| GENE EXPRESSION ASSOCIATED
WITH OXIDATIVE DNA AND PROTEIN DAMAGE IN THE PATHOGENESIS OF
PROSTATIC CANCER Dr R A Sharma1, Prof A J Gescher1, Prof J K Mellon2, Prof W P Steward1 1 Cancer Biomarkers and Prevention
Group, Oncology Department, University of Leicester |
Cancer of the prostate is the most commonly diagnosed solid malignancy in men living in developed countries. With increasing public awareness and greater screening by doctors, more patients are being referred to urologists with suspected early prostate cancer. Most of these patients will have benign pathologies detected on prostatic biopsy, but many will have localised cancer or will go on to develop prostate cancer subsequently. There is currently no means of predicting which patients are at greater risk of developing the disease or which patients with localised cancer will benefit from treatment.
One way of predicting an individual's risk of developing prostate cancer, and to monitor that risk before invasive cancer develops, may be to assess levels of protein and DNA damage in cells, particularly in patients known to be more susceptible to such damage on account of abnormalities in their gene expression. Over the past decade, it has become apparent that oxidative stress is a factor in the development of prostate cancer, and consumption of antioxidants present in many vegetables is thought to protect men from this disease. Oxidative stress continually occurring within our bodies produces chemicals that not only bind to DNA but also damage proteins vital to signalling mechanisms within cells. Such DNA and protein damage appears to be important in the development of many cancers, including prostate cancer.
The aim of this project is to study the levels of DNA or protein damage in human prostate cells grown in vitro and in biopsies from patients, and to study in more detail the cells with high levels of such damage by analysis of which genes are overexpressed or underexpressed. The use of such methods to analyse apparently benign tissue may predict the presence of cells that may become cancerous in the future. They may also be useful in monitoring the effectiveness of drugs or dietary supplements that aim to prevent or delay the onset of prostate cancer in clinical trials.
BACKGROUND
Cancer of the prostate is the most commonly diagnosed solid malignancy in men living in developed countries. As a consequence of greater public awareness of the disease and more widespread use of blood tests such as prostate specific antigen (PSA), significant numbers of cases are now referred to urologists with suspected prostatic cancer, in whom no cancer is identified histologically. Although the presence of high grade prostatic intraepithelial neoplasia (PIN) is a predictor of prostate cancer, the majority of tissue biopsies taken from patients referred to urologists on account of a raised PSA appear hyperplastic. Although such hyperplasia is considered benign (BPH), early clonal evolution of dysplastic cells derived from stem cells may be occurring, not detected by the traditional histology (e.g. cells deficient in the detoxification isoenzyme, glutathione S-transferase PI, may be more likely to develop into PIN). Moreover, an increasing number of patients with localised prostate cancer are being diagnosed, for whom management strategies are controversial and in whom biomarkers of prognosis or likely response to treatment do not exist. An intense need is thus emerging to accurately predict an individual's risk of developing PIN on the basis of apparently benign biopsy tissue or to identify patients with localised prostatic cancer who are likely to benefit from treatment, and to monitor preneoplastic or neoplastic behaviour during pharmacological intervention.
Over the past decade, it has become apparent that chronic oxidative stress is an important aetiological factor in prostate carcinogenesis from its earliest stages. Oxyradicals result in DNA damage, at least in part via formation of covalent adducts (e.g. M1G) to the bases that comprise DNA; such adducts can be measured in biopsy tissue by immunoassay. Similarly, reactive oxygen and nitrogen species within cells damage proteins, particularly tyrosine residues, thus altering their normal function such as cell signalling. It is becoming apparent that some cells are more susceptible to the damaging effects of oxyradicals than others on account of differences in the expression of certain genes.
Aim of Project
The aim of this project is to identify abnormalities of gene expression that are associated with high levels of DNA adducts in non-malignant and malignant prostate cells grown in vitro and in human prostate tissue following laser-capture microdissection.
A method for identifying cells with high levels of oxidative damage will be developed and levels of DNA adducts will be measured in prostate cancer cell lines grown in vitro and in prostatic biopsies from patients with BPH, PIN and cancer.
The hypothesis tested in this project is that the analysis of apparently benign tissue for levels of DNA damage, in conjunction with the expression of genes critical in the body's defence against oxidative insults, will identify individuals at risk of subsequently developing malignancy. Moreover, measurements of oxidative DNA or protein damage may act as intermediate biomarkers of the success of failure of intervention with antioxidants in future clinical trials aiming to prevent the development of prostate cancer or modify the malignant behaviour of early prostate cancer.
RESEARCH PROPOSAL
Aims of Project
This project focuses on the elucidation of detrimental oxidative changes in prostate tissue related to malignancy.
The overall aim of the project is to help:
-
with the identification of individuals at risk of developing prostate cancer, and
-
with the formulation of novel strategies for chemoprevention of
prostate cancer in individuals with preneoplasia or intraepithelial neoplasia.
To achieve this end the following specific questions will be addressed in experiments using normal and malignant prostate cells grown in vitro and human- derived prostate specimens:
1. What are the background levels of DNA adducts indicative of changes induced by reactive oxygen species in normal and malignant prostate cells. Such levels have not been described previously. They are likely to reflect mutagenic and potentially carcinogenic alterations in the prostate.
2. How susceptible are normal and malignant prostate cells towards infliction of damage by oxidants to DNA. These studies, in which cells will be exposed to oxidants, aim to identify prostate-derived cell populations characterised by a particular susceptibility to the damaging effects of oxidative stress. As an adjunct, the hypothesis that androgens induce oxidative protein and DNA damage in hormone-sensitive prostate cancer cells will be tested.
3. Which changes in gene expression accompany, or occur subsequent to, oxidative stress in prostate cells. These experiments aim to identify mechanisms by which oxidative stress in cells can elicit damage leading to malignancy.
4. How do levels of DNA adducts and concomitant changes of gene expression determined in prostatic biopsies from patients with BPH, PIN and cancerous tissues compare with those observed in cells in culture? These experiments will assess the relevance to humans in vivo of the results obtained in cells in vitro.
The mechanistic data obtained in this project, which will describe DNA and protein damage induced by oxidative stress and its gene expression sequelae, may be exploitable in terms of furnishing new targets for chemoprevention or early treatment. The DNA adducts to be studied, and/or the expression of genes identified, may serve as biomarkers of the efficacy of preventive strategies in individuals at high risk of developing prostate cancer and in patients with early neoplasia. Such biomarkers do not currently exist. The methods developed in this project may be used in the future to identify areas of prostatic tissue that may undergo cancerous transformation, or isolate early malignant clones.
Approved by Prostate Research Campaign UK, Medical and Scientific Advisory Panel, and Principal Committee.
"One of the most favoured theories for the initiation process of cancer is tissue damage by Free Radicals. Free Radicals are positively charged particles that can damage tissues and have a potential role in the cause and development of cancer. Anti-oxidants are now quite widely recommended as preventative measures, particularly in relation to the pre-cursor of high grade PIN (prostatic intraepithelial neoplasia). We have very little evidence as yet in relation to this, and we this is an important and quite exciting area of research."
Mr Neil O' Donoghue FRCS, Prostate Research Campaign UK, Medical and Scientific Advisory Panel.