Henri P.J. Bloemers

Activated Leukocyte Cell Adhesion Molecule/CD166, a marker of tumor progression in primary malignant melanoma of the skin

Expression of activated leukocyte cell adhesion molecule (ALCAM)/CD166 correlates with the aggregation and metastatic capacity of human melanoma cell lines (Am J Pathol 1998, 152:805-813). Immunohistochemistry on a series of human melanocytic lesions reveals that ALCAM expression correlates with melanoma progression. Most nevi (34/38) and all thin melanomas studied (Clark levels I and II) did not express ALCAM. In contrast, immunoreactivity was detected in the invasive, vertical growth phase of 2 of the 13 Clark level III lesions tested. The fraction of positive lesions further increased in Clark level IV (13/19) and in Clark level V (4/4) lesions. ALCAM expression was exclusively detectable in the vertical growth phase of the primary tumor. In melanoma metastases, approximately half of the lesions tested (13/28) were ALCAM positive. According to the Breslow-thickness, ALCAM expression was observed in less than 10% of the lesions that were thinner than 1.5 mm and in over 70% of the lesions that were thicker than 1.5 mm. Our results strongly suggest that ALCAM plays an important role in melanocytic tumor progression and depict it as a new molecular marker for neoplastic progression of primary human melanoma.

Evolutionary conserved close linkage of the c-fes/fps proto-oncogene and genetic sequences encoding a receptor-like protein.

Recently we described that genetic sequences in the immediately upstream region of the c‐fes/fps proto‐oncogene, designated fur, constituted a transcription unit for a 4.5‐kb mRNA. Here we present characteristics of the genetic organization of fur and some features of its putative translation product which we call furin. The nucleotide sequence of a 3.1‐kbp fur‐specific cDNA isolated from a human cDNA library revealed an open reading frame of 1,498 bp from which the 499 carboxy‐terminal amino acids of the primary fur translational product could be deduced. Computer analysis indicated that furin contained a possible transmembrane domain which resembled that of class II MHC antigens. Furthermore, a cysteine‐rich region was present. Significant homology, especially with respect to the topography of cysteine residues, was found between the cysteine‐rich regions of the human insulin receptor, the human epidermal growth factor receptor and furin. From the data presented here we deduce that fur may encode a membrane‐associated protein with a recognition function.

fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas.

The recently discovered fur gene encodes a membrane-associated protein with a recognition function. To further characterize the gene, we studied its expression by Northern blot analysis using poly(A)-selected RNA from a variety of organs of African green monkey and rat. The fur gene appeared to be differentially expressed, relatively high levels of fur mRNA being present in specimens of liver and kidney, low levels in brain, spleen, and thymus, and very low levels in heart muscle, lung, and testis. mRNA levels in specimens of human lung tissue without neoplastic lesions were also very low. Similar analyses of primary human lung carcinomas of different histopathological types revealed a highly selective and strong elevation of fur expression in nonsmall cell lung carcinomas, but not in small cell lung carcinomas. These results indicate that fur expression can be used to discriminate between these two types of human lung cancer.

Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus

Abstract Virus-specific protein synthesis in JLS-V9 cells infected with Rauscher leukemia virus (R-MuLV) was studied by an immunoprecipitation technique. One low molecular weight virion protein (p15) could be detected intracellularly and two nonvirion polypeptides with molecular weights of 82,000 and 65,000, respectively. These high molecular weight polypeptides were converted into the virion proteins p30, p15, and p12b, as shown by incubation of the cell lysate. The results suggest that the conversion took place on membrane structures; it was not inhibited by three different protease inhibitors. However, growth of cells in the presence of the arginine analog canavanine prevented formation of the 65,000-dalton polypeptide and the p15 virion polypeptide. From our observations it is concluded that the 82,000- and 65,000-dalton polypeptides are precursors of virion proteins.

Expression of nma, a novel gene, inversely correlates with the metastatic potential of human melanoma cell lines and xenografts.

nma, a novel gene, was isolated by using a subtractive hybridization technique in which the gene expression was compared in a panel of human melanoma cell lines with different metastatic potential. nma mRNA expression (1.5 kb) is high in poorly metastatic human melanoma cell lines and xenografts and completely absent in highly metastatic human melanoma cell lines. Fluorescence in situ hybridization combined with the analysis of a panel of human‐rodent somatic cell hybrids indicated that the nma gene is located on human chromosome 10, in the region p11.2–p12.3. Sequence analysis of nma showed no homologies with other known genes or proteins, except for several partially sequenced cDNAs. The predicted amino acid sequence suggests that the protein encoded by nma contains a transmembrane domain. Expression of nma is high in human kidney medulla, placenta and spleen, low in kidney cortex, liver, prostate and gut and absent in lung and muscle. Whereas nma is not expressed in normal skin tissue, expression is high in melanocytes and in 3 out of 11 melanoma metastases tested.

Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: Synthesis, identification, and processing

Abstract The synthesis of virus-specific polypeptides in JLS-V9 and JLS-V5 cells infected with Rauscher leukemia virus (R-MuLV) was studied in pulse-chase experiments, followed by radioimmunoprecipitation analysis with polyvalent and monospecific antisera against R-MuLV proteins. Two glycosylated polypeptides with molecular weights of about 8 ( env -pr82) were identified as precursors of the virion envelope polypeptides gp69/71 and p15(E). On the other hand, virion polypeptides p30 and pl5 are derived from a 75,000-( gag -pr75) and a 65,000-dalton ( gag -pr65) precursor polypeptide. These precursor-product relations were confirmed by analysis of chymotryptic digests of virion polypeptides and their precursors. In the presence of the arginine analog canavanine two polypeptides with molecular weights of 82,000 and 72,000 ( gag -pr82 and gag -pr72, respectively) were synthesized instead of gag -pr75 and gag -pr65. Processing of precursor polypeptides is reduced in the presence of canavanine. From these results, we conclude that gag -pr82 is possibly the primary gag -gene product and is cleaved into gag -pr75. These studies provided the following additional information: First, we established that immediately after cleavage of their precursors, gp69/71 is found on the outer surface of the cell and p30, p15, and p12 leave the cell as components of budding virions. Therefore, these polypeptides were detected intracellularly in very small amounts only. Polypeptide p15(E) was present within the cell as well as on its outer surface. Second, despite a great similarity in virus-specific (precursor) polypeptides detected in JLS-V9 and JLS-V5 cells, small differences in molecular weights of some of these polypeptides were observed after SDS-PAGE.

Characterization of human c-fes/fps reveals a new transcription unit (fur) in the immediately upstream region of the proto-oncogene

Comparison of nucleotide sequence data of the 5′ region of a fes/fps viral oncogene with those of the v-fes/fps homologous regions of man and cat revealed the position of the 3′ portion of an as yet unidentified c-fes/fps exon. Comparative Southern blot and heteroduplex analysis of human and feline DNA immediately upstream of the v-fes/fps homologous regions showed extensive but discontinuous homology over a 9 kbp DNA stretch, which we have designated as fur. Northern blot analysis of mRNA from KG-1 myeloid cells with fes/fps-or fur-specific probes revealed a 3.0 kb fes/fps and a 4.5 kb fur transcript. Analysis of a number of tissues of an adult Wistar Lewis rat for the presence of fur transcripts revealed its differential expression pattern. An 0.95 kbp fes/fps-related and a 2.2 kbp fur-related cDNA recombinant clone were isolated from an oligo(dT)-primed KG-1 cDNA library. Comparative nucleotide sequence analysis of the fes/fps cDNA and its human genomic counterpart indicated that the cDNA contained genetic sequences that were identical to and colinear with exon 15–19 and , furthermore, that the poly(A) addition signal near the 3′ end of exon 19 was functional. Similar analysis of the 2.2 kbp fur cDNA indicated that the poly(A) addition signal of the fur transcript was in close proximity of the newly discovered fes/fps exon. The region in between contained a CATT sequence but no ‘TATA’ box. The fur transcript was characterized by a long noncoding region at its 3′ end.

The structure of the human c-fes/fps proto-oncogene.

We have determined the complete nucleotide sequence of a human DNA fragment of approximately 13 kbp, which was shown by Southern blot analysis to contain the entire v‐fes/fps cellular homolog. The v‐fes/fps homologous sequences were dispersed over 11 kbp in 18 interspersed segments which were flanked by splice junctions. Fusion of these segments created a DNA fragment in which coding regions similar to those observed in the viral oncogenes v‐fes of the Gardner‐Arnstein (GA) and Snyder‐Theilen (ST) strains of feline sarcoma virus and v‐fps found in Fujinami sarcoma virus could be identified. A potential initiation site in the first exon was found. About 200 nucleotides downstream of a translational stop codon in the v‐fes/fps homologous region, a poly(A) addition signal was identified. The deduced amino acid sequence has a molecular weight of 93 390 dalton resembling NCP92, the recently described human c‐fes/fps product. The topography of human c‐fes/fps appeared to resemble that of chicken c‐fps.

Assessment of the Functional Affinity Constant of Monoclonal-Antibodies Using an Improved Enzyme-Linked-Immunosorbent-Assay

The use of an enzyme-linked immunosorbent assay (ELISA) for the determination of affinity constants implies heterogeneous measurements. Therefore, despite their simplicity, direct solid-phase binding assays are not common. Many investigators have serious, and mostly justified, reservations about the application of solid-phase affinity methods. They refer to problems such as diffusion effects and difficulties in reaching equilibrium due to heterogeneous binding and co-operativity. Accordingly, functional affinity determinations are often described as meaningless. These objections apply to the measurement of the affinity of a monoclonal antibody using the enzyme-linked immunosorbent assay of Beatty et al. (J. Immunol. Methods (1987) 100, 173), which is based on the effect of antibody affinity on the sigmoidal dose response curve. The affinity constant is calculated by mathematical equations, based on the Law of Mass Action and the authors made a number of important assumptions--avoiding the above mentioned problems--in order to justify the use of the Law of Mass Action. By carefully examining these assumptions we have developed an improved ELISA procedure for functional affinity determinations on the basis of a primary coating with the antigen only. the coating conditions were validated by employing gold labelled colloidal particles and physical counting of the bound particles under the scanning electron microscope. Since monovalent binding between human chorionic gonadotropin and its monoclonal antibody could be achieved under equilibrium conditions, the application of the Law of Mass Action and hence of the Beatty formula became possible. We conclude that under these conditions functional affinity determinations are appropriate.

Hydroxylamine cleavage of proteins in polyacrylamide gels

A modification of the hydroxylamine cleavage of proteins is presented in which proteins were cleaved while immobilized in the matrix of a polyacrylamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier, facilitates simultaneous treatment of many samples, and contributes to a high recovery efficiency (60-90%) of the cleavage products. The cleavage is performed with individual protein bands excised from dried slab gels after detection by staining, autoradiography, or fluorography. The procedure can be easily combined with other techniques to further characterize the cleavage fragments. Also a two-dimensional version of the cleavage method was developed, which allows rapid recognition of interrelationships between proteins in a complicated mixture. The versatility of the procedure is demonstrated in a number of applications. Highly related strains of murine leukemia viruses were easily distinguished from one another by the unique cleavage patterns of their gag- and env-precursor polypeptides. Comparing the env-precursor gPr82env synthesized in the presence or absence of tunicamycin with its cell-free synthesized counterpart, revealed the presence of an amino-terminal signal sequence. Cleavage patterns of pro-opiomelanocortin (POMC) from three different species revealed a high degree of homology between rat and mouse POMC, whereas Xenopus POMC was very different. Regions to which carbohydrates are attached could be identified by comparing glycosylated and unglycosylated forms of POMC. Combining the hydroxylamine cleavage procedure with immunological characterization of the fragments showed a small but significant difference between the amino-terminal sequences of the recombinant transforming protein P120 of Abelson murine leukemia virus and of its parent molecule Pr65gag of Moloney murine leukemia virus.