RKO-E6 (ATCC® CRL-2578)

Organism: Homo sapiens, human  /  Tissue: colon  /  Disease: Carcinoma, Papilloma­

Organism Homo sapiens, human
Tissue colon
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2  [Contains HPV6 and CMV viral sequences]
Disease Carcinoma, Papilloma­
Applications
The RKO-E6 cell line can be used together with its parental cell line, RKO (ATCC CRL-2577) to investigate the effects of p53 loss on cellular parameters such as p53 mediated transcription and apoptosis.

Derivation The RKO-E6 cell line was generated from the colon carcinoma RKO (ATCC CRL-2577) cell line by transfection with pCMV-E6 using Lipofectin.
Comments
The cells contain a stably integrated human papilloma virus (HPV) E6 oncogene under control of the cytomegalovirus (CMV) promoter. The HPV E6 oncogene causes a decrease in normal p53 levels and functions.

The RKO-E6 cell line lacks appreciable functional p53.

Disruption of normal p53 function in human colon carcinoma RKO cells with the human papillomavirus E6 oncoprotein results in reduced repair of u.v.-induced DNA damage and also loss of induced repair following cellular u.v.-irradiation.

RKO cells contain wild-type p53 but lack endogenous human thyroid receptor nuclear receptor (h-TRbeta1). The level of p53 protein is higher in RKO (ATCC CRL-2577) cells than in RKO-E6 (ATCC CRL-2578) cells.

The RKO-E6 cell line can be used together with its parental cell line, RKO (ATCC CRL-2577) to investigate the effects of p53 loss on cellular parameters such as p53 mediated transcription and apoptosis.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Subculturing 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-0.53% (w/v) EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. 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).
    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 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C

Subcultivation Ratio: 1:4 to 1:12
Medium Renewal: Every 2 to 3 days.
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

Cryopreservation
culture medium 95%; DMSO, 5%
Culture Conditions
Temperature: 37°C
Name of Depositor MC Hollander, AJ Fornace
References

Smith ML, et al. Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene 10: 1053-1059, 1995. PubMed: 7700629

Smith ML, et al. Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene 13: 2255-2263, 1996. PubMed: 8950993

Bhat MK, et al. Tumor suppressor p53 is a negative regulator in thyroid hormone receptor signaling pathways. J. Biol. Chem. 272: 28989-28993, 1997. PubMed: 9360971

Kessis TD, et al. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc. Natl. Acad. Sci. USA 90: 3988-3992, 1993. PubMed: 8387205

Basic Documentation
References

Smith ML, et al. Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene 10: 1053-1059, 1995. PubMed: 7700629

Smith ML, et al. Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene 13: 2255-2263, 1996. PubMed: 8950993

Bhat MK, et al. Tumor suppressor p53 is a negative regulator in thyroid hormone receptor signaling pathways. J. Biol. Chem. 272: 28989-28993, 1997. PubMed: 9360971

Kessis TD, et al. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc. Natl. Acad. Sci. USA 90: 3988-3992, 1993. PubMed: 8387205