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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Mayne, Cerys Mary (2011)
Languages: English
Types: Unknown
Subjects:

Classified by OpenAIRE into

mesheuropmc: hormones, hormone substitutes, and hormone antagonists, digestive, oral, and skin physiology, digestive system
The gastrointestinal (GI) hormone, gastrin, promotes cancer progression and its down-regulation has been linked to reduced cancer stem cell numbers. Gastrin acts through the cholecystokinin-2 receptor (CCK-2R) and its biological effects are blocked by CCK-2R inhibitors. We investigated the regulation of the CCK-2R and its potential role in promoting survival of cancer stem cells (CSC). A panel of cancer cell-lines, including GI, glioblastoma and lung, with CCK-2R-transfected cells as a positive control, were grown either as monolayers, or, to provide a 3D in vitro tumour model, as spheres. Linear-after-the-Exponential (LATE)-PCR was used to quantify CCK-2R gene expression and this was validated using siRNAs. Flow cytometry was used to investigate receptor protein expression. Activity of CCK-2R promoter reporters was quantified using luciferase assays. LATE-PCR for CCK-2R gene expression is 10,000-fold more sensitive than the Taqman-based assay, and provides a highly precise method for detection of genes which have important biological functions but low expression. This assay showed that primary non-small-cell lung tumours have significantly more expression than normal lung tissue, indicating a potential therapeutic marker. CCK-2R siRNAs resulted in up to 97% (p<0.05) knockdown of the receptor in cancer cells, confirming the specificity of LATE-PCR and offering a therapeutic possibility. The CCK-2R promoter constructs were active in lung, glioma and colorectal cancer cell-lines, demonstrating a potential drug target; however, transcriptional activity did not correlate with gene expression suggesting post-transcriptional or translational regulation is a factor affecting CCK-2R expression. Flow cytometry suggests the presence of a small population of cells within each of these cell-lines which expresses CCK-2R very highly, which was not correlated to CSC markers. However, CCK-2R expression was enriched when cells were grown as spheres, and inhibition caused a delay in sphere-forming, implying that the CCK-2R may play a role in tumour, and CSC, expansion. Thus, CCK2R provides a potential target for therapeutic intervention in cancer.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1.1.1. Gastric adenocarcinoma ................................................ 16
    • 1.1.2. Oesophageal carcinoma ................................................ 18
    • 1.1.3. Colorectal carcinoma...................................................... 19
    • 1.1.4. Pancreatic carcinoma..................................................... 20
    • 1.1.5. Lung carcinoma.............................................................. 21
    • 1.1.6. Glioblastoma .................................................................. 22 1.2. Cancer stem cells................................................................... 22
    • 1.2.1. Cancer stem cells in tumour formation and growth ........ 24
    • 1.2.2. Cancer stem cell markers............................................... 25 1.3. Hormones in cancer ............................................................... 27
    • 1.4.1. Gastrin in cancer tissues ................................................ 30
    • 1.4.2. Gastrin peptides in carcinogenesis................................. 32 1.5. Regulation of gastrin expression ............................................ 34 1.9. Other gastrin receptors........................................................... 43
    • CCK-2Ri4sv.................................................................... 43
    • Other CCK-2R isoforms ................................................. 44
    • Heterodimerisation of CCK-1R and CCK-2R.................. 45
    • Annexin II ....................................................................... 45 Other GPCRs in relation to the CCK-2R ............................. 46 1.10.1. Orexins ........................................................................... 47 1.10.2. Neuropeptide FF receptors............................................. 47 1.13. Hypothesis and aims........................................................... 51
    • 2.2.1. siRNA design.................................................................. 60
    • 2.2.2. siRNA synthesis ............................................................. 60
    • 2.2.3. Transfection with siRNA ................................................. 61
    • 2.2.4. Reverse transfection with siRNA .................................... 62
    • 2.2.5. Harvesting siRNA-transfected cells for RNA extraction.. 62
    • 2.2.6. Harvesting siRNA-transfected cells for cytospins ........... 62 2.3. Histology................................................................................. 63
    • 2.3.1. Cytospins........................................................................ 63
    • 2.3.2. Live cell staining ............................................................. 63
    • 2.3.3. Flow cytometry ............................................................... 65
    • 2.3.4. Paraffin staining.............................................................. 65 2.4. Molecular biology ................................................................... 66 2.7. Table of materials................................................................... 79 Chapter 3 Detection of the CCK-2R ................................................. 82 3.2. CCK-2R expression in a panel of cell-lines ............................ 83 3.3. CCK-2R Taqman assay sensitivity studies............................. 85 3.4. The LATE-PCR assay ............................................................ 86
    • 3.4.1. Primer design for LATE-PCR ......................................... 88
    • 3.4.2. Design of the molecular probe........................................ 88
    • 3.4.3. Optimisation of the primers (and probe) ......................... 89 3.5. Determining the sensitivity of the LATE-PCR assay............... 92 3.7. CCK-2R expression in primary tumour samples..................... 97
    • 3.7.1. Gastrin expression ....................................................... 100
    • 3.7.2. The correlation between CCK-2R and gastrin expression
    • …………………………………………………...……………………….100 3.8. Validation of LATE-PCR using siRNA molecules directed towards the CCK-2R ...................................................................... 102
    • 3.8.1. siRNA design................................................................ 102
    • 3.8.2. siRNA transfection efficiency........................................ 103 3.9. Knockdown of the CCK-2R in AGS-CCK-2R cells................ 104
    • 3.9.1. Knock-down of the CCK-2R in other cells .................... 106 3.10. The effect of CCK-2R knock-down on expression of other 3.11. CCK-2R expression at the single cell level ....................... 110 3.12. CCK-2R protein expression .............................................. 113
    • 4.1.1. Serum-starvation .......................................................... 122
    • 4.1.2. Hypoxia ........................................................................ 123
    • 4.1.3. Cytotoxic drug treatment .............................................. 124 4.2. Studies with the CCK-2R putative promoter ......................... 127
    • 4.2.1. Sequencing the CCK-2R promoter constructs.............. 129
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