Dunedin School of Medicine

More projects

The Role of PAX3 in Melanoma

It is rare to identify factors that are important for cell proliferation and survival, but which may also be excellent targets for the design of cancer therapies. In this project we are investigating whether PAX3 gene is an important factor required for the survival of melanoma cells. If this is true then PAX3 would be an excellent strategic target for melanoma therapy because it is minimally expressed in normal adult tissue. Malignant melanoma is a notoriously aggressive malignant neoplasm of melanocytes, arising de novo, or from a pre-existing benign dysplastic nevus. The incidence of melanoma is rising at an alarming rate, and is of special significance in New Zealand and Australia, where the incidence is the highest in the world. In New Zealand, the solar ultraviolet radiation is high and levels are increasing as a result of depletion of the ozone layer over Antarctica. Reliable incidence and mortality figures from the United States, Australia, New Zealand, and Scotland indicate that the incidence and mortality of malignant melanoma is increasing at a rate faster than our ability to treat advanced disease.

The Role of PAX2 in Cancers of the Urogenital Tract

Enhanced cell survival and/or proliferation leads to cancer, and other hyperproliferative diseases. Indeed, gene mutations play central roles in these diseases, affecting outcomes such as cell survival, attachment, cell-cell contact, differentiation status, metastasis, and many other changes. However, the importance of changes in gene expression, many of which are epigenetic, has only just recently been seriously addressed. Changes in gene expression act alongside genetic mutations to change the cell's internal milieu, and clearly also play an important role in cancer. The present challenge is to identify changes in gene expression that are associated with the cause of cancer, rather than being a consequence of the cancerous phenotype.

Rearrangements of PAX3, PAX5 and PAX8 have been implicated in cancers, such as alveolar rhabdomyosarcoma, lymphoma, acute lymphocytic leukaemia, and thyroid carcinoma. The PAX3 gene, for example, is fused to the FKHR gene in translocations involving chromosomes 2 and 13; t(2;13)(q35;q14) found in alveolar rhabdomyosarcoma. Treatment of rhabdomyosarcoma, and melanoma cell lines, which express PAX3, with PAX3 antisense oligonucleotides decreases PAX3 expression, and induces apoptosis. We have been studying the role of PAX2 in Wilms tumours, and renal cell carcinomas. Antisense PAX2 oligonucleotides have been shown to inhibit the proliferation of renal cell carcinoma cells, and the over-expression of PAX genes causes malignant transformation of several rodent cell types (NIH3T3 and 208 cells). In this project, on-going studies will determine whether PAX2 plays an important role in urogenital tract cancers. We have demonstrated that PAX2 expression promotes the survival of cells in culture, and on this basis we predict that PAX2 expression promotes the survival of cancer cells.

Role of PAX2 in Renal-coloboma Syndrome/ the Role of PAX Genes in Eye, Urogenital Tract, and Central Nervous System Development

Congenital abnormalities of the kidney and urinary tract are common in the human population, occurring at a frequency of 1-2 per hundred live births. It is thought that a high proportion of these congenital abnormalities occur as the result of a breakdown in the mechanisms regulating development. In 1995 we showed that PAX2 mutations cause renal-coloboma syndrome, and since then we have characterized a number of different PAX2 mutations and analyzed the role of PAX2 in mammalian development. Abnormal regulation of cell growth and survival underlies many human diseases, including cancer, developmental abnormalities, and degenerative diseases. Therefore the major aim of this project is to uncover the cellular pathways regulated by PAX genes that dictate the growth or survival of cells during development and disease.

In humans and mice, PAX2 mutations cause kidney, eye, and central nervous system abnormalities. It appears that the PAX2 protein coordinates normal developmental pathways by regulating the expression of downstream genes. Aberrant PAX2 expression consequently disrupts normal gene expression resulting in abnormal development.

Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common, potentially lethal, monogenic disease in humans. The incidence is about 1:500, affecting approximately 4-7,000 individuals in New Zealand and 10 million worldwide. In most families, ADPKD is caused by heritable mutations of either the PKD1 (85%) or PKD2 (10-15%) genes. 95% of ADPKD carriers experience renal failure by age 70, and there is no effective treatment. Moreover, few pathways for designing drug-treatments are known. In the present project we are investigating the role of PAX2 in polycystic kidney disease.

Urogenital Tract Development and Vesicoureteric Reflux

Vesico ureteric reflux (VUR) is a common disorder in humans (1-2%) resulting from a congenital abnormality of the ureter, causing reflux of urine into the kidney. It has been suggested that reflux is a major cause of childhood urinary tract infection. VUR may lead to reflux nephropathy, renal scarring, proteinuria, and is the most significant cause of renal failure in childhood. VUR is graded, depending on severity, from grade I to V. Severity of VUR is the most important factor determining whether renal damage will occur. Generally, grades I and II result in no renal damage. As a child grows the ureter lengthens, and reflux tends to diminish or disappear with age. Although most cases of VUR are sporadic, there is clear evidence for hereditary transmission, but the genetic cause of VUR is unknown. It is thought that VUR is caused by an autosomal dominant gene with variable penetrance.

Gene-targeted Therapies for Cancer and Polycystic Kidney Disease/Gene Delivery Systems

Recently a naturally occurring process was discovered in fungi and plants that involves sequence-specific post-transcriptional silencing of gene expression. Subsequently this process was shown to occur in bacterial and animal cells and the term RNA interferance (RNAi) was coined for this phenomenon. The silencing "triggers" for RNAi are long double stranded RNA (dsRNA) molecules complementary to mRNA sequences that are introduced into cells either experimentally or are derived from endogenous sources such as viruses, transgenes or cellular genes (transposons). These dsRNA molecules are processed into discrete short interfering RNA (siRNA) fragments that are 21- to 25-nucleotides in length. The processing of dsRNAs into siRNAs is carried out by a 500 kDa ribonuclear protein complex known as the RNA-induced silencing complex (RISC). The exact identity of the RISC proteins and its mechanism of action is still under investigation, but the process of RNAi has spurred interest in the field of biotechnology.

When introduced in mammalian cells long dsRNA sequences cause a global shut down of protein synthesis but the introduction of siRNA sequences complementary to an mRNA of interest causes sequence-specific degradation of only that mRNA. This sequence-specific suppression of gene expression is mediated by 21-nucleotide long siRNAs. RNAi is effective in mammalian cells but gene expression is not completely removed and the degree of inhibition varies from low to complete eradication of gene expression. SiRNAs are effective at concentrations that are several orders of magnitude below the concentration typically used in antisense experiments as this effect is amplified within cells. Furthermore some recent research suggests that it might be inherited in the next generation. The RNAi technique has had a huge impact in molecular studies. The use of RNAi in mammalian cells could be just as useful particularly for functional genomics and therapeutics. However many technical issues must be addressed. The success of RNAi depends on cell type, the level of expression of the targeted gene and how optimal the siRNA sequence is for targeting of its complementary mRNA.

The delivery of siRNAs across lipid bilayers of cells presents great challenges because of their membrane impermeance. SiRNAs are polyanions and unassisted permeation across lipid bilayers is negligable. Double stranded RNA can be delivered to C.elegans by feeding or soaking, but effective delivery of siRNAs to mammalian cells is still under investigation. Typically siRNAs are introduced into mammalian cells in conjunction with cationic liposomes that mask their negative charge and "smuggle" them into the cytoplasm.

The longer term aim of this project is to use antisense and RNAi to silence PAX genes and elucidate their exact role and importance during development.