Radiation studies on B cell differentiation marker CD24/SCLC Cluster-4 antigen expressing and non-expressing lung cancer cell lines and mouse fibroblasts.

A correlation between CD24 expression and higher intrinsic radiation sensitivity has been described in B-lineage acute lymphoblastic leukaemia (B-ALL). We recently identified the SCLC surface antigen Cluster-4 (CL-4) to be identical to the B cell differentiation marker CD24, except for one amino acid residue. The CD24/CL-4 antigen is highly expressed on SCLC, but rarely on NSCLC cells. In order to investigate the influence of the expression of CD24/CL-4 on the radiation sensitivity in a non-leukaemic cell system, sublines of the human SCLC H249 cell line transfected with mutated ras oncogene, and differing in their CD24/CL-4 expression, were studied. In addition, we stably transfected the NSCLC A125 cell line and the mouse fibroblast NIH3T3 cell line with the CL-4 cDNA. The differential expression of CD24/CL-4 on the cells had no influence on morphology, proliferation and cloning efficiency. Radiation studies were done with cells in exponential growth phase. In the highly resistant NSCLC A125 cells no difference in radioresponsiveness was observed between CD24/CL-4 expressing and non-expressing cells. In the rather radiosensitive cells, similar responses to radiation were observed between CD24/CL-4 expressing and non-expressing SCLC H249-ras cells, whereas the CL-4 transfected NIH3T3 mouse fibroblasts showed a substantially higher radioresistance than the CD24/CL-4 non-expressing control cells. In conclusion, the correlation between CD24/CL-4 expression and radiation sensitivity is controversial and depends on the cell type.

A mutant of human insulin-like growth factor II (IGF II) with the processing sites of proinsulin. Expression and binding studies of processed IGF II.

A mutant of human insulin-like growth factor II (IGF II) was constructed by site-directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg-Arg and Lys-Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH-3T3 cells as a processed two-chain peptide with a deletion of amino acid residues 37-40 and crosslinked by three disulfide bonds. This des(37-40)[Arg33]IGF II showed 3.6-fold and 7.4-fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild-type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1-fold reduced affinity of the expressed mutant to the co-expressed IGF binding protein 3 (IGFBP-3). The results suggest that des(37-40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.

Association of CD24 with the kinase c-fgr in a small cell lung cancer cell line and with the kinase lyn in an erythroleukemia cell line.

Human CD24 is a highly glycosylated glycosylphosphatidylinositol-linked (GPI-linked) cell surface protein. GPI-linked proteins are involved in signal transduction mediated by members of the protein tyrosine kinase (PTK) family. Therefore we studied associated molecules providing the signaling capacity of CD24. Lysates of SW2 and K562 cells were analysed for expression of PTK of the c-src family by Western blotting. We identified c-fgr in SW2 lysates and c-fgr and also lyn in K562 lysates. To study a putative association of these PTK with CD24 we performed immunoprecipitations with the mAb SWA11 directed against CD24. Western analysis of the precipitates showed an association of c-fgr with CD24 in SW2 cells and lyn in K562 cells. We conclude that either c-fgr or lyn is physically associated with CD24 in a cell-type depending manner. An involvement of these complexes in signaling phenomenons of CD24 in small cell lung cancer and in leukaemias is discussed.

The 5'-flanking region of human CD24 gene has cell-type-specific promoter activity in small-cell lung cancer.

The human surface antigen CD24 is over-expressed in small-cell lung cancer. Here we describe the isolation, sequencing and functional characterization of the 5'-flanking region of the human CD24 gene. A sequence (accession number: Y14692) of 3.4 kb regulates the activity of a luciferase reporter gene in CD24-expressing small-cell-lung-cancer cell lines up to 1.6-fold more than the control SV40 promoter and enhancer. In contrast, little or no luciferase activity was detected in 4 non-small-cell-lung-cancer cell lines. A deletion fragment of 269 bp had maximal activity in small-cell-lung-cancer cell lines (15- to 20-fold higher than control), while activity remained 2- to 10-fold below control in non-small-cell-lung-cancer cell lines. We conclude that the CD24 promoter has a strong and cell-type-specific activity, and propose its exploitation to drive the expression of therapeutic genes in small-cell lung cancer.

The small cell lung cancer antigen cluster-4 and the leukocyte antigen CD24 are allelic isoforms of the same gene (CD24) on chromosome band 6q21.

Cluster-4 and CD24 cDNA's have recently been cloned from the small cell lung carcinoma (SCLC) cell line SW2 and from the erythroleukemia cell line K562, respectively. The only difference in the coding sequence, between cluster-4 and CD24 antigens is the substitution of a single base pair leading to a substitution of Val by Ala near the putative glycosylphosphatidylinositol (GPI) anchorage sites of the mature protein. Here we demonstrate that the nucleotide substitution which distinguishes the cluster-4 and CD24 antigen genes is due to an allelic polymorphism on chromosome band 6q21. In addition, we identified by Southern blotting and PCR of DNA from somatic human x hamster hybrid cell lines homologues of cluster-4/CD24 on the Y chromosome and chromosome 15. We suggest, however, that the gene on 6q21 is the active locus since the mRNA of cell lines always represents the allelic variants found on chromosome 6. The distribution pattern of this allelic polymorphism in SCLC cell lines and leukocytes of healthy donors did not reveal any obvious relationship with disease. However, it is noteworthy that homozygosity for cluster-4 was found in only one case whereas heterozygosity and homozygosity for CD24 both contribute up to 50% of the samples examined.