The interacting RNA polymerase II subunits, hRPB11 and hRPB3, are coordinately expressed in adult human tissues and down-regulated by doxorubicin.
Fanciulli M. Bruno T. Di Padova M. De Angelis R. Lovari S. Floridi A. Passananti C.
Cell Metabolism and Pharmacokinetics Laboratory, Regina Elena Cancer Institute, Rome, Italy. firstname.lastname@example.org
We previously isolated the human RPB11 cDNA, encoding the 13.3 kDa subunit of RNA polymerase II, and demonstrated that expression of this subunit is modulated by doxorubicin. Using hRPB11 as bait in a yeast two-hybrid system, two cDNA variants encoding a second RNA polymerase II subunit, hRPB3, have now been isolated and characterized. These two hRPB3 mRNA species differed in 3' UTR region length, the longer transcript containing the AU-rich sequence motif that mediates mRNA degradation. Both hRPB11 and hRPB3 transcripts share a similar pattern of distribution in human adult tissues, with particularly high levels in both heart and skeletal muscle, and the expression of both is down-regulated by doxorubicin as found previously for the hRPB11 subunit. Taken together, these findings suggest that the interaction between hRPB3 and hRPB11 is fundamental for their function and that this heterodimer is involved in doxorubicin toxicity.
Levels of expression of hRPB11, a core subassembly subunit of human RNA polymerase II, affect doxorubicin sensitivity and cellular differentiation.
Bruno T. Leonetti C. Aloe S. Iacobini C. Floridi A. Di Tondo U. Punturieri A. Fanciulli M.
Cell Metabolism and Pharmacokinetics Laboratory, Regina Elena Cancer Institute, Rome, Italy.
We have previously shown that the human RNA polymerase II subunit 11 (hRPB11) is among the proteins specifically downregulated upon Doxorubicin (Dox) treatment of human cancer cell lines, and that Dox resistant clones derived upon drug selection express about 20% of the protein present in the original parental cell line. Given the prominent role that this subunit appears to have in eukaryotic cells, and the fact that its deletion causes lethality in yeast, we wanted to test the effect of the reintroduction of parental cell line levels of this subunit in Dox resistant colon cancer cells (LoVoDX). Stable transfectants of LoVoDX expressing parental (LoVoH) levels of hRPB11 showed a reduced sensitivity to the drug without changing the response of these cells to other chemotherapeutic agents, confirming a specific inverse correlation between cellular Dox sensitivity anti-hRPB11 levels of expression. In addition we show here that the levels of expression of this same RNA polymerase II subunit directly affect cellular differentiation, reducing the rate of cell proliferation, clonogenicity and increasing the expression of E-cadherin, a marker of epithelial cell differentiation. As expected from cells with these characteristics, upon in vivo administration of these clones in nude mice, we detected a significant reduction in the size and time of appearance of the primary tumors and overall metastatic capability. Finally, the role played by hRPB11 in regulating the transcription of specific genes is underlined by transient transfection experiments that show transactivation of the E-cadherin promoter by this protein.
Transcriptional regulation of the heavy subunit chain of gamma-glutamylcysteine synthetase by ionizing radiation.
Morales A. Miranda M. Sanchez-Reyes A. Colell A. Biete A. Fernandez-Checa JC.
Instituto de Investigaciones Biomedicas, August Pi i Sunyer, CSIC-UB, Hospital Clinic i Provincial, Barcelona, Spain.
Since glutathione (GSH) protects against oxidative stress, we determined the regulation of cellular GSH by ionizing radiation in human hepatoblastoma cells, HepG2. The levels of GSH increased in irradiated HepG2 due to a greater gamma-glutamylcysteine synthetase (gamma-GCS) activity, which was paralleled by gamma-GCS heavy subunit chain (gamma-GCS-HS) mRNA levels. Transcription of deletion constructs of the gamma-GCS-HS promoter cloned in a reporter vector was associated with activator protein-1 (AP-1), consistent with the DNA binding of AP-1 in nuclear extracts of irradiated HepG2. Hence, the transcriptional regulation of gamma-GCS by ionizing radiation emerges as an adaptive mechanism, which may be of significance to control the consequences of the oxidative stress induced by radiation.
Beta structure motif recognition by anti-gliadin antibodies in coeliac disease.
Alfonso P. Soto C. Albar JP. Camafeita E. Escobar H. Suarez L. Rico M. Bruix M. Mendez E.
Structural Analysis of Proteins Unit, Centro Nacional de Biotecnologia, CSIC, Cantoblanco, Madrid, Spain.
A 20-amino acid synthetic peptide from the N-terminal region of gamma3 avenin yields a surprisingly strong reactivity with anti-gliadin antibodies (AGA) of coeliac sera, comparable to that of a gliadin extract. In contrast, a low reactivity is observed with five similar peptides derived from alpha-gliadin, gamma70 and omega1 secalins. Circular dichroism studies of these peptides show that the avenin peptide displays the highest beta-turn content (30%), while other peptides yield much lower values. In agreement with circular dichroism data, nuclear magnetic resonance data point to the presence of a beta-turn in the avenin peptide DPSEQ segment, a sequence with a high statistical beta-turn preference. A strong linear dependence between AGA reactivity and beta-turn content was observed for these peptides, indicating for the first time a role of beta-turn motifs in anti-gliadin antibodies recognition in coeliac disease. This suggests that circulating AGA in coeliac patients comprise not only linear but also conformational antibodies against beta-turn motifs. Polyclonal antibodies raised against the avenin peptide containing beta-turn motifs react by immunoblotting with all gliadin, hordein and secalin proteins, which are rich in beta-turn conformations, despite that their primary structures are unrelated to that of the peptide.