RESUMEN
Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Benzodiazepinas/química , Inmunoconjugados/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de Microfilamentos/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Pirroles/química , Animales , Antineoplásicos/química , Apoptosis , Proliferación Celular , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inmunoconjugados/química , Macaca fascicularis , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Microfilamentos/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background Heart failure is one of the leading causes of death in Western countries, and there is a need for new therapeutic approaches. Relaxin-2 is a peptide hormone that mediates pleiotropic cardiovascular effects, including antifibrotic, angiogenic, vasodilatory, antiapoptotic, and anti-inflammatory effects in vitro and in vivo. Methods and Results We developed RELAX10, a fusion protein composed of human relaxin-2 hormone and the Fc of a human antibody, to test the hypothesis that extended exposure of the relaxin-2 peptide could reduce cardiac hypertrophy and fibrosis. RELAX10 demonstrated the same specificity and similar in vitro activity as the relaxin-2 peptide. The terminal half-life of RELAX10 was 7 days in mouse and 3.75 days in rat after subcutaneous administration. We evaluated whether treatment with RELAX10 could prevent and reverse isoproterenol-induced cardiac hypertrophy and fibrosis in mice. Isoproterenol administration in mice resulted in increased cardiac hypertrophy and fibrosis compared with vehicle. Coadministration with RELAX10 significantly attenuated the cardiac hypertrophy and fibrosis compared with untreated animals. Isoproterenol administration significantly increased transforming growth factor ß1 (TGF-ß1)-induced fibrotic signaling, which was attenuated by RELAX10. We found that RELAX10 also significantly increased protein kinase B/endothelial NO synthase signaling and protein S-nitrosylation. In the reversal study, RELAX10-treated animals showed significantly reduced cardiac hypertrophy and collagen levels. Conclusions These findings support a potential role for RELAX10 in the treatment of heart failure.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Miocardio/patología , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/prevención & control , Fibrosis/inducido químicamente , Fibrosis/tratamiento farmacológico , Fibrosis/prevención & control , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.
Asunto(s)
Antineoplásicos , Benzodiazepinas/química , Inmunoconjugados , Pirroles/química , Receptor ErbB-2/antagonistas & inhibidores , Neoplasias Gástricas/tratamiento farmacológico , Trastuzumab , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Células MCF-7 , Ratones Desnudos , Ratas , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Trastuzumab/química , Trastuzumab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Inmunotoxinas/uso terapéutico , Maitansina/análogos & derivados , Receptor ErbB-2/inmunología , Trastuzumab/uso terapéutico , Ado-Trastuzumab Emtansina , Animales , Neoplasias de la Mama/inmunología , Femenino , Humanos , Maitansina/uso terapéutico , Ratones , Resultado del TratamientoRESUMEN
Wnt signaling controls various aspects of developmental and cell biology, as well as contributing to certain cancers. Expression of the human Rho family small GTPase Wrch/RhoU is regulated by Wnt signaling, and Wrch and its paralog Chp/RhoV are both implicated in oncogenic transformation and regulation of cytoskeletal dynamics. We performed developmental genetic analysis of the single Caenorhabditis elegans ortholog of Chp and Wrch, CHW-1. Using a transgenic assay of the distal tip cell migration, we found that wild-type CHW-1 is likely to be partially constitutively active and that we can alter ectopic CHW-1-dependent migration phenotypes with mutations predicted to increase or decrease intrinsic GTP hydrolysis rate. The vulval P7.p polarity decision balances multiple antagonistic Wnt signals, and also uses different types of Wnt signaling. Previously described cooperative Wnt receptors LIN-17/Frizzled and LIN-18/Ryk orient P7.p posteriorly, with LIN-17/Fz contributing approximately two-thirds of polarizing activity. CHW-1 deletion appears to equalize the contributions of these two receptors. We hypothesize that CHW-1 increases LIN-17/Fz activity at the expense of LIN-18/Ryk, thus making the contribution of these signals unequal. For P7.p to polarize correctly and form a proper vulva, LIN-17/Fz and LIN-18/Ryk antagonize other Wnt transmembrane systems VANG-1/VanGogh and CAM-1/Ror. Our genetic data suggest that LIN-17/Fz represses both VANG-1/VanGogh and CAM-1/Ror, while LIN-18/Ryk represses only VANG-1. These data expand our knowledge of a sophisticated signaling network to control P7.p polarity, and suggests that CHW-1 can alter ligand gradients or receptor priorities in the system.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Polaridad Celular , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Acoplados a Proteínas G/genética , Alineación de Secuencia , Vía de Señalización WntRESUMEN
Activation of seven-transmembrane (7TM) receptors by agonists does not always lead to uniform activation of all signaling pathways mediated by a given receptor. Relative to other ligands, many agonists are "biased" toward producing subsets of receptor behaviors. A hallmark of such "functional selectivity" is cell type dependence; this poses a particular problem for the profiling of agonists in whole cell test systems removed from the therapeutic one(s). Such response-specific cell-based variability makes it difficult to guide medicinal chemistry efforts aimed at identifying and optimizing therapeutically meaningful agonist bias. For this reason, we present a scale, based on the Black and Leff operational model, that contains the key elements required to describe 7TM agonism, namely, affinity (K(A) (-1)) for the receptor and efficacy (τ) in activating a particular signaling pathway. Utilizing a "transduction coefficient" term, log(τ/K(A)), this scale can statistically evaluate selective agonist effects in a manner that can theoretically inform structure-activity studies and/or drug candidate selection matrices. The bias of four chemokines for CCR5-mediated inositol phosphate production versus internalization is quantified to illustrate the practical application of this method. The independence of this method with respect to receptor density and the calculation of statistical estimates of confidence of differences are specifically discussed.
Asunto(s)
Íleon/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Células CHO , Carbacol/química , Carbacol/farmacología , Cricetinae , Cricetulus , Cobayas , Íleon/efectos de los fármacos , Técnicas de Cultivo de Órganos , Oxotremorina/química , Oxotremorina/farmacologíaRESUMEN
Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science 307: 1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.
Asunto(s)
Inhibidores de Fusión de VIH/farmacología , Inhibidores de Fusión de VIH/uso terapéutico , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Receptores CCR5/metabolismo , Internalización del Virus/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores de Fusión de VIH/química , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virologíaRESUMEN
Mutational activation of Ras and a key downstream effector of Ras, the B-Raf serine/threonine kinase, has been observed in melanomas and colorectal carcinomas. These observations suggest that inhibition of B-Raf activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) and the extracellular signal-regulated kinase MAPK cascade may be an effective approach for the treatment of RAS and B-RAF mutation-positive melanomas and colon carcinomas. Although recent studies with interfering RNA (RNAi) and pharmacologic inhibitors support a critical role for B-Raf signaling in melanoma growth, whether mutant B-Raf has an equivalent role in promoting colorectal carcinoma growth has not been determined. In the present study, we used both RNAi and pharmacologic approaches to further assess the role of B-Raf activation in the growth of human melanomas and additionally determined if a similar role for mutant B-Raf is seen for colorectal carcinoma cell lines. We observed that RNAi suppression of mutant B-Raf(V600E) expression strongly suppressed the anchorage-dependent growth of B-RAF mutation-positive melanoma, but not colorectal carcinoma, cells. However, the anchorage-independent and tumorigenic growth of B-RAF mutation-positive colorectal carcinomas was dependent on mutant B-Raf function. Finally, pharmacologic inhibition of MEK and Raf was highly effective at inhibiting the growth of B-RAF mutation-positive melanomas and colorectal carcinoma cells, whereas inhibitors of other protein kinases activated by Ras (AKT, c-Jun NH(2)-terminal kinase, and p38 MAPK) were less effective. Our observations suggest that Raf and MEK inhibitors may be effective for the treatment of B-RAF mutation-positive colorectal carcinomas as well as melanomas.
Asunto(s)
Neoplasias Colorrectales/patología , Melanoma/patología , Proteínas Mutantes/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal , Neoplasias Cutáneas/patología , Animales , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/enzimología , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Melanoma/enzimología , Ratones , Ratones Desnudos , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/enzimologíaRESUMEN
Vascular endothelial growth factor (VEGF) is a proangiogenic factor upregulated in many tumors. The alternative splicing of VEGF mRNA renders 3 major isoforms of 121, 165 and 189 amino-acids in humans (1 less amino-acid for each mouse VEGF isoform). We have designed isoform specific real time QRT-PCR assays to quantitate VEGF transcripts in mouse and human normal and malignant prostates. In the human normal prostate, VEGF(165) was the predominant isoform (62.8% +/- 5.2%), followed by VEGF(121) (22.5% +/- 6.3%) and VEGF(189) (p < 0.001) (14.6% +/- 2.1%). Prostate tumors showed a significant increase in the percentage of VEGF(121) and decreases in VEGF(165) (p < 0.01) and VEGF(189) (p < 0.05). However, the amount of total VEGF mRNA was similar between normal and malignant prostates. VEGF(164) was the transcript with the highest expression in the mouse normal prostate. Unlike human prostate cancer, tumors from TRAMP mice demonstrated a significant increase in total VEGF mRNA levels and in each of the VEGF isoforms, without changes in the relative isoform ratios. Morpholino phosphorodiamide antisense oligonucleotide technology was used to increase the relative amount of VEGF(121) while proportionally decreasing VEGF(165) and VEGF(189) levels in human prostate cell lines, through the modification of alternative splicing, without changing transcription levels and total amount of VEGF. The increase in the VEGF(121)/VEGF(165-189) ratio in PC3 cells resulted in a dramatic increase in prostate tumor angiogenesis in vivo. Our results underscore the importance of VEGF(121) in human prostate carcinoma and demonstrate that the relative expression of the different VEGF isoforms has an impact on prostate carcinogenesis.
Asunto(s)
Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Empalme Alternativo , Animales , Secuencia de Bases , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Morfolinas/administración & dosificación , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Oligonucleótidos/administración & dosificación , Oligonucleótidos/genética , Neoplasias de la Próstata/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Xeroderma pigmentosum group C (XP-C) is a rare autosomal recessive disorder. Patients with two mutant alleles of the XPC DNA repair gene have sun sensitivity and a 1000-fold increase in skin cancers. Clinically normal parents of XP-C patients have one mutant allele and one normal allele. As a step toward evaluating cancer risk in these XPC heterozygotes we characterized cells from 16 XP families. We identified 15 causative mutations (5 frameshift, 6 nonsense and 4 splicing) in the XPC gene in cells from 16 XP probands. All had premature termination codons (PTC) and absence of normal XPC protein on western blotting. The cell lines from 26 parents were heterozygous for the same mutations. We employed a real-time quantitative reverse transcriptase-PCR assay as a rapid and sensitive method to measure XPC mRNA levels. The mean XPC mRNA levels in the cell lines from the XP-C probands were 24% (P<10(-7)) of that in 10 normal controls. This reduced XPC mRNA level in cells from XP-C patients was caused by the PTC that induces nonsense-mediated mRNA decay. The mean XPC mRNA levels in cell lines from the heterozygous XP-C carriers were intermediate (59%, P=10(-4)) between the values for the XP patients and the normal controls. This study demonstrates reduced XPC mRNA levels in XP-C patients and heterozygotes. Thus, XPC mRNA levels may be evaluated as a marker of cancer susceptibility in carriers of mutations in the XPC gene.
Asunto(s)
Codón sin Sentido/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Mutación/genética , ARN Mensajero/genética , Xerodermia Pigmentosa/genética , Adolescente , Adulto , Western Blotting , Niño , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Heterocigoto , Humanos , Lactante , Recién Nacido , Masculino , Padres , Reacción en Cadena de la Polimerasa , Sitios de Empalme de ARN , ARN Mensajero/metabolismo , Xerodermia Pigmentosa/metabolismoRESUMEN
The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.
Asunto(s)
Apoptosis , Azacitidina/análogos & derivados , Metilación de ADN , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal , Quinasas raf/fisiología , Agar/química , Alelos , Animales , Proteínas Reguladoras de la Apoptosis , Azacitidina/farmacología , Northern Blotting , Western Blotting , Butadienos/farmacología , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , ADN Complementario/metabolismo , Decitabina , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/metabolismo , Flavonoides/farmacología , Vectores Genéticos , Humanos , Ratones , Mutación , Células 3T3 NIH , Nitrilos/farmacología , Ovario/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena de la Polimerasa , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Regulación hacia ArribaRESUMEN
The lariat branch point sequence (BPS) is crucial for splicing of human nuclear pre-mRNA yet BPS mutations have infrequently been reported to cause human disease. Using an inverse RT-PCR technique we mapped two BPS to the adenosine residues at positions -4 and -24 in intron 3 of the human XPC DNA repair gene. We identified homozygous mutations in each of these BPS in two newly diagnosed Turkish families with the autosomal recessive disorder xeroderma pigmentosum (XP). Cells from two severely affected children in family A harbor a homozygous point mutation in XPC intron 3 (-9 T to A), located within the downstream BPS. Using a real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) assay, these cells expressed no detectable (<0.1%) normal XPC message. Instead they expressed an XPC mRNA isoform with deletion of exon 4 that has no DNA repair activity in a host cell reactivation (HCR) assay. In contrast, in cells from three mildly affected siblings in family B, the BPS adenosine located at the -24 position in XPC intron 3 is mutated to a G. Real-time QRT-PCR revealed 3-5% of normal XPC message. These cells from family B had a higher level of HCR than cells from the severely affected siblings in family A, who had multiple skin cancers. Mutations identified in two BPS of the XPC intron 3 resulted in alternative splicing that impaired DNA repair function, thus implicating both of these BPS as essential for normal pre-mRNA splicing. However, a small amount of normal XPC mRNA can provide partial protection against skin cancers.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Sitios de Empalme de ARN , ARN Mensajero/metabolismo , Xerodermia Pigmentosa/genética , Adolescente , Adulto , Niño , Reparación del ADN/fisiología , Proteínas de Unión al ADN/biosíntesis , Femenino , Humanos , Intrones , Masculino , Mutación , Neoplasias/genética , Linaje , Isoformas de Proteínas , Análisis de Secuencia de ADN , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/fisiopatologíaRESUMEN
XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82-882 bp) and 15 introns (0.08-5.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcription-polymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in approximately 0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in approximately 0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (-0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (approximately 2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility.