Twenty years of breast cancer in Iran: downstaging without a formal screening program.

BACKGROUND: breast cancer (BC) is the most common malignancy among Iranian women. Although the relative incidence of BC is low, the cause-specific mortality is much higher than developed countries. The present study surveyed the overall trend of BC in Tehran. PATIENTS AND METHODS: all breast pathologic records were studied in five major hospitals in Tehran during three phases (1: 1985-1995; 2: 1996-2000; and 3: 2001-2005). Malignant cases were classified according to the tumor-node-metastasis classification. Data were compared across the study. RESULTS: Of 9050 medical records from male and female patients with 'breast disease', 2946 females with BC were included. A significant increase in the diagnosis of palpable early BCs (stage II increased, stage III decreased) was observed between phases 1 and 2. A relative increases in stages 0 and I were noted between phases 3 and 2. Nevertheless, 76.8% of cases were T2 or higher and 65.3% had positive lymph nodes in the last phase. The proportion of patients with stage IIIb was increased in phase 3, despite the reduction in phase 2. CONCLUSIONS: despite the relative improvement in the status of BC patients, the vast majority are diagnosed in advanced stages. Specific screening measures should be implemented in Iran.

Evaluation of the Effect of Source Geometry on the Output of Miniature X-ray Tube for Electronic Brachytherapy through Simulation.

OBJECTIVE: The use of miniature X-ray source in electronic brachytherapy is on the rise so there is an urgent need to acquire more knowledge on X-ray spectrum production and distribution by a dose. The aim of this research was to investigate the influence of target thickness and geometry at the source of miniature X-ray tube on tube output. METHOD: Five sources were simulated based on problems each with a specific geometric structure and conditions using MCNPX code. Tallies proportional to the output were used to calculate the results for the influence of source geometry on output. RESULTS: The results of this work include the size of the optimal thickness of 5 miniature sources, energy spectrum of the sources per 50 kev and also the axial and transverse dose of simulated sources were calculated based on these thicknesses. The miniature source geometric was affected on the output x-ray tube. CONCLUSION: The result of this study demonstrates that hemispherical-conical, hemispherical and truncated-conical miniature sources were determined as the most suitable tools.

Detection of ebaf, a novel human gene of the transforming growth factor beta superfamily association of gene expression with endometrial bleeding.

Human endometrium is unique since it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. Here, we report on a novel human gene, endometrial bleeding associated factor (ebaf), whose strong expression in endometrium was associated with abnormal endometrial bleeding. In normal human endometrium, this gene was transiently expressed before and during menstrual bleeding. In situ hybridization showed that the mRNA of ebaf was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells. The predicted protein sequence of ebaf showed homology with and structural features of the members of TGF-beta superfamily. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Thus, ebaf is a novel member of the TGF-beta superfamily and an endometrial tissue factor whose expression is associated with normal menstrual and abnormal endometrial bleeding.

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

Quantitative light microscopic autoradiographic study on [3H]leukotriene C4 binding to nonpregnant bovine uterine tissue.

Mammalian uteri contain both lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism. Sulfidopeptidyl leukotrienes formed by the lipoxygenase pathway can stimulate uterine contractions and play a role in uterine preparation for implantation. These actions of leukotrienes are perhaps mediated by binding to specific receptors. To understand the cellular basis of leukotriene C4 action, the present quantitative light microscopic autoradiographic study was undertaken on nonpregnant bovine uterine tissue. The results demonstrated that the circular and elongated myometrial smooth muscle, uterine vascular smooth muscle, stromal cells of endometrium, and fibroblasts of perimetrium, but not the endometrial glands, vascular endothelium, and erythrocytes in lumen of arterioles, contained specific silver grains after incubation with [3H]leukotriene C4. The number of grains per 100-micron2 areas were similar in circular and elongated myometrial smooth muscle (P greater than 0.05), which was higher than in other uterine cells (P less than 0.05-0.01). The grains in all cells were greatly reduced after coincubation with excess unlabeled leukotriene C4, but not with leukotriene A4, leukotriene B4, leukotriene D4, leukotriene E4, prostaglandin E2, prostaglandin F2 alpha, or prostacyclin. In conclusion, leukotriene C4 may regulate both uterine cells and uterine vasculature and exert contractile and noncontractile actions via the specific leukotriene C4-binding sites present in different cell types.

Presence of transforming growth factor-beta and their selective cellular localization in human ovarian tissue of various reproductive stages.

Immunohistochemical studies were performed using specific polyclonal antibodies to transforming growth factor (TGF)-beta 1 and TGF-beta 2 to determine their presence and cellular localization in human ovarian tissues of various reproductive states. In the small ovarian follicles, the immunostaining for TGF-beta 1 was present in oocytes, follicle cells, and granulosa and theca cell layers. The level of immunostaining associated with granulosa and theca cell layers intensified as the size of the follicles increased. In the luteal tissue, both the small and large luteal cells immunostained for TGF-beta 1 and their intensities were similar to theca and granulosa cell layers, respectively. The patterns of immunostaining were similar in early (days 14-19), mid (days 22-25), and late (days 26-29) luteal phases; however, the intensity was highest at mid and decreased at late luteal phase. Corpus albicans showed a very weak immunostaining for TGF-beta 1, whereas ectopic pregnancy small luteal cells immunostained relatively intensely. The ovarian stromal, luteal tissue fibroblasts, and arterioles endothelial and smooth muscle cells were also immunostained for TGF-beta 1. The immunostaining of the ovarian tissues for TGF-beta 2 indicated that the theca cell layers were the exclusive cells in the follicles with intense immunostaining, which increased in the larger follicles. A low immunostaining was also observed in granulosa cell of the large follicles. In the luteal tissues, only small luteal cells showed intense immunostaining for TGF-beta 2, which was similar in intensity to that in the theca cells; however, the large luteal cells showed a low level of immunostaining at midluteal phase. The small luteal cells in corpus albicans and ectopic pregnancy luteal tissues retained their immunostaining for TGF-beta 2, but with lower intensity. Endothelial and smooth muscle cells of arterioles also immunostained for TGF-beta 2, but not ovarian stromal cells. Atretic follicles showed very low or no detectable immunostaining for TGF-beta 1 or TGF-beta 2. The results of present studies show that human ovarian tissue at all the reproductive states locally produces TGF-beta 1 and TGF-beta 2, and although TGF-beta 1 is present in most major ovarian cell types, TGF-beta 2 is only produced by theca cells in the follicles and small luteal cells in luteal tissues.

Dynamics of nuclear associated granules in bovine luteal cells after treatment in vitro with prostaglandin F2 alpha.

A group of electron-dense granules of 150-300 nm in diameter, with a single limiting membrane, were found adjacent to the nuclei of large (25-50 microns in diameter), but not small (15-22 microns), bovine luteal cells of pregnancy. These granules were quite heterogeneous with respect to size, shape, content, etc. Most granules contained varying amounts of light and dark electron-dense material, and very few contained only light or dark material in their core. These granules occupied 160.4 +/- 7.8 microns3 cytoplasmic volume, which did not significantly decrease until 2 h of incubation without any hormone at 22 or 38 C (P greater than 0.05). Subsequently, however, volume occupancy of these granules decreased to 127.3 +/- 6.4 and 117.6 +/- 8.0 microns3 (P less than 0.05) by 4 and 12 h of incubation, respectively. Incubation of luteal tissue with 10 nM prostaglandin F2 alpha (PGF2 alpha), on the other hand, resulted in a decrease to 124.4 +/- 8.0 microns3 of volume occupancy of granules by 10 min (P less than 0.05). The volume occupancy further decreased to 46.2 +/- 4.8 microns3 by 2 h (P less than 0.01), and then the granules virtually disappeared from luteal cells. While PGF2 alpha decreased volume occupancy of granules similarly at 22 and 38 C, it had no effect at 4 C. PGF2 alpha decreased volume occupancy of granules at 0.01 nM (P less than 0.05), which continued to decrease with increasing PGF2 alpha concentrations, reaching a maximal decrease at 10 nM PGF2 alpha (P less than 0.05). PGE1, hCG, human LH, human PRL, or (Bu)2cAMP had no effect on the granules, suggesting that the PGF2 alpha effect was hormone specific. PGE1 and hCG, which protect corpus luteum from the luteolytic action of PGF2 alpha, could not inhibit degranulation by PGF2 alpha. These results demonstrate that treatment with PGF2 alpha in vitro results in a decrease in volume occupancy of nuclear associated granules in bovine large luteal cells of pregnancy in a time-, temperature-, and concentration-dependent and hormone-specific manner.

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF beta-receptors in human endometrial tissue: localization and in vitro action.

Human endometrial tissue and primary stromal cell culture contain immunoreactive epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB as well as EGF and PDGF-beta receptors. The immunostaining for EGF, EGF receptor, and PDGF beta-receptor were associated with endometrial luminal and glandular epithelial and stromal cells, whereas only the stromal cells contain immunoreactive PDGF-AB. The immunostaining intensity of EGF, EGF receptor, and PDGF-AB was similar in both phases of the menstrual cycle, whereas, PDGF-beta receptor immunostaining was highest in proliferative phase and considerably reduced, particularly in luminal and glandular epithelial cells in the secretory phase. In addition primary stromal cell cultures express EGF, PDGF-AB, and contain EGF and PDGF-beta receptors, and very low levels of PDGF-alpha receptor. 3H-Thymidine incorporation indicate that after 48 h of incubation in serum-free medium approximately 75-80% of stromal cells are quiescent. Incubation of quiescent stromal cells with 10% fetal bovine serum stimulate 3H-thymidine incorporation in a time-dependent manner reaching maximal after 30-48 h, with a doubling time of 38.2 h. EGF (1.5-15 ng/ml) stimulates 3H-thymidine incorporation by quiescent stromal cells (P less than 0.001). This effect was significantly reduced at concentrations above 15 ng/ml (P less than 0.005). PDGF-AB (3-10 ng/ml) and PDGF-BB (0.5-10 ng/ml) also stimulate 3H-thymidine incorporation in quiescent stromal cells compared to controls (P less than 0.005). The action of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) was time dependent, reaching maximal after 36 and 48 h of incubation (P less than 0.002). Addition of PDGF-AB (10 ng/ml) to EGF (15 ng/ml) significantly enhanced the action of EGF or PDGF-AB used individually (P less than 0.001). 17 beta-estradiol or progesterone at 1 microM did not stimulate 3H-thymidine incorporation, although they were stimulatory in combination (P less than 0.001), they did not alter the action of EGF or PDGF when added in combination. These observations provide further evidence that human endometrial tissue contains specific immunoreactive EGF receptors. It also demonstrates the presence of immunoreactive EGF, PDGF-AB, and PDGF-beta receptors in endometrial tissue as well as stromal cells in primary culture. Both EGF and PDGF are mitogenic for endometrial stromal cells, suggesting an autocrine/paracrine role in modulation of endometrial cell growth and differentiation.

Human uterine tissue throughout the menstrual cycle expresses transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, TGF beta 3, and TGF beta type II receptor messenger ribonucleic acid and protein and contains [125I]TGF beta 1-binding sites.

The use of isoform-specific transforming growth factor-beta (TGF beta) primers, 35S-labeled 40-mer oligonucleotide probes and polyclonal antibodies, reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemical observations has revealed that human uterine tissue at various reproductive stages expresses TGF beta s and TGF beta type II receptor messenger RNAs (mRNAs) and proteins. The reverse transcription-polymerase chain reaction revealed the predicted 443-, 310-, 524-, and 431-basepair fragments for TGF beta 1, TGF beta 2, TGF beta 3, and TGF beta type II receptor, respectively, in both endometrial and myometrial tissues, which were further verified by restriction enzyme analysis. In situ hybridization and immunohistochemical observations indicated that all uterine cell types express TGF beta s mRNAs and proteins. In the functionalis region, endometrial luminal and glandular epithelial cells are the primary cell types expressing TGF beta s mRNAs and proteins, with lesser expression in stromal cells, whereas in the basalis region, they are equally expressed in both cell types. In myometrium, TGF beta mRNA and protein expression in smooth muscle cells occurs at a substantially lower level than in endometrial tissue. In endometrial tissue, the highest level of TGF beta mRNA and protein expression appeared in the late proliferative and early to midsecretory phases of the menstrual cycle, with a considerable reduction during the late secretory and postmenopausal periods. The pattern and cellular distribution of TGF beta type II receptor protein were similar to those seen with TGF beta isoforms in both endometrial and myometrial tissues. Quantitative autoradiography (net grain density per 100 microns 2) of specific binding of [125I]TGF beta 1 for different uterine cell types indicated that the stromal cells contain a higher grain density than other uterine cell types (P < 0.05), without a significantly different density in the proliferative, compared with the secretory, phase of the menstrual cycle. These data suggest that TGF beta s acting through their specific receptors may play an important role in a variety of uterine functions in an autocrine/paracrine manner, and ovarian steroids may also regulate their expression in endometrial tissue.

Characterization of the synthesis and secretion of transforming growth factor-alpha from salivary glands and saliva.

Whole saliva collected from rat, mouse, and human sources was found to contain high concentrations of transforming growth factor-alpha (TGF alpha) when analyzed by RIA. The concentrations of TGF alpha in unstimulated human saliva (age, 30-45 yr; n = 10; 1.5 +/- 3.1 nM) was reduced with age (age, 55-70 yr; n = 10; 0.4 +/- 0.1 nM), but increased in oral pathologies manifested in xerostomia (age, 57-70; n = 6; 0.8 +/- 0.2 nM) and Paget's disease (age, 58-76; n = 8; 2.0 +/- 0.6 nM). Immunohistochemical localization of TGF alpha in the salivary glands of rats and mice revealed specific immunostaining of the granular ductal cells of the parotid and submandibular glands. Reverse transcription followed by polymerase chain reaction amplification of total RNA from the parotid and submandibular glands of rats and mice demonstrated the presence of TGF alpha mRNA, suggesting endogenous synthesis by the salivary glands. Thus, salivary glands appear to be an exocrine source for a second member of the epidermal growth factor-like growth factor family in the oral cavity.

Expression of transforming growth factor-beta (TGF beta) isoforms and TGF beta type II receptor messenger ribonucleic acid and protein, and the effect of TGF beta s on endometrial stromal cell growth and protein degradation in vitro.

Reverse transcription-polymerase chain reaction analysis of total RNA and immunocytochemical observations revealed that human endometrial glandular epithelial and stromal cells in primary culture express messenger RNAs and proteins for transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 as well as TGF beta type II receptor. The epithelial and stromal cells synthesize and secrete into their culture-conditioned medium 2.6 +/- 0.3 and 1.4 +/- 0.2 ng TGF beta 1/10(6) cells, respectively; after transient acidification of the medium, the TGF beta 1 levels were 18.1 +/- 0.4 and 7.8 +/- 0.7 ng/10(6) cells. These cells also contain specific binding sites for [125I]TGF beta 1, indicated by light microscope autoradiography. TGF beta s at 0.01-10 ng/ml neither stimulated or inhibited subconfluent quiescent stromal cells under serum-free condition nor altered the mitogenic action of 10% fetal bovine serum. However, in the presence of 2% fetal bovine serum, which induced half-maximal stimulation of [3H]thymidine incorporation, TGF beta 1 and TGF beta 2 at 0.1-0.5 ng/ml and TGF beta 3 at 0.1-2.5 ng/ml significantly stimulated the rate of [3H]thymidine incorporation into quiescent stromal cells (P < 0.005); they were ineffective at higher concentrations. TGF beta s did not have any effect on cell proliferation, as determined by cell counting; however, at 0.1 ng/ml and higher concentrations, TGF beta s significantly reduced the metabolic activity of stromal cells, as determined by colorimetric 3-(4,5-dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromide assay (P < 0.05). The stimulatory and inhibitory actions of TGF beta s in both assays were reversible using 5-10 micrograms/ml TGF beta 1- and TGF beta 2- and 3-6 micrograms/ml TGF beta 3-specific neutralizing antibodies. TGF beta 1 at 1 ng/ml had no significant effect on long-lived protein degradation, assayed by incorporation of [14C]valine into newly synthesized protein by stromal cells, and was similar to the effect of epidermal growth factor or platelet-derived growth factor-BB (10 ng/ml). The data suggest that the TGF beta expression by various endometrial cell types in an autocrine/paracrine manner acts as a negative regulator essential for restraining endometrial growth and transition from proliferation to differentiation stages during the secretory phase after mitogenic stimulation during the proliferative phase of the menstrual cycle.

Human fallopian tube expresses granulocyte-macrophage colony stimulating factor (GM-CSF) and GM-CSF alpha and beta receptors and contain immunoreactive GM-CSF protein.

Using specific primers, 35S-40mer oligonucleotide probe and monoclonal antibody, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical observations respectively revealed that human fallopian tube expresses granulocyte macrophage colony stimulating factor (GM-CSF) mRNA and protein, as well as GM-CSF alpha and beta receptors mRNA. The RT-PCR products revealed the predicted 286, 546 and 380 bp fragments for GM-CSF, GM-CSF alpha receptor and GM-CSF beta receptor respectively, which were further verified by restriction enzyme digestion analysis. Tubal epithelial cells in the ampullary and isthmus regions (ciliated and nonciliated) are the primary site of GM-CSF mRNA expression and its immunoreactive gene product, and present to a lesser extent in tubal stromal, smooth muscle, and arterial endothelial and smooth muscle cells. The in situ hybridization and immunostaining observations indicated that the expression of GM-CSF mRNA and protein appeared to be cycle dependent and considerably higher during mid-late proliferative and early-mid secretory, than early proliferative and reduced at late secretory phases of the menstrual cycle and postmenopausal period. The results demonstrate for the first time that human fallopian tube expresses GM-CSF and its alpha and beta receptors mRNA and contains the immunoreactive gene product for GM-CSF, and may be potentially regulated by ovarian steroids. The results imply the importance of GM-CSF in a variety of tubal functions and possibly the early embryonic development.

Epidermal growth factor binding to human amnion, chorion, decidua, and placenta from mid- and term pregnancy: quantitative light microscopic autoradiographic studies.

All of the cell types, but not noncellular elements, found in amnion, chorion, decidua, and placenta of mid- and term pregnancy contained numerous silver grains after incubation with 1 nM [125I]epidermal growth factor ([125I]EGF). These grains completely disappeared when excess unlabeled EGF, but not unlabeled insulin, was added with [125I]EGF, suggesting the presence of specific EGF receptors. Light cells of amnion contained more EGF receptors than dark cells of amnion, and the number of light cells decreased from mid- to term pregnancy. Numerous syncytiotrophoblasts containing a greater number of receptors than darkly stained cells in connective tissue were found in chorion from midpregnancy. The chorionic syncytiotrophoblasts were considerably reduced at term. Dark cells of decidua contained more receptors than light cells of decidua, and these cell numbers did not change during pregnancy. Placental syncytiotrophoblasts contained the EGF receptors. At mid- and term pregnancy, placenta contained the highest number of EGF receptors, followed by chorion greater than decidua greater than amnion. The receptor number in all tissues was higher at mid-compared to term pregnancy. A decrease in number of cells as well as a decrease or fewer receptors per cell during pregnancy may explain the tissue and mid- vs. term pregnancy differences. The higher number of EGF receptors in amnion, chorion, decidua, and placenta at midpregnancy, at a time when the maternal and fetal blood EGF levels are at their peak, suggests that EGF may exert maximal biological effects on the feto-placental unit at midpregnancy.

The presence of leukotriene C4- and prostacyclin-binding sites in nonpregnant human uterine tissue.

Human uterine tissue can synthesize leukotrienes and prostacyclin (PGI2) from arachidonic acid via the lipoxygenase and cyclooxygenase pathways, respectively. Leukotriene C4 stimulates myometrial and vascular smooth muscle contractions, whereas PGI2 inhibits them. In addition, these eicosanoids have other actions in uterine tissue. All of these actions are possibly mediated by specific receptors, but such receptors have not been demonstrated in human uterine tissue. Therefore, these quantitative light microscopic autoradiographic studies were undertaken to determine whether nonpregnant human uterine tissue contains specific leukotriene C4- and PGI2-binding sites. Studies with [3H]leukotriene C4 indicated that luminal epithelial cells of the endometrium, stromal cells, elongated and circular myometrial smooth muscle, and arteriolar smooth muscle contained numerous binding sites. Glandular epithelium, vascular endothelium, and erythrocytes, on the other hand, contained few or no leukotriene C4-binding sites. The number of binding sites in luminal epithelial cells of the endometrium was as high as that in lung, which is a rich source of these binding sites. The number of binding sites in these epithelial cells was higher (P less than 0.05) than that in stromal cells, circular myometrial smooth muscle, and arteriolar smooth muscle, but there were no significant (P greater than 0.05) differences among other cells. Maximal binding occurred at 5 min of incubation at 22 or 38 C. The presence of serine borate, a gamma-glutamyl transpeptidase inhibitor, in the incubation medium resulted in a small to moderate increase in leukotriene C4 binding to uterine cells. Binding to the cells was significantly (P less than 0.001) reduced after coincubation with excess unlabeled leukotriene C4, but not with excess unlabeled leukotriene A4, leukotriene B4, leukotriene D4, leukotriene E4, prostaglandin (PG) E1, PGF2a, or PGI2. Studies with [3H]PGI2 demonstrated that while myometrium and vascular smooth muscle contained PGI2-specific binding sites, endometrium and vascular endothelium contained very few or no binding sites. Elongated myometrial smooth muscle contained a higher number of binding sites than circular myometrial smooth muscle and vascular smooth muscle (P less than 0.01). PGI2 binding to the uterine cells was dependent on the time and temperature of incubation, and Ca2+ was not required for binding. Coincubation with unlabeled PGI2, but not with its stable metabolite 6-keto-PGF1a, resulted in a significant reduction (P less than 0.001) of binding to uterine cells. PGE2, PGF2a, and leukotriene C4 also had no effect on [3H]PGI2 binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of transforming growth factors (TGFs) TGF-alpha and TGF-beta in human ovarian tissues.

Light microscopic immunocytochemical technique was utilized in order to elucidate the presence and cellular distribution of transforming growth factors alpha and beta (TGF-alpha and TGF-beta) in human ovarian tissues in different reproductive states using polyclonal antibodies to TGF-alpha and TGF-beta. The results indicated that the ovarian tissue immunostained for both TGF-alpha and TGF-beta. The immunostaining for TGF-alpha was associated with oocytes in the primary follicles, granulosa and theca cell layers, as well as small and large luteal cells. The granulosa cell layers in the small follicles were immunostained for TGF-alpha, but there was very little staining association to the theca cell layers. However, the immunostaining intensity increased in both granulosa and theca cell layers as the size of the follicles increased. The ovarian stroma cells always showed moderate immunostaining. In luteal tissue, both small and large luteal cells immunostained for TGF-alpha and the intensity was similar in both cell types. The immunostaining intensity was similar in both early and midluteal phases and was less than that seen in the granulosa-theca cell layers of the large follicles, and decreased in the late luteal phase. Both corpora albicans and ectopic pregnancy corpora lutea showed a weak immunostaining. Immunostaining of ovarian tissues for TGF-beta indicated that the oocytes of the small preantral follicles stained faintly in the cytoplasm; however, they stained strongly around the nuclear periphery. In small, medium, and large follicles granulosa and theca cell layers immunostained with similar intensity for TGF-beta and its intensity increased with follicular size. The ovarian stroma cells also immunostained for TGF-beta, but with a moderate to low intensity compared with other regions. In luteal tissue, both small and large luteal cells immunostained for TGF-beta and its intensity was similar in early and midluteal phases and reduced during the late luteal phase. This immunostaining was reduced as compared to the granulosa-theca cell layers of the follicles. Corpora albicans and ectopic pregnancy corpora lutea also immunostained for TGF-beta; however, the immunostaining intensity was lower than that seen in luteal tissues of early, mid, or late luteal phases. There was strong immunostaining of the luteal fibroblasts and, interestingly, in small and medium size arterioles; endothelial and smooth muscle cells also immunostained strongly for TGF-beta, whereas the large arterioles showed very little or no immunostaining.(ABSTRACT TRUNCATED AT 400 WORDS)

Human fallopian tube expresses transforming growth factor (TGF beta) isoforms, TGF beta type I-III receptor messenger ribonucleic acid and protein, and contains [125I]TGF beta-binding sites.

Reverse transcription-polymerase chain reaction analysis of total ribonucleic acid (RNA) from human fallopian tubes revealed that transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 as well as TGF beta type I-III messenger RNA (mRNA) are expressed in this tissue. In situ hybridization and immunohistochemical observations using TGF beta isoform-specific [35S]40-mer oligonucleotide probes and polyclonal antibodies indicate that all the tubal cell types express TGF beta isoforms and TGF beta type II receptor mRNA and protein. The tubal epithelial cells appeared to express more TGF beta 1 mRNA and TGF beta 1-3 proteins than other cell types, whereas TGF beta 2 and TGF beta 3 mRNA appeared to be equally expressed in the epithelial and other tubal cell types. In the epithelial lining, both ciliated and nonciliated cells in the ampullary and isthmus regions appeared to express mRNA and protein for TGF beta s and TGF beta type II receptor at a similar level. The intensity of immunostaining of TGF beta s in tubal epithelial cells was lower during the early proliferative and late secretory than the mid- to late proliferative and early to midsecretory phases of the menstrual cycle and reduced during the postmenopausal period. However, the intensity of immunoreactive TGF beta type II receptor in these cells did not vary during the cycle as much as that seen with TGF beta s. Quantitative autoradiography of [125I]TGF beta 1 indicates that fallopian tubes contain specific binding sites for TGF beta 1. Net grain density per 100 microns 2, calculated for different cell types, indicates that the epithelial cells had a significantly higher grain density than other tubal cell types (P < 0.05), with similar densities in the late proliferative and early secretory phases of the cycle. These results provide the first evidence that human fallopian tubes express mRNA and protein and contain specific binding sites for TGF beta system, suggesting an autocrine/paracrine role for TGF beta in a variety of tubal functions.

Regulation of transforming growth factor-beta1 expression by granulocyte macrophage-colony-stimulating factor in leiomyoma and myometrial smooth muscle cells.

Human myometrium and leiomyomas express granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGFbeta), and their receptors. Overexpression of TGFbeta and, to a limited extent, GM-CSF has been associated with tissue fibrosis throughout the body, including leiomyomas. The objective of the present study was to determine the action of GM-CSF on leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and examine whether the action of GM-CSF is mediated through the induction of TGFbeta1 expression. Using competitive quantitative RT-PCR and enzyme-linked immunosorbent assay, we found that LSMC express significantly higher GM-CSF messenger ribonucleic acid (mRNA; 0.6 +/- 0.1 x 10(3) copies of mRNA/microg total RNA) and protein (0.75 +/- 0.2 ng/mL) than MSMC (0.5 +/- 0.1 x 10(2) copies of mRNA and 0.45 +/- 0.07 ng/mL protein; P < 0.05). In addition, LSMC expressed significantly higher TGFbeta1 mRNA (1.6 +/- 0.3 x 10(4) copies of mRNA/microg total RNA) than MSMC (2.4 +/- 0.4 x 10(3) copies) and synthesized and secreted more TGFbeta1 protein (1.7 +/- 0.2 vs. 0.5 +/- 0.02 ng/mL); whereas MSMC contained more cell-associated TGFbeta1 (56.2 +/- 1.2 ng/mL) than LSMC (35.2 +/- 1.2 ng/mL; P < 0.05). We found that GM-CSF (0.01-100 ng/mL) has limited mitogenic activity for LSMC but not for MSMC determined by the rate of [3H]thymidine incorporation and cell proliferation assay. However, GM-CSF at 1 ng/mL increased its own production, the expression of TGFbeta1 mRNA, the cell-associated TGFbeta1 protein content in both cell types, and TGFbeta1 released into the culture-conditioned medium of LSMC (P < 0.05). TGFbeta1 also increased its own mRNA and protein expression, but had no effect on cell-associated TGFbeta1 in both cell types (P < 0.05). Cotreatment of LSMC and MSMC with GM-CSF and TGFbeta1 induced changes similar to those produced by GM-CSF in both cells. In conclusion, our data suggest that GM-CSF is not a mitogen for MSMC and LSMC, but it regulates its own expression and the expression of TGFbeta1 by these cells, a regulatory interaction that may account for the GM-CSF-induced tissue fibrosis that occurs in leiomyomas.

Binding sites for epidermal growth factor in human fetal membranes.

The present study demonstrates that human fetal membranes bind 125I-epidermal growth factor (125I-EGF), with chorion binding more than amnion. The chorion binding was also higher than that by decidua, but lower than in placenta. The lower binding by chorion compared to placenta was entirely attributable to a lower number of available EGF receptors and not to lower affinity. Chorion and placenta , but not amnion or decidua, from Cesarean section bound significantly more 125I-EGF when compared to tissues obtained after vaginal delivery. The binding of 125I-EGF to chorion exhibited dependency on time, temperature of incubation, pH of the incubation media, and amount of chorion protein. The 125I-EGF was not degraded during the binding reaction with chorion. The binding was specific in that unlabeled EGF inhibited 125I-EGF binding in a dose-dependent manner, whereas very high concentrations of other unlabeled hormones and growth factors had minimal effects on 125I-EGF binding. When some of these other hormones were tested for binding as tracers (125I-hCG, [3H]prostaglandin E1 and F2 alpha), neither chorion, amnion, decidua, nor placenta specifically bound these ligands. The 125I-EGF specific binding to chorion was saturable and a Scatchard plot of this data was curvilinear, which appears to be due to negative cooperativity. The apparent dissociation constant calculated from the initial slope of the plot was 0.20 nM, which is in excellent agreement with the concentration of unlabeled EGF required for half maximal inhibition of 125I-EGF binding, 0.26 nM. Autoradiography at the light microscope level revealed the presence of silver grains in amnion and chorion only when excess unlabeled EGF addition was withheld. The quantification of grains revealed the presence of a significantly (P less than 0.01) greater number of grains in chorion than in amnion, supporting the conclusion of the binding data. The physiological significance of EGF binding to fetal membranes and decidua is not known, but the considerable amount of EGF binding reported here suggests that they are target tissues for EGF.

Gonadotropin-releasing hormone (GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro.

The objective of the present study was to determine whether GnRH and GnRH receptor are expressed in myometrium and leiomyomata, and if GnRH analogs alone or in the presence of ovarian steroids can modulate the rate of DNA synthesis, proliferation, and transforming growth factor-beta 1 (TGF beta 1) production in myometrial smooth muscle cells in vitro. Reverse transcription-PCR revealed that leiomyomata, unaffected myometrium, and isolated myometrial smooth muscle cells express GnRH and GnRH receptor messenger ribonucleic acid. Furthermore, in a dose-dependent manner, GnRH agonist (leuprolide acetate) inhibited, but GnRH antagonist [D-pGlu1,D-Phe2,D-Trp3.6] (GnRH-Ant1) stimulated, the rate of [3H]thymidine incorporation into myometrial smooth muscle cells (P < 0.05), whereas GnRH-Ant2 (Ac-D-P-Cl-Phe1.2,D-Trp3,D-Arg6,D-Ala10) had no effect. 17 beta-Estradiol (E2) medroxyprogesterone acetate (MPA), and E2 plus MPA (1 micromol/L) stimulated the rate of DNA synthesis by smooth muscle cells (P < 0.05), which was inhibited by GnRH analogs used at 5 micromol/L (P < 0.05). GnRH analogs had no significant effect on myometrial smooth muscle cell proliferation, with the exception of GnRH-Ant1; however, they inhibited the stimulatory action of E2, MPA, and E2 plus MPA in a time-dependent manner (P < 0.05). These cells also synthesized and released approximately 1.32 +/- 0.02 ng/mL total (active plus latent) TGF beta 1, of which 0.73 +/- 0.02 ng/mL was in an active form. E2, MPA, E2 plus MPA, and GnRH analog treatments resulted in an increase in total TGF beta 1 production, whereas GnRH agonist and GnRH-Ant2, but not GnRH-An1, inhibited active TGF beta 1 (P < 0.05). GnRH analogs also inhibited the action of E2 plus MPA on total and active TGF beta 1 production, whereas GnRH-Ant1 further stimulated E2, MPA, or E2 plus MPA action on active TGF beta 1 production (P < 0.05). The data demonstrate for the first time that GnRH and GnRH receptor messenger ribonucleic acid are expressed in myometrium, leiomyomata, and myometrial smooth muscle cells. The local expression of GnRH and receptor along with the direct action of GnRH analogs on the smooth muscle cell DNA synthesis and TGF beta 1 production suggest an autocrine/paracrine role for GnRH in these tissues, a mechanism that may be involved in leiomyomata regression in women receiving GnRH agonist therapy.