A cancer derived mutation in the Retinoblastoma gene with a distinct defect for LXCXE dependent interactions
© Henley et al; licensee BioMed Central Ltd. 2010
Received: 18 January 2010
Accepted: 18 March 2010
Published: 18 March 2010
The interaction between viral oncoproteins such as Simian virus 40 TAg, adenovirus E1A, and human papilloma virus E7, and the retinoblastoma protein (pRB) occurs through a well characterized peptide sequence, LXCXE, on the viral protein and a well conserved groove in the pocket domain of pRB. Cellular proteins, such as histone deacetylases, also use this mechanism to interact with the retinoblastoma protein to repress transcription at cell cycle regulated genes. For these reasons this region of the pRB pocket domain is thought to play a critical role in growth suppression.
In this study, we identify and characterize a tumor derived allele of the retinoblastoma gene (RB1) that possesses a discrete defect in its ability to interact with LXCXE motif containing proteins that compromises proliferative control. To assess the frequency of similar mutations in the RB1 gene in human cancer, we screened blood and tumor samples for similar alleles. We screened almost 700 samples and did not detect additional mutations, indicating that this class of mutation is rare.
Our work provides proof of principal that alleles encoding distinct, partial loss of function mutations in the retinoblastoma gene that specifically lose LXCXE dependent interactions, are found in human cancer.
Results and Discussion
Summary of mutation detection.
In this study we sought to identify cancer-associated mutations that specifically target the region of RB1 encoding the LXCXE binding cleft. In this way, our goal was to search for distinct RB1 alleles that are compromised for just one aspect of its function. We characterized an ovarian cancer derived variant of RB1 that possesses biochemical properties that are comparable to our previously published Rb1 ΔL mutant allele. We also showed that a single mutant allele of Rb1 can significantly decrease the response to TGF-β mediated growth arrest, indicating that the retinoblastoma gene displays haploinsufficiency for growth control. While we were unable to find more examples of this type of allele, our work suggests that cancer types that are characterized by resistance to TGF-β growth arrest and maintain heterozygosity for RB1 may possess this type of mutant allele as a means of overcoming growth inhibitory mechanisms that limit tumor formation.
Protein interaction analysis
Glutathione S-transferase (GST) pull-down experiments were performed as described in Dick et al. . The GST-RB constructs contain the large pocket of pRB, encompassing amino acids 379 to 928. The GST-RBΔL and GST-RBM704V contain I753A, N757A and M761A or M704V substitutions, respectively. In each case, recombinant proteins (or GST as a control) were incubated with whole-cell extracts from C33A cells transfected with either CMV-HA-E2F3 and CMV-HA-DP1, or CMV-TAg. Bound proteins were detected by western blotting using 12CA5 hybridoma supernatant for HA-tagged E2F3/DP1, or mouse monoclonal antibody PAb419 (CalBiochem) for SV40-TAg, followed by a peroxidase conjugated anti-mouse IgG secondary antibody. Input levels of GST-fusions were detected by Coomassie staining.
Immunoprecipitations were performed essentially as described in Siefried et al. . The pRB constructs tested contain either the full-length wild type RB1 cDNA (CMV-RB) or RB1 cDNA that has been mutated to create the desired amino acid substitutions (CMV-RBΔL and CMV-RBM704V). In brief, C33A cells were transfected with 10 μg of each CMV-RB construct in addition to either 5 μg of CMV-TAg or 5 μg each of CMV-HA-E2F3 and CMV-HA-DP1. To precipitate TAg immune complexes, the PAb419 antibody (CalBiochem) was used, whereas 12CA5 hybridoma supernatant was used to pull down HA-containing immune complexes. In each case, bound pRB was detected by western blotting using the G3-245 α-pRB antibody (BD Pharmingen), as above. HA-E2F3, HA-DP1 and TAg input levels were also detected by western blotting.
Protein stability assay
To assess protein stability, 2.6 × 106 C33A cells were transfected by Ca2PO4 with 45 μg of CMV-RB, CMV-RBΔL or CMV-RBM704V in a 15 cm plate. Twenty-four hours post-transfection cells were replated into 6 replicate 6 cm plates. Twenty-four hours after replating, cells were treated with 100 μg/mL cycloheximide and harvested after 0, 3, 6, 9, 12 and 15 hours. To detect pRB, 10 μg of total protein was analyzed by western blotting as described above. Protein levels were quantified by densitometry using a Bio Rad Gel Doc XR gene imager and half-lives were calculated accordingly. To assess the expression of pRB in vivo, asynchronously proliferating Rb1 + /+ , Rb1 ΔL/ + and Rb1 ΔL/ΔL MEFs were harvested and 50 μg of total protein was analyzed by western blotting using the Rb1 4.1 hybridoma, followed by a peroxidase conjugated anti-mouse IgG secondary antibody. The hybridoma developed by Julien Sage was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
TGF-β growth arrest assays
Asynchronously proliferating Rb1 + / + , Rb1 ΔL/+ and Rb1 ΔL/ΔL MEFs were treated with 100 pM TGF-β1 (R&D systems) for 24 hours. Cells were then pulse labelled with BrdU (RPN201V1, Amersham Biosciences) for 1.5 hours. BrdU incorporation was quantified using flow cytometry on a Beckman-Coulter EPICS XL-MCL instrument, as previously described .
Peripheral blood DNA samples (627 specimens) from human breast and/or ovarian cancer patients were obtained from the London Health Sciences Centre, Molecular Diagnostics Laboratory (London, ON). All samples were post-testing material from individuals that meet Ontario provincial referral criteria because they are under the age of 35 (173), have three or more cases of breast or ovarian cancer on the same side of the family (155) or otherwise have a pedigree that is strongly suggestive of hereditary breast/ovarian cancer. Frozen breast tumor samples were obtained from the Ontario Tumour Bank. Samples were chosen at random (not based on histological characteristics) from patients between 38 and 87 years of age. All tumor material was derived from the primary site. DNA was isolated from these samples using standard techniques. DNA from ovarian tumor samples was prepared from passage two cells that were isolated from patient ascites. Ovarian samples were from patients between 25 and 85 years of age. DNA was provided courtesy of the Translational Ovarian Cancer Program at the London Health Sciences Centre.
High resolution melting (HRM) analysis
HRM analysis was conducted using the Roche Lightcycler 480 HRM kit. In brief, each 20 μL reaction consisted of 10 μL of 2× Master Mix, 0.1875 μM of each primer, 3.0 to 4.0 mM of MgCl2 and 50 ng of genomic DNA. As a positive control, test constructs encoding individual exons with either a single nucleotide change (mutant) or without (wild type) were mixed in equal quantities and 0.1 pg of the resulting mixture was used in place of a genomic DNA sample. The mutant constructs for exon 21 and exon 22 each contain a single G to T substitution (g.160839G>T and g.162027G>T) from previously reported cancer-causing RB1 alleles , whereas the construct for exon 23 contained a previously reported C to G substitution (g.162241G>C). To amplify each exon the following primer pairs were used: exon 21 cagtatggaaagaaataactctgtag and gtgaatttacataataaggtcagacag, exon 22 gcccccgccgttactgttcttcctcagacattcaa and cccccgcccgaatgttttggtggacccatt and exon 23 gcggcccgccgcccccgccgcttccaccagggtaggtcaa and gccgggcgcgcccccgcccgggatcaaaataatccccctctcat. The amplification and melt analysis were conducted sequentially in the Roche Lightcycler 480. First, samples were incubated at 95°C for 10 minutes, followed by 50 cycles of: 95°C for 10 seconds, a touch down of 65 to 55°C (exon 21) or 70 to 65°C (exons 22 and 23) for 20 seconds, 72°C for 10 seconds. The samples were then heated to 95°C to generate a melt curve. All samples were run in duplicate and each plate contained duplicate positive controls. Lightcycler 480 Gene Scanning software was used to normalize the data and generate difference plots.
To detect sequence changes in tumor DNA samples, PCR products were generated and directly sequenced (McGill University and Genome Quebec Innovation Centre). Exon 21 products were obtained and sequenced using primers 21F (ttgggttaaacacttcatgtagac) and 21R (cctatgttatgttatggatatggatt). Exons 22 and 23 were amplified in a single reaction using primers 22F (tataatatgtgcttcttaccagtcaa) and 23R (aagcaaatatgagtttcaagagtctagc) and sequenced using primers 22F and 23R2 (gcgttgcttaagtcgtaaatagatt).
- RB1 :
human retinoblastoma gene
- Rb1 :
murine retinoblastoma gene
transforming growth factor-beta
high resolution melt.
We would like to thank many past and present laboratory members for contributions during the course of this work. In particular, we are grateful to Alan Stuart, Lindsay Jordan, Steven Russell, and Janet Lew for assistance with plasmid constructions and mutation screening. We are thankful for the generosity of Drs. Trevor Shepherd and Gabe DiMattia, and the Translational Ovarian Cancer Program for providing ovarian cancer derived patient DNA. SMF is supported by a Translational Breast Cancer Studentship from the LRCP. SAH is supported by a post-doctoral fellowship from the Canadian Breast Cancer Foundation, Ontario Chapter. SMF and SAH are thankful for previous stipend support from the Strategic Training Program in Cancer Research (CaRTT). FAD is a Research Scientist of the Canadian Cancer Society. Grant funding from the Cancer Research Society supported this project.
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