Biochemical and Biophysical Research Communications 289, 1180–1187 (2001)

doi:10.1006/bbrc.2001.6131, available online at http://www.idealibrary.com on

GnRH Agonist Buserelin Affects Colony-FormingEfficiency of HHUA and Jurkat Cells

Masahiro Enomoto,1 Takao Mori, and Min Kyun ParkDepartment of Biological Sciences, Graduate School of Science, University of Tokyo,7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received November 19, 2001

26, 28, 29), the rate of cell proliferation (20, 21, 23, 31)

In this study, cell proliferation was examined at low

and high cell densities, using HHUA and Jurkat celllines as experimental models for the antiproliferativeand proliferation-enhancing effects of GnRH agonist,Buserelin, respectively. For efficient evaluation ofBuserelin activity at low cell density, the colony-forming efficiency assay was adopted. Buserelin mark-edly affected colony-forming efficiency in a dose-dependent manner at low cell density; however,Buserelin had no effect at high cell density. The con-ditioned medium of HHUA cells inhibited the Busere-lin action, whereas that of Jurkat cells mimicked it.These results suggest that each cell line secretes somesubstances which regulate cell proliferation, and thatthese substances can also change the effects of Buse-relin. The measurement of colony-forming efficiency isa very effective way of eliminating autocrine and/orparacrine effects, and is a highly sensitive method formeasuring GnRH activity. © 2001 Elsevier Science

Key Words: GnRH; Buserelin; cell proliferation;colony-forming efficiency; conditioned medium; auto-crine; paracrine.

Gonadotropin-releasing hormone (GnRH) is a hypo-thalamic decapeptide that promotes gonadotropin(GTH) secretion from the pituitary. However, there isincreasing evidence for extrapituitary effects of GnRHon a number of peripheral tissues, such as ovary (1–5),testis (6, 7), placenta (8, 9), thymus (10–13), and adre-nal cortex (14–16), suggesting that GnRH has novelphysiological functions. Furthermore, a number ofstudies have demonstrated that various carcinomas ofbreast (17–20), ovary (21, 22), endometrium (22, 23),prostate (24–26), pancreas (27–30) and liver (31) originrespond to GnRH or GnRH analogues, and in thesestudies the suppression of cell proliferation was inves-tigated by measuring tumor weight and/or volume (17,

1 To whom correspondence and reprint requests should be ad-dressed. Fax: 03-5841-4439. E-mail: m-enmt@eurus.dti.ne.jp.

11800006-291X/01 $35.00© 2001 Elsevier ScienceAll rights reserved.

or [3H]thymidine incorporation (19, 20, 31). Recent re-ports have demonstrated that GnRH, in addition tobeing a key hormone of reproductive function in verte-brates, is also an important immunomodulator (32–34). For example, several reports have demonstratedthat in vitro treatment of splenic and thymic cells withGnRH and GnRH analogues increased their prolifera-tive activity (32, 35). In addition, it was demonstratedthat in Jurkat cells, a human mature leukemic cellline, GnRH promotes cell proliferation as measured by[3H]thymidine incorporation (36).

As described above, a number of studies have dem-onstrated that GnRH directly effects cell proliferationin vitro. However, the mechanism by which GnRHregulates cell proliferation is still unclear. Therefore,we tried to establish a new method, a colony-formingefficiency assay, that would make it easy to detect bothpositive and negative effects of the GnRH agonist,Buserelin on cell proliferation. In this report, we de-scribe the effects of Buserelin on colony-forming effi-ciency and the effects of conditioned media on theBuserelin activity.

MATERIALS AND METHODS

GnRH agonist. A GnRH agonist, Buserelin ([D-Ser(But)6, Pro9-NHET]GnRH), was purchased from Sigma (St. Louis, MO). Busere-lin was dissolved at 1 mg/ml in 0.1 M HCl and stored at 280°C.

Cell cultures. The human endometrial carcinoma cell line(HHUA), human mature leukemic cell line (Jurkat), and Chinesehamster ovary cell line (CHO-K1) were purchased from Riken CellBank (Saitama, Japan). Each cell line was maintained at 37°C in theappropriate medium (F12 for HHUA and CHO-K1, RPMI 1640 forJurkat), containing 10 Nu-serum I (Collaborative Biomedical Prod-ucts, Bedford, MA) in a humidified atmosphere of 5% CO2/95% air.

Preparation of conditioned media. Conditioned medium is me-dium in which cells have been cultured for several days, and thus itcontains substances secreted from cells during the culture period.Conditioned media were collected after 3 days of culturing of cellsplated at 50,000 cells/ml (HHUA) or 200,000 cells/ml (Jurkat). Afterbrief centrifugation (1000 rpm, 10 min) the supernatant was filteredusing Durapore Membrane Filters (0.22 mm) purchased from Milli-pore Co. (Bedford, MA) and stored at 280°C.

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Colony-forming efficiency assay. The colony-forming efficiencywas measured based on limiting dilution analysis (37) with minormodifications. At first, cells were plated in flat-bottomed 96-wellplates at four levels of cell density, for example, 1, 2, 4, and 8cells/well in 100 ml of medium. The total well number was 48 wellsfor each cell density. After a 1-day incubation, either 100 ml of asolution of Buserelin in fresh medium or 100 ml of fresh medium(control) was added (final Buserelin concentration, 5 pg/ml (about 4pM)). Then plates were incubated at 37°C for 3 days and colony-containing wells were counted by examining the plates under amicroscope. Moreover, the ratio of the number of non-colony-containing wells to the total well number was calculated at each celldensity (designated as F0). After conversion to 2ln F0, linear regres-sion was done. The slope of the equation describing the lines thusobtained reflects the colony-forming efficiency, and a higher slopevalue means higher colony-forming efficiency.

Measurement of the rate of cell proliferation. Cell proliferationassays were done as follows. At first, cells in 2 ml of medium wereplated in flat-bottomed 24-well plates (20,000 cells/ml). The daywhen cells were plated was defined as Day 0. After a 1-day incuba-tion, either 20 ml of a solution of Buserelin in fresh medium or 20 mlof fresh medium (control) was added (final Buserelin concentration,5 pg/ml). Buserelin was freshly added to the treatment group every24 h during the culture period. The number of cells was countedusing a hemacytometer.

Effects of the medium exchange on the GnRH activity. To inves-tigate the effect of autocrine and/or paracrine activity of each cellline, the GnRH activity was also examined in cultures subjected to adaily medium exchange. In HHUA cells, cells were plated in 24-wellplates and the medium was then replaced by fresh medium andBuserelin was added (final concentration, 5 pg/ml). Since Jurkat cellswere cultured in suspension cultures, these cells were cultured in

FIG. 1. Effects of Buserelin (final concentration, 5 pg/ml) on the cThe number of cells plated per well was 1, 2, 4, and 8 cells in the caof Jurkat cells. The regression equation and correlation coefficient (rr 5 0.9948, E: y 5 0.050x 2 0.043, r 5 0.9972; (B) Œ: y 5 0.029x 2 0.0.120, r 5 0.9986, E: y 5 0.133x 2 0.110, r 5 0.9925.

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two-way-cap 10-ml tubes (Corning Inc., Corning, NY) and the me-dium was exchanged after brief centrifugation (1000 rpm, 10 min).To prevent loss of the cells, only 85% of the medium was exchangedfor fresh medium.

Dose–response relationship between Buserelin and colony forma-tion. The dose–response relationship between Buserelin and colonyformation was examined by analyzing the number of colony-containing wells when cells were plated at the density of 8 cells/well(HHUA cells) or 20 cells/well (Jurkat cells). The effect of Buserelinwas tested at six doses: 0.625 fg, 12.5 fg, 250 fg, 5 pg, 100 pg, and2 ng/ml (about 0.5 fM–1.6 nM).

RESULTS

Effects of GnRH on Colony-Forming Efficiency

The measurement of colony-forming efficiency wasdone as described under Materials and Methods. InJurkat cells, preliminary experiments showed thatcolony-forming efficiency was very low when cells wereplated at a density of 1, 2, 4, or 8 cells/well (data notshown). Therefore, in Jurkat cells, colony-forming effi-ciency was measured at densities of 5, 10, 20, and 40cells/well. As shown in Fig. 1A, Buserelin (5 pg/ml)decreased the colony-forming efficiency of HHUA cells.In Jurkat cells, it had the opposite effect (Fig. 1B). InCHO-K1 cells, used as a control cell line, Buserelin hadno effect (Fig. 1C).

y-forming efficiency of HHUA (A), Jurkat (B), and CHO-K1 cells (C).of HHUA and CHO-K1 cells and 5, 10, 20, and 40 cells in the caseseach experimental group were as follows: (A) Œ: y 5 0.107x 2 0.096,, r 5 0.9930, E: y 5 0.067x 2 0.184, r 5 0.9980; (C) Œ: y 5 0.136x 2

olonses) of106

Day 3 and gradually decreased thereafter until Day 5

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Effects of GnRH on the Rate of Cell Proliferation

In contrast to its effects on colony-forming efficiency,Buserelin (5 pg/ml) had no significant effect on the rateof cell proliferation of either HHUA or Jurkat cellsregardless of the frequency of Buserelin treatment(Fig. 2).

Effects of Replacement of the Medium on the Rateof Cell Proliferation

Figure 3 shows the effect of daily exchange of themedium on the rate of proliferation. In HHUA cells, therate of cell proliferation was significantly reduced on

FIG. 2. Effects of Buserelin on the rate of cell proliferation ofHHUA (A) and Jurkat cells (B). Each cell line was plated at 20,000cells per milliliter, and the number of cells was counted after theindicated culture period. Results are expressed as means 6 SE (n 54). The schedule of Buserelin treatment (final concentration, 5 pg/ml)is described in C.

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(Fig. 3A). In contrast, the rate of cell proliferation ofJurkat cells was significantly increased from Day 3.

Effects of Conditioned Media on Colony-FormingEfficiency

To examine the effects of conditioned medium on thecolony-forming efficiency, we measured the efficiencyunder the following conditions: control (fresh mediumonly), control 1 Buserelin (5 pg/ml), control 1 50%(v/v) conditioned medium, and control 1 50% (v/v) con-ditioned medium 1 Buserelin (5 pg/ml). In the pres-

FIG. 3. Effects of medium-exchange on the rate of cell prolifera-tion of HHUA (A) and Jurkat cells (B). Each cell line was plated at20,000 cells per milliliter, and the number of cells was counted afterthe indicated culture period. Results are expressed as means 6 SE(n 5 4; *P , 0.01; **P , 0.001) The schedule of each experimentalgroup is described in C.

ence of the conditioned medium of HHUA cells, the As for GnRH, it is well-known that substitution in

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colony-forming efficiency was nearly equal to that ofthe control, independent of the addition of Buserelin(Figs. 4A and 4B). In contrast, the colony-forming effi-ciency of cells in the conditioned medium of Jurkatcells was nearly equal to that of the control 1 Busere-lin group, and was independent of the addition of Buse-relin (Figs. 4C and 4D). The conditioned medium ofCHO-K1 cells did not change the effects of Buserelin(Figs. 4E and 4F).

Dose–Response Relationship between Buserelin andColony Formation

Figure 5 shows the ratio of the colony-containingwell number to the total well number for HHUA andJurkat cells. The ratios were decreased for HHUA cellsand increased for Jurkat cells over the range of 6.25fg–5 pg/ml Buserelin in a dose-dependent manner.

DISCUSSION

It is well-known that GnRH has a central role in thehypothalamus–pituitary–gonadal axis. In addition,there have been a number of studies suggesting thatGnRH has novel physiological functions in peripheraltissues (1–16). Our group also demonstrated that au-thentic GnRH receptor mRNA is expressed in a certaincell population in the rat ovary, which suggests thatthe receptor is involved in the control of various ovar-ian functions including follicular development, atresia,ovulation, and luteinization after ovulation and follic-ular atresia (4, 5). Furthermore, a number of studieshave demonstrated that GnRH and GnRH analoguesdirectly effects the proliferation in vitro of some tumorcells and lymphocytes (17–22, 31, 34, 36). It has beenreported that in some tumors and non-cancerous tis-sues, the expression of factors related to apoptosis,such as Fas, Fas ligand, Bcl-family member and mito-chondrial PBR, is affected and the number of apoptoticcells is increased by GnRH and GnRH analogues eitherin vitro or in vivo (22, 38–40). Therefore GnRH andGnRH analogues have been thought to induce apopto-sis directly in some kinds of cells, especially in sometumor cells. However, the range of GnRH concentra-tion used in these studies was higher than the GnRHconcentration in peripheral tissues (41–43). Therefore,it is still unknown whether GnRH directly inducesapoptosis under physiological conditions. On the otherhand, it has been reported that GnRH agonist in-creases [3H]thymidine incorporation in T-cell leukemiccells (36) and interleukin-2 receptor expression in lym-phocytes derived from female rats and human periph-eral blood mononuclear cells (34, 44). However, rela-tively little attention has been paid to the positiveeffect of GnRH on the proliferation of lymphocytescompared with the antiproliferative effects of GnRH.

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position 6, 10 or both can lead to superactive peptides;such a substitution is present in Buserelin (45). In anumber of studies on the tumor-suppressing effects ofGnRH and GnRH analogues, Buserelin has beenwidely used (20, 22, 23, 45), therefore, the GnRH ago-nist, Buserelin was employed in this study and weexamined the effect of Buserelin on colony-forming ef-ficiency, using HHUA and Jurkat cell lines. The mea-surement of colony-forming efficiency is especially suit-able for detecting cell viability under extremely low celldensity conditions. At the same time, we measured therate of cell proliferation at higher cell densities, anassay method adopted in past studies and comparedthe result of these two methods. At low cell density, wewere able to detect effects of Buserelin on cell growthas changes of colony-forming efficiency. In HHUA cellscolony-forming efficiency was decreased, while in Jur-kat cells it was increased in response to Buserelintreatment (Fig. 1). These results are compatible withthose of past studies which showed that GnRH sup-presses the cell proliferation of HHUA cells and pro-motes that of Jurkat cells (22, 36). On the other hand,Buserelin had no significant effect on the rate of cellproliferation at high cell density (Fig. 2). This resultsuggests that the cause of these apparently contradic-tory results at the two cell densities may be the accu-mulation of substances secreted from the cells into themedium. Therefore the effect of daily replacement ofthe culture medium with fresh medium on the action ofBuserelin was also examined. As shown in Fig. 3, fre-quent medium changes resulted in the ability to detectthe effect of Buserelin even at high cell density: Buse-relin inhibited the cell proliferation of HHUA cells andstimulated that of Jurkat cells (Figs. 3A and 3B, respec-tively). These results strongly support the idea that theeffect of Buserelin on cell proliferation in these celllines is markedly affected by the cells’ autocrine and/orparacrine activity. So far, there have been a number ofreports that GnRH or GnRH analogues reduce the rateof cell proliferation in human cancer cell lines (20, 21,23, 31). However, the effect of medium-exchange hasnot been described in detail. Next, to investigate theeffect of substances accumulated in the medium, wemeasured the colony-forming efficiency in the presenceof conditioned media. In the presence of the condi-tioned medium of HHUA cells, the colony-forming ef-ficiency was nearly equal to that of the control, and wasindependent of the addition of Buserelin (Figs. 4A and4B). In contrast, the colony-forming efficiency in thepresence of the conditioned medium of Jurkat cells wasnearly equal to that of the control 1 Buserelin group,and was independent of the addition of Buserelin (Figs.4C and 4D). These results indicate that the conditionedmedium of HHUA cells inhibited the Buserelin activ-ity, whereas the conditioned medium of Jurkat cellsmimicked it. In other words, HHUA cells secrete a

GnRH-inhibiting substance and Jurkat cells secret a

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GnRH-like substance in terms of the effects on cellproliferation, and the effects of GnRH on cell prolifer-ation can be detected when the concentrations of thesesubstances are very low. As far as we know, this is thefirst study to show the effect of conditioned medium onGnRH activity. There is evidence suggesting the pres-ence of GnRH-binding sites in lymphoid tissues, suchas rat spleen (34), rat thymus (11, 34, 46) and porcinelymphocytes (47), and the secretion of a GnRH-likesubstance from lymphoid cells (13, 36, 44). It was alsoreported that both GnRH and GnRH agonists stimu-late interleukin-2 receptor expression in female ratlymphocytes (34). In addition, it has been demon-strated that both interleukin-2 and its receptor areexpressed (48) and that interleukin-2 suppresses cellgrowth in a mouse uterine cervical carcinoma (49).Moreover, the regulation of apoptotic susceptibility ofHHUA cells by several cytokines and growth factorshas also been reported (50–52). To account for thesefacts, one can suggest the possibility that the GnRH-like substance secreted by Jurkat cells is GnRH itselfor a substance, probably one of cytokines, that acts likeGnRH in terms of cell proliferation. On the other hand,our results suggest that HHUA cells secrete anothersubstance that can inhibit the activity of GnRH. Atpresent, there is little information to help us predictwhat these substances are; thus, further investigationis necessary to identify them.

In this study, we also determined the dose-dependency of colony formation on Buserelin. So far, anumber of studies have demonstrated that GnRH in-creases [3H]thymidine incorporation in Jurkat cells(36), Fas ligand expression in reproductive tract tu-mors (22) and interleukin-2 receptor expression in lym-phocytes (34) in a dose-dependent manner in the rangeof 10210–1024 M. However, our results showed that the

Jurkat and CHO-K1 cells, respectively. The left-hand panels (A, C,and E) show the colony-forming efficiency of HHUA cells, and theright-hand panels (B, D, and F) show that of Jurkat cells. Theconditions of each experimental group are described in G. The num-ber of cells plated per well was 1, 2, 4, and 8 cells in the case of HHUAand CHO-K1 cells and 5, 10, 20, and 40 cells in the case of Jurkatcells. The regression equation and correlation coefficient (r) for eachexperimental group are as follows: (A) Œ: y 5 0.091x 1 0.019, r 50.9978, E: y 5 0.037x 1 0.031, r 5 0.9802, L: y 5 0.089x 1 0.037, r 50.9948, h: y 5 0.091x 1 0.025, r 5 0.9932; (B) Œ: y 5 0.027x 2 0.110,r 5 0.9925, E: y 5 0.068x 2 0.257, r 5 0.9992, L: y 5 0.027x 2 0.122,r 5 0.9935, h: y 5 0.027x 2 0.120, r 5 0.9917; (C) Œ: y 5 0.098x 20.059, r 5 0.9963, E: y 5 0.032x 1 0.007, r 5 0.9992, L: y 5 0.032x 10.017, r 5 0.9914, ■: y 5 0.035x 1 0.015, r 5 0.9988; (D) Œ: y 50.033x 2 0.132, r 5 0.9950, E: y 5 0.067x 2 0.212, r 5 0.9989, L: y 50.066x 2 0.212, r 5 0.9958, h: y 5 0.067x 2 0.238, r 5 0.9962; (E) Œ:y 5 0.109x 1 0.014, r 5 0.9990, E: y 5 0.040x 1 0.035, r 5 0.9961, L:y 5 0.100x 1 0.042, r 5 0.9959, h: y 5 0.038x 1 0.044, r 5 0.9927;(F) Œ: y 5 0.027x 2 0.110, r 5 0.9925, E: y 5 0.068x 2 0.257, r 50.9992, L: y 5 0.026x 2 0.108, r 5 0.9917, h: y 5 0.068x 2 0.282, r 50.9973.

FIG. 4. Effects of the conditioned media on colony-forming effi-ciency. The upper (A and B), middle (C and D), and lower panels(E and F) show the results of the conditioned media of HHUA,

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range of Buserelin concentration at which colony for-mation is affected in a dose-dependent manner is10215–10212 M. These results indicate that the mea-surement of colony-forming efficiency is a more sensi-tive method to detect the effect of GnRH on cell prolif-eration. Moreover, the effect of Buserelin on cellproliferation at such a low concentration, close to thatpresent in peripheral tissues, strongly suggests thatGnRH has novel physiological functions.

In conclusion, our results demonstrated that markeddose-dependent effects of Buserelin on cell prolifera-tion were detected by measuring colony formation ofcells and that the Buserelin activity on cell prolifera-tion is closely related to the autocrine and/or paracrineactivity of cells. In a number of studies, GnRH havebeen identified as a decisive modulator that reducesthe cellular proliferation of some tumors. However, ourresults strongly suggest the possibility that the effectof GnRH on cell proliferation may be markedly affectedby other substances which regulate cell proliferation.Concretely, following physiological and pathologicalsettings can be presumed. GnRH stimulates lymphoidcells to proliferate as an initiator, however, when cellsfully proliferate and cell density lead to a certain level,some autocrine and/or paracrine factors, which are se-

FIG. 5. The dose-dependency of the effect of Buserelin on colonyHHUA (A) and Jurkat cells (B) at 0.625 fg, 12.5 fg, 250 fg, 5 pg, 100well for HHUA cells and 20 cells per well for Jurkat cells. The columnumber when Buserelin was not added (control). Results are expres

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creted from cells themselves, act as a decisive regulatoron cell proliferation and conceal the effect of GnRH.While, GnRH also has a property to suppress somekinds of tumors growth, however, tumor cells secretsome substances that can inhibit the tumor-suppressing effect of GnRH and when tumors spread ina wide range and high density, this GnRH activitydisappears. Moreover, from the reports suggesting thesecretion of a GnRH-like substance from lymphoid cells(13, 36, 44) it is also supposed that GnRH itself is oneof autocrine and/or paracrine factors of lymphoid cellsand has a certain role in the immune system to resistsome tumors. Our findings in this study can be trans-lated as described above, however, further investiga-tion is necessary to demonstrate these suppositions.Thus, the colony-forming efficiency assay will be a veryimportant tool for elucidating the mechanism of theeffect of GnRH on cell proliferation.

ACKNOWLEDGMENTS

We are grateful to Dr. M. Matsuda, Dr. Y. Akazome, and Ms. Y.Fujii, Department of Biological Sciences, Graduate School of Science,University of Tokyo, for valuable advice regarding our study. Thiswork was supported by a grant-in-aid for scientific research from the

mation. The colony-forming activity of Buserelin were examined in, and 2 ng/ml final concentration. The cell densities were 8 cells per

each panel shows the ratio of colony-forming wells to the total wellas means 6 SE (n 5 4).

forpg

n insed

Ministry of Education, Science, Spots and Culture of Japan 16. Andreis, P. G., Neri, G., and Nussdorfer, G. G. (1997) Effect of

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(12640645 to M.K.P.).

REFERENCES

1. Clayton, R. N., Harwood, J. P., and Catt, K. J. (1979)Gonadotropin-releasing hormone analogue binds to luteal cellsand inhibits progesterone production. Nature 282, 90–92.

2. Knecht, M., Ranta, T., Feng, P, Shinohara, O., and Catt, K. J.(1985) Gonadotropin-releasing hormone as a modulator of ovar-ian function. J. Steroid. Biochem. 23, 771–778.

3. Billig, H., Furuta, I., and Hsueh, A. J. (1994) Gonadotropin-releasing hormone directly induces apoptotic cell death in the ratovary: Biochemical and in situ detection of deoxyribonucleic acidfragmentation in granulose cells. Endocrinology 134, 245–252.

4. Kogo, H., Kudo, A., Park, M. K., Mori, T., and Kawashima, S.(1995) In situ detection of gonadotropin-releasing hormone(GnRH) receptor mRNA expression in the rat ovarian follicles. J.Exp. Zool. 272, 62–68.

5. Kogo, H., Fujimoto, T., Park, M. K., and Mori, T. (1999)Gonadotropin-releasing hormone receptor mRNA expression inthe ovaries of neonatal and adults rats. Cells Tissues Organs164, 14–22.

6. Clayton, R. N., Katikineni, M., Chan, V., Dufau, M. L., and Catt,K. J. (1980) Direct inhibition of testicular function by gonado-tropin-releasing hormone: Mediation by specific gonadotropin-releasing hormone receptors in interstitial cells. Proc. Natl.Acad. Sci. USA 77, 4459–4463.

7. Bourne, G. A., Regiani, S., Payne, A. H., and Marshall, J. C.(1980) Testicular GnRH receptors—Characterization and local-ization on interstitial tissue. J. Clin. Endocrinol. Metab. 51,407–409.

8. Iwashita, M., Evans, M. I., and Catt, K. J. (1986) Characteriza-tion of a gonadotropin-releasing hormone receptor site in termplacenta and chorionic villi. J. Clin. Endocrinol. Metab. 62, 127–133.

9. Belisle, S., Guevin, J. F., Bellabarba, D., and Lehoux, J. G. (1984)Luteinizing hormone-releasing hormone binds to enriched hu-man placental membranes and stimulates in vitro the synthesisof bioactive human chorionic gonadotropin. J. Clin. Endocrinol.Metab. 59, 119–126.

10. Ataya, K. M., Sakr, W., Blacker, C. M., Mutchnick, M. G., andLatif, Z. A. (1989) Effect of GnRH agonisits on the thymus infemale rats. Acta Endocrinol. 121, 833–840.

11. Marchetti, B., Guarcello, V., Morale, M. C., Bartoloni, G.,Farinella, Z., Cordaro, S., and Scapagnini, U. (1989) Luteinizinghormone-releasing hormone-binding sites in the rat thymus:Characteristics and biological function. Endocrinology 125,1025–1036.

12. Marchetti, B., Guarcello, V., Morale, M. C., Bartoloni, G., Raiti,F., Palumbo, G., Jr., Farinella, Z., Cordaro, S., and Scapagnini,U. (1989) Luteinizing hormone-releasing hormone (LHRH) ago-nisit restoration of age-associated decline of thymus weight,thymic LHRH receptors, and thymocyte proliferative capacity.Endocrinology 125, 1037–1045.

13. Maier, C. C., Marchetti, B., LeBoeuf, R. D., and Blalock, J. E.(1992) Tymocytes express a mRNA that is identical to hypotha-lamic luteinizing hormone-releasing hormone mRNA. Cell. Mol.Neurobiol. 12, 447–454.

14. Rosa-e-Silva, A. A., Lamano-Carvalho, T. L., and Queiroz, A. N.(1991) Effect of LH-RH analog on cat testis and adrenal cortexfunction. Braz. J. Med. Biol. Res. 24, 1107–1111.

15. Chieffi, G., Pierantoni, R., and Fasano, S. (1991) Immunoreac-tive GnRH in hypothalamic and extrahypothalamic areas. Int.Rev. Cytol. 127, 1–55.

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luteinizing hormone-releasing hormone on rat adrenocorticalcells. J. Steroid. Biochem. Mol. Biol. 63, 17–19.

17. Redding, T. W., and Schally, A. V. (1983) Inhibition of mammarytumor growth in rats and mice by administration of agonisticand antagonistic analogs of luteinizing hormone-releasing hor-mone. Proc. Natl. Acad. Sci. USA 80, 1459–1462.

18. Miller, W. R., Scott, W. N., Morris, R., Fraser, H. M., and Sharpe,R. M. (1985) Growth of human breast cancer cells inhibited by aluteinizing hormone-releasing hormone agonist. Nature 313,231–233.

19. Eidne, K. A., Flangan, C. A., Harris, N. S., and Miller, R. P.(1987) Gonadotropin-releasing hormone (GnRH)-binding sites inhuman breast cancer cell lines and inhibitory effects of GnRHantagonists. J. Clin. Endocrinol. Metab. 64, 425–432.

20. Sharoni, Y., Bosin, E. Miinster, A., Levy, J., and Schally, A. V.(1989) Inhibition of growth of human mammary tumor cells bypotent antagonists of luteinizing hormone-releasing hormone.Proc. Natl. Acad. Sci. USA 86, 1648–1651.

21. Emons, G., Ortmann, O., Becker, M., Irmer, G., Springer, B.,Laun, R., Holzel, F., Shulz, K. D., and Schally, A. V. (1993) Highaffinity binding and direct antiproliferative effects of LHRHanalogues in human ovarian cancer cell lines. Cancer Res. 53,5439–5446.

22. Imai, A., Takagi, A., Horibe, S., Takagi, H., and Tamaya, T.(1998) Evidence for tight coupling of gonadotropin-releasing hor-mone receptor to stimulated Fas ligand expression in reproduc-tive tract tumors: Possible mechanism for hormonal control ofapoptotic cell death. J. Clin. Endocrinol. Metab. 83, 427–431.

23. Kleinman, D., Douvdevani, A., Schally, A. V., Levy, J., andSharoni, Y. (1994) Direct growth inhibition of human endome-trial cancer cells by the gonadotropin-releasing hormone antag-onist SB-75: Role of apoptosis. Am. J. Obstet. Gynecol. 170,96–102.

24. Redding, T. W., Schally, A. V., Tice, T. R., and Meyers, W. E.(1984) Long-acting delivery systems for peptides: Inhibition ofrat prostate tumors by controlled release of [D-Trp-6]luteinizinghormone-releasing hormone from injectable microcapsules. Proc.Natl. Acad. Sci. USA 81, 5845–5848.

25. Fekete, M., Redding, T. W., Comaru-Shally, A. M., Pontes, J. E.,Connelly, R. W., Srkalovic, G., and Schally, A. V. (1989) Recep-tors for luteinizing hormone-releasing hormone, somatostatin,prolactin, and epidermal growth factor in rat and human pros-tate cancers and in benign prostate hyperplasia. Prostate 14,191–208.

26. Dondi, D., Moretti, R. M., Marelli, M. M., Pratesi, G., Pollizi, D.,Milani, M., Motta, M., and Limonta, P. (1998) Growth-inhibitoryeffects of luteinizing hormone-releasing hormone (LHRH) ago-nisits on xenografts of the DU 145 human androgen-independentprostate cancer cell line in nude mice. Int. J. Cancer 76, 506–511.

27. Gonzales-Barcena, D., Ibarra-Olmons, M. A., Garcia-Carrasco,F., Gutierrez-Samperio, C., Comaru-Shally, A. M., and Shally,A. V. (1989) Influence of D-Trp6-LH-RH on the survival time inpatients with advanced pancreatic cancer. Biomed. Pharmaco-ther. 43, 313–317.

28. Szende, B., Zalatnai, A., and Schally, A. V. (1989) Programmedcell death (apoptosis) in pancreatic cancers of hamsters aftertreatment with analogs of both luteinizing hormone-releasinghormone and somatostatin. Proc. Natl. Acad. Sci. USA 86, 1643–1647.

29. Szende, B., Srkalovic, G., Schally, A. V., Lapis, K., and Groot, K.(1990) Inhibitory effects of analogs of luteinizing hormone-releasing hormone and somatostatin on pancreatic cancers inhamsters. Events that accompany tumor regression. Camcer 65,2279–2290.

30. Radulovic, S., Comaru-Schally, A. M., Milovanovic, S., and 42. Aksel, S. (1979) Luteinizing hormone-releasing hormone and the

Vol. 289, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Schally, A. V. (1993) Somatostatin analogue RC-160 and LH-RHantagonist SB-75 inhibit growth of MIA PaCa-2 human pancre-atic cancer xenografts in nude mice. Pancreas 8, 88–97.

31. Pati, D., and Habibi, H. R. (1995) Inhibition of human hepato-caricinoma cell proliferation by mammalian and fish gonado-tropin-releasing hormones. Endocrinology 136, 75–84.

32. Marchetti, B., Guarcello, V., Morale M. C., Bartoloni, G.,Palumbo, G., Raiti, F., Cutuli, N., Farinella, Z., and Scapagnini,U. (1990) A physiological role for the neuropeptide luteinizinghormone-releasing hormone (LHRH) during the maturation ofthymus gland function. Int. J. Neurosci. 51, 287–289.

33. Blacker, C. M., Ataya, K. M., Savoy-Moore, R. T., Subramanian,M. G., Mutchnick, M. G., and Dunbar, J. C. (1991) Thegonadotropin-releasing hormone agonist leuprolide affects thethymus and other non-reproductive systems of female rats. Acta.Endocrinol. 125, 581–589.

34. Batticane, N., Morale, M. C., Gallo, F., Farinella, Z., and Mar-chetti, B. (1991) Luteinizing hormone-releasing hormone signal-ing at the lymphocyte involves stimulation of Interleukin-2 re-ceptor expression. Endocrinology 129, 277–286.

35. Rao, L. V., Cleveland, R. P., Kimmel, R. J., and Ataya, K. M.(1994) Effects of GnRH antagonist on lymphocyte subpopula-tions in primary and secondary lymphoid tissues of female mice.Am. J. Reprod. Immunol. 32, 238–247.

36. Azad, N., Lapaglia, N., Kirsteins, L., Uddin, S., Steiner, J.,Williams, D. W., Lawrence, A. M., and Emanuele, N. V. (1997)Jurkat cell proliferative activity is increased by luteinizinghormone-releasing hormone. J. Endocrinol. 153, 241–249.

37. Waldmann, H., Lefkovits, I., and Quintans, J. (1975) Limitingdilution analysis of helper T-cell function. Immunology 28,1135–1148.

38. Papadopoulos, V., Dharmarajan, A. M., Li, H. Culty, M., Lemay,M., and Sridaran, R. (1999) Mitochondrial peripheral-type Ben-zodiazepine receptor expression. Correlation with gonadotropin-releasing hormone (GnRH) agonist-induced apoptosis in the cor-pus luteum. Biochem. Pharmacol. 58, 1389–1393.

39. Mizutani, T., Sugihara, A., Nakamuro, K., and Terada, N. (1998)Suppression of cell proliferation and induction of apoptosis inuterine leiomyoma by gonadotropin-releasing hormone agonist(leuprolide acetate). J. Clin. Endocrinol. Metab. 83, 1253–1255.

40. Zhao, S., Saito, H., Wang, X., Saito, T., Kaneko, T., and Hiroi, M.(2000) Efffects of gonadotropin-releasing hormone agonist on theincidence of apoptosis in porcine and human granulosa cells.Gynecol. Obstet. Invest. 49, 52–56.

41. Sarkar, D. K., Chiappa, S. A., Fink, G., and Sherwood, N. M.(1976) Gonadotropin-releasing hormone surge in pro-oestrousrats. Nature 264, 461–463.

1187

human menstrual cycle. Am. J. Obstet. Gynecol. 135, 96–101.43. Sarda, A. K., Barnes, M. A., and Nair, R. M. (1981) Inter-

relationship between changing patterns of LHRH and gonado-tropins in the menstrual cycle. Clin. Endocrinol. (Oxford) 15,265–273.

44. Chen, H. F., Jeung, E. B., Stephenson, M., and Leung P. C.(1999) Human peripheral blood mononuclear cells expressgonadotropin-releasing hormone (GnRH), GnRH receptor, andinterleukin-2 receptor g-chain messenger ribonucleic acids thatare regulated by GnRH in vitro. J. Clin. Endocrinol. Metab. 84,743–750.

45. Schally, A. V. (1999) Luteinizing hormone-releasing hormoneanalogs: Their impact on the control of tumorigenesis. Peptides20, 1247–1262.

46. Morale, M. C., Batticane, N., Bartoloni, G., Guarcello, V.,Farinella, Z., Galasso, M. G., and Marchetti, B. (1991) Blockadeof central and peripheral luteinizing hormone-releasing hor-mone (LHRH) receptors in neonatal rats with a potent LHRH-antagonist inhibits the morphofunctional development of thethymus and maturation of the cell-mediated and humoral im-mune responses. Endocrinology 128, 1073–1085.

47. Standaert, F. E., Chew, B. P., De Avila, D., and Reeves, J. J.(1992) Presence of luteinizing hormone-releasing hormone bind-ing sites in cultured porcine lymphocytes. Biol. Reprod. 46, 997–1000.

48. Reichert, T. E., Nagashima, S., Kashii, Y., Stanson, J., Gao, G.,Dou, Q. P., and Whiteside, T. L. (2000) Interleukin-2 expressionin human carcinoma cell lines and its role in cell cycle progres-sion. Oncogene 19, 514–525.

49. Wang, L., Wu, Y., and Zhang, Y. (1996) In vivo antitumor effectsof polyethylene glycol-modified recombinant human interleu-kin-2 on mouse uterine cervical carcinoma. Zhonghua. Zhong.Liu. Za. Zhi. 18, 253–255.

50. Tanaka, T., Umesaki, N., Mizuno, K., Chang, L., Miyama, M.,Ohtaki, S., and Ogita, S. (1998) Enhancement of apoptotic sus-ceptibility in human endometrial epithelial cell line HHUA bytransforming growth factor-beta 1. Horm. Metab. Res. 30, 61–65.

51. Tanaka, T., Mizuno, K., Miyama, M., Chang, L., Chen, H.,Ohtaki, S., Umesaki, N., and Ogita, S. (1999) Enhanced Fas/CD95-mediated apoptosis by epidermal growth factor in humanendometrial epithelial cells. Eur. J. Obstet. Gynecol. Reprod.Biol. 86, 189–194.

52. Tanaka, T., and Umesaki, N. (2000) Cytokine regulation of ap-optotic susceptibility in a human endometrial epithelial cell line.J. Reprod. Immunol. 47, 105–119.