Ulrich Rodeck

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide☆

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.

WT1 MUTATION IN MALIGNANT MESOTHELIOMA AND WT1 IMMUNOREACTIVITY IN RELATION TO p53 AND GROWTH FACTOR RECEPTOR EXPRESSION, CELL‐TYPE TRANSITION, AND PROGNOSIS

The Wilms tumour 1 (WT1) gene is believed to contribute to the growth and differentiation of certain tissues, including mesothelium. This study assessed WT1 gene status by mutational screening in 42 malignant mesotheliomas (MMs) and 3 MM cell lines and detected two tumours with identical heterozygous single nucleotide deletions in intron 7, with no apparent consequence for WT1 function. Furthermore, the expression pattern of the WT1 gene was studied in MMs and related lesions using three anti‐WT1 monoclonal antibodies (MAbs). Strong to moderate nuclear immunoreactivity was noted in MM in situ (54/56), cultured mesothelioma cells (4/5), and hyperplastic and normal pleural (non‐neoplastic, NNM) specimens. WT1 immunoreactivity was absent in all primary tumours of lung and in pleural metastases from adenocarcinomas of breast and colon; immunoreactivity was present in pleural metastases from renal carcinomas, melanomas, and papillary carcinomas of the ovary. Expression of the WT1 protein in MM was not correlated with survival. Coordinate expression of the WT1 protein and its putative transcriptional target genes was determined by correlating WT1 immunostaining with epidermal growth factor receptor (EGF‐R) and insulin‐like growth factor 1 receptor (IGF‐1R) expression on MM and NNM; no significant correlation was found, irrespective of p53 expression status. Finally, the putative involvement of WT1 in cell‐type transition was supported by this study, in that epithelial mesothelioma showed the strongest WT1 immunoreactivity while sarcomatous mesothelioma showed the least.

Growth stimulation of colorectal carcinoma cells via the c‐kit receptor is inhibited by TGF‐β1

Activation of the receptor tyrosine kinase c‐kit by the kit‐ligand, also known as stem cell factor (SCF), is essential to melanocyte and germ cell development and during the early stages of hematopoiesis. Deregulated expression of c‐kit has been reported in malignancies affecting these lineages, i.e., myeloid leukemias, melanomas, and germ cell tumors. In addition, c‐kit and SCF are coexpressed in some breast and colorectal cancer (CRC) cells, raising the question of whether c‐kit serves an autocrine role in normal or malignant epithelial tissues. In this study, we demonstrate that human colorectal carcinomas, but not normal colorectal mucosa cells, coexpress SCF and c‐kit in situ. Expression of c‐kit was also observed in mucosa adjacent to colorectal tumor tissue. Consistent with a growth‐regulatory role of SCF in CRC cells, exogenous SCF stimulated anchorage‐dependent and anchorage‐independent growth in four out of five CRC cell lines. Exogenous transforming growth factor (TGF)‐β1 added at nanomolar concentrations to HT‐29 CRC cells, which express the type I, II, and III TGF‐β receptors, downregulated c‐kit expression to background levels and inhibited c‐kit–dependent proliferation. Similarly, TGF‐β1 inhibited SCF‐dependent proliferation of three first‐passage CRC cell lines. In summary, expression of the potential autocrine SCF/c‐kit axis is a tumor‐associated phenomenon in colorectal cancer that can be suppressed by TGF‐β1 in TGF‐β–responsive CRC cells. J. Cell. Physiol. 172:1–11, 1997.

Growth-regulatory factors for normal, premalignant, and malignant human cells in vitro

Normal human cells, cells from nonmalignant proliferative lesions, and primary and metastatic tumor cells can be maintained in vitro and analyzed for requirements for growth in chemically defined media. The human melanocytic cell system with normal melanocytes, precursor nevus cells, and primary and metastatic melanoma cells has been extensively studied for the phenotypic properties of the cells, including their requirements for exogenous growth factors and other mitogens. In high calcium-containing W489 medium, normal melanocytes require four supplements: IGF-I (or insulin); bFGF, TPA, and alpha-MSH. Nevus cells are largely independent of bFGF. Depletion of TPA from medium is not as detrimental to nevus cells as it is to melanocytes, but the phorbol ester is still essential for maintenance of the typical nevic phenotype. Primary melanoma cells require at least one growth factor, IGF-I (or insulin), for continuous proliferation. On the other hand, metastatic cells of melanoma as well as of carcinomas of colon and rectum, bladder, ovary, and cervix are able to proliferate after a short adaptation period in medium depleted of any growth factors and other proteins. Doubling times of metastatic tumor cells in protein-free medium are only 30-60% longer than in FCS-containing medium. The growth autonomy of human tumor cells is apparently due to the endogenous production of growth factors. Likely candidates for autocrine growth stimulation of human tumor cells are TGF-alpha, TGF-beta, and PDGF. Melanoma and colorectal carcinoma cells express functional EGF/TGF-alpha receptors, and produce TGF-alpha, indicating that this growth factor is produced for autocrine stimulation. In addition to the use of anti-growth factor antibodies, other strategies for the inhibition of autocrine growth stimulation include mAbs to growth factor receptors, soluble receptors, receptor-mimicking antiidiotype antibodies, and active immunization against growth factors. Whether any of these therapeutic approaches is clinically feasible will need to be determined in extensive preclinical investigations.

Growth factors in melanoma.

Human melanoma cells in culture are the source of a wide variety of polypeptide growth factors. Melanoma-derived basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-A and PDGF-B chains, transforming growth factor (TGF)-α and TGF-β, interleukin (IL)-1α and IL-1β, and melanoma growth stimulatory activity (MGSA) have similar biochemical and functional properties when compared to their counterparts produced by untransformed cells. In contrast to melanoma cells, normal melanocytes, even under optimal growth conditions, express only TGF-β1 and MGSA at detectable levels suggesting that production of the other growth factors is a tumor-associated phenomenon. Recent evidence suggests that at least two of the growth factors, bFGF and MGSA, contribute to autocrine growth stimulation of melanoma cells. Whether PDGF, TGF-α, IL-1, and TGF-β act in an autocrine mode is unclear at present. However, these four growth factors are among those secreted by melanoma cells and, therefore, can be expected to interact with normal cells of the tumor stroma in vivo. Such paracrine effects include not only growth modulation in the context of angiogenesis and stroma formation, but also tissue degradation by proteolytic enzymes, the modification of extracellular matrix composition, and expression of adhesion receptors.

Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1-680 of gB, and lacking the fragment encoding amino acids 681 907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.

Detection by monoclonal antibodies of the Wilms' tumor (WT1) nuclear protein in patients with acute leukemia

The WT1 gene encodes a transcriptional regulator which during embryogenesis is involved in growth control and differentiation of diverse tissues. It is also expressed in few human malignancies, including acute leukemia. We tested 3 different monoclonal antibodies (MAbs H2, H7, HC17) and the polyvalent serum WTC‐19 for WT1 protein detection in mononuclear cell (MNC) preparations of 104 newly diagnosed acute leukemia patients. Using RT‐PCR, these MNC preparations were also analyzed for WT1 gene expression. MAbs H2, H7 and HC17 and the polyclonal WTC‐19 exhibited nuclear immunoreactivity in 63 of 99, 28 of 56, 38 of 60 and 22 of 43 WT1 gene‐expressing leukemia samples, respectively. With these antibodies, no WT1 immunoreactivity was found in MNCs from blood of healthy volunteers, from CD34+progenitor cell‐enriched leukapheresis products of patients conditioned for peripheral stem cell harvest or from reactive bone marrow. Contrary to WTC‐19, all MAbs reacted highly specifically with the WT1 protein (0.71 vs. 1.0). The WT1 protein was heterogeneously detected in leukemia blast preparations by all antibodies, irrespective of cell morphology. Very few HL60 cells and blasts from newly diagnosed leukemia patients interspersed among normal blood MNCs (50 blasts among 5 × 105MNCs) were easy to identify by indirect immunofluorescence using MAbs H2 and HC17. Taken together, MAbs H2 and HC17 were superior to MAb H7 and the polyvalent WTC‐19 in detecting the WT1 nuclear protein. Int. J. Cancer 70:518–523.

Epidermal growth factor (EGF) and monoclonal antibody to cell surface EGF receptor bind to the same chromatin receptor

Cellular uptake, nuclear translocation, and chromatin binding of epidermal growth factor (EGF) and monoclonal antibodies (MAbs) against the protein domain of the EGF surface receptor (MAb 425) and against the carbohydrate Y determinant on the EGF receptor (MAb Br 15-6A) were analyzed in cell lines that express surface EGF receptor. Both EGF and MAb 425 were translocated to the nucleus and bound in nondegraded form to the chromatin of all cells tested. MAb Br 15-6A was taken up only by SW 948 colorectal carcinoma cells which express EGF receptor whereas neither EGF nor MAb 425 was taken up by SW 707 colorectal carcinoma cells which do not express EGF receptor. MAb 425 immunoprecipitated a 230- to 250-kDa chromatin protein, which appears to be the EGF chromatin receptor. EGF was localized in a single EcoRI DNA fragment suggesting that the chromatin binding was highly specific. Binding of EGF to primarily DNase II-sensitive chromatin regions protected these regions from nuclease action. The role of growth factor binding to chromatin in neoplastic transformation is discussed.

Growth factor independence and growth regulatory pathways in human melanoma development.

SummaryThis review concentrates on growth autonomy of tumor cells in relation to tumor progression. Human malignant melanoma serves as an example for progressive growth factor independence at subsequent stages of tumor progression. Mechanisms by which malignant cells acquire growth factor independence are discussed. In melanoma, deregulation of growth regulatory pathways has been described on four levels: 1) aberrant production of autocrine growth factors that substitute for exogenous growth factors (basic fibroblast growth factor [bFGF]); 2) alterations in the response to negative autocrine growth factors (interleukin [IL]-6 and transforming growth factor [TGF]-β); 3) overexpression of epidermal growth factor receptors (EGF-R); and 4) alterations of cellular protooncogenes involved in signal transduction (RAS, MYB) and growth suppression (p53). In addition to bFGF and IL-6, multiple other growth factor genes are activated in malignant melanoma cells but not normal melanocytes. These include both chains of platelet-derived growth factor (PDGF), TGF-α, IL-1, IL-8, and tumor necrosis factor (TNF)-α. Of these, PDGF-B has been investigated in more detail. Melanoma-derived PDGF clearly does not act in a direct autocrine mode, but has important paracrine effects on normal tissue constituents, notably fibroblasts and endothelial cells, that are essential for tumor developmentin vivo. It is speculated that other melanoma-derived growth factors with as yet undefined functions similarly exert such paracrine or ‘indirect’ autocrine effects that cannot be sufficiently addressed in studies on cultured cells.

Shedding Of Human Tumor-Associated Antigens in Vitro and in Vivo

Publisher Summary This chapter focuses on the tumor-associated antigens (TAAs) and monoclonal antibodies (MAbs) that are shed in vitro and in vivo. TAAs are those glycoproteins and glycolipids that are expressed on the surface or in the cytoplasm of tumor cells and more often than not are detected on either the normal counterpart of tumor cells or on fetal cells. The term shedding implies the release of antigenic components, in soluble or particulate form, from cells into the tissue culture medium or into blood or other human secretions. Antigens can be released by cells as a function of active metabolic turnover or as a result of cell death. MAbs are undefined circulating TAAs in sera of patients with various solid human tumors. For established tumor markers, MAbs are replacing the use of polyclonal antisera. It is expected that new tumor markers become available that may serve, when necessary, as additional diagnostic tools for each tumor type. MAb-defined tumor markers are available for pancreatic and ovarian carcinoma, which are elevated in more than 80% of patients sera. For other tumors, combinations of assays need to be developed.