Stephen A. Butler

The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation

Ectopic production of free beta human chorionic gonadotrophin (hCGβ) by bladder carcinoma is well described and occurs in approximately 35% of cases. hCGβ secreting tumours are more aggressive, radioresistant and have a greater propensity to metastasize. We proposed that the ectopic production of hCGβ was contributing in an autocrine fashion to the radioresistance and metastatic potential of such secreting tumours. Though we demonstrated that the addition of hCGβ to the culture media of bladder, cervical and endometrial carcinoma cell lines brought about an increase in cell populations this was not accompanied by a significant increase in the rate of replication. Since a cell population size is a balance of mitosis and mortality, we proposed that hCGβ was inhibiting apoptosis. Here we have demonstrated that following incubation with recombinant hCGβ, bladder carcinoma cells refrain from undergoing apoptosis. Quantitation of apoptotic bodies was carried out by immunoassay and corrected to cell number as determined by MTT assay. In each cell line, addition of hCGβ reduced the number of apoptotic bodies dose-dependently, indicating a diminished apoptotic rate. Furthermore, TGFβ1-induced apoptosis could be dose-dependently inhibited by co-incubation with hCGβ. We propose, therefore, that such a decline in apoptosis may account for the cell population increase previously reported. It may also explain the radioresistance and aggressive nature of hCGβ-secreting tumours and the poor prognosis associated therein.

Hyperglycosylated hCG, hCGβ and Hyperglycosylated hCGβ: Interchangeable cancer promoters

INTRODUCTION Several groups are researching cancers, and showing that hCGβ is a promoter of cancer growth and malignancy. Recent research shows that some hCGβ is present as Hyperglycosylated hCGβ. Other groups studied Hyperglycosylated hCG as a promoter of choriocarcinoma and germ cell malignancies. The question therefore arises, are Hyperglycosylated hCG, hCGβ and Hyperglycosylated hCGβ interrelated or interchangable promoters of cancer? METHODS The actions of Hyperglycosylated hCGβ, hCGβ and Hyperglycosylated hCG are investigated in 7 cell lines, Jar and JEG-3 choriocarcinoma cell lines, NTERA germ cell cancer line, SCaBER and T24 bladder epithelial carcinoma lines, KLE and Hec-1-a endometrial adenocarcinoma and epithelial carcinoma cell lines. Actions of promoters on cell growth are investigated. RESULTS The actions of Hyperglycosylated hCG, hCGβ and Hyperglycosylated hCGβ appear to be interchangeable in all cell lines investigated. DISCUSSION All hCG-related cancer promoters seem interrelated, working through a similar mechanism, antagonism of apoptosis through know receptors such as TGFβ receptors in all cancers studied.

Does hCG or hCGβ play a role in cancer cell biology

The role that hCG might play in the oncogenic process in cancer is certainly complex. We know that the expression of hCG and its beta subunit is a widespread phenomenon which has been described in many cancer subtypes. However, hCGs involvement in breast cancer has been antithetical: the detection of ectopically expressed hCG(β) by breast tumors has been employed as a biomarker of malignancy, and hCG has been proposed as a ligand vehicle for toxic drugs, with the aim of targeting the LH/hCG receptor which is reported to be expressed by malignant breast tissue. However, it has also been proposed that hCG is a protective agent against the development of breast cancer, leading some to advocate hCG administration to non-pregnant women as a prophylactic measure against cancer. Nevertheless, suggestions that hCG is involved in the angiogenesis, metastasis and immune escape that are central to cancer progression - are phenomena which clearly apply to breast cancer. Indeed, a tumor vaccine based upon hCG has very recently been shown to protect against mammary tumors in mice. We propose that this apparent paradox is resolved if the free beta subunit of hCG produced by tumors acts as an autocrine anti-apoptotic and angiogenic growth factor, whilst intact heterodimeric hCG, as in pregnancy, is part of developmental signaling that initiates tissue differentiation (including breast ductal tissue development), and hence reduces the population of stem-like cells which are susceptible to oncogenic factors.

Human Chorionic Gonadotropin (hCG) in the Secretome of Cultured Embryos Hyperglycosylated hCG and hCG-Free Beta Subunit Are Potential Markers for Infertility Management and Treatment

Human chorionic gonadotropin (hCG) is produced by trophoblast cells throughout pregnancy, and gene expression studies have indicated that hCG-beta subunit (hCGβ) expression is active at the 2 blastomere stage. Here, we investigated the qualitative hCG output of developing embryos in culture and hCG isoforms expressed in the secretome as a novel sensitive method for detecting hCG. Culture media was collected from the culture plates of 118 embryos in culture (including controls and embryos at different stages of culture) from 16 patients undergoing routine fertility treatment. The hCGβ was detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage. The hCGβ was absent in 1PN and arrested embryos as well as all media controls. Prior to hatching, hyperglycosylated hCG (hCGh) was observed selectively in 3PN embryos, but after hatching, along with hCG, became the dominant hCG molecule observed. We have reported at the 2PN stage the earliest evidence of hCGβ expression in embryos. There is a suggestion this may be indicative of quality in early embryos, and hCGh seen at the pronuclear stage may suggest triploid abnormality. The dominance of hCG, and hCGh expression, seen after blastocyst hatching may be indicative of potential implantation success. Thus, hCG isoforms have potential roles as biomarkers of embryo viability for embryo/blastocyst transfer.

Urinary hyperglycosylated hCG in first trimester screening for chromosomal abnormalities.

Hyperglycosylated human chorionic gonadotrophin (H‐hCG), also known as Invasive Trophoblast Antigen or ITA, is a unique metabolic variant of hCG with more complex oligosaccharide side chains. Concentrations are independent of regular hCG. Urine H‐hCG has recently proved to be a highly sensitive marker for Down syndrome screening in the second trimester of pregnancy. We evaluated H‐hCG as a potential marker in the first trimester of pregnancy. Maternal urine samples were collected from 10+0 to 11+6 weeks of gestation prior to genetic analysis and stored in frozen form. Samples from eight cases of Down syndrome, two cases of trisomy 13, one case of trisomy 18, and 55 control pregnancies were hand‐carried frozen to the USA and tested blindly. Samples were tested in a specific H‐hCG immunoassay and values were normalized to creatinine concentration. Values were plotted against gestational age, and multiples of control pregnancy median (MoM) calculated. The median level of the MoMs of the eight Down syndrome cases was 3.6 MoM. Five of the eight Down syndrome cases exceeded the 90th centile of the 55 unaffected cases. The MoMs of the trisomy 13 and 18 pregnancies were 0.2, 0.2 and 0.3. All three cases were under the 10th centile of unaffected pregnancies. The results of this study indicate that H‐hCG testing may be useful in screening for Down syndrome in the first trimester of pregnancy. Further studies are needed to assess the potential screening values of urine H‐hCG and the combination of this test with free β‐subunit, PAPP‐A and other markers for Down syndrome in the first trimester of pregnancy. Copyright

Dissociation of Human Chorionic Gonadotrophin into its Free Subunits is Dependent on Naturally Occurring Molecular Structural Variation, Sample Matrix and Storage Conditions

Measurement of human chorionic gonadotrophin (hCG) is used in many areas of clinical medicine. Recent interest has focused on the stability of this molecule and its fragments during sample storage. In the body hCG is degraded and removed by specific metabolic processes which begin while the molecule is in the bloodstream. In particular, the receptor binding loop of the β subunit is cut or ‘nicked’, initiating a catabolic cascade. Furthermore, the extent and nature of glycosylation is believed to have a significant influence on this process. In these studies we incubated seven glycoforms of hCG, each with different degrees of ‘nicking’, in phosphate-buffered saline, serum, defibrinated blood and urine from healthy non-pregnant women, under varying conditions. Degradation was expressed as the molar increase in free β subunit. Under all conditions there was a steady dissociation of hCG over time, the process being more rapid at higher temperatures. ‘Nicked’ hCG dissociated more rapidly than did non-‘nicked’ hCG. Glycosylation reduced the rate of dissociation. Dissociation was most rapid in urine and buffer solutions, and slowest in serum and defibrinated blood.

Abnormal secretion of melatonin and cortisol in relation to sleep disturbances in children with Williams syndrome

OBJECTIVE A high rate of sleep disturbances has been reported in individuals with Williams syndrome (WS) but the underlying aetiology has yet to be identified. Melatonin and cortisol levels display circadian rhythmicity and are known to affect and regulate sleep/wake patterns. The current study examined the levels of these two endocrine markers and explored a possible relationship with sleep patterns in children with WS. METHODS Twenty-five children with WS and 27 typically developing age- and gender-matched comparison children were recruited. Saliva was collected from each child at three time points: 4-6 pm, before natural bedtime, and after awakening. The levels of salivary melatonin and cortisol were analysed by specific enzyme-linked immunoassays. Sleep patterns were examined using actigraphy and the Childrens Sleep Habit Questionnaire. RESULTS The WS group had shallower drops in cortisol and less pronounced increase in melatonin at bedtime compared to the controls. Furthermore, they also had significantly higher levels of cortisol before bedtime. CONCLUSIONS Increased bedtime cortisol and less pronounced rise in melatonin levels before sleep may play a role in the occurrence of sleep disturbances, such as delayed sleep onset, observed in children with WS. As both markers play a significant role in our circadian rhythm and sleep/wake cycle, it is necessary to examine sleep using multi-system analysis.

Determination of urinary cortisol, cortisone and 6-sulfatoxymelatonin using dilute and shoot ultra-high pressure liquid chromatography–tandem mass spectrometry

Human sleep is a natural part of every individuals life. Clear relationship between sleep and endocrine system has been already established. In particular, melatonin and cortisol are known to affect and regulate sleep/wake patterns. Here we report the development of an ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of 6-sulfatoxymelatonin (MT6s), cortisol and cortisone in urine. A separate method was developed for measurement of creatinine in urine. These levels were used to normalise the levels of analytes. First void morning urine samples were collected from 24 healthy volunteers. Samples were diluted 1:1 in water prior to injection onto reversed-phase C18 column and analysed using UHPLC-MS/MS method. Linear calibrations were obtained for all analytes with correlation coefficient in the range 0.998-0.999. The observed concentration was found to be in the range 92-105% for cortisol, 92-107% for cortisone and between 93 and 120% for MT6s of the reference levels. The total run time of 6 min with all peaks of interest eluting within 3 min was obtained. This demonstrates the feasibility of utilising the method for large multi-scale studies, where high throughput is required for studying the circadian rhythm of melatonin and cortisol secretion. These hormones play significant role in circadian rhythm and sleep/wake cycle; therefore it is important to monitor the levels of these endocrine markers in individuals suffering from sleep disorders. It is also beneficial with clinical applications to analyse melatonin and cortisol simultaneously in order to assess their interrelationships of these substances, such as their effect on diurnal rhythm and sleep.

Single point biochemical measurement algorithm for early diagnosis of ectopic pregnancy.

OBJECTIVES Tubal rupture as a result of an ectopic pregnancy is the leading cause of first trimester maternal mortality. Currently, the diagnosis of ectopic pregnancy depends on transvaginal ultrasound and serial serum measurements of human chorionic gonadotrophin (hCG), which requires follow up. The objective of this study was to examine whether single point measurements at presentation could distinguish between women with ectopic pregnancy, viable pregnancy, and spontaneous miscarriage. DESIGN AND METHODS Serum total hCG (hCGt), hyperglycosylated hCG (hCGh), free beta subunit of hCG (hCGβ), progesterone (P), and CA-125 were measured by chemiluminescence immunoassay over a 3 month period in 441 women presenting at the emergency room with abdominal pain and a positive pregnancy test. Patient outcomes were followed and confirmed by histology. 65 samples were excluded due to poor sample storage, or lost to follow up. RESULTS The pregnancy outcomes were 175 viable pregnancies, 175 spontaneous miscarriages, and 26 ectopic pregnancies. A serum hCGt <3736 mIU/mL cut off was 100% sensitive, with 76% specificity, for distinguishing ectopic pregnancy from viable pregnancy; but did not differentiate spontaneous miscarriage. Serum CA125 <41.98 U/mL produced 100% sensitivity and 43% specificity in distinguishing ectopic pregnancy from spontaneous miscarriage. Sequential application of hCGt and CA-125 cut off followed by ultrasound could detect 100% of ectopic pregnancies with 87% specificity for all intrauterine pregnancies. CONCLUSION The combination of serum hCGt <3736 mIU/mL, followed by CA125 <41.98 U/mL is a promising algorithm for detecting all ectopic pregnancy at initial presentation.

Biological Function of the Free β-Subunit: Expression and Treatment Target in Cancer

This chapter reviews and reinforces the collective data supporting the notion that the ectopic secretion of hCGβ by common epithelial tumors is an epiphenomenon. Indeed, expression of hCGβ by epithelial cancer is approximately 30%, and in bladder cancer, pancreatic cancer, and colorectal cancer, per se, may be as high as 50%. The expression is associated with resistance to radiotherapy, rapid development of metastasis, and thus poor prognosis. hCGβ influences cell population growth by inhibiting apoptosis in culture—an indirect molecular mechanism consistent with the clinical finding of resistance to radiotherapy. Structural biophysical studies and bioinformatics analysis show that hCGβ can homodimerize; it shares topological and homological features with the cystine-knot family of growth factor proteins. The topological resemblance of TGFβ and VEGF to hCGβ is particularly striking, and hCGβ has been shown to reverse TGFβ-induced apoptosis. We hypothesize that any ectopic hCGβ effect is modulated via the TGFβ-RII receptor and/or the VEGF KDR receptor, bringing about multiple responses associated with the oncogenic process. Whatever the precise mechanism, hCGβ has a growth-modulating function in tumorigenesis which can be removed using specific antibodies. This presents a new, adjuvant approach to treatment strategies pertaining to hCGβ-expressing cancers. One such potential approach is immunodepletive therapy in the form of an anti-hCGβ vaccine.