1-specific siRNAs did not affect ERCC1 expression. (b) IC50 values of gemcitabine cytotoxicity in cell lines A549, H292, and H460. Wt, wild-type cell lines; si-control, cell lines transfected with nonspecific siRNA; si-ERCC1; cell lines transfected with ERCC1-specific siRNA; si-RRM1, cell lines transfected with RRM1-specific siRNA.
Since tumoral RRM1 levels and therapeutic efficacy of chemotherapy vary widely [
Endogenous RRM1 levels and gemcitabine, pemetrexed, and carboplatin efficacy in 26 NSCLC cell lines.
Cell Line | ATCC # | Histological Subtype | p53 mutation ( | K-ras mutation | Doubling Time (hr) | RRM1 | RRM2a | RRM2b | ERCC1 | TS | TP53 | P38 | MCM2 | Gem IC50 (uM) | Pem IC50 (uM) | Carbo IC50 (uM) |
A549 | CLL-185 | AD-BAC | wt | G12S | 48 | 2.30 | 1.08 | 1.23 | 2.49 | 0.90 | 0.53 | 0.91 | 0.83 | 0.010 | 0.103 | 41.003 |
ADLC5M2 | — | AD | wt | wt | 24 | 2.42 | 1.79 | 0.30 | 2.40 | 0.66 | 0.12 | 0.58 | 1.22 | 0.079 | 0.044 | 6.678 |
EPLC65H | — | SQ | wt | wt | 47 | 2.83 | 1.07 | 0.55 | 3.92 | 1.23 | 0.12 | 1.22 | 0.97 | 0.019 | 0.039 | 7.590 |
H125 | CRL-5801 | AD/SQ | wt | 70 | 1.65 | 2.53 | 1.05 | 3.64 | 0.60 | 0.00 | 0.92 | 0.87 | 0.374 | 39.678 | ||
H1299 | CRL-5803 | NSCLC-NOS | homozygous deletion | wt | 46 | 1.92 | 2.43 | 2.18 | 1.31 | 0.91 | 0.00 | 2.01 | 1.37 | 0.024 | 0.236 | 27.273 |
H1355 | CRL-5865 | AD | E258K | G13C | 36 | 2.04 | 2.32 | 1.08 | 3.68 | 1.12 | 0.58 | 0.54 | 0.94 | 0.006 | 0.355 | 30.651 |
H157 | CRL-5802 | SQ | G12R | 58 | 2.22 | 2.80 | 1.16 | 0.73 | 0.73 | 0.03 | 1.65 | 1.01 | 0.036 | 0.433 | 21.766 | |
H1648 | CRL-5882 | AD | frame shift codon 35; | wt | 57 | 2.26 | 1.14 | 1.32 | 0.27 | 0.08 | 0.00 | 1.55 | 1.58 | 19.730 | ||
H1650 | CRL-5883 | AD | wt | wt | 49 | 0.83 | 1.58 | 1.07 | 1.09 | 0.08 | 0.02 | 0.97 | 0.81 | 0.020 | 0.053 | 14.535 |
H1703 | CRL-5889 | AD | wt | wt | 42 | 0.91 | 2.45 | 1.45 | 0.73 | 0.66 | 0.04 | 0.64 | 1.02 | 0.002 | 0.173 | 28.421 |
H1975 | CRL-5908 | AD | R273H | wt | 39 | 1.94 | 2.24 | 1.05 | 0.13 | 0.15 | 1.01 | 1.25 | 1.05 | 0.145 | 0.051 | 19.250 |
H2122 | CRL-5985 | AD | Q16L; C176F | G12C | 72 | 1.04 | 1.42 | 0.61 | 0.90 | 0.05 | 0.37 | 0.95 | 0.92 | 0.054 | 56.217 | |
H2172 | CRL-5930 | NSCLC-NOS | frame shift codon 72; | wt | 45 | 1.99 | 0.29 | 2.20 | 1.21 | 0.00 | 0.00 | 0.94 | 1.00 | 0.435 | 0.533 | 10.800 |
H2228 | CRL-5935 | AD | wt | 50 | 0.68 | 0.12 | 0.25 | 1.53 | 0.09 | 0.55 | 0.63 | 1.18 | 0.023 | 0.039 | 11.036 | |
H226 | CRL-5826 | SQ | wt | wt | 76 | 2.30 | 0.45 | 3.28 | 0.79 | 0.24 | 0.45 | 1.63 | 0.96 | 7.491 | 37.317 | |
H23 | CRL-5800 | AD | M246I | G12C | 45 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.008 | 0.125 | 21.220 |
H292 | CRL-1848 | ME | wt | wt | 31 | 2.11 | 1.90 | 1.13 | 2.08 | 0.93 | 0.61 | 1.26 | 0.73 | 0.011 | 0.026 | 30.326 |
H322 | CRL-5806 | AD-BAC | R248L | wt | 36 | 1.26 | 1.65 | 1.06 | 1.65 | 0.38 | 0.72 | 0.76 | 1.02 | 0.016 | 0.118 | 41.434 |
H358 | CRL-5807 | AD-BAC | homozygous deletion | G12C | 74 | 1.61 | 1.36 | 1.22 | 3.80 | 0.29 | 0.00 | 2.02 | 1.20 | 0.135 | 0.030 | 23.634 |
H441 | HTB-174 | AD | R158L | G12V | 58 | 1.86 | 1.91 | 4.49 | 0.99 | 0.05 | 0.47 | 2.77 | 1.87 | 0.033 | 0.093 | 23.545 |
H460 | HTB-177 | LC | wt | Q61H | 35 | 2.47 | 2.13 | 1.30 | 1.15 | 0.84 | 0.76 | 0.79 | 0.94 | 0.053 | 0.183 | 85.373 |
H522 | CRL-5810 | AD | frame shift codon 191 with deletion of P | wt | 108 | 1.08 | 3.17 | 0.73 | 7.04 | 1.10 | 0.22 | 1.74 | 0.89 | 21.298 | ||
H596 | HTB-178 | AD/SQ | G245C | wt | 49 | 1.61 | 1.98 | 1.00 | 3.00 | 0.53 | 0.68 | 1.25 | 0.93 | 0.031 | 0.012 | 28.626 |
H650 | CRL-5835 | AD | K164N | wt | 83 | 1.25 | 1.34 | 1.57 | 1.55 | 0.13 | 0.66 | 0.77 | 0.91 | 32.422 | ||
H661 | HTB-183 | LC | R158L; S215I | wt | 47 | 1.34 | 1.05 | 0.86 | 2.06 | 0.88 | 0.54 | 0.88 | 1.23 | 0.024 | 57.253 | |
H820 | HTB-181 | AD | T284P | wt | 68 | — | — | — | — | — | — | — | — | 6.883 | 32.116 |
We did not observe a statistically significant correlation between RRM1 levels and the gemcitabine
However, the median
The doubling time of cell lines was significantly correlated with gemcitabine
In order to study if wild-type p53 contributes to gemcitabine efficacy, we delivered p53-specific siRNA and nonspecific random siRNA to cell lines H23, A549, H292, and H460. We obtained near complete knock down in A549 and H292, a partial reduction in H460, and a minimal reduction in H23 using 20 nM siRNA concentrations and 24 hrs of exposure (Figure
Western blots of knock-down of TP53 expression in four NSCLC cell lines by RNA interference. TP53-specific siRNA reduced TP53 protein expression to undetectable levels in A549 and H292 and greater then 10-fold in H23 and H460, while random control siRNA did not affect TP53 or RRM1 expression.
To corroborate this result, we used the p53-null cell line H358 and its stably transfected and wild-type p53-expressing counterpart H358p53
Western blots of cell line H358. There is no detectable TP53 expression in wild-type cells; while transfected cells clearly show TP53 expression.
The use of unselected double-agent chemotherapy has resulted in an approximately 50% improvement in overall median survival of patients with advanced NSCLC [
The antitumoral activity of gemcitabine is a result of at least two separate actions. One is a presumed direct interaction with RRM1, with a resulting reduction of ribonucleotide reductase function and deoxynucleotide levels, and the other is incorporation into newly synthesized DNA, with a resulting chain termination. It is the presumed interaction with RRM1 that explains the direct and linear association between RRM1 levels and gemcitabine
Given the molecular complexity of NSCLC, this is not surprising and strongly suggests that a variety of other tumor-specific and host-specific parameters substantially impact the gemcitabine-RRM1 interaction. Our results in a lung, breast, and colon cancer cell line with genetically modified RRM1 levels demonstrate that RRM1 expression levels are the dominant determinant of gemcitabine efficacy despite diverse molecular backgrounds. This result is consistent with prior reports of increased RRM1 levels in pancreatic and colon cancer models upon induction of gemcitabine resistance [
Since RRM1 is often combined with ERCC1 in trials seeking to enhance therapeutic efficiency through agent selection and because both molecules are frequently coexpressed [
Using a panel of lung cancer cell lines with diverse features and molecular characteristics, we identified two variables that significantly impacted gemcitabine efficacy without being associated with RRM1 levels. We found that cell lines with functional p53-impairing mutations, that is, null, truncation, and deletion mutations, displayed a pattern of resistance to gemcitabine. We corroborated this result through transfection of wild-type p53 into a null cell line (H358), which resulted in a statistically significant improvement in gemcitabine efficacy, and through abrogation of p53 by RNA interference in other cell lines, which resulted in gemcitabine resistance. This was not explained by RRM1 expression levels, since no association between RRM1 levels and p53 was observed. In contrast, pemetrexed and platinum efficacy did not seem to be influenced by p53 expression modulation. To our knowledge, the impact of p53 mutations on gemcitabine efficacy has not been studied in clinical trials. It is important to corroborate these results in prospective trials since a potential clinical implication might be that tumoral RRM1 levels may not be predictive of gemcitabine efficacy in patients whose tumors harbor functionally significant p53 mutations.
Finally, our
We demonstrated a dominant role for RRM1 in gemcitabine efficacy and also a role in efficacy of other antimetabolites in selected cell lines. RRM1 did not disrupt the interaction between gemcitabine and other cytotoxic agents when combined. The mutational status of p53 and cell line doubling time were significant and independent determinants of gemcitabine efficacy. Their impact on gemcitabine efficacy in patients with lung cancer in the context of RRM1 expression requires investigations in prospective clinical trials.
Ribonucleotide reductase
RR subunit M1
RR subunit M2a
p53-inducible RR subunit M2b, encoded by the p53R2 gene
Excision repair cross-complementation group 1
p53 tumor suppressor gene
Thymidylate synthase
Minichromosome maintenance protein 2
3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
Nonsmall cell lung cancer.
This work was supported by R01-CA129343 from the NCI.