engleski

CALLA (CD10) molecule and gene expression in human keratinocytes

Introduction. CALLA (common acute lymphoblastic leukemia antigen) has been described as the marker of lymphoid malignancies. Later on, that membrane glycoprotein has been detected as the CD10 marker of normal lymphoid and hematopoietic cells, suggesting a role in cellular differentiation and/or function. In addition, CALLA/CD10 was recognized as the neutral endopeptidase (NEP), a cell membrane enzyme that cleaves enkephalins, substance P, bombesin, atrial natriuretic factor and other neuropeptides, thus regulating their local actions in the central nervous system, lungs, kidney, cardiovascular system etc. We have studied the expression of CALLA/CD10/NEP on human keratinocytes. Material and methods. Normal foreskins were obtained from routine pediatric circumcisions. The skin samples were enzymatically processed to the stage of keratinocytes which were used for further tests. 1. The expression of CD10 on keratinocyte suspensions was determined by means of antibody labeling (Anti - CD10 FITC, DAKO A/S Denmark) and flow cytometry analysis (FACS Calibur). 2. DNA was extracted by phenol-chloroform-isoamylalcohol and precipitated with sodium acetate and absolute ethanol. The quality was verified by electrophoresis in 0.8% agarose gel containing ethidium bromide. 3. RNA was isolated with RNAzol TM B (a modified guanidium-isothyocyanate reagent) and precipitated with isopropanol. The isolates were verified by electrophoresis in 1% agarose gel containing formaldehyde and ethidium bromide. 4. Reverse transcription of RNA into cDNA was carried out by means of M-MuLV reverse transcriptase in the presence of Rnase inhibitor, DTT, random hexamers and dNTPS (all from Boehringer). 5. CALLA/CD10 genomic DNA was demonstrated by PCR using primer pairs CALLA and recommended by the Gene Data bank. 6. CALLA/CD10 specific RNA was detected by RT-PCR and the PCR Core Kit (Boehringer) using the cDNA and the primer pair CALLA. Expression of house-keeping genes b-actin, ABL 2/1-S and ABL 3-S served as positive control. Results. CALLA/CD10+ was detected on 30-41% keratinocytes in 3 samples tested. DNA was isolated from 2, and RNA from 7 samples. The quality of the isolates was satisfactory. cDNA was obtained from 4 of 7 RNA isolates. Presence of the house-keeping genes was confirmed in all 4. Genomic CALLA was demonstrated in the 2 DNA isolates and in all 4 cDNA preparations. Conclusion. Human keratinocytes express CALLA/CD10 cell surface marker and contain CALLA/CD10 gene sequence and the specific RNA. A similar finding has been reported while this work was in progress (Olerud ET et al., J. Invest. Dermatol. 112(6) 873-881, 1999). Ubiquitous expression of CALLA/CD10 in many types of cells (including the keratinocytes) indicates participation of that membrane metalloproteinase in basic cellular processes regulated by neuropeptide signal molecules, e.g. in cell proliferation and differentiation. Acknowledgement. The authors appreciate the gift of anti-CD10 antibody by Dr. T. Marotti (Rudjer Bošković Institute) and of the components for the RT-PCR assay by Dr. R. Spaventi and Mr. K. Brajša (Pliva Pharmaceutical Industry, Zagreb).

CALLA (CD10); RT-PCR; FACS analysis; human keratinocytes

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