Modulation of MUC1 mucin as an escape mechanism of breast cancer cells from autologous cytotoxic T-lymphocytes.

MUC1 mucin is known to serve as a target molecule in the killing of breast cancer cells by cytotoxic T-lymphocytes (CTLs). We searched for a possible mechanism allowing tumour cells to escape from autologous CTLs. When the killing of breast cancer cells by autologous lymphocytes was examined in 26 patients with breast cancer, significant tumour cell lysis was observed in 8 patients, whereas virtually no autologous tumour cell lysis was detected in as many as 18 patients. In the patients who showed negligible tumour cell lysis, the autologous tumour cells expressed MUC1-related antigenic epitopes much more weakly than the tumour cells in the patients who exhibited strong cytotoxicity (significant statistically at P< 0.0005-0.0045), suggesting that the unresponsiveness of cancer cells to CTLs observed in these patients was mainly due to loss of MUC1 expression or modulation of its antigenicity. A breast cancer cell line, NZK-1, established from one of the cytotoxicity-negative patients, did not express MUC1 and was resistant to killing by CTLs, while control breast cancer cell lines expressing MUC-1 were readily killed by CTLs. Transfection of NZK-1 cells with MUC1 cDNA induced significant lysis by autologous T-lymphocytes. These results supported the importance of MUC1 mucin in autologous anti-tumour immunity, but suggested that the major escape mechanism of tumour cells from autologous T-lymphocytes is the loss and/or modulation of MUC1 antigenicity on tumour cells, which would limit the effectiveness of possible immunotherapy designed to target the MUC1 mucin.

The association of sialyl Lewis(a) antigen with the metastatic potential of human colon cancer cells.

BACKGROUND: The vascular endothelium plays a critical role in cancer metastasis by directing circulating cancer cells into extravascular tissues and by expressing cell adhesion molecules. The authors investigated the interaction between a cell line derived from human colon cancer and cultured murine endothelial cells, in vitro, and in vivo. MATERIALS AND METHODS: Variant cell lines with high (WiDr-HA) or low (WiDr-LA) levels of cell surface sialyl Lewis(a) antigen (s-Le(a)) were isolated from the heterogeneous WiDr cells (WiDr-P) in order to characterize the biological behavior of colon cancer cells having elevated cell surface contents of s-Le(a). RESULTS: A correlation was found to exist between the degree of s-Le(a) expression, and the attachment of cancer cells to activated human umbilical vein endothelial cells, or to F-2 cells isolated from murine vascular endothelial cells. The adhesion of WiDr-P and WiDr-HA to F-2 cells was inhibited by the addition of anti-s-Le(a) antibody (Ab). WiDr-P and WiDr-HA both demonstrated the capacity to metastasize to the liver, by the inoculation of cancer cells to the spleen, while WiDr-LA did not do so. The number of metastatic nodules of WiDr-HA was significantly higher than that of WiDr-P. Treatment with anti-s-Le(a) Ab inhibited the metastasis of WiDr-P and WiDr-HA to the liver, but not with anti-s-Le(x) Ab. CONCLUSIONS: These findings suggest that s-Le(a) plays an important role in cell adhesion molecules, in the metastasis of colon cancer.

Elevation of an alpha(1,3)fucosyltransferase activity correlated with apoptosis in the human colon adenocarcinoma cell line, HT-29.

We studied changes in the carbohydrate expression following apoptotic cell death induced by treatment with interferon (IFN)-gamma and anti-Fas antibody using human colon adenocarcinoma HT-29 cells. An apoptotic cell death of HT-29 accompanied with typical DNA fragmentation was observed when the cells, were cultured sequentially with IFN-gamma and anti-Fas antibody. In flow cytometric analyses, the expression of Le(x) and Le(y) antigen was strongly and slightly enhanced, respectively, on the cell surface in accordance with the apoptosis. When the fucosyltransferase (Fuc-T) activities of the lysates from the treated cells were examined relative to those from untreated cells, a 2.5-fold increase of alpha(1,3)-Fuc-T activities and a slight increase of alpha(1,2)-Fuc-T activities were observed, but little or no increase of alpha(1,4)-Fuc-T activity was detected. In Northern blot analyses using probes for Fuc-T III, IV, V, VI and VII genes, strong RNA messages for Fuc-T III, V and/or VI and a weak RNA message for Fuc-T IV were detected in the untreated HT-29 cells. On the other hand, in the treated cells, the messages for Fuc-T III, V and/or VI were found to almost disappear and the 2.3 kb message for Fuc-T IV was observed to elevate 2.8-fold. Therefore, we suggest that the strongly increased expression of Le(x) antigen found on the HT-29 cell surface might be involved in the process of apoptosis, and that the enhancement of the antigen expression seems to result from the increased activity of alpha(1,3)-Fuc-T encoded mainly by the Fuc-T IV gene.

[Structures, synthesis and functions of sialyl Le(a)/sialyl Le(x) antigens].

Sialyl Lewis a/sialyl Lewis x antigens, which panels of monoclonal antibodies can recognize, are synthesized by a series of glycosyltransferases. Especially, alpha (1,3)/(1,4) fucosyltransferases and/or alpha(2,3)sialyltransferases regulate expression patterns temporally and spatially, such as in epithelium of digestive systems or in leukocytes. The carbohydrate ligands of P-selectin and E-selectin have been identified as sialyl Lewis x expressed on granulocytes, monocytes, and natural killer cells. Expression of sialyl Lewis x on these cells is mainly determined by Fuc-TVII, but as for the counterparts of sialyl-transferases, there remains much uncertainty. P-selectin-dependent adhesion of tumor cells and E-selectin-dependent adhesion of tumor cells to endothelial cells play some role for tumor cell aggregation leading to microembolism and hematogenous metastasis of cancers. We have found expression of some fucosyltransferases (Fuc-TIII, VI, VII) and a sialyltransferase (ST3O) are increased in colon cancer tissues coincidentally with sialyl Lewis a antigens, with which E-selectin can interact. As already started in many laboratories, genetic manipulation of glycosylation pathways in gene-targeted animals has an outstanding potential to yield clues to oligosaccharide function.

Molecular cloning of a cDNA encoding a novel human leukocyte alpha-1,3-fucosyltransferase capable of synthesizing the sialyl Lewis x determinant.

The sialyl Lewis x determinant (NeuAc alpha 2,3Gal beta 1, 4[Fuc alpha 1,3]GlcNAc) is an essential component of leukocyte counterreceptors for E-selectin and P-selectin. The final step in sialyl Lewis x synthesis is catalyzed by alpha-1,3-fucosyltransferases acting on sialylated glycoconjugate precursors. Cultured human leukocytic cell lines express an alpha-1,3-fucosyltransferase gene termed Fuc-TIV or ELFT but do not express the other three cloned human alpha-1,3-fucosyltransferase genes to any significant degree. The physiological role of Fuc-TIV/ELFT in sialyl Lewis x biosynthesis is uncertain, however, since it can catalyze the synthesis of this determinant in some, but not all, transfected cell lines in a manner that is dependent upon the glycosylation phenotype of the host cell. We report here the molecular cloning of a cDNA encoding a new human leukocyte alpha-1,3-fucosyltransferase, termed Fuc-TVII, capable of synthesizing the sialyl Lewis x moiety. The cDNA sequence predicts a 341-amino acid-long type II transmembrane protein typical of mammalian glycosyltransferases. When expressed in mammalian cells, the Fuc-TVII cDNA directs the synthesis of cell surface sialyl Lewis x moieties but not the Lewis x, Lewis a, sialyl Lewis a, or VIM-2 determinants. Fuc-TVII can efficiently utilize alpha-2,3-sialyllactosamine in vitro to form the sialyl Lewis x tetrasaccharide but does not utilize lactosamine to form the Lewis x moiety. Northern blot analyses show that the Fuc-TVII gene is transcribed in HL-60 cells, a human promyelocytic cell line, and in YT cells, a natural killer-like cell line. Fuc-TVII represents a leukocytic alpha-1,3-fucosyltransferase that can participate in selectin ligand synthesis via its ability to catalyze the synthesis of sialyl Lewis x determinants.

Immunoglobulin variable region sequences of two human monoclonal antibodies directed to an onco-developmental carbohydrate antigen, lactotetraosylceramide (LcOse4Cer).

A human monoclonal antibody, 11-50, was generated and was shown to recognize an onco-developmental carbohydrate antigen, LcOse4Cer. The isotype of this antibody was IgM, lambda, similar to the previously known human anti-LcOse4 antibodies, such as IgMWOO and HMST-1. We raised a murine anti-idiotypic antibody G3 (IgG1, kappa) against 11-50, and tested its reactivity towards the affinity purified human polyclonal anti-LcOse4 antibodies prepared from pooled human sera using a Gal beta 1-->3GlcNAc beta-immobilized column. The results indicated that at least a part of the human polyclonal anti-LcOse4 antibodies shared the G3 idiotype with 11-50. We further analyzed the sequence of variable regions of the two anti-LcOse4 antibodies, 11-50 and HMST-1. Sequence analysis of the heavy chain variable regions indicated that the VH regions of these two antibodies were highly homologous to each other (93.5% at the nucleic acid level), and these antibodies utilized the germline genes VH1.9III and hv3005f3 as the VH segments, which are closely related germline genes of the VHIII family. It was noted that these germline VH genes are frequently utilized in fetal B cells. The JH region of both antibodies was encoded by the JH4 gene. For the light chain, the V lambda segments of the two antibodies were 96.3% homologous to each other at the nucleic acid level. The V lambda segments of both antibodies showed the highest homology to the rearranged V lambda gene called V lambda II.DS among reported V lambda genes, while the exact germline V lambda genes encoding the two antibodies were not yet registered in available sequence databanks. The amino acid sequences of the J lambda segments of both antibodies were identical. These results indicate that the two human antibodies recognizing the onco-developmental carbohydrate antigen Lc4 are encoded by the same or very homologous germline genes.

Expression of alpha-(1,3)-fucosyltransferases which synthesize sialyl Le(x) and sialyl Le(a), the carbohydrate ligands for E- and P-selectins,in human malignant cell lines.

Transcripts of alpha-(1,3)-fucosyltransferases in human epithelial cancer and leukemia cell lines were analyzed by Northern blotting and reverse transcriptase mediated-polymerase chain reaction using specific probes and primers which can discriminate between the transcripts derived from the four alpha-(1,3)-fucosyltransferase genes Fuc-TIII, IV, V, and VI. Flow cytometric analysis of the sialyl Le(x) and sialyl Le(a) antigens was also performed on the same cell lines. The sialyl Le(x) antigen was expressed on 14 of 15 epithelial cancer cell lines, and the sialyl Le(a) antigen was detected on 8 of them. The message of Fuc-TIII was detected in most of the epithelial cancer cell lines (14 of 15), which correlated with the surface expression of these carbohydrate determinants. In addition, the messages of Fuc-TIV and Fuc-TVI were detected in most epithelial cancer cell lines, while the message of Fuc-TV was undetectable in most of them. On the other hand, all leukemia cell lines were positively stained for sialyl Le(x), but none of them was stained for sialyl Le(a) in flow cytometry. The messages of Fuc-TIV++ were detected in all leukemia cell lines tested. Small quantities of Fuc-TIII, V, and/or VI messages were also detected in some leukemia cell lines in reverse transcriptase mediated-polymerase chain reaction analysis. These studies indicate that alpha-(1,3)-fucosyltransferase activities in epithelial cancer and leukemia cell lines are mixtures of multiple molecular species of alpha-(1,3)-fucosyltransferases. It is natural that epithelial cancer cells contain a significant amount of Fuc-TIII mRNA and leukemia cell lines contain Fuc-TIV mRNA, since their normal counterparts, normal epithelial cells and leukocytes, respectively, are known to contain these fucosyltransferases. The unexpectedly frequent occurrence of Fuc-TIV mRNA in epithelial cancer cell lines may be related to their retro-differentiation associated with tumorigenesis. Another unexpected finding was a weak but significant expression of the alpha-(1,3)-fucosyltransferases Fuc-TIII, V, and/or VI in leukemia cell lines detected by reverse transcriptase mediated-polymerase chain reaction analysis. Since these enzymes are known to be capable of synthesizing the sialyl Le(x) determinant, this finding implies a possibility that some of them may be involved in the synthesis of sialyl Le(x) in leukemia cells.

Preparation of mouse-human chimeric antibody to an embryonic carbohydrate antigen, Lewis Y.

Stage-specific embryonic antigen-1 (SSEA-1) is a well-known carbohydrate antigen that is specifically expressed on the surface of cancer cells as well as embryonic cells. In this study, starting with a previously established hybridoma producing a monoclonal antibody (named H18A) to Lewis Y antigen, which is closely related to SSEA-1, we cloned genomic DNA encoding active variable regions both of heavy and light chains of the antibody. Sequence analysis showed that VH and V kappa genes of H18A were in the VH 7183 family and V kappa C1 family, respectively. A transfected cell line named HC-H18A-7 expressing a recombinant chimeric H18A composed of mouse-derived antigen-binding variable regions and human-derived constant regions was established. The chimeric H18A was purified to homogeneity and shown to bind purified Lewis Y antigen with the same dose-response curve as the original H18A. The chimeric H18A looks more promising for clinical application than the original mouse-derived H18A because its antigenicity is expected to be reduced.

Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A.

Recently the lectin-like domain on ELAM-1 (endothelial leukocyte adhesion molecule-1) was shown to recognize a carbohydrate antigen, sialyl Lewis x. In this paper we demonstrate, by a series of inhibition experiments utilizing specific monoclonal antibodies and pure glycolipid preparations, that the sialyl Lewis a antigen serves as a specific ligand for ELAM-1 as well as sialyl Lewis x and plays a significant role in the ELAM-1-mediated binding of human cancer cells to activated endothelial cells.

[Carbohydrate auto-antigens in auto-immune hemolytic anemia].

Anti-erythrocyte antibodies which appear in the sera of patients with auto-immune hemolytic anemia frequently recognize carbohydrate auto-antigens. Most of cold agglutinins are known to recognize the Ii-antigens, and Donath-Landsteiner antibodies which appear in patients with paroxismal cold hemoglobinuria are almost exclusively directed to the P-antigen. These carbohydrate auto-antigens are strongly expressed in various tissues and organs other than erythrocytes, and behave as differentiation- or developmental-antigens, in both humans and mice. The study of nucleotide sequences of human and murine anti-Ii antibodies shows that these antibodies share a highly homologous antigen-binding site in their VH regions. These results indicate that the carbohydrate auto-antigens in autoimmune hemolytic anemia are evolutionally conserved developmental antigens, and suggest that the immunoglobulin genes which encode variable regions of the auto-antibodies directed to these antigens are also conserved evolutionally.

High idiotypic connectivity of the VH7183-encoded antibodies directed to a murine embryonic carbohydrate antigen, Lewis Y, as ascertained by syngenic anti-idiotype monoclonal antibodies.

The Lewis Y Ag is a carbohydrate Ag which is closely related to a well-known murine embryonic Ag, the stage-specific embryonic Ag-1 (SSEA-1), in its biochemical structure. It is expressed at the surface of murine embryonic cells as well as many murine cancer cells. For the analysis of idiotopes carried by the anti-Lewis Y antibodies, we generated two syngenic anti-idiotypic mAb, Id-A1 and Id-B4 (both BALB/c IgG1), which are directed to the idiotypic determinants carried by the anti-Lewis Y mAb, AH-6 (BALB/c IgM). Both Id-A1 and Id-B4 (Ab2) recognized paratope-related idiotopes carried by the AH-6 antibody (Ab1); they specifically inhibited the binding of AH-6 to the Lewis Y Ag. The high idiotypic connectivity of anti-Lewis Y antibodies was noted; the polyclonal anti-idiotype antibody, produced in the sera of BALB/c mice by immunizing AH-6 antibody, cross-reacted with several anti-Lewis Y mAb which has been established in different laboratories. Id-B4 and Id-A1 seem to represent such cross-reactive anti-idiotypic antibodies. Id-A1 recognized an idiotope carried by two out of six panel Ab1 mAb directed to the Lewis Y Ag. Id-B4 reacted with four out of the six panel antibodies, and was considered to recognize a recurrent idiotope of anti-Lewis Y antibodies which occurs more commonly than the idiotope recognized by Id-A1. All of the anti-Lewis Y antibodies which carry idiotopes that react with Id-A1 or Id-B4 were encoded by the VH genes of the VH7183 family; the most D-J proximal VH gene family in BALB/c mice, which is known to be preferentially expressed in embryonic B cells. Immunization of BALB/c mice with keyhole limpet hemocyanin-conjugated Id-B4 and/or Id-A1 induced a significant titer of anti-Lewis Y antibodies (Ab1-like Ab3) in the sera.

[Immunoglobulin genes encoding antibodies directed to oncodevelopmental carbohydrate antigens].

We investigated the immunoglobulin genes which encode the variable region of the monoclonal antibodies directed to the onco-developmental carbohydrate antigens such SSEA-1, fucosyl SSEA-1, SSEA-3 and SSEA-4. The VH region of these antibodies was preferentially encoded by the gene members of the X24, VH7183 and Q52 families, the families which are known to be located at the 3'-end region of the murine germ line VH gene. This result is interesting particularly when considering that the members of the 3'-end VH families are known to be preferentially expressed in embryonic B lymphocytes by an intrinsic genetic program. The comparative study of the nucleic acid sequences of mRNAs encoding these antibodies and the sequences of the corresponding germ line VH genes disclosed that the sequences encoding the antibodies contain no mutation from the germ line VH genes, or contain only a few somatic mutations, which are thought to be insignificant for the reactivity of the antibodies to the nominal antigens. These results imply that some of the embryonic B lymphocytes that express the unmutated germ line VH genes of the 3'-end families can be reactive with embryonic carbohydrate antigens, albeit rearranged with appropriate D-JH gene segments, and coupled with proper light chains. The VH region of the syngenic monoclonal anti-idiotypic antibodies directed to these anti-carbohydrate antibodies were also encoded preferentially by the members of the 3'-end VH families. We propose here that a part of the virgin embryonic B lymphocytes, which express the antibody encoded by the gene members of the 3'-end VH families at the cell surface, will be stimulated by the embryonic carbohydrate antigens which are abundantly present in the internal milieu of the embryo. The clonally expanded B lymphocytes, in turn, will facilitate the proliferation of other populations of embryonic B lymphocytes expressing the corresponding anti-idiotypic antibodies, which are also encoded by the gene members of the 3'-end VH families. This process will lead to the formation of the primitive immune idiotype network system directed to the embryonic carbohydrate auto-antigens in the embryo, which is rarely exposed to the external antigens.

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages.

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.

Characterization of a CD4(L3T4)-positive cytotoxic T cell clone that is restricted by class I major histocompatibility complex antigen on FBL-3 tumor cell.

An L3T4(CD4)+ CTL clone specific for Friend virus-induced tumor FBL-3 was isolated, characterized and compared with a conventional Lyt-2(CD8)+ CTL clone. This clone L3.1 was obtained from the limiting dilution culture of splenic MLTC cells from a CB6F1 mouse whose CD8+ T cells had been suppressed by an in vivo injection of anti-Lyt-2.2 mAb. The phenotype of clone L3.1 was sIg-, Thy-1.2+, L3T4(CD4)+, Lyt-2 (CD8)-, and Ia- as determined by flow-cytometry. Northern blot analysis also confirmed that mRNA for L3T4(CD4), but not for Lyt-2 (CD8) was present in the total RNA of L3.1. The FBL-3-specific killing activity of L3.1 was inhibited by anti-H-2D6 mAb, and the tumor cells did not express class II MHC antigen, indicating that the recognition of tumor antigen by this CD4+ CTL clone was restricted by the class I MHC molecule on the tumor cells. Furthermore, the finding that anti-L3T4(CD4) mAb GK1.5 inhibited the specific and lectin-dependent non-specific cytotoxicity of L3.1 suggested that CD4 molecules on this CTL clone are not ligand (MHC class II)-binding proteins, but are involved in signal transduction.

[Analysis of VH genes which encode the variable region of monoclonal antibodies directed to cancer-associated carbohydrate antigens].

Immune responses against cancer-associated, carbohydrate antigens are investigated by studying idiotypic determinants of specific antibodies with monoclonal anti-idiotypic antibodies, and by analyzing the structure of VH genes which encode the V region of the anti-carbohydrate antibodies. Four syngenic anti-idiotypic antibodies towards monoclonal antibodies which are specific to the sialyl Lewis A antigen and two kinds of SSEA-1 related antigens (sialyl SSEA-1 and fucosul SSEA-1) were obtained. Antibodies directed to carbohydrate antigens were mostly of IgM isotype, indicating these antigens are T-independent antigens, while anti-idiotypic monoclonal antibodies directed to those antibodies were mostly of IgG isotype, suggesting that T cells participate actively in the anti-idiotypic response. The Northern blotting analysis of VH genes of monoclonal antibodies directed to negatively-charged carbohydrate antigens such as gangliosides or sulfated glycolipids expressed the VH gene family J558 (group 1), followed by J606 (group 6) and Q52 (group 2) families. On the other hand, monoclonal antibodies directed to SSEA-1 related neutral carbohydrate antigens expressed VH genes of a minor family such as X24 (group 4), V31 (group 9), or 7183 (group 5). The same VH family as expressed in anti-SSEA-1 antibody (x24) was also expressed in the antibodies such as anti-i and anti-I antibodies, which are directed to the synthetic precursors of the SSEA-1 antigen. In either case, the antibodies directed to one particular carbohydrate antigen tended to express the VH gene of one particular family exclusively. This suggests idiotypic homogeneity of the anti-carbohydrate antibodies.

Stage-specific expression of SSEA-1-related antigens in the developing lung of human embryos and its relation to the distribution of these antigens in lung cancers.

The localization of three carbohydrate antigens, Lex, Ley, and sialylated Lex-i, which are closely related to stage-specific embryonic antigen 1, in the lung of developing human embryos was investigated using specific monoclonal antibodies. In the 38-day-old embryo, when the primitive lung bud has appeared and developed into two lung sacs, only Ley antigen was specifically positive in the proliferating cells in the terminal portion of lung bud. In the 50-53-day-old embryos, the future bronchi were actively developing from the bronchial buds. At this stage, the Ley antigen was maximally expressed and the Lex antigen appeared in the bud cells. In the lung of the 12-week-old embryo, buds for the future bronchioles were lined by simple cuboidal epithelial cells, which were strongly positive for Lex antigen, weakly positive for Ley antigen, and still completely negative for sialylated Lex-i antigen. Sialylated Lex-i antigen finally appeared in 18-week-old embryos, in the cells of the terminal buds for the future alveoli. At this stage, the Lex and Ley antigens were already beginning to disappear and were only weakly positive in cells of terminal buds. At 20 weeks, only sialylated Lex-i antigen was weakly detected in the cells in the terminal buds; after 8 months, all three antigens were essentially not detected in the respiratory cells in most of the embryos examined in this study. Formation of bronchial glands was detected at 18 weeks, where the developing gland cells were specifically positive for sialylated Lex-i antigen. Ciliation of the bronchial epithelial cells started at 12 weeks and propagated thereafter. The ciliation was accompanied by the reappearance of Ley and Lex antigen in the epithelial cells. These findings collectively indicated that the three antigens all have a physiological significance as stage-specific developmental antigens of the human lung; those antigens were specifically present in the bud cells at each important step of the morphogenesis of the human lung, such as cells in the lung buds, bronchial buds, and terminal buds for the formation of the alveolus, and cells differentiating into bronchial gland cells. The three antigens gradually disappear in the later stage of development along with the maturation process of the lung. Stage-specific embryonic antigen 1 and related antigens are known to be associated with various human cancers, including lung cancers. We suggest that the expression of these antigens in the lung cancer cells is the result of the retrodifferentiation of the cancer cells to the stages of immature embryonic lung cells.

Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing epitopes consisting of sialyl or sialyl-fucosyl type 1 chain.

The levels of carbohydrate antigens having epitopes consisting of type 1 chain (R----Gal beta 1----GlcNAc beta 1----3Gal beta 1----R) in the sera of patients with various malignant and nonmalignant disorders have been investigated with the use of three monoclonal antibodies, N-19-9, FH-7, and FH-9. Serum levels of 2----3 sialylated Lea antigen and 2----6 sialylated Lea antigen, defined respectively by antibodies N-19-9 and FH-7, were found to be frequently high in patients with cancer of the digestive system, particularly pancreatic cancer. High levels of 2----3,2----6 disialylated Lc4 antigen, defined by antibody FH-9, were less frequent in cancer patients when compared with the other two antigens. In patients with nonmalignant disorders, especially renal and autoimmune diseases, serum levels of the two type 1 chain antigens defined by FH-7 and FH-9 were more frequently high than that defined by N-19-9. Molecular weights and other general biochemical characteristics of serum mucin carrying the type 1 chain determinants were not significantly different in cancer patients as compared with patients with nonmalignant disorders. However, the degree of glycosylation of the antigen, as assessed by its solubility in perchloric acid, showed significant differences; i.e., the mucin antigen carrying 2----6 sialylated Lea determinant in the sera of patients with nonmalignant disorders had the highest carbohydrate/protein ratio, followed by the mucin carrying the same determinant in the sera of cancer patients. Mucin antigen carrying 2----3 sialylated Lea antigen or 2----3, 2----6 disialylated Lc4 antigen in cancer patients had the lowest carbohydrate/protein ratio among the four groups tested. Thus, the carbohydrate/protein ratio in the type 1 chain mucin antigens in sera of normal subjects is higher than that in sera of cancer patients (P less than 0.05). This finding is in contrast to previous findings on the mucin antigens carrying the type 2 chain determinant (R. Kannagi et al., Cancer Res., 46: 2619-2626, 1986), in which the mucin antigen in cancer patients was found to have a much higher carbohydrate/protein ratio than that carrying the same antigenic determinants in patients with nonmalignant disorders.