RESUMEN

INTRODUCTION: Insulin icodec is a novel, long-acting insulin analog designed to cover basal insulin requirements with once-weekly subcutaneous administration. Here we describe the molecular engineering and the biological and pharmacological properties of insulin icodec. RESEARCH DESIGN AND METHODS: A number of in vitro assays measuring receptor binding, intracellular signaling as well as cellular metabolic and mitogenic responses were used to characterize the biological properties of insulin icodec. To evaluate the pharmacological properties of insulin icodec in individuals with type 2 diabetes, a randomized, double-blind, double-dummy, active-controlled, multiple-dose, dose escalation trial was conducted. RESULTS: The long half-life of insulin icodec was achieved by introducing modifications to the insulin molecule aiming to obtain a safe, albumin-bound circulating depot of insulin icodec, providing protracted insulin action and clearance. Addition of a C20 fatty diacid-containing side chain imparts strong, reversible albumin binding, while three amino acid substitutions (A14E, B16H and B25H) provide molecular stability and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro cell-based studies showed that insulin icodec activates the same dose-dependent IR-mediated signaling and metabolic responses as native human insulin (HI). The affinity of insulin icodec for the insulin-like growth factor-1 receptor was proportionately lower than its binding to the IR, and the in vitro mitogenic effect of insulin icodec in various human cells was low relative to HI. The clinical pharmacology trial in people with type 2 diabetes showed that insulin icodec was well tolerated and has pharmacokinetic/pharmacodynamic properties that are suited for once-weekly dosing, with a mean half-life of 196 hours and close to even distribution of glucose-lowering effect over the entire dosing interval of 1 week. CONCLUSIONS: The molecular modifications introduced into insulin icodec provide a novel basal insulin with biological and pharmacokinetic/pharmacodynamic properties suitable for once-weekly dosing. TRIAL REGISTRATION NUMBER: NCT02964104.

Asunto(s)

Diabetes Mellitus Tipo 2 , Insulina , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Hipoglucemiantes/farmacología , Insulina de Acción Prolongada , Insulina Regular Humana

RESUMEN

Insulin analogue X10 has a higher mitogenic potency than native human insulin in vitro and supra-pharmacological doses of insulin X10 increased the incidence of mammary tumours in rats. Compared to native human insulin, insulin X10 has increased binding affinity to the insulin receptor and the IGF-1 receptor, but it is not known whether either or both characteristics are important for stimulation of cell proliferation in vivo. The aim of this study was to explore how increased binding affinity to the insulin receptor or the IGF-1 receptor contributes to stimulation of cell proliferation in vivo. A mouse xenograft model was established with rat L6 myoblast cells transfected with the human insulin receptor (L6hIR cells) and effects of supra-pharmacological doses of native human insulin, insulin X10 or novel insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor were examined. Treatment with insulin X10 and insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor increased growth of L6hIR cell xenografts significantly compared to native human insulin. Thus, increased binding affinity to the insulin receptor and the IGF-1 receptor are each independently linked to increased growth of L6hIR cell xenografts in vivo.

Asunto(s)

Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animales , Xenoinjertos , Humanos , Insulina/metabolismo , Ratones , Unión Proteica , Ratas

RESUMEN

Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples. Using these strategies, we were able to identify highly specific insulin receptor monoclonal antibodies that lack cross-reactivity to the IGF1 receptor using the Adimab platform and a highly specific IGF1 receptor monoclonal antibody that lacks cross-reactivity to the insulin receptor using the rabbit antibody platform. Unlike earlier monoclonal antibodies reported in the literature, these antibodies show cross-species reactivity to the extracellular domains of mouse, rat, pig, and human receptors, indicating that they bind conserved epitopes. Furthermore, the antibodies work well in several different assay formats, including ELISA, flow cytometry, and immunoprecipitation, and therefore provide new tools to study insulin and IGF1 receptor biology with translation across several species and experimental model systems.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Receptor IGF Tipo 1/inmunología , Receptor de Insulina/inmunología , Animales , Anticuerpos Monoclonales/química , Reacciones Cruzadas , Células HCT116 , Humanos , Ratones , Conejos , Ratas , Especificidad de la Especie , Porcinos

RESUMEN

AIMS/HYPOTHESIS: Recent studies with normal rats and mouse allograft models have reported that insulin and insulin analogues do not activate the IGF-1 receptor in vivo, and that this characteristic therefore cannot be responsible for the increased incidence of mammary tumours observed for the insulin analogue X10 in chronic toxicity studies with Sprague Dawley rats. This is in clear contrast to reports of insulin and insulin analogues in vitro. Clarification of this is important for understanding the mechanisms behind possible growth-promoting effects of insulin analogues, and will have implications for the development of novel insulin analogues. METHODS: We established a xenograft model in BALB/c nude mice with the human colon cancer cell line COLO-205, which expresses human insulin and IGF-1 receptors, and explored the acute and chronic effects of treatment with supra-pharmacological doses of human insulin, insulin analogue X10 and human IGF-1. With a novel antibody, acute IGF-1 receptor activation was also examined in various tissues from normal rats treated with human insulin, insulin analogue X10 or human IGF-1. Finally, the effects of pharmacologically relevant doses of human insulin and insulin analogue X10 on receptor activation and growth of COLO-205 xenograft were explored in BALB/c nude mice with alloxan-induced hyperglycaemia. RESULTS: In normal rats and in BALB/c nude mice bearing a COLO-205 cell xenograft, treatment with supra-pharmacological doses of human insulin, insulin analogue X10 or human IGF-1 resulted in activation of insulin receptors as well as IGF-1 receptors. Treatment of diabetic nude mice with pharmacologically relevant doses of human insulin or insulin analogue X10, which decreased blood glucose from hyperglycaemic levels to the normoglycaemic range, did not increase IGF-1 receptor activation. Furthermore, repeated treatment with supra-pharmacological as well as pharmacological doses of human insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts. CONCLUSIONS/INTERPRETATION: This study demonstrates that activation of IGF-1 receptors in cancer cells by insulin and insulin analogues cannot be considered as a purely in vitro phenomenon. It does occur in vivo in animal models, although only after treatment with supra-pharmacological doses. Furthermore, treatment with insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts under normo- or hypoglycaemic conditions. Further studies are needed before a conclusion can be reached on whether IGF-1 receptor activation by insulin analogues correlates with increased growth in vivo.

Asunto(s)

Hipoglucemia/tratamiento farmacológico , Hipoglucemia/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Aloxano/toxicidad , Animales , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante Heterólogo

RESUMEN

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.

Asunto(s)

Reparación de la Incompatibilidad de ADN , ADN Polimerasa III/metabolismo , Mutación , Metilación de ADN/efectos de los fármacos , ADN Polimerasa III/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Células HeLa , Humanos , Metilnitronitrosoguanidina/farmacología

RESUMEN

Epidemiological and laboratory studies raise the possibility of a link between clinically prescribed insulin analogues and increased cancer risk. Accordingly, there is a regulatory mandate for cancer-related pre-clinical safety evaluation during insulin analogue development, but currently, there is no standardized framework for such in vitro evaluation. We tested human insulin; the super-mitogenic insulin, X10 and insulin-like growth factor I, in four cancer cell lines with a range of insulin-like growth factor-I receptor (IGF-IR)/IR (insulin receptor) ratios (HCT 116, HT-29, COLO 205 and MCF7) and related these to IGF-IR and IR expression in 17 human adenocarcinomas. All cell types were IR-A isoform dominant. We determined IGF-IR/IR signalling pathway endpoints in dose- and time-varying experiments, and performed mitogenic dose-response equivalent assays to derive EC50 values, and correlated these with IGF-IR/IR ratios. We superimposed relative EC50 values onto data from the literature in a meta-analysis. The IGF-IR/IR ratios varied from <1 to 12 in the selected cell lines; similar pattern ranges were observed in human adenocarcinomas. The three ligands demonstrated differential IR/IGF-IR and Akt phosphorylation, which correlated with cell-specific IGF-IR/IR ratios. Mitogenic profiles of X10 mimicked those for insulin-like growth factor I (IGF-I) and correlated with IGF-IR/IR ratios. The meta-analysis, adding data from five additional studies, supported the hypothesis that ligand mitogenic potency, relative to human insulin, increases with increasing cell-specific IGF-IR/IR ratio. This study established a framework for the in vitro evaluation of cancer-relevant bioassays for comparisons of insulin analogues, and specifically consolidated earlier studies that determination of the cell-specific IGF-IR/IR ratio is crucial for the interpretation of ranking relative biological activities.

Asunto(s)

Neoplasias de la Mama/patología , Neoplasias del Colon/patología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/análogos & derivados , Receptor de Insulina/metabolismo , Adenocarcinoma/patología , Apoptosis/genética , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Neoplasias del Colon/epidemiología , Neoplasias del Colon/genética , Femenino , Células HCT116 , Células HT29 , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Células MCF-7 , Fosforilación , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor de Insulina/biosíntesis , Receptor de Insulina/genética , Transducción de Señal

RESUMEN

There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF-I receptors (IGF-IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF-I caused phosphorylation of the IR as well as IGF-IR. Insulin exhibited mitogenicity EC(50) values in the single-digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF-IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA-mediated knockdown of IR and IGF-IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF-IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF-IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF-I in cells expressing more IR than IGF-IR, the hyper-mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper-mitogenic effect of X10 involves the IR as well as the IGF-IR. These results are relevant for preclinical safety assessment of developmental insulin analogues.

Asunto(s)

Insulina/farmacología , Mitosis/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Células HCT116/efectos de los fármacos , Células Hep G2/efectos de los fármacos , Humanos , Insulina/análogos & derivados , Células MCF-7/efectos de los fármacos , Ratas

RESUMEN

In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare.

Asunto(s)

Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética , Modelos Moleculares , Proteína 2 Homóloga a MutS/genética , Sustitución de Aminoácidos/genética , Secuencia de Bases , Pruebas Genéticas , Humanos , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/deficiencia , Mutagénesis , Mutación/genética , Genética Inversa/métodos , Análisis de Secuencia de ADN , Tioguanina , Virulencia/genética

RESUMEN

Germline mutations in the human DNA mismatch repair (MMR) genes MSH2 and MLH1 are associated with the inherited cancer disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer or HNPCC. A proportion of MSH2 and MLH1 mutations found in suspected LS patients give rise to single amino acid substitutions. The functional consequences in regard to pathogenicity of many of these variants are unclear. We have examined the functionality of a panel of MLH1 missense mutations found in LS families, by testing the variant proteins in functional assays, addressing subcellular localization, and protein-protein interaction with the dimer partner PMS2 and the MMR-associated exonuclease 1. We show that a significant proportion of examined variant proteins have functional defects in either subcellular localization or protein-protein interactions, which is suspected to lead to the cancer phenotype observed in patients. Moreover, the obtained results correlate well with reported MMR activity and with in silico analysis for a majority of the variants.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Mutación Missense , Proteínas Nucleares/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/química , Animales , Enzimas Reparadoras del ADN/química , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Ratones , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Modelos Moleculares , Homólogo 1 de la Proteína MutL , Proteínas MutL , Mutagénesis Sitio-Dirigida , Células 3T3 NIH , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Homología Estructural de Proteína

RESUMEN

AIMS/HYPOTHESIS: There is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10. METHODS: We measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types. RESULTS: Detemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤ 1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs. CONCLUSIONS: Detailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.

Asunto(s)

Hipoglucemiantes/farmacocinética , Insulina de Acción Prolongada/farmacocinética , Insulina Regular Humana/farmacocinética , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Receptor IGF Tipo 1/metabolismo , Células Cultivadas , Femenino , Humanos , Hipoglucemiantes/farmacología , Insulina Detemir , Insulina Glargina , Insulina de Acción Prolongada/farmacología , Insulina Regular Humana/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Factor I del Crecimiento Similar a la Insulina/farmacología , Mitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica , Receptor IGF Tipo 1/genética

RESUMEN

DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3-34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89-108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.

Asunto(s)

Núcleo Celular/metabolismo , Reparación del ADN , ADN/genética , Transporte Activo de Núcleo Celular , Núcleo Celular/genética , Humanos , Transporte de Proteínas

RESUMEN

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.

Asunto(s)

Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteína 2 Homóloga a MutS/genética , Mutación Missense , Animales , Secuencia de Bases , Neoplasias Colorrectales Hereditarias sin Poliposis/metabolismo , Reparación de la Incompatibilidad de ADN/genética , ADN de Neoplasias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ratones , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/deficiencia , Proteína 2 Homóloga a MutS/metabolismo , Mutagénesis Sitio-Dirigida , Células 3T3 NIH , Fenotipo , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome is caused by DNA variations in the DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and PMS2. Many of the mutations identified result in premature termination of translation and thus in loss-of-function of the encoded mutated protein. These DNA variations are thought to be pathogenic mutations. However, some patients carry other DNA mutations, referred to as unclassified variants (UVs), which do not lead to such a premature termination of translation; it is not known whether these contribute to the disease phenotype or merely represent rare polymorphisms. This is a major problem which has direct clinical consequences. Several criteria can be used to classify these UVs, such as: whether they segregate with the disease within pedigrees, are absent in control individuals, show a change of amino acid polarity or size, provoke an amino acid change in a domain that is evolutionary conserved and/or shared between proteins belonging to the same protein family, or show altered function in an in vitro assay. In this review we discuss the various functional assays reported for the HNPCC-associated MMR proteins and the outcomes of these tests on UVs identified in patients diagnosed with or suspected of having HNPCC. We conclude that a large proportion of MMR UVs are likely to be pathogenic, suggesting that missense variants of MMR proteins do indeed play a role in HNPCC.

Asunto(s)

Disparidad de Par Base , Reparación del ADN/genética , Humanos , Polimorfismo Genético

RESUMEN

The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.

Asunto(s)

Disparidad de Par Base/efectos de los fármacos , Cloruro de Cadmio/toxicidad , Reparación del ADN/efectos de los fármacos , Alquilación , Secuencia de Bases , Carcinógenos/toxicidad , Cationes Bivalentes/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular , ADN/efectos de los fármacos , ADN/genética , Daño del ADN , Enzimas Reparadoras del ADN , Desoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Humanos , Riñón , Cinética , Oligodesoxirribonucleótidos/química

RESUMEN

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.

Asunto(s)

Disparidad de Par Base , Ciclo Celular , Ciclina D1/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Metilación de ADN , Alquilantes/toxicidad , Alquilación , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Cisteína Endopeptidasas/metabolismo , Daño del ADN/genética , Reparación del ADN , ADN de Neoplasias/análisis , Humanos , Masculino , Metilnitronitrosoguanidina/toxicidad , Complejos Multienzimáticos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Complejo de la Endopetidasa Proteasomal , Factores de Tiempo , Rayos Ultravioleta/efectos adversos

RESUMEN

Human exonuclease 1 (hEXO1) has been implicated in DNA mismatch repair (MMR), replication, and recombination, but the nature of its interaction with these cellular processes is still ambiguous. We show that hEXO1 colocalizes with proliferating cell nuclear antigen (PCNA) at DNA replication sites and that the C-terminal region of hEXO1 is sufficient for this localization. We also show that both hMLH1-hPMS2 (MutLalpha) and hMLH1-hEXO1 complexes are formed in a reaction mixture containing all three proteins. Moreover, hEXO1 5' double-stranded exonuclease activity on a homoduplex substrate but not on a substrate containing a G/T mismatch was inhibited by complex formation with hMSH2-hMSH6 (MutSalpha) or MutLalpha. Taken together, the results support a model in which hEXO1 plays a role in events at the replication sites as well as a functional role in the MMR and/or recombination processes.

Asunto(s)

División Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Reparación del ADN/fisiología , Enzimas Reparadoras del ADN , Humanos , Homólogo 1 de la Proteína MutL , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Regulación hacia Arriba