Library

Notes: Summary In this study we have treated three malignant (TGrIII) and two pre-malignant (TGrII) urothelial cell lines with recombinant human interferon γ (rHu-INFγ). The malignant cells (HCV29-T112C1, Hu1703He and T24) were inhibited in growth by more than 50% after treatment with 100–1000 units of rHu-INFγ/ml for 4 days as compared to untreated controls. The growth of the pre-malignant cell lines (HCV29 and Hu609) was not influenced to the same extent in the presence of rHu-INFγ in the culture medium. Treatment with rHu-INFγ increased the expression of monomorphic human leukocyte antigens (HLA) A,B,C as well as β2-microglobulin in all the cell lines tested, as demonstrated using a quantitative immunofluorescence assay. The tumourigenic cell lines increased their expression of HLA in a dose-dependent way, whereas treatment of the non-tumourigenic cells with higher concentrations of rHu-INFγ than 10 units/ml, did not increase the HLA-A,B,C expression further. None of the cell lines expressed HLA-DR unless treated with rHu-INFγ. No correlation between tumourigenicity and the dose of rHu-INFγ required for “de novo” induction of HLA-DR could be demonstrated. After removal of rHu-INFγ from the medium, the expression of HLA-DR gradually decreased in less than 14 days, indicating that the expression of HLA-DR is not constitutive but dependent upon the presence of rHu-INFγ. We conclude that human urothelial cells grown in vitro are sensitive to the anti-proliferative and major-histocompatibility-complex-modulating effects of rHu-INFγ, and that malignant urothelial cells are more sensitive than pre-malignant cells. Finally, our data indicate a possible role for rHu-INFγ in the management of human bladder cancer.

Notes: Summary Quantitative changes in major histocompatibility class I antigen expression in tumour cells are believed to affect the host immune response against the tumour. In tumourigenic (TGrIII) human urothelial cell lines the apparent loss of polymorphic HLA-A,B epitopes has previously been demonstrated. In the present study, 3 non-tumourigenic (TGrII) and 6 tumourigenic (TGrIII) human urothelial cell lines have been investigated for their quantitative expression of monomorphic HLA-A,B,C and B2-microglobulin. Evidence is provided that an inverse correlation exists between tumourigenicity and HLA-A,B,C and B2-microglobulin expression. Furthermore, treatment of the cells with neuraminidase partly restored the expression of monomorphic HLA-A,B,C suggesting that at least some of the observed quantitative differences could be due to masking of the membrane bound HLA antigens by sialic acid-containing glycoconjugates.

Notes: Summary In order to evaluate in mathematical terms the morphological changes occurring in the course of cell spreading, Fourier analysis of shape was applied. Human urothelial Hu 961 b cells plated on type IV collagen, fibronectin, laminin, glass and bovine serum albumine (BSA) were studied. Fourier parameters describing cell shape as well as surface areas covered by the cells on the substrate were subjected to statistical analysis. Using analysis of variance and discriminant analysis it was found that parameters describing cell shape (both gross shape of cells and their fine scale contour foldings) possessed a higher power of discrinunation between the cells spread on various substrates than the differences in cell surface areas. In the course of observation (75 and 150 min) the highest number of attached cells and highest degree of spreading were found when cells were plated on type IV collagen. Moderate alterations in cell shape and moderate increase of surface area were seen in the group of cells seeded on fibronectin, whereas the cells plated on laminin, glass and BSA revealed a moderate increase of surface area, but no changes in their shape were observed. The differences in attachment of cells and in the degree of their spreading might be due to the variation in expression of plasma membrane receptors for various substrates. The Fourier analysis of cell shape coupled with measurement of surface area is a good tool for quantitative evaluation of cell spreading and can be used for discrimination between cells spread on different substrates.

Notes: Summary The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I=nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II=nonmalignant cell lines with an infinite life span. TGr III=malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs-each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)-were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by “spontaneous” transformation during propagation in vitro of the respective maternal TGr II-cell lines. One of these TGr II cell lines (HCV-29) originated from the histologically normal bladder mucosa obtained from a patient with a previous history of bladder papillomata treated with irradiation (Fogh, personal communication). The other TGr II cell line (Hu 609) was derived from the normal ureter of a patient with renal carcinoma. In each of these 8 cell lines, the shape of 100 cells chosen at random were subjected to Fourier analysis of shape. Each of the particular harmonic amplitude values studied was used as an individual parameter for the evaluation of differences between compared cell lines, using Chi-Square test and discriminant analysis. It was found that in two of four analysed pairs of cell lines, i.e. Hu 1703S (TGr I) vs Hu 1703He (TGr III) and HCV-29 (TGr II) vs HCV-29T (TGr III), the differences in cell shape between the two populations were very well pronounced, as was shown by several statistical parameters. In the two other pairs of cell lines, i.e. Hu 961b (TGr I) vs Hu 961a (TGr III) and Hu 609 (TGr II) vs Hu 609T (TGr III) significant differences in cell shape were also found, but they were less pronounced. The conclusion is, that differences in cell shape in vitro may reveal the cellular heterogeneity of the original transitional cell carcinoma and/or the progression of in vitro propagated urothelial cells from one grade of transformation into another.

Notes: Summary The aim of this paper is to show the possibility of objective mathematical description of changes occurring in the shape of cells in the process of transformation. The evaluation of the changes in cell shape of the chosen cell lines differing in transformation grade was performed by the use of Fourier analysis of the shape. Any two-dimensional contour can be described with specific accuracy in a mathematical manner using the closed form Fourier series of cosines. The components forming the analysed shape, called harmonics, are independent and uncorrelated measures of their contribution to the total shape. The shape of each cell can be represented by the spectrum of harmonic amplitudes. To quote the paper by Healy-Williams and Williams (1981): “The observed shape is partitioned into series, where gross shape, as elongation or triangularity, is measured by the harmonic amplitudes of the lower harmonic order and increasingly fine scaled surface sculpture is measured at higher orders”. The statistically evaluated results allow the objective comparison of the cell shapes of several compared cell lines differing in transformation grades. Malignant transformation is supposed to be a multistep process. The different grades of transformation could be defined by several parameters as changes in the morphology of the cells, their ability to compete with fibroblasts, their life span, their angiogenic potency, their invasiveness in vitro and their tumorigenicity in nude mice. In this paper several human urothelial cell lines of normal and tumor origin differing in their transformation grade (TGr I–III) were compared by the use of Fourier analysis of their shape. TGr I cultures have finite life span but do not need intermittent collagenase treatment to prevent fibroblast overgrowth. TGr II cultures acquire infinite growth potential, here defined as capacity to survive at least 70 passages. They are neither tumorigenic nor invasive. TGr III cultures show infinite growth transformation, increased angiogenicity and ability to invade normal host tissue in vitro. They produce progressively growing tumors in nude mice. The following human uroepithelial cell lines differing in the degree of transformation were studied and compared by statistical evaluation of the harmonic amplitudes deseribing mathematically the cell shape: Two cell lines derived from human transitional cell carcinoma (TCC): 1. Hu 1703S classified as TGr I, 2. Hu 1703He classified as TGr III. It was found that these two cell lines differ in all harmonics. Two cell lines derived from morphologically normal human bladder epithelium: 3. HCV-29 classified as TGr II. The confluent (HCV-29 confl) and peripherial (HCV-29 periph) parts of the HCV-29 cultures were studied separately. 4. HCV-29T “spontaneously” transformed in culture, subline of HCV-29 classified as TGr III. 5. Normal human fibroblast line was used for comparison and control. — It was shown that the confluent part of the epithelial HCV-29 line (HCV-29 confl) which is not tumorigenic was identical in shape to the cells of HCV-29T tumorigenic cultures. The parameters describing the shape of cells in the periphery of HCV-29 cultures (HCV-29 periph) were nearly identical to those of human fibroblasts. Both HCV-29 lines are statistically different from the Hu 1703He line. The applied technique allows for the numerical description of cell shape — the one of many changeable parameters in the process of transformation. These numerical data could be analysed by statistical methods, which allow to define the similarities or differences between the compared cell lines in a quantitative way.

Notes: Summary It was found that a decrease in electrophoretic mobility of pyruvate kinase (PK) isoenzyme, and an increase of the sensitivity of this enzyme to L-cysteine, were markers of immortalization and tumorigenic properties, respectively, in human urothelial cell lines characterized by different grades of transformation (TGr) in vitro.

Notes: Abstract The presence of specific binding sites for phorbol esters was studied in a transformed but non-tumorigenic human urothelial cell line HCV-29 by assay of specific binding of3H-phorbol-12,13-dibutyrate (3H-PDBu) to intact living cells.3H-PDBu bound specifically to HCV-29 cells in a saturable and competitive manner. Scatchard plot analysis of specific binding yielded a curved plot consistent with two binding sites with Kd of 11 nM and 102 nM, respectively. At saturation the corresponding PDBu binding capacities (Bmax) were 8.8 pmol/106 cells (5.2×106 molecules bound per cell) and 2.8 pmol/106 cells (1.7×106 molecules bound per cell).3H-PDBu binding was displaced by biologically active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein,but not by tumor promoters such as L-tryptophan, anthranilic acid and sodium saccharin. In cells desensitized by pretreatment with 1 μg/ml (2μM) TPA or PDBu for 24 h the level of binding was reduced to 28% of the level in non-exposed cells. The ability of desensitized cells to bind3H-PDBu was gradually restored within 5–6 days. At the same time the cells became sensitive to the morphological alteration induced by PDBu. This suggests that desensitization of HCV-29 cells is due to a decreased receptor-ligand binding capacity probably associated with down regulation of the phorbol ester receptors.