Product Information Sheet for HTB-38

Cell Line Designation: HT-29 ATCC Catalog No. HTB-38

Table of Contents:
Cell Line Description Biosafety Level Use Restrictions Handling Procedure for Frozen Cells Handling Procedure for Flask Cultures Subculturing Procedure Medium Renewal Complete Growth Medium Cryoprotectant Medium References Replacement Policy Specific Batch Information
The line is positive for expression of c-myc, K-ras, H-ras, N-ras. Myb, sis and fos oncogenes. N-myc oncogene expression was not detected. Karyology: The stemline chromosome number is hypertriploid with the 2S component occurring at 2.4%. Seventeen marker chromosomes are found in most metaphases, generally in single copy per chromosome. The marker designations are: M1p-(=t(3p-;?) with a deleted short arm), t(7q;?), t(10q;?), i(13q), 19q+a; M6, ?t(8q;9q-), ?Xp, M9, 6q+, t(13;?)a, t(13;?)b, 19q+b, M14, M15, 15p+, and Xq-. Chromosome 13 is nullisomic and chromosomes 8 and 14 are generally monosomic. No Y chromosome was detected by QM band analysis. Note: Cytogenetic information is based on initial seed stock at ATCC. Cytogenetic instability has been reported in the literature for some cell lines. Derivative -- ATCC CCL-218 Purified DNA from this line is available as ATCC HTB-38D (10g)

Cell Line Description

Organism: Homo sapiens (human) Tissue: colorectal adenocarcinoma Age: 44 years Gender: female Ethnicity: Caucasian Receptors expressed: human adrenergic alpha2A urokinase receptor (u-PAR); vitamin D (moderate expression) Morphology: epithelial Growth properties: adherent VirusSuscept: human immunodeficiency virus (HIV, LAV) Tumorigenic: yes, in nude mice; forms well differentiated adenocarcinoma consistent with colonic primary (grade I); tumors also form in steroid treated hamsters Oncogene: myc +; ras +; myb +; fos +; sis +; p53 +; abl -; ros -; src AntigenExp: Blood Type A; Rh+; HLA A1, A3, B12, B17, Cw5 Products: secretory component of IgA; carcinoembryonic antigen (CEA); transforming growth factor beta binding protein; mucin DNA profile (STR analysis): Amelogenin: X CSF1PO: 11,12 D13S317: 11,12 D16S539: 11,12 D5S818: 11,12 D7S820: 10 TH01: 6,9 TPOX: 8,9 vWA: 17,19 Depositors: J. Fogh Comments: Ultrastructural features include microvilli, microfilaments, large vacuolated mitochondria with dark granules, smooth and rough ER with free ribosomes, lipid droplets, few primary and many secondary lysosomes;. no virus particles HT-29 cells express urokinase receptors, but do not have detectable plasminogen activator activity. It produces carcinoembryonic antigen and tumors in nude mice. There is a G -> A mutation in codon 273 of the p53 gene resulting in an Arg -> His substitution. The p53 antigen is overproduced. The cells are negative for CD4, but there is cell surface expression of galactose ceramide (a possible alternative receptor for HIV). American Type Culture Collection P.O. Box 1549 Manassas, VA 20108 USA www.atcc.org

Biosafety Level: 1
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 4th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 1999. The entire text is available online at www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm.

Use Restrictions
These cells are distributed for research purposes only. HT-29 was deposited in the ATCC by J. Fogh, Memorial Sloan-Kettering Cancer Center, New York, NY. The cells are distributed for research purposes only. The Memorial Sloan-Kettering Cancer Center releases the line subject to the following: 1.) The HT-29 cells or their products must not be distributed to third parties. Commercial interests are the exclusive property of Memorial Sloan-Kettering Cancer Center. 2.) Any proposed commercial use of these cells must first be negotiated with The Director, Office of Industrial Affairs, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; phone (212) 639-3620; FAX (212) 753-5764.

Handling Procedure for Frozen Cells

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at 70C. Storage at 70C will result in loss of viability. SAFETY PRECAUTION: ATCC highly recommends that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris. 1. Thaw the vial by gentle agitation in a 37C water bath. To reduce the possibility of contamination, keep the O-ring and 800-638-6597 (U.S., Canada, and Puerto Rico) 703-365-2700 Fax: 703-365-2750 E-mail: tech@atcc.org

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Product Information Sheet for HTB-38

cap out of the water. Thawing should be rapid (approximately 2 minutes). 2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions. Transfer the vial contents to a centrifuge tube containing 9.0 ml complete culture medium. and spin at approximately 125 xg for 5 to7 minutes. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm2 culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6). Incubate the culture at 37C in a suitable incubator. A 5% CO 2 in air atmosphere is recommended if using the medium described on this product sheet. 3. EDTA solution to remove all traces of serum, which contains trypsin inhibitor. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

3.

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37C to facilitate dispersal. 4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. Add appropriate aliquots of the cell suspension to new culture vessels. Subcultivation Ratio: 1:3 to 1:8. Incubate cultures at 37C.

4.

5.

6.

5.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Handling Procedure for Flask Cultures

The flask was seeded with cells (see specific batch information) grown and completely filled with medium at ATCC to prevent loss of cells during shipping. 1. Upon receipt visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination. Also check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable). If the cells are still attached, aseptically remove all but 5 to 10 ml of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37C in a 5% CO 2 in air atmosphere until they are ready to be subcultured. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 xg for 5 to 10 minutes. Remove shipping medium and save. Resuspend the pelleted cells in 10 ml of this medium and add to 25 cm2 flask. Incubate at 37C in a 5% CO 2 in air atmosphere until cells are ready to be subcultured.

Medium Renewal
Two to three times per week.

Complete Growth Medium

The base medium for this cell line is ATCC-formulated McCoys 5a Medium Modified, Catalog No. 30-2007. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10% This medium is formulated for use with a 5% CO 2 in air atmosphere. ATCC tested fetal bovine serum is available as ATCC Catalog No. 30-2020.

Cryoprotectant Medium
Complete growth medium described above supplemented with 5% (v/v) DMSO. Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

2.

3.

Additional Information
Additional product and technical information can be obtained from the catalog references and the ATCC Web site at www.atcc.org, or by e-mail at tech@atcc.org.

References
(additional references may be available in the catalog at www.atcc.org) Fogh, J., ed., Human tumor cells in vitro. New York: Plenum Press; 1975:pp. 115-159 Fogh J et al. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J. Natl. Cancer Inst. 58: 209214, 1977 PubMed: 77097006

Subculturing Procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 1. Remove and discard culture medium. 2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM

American Type Culture Collection P.O. Box 1549 Manassas, VA 20108 USA www.atcc.org

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Product Information Sheet for HTB-38

Fogh J et al. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59: 221-226, 1977 PubMed: 77210034 Didier ES et al. Characterization of Encephalitozoon (Septata) intestinailis isolates cultured from nasal mucosa and bronchoalveolar lavage fluids of two AIDS patients. J. Eukaryot. Microbiol. 43: 34-43, 1996 PubMed: 96151459 Chen TR et al. WiDr is a derivative of another colon adenocarcinoma cell line, HT-29. Cancer Genet. Cytogenet. 27: 125-134, 1987 PubMed: 87215536 Adachi A et al. Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus. J. Virol. 61: 209-213, 1987 PubMed: 87061242 Fantini J et al. Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion. J. Virol. 66: 580-585, 1992 PubMed: 92085432 Butzow R et al. A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans. J. Cell Biol. 122: 721-727, 1993 PubMed: 93328773 Trainer DL et al. Biological characterization and oncogene expression in human colorectal carcinoma cell lines. Int. J. Cancer 41: 287-296, 1988 PubMed: 88114170 Hanski C et al. Tumorigenicity, mucin production and AM-3 epitope expression in clones selected from the HT-29 colon carcinoma cell line. Int. J. Cancer 50: 924-929, 1992 PubMed: 92210223 Reiter LS et al. The role of the urokinase receptor in extracellular matrix degradation by HT29 human colon carcinoma cells. Int. J. Cancer 53: 444-450, 1993 PubMed: 93154838 Barnett SW et al. Characterization of human immunodeficiency virus type 1 strains recovered from the bowel of infected individuals. Virology 182: 802-809, 1991 PubMed: 91220730 Shabahang M et al. 1,25-Dihydroxyvitamin D3 receptor as a marker of human colon carcinoma cell line differentiation and growth inhibition. Cancer Res. 53: 3712-3718, 1993 PubMed: 93338694 Lesuffleur T et al. Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT- 29 cell subpopulations. J. Cell Sci. 106: 771-778, 1993 PubMed: 94140929 Pollack MS et al. HLA-A, B, C and DR alloantigen expression on forty-six cultured human tumor cell lines. J. Natl. Cancer Inst. 66: 1003-1012, 1981 PubMed: 81218998 Fantini J et al. Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor. Proc. Natl. Acad. Sci. USA 90: 2700-2704, 1993 PubMed: 93219351 Devedjian JC et al. Regulation of the alpha 2A-adrenergic receptor in the HT29 cell line. Effects of insulin and growth factors. J. Biol. Chem. 266: 14359-14366, 1991 PubMed: 91317789 Santoro IM and Groden J. Alternative splicing of the APC gene and its association with terminal differentiation. Cancer Res. 57: 488-494, 1997 PubMed: 97164672 Bermudez LE et al. Exposure to low oxygen tension and increased osmolarity enhance the ability of Mycobacterium American Type Culture Collection P.O. Box 1549 Manassas, VA 20108 USA www.atcc.org avium to enter intestinal epithelial (HT-29) cells. Infect. Immun. 65: 3768-3773, 1997 PubMed: 97427962 Tsao H et al. Novel mutations in the p16/CDKN2A binding region of the Cyclin-dependent Kinase-4 gene. Cancer Res. 58: 109-113, 1998 PubMed: 98086035 Qian XC and Brent TP. Methylation hot spots in the 5' flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene. Cancer Res. 57: 3672-3677, 1997 PubMed: 97433101 Morin PJ et al. Apoptosis and APC in colorectal tumorigenesis. Proc. Natl. Acad. Sci. USA 93: 7950-7954, 1996 PubMed: 96353925 White LJ et al. Attachment and entry of recombinant norwalk virus capsids to cultured human and animal cell lines. J. Virol. 70: 6589-6597, 1996 PubMed: 96386543 Kolanus W et al. alphaLbeta2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1 a cytoplasmic regulatory molecule. Cell 86: 233-242, 1996 PubMed: 96319726 Wang R et al. Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor. Proc. Natl. Acad. Sci. USA 93: 8425-8430, 1996 PubMed: 96323238 : Young SW et al. Gadolinium(III) texaphyrin: a tumor selective radiation sensitizer that is detectable by MRI. Proc. Natl. Acad. Sci. USA 93: 6610-6615, 1996 PubMed: 96270589 Groh V et al. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc. Natl. Acad. Sci. USA 93: 12445-12450, 1996 PubMed: 97057262 Takahashi K et al. Keratan sulfate modification of CD44 modulates adhesion to hyaluronate. J. Biol. Chem. 271: 94909496, 1996 PubMed: 96199206 Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC. Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988. Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

ATCC Warranty
The viability of ATCC products is warranted for 30 days from the date of shipment. If you feel there is a problem with this product, contact Technical Services by phone at 800-638-6597 (U.S., Canada, and Puerto Rico) or 703-365-2700 (elsewhere) or by email at tech@atcc.org.

Disclaimers
This product is intended for laboratory research purposes only. It is not intended for use in humans. While ATCC uses reasonable efforts to include accurate and up-todate information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, and use. ATCC is not liable for any damages or injuries arising from receipt and/or use of this product. 800-638-6597 (U.S., Canada, and Puerto Rico) 703-365-2700 Fax: 703-365-2750 E-mail: tech@atcc.org

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Product Information Sheet for HTB-38

While reasonable effort is made to insure authenticity and reliability of strains on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of cultures. Please see the enclosed Material Transfer Agreement (MTA) for further details regarding the use of this product. The MTA is also available on our Web site at www.atcc.org. ATCC 2007. All rights reserved.

ATCC is a registered trademark of the American Type Culture Collection. 09/07

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