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MEK1 Mutant-A375 Isogenic Cell Line (ATCC® CRL-1619IG-3™)

Organism: Homo sapiens, human  /  Tissue: skin  /  Disease: malignant melanoma

Permits and Restrictions

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Organism Homo sapiens, human
Tissue skin
Product Format frozen 1.0 mL
Morphology epithelial
Culture Properties adherent
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Disease malignant melanoma
Age 54 years
Gender female
Applications

Anti-cancer drug screening, BRAF drug resistant melanoma model, tumor-associated MEK1Q56P/BRAFV600E research, MAPK/ERK signaling pathway. Ideally the parental cell line (ATCC® CRL-1619™) is included as a control for drug-sensitive response in experiments with this cell line.
Storage Conditions liquid nitrogen vapor phase
Images
Clinical Data 54 years
female
Comments

This is a malignant BRAFV600E/MEK1Q56P mutant isogenic line derived from the parental A375 (ATCC CRL-1619) cell line. The c.167A>C knock-in mutation encoding MEK1 p.Q56P protein expression was generated at ATCC by utilizing the CRISPR/Cas9 gene editing technology. This is a heterozygous mutation expressing the MEK1 wild-type and the c.167A>C mutant alleles.

BRAF is a proto-oncogene encoding B-RAF, a serine/threonine kinase of the RAF family that acts downstream of RAS and upstream of MEK in the MAPK/ERK signaling pathway. Mutations in BRAF lead to excessive cellular proliferation, differentiation, and survival. BRAF V600E mutations are present in 50% of melanomas and although there are current BRAF inhibitors used as successful therapeutics, patients often become resistant to drugs several months following treatment. One mechanism of resistance to these inhibitors is caused by upstream secondary RAS acquired mutations. The MEK1 Q56P mutant isogenic line, ATCC CRL-1619IG-3 has been validated at the genomic, transcript, and protein bio-functional levels and exhibits significant resistance to the BRAF inhibitors Dabrafineb and Vemurafenib, as well as the MEK inhibitor trametinib when compared to its parental cell line. Furthermore, A-375 MEK1Q56P (ATCC CRL-1619IG-3™) cells display increased sensitivity to combination MEK/BRAF inhibitor treatments, making this line an ideal model system for the development of novel combination therapies targeting multiple points in the RAS-RAF-MEK-ERK-MAPK signaling pathway, as well as for the screening of new potential BRAF and MEK inhibitors.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco’s Modified Eagle’s Medium (DMEM; ATCC 30-2002). To make the complete medium add the following component to the base medium: Fetal bovine serum (FBS; ATCC 30-2020) 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. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM 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.
    Cultures can be established between 1.0 x 104 and 4 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 8 X 103 and 4 X 104 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:6 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation Complete growth medium supplemented with 5% (v/v) DMSO (ATCC 4-X)
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial ≥ 1 x 106
Volume 1.0 mL
STR Profile
Amelogenin: X
CSF1PO: 11,12
D13S317: 11,14
D16S539: 9
D5S818: 12
D7S820: 9
TH01: 8
TPOX: 8,10
vWA: 16,17
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human immunodeficiency virus: None detected
Epstein-Barr virus: None detected
Human papillomavirus: None detected
Viability ≥ 50%
Functional Tests Genotype testing for knock-in mutation - heterozygous DNA change: c.167A>C mutation (correlates to protein sequence p.Q56P.
Resistance to BRAF-specific inhibitor drugs during product development and validation.
Population Doubling Time Approximately 19 hours
Year of Origin 2018
References

Wagle N, et al. MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition. Cancer Discov 4(1):61-68, 2014. PubMed: 24265154

Emery CM, et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc Natl Acad Sci USA 106(48): 20411-20416, 2009. PubMed: 19915144

Sharma SD, et al. Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors. Proc. Natl. Acad. Sci. USA 93: 13715-13720, 1996. PubMed: 8943000

Gershwin ME, et al. Immunobiology of heterotransplanted human tumors in nude mice. J. Natl. Cancer Inst. 58: 1455-1463, 1977. PubMed: 857033

Giard DJ, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51: 1417-1423, 1973. PubMed: 4357758

Cormier JN, et al. Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo. Int. J. Cancer 75: 517-524, 1998. PubMed: 9466650

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
SDS
SDS
Other Documentation
Other Document
Application Data Other Document
Application Data Other Document
Application Data Other Document
Application Data
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103F™).

Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development.

If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.

In addition to the foregoing, this product's use is governed by the CRISPR Label License Agreement. For information on purchasing a license to use this product for purposes other than those permitted in the CRISPR Label License Agreement, please contact The Broad Institute at partnering@broadinstitute.org.
References

Wagle N, et al. MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition. Cancer Discov 4(1):61-68, 2014. PubMed: 24265154

Emery CM, et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc Natl Acad Sci USA 106(48): 20411-20416, 2009. PubMed: 19915144

Sharma SD, et al. Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors. Proc. Natl. Acad. Sci. USA 93: 13715-13720, 1996. PubMed: 8943000

Gershwin ME, et al. Immunobiology of heterotransplanted human tumors in nude mice. J. Natl. Cancer Inst. 58: 1455-1463, 1977. PubMed: 857033

Giard DJ, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51: 1417-1423, 1973. PubMed: 4357758

Cormier JN, et al. Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo. Int. J. Cancer 75: 517-524, 1998. PubMed: 9466650