Interleukin 1beta‐converting enzyme‐like (ICE‐like) proteases are important mediators of apoptosis in diverse cell types and organisms. However, the role of these proteases in apoptosis cannot be satisfactorily explained on the basis of the physiological functions of their known substrates. Here we show that the C‐terminal 42 amino acid peptide of the retinoblastoma (Rb) protein, an important cell cycle regulator with a known anti‐apoptotic function, is specifically cleaved off by an ICE‐like protease in tumour necrosis factor (TNF)‐ and staurosporine‐induced apoptosis. Cleavage of Rb induced by TNF was blocked in vivo and in vitro by two specific inhibitors of ICE‐like proteases, and in vitro by a point mutation (Asp886 to Ala) within the ICE‐like protease cleavage site of Rb, (883)DEAD(886). An antibody raised against the C‐terminal 15 amino acid peptide of Rb recognized the full‐length but not the cleaved form of Rb. The extent of Rb cleavage correlated directly with TNF‐induced apoptosis in all tumour cell lines examined. Cleaved Rb bound cyclin D3 and inhibited the transcriptional activity of E2F‐1, but failed to bind to the regulatory protein MDM2, which has been implicated in apoptosis. As Rb suppresses cell death and its C‐terminus has important regulatory functions, our results suggest that Rb cleavage is an important event in apoptosis.

The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1b in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1␤ and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes.We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-␥ strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-␥ up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1␤. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-␥ and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

There is growing interest in pharmacological interventions directly targeting the aging process. Pharmacological interventions against aging should be efficacious when started in adults and, ideally, repurpose existing drugs. We show that dramatic lifespan extension can be achieved by targeting multiple, evolutionarily conserved aging pathways and mechanisms using drug combinations. Using this approach in C. elegans, we were able to slow aging and significantly extend healthy lifespan. To identify the mechanism of these drug synergies, we applied transcriptomics and lipidomics analysis. We found that drug interactions involved the TGF-b pathway and recruited genes related with IGF signaling. daf-2, daf-7, and sbp-1 interact upstream of changes in lipid metabolism, resulting in increased monounsaturated fatty acid content and this is required for healthy lifespan extension. These data suggest that combinations of drugs targeting distinct subsets of the aging gene regulatory network can be leveraged to cause synergistic lifespan benefits. Agilent RNA 6000 Nano reagents Agilent Technologies 5067-1511 Deposited Data Raw and Processed RNA-Seq data This paper GEO: GSE108263 Raw and Processed Lipidomics data This Paper MetaboLights: MTBLS648 Experimental Models: Organisms/Strains C. elegans: Strain Bristol wild-type N2 Caenorhabditis Genetics Center N2 C. elegans: Strain daf-2(e1370)III Caenorhabditis Genetics Center CB1370 C. elegans: Strain daf-16(mu86) I Caenorhabditis Genetics Center CF1038 C. elegans: Strain daf-7(e1372) III Caenorhabditis Genetics Center CB1372 C. elegans: Strain eat-2 (ad1116) II Caenorhabditis Genetics Center DA1116 C. elegans: Strain sbp-1 (ep79) III Caenorhabditis Genetics Center CE541 D. melanogaster: Male Bloomington Drosophila Stock Center Oregon-R Oligonucleotides Primer: sbp-1 Forward: gca cgt tct gac atg tgg aa This paper N/A Primer: sbp-1 Reverse: ccg cca aac cca gat tgt ct This paper N/A

c-Myc is an important mediator of apoptosis in cytokine- or serum-deprived cells and sensitizes various cell types to tumor necrosis factor alpha (TNF) cytotoxicity. However, downstream mediators of c-Myc-dependent apoptosis are largely unknown. In this study, we investigated whether one or more cyclins which, like c-Myc, are important regulators of the cell cycle are involved in TNF-induced apoptosis downstream of c-Myc. Cyclin D3 and c-Myc levels in HeLa and fibrosarcoma cells correlated with sensitivity of these cells to TNF-induced apoptosis, as both proteins were highly expressed in TNF-sensitive HeLa D98 cells and HT-1080 fibrosarcoma cells but not in their TNF-resistant counterparts, HeLa H21 and SS-HT-1080 cells, respectively. All other cyclins tested were equally expressed in all tumor cell lines. Reduction in the expression of c-Myc by dexamethasone or inhibition of the transcriptional activity of c-Myc by introduction of a dominant negative form of c-Myc into TNF-sensitive HeLa D98 cells strongly suppressed the expression of cyclin D3 (but none of the other cyclins) and rendered the cells resistant to TNF-induced apoptosis. Conversely, introduction of the c-myc gene into TNF-resistant, c-Myc- and cyclin D3-deficient HeLa H21 cells resulted in enhanced cyclin D3 expression and TNF killing. When cyclin D3 expression in HeLa cells was altered by sense or antisense cyclin D3 cDNA, there was a concomitant alteration in their susceptibility to TNF-induced apoptosis without any change in c-Myc levels. Overall, our results show that cyclin D3 sensitizes tumor cells to TNF-induced apoptosis and indicate that the expression of c-Myc and expression of cyclin D3 in HeLa and in HT-1080 fibrosarcoma cells are closely linked.

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NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis.

Background: UVB light can generate potentially harmful hydrogen peroxide (H₂O₂) in vivo, but it can also promote the beneficial proliferation and migration of melanocytes. The successful use of UVB monotherapy for treatment of vitiligo suggests that H₂O₂ may have a biphasic effect on melanin synthesis and melanosome transfer. Objective: To study the beneficial role of H₂O₂ on melanogenesis and melanosome transport in living melanocytes and keratinocytes. Methods: A co-culture system model was constructed using the primary human melanocytes and keratinocytes. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell proliferation, NaOH was used to determine the melanin content, and real-time PCR was used to determine tyrosinase expression. Western blot was used to determine Rab-27A and protease-activated receptor 2 (PAR-2) expression. Results: This study demonstrated that tyrosinase was activated by low concentrations of H₂O₂ (≤0.3 mM); however, this activity was downregulated by high concentrations of H₂O₂ (>0.3 mM). Activation of high levels of melanin synthesis was induced when cells were treated with low concentrations of H₂O₂ (0.3 mM). Further observation using an in vitro co-culture system of fluorescein (carboxyfluorescein diacetate succinimidyl ester, CFDA-SE)-labeled melanocytes and keratinocytes indicated that melanosome transfer occurred in normal human epidermal melanocytes. Fluorescence microscopy revealed increased melanosome transfer into keratinocytes treated with 0.3 mM H₂O₂ in the co-culture compared to the control. Examination of melanosomes in the keratinocytes by flow cytometry confirmed these results. Furthermore, treatment with H₂O₂ (0.3 mM) upregulated the expression of Rab-27A and PAR-2, significant proteins involved in melanosome transfer, according to Western blot. Conclusion: These results confirmed that low concentration levels of H₂O₂ play a major role in the regulation of human pigmentation by increasing melanin synthesis and melanosome transfer.