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Biosynthetic Cell and Organ Culture Methods 21921918Biosynthesis of Mucin Cell and Organ Culture Methodsfor Biosynthetic StudyAnthony P. Corfield, Neil Myerscough,Alexandra W. C. Einerhand, B. Jan-Willem Van Klinken,Jan Dekker, and Christos Paraskeva1. IntroductionThe study of the biosynthesis has been greatly assisted by the use of cultured cellsand tissue explants in short-term culture. Cells are available from a wide range oftissue sources, and this chapter focuses on the use of intestinal cells and tissue. Humancolonic cell lines have been widely used in biosynthetic studies and the relationship ofsome lines to stages in the adenoma-carcinoma sequence is of particular interest,allowing study of the expression of mucin during the development and progression ofdisease (1–3). Recently the importance of proliferation, differentiation, and apoptosishas attracted attention to the use of culture systems for the study of cell behavior innormal and disease processes (4,5). In the same way, tissue obtained from patients atsurgery or as biopsies can be placed in short-term primary or organ culture to studysimilar changes in disease (6,7).Improvements in the study of glycoproteins, especially mucins, have been achievedthrough the use of defined human mucosal cells that can be grown in long-term culture(1,2). Radioactive tracer methods allow relatively small numbers of cells and tissuefragments (biopsies) to be analyzed, and cell culture also gives access to larger amountsof the mucins produced by the individual cell lines (8,9).Each of the model systems described for the study of the synthesis and secretion ofmucin has distinct advantages and drawbacks. Ideally we would wish for a clonal cellline, which has all the typical characteristics of the mucin-producing cells in vivo.Because no such cell line exists for any of the mucin producing cells, we must settlefor either tissue explants, in which the mucin-producing cells have retained their natu-ral tissue context, or isolated clonal cells from carcinomas. The first system is natu-rally short-lived, which affects reproducibility, whereas the latter system is far morereproducible; however, these cells will have irreversible genetic changes that distin-From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 220 Corfield et al.guish them from the cells in vivo. On the other hand, the cell lines may consist of onlyone cell type, which can be an advantage.Study of the colon has the advantage that the sequence of changes during carcino-genesis in the colonic epithelial cells has been documented particularly well. Thisimplies that cells can be isolated from each stage of malignant growth: starting fromhealthy tissue, from which normal epithelial cells can be brought into primary cultureand display only minimal growth in vitro, via the adenoma stage that maintains inter-mediary characteristics to the full blown carcinoma cells, which usually grow quitewell (10). This sequence of changes in the epithelium allows precise studies of howthe changes in cells influence the mucins that are produced. Several of the end prod-ucts of malignant transformation of colonic epithelium have yielded valuable cell lines,such as the goblet cell-like LS174T and the enterocyte-like Caco-2 cell lines, whichare widely used to study the synthesis and regulation of mucin in detail (11,12). ThePC/AA cell lines, originally derived from a single, large, colonic tubular adenoma,have been used at early premalignant, intermediate premalignant, adenocarcinoma,and mucinous carcinoma stages (1,2,8,9,13). Furthermore, the HRA-19 colorectal cellline (3) is valuable because it can be cloned to give all three differentiation pathwaysto columnar, goblet, and endocrine cells.The methods described here cover the use of cellular systems for the study of thebiosynthesis of mucin which includes cultured cells (primary cultures and cell lines) andorgan culture. The data refer to human colorectal cells and tissue, but similar systemsand principles apply to a wide range of other tissues where mucins are produced.2. Materials1. Human gastrointestinal (GI) biopsies or surgical tissue from stomach (corpus and an-trum), duodenum, jejunum, ileum, colon (ascending, transverse, descending, sigmoid,and rectum), or gallbladder. Tissue obtained at surgery is dissected to isolate the mucosallayer and cut into small sections of approx 2–4 mm3. Endoscopically obtained biopsiesare used without further manipulations.2. GI tissue explants of rat or mouse from stomach (corpus and antrum), duodenum, jejunum,ileum, colon (proximal, mid, and distal), or gallbladder (only in mouse). Full thicknessexplants, including the muscle layers are used cut to give segments of 2–4 mm2of mu-cosal surface.3. Cell lines:a. Swiss 3T3 cells obtained from the American Tissue Type Culture Collection (ATCC,Manassas, VA, cat. no. CCL92).b. LS174T, colonic adenocarcinoma cell line (the parental line obtained from ATCC,cat. no. CL 188).c. Caco-2, colonic adenocarcinoma cell line (from Prof. G. J. Strous, Laboratory forCell Biology, Utrecht, The Netherlands).d. A431, epidermoid carcinoma cell line (ATCC, cat. no. CRL 1555).4. Fetal bovine serum (FBS). Batch testing is essential.5. Cell culture mediaa. Standard growth medium: Dulbecco’s modified Eagles medium (DMEM) containing2 mM glutamine, 1 µg/mL of hydrocortisone sodium succinate, 0.2 U/mL of insulin,100 IU/mL of penicillin, 100 mg/mL of streptomycin, and 20% of FBS. Biosynthetic Cell and Organ Culture Methods 221b. Washing medium: The same as the standard growth medium, but with 5% FBS, doublethe concentration of penicillin and streptomycin, and 50 µg/mL of gentamicin.c. Digesting solution: The same as the washing medium but containing 5% FBS togetherwith 1.5 mg/mL of collagenase (Worthington type 4, Lorm Diagnostics, Reading,UK) and 0.25 mg/mL of hyaluronidase (type 1; Sigma, Poole, UK).d. 3T3 Conditioned medium: DMEM is supplemented with 10% FBS, 2 mM glutamine,100 IU/mL of penicillin, 100 µg/mL of streptomycin, and put onto 24-h postconfluent3T3 cell layers for 24 h. After conditioning, the medium is filtered through a 0.2-mmfilter (Nalgene, Milton Keynes, UK) and further supplemented to give 20% FBS,1 µg/mL of hydrocortisone sodium succinate, and 0.2 U/mL of insulin.e. Cell growth medium: Dulbecco’s modified Eagles medium (DMEM, Gibco/BRL,Parsley, Scotland) containing 100 IU/mL of penicillin, 100 µg/mL of streptomycin,3.7 g/L of NaHCO3, and nonessential amino acids (sterile 100X stock solution, Gibco/BRL), supplemented with 20% of FBS (LS174T cells) or 10% of FBS for Caco-2 andA431 cells.f. Mouse thymocyte culture medium: RPMI medium (Gibco/BRL) supplemented with10% FBS, 2 mM of glutamine, 100 IU/mL of penicillin, 100 µg/mL of streptomycinand 2 mM of glutamine.6. Collagen (human placental type 4; Sigma) at 1 mg/mL is prepared in 1 part glacial aceticacid to 1000 parts sterile tissue culture grade distilled water and, and stored at 4°C.7. Dispase solution: Dispase (grade 1; Boehringer, Lewes, UK) is prepared in DMEM con-taining 10% FBS, sterile filtered, and stored at –20°C.8. MF-1 mice (Olac, Bicester, UK).9. Trypsin 0.1% by weight in 0.1% EDTA in PBS.10. Acridine orange (Sigma).11. Organ culture medium:a. Grid cultures: DMEM, containing 10% FBS, 10 mM of sodium bicarbonate, 2 mM ofglutamine, 100 µg/mL of streptomycin, 50 IU/mL of penicillin, 50 µg/mL of gentami-cin, and 20 mM of HEPES, pH7.2b. Submerged cultures: Eagle’s minimal Essential medium (EMEM, Gibco/BRL),supplemented with: non-essential amino acids (sterile 100X stock solution, Gibco/BRL), 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 2 mM ofL-glutamine.Incubate under 95% O2/5% CO2.12. Carbogen gas: 5% O2/5% CO2.13. Culture dishes (Costar, Cambridge MA).14. Airtight capped, transparent tubes (3–5 mL) with round bottom (Sarstedt, Nümbrecht,Germany).15. Water bath at 37°C.3. Methods3.1. Preparation of Collagen Coated Culture Flasks and Swiss 3T3Feeder Cells (see Notes 1–3)1. To prepare collagen-coated flasks, coat tissue culture flasks (T2525 cm2) with a thin layerof collagen solution (Subheading 2., item 6; 0.2 mg/flask), and allow to dry at roomtemperature in a laminar flow hood for 2–4 h (see Note 4).2. Grow Swiss 3T3 cells (Subheading 2., item 1) on collagen on plastic tissue culture flasksin DMEM containing 10% fetal calf serum until they are 24 h postconfluent. 222 Corfield et al.3. Lethally irradiate the cells with 60 Kgray (6 mrad) of radiation, or treat with 10 µg/mL ofmitomycin C (Sigma) for 2 h.4. Wash the cells and produce a single-cell suspension using a Pasteur pipet (wide apertureto avoid shearing of cells). The cells can either be used immediately as feeders or bestored at 4°C as a single-cell suspension for up to 4 d (see Note 5).3.2. Primary Culture-Enzyme Digestion (see Notes 3, 6, and 7)1. Wash the tumor specimens (adenoma and carcinoma) four times in washing medium(Subheading 2., item 5b) and cut with surgical blades to approx 1 mm3in a small volumeof the same medium.2. Wash the tissue four times in washing medium, and place in digestion solution(Subheding 2., item 5c). Pellet cells by centrifugation at 300g for 5 min. Roughly put 1 cm3in 20–40 mL of solution.3. Rotate at 37°C overnight (12–16 h).4. Mix the suspension by using a Pasteur pipet to improve the separation of the epithelialelements from the stroma resulting from enzymatic digestion.5. Filter the suspension through 50-mm mesh nylon gauze, or repeatedly allow to settle outunder gravity and collect the pellets. The large clumps of cells and epithelial tubules(organoids that contain the majority of the epithelial cells) are separated from the singlecells (mostly from the blood and stroma) and cell debris.6. Wash the cell pellets three times, and place in culture on collagen-coated T25 flasks inthe presence of Swiss mouse 3T3 feeders (approx 1 × 104cells/cm2) at 37°C in a 5% CO2in air incubator (13). In some situations, 3T3-conditioned medium (Subheading 2., item5d) can be used instead of adding mouse 3T3 cells directly to cultures (see Note 5).3.3. Long-Term Culture of Adenoma Cell Lines (see Note 8)1. Prepare culture conditions for adenoma cell lines as previously described for primarycultures.2. Carry out passage of adenoma cells as clumps of cells using sufficient dispase just tocover the cells, and incubate for approx 30 min at 37°C. Remove the cells as a sheet, andpipet with a Pasteur pipet to remove them from the flask and to break up the sheets intosmaller clumps of cells (13).3. Wash the clumps of cells and replate under standard culture conditions. Reattachment ofcells may take several days, and during medium changing, any floating clumps of cellsmust be centrifuged and replated with fresh medium.3.4. Long-Term Culture of Clonal Carcinoma Cell Lines, IncludingLS174T, Caco-2, and A431 Cell Lines (see Notes 8–10)1. Grow the carcinoma cell lines in tissue culture plastics without collagen coating and 3T3 feedersin DMEM supplemented with 10% FBS and 1 mM of glutamine (Subheading 2., item 5e).2. Carry out passage as single cells using 0.1% trypsin in 0.1% EDTA (see Notes 9 and 10).3.5. Apoptotic and Differentiating Cells3.5.1. Isolation and Identification of Apoptotic and Differentiating Cells(seeNote 11)1. During routine culture of cell lines, remove floating cells with the medium and pellet bycentrifugation. Most cell lines give rise to floating cells, the majority of which undergoapoptosis. Biosynthetic Cell and Organ Culture Methods 2232. Stain the cells with acridine orange at 5 µg/mL in PBS, which stains the DNA and allowsvisualization of the condensed chromatin of apoptotic cells. Stain cells for 10 min andthen observe with a fluorescent microscope using narrow-band FITC excitation (excita-tion wavelength, 450–490 nm; and barrier filter, 520–560 nm). Count at least 300 cells.3. Extract DNA from 106cells and electrophorese on 2% (w/v) agarose gel containing 0.1mg/mL of ethidium bromide at 40 V until the dye front has migrated 3 to 4 cm. Run theDNA from an equivalent number of attached cells as a control, and use dexamethasonetreated mouse thymocytes as a positive control for DNA laddering (see Subheading 3.5.2.and Note 11).3.5.2. Preparation of Thymocyte Cell Cultures (seeNote 11)1. Sacrifice MF-1 mice (Subheading 2., item 8) at 2–3 mo, dissect out the thymus andrelease thymocytes by pressing through sterile gauze.2. Suspend the thymocytes in thymocyte culture medium (Subheading 2., item 5f) at adensity of 106/mL and treate with 10–7M of dexamethasone.3. Collect samples after 18 h incubation with dexamethasone.3.6. Organ Culture of GI Tissue3.6.1. Grid Cultures (6) (seeNote 2)1. Place the biopsies or explants, singly or up to six per dish, on lens tissue placed over astainless steel grid in culture dishes with a central well containing 2 mL of medium (Sub-heading 2., item 11a). The orientation of the tissue is with the luminal surface uppermost.2. Place dishes in an incubation oven connected with a continuous supply of carbogen gas(Subheading 2., item 12) at 37°C.3.6.2. Submerged Cultures (14,15) (seeNotes 2, 12–15)1. Place the biopsy or explants, up to a maximum of six, in a small, airtight capped tube in100 µL of EMEM (Subheading 2., item 11b) (see Notes 16 and 17).2. Blow carbogen gas into the tube and seal the carbogen gas atmosphere by replacing the cap.3. Place the tube in a 37°C water bath.4. Notes1. The production and secretion of mucins by cultured colonic cells should be examined usingcells at different stages of confluency, because the stages of growth may alter the differen-tiation properties of the cells and therefore the amount and type of mucin produced.2. When using organ and primary cultures, it is important to consider the heterogenous natureof the cell types in the tissue (i.e., stromal elements and lymphoid cells in addition to thecolonic epithelial cells). It may not be clear which cell type is producing the glycopro-teins. Identification of specific cell-located glycoprotein expression can be further exam-ined using histological methods with chemical, lectin, or antibody stains or in situhybridization to identify the cellular origin of the mucin of interest (Chapters 3 and 27).3. The primary culture techniques described can be used for normal adult colon. However,these are not as reproducible as those with the adenomas and carcinomas, and there aremore problems from contaminating stromal elements. There are at present no normaladult colonic epithelial cell lines, only adenoma (1) and carcinoma cell lines such as PC/JW/F1 (13), HT29 (10), LS174T (goblet cell like), and Caco-2 (enterocyte like) (11,12).4. Collagen-coated flasks are necessary to obtain efficient attachment of primary culturesand some adenomas and carcinomas to the flasks, and to retain the optimum differenti- 224 Corfield et al.ated characteristics of the cells (13). “Tissue culture-treated” culture plates (Costar) (Sub-heading 2., item 13) can be used successfully for established carcinoma cell lines with-out collagen coating.5. The use of 3T3 cell feeders requires controls to determine which cell type is producingthe glycoproteins of interest. This can be achieved using 3T3-conditioned medium inwhich the production of mucin is being assessed, or removing the 3T3 feeders from theflask once the epithelium has grown. In addition, the 3T3 cells should be examined forthe production of mucin under the culture conditions used.6. Colorectal adenomas invariably need digestion with enzymes because of their organiza-tion into well-differentiated glandular structures. With carcinomas, it is possible to adopta nonenzymatic approach with surgical blades to release small clumps of tumour cellsthat can be cultured (16).7. When using all colonic cell lines, it is important to check the true colonic epithelial natureusing a battery of markers, including antikeratin antibodies, ultrastructural analysis show-ing the presence of desmosomes, and other colonic differentiation markers (10). Thisverification is especially important with primary cultures and newly derived lines, butmay also be important when culture conditions induce changes in cell behavior.8. Although many tumor cell lines, especially colon carcinomas, can be grown in simple mediawithout 3T3 feeders and collagen coating, the colonic cells retain better differentiated pheno-types when using the more complex culture conditions described for primary cultures.9. LS147T, Caco-2, and A431 cells are cultured in DMEM as described under Subheading2., item 5e at 37°C and 5% CO2. Caco-2 and A431 cells are passaged (trypsinised) andsplit 1:4 and the medium is changed every 2 to 3 d. LS174T cells are split 1:2 and themedium changed daily.10. The three cell lines LS174T, Caco-2 and A431, together produce all gastrointestinalmucins known at present: LS174T produces MUC1, -2, -5AC, -5B, and -6 (12); Caco-2produces MUC1 and -3 (12) and A431 produces MUC4 (Van Klinken, Einerhand, andDekker, unpublished results). They serve as excellent cell lines for the isolation of thecorresponding mucin precursors as detailed in chapters 20 and 21. In addition, the PC/AAcell lines produce only MUC 1 and MUC2 at early passage, but at later passage and inlater premalignant and malignant stages, they show de novo expression of MUC5AC,MUC5B, and MUC6 (Corfield, Myerscough, and Paraskeva, unpublished results).11. Proliferating cells in the bottom of the colonic crypts migrate to the upper half of thecrypts, where they differentiate. These differentiated cells migrate to the top of the crypt,and there is evidence that they die by apoptosis and that apoptosis may be the terminalstage of differentiation. The relationship among proliferation, differentiation andapoptosis may be studied using the culture system described here. We have shown thatcultured colonic normal adenoma and carcinoma cells spontaneously die by apoptosis invitro and that the levels of apoptosis can be modulated by dietary short-chain fatty acids(butyrate, acetate, and propionate) and bile acids (5). During routine culture of colonicepithelial cells, some cells detach from the flask and float in the medium (4). These cellscontain condensed chromatin, which can be detected with acridine orange staining. Inaddition, characteristic DNA laddering resulting from internucleosomal fragmentationcan be seen after analysis of total cellular DNA. Dexamethasone treated mouse thy-mocytes are a convenient source of cells to use as a positive control for DNA laddering.12. A mucosal biopsy is taken by endoscopy from a human individual with an otherwiseintact organ. A tissue explant is obtained from surgically resected human or animal tis-sue. For human tissue, the mucosa can be relatively easily dissected from the muscle Biosynthetic Cell and Organ Culture Methods 225layer due to its size. In animal tissues, smaller sections of the entire wall of the organ areused, because these tissues are more fragile and dissection is difficult. The presence ofthe muscle layer does not appear to affect the synthesis of epithelial mucins.13. Submerging the tissue segments in the culture medium is particularly advantageous whenthe secretion of mucin is studied. The grid technique results in the accumulation of amucous gel layer on the top of the tissue during prolonged incubations and requires care-ful collection as a separate fraction. Upon submersion, the mucins are secreted into themedium, although some of the mucin may remain adherent to the epithelial tissue. Thesubmerged culture system, under basic nonstimulated conditions, leads to the recovery of20% of the total MUC2 in the medium during a 4-h culture of human colonic biopsies.This percentage is very reproducible when compared for a large number of patients (VanKlinken, Einerhand, and Dekker, unpublished results).14. The submerged tissue culture technique has the advantage that the mucins are more efficientlylabeled during metabolic labeling experiments compared to the grid culture system. In otherwords a higher incorporation of radioactive precursor is found in the mucins prepared usingthe submerged technique if the same concentration of precursor is used in both culture sys-tems. Moreover, the labeling is more economical because similar labeling levels can beachieved with smaller (5%) amounts of precursor owing to the smaller volume (0.1 vs 2 mL).Because prolonged incubations will certainly deplete the medium of nutrients the submergedtechnique in small volumes (0.1 mL/explant) is only applied to the metabolic pulse labeling oftissue. The grid culture system is thought to mimic the in vivo state more closely, with reten-tion of the secreted mucus at the tissue surface. Although lower levels of incorporation intomucins are found, these are quite adequate for comparative studies in disease (6,7).15. Standard media usually contain sodium bicarbonate as primary buffer (2.2 g/L), and areused only with a 5% CO2atmosphere. Alternatively, the bicarbonate buffer can bereplaced with the nontoxic buffer HEPES, which enables incubation under an air atmo-sphere and does not require CO2. GI tissue is extremely sensitive to hypoxic conditions;therefore, the use of an oxygen atmosphere (carbogen gas: 95% O2/5% CO2) is preferredto the 95% air atmosphere.16. Temperature control by a water bath is preferred to an incubator because the heat exchangeis much quicker and the temperature is prone to only very slight fluctuations. This isparticularly relevant for pulse/chase experiments because these involve relatively shortincubation periods and require rapid temperature equilibration (see Chapters 20 and 21).17. If the incubation is part of a pulse/chase experiment, the EMEM should be depleted of thecompound used as a precursor to label the mucin (see Chapter 19, Subheading 3.1., andChapters 20 and 21).References1. Williams, A. C., Browne, S. J., Manning, A. M., Hague, A., van der Stappen, J. W. J., andParaskeva, C. (1993) Biological consequences of the genetic changes which occur duringhuman colorectal carcinogenesis. Sem. Cancer Biol. 4, 153–159.2. Williams, A. C., Harper, S. A. and Paraskeva, C. (1990) Neoplastic transformation of ahuman colonic epithelial cell line: Experimental evidence for the adenoma to carcinomasequence. Cancer Res. 50, 4724–4730.3. Kirkland, S. (1988) Clonal origin of columnar, mucous and endocrine lineages in humancolorectal epithelium. Cancer 61, 1359–1363.4. Hague, A., Manning, A. M., Hanlon, K., Huschtscha, L. I., Hart, D. and Paraskeva, C.(1993) Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53 226 Corfield et al.independent pathway: Implications for the possible role of dietary fibre in the preventionof large bowel cancer. Int. J. Cancer 55, 498–505.5. Hague, A., Elder, D. J. E., Hicks, D. J. and Paraskeva, C. (1995) Apoptosis in colorectaltumour cells: Induction by the short chain fatty acids butyrate, propionate and acetate andthe bile salt deoxycholate. Int. J. Cancer 60, 400–406.6. Corfield, A. P., Warren, B. F., Bartolo, D. C. C., Wagner, S. A., and Clamp, J. R. (1992)Mucin changes in ileoanal pouches monitored by metabolic labelling and histochemistry.Brit. J. Surgery 79, 1209–1212.7. Probert, C. S. J., Warren, B. F., Perry, T., Mackay, E. H., Mayberry, J. F., and Corfield, A.P. (1995) South Asian and European colitics show characteristic differences in colonicmucus glycoprotein type and turnover. Potential identification of a lower risk group forsevere disease and cancer. Gut 36, 696–702.8. Vavasseur, F., Dole, K., Yang, J., Matta, K. L., Corfield, A. P., Myerscough, N., Paraskeva,C., and Brockhausen, I. (1994) O-glycan biosynthesis in human colonic cells during pro-gression to cancer. Eur. J. Biochem. 222, 415–424.9. Corfield, A. P., Clamp, J. R., Casey, A. D., and Paraskeva, C. (1990) Characterization of asialic acid-rich mucus glycoprotein secreted by a premalignant human colorectal adenomacell line. Int. J. Cancer 46, 1059–1065.10. Laboisse, C. L. (1989) Differentiation of colon cells in culture, in The Cell and MolecularBiology of Colon Cancer (Augenlicht, L. H., eds.), CRC Press, Boca Raton, FL, pp. 27–43.11. Van Beers, E. H., Al, R. H., Rings, E. H. H. M., Einerhand, A. W. C., Dekker, J., andBüller, H. A. (1995) Lactase and sucrase-isomaltase gene expression during Caco-2 celldifferentiation. Biochem. J. 308, 769–775.12. Van Klinken, J.-W., Oussoren, E., Weenink, J.-J., Strous, G. J., Büller, H. A., Dekker, J.and Einerhand, A. W. C. (1996) The human intestinal cell lines Caco-2 and LS174T asmodels to study cell-type specific mucin expression. Glycoconjugate J. 13, 757–768.13. Paraskeva, C., Buckle, B. G., Sheer, D. and Wigley, C. B. (1984) The isolation and char-acteristics of colorectal epithelial cell lines at different stages in malignant transformationfrom familial polyposis coli patients. Int. J. Cancer 34, 49–56.14. Dekker, J., Van Beurden-Lammers, W. M. O., and Strous, G. J. (1989) Biosynthesis ofgastric mucus glycoprotein of the rat. J. Biol. Chem. 264, 10,431–10,437.15. Van Klinken, B.J.W., De Bolos, C., Büller, H.A., Dekker, J. and Einerhand, A.W.C.(1997) Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the gastrointesti-nal tract. Am. J. Physiol. 273, G296-G30216. Leibovitz, A., Stinson, J. C., McComb, W. B., McCoy, C. E., Mazur, K. C., and Mabry, N.D. (1976) Classification of human colorectal adenocarcinoma cell lines. Cancer Res. 36,3562–3569. . LS174T produces MUC1, -2 , -5 AC, -5 B, and -6 (12); Caco-2produces MUC1 and -3 (12) and A431 produces MUC4 (Van Klinken, Einerhand, andDekker, unpublished. Caco-2, and A431 cells are cultured in DMEM as described under Subheading2., item 5e at 37°C and 5% CO2. Caco-2 and A431 cells are passaged (trypsinised) andsplit