cgb2 And cgb1 Genes; Diagnosis, Monitoring And Treatment Of Cancer

Abstract

The present invention relates to methods for screening human tissue or fluid samples for changes in the expression of genes, in particular CGB2 and CBG1, characteristic of poor prognosis in cancer such as bladder cancer. The methods have application for the screening, diagnosis or monitoring of other common epithelial cancers including those of the bladder, breast, cervix, colon, endometrium, kidney, lung (including small cell lung carcinomaSCLC), nasal/pharynx, oro/facial, ovary, prostate, pancreas, vagina and vulva. Methods of treating cancers by reducing the level of expression of CGB2 and CGB1 or products thereof are also disclosed.

Claims

1-34. (canceled)

35. A kit comprising an agent for detecting and/or measuring the expression product of the CGB2 and/or CGB1 gene(s), wherein the agent is a forward primer which hybridises to a sequence within Exon 1 of the CGB2 and/or CGB1 gene, and/or a reverse primer which hybridises to a sequence after the initiation site in Exon 2 of the CGB2 and/or CGB1 gene.

36. The kit of claim 35, wherein the forward primer hybridises to a sequence at the Exon1/Exon2 boundary the CGB2 and/or CGB1 gene.

37. The kit of claim 35, wherein the forward primer is 5-CGTCCAACACCCCTCACTCC-3 (SEQ ID NO:4).

38. The kit of claim 35, wherein the reverse primer is 5-GGCAGCCCTCCTTCTCCAC-3 (SEQ ID NO:5).

39. The kit of claim 35, wherein the kit comprises a set of primers comprising the forward primer 5-CGTCCAACACCCCTCACTCC-3 (SEQ ID NO:4) and the reverse primer 5-GGCAGCCCTCCTTCTCCAC-3 (SEQ ID NO:5).

40. The kit of claim 35, further comprising an agent for detecting and/or measuring the expression of CGB3, CGB5, CGB7 and/or CGB8.

41. The kit of claim 40, wherein the agent for detecting and/or measuring the expression of CGB3, CGB5, CGB7 and/or CGB8 comprises a set of primers capable of hybridising to the mRNA generated from CGB3, CGB5, CGB7 and/or CGB8.

42. The kit of claim 41, wherein the set of primers comprises the forward primer 5-CAGCACCTTTCTCGGGTCAC-3 (SEQ ID NO:6.) and/or the reverse primer 5-CAGGGAGTAGGGTGTAGGAAGG-3 (SEQ ID NO:7).

43. The kit of claim 35, further comprising primers or hybridisation probes capable of hybridising with a housekeeping gene, used as a control.

44. The kit of claim 43, wherein the housekeeping gene is glyceraldehyde-3-phosphate dehydrogenase (GaPDH).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. An embodiment of the invention will now be described in the following examples which refer to the following figures:

[0107] FIG. 1 provides a Diagrammatic representation of the hCG gene cluster and comparison of different hCG genes.

[0108] A. hCG-LH gene cluster; diagrammatic representation showing relative positions of the CGB genes, LHB and SNARs. Straight arrows show direction of transcription, and open arrowheads represent SNAR-G genes. Start codonsATG.sub.1 and ATG.sub.2 correspond to alternative open reading frame.

[0109] B: Schematic of CGB1 and -2 genes compared to CGB, CGB5, CGB7, CGB8 gene structures, illustrating exon and the intron regions of the CGB genes and indicating the potential three main splice variants reading through into intron 1 (+47 bp, +166 bp, +167 bp). Translated regions are depicted as black boxes; open reading frames are shown as open boxes. SNAR-Gs are located in the upstream region of exon 1 of genes CGB1 and -2 and are shown as a smaller black box along the gene line.

[0110] FIG. 2 shows Electrophoresis and elecropherogram of qPCR products on Microchip Electrophoresis System MultiNA. Separation of real-time PCR products and analysis by electrophoresis system MultiNA. A: Amplification of classical CGB genes by quantitative PCR. The desired product is seen in appropriate lanes: placenta, SCaBER, RT112, T24, C2235 and C2238. B: PCR products obtained during amplification of genes CGB1 and -2. The product 231 bp corresponds to calculated specific product of CGB1 and -2, whilst the primers are the bands below 25 bp. In addition +166 bp splicing variant was amplified (with calculated by MultiNa software mass 396 bp). The position of upper (UM) and lower (LM) marker dyes of the MultiNA separation are indicated, as is the position of the base pair reference mass markers. The ladder (Invitrogen) shows the bands between 25 bp and 450 bp. Any slight difference in size of product is caused by variations in separation on different microchips. Non template control (NTC) and negative control (Neg ctrl) are indicated. Representative electropherograms from MultiNA electrochip system of separated PCR products amplified (A) with CGB, CGB5, -7 and -8 primers with a mass of 81 bp (B) and with CGB1 and -2 specific primers with predominant product with a mass of 231 bp (calculated 229 bp), and less abundant 396 bp. LM and UM were used for calibration according to the manufacturer. The axes of the plot indicates migration index (x) and fluorescence intensity (y). Each peak in the electropherogram represents a DNA fragment of PCR product or primer which migrates according to molecular weight. Migration index is inversely proportional to the length of DNA fragments and is used by software to calculate exact mass.

[0111] FIG. 3 shows Sequencing analysis of CGB1 and -2 qPCR products. Sections (SEQ ID NO:34 and SEQ ID NO:35, respectively) of specific sequencing of CGB1 and -2 PCR products (both 231 bp and 396 bp) were sequenced indicating the single marker nucleotide differences between CGB1 and CGB2 mRNA (at nucleotides 185 and 223-accession numbers NM_033377.1 and NM_033378.1 respectively). This demonstrates that expression of CGB2 mRNA is the predominant source of CGB1/2 RT-PCR product detected in qPCR with CGB-specific primers.

[0112] FIG. 4 shows the Real-time PCR amplification curves to establish crossing points (Cp). Amplification of control clone SCaBER (CGB) gene expression following transfection with Non-Target shRNA Control Vector in comparison to the SCaBER clones stably transfected with shRNA targeting CGB gene. The control clone corresponds to Cp of 16.44, The lowest Cp (23.95) of the CGB-targeted clones corresponds to SCaBER clone 1 and the highest Cp (27.71) corresponds to SCaBER clone 2.

[0113] FIG. 5 shows the Effect of human chorionic gonadotropin (CGB) knockdown by gene-specific short hairpin RNA (shRNA) lentiviral particles. Relative level of (CGB) mRNA measured by real-time PCR (qPCR) after silencing with (shRNA) target construct, amount of hCG in the culture medium estimated by ELISA and number of viable cells measured by MTS. Data were normalised to the level set at 100% measured for non-target shRNA transduced cells. A decreased mRNA level of (CGB) transcript to 38% in clone 1 and 21% in clone 2 was detected by real-time PCR. Level of hCG was reduced for clone 1 (to 20.45% of control) and clone 2 (to 9.09% of control). Cancer cell number was reduced in clone 1 by 55% and in clone 2 by 40%.

[0114] FIG. 6 shows the Effect of silencing of CGB1 and CGB2 and classical CGB genes on bladder cancer cell line SCaBER.

[0115] 6A: Secretion of hCG into culture media of SCaBER cancer cells treated with increasing concentrations of siRNA (targeting CGB1 and CGB2, or CGB, CGB5, -7 and -8). Values are expressed as percentage of untreated control and each point is the mean of six replicates of the assay run in duplicate; the bars represent standard deviation about the mean. EGFP silencing represents non-specific effects of introducing a non-functioning siRNA.

[0116] 6B: Influence of RNA silencing on SCaBER cell viabilityincreasing concentration of siRNA (nmol/l) and relative level of viable cells are shown. MTS assay values are expressed as a percentage that of untreated control. Results are the mean from six replicates of the assay. The bars represent standard deviation about the means. EGFP silencing represents non-specific effects of introducing a non-functioning siRNA.

[0117] It is to be understood that the various features that are described in the following and/or illustrated in the drawings are preferred but not essential. Combinations of features described and/or illustrated are not considered to be the only possible combinations. Unless stated to the contrary, individual features may be omitted, varied or combined in different combinations, where practical.

Example 1: CGB2 and CGB1 Gene Expression

[0118] The human bladder cancer cell lines RT112, SCaBER and T24; breast cancer cell lines C2235 and C2238; prostate cancer LN-CAP and PC-3, were used. Third trimester placenta was used as a positive control.

[0119] The cell lines were obtained from American Type Culture Collection (Manassas, USA) and from the European Collection of Animal Cell Cultures (Porton Down, Dorset, UK). Cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) with 5% antibiotics at 37 C. in 5% CO.sub.2.

[0120] Total RNA was isolated from all cell lines as well as normal term placental tissue (placenta obtained after vaginal delivery in 38th week of pregnancypositive control), and fresh water shrimp (negative control) using SV Total RNA Isolation System (Promega, UK). 1 g of total mRNA was used for first-strand synthesis cDNA by Verso cDNA Kit using oligo-dT primer and random hexamers in a 1:3 ratio (Thermo Scientific, UK).

[0121] Relative quantification of CGB gene expression for the selected breast, bladder and prostate cancer cells lines and placental tissues was carried out in Quantica thermocycler (Techne, Barloworld Scientific Ltd) using ABsolute Blue QPCR SYBR Green Mix kit (Thermo Scientific) with ROX passive reference dye as recommended by the manufacturer. PCR amplification was carried out in a total volume of 25 l, containing 1SYBR Green mix (3 mM MgCl.sub.2, 100 nm ROX, 0.2 mM dNTPs, 1 U Taq DNA polymerase), 70 nM of specific forward and reverse primers (Table 1) and 2 l of cDNA sample. The amplification program consisted of 1 cycle of 95 C. 10 min, followed by 40 cycles of 95 C. for 10 sec, annealing 60 C. for 5 sec, and 72 C. for 9 sec. After termination of 45 cycles, a dissociation curve analysis was performed to confirm melting temperatures of expected products. All primers were designed in NCBI/Primer3BLAST tool and purchased from Sigma (Sigma Life Science). One set of primers were specific for CGB1/2:

forward primer (5-CGTCCAACACCCCTCACTCC-3) (SEQ. ID NO: 4) designed to anneal to nucleotides 118-137 in mRNAs of human chorionic gonadotropin, beta polypeptide 1 (CGB1) and mRNA of human chorionic gonadotropin, beta polypeptide 2 (CGB2) (GeneBank accession no. NM_033377 and NM_033378, respectively), and reverse primer (5-GGCAGCCCTCCTTCTCCAC-3) (SEQ. ID NO: 5) to nucleotides 329-347 of mRNAs. This set of primers span intron 1 and all major splicing variants, which have been described before in placenta, should be detected.

[0122] The second set of primers (F 5-CAGCACCTTTCTCGGGTCAC-3 (SEQ. ID NO: 6) and R 5-CAGGGAGTAGGGTGTAGGAAGG-3) (SEQ. ID NO: 7) were specific for CGB, CGB5, CGB7 and CGB8 mRNAs designed to anneal to nucleotides 10-30 and nucleotides 73-95 in mRNA (GeneBank accession no. NM_033183).

[0123] In order to check the integrity of the reverse transcriptase reaction the expression of the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplified using a specific primer set (F 5-CATGGGTGTGAACCATGAGAAG-3 (SEQ. ID NO: 8) and R 5-GTGCTAAGCAGTTGGTGGTGC-3 (SEQ. ID NO: 9)) was used as an endogenous control (GeneBank accession no. NM_002046). NTC (Non Template Control) contained complete qPCR master mix but no cDNA. As a negative control we used non-human cDNA from shrimp. All experiments were performed in triplicates and mean 20 crossing point (Cp) values were calculated.

[0124] Real-time PCR products we detected by Microchip Electrophoresis System MCE202 MultiNA (Shimadzu) according to manufacturer instructions for DNA-500 kit (Shimadzu). The length of DNA products was estimated based on separation of 25 bp DNA ladder (Invitrogen). Applying MultiNA electrochip system enabled very precise size estimation of qPCR products by MultiNA Viewer software. In addition, to fully identify the qPCR products, they were separated on 1% agarose gel, cut out and purified with PureLink Quick Gel Extraction Kit (Invitrogen), followed by sequencing (forward and reverse direction) by GATC Biotech (London, UK). Blast sequence analysis was performed on generated sequences to identify homologies with those deposited in NCBI gene databases.

[0125] For the calculation of relative quantity of CGB genes we applied Cp method described by Livak and Schmittgen. The level of transcription of CGB1/2 and CGB, CGB5, CGB7, CGB8 in studied cancer cell lines was normalised on the basis of the level of its housekeeping gene GAPDH content and relatively calculated to the level of the gene expression in placental tissue (calibrator). Mean crossing point value was used in calculations. Final results are expressed as N-fold differences in CGB expression relative to calibrator gene expression level equal to 1.0.

[0126] The media from confluent cell cultures grown on 75 cm.sup.2 flasks was collected and assayed to estimate the amount of secreted protein by free hCG assay. Cell numbers were estimated using a haemocytometer following trypsin-EDTA liberation from the flask. A two-site FBT-11 immunoenzymometric assay, described previously, for the highly specific detection of free beta subunit of hCG was used. This system utilizes a unique mouse monoclonal anti-hCG antibodyFBT11directed against epitope 6/7 of hCG as capture antibody and rabbit anti-hCG conjugated with horseradish peroxidise (4001-POD), which is a core antibody recognising the 1 epitope, for detection. A solution of TMB Reagent (Sigma) diluted in deionised water (1:1), was used for quantification of peroxidase and produced a blue product. The reaction was stopped with the addition of 2N HCl and absorbance was measured spectrophotometrically at 450 nm on a FLUOstar OPTIMA (BMG Labtech). The assay was calibrated each time against a standard curve of recombinant hCG (Sigma) at serially diluted concentration range of 50 ng/ml to 0.5 ng/ml, which had been calibrated against the international reference preparation of hCG (NIBSC, Potters Bar, UK). Intact hCG concentrations were quantified using the USA hCG Reference Service in house intact hCG ELISA utilising the antibody combination of McAb 2119 with 4001-POD.

[0127] Results

[0128] The hCG gene cluster was separated into two classes of genes: CGB, CGB 5, 7, 8 and CGB 1/2 using sequence specific primers we amplified genes independently to quantify expression levels. No product appeared to be derived from genomic DNA as no band corresponding to predicted products of 481 or 629 bp (containing the entire intronic sequence) respectively, were amplified.

[0129] In all bladder cancer (SCaBER, T24, RT112); breast cancer (C2235, C2238) and prostate cancer (LN-CAP, PC-3) cell lines tested, transcripts for CGB, CGB5, 7 & 8 were detected (FIG. 2-A). The placental control tissue also revealed the presence of CGB, CGB5, 7, 8 transcripts. The normalised values of average crossing point (Cp) differ between cancer cells and indicates different expression level of CGB genes in selected cancer cell lines. Separation of the qPCR products on the MultiNA system confirmed the presences of the predicted PCR products with calculated mass (FIG. 2-B). The transcripts of CGB1, 2 genes were found only in bladder cancer cell lines SCaBER and RT112 and breast cancer cell line C2235 (FIG. 2-A). All qPCR products were separated by MultiNA electrophoresis, where a 231 bp product was detected (a 229 bp product was predicted). In addition in these cell lines a less abundant high molecular size product of 396 bp was detected for CGB1/2 qPCR (FIG. 2-B). The products (81 bp, 229 bp, 396 bp) were sequenced and align with known CGB GenBank sequences database.

[0130] The 81 bp product arising from CGB(3), 5, 7, 8 qPCR was aligned with the known mRNA and predicted sequences for these CGB genes. For the 229 bp and 396 bp qPCR product for CGB1/2 amplification sequence analysis identified that the product was arising from expression of CGB2 (see FIG. 3) as these vary by two nucleotides at positions 185 and 223 (accession numbers NM_033377.1 & NM_033378.1 respectively for genes 1 and 2). The predominant nucleotides detect at these sequence positions were C and G of CGB2 (GeneBank accession no. NM_033378.1).

[0131] The less abundant high molecular sized qPCR product (396 bpFIG. 2-B), correspond to one of the splicing variant of genes CGB1/2 described previously in testes and placenta. The presence of this splicing variant includes an additional 166 base pairs from intron 1 (165 as determined by the MultiNA separation system). This larger fragment revealed base pair coding corresponding to the predicted inclusion region of intron-1 nucleotide sequences of the CGB genes.

[0132] The level of CGB genes was quantified and compared to the expression level of CGB genes in term placenta. CGB, CGB5, 7, 8 genes among our tested samples were the highest in the placental tissue. Expression of CGB, CGB5, 7, 8 mRNA by cancer cell lines varied but was in most cases was less than 1% of that seen by placental expression. Expression by the bladder cancer cell line T24 was 1.6%, SCaBER 3.1% and by the two breast cancer cell lines 6% (C2335) and 25% (C2238), of that seen in the placental control tissue (See table). mRNA from genes CGB1/2 was detected in two out of three bladder cancer cell lines; SCaBER and RT112 showed significantly higher level of transcripts in comparison to term placenta, both 128 fold higher. The breast cancer cell line C2335 demonstrated a 32 fold higher expression of CGB1/2 mRNA compared to placenta control sample (See table 1). The presence and amount of free hCG protein secreted into the culture medium was measured by specific hCG ELISA. The highest level of hCG protein in medium was seen in the bladder cancer cell line SCaBER (4.4 ng/106 cells/24h), breast cancer cell line C2235 (2.3 ng/106 cells/24h) and bladder cancer cell line RT112 (0.78 ng/106 cells/24h). In the remaining cancer cell lines (T24, PC-3, LN-CAP, C2238) no hCG could be detected in the medium (<0.5 ng/106 cells/24h) using this method. No intact hCG was detected in any culture medium (see Table 1).

Example 2siRNA Directed Silencing of hCG Expression

[0133] Method 1all hCG Approach, Stable Protein Knockdown

[0134] Lentiviral-based shRNAs that generate small interfering RNAs (siRNAs) were used to transduce hCG secreting cancer cells. Two different shRNA gene specific constructs targeting different exonic regions of CGB genes were used in these studies. Stable gene silencing was established by puromycin resistance clone selection. Reduction in the level of CGB mRNA was measured by real-time polymerase chain reaction (PCR) and protein detection by enzyme-linked immunosorbent assay (ELISA). The effect on cell population growth was then estimated by MTS (tetrazolium salt reduction) assay.

shRNA lentiviral transduction. MISSION shRNA Lentiviral Transduction Particles (Sigma Aldrich, Pool, UK) were used to knockdown the CGB in hCG-expressing bladder cancer cell line SCaBER, derived from squamous cell carcinoma of the human urinary bladder (ATCC, Rockville, Md., USA) which have been studied previously. TRC1-pLKO.1-puro vector containing a hairpin insert with gene specific sequence was used for cancer cell transduction according to the manufacturers' protocol in addition to hexadimethrine bromide (8 g/ml) to enhance transduction efficiency. Sequences of inserts in shRNA constructs targeting CGB gene (exon 2 or 3) (Acc. No. NM033043) are shown in Table 3. Stable gene knockdown was established by cellular resistance to puromycin (500 ng/ml). Clones were isolated and several sub-clone cell lines were established. The Non-Target shRNA Control Vector (Sigma Aldrich, Pool, UK) containing an insert sequence that does not target any human gene but can activate RNA-induced silencing complex (RISC) and the RNAi pathway served as a negative control. In addition, MISSION Control Vector pLKO.1-puro (no shRNA insert) (Sigma Aldrich, Pool, UK) along with untreated wild-type cells were used. RNA extraction and cDNA synthesis. Quantitative real-time PCR Total RNA was extracted from the selected cell clones using SV Total RNA Isolation System (Promega, Southhampton, UK). One microgramme of total mRNA was used for first-strand synthesis cDNA by Verso cDNA Kit (Thermo Scientific, Leicestershire, UK). CGB transcript quantity was determined by real-time PCR (Quantica, Techne, Stone, UK); using specific primers designed for amplification of any CGB genes coding hCG protein (NM_033043): forward primer 5-TCACCGTCAACACCACCATCT-3 SEQ. ID NO: 32 and reverse primer 5-ATGGAGTCGAAGCGCACATC-3 SEQ. ID NO: 33 and the level of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; NM_002046) forward primer 5-CATGGGTGTGAACCATGAGAAG-3 (SEQ. ID NO:8) and reverse primer 5-GTGCTAAGCAGTTGGTGGTGC-3 (SEQ. ID NO:9). Real-time PCR was carried out according to the protocol for ABsolute Blue QPCR SYBR Green Mix kit (Thermo Scientific). NTC (Non Template Control) contained complete qPCR master mix but no cDNA template. As a negative control we used non human cDNA from shrimp. All experiments were performed in triplicates and mean crossing point (Cp) values were calculated.

[0135] Relative quantification of the level of CGB mRNA transcripts. Relative quantification of CGB gene expression was calculated using the Cp method described by Livak and Schmittgen. The level of transcription of CGB genes was normalised against the level for the housekeeping gene GAPDH and calculated relatively to the level of the gene expression in the cells transduced with Non-Target shRNA Control Vector (calibrator) using average crossing point values. Final results are expressed as percentage differences in CGB expression relative to calibrator gene expression set at 100%.

[0136] hCG-specific ELISA. Synthesis of hCG and release into culture media was determined by specific free hCG ELISA utilising monoclonal antibody FBT11 directed against epitope 6/7 of hCG and capture antibodyrabbit anti-hCG conjugated with horseradish peroxidise (4001-POD), which is a core antibody recognising 1 epitope. The assay has been validated and described previously. Standards used here were recombinant hCG (Sigma) these were calibrated against 1st International Reference Preparation (IRP) for hCG(NIBSC, Potters Bar, UK) in a concentration range from 0.5 ng/ml to 50 ng/ml. The media from confluent cell cultures grown on 75 cm.sup.2 flasks were collected and assayed to estimate the amount of secreted protein normalised to 110.sup.6 cells.

[0137] Cell proliferation (MTS) assay. For viability assays 100 l of complete growth medium in the cell culture wells was replaced with 20 l of CellTiter 960 AQueous One Solution Cell Proliferation Assay reagent (Promega). The plate was incubated at 37 C. in humidified atmosphere with 95% air, 5% CO.sub.2 for 1-4 hours until colour was well developed and the absorbance was then measured at 490 nm on a Fluostar OPTIMA (BMG Labtech, Aylesbury, UK). Data was normalised against the optical density achieved for the control (set at 100%) and expressed as a percentage change in cell number.

[0138] Results

[0139] Effect of stable CGB gene silencing on protein expression and cell growth. Real-time PCR analysis of clones showed about 60%-80% (clone 1 and clone 2 respectively) reduction in the level of hCG mRNA transcripts when compared to SCaBER cells transduced with the Non-Target shRNA Control Vector (FIG. 4). Similarly, the hCG protein concentration in culture media for clone 1 and clone 2 was reduced to 20% and 9% respectively. hCG concentration in exhausted culture media was reduced to 0.4 and 0.9 ng/ml compared to 4.4 ng/ml in medium from wild type SCaBER cells over the same period and under the same conditions. After 72 hours in culture, populations of sub clone cells, with stable knockdown of CGB expression, were reduced when compared with wild-type SCaBER cells as measured by MTS assay (FIG. 5). There was less than 5% difference in the level of hCG transcripts, protein secretion and growth of cells between control (non-infected cells) and cells exposed to control vector non-target shRNA (see FIG. 5).

[0140] Method 2Specific CGB1/2 Approach, Transient Protein Knockdown

[0141] The siRNA duplexes (Sigma-Aldrich, Gillingham, Dorset, UK) were designed primarily to target mRNA arising from the published sequences for CGB2 (GenBank accession no. NM_033378) and CGB8 (GenBank accession no. NM_033183). These duplex sets were distinct in target but CGB2 siRNA duplexes cross-hybridized significantly (100%) with CGB1, and the CGB8 siRNA duplexes significantly cross-hybridized (>95%) with all classical CGB genes (Table 5). Bladder cancer cell line SCaBER cells were transfected with siRNA duplexes using CodeBreaker siRNA Transfection Reagent (Promega, UK) according to the manufacturer's protocol. A total of 510.sup.3 cells per well were plated into 100 l complete growth medium in 96-well plates (BD Falcon, Oxford, Oxfordshire, UK) and allowed to grow for 24 hours (until they were 50% confluent). Cells were then transfected with siRNA at a final concentration of 15 nM, 20 nM and 25 nM of siRNA in sets of six replicates. Plates were incubated for a further 72 hours at 37 C. and 5% CO.sub.2. A non-specific siRNA targeting enhanced green fluorescent protein (EGFP) was included as a transfection response control. Negative controls were cells which had been treated with the transfection reagents (CodeBreaker siRNA; Promega), but without any siRNA.

[0142] The media from confluent cell cultures grown in 75 cm.sup.2 flasks from before and after specific CGB gene silencing was harvested for hCG protein quantification. hCG concentration was estimated using a two-site FBT-11 immunoenzymetric assay, described previously. Cell numbers, post culture, were estimated using a haemocytometer and used to normalise protein concentrations (110.sup.6 cells). The assay was calibrated against a standard curve of recombinant hCG (Sigma) (range of 50 pg/l to 0.5 pg/l) which had been calibrated against the first international reference preparation of hCG (batch 75/551; NIBSC, Potters Bar, UK). Intact hCG concentrations were quantified using the USA hCG Reference Service in-house intact hCG ELISA, utilising the antibody combination of McAb2119 with 4001-POD. This was calibrated against the 3rd International Standard preparation of hCG (batch 75/589; NIBSC). The limit of detection (lowest standard) for the intact hCG assay was 0.75 pg/I.

[0143] For viability assays, cells were seeded in complete growth medium as described above. Growth medium was replaced with 20 l of CellTiter 960 AQueous One Solution Cell Proliferation Assay reagent (Promega) and cells were incubated at 37 C. for 1-4 hours until colour was well developed in a humidified atmosphere with 95% air, 5% CO2. 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS) is a tetrazolium salt reduction-type assay methodabsorbance at 490 nm was measured on a Fluostar OPTIMA (BMG Labtech, Aylesbury, Buckinghamshire, UK), and optical density was measured and expressed as percentage change relative to control cultures in the 96-well plates. Data were normalised against the optical density achieved for the untreated controls, and expressed as a percentage change in cell number. Data were then pooled between plates and expressed as mean and standard deviation change in normalised cell populations from quintuplicate experiments. Statistical significance was conducted using Stats Direct (Altrincham, Cheshire, UK) software in ANOVA, or Friedman's two-way analysis (data were not normally distributed).

[0144] ResultsExample 2 Method 2

[0145] Following siRNA control transfection, secreted hCG protein levels ranged from 3-4.6 pg/l for bladder cancer cells. siRNA CGB2 oligo duplex (25 nM) reduced secretion by 96%, while CGB8 oligo duplex reduced secretion by 42% of controls. At all concentrations, we observed that siRNA duplexes effectively inhibited CGB expression (FIG. 6A). We further examined whether viability of cells in culture could be changed after silencing to determine the impact of hCG secretion on survival. At all concentrations of siRNA, CGB2 oligo-duplex reduced cell numbers significantly: 25 nM by 44% (p<0.0001), 20 nM by 24% (p=0.0011), and 15 nM by 19% (p=0.02) that of the control (FIG. 6B). In contrast, the control siRNA directed against the sequence of an unrelated protein (EGFP) failed to produce any significant reduction in relative cell numbers, with reduction of only 13% (p=0.22). When CGB8 gene was targeted by siRNA oligo-duplex, only the highest concentration (25 nM) resulted in a significant reduction of cell growth by 23% that of the control (p=0.0004) (FIG. 6B). Non-specific silencing of control EGFP sequence had no effect on cell numbers (1.01%, p=0.42).

[0146] Table 4 provides detail of siRNA sequences used for silencing CGB genes. siRNA sequences used for silencing CGB2 (and 1) and CGB8 (and CGB, CGB5, -7 and -8); their location on mRNA mapping to base-pairs on exon 1 (according to GenBank accession number NM_033378 for CGB2 and NM_033183 for CGB8, respectively) and the degree of homology between the siRNA duplexes and the different CGB gene sequences.

[0147] In this specification, the word preferable (or any of its derivatives) indicates one feature or more that is preferred but not essential.

[0148] All or any of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all or any of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0149] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0150] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

TABLE-US-00001 TABLE 1 Placental Placental Secretion Secretion Normalised Normalised Relative Relative Immuno- of hCG of hCG Cell line Cp CGB(3), Cp Level CGB(3), Level staining (ng/1 10.sup.6 (ng/1 10.sup.6 or tissue 5, 7, 8 CGB1/CGB2 5, 7, 8 CGB1, 2 for hCG cells/24 h) cells/24 h) Placenta 22 34 1 1 + + + SCaBER 27 27 0.0313 128 + 4.4 <0.5 RT112 30 27 0.0039 128 + + 0.78 <0.5 T24 28 ND 0.0156 ND + + + <0.5 <0.5 PC-3 31 ND 0.002 ND + + + <0.5 <0.5 LN-CAP 30 ND 0.0039 ND + + <0.5 <0.5 C2235 26 29 0.0625 32 + 2.3 <0.5 C2238 24 ND 0.25 ND + + + <0.5 <0.5

TABLE-US-00002 TABLE2 Peptidesequencedifferences(italics)andhomology(bold) betweenCGB3,CGB5CGB7,CGB8mRNACGB2/1230splicevariantiftranslated. Genevariant Prepeptide Maturepeptide CB5,CGB3,CGB7and MEMFQGLLLLLLLSMGGTWA SKEPLRPRCRPINATLAVEKEGC...(remainingmature CGB8correctlysplicedat peptidesequence- exon1toexon2boundary. PVCITVNTTICAGYCPTMTRVLQGVLPALPQVVCNYRDVRFESIRLP Peptidesequence GCPRGVNPVVSYAVALSCQCALCRRSTTDCGGPKDHPLTCDDPRFQD SSSSKAPPPSLPSPRLPGPSDTPILPQ) CGB2230bpsplicevariant MSTSPVLAEDIPLRERHVKGAAAVAAAEHGRDMGIQGAASATVPPHQCHPGCGEGGL... identified:peptidesif (continuedmismatchduetoframeshift)... translationinitiatedat firstATG CGB2230bpsplicevariant MSKGLLLLLLLSMGGTWA SKEPLRPRCRPINATLAVEKEGC...(Remainingmature identified:Peptidesif peptidesequencematch)... translationinitiatedat 2.sup.nd(consensus)ATG

TABLE-US-00003 TABLE3 AshorthairpinRNA(shRNA)whichtargetsallhumanchorionicgonadotropin (CGB)genesdesignedagainstaCGB5wasconstructedwithinalentivirus vectorpLKO.1-Puro.Theinsertedhairpinsequencecontainsgene-specifictarget sequence(highlightedingrey)andloopregionwhichcorrespondtosequenceCTCGAG. Table3 Targetgeneandexon shRNAlentiviralparticles SequencetargetingallCGBgenes positionofsense andclonenumber insertedintopLKO.1-purovector andantisensesequences TRCN0000082824 GTGGTGTGCAACTACCGCGATCTCGAGATCGCGG CGB5mRNANM_033043exon3 clone1 TAGTTGCACACCAC Sensestrand:nt588-608 Antisensestrand:nt588-608 TRCN0000082826 CCGTGTGCATCACCGTCAACACTCGAGTGTTGAC CGB5mRNANM_033043exon2 clone2 GGTGATGCACACGG Sensestrand:nt496-515 Antisensestrand:nt517-503

TABLE-US-00004 TABLE4 Targetgene Nucleotideposition Between-genehomology(%) inexon1 siRNAduplexSequence CGB1 CGB2 CGB3 CGB5 CGB7 CGB8 CGB2 Sense 1: 100 100 37 37 37 42 CAGUGCUUGCGGAAGAUAUdTdT position:188 Antisense 1: AUAUCUUCCGCAAGCACUGdTdT CGB2 Sense 2: 100 100 42 37 37 37 CACGGAGACUCAAUUUACUdTdT position:152 Antisense 2: AGUAAAUUGAGUCUCCGUGdTdT CGB2 Sense 3: 100 100 42 42 42 42 CGCUAAGAGAGAGACAUGUdTdT position:209 Antisense 3: ACAUGUCUCUCUCUUAGCGdTdT CGB8 Sense 1: 28 28 84 95 100 100 UCACUCCAGCAUCCUACAAdTdT position:280 Antisense 1: UUGUAGGAUGCUGGAGUGAdTdT CGB8 Sense 2: 42 42 95 95 95 100 AGGUUUAAAGCCAGGUACAdTdT position:322 Antisense 2: UGUACCUGGCUUUAAACCUdTdT CGB8 Sense 3: 0 0 100 95 95 100 AGUCUCUGAGGUCACUUCAdTdT positionl135 Antisense 3: UGAAGUGACCUCAGAGACUdTdT EGFP Sense 1: 0 0 0 0 0 0 GCAAGCUGACCCUGAAGUUCAUdTdT Antisense 1: GAACUUCAGGGUCAGCUUGCCGdTdT