Molecular mechanism of caffeine-induced expression of two cytochrome P450 genes, \u3cem\u3eCyp6a2\u3c/em\u3e and \u3cem\u3eCyp6a8\u3c/em\u3e, in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Cytochrome P450 monooxygenases or CYPs comprise a large family of enzymes that are found in all classes of living organisms, from bacteria to man. These enzymes are involved in the metabolism of many endogenous and xenobiotic (foreign) compounds. In insects, CYPs confer resistance to various insecticides, and resistance-associated overexpression of multiple CYP genes in resistant insects is a common phenomenon. In Drosophila, multiple Cyp genes including Cyp6a2 and Cyp6a8 show higher level of expression in resistant strains than in the susceptible ones. To date, molecular basis of CYP gene overexpression has not been examined in detail. Barbiturate compounds such as phenobarbital and barbital induce both these genes. An unpublished observation from our laboratory showed that Cyp6a2 as well as Cyp6a8 are induced by over-the-counter caffeine tablet, Vivarin. In the present study, I used pure caffeine as a tool to better understand the mechanism of Cyp6a2 and Cyp6a8 gene regulation in Drosophila. The specific objectives of this project has been to (1) map the upstream DNA of both genes for the sequences responsible for caffeine-induction; (2) examine whether adenosine receptors and/or cAMP-specific phosphodiesterase (cPDE) are involved in transduction of caffeine signal to induce the two Cyp6 genes; and (3) investigate whether Drosophila AP-l transcription factors (D-JUN and D-FOS) playa role in caffeine induction of Cyp6a8 gene. For these objectives, several Cyp-luc reporter plasmids carrying firefly lueiferase (luc) reporter gene under the control of different regions of Cyp6a8 and Cyp6a2 upstream DNAs were constructed to transfect Drosophila embryonic cells, Schneider line 2 (SL-2). Two transgenic reporter strains carrying firefly lueiferase (luc) reporter gene under the control of O.8-kb or 0.2-kb upstream DNA of Cyp6a8 gene were also used to study the mechanism of caffeine induction in vivo. Results of Northern blot analysis showed that caffeine induces endogenous Cyp6a2 and Cyp6a8 genes at the steady-state mRNA levels both in reporter transgenic and wild-type flies. Transfection experiments with SL-2 cells showed that -983/-1 and 7611-11 upstream DNAs of Cyp6a2 and Cyp6a8, respectively, have sequences for caffeine-induced expression. Further transfection experiments with reporter plasmids carrying luc reporter gene attached to truncated upstream DNAs of Cyp6a2 and Cyp6a8 genes showed that the regions between -265/-129 of Cyp6a2 and -199/-109 of Cyp6a8 have sequences that confer caffeine-induced expression. However, the level of both constitutive and induced expression was highest with -981/-1 DNA of Cyp6a2and -761111 DNA of Cyp6a8genes. Sequence analysis identified several putative binding sites for Activator Protein -1 (AP-l) and cyclic AMP response element CRE binding protein. (CREB) motifs in the upstream DNA of both genes. Moreover, when the four core bases of the single AP-l site present in the -109/-11 DNA of Cyp6a8 were mutated, constitutive expression decreased by 8-fold, suggesting the positive role of AP-l in Cyp6a8 gene expression. To examine whether caffeine signaling is mediated via adenosine receptor (AdoR) and/or via cPDE inhibition, SL-2 cells transfected with Cyp6a2 and Cyp6a8 reporter constructs were treated with AdoR agonists or with antagonists or with cPDE inhibitors. The Cyp6a8-luc reporter transgenic lines were also treated with these chemicals. The results showed that caffeine signaling is mediated by PDE inhibition and via increase in the intracellular cAMP level. Indeed, treatment with dibutyryl cAMP induces Cyp6a2 as well as Cyp6a8 promoters. Since induction of cAMP pathway is known to upregulate AP-I transcription factors, effect of overexpression of Drosophila D-FOS and (or) DJUN (components of AP-I) on Cyp6a8 promoter activity in SL-2 cells was examined. Surprisingly, activity of Cyp6a8-1uc reporter construct was inhibited when D-FOS or DJUN proteins were overexpressed, suggesting that AP-I proteins are inhibitory for Cyp6 gene expression. In contrast, the Cyp6a8 promoter activity was upregulated, when cells were transfected with anti-D-JUN plasmid or when cells transfected with D-JUN or DFOS sense construct were treated with caffeine. When relation between caffeine and the two AP-I proteins was examined, it was found that caffeine treatment significantly lowers the D-JUN protein level both in SL-2 cells as well as in adult flies. Reporter gene assays and Northern blot analysis showed that caffeine treatment has no effect on the transcriptional activity of the D-jun and D-fos genes. Taken together, it might be concluded that caffeine induction of Cyp6a2 and Cyp6a8 genes is mediated via degradation of D-JUN that acts as a repressor for the promoter of Cyp6a8. Induction of the cAMP pathway and subsequent phosphorylation of the AP-l proteins may relieve the AP-l mediated-repression by promoting the degradation of these proteins. Further investigation is required to resolve these possibilities

Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans

Importance: Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. Objective: To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). Design, Setting, and Participants: A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. Exposures: Ionizing radiation and doxorubicin. Main Outcomes and Measures: Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. Results: Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional to TP53 status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%]; P \u3c .001; doxorubicin exposure: human controls, 8.10% [95% CI, 6.55%-9.66%] vs elephants, 24.77% [95% CI, 23.0%-26.53%]; P \u3c .001). Conclusions and Relevance: Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies of TP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression

The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-κB

Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical us

The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-kappaB

Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-?B p65 and its deacetylation at various lysines. NF-?B p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-?B p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use

Histone deacetylase 11 as a key regulator of metabolism and obesity

In this thought commentary, I highlight the discoveries made by Seto and colleagues related to HDAC11 and obesity. I discuss how their reported work fills a gap in the HDAC field and comment on the clinical implications of their findings. Overall, selective inhibition of HDAC11 could be a novel potential therapeutic avenue for both obesity and diabesity, the diabetes caused by obesity. Future studies to further dissect this mechanistic link between HDAC11 and metabolic programs will pave the way for designing mechanism-based combination therapeutic strategies for these two life style diseases

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer

Analysis of protein dynamics at active, stalled, and collapsed replication forks

Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation