The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.

Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor.

Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III and art genes. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-kappa B, with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-kappa B acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

An inducible transcription factor activates expression of human immunodeficiency virus in T cells

Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements

Binding of peptide hormones to surface membrane receptors leads to the transcription of specific genes within relevant target cells. How these signals are transduced to alter gene expression is largely unknown, but this mechanism probably involves a sequence of enzymatic steps that activate factors in the nucleus that modulate transcription. We now demonstrate that two different peptide hormones, or cytokines, stimulate the human immunodeficiency virus enhancer, and this effect is mediated by nuclear factor (NF) kappa B (nuclear factor that binds the kappa immunoglobulin light chain gene enhancer). These cytokines, tumor necrosis factor alpha and interleukin 1, act on multiple cell types and represent the only naturally occurring activators of this transcription factor among eight cytokines examined. Although NF-kappa B binding can be stimulated by phorbol 12-myristate 13-acetate, tumor necrosis factor alpha acts through an independent mechanism, inducing NF-kappa B binding in HT-2 cells, which did not show increased binding in response to phorbol 12-myristate 13-acetate, and causing superinduction in Jurkat T-lymphoma cells. Tumor necrosis factor alpha is also a more selective activator of T cells than phorbol 12-myristate 13-acetate, having no effect on lymphokine production in EL-4 cells at the same time it induces NF-kappa B. These findings suggest that human immunodeficiency virus gene expression can be induced in T cells without activating lymphokine secretion and that the role of these cytokines in the activation of latent human immunodeficiency virus infection deserves further clinical evaluation. Finally, this link between binding at the surface membrane and stimulation of a specific transcription factor should help define intermediates for these cytokine activation pathways.

Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B

Protein kinase C is important in the transduction of signals generated at the plasma membrane. The physiological activators of protein kinase C are diacylglycerols1, and the tumour-promoting phorbol esters, such as 12-O-tetradecanoyI-phorbol-14 acetate (TPA), constitute another group of specific activators1,2. Many cellular substrates for phosphorylation by protein kinase C have been described1,3, but proteins that directly control transcription in response to protein kinase C activation are yet to be identified. TPA treatment leads to induction of various proto-oncogenes4–7, growth factor genes7,8, and genes encoding secreted proteases9. In addition. TPA increases the activity of viral enhancer elements10–13. To identify trans-acting factors that mediate the transcriptional response to TPA we chose the simian virus 40 (SV40) enhancer14 as a model, because it is known to be composed of several discrete cis-acting elements15–18 which are recognized by multiple trans-acting factors19–21. We report here that the SV40 enhancer contains at least four different TPA responsive elements whose activity is dependent on cell-type. The induction response is likely to involve at least two distinct post-translational steps which modulate the activity of the proteins that recognize these elements.

Multiple cis- and trans-acting elements mediate the transcriptional response to phorbol esters.

Phorbol ester tumour promoters can induce the transcription of a number of genes1, including c-myc and c-fos2. These genes are part of a group referred to as ‘competence’ genes3,4 because they are expressed very early after quiescent cells are stimulated to enter the cell cycle5–7. The ‘competence’ genes are coordinately induced by serum and by factors such as platelet-derived growth factor and fibroblast growth factor1,2,8–12. These factors, as well as the tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), are thought to exert their action by a mechanism involving the activation of protein kinase C13–16. It is likely that these factors induce the transcription of the ‘competence’ genes either by activating specific transcription factors or by increasing their intracellular concentration; either mechanism may be mediated by protein kinase C. One approach to identifying such a putative transcription factor is to characterize the cis-acting transcriptional control elements that serve as a target site for the factor. Here we report that, in a human hepatoma cell line, TPA can specifically induce the activity of the simian virus 40 (SV40) transcriptional enhancer element. Since the SV40 enhancer is a thoroughly characterized cis-actitng element17–19, this system may facilitate the eventual identification of the trans-acting factor(s) whose activity is modified by TPA treatment.

Phorbol ester induces the transcriptional stimulatory activity of the SV40 enhancer.

BALB/c-3T3 cells have been isolated that are resistant to NiCl2. The degree of resistance is directly dependent upon the NiCl2 exposure concentration and ranges from 6- to 11-fold. Resistance to NiCl2 does not appear to be due to alterations in cellular uptake, since the entry of Ni(II) into wild-type or resistant cells was similar. Resistance does not appear to be due to alterations in metallothionein expression. Resistant cells have a high incidence of heterochromatic abnormalities involving fusions at the centromeres as determined by C-banding and in situ hybridization utilizing a cloned mouse satellite DNA probe. Cells retain nickel resistance for many generations in the absence or presence of NiCl2 selection; however, with time in the absence of NiCl2, the level of resistance decreases. This loss of resistance is associated with a decreased number of centromeric fusions. These results indicate that nickel resistance is involved with changes in heterochromatin and suggest that this effect of nickel on heterochromatin may be important as an early step in nickel carcinogenesis.

https://cancerres.aacrjournals.org/content/canres/48/23/6850.full.pdf

Characterization of mouse cell lines resistant to nickel(II) ions.

Alterations in DNA-protein interactions (DPI) may play an important role in carcinogenesis. Although the mechanism of nickel carcinogenesis is unknown, nickel reportedly affects DPI. A microfiltration, nitrocellulose filter assay was utilized to study DPI in intact Chinese hamster ovary (CHO) cells and in isolated nuclei. Prior to exposure of CHO cells or isolated CHO cell nuclei, DNA and proteins were radiolabeled using3H-thymidine and35S-methionine, respectively. Nuclei were exposed to NiCl2 in 10 mM HEPES buffer (pH 6.8). CHO cells were exposed in either complete or a salts-glucose medium. Following exposure, nuclei or cells were incubated at 37°C for 20 min in a high salt lysis solution; aliquots were loaded onto nitrocellulose filters and washed with a low salt solution. DNA (3H) retained on each filter was normalized to protein (35S) bound on the filter. Exposure of either whole cells or isolated nuclei to increasing, noncytotoxic concentrations of NiCl2 resulted in a dose dependent decrease in DPI. The effect of nickel on specific DNA-protein interactions was examined using a band shift assay and a cloned satellite DNA sequence. Nickel inhibited specific protein binding to the satellite DNA probe. The results of these two independent assays, which were conducted at physiological pH, indicate that NiCl2 inhibits specific DNA-protein interactions.

Effect of nickel(II) on DNA-protein interactions.

Nickel ions selectively damage heterochromatin. Differences in the unique association of DNA and protein in heterochromatin compared to euchromatin led us to investigate a possible mechanism for this selective damage by examining the in vitro effect of nickel chloride on specific DNA‐protein interactions using a radiolabeled mouse satellite DNA sequence, which comprises the heterochromatic centromeres in mouse chromosomes. Our results indicate that nickel ions, as well as several other metal ions, inhibit specific protein binding to this DNA sequence. Inhibition of protein binding occurs at different concentrations for each metal ion but, relative to its cytotoxicity, nickel chloride seems to be a most potent inhibitor of DNA‐protein interaction. However, it should be noted that nickel ions also inhibit specific protein binding to the metallothionein promoter sequence. Magnesium ion increases the apparent affinity of proteins to the satellite DNA but not to the metallothionein promoter and decrea...

Richard J. Imbra

Papers

Multiple cis- and trans-acting elements mediate the transcriptional response to phorbol esters.

Phorbol ester induces the transcriptional stimulatory activity of the SV40 enhancer.

Characterization of mouse cell lines resistant to nickel(II) ions.

Effect of nickel(II) on DNA-protein interactions.

Studies on the mechanism of nickel‐induced heterochromatin damage; effect on specific DNA‐protein interactions