Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors.

Abstract

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytesmacrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and SalmoneUla typhimurum histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-KB. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding 'y-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-KB pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

2-Aminopurine Selectively Inhibits Splicing of Tumor Necrosis Factor Alpha mRNA

“…5). In certain cell lines, 2-AP induces a reversible G 2 arrest associated with the induction of expression of transfected genes (5,38). In the U937 human promonocytic cell line, increased levels of transcription and stabilization of mRNA accompanied the induction of exogenous gene expression by 2-AP; the stability of histidinol dehydrogenase mRNA and c-myc mRNA increased 15-and 2-fold, respectively (38).…”

The Interferon-inducible Protein Kinase PKR Modulates the Transcriptional Activation of Immunoglobulin κ Gene

“…Furthermore, cells depleted of PKR activity were unresponsive to activation of NF-B by dsRNA (29). Other mechanisms which are NF-B independent cannot be excluded, however (27).…”

“…In addition to its role in translational control, several reports have suggested a role for PKR in regulation of gene transcription (21)(22)(23)(24). For example, the PKR inhibitor 2-aminopurine inhibits gene transcription that is induced by virus infection or dsRNA treatment (25)(26)(27). Moreover, PKR activation by dsRNA results in phosphorylation of IB␣ leading to activation of NF-B (28).…”

The Transcriptional Inhibitors, Actinomycin D and α-Amanitin, Activate the HIV-1 Promoter and Favor Phosphorylation of the RNA Polymerase II C-terminal Domain

“…However, stimulation of the HIV LTR by Tat is NFB-independent in the J-Jhan lymphoblastoid cells (61). Furthermore, an NFB-independent activation of the HIV LTR has also been reported after treatment with phorbol ester (62,63), UV irradiation (64), histone deacetylase inhibitors (65), protein kinase inhibitors (such as 2-aminopurine) (66), okadaic acid treatment (67), heat shock (68), and peroxovanadium compounds (69). Similarly, the NFB-binding sequences are not required for actinomycin stimulation.…”