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B Canonico

Publications and source records attributed to B Canonico.

8 recordsLinked to original sources
PubMed2003

Hyperthermia triggers apoptosis and affects cell adhesiveness in human neuroblastoma cells.

Hyperthermia is a known apoptotic inducer and has been recently utilized in combination with chemo-and/or radiotherapy in cancer treatment. In this study we have described its effect on SK-N-MC human neuroblastoma tumor cells, a line which grows as a double adherent and floating population. Considering this particular culture behavior, we also investigated the relationship between hyperthermia and cell adhesiveness by evaluating integrin expression, namely CD11a, which is, as known, closely correlated to cell adhesion properties. By a multiple, ultrastructural and flow cytometrical approach, we have demonstrated that hyperthermia, while triggering apoptosis, also determines a CD11a surface expression decrease in apoptotic and living cells. We thus suggest a further role for this treatment, which, affecting adhesion mechanisms, could down-regulate metastatic diffusion.

Apoptosis↗
PubMed2001

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Annexin A5↗
PubMed2000

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia.

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH2, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH2 cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH2 cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies.

Apoptosis↗
PubMed1998

The K562 chronic myeloid leukemia cell line undergoes apoptosis in response to interferon-alpha.

BACKGROUND AND OBJECTIVE: The K562 cell line, derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 bcr-abl hybrid gene, is known to be particularly resistant to apoptotic death. IFN-alpha treatment of CML patients impairs malignant cell clone, apparently protecting from progression to terminal blast crisis. The mechanisms underlying this kind of cell deletion are analyzed here by multiple technical approaches. DESIGN AND METHODS: K562 cells, variably treated with IFN-alpha, were examined by agarose gel DNA electrophoresis, light and electron microscopy. The presence of bcr-abl rearrangement was revealed by RT-PCR. RESULTS: At 4 day treatment both DNA ladder and apoptotic nuclear changes were identified, consistently in the presence of bcr-abl expression. INTERPRETATION AND CONCLUSIONS: Even cells expressing bcr-abl, such as K562, can be triggered to apoptosis. Therefore, this genetic condition, commonly preventing PCD, does not prevent IFN-alpha-mediated apoptosis. PCD seems thus to be the mechanism underlying IFN-alpha-treated K562 cell deletion and it could be the basis of malignant clone reduction in IFN-alpha treated CML patients.

Apoptosis↗
PubMedDate not supplied

CD90.

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Amino Acid Sequence↗
PubMedDate not supplied

CD117.

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Amino Acid Sequence↗
PubMedDate not supplied

Intracellular detection of Bcl-2 and p53 proteins by flow cytometry: comparison of monoclonal antibodies and sample preparation protocols.

Several techniques have been proposed for flow cytometric evaluation of intracellular antigens. This approach is particularly important for detection at the single cell level of proteins which correlate to tumour progression. Bcl-2 and p53 are two of the most relevant proteins. In the present study we have compared five different cell fixation-permeabilisation protocols and nine fluorochrome-conjugated (FITC or PE) monoclonal antibodies (mAb): four mAb directed against Bcl-2 and five against p53. For detection of Bcl-2 we have analysed three Bcl-2 positive cell lines (K562, Daudi and MCF-7), and peripheral blood samples obtained from nine healthy subjects. To distinguish internal positive (lymphocytes) and negative control cells (granulocytes), it was necessary to perform simultaneous detection of surface and intracellular antigens. For detection of p53 three cell lines, two p53 positive (Raji and CEM) and one p53 negative (HL-60), were analysed. Using these cells we have performed a combined analysis of the efficiency of monoclonal antibodies and sample preparation techniques. In conclusion, clones 124-FITC and Bcl-2/100-PE (Bcl-2), and clones BP53,12-FITC and G59-12-PE (p53) provided the highest specific fluorescence intensity of the respective markers independent of cell preparation protocols. Importantly, our results show that mAb background may depend on the specific fixation/permeabilisation kit and that mAb titration using negative and positive control cells is essential to determine the specificity and the sensitivity of the mAb used.

Antibodies, Monoclonal↗