Healing venom.

Neuroblastoma (NB) cells possess morphological and biological properties in vivo and in vitro, the cell type having the characteristics of primitive 1981). NB cell lines established in vitro have been shown to consist of two morphologically distinct types of cells: neuroblast-like and epithelial-like These two cell types can undergo bidirectional phenotypic inter-conversion and only neuroblast-like cells contain activities for enzymes unique to catecholamine neurons (Ross et al., 1983). Recently it has been demonstrated that several monoclonal antibodies (Moabs) primarily developed against haemato-poietic cells cross-react with NB cells, and the analysis of surface membrane antigen expression may be utilized for clinical diagnosis and sub-classification of this tumour (Sugimoto et al., 1984). However it remains to be determined whether sub-grouping of NB cells by surface antigen phenotype may be associated with any histological, biological and clinical characteristics. In the present study we examined the reactivity of a panel of 37 Moabs against 10NB cell lines (5 neuroblast-like and 5 epithelial-like) to determine whether there were any differences in phenotypic patterns of Moab binding between neuroblast-like and epithelial-like cell lines.

Neuroblastoma (NB) cells possess morphological and biological properties in vivo and in vitro, the cell type having the characteristics of primitive sympathetic neuroblasts (Beckwith et al., 1963;Biedler et al., 1981;Kissel et al., 1981;Ross et al., 1981). NB cell lines established in vitro have been shown to consist of two morphologically distinct types of cells: neuroblast-like and epithelial-like (Tumilowicz et al., 1970;Weston, 1970;Biedler et al., 1973;Bernal et al., 1983). These two cell types can undergo bidirectional phenotypic interconversion and only neuroblast-like cells contain activities for enzymes unique to catecholamine neurons (Ross et al., 1983). Recently it has been demonstrated that several monoclonal antibodies (Moabs) primarily developed against haematopoietic cells cross-react with NB cells, and the analysis of surface membrane antigen expression may be utilized for clinical diagnosis and subclassification of this tumour (Sugimoto et al., 1984). However it remains to be determined whether subgrouping of NB cells by surface antigen phenotype may be associated with any histological, biological and clinical characteristics.
In the present study we examined the reactivity of a panel of 37 Moabs against 10NB cell lines (5 neuroblast-like and 5 epithelial-like) to determine whether there were any differences in phenotypic patterns of Moab binding between neuroblast-like and epithelial-like cell lines.
A total of 10 human NB cell lines (SK-N-SH, SJ-N-SD, SJ-N-KS, IMR32, NB 1, SJ-N-CG, SJ-N-*JF, SJ-N-KP, Goto, Nb/1-M) were maintained in RPMI 1640 medium with 10% foetal calf serum (FCS) at 37°C in 5% humidified CO2. Adherent NB cells were grown on glass slides and their surface membrane antigen expression was determined using an indirect immunofluorescence assay. Briefly NB cells grown on glass slides were incubated with Moab at room temperature for 30 min. The slides were then washed with RPMI 1640 medium containing 10% FCS and subsequently incubated with fluorescein-conjugated goat anti-mouse immunoglobulins diluted 1:20 for 30min. The slides were again washed and mounted in PBS containing 60% glycerol. All samples were examined for fluorescence by two investigators and at least 200 cells per sample were examined. Results were expressed as percentage of fluorescencepositive cells. For the antibody controls, slides were stained with a negative primary antibody of the same immunoglobulin isotype.
Ten human NB cell lines were analyzed for the surface antigen expression using a large panel of Moabs. Each cell line displayed a distinct morphology. SK-N-SH, SJ-N-SD, SJ-N-KS, IMR 32 and NB 1 cells were neuroblast-like in appearance, with small, round cell bodies, scant cytoplasm and small-to-medium length neurites that extended radially from the cell body ( of the 10 NB cell lines assayed in this study. One (OKB 2) of six anti-B-cell antibodies strongly cross-reacted with all 10 NB cell lines, but HLA-DR antigens could not be detected on the NB cell lines with three anti-HLA-DR antibodies (OKIal, MAb-BI and SJ-7B9).
Both HNK-1 and Leu 1lb showed distinct crossreactivity with NB cells in culture. The analysis of 5 epithelial-like NB cell lines revealed that all 5 cell lines uniformly bound Leu llb antibody and 4 of 5 lines reacted with HNK-1 antibody. In contrast all   5 neuroblast-like cell lines had consistent reactivity with HNK-l antibody, but none of the 5 lines were reactive to Leu 1 lb antibody (Figures 2a & 2b). Out of 6 anti-leukaemia-associated Moabs, 2 Moabs were found to cross-react with NB cells. The SJ-9A4 antibody  recognizing a cell surface glycoprotein, p24 present on common acute lymphoblastic leukaemia (cALL) cells, platelets and subpopulation of immature T and B-lymphocytes, reacted with all 10 NB cell lines. BA-1 antibody (Abramson et al., 1981), which bound to cALL cells, cells of normal and malignant B lymphocyte origin and granulocytes, was uniformly reactive with all 10 NB cell lines. The J 5 (Ritz et al., 1980) and NL-1 (Ueda et al., 1982) antibodies recognizing common ALL antigen, bound to 1 (SJ-N-CG) of the 5 epithelial-like NB cell lines as previously reported (Sugimoto et al., 1984).
All 9 anti-myeloid-monocyte-associated Moabs were totally unreactive with 10 NB cell lines assayed in this study.
P1153/3 antibody (Kennett et al., 1979) primarily raised against NB cells, recognizing a cell surface glycoprotein, p20 present on cALL cells, early and mature B lymphocytes and foetal brain cells, showed strong reactivity with all 10 NB cell lines.
In the present study we have demonstrated that two morphologically distinct types of human NB cell lines (neuroblast-like and epithelial-like) showed a similar phenotypic profile, except for the reactivity of one Moab, Leu 1lb. It is noteworthy that Leu 1lb antibody reacts with all 5 epitheliallike lines but not with any neuroblast-like line assayed in this study.
The SJ-9A4, BA 1, PI153/3 and HNK-1 Moabs have been reported to react with human NB cells Sugimoto et al., 1984;Greaves et al., 1980;Caillaud et al., 1984). In this study we found that 2 additional Moabs (OKB 2 and Leu 1 lb) were reactive with human NB cell lines. The OKB 2 antibody reacted with all 10 NB cell lines analyzed. This Moab (Mittler et al., 1983) was originally raised against Burkitt's lymphoma cells and reported to recognize a cell surface structure present on cALL cells, cells of normal and malignant B lymphocyte origin and granulocytes. In contrast with the fact that the majority of Moabs reactive with NB cells appear to recognize antigens which are universally expressed on all cells within the culture irrespective of the cell morphology, the Leu 1lb antibody recognizes an antigen which appears to be expressed only on a proportion of cells in epithelial-like cultures as shown in Table II. The Leu llb antibody (Thompson et al., 1982) originally raised against granulocytes, detected a cell surface structure present on peripheral blood lymphocytes containing large azurophilic granules and neutrophils. In vitro study (Ross et al., 1983) demonstrated a coordinate morphological and biological interconversion of NB cells (SK-N-SH) and a plasticity in morphological expression in malignant neuronal cells. Only neuroblast-like clones contained activities for tyrosine hydroxylase and dopamine hydroxylase, enzymes unique to catecholamine neurons; epithelial-like cells lacked activities for these enzymes. Similarly the antigen recognized with the Leu 1lb antibody might be expressed in association with the change in cell morphology, and the synthesis of this cell surface antigen could be newly induced and significantly increased to the detectable level only on a proportion of epithelial-like NB cells. The surface phenotypes of NB cells could be associated with morphological characteristics. The present study has revealed that several antigenic regions are common to the cells of different histological lineages: haematopoietic cells and neuroblasts. The cross-reactivity of certain Moab may not necessarily recognize an identical gene product. For instances the difference in the glycosylation of leukaemia-associated antigen p24 from cALL cells and neuroblasts has been already reported . Further biochemical analysis of the cell surface antigens thought to be shared between haematopoietic cells and neuroblasts needs to be undertaken.