Isolation and partial characterization of macromolecular urinary aggregates containing carcinoembryonic antigen-like activity

Carcinoembryonic antigen-like substances in the urine of patients with bladder carcinoma and of healthy male subjects occur in a wide range of average molecular sizes. Some of these substances are glycoproteins giving reactions of antigenic identity with carcinoembryonic antigen (CEA) derived from colorectal carcinoma and occur in aggregation with proteins showing antigen specificities of albumin, haptoglobin and the heavy chains of immunoglobulins G, A and M. Recoveries of CEA-like activities, following Sepharose 4B chromatography of urinary materials of molecular size > 3 × 10⁴, varied from 40 to 1530%. Treatment with 1 mol/1 HClO₄ caused an apparent solubilization of 85% of the CEA-like activity of such materials from the urine of a patient with bladder carcinoma and raised the specific CEA-like activity of the solubilized product to 379% of that of the untreated materials.     

Isolation and partial characterization of plasma membranes bearing human fetal-associated antigens.

A rabbit antiserum to first-trimester human fetal tissue had greater reactivity in complement fixation and saturation binding assays with fetal tissues than with both a pool of normal adult lung, liver, and kidney and pools of the individual organs. This anti-fetal membrane reactivity was only partially inhibited by carcinoembryonic antigen. The serum still reacted strongly with human fetal and tumor cells after rendering it specific for plasma membrane components by adsorption to and elution from intact human fetal tissue culture cells. This plasma membrane-specific serum was then used to monitor the purification of the fetal membrane-associated antigens. The fetal antigens copurified with the putative plasma membrane enzymatic markers 5'-nucleotidase and Mg2+-adenosinetriphosphatase through differential and density gradient centrifugation. Insulin-binding activity only partially copurified with the antigenic activity. Little antigenic activity was found in nuclear and mitochondrial fractions. The isolation protocol gives fetal plasma membrane-associated antigens in approximately 50% yield with moderate purification. The sera and isolation procedures described should have general utility for the detection of human oncofetal antigens.     

Isolation and partial characterization of a transformation-associated sequence from human nasopharyngeal carcinoma.

A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion-sensitive mouse JB6(P+) C141 cells. To clone this transformation-associated sequence, we carried out three cycles of transfection, followed by cloning of anchorage-independent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in both soft-agar assay and nude-mice implantation, was used to make a genomic library in the vector lambda dash. Using the human repeated sequence Blur 8 to screen the library, we obtained 10 human Alu-positive clones. A cloned Alu-positive insert of 16 kbp, CNE 323, was characterized in detail. CNE 323 transferred moderate transforming activity when introduced into JB6 P+ cells and showed no homology to Ha-, Ki-, or N-ras genes; human promotion sensitivity genes; src, myb, jun, myc, fos, raf, or int-2 oncogenes; or epidermal growth factor receptor. The isolated CNE 323 DNA sequence appeared to preserve the genomic structure of the original sequence found in CNE2 cells and in nude mouse tumors induced by CNE2 cells or by CNE/JB6 transfectant cells, indicating that the cloned NPC sequence was activated during NPC carcinogenesis and not during transfection or construction of the library, and that the cloned sequence or a larger sequence of which it was part played a role in tumor formation. Finally, we identified a 1.3-kb mRNA that hybridizes to a subclone of the 16-kb NPC sequence in CNE2 cell poly (A)+ RNA.     

Isolation and partial characterization of a soluble oncofetal antigen from murine and human amniotic fluids.

A soluble (cell-free) oncofetal antigen (OFA) was detected in murine and human amniotic fluids by immunostaining with the murine monoclonal antibody (MAb 115) produced by syngeneic immunization with mid-gestational mouse fetal cells. OFA was purified from the amniotic fluids by ammonium sulfate precipitation at 30-70% saturation, followed by successive gel chromatography of the OFA-containing fraction on Sephacryl-S300 HR, Q- and S-Sepharoses and lentil lectin agarose. The fraction eluted from the lentil lectin column gave a single band on SDS-PAGE of the same molecular weight as the membrane-bound OFA found on both fetal and tumor tissues of humans and several rodents. Both soluble and membrane-bound OFAs share several chemical characteristics, including binding to lentil lectin and wheat-germ agglutinin, molecular weight (44 kDa) and pI (6.8). Mild periodate oxidation of OFA did not affect its binding to MAb 115 in an enzyme-linked immunosorbent assay, indicating that the reactive epitope is a peptide.     

Isolation and partial characterization of radioiodinated myeloblastic leukemia-associated cell surface glycoprotein antigen.

Peripheral blood myeloblasts from five patients with acute myeloblastic leukemia and peripheral remission leukocytes from two of these patients were radiolabeled by the lactoperoxidase-catalyzed surface radioiodination technique and incubated in a nutrient medium at 37 degrees. Radioactive materials shed from viable cells into the supernatant at 24 hr were purified by gel filtration and by DEAE-cellulose chromatography. The radiolabeled leukemic cells shed relatively few molecular species into the culture medium. The DEAE-cellulose eluate usually contained one major peak in which radioactivity and protein levels were coincident; the molecular weight of this compound was 350,000 to 400,000, and it contained carbohydrate as well as protein. Glycoprotein shed from leukemic cells was specifically reactive in a coprecipitation assay with defined antimyeloblast alloantisera obtained from leukemic patients receiving immunotherapy. No reaction was seen with antisera directed against HLA or B-cell antigens. Material shed from remission cells did not coprecipitate with antileukemic antisera. The isolation of radioactively labeled antigen derived from myeloblasts may ultimately allow the monitoring of human antigen levels in leukemic blood by radioimmunoassay.     

Isolation and characterization of thomsen-friedenreich-specific antibodies from human serum.

The Thomsen-Friedenreich (TF) disaccharide, galactose (Gal)beta1-3GalNAcalpha-, is a blood group-related oncofetal antigen with remarkable tumor specificity. Postpartum, carbohydrate structures on the cell walls of the gastrointestinal flora evoke natural antibodies of presumed TF specificity. These antibodies may provide an early barrier against TF-carrying tumor cells. Their possible role, however, has been difficult to assess, since so far only a multivalent immunosorbent, asialoglycophorin (aGP), has been employed for their preparation, and therefore their fine specificities have been only insufficiently defined. We have used a novel immunosorbent consisting of synthetic TFalpha disaccharides (Galbeta1-3GalNAcalpha-) coupled to polyacrylamide (PAA), which itself was covalently bound to cross-linked sepharose. For specificity analyses, aGP and a panel of PAA-conjugated mono- and oligosaccharides were employed. Binding to the PAA moiety was excluded. The affinity-purified anti-TFalpha antibodies were of the IgM (> or =0.5 mg/100 ml of serum) and IgG (approximately 0.05 mg/100 ml of serum) classes. They did partially cross-react with TFbeta, although we detected a second group of anti-TFbeta antibodies (both IgM and IgG) which did not cross-react with TFalpha. The affinity-purified TFalpha antibodies showed only marginal cross-reactivity with the related antigens lactose, Gal, Tn or the Forssman antigen. Besides TF-specific antibodies, we found antibodies to the carbohydrate antigens Tn, Forssman and beta-D-Gal as well as to noncarbohydrate epitopes of glycophorin in human serum.     

Carcinoembryonic antigen in human ovarian neoplasms.

The presence and location of carcinoembryonic antigen (CEA) was examined in tissue sections of 14 mucinous and 13 serous cystadenomas and cystadenocarcinomas of the ovary. CEA was demonstrated in the mucinous tumors but was not present in the serous tumors. In the mucinous tumors, CEA was located in the glycocalyx and apical portions of the absorptive-type epithelium, with only trace quantities in goblet cells, a pattern identical to that seen in colonic neoplasms. Endocervical-type epithelium in the mucinous tumors contained little or no CEA.     

Carcinoembryonic antigen: characterization of binding with insoluble lectins.

Insoluble conanavalin A and wheat germ agglutinin (WGA) were found to bind to carbohydrates on radio-labelled carcinoembryonic antigen (CEA). Binding by WGA was inhibited both by N-acetyl D-glucosamine and fragments of antibody to CEA, but was increased by intact antibody to CEA. This suggests that WGA binds to exposed N-acetyl D-glucosamine determinants on 125I CEA and also on antibody molecules. It also suggests that 125I CEA contains binding sites for anti-CEA which contain N-acetyl D-glucosamine as well as others which do not. Molecules of 125I CEA which bound to the insoluble lectins were more antigenic for anti-CEA than unbound molecules. These results suggest that the principal antigenic site on CEA contains N-acetyl D-glucosamine and may help to explain the agglutination of tumour cells by lectins.     

Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties.

A cDNA encoding murine O6-alkylguanine-DNA-alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence. Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa). In liquid hybridization experiments, anti-human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti-rat polyclonal antibodies inhibited rat and mouse but not human ATase. Both antibodies detected all mammalian ATases tested by western analysis so far. These results indicate some common epitopes and at least one unique human epitope. We compared the amino-acid sequence of the murine ATase with those of other mammalian and bacterial ATases. The proteins of this family all have a large domain (approximately 70 amino acids) of highly conserved residues flanking the sequence PCHRV, which contains the alkyl-accepting cysteine residue of the active site. No evidence was found in the sequences for helix-turn-helix, leucine-zipper, or zinc-finger motifs for DNA recognition and binding. Nuclear localization signals (basic-residue-rich regions) could not be uniquely identified in the mammalian members of the family. Outside of the conserved PCHRV region, there were major differences between prokaryotic and eukaryotic proteins at the primary structure level: there was a series of proline-rich motifs, but these also varied between sequences.     

Isolation and partial characterization of surface components of cell line MDA-MB-231 derived from a human metastatic breast carcinoma

Neuraminidase (Vibrio cholerae) treatment of human metastatic mammary carcinoma MDA-MB-231 cells grown in culture released 0.60-0.63 mg of N-acetylneuraminic acid from 10(9) cells. Incubation of intact cells with a modified trypsin and fractionation by gel filtration gave mainly O-glycopeptides. The presence of O-glycosyl-linked chains having one or two carbohydrate residues was confirmed by treatment of the glycopeptide fractions with galactose oxidase, followed by reduction with alkaline sodium borotritide and fractionation. The major glycopeptide fraction, which consisted of 53% carbohydrate and 47% protein, and a minor glycopeptide fraction each inhibited hemagglutination by peanut lectin. These results suggest the presence of O-beta-D-galactopyranosyl-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 leads to 3)-L-serine (threonine) residues. The absorptive capacities for anti-HLA-A2 and anti-HLA-B8 antisera were slightly greater for intact than for lyophilized cells, which suggested that masking of these major histocompatibility antigens did not occur in intact cells.     

Isolation and characterization of human breast tumor-derived endothelial cells

Increasing evidence indicates that tumor-derived endothelial cells (TEC) possess a distinct and unique phenotype in respect to normal endothelial cells and may be able to acquire resistance to drugs. However, few functional studies are available on cultured TEC. In the present study, we obtained TEC from human breast carcinomas and, to dispel the possibility of contaminating tumor cells, we established six different clones that we characterized at a functional level. Breast TEC cell lines and clones did not undergo normal cell senescence in culture and showed constant expression of markers of endothelial activation and angiogenesis. These cells showed increased apoptosis resistance to vincristine and doxorubicin and increased random cell motility, as compared to normal micro-endothelial cells. In addition, breast TEC, at variance to normal endothelial cells, were able to grow and to organize in the absence of serum in capillary-like structures on Matrigel that persisted up to one week. These functional characteristics of breast TEC may be relevant for tumor angiogenesis and may indicate an increased pro-angiogenic activity of endothelial cells within the tumor. Moreover, our data suggest that TEC might be more appropriate for screening antiangiogenic drugs than normal endothelial cells.     

Isolation and characterization of proteoglycans from human nonepithelial tumors

Proteoglycans (PGs) and glycosaminoglycans (GAGs) were identified in myogenic and fibrogenic tumors. More PGs containing mainly chondroitin sulfate could be detected in malignant tumors (leiomyosarcomas) than in benign tumors (leiomyomas and fibromas). Two groups of PGs were detected in the malignant tumors by ion-exchange chromatography and gel chromatography. One group was a large molecule with chondroitin sulfate side chains, seemingly composed of two or more subpopulations that were eluted from Sepharose CL-4B with a kav of 0.45. After removal of GAG side chains from the PG by chondroitinase AC digestion, core molecules with molecular weights greater than 200,000 were obtained. Another PG detected was a fraction of small PG eluted from Sepharose CL-4B with a kav of 0.45. It consisted of a core molecule with a molecular weight approximately equal to 48,000 and GAG side chains containing chondroitin sulfate-dermatan sulfate hybrids. The mixed sequence of L-iduronic acid with D-glucuronic acid in the same GAG chain was demonstrated by the formation of a small proportion of tetrasaccharide after chondroitinase AC digestion. In the benign tumors, the large PG was found only in very small amounts, and PG detected was composed mainly of the small one eluted from Sepharose CL-4B with a kav of 0.45. Its core protein had a molecular weight of approximately equal to 46,000, which was similar to that of small PG obtained from leiomyosarcomas, but its GAG side chains were composed mainly of dermatan sulfate containing small amounts of glucuronic acid. The results suggest that the core molecules of small PGs from both benign and malignant tumors are the products of the same gene but that they are processed in a different manner to form proteoglycans with different types of GAG chains.     

Isolation and characterization of lymphocytes and macrophages from solid, malignant human tumours.

In mechanically prepared cell suspensions from 17 solid, malignant human tumours, 0.5-5.0% (mean 2.0%) lymphocytes and 1.0-28.0% (mean 7.4+) macrophages were found. Mononuclear cells (MC) were isolated using the B?yum technique. From each biopsy weghing 1-4 g, on average, 1.3 x 10(6) lymphocytes and 0.8 x 10(6) macrophages were recovered. The tumour-infiltrating lymphocytes (TIL) were characterized with regard to T and B markers. The proportion of TIL-forming rosettes with SRBC (T cells) was 43%, which was significantly less than for peripheral blood lymphocytes (PBL) from cancer patients (58%), or normal controls (80%), On average, 15% of the TIL were B cells, whereas 42% had no T- or B-cell markers. Macrophages (TIM) were identified by non-specific esterase staining and phagocytosis. In four cases the tumour cells were also stained with alpha-naphthyl butyrate. Corresponding findings were made on esterase-stained cryostat sections from four tumours. Macrophages were found within and around the tumour tissues, occasionally localized to necrotic areas, but in most cases with no sign of necrosis of the surrounding cells. In some tumour cell suspensions typical clusters of lymphocytes and macrophages were seen. Total lymphocytes and T-cell were markedly reduced inthe peripheral blood of cancer patients, whereas total monocyte counts were within the normal range.     

Isolation and characterization of arsenite-resistant human epidermoid carcinoma KB cells

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein 1 (MRP1) in KAS cells was confirmed by quantitative RT-PCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.     

Isolation and characterization of an anthracycline-resistant human leukemic cell line

An anthracycline-resistant subline of HL-60 promyelocytic leukemia cells (HL-60/AR) has been isolated in vitro by subculturing in progressively higher concentrations of Adriamycin. The resistant cells are capable of sustaining continuous growth in 10(-6) M Adriamycin which is more than 50 times the 50% inhibitory dose for the parent line. HL-60/AR expressed variable degrees of cross-resistance to daunorubicin, dihydroxyanthracenedione, vincristine, vinblastine, and actinomycin D, but it remained sensitive to methotrexate and 1-beta-D-arabinofuranosylcytosine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of glycoproteins of HL-60/AR revealed two prominent glycoproteins with molecular weights of 160,000 +/- 10,000 and 110,000 +/- 10,000 which were not detected in the sensitive cells. Cellular uptake and retention of daunorubicin was studied in the resistant and sensitive cells utilizing digitized video fluorescence microscopy. The sensitive cells accumulated more drug and showed at least 2-fold greater levels of brightness than the resistant cells. Studies of total intracellular accumulation, utilizing 10(-6) M [14C]-daunorubicin as a marker, showed a 1-h accumulation of 98 +/- 20 pmol/10(6) cells in HL-60/AR versus 255 +/- 25 pmol/10(6) cells in HL-60. Exposure to nontoxic concentrations of the calcium channel blocker Verapamil (10(-5) M) led to enhanced accumulation (175 +/- 8 pmol/10(6) cells) and retention of the drug in HL-60/AR, resulting in increased cytotoxicity in HL-60/AR. These anthracycline-resistant leukemic cells may serve as a valuable experimental model in studying the phenomenon of multiple drug resistance as well as strategies to circumvent it in human myeloid leukemia.     

Isolation and characterization of tumor stem-like cells from human meningiomas

Recent advances in research have found that tumor stem-like cells are resistant to surgery, radiotherapy, and chemotherapy. Tumor stem-like cells play critical roles in tumor recurrence, angiogenesis, and invasion in malignant brain tumors. However, the identification of tumor stem-like cells in meningiomas has not been clarified. In this study, we identified the stem-like features of sphere-forming cells in human meningiomas. The results showed that meningioma stem-like cells possess the ability to form spheres in identical stem cell culture condition. These meningioma sphere cells (MgSCs) expressed progenitor cell marker, CD133, but not differentiated cell marker, epithelial membrane antigen (EMA) on immunofluorescence staining. Importantly, the mRNA expression of ABCG, and CD133 was higher in MgSCs than daughter meningioma adherent cells (MgACs). In addition, cells displayed chemotherapeutic resistance to vincristine more in MgSCs than MgACs. This phenomenon was also found in single cell form from dissociated spheres than MgACs. Moreover, MgSCs are more resistant to irradiation treatment than MgACs. Furthermore, MgSCs revealed high tumorigenicity in vivo following orthotopic inoculation into the brains of immunodeficient mice. The corresponding immunohistochemical (IHC) staining found that MgSCs are positive for EMA, vimentin, and CD133, consistent with IHC of primary human meningiomas. These findings have provided better understanding of meningioma cell biology and suggested that meningioma stem-like cells may serve as a novel target in therapeutic resistant meningiomas.