Quantitative expression patterns of multidrug‐resistance P‐glycoprotein (MDR1) and differentially spliced cystic‐fibrosis transmembrane‐conductance regulator mRNA transcripts in human epithelia

Abstract

P‐glycoprotein (MDR1), that confers multidrug resistance in cancer, and the cystic‐fibrosis transmembrane‐conductance regulator (CFTR), that is causative defective in cystic fibrosis, belong to the family of ATP‐binding transport proteins. The expression of MDR1 and CFTR in human epithelial tissues and the cell lines T84 and HT29 was estimated by primer‐directed reverse transcription (RT) and subsequent monitoring of the kinetics of cDNA product formation during the polymerase chain reaction (PCR), MDR1 mRNA was found in high levels, 15–50 amol mRNA/μg RNA, in the intestine, kidney, liver and placenta, and in low levels, 0.2 amol/μg RNA, in respiratory epithelium. Large amounts of CFTR mRNA were measured in the gastrointestinal tract, whereas the kidney, as the phenotypically normal organ, and the lung, as the most severely affected organ in cystic fibrosis, both contained low amounts, 3 amol CFTR/μg RNA. CFTR transcript levels of 1–5 amol/μg RNA were determined in lymphocytes and lymphoblast cell lines, suggesting that lymphoblasts are an accessible source for the study of the molecular pathogenesis of cystic fibrosis. When transcripts were scanned by overlapping RT/PCR analyses, only transcript of expected size was detected for MDR1 mRNA, whereas variable in‐frame deletions of either exon 4, 9 or 12 were observed in CFTR mRNA. The complete loss of single exons was seen at proportions of 1–40% in all investigated tissues and cell lines with large donor‐to‐donor variation. Exons 9 and 12 of the CFTR gene encode parts of the evolutionarily well‐conserved first nucleotide‐binding fold including the two Walker motifs. Alternative splicing may give rise to various CFTR forms of different function and localization.

A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis.

“…There was no evidence for use of the cryptic splice sites in CFTR transcripts of lymphocytes from patients and several controls. Furthermore, the cryptic exon has never been detected in kidney, liver, jejunum, placenta, lung, and nasal polyps from non-CF controls (21 ).…”

“…Human pancreas total RNA was obtained from Clontech (Palo Alto, CA). Colon cell lines T84 and HT-29 were cultured as described previously (21 ).…”

Examination of the Functional Activity of P-glycoprotein in the Rat Placental Barrier Using Rhodamine 123

“…High levels of P-gp have been detected in human and murine syncytiotrophoblast layers, which are the crucial parts of the hemochorial placental barrier (Cordon-Cardo et al, 1990;Sugawara, 1990;Bremer et al, 1992;Trezise et al, 1992;MacFarland et al, 1994;Nakamura et al, 1997;Lankas et al, 1998;Myloma et al, 1999;Smit et al, 1999;Ushigome et al, 2000;Tanabe et al, 2001). P-gp has been demonstrated to be integrated in the microvillous membrane of the human syncytiotropho-blast that faces directly maternal blood (MacFarland et al, 1994;Ushigome et al, 2000).…”

Transcript profiling of the Pseudomonas aeruginosa genomic islands PAGI-2 and pKLC102

“…The mRNA concentration was determined from the kinetics of amplification of cDNA during PCR by titration for the first reaction cycle with detectable fluorescence of gelseparated PCR product with the ethidium bromide stain (Bremer et al, 1992;Hoof et al, 1991). A PCR product becomes visible by ethidium fluorescence during the late-exponential phase of PCR.…”