Background
The retinoblastoma gene
(RB1) is a tumor suppressor gene. pRb, the protein coded for by the
RB1 gene, plays a pivotal role in cell cycle regulation, promoting G1/S arrest and growth restriction through inhibition of the E2F transcription factors [
1]. Germline mutations affecting the
RB1 gene are strongly associated with retinoblastoma development in children, and recent evidence has revealed an increased risk of different malignancies, including breast cancers, among patients cured from hereditary retinoblastoma [
2].
Somatic alterations of the
RB1 gene have been detected in different malignancies [
3‐
5]. Previous studies have reported allelic imbalance (AI), loss of pRb protein expression [
3], hypermethylation of the
RB1 promoter [
6] and, in some rare cases, large intragenic deletions [
7] in the
RB1 gene in primary breast cancer. However, point mutations (1163T>C and 1544C>T) have, so far, only been detected in a single breast cancer cell line (BT20) [
8]. To the best of our knowledge, no point mutations have previously been reported in biopsies from breast carcinomas.
While the cellular functions of pRb are well characterized, the effect of disturbances in the
RB1 gene on tumor growth and response to systemic therapy in breast cancer is incompletely understood. Lack of pRb protein and loss of heterozygosity (LOH) at the
RB1 locus have been related to triple negative (TNBC) or basal cell-like breast cancer [
9,
10]. Absence of pRb expression has been linked to poor prognosis in breast cancer patients receiving adjuvant endocrine therapy [
11,
12]. In contrast, loss of expression has been associated with good prognosis in patients receiving chemotherapy [
10,
12]. However, these findings may not be interpreted as direct evidence that alterations in
RB1 predict chemosensitivity [
13]. Breast cancer patients are selected for systemic treatment options based on tumor characteristics like histological grading, estrogen receptor expression, and Her-2 status, thus, the patient cohorts referred to above may differ with respect to key biological parameters. Experimental studies have provided contradictory results, revealing loss of pRb function to enhance [
11,
14‐
17] as well as to reduce [
18,
19] cell death and sensitivity to chemotherapeutic agents.
In the current study, we analyzed 73 breast cancers undergoing pre-surgical treatment with doxorubicin or mitomycin with 5-FU for genetic and epigenetic changes in the RB1 gene. We report for the first time point mutations affecting RB1 in breast cancer tissue. Each mutation lead to amino acid substitution (Leu607Ile, Arg698Trp, and Arg621Cys) in pRb. The mutated pRb variants were all located to the nuclear compartment and expressed reduced apoptotic capacity compared to wild-type pRb. Furthermore, MLPA unveiled two large multiexon deletions (exons 13 to 27 and exons 21 to 23). Most interesting, three out of four tumors harboring RB1 mutations expressed resistance to chemotherapy. Our data provide the first indication that RB1 might be a candidate gene involved in drug resistance.
Discussion
The present work is the first study reporting RB1 point mutations in primary breast carcinomas. Based on our findings that all three point mutated pRb proteins expressed reduced pro-apoptotic effect in vitro, and three out of four tumors harboring RB1 mutations were resistant to chemotherapy, our data provide the first indication that RB1 alterations could influence breast cancer chemosensitivity in vivo.
Somatic point mutations in
RB1 have been detected in different malignancies including bladder [
4] and prostate [
5] cancer. While previous studies have examined breast cancer samples with respect to
RB1 LOH, loss of pRb immunostaining [
3] and chromosomal rearrangements [
7], except for one study reporting no mutations when sequencing the exon 21 only [
27] and the finding of point mutations in a single breast cancer cell line [
8], we are not aware of any study reporting
RB1 point mutations in breast cancer tissue
http://rb1-lsdb.d-lohmann.de/.
In this study, we evaluated potential point mutations through gene sequencing (N = 73) and intragenetic deletions by use of MLPA (N = 71) across the whole coding region of the RB1 gene in stage III breast cancers. We identified three novel single nucleotide mutations in RB1, each leading to amino acid substitutions. While one of the point mutations was germline, the patient harboring this alteration revealed no family history of retinoblastoma or family clustering of either breast cancer or any other malignancy.
Each of the detected point mutations was located in the functionally important pocket domain. The pocket is essential for pRb's interaction with other proteins [
28]. It consists of the so-called A and B boxes [
23], forming a tight hydrophobic interface, with the two parts being covalently linked by the spacer region (Figure
1). Thus, mutations affecting the A [
29] and B [
30] boxes of pocket domain are known to give rise to pRb proteins with disturbed function. Alterations located in both of these parts have been detected in human sporadic cancers [
4,
5], but also as germline mutations in patients with retinoblastoma [
31,
32]. One of the point mutations identified here (Arg698Trp) is located in the B box, and
in silico structural analysis suggested this mutation to disrupt a hydrogen-bond network. Furthermore, our findings indicate this mutant to be less stable than the wild-type pRb protein (Figure
7). Taken together, these findings suggest that mutations in this location might significantly affect pRb function and stability. In contrast, mutations Leu607Ile and Arg621Cys locate to the spacer region. While germline [
31‐
33] as well as somatic [
5] mutations within the spacer region have been identified, many authors have considered this domain non-essential to peptide-binding activity of the pRb pocket [
23]. Our alignment analysis confirmed the spacer domain to be well conserved across different species (Figure
3), and the Leu607Ile mutant protein was considerably less stable compared to pRb wild-type These findings, in addition to our results revealing both of these mutations, similar to Arg698Trp, to express a reduced apoptotic function, further indicates a functional role for this region of the pRb pocket.
Considering the intragenic deletions (exon 21-23 and exon 13-27), these deletions result in truncated proteins missing half of the B box and both A and B boxes respectively, most likely abolishing the function of the pRb pocket in both cases.
The retinoblastoma gene encodes a nuclear phosphoprotein which in its unphosphorylated state binds to and inactivates E2F1, causing G1-S phase arrest [
1]. Potential additional roles, including pro-apoptotic functions of pRb have been suggested [
26]. While most studies have indicated an anti-apoptotic role of pRb [
34], some studies have shown pRb to enhance apoptosis following γ-irradation [
25] as well as doxorubicin-induced DNA damage [
19] which is more in accordance with its role as a tumor suppressor. Here we found the apoptotic response to doxorubicin treatment to be restored by transfecting the
RB1-deficient C-33 A and Saos-2 cell lines with wild-type pRb, but only to a minor degree when transfecting the pRb point mutants, supporting a pro-apoptotic function of pRb in response to anthracycline therapy (Figure
6a and
6b). However, the corresponding protein level controls and subsequent stability assays showed that the Leu607Ile and Arg698Trp pRb mutants were less stable than the mutant Arg621Cys pRb and the pRb wild-type proteins which harbored similar protein levels and stabilities. Thus, we believe the relatively low percentage of apoptotic cells observed for all the 3 mutants is due to reduced protein stability in the case of Leu607Ile and Arg698Trp, and due to impaired ability to induce apoptosis in the case of the more stable pRb mutant Arg621Cys.
Further, in support of a pro-apoptotic role of the pRb protein in breast cancer, three out of four tumors harboring
RB1 mutations expressed resistance to doxorubicin or mitomycin treatment
in vivo. The two drugs have similarities with respect to their mechanisms of antitumor actions. Thus, the
in vitro findings on apoptotic response are consistent with our observations on patient drug resistance
in vivo. The fact that one of the tumors (Dox 111 harboring Arg698Trp) responded to chemotherapy do not refute such a hypothesis; while we previously reported mutations affecting
TP53 [
20,
21] to be associated with chemoresistance
in vivo, the predictive power was not 100%; indicating alternative mechanisms may act in concert [
35].
Methods
Patients
This study included patients from two prospective studies addressing the potential role of mutations in
TP53 and other genes regarding resistance to treatment with doxorubicin [
20] or mitomycin and 5-fluorouracil [
21] in locally advanced breast cancer. Both studies were approved by the Regional Ethical Committee, and each patient gave written informed consent to the procedure. Because these studies were designed to explore causes of chemoresistance, we focused on comparing tumors that showed primary drug resistance (progressive disease within 12 weeks) with the combined group of tumors showing stable disease or objective response [
22,
36]. Thus, for the patients treated with doxorubicin, we analyzed
RB1 mutational status in all nine tumors that were resistant to therapy together with a randomly selected subgroup of 27 responding tumors. Regarding the group of patients treated with mitomycin and 5-fluorouracil, we analyzed 37 tumors for the presence of
RB1 mutations, including eight patients resistant to therapy [
21] and four none evaluable samples. Included were three patients with locally advanced breast cancer, treated with mitomycin and 5-FU, not participating in these studies. Thus, a total of 73 patients were included (Additional file
1).
Control subjects
Due to the fact that we discovered a novel germline base substitution C1861T (Arg621Cys) in one breast cancer patient, we sought to evaluate its frequency in the general Norwegian population. Thus, we examined blood DNA from 231 healthy women recruited from the national mammographic program into other studies described elsewhere [
37].
RNA Purification
RNA was purified by Trizol (life Technologies, Inc.) extraction from snap-frozen tissue samples according to the manufacturer's instructions. After extraction, the RNA was dissolved in diethyl pyrocarbonate-treated double-distilled, deionized H 2O. cDNA was synthesized by reverse transcription using Superscript II reverse transcriptase (Invitrogen)
DNA Purification
Genomic DNA was purified from tumor tissue or lymphocytes using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions.
Multiplex ligand probe amplification (MLPA)
MLPA analysis of genomic DNA from 71 patients was performed using the SALSA MLPA RB1 kit (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer's instructions. In the patient samples, the peak areas of all MLPA products resulting from RB1 specific probes were first normalized by the average of peak areas resulting from control probes specific for locations other than on chromosomes 13. A ratio was then calculated where this normalized value was divided by the corresponding value from a sample consisting of pooled DNA from 10 healthy individuals. A sample was scored as having a reduced copy number at a specific location if this ratio was below 0.75, and as having an increased copy number if the ratio was above 1.25.
LOH analysis
Samples shown by MLPA analysis to harbor AI at the
RB1 locus were subsequently analyzed by LOH. Three markers were applied spanning 13q14.1-3: D13S263, centromeric to
RB1, D13S153, and RB1, the two latter located within intron 2 and intron 20 of the
RB1 gene, respectively. Both D13S263 and D13S153 are microsattelite markers; while RB1 is a variable number tandem repeat (VNTR). All three markers were amplified by PCR using primers as specified in Table
2 and the PCR products were analyzed on an automated DNA sequencer (ABI 3700). Data were analyzed by comparing normal and tumor tissue allele peak-height ratios. A sample was scored as having AI when the ratio between height of tumor and normal sample was less than 0.84 [
38].
Table 2
PCR and DNA sequencing primers
RB1M | 5'-GGG AGT TTC GC G GAC GTG AC-3' | 5'-ACG TCG AAA CAC G CC CCG-3' | 172 | 65.0 |
RB1U | 5'-GGG AGT TTT GT G GAT GTG AT-3' | 5'-ACA TCA AAA CAC A CC CCA-3' | 172 | 61.0 |
RB1 promoter | 5'-CGC CCC AGT TCC CCA CAG A-3' | 5'-GGC AAC TGA GCG CCG CGT-3' | 163 | 53.0 |
RB1 exon 1 | 5'-AAC GGG AGT CGG GAG AG-3' | 5'-AAT CCT GTC ACC ATT CTG C-3' | 412 | 55.2 |
RB1 exon 2 | 5'-GAT TAT TTT CAT TTG GTA GGC-3' | 5'-AAA GTG GTA GGA TTA CAG GC-3' | 351 | 51.3 |
RB1 exon 3 | 5'-TTT TAA CAT AGT ATC CAG TGT GTG-3' | 5'-TAC ACT TTC ATA ACG GCT CC-3' | 350 | 54.4 |
RB1 exon 4 | 5'-GAC CCT TCG TTT TCT TAT ATT CTC-3' | 5'-ATC AGA GTG TAA CCC TAA TAA AAT G-3' | 390 | 55.2 |
RB1 exon 5 | 5'-ATT GGG AAA ATC TAC TTG AAC-3' | 5'-TCA AAC TAA CCC TAA CTA TCA AG-3' | 265 | 54.2 |
RB1 exon 6 | 5'-CAT TCT ATT ATG CAT TTA ACT AAG G-3' | 5'-CTA ACA GTT AAT AAG CCA AGC AG-3' | 340 | 53.6 |
RB1 exon 7 | 5'-ATG GAT ATA CTC TAC CCT GCG-3' | 5'-ATC CTG TCA GCC TTA GAA CC-3' | 291 | 55.2 |
RB1 exon 8 | 5'-TAA AAG TAG TAG AAT GTT ACC AAG-3' | 5'-CAG TGA TTC CAG AGT GAG G-3' | 470 | 55.2 |
RB1 exon 9 | 5'-TTG ACA CCT CTA ACT TAC CCT G-3' | 5'-TTG GCT AGA TTC TTC TTG GG-3' | 301 | 55.7 |
RB1 exon 10 | 5'-GAA ATC TGT GCC TCT GTG TG-3' | 5'-AAA GGT AAC TGT TAT AGG ACA CAC-3' | 200 | 52.9 |
RB1 exon 11 | 5'-GTT ATC AAT ACC ACC AGG GAG-3' | 5'-CAA ATC TGA AAC ACT ATA AAG CC-3' | 443 | 53.0 |
RB1 exon 19 | 5'-AGA CAA GAT GTA TCT GGG TGT AC-3' | 5'-CAT GAT TTG AAC CCA GTC AG-3' | 306 | 53.6 |
RB1 exon 20 | 5'-CTT ATT CCC ACA GTG TAT GCC-3' | 5'-AGC CTG GGT AAC AGA GTG AG-3' | 341 | 47.5 |
RB1 exon 21 | 5'-ATT CTG ACT ACT TTT ACA TC-3' | 5'-TTA TGT TAT GGA TAT GGA T-3' | 192 | 58.0 |
RB1 exon 22.1 | 5'-ATA TGT GCT TCT TAC CAG T-3' | 5'-CAC GTT TGA ATG TCT GAG GA-3' | 148 | 53.5 |
RB1 exon 22.2 | 5'-CCT CAG ACA TTC AAA CGT GT-3' | 5'-TTG GTG GAC CCA TTA CAT TA-3' | 175 | 54.1 |
RB1 exon 23 | 5'-TAA TGT AAT GGG TCC ACC AA-3' | 5'-TCA AAA TAA TCC CCC TCT CA-3' | 277 | 55.6 |
RB1 exon 24 | 5'-GAA TGA TGT ATT TAT GCT CA-3' | 5'-TTC TTT TAT ACT TAC AAT GC-3' | 165 | 46.1 |
RB1 exon 25 | 5'-CTT TGC CTG ATT TTT GAC AC-3' | 5'-CAG TGC TGA GAC TCT GGA TTC-3' | 270 | 56.3 |
RB1 exon 26 | 5'-CAT TTA TGT TTT AGA TGG TTA G-3' | 5'-GTT TAT TTC GTT TAC ACA AG-3' | 318 | 46.8 |
RB1 exon 27 | 5'-CAG CCA CTT GCC AAC TTA C-3' | 5'-CAT AAA CAG AAC CTG GGA AAG-3' | 230 | 53.5 |
RB1-1.r-S2/AS4 | 5'-AAC GGG AGT CGG GAG AG-3' | 5'-GAA TTA CAT TCA CCT CTT CAT CAA G-3' | 1204 | 45.0 |
RB1-1.r-S3/AS2 | 5'-ATG ATA AAA CTC TTC AGA CTG ATT C-3' | 5'-TGT CCA CCA AGG TCC TGA G-3' | 1794 | 45.0 |
RB1-2.r-frag1-S/AS | 5'-AGG AGG ACC CAG AGC AGG AC-3' | 5'-CCA AGA AAC TTT TAG CAC CAA TG-3' | 496 | 45.0 |
RB1-2.r-frag2-S/AS | 5'-CTA CTG AAA TAA ATT CTG CAT TGG T-3' | 5'-CTC TTC ATC AAG GTT ACT TTT TCG T-3' | 528 | 45.0 |
RB1-2.r-frag3-S/AS | 5'-GAA ACA CAG AGA ACA CCA C-3' | 5'-ATT CTG AGA TGT ACT TCT GCT A-3' | 461 | 45.0 |
RB1-2.r-frag4-S/AS | 5'-AGC AAA CTT CTG AAT GAC AAC-3' | 5'-GAG AGG TAG ATT TCA ATG G-3' | 518 | 45.0 |
RB1-2.r-frag5-S/AS | 5'-CTC CAA AGA AAA AAG GTT CAA-3' | 5'-GGT ATT GGT GAC AAG GTA GG-3' | 512 | 45.0 |
RB1-2.r-frag6-S/AS | 5'-GTA TTC TAT AAC TCG GTC TTC A-3' | 5'-CAT TTC TCT TCC TTG TTT GA-3' | 526 | 45.0 |
RB1-plasmid-S1/AS1 | 5'-GGT TTT TCT CAG GGG ACG-3' | 5'-GTG AGA GAC AAT GAA TCC AGA G-3' | | 45.0 |
RB1-plasmid-S/AS-STOP | 5'-CAC AGC TCG CTG GCT CCC-3' | 5'-TTT CTC TTC CTT GTT TGA G-3' | | 45.0 |
RB1 (Leu607Ile) | 5'-GCA GCA GAT ATG TAT ATT TCT CCT GTA AGA TCT CC-3' | | | |
RB1 (Arg621Cys) | 5'-AAA GGT TCA ACT ACG TGT GTA AAT TCT ACT GC-3' | | | |
RB1 (Arg698Trp) | 5'-GAA CTC ATG AGA GAC TGG CAT TTG GAC CAA ATT ATG-3' | | | |
D13S153 F/R | 5'-TTG CAC TGT GGA GAT AAA CAC ATA G-3' | 5'-TCA CAT TGT CTT TTA AGG CAG GAG-3' | | |
D13S263 F/R | 5'-CCT GGC CTG TTA GTT TTT ATT GTT A-3' | 5'-CCC AGT CTT GGG TAT GTT TTT A-3' | | |
RB1 | 5'-TGT ATC GGC TAG CCT ATC TC-3' | 5'-AAT TAA CAA GGT GTG GTG G-3' | | |
PCR Amplification of RB1 from cDNA
Six fragments encompassing the
RB1 reading frame starting from nucleotide 89 to 2749 (TGA) were amplified by nested PCR using the primers listed in Table
2. The nucleotides 1 (starting from ATG) to 88 were not covered by our analysis. The frequency of published mutations in this region is approximately 2% for patients with retinoblastoma
http://rb1-lsdb.d-lohmann.de/. It was therefore concluded that the risk of missing somatic alterations was acceptably low. Observed mutations were verified by PCR amplification of the affected exons from genomic DNA. In some tumors, no cDNA-based products were detected for parts of the
RB1 gene. To verify the integrity of the
RB1 gene in these patients, we amplified the exons of interest using genomic DNA.
PCR was carried out with Dynazyme EXT DNA polymerase (Dynazyme) in a 50 μl solution containing 1× PCR buffer, 1.5 mM MgCl 2, 0.5 mM of each deoxynucleotide tri-phosphate, 5% DMSO, 0.2 μM of each primer, and 0.5 μl of cDNA, 1 μl of first-round PCR-product or ~50 ng genomic DNA was used as template. The RB1 PCR conditions for the first round of the nested PCR for fragments one and two were: An initial 5 min denaturation at 94°C, 40 cycles of 30 sec at 94°C, 30 sec at 52.5°C, and 120 sec at 72°C, and a final 7 min extension at 72°C. The second round of the nested PCR for fragments one and two was conducted as follows: An initial 5 min denaturation at 94°C, 40 cycles of 30 sec at 94°C, 30 sec at 55.6°C, and 60 sec at 72°C, and a final 10 min extension at 72°C.
The RB1 PCR conditions for both rounds of the nested PCR for fragments three to six were identical and consisted of: An initial 5 min denaturation at 94°C, 40 cycles of 30 sec at 94°C, 30 sec at 45°C, and 120 sec at 72°C, and a final 7 min extension at 72°C.
PCR on genomic DNA was done with an initial 5 min denaturation at 94°C, 40 cycles of 60 sec at 94°C, 30 sec at an annealing temperature optimized for each exon (Table
2), and 45 sec at 72°C, and a final 7 min extension at 72°C.
Finally, the RB1 promoter PCR was done with an initial 5 min denaturation at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at 53°C, and 45 sec at 72°C, and a final 5 min extension at 72°C.
DNA Sequencing
Sequencing was performed as described previously [
39] using primers specific for the different
RB1 fragments (Table
2). The sequences generated were compared with wild-type
RB1 (GenBank accession number: L11910) for sequence alterations.
RB1 promoter methylation status was analyzed by methylation-specific PCR in 71 patients. Genomic DNA from patients was modified using the CpGenome DNA Modification Kit (Intergen), and primers designed specific for methylated (RB1M-S and RB1M-AS, Table
2) and unmethylated (RB1U-S and RB1U-AS, Table
2) DNA [
40]. Methylation- and unmethylation-specific PCRs (MSP and USP) were done with AmpliTaq Gold DNA Polymerase (Applied Biosystems) in a 50 μl solution containing 1× PCR buffer, 1.5 mM MgCl
2, 0.5 mM of each deoxynucleotide triphosphate, 0.2 μM of each primer and 25 ng of modified genomic DNA. The methylation-specific PCR was carried out for 35 cycles of 60 sec at 95°C, 45 sec at 65°C, and 1 min at 72°C. The unmethylation-specific PCR was carried out for 35 cycles of 60 sec at 95°C, 45 sec at 61°C, and 1 min at 72°C. Both PCRs were performed with an initial 5 min of denaturation at 95°C and concluded with 2 min at 72°C. After amplification, the PCR products were visualized on a 3% agarose gel. Included in each run was a positive control (CpGenome Universal Methylated DNA, Millipore) and two negative controls (modified DNA from healthy donors and water).
Plasmid constructs
RB1 wild-type was amplified from cDNA by nested PCR using the primers RB1-plasmid-S1 and RB1-plasmid-AS1 in the first, and RB1-plasmid-S and RB-AS-STOP in the second PCR (Table
2). The first PCR was carried out in a 50 μl reaction mixture of 2.5 U KOD XL DNA polymerase (Novagen), 1× PCR buffer, 0.2 mM of each deoxynucleotide triphosphate, 0.2 μM of each primer and 10 μl of cDNA. The thermal conditions for the first PCR were as follows: 94°C for 5 min, 30 cycles of 30 sec at 94°C, 2 sec at 45°C and 180 sec at 70°C and a final extension at 74°C for 10 min. For the second PCR, a 50 μl reaction solution was made consisting of 0.5 U Dynazyme EXT DNA polymerase, 1× PCR buffer, 5% DMSO, 0.2 mM of each deoxynucleotide triphosphate, 0.2 μM of each primer and 1 μl of first-round PCR product. The amplification conditions were: 5 min denaturation at 94°C followed by 40 cycles of 30 sec at 94°C, 30 sec at 45°C and 180 sec at 72°C before a final step at 72°C for 7 min.
The final PCR product was TA-cloned into the expression vector pcDNA3.1/V5-His TOPO (Invitrogen) according to the manufacturer's instructions. Using the resulting construct
RB1wild-type-V5, corresponding primers (Table
2), and QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene), the following constructs were made;
RB1Arg621Cys-V5, RB1Leu607Ile-V5, and RB1Arg698Trp-V5.
Cell culture and transfection
C-33 A cells and Saos-2 cells were cultured in EMEM and McCoys 5A, respectively, supplemented with 10% FBS. Both cell lines were purchased from ATCC (American Type Culture Collection). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction.
Western blot analysis
Protein samples were separated on 8% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes (Whatman). The membranes were probed with the following antibodies: anti-V5 (Invitrogen) and anti-actin (Santa Cruz Biotechnology), and the HRP-conjugated 2. antibodies sheep anti-mouse (GE Healthcare) and mouse anti-goat (Santa Cruz Biotechnology). Signals were detected using ECL Western blotting Detection Reagents (GE Healthcare).
Protein stability
Analyses of protein stability using cycloheximide was performed as previously described [
41]. After 0, 2, 4, and 6 h, the cells were harvested and lysed in 1 X SDS lysis buffer (0.075 M Tris pH 6.8, 2% SDS, 20% glycerol, 0.1 M β-mercaptoethanol, 0.01% bromphenol blue). The samples were analyzed by SDS-PAGE and western blot using anti-V5 and anti-actin (loading control).
Immunofluorescence
Cells grown on cover slips were fixed in 3.7% formaldehyde for 20 minutes and washed with 1× PBS. The cells were permeabilized for 12 minutes using 0.1% triton X100 in 1× PBS before blocking with 1% BSA in 1× PBS. Detection of the corresponding proteins was performed using monoclonal mouse anti-V5 and TXR-conjugated goat anti-mouse Ig secondary antibody. The slides were mounted with Vectashield (Vector Laboratories, USA) and examined using a confocal laser scanning microscope (Leica TCS Confocal System attached to a Leica DM RXA microscope).
Statistical Analysis
Alterations in the
RB1 gene were correlated to response to chemotherapy by use of Fisher exact test (p-value given as cumulative, two-sided). Differences between pRb wild-type and mutants with respect to induction of apoptosis were evaluated by analyses of variance (ANOVA). Subject to statistical significance, the efficacy of each mutant was compared to pRb wild-type with use of the Student test for 2 samples. Statistical calculations were performed using the SPSS 15.0 software and
http://www.quantitativeskills.com/sisa/.
TUNEL assay
RB1-deficient C-33 A cells [
26] were transfected with pcDNA3.1/V5-His-TOPO vector or one of the four constructs
RB1wild-type-V5,
RB1Arg621Cys-V5, RB1Leu607Ile-V5, or RB1Arg698Trp-V5. After 24 hours, the cells were treated with 5 μM doxorubicin (adriamycin) for 6 hours, and washed with 1× PBS before cytospins were prepared. The cells were fixed with 3.7% paraformaldehyde o.n at room temperature and permabilized with 0.1% Triton X-100 in sodium citrate for 2 min on ice. TUNEL reaction mixture (In situ Cell Death Detection Kit, TMR red, Roche) was applied for 2.5 h in dark at 37°C. Controls were either treated with DNaseI (positive control) or with a TUNEL reaction mixture not containing enzyme. Hoechst was used to visualize the nucleus and the cells were analyzed using Leica DMI6000B epifluorescence microscope (Leica Microsystems). A minimum of 1000 cells for all samples were counted in each of three independent experiments. The same experiment was repeated in Saos-2 cells. Here, the optimal conditions for detection of changes in pro-apoptotic function differed from C-33 A cells. Saos-2 cells were treated with 0.5 μM doxorubicin (adriamycin) for 12 hours before harvest.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EOB prepared the manuscript and was responsible for the immunofluoresence experiment. EOB and SK carried out the LOH, MLPA and TUNEL assays and the statistical calculations. SG performed the nucleic acids extraction, sequencing, and methylation experiments supported by VS and EOB. PP was responsible for the multiple sequence alignment and the in silico structural modeling analysis. MP contributed to the design of plasmid constructs, and ALBD was involved in the LOH analysis and helped drafting the manuscript. JRL supervised the laboratory analyses and participated in writing the paper. PEL directed the clinical study and provided the tumor samples. In addition PEL conceived and directed the project, and contributed to the writing of the manuscript. All authors have read and approved the manuscript.