Cyclin-dependent kinase inhibitor p27Kip1 controls growth and cell cycle progression in human uterine leiomyoma

Abstract

The molecular mechanism of the cell-cycle machinery in uterine leiomyoma has not yet been fully elucidated. Among the various types of cell-cycle regulators, p27(Kip1) (p27) is considered to be a potent tumor suppressor. To provide further molecular basis for understanding the progression of uterine leiomyoma, our objective was to evaluate the expression level of p27 in normal myometrium and uterine leiomyoma tissue and its effect on cytogenic growth. Western blot analysis, real-time polymerase chain reaction (PCR) and immunohistochemical staining revealed that p27 protein and messenger RNA were down-regulated in uterine leiomyoma tissue and cultured cells compared to normal myometrium. Full-length human p27 cDNA was transferred using a replication-deficient recombinant adenoviral vector (Ad.p27) into uterine leiomyoma cells and evaluated the effect on cell proliferation. Transfection of Ad.p27 into uterine leiomyoma cells resulted in the induction of apoptosis, reduction in viability and proliferation of uterine leiomyoma cells. Our results suggest a new paradigm that down-regulated p27 protein expression is the possible underlying mechanism for the growth of uterine leiomyoma and over-expression of p27 induces cell death. This study provides better understanding of the control exerted by p27 in regulating growth and disease progression of uterine leiomyoma.

Fig. 1

Decreased p27 protein expression levels in human uterine leiomyoma tissue.Total protein was isolated from uterine leiomyoma and normal myometrial tissues. Nine pairs of tumor and normal tissues; i.e., each pair of samples was obtained from same patient - M; uterine leiomyoma, N; normal myometrium, 5 of the tissue samples belonged to the proliferative phase (#1 to #5) and the rest to the secretory phase (#6 to #9). Fifty micrograms of the total isolated protein were resolved by SDS-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. The blot was incubated with antibody against p27. Reactive bands were visualized with an ECL labeling and detection system. β-tubulin was used as the loading control. Densitometric analysis was also done to the western blots to determine the relative protein expression levels.

Fig. 2

Decreased p27 protein expression levels in cultured human uterine leiomyoma tissue. Total protein was isolated from cultured, well-grown, confluent uterine leiomyoma and normal myometrium tissue samples (3 pairs of tumor and normal tissue, obtained from same patient - M; uterine leiomyoma, N; normal myometrium, 2 of the cultured cells were from proliferative phase tissue samples (#1 and #2) and #3 was cultured cells of the secretory phase tissue sample). Fifty micrograms of the total isolated protein were resolved by SDS-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. The blot was incubated with antibody against p27. Reactive bands were visualized with an ECL labeling and detection system. β-tubulin was used as the loading control. Densitometric analysis was also done to the Western blots to determine the relative protein expression levels.

Fig. 3

Comparison of immunohistochemical staining for p27 protein. Immunohistochemical staining for p27 formalin-fixed paraffin-embedded sections of uterine leiomyoma (LM) and myometrial tissues (NM). (A) Myometrial smooth muscle cells showed more predominant immunostaining for p27 protein than the leiomyoma cells (magnification ×40), (B). Normal myometrial cells showed positive for p27 protein (magnification ×100), (C). Leiomyoma cells showed negative for p27 protein (magnification ×100), (D). Cytoplasmic and nuclear brown-staining cells are positive for p27 protein expression (magnification ×400).

Fig. 4

Decreased p27 mRNA levels in human uterine leiomyoma tissue. Total RNA was isolated from uterine leiomyoma and normal myometrial tissues (4 pairs of tumor and normal tissues; i.e., each pair of samples was obtained from same patient; tissue samples #1 and #2 were isolated at the proliferative phase and tissue samples #3 and #4 were isolated at the secretory phases of the menstrual cycle). cDNA templates for RT were prepared from the total RNA extracted. The level of p27 mRNA was determined by quantitative real-time PCR. Relative p27 mRNA levels (normalized to GAPDH) in paired samples represented as M; uterine leiomyoma, N; normal myometrium. ^*, p<0.05; ^**, p<0.01.

Fig. 5

Effect of Ad.p27 transfection on uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with Ad.null and Ad.p27 recombinant, non-replicating adenoviral vectors. Twenty-four hours after transfection, immunoblotting was performed to detect the expression levels of p27, p21, Rb, cdk2 and cdk4. β-tubulin was used as the loading control. Transfection of Ad.p27 onto uterine leiomyoma cells caused increased expression levels of p27 and dephosphorylation of Rb.

Fig. 6

Inhibition of the synthesis of DNA in uterine leiomyoma cells by Ad.p27. The antiproliferative effects were determined after transfecting uterine leiomyoma cells for 24 hr with Ad.null and Ad.p27 adenoviral vectors and labeling with BrdU (10 µM) for 24 hr. Results are expressed as percentage inhibition of BrdU incorporation relative to control cells. Values are the means (±SD) of three experiments with triplicate determinations. ^*, p<0.05; ^**, p<0.01.

Fig. 7

Effect of Ad.p27 on the cell cycle profile. Ad.null and Ad.p27 transfected uterine leiomyoma cells were harvested, fixed, stained with PI, and analyzed by flow cytometry analysis. The values represent the number of cells in each phase of the cell cycle as a percentage (%) of total cells. Following transfection with Ad.p27, the growth of uterine leiomyoma cells was affected with an increase in the percentage of cells in the sub-G1 phase.