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The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)-associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear-cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up-regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor-associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12-13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti-G250 reactivity is rare., (Copyright 2000 Wiley-Liss, Inc.)

A three-dimensional single-particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA-1 forms a connection to the cytoskeleton after binding to its ligand ICAM-1. LFA-1 is an integrin consisting of an alpha and a beta chain. Thus far, only the cytoplasmic tail of the beta chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete beta cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single-particle tracking measurements using beads coated with the ligand ICAM-1 indicate that this mutant form of LFA-1 does not move freely within the cell membrane, suggesting that LFA-1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA-1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the beta chain.

Most studies concerning a possible protective role of carotenoids against cancer focus on serum carotenoid levels. We have used Raman microspectroscopy to study the intracellular amounts of carotenoids in lymphocytes of lung cancer patients and of healthy individuals. Our results indicate a significant decrease of carotenoids in lung carcinoma patients compared with healthy individuals, particularly in adenocarcinoma patients. Carotenoid supplementation raised the serum concentration in 2 lung cancer patients up to normal levels, whereas intracellular content remained significantly lower. This indicates that carotenoid uptake by lymphocytes is not only dependent on serum carotenoid concentration. Our findings indicate that Raman microspectroscopy, a recently developed technique to measure intracellular levels of drugs, is also well suited to obtain quantitative data on carotenoid amounts inside cells.

The MHC class-I binding affinity of an epitope is an important parameter determining the immunogenicity of the peptide-MHC complex. In order to improve the immunogenicity of an epitope derived from melanocyte lineage-specific antigen gp100, we performed amino-acid substitutions within the epitope and assayed both HLA-A*0201 binding and CTL recognition. Anchor replacements towards the HLA-A*0201 peptide-binding motif gave rise to peptides with higher HLA-A*0201 binding capacity compared to the wild-type epitope. In addition, several of the gp100 154-162 epitope-analogues were more efficient at target-cell sensitization for lysis by anti-gp100 154-162 CTL compared to the wild-type epitope. These altered gp100 154-162 epitopes were subsequently tested for their capacity to induce CTL responses in vivo using HLA-A*0201/Kb transgenic mice, and in vitro using HLA-A*0201 + donor-derived lymphocytes. Interestingly, the peptide-specific CTL obtained, which were raised against the different gp100 154-162 epitope-analogues, displayed cross-reactivity with target cells endogenously processing and presenting the native epitope. These data demonstrate that altered epitopes can be exploited to elicit native epitope-reactive CTL. The use of epitope-analogues with improved immunogenicity may contribute to the development of CTL-epitope based vaccines in viral disease and cancer.

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo) emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism.

Cytotoxic T lymphocytes (CTL) reactive with human melanoma tumor cells occasionally display cross-reactivity with normal melanocytes. Previously, we identified the melanocyte lineage-specific antigen gp 100 that is expressed by both melanoma cells and normal melanocytes, as a target antigen for tumor-infiltrating lymphocytes derived from a melanoma patient (TIL 1200). Here, we demonstrate that the oligoclonal HLA-A2.1-restricted TIL 1200 line is reactive with 2 distinct peptides derived from the gp 100 protein. Apart from the peptide corresponding to gp 100 amino acids 457-466, we identified the gp 100 peptide 154-162 as a second epitope recognized by TIL 1200. A 100-fold lower concentration of this novel gp 100 peptide was required for target-cell sensitization compared to peptide 457-466, indicating that the 154-162 peptide is the dominant gp 100 epitope for TIL 1200. Together with the recently described gp 100 280-288 epitope, 3 distinct CTL epitopes have now been identified in gp 100, all presented in the context of HLA-A2.1. Therefore, gp 100 is an attractive target antigen in the development of immuno-therapeutic protocols against melanoma.

Changes in organization of F-actin during the cytotoxic process between NK and K562 cells have been observed and studied using confocal laser scanning microscopy and quantitative fluorescence microscopy. An increase in F-actin content and orientation of F-actin towards the target cell have been observed in conjugated NK cells. The increase in F-actin content probably reflects activation of the NK cell for the killing process. An increase in F-actin content in the conjugated K562 cell, occurring simultaneously with the appearance of filamentous actin structures that often originated/ended at the contact place with the NK cell, was also observed. These changes were delayed compared to the increase in F-actin content in the NK cell and were accompanied by increasing cytotoxic activity. This indicates that they were results of the interaction of the K562 cell with the activated NK cell. The possible role of target cell microfilaments in the cytotoxic process is addressed.

We compared integrin-mediated adhesion to extracellular matrix (ECM) components of cultured human melanocytes and 6 human melanoma cell lines with different metastatic capacities in nude mice. Cultured melanocytes and most melanoma cell lines adhered strongly to fibronectin (FN), whereas only highly metastatic cell lines adhered to laminin (LM), collagen type I (COI) and type IV (COIV). Adhesion to LM and CO could be blocked by anti-alpha 6 and anti-alpha 2 monoclonal antibodies (MAbs) respectively. This observation is consistent with the finding that expression of LM receptor alpha 6 beta 1 and LM/CO receptor alpha 2 beta 1 was low on melanocytes and non- or poorly metastatic cell lines, whereas these integrins were strongly expressed on highly metastatic cell lines. In addition, immunoprecipitation from [35S]-methionine-labeled cells demonstrated increased synthesis of alpha 6, alpha 2 and beta 1 in highly metastatic cell lines and immunohistochemistry showed expression of alpha 6 beta 1 and alpha 2 beta 1 only in xenograft lesions from highly metastatic cell lines. Furthermore, the observation that adhesion of melanocytes and non- or poorly metastatic cell lines could be stimulated with anti beta 1 MAbs demonstrates that these receptors, on these cells, are expressed in an inactive state. Our results suggest that alpha 2 beta 1 and alpha 6 beta 1 play a role in human melanoma metastasis in nude mice and demonstrate that interactions of these integrins with their ligands can be regulated at the level of surface expression and activation state of the receptor.

The human promyelocytic cell line NB4 exhibited a weak adhesion capacity for bone marrow-derived stromal cells and their extracellular matrices (5-15% of adherent cells). Adhesion was enhanced by pulse-treatment of cells with phorbolester (PMA 10(-7) M). Adhesion was induced within minutes, was fibronectin-specific, and affected up to 100% of the treated cells. This biological response to PMA resulted from the activation of protein kinase C (PKC), since PKC inhibitors (staurosporine, sphingosine, CGP 41251, and calphostin C) prevented the phenomenon. Phenotypical analysis of integrin receptor expression (particularly FN receptors VLA-4 and VLA-5) at the membrane of untreated or PMA-treated cells revealed that PMA induced no significant modification of the level of expression of these receptors. However, inhibition studies carried out with anti-VLA monoclonal antibodies demonstrated that the FN-specific adhesion triggered by PKC involved the alpha 5 beta 1 FN-specific receptors (VLA-5). We showed that the binding of NB4 cells to fibronectin was RGD-dependent. PMA-induced adhesion was not correlated to phosphorylation of the VLA-5 receptor. These findings may partially explain the malignant behaviour of these cells: The loss of their capacity to adhere to stromal cells may arrest differentiation and explain the large number of leukemic cells in the circulation.

We investigated the capacity of 3 major cytokines secreted by activated monocytes, IL-1 beta, TNF alpha and IL-6, to inhibit growth of melanoma tumor cells. Using neutralizing antibodies against IL-1 beta, TNF alpha and IL-6, we observed that the cytostatic activity against A375 melanoma cells is largely due to the presence of IL-6 in culture supernatants of monocytes stimulated with LPS. A375 cells appeared to be extremely sensitive to monocyte-derived cytokines, since in addition to rIL-6 also rIL-1 beta and rTNF alpha displayed cytostatic activity against A375 cells. We observed additive or synergistic cytostatic effects upon use of combinations of these cytokines. When 7 other melanoma cell lines and short-term melanoma cultures were tested and compared with A375, a major difference in their sensitivity to monocyte-derived cytokines were observed. Although 7 out of 8 melanoma cell lines were sensitive to culture supernatants of monocytes stimulated with LPS, significant differences were found when recombinant cytokines were used. The widely used A375 was the only melanoma cell line sensitive to rIL-1 beta, rTNF alpha and rIL-6. The growth of none of the other 7 melanoma cell cultures was significantly affected by rIL-1 beta. Seven out of 8 melanoma cell cultures were sensitive to rTNF alpha and 3 out of 8 to rIL-6. The results of our study indicate that the sensitivity of melanoma cell cultures for different monocyte-derived cytokines is highly variable, and that it is questionable whether the A375 melanoma cell line, sensitive to rIL-1 beta, rTNF alpha and rIL-6, is representative for melanoma.

Recent data provide solid experimental evidence that increase in cell size of human lymphocytes, caused by osmotic stress, is not directly proportional to forward scattering intensity but rather is inversely proportional. Here, we provide arguments that this phenomenon can be quantitatively explained by means of a model based on a modified Rayleigh-Debye-Gans theory. As a consequence, the current view that forward scattering intensities can be used as a measure of gross cell size needs to be reconsidered. In addition, we postulate that structural changes of the cytoplasm, imposed by an osmotic stress, reflect corresponding changes in the nucleus.

Spectral decomposition of flow cytometric datafiles of arbitrary dimension reveal information of both the signal and the noise components that constitute the histograms. This spectral information is used to construct a low-pass digital filter, which removes the high-frequency noise from the actual data. It is shown that this procedure guarantees non-trivial smoothing of the flow cytometric data in accordance with the local experimental situation. As a consequence optimal reconstruction of the signal is possible, which facilitates unambiguous interpretation of the data files and mathematical estimation of the statistical parameters.

A new fast trivariate display technique based on the ratio of three measured variables and of relative cell number, obtained from flow cytometric measurements, is described.

The functional properties of the melanoma-associated antigens detected by monoclonal antibodies (MAbs) AMF-6 and AMF-7 were investigated. These MAbs were selected previously because of their capacity to block the anti-melanoma reactivity of cytotoxic T-lymphocyte clones AMF-6 and AMF-7 detect a melanoma-associated proteoglycan (MW greater than 450-250 kDa) and a molecular complex, which under reducing conditions consists of 4 compounds of 120, 95, 29 and 25 kDa respectively. AMF-6 reacted strongly with all 30 cultured melanomas and all 41 melanomas in frozen tissue sections. Significant cross-reactivity was only observed with nevi and perineurium, whereas normal skin melanocytes were negative. AMF-7 reacted with all 25 cultured melanomas and all 34 melanomas in frozen sections. AMF-7 cross-reacted with a proportion of nevi and endothelial cells from small vessels. The antigen detected by AMF-6 and AMF-7 could not be modulated by retinoic acid or recombinant gamma-IFN, which induced or enhanced the expression of HLA-DR, HLA-DQ and Class-I MHC antigens. In addition, the antigens were not readily modulated when cells were incubated in excess amounts of AMF-6 and AMF-7. Interestingly, the antigen detected by AMF-7 was strongly associated with the adhesion and cytoplasmic spreading of melanoma cells to plastic surfaces and monolayers of vascular endothelial cells. AMF-6 did not block the adhesion of melanoma cells but delayed cytoplasmic spreading. Both AMF-6 and AMF-7 blocked fibronectin-induced chemotaxic motility and chemokinesis of melanoma cells. In addition to their membrane localization, the antigens detected by AMF-6 and AMF-7 were also abundant in extracellular adhesion plaques deposited by cultured melanoma cells. Our results indicate that the high-MW melanoma-associated proteoglycan and the antigen detected by AMF-7 are associated with adhesion and/or cytoplasmic spreading and motility of human melanoma cells, suggesting that these antigens are associated with the (hematogenic) dissemination of human melanoma. This is supported by the finding that AMF-7 stained primary tumors heterogeneously, whereas metastases were homogeneously stained.