Immunoelectron microscopy in mycosis fungoides and benign dermatoses. Expression of CD3, CD4 and CD7 receptors.

UNLABELLED: Mycosis fungoides is an epidermotropic cutaneous T-cell lymphoma (CTCL).Specimens for presented study were taken from sixteen patients with MF confirmed by immunohistochemical methods and PCR and from nine patients with benign dermatoses. To demonstrate CD3, CD4 and CD7 antigens immunogold method was used. We saw morphological differences between lymphocytes from MF and benign dermatoses. In MF, CD3 and CD4 were present rather in form of clusters placed on the surface of cell. On the contrary -CD3 to CD7 distribution analysis showed that these antigens were present rather individually, however there were seen clusters as well. In MF tumor stage labelling decreased in following order: CD7, CD3 and CD4. We also found internalisation of studied antigens via the coated structures of the cell membrane -especially in tumor stage. In benign dermatoses the majority of all receptors was present on the cell membrane. Our work showed differences in localization of studied antigens, between MF stages, what can suggest their possible translocation in cells. We also found loss of CD3 and CD7 antigens in tumor stage what might be use as adiagnosis tool for this disease. KEYWORDS: mycosis fungoides, benign dermatoses, CD3, CD4 and CD7 receptors, immumogold labelling.

Immunogold detection of CD3 and CD4 antigens in patients with Mycosis fungoides.

In this study, we analyzed the distribution of CD3 and CD4 antigens at the ultrastructural level in tissue samples from mycosis fungoides patients using double-immunogold labeling. We observed clusters composed of CD3 and CD4 antigens on the plasma membrane and intracellular. There were also clusters only of one type of the antigen and we could observe more often CD4 than CD3. Labeling of CD3 and CD4 was not found in control cells incubated with non-immune serum. In conclusion, our ultrastructural studies not only visualized pattern distribution and relationship between CD3 and CD4 antigens but might also suggest that the type and form of distribution provides new clues to their possible translocation in mycosis fungoides cells.

Reorganization of actin in K-562 and HL-60 cells treated with taxol.

Influence of taxol, microtubular poison, has been studied on distribution of actin. K-562 and HL-60 cells were treated with taxol in a range of concentrations 0.02-10 microM for 72 hours. The reorganization of F-actin was dependent on its dose. Phalloidin conjugated to TRITC was used to evaluate actin distribution by classical fluorescence and confocal microscopy. Actin was visualized at ultrastructural level by using a postembedding streptavidin-gold method. The treatment of K-562 and HL-60 cells with 2-10 microM of taxol resulted in an increase of F- actin in the cytoplasm, with intense labeling as a ring close to surface of the cell. In HL-60 cells a concentration of F- actin at the site of apoptotic bodies was often observed. Immunogold labeling of actin was localized in the nuclei and cytoplasm in control cells and cells treated with all doses of taxol. At higher doses, compaction of chromatin in the nucleus with strong actin labeling was observed. These observations at the ultrastructural level suggest actin involvement in chromatin reorganization during the process of apoptosis. The present study demonstrated a dose dependent reorganization of actin after treatment with taxol.

The effect of doxorubicin on the expression of cyclin A in K-562 leukemia cell line.

Two opposite processes such as apoptosis and cell division cycle play an important role in cancer biology. During apoptosis cells die by degrading proteins and genome, whereas in the cell cycle cells divide and the genome is duplicated. There are very few studies on the role of cell cycle proteins in apoptosis although their role is distinctly different from that in the cell cycle. The significance of expression of cyclin A and other cyclin cell proteins (eg. Cdk2) in apoptosis remains to be investigated. The aim of this study was to characterize the distribution pattern of cyclin A by using the stereptavidin - biotin - peroxidase technique in K-562 cells treated with doxorubicin. The analysis of cell cycle phases using cytophotometric methods to estimate the cellular response to doxorubicin was also used. Studied cells were treated with doxorubicin in the range 0.5; 5.0 and 10 microM. Expression of cyclin A in K-562 was 32.2; 41.8; 69.9%, respectively, according to doses of doxorubicin. The number of apoptotic cells was increasing together with the increase of doxorubicin doses as well as positive labeling for cyclin A. After doxorubicin treatment decrease of G1/G0 phase and the growth of cell percentage with dose dependent manner at G2/M phase, compared to control was observed. The results allow to suggest that expression of cyclin A may have pro-apoptotic role however more studies are required to clarify whether and what role cyclin A plays in apoptosis.