Molecular mechanisms of expression of Lewis b antigen and other Type I Lewis antigens in human colorectal cancer

Shoko Nishihara, Tsuneo Hiraga, Yuzuru Ikehara, Takashi Kudo, Hiroko Iwasaki, Kyoei Morozumi1, Suguru Akamatsu2, Tetsuya Tachikawa2 and Hisashi Narimatsu3

Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan, 1Department of Surgery, Fussa Hospital, 1-6-1 Kamidaira, Fussa, Tokyo 197-8511, Japan and 2Diagnostic Division, Otsuka Pharmaceutical Co. Ltd., 224-18 Kawauchi-machi, Tokushima 771-0132, Japan

Received on September 7, 1998; revised on November 24, 1998; accepted on November 25, 1998

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Leb amounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with [alpha]2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.

Key words: colon cancer/[alpha]1,2fucosyltransferase/H enzyme/ Leb antigens/Se enzyme

Introduction

The Lewis b (Leb) antigens are expressed throughout the colon during fetal life, but after birth they gradually disappear in the distal colon, there being limited expression in the proximal colon in adults, while Lewis a (Lea) antigens exhibit a pan-colonic distribution even after birth (Yuan et al., 1985; Jass and Roberton, 1994). Human colorectal carcinomas progress through several histopathologically distinct stages, beginning with hyperproliferation of the epithelium with dysplasia, followed by adenoma and carcinoma formation, and ultimately metastasis. Many immunohistochemical studies have revealed that the Leb and Lewis y (Ley) antigens reappear at the adenoma stage of tumor development and then increase with tumor progression, which occurs irrespectively of the secretor status and particularly in all cases in distal colon tissues (Ernst et al., 1984; Yuan et al., 1985; Abe et al., 1986; Sakamoto et al., 1986). Leb reappearance has been observed in distal adenomatous polyps (Aps), but not in distal hyperplastic polyps (Hps) (Itzkowitz, 1992). Aps are considered to have malignant potential, while Hps are not. Therefore, Leb antigen reappearance is the suitable tumor marker for distinguishing malignant tumors from benign ones, and also can be applied to many cases of colon cancers, because more than two-thirds of them occur in the distal colon.

With respect to the biological activities of the [alpha]1,2fucosylated Lewis antigens, many investigators have reported that alterations in Lewis antigen expression affect tumor cell characteristics, as follows. (1) Statistical analyses of the patient prognosis with lung and colon carcinomas demonstrated that a higher level of H, Leb, and Ley antigen expression is correlated with a worse prognosis of the patients (Miyake et al., 1992; Naitoh et al., 1994). These antigens were also proposed to participate in the control of cell motility and tumor cell metastasis (Miyake and Hakomori, 1991). (2) The increased expression of the [alpha]1,2fucosylated structure on cells has been shown to be correlated with their increased tumorigenicity, and their increased resistance to cytotoxicity mediated by NK or LAK cells (Zennadi et al., 1992; Goupille et al., 1997). (3) The [alpha]1,2fucosylation of CD44v in rat colon carcinomas contributes to the tumorigenicity (Labarriere et al., 1994; Goupille et al., 1997).

As shown in Figure 1, the H type I structure is synthesized through the transfer of fucose (Fuc) to the type I structure, which is catalyzed by [alpha]1,2fucosyltransferases ([alpha]1,2Fuc-Ts), and then the Le enzyme sequentially transfers Fuc to the H type I structure to synthesize Leb antigens. Two human [alpha]1,2Fuc-T genes, which encode the H and Se enzymes, have been cloned to date (Larsen et al., 1990; Kelly et al., 1995). We concluded in a series of previous studies that the Se enzyme is solely responsible for Leb expression on erythrocytes and solely determines the secretor status, whereas ABH antigen expression on erythrocytes is solely determined by the H enzyme (Kudo et al., 1996; Kaneko et al., 1997). In colon tissues, both [alpha]1,2Fuc-Ts, i.e., the Se and H enzymes, are expected to be involved in the synthesis of the H-type I structures finally resulting in the synthesis of Leb antigens, although there have been no tangible proofs on their contribution and no detailed studies on the tissue distributions of the H and Se enzymes in colon tissues.


Figure 1. Biosynthetic pathway of Type I Lewis antigens in normal and cancerous colon tissues.

One of the five homologous [alpha]1,3Fuc-Ts (Goelz et al., 1990; Kukowska-Latallo et al., 1990; Koszdin and Bowen, 1992; Weston et al., 1992; Natsuka et al., 1994; Sasaki et al., 1994), Fuc-TIII, has been identified as the Lewis (Le) enzyme that determines the expression of the Lea and Leb antigens on erythrocytes (Koda et al., 1993; Nishihara et al., 1993, 1994; Mollicone et al., 1994). We demonstrated in the previous studies involving Le gene mutant analysis that the Le enzyme is localized in the Golgi area of colon epithelial cells, and is solely responsible for the synthesis of fucosylated type I Lewis antigens, i.e., Lea, Leb, and sialyl Lewis a (sLea) antigens, in colon tissues (Kimura et al., 1995; Narimatsu et al., 1996). A series of mutant analyses of the Le gene revealed that homozygotes with the inactive Le gene (le/le) genetically lack the Le enzyme and never express any of the type I Lewis antigens, i.e., the Lea, Leb, and sLea antigens, on erythrocytes or in colon tissue.

We have established a polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) method for distinguishing three kinds of Se alleles, two active alleles designated as Se1 and Se2, and one less active allele designated as sej (Kudo et al., 1996), and four kinds of Le allele, one active allele designated as Le, and three inactive alleles designated as le1, le2, and le3, which are rather widely distributed in the Japanese population (Nishihara et al., 1994; Narimatsu et al., 1998). Koda et al. recently reported the finding of another inactive se5 allele in the Japanese population, which arose through a crossover between two genes, the Se and Sec1 genes (Koda et al., 1996). In the present study, we determined the Se and Le genotypes of patients by the PCR-RFLP method (Nishihara et al., 1994; Kudo et al., 1996; Narimatsu et al., 1996), because the dosages of the Se and Le genes, i.e., the zygosities of the two genes, are known to profoundly influence the production of the type I Lewis antigens in colon tissues (Ørntoft et al., 1996; Narimatsu et al., 1998).

Carcinomatous rectal tissues have been reported to exhibit significantly higher [alpha]1,2Fuc-T activity, and higher levels of ABH and Leb antigens than noncancerous tissues of the same individuals, irrespective of the secretor status (Sakamoto et al., 1986; Ørntoft et al., 1991; Jass and Roberton, 1994). H gene transcription was recently demonstrated by reverse transcribed-polymerase chain reaction (RT-PCR) to be upregulated in association with human colon adenocarcinoma progression (Sun et al., 1995), although they did not perform quantitative analysis, i.e., H transcript measurement. They did not mention anything at all regarding the Se gene transcription in colon cancers.

In the present study, we determined the actual amounts of both [alpha]1,2Fuc-T transcripts, i.e., the H and Se transcripts, and the Le transcripts expressed in cancer tissues and adjacent noncancerous tissues by the competitive RT-PCR method, and compared them with the level of Leb antigen expression, considering the secretor status, which was determined by Se genotyping. We also examined whether or not on the in vivo syntheses of the Lea, Leb, and sLea antigens there was any competition for the acceptor substrate.

We present here noteworthy results demonstrating upregulated expression of both [alpha]1,2Fuc-Ts in cancer tissues, the Se gene dosage effect on the expression of Leb antigens in noncancerous and cancerous tissues, and the competition between Leb antigen synthesis and Lea or sLea antigen synthesis. Based on the results regarding glycosyltransferase expression, the phenomenon of the Leb-increase in cancer tissues will be discussed in relation with colon tissue development.

Results

Characterization of monoclonal antibodies (mAbs), 2DG8 and TT42.

Specificities of each monoclonal antibody, 2DG8 or TT42, to chemically synthesized oligosaccharides, Lea, Leb, H type I, Lex, Ley, and H type II, were shown in Figure 2a,b. 2DG8 and TT42, which were used in the following experiments, revealed the specificities restricted to Lea and Leb oligosaccharides, respectively.


Figure 2. Specificity of anti-Lea mAb, 2DG8 (a) and anti-Leb mAb, TT42 (b). Specificies of each antibody to synthesized oligosaccarides, Lea, Leb, H type I, Lex, Ley and H type II, was determined by ELISA.

Immunohistochemical staining of various noncancerous colorectal regions with anti-Leb and anti-Lea mAbs

The staining intensity on microscopy observation was classified into six degrees, 0-5, as shown in Figure 3. Only one le/le individual did not show any staining with anti-Leb or anti-Lea mAb throughout the whole colon. The gradient expression of the Leb antigen from the ascending colon to the rectum was observed in almost all the 18 Lewis-positive individuals regardless of the secretor status (Figure 3a). All 15 secretors having at least one of active Se allele (Se/-) apparently showed stronger staining with anti-Leb throughout the whole colon than the three nonsecretors (se/se). As to the Lea staining in Figure 3b, the three nonsecretors apparently showed stronger staining of Lea than the secretors throughout the whole colon, while many of 14 secretors showed a gradual increase in Lea expression from the right hemi-colon to the left hemi-colon, which is the reverse of in the case of the Leb antigen. The positive staining with anti-Lea was colocalized in the cells stained with anti-Leb (data not shown).


Figure 3. Immunohistochemical staining of noncancerous colon tissues with anti-Leb mAb (a), and anti-Lea mAb (b). Open circles, Le/- and Se/-; closed circles, Le/- and sej/sej or se5/se5; closed squares, le/le. The vertical scales indicate the staining intensities determined on microscopic observation. The numbers on each line indicate those of individuals. For example, 2* indicates that there were two individuals who showed the 4th, 3rd and 3rd degrees of positive staining in the ascending, descending and rectum, respectively.

The above staining results indicated that expression of the Leb and Lea antigens is apparently influenced by the Se gene dosage, and the Se enzyme is definitely involved in part in the synthesis of the Leb antigen throughout the whole colon.

Table I. Genotyping and immunohistochemical staining of colorectal tissue
Colon region Genotype Immunohistochemical staining by anti-Leb mAb
  Lewis Secretor Noncancerous Cancerous
- ± + - ± +
Right Le/- Se/- 0/9 0/9 9/9 0/9 0/9 9/9
Le/- se/se 0/3 3/3 0/3 0/3 0/3 3/3
le/le Se/- 1/1 0/1 0/1 1/1 0/1 0/1
Left Le/- Se/- 17/22 5/22 0/22 0/22 0/22 22/22
Le/- se/se 11/11 0/11 0/11 0/11 0/11 11/11
le/le Se/- 4/4 0/4 0/4 4/4 0/4 0/4
Le/- represents Le/Le; Se/- represents Se/Se or Se/se.

Immunohistochemical staining of cancerous colon tissues with anti-Leb and anti-Lea mAbs

As shown in Table I, expression of the Leb antigen in noncancerous and cancerous tissues definitely required the Le enzyme, i.e., five homozygotes with the null Le allele (le/le) showed no staining with anti-Leb mAb. However, all colon cancer tissues from 50 patients with the Lewis-positive genotype (Le/-) showed strongly positive staining with anti-Leb mAb, irrespective of the secretor status or the region of the colon cancer. The results strongly suggested that an [alpha]1,2Fuc-T other than the Se enzyme, probably the H enzyme, contributes to the expression of the Leb antigen in colon cancer tissues.

Western blotting analysis of right and left hemi-colon tissues with anti-Leb mAb

As can be seen in Figure 4a, mucin-like proteins with high molecular weights were mainly detected as smear bands with the anti-Leb antibody. The amounts of Leb antigens are plotted in Figure 4b. The se5 allele was not found in the patients whose specimens were used in the following experiments. The sej allele was the only one negative allele for the Se enzyme that could be detected in these patients. It was clearly demonstrated that the Leb antigens are much more abundant in the right hemi-colon than in the left hemi-colon, and the Se enzyme definitely affects the Leb expression because the secretors (Se/-) obviously exhibited stronger intensities of Leb bands than the nonsecretors (sej/sej), regardless of whether it was the right or left hemi-colon. On comparison of the five right hemi-colon samples from the Se/sej individuals and the five left hemi-colon samples from the Se/sej individuals, the amounts of Leb antigens in the right hemi-colon were found to be almost 10 times greater than those in the left hemi-colon. In the right hemi-colon, the amounts of Leb antigens expressed in the three sej/sej individuals were about one-fourth of that in the five Se/sej individuals. This confirmed that Leb antigens in the noncancerous colon tissues were mainly synthesized by the Se enzyme. The residual expression of Leb antigens in the colon tissues of sej/sej individuals was considered to be synthesized by another [alpha]1,2fucosyltransferase, probably by the H enzyme.


Figure 4. Quantitative analysis of Leb antigens on glycoproteins by Western blotting. The se5 allele was not found in the patients whose specimens were used in the following experiments. The sej allele was the only one negative allele for the Se enzyme that could be detected in these patients. (a) A representative pattern on Western blot analysis of noncancerous colon tissues with anti-Leb mAb. Each point in (b) and (c) indicates a single individual. The intensities of positive bands of each individual were determined by a densitometer to estimate the amounts of Leb antigens. (b) The amounts of Leb antigens in noncancerous right and left hemi-colorectal tissues of the patients who had the Se genotype. (c) The amounts of Leb antigens in cancerous colon tissues of the patients who had the Se genotype. (d) The amount of Leb antigens in noncancerous and cancerous colon tissues of the patients. The noncancerous and cancerous samples from the same patient are connected by solid (left hemi-colon) and dotted (right hemi-colon) lines. Closed circles, left hemi-colorectal tissues of secretor (Se/-) patients; closed triangles, left hemi-colorectal tissues of nonsecretor (sej/sej) patients; open circles, right hemi-colorectal tissues of secretor (Se/-) patients; open triangles, right hemi-colorectal tissues of nonsecretor (sej/sej) patients.

Augmented expression of Leb antigens on glycoproteins in colon cancers

The amounts of Leb antigens in the 14 cancerous tissue specimens from the above patients were also plotted in Figure 4c, in which a Se gene dosage effect on the amount of the Leb antigen was observed in both the left and right hemi-colon cancerous tissues as in the case of noncancerous tissues. This Se gene dosage effect in the cancerous tissues was not demonstrated by the immunohistochemical study in the previous section, since immunohistochemistry is not quantitative.

Figure 4d shows the degree of augmented expression of Leb antigens in cancerous tissues compared to that in the noncancerous tissue in the 14 patients. A dramatic increase in Leb antigens was observed in the secretor patients (Se/-) with left-hemi colon cancers, since the noncancerous tissue of the left hemi-colon originally expressed low amounts of Leb antigens. The left hemi-colon cancerous tissues of the nonsecretors (sej/sej) also showed an increase in Leb antigens, but the extent of the increase was not so great. The right hemi-colon cancerous tissues of the secretors also showed a significant increase, of which the extent was not as high as that in the left hemi-colon cancers of the secretors, since the noncancerous right hemi-colon tissue originally expressed substantial amounts of Leb antigens. The right hemi-colon cancer tissues of the three nonsecretors showed a very small increase.

These results strongly suggested that the Leb antigen in colon cancer tissues was mainly synthesized by the Se enzyme, as in the case of noncancerous colon tissues. The residual expression of the Leb antigen in colon cancer tissues of sej/sej patients should be due to another [alpha]1,2Fuc-T, probably by the H enzyme.

Quantitative analysis by competitive RT-PCR of three glycosyltransferase transcripts for the H, Se, and Le genes

The amounts of the transcripts for three glycosyltransferase genes expressed in the noncancerous right and left hemi-colon tissues were measured and are plotted in Figure 5a-c. The competitive RT-PCR method can reveal the relative amounts of the transcripts as to each other. Note the different scales of the transcript amounts. The amount of Se transcripts was almost 20 times greater than that of H transcripts. The Le transcripts were most abundantly expressed among the three enzymes. The Se and Le transcripts were apparently more abundantly expressed in the noncancerous tissues of the right hemi-colon, approximately from two to three times, than in those in the left hemi-colon. This is in good agreement with the gradient expression of Leb antigen from the proximal colon to the distal colon. In contrast, the H enzyme was constantly expressed in the right and left hemi-colon. The Se and Le enzymes are considered to be responsible for the gradient expression of the Leb antigen in noncancerous colon tissues.


Figure 5. The amounts of three glycosyltransferase transcripts, H, Se and Le, in noncancerous and cancerous colon tissues determined by competitive RT-PCR. (a-c) The amounts of each gene transcript in noncancerous right and left hemi-colorectal tissues. Each point indicates a single individual. (d-f) The amounts of each gene transcript in noncancerous and cancerous colorectal tissues from the same patient are connected by solid (left hemi-colon) and dotted (right hemi-colon) lines. Closed circles, left hemi-colorectal tissues; open circles, right hemi-colorectal tissues.

Figure 5d-f shows upregulation or downregulation of the transcripts of the three genes expressed in the cancerous colon tissues of 16 patients. They were 8 patients with right hemi-colon cancers (open circles with dotted lines) and 8 with left hemi-colon cancers (solid circles with solid lines). The expression of H transcripts was markedly augmented in all 16 cases, in both right and left hemi-colon cancer tissues compared to in the respective noncancerous tissues. On the other hand, the expression of Se transcripts was markedly augmented in all 8 left hemi-colon cancer tissue specimens, however, it was not significantly augmented in the cases of right hemi-colon cancers. The Se transcripts in some right hemi-colon cancers were rather downregulated, in which the original level of Se transcripts in noncancerous tissue was relatively high. The level of Le transcripts was upregulated in neither the right hemi-colon cancer nor the left hemi-colon cancer, except in four cases. The Le enzyme was rather downregulated in some cancer tissues. The H and Se enzymes were considered to be responsible for the augmented expression of the Leb antigen, particularly in left hemi-colon cancers.

Relative activities of H, Se, and Le enzymes toward Type I and Type II acceptor substrates

When examining the correlation of the amount of transcripts for each glycosyltransferase gene with the amounts of Leb antigens, the relative activity of each enzyme toward the acceptor substrates should be taken into consideration. The amount of each recombinant enzyme in Namalwa cells, which stably expressed each enzyme, was determined by measurement of the respective transcripts. Cell lysates containing a fixed amount of each enzyme were subjected to the enzyme activity assay with the fixed amount of each substrate. The results are summarized in Table II, in which the relative activity for H type I chain synthesis by the H enzyme toward lacto-N-tetraose (LNT) is expressed as 100% in comparison with the activities of the other enzymes. The H enzyme exhibited five times stronger activity as to the H-type I chain synthesis toward LNT than the Se enzyme.

Correlation between the amounts of Leb antigens and the amounts of H, Se, and Le transcripts which were normalized as to the relative activity

It is uncertain whether or not the two times larger amounts of the Se and Le enzymes in the right hemi-colon are the only cause of the increased expression of Leb antigens in the right hemi-colon in comparison with that in the left hemi-colon. In fact, we observed that the amount of Leb antigens in the right hemi-colon is almost 10 times greater than that in the left hemi-colon (Figure 4a,b). It is also uncertain whether or not the 3 times augmentation of the H and Se enzymes in cancerous tissues of the left hemi-colon is the only cause of the augmented expression of Leb antigens. In fact, we observed that the amount of Leb antigens in left hemi-colon cancerous tissues was more than 10 times greater than that in noncancerous left hemi-colon tissues (Figure 4c,d). In order to determine the relation between the amounts of transcripts and the amounts of Leb antigens, the relative activities determined in the previous section and the zygosity of the polymorphic Se alleles should be taken into consideration. The RT-PCR-RFLP method was applied to total cellular RNAs to determine whether the inactive sej allele was transcribed or not. The sej allele of all individuals possessing the sej allele was found to be transcribed equally with the active Se allele (data not shown).

Table II. Acceptor substrate specificities of the fucosyltransferases, i.e., the H, Se, and Le enzymes, expressed in Namalwa cells
Substrate Relative activity (%)a
H enzyme Se enzyme Le enzyme
Gal[beta]1-3GlcNAc[beta]1-3Gal[beta]1-4Glc (LNT) 100 21 99
Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc (LNnT) 50 1.5 N.D.
aThe expression levels of three fucosyltransferases relative to the level of [beta]-actin were determined by competitive PCR using the respective cDNAs, which were reverse-transcribed from RNA samples. Each enzyme reaction was carried out using the same amount of each enzyme and the same amount of an acceptor substrate. The values were comparable between different kinds of enzyme reactions.
N.D., Not determined.

The sej enzyme derived from the sej allele is known to exhibit partial activity, i.e., 3% of the activity of the Se enzyme derived from the wild-type Se allele. Two parameters, the Se genotype of each patient, and the relative [alpha]1,2Fuc-T activities of the H and Se enzymes as to H-type I chain synthesis, were taken into consideration to determine [alpha]1,2Fuc-T activity as to the H type I synthesis on the basis of the amounts of the Se and H transcripts. For example, the amount of the Se transcripts in the Se/sej patients was reduced to 103/200 of the original amount, and the amount of the Se transcripts in the sej/sej patients was reduced to 3% of the original amount. The amounts of H transcripts were 5 times higher, because of the 5 times stronger activity than that of the Se enzyme as to the H-type 1 synthesis. The modified amounts of Se and H transcripts were combined and are plotted in Figure 6a as the estimated-[alpha]1,2Fuc-T activity as to H type I synthesis directed by both the H and Se enzymes. The estimated-[alpha]1,2Fuc-T activity was significantly upregulated in both the right and left hemi-colon cancers, particularly in the secretors, in which the upregulation of both enzymes was combined. The upregulation of the estimated-[alpha]1,2Fuc-T activity in the nonsecretors was less than that in the secretors, reflecting the upregulation of the H enzyme alone.


Figure 6. Estimated-[alpha]1,2Fuc-T activity as to synthesis of the H type I structure in noncancerous and cancerous tissues, and the correlation between the estimated-[alpha]1,2Fuc-T activity and the amounts of Leb antigens. The estimated-[alpha]1,2 Fuc-T activity was obtained as ((Se transcripts normalized with Se genotype /[beta]-actin transcripts) + 5 x (H transcripts / [beta]-actin transcripts )). (a) The estimated-[alpha]1,2 Fuc-T activity in noncancerous and cancerous tissues from the same patient is connected by solid (left hemi-colon) and dotted (right hemi-colon) lines. Closed circles, left hemi-colon tissues of secretor (Se/-) patients; closed triangles, left hemi-colorectal tissues of nonsecretor (sej/sej) patients; open circles, right hemi-colorectal tissues of secretor (Se/-) patients; open triangles, right hemi-colorectal tissues of nonsecretor (sej/sej) patients. (b) The correlation between the estimated-[alpha]1,2 Fuc-T activity and the amounts of Leb antigens in noncancerous and cancerous tissues. Closed circles connected with a solid line, left hemi-colorectal noncancerous tissues (NL); closed squares connected with a solid line, left hemi-colorectal cancerous tissues (CL); open circles connected with a dotted line, right hemi-colorectal noncancerous tissues (NR); open squares connected with a dotted line, right hemi-colorectal cancerous tissues (CR).

The estimated-[alpha]1,2Fuc-T activity was correlated with the amounts of Leb antigens. As shown in Figure 6b, linear lines were obtained, indicating that the estimated-[alpha]1,2Fuc-T activity was proportional to the amounts of Leb antigens in each cancerous and noncancerous tissue specimen, irrespective of whether it was the right or left hemi-colon. This strongly indicated that the Se and H enzymes are both key enzymes determining the amounts of Leb antigens. The slopes of the proportional linear lines for the three tissues, i.e., noncancerous right hemi-colon tissues (NR), cancerous tissue of the right hemi-colon (CR), and cancerous tissue of the left hemi-colon (CL), gave almost the same values, while the value of the slope for the noncancerous left hemi-colon tissues was very small (NL).

Effect of the Se genotype on sLea and Lea antigens in colon tissues

Figure 7a shows representative patterns on Western blotting analysis of cancerous tissues with the anti-sLea antibody. Smear bands corresponding to high molecular weights, 200 kDa and 350 kDa, were mainly observed. A Se gene dosage effect on the amounts of the sLea antigens was apparently observed (Figure 7b). The colon cancer tissues from nonsecretor (sej/sej) patients exhibited significantly larger amounts of sLea antigens than those from secretor (Se/-) patients, regardless of whether it was the right or left hemi-colon.


Figure 7. Quantitative analysis of sLea and Lea antigens on glycoproteins by Western blotting. (a) A representative pattern on Western blot analysis of cancerous colon tissues with anti-sLea mAb. Each point in (b-d) indicates a single patient. (b) The amounts of sLea antigens in cancerous right and left hemi-colorectal tissues from the patients who had the Se genotype. (c) The amounts of Lea antigens in noncancerous (c) and cancerous (d) colon tissues from the patients who had the Se genotype.

As can be seen in Figure 7c, the amounts of Lea antigens were greater in the nonsecretors than in the secretors in the noncancerous tissues of both the right and left hemi-colon. The difference in the right hemi-colon was much clearer than that in the left hemi-colon, in which the Leb antigen was only faintly expressed. This indicated that there is competition of the Se enzyme with the Le enzyme for type I chain acceptors, as proposed in the biosynthetic pathways in Figure 1. A Se gene dosage effect on the amounts of the Lea antigens in the cancerous tissues was also observed. The amounts of Lea antigens in cancerous tissues from the sej/sej patients were apparently greater than those in the secretor patients (Se/-; Figure 7d).

Discussion

The results in the present study are summarized in Figure 8, as follows. (1) An immunohistochemical study (Table I, Figure 3) and Western blotting analysis demonstrated that the expression of Leb antigens in noncancerous colon tissues showed a clear gradient from the right to the left hemi-colon, while that of Lea antigens showed the reverse. The Se enzyme mainly contributes to the gradient expression of Leb antigens in the noncancerous colon. Although the transcripts of the Le enzyme are gradiently expressed from the right to the left in the noncancerous colon, the Le enzyme is unlikely to contribute to the gradient Leb expression because of the reason as described in the later section. The H enzyme, which is expressed constantly throughout the whole colon, does not contribute to the gradient expression of the Leb and Lea antigens. (2) The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. This suggests that the Se enzyme competes with [alpha]2,3sialyltransferases and the Le enzyme for the type I acceptor substrates. It is a matter of course that le/le individuals do not express the type I Lewis antigens at all. (3) Considering the Se genotype, and the relative activities of the H and Se enzymes, the amounts of Leb antigens were demonstrated to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme makes a greater contribution to determining the Leb amounts than the H enzyme. (4) In cancerous tissues, the Leb and sLea antigens become strongly expressed throughout the whole colon. The degree of augmentation of Leb expression is much more dramatic in cancerous tissues of the left hemi-colon than in those of the right hemi-colon. The marked augmentation of Leb antigens in the left hemi-colon cancer tissues is caused mainly by the upregulation of the Se enzyme and partly by the H enzyme, while slight augmentation of Leb antigens in right hemi-colon cancer tissues is caused by upregulation of the H enzyme alone. The Se enzyme is already expressed abundantly in the noncancerous tissues of the right hemi-colon, so it is not upregulated in the cancer tissues of the right hemi-colon.


Figure 8. Schematic diagrams showing the distributions of type I Lewis antigens, and the transcripts for the Se, H and Le genes in noncancerous and cancerous colon tissues.

The patterns of Leb and Lea expression observed in this study were consistent with the results of many immunohistochemical analyses previously reported by others (Ernst et al., 1984; Yuan et al., 1985; Jass and Roberton, 1994), although some reports mentioned the constant expression of Lea antigens throughout the whole colon. The reverse-gradient Lea expression, compared with the Leb gradient, suggests that there is competition between Lea and Leb syntheses, probably between [alpha]1,2-fucosylation and [alpha]1,4-fucosylation, when the amounts of acceptor substrates are limited.

The Km values of the purified human H and Se enzymes demonstrated that the Se enzyme preferentially transfers a fucose to the type I precursor, and that the H enzyme can transfer one to the type I and type II precursors almost equally (Sarnesto et al., 1992). However, the relative activity toward the type I precursor of the H enzyme as to that of the Se enzyme has not been reported yet. In the present study, the H enzyme exhibited five times stronger activity than the Se enzyme toward the type I precursor.

Considering the two results, i.e., the presence of Se transcripts, in an almost twenty times higher amount than H transcripts, and the relative [alpha]1,2Fuc-T activity of the Se enzyme, which is one-fifth that of the H enzyme, we concluded that four-fifths of the Leb antigen is synthesized by the Se enzyme and one-fifth by the H enzyme in colon tissues of secretors with the Se/Se genotype. In the case of nonsecretors (se/se) and secretors with the Se/se genotype, the Se gene dosage profoundly affected the amounts of Leb antigens. The residual expression of Leb antigens in the cancerous tissues of nonsecretors was due to the H enzyme, and was enough for the immunohistochemically positive-staining of colon cancers with the anti-Leb mAb.

The [alpha]1,2Fuc-T activities have been measured, using phenyl-[beta]-d-galactose and lacto-N-biose I as acceptor substrates, in colorectal cancer tissues (Ørntoft et al., 1991). The colorectal tissues of nonsecretors were shown to exhibit one-fifth the [alpha]1,2Fuc-T activity toward phenyl-[beta]-d-galactose, and one-third that towards lacto-N-biose I, compared to those of secretors (Ørntoft et al., 1991). The ratios of the activities between secretors and nonsecretors were quite consistent with our results.

The values of the slopes in Figure 6b are considered to be determined by the [alpha]1,4Fuc-T activity of the Le enzyme and the concentrations of type I precursors. The Le enzyme transfers Fuc to the H type I chain to synthesize the Leb antigen as a final step. The null Le alleles, le1 and le2, do not exhibit enzyme activity at all (Nishihara et al., 1993, 1994; Mollicone et al., 1994), and the le1 and le2 alleles were determined to be fully transcribed, like wild type Le allele (Kudo et al., 1998). Considering the Le genotype of the patients, we also normalized the amounts of Le transcripts to obtain the estimated-[alpha]1,4Fuc-T activity as to the Leb synthesis. However, the estimated-Le enzyme activity showed no correlation with the amounts of Leb antigens in any colon tissue (data not shown), suggesting that the Le enzyme is not a key enzyme determining the amounts of Leb antigens, probably because it is abundantly expressed at a saturated level even in the noncancerous left hemi-colon.

The amounts of Le transcripts were almost thirty times and three times greater than those of the H and Se transcripts, respectively. The relative activity toward the type I chain substrate of the Le enzyme was determined to be almost as the same as that of the H enzyme, and five times stronger than that of the Se enzyme (Table II). Nevertheless, the Le enzyme did not participate in determination of the amounts of Leb antigens. This indicated that [alpha]1,2Fuc-Ts, H and Se, transfer Fuc to type I chain acceptors, followed by [alpha]1,4-fucosylation by the Le enzyme, probably because the H and Se enzymes are localized in more proximal Golgi compartments in the cells than the Le enzyme is. Thus, the Golgi subcompartmentation of glycosyltransferases, which is difficult to examine, will become one of the most important factors for elucidating what glycosyltransferase(s) determine the amounts of the respective carbohydrate structures, in addition to the relative activities of the enzymes and the amount of each enzyme expressed.

Thus, the amounts of Leb antigens were found to be determined by two [alpha]1,2Fuc-Ts, the H and Se enzymes, not by the Le enzyme, as shown by the linear proportions in Figure 6b. However, the line for the noncancerous right hemi-colon (NR) was apparently steeper than that for the noncancerous left hemi-colon (NL). This strongly suggested that another factor(s) is involved in determination of the amounts of Leb antigens, which gives rise to the different values of the slopes. One possibility is that the amount of the precursor structure of Lewis antigens, the type I chain, Gal[beta]1,3GlcNAc-R, differs between NR and NL. NR may express more type I chains than NL. Very recently, four kinds of [beta]1,3Gal-T genes, which are candidate genes for type I chain synthesis, were cloned (Amado et al., 1998; Hennet et al., 1998; Kolbinger et al., 1998). A study on [beta]1,3Gal-T gene expression in colon tissue will be performed in the future to answer the above question.

We also aimed to determine which glycosyltransferase is a key enzyme as to the augmented expression of Leb antigens in cancer tissues. The straight lines for right hemi-colon cancers (CR) and left hemi-colon ones (CL) in Figure 6b indicated that the amounts of Leb antigens are determined by the [alpha]1,2Fuc-T activity directed by both the H and Se enzymes. We concluded that the upregulation of both the H and Se enzymes in left hemi-colon cancers and that of the H enzyme alone in right hemi-colon cancers are responsible for the augmented expression of Leb antigens in cancerous tissues. The Se gene was not upregulated in right hemi-colon cancers, it probably already being expressed at saturated levels even in noncancerous right hemi-colon tissues. The value of the slope of the CL straight line was very close to those of the slopes of NR and CR (Figure 6b). This indicated that the type I chains in the left hemi-colon cancers become close to the levels in noncancerous and cancerous right hemi-colons. The synthesis of the type I chain may be upregulated in left hemi-colon cancerous tissues. Recently [beta]1,3-N-acetylglucosaminyltransferase (Sasaki et al., 1997), four [beta]1,3Gal-Ts (Amado et al., 1998; Hennet et al., 1998; Kolbinger et al., 1998), and six [beta]1,4Gal-Ts (Narimatsu et al., 1986; Almeida et al., 1997; Lo et al., 1998; Sato et al., 1998), which would affect the amount of type I chain acceptor substrate, have been cloned. In case of N-linked sugar chains on carcinoembryonic antigen, Seko et al. proposed that the balance between [beta]1,3Gal-T and [beta]1,4Gal-T activities determines the ratio of type I chain and type II chain (Seko et al., 1996). The regulation of type I chain synthesis, the precursor structure for Leb, will be analyzed in the future by examining the multiple [beta]1,3Gal-Ts and [beta]1,4Gal-Ts.

We demonstrated in the previous study that the Se gene dosage profoundly affects the serum CA19-9 (sLea) levels in normal individuals and colorectal cancer patients, suggesting competition between [alpha]1,2-fucosylation and [alpha]2,3-sialylation (Narimatsu et al., 1998). We demonstrated in the present study that this effect was also the case at the tissue level, i.e., the Se gene dosage negatively affected the amounts of the sLea and Lea antigens in both noncancerous and cancerous tissues, irrespective whether it was the right or left hemi-colon. By plotting the amounts of Lea antigens versus either the estimated-[alpha]1,2Fuc-T activity or the amounts of Leb antigens, we obtained linear lines exhibiting the inverse proportion of the former to either of the latter (data not shown). Thus, Leb synthesis actually competed with Lea and sLea syntheses in colon tissues.

Materials and methods

Tissue samples and monoclonal antibodies

We collected biopsy samples of noncancerous colon tissue from 19 colon cancer patients at the time of colon fiber examination for diagnosis, and the samples were subjected to immunohistochemical analysis. Fifty colon cancer tissue specimens were also preserved for immunohistochemical staining. For Western blotting and competitive RT-PCR analysis, cancerous tissues and noncancerous tissues adjacent to the cancerous region, comprising histologically the tunica mucosa with muscularis mucosae and being devoid of the muscularis propria, were prepared from the surgical samples at the Department of Surgery, Fussa Hospital (Tokyo). All cancer specimens were determined to be advanced colon cancers by pathological diagnosis. These samples were also subjected to immunohistochemical analysis. The monoclonal antibodies used in this study were 2DG8 (anti-Lea: IgG3) and TT42 (anti-Leb: IgM).

Characterization of 2DG8 and TT42 mAbs

Chemically synthesized oligosaccharides, Lea, Leb, H type I, Lex, Ley, and H type II, conjugated to bovine serum albumin (BSA) were obtained from Chembiomed (Edmonton, Canada). Microtiter plate wells (Corning Costar, USA) were coated with 100 µl of each oligosaccharide solution at a concentration of 1 µg/ml in phosphate buffer saline (PBS). Unoccupied binding sites were blocked with 1% BSA (Seikagaku Kogyo, Tokyo, Japan) and 5% sorbitol (Wako Pure Chemicals, Osaka, Japan) in PBS. The plates were air-dried after aspiration of the blocking solution and stored at 4°C with desiccant until use.

The plates were incubated with 100 µl of 1000-fold diluted ascites of 2DG8 or TT42 mAb in PBS containing 1% BSA, 0.1% Tween 20 (Wako Pure Chemicals, Osaka, Japan), and 0.1% XL-II (Zeneca, London) at 25°C for 90 min, rinsed with PBS containing 0.05% Tween 20 and 0.05% sodium azide (PBS-T), and then incubated at 25°C for 90 min with 100 µl of horseradish peroxidase (HRP)-conjugated anti-mouse IgG (H+L) (Bio-Rad, Hercules, CA) or HRP-conjugated anti-mouse IgM (Biosource, Camarillo, CA) which was diluted by PBS containing 1% BSA, 0.1% Tween 20, 1% normal goat serum, 0.1% XL-II. The rinsed plates were incubated with 100 µl of 3,3[prime],5,5[prime]-tetramethylbenzidine solution (Scytek Laboratories, Logan, UT) for color development. The reaction was stopped by adding 100 µl of stop solution for TMB (Scytek Laboratories). Optical density was measured at 450 nm using a plate reader (Molecular Devices Vmax, Sunnyvale, CA).

Se and Le genotyping

The Se and Le genotypes were determined by the PCR-RFLP method using genomic DNAs extracted from peripheral blood leukocytes. The methods for Se and Le genotyping were described in detail previously (Nishihara et al., 1994; Kudo et al., 1996; Narimatsu et al., 1998). For detection of the se5 allele, Koda's simple PCR method was employed (Koda et al., 1996).

Immunohistochemical staining

The samples were fixed in 10% formaldehyde and then embedded in paraffin. Deparaffinized 4 µm sections were subjected to immunohistochemical analyses according to the manual of a Vectastain Elite ABC Kit (Vector Laboratories). DAB-4HCl (0.1 mg/ml) (Dojin, Kumamoto, Japan) in 0.1 M Tris-HCl buffer (pH 7.6) was used for the peroxidase reaction.

Western blotting analysis

The tissues were solubilized in HEPES buffer (pH 7.2) containing 2% Triton X-100 by brief sonication, and then finally suspended in Laemmli's sample buffer. The samples containing 30 µg protein were subjected to 6% SDS-PAGE. The separated proteins were transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA) in a Transblot SD cell (Bio-Rad, Richmond, CA). The membrane was stained according to the manual with the ECL Western blotting detection reagents (Amersham, UK). A densitometer (Shimadzu, Japan) was used to determine the intensities of positive bands on the exposed film (Hyper film TM-ECL, Amersham). The experimental conditions were improved for accurate quantitative analysis.

Quantitative analysis of glycosyltransferase transcripts by competitive RT-PCR

We employed the competitive RT-PCR method to determine the amounts of transcripts in the native tissues, because it is impossible to obtain the amount of RNA enough for Northern analysis from the small native tissues. The competitive RT-PCR method which we established was confirmed to precisely determine the amount of transcripts in our previous study (Kudo et al., 1998).

Four human cDNAs, i.e. those of the H, Se, Le, and [beta]-actin genes, in the pBluescript vector were used as standard DNAs (Nishihara et al., 1994; Kudo et al., 1996; Narimatsu et al., 1998). The competitor DNA plasmids encoding the Le and [beta]-actin genes were constructed in the previous study (Kudo et al., 1998). Competitor DNA plasmids encoding the Se and H genes were prepared by deleting the 106-bp Tth111I-BstEII and 123 bp PmlI-BstEII fragments of the Se and H cDNA clones, respectively.

Total cellular RNA isolated by the acid guanidium thiocyanate-phenol chloroform method was treated with 5 units/ml of DNase I (GIBCO Laboratories, NY) before cDNA synthesis. cDNAs were synthesized with an oligo (dT) primer from 5 µg of the total RNA in a 20 µl reaction mixture using a SUPERSCRIPT Preamplification System for First Strand cDNA Synthesis (GIBCO Laboratories). After cDNA synthesis, the reaction mixture was diluted 50-fold with H2O and then stored at -80°C until use.

The competitive PCR was performed with recombinant Taq polymerase (TOYOBO, Japan) in a total volume of 50 µl comprising 10 µl of standard plasmid DNA or sample cDNA synthesized from colon tissue, PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1% Triton X-100), 2.5 mM MgCl2, 10 µl of competitor DNA at the optimal concentration, which differs depending on the glycosyltransferase, and 0.2 µM of each primer of the following primer-sets, i.e., 5[prime]-TGAGAGATCCTTTCCTGAAGCTCT-3[prime] and 5[prime]-TTGGCCAGGTAGACAGTGTCTCC-3[prime], 5[prime]-TCCCGGGGGAGTACGTCCGCTT-3[prime] and 5[prime]-ATTGGCCAGGTAGATGGTGTCTCC-3[prime], and 5[prime]-CCTCCCGACAGGACACCACTCC-3[prime] and 5[prime]-GCGTCCGTACACGTCCACCTTG-3[prime] for the detection of H, Se, and Le transcripts, respectively. The optimal cycle of PCR was 30 sec at 94°C, 1 min at 65°C, and 2 min at 72°C. After the PCR reaction, a 10 µl aliquot was electrophoresed on a 1% agarose gel and the bands were visualized by ethidium bromide staining. The intensities of the amplified fragments were determined. The standard curve for each glycosyltransferase was first established using fixed concentrations of the competitor DNA and the titrated standard DNA. The actual values for the transcripts in colon tissues were obtained by plotting on the standard curves. The amounts of [beta]-actin transcripts in each tissue were also determined by competitive RT-PCR (Saiki et al., 1996) for normalization as to the efficiency of the cDNA preparation.

Establishment of Namalwa cells transfected stably with the H, Se, and Le genes

The DNA fragments encoding the full-length open reading frames of the H, Se and Le genes were cloned in previous studies (Nishihara et al., 1994; Kudo et al., 1996; Kaneko et al., 1997). They were cloned into a pAMo vector for expression in Namalwa cells (Kimura et al., 1995).

Measurement of Fuc-T activity of H, Se, and Le enzymes toward Type I and Type II precursors

Lysates of stable transformant cells were assayed for Fuc-T activities in 0.1 M cacodylate buffer (pH 6.8), 5 mM ATP, 10 mM l-fucose, 75 mM guanosine diphosphate fucose, 25 mM MnCl2, and 25 mM pyridylaminated acceptor. The acceptors, LNT and lacto-N-neotetraose (LNnT), were purchased from Oxford Glycosystems, and pyridylaminated according to the method of Kondo et al. (Kondo et al., 1990). After incubation at 37°C for 2 h, the enzyme reactions were terminated by boiling for 3 min followed by the addition of water. The reaction products were subjected to HPLC analysis on a TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh, Japan), and were eluted with 20 mM ammonium acetate buffer (pH 4.0) at the flow rate of 1.0 ml/min at 35°C, with monitoring with a fluorescence spectrophotometer (JASCO FP-920; Nihon Bunkoh, Tokyo, Japan).

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas, No.10178104, from the Ministry of Education, Science, and Culture, Japan, and by Special Coordination Funds from the Science and Technology Agency of the Japanese Government.

Abbreviations

Aps, adenomatous polyps; bp, base pair(s); cDNA, complementary DNA; Fuc, fucose; Fuc-T, fucosyltransferase; Hps, hyperplastic polyps; kDa, kilodalton; le, le1 or le2; Le/-, Le/Le or Le/le; Leb, Lewis b; Ley, Lewis y; mAb, monoclonal antibody; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; RFLP, restriction fragment length polymorphism; RT-PCR, reverse transcription-polymerase chain reaction; Se, Se1 or Se2; se, sej or se5; Se/-, Se/Se or Se/se; sLex, sialyl Lewis x; sLea, sialyl Lewis a.

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3To whom all correspondence should be addressed


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