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.

Human c-fms proto-oncogene: comparative analysis with an abnormal allele.

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.

Identification of melanoma inhibitory activity and other differentially expressed messenger RNAs in human melanoma cell lines with different metastatic capacity by messenger RNA differential display.

The differential display technique was used to identify mRNAs differentially expressed in human melanoma cell lines with different metastatic capacity. We report the isolation of nine different clones, of which four were uniquely expressed in the highly metastatic human melanoma cell line MV3, whereas the other five clones were uniquely expressed in the poorly metastatic human melanoma cell line 530. The differences in expression identified by differential mRNA display were confirmed by Northern blot analyses. DNA sequencing followed by computer search analyses indicated that of the nine differentially expressed clones, five represented novel gene products. The other four were histocompatibility antigen HLA-DR, laminin B2, melanoma inhibitory activity (MIA), and tissue inhibitor of metalloproteinases 3. MIA was also identified in RNA from human melanoma metastasis lesions in a comparison by differential display with pooled human nevi. Northern blot analysis confirmed MIA mRNA expression in nonmetastasizing melanoma cell lines and in melanoma metastasis lesions, while expression was absent in highly metastasizing cell lines and pretumor stages. In the 11 metastasis lesions examined, MIA mRNA expression was apparently inversely correlated with pigmentation.

Expression of calcyclin in human melanocytic lesions.

When comparing two subsequent stages of melanocytic tumor progression we identified calcyclin as a new potential progression marker, the expression of which was correlated with metastatic behavior of various human melanoma cell lines in nude mice. In this study, we describe a good correlation between RNA and protein levels in the xenografts of these cell lines and extended these experiments to a panel of 120 routinely processed human melanocytic cutaneous lesions. Northern blot analysis demonstrated that calcyclin RNA expression was elevated in melanoma metastases as compared to several types of nevocellular nevi. Calcyclin staining using a specific polyclonal antiserum showed a more complex pattern. A stronger staining in a higher percentage of positive cells was observed in thick primary melanoma (> or = 1.5 mm) as compared to thin primary melanoma (< 1.5 mm). Calcyclin expression was also present in a higher percentage of cells showing a stronger staining in melanomas with higher Clark levels (> II) corresponding to the vertical growth phase of primary melanomas. Protein expression in nevocellular nevi was confined to the dermal part and was highest in the lower parts of the dermis. Remarkably, dysplastic nevi (atypical moles), potential precursors of melanoma, did not show any expression at all, either in junctional or dermal parts. Confinement of the expression to the dermal part of nondysplastic nevi and primary melanomas may reflect interactions with the microenvironment of the reticular dermis that occurs with vertical growth.

Expression of calcyclin in human melanoma cell lines correlates with metastatic behavior in nude mice.

Since our aim was to isolate and identify new progression markers of human cutaneous melanoma, we applied the differential hybridization technique, in which we compared the gene expression in two subsequent stages of this progression. Tumors in nude mice arising after transplantation and serial passage in vivo of either the horizontally and early vertically growing part or the advanced vertically growing part of a primary melanoma of the same patient were used for this assay. This resulted in the isolation of a number of complementary DNA clones that were differentially expressed. Based on the marked difference in expression, one of them, designated pMW1, was chosen for further characterization and appeared to be coding for calcyclin, a cell cycle-regulated protein, belonging to a family of small calcium-binding proteins. Calcyclin expression was elevated in high-metastatic human melanoma cell lines in nude mice compared to low-metastatic ones. Immunoprecipitation of calcyclin showed that the differential expression at the RNA level is also reflected at the protein level. These findings show that expression of calcyclin is related to metastasis of human melanoma cell lines in nude mice and emphasize the role of this family of calcium-binding proteins in neoplastic progression as was reported for the mouse homologue of calcyclin and other members of the same family.

Rat desmin gene structure and expression.

The isolation of two partial genomic clones and a near full length cDNA clone encoding the rat intermediate filament protein desmin is reported. Desmin is a differentiation marker for all types of muscle cells. The nucleotide order of the coding region and 0.1 kb of 5'-flanking sequences of the rat desmin gene has been determined. One genomic clone encompasses exons I-III and approx. 12 kb of 5'-flanking sequences, while the other clone contains exons VII-IX and about 12 kb of 3'-flanking sequences. Northern analysis of RNA from different organs reveals that, as expected, desmin is expressed in striated, heart and smooth muscle cells containing tissues; among other tissues, lung displays relatively high expression levels, while desmin mRNA is barely detectable in spleen, kidney and liver. S1 mapping reveals that the same transcription initiation site is used in all desmin mRNA containing tissues.

Expression of basic fibroblast growth factor and fibroblast growth factor receptor genes in cultured rat aortic smooth muscle cells.

Basic fibroblast growth factor (bFGF) exerts a differential effect on DNA synthesis, bFGF mRNA synthesis, and expression of FGF-receptor genes by cultured smooth muscle cells from aortae of newborn and adult rats (used as a model in atherosclerosis research). Cells from adult animals, are more sensitive to bFGF, and bFGF triggers its own mRNA synthesis. Moreover, the level of the transcript of the FGFR-1 gene (coding for the most abundant FGF-receptor in smooth muscle cells) is higher in smooth muscle cells from adult rats. In contrast, the FGFR-3 gene only is expressed in smooth muscle cells from newborn rats. Crosslinking of [125I]bFGF to its receptor showed 130 kDa and 160 kDa complexes both in newborn and adult smooth muscle cells.

memA/DRS, a putative mediator of multiprotein complexes, is overexpressed in the metastasizing human melanoma cell lines BLM and MV3.

memA was isolated by subtractive hybridization in which the mRNA repertoire was compared in a panel of human melanoma cell lines with different metastasizing potential. Expression of memA mRNA is elevated in the highly metastasizing human melanoma cell lines and derived xenografts, as compared with the non-metastasizing ones. In a collection of human tumor cell lines and melanoma metastasis lesions, memA mRNA expression could be detected in the A-431 (epidermoid carcinoma), HT-1080 (fibrosarcoma), JEG-3 and JAR (choriocarcinomas) cell lines and in three out of 11 melanoma metastasis lesions. The distribution of memA mRNA in a collection of healthy human organs is also tissue restricted. Sequence analysis revealed that the MEMA protein is identical with a 160 kDa nuclear 'domain rich in serines' (DRS) protein occurring free in the nucleoplasm and in U2-ribonucleoprotein structures. MEMA is also homologous to pinin, a 140 kDa protein associated with the desmosome-intermediate filament complex, and to a 32 kDa porcine neutrophilic protein that was copurified with components of the NADPH-oxidase enzyme complex. The encoded amino acid sequence predicts that the MEMA protein has three coiled-coil domains, one glycine loop domain, is very hydrophilic and contains regions rich in glutamine/proline, glutamic acid and serine residues.

Characterization of the feline c-abl proto-oncogene.

Analysis of total feline DNA by genomic blot hybridization, using the viral oncogene of Abelson murine leukemia virus as a specific probe, has led to the identification of multiple v-abl homologous genetic sequences in the cat genome. Upon restriction endonuclease BamHI digestion, the combined size of the v-abl homologous DNA fragments was about 31 kbp. To characterize these sequences further, four independent v-abl homologous cosmid clones with overlapping cellular inserts have been isolated from a gene library of cat lung genomic DNA. These inserts represent a contiguous region of cellular DNA sequences of 56 kbp in length. Within this region of the feline genome, the v-abl homologous sequences are discontinuously dispersed over a region of about 34 kbp. They represent the complete feline v-abl cellular homolog and are colinear with the viral v-abl oncogene. Nine regions of highly repetitive DNA sequences have been mapped in close proximity to v-abl homologous sequences. These results establish the presence of only a single c-abl proto-oncogene in the cat genome and present its genetic organization.

Identification of regulatory sequences in the promoter of the PDGF B-chain gene in malignant mesothelioma cell lines.

Platelet-derived growth factor (PDGF) B-chain mRNA is readily detectable in malignant mesothelioma (MM) cell lines, but not in normal mesothelial (NM) cell lines. The high affinity receptor for PDGF B-chain dimers, the PDGF beta-receptor, is expressed in MM cell lines. NM cell lines predominantly express the PDGF alpha-receptor. Coexpression of the PDGF beta-receptor and its ligand may lead to an autocrine growth stimulating loop in the malignant cell type. In nuclear run off experiments, PDGF B-chain mRNA was detectable in MM cells only, indicating an increased level of transcription in this cell type. The proximal promoter of the PDGF B-chain gene contains DNaseI hypersensitive (DH) sites and mediates reporter gene activation in both normal and malignant cells. Nuclear proteins, extracted from both cell types, interact with DNA sequences within the proximal promoter around bp-64 to -61 relative to the transcription start site. Electrophoretic mobility shift assays (EMSAs) indicate that these factors are more abundantly present in the malignant than in the normal cell type. A DH site around -9.9 kb was found in both cell types. When tested in CAT assays, this region exerted a stimulatory effect on transcription in malignant cells. The elevated level of transcription of the PDGF B-chain gene in malignant cells may well be the result of interaction of regulatory sites in the proximal promoter and an enhancing element located at -9.9 kb from the transcription start site.

Comparative analysis of the human and feline c-sis proto-oncogenes. Identification of 5' human c-sis coding sequences that are not homologous to the transforming gene of simian sarcoma virus.

Feline and human genetic sequences, homologous to the v-sis gene of simian sarcoma virus, have been isolated from cosmid gene libraries and characterized by restriction endonuclease analysis. Comparison of the two loci revealed their related structural organization. In both loci, similar unique genetic sequences were found upstream of the v-sis homologous region and these hybridized to a 4.2 kbp c-sis transcript in human lung tumor cells. These data establish and map as yet unidentified coding sequences at the 5' part of the c-sis proto-oncogene of both species.

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene.

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.

nmd, a novel gene differentially expressed in human melanoma cell lines, encodes a new atypical member of the enzyme family of lipases.

nmd, a novel gene, was isolated by applying the differential mRNA display method to human melanoma cell lines with different metastatic capacity. In a panel of 17 other human tumor cell lines, nmd RNA expression could only be detected at low levels in T24 (bladder carcinoma) and Caco-2 (colon adenocarcinoma). Furthermore, it was found in placenta and liver, but not in skin, colon, spleen, lung, muscle, prostate and kidney. Sequence analysis classified the nmd gene product as a new member of the enzyme family of lipases (almost 30% identity in amino acid sequence with other human lipases). Active site residues of lipases were conserved in NMD, but NMD lacks the regulatory lid domain, which controls entry to the active site in classical lipases. A similar deletion was earlier reported by others in the guinea pig pancreatic (phospho)lipase GPLRP2 and the phospholipase A1 from hornet venom (DolmI).

Effect of ploidy on transcription levels in cultured rat aortic smooth muscle cells.

Aging and hypertension increase the number of polyploid smooth muscle cells (SMC) in a blood vessel. We assessed the effect of ploidy on the transcription of several genes in SMC cultures derived from newborn and adult rats. In diploid and tetraploid subcultures of SMC from newborn rats, RNA expression of the genes assayed is linked with ploidy. However, when phenotypically different SMC cultures derived from newborn and adult rats were compared, transcription levels varied from gene to gene and not linked with the ploidy. Thus, differences in gene expression due to polyploidy are superimposed on those due to other phenotypical features.

Affinity of monoclonal antibodies. Interpretation of the positive cooperative nature of anti-hCG/hCG interactions.

The interaction of some individual MAbs and human chorionic gonadotrophin (hCG) showed apparent positive cooperativity as observed by equilibrium binding studies. This form of cooperative interaction has now been further characterized. The main results were: (1) the apparent positive cooperativity was strongly dependent upon concentration and temperature; (2) the cooperativity was strongly reduced by using peptic F(ab')2 fragments of IgG and became undetectable when the MAb was replaced by the corresponding Fab fragment; (3) the molecular weight of the complex changed from 226 kDa to 450 kDa upon increasing the hCG/MAb ratio. From these and additional results it is hypothesized that the apparent positive cooperativity results from self (Fc-Fc) associations mediated or facilitated by prior antigen binding.

The influence of binding capacity and affinity on the improved performance of N-terminally extended hCG peptides, determined by ELISA-based procedures.

The improvement of peptide-ELISA responses by the use of small synthetic peptides elongated at the N-terminus with an Ata-group or a (Lys)7 extension has been analyzed. For this purpose, binding capacity and affinity were evaluated by specific ELISA procedures. The ELISA experiments on binding capacity, performed with saturating antibody concentrations, revealed a difference of more than three orders of magnitude in binding capacity between the parent peptides and the N-terminally linked peptides, in favor of the latter peptides. Antibody affinity values were determined by a liquid-phase equilibrium method as well as by a solid-phase equilibrium method. N-terminal extension of the peptides had almost no effect on the affinity when equilibrium between the peptide and the antibody was reached in solution. In contrast, solid-phase affinity was greatly enhanced when the N-terminally linked peptides were adsorbed to the polystyrene surface. This enhancement was determined by the N-terminal extension and the peptide amino acid sequence (40 to 600 times higher). Thus, the use of N-terminally extended peptides can greatly increase the performance of a peptide-ELISA through improved surface effects, resulting in higher binding capacity and functional affinity.

Adsorption studies of tritium-labeled peptides on polystyrene surfaces.

In this study, three presentation formats of an epitope peptide (hepta-peptide), derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear (Lys)7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3H-labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mg/m2 when 1.0 mmol/l was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mg/m2 (at 1.5 mmol/l). The lysyl (K7) peptide showed a maximum plateau value of 0.2 mg/m2, and therefore the lysyl (K7) extension reduced the peptide surface coverage at relatively high coat concentrations (above 0.1 mmol/l) compared to the parent peptide. At lower input concentrations (below 0.1 micromol/l), however, the packing density of the lysyl (K7) peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and (functional) affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl (K7) extension.

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.

Reversion to the nontransformed phenotype of Abelson murine leukemia virus-transformed cells and their subsequent retransformation.

Two independent clones of fetal mink lung cells (CCL64) nonproductively transformed by Abelson murine leukemia virus (Ab-MuLV) were used to study spontaneous reversion to the nontransformed phenotype and subsequent retransformation of the revertants. One clone, D62, contained two complete Ab-MuLV proviruses and expressed polyprotein P120. The other clone, K49, contained four proviruses: three of them were complete and one represented a deletion mutant. In addition to P120, a new polyprotein, P60, was expressed in this clone. During the processes of reversion and retransformation proviral DNAs were conserved with respect to size and integration site. In contrast to the transformants, expression of Ab-MuLV P120, and in case of clone K49 also of P60, was blocked in revertant lines as a result of loss of transcription of proviral DNA. In retransformants, expression of Ab-MuLV P120 was found in both clones. However, no expression of P60 was detectable in retransformants of K49-derived revertants. Reversion to the nontransformed phenotype was associated with increased cytosine methylation in proviral DNA sequences, whereas in spontaneous retransformants methylation tended to resume control levels. These findings demonstrate regulation of viral oncogene mediated transformation by cytosine methylation and suggest that transcription of proviral DNA is under both viral and cellular control. They furthermore suggest that processes involved in regulation of proviral expression do not affect all such proviruses simultaneously in the same way.

Structural analysis of a variant clone of Snyder-Theilen feline sarcoma virus.

A variant clone of Snyder-Theilen feline sarcoma virus (ST-FeSV) encoding a polyprotein with a molecular weight of approximately 104 kDa (P104) was compared to the P85 encoding prototype clone of ST-FeSV. Analysis of chimeric genes constructed with the viral oncogenes of the two clones indicated that the variant clone coded for a larger polyprotein than the prototype clone because of genetic differences in its 3' portion. Comparative DNA sequence analysis revealed that one nucleotide just upstream of the termination condon TGA in the prototype proviral DNA was deleted from the variant clone resulting in a 468-bp larger open reading frame. Furthermore, it appeared that the U3 regions of the long terminal repeats (LTRs) of the variant clone contained an insertion of 71 bp as compared to the LTRs of the prototype clone. In addition, both clones differed also from each other with respect to genetic sequences deleted from their env gene regions.