CpG island methylation and promoter usage in the parathyroid hormone-related protein gene of cultured lung cells.

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.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

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).

Increased upstream methylation has no influence on the overexpression of the parathyroid hormone-related protein gene in squamous cell carcinoma of the lung.

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.

Patterns of DNA methylation of the parathyroid hormone-related protein gene in human lung carcinoma.

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.

Psychosis and aggression in Alzheimer's disease: the effect of dopamine receptor gene variation.

This study investigated possible associations between selected polymorphisms in the dopamine receptor genes DRD1 and DRD3 with the presence of psychotic phenomena or aggressive behaviour in a community based cohort of 134 patients with late onset Alzheimer's disease. An association was found between the presence of psychotic symptoms and aggressive behaviour and the DRD1 polymorphism and between the presence of psychosis, but not aggression, and the DRD3 polymorphism. Specifically, carriers of the DRD1 B2 allele were more likely to be aggressive or experience hallucinations whereas homozygous carriers of the DRD3 1 allele were more likely to experience delusions.

A monoclonal anti-peptide antibody reacting with the insulin receptor beta-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors.

A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.