|Year : 2004 | Volume
| Issue : 2 | Page : 75-78
Molecular biology in urology
Department of Urology, AIIMS, New Delhi, India
Department of Urology, All India Institute of Medical Sciences, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Molecular biologic techniques form the cornerstone of progress in the field of medicine in the next millennium. These techniques are useful in diagnosis, prognosis, management and follow-up of patients.
Methods: Recent publications in the field of molecular biology and its use in urology were reviewed. The material was then edited to produce this article.
Results: There has been a tremendous interest in this field in the last decade. Over 50 articles in indexed journals and edited text books were found. Literature relating to its use in urology is less common but seems to be increasing in the last few years.
Conclusions: Molecular diagnosis is the identification of presence of disease or susceptibility to disease based on the presence of abnormal genes or their products. Molecular therapy is the development of rational therapeutic strategies aimed at correcting the basic genomic abnormality using molecular biologic techniques. These two applications of molecular biology shall be the focus of advance in urology in the next millennium.
Keywords: Gene therapy, nucleotides, genetic engineering, FISH.
|How to cite this article:|
Kumar R. Molecular biology in urology. Indian J Urol 2004;20:75-8
| Introduction|| |
Molecular biology is a field that aims to find the molecular basis for all processes that occur in the body-both physiologic and pathologic. After defining these molecular changes, it helps develop clinical tools to make early diagnosis of pathologic changes and also ways to reverse them. In this review, the role of molecular biology role in clinical urology will be discussed.
Molecular diagnostics involves the detection of a DNA, protein or gene sequence specific to a neoplasm or disease. Neoplastic cells often acquire genetic sequences different from the host cell. These sequences may be responsible for conferring immortality to neoplastic cells. The detection of these specific genetic abnormalities may help not only identify but also prognosticate the course of the disease. Manipulation of the genetic make up of experimental animals or cell lines using genetic engineering can help understand the effect of natural mutations in disease causation and termination. The most important therapeutic use of molecular biology techniques lies in gene therapy whereby defective genes may be removed and tumor suppressor or drug susceptibility inducing genes may be selectively introduced into diseased cells.
| Methods|| |
A literature search on the Pubmed was done using the key words molecular biology, urology, techniques, genetic engineering, FISH, PCR and blotting. Relevant articles were accessed and reviewed. Current textbooks on urology were also reviewed for relevant topics and the material was edited to suit the requirements.
| Detection of Genetic Disorders|| |
Prostate cancer: Heredity and polvmorphisms: Up to 10% of prostate cancer may be linked to heredity and some of the high-risk alleles have been located on chromosome 1 and X chromosome. Genetic polymorphism at the Xgl1Xg12 locus is the most common abnormality localized in carcinoma prostate cells. This gene codes for the androgen receptor and the length of the CAG nucleotide repeat sequences at this locus correlates with the risk for cancer  . Races such as Asians with longer repeat sequences have been shown to have a lower cancer risk. Another important polymorphism identified is a single nucleotide polymorphism (SNP) at the Glutathione Transferase (GST) pi gene which may be associated with an increased cancer risk. 
Growth factors: A large number of growth factors such as epidermal growth factor (EGF), transforming growth factor (TGF) and insulin like growth factors (IGF) have been identified whose receptors may be over expressed in certain certain high-grade prostate cancers, indicating their dependence on these factors.  This may help decipher the stimulus necessary for cancer progression.
Apoptosis: Normal cells undergo programmed cell death known as apoptosis. One of the major mechanisms of carcinogenesis is loss of this apoptosis leading to an immortal cell. Certain proteins within cells may act as inhibitors of apoptosis. Levels of anti-apoptosis proteins bcl-2 and mcl-1 are raised in cancers of the prostate.  These raised protein levels also predict a poorer outcome of therapy and is associated with a higher growth in artificial tumor cell lines such as LnCAP. Insertion of antisense bcl-2 oligonucleotides that block the natural bcl-2 protein can inhibit the growth of LnCAP and increase tumor sensitivity to Taxol and Mitoxantrone.  Phase 1 clinical trials are underway with the use of bcl-2 oligonucleotides with or without mitoxantrone in treatment of clinical cancer prostate highlighting the most important role of molecular biology in urology. 
Tumor suppressor genes: Two oncogenes are currently under investigation in prostate cancer. PTEN is inactivated by mutation in certain prostate cancers especially high grade metastatic ones.  It may be an apoptosis promoting gene similar to p53, the other important oncogene. The product of the p53 gene is essential in diverting the cell towards apoptosis.  Mutated p53 gene is found in high-grade metastatic prostate cancer and its presence is associated with poorer results following surgery or chemotherapy.
Other molecular abnormalities: The most commonly described molecular abnormality in prostate cancer is the aberrant expression of the cell adhesion molecule ECadherin coded by the GSTP1 gene that is over expressed. Prostate cancer cells can be detected in the peripheral blood using a PCR technique for PSA within these cells.  This is one of the most sensitive staging methods for prostate cancer and can be used to predict failure in patients undergoing radical prostatectomy.
Renal Cancer: 4% of renal cell cancer has been shown to be hereditary and in Von-Hippel Lindau (VHL) disease, upto 45% of the affected individuals may develop renal cancer. The gene causing VHL has been identified on chromosome 3 (3p25). This allows antenatal diagnosis and genetic counselling. Genetic changes responsible for the development of different histologic subtypes of renal cancer include 3p LOH and VHL gene in RCC-clear cell type; 9p-, 17p-, 21p- and 17p+ in papillary RCC; C-erb-1 oncogene expression in Bellini's Ductal carcinoma and l lp13 mutation in Wilms' tumor. This knowledge may be useful in diagnosis of the primary or the metastasis and allow gene therapy.
The presence of lAl allele of the cytochrome p450 (CYP) gene along with the TT1 or TPI allele of GST or the NAT-2 polymorphism (N acetyl transferase) is associated with an increase in risk for developing renal cancer. Loss of the mul allele of GST (null polymorphism) and the presence of the pil GST polymorphism indicate a higher risk if the CYP lA 1 allele of NAT-2 allele is also present. 
Urothelial cancer: Induction of oncogenes is one of the potential mechanisms responsible for urothelial cancers. The ras oncogene on chromosome p 21 has been found to be present in upto 10% cancers with single strand conformation polymorphism analysis and restricted fragment length polymorphisms showing some correlation with the grade of the tumor. , Abnormality in another oncogene, c-myc, is also commonly seen in bladder cancers and c jun may also serve as a marker for bladder cancer as its abnormal expression may indicate aggressive growth and increased expression of EGF receptor, which in itself has a poor prognosis. 
The p53 gene is one of the most commonly altered suppressor oncogene in human cancers. However, despite this frequency, it is not essential that every mutation in the gene will be significant. It is now possible using crystallography to predict how the genetic mutation will affect the structure of p53 gene product and thus identify which mutation is likely to have functional consequences.  In a study of 243 patients, organ confined bladder cancer with p53 positivity had a significantly worse 5 year survival and relapse free rate compared to p53 negative tumors.'' However, this data, and that similar to it, cannot still be used to guide therapy. Studies are currently underway to help establish the specific role for p53 as a prognostic indicator to dictate therapy especially in cases of high-grade superficial tumors where it may guide the choice between bladder salvage and more aggressive therapy.
Another tumor suppressor gene, Rb, also codes for proteins responsible for diverting the cell into an apoptotic cycle. This was demonstrated by the non-random deletions of chromosomes 13q and 9 in bladder cancers.  Several groups have also demonstrated chromosome 9 deletions as the primary event in superficial bladder cancer and have shown that in these cases, p53 may be normal. This separation of effects of chromosome 9 and p53 would be invaluable in predicting the outcome of clinically superficial bladder cancer and choosing aggressive therapy for p53 positive superficial tumors. 
The third important genetic mechanism for tumorigenesis in bladder cancer is over expression of growth factors and their receptors. The gene for EGF receptor is located on chromosome 7 and trisomy 7 is associated with a particularly aggressive bladder cancer. In fact, this is the only other molecular marker that has approached the point of clinical usefulness.  Positive tumors have a significantly poorer prognosis but identification techniques are currently not standardised. Erb-2 oncogene that codes for a growth factor similar to EGF is also a potential marker for aggressive bladder cancer. 
| Genetic Engineering and Gene Therapy|| |
An important development in molecular biologic techniques is the ability to introduce selected DNA fragments into recipient cells. DNA fragments can be obtained from endonuclease action on pre-existing DNA or through laboratory synthesis by using a known RNA template. Introduction of this selected fragment into cells can be done through the use of vectors such as viruses, plasmids, microinjection, liposomes or electric gradient. An animal which gains this new genetic information is termed transgenic. This technology forms the backbone of development of monoclonal antibodies and gene therapy.
Gene therapy is one of the prime goals of all molecular biologic techniques. There are over twelve trials currently underway in the USA on the role of gene therapy in urologic malignancies.  Gene therapy is defined as the introduction of normal or modified genetic code into human cells to reverse a genetic or acquired disease. This therapy is based on the knowledge that most diseases, particularly cancer, result from aberration in normal genetic sequences. Once these abnormalities have been identified, it may be possible to correct them using genetic engineering. There are two basic mechanisms of performing gene therapy. These are corrective gene therapy and cytoreductive gene therapy. Corrective gene therapy involves replacement of a defective gene in the cancer cells while cytoreductive therapy involves the addition of a gene that promotes apoptosis in the cancer cells. Another strategy in the cytoreductive model is the introduction of certain genes that will potentiate the action of chemotherapeutic drugs or prevent the normal cells such as the bone marrow from the adverse effects of these drugs, allowing higher doses to be used.
Corrective gene therapy is used to introduce the normal or wild type gene into deficient cells. The correct identification of defective genes in certain urologic cancers (p53 and 9p in bladder cancer; GST-pi and p53 in prostate cancer; VHL gene in RCC and WT1 gene in Wilm's tumor) has opened the potential for corrective gene therapy. p53 gene replacement has been attempted using viral vectors in vitro in bladder cancer biopsies.  Reintroduction of the GST-pi gene in prostate cancer hasa similar therapeutic potential. Another corrective option is the prevention of emergence of metastatic clones through over expression of oncogenes. Antisense sequences to oncogenes such as c-myc can be developed to prevent their protein synthesis. The KAI-1 gene is an anti metastasis gene that is lost in prostate cancer.  This may be replaced using gene therapy.
There is a potential to develop PCR based diagnostic kits for p53, GST-pi and VHL mutations in urine or expressed prostatic secretions. In bladder cancer, there is further therapeutic potential for delivering gene therapy vectors through bladder instillations. Corrective wild type VHL gene may be delivered into renal epithelium through renal artery catheterization.
The most thoroughly evaluated form of cytoreductive gene the rap, is the development of tumor vaccines which contain genetically engineered cells that provide immunostimulatory signals against tumor antigens. Tumor cells are removed during surgery and, in tissue culture, are transduced genetically with a cytokine-producing gene that stimulates the immune response to antigens present on the tumor cells. This modified cell is irradiated to prevent its neoplastic growth and reintroduced into the patient. There are trials already underway using this modality for renal cancers. 
| Conclusions|| |
Disease diagnosis has evolved through symptom identification, evaluation of signs, basic laboratory tests and microscopy to the current molecular levels. Therapy has also progressed from symptomatic, empirical and specific to gene therapy. Identification of basic cellular codes for the normal and abnormal cell function promises to be the cornerstone for all rational therapy in the future. This also has the potential for primary prophylaxis and early intervention, preventing any significant morbidity. The challenge lies in the ability to make these tools fool proof and cost effective.
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