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Comparative Messenger Ribonucleic Acid Analysis of Immediate Early Genes and Sex Steroid Receptors in Human Leiomyoma and Healthy Myometrium

Research Laboratories of Schering AG (M.L., M.K., S.S., K.S., U.F.), 13342 Berlin, Germany; and Virchow Klinikum (A.R.), 13353 Berlin, Germany

Address all correspondence and requests for reprints to: Dr. Monika Lessl, Research Laboratories of Schering AG, Strategic Business Unit Fertility Control and Hormone Therapy, Müllerstr. 170–78, 13342 Berlin, Germany.

	Abstract

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To shed light on the molecular mechanisms involved in the pathogenesis of uterine leiomyomas, transcript levels of the immediate early genes c-fos, c-myc, and c-jun and of the estrogen receptor (ER) and progesterone receptor (PR) were determined in tissue samples of human myometrium and leiomyoma. The messenger RNA (mRNA) content was analyzed by RT-PCR. mRNAs for c-fos, c-myc, c-jun, ER, and PR were detected in all 18 samples of leiomyoma and corresponding myometrial tissue collected in this study. Interestingly, in contrast to healthy tissues, we found a distinct and significant reduction of c-fos mRNA in the tumor. These data were substantiated by the finding of lowered c-Fos protein levels in leiomyomas tissues. Moreover, transcripts of c-jun and c-myc were less abundant in most of the leiomyomas than in the myometrium. This different expression of the protooncogenes in leiomyomas and myometrium was independent of the phase of the menstrual cycle in which samples were collected. In contrast to the reduced transcript levels observed for the immediate early genes, the ER and PR mRNA contents of the leiomyomas and myometrium did not differ. These results were confirmed by immunohistochemical studies for ER and PR protein.

In conclusion, our data show that the deregulated expression of protooncogenes, especially of c-fos, is linked to the pathogenesis of leiomyomas. Confirmation of a potential role of downregulated c-fos levels for the benign character of these tumors requires further investigation. Additionally, the findings suggest that sex steroids do not influence the different expression patterns of c-fos, c-myc, and c-jun in leiomyomas, as compared with myometrium.

	Introduction

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UTERINE LEIOMYOMAS (fibroids) are the most common solid benign tumors in the female genital tract and occur in 20–30% of all women of reproductive age. Mostly, multiple fibroids are found within the same uterus. Women with leiomyomas complain of fibroid-related symptoms, e.g. abnormal uterine bleeding and pelvic pain. Fibroids also may interfere with pregnancy, leading to fetal wastage. Although these benign tumors are very common, their pathogenesis is not well understood. Steroids seem to influence the growth of leiomyomas, because they are only observed in women of reproductive age and regress after the menopause (1). The effectiveness of GnRH agonists in shrinking these tumors also militates in favor of their growth being modulated by steroid hormones. However, the mediators of steroid hormone action in leiomyomas have not yet been identified.

Uterine fibroids are believed to be monoclonal in origin and arise from a single myometrial cell. From karyotyping of over 800 leiomyomas, it is known that deletion of the long arm of chromosome 7 and chromosomal translocation, involving region q14–15 of chromosome 12, are the most frequent alterations in uterine leiomyomas. Recently, breakpoint mapping of this region on chromosome 12 led to the identification of a gene called HMGI-C (a high-mobility group gene) (2, 3), suggesting a causal relationship between mutations of the HMGI-C gene and the development of uterine leiomyomas.

To identify additional genes involved in the development or growth regulation of uterine leiomyomas and to elucidate the role of potential mediators of steroid hormone action in the pathogenesis of leiomyomas, we determined messenger RNA (mRNA) levels for selected protooncogenes and for the estrogen receptor (ER) and progesterone receptor (PR). Studies were done with mRNA obtained from human leiomyomas and corresponding healthy myometrial tissue. Because the immediate early genes c-jun, c-myc, and c-fos are known to be sex steroid hormone-regulated in the rat uterus (4, 5, 6) and play crucial roles in proliferating and differentiating processes (7), an intriguing question was whether these factors are somehow involved in the pathogenesis of this disease. Because human tissue material available for this study was limited, RT-PCR analyses were performed. In addition to the RNA analyses, the protein content of the ER and PR was determined immunohistochemically in the biopsy material. Furthermore, the level of c-Fos protein was determined by Western blot analysis in human leiomyoma and myometrial tissue. To assess the proliferative activity of the material investigated, slides of samples were reacted with antibodies against the proliferating cellular nuclear antigen (PCNA), a protein expressed in the nucleus of dividing cells.

	Materials and Methods

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Tissues

Samples of leiomyoma and corresponding myometrial tissue were obtained from 18 women undergoing hysterectomy for uterine leiomyomas. The donors had regular menstrual cycles (20–30 days) and had received no exogenous hormones for at least 2 cycles before surgery. Immediately upon receipt, part of each sample was frozen in liquid nitrogen and stored at -70 C for mRNA or protein studies. A second part was fixed in buffered formalin for immunohistochemical analysis. All samples were collected with the approval of the local ethical committee. The stage of the menstrual cycle was determined by histological dating (8) and from the date of the last menstrual period.

RNA isolation

Total RNA was obtained from tissues using guanidinium isothiocyanate extraction followed by CsCl centrifugation (9). The quality of the RNA was checked by agarose gel electrophoresis under denaturing conditions.

RT-PCR

To avoid contamination of the RNA by DNA, RNA was treated with deoxyribonuclease for 15 min and then phenolized. For the first strand complementary DNA synthesis, 5 µg of total RNA and oligo deoxythymidine primer were used. To obtain full-length complementary DNAs, Superscript polymerase was applied. Reactions were carried out according to the manufacturer’s protocol (Life Technologies, Eggenstein, Germany). One microliter of the cDNA mixture and gene-specific primers (Table 1) was used for amplification. Concentrations in the PCR reaction mixture were as follows: 0.1 µmol/L specific primers, 0.1 mmol/L each deoxynucleotide triphosphate, 0.05 µL [p³²]deoxycytidine phosphate (3000 mCi/mmol), 2 mmol/L MgCl₂, and 1.25 U Taq polymerase. The number of cycles resulting in PCR products in the linear logarithmic phase of the amplification curve was determined for each pair of primers. Thirty cycles were applied for analysis of c-jun, c-myc, and c-fos transcripts; 28 for ER and PR and 18 for 1A primers. The identity of bands obtained by the PCR was checked by nucleotide sequencing. After amplification, 20 µL of the reaction mixture was loaded onto a 6% polyacrylamide gel. After electrophoresis, the gel was dried and exposed to a Phosphor-Imager screen (Molecular Dynamics). The intensity of the bands was quantified using Molecular Dynamics Imagequant software.

Statistical analysis of differences in mRNA levels for c-jun, c-myc, c-fos, ER, and PR in leiomyoma and myometrial tissue was done by the Wilcoxon matched-pair signed rank test. Results are expressed as mean ± SE. For each pair of samples, the Phosphor-Imager reading of the myometrium was normalized to 1 to allow comparisons between different samples and different experiments. To assess the intraassay variability, each experiment was repeated three times.

Western blotting

For Western blot analysis, tissue samples frozen in liquid nitrogen were pulverized and homogenized in SDS loading buffer (100 mmol/L Tris pH 6.8, 4% SDS, 0.1% Bromphenol-blue, 20% glycerol, 0.2% dithiothreitrol). Samples were boiled for 10 min at 95 C, cleared by centrifugation (10 min, 11 C, 100,000 x g) and stored at -80 C. The extracted proteins were separated on a 10% SDS polyacrylamide gel. To check equal protein loading, the separated proteins were visualized by coomassie staining. For c-Fos detection, the proteins were electrotransferred to a nitrocellulose membrane (Amersham-Buchler, Braunschweig, Germany). The blots were incubated at room temperature in 1.0% blocking reagent (Boehringer Mannheim, Mannheim, Germany) for 1 h, then with primary antibody ICSM47Q (IC Chemikalien, Ismaning, Germany; 1:500 in 1% blocking reagent) overnight and with horse radish peroxidase-conjugated rabbit antimouse IgG (Amersham-Buchler, 1:3000 in 1% blocking reagent) for 4 h. Bound antibodies were visualized using the ECL detection system (Amersham-Buchler).

Immunohistochemistry

If sufficient material was available, one portion of each tissue specimen was processed for histological analyses. Tissue was fixed in 4% formaldehyde in phosphate-buffered saline and embedded in paraffin. An antibody distributed by DIANOVA (Hamburg, Germany) was used for the detection of the PCNA. The staining procedure for PR was performed using a PR-immunocytochemical assay monoclonal kit (Abbott, Chicago, IL) according to the manufacturer’s instructions. For staining of ER (Biogenex antibodies, San Ramon, CA) slides were deparaffinized, boiled in 10 mmol/L citrate buffer (pH 6.0) for 10 min, and rinsed with phosphate-buffered saline. Slides were incubated with normal goat serum to reduce nonspecific binding of the primary antibody. After incubation with the specific primary antibody, specimens were treated with biotinylated antirat or antirabbit IgG antiserum, then with avidin-biotin-peroxidase complexes, and finally stained with diaminobenzidine for 5 min. Specific staining was observed as brown granules, and control slides treated with control antibodies yielded negative results. The intensity of staining for ER, PR, and PCNA was evaluated by two independent observers. Intensity was graded as - for no immunostaining, + for weak, ++ for moderate, and +++ for strong signals.

	Results

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Levels of c-fos mRNA are much lower in leiomyoma than in normal myometrium, irrespective of the phase of the menstrual cycle

The goal of our experiments was to analyze relative mRNA levels of selected genes in human leiomyoma and adjacent myometrial tissue. Because the amount of material available was limited and we aimed to analyze a number of tissues from different patients, the semiquantitative RT-PCR method was chosen. The validity of this method was proven in recent experiments by Knauthe et al. (10), which revealed identical results by this RT-PCR technique and a competitive RT-PCR approach.

Using this quantitative RT-PCR approach, we were able to detect mRNAs for the protooncogenes c-jun, c-myc, and c-fos in all of the biopsy samples. The identity of the PCR products was checked by nucleotide sequencing. Measurement of protooncogene transcripts was done in 18 leiomyoma and 18 corresponding myometrial samples collected from 9 women in the proliferative and 9 in the secretory phase of the menstrual cycle. Quantification of the radiolabeled PCR signals was done by a Phosphor-Imager. Because mRNA expression of genes widely used standards, like glycerinaldehyde-phosphate dehydrogenase and ß-actin, are known to be steroid hormone-regulated in uterine tissues (11; U. F. personal communication), 1A mRNA was chosen as the internal control to normalize the mRNA content. 1A mRNA was originally identified by Hsu et al. (12) as an mRNA not regulated by sex steroids in the rat uterus. Nucleotide sequencing of this 1A clone revealed that it encodes a section of subunit I of cytochrome c oxidase. On the basis of this information, we constructed primers specific for subunit I of human cytochrome c oxidase (Table 1 and 13 and applied them as internal standards. Comparison of 1A PCR signals with the content of 18SrRNA in various samples revealed a good correlation between the two signals, indicating that 1A mRNA expression is not influenced by sex steroids in the human uterus either.

After quantification of PCR products for the protooncogenes, we found distinctly lower amounts of c-fos mRNA in leiomyoma than in myometrial tissue (see Figs. 1 and 3). To allow comparisons of data obtained from samples from different patients and generated in different experiments, mRNA levels of the myometrium of each patient were normalized to 1 (see Fig. 3), allowing the detection of relative mRNA differences between the two tissues. An average reduction of c-fos mRNA to 40% of the level in normal myometrium was observed in leiomyoma tissue from all patients (see Figs. 1 and 3). mRNA levels for c-jun and c-myc also were slightly reduced in the tumor material. However, it has to be noted that differences were not as distinct as for c-fos and just failed to reach statistical significance (Fig. 1). As far as the relative mRNA values for the individual patients (data not shown) are concerned, a distinct overexpression of c-myc in leiomyoma tissue was observed for only one patient, harboring a tumor with a mass of 30 kg. Levels of c-jun were not elevated in any of the tumor samples.

View larger version (35K): [in this window] [in a new window]   Figure 1. Relative mRNA levels in human leiomyoma (L) and myometrial (M) tissue of c-fos (A), c-myc (B), and c-jun (C). In all, 32 samples were analyzed; 18 samples were obtained from the myometrium and 18 of leiomyoma tissue. The bars represent the mean of 9 samples (± SE) collected in the proliferative (9 samples) and secretory (9 samples) phases of the menstrual cycle, respectively. To allow comparisons of data obtained from samples from different patients and generated in different experiments, the Phosphor-Imager reading of the myometrium was normalized to 1. The asterisk (*) indicates a statistical significance of difference of P < 0.05.

View larger version (59K): [in this window] [in a new window]   Figure 3. Representative autoradiograms of P32-labeled RT-PCR products from leiomyoma (L) and myometrial (M) tissue of four patients.

Relative mRNA levels of ER and PR did not change in leiomyoma, as compared with myometrial tissue, irrespective of the menstrual cycle stage

RT-PCR products corresponding to the ER and PR mRNA could be identified in all biopsies. In contrast to the situation observed for the protooncogenes, for both receptors, no differences in their relative transcript levels regarding leiomyoma and myometrial tissue (Figs. 2 and 3) were found. As for c-jun, c-fos, and c-myc, the stage of the menstrual cycle did not influence the relative rates of mRNA measured (Fig. 2), comparing myometrial and leiomyomal tissue.

View larger version (37K): [in this window] [in a new window]   Figure 2. Relative mRNA levels in human leiomyoma (L) and myometrial (M) tissue of ER and PR. Collection of samples and analysis of data were performed as described for Fig. 1.

In consequence of our finding of lower c-fos mRNA levels in leiomyomas than in myometrium, we aimed to determine the expression pattern of the c-Fos protein in these tissues. Therefore, we analyzed leiomyoma and myometrial tissue from five patients by Western blot studies (Fig. 4). The Western blot data clearly revealed less abundant c-Fos protein in the tumor than in healthy tissue, paralleling the findings for the mRNA transcripts.

View larger version (24K): [in this window] [in a new window]   Figure 4. Western blot analysis for c-Fos protein in leiomyoma (L) and myometrial (M) tissue of five patients.

In addition to the mRNA analyses, the protein content of ER and PR in leiomyoma and corresponding myometrial tissue was evaluated immunohistochemically. Expression of the receptors was confined exclusively to the nucleus, but was very heterogeneous in the tumor tissue, making quantification difficult. Although the immunostaining did not allow an accurate quantitative comparison of sex steroid receptor proteins between the two tissues, our results suggest no significant differences in the average amount of protein present in tumor and normal tissues (Table 2). As for mRNA, the relative content of ER and PR protein did not change with the phase of the menstrual cycle, assuming no direct influence of sex steroids on the relative expression of ER and PR in leiomyomas and myometrium (Table 2). Addressing the overall content of ER and PR in the myometrium in the proliferative and secretory cycle phases, corresponding to published data, reduced levels for ER were seen in the secretory myometrium (14).

Detection of a protein present in dividing cells was used to assess proliferative activity. Tissue sections were treated with a monoclonal antibody against PCNA, a protein expressed in the nucleus of cycling cells, but not, or only at a low level, in resting cells. As for steroid hormone receptors, quantitation of positive nuclei was difficult, because the tumor tissue exhibited a large degree of variability in the number of positive cells in different tissue sections. This might indicate that some regions of the tumor may be more actively dividing than others. However, if the overall count of positive staining was assessed, no significant differences between tumor and myometrial tissue were observed (Table 2). Therefore, it might be assumed that proliferation in the tumor tissue takes place only locally, potentially reflecting the focal growth character of these tumors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The goal of our study was to identify crucial genes involved in the molecular mechanisms of the development of uterine leiomyomas. The protooncogenes c-jun, c-fos, and c-myc, as well as the ER and PR, were selected as initial candidate genes. As a result, the present study shows for the first time that leiomyomas and normal myometrial tissue express mRNAs for the protooncogenes c-jun, c-myc, and c-fos. None of these factors exhibit elevated transcript levels in the tumor tissue. By contrast, a distinct downregulation of c-fos mRNA was observed. This finding was strengthened by lower c-Fos protein levels in the leiomyomas. Interestingly, c-fos underexpression was described for other tumors too, namely pleomorphic adenomas of the salivary gland (15), in adenomatous polyps and adenocarcinomas in patients with familial polyposis coli (16). A recent report of Saez et al. (17) describes the essential role of c-fos for the malignant progression of skin tumors. They showed that c-fos-deficient mice were able to develop benign papillomas after stimulation with tumor-promoting agent at the same rate as wild-type animals. However, these tumors failed to undergo malignant transformation like that seen for the c-fos wild-type animals. The authors concluded that c-fos is not necessary for tumor promotion but seems to play a pivotal role in the progression of benign to malignant tumors. As regards these results, we might speculate that the lower c-fos levels observed in leiomyomas might be involved in preventing the tumor from undergoing malignant progression. Because low levels of c-fos mRNA were observed in tumors of all patients in this study, this gene seems to be linked to the pathogenesis of leiomyomas. However, further studies are necessary to elucidate whether c-fos deregulation is the cause, or consequence, or merely a coincidental factor in the development of leiomyomas. Lowered mRNA levels measured for c-jun in leiomyomas might enhance the effect of c-fos reduction by leading to reduced amounts of AP-1 complexes, consisting of the proteins c-Jun and c-Fos.

Extensive cytogenetic studies have firmly implicated region q14–15 of human chromosome 12 in the development of a variety of benign tumors. Using positional cloning, Schoenmakers et al. (2) and Ashar et al. (3) very recently identified a gene in this region of chromosome 12 named HMGI-C, which is rearranged in a number of mesenchymal tumors, including uterine leiomyomas, lipomas, or pleomorphic adenomas. HMGI-C belongs to a subclass of the high-mobility group of DNA-binding proteins which encode for so-called architectural transcription factors. Interestingly, lowered levels of c-fos transcripts were found not only in uterine leiomyomas (this study) but also in pleomorphic adenomas (15). As mentioned, both of these tumors show aberration in the HMGI-C gene, potentially indicating a link between reduced c-fos levels and rearrangement of HMGI-C.

Interestingly, c-myc levels also were not elevated in leiomyoma tissues, with the exception of one. In this case, the biopsy material was obtained from a women harboring a highly proliferating tumor with a mass of 30 kg. Because enhanced expression c-myc has been associated with malignant progression in a variety of human tumors (18), c-myc expression at a reduced level, as found in our study, might underscore the benign character of these tumors.

As far as the ER and PR are concerned, differences in the relative expression were observed neither on the mRNA level nor on the protein level in leiomyoma and myometrial tissue. The literature reveals conflicting data on this point. Vollenhoven et al. (19) confirm our results, presenting no difference in mRNA abundance for either receptor between fibroids and myometrium. By contrast, Brandon et al. (20, 21) reported elevated PR and ER levels in leiomyomas. However, they used ß-actin transcript levels to normalize the mRNA content. Because ß-actin is known to be regulated by sex steroids (11), differences in data obtained might be caused by this fact.

Nevertheless, the question remains as to how steroids influence the growth and development of leiomyomas. Because the relative mRNA expression levels in tumor and healthy tissue of c-jun, c-myc, and c-fos and of the steroid hormone receptors are similar during the menstrual cycle, we can assume that relative differences measured are not influenced by sex steroids. Therefore, further studies will have to be performed to define the targets of steroid hormone action in leiomyomal tissue.

	Acknowledgments

 
We thank A. Weisz for kindly providing the rat 1 A clone, and G. Repenthin and K. Ulrich for expert technical assistance. Furthermore, we thank Dr. Huber-Schumacher, Dr. Minguillon, and Prof. Lichtenegger from the Virchow Klinikum for their help in the collection of tissue material.

Received January 2, 1997.

Revised April 18, 1997.

Accepted April 29, 1997.

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