Dataset Information

Use of viral promoters in mammalian cell-based bioassays: How reliable?

ABSTRACT: Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalian genes are expressed in a cell/tissue specific manner, which makes them (i.e. cells/tissues) specialized for specific function in vivo. Therefore, if the receptor/reporter system is to be used as a cell-based screen for testing of hormones and drugs for human therapy then the choice of cell line as well as the promoter in the reporter module is of prime importance so as to get a realistic measure of the bioactivities of 'test' compounds. We evaluated two conventionally used viral promoters and a natural mammalian promoter, regulated by steroid hormone progesterone, in a cell-based receptor/reporter system. The promoters were spliced into vectors expressing enzyme CAT (chloramphenicol acetyl transferase), which served as a reporter of their magnitudes and consistencies in controlling gene expressions. They were introduced into breast cell lines T47D and MCF-7, which served as a cell-based source of progesterone receptors. The yardstick of their reliability was highest magnitude as well as consistency in CAT expression on induction by sequential doses of progesterone. All the promoters responded to induction by progesterone doses ranging from 10-12 to 10-6 molar by expressing CAT enzyme, albeit with varying magnitudes and consistencies. The natural mammalian promoter showed the most coherence in magnitude as well as dose dependent expression profile in both the cell lines. Our study casts doubts on use of viral promoters in a cell-based bioassay for measuring bioactivities of drugs and hormones for human therapy and suggests caution regardingtranslation in toto, of a research technique as a cell-based bioassay for drug screening.

SUBMITTER: Betrabet SS

PROVIDER: S-EPMC331422 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Use of viral promoters in mammalian cell-based bioassays: How reliable?

Betrabet Shrikant S SS Choudhuri Jyoti J Gill-Sharma Manjit M

Journal of translational medicine 20040114 1

Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalia ...[more]

PMID: 14720299

Similar Datasets

Project description:BackgroundMachine learning approaches are emerging as a way to discriminate various classes of functional elements. Previous attempts to create Regulatory Potential (RP) scores to discriminate functional DNA from nonfunctional DNA included using Markov models trained to identify sequences from promoters and enhancers from ancestral repeats. We proposed that knowledge gleaned from those methods could be further refined using a multiple class predictor to separate classes of promoter elements from enhancers or nonfunctional DNA.ResultsWe extended our previous work, which identified over 5,000 candidate bidirectional promoters in the human genome, to map the orthologous promoter regions in the mouse genome. Our algorithm measured the robustness of evidence provided by the spliced EST annotations and incorporated evidence from annotations of UCSC Known Genes and GenBank mRNA. In preparation for de novo prediction of this promoter type, we examined characteristic features of the dataset as a whole. For instance, bidirectional promoters score very highly among all functional elements for Regulatory Potential Scores. This result was unexpected due to the limited sequence conservation found in these noncoding regions. We demonstrate that bidirectional promoters can be classified apart from other genomic features including non-bidirectional promoters, i.e. those promoters having no nearby upstream genes. Furthermore bidirectional promoters consistently score at the level of very highly conserved functional elements in the genome- developmental enhancers. The high scores are due to sequence-based characteristics within the promoters, not the surrounding exons. These results indicate that high-scoring RP regions can be deconvoluted into various functional classes of genomic elements. Using a multiple class predictor we are able to discriminate bidirectional promoters from enhancers, non-bidirectional promoters, and non-promoter regions on the basis of RP scores and CpG islands.ConclusionsWe examine orthology at bidirectional promoters, use discriminatory machine learning approaches to differentiate multiple types of promoters from other functional and nonfunctional features in the genome and begin the process of deconvoluting classes of functional regions that score well with RP scores. These types of approaches precede supervised learning techniques to discover unannotated promoter regions.

Dataset Information

Use of viral promoters in mammalian cell-based bioassays: How reliable?

Publications

Use of viral promoters in mammalian cell-based bioassays: How reliable?

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets