Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors

AbstractDamnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis

Proteolytic Processing of Stat6 Signaling in Mast Cells as a Negative Regulatory Mechanism

Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases. In this study, we show a novel regulatory mechanism of Stat6-mediated signaling in mast cells. When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells. The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription. The retrovirus-mediated expression of cleavage-resistant Stat6 mutants prolongs the nuclear accumulation of Stat6 upon IL-4 stimulation and enhances IL-4–induced gene expression and growth inhibition in mast cells. These results indicate that the proteolytic processing of Stat6 functions as a lineage-specific negative regulator of Stat6-dependent signaling in mast cells, and thus suggest that it may account for the limited role of Stat6 in IL-4 signaling in mast cells

Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Diagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available.</p> <p>Results</p> <p>Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (esophageal carcinoma SEREX antigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues.</p> <p>Conclusions</p> <p>We have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.</p

Protein assay analysis of colorectal cancer

Protein microarray screening We performed the screening using the ProtoArray v5.1 human protein microarray system (Thermo Fisher Scientific, Waltham, MA), which comprises 9375 protein species. We employed Alexa Fluor 647-anti-human IgG detection reagent to quantify the serum IgG bound to immobilized proteins and made pairwise comparisons between the 2 sample populations. The algorithm to select candidate antigens in our study determined that the antigen positivity rate in the sera of patients with CRC was more than 60%, and the positivity rate in the sera of HDs was less than 20% as described [1, 2]. Ref. 1.  Naito A, Hiwasa T, Tanabe N, Sanada TJ, Sugiura T, Shigeta A, Terada J, Takizawa H, Kashiwado K, Sakao S, et al. Elevated levels of autoantibodies against EXD2 and PHAX in the sera of patients with chronic thromboembolic pulmonary hypertension. PLoS One. 2019;14:e0211377. PMID: 30759165 Ref. 2. Hiwasa T, Wang H, Goto K, Mine S, Machida T, Kobayashi E, Yoshida Y, Adachi A, Matsutani T, Sata M, et al. Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke. BMC Medicine. 2021;19:131. doi:10.1186/s12916-021-02001-9. PMID:34103026</p

Protein Array-ATS_110526.xls

  ProtoArray® screening. The initial screening was performed using ProtoArray® Human Protein Microarrays v.4.0 (Thermo Fisher Scientific, Waltham, MA, USA), which are loaded with 9480 proteins, as described previously (1). A total of 20 serum samples (10 each from HDs and patients with atherosclerosis) were used to select antigenic proteins specifically recognized by serum immunoglobin G (IgG) antibodies. The results were analyzed using Prospector (Thermo Fisher Scientific) software based on M-statistics. When comparing the two groups, a positive cutoff value was calculated for each protein using M-statistics. The percentage of the samples in each group that showed an immune response above the cutoff value was determined and the P-value for the significance of the difference between the two groups was calculated, as previously described (2). Ref. 1. Hamanaka S, Nakagawa T, Hiwasa T, Ohta Y, Kasamatsu S, Ishigami H, Taida T, Okimoto K, Saito K, Maruoka D, et al.: Investigation of novel biomarkers for predicting the clinical course in patients with ulcerative colitis. J Gastroenterol Hepatol 33: 1975-1983, 2018. doi: 10.1111/jgh.14297. PMID:29869393 Ref. 2. Vermeulen N, de Béeck KO, Vermeire S, Van Steen K, Michiels G, Ballet V, Rutgeerts P and Bossuyt X: Identification of a novel autoantigen in inflammatory bowel disease by protein microarray. Inflamm Bowel Dis 17: 1291-1300, 2011. doi: 10.1002/ibd.21508. PMID:21560193</p

Protoarray-ATS_110525.xlsx

ProtoArray® screening. The initial screening was performed using ProtoArray® Human Protein Microarrays v.4.0 (Thermo Fisher Scientific, Waltham, MA, USA), which are loaded with 9480 proteins, as described previously (1). A total of 20 serum samples (10 each from HDs and patients with atherosclerosis) were used to select antigenic proteins specifically recognized by serum immunoglobin G (IgG) antibodies. The results were analyzed using Prospector (Thermo Fisher Scientific) software based on M-statistics. When comparing the two groups, a positive cutoff value was calculated for each protein using M-statistics. The percentage of the samples in each group that showed an immune response above the cutoff value was determined and the P-value for the significance of the difference between the two groups was calculated, as previously described (2).    Ref. 1. Hamanaka S, Nakagawa T, Hiwasa T, Ohta Y, Kasamatsu S, Ishigami H, Taida T, Okimoto K, Saito K, Maruoka D, et al.: Investigation of novel biomarkers for predicting the clinical course in patients with ulcerative colitis. J Gastroenterol Hepatol 33: 1975-1983, 2018. doi: 10.1111/jgh.14297. PMID:29869393 Ref. 2. Vermeulen N, de Béeck KO, Vermeire S, Van Steen K, Michiels G, Ballet V, Rutgeerts P and Bossuyt X: Identification of a novel autoantigen in inflammatory bowel disease by protein microarray. Inflamm Bowel Dis 17: 1291-1300, 2011. doi: 10.1002/ibd.21508. PMID:21560193</p