Antiproliferative Signaling of Luteinizing Hormone- Releasing ...

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 6 Printed in U.S.A.

Antiproliferative Signaling of Luteinizing HormoneReleasing Hormone in Human Endometrial and Ovarian Cancer Cells through G Protein ␣I-Mediated Activation of Phosphotyrosine Phosphatase* ¨ NDKER, PETER VO ¨ LKER, CARSTEN GRU

AND

¨ NTER EMONS GU

Department of Gynecology and Obstetrics, Georg August University, D-37070 Gottingen, Germany ABSTRACT The signaling pathway through which LHRH acts in endometrial and ovarian cancers is distinct from that in the anterior pituitary. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors, resulting in down-regulation of expression of c-fos and proliferation. Only limited data are available on the crosstalk between LHRH receptor signaling and inhibition of mitogenic signal transduction. The present experiments were performed to analyze in endometrial and ovarian cancer cells: 1) whether mutations or splice variants of the LHRH receptor are responsible for differences in LHRH signaling, 2) the coupling of G protein subtypes to LHRH receptor, 3) the phosphotyrosine phosphatase (PTP) activation counteracting growth factor receptor tyrosine kinase activity. For these studies, the well characterized human Ishikawa and Hec-1A endometrial cancer cell lines and human EFO-21 and EFO-27 ovarian cancer cell lines were used, which express LHRH and its receptor. 1) Sequencing of the complementary DNA of the LHRH receptor from position 31 to position 1204, covering the complete coding region (position 56 to position 1042) showed that there are neither mutations nor splice variants of the LHRH receptor transcript in Ishikawa and Hec-1A endometrial cancer cells or in EFO-21 and EFO-27 ovarian cancer cells. 2) All analyzed cell lines except for the ovarian cancer cell line EFO-27 expressed both G proteins, ␣i and ␣q, as shown by RTPCR and Western blotting. In the EFO-27 cell line only G protein ␣i, not G protein ␣q, expression was found. Cross-linking experiments using disuccinimidyl suberate revealed that in the cell lines express-

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HE HYPOTHALAMIC decapeptide LHRH plays a key role in the control of mammalian reproduction (1–3 ). The neurohormone specifically binds to high affinity receptors in pituitary gonadotrophs that are coupled to the pertussis (PTX)-insensitive G protein ␣q/11 and to the phospholipase C signaling pathway (4 – 6 ). In addition to this well documented classic hypophysiotropic actions, LHRH is present in the brain and a variety of peripheral organs, both normal and tumoral, where it might act in an autocrine/ paracrine fashion (7–16). Expression of LHRH and its receptor has been detected in most endometrial and ovarian cancer cell lines and in over 80% of biopsy specimens of these Received November 27, 2000. Address all correspondence and requests for reprints to: Prof. Dr. Gu¨nter Emons, Department of Gynecology and Obstetrics, Robert Koch Street 40, D-37075 Gottingen, Germany. E-mail: [email protected]. * This work was supported by the Bundesministerium fu¨r Bildung und Forschung (Berlin, Germany) and Asta Medica (Frankfurt, Germany).

ing G protein ␣i and G protein ␣q, both G proteins coupled to the LHRH receptor. Inhibition of epidermal growth factor (EGF)-induced c-fos expression by LHRH, however, was mediated through pertussis toxin (PTX)-sensitive G protein ␣i. Moreover, LHRH substantially antagonized the PTX-catalyzed ADP-ribosylation of G protein ␣i. 3) Using a phosphotyrosine phosphatase assay based on molybdate-malachite green, treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and quiescent Ishikawa and Hec-1A endometrial cancer cells with 100 nM of the LHRH agonist triptorelin resulted in a 4-fold increase in PTP activity (P ⬍ 0.001). This effect was completely blocked by simultaneous treatment with PTX, supporting the concept of mediation through G protein ␣i. As shown by quantitative Western blotting, EGF-induced tyrosine autophosphorylation of EGF receptors was reduced 45– 63% after LHRH (100 nM) treatment (P ⬍ 0.001). This effect was completely blocked using the PTP inhibitor vanadate (P ⬍ 0.001). These results demonstrate that mutations or splice variants of the LHRH receptor in human endometrial and ovarian cancer cells are not responsible for the different signal transduction compared with that in pituitary gonadotrophs. We provide evidence that the tumor LHRH receptor couples to multiple G proteins, but the antiproliferative signal transduction is mediated through the PTX-sensitive G protein ␣i. The tumor LHRH receptor activates a PTP counteracting EGF-induced tyrosine autophosphorylation of EGF receptor, resulting in down-regulation of mitogenic signal transduction and cell proliferation. (Endocrinology 142: 2369 –2380, 2001)

cancers (11). The proliferation of the cancer cell lines that express LHRH receptors, was inhibited by both agonistic and antagonistic analogs of LHRH, indicating that the dichotomy of LHRH agonists and antagonists does not exist in tumor cells (11). These antiproliferative effects were evident at nanomolar concentrations of the LHRH analogs, suggesting that they are mediated through the LHRH receptors in the tumor cells (11). The classical LHRH receptor signal transduction mechanisms known to operate in the pituitary (6, 17) are not involved in the mediation of antiproliferative effects of LHRH analogs in cancer cells (18). LHRH analogs instead interfere with the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity, resulting in a down-regulation of growth factor-induced c-fos expression (18, 19). The reasons for LHRH inducing this specific signaling in cancer cells, however, are still obscure. The present experiments were performed to clarify the

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LHRH SIGNALING IN OVARIAN AND ENDOMETRIAL CANCER

reasons for the differences in LHRH signal transduction in endometrial and ovarian cancer cells compared with that acting in pituitary gonadotrophs. Both endometrial and ovarian epithelial cancers develop from the Mu¨llerian epithelium and have several features in common, in particular regarding their LHRH system (11). As this system has been well characterized in these cancers (11) we choose them as a model for our present experiments. Experimentally induced mutations of the LHRH receptor have altered LHRH binding, G protein-receptor interaction, or proper membrane incorporation (20 –25). Some normal and neoplastic human tissues were found to express differential splice variants of the LHRH receptor gene in a tissuedependent manner (26). Therefore, it seemed reasonable to check whether mutations or splice variants of LHRH receptors expressed in human cancers are responsible for the signaling mechanisms different from that acting in pituitary gonadotrophs. If this was not the case, it might be possible that a different coupling to G proteins is responsible for the distinct LHRH signal transduction in endometrial and ovarian cancer cells. To check whether a different cellular equipment of G proteins exists in the tumor cells, we analyzed which subtypes of G proteins are expressed in ovarian and endometrial cancer cells. To show directly which subtype of G proteins couples to LHRH receptor in the tumor cells, we performed cross-linking experiments using disuccinimidyl suberate (DSS). To confirm the hypothesis that LHRH analogs act through G protein ␣i, we investigated whether LHRH affects PTX-induced ADP-ribosylation of the G protein ␣i. In addition, we analyzed whether PTX inhibits LHRH-induced down-regulation of epidermal growth factor (EGF)-induced c-fos expression. It has been speculated that LHRH activates a phosphotyrosine phosphatase (PTP) and thus antagonizes growth factor-induced tyrosine phosphorylation (11). The antiproliferative effects of LHRH analogs might be directly mediated through inhibition of growth factor signaling on its first step, the autophosphorylation of tyrosine residues of growth factor receptors. Some indirect evidence that LHRH activates a PTP was found by Imai et al. (27), showing that LHRH reduces the net tyrosine phosphorylation of membrane proteins. Direct proof of the G protein ␣i-mediated activation of a PTP and the reduction of the EGF receptor tyrosine phosphorylation by LHRH has not been provided to date. To assess whether LHRH activates a PTP that counteracts EGF receptor tyrosine kinase activity in endometrial and ovarian cancer cells, we analyzed whether LHRH activates a PTP and reduces EGF receptor phosphorylation. In addition, we analyzed whether activation of PTP is mediated through the PTX-sensitive G protein ␣i. Materials and Methods Cell lines and culture conditions The human endometrial cancer cell lines used were derived from an endometrial adenocarcinoma (Ishikawa) (28) or a moderately differentiated papillary adenocarcinoma (Hec-1A) (29). The human ovarian cancer cell lines used were derived from a poorly differentiated serous adenocarcinoma (EFO-21) (30) or a mucinous papillary adenocarcinoma of intermediate differentiation (EFO-27) (30). The cells were cultured as described in detail previously (31).

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LHRH analogs The LHRH agonist [d-Trp6]LHRH (triptorelin) was provided by Ferring Pharmaceuticals Ltd. Arzneimittel (Kiel, Germany).

Isolation of RNA and complementary DNA (cDNA) synthesis Total RNA was prepared from cells grown in monolayer using the RNeasy protocol (QIAGEN, Hilden, Germany). The concentration of RNA in each sample was determined by photospectroscopy. First strand cDNA was generated by RT of 4 ␮g total RNA using p(deoxythymidine)15 primers (Roche Molecular Biochemicals, Mannheim, Germany) with Moloney murine leukemia virus reverse transcriptase according to the instructions of the suppliers (Life Technologies, Inc., Karlsruhe, Germany). After determining the concentrations of the cDNAs, the samples were used for PCR analysis. The integrity of the samples was tested by RT-PCR of the housekeeping gene GAPDH (forward primer, 5⬘-CAT CAC CAT CTT CCA GGA GCG AGA-3⬘; backward primer, 5⬘-GTC TTC TGG GTG GCA GTG ATG G-3⬘).

RT-PCR of G proteins The cDNAs (2 ng) were amplified in a 50-␮l reaction volume containing 10 mm Tris-HCl (pH 8.3), 50 mm potassium chloride, 1.5 mm magnesium chloride, 200 ␮m of each of the deoxy-NTPs, 1 ␮m of the appropriate primers (G protein ␣i: forward primer, 5⬘-CAG TCC ATC ATT GCA ATC ATA AGA-3⬘; backward primer, 5⬘-CTC AGC CAG AAC AAG GTC ATA ATC-3⬘; G protein ␣q: forward primer, 5⬘-ATG ACT TGG ACC GTG TAG CCG ACC-3⬘; backward primer, 5⬘-CCA TGC GGT TCT CAT TGT CTG ACT-3⬘), and 1.25 U Taq polymerase (Roche Molecular Biochemicals) in a Perkin-Elmer Corp. DNA thermal cycler 2400 (Weiterstadt, Germany). Twenty-five cycles of amplification were carried out: denaturation at 94 C for 30 sec, annealing at 55 C (G protein ␣i) or 60 C (G protein ␣q) for 30 sec, followed by extension at 72 C for 60 sec. The PCR product amplified with the G protein ␣i primers has a total length of 474 bp. The PCR product amplified with the G protein ␣q primers has a total length of 260 bp. The respective DNA products were run on 1.5% agarose gels, and bands were visualized by ethidium bromide staining on an UV transilluminator.

Sequence analysis of LHRH receptor messenger RNA (mRNA) For LHRH receptor sequencing and splice variant analysis, the following oligonucleotide primers (Fig. 1) were designed according the sequence found by Kakar et al. (32): LHRH-R-1: forward primer, 5⬘-GCT TGA AGC TCT GTC CTG GG-3⬘; backward primer, 5⬘-CAG GCT GAT CAC CAC CAT CAT-3⬘ (positions 31– 466); LHRH-R-2: forward primer, 5⬘-AGT CCA ATG GTA TGC TGG AGA-3⬘ (positions 367–777); backward primer, 5⬘-ACC CGT GTC AGG GTG AAG AT-3⬘; and LHRH-R-3: forward primer, TCA TGC TGA TCT GCA ATG CAA-3⬘; backward primer, AAT TGA GGC TCT GAA GAC TGA GT-3⬘ (positions 732-1204). The PCR runs were carried out under the same conditions as those described above: denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, followed by extension at 72 C for 60 sec. The PCR product amplified with the LHRH-R-1 primers (positions 31– 466) has a total length of 436 bp, the PCR product amplified with the LHRH-R-2 primers (positions 367–777) has a total length of 411 bp, and the PCR product amplified with the LHRH-R-3 primers (positions 732-1204) has a total length of 473 bp. Direct fluorescence sequence analysis of LHRH receptor was performed on PCR products using oligonucleotide primers designed to yield approximately 450 nucleotides between primers and approximately 100 nucleotides of overlap. The PCR products were purified from low melting point agarose by phenol extraction and precipitation in ethanol. Sequences were determined using the dideoxynucleotide chain terminating system from Perkin-Elmer Corp. Reactions used the ABI PRISM Dye Terminator cycle sequencing ready reaction kits (PerkinElmer Corp.). Sequence reactions were fractionated on an ABI PRISM 310 DNA sequencer equipped with a 43-cm microcapillary (PerkinElmer Corp.). All sequences were determined from both directions, and sequence data were compiled manually.


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FIG. 1. Oligonucleotide primers designed for LHRH receptor sequence analysis according to the sequence found by Kakar et al. (32). LHRH-R-1 (A): forward primer, 5⬘-GCT TGA AGC TCT GTC CTG GG-3⬘; backward primer, 5⬘-CAG GCT GAT CAC CAC CAT CAT-3⬘ (positions 31– 466); LHRH-R-2 (B): forward primer, 5⬘-AGT CCA ATG GTA TGC TGG AGA-3⬘ (positions 367–777); backward primer, 5⬘-ACC CGT GTC AGG GTG AAG AT-3⬘; LHRH-R-3 (C): forward primer, TCA TGC TGA TCT GCA ATG CAA-3⬘; backward primer, AAT TGA GGC TCT GAA GAC TGA GT-3⬘ (positions 732-1204).

Plasma membrane isolation Cells were collected by centrifugation at 200 ⫻ g and washed twice with PBS/BSA. After counting aliquots, cells were suspended and homogenized using an all-glass Potter homogenizer (Braun, Melsungen, Germany) in 10 mmol/liter Tris-HCl buffer, pH 7.6, containing 2 g BSA/liter, 2 g NaN3/ liter, and 1 mmol/liter dithiothreitol (DTT; Merck & Co., Darmstadt, Germany). After removing nuclei and debris by centrifugation at 200 ⫻ g, plasma membranes were collected at 70,000 ⫻ g. Aliquots of the membrane preparations, equivalent to 300,000 – 400,000 cells, were resuspended in lysis buffer (1 mmol/liter EGTA, 1 mmol/liter DTT, and 10 mmol/liter Tris-HCl, pH 7.4).

Western blotting of G proteins Cell membranes were electrophoresed on SDS-PAGE (7.5%) under reducing conditions and transferred to nitrocellulose. The nitrocellulose membranes were blocked in 3% BSA (Sigma, St. Louis, MO) in TBST [10 mm Tris (pH 8), 500 mm NaCl, and 0.1% Tween 20] for 2 h, incubated with polyclonal rabbit antihuman G protein ␣i or G protein ␣q antibodies (a gift from Dr. Hinsch, Giessen, Germany) in a 1:1,000 dilution in 1% BSA in TBST for 1 h, and then, after washings, incubated with horseradish peroxidase-conjugated antirabbit IgG in an 1:10,000 dilution in 1% BSA in TBST (Amersham Pharmacia Biotech, Aylesbury, UK) for 1 h. After washings, specifically bound antibody was detected using the enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech).

ADP-ribosylation ADP-ribosylation was carried out as described previously (21). Briefly, isolated plasma membranes (0.5 mg/ml) were incubated with PTX (2 ␮g/ml) in 20 mmol/liter Tris-HCl, pH 7.5, containing 1 mmol/ liter ATP, 1 mmol/liter EDTA, 1 mmol/liter DTT, 10 mmol/liter thymidine, 10 ␮mol/liter [32P]NAD (5 ⫻ 106 cpm/nmol), and the ligand tested in a final volume of 200 ␮l. After incubation for 30 min at 37 C, the reaction was quenched by adding 1 ml ice-cold 20 mmol/liter TrisHCl, pH 7.5, containing 1 mmol/liter EDTA. Membranes were pelleted by centrifugation and washed twice in the same buffer. Membrane proteins were solubilized in Laemmli’s SDS sample buffer and resolved by 15% PAGE as described above (Western blotting). ADP-ribosylated proteins were detected by autoradiography.

Cross-linking analysis After washing in PBS containing 0.1% BSA (three times, 10 min each time), the cells were placed in PBS containing 0.75 mm disuccinimidyl suberate (DSS), diluted from a 25-mm stock in dimethylsulfoxide. Crosslinking was allowed to proceed at 25 C for 15 min and was terminated by placing the cells in 10 mm Tris-HCl, pH 7.4, containing 1 mm EDTA and 0.15 m NaCl for 30 min at 4 C. The cells were then lysed as described

above. Protein separation was performed by SDS-PAGE in 7.5% gels. The mol wt standard was obtained from Pharmacia Biotech (Freiburg, Germany). After electrophoresis, the proteins were transferred to nitrocellulose. The nitrocellulose membranes were blocked in 3% bovine serum as described above and then, after washings, were incubated with a monoclonal antibody raised against the human pituitary LHRH receptor (provided by Dr. A. A. Karande, Bangalore, India) in a 1:500 dilution in 1% BSA in TBST for 1 h or with polyclonal rabbit antihuman G protein ␣i or G protein ␣q antibodies in a 1:1,000 dilution in 1% BSA in TBST for 1 h, and then, after washings, incubated with horseradish peroxidase-conjugated antimouse IgG (LHRH receptor) or antirabbit IgG (G proteins) in a 1:10,000 dilution in 1% BSA in TBST (Amersham Pharmacia Biotech) for 1 h. After washings, specifically bound antibody was detected using the ECL kit (Amersham Pharmacia Biotech).

Quantification c-fos mRNA expression Semiquantitative RT-PCR of c-fos was carried out as described in detail previously (19). Briefly, a 161-bp internal standard was generated by PCR containing synthetic DNA and c-fos-specific primer sites. The PCR product amplified with the c-fos primers (forward primer, 5⬘-GAG ATT GCC AAC CTG CTG AA-3⬘; backward primer, 5⬘-AGA CGA AGG AAG ACG TGT AA-3⬘) has a total length of 483 bp. For determining the optimal concentration of internal standard used in semiquantitative PCR, internal standard and target cDNA were added to the PCR tubes in inverse serial dilutions. PCR products were separated by gel electrophoresis in 1.5% agarose. PCR reactions yielding standard and target signals of identical intensity were used for PCR analysis for determination of c-fos expression levels. The respective DNA products were run on 1.5% agarose gels, and bands were visualized by ethidium bromide staining on an UV transilluminator. The bands were quantified using the Kodak 1D image system (Kodak, New Haven, CT) in comparison with basal c-fos expression levels.

PTP assay The cells were plated at a density of 106 cells in 100-mm dishes and grown under standard conditions. After 2 days, the cells (⬃90% confluence) were incubated in serum-free medium for 24 h before treatment with or without 100 nm of the LHRH agonist triptorelin with or without PTX for 15 min. The stimulation reactions were stopped by washing with ice-cold PBS. After washing the cells were detached immediately with 1 ml of a solution containing 0.5 g trypsin (Biochrom, Berlin, Germany) and 5 mmol EDTA in 1 liter PBS/BSA, counted, and lysed in 50 mm ␤-glycerophosphate (pH 7.3), 2 mm EDTA, 1 mm EGTA, 5 mm ␤mercaptoethanol, 1% Triton X-100, 1.0 mm benzamidine, 0.1 mm phenylmethylsulfonylfluoride, 20 ␮g/ml leupeptin, 1 ␮m pepstatin A, and 1 ␮g/ml aprotinin. Endogenous phosphate was removed using Sephadex G-25 spin columns (Promega Corp., Madison, WI). Tyrosine phos-

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phatase activity was measured using the malachite green detection assay (Promega Corp.), initially described by Harder et al. (33). The synthetic PTP-specific phosphopeptide used in this assay was END(pY)INASL (34). The assay was performed with 5–15 ␮g protein, initiated by the addition of enzyme sample, and incubated at 23 C for 15 min. Reaction was stopped by adding molybdate-malachite green. Data resulting from this assay are expressed as a percentage of the untreated controls (⫽100%).

Quantification of phosphorylated EGF receptor The cells were plated at a density of 106 cells in 100-mm dishes and grown under standard conditions. After 2 days, culture media were changed to FCS-free and phenol red-free medium for 72 h. The quiescent cells were incubated with 100 nm bovine EGF (Sigma) for 30 min with or without 10 ␮m of the LHRH agonist triptorelin in the absence or presence of 100 ␮m sodium vanadate (Sigma). After incubation the cells were detached immediately with 1 ml of a solution containing 0.5 g trypsin (Biochrom) and 5 mmol EDTA in 1 liter PBS/BSA and counted. Plasma membranes were isolated as described above and than lysed using a buffer containing 9.5 m urea, 2.0% Nonidet P-40, and 5.0% ␤-mercaptoethanol. The cell lysates were electrophoresed on SDS-PAGE (7.5%) under reducing conditions and transferred to nitrocellulose. The nitrocellulose membranes were blocked in 3% BSA (Sigma) in TBST [10 mm Tris (pH 8), 500 mm NaCl, and 0.1% Tween 20] for 2 h, incubated with polyclonal rabbit antihuman phosphotyrosine (Promega Corp., Mannheim, Germany) in a 1:100 dilution in 1% BSA in TBST for 1 h, and then, after washings, incubated with horseradish peroxidase-conjugated antirabbit IgG in a 1:1500 dilution in 1% BSA in TBST (Amersham Pharmacia Biotech) for 1 h. After washings, specifically bound antibody was detected using the ECL kit (Amersham Pharmacia Biotech). The bands were analyzed using the Kodak 1D image system.

Statistical analysis All experiments were reproduced three times in different passages of the respective cell lines. Data were tested for significant differences using the Mann-Whitney U test. The data from the phosphatase experiments were tested for significant differences by one-way ANOVA, followed by Student-Newman-Keuls test for comparison of individual groups, after a Bartlett test had shown that variances were homogenous.

FIG. 2. Nucleotide sequence of human LHRH receptor mRNA in EFO-21 and EFO-27 ovarian cancer cells and in Hec-1A and Ishikawa endometrial cancer cells. The coding sequence is shown in bold letters.

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Results Sequence analysis and splice variants of LHRH receptor

To analyze whether LHRH receptor mutations or LHRH receptor splice variants are responsible for the LHRH signal transduction different from that found in the pituitary, we checked ovarian cancer cell lines EFO-21 and EFO-27 and endometrial cancer cell lines Ishikawa and Hec-1A for the expression of mutated or alternatively spliced LHRH receptors. Using RT-PCR we could not detect any sign of alternatively spliced LHRH receptors in any of the analyzed cell lines. Figure 1 shows the oligonucleotide primers pairs used for LHRH receptor sequence analysis and the respective PCR products. The primers were designed according to the sequence found by Kakar et al. (32), taking care that the cDNA fragments overlapped. Figure 2 shows the nucleotide sequence of the human LHRH receptor mRNA in EFO-21 and EFO-27 ovarian cancer cells and in Hec-1A and Ishikawa endometrial cancer cells. Sequencing of the cDNA of the LHRH receptor from position 31 to position 1204, covering the complete coding region (positions 56 –1042) showed no point mutations, deletions, or insertions in any of the analyzed cell lines (Figs. 1 and 2). Expression of G proteins

To check whether the ovarian cancer cell lines EFO-21 and EFO-27 and the endometrial cancer cell lines Ishikawa and Hec-1A express G protein ␣i, G protein ␣q, or both, we assessed the presence of G protein ␣i and G protein ␣q mRNA (Fig. 3) and immunoreactivity (Fig. 4). Both G proteins were expressed in the ovarian cancer cell line EFO-21 and the endometrial cancer cell lines Ishikawa and Hec-1A, but not


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FIG. 3. Expression of G protein ␣i and ␣q mRNA in EFO-21 and EFO-27 ovarian cancer cells and in Hec-1A and Ishikawa endometrial cancer cells by RTPCR using specific primers for human G protein ␣i (A) and human G protein ␣q (B), respectively. The cell lines EFO-21, Hec-1A, and Ishikawa were shown to express G proteins ␣i and ␣q. In the EFO-27 cell line only G protein ␣i, not G protein ␣q, was expressed.

FIG. 4. Immunoblot of G protein ␣i and ␣q proteins in EFO-21 and EFO-27 ovarian cancer cells using specific antibodies for human G protein ␣i (left) and human G protein ␣q (right), respectively. In the cell line EFO-21, both G proteins ␣i and ␣q could be detected. In the EFO-27 cell line, only G protein ␣i, not G protein ␣q, was detectable. Experiments using endometrial cancer cell lines Hec-1A and Ishikawa gave identical results as those using ovarian cancer cell line EFO-21.

in the ovarian cancer cell line EFO-27. In this cell line only the G protein ␣i was detectable. Effects of LHRH on PTX-catalyzed ADP-ribosylation

Our previous observations that PTX significantly inhibits LHRH-induced reduction of cancer cell proliferation (unpublished results) suggest that LHRH analogs might act through G protein ␣i. To further confirm this hypothesis, we investigated whether LHRH agonists might affect PTXinduced ADP-ribosylation of the G protein ␣i in the ovarian cancer cell lines EFO-21 and EFO-27 and the endometrial cancer cell lines Ishikawa and Hec-1A (Fig. 5). G Proteins in the cell membranes, which are isolated in the absence of GTP, GTP analogs, or other activators, are in their inactive trimeric form and are good substrates for PTX-catalyzed ADP-ribosylation. Incubation of membranes of the above-mentioned cell lines with [32P]NAD and PTX in the absence of GTP, GTP analogs, or other activators resulted in a marked PTX-catalyzed ADP-ribosylation in the 41-kDa protein (Fig. 5, lane 1). G protein ␣i becomes a poor substrate for PTX-catalyzed ADP-ribosylation when the trimeric G protein has become dissociated upon LHRH receptor activation. Incubation with the LHRH analog triptorelin in the presence of GTP remarkably reduced ADP-ribosylation in the 41-kDa protein (Fig. 5, lane 4). Incubation with triptorelin and a nonhydrolysable GTP analog, GTP-␥-S, produced a further reduction of ribo-

sylation of this protein, bringing about a loss of greater than 90% of the 32P count (Fig. 5, lane 2). The inhibitory action of triptorelin on the transfer of [32P]ADP-ribose to 41-kDa protein revealed a dose dependency (not shown). The G protein ␣i-subunit is a better substrate for ADP-ribosylation when the nucleotide binding site is free of GDP and GTP, and GDP dissociation is stimulated by triptorelin. In the absence of GTP, but in the presence of triptorelin, ADP-ribosylation of the G protein ␣i was increased (Fig. 5, lane 3). GTP alone had no effect (Fig. 5, lane 5). These results support the concept that the G protein ␣i is activated by the tumor LHRH receptor. Cross-linking of G proteins to LHRH receptor

To analyze whether the LHRH receptors in ovarian and endometrial cancer cells are able to couple to G protein ␣q in addition to G protein ␣i, we performed cross-linking experiments (Fig. 6). Using a G protein ␣i antibody, two protein complexes could be distinguished, migrating at about 41 and 103 kDa. Assuming a stoichiometric binding of 1:1, subtraction of the molecular mass of G protein ␣i (41 kDa) yielded a protein of about 62 kDa. This corresponds to the molecular mass of LHRH receptor. The band of 41 kDa corresponds to the molecular mass of G protein ␣i. Using the G protein ␣q antibody, the results were similar, except for membranes from the ovarian cancer cell line EFO-27, which does not

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FIG. 5. Effects of the LHRH agonist triptorelin (100 nM) on the PTX-induced ADP-ribosylation in membranes from EFO-21 ovarian cancer cells. G proteins in the cell membranes, which are isolated in the absence of GTP, GTP analogs, or other activators, are in their inactive trimeric form. Incubation of the membranes of these cell lines with [32P]NAD and PTX resulted in a marked PTX-catalyzed ADP-ribosylation in the 41-kDa protein (lane 1). In the absence of GTP, but in the presence of triptorelin, ADP-ribosylation of G protein ␣i was increased (lane 3). The G protein ␣I-subunit is a better substrate for ADP-ribosylation when the nucleotide-binding site is free of GDP and GTP, and GDP dissociation is stimulated by triptorelin. Incubation with the LHRH analog triptorelin in the presence of GTP remarkably reduced ADP-ribosylation in the 41-kDa protein (lane 4). G protein ␣i becomes a poor substrate for PTX-catalyzed ADP-ribosylation when the trimeric G protein has become dissociated upon LHRH receptor activation. Therefore, addition of triptorelin in the presence of GTP diminished ADP-ribosylation of G protein ␣i coupled to the LHRH receptor. Incubation with triptorelin and a nonhydrolysable GTP analog, GTP-␥-S, produced a still more pronounced reduction of ribosylation of this protein (lane 2). GTP alone had no effect (lane 5). The figure shows a representative profile of four independent experiments performed in duplicate in four different passages of the cell lines that gave identical results. Experiments using endometrial cancer cell lines Hec-1A and Ishikawa or ovarian cancer cell line EFO-27 gave comparable results.

express G protein ␣q. In this cell line neither the 41-kDa band (G protein ␣q) nor the 103-kDa band (G protein ␣q plus LHRH receptor) could be detected (Fig. 6). Using a LHRH receptor antibody, we could also detect the 103-kDa band, indicating that the 62-kDa part of the 103-kDa complex after subtraction of the molecular mass of the G proteins seems to be the LHRH receptor protein. Additional use of LHRH receptor antibody lead to the detection of a lower band at about 62 kDa, representing the unbound LHRH receptor protein. In addition, some other inconsistent bands were observed that we consider to be nonspecific artifacts. These findings demonstrate in addition to the findings of the ADP-ribosylation experiments, that the LHRH receptor couples to G protein ␣i. Furthermore LHRH receptor couples to G protein ␣q. Effects of PTX on LHRH-induced inhibition of EGFinduced c-fos expression

Previous experiments indicated that treatment of ovarian and endometrial cancer cells with LHRH agonists does not affect signaling mechanisms of LHRH receptors known to operate in the pituitary [phospholipase C, protein kinase C (PKC), and adenylyl cyclase] (35), suggesting that LHRH receptor signaling in these cells might not be mediated through G protein ␣q. Therefore, we hypothesized that in these cells the effects induced by LHRH agonists might be mediated by G protein ␣i. PTX impairs the receptor-effector interaction through ADP-ribosylation of G protein ␣i. Therefore, we examined LHRH-induced inhibition of EGFinduced c-fos expression in the cell lines treated with PTX (Fig. 7). The LHRH-induced inhibition of EGF-induced c-fos

expression was blocked completely by PTX in ovarian cancer cell lines EFO-21 and EFO-27 and in endometrial cancer cell lines Ishikawa and Hec-1A (Fig. 7). This demonstrates that LHRH-induced inhibition of EGF-induced c-fos expression, in these cell lines is mediated through the G protein ␣i. As mentioned, PTX also impaired LHRH-induced reduction of proliferation (not shown). Effects of LHRH on PTP activity

To assess whether LHRH agonist binding to tumor LHRH receptors activates PTP through a PTX-sensitive G protein ␣i, the next set of experiments was performed. Treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and quiescent Ishikawa and Hec-1A endometrial cancer cells with 100 nm of the LHRH agonist triptorelin resulted in a significant increase in PTP activity (Fig. 8). This effect was completely blocked by simultaneous treatment with PTX. PTP activity is expressed as a percentage of the control value (no treatment ⫽ 100%). In the ovarian cancer cell lines EFO-21 (Fig. 8A) and EFO-27 (Fig. 8B), PTP activity was increased to 397.5 ⫾ 47.2% or 372.5 ⫾ 36.1%, respectively (P ⬍ 0.001 vs. control). Simultaneous treatment with PTX resulted in a decrease to 77.5 ⫾ 7.5% or 80.2 ⫾ 9.1% of the control value, respectively (P ⬍ 0.001 vs. triptorelin). In the endometrial cancer cell lines Ishikawa (Fig. 8C) and Hec-1A (Fig. 8D), PTP activity was increased to 442.5 ⫾ 27.8% or 417.5 ⫾ 26.3%, respectively (P ⬍ 0.001 vs. control). Simultaneous treatment with PTX resulted in a decrease to 91.3 ⫾ 10.1% or 90.1 ⫾ 10.8%, respectively (P ⬍ 0.001 vs. triptorelin).


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FIG. 6. Cross-linking of G protein ␣-subunits and LHRH receptor in ovarian cancer cell lines EFO-21 (A) and EFO-27 (B). Detection of the LHRH receptor protein (62 kDa) and the cross-linked LHRH receptor-G protein ␣ complex using an anti-LHRH receptor antibody (left). Detection of G protein ␣i and cross-linked LHRH receptor-G protein ␣i complex using an anti-G protein ␣i antibody (middle). Detection of G protein ␣q and cross-linked LHRH receptor-G protein ␣q complex using an anti-G protein ␣q antibody (right). Assuming a stoichiometric binding of 1:1, subtraction of the molecular mass of G protein ␣i (41 kDa) or G protein ␣q (41 kDa) from the cross-linked complex (103 kDa) yielded a protein of about 62 kDa. This corresponds to the molecular mass of LHRH receptor. Both G protein ␣i and ␣q were cross-linked to LHRH receptor in the ovarian cancer cell line EFO-21 (A). In the EFO-27 cell line, which does not express G protein ␣q, only G protein ␣i was cross-linked to LHRH receptor (B). The figure shows is a representative example of three independent experiments performed in duplicate in three different passages of the cell lines that gave identical results. Experiments using endometrial cancer cell lines Hec-1A and Ishikawa gave identical results as ovarian cancer cell line EFO-21.

Effects of LHRH on EGF-induced EGF receptor tyrosine autophosphorylation

Having shown that LHRH agonist binding to tumor LHRH receptor significantly activates PTP through coupling to PTX-sensitive G protein ␣i, we now assessed whether this leads to a relevant reduction of EGF-induced autophosphorylation of EGF receptors. The amount of phosphorylated EGF receptors in quiescent cells was below the detection limit of our assay (not shown). Treatment of quiescent EFO-21 (Fig. 9, A and B) and EFO-27 (Fig. 9C) ovarian cancer cells and quiescent Ishikawa (Fig. 9D) and Hec-1A (Fig. 9E) endometrial cancer cells with 100 nm EGF resulted in a dramatic increase in tyrosine-phosphorylated EGF receptors (⫽100%). This effect of EGF was antagonized by simultaneous treatment with LHRH agonist triptorelin (10 ␮m). In the ovarian cancer cell lines EFO-21 and EFO-27 the amount of phosphorylated EGF receptors was decreased to 63 ⫾ 2.1% or 44.5 ⫾ 9.6% of the control value, respectively (P ⬍ 0.001). In the endometrial cancer cell lines Ishikawa (Fig. 9D) and Hec-1A (Fig. 9E), the amount of tyrosine-phosphorylated EGF receptors was decreased to 58 ⫾ 6.8% or 59 ⫾ 2.4% of the control value, respectively (P ⬍ 0.001). When the PTP inhibitor vanadate (100 ␮m) was present in these experiments, this antagonism was completely blocked (P ⬍ 0.001). Figure 9 shows the relative changes in the amount of phosphorylated EGF receptors in the absence or presence of vanadate in terms of the percentage of phosphorylated EGF receptors after EGF treatment.

Discussion

LHRH receptors in the human pituitary, human normal extrapituitary tissues, and human cancers appear to be quite similar as far as their ligand binding properties are concerned (31, 36). Their signal transduction mechanisms, however, are different (6 –18). The LHRH signal transduction pathway operating in normal tissues is not essential in cancer cells (18). In pituitary gonadotrophs, LHRH receptor signaling is essentially mediated through G protein ␣q, leading to activation of PLC, rapid hydrolysis of membrane phospholipids, liberation of inositol phosphates, subsequent mobilization of intracellular Ca2⫹, and activation of PKC (5, 6, 17). We clearly demonstrated in earlier studies the activation of phospholipase C, PKC, and adenylyl cyclase in the tumor cells by pharmacological stimuli. The LHRH agonist triptorelin, at concentrations that are clearly inhibitory to proliferation, however, had no effect on the activities of these signaling systems (18). Instead, we found that the antiproliferative effects of LHRH analogs are mediated through interaction with growth factor-induced mitogenic signaling, as LHRH analogs antagonized growth factor-induced proliferation, mitogen-activated protein kinase activity, and c-fos expression (18, 19). Comparable data were obtained by Moretti et al. (38) in the human prostatic cancer cell lines LNCaP and DU 145. In addition, the proliferation of endometrial and ovarian cancer cells was inhibited by both agonistic and antagonistic analogs of LHRH, indicating that the di-

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FIG. 7. Effects of PTX on LHRH agonist triptorelin-induced inhibition of EGF-induced c-fos expression. c-fos expression of quiescent EFO-21 ovarian cancer cells (first bar; control) and after treatment with 100 nM EGF (10 min) without (second bar) or with (third bar) previous treatment (15 min) with the LHRH agonist triptorelin (100 nM) or with previous treatment with LHRH agonist triptorelin and with (2 ng/ml) PTX (fourth bar). After treatment with EGF, a significant increase in c-fos expression was observed (P ⬍ 0.001; second bar). After treatment with triptorelin followed by EGF, no increase in c-fos expression was observed (third bar). After treatment with PTX, triptorelin-induced inhibition of EGF-induced c-fos expression was blocked (P ⬍ 0.001; fourth bar). Columns represent the mean ⫾ SE of data obtained from four independent experiments performed in duplicate in four different passages of each cell line. Experiments using endometrial cancer cell lines Hec-1A and Ishikawa or ovarian cancer cell line EFO-27 gave similar results. a, P ⬍ 0.001 vs. control; b, P ⬍ 0.001 vs. EGF; c, P ⬍ 0.001 vs. EGF/TRP.

chotomy of LHRH agonists and antagonists does not exist in tumor cells (11). Looking for reasons for the differences between LHRH signaling in pituitary gonadotrophs and human cancers, we first focused on putative mutations or splice variant formation as a possible explanation. We could not find any mutations or splice variants of the LHRH receptor transcripts in the endometrial and ovarian cancer cell lines analyzed. The LHRH receptor sequence found in Ishikawa and Hec-1A endometrial cancer cell lines and in EFO-21 and EFO-27 ovarian cancer cells lines was identical to that found in the pituitary gonadotrophs by Kakar et al. (32). In agreement with our results, Kakar and co-workers (39) have shown that in a not well characterized ovarian tumor biopsy, composed of malignant cells and desmoplastic fibrous tissue, that the LHRH receptor transcript was identical to that found in pituitary gonadotrophs. In contrast to this biopsy we show here for the first time that in well characterized human epithelial ovarian cancer cells lines obtained from a poorly differentiated serous ovarian adenocarcinoma and a mucinous papillary ovarian adenocarcinoma of intermediate differentiation, respectively, the LHRH receptor transcript was identical to that found in pituitary gonadotrophs. LHRH receptor sequence data obtained from endometrial cancer cells are shown for the first time here. It cannot be ruled out, however, that the LHRH receptor in tumors might differ in

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its posttranslational processing compared with the pituitary LHRH receptor. In the next step we studied whether the G proteins might be responsible for the different LHRH signal transduction pathway. Imai et al. (40) speculated that the G protein ␣i that possibly couples the LHRH receptor to the effector may be responsible for difference in response in peripheral tumors and the anterior pituitary. Coupling of LHRH receptor to G protein ␣q in cancer cells was not shown (40). As we found both G protein subunits ␣i and ␣q to be highly expressed in endometrial and ovarian cancer cells, this theory cannot be explained by a lack of G protein ␣q expression. Our study provides evidence for LHRH receptor coupling to both G protein ␣i and ␣q in human endometrial and ovarian cancer cells. Our data demonstrate that tumor cell LHRH receptor couples to both G proteins in a single cell type. However, the antiproliferative actions of LHRH analogs were predominantly mediated through the PTX-sensitive G protein ␣i. EGF-induced expression of the immediate early gene c-fos is reduced by binding of LHRH to its receptors in endometrial and ovarian cancer cell lines (19). This effect was completely blocked by PTX, indicating that it is mediated by G protein ␣i. Recently and in the present paper we showed that in the ovarian cancer cell line EFO-27, which does not express G protein ␣q, the EGF-induced c-fos expression (19) as well as the EGF-induced cell proliferation (18) were antagonized by LHRH agonist as effectively as in other tumor cell lines expressing both G proteins, ␣q and ␣i. These findings support the concept that the antiproliferative effects of LHRH agonists in endometrial and ovarian cancer cells are mediated through G protein ␣i. We recently found that LHRH induces activation of nuclear factor ␬B in ovarian cancer cells (41). This effect was inhibited by PTX, indicating that LHRHinduced activation of nuclear factor ␬B is also mediated through PTX-sensitive G protein ␣i (unpublished results). Moreover, the LHRH agonist triptorelin substantially antagonized the PTX-catalyzed ADP-ribosylation of G protein ␣i. Limonta et al. (42) found a comparable mechanism in prostate cancer cells, suggesting that LHRH receptor seems to be coupled to the G protein ␣i-cAMP signal transduction pathway rather than to the G protein ␣q/11-PLC signaling system (42). In endometrial and ovarian cancer cells, however, cAMP is not involved in LHRH signaling (18). It is true that the LHRH receptor in endometrial and ovarian cancer cells couples additionally to G protein ␣q as in the pituitary gonadotrophs; the signaling mechanisms induced by G protein ␣q in the pituitary, however, are not induced by LHRH receptor in these cancer cells (18). In addition, the antiproliferative effects of LHRH analogs are not mediated through G protein ␣q. It might be speculated that minor mutations of the G protein ␣q to which LHRH receptors are coupled in the endometrial and ovarian cancer cells could be responsible for the phenomenon that G protein ␣q is able to couple the LHRH receptor, but that a signal transduction comparable to that in pituitary gonadotrophs is not activated. In addition, it is possible that the three-dimensional structures of the LHRH receptor might be different in pituitary gonadotrophs and peripheral cancers. This speculation will be the subject of further investigations. The ability of the LHRH receptor to couple to multiple G


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FIG. 8. Effects of the LHRH agonist triptorelin on PTP activity of EFO-21 (A) and EFO-27 (B) ovarian cancer cells and of Ishikawa (C) and Hec-1A (D) endometrial cancer cells. Quiescent cells were incubated for 15 min in the absence (control) or presence of triptorelin (TRP) with or without PTX, before PTP activity was measured. PTP activity is expressed as a percentage of the control value (control ⫽ no treatment ⫽ 100%). Columns are the mean ⫾ SE of data obtained from three independent experiments performed in duplicate in three different passages of each cell line. a, P ⬍ 0.001 vs. control; b, P ⬍ 0.001 vs. TRP.

proteins seems not to be specific to endometrial and ovarian cancer cells. Stanislaus and co-workers recently reported that the LHRH receptor in pituitary gonadotrophs and in the lactotroph cell line GGH3 couples to multiple G proteins (43). The ability of seven-transmembrane domain receptors to couple to multiple G proteins is documented for other members of this family (44, 45). In contrast, Grosse et al. (46) reported that in gonadotropic ␣T3–1 cells and in CHO-K1 cells transfected with the human LHRH receptor cDNA LHRH receptors couple exclusively to G proteins of the G␣q/11 family. However, in endometrial and ovarian cancer cells the picture is different. The mechanism through which multiple G proteins interact with the LHRH receptor is unknown. The second and third intracellular loops appear to be involved in signal transduction, suggesting that multiple sites on the receptor may interact with G proteins. Having shown that LHRH agonists inhibit EGF-induced net tyrosine phosphorylation in endometrial and ovarian cancer cells (18), we checked whether this effect might be due to LHRH-induced activation of a PTP. In addition, we

analyzed whether this effect might be mediated through PTX-sensitive G protein ␣i. To specify PTP activity we used the enzyme-specific phosphopeptide END(pY)INASL (34) to exclude interference with other phosphatase enzymes. The system used determines the amount of free phosphate generated in a reaction by measuring the absorbance of molybdate-malachite green-phosphate complex (33). LHRH treatment resulted in a significant increase in PTP activity. This effect was completely blocked by simultaneous treatment with PTX, indicating the involvement of PTX-sensitive G protein ␣i in LHRH-induced PTP activation. As a final step we checked, whether the LHRHinduced activation of PTP really results in a reduction of EGF-induced tyrosine autophosphorylation of EGF receptors. LHRH treatment resulted in a marked decrease in tyrosine-phosphorylated EGF receptors. When the experiments were performed in the presence of vanadate, an inhibitor of PTP, the reduction of EGF-induced tyrosine autophosphorylation of EGF receptors by treatment with triptorelin was completely blocked. Both activation of PTP

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FIG. 9. Quantification of tyrosine-phosphorylated EGF receptors by quantitative Western blotting of membranes obtained from ovarian cancer cell line EFO-21 (A and B) and EFO-27 (C) and endometrial cancer cell lines Ishikawa (D) and Hec-1A (E). In the absence of EGF, no phosphorylated EGF receptors were detectable. Relative changes in the amount of phosphorylated EGF receptors after treatment with EGF (100 nM) are shown in the absence (control ⫽ 100%) or presence (⫹TRP) of the LHRH agonist triptorelin (10 ␮M) with or without vanadate (VAN). Columns are the mean ⫾ SE of data obtained from three independent experiments performed in duplicate in three different passages of each cell line. a, P ⬍ 0.001 vs. control; b, P ⬍ 0.001 vs. TRP.

and reduction of EGF-induced phosphorylation of EGF receptors were induced in EFO-27 cells, which do not express G protein ␣q as effectively as other cell lines expressing this G protein-coupled receptor. These data also

support the concept that the antiproliferative activity of LHRH in endometrial and ovarian cancer cells is not mediated through G protein ␣q. These findings suggest that the link between LHRH receptor activity and growth factor-


induced cell proliferation is a PTP. PTP activation by LHRH analogs counteracts EGF-induced tyrosine autophosphorylation of EGF receptor. This results in downregulation of mitogenic signal transduction and cell proliferation. In summary, this study provides evidence that in human endometrial and ovarian cancers, LHRH receptor gene mutations as well as splice variants are not responsible for the signaling different from that acting in the pituitary. We could show that the tumor LHRH receptor instead activates a PTP mediated through the PTX-sensitive G protein ␣i counteracting EGF-induced tyrosine autophosphorylation of EGF receptors and resulting in inhibition of mitogenic signal transduction and reduction of cell proliferation. In addition, the present data indicate that in endometrial and ovarian cancer cells, the LHRH receptor couples to G protein ␣i and G protein ␣q in a single cell type in endometrial and ovarian cancer cells. Nevertheless, the antiproliferative signal transduction seems to be mediated predominantly through PTXsensitive G protein ␣i. Whether the coupling of G protein ␣q to the LHRH receptor, as in the pituitary gonadotrophs, induces any intracellular activities in tumor cells is not known and will be the subject of further investigations.

14.

15. 16.

17. 18.

19.

20. 21.

22.

Acknowledgments We are grateful to Ferring Pharmaceuticals Ltd. Arzneimittel (Kiel, Germany) for the gift of the LHRH agonist triptorelin. We thank Dr. A. A. Karande (Indian Institute of Science, Bangalore, India) for her gift of the F1G4 monoclonal antibody to the LHRH receptor. We thank Katharina Schneider and Katja Schulz for excellent technical assistance.

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Charles E. Culpeper Scholarships in Medical Science The Rockefeller Brothers Fund is currently accepting applications for its 2002 Charles E. Culpeper Scholarships in Medical Science Program designed to support the career development of academic physicians. Up to four awards of $100,000 per year for three years will be made to United States medical schools or equivalent United States educational institutions on behalf of candidates who are U.S. citizens or aliens who have been granted permanent U.S. residence (proof required); who have received their M.D. degree from a U.S. medical school or the equivalent of an M.D. degree from an educational institution equivalent to a United States Medical School in 1993 or later (except under extraordinary circumstances, as approved by the Fund before submittal); and who are judged worthy of support by virtue of the quality of their research proposals. All scientific research relevant to human health is eligible for consideration. No institution may nominate more than one candidate. In selecting awardeees, emphasis will be on identifying young physicians with clear potential for making substantial contributions to science as academic physicians. Since January 1988, 45 physicians have been selected as Charles E. Culpeper Medical Scholars. Deadline for applications is August 15, 2001. Awards will be announced in January 2002, for activation on or about July 1, 2002. Application forms and instructions may be obtained on the Web at www.rbf.org or by contacting the Rockefeller Brothers Fund, 437 Madison Avenue, 37th floor, New York, NY 10022-7001, telephone: 212/812-4200, fax: 212/812-4299.