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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 10 5009-5016
Copyright © 2003 by The Endocrine Society

Enhanced Polyadenosine Diphosphate-Ribosylation in Gonadotropin-Releasing Hormone Agonist-Treated Uterine Leiomyoma

Soon-Cen Huang, Ming-Jer Tang, Ya-Min Cheng, Keng-Fu Hsu, Chung-Liang Ho and Cheng-Yang Chou

Department of Obstetrics and Gynecology (S.-C.H., Y.-M.C., K.-F.H., C.-Y.C.), Institute of Clinical Medicine (S.-C.H., M.-J.T., Y.-M.C., K.-F.H., C.-Y.C.), Department of Physiology (M.-J.T.), and Department of Pathology (C.-L.H.), College of Medicine, National Cheng Kung University, Tainan 704, Taiwan

Address all correspondence and requests for reprints to: Cheng-Yang Chou, M.D., Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan 704, Taiwan. E-mail: chougyn{at}mail.ncku.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
This study aimed to examine the activation of poly(ADP-ribose) polymerase (PARP) and the accumulation of its end product, poly(ADP-ribose) (PAR), in uterine leiomyoma specimens obtained from 25 patients receiving Leuplin depot [leuprorelin acetate, depot (LA)] treatment and 46 control patients and explore their correlation with tumor shrinkage and degeneration caused by the therapy. Immunoblotting analysis showed that specimens from LA-treated patients had higher PARP expression. The numbers of both PARP- and PAR-immunolabeled cells were higher in leiomyoma with LA treatment. This was correlated with the clinical response of LA therapy that LA induced more leiomyoma degeneration. The analysis of power Doppler sonography indicated a progressive decrease in blood supply to tumor following LA treatment. In vitro experiments using primarily cultured leiomyoma cells exhibited that the deprivation of serum or ovarian hormones or LA directly failed to induce PARP and PAR production. Our results suggested that reduced blood flow and subsequent ischemic damages in leiomyoma could be responsible for PARP overexpression and PAR accumulation, clinical response, and tumor degeneration caused by LA treatment.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
UTERINE LEIOMYOMA, A common pelvic tumor of reproductive age that sometimes causes significant morbidity, is a smooth muscle tumor with increased cellularity and hypertrophy of the cells (1). Its growth depends on ovarian steroid hormones and growth factors. Studies have demonstrated that monthly administration of a long-acting GnRH agonist (GnRH-a) on patients with uterine leiomyomas reduces tumor sizes and the levels of ovarian hormones and some growth factors that are required for tumor growth (2, 3, 4).

Although the molecular mechanism by which GnRH-a acts on uterine leiomyomas has not been fully resolved, it is suggested that the effect of GnRH-a on reducing tumor volume can be mediated by the induction of cellular atrophy of leiomyoma cells (5) and/or the decrease of the tumor cell number (probably resulting from apoptotic/necrotic cell death and mitosis suppression) (6, 7). Our group and others (8, 9, 10) have demonstrated that GnRH-a treatment of the uterine leiomyoma did not increase apoptotic cell death involving the interaction of Fas and its ligand, caspases, and Bcl2. In addition, accumulating evidence points to subsequent cell damage (5), degeneration, and necrosis (7) that contribute to tumor shrinkage in the aftermath of GnRH-a therapy.

One reported event following GnRH-a therapy is the reduction of blood flow to the uterus (11, 12). It is because a hypoestrogenic environment caused by GnRH-a has increased vascular resistance, leading to the subsequent decrease of blood supply to leiomyoma nodules (12, 13). Although ischemia causes oxidative stress that in turn damages DNA, DNA repair and other protective mechanisms in the cells would be turned on to avoid the replication of impaired DNA (14). For most of the cells, such damage can be just sublethal and reversible. For injured cells with energy supply depletion, however, mortality ensues reasonably.

Poly(ADP-ribose) (PAR) polymerase (PARP), a nuclear enzyme relating to DNA repair, is activated by and binds single- or double-strand DNA breaks (15). Experimental studies indicate that PARP overactivation in response to oxidative damage of DNA can stimulate DNA repair, silence gene transcription for cell growth when ADP-ribose polymers accumulate, and enhance ATP generation from NAD+ to maintain cell survival (16, 17).

Because a PARP requires one molecule of NAD+ to transfer each ADP-ribose unit and NAD+ regeneration demands four molecules of ATP, thereafter prolonged PARP activation in injured cells therefore causes a massive consumption of NAD+ and ATP and leads to cell death by necrosis as a result of energy depletion (18, 19, 20, 21).

In this article, we investigated the expression profiles of PARP and PAR in leiomyoma cells as well as control cells with or without GnRH-a therapy and then explored how these expression profiles correlated with tumor responses to the therapy and some pathological findings relating to cell injury such as degeneration and necrosis. The changes of blood supply to the leiomyoma were assessed by three- dimensional power Doppler ultrasonography. In addition, in vitro primary cultures of leiomyoma were exploited to study the effect of ovarian hormones, LA, or hypoxic insults on the expression of PARP and PAR.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Tissue collection, characterization, and tissue cell lysate preparation

From January 1998 to December 2001, we prospectively collected patients who were treated with three doses of Leuplin Depot [leuprorelin acetate, depot (LA), Takeda Chemical Industries, Ltd., Tokyo, Japan], a long-acting GnRH-a, for approximately 2–3 wk before myomectomy at the National Cheng Kung University Hospital (Tainan, Taiwan) as the LA-treated group. Women with regular menstrual cycle undergoing hysterectomy or myomectomy during the follicular phase of the cycle without previous exogenous hormonal or GnRH-a therapy were selected as the control group. A specimen pair of leiomyoma and myometrium belonging to each patient of either the LA-treated group or the control group was obtained from the surgeries in the respective conditions mentioned above. The works of these investigations were conducted after obtaining informed consent from patients and approval from the institutional review board at National Cheng Kung University Hospital. The representative tissue samples were taken from distinct nondegenerating leiomyomas and a separate site 1 cm away from the tumor as normal myometrium. The final diagnosis of uterine leiomyomas was confirmed by the pathologic findings in each case. The specimens of tissue lysate from frozen tissues were prepared for further analysis as described previously (10).

Ultrasonographic examination

All of the patients of both groups underwent ultrasonographic examination using an end-viewing 90 degree-angled real-time convex 5-MHz transvaginal transducer or a 3.5-MHz transabdominal probe as an adjunct to the transvaginal probe (Aloka SSD-680, Aloka, Tokyo, Japan) by one investigator 1 d before surgery. For the LA-treated patients, additional scans were performed before and during LA treatment. The details on the method of estimating tumor size and response after treatment were described previously (9). In brief, the volumes of leiomyomas were calculated by the formula for the volume of an ellipse ({pi} x R1 x R2 x R3/6), in which R1, R2, and R3 were the maximal transverse, anteroposterior, and longitudinal lengths of tumor, respectively. In patients with more than one leiomyoma nodule, only the largest nodule detected was selected. The percentage of tumor volume reduction was calculated by 1 (volume before surgery/volume before LA treatment). Tumor volume reduction of less than 20% was deemed as a poor response to therapy.

During the study period, 16 of the LA-treated patients were also arranged to receive three-dimensional (3-D) power Doppler sonography before and during LA treatment to assess the spatial blood flow distribution in uterine leiomyomas. This technique would provide direct visualization of a serial blood flow change in leiomyoma. The performance of 3-D ultrasonic scans was served by a 3-D power Doppler ultrasound (Voluson 530D; Medison-Kretz, Seoul-Zipf, Korea-Austria) attached with a 4.5-MHz abdominal transducer (S-VAW4–7) performing at a switchable scan angle from 31 degrees to 87 degrees (2 degrees per step). The direction of insonation was extending forward in relation to transducer’s longitudinal axis. When performing Doppler angiography, the equipment was set as the following conditions according to user’s manual: angio mode, center; frequency, low; persistence filter, 0.3/0.4; quality, 9; density, 8; enhance, 3; balance, G>192; reject, 81; color flow velocity range, 1.0; color flow transmit power, 5dB; wall motion filter, 115; frequency range, 7.3.

After the total color percentage and the flow amplitude in the total volume of interest were estimated, the 3-D Doppler angiography and the VOCAL (visual organ computer aided analysis) software (Medison-Kretz) were applied to make a 3-D power Doppler histogram analysis to determine the indices of blood flow and the vascularization of tissues through computer algorithms. The vascularization index (VI) indicates the proportion of the volume showing a flow signal in the total volume of the uterine myoma. The flow index (FI) is an average of the intensity of flow signal inside the uterine myoma. The vascularization flow index (VFI) is a combination of the information of vessel presence and amount of flow made by multiplying the FI and VI. All the qualitative and quantitative measurements were performed by Y.-M.C.

Primary culture of leiomyoma cells

Primary explant cultures of leiomyoma were isolated from leiomyoma tissue and maintained as described previously (22). In brief, the leiomyoma tissue was minced into 1- to 2-mm3 explants, placed in 6-cm petri dishes and cultured with DMEM (Life Technologies, Grand Island, NY) plus 20% fetal bovine serum (FBS). Dishes were left undisturbed for 1 wk and then examined for the evidence of cell outgrowth. After cell confluence, cells were transferred to culture flasks containing DMEM plus 10% FBS. These smooth muscle-like cells were confirmed to have the characteristic features of uterine muscle cells as follows: fusiform shape, expression of smooth muscle-specific {alpha}-actin, and estrogen responsiveness. All the cultured cells harvested for in vitro experiments were either nonpassaged cells or those in two to four subcultures. The growth medium in the culture dishes with leiomyoma cells was replaced with serum-free and phenol red-free DMEM 24 h before experiments.

Immunoblotting analysis

Because the tissue lysate might contain a various proportion of extracellular protein, intracellular ß-actin was used to ensure the even loading of protein samples on SDS-polyacrylamide gel. The protein samples separated by SDS-PAGE were then transferred onto nitrocellulose membranes by a semidry blotting system. The membranes were blocked in 1 x PBS containing 5% (wt/vol) skim milk at room temperature for 1 h, washed in a mixture of PBS and 0.05% Tween 20 (Tween-PBS; Sigma Chemical, St. Louis, MO) and then incubated overnight at room temperature with PARP antibody (Transduction Laboratories, Lexington, KY) for 1 h. After being washed in Tween-PBS, the membranes were incubated with 1000-fold diluted, biotinylated antimouse IgG antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech Inc., Piscataway, NJ) at room temperature for 1 h and then developed by the enhanced chemiluminescence system (Amersham Pharmacia Biotech Inc.). The intensity of each band in Western blot was then analyzed by a laser densitometer (PD-120, Molecular Dynamics, Sunnyvale, CA). To obtain comparable data from different films, relative intensity was used and defined as the ratio of band intensity of each lane to that of its corresponding ß-actin.

PARP and PAR immunostaining of surgical specimens

After surgery the surgical specimens were sent for histological examination as soon as possible. To reduce the heterogeneity of clinical specimens, sections of 5 µm in thickness were cut from each formalin-fixed, paraffin-embedded tissue block that contained the largest tumor nodule of each patient. For each tumor, one section was taken and stained with hematoxylin-eosin for histological confirmation of tumor presence and evaluation of histological changes after LA treatment. The adjacent sections were stained with PARP or PAR antibody according to the standard immunoperoxidase staining methods. The presence of leiomyomas and histologic findings such as hyaline and hydropic degeneration on the slides were reviewed by one investigator (C.-L.H.), who was blind to patient status of each sample. Hyaline degeneration was defined as a nodular confluent acellular area, and hydropic degeneration is a geographically distributed edematous zone that probably preceded hyaline degeneration (7). The pathologic changes were graded according to the percentage of degeneration area in a x100 field and designated as follows: grade 1, 0–5%; grade 2, 5–20%; grade 3, more than 20%.

Monoclonal antibodies against PARP (Transduction) and PAR (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) were used to stain the histological slides from the paraffin blocks as described elsewhere with minor modifications (23). Briefly, sections were deparaffinized and treated with methyl alcohol and hydrogen peroxide to block endogenous peroxidase activity. Section-bearing slides placed in 0.01 mmol/liter, pH 6.0 citrate buffer (Antigen Retrieval Citra, BioGenex, San Ramon, CA) were heated in a microwave oven (Bio-Rad H2500 microwave, Energy Beam Science, Richmond, CA), set at 97% maximal power level, at 97 C for two 5-min cycles with an interval of 2 min. The slides were then treated with anti-PARP and anti-PAR antibodies diluted with antibody diluent (DAKO, Santa Barbara, CA), a buffer containing background-reducing components, to a concentration of 5 µg/ml and 1 µg/ml, respectively. After processed with prediluted goat antimouse biotinylated antibody (DAKO) and incubated with peroxidase-conjugated streptavidin (DAKO), sections were incubated with reagents of an aminoethyl carbazole substrate kit (Zymed, San Francisco, CA). Brain slices from a patient with Alzheimer’s disease were used as positive controls of both PARP and PAR (23), and negative controls were prepared by replacing the primary antibodies with nonspecific isotype antibodies of the same concentrations (mouse IgG1 for PARP, mouse IgG3 for PAR; both were purchased from PharMingen, San Diego, CA). The quantification of PARP- or PAR-positive staining cells was performed by two investigators (S.-C. H. and K.-F. H.) with a multiheaded microscope. Both investigators had to concur with each other on the outcomes of analyzed areas and were blind to patients’ preoperative treatments the samples corresponding to. This quantification of each case was performed on an average of three blocks.

All available slides were examined at a low-power magnification (x40) to identify the areas with the highest number of positive-staining cells within the tumor. In each slide, five areas considered to have the highest number of positive-staining cells were selected. The cells of each area were then assessed in a x200 field. A semiquantitative scoring system was established and used to determine the proportions of PARP- and PAR-positive cells in leiomyoma. That was grade 1 (<1% positive cells), grade 2 (1–20%) and grade 3 (>20%) for PARP staining; and grade 1 (<50%) and grade 2 (>50%) for PAR staining. We reassessed the slides if there were differences in the grade assignment between these two observers.

PARP or PAR immunostaining of cultured leiomyoma cells

Primarily cultured leiomyoma cells seeded on slides were fixed with 4% paraformaldehyde and 0.2% Triton X-100 solution for 1 h and washed in PBS for 5 min twice. The cells were then incubated for 30 min with PBS containing 3% FBS for blocking. In the primary antibody staining, cells with anti-PARP antibody in a 1:50 dilution and anti-PAR antibody in a 1:200 dilution were stayed at 4 C overnight. In the secondary antibody staining, cells were incubated with prediluted goat antimouse biotinylated antibody (DAKO). After the cells were treated with peroxidase-conjugated streptavidin (DAKO), the enzymatic staining was developed with the chromogen supplied by an aminoethyl carbazole substrate kit (Zymed). Leiomyoma cells cultured in 10% FBS medium with or without hydrogen oxide, which could cause DNA damage, were also prepared to go through the immunostaining procedures to serve as positive and negative control, respectively (24).

Statistical analysis

Numeral data were expressed as mean ± SD. Paired or unpaired t, Pearson correlation, Spearman’s {rho} correlation, Mann-Whitney U, or Wilcoxon signed ranks tests (SPSS for Windows 11.0.1, SPSS Inc., Chicago, IL), as appropriate, were used to analyze the results. A value of P < 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Clinical findings

During the study period, a total of 25 consecutive patients receiving preoperative LA treatment were collected. The patient number of control group (without preoperative LA treatment) collected in the same study session was 46. The patients of both groups were normally distributed in tumor volumes. The age of patients was 29–37 yr (mean = 33) in the LA-treated group and 32–43 yr (mean = 37) in the control group. The mean tumor volume before LA treatment was 187.4 ± 94.2 ml (ranging from 33–413 ml), whereas the mean volume after therapy was 88.4 ± 50.8 ml (ranging from 16–203 ml). LA significantly reduced the leiomyoma tumor volume (P < 0.001, paired t test). The median volume of tumor reduction was 76 ml, and the volume reduction rate of individual tumor nodule varied from 8–97%. Five patients had a tumor volume reduction less than 20% and were deemed as poor responders. The mean volume of leiomyomas in LA-naïve patients was 150.3 ± 71.8 ml (ranging from 14–323 ml). There was no significant difference between the two groups on the aspect of age (P > 0.05, unpaired t test) and tumor size (P > 0.05, unpaired t test) before LA treatment.

Expression of PARP and PAR and their correlations with degeneration of leiomyomas

Figure 1Go shows the representative results of four pairs of myometria and leiomyomas from two LA-treated patients and two LA-untreated patients in Western blot analysis for PARP, in which significant bands referring PARP are noted in relative intensities. The statistical data of all these 25 LA-treated patients and 46 LA-untreated patients are also summarized in Fig. 1Go. The LA-untreated leiomyomas had an up-regulated expression of PARP as compared with their homologous myometria (P < 0.05, paired t test). The expression levels of PARP in the LA-treated leiomyomas were observed significantly higher than those in the nontreated controls (P < 0.001, unpaired t test).



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  		FIG. 1. A, Representative PARP expressions in the pairs of leiomyomas (T) and myometria (N) from two patients without prior Leuplin depot therapy [LA(-)] and two patients with prior LA treatment [LA(+)]. Western blots showed that LA-treated leiomyomas expressed more PARP than homologous myometria or LA-untreated leiomyomas. ß-Actin was used to ensure the even loading of each specimen. B, PARP expressions in the pairs of leiomyomas and myometria from 25 LA-treated patients and 46 LA-untreated patients. PARP levels were measured by densitometry, and each column was presented in mean ± SD. Relative intensity was defined as the ratio of band intensity in each lane to the corresponding intensity of ß-actin by densitometric measurement. The asterisk indicates a significant difference in the comparison between LA-untreated leiomyomas and paired myometria using paired t test. The double asterisk indicates a significant difference in the comparison between LA-treated and nontreated leiomyomas using unpaired t test.

 
Immunohistochemical staining of PARP (Fig. 2AGo) and PAR (Fig. 2CGo) found that almost all of the stain was confined in cell nucleus of leiomyoma. Compared with myometria, leiomyomas were more frequently detected as PARP-positive cells in particular around the degenerative areas of the tumors. Besides, PAR-staining positive cells far exceeded PARP-staining positive cells in number.



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  		FIG. 2. Representative immunohistochemical pictures (x400) of a grade 3 PARP (A) and grade 2 PAR (C) in an LA-treated leiomyoma. Both PARP and PAR had nuclear staining, and PAR-positive cells are more frequently detected than those of PARP-positive cells. Negative controls for PARP (B) and PAR (D) were prepared by replacing the primary antibodies with nonspecific isotype antibodies.

 
Among the 25 LA-treated patients, the PARP-staining grades and the patient numbers of particular grades are described as follows: grade 3 (seven patients), grade 2 (11 patients), and grade 1 (seven patients); and the PAR-staining grades and their patient numbers: grade 2 (16 patients) and grade 1 (nine patients). It was seemly that the results of PARP staining were parallel with those of PAR staining. Among the 46 LA-untreated patients, the PARP-staining grades and the corresponding patient numbers were: grade 3 (three patients), grade 2 (13 patients), and grade 1 (30 patients); and the PAR-staining grades and their patient numbers: grade 2 (12 patients) and grade 1 (34 patients). Statistical analyses revealed that leiomyomas with LA treatment expressed more PARP and PAR as compared with nontreated controls (P < 0.001 and P < 0.001, respectively; Mann-Whitney U test). In addition, the extent of tumor shrinkage was positively correlated with PARP and PAR grades (Pearson correlation coefficients were 0.881, P < 0.001 and 0.701, P < 0.001, respectively) (Fig. 3Go). The degeneration grades and the corresponding patient numbers in LA-treated group were: grade 3 (four patients), grade 2 (five patients) and grade 1 (16 patients). Those in LA-untreated group were: grade 3 (two patients), grade 2 (nine patients) and grade 1 (35 patients). Of note, more nodular hyalinization or geographic hydropic degeneration was found in LA-treated leiomyoma (P < 0.05; Mann-Whitney U test) as compared with nontreated controls. Moreover, tumors with higher PARP or PAR production were more likely to have nodular hyalinization and/or geographic hydropic degeneration (Spearman’s {rho} correlation coefficients were 0.704, P < 0.001 and 0.407, P < 0.05, respectively).



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  		FIG. 3. Correlation between the percentage decrease in the volume of the largest myoma after three doses of LA therapy and the grades of PARP (A) or grades of PAR (B).

 
3-D power Doppler sonography

We used 3-D color Doppler sonography examination to measure the change of blood flow to tumor before and during LA treatment. We found that more than 90% of leiomyomas in the 16 LA-treated patients had a peripheral distribution of vascular supply and that a progressive decrease of the blood flow to tumor could be observed in the serial follow-up examination. Figure 4Go shows the serial changes of blood flow patterns, tumor angiography, and blood-flowing parameters in tumor detected by 3-D power Doppler sonography in a representative patient receiving LA treatment. The preliminary analysis on the changes of blood flow parameters in uterine leiomyoma revealed that average VI decreased from 1.14 ± 0.76 to 0.46 ± 0.31 (P < 0.001; Wilcoxon signed ranks test), FI decreased from 50.77 ± 5.00 to 38.48 ± 8.98 (P < 0.001; Wilcoxon signed ranks test), and VFI decreased from 0.59 ± 0.39 to 0.23 ± 0.17 (P < 0.001; Wilcoxon signed ranks test) after three doses of LA treatment.



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  		FIG. 4. Representative sonograms of a serial follow-up for the blood flow changes on a patient with one solitary leiomyoma with 3-D power Doppler sonography. A progressive decrease in tumor blood flow was observed during the treatment period. A, Before the first shot of LA injection, the power Doppler signal parameters were VI: 1.424, FI: 53.11, VFI: 0.756. B, Before the third shot of LA injection, the power Doppler signal parameters were VI: 0.756, FI: 24.36, VFI: 0.315. C, Before myomectomy, the power Doppler signal parameters were VI: 0.012, FI: 3.654, VFI: 0.047. D, Schematic illustration of the 3-D power Doppler sonograms. Left upper, Sagittal plane of uterine myoma; right upper, coronal plane of uterine myoma; left lower, transverse plane of uterine myoma; right lower, power angiogram of the uterine myoma.

 
Effects of sexual steroids, LA, and cobalt chloride on expression of PARP and PAR

Regarding the factors that might be involved in PARP up-regulation and PAR accumulation in LA-treated leiomyomas, the reduction of both blood flow and sex steroid hormones could have played some roles. We used primary cultures of leiomyoma to investigate the effects of serum, LA, estrogen, or cobalt chloride (CoCl2) known as a hypoxia-mimicking agent on the expressions of PAR and PARP. It had been suggested that a substitution of ferrous ions in heme by cobalt ions would cause a conformational change in a heme protein O2 sensor and induce hypoxic effects (25). When leiomyoma cells were cultivated in medium with 10% FBS supplement, PARP and PAR expression were relatively low as shown in the results of Western blot (Fig. 5AGo) and immunostaining (Fig. 5BGo). This was in direct contrast to the positive control that exposed to 1 mM H2O2 and exhibited an elevated PARP level in Western blot analysis and more intense stain in the augmented nuclei in the immunostaining results of PARP and PAR. When the growth medium was replaced with serum-free, phenol red-free medium for 5 d, the PARP and PAR expression of leiomyoma cells remained unchanged. Furthermore, cells serum-starved for 5 d were treated with 250 nM 17 ß-estradiol or 1 nM LA and incubated for another 24 h. The PARP levels in leiomyoma cells were not affected. These indicated that PARP and PAR expressions were dependent on neither GH nor estrogen supplement and LA had no direct effects on their expressions. Of note, the treatment of 200 µM CoCl2 did not induce any elevated PARP levels in the serum-starved leiomyoma cells as long as 5 d. This reflected the fact that hypoxia induced by CoCl2 alone in this study condition was not sufficient to activate PARP, which as widely known could be activated by broken nuclear DNA (26).



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  		FIG. 5. Sexual steroid depletion, GnRH-a or hypoxia-mimicking agent CoCl2 did not increase cellular expression of PARP or PAR in primary-cultured leiomyoma cells. A, Western blot analysis showed that leiomyoma cells cultured in phenol red-free DMEM containing 0% FBS for 5 d with or without the addition of 250 pM 17 ß-estradiol (E2) or 1 nM LA for an additional 24 h or 200 µM CoCl2 in various durations did not express more PARP than those cultured in 10% FBS. Cells cultured in medium containing 10% FBS and treated with 1mM H2O2 were used as positive control. ß-Actin was used to ensure the even loading of each specimen. B, Immunocytochemistry of leiomyoma cells cultured in medium containing 10% FBS, 10% FBS plus 1mM H2O2, and 0% FBS plus PARP or PAR antibody revealed that serum starvation did not induce increased nuclear staining intensity or nuclear morphologic changes, compared with those maintained in 10% FBS. In contrast, cells treated with 1mM H2O2 showed a slight increase in nuclear staining intensity and enlarged nuclei.

 
Geographical distribution of PARP and PAR in LA-nontreated leiomyomas

The observation that hormonal depletion failed to induce PARP activation led us to suspect that PARP overexpression after LA treatment is more likely to be related to a diminished vascular supply than hormonal depletion. Therefore, we hypothesized that leiomyoma cells in the central part of tumor might be more vulnerable to ischemic damage and should have higher PARP expression than those in the peripheral region because leiomyomas have a peripheral distribution of blood supply. We had assessed the PARP expression of both peripheral and central parts for 10 specimen pairs of leiomyoma and myometrium of patients whose leiomyoma nodules were larger than 5 cm in diameter. Representative results shown in Fig. 6Go indicated that leiomyoma cells at the central part of the tumor had the highest extent of PARP expression, and myometrium had the least PARP expression. These results provide further support for our supposition that PARP expression in leiomyomas is related to tissue ischemia.



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  		FIG. 6. Representative PARP expressions in the pairs of the peripheral (P) and central (C) parts of uterine leiomyomas and myometria (N) from two patients without prior LA therapy. The results revealed a geographic difference in PARP distribution that leiomyoma tissues have more DNA damage than their normal counterpart and their central portion is more severely damaged.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The growth of uterine leiomyoma depends on growth-supporting hormones and adequate blood supply. GnRH-a is able to bring volume shrinkage to this tumor, although the molecular mechanisms by which GnRH-a acts on uterine smooth muscle cells requires further experimental elucidation. Accumulating evidence has demonstrated that the cellular and pathological changes underlying the mechanism of tumor shrinkage can be mediated by the induction of cellular damage and atrophy (5), the decrease of tumor cell number (6, 7), and the subsequent development of tumor degeneration or necrosis (7). Our previous work has pointed out that GnRH-a-induced leiomyoma shrinkage was due in part to a mechanism involving DNA damage (22) but not to apoptotic signal pathway initiated by Fas/Fas-L activity (9, 10). However, the questions on the molecular mechanism of DNA damage and its link to tissue ischemia or hormonal depletion as well as subsequent tumor degeneration still have no answers. In this article, we provide evidence on the expression of PARP, a nuclear protein playing a prominent role in the regulation of DNA repair and relevant gene transcription, to see how it reacts to GnRH-a effect.

The results revealed that the blood supply to uterine leiomyoma was geographically uneven, i.e. blood flow preferentially distributed to the peripheral part of tumor. The uneven blood supply to leiomyomas is in agreement with a study of Chiang et al. (27). The activation or up-regulation of PARP protein and the accumulation of PAR were more frequently detected in leiomyomas as compared with myometria. LA treatment further accentuated PARP production in leiomyomas, especially at the central portion of tumor. This was inversely correlated with the vascular supply to tumor tissue. Leiomyomas with high PARP and PAR grades following LA therapy were found to show greater tumor shrinkage. These results therefore support the findings that hyaline degeneration and geographic hydropic degeneration were found in a higher proportion of LA-treated patients (7) and further suggest to associate positively the degenerations of tumor with PARP and PAR grades. Moreover, in vitro studies pointed out that PARP expression had no dependence on hormonal depletion. Altogether, we suggest that it is not hormonal depletion but blood flow reduction that accounts for the tumor shrinkage after LA treatment.

Conventional color Doppler sonography examination could provide limited information regarding the qualitative and quantitative blood flow changes in a follow-up examination. In contrast, 3-D power Doppler sonography is more sensitive, less angle dependent, and not susceptible to aliasing. It is capable of the visualization of small vessels and the quantification of Doppler signal with a histogram software (28). Our power Doppler flow mapping indicated a progressive decrease in the blood flow to uterine leiomyoma following LA treatment. Because the blood supply was geographically partial to the peripheral region, the center of tumor would be more vulnerable to ischemia. Ischemia and the resultant oxidative stress can induce extensive subcellular damages. For instance, mitochondrial swelling is one of the common changes appearing in the ischemic cells (29) and observed in more than half of the uterine leiomyomas treated with GnRH-a (5). The swelling of mitochondria could not be reproduced in vitro by the deprivation of ovarian steroid hormones in culture medium, suggesting that hormonal depletion might not directly be responsible for the morphologic changes in mitochondria (30).

DNA is a target of oxidative stress prompted by ischemia. Impaired DNA strands can activate DNA repair and other protective mechanisms of the cell to maintain genome integrity (14, 15). In this study, the existence of PAR, indicating de novo PARP activation and NAD+ consumption, was a common phenomenon in uterine leiomyoma. In addition to activation, the up-regulation of PARP was also found prevalently in leiomyomas, especially in the central portion of the tumor. These results suggest that GnRH-a may cause a progressive decrease and deterioration of uneven blood supply to tumor centers and accentuate DNA damage of the cells in the regions. Activated PARP functions to catalyze poly(ADP-ribosyl)ation of nuclear proteins, which has been suggested to have some roles of importance in cellular functions, such as DNA synthesis, DNA repair, gene transcription, and cell differentiation (29). In addition to stimulating DNA repair, PARP is stated to be capable of modifying several transcriptional factors by poly(ADP-ribosyl)ation and preclude their binding to DNA (17, 31). Transcription of genes encoded in damaged DNA is thereby prevented, and those required for cell growth and proliferation also can be inhibited. Therefore, in the presence of sublethal DNA damage, increased PAR by GnRH-a treatment could have played roles in size reduction of leiomyoma cells.

In contrast, in the presence of severe DNA damage, PARP overactivation could deplete the intracellular stores of NAD+, in turn slowing the rate of glycolysis, mitochondrial electron transport, and ATP formation. The consequence is a severe energy crisis, leading to acute cell dysfunction and necrotic cell death. Therefore, despite playing an important role in DNA repair, PARP has long been recognized to induce cell death if overactivated under some conditions (18). Further evidence shows that genetic disruption or pharmacological blockage of the PARP pathway can effectively protect cells from necrosis (32). Actually, whether PARP activation leads to cell death depends on intracellular NAD+ levels. For instance, PARP activation without subsequent NAD+ depletion following mild transient ischemia is regarded as neuroprotective in the brain (14). Increasing PARP levels, however, would deplete energy ultimately and thus end up in cell death. Such neurons doomed to death, e.g. secondary retinal ganglion cells following rat optic nerve transection (33) and brain cells in Alzheimer’s disease (23), were reported high in PARP levels. Our result that LA-induced tumor shrinkage is correlated with the increased production of PARP protein and PAR as well as some pathological findings such as various types of tissue degeneration supports the notion that an increase of PARP level would in turn deplete energy store and be more likely to cause tissue degeneration and cell necrosis.

It is noteworthy that the effects of GnRH-a therapy on reducing blood flow to tumor and lowering growth- supporting hormones may not be mutually exclusive. Estrogen has been demonstrated to have a vasodilatory effect (34) on uterine artery that expresses estrogen receptors on the vascular wall (35). An increase of vascular resistance in the hypoestrogenic environment caused by GnRH-a can lead to a subsequent decrease of blood supply to leiomyomas (12, 13). Moreover, there is a direct correlation between the fall in serum estradiol and the increase in the pulsatile index of the uterine artery (12). In vivo studies suggest that menopause-induced ovarian hormonal depletion could increase the vascular bed resistance of the arterial vessels in uterus (36, 37), whereas hormone replacement therapy inverts the menopause effect with time. Therefore, GnRH-a is likely to induce the subcellular changes in leiomyoma via reducing growth-supporting hormones and diminishing vascular supply to tumor simultaneously.

Both generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of CoCl2. Accumulation of excessive ROS is one of the mechanisms to form strand breaks in nuclear DNA. Our in vitro experiment showed that H2O2, a representative ROS, could elevate PARP expression in cultured cells, but CoCl2 could not. Probably, in our study condition, the hypoxia-induced cell damages caused by CoCl2 were not severe enough to up-regulate PARP expression. H2O2 induces an excessive PARP activation that may result in a depletion of intracellular NAD+ and ultimately cell death. On the other hand, in rat neuronal PC12 cells, CoCl2 caused DNA damages in mitochondrias but not in nuclei (26). Mitochondrial DNA damage is accompanied by some changes in both cellular and molecular levels (26), and our results from CoCl2 studies suggest that mitochondrial DNA damage alone may not be sufficient to induce significant changes in PARP expression. It deserves further studies to dissect whether the cellular and molecular changes in leiomyoma caused by LA treatment are attributable to hypoxia, DNA damage, and sex hormone/growth factor depletion.

In conclusion, the cytopathological effects of GnRH-a on uterine leiomyomas could be attributed to blood flow reduction and subsequent oxidative stress, DNA damage, and cell necrosis. We have provided a molecular basis for uterine artery embolization (38) that is probably a similar but more aggressive mechanism as compared with GnRH-a-induced reduction of tumor blood flow. It offers a contribution for a better understanding on the real mechanism by which GnRH-a causes the degeneration of leiomyoma, which is the key of this therapy to manage the disease.


   Acknowledgments
 
We thank Dr. Ko-Hung Lee for his assistance with the statistical analysis and discussion.


   Footnotes
 
This work was supported by grants from the National Science Council, Taiwan (NSC89-2314-B-006-102, NSC90-2314-B-006-169); National Cheng Kung University Hospital (NCKUH 89-028, NCKUH 90-045); and MOE Program for Promoting Academic Excellent of Universities (91-B-FA09-1-4).

Abbreviations: 3-D, Three-dimensional; FBS, fetal bovine serum; FI, flow index; GnRH-a, GnRH agonist; LA, leuprorelin acetate; PAR, poly(ADP-ribose); PARP, PAR polymerase; ROS, reactive oxygen species; VFI, vascularization FI; VI, vascularization index.

Received February 3, 2003.

Accepted July 2, 2003.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Buttram Jr VC, Reiter RC 1981 Uterine leiomyomata: etiology, symptomatology and management. Fertil Steril 36:433–445[Medline]
  2. Liu CH, Lin YS, Lin CC, Tzeng CC, Chou CY 1993 Medical treatment of uterine myoma with long-acting gonadotropin-releasing hormone agonist prior to myomectomy. J Formos Med Assoc 92:650–654
  3. West CP, Lumsden MA, Lawson S, Williamson J, Baird T 1987 Shrinkage of uterine fibroids during therapy with goserelin (Zoladex), a luteinizing hormone-releasing hormone agonist administered as a monthly subcutaneous depot. Fertil Steril 48:45–51[Medline]
  4. Costatini S, Anserini P, Valenzano M, Remorgida V, Venturini PL, De Cecco L 1990 Luteinizing hormone-releasing hormone analog therapy of uterine fibroid: analysis of results obtained with buserelin administered intranasally and goserelin administered subcutaneously as a monthly depot. Eur J Obstet Gynecol Reprod Biol 37:63–69[CrossRef][Medline]
  5. Ito F, Kawamura N, Ichimura T, Tsujimura A, Ishiko O Ogita S 2001 Infrastructural comparison of uterine leiomyoma cells from the same myoma nodule before and after GnRH-agonist treatment. Fertil Steril 75:125–130[CrossRef][Medline]
  6. Mizutani T, Sugihara A, Nakamuro K, Terada N 1998 Suppression of cell proliferation and induction of apoptosis in uterine leiomyoma by gonadotropin-releasing hormone agonist (leuprolide acetate). J Clin Endocrinol Metab 83:1253–1255[Abstract/Free Full Text]
  7. Deligdisch L, Hirschmann S, Altchek A 1997 Pathologic changes in gonadotropin releasing hormone agonist analogue treated uterine leiomyomata. Fertil Steril 67:837–841[CrossRef][Medline]
  8. Burroughs KD, Kiguchi K, Howe SR, Fuchs-Young R, Trono D, Barrett JC, Walker C 1997 Regulation of apoptosis in uterine leiomyomata. Endocrinology 138:3056–3064[Abstract/Free Full Text]
  9. Huang SC, Chou CY, Lin YS, Tsai YC, Hsu KF, Liu CH, Huang KE 1997 Enhanced deoxyribonucleic acid damage and repair but unchanged apoptosis in uterine leiomyomas treated with gonadotropin-releasing hormone agonist. Am J Obstet Gynecol 177:417–424[CrossRef][Medline]
  10. Huang SC, Tang MJ, Hsu KF, Cheng YM, Chou CY 2002 Fas and its ligand, caspases, and Bcl-2 expression in GnRH agonist-treated uterine leiomyoma. J Clin Endocrinol Metab 87:4580–4586[Abstract/Free Full Text]
  11. Matta WHM, Stabile I, Shaw RW, Campbell S 1988 Doppler assessment of uterine blood flow changes in patients with fibroids receiving gonadotropin-releasing hormone agonist buserelin. Fertil Steril 46:1083–1085
  12. Weeks AD, Duffy SR, Walker JJ 1999 Uterine ultrasonographic changes with gonadotropin-releasing hormone agonists. Am J Obstet Gynecol 180:8–13[CrossRef][Medline]
  13. Spong CY, Sinow R, Renslo R, Cabus E, Rutgers J, Kletzky OA 1995 Induced hypoestrogenism increases the arterial resistance index of leiomyomata without affecting uterine or carotid arteries. J Assist Reprod Genet 12:338–341[CrossRef][Medline]
  14. Nagayama T, Simon RP, Chen D, Henshall DC, Pei W, Stetler RA, Chen J 2000 Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia. J Neurochem 74:1636–1645[CrossRef][Medline]
  15. Chiarugi A 2002 Poly(ADP-ribose) polymerase: killer or conspirator? The ‘suicide hypothesis’ revisited. Trends Pharmacol Sci 23:122–129[CrossRef][Medline]
  16. De Murcia G, Schreiber V, Molinete M, Saulier B, Poch O, Masson M, Niedergang C, Menissier de Murcia J 1994 Structure and function of poly(ADP-ribose) polymerase. Mol Cell Biochem 138:15–24[CrossRef][Medline]
  17. Ziegler M, Oei SL 2001 A cellular survival switch: poly(ADP-ribosyl)ation stimulates DNA repair and silences transcription. Bioessays 23:543–548[CrossRef][Medline]
  18. Berger NA 1985 Poly(ADP-ribose) in the cellular response to DNA damage. Radiat Res 101:4–15[Medline]
  19. Zhang J, Dawson VL, Dawson TM, Snyder SH 1994 Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263:687–689[Abstract/Free Full Text]
  20. Eliasson MJ, Sampei K, Mandir AS, Hurn PD, Traystman RJ, Bao J, Pieper A, Wang ZQ, Dawson TM, Snyder SH, Dawson VL 1997 Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med 10:1089–1095
  21. Endres M, Wang ZQ, Namura S, Waeber C, Moskowitz MA 1997 Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase. J Cereb Blood Flow Metab 1711:1143–1151
  22. Cheng YM, Chou CY, Huang SC, Lin HC 2001 Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists. Br J Obstet Gynaecol 108:95–102[CrossRef]
  23. Love S, Barber R, Wilcock GK 1999 Increased poly(ADP-ribosyl)ation of nuclear proteins in Alzheimer’s disease. Brain 122:247–253[Abstract/Free Full Text]
  24. Ha HC, Snyder SH 1999 Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci USA 96:13978–13982[Abstract/Free Full Text]
  25. Goldberg MA, Dunning SP, Bunn HF 1988 Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242:1412–1415[Abstract/Free Full Text]
  26. Wang G, Hazra TK, Mitra S, Lee HM, Englander EW 2000 Mitochondrial DNA damage and a hypoxic response are induced by CoCl2 in rat neuronal PC12 cells. Nucleic Acids Res 28:2135–2140[Abstract/Free Full Text]
  27. Chiang CH, Chang MY, Hsu JJ, Chiu TH, Lee KF, Hsieh TT, Soong YK 1999 Tumor vascular pattern and blood flow impedance in the differential diagnosis of leiomyoma and adenomyosis by color Doppler sonography. J Assist Reprod Genet 16:268–275[CrossRef][Medline]
  28. Pairleitner H, Steiner H, Hasenoehrl G, Staudach A 1999 Three-dimensional power Doppler sonography: imaging and quantifying blood flow and vascularization. Ultrasound Obstet Gynecol 14:139–143[CrossRef][Medline]
  29. Schaper J, Schwarz F, Kittstein H, Stammler G, Winkler B, Scheld H, Hehrlein F 1982 The effects of global ischemia and reperfusion on human myocardium: quantitative evaluation by electron microscopic morphometry. Ann Thorac Surg 33:116–122[Abstract]
  30. Kawaguchi K, Fujii S, Konishi I, Okamura H, Mori T 1985 Ultrastructural study of cultured smooth muscle cells from uterine leiomyoma and myometrium under the influence of sex steroids. Gynecol Oncol 21:32–41[CrossRef][Medline]
  31. Hayaishi O, Ueda K 1977 Poly(ADP-ribose) and ADP-ribosylation of proteins. Annu Rev Biochem 46:95–116[CrossRef][Medline]
  32. Szabo G, Bahrle S, Stumpf N, Sonnenberg K, Szabo EE, Pacher P, Csont T, Schulz R, Dengler TJ, Liaudet L, Jagtap PG, Southan GJ, Vahl CF, Hagl S, Szabo C 2002 Poly(ADP-Ribose) polymerase inhibition reduces reperfusion injury after heart transplantation. Circ Res 90:100–106[Abstract/Free Full Text]
  33. Weise J, Isenmann S, Bahr M 2001 Increased expression and activation of poly(ADP-ribose) polymerase (PARP) contribute to retinal ganglion cell death following rat optic nerve transection. Cell Death Differ 8:801–807[CrossRef][Medline]
  34. Killam AP, Rosenfeld CR, Battaglia FC, Makowski EL, Meschia G 1973 Effect of estrogens on the uterine blood flow of oophorectomized ewes. Am J Obstet Gynecol 115:1045–1052[Medline]
  35. Lantta M, Karkkainen J, Lehtovirta P 1983 Progesterone and estradiol receptors in the cytosol of the human uterine artery. Am J Obstet Gynecol 147:627–633[Medline]
  36. Pirhonen JP, Vuento MH, Makinen JI, Salmi TA 1993 Long-term effects of hormone replacement therapy on the uterus and on uterine circulation. Am J Obstet Gynecol 168:620–630[Medline]
  37. Kurjak A, Kupesic S 1995 Ovarian senescence and its significance on uterine and ovarian perfusion. Fertil Steril 64:532–537[Medline]
  38. Watson GM, Walker WJ 2002 Uterine artery embolisation for the treatment of symptomatic fibroids in 114 women: reduction in size of the fibroids and women’s views of the success of the treatment. Br J Obstet Gynaecol 109:129–135




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