Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.     

Phyllanthus urinaria triggers the apoptosis and Bcl-2 down-regulation in Lewis lung carcinoma cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect in its water extract for the first time. The water extract of P. urinaria significantly decreased the number of Lewis lung carcinoma cells in a dose-and time-dependent manner as determined by MTT assay. However, the water extract of P. urinaria did not exert any cytotoxic effect on normal cells such as endothelial cells and liver cells. Result from flow cytometry revealed a dose-dependent increase of dead cells 24 hours after treating Lewis lung carcinoma cells with P. urinaria extract. The anticancer activity of P. urinaria extract was due to the apoptosis induced in Lewis lung carcinoma cells, which was demonstrated by DNA fragmentation analysis and increased caspase-3 activity. The apoptosis triggered by P. urinaria extract in Lewis lung carcinoma cells was associated with the down-regulation of Bcl-2 gene expression, but not with p53, p21 and Bax. Furthermore, the partial inhibition of P. urinaria-induced apoptosis in Lewis lung carcinoma cells by pretreatment with cyclosporin A, a mitochondria permeability transition pore inhibitor, suggesting that P. urinaria extract induced the apoptosis of Lewis lung carcinoma cells, at least in part, through a mitochondria-associated intrinsic pathway.     

Glucose-6-phosphate dehydrogenase and succinate dehydrogenase in metastatic cells of Lewis lung carcinoma

By the experimental model of the Lewis lung carcinoma we studied the activity of glucose-6-phosphate-dehydrogenesis (G6PDH) and succinic-dehydrogenesis (SDH) in the carcinoma itself, the lung metastases, the peripheral blood. A set of carcinoma, lung sections, peripheral blood smears were studied by histochemical techniques day by day since the 3rd day to the 15th one after the carcinoma grafting. The reported results allow us to conclude that G6PDH looks like a determinant of metastatic cell survival and growth and can be regarded as a marker of metastatic cells in the present model.     

CXCR4 positive cells from Lewis lung carcinoma cell line have cancer metastatic stem cell characteristics

There is increasing evidence that cancer stem cells contribute to the initiation and propagation of many tumor. Therefore, to find out and identify the metastatic tumor stem-like cells in Lewis lung cancer cell line (LLC), the expression of CXCR4 was measured in LLC by flow cytometry and observed by laser scanning confocal microscope (LSCM). After the CXCR4(+) LLC cell was isolated from LLC by magnetic cell sorting, its properties were evaluated by their tumorigenic and metastatic potentials. CXCR4(+) cells were counted for 0.18% of the total number of LLC, and immunofluorescent staining cells were identified by LSCM. CXCR4(+) LLC suspension cultured in a serum-free medium, cell spheres expressed a high level of Sca-1. The chemotherapy sensitivity to cisplatin of CXCR4(+) LLC was lower than that of CXCR4(-) LLC. The expression of ABCG2 and IGF1R mRNA in CXCR4(+) LLC was higher than that in CXCR4(-) LLC (P < 0.01). Most of CXCR4(+) LLC cells were close to vascular endothelial cells, aberrant vasculature around it was forming. The expression of VEGF and MMP9 mRNA in CXCR4(+) LLC was higher than that in CXCR4(-) LLC (P < 0.05), the microvessel density (MVD) of CXCR4(+) subsets growing were higher than that of CXCR4(-) subsets growing tumor tissue (P < 0.01). The tumor size, volume, and metastatic foci in the lungs of CXCR4(+) LLC was significantly higher than that in CXCR4(-) LLC (P < 0.001). Similarly, elevated expression of MMP9 and VEGF was also positively associated with CXCR4(+) LLC. Our results demonstrated that CXCR4(+) cells from Lewis lung carcinoma cell line exhibit cancer metastatic stem cell characteristics.     

肺癌组织中岩藻糖化糖链结构免疫组化研究

Lewis X(Le~x)、唾液酸化的Lewis X(Sialyl Lewis X,SLe~x)和唾液酸化的双岩藻糖Lewis X(Sialyl Dimeric Lewis X,SDLe~x)是细胞表面外侧带α1,3岩藻糖的糖链结构。本文用免疫组化ABC法研究了肺癌原发灶、转移灶和癌旁组织中这三种抗原结构的表达。结果发现这三种抗原在肺癌细胞表面及胞浆中均有不同程度的表达,而在肺癌癌旁组织及正常肺组织中未见表达。有转移的肺癌和(或)低分化肺癌中这三种抗原结构的表达要明显高于未发生转移和高、中分化肺癌中相同抗原结构的表达。其中以SLe~x的表达与肺癌细胞的转移能力和分化程度关系最为密切。另外,肺癌浸润转移的淋巴结中也有Le~x、SLe~x的明显表达和SDLe~x的少量表达,而未被肺癌浸润转移的淋巴结中就没有它们的表达。     

Immunohistochemical study of the expression of fucosylated glycoconjugates in human lung carcinoma]

Lewis X, Sialyl Lewis X and Sialyl Dimeric Lewis X are three fucosylated glycoconjugates on cell surface. With immunohistochemical method, the expression of the three structures in the original lung cancer tissues (with or without metastasis), adjacent tissues and metastatic lesions of lung carcinoma were studied. It was found that the three antigens were expressed with different intensity on the cell surface and in the cytoplasm of the lung carcinoma cell. However, there was no or only trace expression of these antigens in the adjacent tissues of lung carcinoma and normal lung tissues. Moreover, the original lesions of lung carcinoma with metastasis and/or poor differentiation expressed higher level of the three antigens than those without metastasis and/or with well or medium differentiation. Sialyl Lewis X was considered to be more closely related to the metastatic ability and differentiation of the lung carcinoma cell than the other two antigens, Lewis X and Sialyl Dimeric Lewis X. Furthermore, in the lymph nodes with lung carcinoma cell metastasis, there were expression of the three antigens with different degree, while in those lymph nodes without lung carcinoma cell metastasis, there was no expression of the three antigens.     

Tumor immunity in perforin-deficient mice: a role for CD95 (Fas/APO-1)

CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39. 5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF-, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.     

IL-10 promotes resistance to apoptosis and metastatic potential in lung tumor cell lines

Treatment of non-small cell lung carcinoma (NSCLC) remains at a disappointingly low success rate. Not only is metastatic spread common in NSCLC, but therapeutic success decreases dramatically once metastases are present. Understanding factors which contribute to poor prognosis in NSCLC is critical for development of more successful therapeutic approaches. Interleukin-10 (IL-10) expression has been shown in several studies to correlate with a poorer prognosis in NSCLC; however, the mechanisms by which IL-10 affects lung tumor growth and metastases are unclear. The goal of this study was to evaluate the effects of tumor-derived IL-10 on the growth and metastasis of lung cancer cells in a murine model. Lewis lung carcinoma cells were stably transfected with the chicken ovalbumin gene (cOVA) as a model tumor antigen (LL43 tumor cells) and subsequently transfected with the murine IL-10 gene (LL43-10 tumor cells). Subcutaneous growth of the LL43 tumor cells was not affected by expression of IL-10. However, LL43-10 tumors had a fourfold increase in tumor microvessel density, as indicated by CD31 staining. Metastatic potential was also increased in IL-10-expressing lung tumor cells, leading to a greater number of tumor cells in lymph nodes draining the primary tumor site. Finally, exposure of Lewis lung tumor cells in vitro to exogenous IL-10 dramatically increased their resistance to UV-induced apoptosis. These results indicate that a primary effect of IL-10 on lung cancer cells may be to increase their metastatic potential by promoting angiogenesis and resistance to apoptosis.     

Cross Talk between the TM4SF5/Focal Adhesion Kinase and the Interleukin-6/STAT3 Pathways Promotes Immune Escape of Human Liver Cancer Cells

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway.     

Factors Affecting Phage D29 Infection: A Tool to Investigate Different Growth States of Mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.     

Inhibition of spontaneous pulmonary metastases of Lewis lung carcinoma by oral treatment with Respivax and Broncho-Vaxom

Summary The antimetastatic activity of orally administered polybacterial vaccines, Broncho-Vaxom (BV) and Respivax (RV) was examined in C57BL/6 mice, bearing implants of Lewis lung carcinoma (3LL) in the footpad. The oral administration of BV or RV for 10 consecutive days before or after surgery caused significant reduction of the number and volume of lung metastases. In addition, the therapeutic potential of BV and RV was examined in combination with chemotherapy to determine if there is additive activity. In animals bearing pulmonary micrometastases, treatment with a combination of cyclophosphamide at 50–150 mg/kg with BV or RV was found to be more effective than each of these treatments alone. In immune function studies it was established that the oral administration of BV and RV induced an increase in the number of cells, recovered by broncho-alveolar lavage, and alveolar macrophages were dominant in these cell populations. Furthermore, oral treatment of mice with these vaccines rendered their alveolar macrophages tumoricidal for syngeneic metastatic 3LL cells in vitro. These results show that pulmonary macrophages induced by oral administration of BV and RV played a key role in the inhibition of metastasis in 3LL-bearing mice.     

Study of DNA synthesis in short-term cultures of mammalian cells in the evaluation of the reaction of tumor and normal tissues to irradiation and hyperthermia

The effect of radiation of hyperthermia was estimated with a reference to the degree and duration of inhibition of DNA synthesis in the primary suspension cell cultures of Lewis lung carcinoma and bone marrow carcinoma of mice in vitro. The optimum conditions were chosen for cultivation of the primary suspension cell cultures according to the DNA synthesis. A study was made of the peculiarities of suppression and recovery of DNA synthesis in cells of Lewis lung carcinoma and bone marrow carcinoma of mice exposed to different gamma-radiation doses and hyperthermia.     

The effect of psychoemotional state on Lewis lung carcinoma metastasis

The effect of a depression-like status formed by chronic stress on development of Lewis lung carcinoma metastases in C57Bl/6J mice was investigated. Two types of acute stress (restraint and social stress) were used for comparison. The depression-like status was induced by eight-week exposure to repeated but unpredictable stressors (chronic mild stress model) and was assessed in the forced swim test. Tumor cells were inoculated an hour after the onset of social stressor or immediately after physical or chronic stressor impacts. The number of metastases was counted 17 days after the inoculation. The results indicate that chronic mild stress provokes the development of a depression-like state in mice and causes a twofold increase in the number of metastases in the lungs, while both types of acute stress have no such effects. Thus, a depression-like psychoemotional status of animals enhances the metastasis of Lewis lung carcinoma.     

Anti-apoptotic protein BRE/BRCC45 attenuates apoptosis through maintaining the expression of caspase inhibitor XIAP in mouse Lewis lung carcinoma D122 cells

Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.     

Metastatic growth of lung cancer cells is extremely reduced in Vitamin D receptor knockout mice

Lung metastatic neoplasms are the major cause of cancer mortality. Despite the progress of diagnostic techniques and improvements in surgical procedures, the prognosis of patients with lung cancer is generally poor, even in the early stages of cancer [Cancer: Principles and Practice of Oncology, vol. 1, fifth ed., Lippincott-Raven, New York, 1997, p. 849]. Epidemiological studies indicate a positive correlation with the prevalence of cancers and low serum levels of Vitamin D metabolites [Am. J. Clin. Nutr. 54 (1991) 193s; Cancer Epidemiol. Biomark. Prev. 9 (2000) 1059]. 1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is a potent inhibitor of cancer cell proliferation in vitro [Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 4990; Endocrinol. 139 (1998) 1046; Mol. Endocr. 15 (2001) 1127]. There is, however, no report demonstrating that 1alpha,25(OH)(2)D(3) is operative in vivo to inhibit metastatic growth of cancer cells. To verify this possibility, we generated a stable transfectant of the Lewis lung carcinoma (LLC) cell expressing green fluorescent protein (GFP) and examined its metastatic activity in wild-type mice and Vitamin D receptor (VDR) knockout mice that exhibit no Vitamin D-dependent calcemic activity and extremely high serum levels of 1alpha,25(OH)(2)D(3) due to the overexpression of the 1alpha-hydroxylase gene [Nat. Genet. 16 (1997) 391; Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 9831]. Here, we show that 1alpha,25(OH)(2)D(3) inhibits metastatic growth of lung cancer cells in the defined animal model and may work as an intrinsic factor for prevention of metastasis in intact animals. These findings establish a critical role for 1alpha,25(OH)(2)D(3) in lung metastatic neoplasms and provide a new model for metastasis of malignant cells.     

Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer vaccine induces apoptosis and antiangiogenic effect in a metastatic lung carcinoma

Anti-idiotype monoclonal antibody (mAb) 1E10 was generated by immunizing BALB/c mice with an Ab1 mAb which recognizes NeuGc-containing gangliosides, sulfatides and some tumor antigens. 1E10 mAb induces therapeutic effects in a primary breast carcinoma and a melanoma model. However, the tumor immunity mechanisms have not been elucidated. Here we show that aluminum hydroxide-precipitated 1E10 mAb immunization induced anti-metastatic effect in the 3LL-D122 Lewis Lung carcinoma, a poorly immunogenic and highly metastatic model in C57BL/6 mice. The therapeutic effect was associated to the increment of T cells infiltrating metastases, the reduction of new blood vessels formation and the increase of apoptotic tumor cells in lung nodules. Interestingly, active immunization does not induce measurable antibodies to the 1E10 mAb, the NeuGc-GM3 or tumor cells, which may suggest a different mechanism which has to be elucidated. These findings may support the relevance of this target for cancer biotherapy.     

Development of a metastatic fluorescent Lewis Lung carcinoma mouse model: Identification of mRNAs and microRNAs involved in tumor invasion

Cancer metastasis is the foremost cause of death in cancer patients. A series of observable pathological changes takes place during progression and metastasis of cancer, but the underlying genetic changes remain unclear. Therefore, new approaches are required, including insights from cancer mouse models. To examine the mechanisms involved in tumor metastasis, we first generated a stably transfected Lewis Lung carcinoma cell line expressing a far-red fluorescent protein, called Katushka. After in vivo growth in syngeneic mice, two fluorescent Lewis Lung cancer subpopulations were isolated from primary tumors and lung metastases. The metastasis-derived cells exhibited a significant improvement in in vitro invasive activity compared to the primary tumor-derived cells, using a quantitative invasion chamber assay. Moreover, expression levels of 84 tumor metastasis-related mRNAs, 88 cancer-related microRNAs as well as Dicer and Drosha were determined using RT-qPCR. Compared to the primary Lewis Lung carcinoma subculture, the metastasis-derived cells exhibited statistically significantly increased mRNA levels for several matrix metalloproteinases as well as hepatocyte growth factor (HGF) and spleen tyrosine kinase (SYK). A modest decrease in Drosha and Dicer mRNA levels was accompanied by significant downregulation of ten microRNAs, including miR-9 and miR-203, in the lung metastatic Lewis Lung carcinoma cell culture. Thus, a tool for cancer metastasis studies has been established and the model is well suited for the identification of novel microRNAs and mRNAs involved in malignant progression. Our results suggest that increases in metalloproteinase expression and impairment of microRNA processing are involved in the acquirement of metastatic ability.     

Sugar-specific endocytosis of glycoproteins by Lewis lung carcinoma cells

Lewis lung carcinoma cells from tumors, metastasis nodules, or from culture bind fluorescent derivatives of neoglycoproteins containing α-D -glucose residues: This binding is competitively inhibited by neoglycoproteins containing α-D -glucose, by mannan, and by several other neoglycoproteins. Cell binding and uptake of the fluorescent derivatives of the neoglycoproteins was quantified by lysing the cells with an alkylpolyol (MAC 19 or MAC 18) and measuring the fluorescence intensity of the supernatant. The amount of cell-associated neoglycoprotein was higher at 37°C than at 4°C with LLC from tumor. The binding and uptake were inhibited by glycoconjugates containing α-D -glucose. These results suggest the presence of sugar specific receptors in Lewis lung carcinoma cells which are involved in a sugar-specific binding and endocytosis phenomenon. The implication of the existence of a carbohydrate-binding protein on the surface of Lewis lung carcinoma cells are discussed with regard to the in vivo behaviour of these cells, especially in relation to their metastatic properties and to the possibility of using neoglycoproteins as specific carriers of cytotoxic drugs. Hybrid molecules of gelonin and a neoglycoprotein containing α-D -glucose were used as targetted toxin: The targetted toxin was found to bind to and to enter the intact cells and was 100 times more toxic than free drug.     

Altered expression of sialylated carbohydrate antigens in HT29 colonic carcinoma cells

To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.