Oligosaccharide Le(a)-Le(x): a cell surface marker for carcinomas derived from embryonic endoderm.

An immunohistochemical evaluation of Le(a)-Le(x) expression by adenocarcinomas of the biliary tree, pancreas, colon and stomach was undertaken as examples of epithelial tumors derived from embryonic endoderm. This complements previous studies showing that Lea-Lex was present on the cell surface of non-small cell lung carcinomas, some non-lung carcinomas, and is a prognostic marker for squamous cell lung carcinomas. All of the tumor specimen evaluated were positive and no expression of Le(a)-Le(x) was detected in derivatives of neural, connective or muscle tissues. These findings indicate that it could be informative to examine the biological significance of Le(a)-Le(x) not only in carcinogenesis but during embryogenesis, as well.

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins.

Intratumoral phenotypic diversity is well documented with regard to tumor associated carbohydrate antigens (TACA). The factors which control the expression of these cell-surface oligosaccharides on different cells of the same tumor are not understood. We investigated the expression of a panel of mucin associated oligosaccharides in cell lines growing at different surface densities (number of cells per cm2 of growth flask). Results show that the apparent expression of extended Lea-Lex, Lea and Lex, sialyl Lea, Tn and sialyl Tn varies with density of growth by an invasive human squamous cell lung carcinoma cell line (NU6-1), a benign variant (NE-18) and the human lung epithelial cell line BEAS-2B. The results indicate that one of the factors influencing the apparent expression of mucin-associated oligosaccharides is cell-cell interactions.

Theory of the mobility-shift assay of nonspecific protein-DNA complexes governed by conditional probabilities: the HU:DNA complex.

Complexes of an 88 bp DNA and the HU protein were studied by both experimental and theoretical electrophoretic mobility-shift analyses. Experimental analysis defined the stoichiometry of binding and estimated an apparent intrinsic dissociation constant (Kd = 1 to 3 x 10(-7) M) for the HU:DNA complexes. The theory of conditional probabilities was applied to the binding of HU to DNA in order to fix the initial equilibrium composition of mixtures to be assayed theoretically by the mobility-shift procedure. Electrophoretic mobility-shift patterns were obtained by numerical solution of a set of simultaneous transport-reaction equations, in which the chemical kinetic term is formulated in terms of dissociation of the different DNA:HU complexes in gel cages. The computed patterns simulated the experimental patterns describing the titration of a fixed concentration of an 88 bp DNA fragment with dimeric HU. These insightful results provide guidelines for interpretation of the electrophoretic behavior of systems in which a ligand binds nonspecifically to DNA. In particular, the narrow unresolved zone observed both experimentally and theoretically beyond 50-60% saturation is a reaction zone characteristic of noncooperative ligand-binding governed by conditional probabilities. The discrepancy between the theoretically assigned and experimental values of the intrinsic binding constant is attributed to an HU-induced change in the conformation of DNA.

Increased DNA-bending activity and higher affinity DNA binding of high mobility group protein HMG-1 prepared without acids.

Recently, DNA ring closure assays showed that high mobility group protein HMG-1 and its close homolog HMG-2 mediate sequence-independent DNA flexion. This DNA-bending activity appears to be central to at least some of the recently elucidated functions of HMG-1/2, such as the enhancement of progesterone receptor DNA binding. Here we show that standard purification procedures utilizing perchloric and trichloroacetic acid can produce HMG-1 significantly deficient in its abilities to bind and bend double-stranded DNA, while acid-independent methods purify HMG-1 that is superior in these respects. Significant losses of DNA ring closure activity were seen upon limited 2-5-h exposures of nonacid-purified HMG-1/2 to perchloric acid and/or trichloroacetic acid. Measurements of the apparent DNA dissociation binding constant (Kd(app)) of acid-extracted preparations of HMG-1 gave a wide range of values, and only those preparations demonstrating little DNA ring closure activity had Kd values near the previously published value (approximately 10(-6) M). The highest ring closure activities and lowest Kd(app) (< 3 x 10(-9) M) were obtained for HMG-1 purified without acids. These combined results support the use of alternative, non-acid purification procedures for preserving the DNA-bending activity of HMG-1/2 and suggest that past procedures utilizing acids have led to an underestimation of the affinity of HMG-1 for DNA.

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.

Tumor-associated antigen 43-9F is of prognostic value in squamous cell carcinoma of the lung. A retrospective immunohistochemical study.

BACKGROUND: Squamous cell lung carcinoma (SLC), the most frequent type of lung cancer, generally is treated surgically and its prognosis is poor. The only current clinically useful prognostic criterion is lymph node staging (TNM classification). Expression of a novel tumor-associated carbohydrate epitope Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3 Gal beta 1-4Glc identified by the 43-9F monoclonal antibody (MoAb) is associated with the growth pattern of SLC cell lines in athymic mice and in vitro. This implies that the 43-9F epitope may be related to tumor progression in patients with SLC and that, as such, it could be of prognostic value. METHODS: Primary tumor specimens from 231 patients with lung carcinoma (130 with SLC, 64 with adenocarcinoma, 10 with small cell carcinoma, 16 with large cell carcinoma, and 11 with adenosquamous carcinoma) were examined by immunohistochemical studies on formalin-fixed, paraffin-embedded tissue samples for immunoreactivity with an MoAb to the 43-9F antigen. Univariate and step-wise Cox regression analyses were used to compare survival time by histopathologic diagnosis, smoker status, TNM classification, and type of surgical treatment. RESULTS AND CONCLUSIONS: Patients with 43-9F epitope-positive SLC tumors had a significantly (P less than 0.01) better prognosis than patients with epitope-negative tumors. In contrast, no association was seen between 43-9F epitope expression and survival time for patients with lung adenocarcinomas. Further, the prognostic value of 43-9F expression in SLC was found to be superior to the N-classification with the added advantage that it requires access only to primary tumor tissue and thus is available before therapy.

Mucin gel formed by tumorigenic squamous lung carcinoma cells has Le(a)-X oligosaccharides and excludes antibodies from underlying cells.

Cells of cloned lines of human squamous lung carcinomas elaborate large glycoproteins that are associated with their tumorigenic potential. Two groups of clones (called Le(a)-X-positive and Le(a)-X-negative) were studied that either do or do not express the Le(a)-X oligosaccharide associated with large glycoproteins and mucins secreted by these clones. Le(a)-X-positive cells elaborate a mucin gel complex associated with their apical surfaces, which appears as a mosaic of extracellular plates. Clones of this type are tumorigenic in nude rodents when injected s.c. or when introduced into the lungs via intrabronchial aerosol. By contrast, the Le(a)-X-negative clones do not form extracellular plates and are not tumorigenic in the lungs or subcutaneously. We demonstrate that the extracellular plates of Le(a)-X-positive cells exclude antibodies from interacting with the underlying squamous lung carcinoma cells and may therefore exert an immunoprotective effect. In support of this possibility it was found that: (a) There is a substantial inflammatory cell infiltrate associated with regressing nodules of Le(a)-X-negative cells in nude rodent lung and subcutaneous nodules, while there is no observable infiltration associated with progressing Le(a)-X-positive tumors. (b) In the brain (an immunoprivileged site) tumors develop and progress when either Le(a)-X-negative or -positive cells are introduced.

Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli.

Growing bacterial cells forming division septa have sites near the septa that are sensitive to EDTA shock. Cells treated with EDTA incorporate proteins and other molecules from the surrounding medium, probably via vesiclelike lesions at the septa that are induced by EDTA. The amount of protein taken up is proportional to the protein concentration in the permeabilization medium. Incorporated molecules equilibrate throughout the cytoplasm, and those with affinity for DNA bind to the nucleoid. Conditions that promote the viability of permeabilized cells and help to avoid otherwise irreversible effects of EDTA are defined. Procedures for selecting cells that have incorporated protein and for studying the distribution of the protein and its effects in growing-dividing cells are described. The procedure may have several applications to molecular and cellular biology; however, we describe here the localization in living cells of the histonelike protein HU. Fluorescence microscopy of cells containing different amounts of fluorescein-labeled HU (varied from approximately 10(3) to 10(5) molecules per cell) showed that the HU concentrates in the nucleoid and is uniformly distributed throughout this structure. Control experiments demonstrated that unlabeled interior parts of the nucleoid can be resolved when labeled proteins that do not bind DNA or enter the nucleoid are introduced into living cells. It was concluded that in vivo added HU binds primarily DNA and that there are no intrinsic restrictions on major regions of the nucleoid to which the added HU protein may bind.

DNA ring closure mediated by protein HU.

The histone-like protein HU serves as an accessory factor that can facilitate the interaction of certain proteins with their specific DNA binding sites. Examples occur in different systems for prokaryotic DNA replication, transcription, and gene regulation. The protein-DNA interactions that are stimulated by HU generally involve coiling or looping of the DNA, and the possibility has been considered that HU exerts its effect by contributing flexibility to different DNA binding sites, but there has been no direct demonstration of this. To explore the possibility that HU can mediate tight DNA curvatures, we studied its effect on the formation of DNA circles when DNA ligase cyclizes short linear DNA fragments. It is demonstrated that HU greatly increases the cyclization rates of all fragments that were examined having lengths greater than 98 base pairs. Fragments of 99, 108, 120, or 126 base pairs could not cyclize in the absence of HU, but cyclization went rapidly with HU, showing that HU can mediate very tight DNA curvatures.

Relaxation of DNA torsional tension in defined domains of bacterial chromosomes in vivo.

A procedure is described for selectively relaxing the DNA torsional tension in defined regions of the chromosome of living bacterial cells. Regions of the chromosomal DNA labelled with bromodeoxyuridine are selectively nicked by irradiation of the cells with long-wavelength ultraviolet light and then trimethylpsoralen residues are photobound to the chromosome in vivo. It is demonstrated that the rate of photobinding to the bromouridine-labelled parts of the chromosomes declines relative to the unlabelled parts of the same chromosomes as nicks are introduced into the former regions. The maximal difference in photobinding rates is that expected for the difference between relaxed and negatively supercoiled DNA. Analysis of the number of DNA breaks required for minimizing the photobinding rates permits a calculation of the number of domains of supercoiling per Bacillus subtilis chromosome.

Tumorigenic human squamous lung cancer cells have defined cell surface carbohydrates that are absent from nontumorigenic cells.

Cells of cloned lines derived from human squamous lung carcinomas spontaneously become heterogeneous with respect to several tumor-associated cell surface carbohydrates such as the sialosyl-Lea oligosaccharide antigen or the recently described oligosaccharide recognized by monoclonal antibody 43-9F. Subclones derived from these cultures are initially homogeneous with respect to the presence or absence of a specific cell surface carbohydrate but gradually revert back to a heterogeneous population. Cells of homogeneous subclones having both the sialosyl-LEa and 43-9F cell surface antigens and other subclones lacking them were injected subcutaneously in nude mice. All clones expressing these tumor-associated cell surface carbohydrates were found to be highly tumorigenic, whereas those lacking them were nontumorigenic or, at most, weakly tumorigenic. Clones having the tumor-associated cell surface carbohydrates were more resistant to cytotoxic attack by purified mouse natural killer cells than those clones lacking these carbohydrates, suggesting that the tumorigenicity of the former clones may be influenced by immunoprotective effects of these novel carbohydrates.

In vitro B-lymphocyte antigen priming against both non-immunogenic and immunogenic molecules requiring low amounts of antigen and applicable in hybridoma technology.

A method to efficiently antigen-prime B-lymphocytes with low amounts (less than 1 microgram/10(8) cells) of either immunogenic or non-immunogenic molecules is described. Keyhole limpet hemocyanin (KLH) and histone were used as prototypes for strongly immunogenic and for phylogenetically conserved non-immunogenic epitopes, respectively. Several modifications of previously reported methods were applied to the system and resulted in the requirement of antigen amounts sufficiently low to be obtainable by elution of proteins from electrophoretic gels. Antigen priming against highly purified antigen preparations is thereby feasible even when purified material cannot be obtained by conventional biochemical procedures. The amount of T- and B-lymphocytes and interleukin-2 production was estimated under various conditions during the priming procedure, and those optimal for generation of a high number of antigen-specific B-lymphocytes determined. In vitro antigen-primed B-lymphocytes were immortalized by conventional hybridoma technology. By fusion of lymphoid cells with myeloma cells at each day during the antigen-priming period, the optimal day of fusion to generate antigen-specific hybridomas was determined. Further, in 12 experiments with different antigens, 11 monoclonal antibodies to histones H3 and H4, two to the murine glucose transporter, 17 to trinitrophenyl-sheep red blood cells, and 20 to KLH were obtained. All specific hybridomas produced IgMs, as the antigen-priming period could not be extended for more than 9-10 days, whereafter a rapid decay in B-lymphocytes occurred.

Glycoproteins distinguishing non-small cell from small cell human lung carcinoma recognized by monoclonal antibody 43-9F.

Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6] and mucin-like properties. A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate. The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells. A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell. This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen. In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands. In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells. Other organ systems lack any reactive cells. The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope. The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.

Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Properties of purified NuMA protein.

Monoclonal antibodies and human autoimmune sera specific for the nuclear mitotic apparatus protein (NuMA protein) were applied to study the structure of this protein and its intracellular distribution. The NuMA protein was purified using immuno-affinity columns. Studies on this large (250 kD) nuclear protein indicated that it is a highly asymmetric phosphoprotein. It is present in all mammalian cells examined and in those of some non-mammals. Immunofluorescence studies on fixed cells demonstrated that its intracellular distribution is essentially the same in all species at all stages of the cell cycle. Immunoblot (western blot) analysis showed that the size of the NuMA protein varies slightly in different species. At the onset of mitosis the NuMA protein redistributes from the nucleus to two centrosomal structures that later will become part of the mitotic spindle pole. This occurs at the time of nuclear breakdown and eventually leads to an accumulation of the NuMA protein at the polar region of the mitotic spindle. After anaphase the protein redistributes from the spindle polar region into the reforming nucleus and concentrates initially at the site where nuclear lamins and perichomatin have been reported to assemble. Living cells microinjected with fluorescent anti-NuMA antibodies were studied to examine parameters that effect the redistribution of the NuMA protein in vivo. These experiments indicate that microtubule assembly is essential for the NuMA protein to accumulate in the polar region.

Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered DNA helical pitch.

Nuclease digestion studies of DNA bound to the histone-like protein HU show that cuts in each strand of the DNA double helix are made with a periodicity of 8.5 base-pairs. By contrast, similar digestions of DNA in eukaryotic nucleosomes show a repeat of 10.4 base-pairs. This and other results (including circular dichroism studies) are consistent with the proposal that the pitch of the DNA double helix in the HU complex is reduced from a repeat length of 10.5 to 8.5 base-pairs per helical turn. Simultaneously, the DNA in the HU-DNA complex containing two dimers of HU per 60 base-pairs has its linking number decreased by 1.0 turn per 290 base-pairs. From these changes it is calculated that HU imposes a DNA writhe of 1.0 per three to four monomers of HU. The results suggest a model in which DNA is coiled in left-handed toroidal supercoils on the HU complex, having a stoichiometry resembling that of the half-nucleosome of eukaryotic chromatin. An important distinction is that HU complexes can restrain the same number of DNA superhelical turns as eukaryotic nucleosomes, yet the DNA retains more negative torsional tension, just as is observed in prokaryotic chromosomes in vivo. Another distinction is that HU-DNA complexes are less stable, having a dissociation half-life of 0.6 min in 50 mM-NaCl. This last property may explain prior difficulties in detecting prokaryotic nucleosome-like structures.

Immunization in vitro and production of monoclonal antibodies specific to insoluble and weakly immunogenic proteins.

A procedure is described for immunizing in vitro and stimulating proliferation of specific B-cell lymphocytes. The method is applicable to production of monoclonal antibodies against proteins that are soluble only in denaturing solvents. An induction period is described in which antigen is presented to the B-cell population in the absence of serum. Also, antigen is coupled to mitogenic silica, which allows the effective presentation of both soluble and insoluble antigens. The results indicate hybridomas can be obtained that secrete IgMs directed against highly conserved or weakly immunogenic antigens.

NuMA protein is a human autoantigen.

Routine examination of sera from patients with suspected or confirmed connective tissue disease has revealed the presence of autoantibodies directed against an unusual nuclear antigen. As characterized by immunofluorescence studies, the antigen is found exclusively in the nuclei of interphase cells, but appears to be part of the spindle pole in mitotic cells. Similar distributions in interphase and mitotic cells have been reported for the recently discovered nuclear mitotic apparatus (NuMA) protein. Using immunoblot analysis we have demonstrated that the autoantibodies that decorate the mitotic spindle poles are specific for the NuMA protein. Therefore, we conclude that the NuMA protein is a human autoantigen.

Cruciform transitions in DNA.

The rates of transition between the cruciform and linear conformations of a perfectly inverted repeated lac operator DNA sequence have been measured using a trimethylpsoralen intrastrand cross-linking assay. The rate and extent of the linear to cruciform transition were dependent on temperature and on the superhelical density of the DNA. Apparent half-lives for this transition were between 4-9 min at 37 degrees C for supercoiled DNAs as isolated from cells. The half-life for the cruciform to linear transition in relaxed DNA was about 30 s at 37 degrees C. Mg2+ stabilized both conformations but stabilized the linear form to a greater degree than the cruciform. The rates of transition were temperature dependent suggesting enthalpies of activation of 26.3 kcal mol-1 for the cruciform to linear transition and 33.4 kcal mol-1 for the linear to cruciform transition. The rate of the linear to cruciform transition was slower at 50 than 37 degrees C. Heating above 70 degrees C resulted in the loss of the cruciform structure.

Nuclear proteins that become part of the mitotic apparatus: a role in nuclear assembly?

A structure located at the poles of the mitotic spindle is described, which may function as a centre for post-mitotic nuclear assembly. Evidence in support of this function is incomplete, but comes from two different kinds of experiments, which are reviewed here. First, fluorescence microscopy studies show that mitotic chromosomes at telophase or late anaphase are drawn into juxtaposition with this polar structure and second, the structure is made up in part of a non-histone chromosomal protein that in interphase cells can be detected only in the nucleus. Studies of this nuclear-mitotic apparatus protein (NuMA protein) are reported here. Monoclonal antibodies specific for the NuMA protein have been used in immunofluorescence studies to visualize the prenucleus-like polar structure and to identify the NuMA protein by immunoblotting after electrophoretic separation. The NuMA protein is a non-histone chromosomal protein of molecular weight 250000 relative to standard protein molecular weight markers in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Experiments are described that indicate several difficulties in studying the possible affinity and association of NuMA protein with mitotic chromosomes. Metaphase chromosomes isolated by the polyamine procedure of Lewis and Laemmli have bound NuMA protein detectable by immunofluorescence or by immunoblotting, but measurements made at different stages of chromosome purification show that most of the NuMA protein is separated from the chromosomes using this purification procedure. Chromosomes purified from mixtures of human and Chinese hamster cells (the latter have none of the human form of NuMA recognized by a monoclonal antibody) have human NuMA protein bound to the hamster chromosomes. Results suggest that in cell extracts exchange reactions of NuMA protein can occur, which must be avoided in the study of its natural function.