Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.

Laminin, a non-collagenous extracellular connective tissue glycoprotein, was detected on the surface of mouse peritoneal macrophages. As determined by indirect immunofluorescence, as many as 60% of peritoneal macrophages elicited with thioglycollate expressed cell surface laminin. Only 14% of resident cells displayed detectable laminin. The expression of laminin increased with time post-injection. Concomitant with laminin expression, macrophages also displayed a receptor for the IB4 isolectin from Griffonia simplicifolia. This lectin, which binds methyl-alpha-D-galactopyranoside, may also react with the carbohydrate moeity of laminin. A small population of macrophages displayed both laminin and surface fibronectin. Unlike the difference in laminin expression between resident and thioglycollate-stimulated cells, there was no difference in cell surface fibronectin between these cell populations. Since laminin has been found to mediate cell attachment in other systems, expression of this molecule on the surface of stimulated macrophages may be important in cell-cell and cell-matrix adhesive properties of these cells.

Purified 125I-labeled lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia (I-B4 isolectin) were used to analyze changes in the expression of carbohydrates on the surface of resident (PC) and thioglycollate-stimulated murine (C57B/6J) peritoneal exudate cells (PEC). The lectins from D. stramonium, E. europaea, and G. simplicifolia I-B4 bind specifically to PEC with relatively high affinity (Kd = 5.65 +/- 1.08 X 10(-7) M, 1.08 +/- 0.12 X 10(-8) M, and 1.33 +/- 0.15 X 10(-7) M, respectively). Assuming a single lectin molecule binds to each cell surface saccharide, the number of receptor sites per cell ranged for different cell samples from 22.3 to 50.0 X 10(6), from 3.8 to 4.8 X 10(6), and from 2.0 to 16.8 X 10(6) for D. stramonium, E. europaea, and G. simplicifolia I-B4 lectins, respectively. There were approximately 3- to 7-fold, 16- to 20-fold, and 2- to 20-fold increases in binding capacity for D. stramonium, E. europaea and G. simplicifolia I-B4, respectively, compared to the binding to resident, peritoneal cells. Scatchard plots of the binding of all three lectins to PEC were linear, suggesting that the receptor sites for these lectins are homogeneous and noninteracting. The binding capacity of these lectins to PEC was unchanged after trypsin digestion of cells. The expression of carbohydrates on the surface of PEC was also monitored by an agglutination assay. PEC were agglutinated by all three lectins whereas PC either were not agglutinated or were agglutinated only at high lectin concentrations. On the basis of our knowledge of the carbohydrate binding specificity of the D. stramonium and G. simplicifolia I-B4 lectins, we postulate that, parallel with thioglycolate stimulation, there is an increase in the number of N-acetyllactosamine residues and terminal alpha-D-galactosyl end groups. The blood group B, and H type 1 determinants--DGa1 alpha 1,3[LFuc alpha 1,2]DGa1 beta 1,3(or 4)DGlcNAc and LFuc alpha 1,2DGa1 beta 1,3DG1cNAc, respectively, as well as DGa1 alpha 1,3DGa1 beta 1,3(or 4)DGlcNAc--may be considered to be possible receptors for the E. europaea lectin. These glycoconjugates, present on the surface of peritoneal exudate cells, provide new chemical markers for studying the differentiation of resident peritoneal cells.

Hybridoma cells (S3H5/gamma 2bA2) were cultivated using RPMI 1640 media with different serum concentrations at various initial cell densities. It was found that the serum requirement for cell growth was population-dependent. At an initial cell density of 1.10(4) cells/ml, the minimum serum concentration for cell growth was 5% (v/v) and reduced to 1% at the initial cell density of 1.10(5) cells/ml. When the initial density was increased to 1.10(6) cells/ml, serum was not needed for cell growth. The maximum cell density and the integral of viable cells over cultivation time increased as serum concentration or initial cell density was increased. However, specific antibody production rate was almost constant at 0.16 pg/cell.hr irrespective of serum concentration or initial cell density.