HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

Carcinogenesis is determined based on both cell proliferation and death rates. Recent studies demonstrate that heat shock proteins (HSPs) regulate apoptosis. HLJ1, a member of the DnaJ-like Hsp40 family, is a newly identified tumor suppressor protein closely related to relapse and survival in non-small cell lung cancer (NSCLC) patients. However, its role in apoptosis is currently unknown. In this study, NSCLC cell lines displaying varying HLJ1 expression levels were subjected to ultraviolet (UV) irradiation, followed by flow cytometry. Interestingly, the percentages of apoptotic cells in the seven cell lines examined were positively correlated with HLJ1 expression. Enforcing expression of HLJ1 in low-HLJ1 expressing highly invasive cells promoted UV-induced apoptosis through enhancing JNK and caspase-3 activation in NSCLC. Additionally, UV irradiation led to reduced levels of HLJ1 predominantly in apoptotic cells. The pan-caspase inhibitor, zVAD-fmk and caspase-3-specific inhibitor, DEVD-fmk, prevented UV-induced degradation of HLJ1 by the late stage of apoptosis. Further experiments revealed a non-typical caspase-3 cleavage site (MEID) at amino acid 125–128 of HLJ1. Our results collectively suggest that HLJ1 is a novel substrate of caspase-3 during the UV-induced apoptotic process

Longitudinal assessment of cognitive and psychosocial functioning after Hurricanes Katrina and Rita: Exploring disaster impact on middle-aged, older, and oldest-old adults

The authors examined the effects of Hurricanes Katrina and Rita on cognitive and psychosocial functioning in a lifespan sample of adults 6-14 months after the storms. Participants were recruited from the Louisiana Healthy Aging Study. Most were assessed during the immediate impact period and retested for this study. Analyses of pre- and post-disaster cognitive data confirmed that storm-related decrements in working memory for middle-aged and older adults observed in the immediate impact period had returned to pre-hurricane levels in the post-disaster recovery period. Middle-aged adults reported more storm-related stressors and greater levels of stress than the two older groups at both waves of testing. These results are consistent with a burden perspective on post-disaster psychological reactions. © 2012 Wiley Periodicals, Inc

Mactinin, a fragment of cytoskeletal α-actinin, is a novel inducer of heat shock protein (Hsp)-90 mediated monocyte activation

<p>Abstract</p> <p>Background</p> <p>Monocytes, their progeny such as dendritic cells and osteoclasts and products including tumor necrosis factor (TNF)-α, interleukin (IL)-1α and IL-1β play important roles in cancer, inflammation, immune response and atherosclerosis. We previously showed that mactinin, a degradative fragment of the cytoskeletal protein α-actinin, is present at sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. We therefore sought to determine the mechanism by which mactinin stimulates monocytes.</p> <p>Results</p> <p>Radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least 3 proteins of molecular weight 88 kD, 79 kD and 68 kD. Affinity purification, mass spectroscopy and Western immunoblotting identified heat shock protein (Hsp)-90 as the 88 kD component of this complex. Hsp90 was responsible for mediating the functional effects of mactinin on monocytes, since Hsp90 inhibitors (geldanamycin and its analogues 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG]) almost completely abrogated the stimulatory activity of mactinin on monocytes (production of the pro-inflammatory cytokines IL-1α, IL-1β and TNF-α, as well as monocyte chemotaxis).</p> <p>Conclusion</p> <p>Mactinin is a novel inducer of Hsp90 activity on monocytes and may serve to perpetuate and augment monocytic activation, thereby functioning as a "matrikine." Blockage of this function of mactinin may be useful in diseases where monocyte/macrophage activation and/or Hsp90 activity are detrimental.</p

Targeting Toll-like receptor 7/8 enhances uptake of apoptotic leukemic cells by monocyte-derived dendritic cells but interferes with subsequent cytokine-induced maturation

Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE2) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20–30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation

Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation

Introduction The prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook, to the best of our knowledge, the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesise that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression, and possibly how it regulates normal mammary gland development. Methods We used subtractive hybridisation to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern blotting analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterised using apoptosis assays. Results We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90&#945;, promote survival, as shown by apoptosis assays and by the addition of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitised differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90&#945; promoted survival in the presence of serum but appeared to have little effect during starvation. Conclusions In addition to identifying new prolactin-regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased expression of HSP90A in breast cancer is correlated with increased cell survival and poor prognosis and HSP90&#945; inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90&#945; promotes survival depending on the cellular conditions and state of cellular transformation

The expression of CD11/CD18 molecules on rabbit leucocytes: identification of monoclonal antibodies to CD18 and their effect on cellular adhesion processes.

The monoclonal antibodies, L13/64 and RCN1/21, raised against rabbit leucocytes, have been shown, by sequential immunoprecipitation and immunoblotting, to react with the rabbit CD18 molecule. They recognise not only surface-expressed CD18 but also an intracellular form which appears to be partially glycosylated. The expression of the CD11 and CD18 glycoproteins on a wide variety of rabbit leucocyte populations has been investigated by flow cytometry, using these two monoclonal antibodies (Mabs), together with others which recognise human CD11 and CD18 proteins but cross-react with rabbit tissues. The distribution of these leucocyte integrin molecules has been shown to be similar to that observed in humans and determination of the N-terminal sequence of rabbit CD11b shows strong homology with human and mouse sequences. Of four anti-rabbit CD18 Mabs tested, only one, L13/64, has been shown to be capable of inhibiting the adhesion of fMLP-stimulated neutrophils to gelatin coated plastic and the homotypic aggregation of PMA-stimulated T cells, both of which assays have been shown to be CD18-dependent. RCN1/21 causes aggregation of unstimulated neutrophils, but it is not known whether this is due to cellular activation or agglutination

Novel costimulators in the immune gene therapy of cancer.

One of the major goals of cancer immunotherapy is the induction of tumour-specific T-lymphocyte responses that will be effective in the rejection of established tumours. The prospects for such therapy rely on the identification of tumour antigens, and although there is persuasive evidence for the presence of such antigens,1,2 the occurrence of the disease does illustrate that the immune system is at least, on some occasions, unable to recognise and destroy these targets. Tumour antigens may be novel proteins (from genetic lesions or viral infections), modified existing antigens (eg, abnormally glycosylated cell surface proteins), or inappropriately expressed normal gene products (eg, CA125, carcinoembryonic antigen, and alpha-fetoprotein).1 Involvement of the immune system in the normal surveillance and suppression of cancer is further suggested by the increased incidence of tumours in immunocompromised patients.3 However, recent evidence has shown that, at least in model systems, cancer cells can be modulated in such a way that they stimulate cells of the immune system to recognise and destroy these malignant cells. This review summarizes the costimulatory molecules involved in the activation of such cells, the principles and mechanisms underlying their activation, and how such knowledge can be used to persuade the immune system to challenge cancer

Novel costimulators in the immune gene therapy of cancer.

One of the major goals of cancer immunotherapy is the induction of tumour-specific T-lymphocyte responses that will be effective in the rejection of established tumours. The prospects for such therapy rely on the identification of tumour antigens, and although there is persuasive evidence for the presence of such antigens,1,2 the occurrence of the disease does illustrate that the immune system is at least, on some occasions, unable to recognise and destroy these targets. Tumour antigens may be novel proteins (from genetic lesions or viral infections), modified existing antigens (eg, abnormally glycosylated cell surface proteins), or inappropriately expressed normal gene products (eg, CA125, carcinoembryonic antigen, and alpha-fetoprotein).1 Involvement of the immune system in the normal surveillance and suppression of cancer is further suggested by the increased incidence of tumours in immunocompromised patients.3 However, recent evidence has shown that, at least in model systems, cancer cells can be modulated in such a way that they stimulate cells of the immune system to recognise and destroy these malignant cells. This review summarizes the costimulatory molecules involved in the activation of such cells, the principles and mechanisms underlying their activation, and how such knowledge can be used to persuade the immune system to challenge cancer