MUC4 regulates cellular senescence in head and neck squamous cell carcinoma through p16/Rb pathway.

The limited effectiveness of therapy for patients with advanced stage head and neck squamous cell carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. MUC4, a high molecular weight glycoprotein, is differentially overexpressed in many human cancers and implicated in cancer progression and resistance to several chemotherapies. However, its clinical relevance and the molecular mechanisms through which it mediates HNSCC progression are not well understood. This study revealed a significant upregulation of MUC4 in 78% (68/87) of HNSCC tissues compared with 10% positivity (1/10) in benign samples (P=0.006, odds ratio (95% confidence interval)=10.74 (2.0-57.56). MUC4 knockdown (KD) in SCC1 and SCC10B HNSCC cell lines resulted in significant inhibition of growth in vitro and in vivo, increased senescence as indicated by an increase in the number of flat, enlarged and senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. Decreased cellular proliferation was associated with G0/G1 cell cycle arrest and decrease expression of cell cycle regulatory proteins like cyclin E, cyclin D1 and decrease in BrdU incorporation. Mechanistic studies revealed upregulation of p16, pRb dephosphorylation and its interaction with histone deacetylase 1/2. This resulted in decreased histone acetylation (H3K9) at cyclin E promoter leading to its downregulation. Orthotopic implantation of MUC4 KD SCC1 cells into the floor of the mouth in nude mice resulted in the formation of significantly smaller tumors (170±18.30 mg) compared to those (375±17.29 mg) formed by control cells (P=0.00007). In conclusion, our findings showed that MUC4 overexpression has a critical role by regulating proliferation and cellular senescence of HNSCC cells. Downregulation of MUC4 may be a promising therapeutic approach for treating HNSCC patients.

PTTG induces EMT through integrin αVß3-focal adhesion kinase signaling in lung cancer cells.

Pituitary tumor transforming gene (PTTG) is a well-studied oncogene for its role in tumorigenesis and serves as a marker of malignancy in several cancer types including lung. In the present study, we defined the role of PTTG in actin cytoskeleton remodeling, cell migration and induction of epithelial mesenchymal transition (EMT) through the regulation of integrin α(V)ß(3)-FAK (focal adhesion kinase) signaling pathway. Overexpression of PTTG through an adenovirus vector resulted in a significant increase in the expression of integrins α(V) and ß(3), a process that was reversed with the downregulation of PTTG expression through the use of an adenovirus expressing PTTG-specific small interfering RNA (siRNA). Western blot analysis of cells infected with adenovirus PTTG cDNA resulted in increased FAK and enhanced expression of adhesion complex molecules paxillin, metavincullin, and talin. Furthermore, downstream signaling genes Rac1, RhoA, Cdc42 and DOCK180 showed upregulation upon PTTG overexpression. This process was dependent on integrin α(V), as blockage by antagonist echistatin (RGD peptide) or α(V)-specific siRNA resulted in a decrease in FAK and subsequent adhesion molecules. Actin cytoskeleton disruption was detected as a result of integrin-FAK signaling by PTTG as well as enhanced cell motility. Taken together, our results suggest for the first time an important role of PTTG in regulation of integrins α(V) and ß(3) and adhesion-complex proteins leading to induction of EMT.

LHRH receptor targeted therapy for breast cancer.

Breast cancer remains the most common cancer among women, with an estimated 212,920 new cases and 40,970 deaths in the United States in 2006. The present work extends the studies of nanoparticles targeted to the luteinizing hormone-releasing hormone (LHRH) receptor which is overexpressed in breast, ovarian, endometrial and prostate cancer cells. In contrast, LHRH receptors are not expressed, or expressed at a low level in most visceral organs. In our studies, we conjugated Fe3O4 nanoparticles (20-30 nm) with [D-Trp6]LHRH (Triptorelin), a decapeptide analog of LHRH currently used for treatment of sex-hormone-dependent tumors. Conjugation of [D-Trp6]LHRH to Fe3O4 particles retained its binding affinity and biological activity for the LHRH receptor. Treatment of two separate breast tumor cell lines (MCF-7 and MDA-MB231) with these conjugated nanoparticles resulted in 95-98% cell death and loss of viability within 24 h whereas no change in cell proliferation or cell apoptosis was observed in cells treated with equal amounts of either [D-Trp6]LHRH or unconjugated Fe3O4 nanoparticles. These studies provide critical and important information regarding use of LHRH receptor targeted therapy for breast cancer.

Pituitary tumor transforming gene: an important gene in normal cellular functions and tumorigenesis.

Pituitary tumor transforming gene (PTTG) is an oncogene which is found to be highly expressed in proliferating cells and in most of the tumors analyzed to date. Overexpression of PTTG induces cellular transformation and promotes tumor development in nude mice. PTTG is regulated by various growth factors including insulin and IGF-1. PTTG is a multifunctional and multidomain protein. Some of the functions of PTTG include inhibition of separation of sister chromatids, expression and secretion of angiogenic and metastatic factors. In this review we focus on expression of PTTG in normal and tumor tissues, define its biological function, its role in tumorigenesis, and its interaction with other proteins that may play important role in mediating tumorigenic function of PTTG.

Identification of gene networks modulated by activin in LbetaT2 cells using DNA microarray analysis.

Activins, members of the TGFbeta family of proteins, are widely expressed in a variety of tissues. First identified based on their ability to regulate biosynthesis and secretion of follicle-stimulating hormone (FSH), activins have also been shown to modulate development, cell growth, apoptosis, and inflammation. Despite their many known functions, the precise mechanisms and downstream signaling pathways by which activins mediate their diverse effects remain unknown. We have used a DNA microarray assay to identify genes that are regulated by activin, alone or in combination with gonadotropin-releasing hormone (GnRH), another major regulator of FSH, in a murine gonadotrope-derived cell line (LbetaT2). We used mRNA from these cells to screen Affymetrix Mu74av2 mouse Gene Chip oligonucleotide microarrays, representing approximately 12,400 mouse genes. Treatment of LbetaT2 cells with activin A, a gonadotropin-releasing hormone agonist (GnRHA) or activin A plus GnRHA resulted in alterations in levels of gene expression that ranged in magnitude from 15 to 67-fold. Data analysis identified 268 transcripts that were up- or down-regulated by two-fold or more. Distinct sets of genes were affected by treatment with activin, GnRHA and activin plus GnRHA, suggesting interactions between activin and GnRHA. Changes in expression of seven randomly selected representative genes identified by the microarray technique were confirmed by real-time quantitative PCR and semi-quantitative reverse transcription/PCR (RT/PCR). Modulation of expression of genes by activin suggests that activin may mediate its effects through a variety of signaling pathways.

PTTG and cancer.

Pituitary tumor transforming gene (pttg) is a recently isolated oncogene that is expressed in most of the tumors. Overexpression of pttg results in an increase in cell proliferation, induces cell transformation in vitro, and promotes tumor formation in nude mice. The gene encodes a protein of 202 amino acids with no significant homology with other known proteins. The protein is a multi domain consisting of a transactivation domain, domain required for ubiquitin-mediated proteolysis and a DNA binding domain. pttg protein is bestowed with a multitude of functions and seems to be involved in most of the important mechanisms of cell proliferation, differentiation and signaling. Given the number of processes that are involved in the manifestation of cancer, it thus becomes mandatory to study the role of this potent oncogene in relation to the processes of cell survival, death and functioning.

Characterization of a polyclonal antibody to human pituitary tumor transforming gene 1 (PTTG1) protein.

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His(6) tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.

Molecular cloning of pituitary tumor transforming gene 1 from ovarian tumors and its expression in tumors.

Pituitary tumor transforming gene 1 (PTTG1) recently cloned from human testis is a potent oncogene and is highly expressed in all the tumors analyzed to date. However, primary structure of PTTG1 and the cell types that express PTTG1 in tumors remained undescribed. We have used the reverse transcriptase-polymerase chain reaction technique to clone PTTG1 from ovarian tumors. Nucleotide sequencing of the PTTG1 cDNAs from various ovarian tumors showed identity with that of the human testis PTTG1. To determine the cell types that express PTTG1 in normal and tumor tissues, we performed in situ hybridization using digoxigenin-labeled cRNA as a probe. Our studies revealed a high level of expression of PTTG1 mRNA in both seminomatous and non-seminomatous testicular tumors; epithelial, sex-cord and stromal cell, and germ cell tumors of the ovary; and invasive ductal, ductal in situ and infiltrating ductal carcinoma of the breast. In normal tissues, expression of PTTG1 mRNA was very low or undetectable except in testis, where PTTG1 mRNA was found to be localized to spermatocytes and spermatids. Tumors that expressed high levels of PTTG1 mRNA also exhibited high levels of expression of basic fibroblast growth factor (bFGF), suggesting a correlation between PTTG1 and bFGF expression, and further suggesting that the PTTG1 protein may be involved in tumor angiogenesis and mitogenesis.

Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization.

In an attempt to determine the mechanism of human tumorigenesis, we have searched for oncogenes and recently reported the molecular cloning of a potent oncogene (hPTTG) from human testis. hPTTG mRNA is expressed at high levels in various human tumors and tumor cell lines. Overexpression of hPTTG in the mouse fibroblast cell line (NIH 3T3) results in an increase in cell proliferation, induces cellular transformation in vitro, and promotes tumor formation in nude mice. The hPTTG gene isolated from the human genomic library consists of five exons and four introns and spans over 10kb. In the studies reported here, we further investigated the possibility of the presence of additional genes homologous to hPTTG in the human genome, which was first indicated by Southern blot analysis of the human genomic DNA and chromosomal mapping of the hPTTG gene using DNA from humanxhamster hybrid cell lines in PCR. Sequencing and restriction map analysis of the additional genomic clones identified two intronless genes homologous to hPTTG. This finding was confirmed by the chromosomal location of the second gene to chromosome 4p15.1 and the third gene to chromosome 8q13.1. Based on the similarity in sequences, we proposed that hPTTG be renamed hPTTG1 and the new genes be named hPTTG2 and hPTTG3. hPPTG2 was found to be 91% identical and hPPTG3 89% identical with hPPTG1 at the amino acid level. Northern blot and reverse transcriptase/polymerase chain reaction (RT/PCR) analyses of the mRNA from various human tissues revealed differential expression of the hPTTG2 and hPTTG3 genes in normal and tumor tissues, suggesting that these genes may be associated with tumorigenesis.

Molecular cloning, genomic organization, and identification of the promoter for the human pituitary tumor transforming gene (PTTG).

Recently, we cloned and sequenced cDNA of a potent pituitary tumor transforming gene (PTTG) from human testis and showed that this gene is expressed highly in various human tumors, including tumors of the pituitary and adrenal glands, and the liver, kidney, endometrium, uterus, and ovary. To determine the genomic organization of the PTTG and its transcriptional regulation in tumors, we isolated the gene. The PTTG spans more than 10kb and contains five exons and four introns. Primer extension and RNA protection assays indicated a transcription start site at an adenine residue at 37 bases upstream of the translation start site (ATG). Analysis of the 5' flanking region of the gene revealed the existence of three SP1/GC boxes, three AP1 and one AP2 binding sequences, a cyclic AMP response element sequence, and an insulin response element sequence. The promoter activity of the PTTG was evaluated by transfecting a human ovarian tumor cell line (SKOV3) and a mouse fibroblast cell line (NIH 3T3) with a chimeric fusion construct containing the 5' flanking sequence (nucleotide from -1336 to +34) and luciferase reporter gene (pluc 1370). The promoter activity of this construct was 210-fold higher in SKOV3 and 20-fold higher in NIH 3T3 cells than the promoterless vector. Deletion of sequences at the 5' end of the pluc 1370 construct from nucleotide -1336 to -1157 (pluc 1190), from nucleotide -1336 to -977 (pluc 1010) and from nucleotide -1336 to -707 (pluc 740) further increased luciferase activity. Further deletion of the 5' sequence from nucleotide -1336 to -407 (pluc 440), and from nucleotide -1336 to -127 (pluc 160) decreased activity by 95%. These results suggest that the sequence from nucleotide -126 to +34 is sufficient for PTTG promoter activity and that the sequence between nucleotide -706 and -407 contains an enhancer element. PTTG promoter activity was eight- to ten-fold higher in SKOV3 cells than NIH 3T3 cells, suggesting a basis for the tumor-specific expression of the PTTG. Knowledge of the genomic organization and the promoter region of the human tumor transforming gene will allow further studies of possible disorders of the PTTG as well as facilitate elucidation of the transcriptional control of PTTG expression in human tumors.

Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis.

We cloned and sequenced the cDNA of a potent tumor transforming gene (TUTR1) from human testis and determined its primary structure. The TUTR1 cDNA is composed of 656 nucleotides and encodes a novel protein of 202 amino acids. The predicted TUTR1 protein is extremely hydrophilic and contains two proline-rich motifs at its C-terminus. Northern blot analysis of the mRNA from various human tissues and tumors revealed that TUTR1 mRNA is highly expressed in tumors of the pituitary gland, adrenal gland, ovary, endometrium, liver, uterus, and kidney as well as in cell lines derived from tumors of the pituitary, breast, endometrium, and ovary. With the exception of the testis, the levels of TUTR1 mRNA were either very low or undetectable in normal human tissues. Overexpression of TUTR1 in mouse fibroblasts (NIH 3T3) cells resulted in an increase in cell proliferation, induced cellular transformation in vitro, and promoted tumor formation in nude mice. These results suggest that TUTR1 is a novel and potent transforming gene, which may be involved in tumorigenesis in numerous different human tumors.

Inhibition of growth and proliferation of EcRG293 cell line expressing high-affinity gonadotropin-releasing hormone (GnRH) receptor under the control of an inducible promoter by GnRH agonist (D-Lys6)GnRH and antagonist (Antide).

The mechanism by which gonadotropin-releasing hormone (GnRH) agonists and antagonists inhibit tumor cell growth and proliferation is controversial. Direct mediation of the antitumor effects through the high-affinity GnRH receptors has been questioned because of the low level of expression of receptors on the tumor cells. We have developed a human kidney embryonic cell line (EcRG293) that expresses high-affinity GnRH receptor under the control of an inducible promoter activated by muristerone A. Treatment of this cell line with either GnRH agonist (D-Lys6)GnRH or GnRH antagonist (Antide) resulted in a significant, time-dependent decrease in cell proliferation in muristerone A-induced cells but not in the uninduced cells, which do not express the GnRH receptor. These data suggest strongly that the antitumor effect of GnRH agonists and antagonist is specific, direct, and mediated through high-affinity GnRH receptors present on the cell membranes of tumor cells.

Molecular structure of the human gonadotropin-releasing hormone receptor gene.

GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent tumor tissues. Recently, cDNAs for the GnRH receptors of several species including the human have been cloned. To determine the structure of the gene encoding the human GnRH receptor, we isolated the receptor gene clones from the human genomic libraries. Comparison of the genomic and cDNA sequences revealed that the human GnRH receptor gene is composed of three exons and two introns and spans over 20 kb in size. Exon 1 encodes the 5' untranslated sequence and nucleotide +1 to +522 in the open reading frame, exon 2 encodes nucleotide +523 to +742 and exon 3 encodes nucleotide +743 to +987 in the open reading frame as well as the 3' untranslated sequence. Southern blot analysis of genomic DNA and localization of the GnRH receptor gene to a single site on human chromosome 4 (4q13) indicate the presence of a single copy of the gene in the human genome. Several regulatory sequences for various hormones and other regulatory factors were identified, including PEA-3, AP-1, AP-2, and Pit-1 sites. In addition, glucocorticoid/progesterone response element thyroid hormone response element, and cAMP response element sequences were identified. Reverse transcriptase-primer extension and 5' RACE analysis of the human pituitary RNA demonstrated the presence of multiple transcriptional start sites upstream of the translational start site. Analysis of the 5' flanking region of the gene also revealed the presence of multiple TATA and CAAT sequences. The finding of multiple transcriptional start sites raises the possibility of tissue-specific regulation and the existence of variable size transcripts. Chimeras containing 1.26 kb (-534 to 728) of the 5' flanking region of the receptor gene and the luciferase (Luc) gene expressed a significant luciferase activity when transfected into a human endometrial tumor cell line (HEC-1A) and a breast tumor cell line (MCF-7) but not in a mouse pituitary gonadotrope cell line (alpha T3-1), suggesting the existence of multiple promoter elements in the gene. These findings indicate a multiplicity of regulation of expression of the GnRH receptor and provide the substrate for detailed investigation in the reproductive system.

The inhibition of growth and down-regulation of gonadotropin releasing hormone (GnRH) receptor in alphaT3-1 cells by GnRH agonist.

Gonadotropin releasing hormone (GnRH) and its analogs inhibit the growth of hormone-dependent tumors in vivo and in vitro. The inhibition of growth and proliferation of tumor cells in vitro by GnRH and its analogs indicates that the tumor suppressing effect of the hormone is only partially due to suppression of pituitary gonadotropin release which reduces circulating steroid levels that are required for proliferation. Demonstration of GnRH-binding sites on some tumors suggests a direct inhibitory effect of GnRH and its analogs. However, the mechanism by which GnRH and its analogs inhibit tumor cell growth is not known. Our hypothesis is that the inhibition of growth and proliferation of tumor cells by GnRH and its analogs are mediated through down-regulation of its receptor expression. To test this hypothesis, mouse pituitary gonadotrope cell line (alphaT3-1) was selected as a model since this is the only cell line which expresses a sufficiently high level of GnRH receptors for precise measurements of the mRNA for the receptor. Addition of GnRH agonist (D-Lys6)GnRH to the cell cultures caused a time-dependent decrease in both cell growth, as measured by cell number, and cell proliferation, as measured by [3H]thymidine incorporation into DNA. After 1 h of treatment of alphaT3-1 cells with 1 microM of (D-Lys6)GnRH, the cell number was reduced to 83.0 +/- 13.4 compared to control, decreased to 75.1 +/- 3.2 at 2 h, 63.2 +/- 0.66 at 4 h and 52.2 +/- 0.87 at 24 h. This decrease in cell number was accompanied by a parallel decrease in [3H]thymidine incorporation into DNA. The inhibition of cell growth and [3H]thymidine incorporation by treatment with 1 microM of (D-Lys6)GnRH was sustained for at least 72 h. Inhibition of alphaT3-1 cell growth and [3H]thymidine incorporation was dose-dependent; thus 10(-9) M (D-Lys6)GnRH resulted in about 30% inhibition within 4h which was comparable to 10(-6) M (D-Lys6)GnRH, whereas 10(-12) M (D-Lys6)GnRH was ineffective. Measurement of mRNA for the GnRH receptor by Northern blot analysis showed a decrease in levels of mRNA by 5% within 2 h of treatment of alphaT3-1 cells with 1 microM (D-Lys6)GnRH, by 30% at 4 h and by 50% at 24 h. In conclusion these data demonstrate that treatment of alphaT3-1 cells with (D-Lys6)GnRH causes an inhibition of cell growth and proliferation, and down-regulates the GnRH receptor mRNA levels.

Chronic administration of the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix decreases gonadotrope responsiveness and pituitary LHRH receptor messenger ribonucleic acid levels in rats.

Continuous exposure to LHRH or its agonistic analogs results in a reduction of LHRH receptor sites and messenger RNA (mRNA) transcripts as well as in desensitization of the pituitary gonadotropes. To determine, whether LHRH antagonists might be similar in this respect to the agonists, we treated male rats for 4 weeks with daily sc injections of LHRH antagonist [Ac-D-Nal2,Phe(4Cl)2,D-Pal(3)3, D-Cit6,D-Ala10]LHRH (Cetrorelix acetate) or LHRH agonist, [D-Trp6]LHRH, in doses of 100 micrograms/animal-day. Another group of rats received a single im injection of 4.5 mg Cetrorelix pamoate depot, a sustained delivery formulation of the LHRH antagonist. An iv stimulation test with LHRH (200 ng/rat) was performed after 4 weeks of treatment. The rats were killed, and pituitary LHRH receptor characteristics were measured by RRA. To examine the effect of LHRH antagonist treatment on the expression of the pituitary LHRH receptor gene, some of the rats injected with Cetrorelix pamoate depot were killed after 2 weeks, and levels of LHRH receptor mRNA were determined by Northern blot and dot blot hybridization to a 32P-labeled rat complementary DNA probe. Our data show that LHRH-stimulated LH secretion at 30 min was suppressed by approximately 33% (P < 0.01) in rats pretreated with [D-Trp6]LHRH compared to that in animals injected with LHRH alone. Pretreatment of the rats with the LHRH antagonist suppressed the LH response to LHRH more markedly, the LH levels at 30 min were decreased by 89.8% and 96% in groups treated with Cetrorelix acetate and Cetrorelix pamoate depot, respectively. The testosterone response was virtually abolished in groups receiving Cetrorelix. The concentration of pituitary receptors for LHRH fell by 69% in the [D-Trp6]LHRH group, whereas the reductions in the Cetrorelix acetate group and in the group that received Cetrorelix pamoate depot were 77% and 82%, respectively. Treatment with Cetrorelix pamoate depot led to a 75-80% decrease in the levels of 5.0- and 4.5-kilobase forms of LHRH receptor mRNA compared to those in the control group. Dot blot analysis also showed 83% reduction in the mRNA for LHRH receptor. In conclusion, these data demonstrate that prolonged administration of LHRH antagonists such as Cetrorelix causes an impairment of gonadotropin secretion and a marked decrease in the levels of LHRH receptors as well as in the expression of the LHRH receptor gene. Thus, the down-regulation of pituitary LHRH receptors produced by LHRH antagonists appears to be similar to that resulting from the agonists.

Expression of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor mRNAs in various non-reproductive human tissues.

Recently, cloning of the gonadotropin-releasing hormone (GnRH) receptor from the human breast tumor cell line (MCF-7) and from an ovarian tumor, and its expression in various other human tumors, tumor cell lines and reproductive organs have been reported (Kakar et al., Mol. Cell. Endocrinol., 106 (1994) 145-149). In the present studies, we investigated the expression of GnRH and GnRH receptor mRNAs in normal human non-reproductive tissues. Using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques and specific oligonucleotide primers derived from the placental GnRH cDNA sequence, PCR products of the expected size were obtained from human liver, heart, skeletal muscle, kidney, placenta, and pituitary. The authenticity of the PCR products was confirmed by Southern blot analysis with an internal oligonucleotide primer as probe. Similarly, using specific oligonucleotide primers for the GnRH receptor selected from the human pituitary GnRH receptor cDNA sequence, PCR products of the expected size were amplified from human liver, heart, skeletal muscle, kidney, placenta, and pituitary, and these strongly hybridized with the human GnRH receptor cDNA on Southern blot. Cloning and nucleotide sequencing of the PCR products for the GnRH and GnRH receptor from heart revealed identical sequences when compared to the human placental GnRH and pituitary GnRH receptor cDNAs, respectively. These data demonstrate for the first time the existence of GnRH and GnRH receptor mRNAs in normal human non-reproductive tissues and suggest that GnRH and its receptor may play an important role in the regulation of cellular functions in an autocrine or paracrine manner, in addition to regulating the secretion of gonadotropins from the anterior pituitary.

Expression of the messenger RNAs for luteinizing hormone-releasing hormone (LHRH) and its receptor in human ovarian epithelial carcinoma.

Recently we reported the presence of specific high affinity binding sites for luteinizing hormone-releasing hormone (LHRH) and its analogues (Kd = 1.5 or 1.7 nM) in the human epithelial ovarian cancer cell lines EFO-21 and EFO-27. The proliferation of these cell lines was inhibited by nM concentrations of a LHRH agonist. This study was performed to ascertain whether these ovarian cancer cell lines produce LHRH and whether the high affinity LHRH binding site found previously was identical to the pituitary LHRH receptor. Significant amounts of immunoreactive LHRH were found in the extracts of both the EFO-21 cell line (449 +/- 56 fmol/10(6) cells) and the EFO-27 line (409 +/- 76 fmol/10(6) cells). LHRH bioactivity of these extracts, assessed in terms of release of luteinizing hormone by rat pituitary cells, was comparable to that of authentic LHRH. EFO-21 and EFO-27 cells expressed the mRNAs for both human LHRH and human LHRH receptor as assessed by reverse transcriptase-PCR using oligonucleotide primers according to published sequences. In addition, in eight of eight biopsy samples of human epithelial ovarian cancers we detected mRNA for LHRH, six of these specimens expressed the mRNA representing the LHRH receptor. These data support the concept that human epithelial ovarian cancers might have a local system based on LHRH to regulate cell proliferation. It is still obscure at present whether LHRH produced locally has a stimulatory, inhibitory, or no impact on the proliferation of ovarian cancer cells. However, exogenous LHRH agonists seem to have clear antiproliferative activity, probably mediated through LHRH receptors. This finding might provide the base for novel approaches in the therapy of epithelial ovarian cancer.

The human gonadotropin-releasing hormone receptor gene (GNRHR) maps to chromosome band 4q13.

A cDNA representing the high-affinity gonadotropin-releasing hormone (GnRH) receptor has been molecularly cloned from the human pituitary gland, a breast tumor cell line (MCF 7), and an ovarian tumor. The nucleotide sequence of this cDNA was determined, and its expression in various human tumors and tumor cell lines was demonstrated. In this study, we localized the gene encoding the GnRH receptor to human chromosome 4, using polymerase chain reaction (PCR) analysis of genomic DNA from human x hamster somatic cell hybrids. The gene was sublocalized to chromosome band 4q13 using fluorescence in situ hybridization with the GnRH receptor gene (GNRHR).