Potential link between estrogen receptor-alpha gene hypomethylation and uterine fibroid formation

Uterine leiomyomas are the most common uterine tumors in women. Estrogen receptor-alpha (ER-alpha) is more highly expressed in uterine leiomyomas than in normal myometrium, suggesting a link between uterine leiomyomas and ER-alpha expression. DNA methylation is an epigenetic mechanism of gene regulation and plays important roles in normal embryonic development and in disease progression including cancers. Here, we investigated the DNA methylation status of the ER-alpha promoter region (-1188 to +229 bp) in myometrium and leiomyoma. By sodium bisulfite sequencing, 49 CpG sites in the proximal promoter region of ER-alpha gene were shown to be unmethylated in both leiomyoma and normal myometrium. At seven CpG sites in the distal promoter region of the ER-alpha gene, there was a variation in DNA methylation status in myometrium and leiomyoma. Further analysis of the DNA methylation status by bisulfite restriction mapping among 11 paired samples of myometrium and leiomyoma indicated that CpG sites in the distal region of ER-alpha promoter are hypomethylated in leiomyomas of nine patients. In those patients, ER-alpha mRNA levels tended to be higher in the leiomyoma than in the myometrium. In conclusion, there was an aberrant DNA methylation status in the promoter region of ER-alpha gene in uterine leiomyoma, which may be associated with high ER-alpha mRNA expression.     

HMGI(Y) expression in human uterine leiomyomata. Involvement of another high-mobility group architectural factor in a benign neoplasm.

Chromosomal rearrangements involving 6p21 have been observed in uterine leiomyomata and a variety of other benign tumors. The gene for HMGI(Y), a member of the high-mobility group (HMG) family of proteins, has been localized to 6p21. To determine whether rearrangements observed in this area alter HMGI(Y) expression, we analyzed HMGI(Y) DNA-binding activity in protein extracts from uterine leiomyoma and normal myometrium tissues. This report describes a uterine leiomyoma specimen with an inv(6)(p21q15). A genomic P1 clone that contains the HMGI(Y) region of chromosome 6 is found to span the inversion breakpoint by fluorescent in situ hybridization of metaphase chromosomes. Expression of HMGI(Y) protein in this leiomyoma specimen is increased dramatically as compared with the matching normal myometrial tissue. Elevated HMGI(Y) expression was also found in 8 of 16 leiomyomas without cytogenetically detectable chromosome 6p21 aberrations but not in any of the 9 matching myometrial tissues. Analysis of the genetic events involved in the pathobiology of these benign tumors will provide a basis for understanding the process of improper cellular growth and might be important in deciphering the multistep pathway of tumorigenesis.     

The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells

Uterine fibroids (leiomyomas) are a major women's health problem. Currently, the standard for treatment remains hysterectomy, since no other treatment modalities can reduce both symptoms and recurrence. As leiomyomas are benign neoplasias of smooth muscle cells, we sought to understand the regulation of uterine smooth muscle cell mitogenesis by CCN5, a growth arrest-specific gene in vascular smooth muscle cells which is induced and maintained by heparin treatment. Using autologous human myometrial and leiomyoma smooth muscle cells, we demonstrate that the proliferation and motility of both cell types are inhibited by the overexpression of CCN5. Surprisingly, we show that even though CCN5 is induced by heparin in vascular smooth muscle cells, treatment with heparin does not induce CCN5 expression in human uterine smooth muscle cells. Furthermore, we examine CCN5 mRNA expression in 10 autologous pairs of human myometrial and leiomyoma tissues and determine that CCN5 is down-regulated in 100% of the leiomyoma tissues analysed when compared to their normal myometrial counterparts. Thus, our data strongly suggest that CCN5 may exert an important function in maintaining the normal uterine phenotype and that loss of the anti-proliferative protein CCN5 from normal myometrium may account, at least in part, for tumorigenesis.     

Heparin inhibits proliferation of myometrial and leiomyomal smooth muscle cells through the induction of alpha-smooth muscle actin, calponin h1 and p27

Mast cells are widely distributed in human tissues, including the human uterus. However, the function of mast cells in uterine smooth muscle has not been clearly established. Mast cells possess secretory granules containing such substances as heparin, serotonin, histamine and many cytokines. To help establish the role of mast cells in the human myometrium, the action of heparin was investigated using smooth muscle cells (SMC) from normal myometrium and from leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by heparin treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under heparin treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by heparin, whereas cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not changed. Taken together, these results indicate that heparin inhibits the proliferation of myometrial and leiomyomal SMC through the induction of alpha-SMA, calponin h1 and p27. We suggest that heparin from mast cells may induce differentiation in uterine SMC and may influence tissue remodelling and reconstruction during physiological and pathophysiological events.     

子宫肌瘤组织EGFR受体与IGF-1R的定位及定量分析

目的 :探讨表皮生长因子受体与胰岛素样生长因子受体在子宫肌瘤发病机理中的作用。方法 :应用流式细胞术定量分析法及免疫组化S -P法分析比较 4 0例子宫肌瘤患者子宫肌层和子宫肌瘤组织表皮生长因子受体 (EGFR)和胰岛素样生长因子受体 (IGF - 1R)的含量及表达水平 ,月经周期根据月经史及子宫内膜组织来判断。结果 :子宫肌瘤组织EGFR ,IGF - 1R的表达及含量显著高于同一子宫邻近正常肌层组织 (p <0 0 5 ,p <0 0 1 ) ;子宫肌瘤组织与其邻近肌层组织相比较 ,EGFR的含量及表达在分泌期无显著性差异 (p >0 0 5 ) ,而在增生期含量明显增高 (p <0 0 5 ) ;而IGF - 1R的表达与月经周期无关。 结论 :EGFR ,IGF - 1R在子宫肌瘤组织中高表达与其发生、发展有关     

Clinical implications of coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine leiomyoma

The expression of Gas6, the protein product of the growth arrest-specific gene 6 (gas6), a member of the vitamin K-dependent protein family, and the receptor tyrosine kinases Axl and Sky and their mRNAs in uterine leiomyoma and normal uterine myometrium tissues were investigated by competitive RT-PCR-Southern blot analysis using recombinant RNA and immuno histochemical analysis respectively. There was no significant difference between the histoscores and levels of Sky mRNA in uterine leiomyoma and normal uterine myometrium, although the levels of Gas6 and Axl mRNAs in uterine leiomyoma were significantly higher than in normal uterine myometrium in each case. It is suggested that Gas6 and Axl signal transduction is aberrantly stimulated in uterine leiomyoma, possibly related to its growth.     

Cell proliferation and apoptosis in human uterine leiomyomas and myometria

To determine the role of cell proliferation and apoptosis in uterine leiomyoma growth, we studied protein expression of two major regulatory proteins of apoptosis -- Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) -- and two endogenous markers of cell replication - proliferating cell nuclear antigen (PCNA) and Ki-67 - in tumors and matched myometrium from premenopausal women. Conventional mitotic indices also were determined, and all proliferation data were correlated to tumor size. In situ end-labeling of fragmented DNA and routine histology were used to assess apoptosis. Our results showed that the apoptosis-regulating proteins (Bcl-2 and Bax) were expressed in the cytoplasm of the leiomyoma and myometrial smooth muscle cells throughout the menstrual cycle. Bax expression differed from Bcl-2 in that it also was found in the cytoplasm of vascular smooth muscle cells of the myometria and tumors. Both tumors and myometrial samples expressed 26-kDa and 21-kDa proteins that reacted with antibodies directed towards Bcl-2 and Bax, respectively. Apoptosis was not a prominent feature of uterine leiomyomas or myometrium. PCNA- and Ki-67-labeling and mitotic counts were significantly ( P<0.05) higher in leiomyomas than in matched myometrial samples. Proliferative activity was variable for individual tumors of the same patient and independent of tumor size. Our results suggest that altered apoptosis by overexpression of Bcl-2 or by decreased expression of Bax does not appear to be a major factor in uterine leiomyoma growth. We conclude that increased cell proliferation is the most significant contributor to growth and that the proliferative state is autonomous for each tumor in a given patient and is independent of tumor size.     

Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics

In vitro model systems for studying uterine leiomyomas are limited in that human-derived leiomyoma cells grow poorly in culture compared with normal myometrial cells and begin to senesce early, at approximately passage 10 in our studies. To our knowledge, a good in vitro human-derived cell culturing system for leiomyomas does not exist. In an attempt to fill this void, we have immortalized a uterine leiomyoma cell line by inducing telomerase activity, which allows cells to bypass their normal programmed senescence. Telomerase activity was induced by infecting the target (uterine leiomyoma and normal myometrial) cells with a retroviral vector containing hTERT, the gene for the catalytic subunit of telomerase. Subsequent analysis by RT-PCR and the telomeric repeat amplification protocol assay confirmed expression of the inserted gene and induction of telomerase activity in leiomyoma and myometrial cells. Analysis of cells for estrogen receptor-alpha and progesterone receptor proteins by Western blotting showed no change in expression of these proteins between the immortalized and parental leiomyoma and myometrial cells. Both immortalized and parental myometrial and leiomyoma cells expressed the smooth muscle-specific cytoskeletal protein alpha-actin and were negative for mutant p53 protein as evidenced by immunocytochemical staining. The immortalized leiomyoma and myometrial cells showed no anchorage-independent growth, with the exception of a small subpopulation of immortalized leiomyoma cells at a higher passage that did form two to three small colonies (per 50,000 cells) in soft agar. None of the immortalized cells were tumorigenic in nude mice. In conclusion, our data show the successful insertion of the hTERT gene into leiomyoma and myometrial cells and the immortalization of these cell lines without phenotypic alteration from the parental cell types (up to 200 population doublings). These cells should help to advance research in understanding the molecular pathways involved in the conversion of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of uterine leiomyomas. Answers to these questions will undoubtedly lead to the development of more effective treatment and intervention regimens for clinical cases of uterine leiomyoma.     

Increased expression of calcium-binding protein S100 in human uterine smooth muscle tumours

S100 proteins belong to the EF-hand Ca(2+ )-binding protein family and regulate a variety of cellular processes via interaction with different target proteins. Several diseases, including cancer and melanoma, are related to the abnormal expression of S100 proteins, which are expressed in cell- and tissue-specific manners. We investigated the expression of S100 family members in human uterine smooth muscle tumours. Expression of six members of the S100 protein family: S100A1, A4, A6, A7, A10 and A11, was found in human uterine leiomyoma and myometrium tissue, but expression of other members was not detected by RT-PCR. Real-time PCR showed that S100A11 expression was significantly increased in leiomyoma compared with myometrium. Suppression of S100A11 by small interfering RNA (siRNA) led to apoptosis, and the overexpression of S100A11 inhibited apoptosis in human uterine smooth muscle tumour cells. These findings suggest that S100A11 has an anti-apoptotic function and is related to the process of growth of human uterine leiomyoma.     

Evaluation of nucleolar organizer region (NOR) parameters in the uterine leiomyoma

Nucleolar organizer regions (NORs) were assessed in 27 women affected by uterine leiomyoma. Tissue samples obtained during surgery were silver-stained according to the method of Ploton et al. The assessed parameters were as follows: the number of argyrophylic nucleolar organizer regions (AgNORs) per nucleus, single AgNOR area, AgNOR intranuclear distribution, and AgNOR coefficient. The parameters were assessed quantitatively. It was found that the AgNOR coefficient was higher in uterine leiomyoma compared to the normal smooth muscle cells. The development of leiomyoma is associated with a marked decrease in myocyte nucleolar area, which accounts for 30% of the nucleus in the normal myometrium. With the comparable AgNOR number in the single nucleus, there were no differences in the single AgNOR granule area (1.21 microm2 +/- 0.047 and 1.11 microm2 +/- 0.025 in normal myometrium and in leiomyoma, respectively). In the normal myometrium, there was a positive correlation between nuclear area and the single AgNOR granule area, as well as between the AgNOR coefficient and the single AgNOR granule area. There was also a negative correlation between the number of granules per nucleus and their central and peripheral intranuclear distribution. The development of leiomyoma was associated with loss of all correlations observed in the control group.     

Transforming growth factor-alpha, epidermal growth factor receptor, and PCNA immunoexpression in uterine leiomyosarcomas and leiomyomas in B6C3F1 mice.

The role of growth factors in the development of murine uterine mesenchymal tumors is unknown. In this study, immunohistochemical expression of transforming growth factor alpha (TGF-alpha) and its receptor epidermal growth factor (EGF-R) was assessed in spontaneous uterine leiomyomas and leiomyosarcomas in B6C3F1 mice. Cell proliferation, which has been induced by some growth factors, was evaluated by immunohistochemical detection of an endogenous marker of cell proliferation, proliferating cell nuclear antigen (PCNA). PCNA labeling indices were determined and compared to the intensity and distribution of TGF-alpha staining in sequential sections of control myometrium or tumor tissue. Results showed uterine leiomyosarcomas had positive cytoplasmic staining for TGF-alpha; however, all uterine leiomyomas evaluated were negative. Positive EGF-R staining was also observed in the uterine leiomyosarcomas, but not in the leiomyomas. EGF-R immunoexpression was detected primarily within the cytoplasm of the leiomyosarcoma cells, with occasional nuclear immunoreactivity. Immunohistochemical staining for PCNA was more intense and there were increased numbers of positively staining nuclei in the leiomyosarcomas compared to samples of control myometrium or leiomyomas. The mean labeling index for the uterine leiomyosarcomas (7.40%) was significantly (p < 0.01) higher than that of leiomyomas (0.29%) and control uterine myometrium (0.13%). We conclude, that TGF-alpha and its receptor, EGF-R, are expressed more intensely in uterine leiomyosarcomas, compared to leiomyomas in B6C3F1 mice. Immunoexpression of TGF-alpha may be an important biomarker of malignancy in uterine smooth muscle tumors in mice. Futhermore, TGF-alpha may play a critical role in increased proliferation of uterine smooth muscle tumor cells as suggested by increased immunolocalization of PCNA in rodent leiomyosarcomas expressing TGF-alpha, although other factors regulating cell replication can not be ruled out.     

Immunohistochemical analysis of bone morphological protein signaling pathway in human myometrium

We assessed by immunohistochemistry the expression of the phosphorylated (activated) form of Smad1 and 5 (P-SMAD1/5), of Noggin and of two smooth muscle cell markers (α-SMA and SM22) in a series of human myometrium samples and in a smooth muscle cell line derived from human myometrium (HUt-SMC, PromoCell, USA). Myometrium samples were removed from two cadavers (a fetus at 26 weeks of gestation and a neonate) and from ten non-menopausal women who underwent hysterectomy for adenomyosis and leiomyoma. P-SMAD1/5 expression was never detected in myometrium (both normal and pathological specimens), but only as a nuclear positive staining in glandular and luminal epithelial cells in sections in which also the endometrial mucosa was present. Noggin was strongly expressed especially in myometrium and adenomyosis samples from non-menopausal patients in comparison to the neonatal and fetal myometrium specimens in which muscle cells were less positive. In more than 95% of HUt-SMCs, α-SMA and Desmin were co-expressed, indicating a pure smooth muscle phenotype. When progesterone was added to the culture medium, no P-SMAD1/5 expression was detected, whereas the expression Noggin and SM22, a marker of differentiated smooth muscle cells, increased by 3 fold (p=0.002) and 4.3 fold (p=0.001), respectively (p=0.002). Our results suggest that, in non-menopausal normal human myometrium, the BMP pathway might be inhibited and that this inhibition might be enhanced by progesterone, which increases the differentiation of smooth muscle cells (SM22 levels). These findings could help in the identification of new mechanisms that regulate uterine motility.