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Diaa M. El-Mowafi - Zagagig University, Egypt

Bcl-2 Oncoprotein as a Determinant to Apoptosis in Uterine Myoma

Diaa M. El-Mowafi, MD1 and Umnia A. El-Hendy, MD2
Obstetrics and Gynecology1 and Microbiology & Immunology2 Departments, Benha Faculty of Medicine, Egypt

Correspondence: Dr. Diaa El-Mowafi, 4 Ghazza St., El-Hossania, El-Mansoura 35111, Egypt.
Fax +2 (050) 2332771

Abstract

Apoptosis is a natural physiological mechanism that leads to death of unwanted cells. Bcl-2 is a protein that inhibits apoptosis. Recent studies have shown a strong correlation between bcl-2 and estrogen-receptor expression in both normal and neoplastic breast epithelium. Many investigators have suggested that estrogen plays an important role in the development of uterine leiomyoma.

In our study, we validated and evaluated ELISA to quantify bcl-2 protein in tissues of leiomyoma, the myometrium from which it developed and the control myometrium. The results were compared with that of western blot technique. We found that bcl-2 concentration in myomatous tissues was statistically higher than that of the control myometrium group (P < 0.001). Bcl-2 was also statistically higher in leiomyoma when compared to that in the myometrium from which it developed (P < 0.001). The sensitivity of ELISA in picking up the positive cases was more than the western technique.

It is concluded that the abundant expression of bcl-2 oncoprotein in leiomyoma relative to that of normal myometrium may be one of the molecular bases for the enhanced growth of leiomyoma. Anti-bcl-2 therapy may be used in selected cases to avoid surgical treatment of fibroids particularly if multiple and/or huge especially in infertile patients. It is also recommended to use ELISA for quantitative determination of bcl-2 concentration.

Introduction

Apoptosis or programmed cell death, is important mechanism of maintaining homeostasis during development (Jacobson et al., 1997). Apoptosis is the mode of cell deletion in hormone dependent atrophy, metamorphosis, embryological development, normal cell turnover, and the targets of cell killing by cytotoxic T-cells and probably also killer and natural killer cells (Duval and Wyllie, 1986). Apoptosis is responsible for substantial proportion of the total cell loss from the tumor and undergoes dramatic changes in frequency during early tumor growth (Sarraf and Bowen, 1986).

The protein encoded by bcl-2 (B-cell lymphoma/ leukemia-2) gene is probably the most well characterized of these genes and data now support a role of bcl-2 protein as a cell death suppressor (Kerr et. al., 1994). Bcl-2 encodes a 26 kilo-dalton (KDa) protein that a novel proto-oncogens being localized to inner mitochondrial membrane (Hackenberg et al., 1990). Several studies have shown over expression of bcl-2 protein in a variety of solid tumors including prostate carcinoma (Mc Donnell et al., 1990), squamous cell carcinoma (Ben Ezra et al., 1994), and breast cancer (Joensuu et al., 1994). Recent studies have shown a strong correlation between bcl-2 and estrogen receptor expression in both normal and neoplastic breast epithelium (Leek et al., 1994).

Uterine leiomyoma is the most common benign smooth muscle tumor occurring as many as 30% of women over 35 years of age (Vollenhoven et al., 1990). Many investigators have suggested that estrogen plays an important role in the development of uterine leiomyoma (Andersen et al., 1993).

In the present study, we validate and evaluate an enzyme-linked immunosorbant assay (ELISA) to quantify bcl-2 protein in the tissues of leiomyoma and in the control myometrium and compare the results with that of western blot technique (WB).

Materials and Methods

Patients and specimens

The present study was performed on 34 women attended Obstetrics and Gynecology Department, Benha Faculty of Medicine. Twenty-two were diagnosed clinically and by ultrasound as uterine fibroids and these fibroids were examined histopathologically after surgery (hysterectomy or myomectomy) to ensure diagnosis. Twelve women with normal myometrium and having other pathology than fibroid. None of the patient received any preoperative hormonal therapy for 6 months prior to surgery. The myometrial and/or leiomyomatus specimens obtained after surgery were carried on ice to the laboratory where they were washed with cold saline and divided into 2 portions; one of which was fixed in 10% formalin for histopathological examination. The other portion of the sample was stored in citrate sucrose dimethylesulphoxide buffer pH 7.4 at - 80oC for subsequent determination of bcl-2 in cell lysate by ELISA and western blotting.

Preparation of cell lysates ( Eissa and Seada, 1998)

All steps were carried at 4oC. Tissues were washed with ice-cold saline and homogenized on ice in 10 mmol/L HEPES buffer (pH 7.5) containing 10 mmol/L K2EDTA, 50 mmol/L NaCl, 5 mmol/L bezamidine, 10 ml/L Triton X-100, 10 mmol/L 2-mercaptoethanol, 0.39 mmol/L phenylmethyle-sulphonyl fluoride (PMSF) and 5mg/L aprotinin with a homogenizer (Art Miccra- D3, Italy) for 60 seconds each, separated by a pause for 1 min. The homogenate was incubated in the lysing buffer on ice for 30 min, with vortex-mixing every 10 min. The homogenate was filtered and then centrifuged at 20 000 g for 20 min with a Hettich (universal 16L-Germany) and the resulting supernatents (lysates) were frozen at - 80oC till time of use. The protein concentration was determined by Bradford's method (1976), using bovine serum albumin as a calibrator.

Quantitative measurement of bcl-2 protein in cell lysate by ELISA (Hockenbery, 1995)

Bcl-2 protein was measured in cell lysate with a monoclonal antibody-based ELISA kit (Oncogene Science Products USA). The results were obtained as units (U) bcl-2/ml and then were expressed in U/mg protein, where one unite of bcl-2 equals 5.6X104 cells of an internal control cell line (HL60).

Detection of bcl-2 by western blot tachnique (Sambrook et al., 1989)

Sixty micrograms of cell lysate proteins in loading buffer (50 mml/L tris, 20g/L sodium dodecyl sulphate, 100 ml/L glycerol, 100 mmol/L beta-mercaptoethanol and 0.05% bromephenol blue solution, pH 6.8) were boiled for 5 min and separated by 10% sodium dodecyle sulphate-polyacrylamide gel electrophoresis. The gels were transblotted to nitrocellulose filters in tris-glycine buffer (25 mmol/L tris, 192 mmol/l glycine, 200 ml/L methanol, pH 7.4) for 5 hours at 60 volts. The nitrocellulose sheets were washed and unoccupied binding sites were saturated with 50g/L bovine serum albumin in tri-buffered saline buffer (50 mmol/L tris, pH 7.5, 150 mmol/L NaCl, 2 mmol/L EDTA) for 1 hour at room temperature. Then the filters were incubated with phosphate buffer saline and mouse monoclonal anti bcl-2 antibody diluted 1:300 (by volume) overnight at 4oC, then with rabbit anti-mouse IgG alkaline phosphatase conjugate diluted 1:500 for 90 min at room temperature. T hese two steps was separ ated by 3-5 min wash in phosphate buffer saline. Finally, the filters were incubated with alkaline phosphatase substrate solution at room temperature until the developed bands were of desired intensity. Then the reaction was stopped by 200 m l of 0.5 mol/L EDTA (pH 8) and 50 ml of phosphate buffered saline. By comparison of the molecular weight marker we could identify the band of bcl-2 protein (M.W.= 26 KDa). Statistical analysis was done using t test.

Results

Table 1 demonstrates the clinical data of the study and the control group. There was a statistically insignificant difference between the study cases and controls as regard age, gravidity, parity and abortions.

Table 1: Clinical data of the studied and control groups

 

Study cases (n=22)

Controls (n=12)

Significance
Mean ± SD Mean ± SD t P
Age 45.23 ± 3.25 46.75 ± 4.25 1.17 > 0.05
Gravidity 5 ± 1.83 5.5 ± 1.62 0.79 > 0.05
Parity 4.32 ± 1.39 4.92 ± 1.44 1.18 > 0.05
Abortions 0.68 ± 0.72 0.58 ± 0.79 0.37 > 0.05

There was a highly significant difference in bcl-2 content of the leiomyomtous tissues and the myometrium from which it developed (studied myometrium) and also between leiomyomatous tissues and the control myometrium (table 3). There was an insignificant difference between bcl-2 content ofthe studied and control myometrium (table 3).

Table 2: Bcl-2 content of leiomyoma, studied and control myometrium

Tissues Bcl-2 (mean ± SD) U/mg protein t P
Leiomyoma (n=22) 296.74± 114.39 6.62 < 0.001
Studied myometrium (n=22) 107.90± 51.59
Control myometrium (n=12) 77.16± 48.06

Table 3: Bcl-2 content of the myometrium of the studied and control groups

Tissues Bcl-2 (mean ± SD) U/mg protein t P
Studied myometrium (n=22) 107.90± 51.59 1.70 > 0.05
Control myometrium (n=12) 77.16± 48.06

There was statistically insignificant difference in bcl-2 contents of proliferative and secretory endometrium in studied and control groups (table 4).

Table 4: Bcl-2 content of proliferative and secretory endometrium in studied and control groups

Group Endometrial pattern Bcl-2 (mean ± SD) U/mg protein t P
Studied Endometrium Proliferative (n=9) 97.94± 45.3 0.74 > 0.05
Secretory (n=13) 114.78± 56.27
Control Endometrium Proliferative (n=3) 45.43± 16.39 1.68 > 0.05
Secretory (n=9) 101.97± 55.31

The thirty-four women (studied and control groups) were analysed by both ELISA and WB techniques where we used the same tissue lysates. Only 3 samples of the control group showed no bcl-2 band (their level by ELISA were less than 40 U/mg protein). The rest of the samples showed the band, which differs in intensity according to the concentration of bcl-2 (Fig.1).

Discussion

Recent evidence suggests that the failure of cells to undergo apoptotic cell death might be involved in the pathogenesis of a variety of human diseases including cancer, autoimmune disease and viral infection (Vaux et al., 1994)þ.

The bcl-2 oncogene may be a generalized " cell death suppressor" gene that directly regulates apoptosis (Korsmeyer, 1992).

A close association between bcl-2 over expression and tumorgenesis has been proposed in a variety of human tissues. This is based on the fact that prolonging life span of the cell increases the risk of secondary genetic alteration resulting in malignant transformation (Saegusa et al., 1995). Recent studies of bcl-2 expression in the endometrium showed that its expression was varied with menstrual cycle (Gompel et al., 1994). The cyclic bcl-2 expression pattern in the endometrial glandular cells is related to those for estrogen and progesterone receptors in normal menstrual cycle, suggesting regulation of bcl-2 expression by ovarian hormones specially estrogen (Tao et al., 1997).

In this study, there were no significant changes in bcl-2 protein expression between proliferative and secretory phases in the studied myomatous group (table 5). This agrees with Compel et al., 1994 and that of Toa et al., 1997 but this may be explained by high estrogen level in myomatous cases where many investigators have suggested that estrogen plays an important role in development of uterine liomyoma (Andersen et al., 1993). In the control group, the insignificant difference in the level of bcl-2 (table 5) may be explained by the relatively small number of cases.

In this study, the concentration of bcl-2 concentration in the myomatous tissue was significantly higher than its concentration in the control myometrium group (p < 0.001). These results agrees with that of Matsuo et al., 1997 who conducted their study to determine the expression of bcl-2 protein in leiomyomas in comparison with that in the normal myometrium by means of immunohistochemical techniques. Our results also agrees with that of Khurana et al., 1999.

In this study, we have tried to find an easier and less time-consuming technique that would enable us to have values for bcl-2 assayed in cell lysates. The performance of the bcl-2 ELISA has been evaluated and shown to be reliable for the quantitation of bcl-2 in minimal amount of cell lysates. Measurement of bcl-2 antigen was confirmed by western blot technique. We found that sensitivity of ELISA in picking up the positive cases is more than the western technique. So we recommended the use ELISA for quantitative determination of bcl-2 concentration if compared to western blotting technique.

We concluded that the abundant expression of bcl-2 oncoprotein in leiomyoma relative to that of normal myometrium might be one of the molecular bases for the enhanced growth of leiomyoma. We have the hope that an anti-bcl-2 treatment can be used in selected cases to avoid surgical treatment of fibroids particularly if multiple and/or huge especially in infertile patients.

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