J Korean Med Sci.  2004 Jun;19(3):321-326. 10.3346/jkms.2004.19.3.321.

Effects of HMGB-1 Overexpression on Cell-Cycle Progression in MCF-7 Cells

• Yoon S
• Lee JY
• Yoon BK
• Bae DS
• Choi DS

• KMID: 1786806
• DOI: http://doi.org/10.3346/jkms.2004.19.3.321

Abstract

High mobility group-1 (HMGB-1) enhances the DNA interactions and possesses a transcriptional activation potential for several families of sequence-specific transcriptional activators. In order to examine the effect of HMGB-1 on the cell cycle progression in MCF-7 cells, the HMGB-1 expression vector was transfected into synchronized MCF-7 cells, and the effect of HMGB-1 overexpression on the cell cycle was examined. The HMGB-1 protein level in the transfected cells increased 4.87-fold compared to the non-transfected cells. There were few changes in the cell cycle phase distribution after HMGB-1 overexpression in the MCF-7 cells. Following the estrogen treatment, the cell cycle progressed in both the HMGB-1 overexpressed MCF-7 and the mock-treated cells. However, a larger proportion of HMGB-1 overexpressing MCF-7 cells progressed to the either S or G2 phase than the mock-treated cells. The mRNA levels of the cell cycle regulators changed after being treated with estrogen in both the HMGB-1 overexpressing MCF-7 and the mock-treated cells, but the changes in the expression level of the cell cycle regulator genes were more prominent in the HMGB-1 overexpressing MCF-7 cells than in the mock-treated cells. In conclusion, HMGB-1 overexpression itself does not alter the MCF-7 cell cycle progression, but the addition of estrogen to the HMGB-1 overexpressing MCF-7 cells appears to accelerate the cell cycle progression.

Keyword

Cell Cycle; HMG-Box Domains; HMGB1 Protein; Receptors, Estrogen

MeSH Terms

Blotting, Western
Cell Cycle
Cell Line, Tumor
Densitometry
Estrogens/metabolism
G2 Phase
Genetic Vectors
HMGB1 Protein/*biosynthesis
Human
Kinetics
Oligonucleotides/chemistry
Plasmids/metabolism
Protein Structure, Tertiary
RNA, Messenger/metabolism
Reverse Transcriptase Polymerase Chain Reaction
S Phase
Support, Non-U.S. Gov't
Time Factors
Trans-Activation (Genetics)
Transfection

Figure

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  Fig. 1 Construction of the HMGB-1 vector. (A) The entire HMGB-1 (nucleotide +51-+744) sequences were based on the GenBank database. Bold ATG was the start codon and TAA was the stop codon. The square box indicates the PCR primer. (B) The entire HMGB-1 was PCR amplified using the primers HMGB-1T primer. PCR products were cloned into the pCIneo vector.

• 

  Fig. 2 Transfection efficiency of the HMGB-1 expression vector in the MCF-7 cells. MCF-7 cells synchronized at the G0/G1 phase were transfected with 1 µg HMGB-1 using the Lipofectamin Plus reagent. (A) After 2 days, the cell lysates were prepared and analyzed by immunoblotted with HMGB-1 antisera. Mock -treated cells were used as the control. (B) The film was scanned with the Gel Doc. System and the relative densities were analyzed.

• 

  Fig. 3 Kinetics of the cell cycle progression in the HMGB-1 overexpressing MCF-7 cells and the mock cells treated with or without estrogen. The MCF-7 cells synchronized at the G0/G1 phase were transfected with HMGB-1, trypsinized, and treated with propidium iodide. The cell cycle distribution was then analyzed by flow cytometry. (A) Before and after synchronization, the cell cycle distribution was analyzed by flow cytometry. (B) Mock (C) Mock with estrogen (10e-7M). (D) HMGB-1 overexpressed MCF-7 cells. (E) HMGB-1 overexpressing MCF-7 cells with estrogen (10e-7M). The results from two independent experiments are represented.

• 

  Fig. 4 mRNA levels of the cell cycle related genes in HMGB-1 overexpressing MCF-7 cells and mock cells treated with or without estrogen. Mock and HMGB-1 overexpressed MCF-7 cells were seeded and treated with or without estrogen (10e-7M) as Fig. 3. At the indicated times, the total RNAs were prepared and analyzed by RT-PCR. (A) Mock (B) Mock with estrogen (10e-7M) (C) HMGB-1 overexpressing MCF-7 cells (D) HMGB-1 overexpressing MCF-7 cells with estrogen (10e-7M) (E) Bands of the RT-PCR product of the mock with estrogen (10e-7M) were scanned and relative densities were analyzed (F). Bands of the RT-PCR products of HMGB-1 overexpressing MCF-7 cells with estrogen (10e-7M) were scanned and the relative densities were analyzed.

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