Characterization of beta subunit variants of recombinant human chorionic gonadotrophin.

Human chorionic gonadotropin (hCG), an endogenous glycoprotein hormone, has been widely used for the treatment of infertility and corpus luteum defect in women. The biological specificity of hCG is essentially determined by its beta (ß-) subunit, whereas the alpha (α-) subunit is a common subunit shared among the gonadotropin family. In development of a therapeutic recombinant hCG, the purity analysis showed that the beta (ß-) subunit has two variants, ß1 and ß2. Structural characterization using a combination of analytical techniques has demonstrated that ß1-subunit is derived from non-glycosylation at Asn 13, whereas ß2-subunit is a normal species with complete N-glycosylation at both Asn 13 and Asn 30. In vivo Bioactivity evaluation of the r-hCG fractions with various ratios of ß1-and ß2-subunits showed that incomplete glycosylation at Asn 13 potentially reduced the biological activity of r-hCG to promote uterus growth. Although hCG has a long history of medicinal use, this is the first report to identify the structural difference of hCG ß-subunit variants, as well as to preliminary establish the structure-activity relationship of this variation. The obtained results also suggest the importance of variant characterization and necessary quality control of product variants during the development of recombinant protein therapeutics.

Hyperglycosylated hCG activates LH/hCG-receptor with lower activity than hCG.

While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it is controversial whether the hyperglycosylated form of hCG (hCG-h), which is the major hCG isoform during the first 4-5 weeks of pregnancy, is able to activate LH/hCG receptor (LHCGR). To address this, we utilized different extensively characterized hCG and hCGß reference reagents, cell culture- and urine-derived hCG-h preparations, and an in vitro reporter system for LHCGR activation. The WHO hCG reference reagent (99/688) was found to activate LHCGR with an EC50-value of 3.3 ±â€¯0.6 pmol/L (n = 9). All three studied hCG-h preparations were also able to activate LHCGR, but with a lower potency (EC50-values between 7.1 ±â€¯0.5 and 14 ±â€¯3 pmol/L, n = 5-11, for all P < 0.05 as compared to the hCG reference). The activities of commercial urinary hCG (Pregnyl) and recombinant hCG (Ovitrelle) preparations were intermediate between those of the hCG reference and the hCG-h. These results strongly suggest that the hCG-h is functionally similar to hCG, although it has lower potency for LHCGR activation. Whether this explains the reduced proportion of hCG-h to hCG reported in patients developing early onset pre-eclampsia or those having early pregnancy loss remains to be determined.

Proteomic Profiling of ß-hCG-Induced Spheres in BRCA1 Defective Triple Negative Breast Cancer Cells.

Previously, we identified that ß-hCG is expressed by BRCA1 mutated but not wild type breast cancers in vitro/in vivo and exhibited a novel event in ß-hCG overexpressing BRCA1 mutated HCC1937 cells where the cells were able to form spheres (HCC1937 ß spheres) in adherent cell culture plates even in the absence of any growth factors. These spheres express stem cell and EMT markers. In the present study, we carried out the total proteomic profiling of these HCC1937 ß spheres obtained from BRCA1 defective ß-hCG expressing stable breast cancer cells to analyze the cell signaling pathways that are active in these cells. Functional annotation revealed proteins (164 cellular and 97 secretory) predominantly involved in oxygen binding, nucleosome assembly, cytoskeleton organization, protein folding, etc. Many of the proteins identified from HCC1937 ß spheres in this study are also up regulated in breast cancers, which are directly linked with poor prognosis in human cancer samples as analyzed using TCGA data set. Survival analysis shows that ß-hCG expressing cancer patients are linked with poor survival rate. Interestingly, hemoglobins were identified at both cellular and secretory level in HCC1937 ß spheres and experiments after treating with ROS inducers revealed that ß-hCG induces hemoglobin and protects the cancer cells during oxidative stress. Our proteomic data strongly propose ß-hCG as an oncogenic molecule associated with BRCA1 mutation, and hence, targeting ß-hCG could be a strategy to treat BRCA1 defective breast cancers.

Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation.

Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.

Oligopeptides of Chorionic Gonadotropin ß-Subunit in Induction of T Cell Differentiation into Treg and Th17.

The role of oligopeptides of chorionic gonadotropin ß-subunit (LQGV, AQGV, and VLPALP) in induction of differentiation into T-regulatory lymphocytes (Treg) and IL-17-producing lymphocytes (Th17) was studied in an in vitro system. Chorionic gonadotropin and oligopeptides promoted CD4(+) cell differentiation into functionally active Treg (FOXP3(+)GITR(+) and FOXP3(+)CTLA-4(+)), while chorionic gonadotropin and AQGV additionally stimulated IL-10 production by these cells. In parallel, chorionic gonadotropin and oligopeptides prevented CD4(+) cell differentiation into Th17 lymphocytes (ROR-gt(+)IL-17A(+)) and suppressed IL-17A secretion. Hence, oligopeptides of chorionic gonadotropin ß-subunit promoted differentiation of CD4(+) cells into Treg and, in parallel, suppress Th17 induction, thus virtually completely reproducing the effects of the hormone, which opens new vista for their use in clinical practice.

Human chorionic gonadotropin ß induces cell motility via ERK1/2 and MMP-2 activation in human glioblastoma U87MG cells.

Human chorionic gonadotropin ß (hCGß) promotes tumorigenesis in a variety of tumors including glioblastoma, breast and prostate cancer cells, etc. However, the involved mechanisms remain elusive. Distinct from the other tumors, glioblastoma is a highly invasive brain tumor; invasion causes high recurrence and mortality. Characterization of hCGß signaling is to determine therapeutic targets to inhibit invasion and lower recurrence. Through both a stable cell line over-expressing hCGß and hCGß standards, we tested hCGß signaling, migration and invasion in human glioblastoma U87MG cells. ELISA showed that hCGß secreted into culture medium at an amount of 237.8 ± 7.8 ng/10(7) cells in hCGß transfected stable cells after the cells were grown for 24 h. Through Western blot and Gelatin zymography, we found that hCGß standards phosphorylated ERK1/2 and upregulated MMP-2 expression in dose- and time-dependent manners. Meanwhile, overexpressed hCGß phosphorylated ERK1/2, and upregulated MMP-2 expression and activity, whereas ERK1/2 blocker PD98059 (25 µM) significantly decreased both ERK1/2 and MMP-2 expression and activity. In addition, in the same conditions as the signaling test, hCGß promoted cell migration and invasion, whereas the PD98059 diminished these effects. These findings demonstrated that hCGß phosphorylated ERK1/2 upregulating MMP-2 expression and activity leading to cell migration and invasion, suggesting that hCGß, ERK1/2 and MMP-2 are the potential targets to inhibit glioblastoma invasion.

Influences of ß-HCG administration on carbon isotope ratios of endogenous urinary steroids.

Several factors influencing the carbon isotope ratios (CIR) of endogenous urinary steroids have been identified in recent years. One of these should be the metabolism of steroids inside the body involving numerous different enzymes. A detailed look at this metabolism taking into account differences found between steroids excreted as glucuronides or as sulphates and hydrogen isotope ratios of different steroids pointed out possibility of unequal CIR at the main production sites inside the male body - the testes and the adrenal glands. By administration of ß-HCG it is possible to strongly stimulate the steroid production within the testes without influencing the production at the adrenal glands. Therefore, this treatment should result in changed CIR of urinary androgens in contrast to the undisturbed pre-treatment values. Four male volunteers received three injections of ß-HCG over a time course of 5 days and collected their urine samples at defined intervals after the last administration. Those samples showing the largest response in contrast to the pre-administration urines were identified by steroid profile measurements and subsequent analysed by GC/C/IRMS. CIR of androsterone, etiocholanolone, testosterone, 5α- and 5ß-androstanediol and pregnanediol were compared. While pregnanediol was not influenced, most of the investigated androgens showed depleted values after treatment. The majority of differences were found to be statistically significant and nearly all showed the expected trend towards more depleted δ(13)C-values. These results support the hypothesis of different CIR at different production sites inside the human body. The impact of these findings on doping control analysis will be discussed.

Chorionic gonadotropin and its receptor are both expressed in human retina, possible implications in normal and pathological conditions.

Extra-gonadal role of gonadotropins has been re-evaluated over the last 20 years. In addition to pituitary secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), the CNS has been clearly identified as a source of hCG acting locally to influence behaviour. Here we demonstrated that human retina is producing this gonadotropin that acts as a neuroactive molecule. Müller glial and retinal pigmented epithelial (RPE) cells are producing hCG that may affects neighbour cells expressing its receptor, namely cone photoreceptors. It was previously described that amacrine and retinal ganglion (RGC) cells are targets of the gonadotropin releasing hormone that control the secretion of all gonadotropins. Therefore our findings suggest that a complex neuroendocrine circuit exists in the retina, involving hCG secreting cells (glial and RPE), hCG targets (photoreceptors) and hCG-release controlling cells (amacrine and RGC). The exact physiological functions of this circuit have still to be identified, but the proliferation of photoreceptor-derived tumor induced by hCG demonstrated the need to control this neuroendocrine loop.

Triggering final oocyte maturation with reduced doses of hCG in IVF/ICSI: a prospective, randomized and controlled study.

OBJECTIVE: To compare the effectiveness of urinary human chorionic gonadotropin (u-hCG) at reduced doses of 4000 IU and 6000 IU in inducing final oocyte maturation during in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles. STUDY DESIGN: 164 patients with an indication for IVF or ICSI recruited in this randomized, single-blinded and controlled study in IVF clinic at the Sun Yat-sen Memorial Hospital. Patients were prospectively randomized to receive 4000 IU (Group A, n=83) and 6000 IU (Group B, n=81) of hCG for triggering final oocyte maturation. Number or percentage of mature oocytes retrieved per patient, fertilization rates, pregnancy rates were the main outcome measures. RESULTS: No evidence of statistically significant difference in the number or proportion of mature oocytes retrieved was observed in both groups. The lower fertilization rate and significantly lower clinical pregnancy rate were observed in Group A. The ovarian hyperstimulation syndrome (OHSS) rates in both groups were also similar. In the subgroup of BMI< 20 kg/m(2), fertilization rate were significantly higher in the administration group of hCG at the dose of 6000 IU when compared with the dose of 4000 IU (82.40% vs. 70.92%, P=0.017); in contrast, no significant difference in clinical pregnancy rates was observed in both groups. In the subgroup of BMI 20-25 kg/m(2), clinical pregnancy rates were significantly higher in patients treated with hCG at dose of 6000 IU than patients treated with hCG at dose of 4000 IU (65.3% vs. 35.0%, P=0.004); however, no significant difference in fertilization rates was observed. CONCLUSION: Both doses of u-hCG revealed an equal effect on the induction of final oocyte maturation in the patients with moderate or high ovarian response; however, the reduced dose of hCG could result in an obvious impact on clinical pregnancy rates and did not exhibit an obvious effect on OHSS rates.

Logistic regression when covariates are random effects from a non-linear mixed model.

In many studies, the association of longitudinal measurements of a continuous response and a binary outcome are often of interest. A convenient framework for this type of problems is the joint model, which is formulated to investigate the association between a binary outcome and features of longitudinal measurements through a common set of latent random effects. The joint model, which is the focus of this article, is a logistic regression model with covariates defined as the individual-specific random effects in a non-linear mixed-effects model (NLMEM) for the longitudinal measurements. We discuss different estimation procedures, which include two-stage, best linear unbiased predictors, and various numerical integration techniques. The proposed methods are illustrated using a real data set where the objective is to study the association between longitudinal hormone levels and the pregnancy outcome in a group of young women. The numerical performance of the estimating methods is also evaluated by means of simulation.

The ß-human chorionic gonadotropin-related peptide LQGV reduces mortality and inflammation in a murine polymicrobial sepsis model.

OBJECTIVE: Mortality in sepsis remains high and efforts to modulate the inflammatory response so far mostly failed to improve survival. The human chorionic gonadotropin-related tetrapeptide LQGV was recently shown to exert anti-inflammatory activity. The aim of this study was to assess the effect of LQGV on cecal ligation and puncture-induced mortality and inflammation. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Male C57BL/6 mice. INTERVENTIONS: To examine the effect of LQGV by itself on cecal ligation and puncture-induced mortality and inflammation, C57BL/6 mice were exposed to a moderate cecal ligation and puncture procedure (40% ligation and double puncture) with a mortality rate of approximately 80% within 5 days in control mice. In addition, to examine whether LQGV was of additive value to standard sepsis care (antibiotics and fluid resuscitation), a more severe cecal ligation and puncture procedure was used (80% ligation and double puncture), yielding approximately 100% mortality within 12 days in control mice. LQGV (5 mg/kg body weight), phosphate-buffered saline (as control), or dexamethasone (2.5 mg/kg body weight) was administered perioperatively. Survival was monitored for 21 days and inflammatory markers were determined in plasma, peritoneal cavity, and lungs. MEASUREMENTS AND MAIN RESULTS: LQGV significantly improved survival from 20% to 50% during the first 5 days after moderate cecal ligation and puncture. This was associated with reduced cytokine and E-selectin levels in peritoneal lavage fluid, lungs, and, to a lesser extent, in plasma. LQGV treatment also reduced pulmonary nuclear factor-κB activation and pulmonary damage. In the severe cecal ligation and puncture model, LQGV combined with fluid resuscitation and antibiotics resulted in significantly better survival (70%) than that observed with fluid resuscitation and antibiotics alone (30%). CONCLUSIONS: LQGV improves survival after cecal ligation and puncture. This is likely established by a modest reduction of the acute inflammatory response through a nuclear factor-κB-dependent mechanism. Furthermore, LQGV may be a valuable additive next to the standard care in polymicrobial sepsis.

The beta-human chorionic gonadotropin-related peptide LQGV exerts anti-inflammatory effects through activation of the adrenal gland and glucocorticoid receptor in C57BL/6 mice.

The systemic inflammatory response syndrome is a complex host response to a variety of clinical insults, generally leading to severe pathology. The human chorionic gonadotropin ß-chain-related tetrapeptide leucine-glutamine-glycine-valine (LQGV) reduces hemorrhagic and LPS-induced systemic inflammatory response syndrome, but its mechanisms of action are not yet fully understood. Through the combination of in vivo, in vitro, and ex vivo approaches, we demonstrate that LQGV actively stimulates corticosterone production in mice and thereby suppresses in vivo TLR4-directed inflammation upon LPS administration. Blocking in vivo glucocorticosteroid receptor signaling reduced the prosurvival effect of LQGV. Also, upon multiple TLR activation by heat-killed Listeria monocytogenes, splenocytes from LQGV-treated mice produced significantly less TNF-α and IL-6, which was absent after in vitro blockage of the glucocorticosteroid receptor. Using adrenal gland and adrenal cell line cultures, we show that LQGV stimulates corticosterone production. Moreover, by using specific pharmacological inhibitors of the adrenocorticotropic hormone (ACTH) and luteinizing hormone receptors as well as of cAMP signaling, we demonstrate that LQGV stimulates the ACTH receptor. These data show that the ß-human chorionic gonadotropin-related tetrapeptide LQGV stimulates adrenal glucocorticosteroid production through activation of the ACTH receptor with consequent glucocorticoid receptor activation and immunosuppression in C57BL/6 mice.

Administration of betaHCG leads to dose-dependent changes of gene expression signature of endometriotic stromal cells.

Preliminary studies have shown that systemic beta-human chorionic gonadotrophin (betaHCG) therapy alleviates endometriosis-related chronic pelvic pain. The underlying mechanism, however, is completely unknown. This study has investigated the dose-dependent alterations in the overall gene expression profile of endometriosis-derived stromal cells under increasing concentrations of betaHCG by using the Affymetrix GeneChip U133 Set. It has been previously shown that betaHCG concentrations of 0.1U/ml and higher lead to a significant and dose-dependent increase in the expression of 68 genes. This study reports on a cluster analysis which identified three clusters of genes with a comparable expression pattern in response to increasing concentrations of betaHCG. Most of the up-regulated genes encoded proteins that are involved in cell adhesion, intercellular communication, extracellular matrix remodelling, apoptosis and inflammation. Stromal monocultures from eight patients, treated with and without 50U/ml of betaHCG, were then incubated and real-time polymerase chain reaction for the highly up-regulated genes PAI2, DUSP6, PLAU and MMP1 performed in order to validate the cDNA array findings in patients with endometriosis. Taken together, this study shows that betaHCG induces dose-dependent characteristic response clusters in the gene expression profile of stromal cells obtained from endometriotic lesions which could explain the differential biological responses of betaHCG in endometriosis.

Amelioration of renal ischaemia-reperfusion injury by synthetic oligopeptides related to human chorionic gonadotropin.

BACKGROUND: We have previously reported that small synthetic oligopeptides related to human beta-chorionic gonadotropin (beta-hCG) can reduce inflammation. Here we investigated whether such oligopeptides can reduce renal ischaemia-reperfusion injury in the mouse. METHODS: Ten different oligopeptides were administered 1 min before induction of renal ischaemia and 1 min before reperfusion. RESULTS: Survival at 72 h post-reperfusion was significantly higher in mice treated with oligopeptides MTRV, LQG, VLPALPQ or AQGV as compared to placebo-treated mice. Some oligopeptides were more effective than others. AQGV completely prevented mortality and best preserved kidney function. Next, AQGV was tested in a dose-escalating study in a range of 0.3-30 mg/kg. A survival gain was observed with all doses. Improvement of kidney function was observed from 1 mg/kg. Highest survival and best preserved kidney function were observed at 3 and 10 mg/kg. Upon treatment with AQGV, a significantly lower influx of neutrophils was found, apoptosis was decreased, whereas tubular epithelial cell proliferation was significantly increased at 24 h post-reperfusion. Serum levels of TNF-alpha, INF-gamma, IL-6 and IL-10 were significantly decreased at 24 h post-reperfusion. E-selectin mRNA levels in kidneys were significantly decreased at 6 h post-reperfusion. AQGV did not reduce mortality when treatment was started after reperfusion. CONCLUSIONS: This study shows that small oligopeptides related to the primary structure of beta-hCG, especially AQGV, are promising potential drugs for preventing the development of renal ischaemia-reperfusion injury.

Targeted therapy for adrenocortical tumors in transgenic mice through their LH receptor by Hecate-human chorionic gonadotropin beta conjugate.

Novel strategies are needed for the treatment of adrenocortical tumors that are usually resistant to chemotherapy. Hecate, a 23-amino acid lytic peptide, was conjugated to the 15-amino acid (81-95) fragment of the human chorionic gonadotropin beta (CGbeta) chain, which would selectively kill cancer cells expressing the LH receptor (LHR) sparing the normal ones with LHR. To prove the principle that Hecate-CGbeta conjugate may eradicate tumors ectopically expressing plasma membrane receptors, transgenic (TG) inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen mice, expressing LHR in their adrenal gland tumors, were used as the experimental model. Wild-type control littermates and TG mice with adrenal tumors were treated with either Hecate or Hecate-CGbeta conjugate at the age of 6.5 months for 3 weeks and killed 7 days after the last treatment. The Hecate-CGbeta conjugate reduced the adrenal tumor burden significantly in TG male but not in female mice, in comparison with Hecate-treated mice. Hecate-CGbeta conjugate treatment did not affect normal adrenocortical function as the serum corticosterone level between Hecate and Hecate-CGbeta conjugate groups were similar. The mRNA and protein expressions of GATA-4 and LHR colocalized only in tumor area, and a significant downregulation of gene expression was found after the Hecate-CGbeta conjugate in comparison with Hecate- and/or non-treated adrenal tumors by western blotting. This finding provides evidence for a selective destruction of the tumor cells by the Hecate-CGbeta conjugate. Hereby, our findings support the principle that Hecate-CGbeta conjugate is able to specifically destroy tumor cells that ectopically express LHR.

Conjugates of lytic peptides and LHRH or betaCG target and cause necrosis of prostate cancers and metastases.

In a series of in vivo and in vitro experiments, it was shown that membrane disrupting lytic peptides (Hecate, Phor14, or Phor21) conjugated to a 15 amino acid segment of the beta chain of CG or to LHRH were able to target and destroy hormone dependent and independent human prostate cancer xenografts in nude mice. In vitro sensitivity of the cells to the drugs was directly related to LH/CG receptor expression, and pretreatment in vitro or in vivo with estrogens or FSH to enhance LH/CG receptor expression capacity and increased sensitivity to the drugs. Administration of unconjugated Hecate and LHRH was ineffective. Most importantly, all of the lytic peptide-betaCG conjugates tested were highly effective in destroying prostate cancer metastatic cells in lymph nodes, bones and lungs.

A delayed gonadotropin-dependent and growth factor-mediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates aromatase expression in granulosa cells.

Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.

LHRH-conjugated magnetic iron oxide nanoparticles for detection of breast cancer metastases.

Targeted delivery of superparamagnetic iron oxide nanoparticles (SPIONs) could facilitate their accumulation in metastatic cancer cells in peripheral tissues, lymph nodes and bones and enhance the sensitivity of magnetic resonance imaging (MRI). The specificities of luteinizing hormone releasing hormone (LHRH) and luteinizing hormone/chorionic gonadotropin (LH/CG)- bound SPIONs were tested in human breast cancer cells in vitro and were found to be dependent on the receptor expression of the target cells, the time of incubation and showed saturation kinetics. In incubations with MDA-MB-435S.luc cells, the highest iron accumulation was 452.6 pg Fe/cell with LHRH-SPIONs, 203.6 pg Fe/cell with beta-CG-SPIONs and 51.3 pg Fe/cell with SPIONs. Incubations at 4 degrees C resulted in 1.1 pg Fe/cell. Co-incubation with the same ligands (betaCG or LHRH) decreased the iron accumulation in each case. LHRH-SPIONs were poorly incorporated by macrophages. Tumors and metastatic cells from breast cancer xenografts were targeted in vivo in a nude mouse model. LHRH-SPION specifically accumulated in cells of human breast cancer xenografts. The amount of LHRH-SPION in the lungs was directly dependent on the number of metastatic cells and amounted to 77.8 pg Fe/metastastic cell. In contrast, unconjugated SPIONs accumulated in the liver, showed poor affinity to the tumor, and were not detectable in metastatic lesions in the lungs. LHRH-SPION accumulated in the cytosolic compartment of the target cells and formed clusters. LHRH-SPIONs did not accumulate in livers of normal mice. In conclusion, LHRH conjugated SPIONs may serve as a contrast agent for MR imaging in vivo and increase the sensitivity for the detection of metastases and disseminated cells in lymph nodes, bones and peripheral organs.

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo.

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.