Fetal gastric and small intestine pattern of intestinal mucus antigens in human gastric carcinomas.

The present immunohistological study was performed to investigate the expression of intestinal mucus-associated antigens in the different histological types of gastric carcinoma according to the classification of Laurèn and the WHO classification. We used the following antigenic markers: M3, present in all the goblet cells of the whole small and large intestine; M3SI, expressed in all the goblet cells of the small intestine, but only in some of them of the large intestine; M3D, mainly produced in the upper small intestine; and M3C, specific for colonic mucus cells. All these antigens were found to be similarly expressed in both Laurèn's intestinal and diffuse types. Nevertheless, M3 and M3C appeared to be more largely produced in carcinomas showing well-differentiated cells (tubulopapillary, mucinous, and signet ring cells according to the WHO classification). Our results evidenced the occurrence of two main fetal antigenic patterns in gastric carcinomas, one of the gastric type (M3SI produced to a larger extent than M3 and M3C) and the other one of the small intestinal type (M3 and M3SI more largely expressed than M3C). On the basis of their similar antigenic pattern, the histogenesis of carcinomas showing the fetal small intestinal antigenic profile may be associated with intestinal metaplasia. On the other hand, carcinomas with the fetal gastric pattern may originate from undifferentiated stem cells of the gastric mucosa. Thus, such immunohistological studies could lead to a better understanding of the histogenesis of gastric carcinomas.

Isolation and characterization of colon cancer mucin from xenografts of LS174T cells.

The structure of colonic mucin, which is thought to be important in several diseases, including ulcerative colitis and colon cancer, is poorly understood. Mucin was isolated from nude mouse xenografts of the LS174T colonic adenocarcinoma cell line by gel filtration and CsCl density gradient centrifugation. The isolated mucin had a high content of threonine, serine, and proline, with 28% of the total amino acids O-glycosylated. The carbohydrates present were fucose, sialic acid, galactose, N-acetyl-glucosamine, and N-acetyl-galactosamine in the ratio of 0.4:1.5:1.0:0.9:1.4. Rabbit antibodies were prepared that recognized primarily protein-dependent determinants. By DEAE-cellulose chromatography, the purified mucin was found to be heterogeneous, with three major components that had small differences in carbohydrate composition. LS174T was antigenically and chromatographically similar to mucins in colon cancer tissue specimens and in nonmalignant colonic mucosae.

Intestinal metaplasia and carcinomas of the human stomach: an immunohistological study.

Rabbit immunization with duodenal or colonic high molecular weight components allowed us to distinguish three new M3 antigens associated with intestinal goblet cells; these were denoted as M3SI, M3D, and M3C. Using immunoperoxidase staining, the anti-M3SI serum labeled all goblet cells in the small intestine, and a certain number of them in the colon, mainly in its proximal part. The anti-M3D serum reacted primarily with the goblet cells of the duodenal villosities, and the anti-M3C serum with all goblet cells of the large, but not the small, intestine. Moreover, the M3C antigen was recovered in some goblet cells of the fetal duodenum, in association with the two other markers. Only the M3D and the M3SI antigens were observed in the normal duodenum and intestinal metaplasia (IM) of benign gastric mucosa. In contrast, IM adjacent to gastric carcinomas, whatever their histological type, contained the M3C antigen in addition to M3D and M3SI antigens, and thus showed an antigenic pattern similar to fetal duodenum. Some gastric carcinomas, especially those with intestinal-like differentiation, produced the colonic M3C antigen as the neighboring IM. Thus, these two tissues displayed common differentiation features, which could be related to fetal duodenum rather than to adult colon.

Pancreatic cancer mucin from xenografts of SW1990 cells: isolation, characterization, and comparison to colon cancer mucin.

Mucin has been purified from nude mouse xenografts of SW1990 human pancreatic cancer cells. The mucin was eluted at the void volume of Sepharose CL-4B and was of density greater than 1.3 in CsCl gradients. The isolated mucin had a high content of threonine, serine, and proline, with 31% of the amino acid residues O-glycosylated. The average oligosaccharide composition was NeuAc1.8Fuc0.7Gal2.0GlcNAc1.7GalNAc1.4. Polyclonal rabbit antibodies prepared against the purified mucin recognized primarily mucin polypeptide, and there was extensive immunological cross-reaction between SW1990 pancreatic cancer mucin and LS174T colon cancer mucin. However, using carbohydrate-specific monoclonal antibodies, the two mucins were found to differ. SW1990 mucin had more Lewis, sialyl Lewis, and sialyl Lewis activity, while the colon cancer mucin had more sialyl T antigen. Since pancreatic mucins, whether from normal pancreas or pancreatic cancer, have not previously been well characterized, the availability of SW1990 pancreatic cancer mucin may be useful as a model for studying the expressing of organ-specific or cancer-associated antigens.

Monoclonal antibodies against human solid tumors.

Many monoclonal antibodies have been generated against human solid tumors. Very often, animals were immunized with long term culture cell lines, or sometimes with membrane preparations, which were obtained from such cell lines or from chirurgically resected tumors. High precautions must be taken to ensure the specificity of the resulting monoclonal antibodies: especially, it must be confirmed that they do not react with lymphocytes or blood group antigens. Monoclonal antibodies allowed investigators to evidence several new antigens. Some of them are organ specific antigens expressed in the tumoral as well as in the normal tissue, while others really look as tumor associated antigens, though such a feature has to be cautiously claimed. Indeed, these antigenic markers are often recovered in some normal cells. Further, the oncofetal pattern of several of these antigens is to be noticed. Human-mouse hybridomas could allow one to immortalize antibodies raised in cancerous patients against components of their own tumor. However, some difficulties do yet persist in the generation of such cell hybrids and furthermore, their stability is not quite satisfactory.

Peanut agglutinin (PNA)-binding properties of murine thymocyte subpopulation.

Surface receptors for peanut agglutinin (PNA), a lectin with D-galactose specificity, were detected on mouse thymocytes using fluorescence microscopy. Depending on mouse strain, 69-85% of unseparated thymocytes could thus be characterized as PNA+. Electrophoretic fractionation of thymocytes from normal or immunosuppressive drug-treated donors revealed an inverse relationship between PNA-binding properties and cell electrophoretic mobility (EPM). Thus, all thymocytes recovered in the lowest EPM fractions were strongly PNA+ whereas those in the highest EPM fractions were in the majority PNA-. Most of the cells collected in the intermediate EPM range were PNA+ but staining with the fluoresceinated lectin appeared weaker than for the low EPM thymocytes. Reciprocal experiments in which thymocytes were separated by PNA-mediated aggregation into fractions with different affinities for the lectin and then subjected to physical analysis, definitely established that PNA+ cells are of lower EPM than PNA- cells and that these two cell types also differ in size distribution. These data show that the four physical subpopulations of thymocytes previously described present distinctive PNA-binding properties: Th1 and Th2 cells can be classified as strongly PNA+, Th3 cells as less intensely PNA+, and Th4 cells as mostly PNA-.

Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis.

Zinc fingers of the Cys2/His2 class are conserved 28-30 amino acid motifs that constitute an important and widespread family of eukaryotic DNA-binding domains. It is therefore of great interest to understand the rules that govern specific recognition of DNA by zinc fingers. The DNA-binding domain of the transcription factor Krox-20 consists of three zinc fingers, each of them making its primary contacts with a three-base pair subsite. We have performed a data base-guided site-directed mutagenesis analysis of Krox-20: nine derivatives were generated, in which one to three amino acid changes had been introduced within finger 2, at positions which were likely to affect the specificity of DNA recognition. The affinities of the different proteins for a panel of potential DNA binding sites were estimated by gel retardation assay. Six of the derivatives bound specific targets with affinities comparable to that of wild type Krox-20 for its consensus binding site. However, the specificity of recognition was dramatically modified at the expected bases, in a manner that could be explained by examining the newly introduced amino acids within the context of the overall finger/triplet interaction. These data provide new insights into the details of zinc finger-DNA interactions and, combined with the modular nature of zinc fingers, illustrate both the potential and the difficulties of utilising these motifs for designing DNA-binding proteins with novel specificities.

Base sequence discrimination by zinc-finger DNA-binding domains.

Zinc fingers constitute important eukaryotic DNA-binding domains, being present in many transcription factors. The Cys2/His2 zinc-finger class has conserved motifs of 28-30 amino acids which are usually present as tandem repeats. The structure of a Cys2/His2 zinc finger has been determined by nuclear magnetic resonance, but details of its interaction with DNA were not established. Here we identify amino acids governing DNA-binding specificity using in vitro directed mutagenesis guided by similarities between the zinc fingers of transcription factors Sp1 and Krox-20. Krox-20 is a serum-inducible transcription activator which is possibly involved in the regulation of hindbrain development; it contains three zinc fingers similar to those of Sp1 and binds to a 9-base-pair target sequence which is related to that of Sp1. Our results show that each finger spans three nucleotides and indicate two positions in Krox-20 zinc fingers that are important for base-pair selectivity. Modelling with molecular graphics suggests that these residues could bind directly with the bases and that other amino acid-base contacts are also possible.

Biochemical characterization of a rat oncofetal colonic antigen defined by a monoclonal antibody raised against gastric surface epithelium.

The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.

Colonic and gastric mucus-associated antigens: a comparative immunohistological study in precancerous and cancerous rat intestinal mucosa.

Gastric M1 antigens were previously shown to be oncofetal markers for the colon in man and in the rat. They were observed very early during carcinogenesis in goblet cells of precancerous colonic mucosa; using an immunohistological method, we found that M1 were produced in 68% (41/60) of colonic adenocarcinomas and in 33% (21/63) of duodenal adenocarcinomas. M3C antigen has been described as being associated with human colonic mucus; in the rat it is restricted to the proximal colon. We found that M3C was produced in 91% (55/60) of colonic adenocarcinomas and in 15% (8/53) of duodenal adenocarcinomas. Before tumor appearance, M3C was sometimes expressed by goblet cells of the distal colon. We could not find it during fetal life. We have concluded that mucus-associated antigens can characterize modifications in cell differentiation in rat colonic carcinomas.

Fetal and ectopic mucous markers expressed by distal and proximal rat colonic carcinomas.

Using immunohistochemistry, seven markers associated with mucous glycoproteins were studied in rat colonic mucosa during fetal and adult life and in carcinomas. These included: blood group A antigen, terminal fucose, sulfated and sialylated groups, M3 intestinal, M3C colonic and M1 gastric antigens. It was found that colonic carcinomas expressed normal adult markers, fetal markers and an ectopic marker. The differentiation of goblet cells was not the same in distal and proximal colon. Finally, this differentiation shared numerous analogies with that of human colonic goblet cells.

Expression and genetic interaction of transcription factors GATA-2 and GATA-3 during development of the mouse central nervous system.

Here we examine the expression of transcription factors GATA-2 and GATA-3 during early stages of embryonic development in the central nervous system (CNS) of the mouse. GATA-2 is expressed as early as 9 dpc in the hindbrain, in ventral rhombomere 4, and transiently in ventral rhombomere 2 (r2). From 9.5 to 11.5 dpc, activation of the gene spreads to many sites of early neuronal differentiation, such as the olfactory bulbs, the pretectum, and the oculomotor nucleus in the midbrain, a thin stripe of cells lining the floor plate from the mesencephalon to the cervical spinal cord and a ventral column of cells spanning the neural tube from rostral hindbrain and including motor neuron as well as ventral interneuron precursors. GATA-3 is expressed in a pattern very similar to that of GATA-2. Distinguishing features are the lack of expression in r2 at 9 dpc and a slight delay in its activation. In addition, GATA-2 is activated in both the ventricular and the subventricular zones of the neural tube, whereas GATA-3 is restricted mainly to the subventricular zone. Expression analyses performed on GATA-2 -/- mouse embryos between E9.5 and 10.5 dpc established that: (i) the expression of GATA-3 in the developing CNS of the mouse embryo is dependent on the presence of GATA-2 and (ii) loss of GATA-2 leads to severe defects in neurogenesis, which strongly suggests that GATA-2 is involved, as in hematopoiesis, in the maintenance of the pool of ventral neuronal progenitors.

Enhanced deoxyribonuclease activity in human transformed cells and in Bloom's syndrome cells.

Human hereditary diseases such as xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome are characterized by a proneness for developing cancer associated with abnormalities in the processing of DNA damage. The molecular defects responsible for predisposing human tissues to cancer are still not well understood, despite the fact that a considerable amount of work has already been done on this problem. In this paper, we show that in human tumor cell lines, in cells transformed by DNA tumor viruses, and in cells derived from certain cancer-prone disorders, the level of activity of a 42-kDa deoxyribonuclease is many times higher than in diploid untransformed control cells. This suggests that this activity is linked to, or may play a role in, malignant transformation.

DNA ligase activity in human cell lines from normal donors and Bloom's syndrome patients.

DNA ligase activity was studied in several untransformed or virus-transformed human cell lines from normal donors and from Bloom's syndrome (BS) patients. This proneness genetic disease is characterized by several cytological abnormalities and cancer proneness and, recently, some transformed cell lines from these patients were described to present a reduced activity of DNA ligase I. Results presented in this work indicate that: (i) the total DNA ligase activity in crude extract from untransformed or transformed cell lines from several BS patients was significantly higher than in control cells; (ii) the partial purification of the enzyme after gel filtration on fast protein liquid chromatography of crude extracts from lymphoblastoid BS cells showed that the enzyme activity was eluted in a major 180 kDa form in which activity was higher than in control cells; (iii) the activity gel analysis of these enzyme fractions revealed that DNA ligase of human cells was correlated to a major 130 kDa polypeptide and, in BS cells, the extent of the activity of this band was equal or higher than that in control untransformed or transformed cells.

Differences in the expression of mucus-associated antigens between proximal and distal human colon adenocarcinomas.

An immunohistological study showed differences in the expression of mucus-associated gastric M1 and intestinal M3 antigens between the proximal (100 cases) and distal (200 cases) colonic adenocarcinomas. Such a regional difference was not observed in the normal colon. A total of 55% and 78% of proximal tumours produced M1 and M3 antigens, respectively (versus 13% and 47% in the distal tumours). The high percentage of M1 positive proximal cancers could be explained by the higher percentage (i) of mucus-producing tumours, such as signet ring cell (6% vs 1%) or mucinous adenocarcinomas (29% vs 11%); and (ii) of M1(+) well-differentiated adenocarcinomas (45% vs 8.5%) and the presence of undifferentiated carcinoma producing M1 antigens (12% vs 0%). These latter carcinomas were found in older patients (mean age 78 years vs 66 years). These results suggest that, on the proximal side, the stem cells were more often engaged in a differentiation process involving the expression of M antigens than were those of the distal side. Moreover, the proximal stem cells more frequently produce a foetal differentiation program showing simultaneous expression of M3 and M1 antigens (in 48% of proximal tumours, vs 11.5% for the distal side). Around 12% of proximal adenocarcinomas (vs 2% of distal tumours) contained stem cells engaged in a cell differentiation program not observed in the normal adult or foetal colon, involving the predominant expression of M1 antigens associated with an undifferential histological pattern.