Prediction of aryl hydrocarbon receptor-mediated enzyme induction of drugs and chemicals by mRNA quantification.

Enzyme-specific testing for drug interactions by in vitro techniques has become a routine practice in drug development. With many drugs, enzyme induction has similar importance for the prediction of drug-drug interactions. We developed a method for recognizing enzyme induction mediated via the aryl hydrocarbon receptor. This type of induction may be clinically important since experimental data suggest a higher rate of toxification in induced subjects. Twenty-four drugs and environmental chemicals, selected as prototype inducers or being chemically related to known inducers, including HIV protease inhibitors nelfinavir, saquinavir, ritonavir, and indinavir, were tested for their potency to induce cytochrome P450 1A1 mRNA in human Hela cell cultures by a quantitative reverse transcriptase polymerase chain reaction. Known prototype inducers such as beta-naphthoflavone and 3-methylcholanthrene exhibited the highest inducing potency quantified with an Imax value (maximal induction of cytochrome P450 1A1 mRNA synthesis) of 5.48 and 10.7 x 10(6) mRNA molecules per 150 ng of total RNA, respectively. The enzyme-inducing efficacy of some compounds such as resveratrol (2.92 x 10(6)) and the protease inhibitors was not much lower (2.23-3.08 x 10(6)). All compounds that were structurally similar to benzimidazoles exhibited some extent of enzyme induction; e.g., Imax values were 0.86 x 10(6), 0.20 x 10(6), and 0.14 x 10(6) for omeprazole, lansoprazole, and losartan, respectively. To predict the clinical relevance of these inducing effects, the concentration at half-maximal induction IM was estimated; the plasma concentrations of these drug substances were within 1 order of magnitude of the IM values, upon usual dosage. In conclusion, cytochrome P450 1A1 enzyme induction by drugs is a common phenomenon, though there is a great range in the inducing efficacy. In vitro prediction of enzyme induction may be useful for explaining or foreseeing drug interactions, drug side effects, or toxicity by xenobiotics.

Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease.

The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively. The RNA sequence surrounding the stop codon in ORF 1a shows structural elements typical of ribosomal frameshifting signals in a number of animal and plant viruses. It is predicted that the ORF 1b product is expressed via a +1 ribosomal frameshifting as the 348K ORF 1a/1b fusion protein. This putative protein contains the array of methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains that is conserved in the Sindbis-like supergroup of positive-strand RNA viruses. The 348K protein of BYV is longer than the putative replicases of the most closely related viruses (tobra- and tobamoviruses) by about 1300 amino acids distributed between two unique regions, one at the N-terminus, and the other in the central portion. The N-terminal domain showed sequence similarity to the helper component papain-like protease of potyviruses. By using in vitro translation of the T7 transcripts encoding the N-terminal 92K peptide of the BYV ORF 1a product, we found that the N-terminal fragment of 588 amino acids is released from the translation product by cleavage at the Gly-Gly dipeptide. Site-directed mutagenesis of either of the predicted catalytic residues Cys-509 and His-569 or of the Gly-588 at the cleavage site completely abolished the cleavage. The central unique region of the 348K protein contains a domain distantly resembling the aspartic protease of HIV and other lentiviruses. As shown previously, the 3'-terminal portion of the BYV genome encompasses seven more ORFs, one of which codes for a protein related to the HSP70 cell heat shock proteins, whereas two others encode the capsid protein and its diverged copy. Thus, despite the apparent evolutionary relationship with Sindbis-like viruses, BYV comprises a collection of genomic modules absorbed from different sources and has a unique expression strategy.

Newly established human retinoblastoma cell lines exhibit an "immortalized" but not an invasive phenotype in vitro.

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.