RESUMO
Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Assuntos
Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Calibragem , Clonagem Molecular , DNA , Proteínas de Escherichia coli/genética , Dosagem de Genes , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Proto-Oncogênicas c-bcr/genética , RNA Mensageiro/metabolismo , Padrões de ReferênciaRESUMO
Humoral hypercalcaemia of malignancy (HHM) commonly results from the excessive production of a parathyroid hormone-related protein (PTHrP) by tumours. We have previously shown malignancy is associated with increased DNA methylation in the 5' region of the PTHrP gene. In a series of patients with lung carcinoma and relatively high serum calcium levels, 3 patients showed substantially increased PTHrP gene methylation while 5 patients showed no change in methylation status in this region. Patients showed marked tumour-specific expression of PTHrP through the P1 and P3 promoters with more general tumour and non-tumour expression through the P2 promoter. The lack of potential key regulatory CpG sites in the P1 promoter and the complete demethylation in the P2 and P3 promoters suggests methylation does not influence tumour-specific expression of PTHrP. Although demethylation may be a prerequisite for P2 and P3 expression, the overexpression of the PTHrP gene in cancer cells must be mediated through mechanisms other than DNA methylation.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Proteínas/genética , Idoso , Carcinoma de Células Escamosas/metabolismo , Metilação de DNA , DNA de Neoplasias/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Relacionada ao Hormônio Paratireóideo , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Excessive production of a parathyroid hormone-related protein (PTHrP) by tumours commonly results in the syndrome of humoral hypercalcaemia of malignancy. We have investigated whether epigenetic changes play a role in over-expression of the PTHrP gene, using cultures lung cells as a model system. Study of the methylation status of CpG dinucleotides in the 5' region of the gene showed that in normal cells the CpG island was completely unmethylated. In the lung squamous cell carcinoma cell line, BEN, two-thirds of the CpG island was substantially methylated. RT-PCR analysis showed that this heavy methylation did not prevent expression of any of the three PTHrP gene promoters. This is a surprising finding, since methylation is usually associated with inhibition of gene activity. Methylation of the 5' non-coding region of the PTHrP gene may not play a role in the regulation of adjacent promoters. Alternatively, maintenance of a demethylated state in the 170 bp at the 3' end of the CpG island may be fundamental for the use of PTHrP promoters.
Assuntos
Ilhas de CpG , Pulmão/metabolismo , Hormônio Paratireóideo/genética , Regiões Promotoras Genéticas , Proteínas/genética , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Metilação , Proteína Relacionada ao Hormônio Paratireóideo , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Humoral hypercalcaemia of malignancy often results from production of parathyroid hormone-related protein (PTHrP) by the tumour. We have investigated whether malignancy is associated with epigenetic changes in the PTHrP gene in lung. In normal and tumour tissue, there was a general background of nonmethylation in the PTHrP gene. In the 5' region, there appeared to be increased methylation of sites upstream of the promoter, P2. The extent of methylation increased from germ line to normal tissue to tumour tissue to tumour cell line, indicating that new methylation events in this region mark neoplastic change in lung cells.