KC-5471

HCC1954-ABCG2 Cell Line

56926

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Background of HCC1954-ABCG2 Cell Line

ABCG2 (ATP Binding Cassette Subfamily G Member 2), also known as BCRP (Breast Cancer Resistance Protein), CD338, and MXR (Mitoxantrone Resistance Protein), is an ATP-binding cassette (ABC) transporter that functions as an efflux pump for xenobiotics and endogenous molecules. It is highly expressed in placental tissue, with lower levels in the small intestine, liver, colon, and blood-brain barrier. Pathologically, ABCG2 is a major mediator of multidrug resistance (MDR) in various cancers, including breast, ovarian, and lung cancer, by actively exporting chemotherapeutic agents such as mitoxantrone, doxorubicin, and topotecan. It also plays a role in physiological processes like urate excretion and porphyrin homeostasis. Drug development efforts have focused on overcoming MDR through ABCG2 inhibition; while many inhibitors have been identified, none have received clinical approval. Recent advances include dynamic inhibitors (e.g., PZ-39) that induce ABCG2 degradation via lysosomal targeting, representing a novel class of targeted protein degradation (TPD) agents for membrane proteins. Other strategies involve targeting upstream regulators like p38 to disrupt ABCG2 oligomerization and function.

Specifications

Catalog Number	KC-5471
Cell Line Name	HCC1954-ABCG2 Cell Line
NCBI/UniProt Accession Number	NM_004827
Clone Number	10#
Host Cell Line	Human HCC1954 Cell Line
Description	Stable HCC1954 cell line expressing exogenous human ABCG2 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 3μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Large epithelial
Subculture	Split saturated culture 1:2 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 45 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

HCC1954-ABCG2 cell line was generated using a lentiviral vector expressing the human ABCG2 sequence.

Characterization

Figure 1: Characterization of human ABCG2 overexpression in the HCC1954-ABCG2 stable clone using FACS.

Figure 2: Characterization of ABCG2 overexpressing in HCC1954-ABCG2 stable clones using PCR sequencing.

Figure 3: HCC1954-ABCG2 were seeded into 96-well plates, treated with compounds for 5 days, and then read out with Cell-Titer Glo system.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:2 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Damiani D, Tiribelli M. ABCG2 in Acute Myeloid Leukemia: Old and New Perspectives. Int J Mol Sci. 2023 Apr 12;24(8):7147. doi: 10.3390/ijms24087147. PMID: 37108308; PMCID: PMC10138346.
2. Robey RW, To KK, Polgar O, Dohse M, Fetsch P, Dean M, Bates SE. ABCG2: a perspective. Adv Drug Deliv Rev. 2009 Jan 31;61(1):3-13. doi: 10.1016/j.addr.2008.11.003. Epub 2008 Dec 16. PMID: 19135109; PMCID: PMC3105088.
3. Chandratre S, Olsen J, Howley R, Chen B. Targeting ABCG2 transporter to enhance 5-aminolevulinic acid for tumor visualization and photodynamic therapy. Biochem Pharmacol. 2023 Nov;217:115851. doi: 10.1016/j.bcp.2023.115851. Epub 2023 Oct 17. PMID: 37858868; PMCID: PMC10842008.
4. Hira D, Terada T. BCRP/ABCG2 and high-alert medications: Biochemical, pharmacokinetic, pharmacogenetic, and clinical implications. Biochem Pharmacol. 2018 Jan;147:201-210. doi: 10.1016/j.bcp.2017.10.004. Epub 2017 Oct 13. PMID: 29031817.