Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice.

Endotoxin-responsive (C3H/HeN) and -hyporesponsive (C3H/HeJ) murine B lymphocytes purified by adherence to anti-immunoglobulin ("antibody panning") possess identical gangliosides but different ganglioside surface accessibilities. We investigated the distribution and surface accessibility of gangliosides of B lymphocytes purified by adherence to plastic ("plastic panning") or by subtraction of non-B-lymphocyte components. As with antibody panning, there were no entirely new or absent gangliosides in plastic-panned or subtraction-purified B lymphocytes of each strain. However, striking changes in relative expression of five gangliosides were detected with each purification protocol. Moreover, five gangliosides of antibody-panned and plastic-panned B lymphocytes but only two gangliosides of subtraction-purified B lymphocytes were inaccessible to surface labeling. Unlike the situation for antibody-panned B lymphocytes, no interstrain (HeN vs. HeJ) surface accessibility differences existed in gangliosides of plastic-panned or subtraction-purified cells. Exposure of subtraction-purified B lymphocytes to anti-immunoglobulin failed to elicit changes in ganglioside expression. Murine B lymphocytes have distinct protocol-dependent differences in glycolipid phenotype which likely denote individual subpopulations.

The biosynthesis and secretion of prostate-specific antigen in LNCaP cells.

Prostate-specific antigen (PSA) has been demonstrated to release the active form of insulin-like growth factor I in vitro (P. Cohen et al., J. Clin. Endocrinol. & Metab., 75: 1046-1053, 1992; P. Cohen et al., J. Clin. Endocrinol. & Metab., 79: 1410-1415, 1994; P. Cohen et al., Horm. Metab. Res., 26: 81-84, 1994) and has significant mitogenic activity on osteoblast cells, fibroblasts, and other cultured cells (C. S. Killian et al., Biochem. Biophys. Res. Commun., 192: 940-947, 1993). Recently, PSA has been found not only in prostate tissues but also in breast, colon, ovarian, and other tissues (E. P. Diamandis and H. Yu, J. Clin. Endocrinol. & Metab., 80: 1515-1517, 1995; E. P. Diamandis and H. Yu, Clin. Chem., 41: 204-210, 1995; A. Clements and A. Mukhtar, J. Clin. Endocrinol. & Metab., 78: 1536-1539, 1994). Therefore, PSA has been proposed as a candidate growth factor, cytokine, or growth factor regulator. In this setting, knowing how to manipulate or block the secretion of PSA by the prostate cancer cells could be a useful approach to controlling the progression of human prostate cancers. Using metabolic labeling experiments, we have studied the biosynthesis and secretion of PSA in LNCaP cells. We have also examined the effects of DTT, tunicamycin, 1-deoxymannojirimycin, pilocarpine, and testosterone on PSA biosynthesis and secretion. The results indicate that the secretion of PSA in LNCaP cells is constitutive instead of regulated and that the disruption of intramolecular disulfide bonds affects the transport of PSA from the endoplasmic reticulum to the Golgi apparatus. The biosynthesis of PSA is potentiated by testosterone and inhibited by brefeldin A and DTT. These results will help us understand PSA biosynthesis and secretion in human prostate cancers.

Gene therapeutic approach to primary and metastatic brain tumors: I. CD44 variant pre-RNA alternative splicing as a CEPT control element.

Our laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible for the generation of CD44 variant (CD44v) isoforms. Based on clear differences in the expression of these CD44v isoforms between normal and malignant tissues, we believe that elucidation of the mechanisms underlying the regulation of CD44 alternative splicing may provide a new gene therapeutic targeting approach based on CD44 pre-mRNA processing in vivo. This strategy incorporates utilization of CD44 alternative splicing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT) approach for the treatment of brain metastases from tumors of systemic origin. As initial steps towards the development of a gene therapeutic approach based on targeting tumor cell expression of specific CD44v alternatively spliced isoforms, we have: (1) developed a novel in vivo assay system that allows the rapid analyses of potentially therapeutic CD44 alternative splicing minigene constructs; and (2) cloned the E. coli cytosine deaminase (CD) gene and fused its enzymatically active domain to alternatively spliced CD44 exons (CD44/CD). Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated in E. coli cell lysates to be equal to that of wild type cytosine deaminase.