| A ROLE FOR THE HEPARIN-BINDING GROWTH-PROMOTING CYTOKINES MIDKINE AND PLEIOTROPHIN IN BPH!
Dr Martin G Rumsby, Professor Norman Maitland University of York, Department of Biology |
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INTERIM PROGRESS REPORT
Duration of project: August 2007 to August 2008.
Timescale of research programme:
August-September 2007: Establish cell cultures of human prostate cells including stromal cells isolated from prostate biopsy samples. Characterise the growth patterns of the prostate cells.
September-November 2007: Analyse cultured prostate cells, especially stromal cells, to determine if they synthesise the two novel proteins of interest (midkine and pleiotrophin) and secrete them to the medium where they could diffuse in the tissue and act on other neighbouring cells to influence their properties such as proliferation.
November-December 2007: Characterise the cells for cell surface receptor proteins which the midkine and/or pleiotrophin might bind to to produce intracellular effects. Start to analyse prostate cancer tissue to determine if midkine and pleiotrophin levels are elevated in the disease compared with control areas of tissue and with tissue sections from benign prostate hyperplasia tissue.
December-March 2008: Dr M Sharrard was co-opted into the lab to undertake the molecular biology needed to establish a cell system to produce midkine and pleiotrophin for purification. Continue to analyse sections of prostate cancer tissue supplied by Dr G Rodrigues, Hull Royal Infirmary.
March-April 2008: Test purified midkine and pleiotrophin on prostate cells to characterise their growth-promoting, motility and anti-cell death effects. Commence analysis of tissue sections from benign prostate hyperplasia tissue to determine if levels of midkine and pleiotrophin are elevated compared with control tissue.
May-project end: Continue analysis of tissue sections from BPH cases to accumulate sufficient numbers to draw reliable conclusions. Determine effects of secretions from prostate stromal cells on epithelial cell growth. Determine if midkine and pleiotrophin protect prostate stromal and epithelial cells from apoptosis (programmed cell death). If time allows examine intracellular pathways by which midkine and pleiotrophin influence cell growth and could prevent programmed cell death.
Specific work carried out during this reporting period:
1. Cultures of prostate epithelial cell lines have been established. These cultures have all been analysed for synthesis and secretion of midkine and pleiotrophin carrying out experiments in triplicate. In addition, the cells have been analysed to determine which surface receptors for pleiotrophin and midkine they contain.
2. Cultures of prostate stromal cells have been set up. We have established that prostate stromal cells synthesise and secrete both of the novel proteins of interest-pleiotrophin and midkine, but only secrete pleiotrophin. This is an interesting observation as it shows that the cells handle the two closely related proteins rather differently. This will be investigated further if time allows.
3. We have established two methods for quantitating levels of midkine and/or pleiotrophin in culture fluid and blood.
4. We have worked up a method to detect pleiotrophin and midkine on tissue sections of prostate biopsy samples using antibodies.
5. Jo Farmery has begun a blind analysis of prostate cancer biopsy tissue sections to determine if levels of pleiotrophin and/or midkine are increased in the disease.
6. We have obtained DNA coding for pleiotrophin and midkine from collaborators overseas.
7. Dr Sharrard has begun to introduce the DNA coding for pleiotrophin and/or midkine into mammalian cells so we can purify the proteins from the culture medium to investigate their effects on prostate cells.
8. We have verified that the cells containing this special DNA are producing the correct proteins and that it is secreted to the medium.
Identify any problems that have arisen:
1. Initially we tried to cut our own sections of biopsy tissue from blocks provided from Hull. It became clear that it was more efficient to have the experts in the Pathology lab in Hull cut the sections for us. This is now our routine practice.
2. It has taken a few weeks to get the technique right for detecting pleiotrophin and midkine on tissue sections. There is a lot of work up in developing such techniques to get the dilution of antibody correct, etc. We were helped here by experts in the Path lab at Hull Royal Infirmary.
3. A technique associated with introducing pleiotrophin DNA into cells resulted in the synthesis of a mutant form of the protein. Dr Sharrard has now identified the problem and pleiotrophin with the correct amino acid sequence is now being synthesised.
4. The method of getting pleiotrophin/midkine DNA into cells gave poor results initially but buying a new reagent has greatly improved the number of cells that the DNA inserts into, resulting in better yields.
How well is objective being attained: progress so far: See separate list of objectives and chart.
What publications have been made: None so far but one paper is being written at the moment for submission to a journal called The Prostate.
Benefit to patients and new therapy or drug possibility: This cannot be determined yet until final results are available.
Estimate of time needed to develop new drugs (if applicable) and for patients to benefit from the results of this project: Have to await final results to decide whether pleiotrophin/midkine might be useful targets for treating prostate cancer/BPH.
Next stage of research on completion of this project:
1. Write up results for publication.
2. Write grant applications for further funding to continue investigating the role of pleiotrophin and midkine in PCa and BPH. In particular determine how pleiotrophin and midkine signal into cells to control effects on proliferation, motility and counter programmed cell death.
Research Interim report, 27 February 2008.
Project 2006/07