Characterisation of a cell-surface marker of apoptosis

The studies undertaken in this research project characterised as fully as possible the antigen identified by the monoclonal antibody BOB78. Earlier studies (Hart, Ross et al. 2000) have linked BOB78 to the identification of apoptotic cells. The present project extended this finding by further studies utilising various antibody -based techniques on cancer -derived cell lines in different stages of apoptosis.The BOB78 antigen was confirmed to be present in normal cells and to be multi -lineage. BOB78 antigen is normally expressed within the cytosol of nucleated cells, but absent in anucleated red blood cells which do not undergo apoptosis. Following depolarisation of mitochondrial transmembrane potential, a key event in the initiation of apoptosis, BOB78 antigen is detected on the surface of the outer cell membrane. This surfacing of the BOB78 antigen parallels that of phosphatidylserine residues, presently the most well characterised marker for apoptosis in analysis with flow cytometry. Immunocytochemistry corroborated this finding by showing the translocation of the BOB78 antigen to membrane blebs of apoptotic cells. Observations using agents which disrupt the endomembrane synthetic pathways suggest that BOB78 is probably not trafficked through the Golgi apparatus or processed for secretion. Although BOB78 is an IgM antibody, it does not identify a carbohydrate epitope and is therefore likely to be specific for a multi -lineage protein.Membrane blebbing in apoptotic cells appears to share certain characteristics with the budding of platelets from megakaryocytes. For example, the production of platelets during the terminal differentiation of megakaryocytes involves activation of caspases. Interestingly, BOB78 was found by flow cytometric analysis to be present in platelets as they develop from the membranes of the MEG -01 megakaryocytes. BOB78 was also expressed on the surface of senescent platelets. These MEG -01 platelets were therefore used for purification of BOB78 through immunoprecipitation. The captured BOB78 antigen was sequenced using MALDI -TOF, which identified it as chaperonin, also known as heat shock protein 60 (hsp60).Heat shock protein 60 is a highly conserved stress protein which has chaperone functions in prokaryotes as well mammalian cells. Expression of hsp60 on the surface of apoptotic cells may serve novel functions or purposes akin to molecular chaperoning. Mechanisms underlying the translocation of hsp60 to the cell membrane have been characterised in various prokaryotes. Homologous pathways of movement for this highly conserved entity are likely to exist in mammalian cells. The observation that hsp60 is expressed on the surface of senescent platelets suggests a possible role as a recognition signal in phagocytosis. Platelets are equipped with caspases and when senescent display membrane changes typical of apoptotic cells. Like apoptotic cells, platelets are normally cleared from tissues by macrophages. Surface expression of hsp60 is probably an essential change marking cellular entities for uptake by phagocytes. It remains to be studied if engulfment of bodies displaying an endogenous moiety like hsp60 might play a key role in mediating a non -inflammatory response observed in apoptosis. It may also be speculated that hsp60 serves the function of molecular mimicry as many intracellular bacteria display surface hsp60, the expression of which has been linked to pathogenic invasiveness. The ability of these microbes to evade immune clearance may be linked to the non -inflammatory apoptotic disguise which surface hsp60 confers on them

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites

Survival Strategies of Klang Valley Malaysian Small and Medium Building Contractors in the Post-Covid-19 Era

The post-COVID-19 era in Malaysia’s busiest area, Klang Valley, necessitates an examination of the survival strategies used by small and medium building contractors in the construction industry, which are numerous in a developing country like Malaysia and contribute significantly to the country’s economy. As a result, the purpose of this study was to investigate the survival strategies and perceived success of Klang Valley Malaysian small and medium building contractors in the post-COVID-19 era, to uncover the strategies that would ensure the small and medium building contractors’ continuous survival. In this study, a survey approach was used, and building contractors in six grades ranging from G1 to G6 of CIDB Malaysia registered building contractors were sampled using a structured questionnaire. Mean ranking and factor analysis were used to analyse the data collected. The top five important survival strategies were as follows: (1) effective financial management, (2) upgrading of the skills of employees for productivity improvement, (3) improvement of service quality performance, (4) improvement of problem-solving capabilities, and (5) efficient marketing. The factor analysis method was used to identify the underlying factors of survival strategies such as diversification, networking, and innovation strategies; workforce management strategies; and financial management strategies. The effects of organisational survival on small and medium building contractors’ perceived success in terms of business growth, profit growth, and employment growth were also investigated. This study raised awareness and deepened our understanding of the survival strategies used by Klang Valley Malaysian small and medium building contractors, as well as how their organisational survival was perceived. Keywords: Survival strategies; Klang Valley Malaysia; Small and medium building contractors; Post-COVID-19 era

Gelsolin immunohistochemistry in human colorectal carcinoma tissues.

<p>Gelsolin is heterogeneously expressed in a number of primary tumors (A) and liver metastases (B), with islands of low (<i>arrowed in blue</i>) and high (<i>arrowed in red</i>) expression observed within a tumor. Gelsolin expression is mainly cytoplasmic but occasionally, nuclear (C) and perinuclear (D) staining are detected (<i>arrowed</i>). Gelsolin is strongly expressed in a mucinous adenocarcinoma (E) and in stroma (A, B).</p

Gelsolin increases uPA secretion of colorectal tumor cells and enhances invasion via the urokinase-type plasminogen (uPA) cascade.

<p>(<b>A</b>) Increased gelsolin in HCT116 cells augmented the secretion and activity of uPA. The levels of secreted uPA by cells cultured for 48 hours in serum-free conditions were detected in the supernatant using ELISA. Gelsolin-overexpressing HCT116 cells secreted significantly higher uPA levels compared to control cells (<i>left</i>), which correlated with higher uPA enzymatic activity, as evident from uPA zymographic analysis using 16-hour serum-free conditioned media of cells (<i>right</i>). (<b>B</b>) Knockdown of gelsolin (KD) reduced the secretion of uPA. Gelsolin-overexpressing HCT116, wildtype HCT116, DLD-1 and Caco-2 cells were treated with gelsolin siRNA or control siRNA for 24 hours prior to culture under serum-free conditions for a further 48 hours. The levels of secreted uPA were detected using ELISA (<i>left</i>). Zymographic analysis indicated that uPA activity in the 3-hour conditioned media of the colorectal tumor cells was reduced 48 hours after gelsolin siRNA knockdown treatment (<i>right</i>). (<b>C</b>) Gelsolin expression affects TNF-α-stimulated uPA secretion in colorectal cancer cells. DLD-1 and Caco-2 cells were treated with gelsolin siRNA (KD) or control siRNA (Cont) for at least 24 hours and serum-starved overnight prior to incubation with 5 ng/mL (DLD-1) and 10 ng/mL (Caco-2) TNF-α for 24 hours. The levels of secreted uPA in the supernatant of cultured cells were detected using ELISA. uPA secretion was effectively stimulated by TNF-α in DLD-1 and Caco-2. However, siRNA knockdown of gelsolin significantly attentuated TNFα-stimulated uPA levels in the colorectal cancer cell lines. (<b>D</b>) Inhibition of uPA cascade attenuates the invasion of gelsolin-overexpressing HCT116 cells through matrigel. Gelsolin-overexpressing cells were treated with either 50 µM amiloride, 200 µg/mL of function-blocking anti-uPA or 80 µg/mL of anti-uPAR antibodies and examined for changes in invasive potential through matrigel. DMSO treatment and mouse IgG antibody at 200 µg/mL were used as controls to amiloride and the function-blocking antibodies and respectively. Untreated vector control HCT116 was included and normalized to DMSO vehicle control. The anti-uPA and anti-uPAR as well as amiloride treatments significantly attenuated invasion of gelsolin-overexpressing HCT116 cells, indicating that the enhanced invasiveness induced by gelsolin is mediated through the uPA cascade. All data shown are the mean ± standard error of triplicate (ELISA) and duplicate (invasion assay) measurements and are representative of at least two independent experiments. P<0.05 (student’s t test).</p

Gelsolin expression is prominent at the invasive front of human colorectal cancer tumors.

<p>Gelsolin expression is high along the tumor periphery in primary tumors and liver metastases. The tumor periphery is outlined as red dotted line. A magnified view of a region of primary tumor edge (boxed) is shown on the bottom panel. The invading liver metastases shown are confirmed by cytokeratin stain, AE1/3, on an adjacent slide (bottom middle panel). Increased gelsolin expression was also detected in less-differentiated tumor cells which appeared to be breaking away from well-formed glandular structures (arrowed, top right panel). Gelsolin expression were scored and represented in the scatter dot plot (bottom). The median scores are represented by the red horizontal bars. Mann-whitney test was to compare the gelsolin expression score between the main tumor bulk and their periphery. Bar: 50 µm.</p

Gelsolin promotes migration and invasion of colorectal cancer cells.

<p>(<b>A</b>) Endogenous gelsolin level was increased in an <i>in vivo</i>-derived metastatic variant of HCT116, E1, which was described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043594#pone.0043594-Tay1" target="_blank">[31]</a>. (<b>B</b>) Gelsolin overexpression plasmid was constructed by cloning human cytoplasmic gelsolin cDNA into pIRES2-EGFP vector (<i>left</i>). HCT116 cells were either transfected with gelsolin-pIRES2-EGFP plasmid or empty pIRES2-EGFP plasmid to establish stable gelsolin-overexpressing cell lines and vector control cell lines respectively. Western blot analysis confirmed increased gelsolin expression in HCT116 cell lines stably-transfected with the gelsolin-overexpression plasmid compared to those transfected with control plasmid. (<b>C</b>) Western blot showing transient gelsolin siRNA knockdown (KD) at 3 days in wild-type HCT116 and gelsolin-overexpressing HCT116 cells, E1, as well as other colorectal cancer cell lines DLD-1 and Caco-2 cells. Control cells (Cont) were treated with control siRNA. (<b>D</b>) Increased gelsolin in HCT116 enhanced tumor cell migration and invasion. The migration of gelsolin-overexpressing HCT116 cells through uncoated transwells and invasion through matrigel-coated transwells were ascertained over 48 hours, and was observed to be increased compared to vector control cells. (<b>E</b>) siRNA downregulation of gelsolin abrogates invasion. Gelsolin knockdown significantly reduced invasion of HCT116 and gelsolin-overexpressing HCT116 through matrigel. A requirement for gelsolin in invasion was also observed in E1 and DLD-1 cells. All data shown are the mean ± standard error of duplicate measurements and are representative of at least three independent experiments. *P<0.05.</p

Integration of antiangiogenic therapy with cisplatin and gemcitabine chemotherapy in patients with nasopharyngeal carcinoma.

10.1158/1078-0432.CCR-20-1727Clin Cancer Resclincanres.1727.2020-clincanres.1727.202