Claus Kristensen

Alanine Scanning Mutagenesis of Insulin

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the α-helix B8–B19 to the β-turn B20–B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.

Impact of HPV-associated p16-expression on radiotherapy outcome in advanced oropharynx and non-oropharynx cancer

BACKGROUND AND PURPOSE HPV is found in head and neck cancer from all sites with a higher prevalence in oropharynx cancer (OPC) compared to non-OPC. HPV/p16-status has a significant impact on radiotherapy (RT) outcome in advanced OPC, but less is known about the influence in non-OPC. We analyzed HPV-associated p16-expression in a cohort of patients with stage III-IV pharynx and larynx cancer treated with primary, curatively intended (chemo-)RT, aiming to test the hypothesis that the impact of HPV/p16 also extends to tumors of non-oropharyngeal origin. MATERIAL AND METHODS 1294 patients enrolled in previously conducted DAHANCA-trials between 1992 and 2012 were identified. Tumors were evaluated by p16-immunohistochemistry and classified as positive in case of staining in >70% of tumors cells. RESULTS Thirty-eight percent (490/1294) of the tumors were p16-positive with a significantly higher frequency in OPC (425/815) than in non-OPC (65/479), p<.0001. In OPC p16-positivity significantly improved loco-regional control (LRC) (adjusted HR [95% CI]: 0.43 [0.32-0.57]), event-free survival (EFS) (HR 0.44 [0.35-0.56]), and overall survival (OS) (HR: 0.38 [0.29-0.49]), respectively, compared with p16-negativity. In non-OPC no prognostic impact of p16-status was found for either endpoint: LRC (HR: 1.13 [0.75-1.70]), EFS (HR: 1.06 [0.76-1.47]), and OS (HR: 0.82 [0.59-1.16]). CONCLUSIONS The independent influence of HPV-associated p16-expression in advanced OPC treated with primary RT was confirmed. However, RT-outcome in the group of non-OPC did not differ by tumor p16-status, indicating that the prognostic impact may be restricted to OPC only.

Expression and Characterization of a 70-kDa Fragment of the Insulin Receptor That Binds Insulin MINIMIZING LIGAND BINDING DOMAIN OF THE INSULIN RECEPTOR

In order to characterize regions of the insulin receptor that are essential for ligand binding and possibly identify a smaller insulin-binding fragment of the receptor, we have used site-directed mutagenesis to construct a series of insulin receptor deletion mutants. From 112 to 246 amino acids were deleted from the α-subunit region comprising amino acids 469–729. The receptor constructs were expressed as soluble insulin receptor IgG fusion proteins in baby hamster kidney cells and were characterized in binding assays by immunoblotting and chemical cross-linking with radiolabeled insulin. The shortest receptor fragment identified was a free monomeric α-subunit deleted of amino acids 469–703 and 718–729 (exon 11); the mass of this receptor fragment was found by mass spectrometry to be 70 kDa. This small insulin receptor fragment bound insulin with an affinity (K d ) of 4.4 nm, which is similar to what was found for the full-length ectodomain of the insulin receptor (5.0 nm). Cross-linking experiments confirmed that the 70-kDa receptor fragment specifically bound insulin. In summary we have minimized the insulin binding domain of the insulin receptor by identifying a 70-kDa fragment of the ectodomain that retains insulin binding affinity making this an interesting candidate for detailed structural analysis.

Specificity of Insulin and Insulin-like Growth Factor I Receptors Investigated using Chimeric Mini-Receptors ROLE OF C-TERMINAL OF RECEPTOR α SUBUNIT

We have investigated the role of the C-terminal of the α-subunit in the insulin receptor family by characterizing chimeric mini-receptor constructs comprising the first three domains (468 amino acids) of insulin receptor (IR) or insulin-like growth factor I receptor (IGFIR) combined with C-terminal domain from either insulin receptor (IR) (residues 704–719), IGFIR, or insulin receptor-related receptor (IRRR). The constructs were stably expressed in baby hamster kidney cells and purified, and binding affinities were determined for insulin, IGFI, and a single chain insulin/IGFI hybrid. The C-terminal domain of IRRR was found to abolish binding in IR and IGFIR context, whereas other constructs bound ligands. The two constructs with first three domains of the IR demonstrated low specificity for ligands, all affinities ranging from 3.0 to 15 nm. In contrast, the constructs with the first three domains of the IGFIR had high specificity, the affinity of the novel minimized IGFIR for IGFI was 1.5 nm,whereas the affinity for insulin was more than 3000 nm. When swapping the C-terminal domains in either receptor context only minor changes were observed in affinities (<3-fold), demonstrating that the carboxyl-terminal of IR and IGFIR α-subunits are interchangeable and suggesting that this domain is part of the common binding site.

The impact of comorbidity on outcome in 12 623 Danish Head and Neck Cancer Patients: A population based study from the DAHANCA database

Abstract Background. Head and neck squamous cell carcinoma (HNSCC) is primarily caused by smoking and alcohol. Besides having a carcinogenic effect, smoking also leads to other diseases and thus contributes to a high prevalence of comorbidities among HNSCC patients. Furthermore, the world population is becoming older resulting in more elderly patients with HNSCC. The aim of this study was to investigate the prevalence and impact of comorbidity in a retrospective nationwide population-based study of all Danish HNSCC patients diagnosed from 1992 to 2008. Material and methods. A total of 12 623 patients diagnosed with HNSCC in the period from 1992 to 2008 were identified through the Danish Head and Neck Cancer group (DAHANCA) database. By linking to the Danish registers, information on somatic comorbidity present prior to the HNSCC diagnosis was obtained and adapted to the Charlson Comorbidity Index (CCI). The influence of comorbidity on overall survival and cancer specific death was evaluated and the type and prevalence of comorbidity described. Results. In total, 36% of patients had comorbidity according to CCI. Increasing age was significantly associated with increasing CCI. In multivariate analyses, the CCI score remained a strong independent prognostic factor for overall survival, the HR being 1.16 (95% CI 1.08; 1.25), 1.34 (1.22; 1.46), 1.63 (1.51; 1.80) for patients with CCI score 1, 2, and 3+, respectively. The CCI score did not influence cancer specific death. Conclusion. Comorbidity is common among HNSCC patients and has a negative prognostic impact on overall survival. Cancer specific death was not affected by comorbidity suggesting that patients die from their comorbidities rather than their cancer. In the future, more elderly patients with comorbidity will require treatment which will demand a change in the healthcare system with a multidisciplinary approach required in order to take care of both their cancer and their comorbidities.

Recurrences after intensity modulated radiotherapy for head and neck squamous cell carcinoma more likely to originate from regions with high baseline [18F]-FDG uptake.

BACKGROUND AND PURPOSE To analyze the recurrence pattern in relation to target volumes and (18)F-fluorodeoxyglucose (FDG) uptake on positron emission tomography in head and neck squamous cell carcinoma (HNSCC) patients treated with definitive chemoradiation. MATERIAL AND METHODS 520 patients received radiotherapy for HNSCC from 2005 to 2009. Among 100 patients achieving complete clinical response and a later recurrence, 39 patients with 48 loco-regional failures had a recurrence CT scan before any salvage therapy. The estimated point of origin of each recurrence was transferred to the planning CT by deformable image co-registration. The recurrence position was then related to the delineated target volumes and iso-SUV-contours relative to the maximum standard uptake value (SUV). We defined the recurrence density as the total number of recurrences in a sub-volume divided by the sum of that volume for all patients. RESULTS 54% (95% CI 37-69%) of recurrences originated inside the FDG-positive volume and 96% (95% CI 86-99%) in the high dose region. Recurrence density was significantly higher in the central target volumes (P<0.0001) and increased with increasing FDG avidity (P=0.036). CONCLUSIONS The detailed pattern-of-failure data analysis suggests that most recurrences occur in the FDG PET positive areas or the solid tumor.

Role of Insulin Receptor Dimerization Domains in Ligand Binding, Cooperativity, and Modulation by Anti-receptor Antibodies

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the α-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between α-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1–593 and 704–719) bound 125I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1–601 and 650–719) and greater than that of dimeric mIR.Fn0 (amino acids 1–601 and 704–719) and monomeric IR473.CT (amino acids 1–473 and 704–719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound125I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471–593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.

Evaluation of comorbidity in 9388 head and neck cancer patients: a national cohort study from the DAHANCA database.

BACKGROUND Comorbidity is common in head and neck squamous cell carcinoma (HNSCC) patients due to the etiology of the disease being primarily smoking. The aim of this study was to investigate the impact of comorbidity on survival in a national population-based cohort study on 9388 HNSCC-patients treated with radiotherapy (RT), to re-evaluate the prognostic impact of individual diseases within the Charlson Comorbidity Index (CCI), and to develop a revised head and neck comorbidity index (HN-CCI). MATERIAL AND METHODS A national cohort of 9388 HNSCC-patients treated with curative intended RT diagnosed from 1992 to 2008 was identified from the DAHANCA-database. Data on comorbidity prior to HNSCC-diagnosis was obtained from the National Patient Registry and adapted to the CCI. RESULTS By dividing the patients into two groups, we tested and validated which type of comorbidities within the CCI affected overall survival (OS) and cancer specific death (CSD). In total, 36% of patients had comorbidity. Six comorbid conditions within the CCI significantly reduced five-year OS probability: congestive heart failure, cerebrovascular disease, chronic pulmonary disease, peptic ulcer disease, liver disease, and diabetes, and based on these conditions the new head and neck specific comorbidity index was developed, the HN-CCI. Comorbidity according to HN-CCI had a highly significant impact on OS, whereas it was not associated with CSD. Chronological age was not associated with increased risk of CSD after controlling for comorbidity. CONCLUSIONS Comorbidity is frequent in HNSCC patients and negatively impacts OS. Therefore assessment of comorbidity will be of great importance, both in order to treat/optimize patients health before radiotherapy, but also in order to be able to stratify/control for comorbidity in randomized trials to avoid bias. Re-evaluation of the CCI revealed that only six conditions had an impact on survival, and a new modified index to assess comorbidity for HNSCC-patients was developed. The performance of HN-CCI to stratify patients on survival was good and HN-CCI is highly recommended for future assessment of comorbidity and prognostic staging of radiotherapy-treated HNSCC-patients.

Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity.

The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the α-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780–17786). To investigate the insulin binding properties of dimerized α-subunits, we have reintroduced the domains containing α-α disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4–8 pm) similar to what was obtained for the solubilized holoreceptor (14–24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the α-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.

Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy

BackgroundThe DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins.ResultsHere we present the genomic sequence of the CHO DXB11 genome sequenced to a depth of 33x. Overall a significant genomic drift was seen favoring GC → AT point mutations in line with the chemical mutagenesis strategy used for generation of the cell line. The sequencing depth for each gene in the genome revealed distinct peaks at sequencing depths of 0x, 16x, 33x and 49x coverage corresponding to a copy number in the genome of 0, 1, 2 and 3 copies. This indicate that 17% of the genes are haploid revealing a large number of genes which can be knocked out with relative ease. This tendency of haploidy was furthermore shown to be present in eight additional analyzed CHO genomes (15-20% haploidy) but not in the genome of the Chinese hamster. The dhfr gene is confirmed to be haploid in CHO DXB11; transcriptionally active and the remaining allele contains a G410C point mutation causing a Thr137Arg missense mutation. We find ~2.5 million single nucleotide polymorphisms (SNP’s), 44 gene deletions in the CHO DXB11 genome and 9357 SNPs, which interfere with the coding regions of 3458 genes. Copy number variations for nine CHO genomes were mapped to the chromosomes of the Chinese hamster showing unique signatures for each chromosome. The data indicate that chromosome one and four appear to be more stable over the course of the CHO evolution compared to the other chromosomes thus might presenting the most attractive landing platforms for knock-ins of heterologous genes.ConclusionsOur studies reveal an unexpected degree of haploidy in CHO DXB11 and CHO cells in general and highlight the chromosomal changes that have occurred among the CHO cell lines sequenced to date.