Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients

1. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients Tanya N. Basu, David H. Gutman Jonathan A. Fletcher,Thomas W.Glover Francis S. Collins, Julian Downard Presented by: Brooke Pinney

2. What is neurofibromatosis? • Neurofibromatosis 1 (NF 1) also called von Recklinghausen NF or Peripheral NF • The most common neurological disorder caused by a single gene • Affects approx. 1 out of 4,000 births throughout the world

3. Autosomal dominant genetic disorder • Inherited from parent or by “spontaneous mutation” in the sperm or egg • Higher risk for rare malignant tumors in brain, nerves, or spinal cord

4. Characteristics of NF • Developmental abnormalities including benign and malignant tumors of neural crest origin • Neurofibromas (benign tumors) • Neurofibrosarcoma (malignant tumors)

5. Characteristics cont’d • Family history of NF1 • 6 or more café-au-lait spots on the skin • Presence of pea-sized bumps on skin • Large areas of the skin that look swollen

6. Freckling under the arms or in the groin • Pigmented bumps on the iris of the eye • Skeletal abnormalities – bowed legs, scoliosis, or thin shin bone • Tumor on the optic nerve that may interfere with vision

7. Disfigurement – large areas of plexiform neurofibromas may enlarge limbs • Scoliosis – lateral curvature of the spine • Learning Disabilities – reading, writing, and use of numbers in “normally” intelligent inds.

8. Large Heads – hydrocephalus • Optic gliomas – causing poor vision or bulging eye • High blood pressure • Renal artery stenosis • pheochromocytoma

9. Defects in the NF1 gene have been implicated in the NF1 disorder • The NF1 gene codes for a protein similar to p120GAP, the GTP-ase activating protein (GAP) for the products of the ras pathway.

10. Tumor cells have wild type p21ras but no NF1 protein See if NF1 is essential for negative regulation of ras proteins, or is a tumor- suppressor gene whose product acts upstream of ras

11. Testing • Activations state of p21ras can be measured by taking the ratio of active GTP to the inactive GDP bound to it • Wild type p21 ras has GDP bound to it • Transforming mutants bind large amounts of GTP

12. General test

13. Testing • Increased activation site of the p21ras in the neurofibrosarcoma cells is due to : • 1. A mutation in the ras gene itself • 2. A defect in the protein normally involved in regulation of p21ras

14. Ras gene mutation • P21ras was immunoprecipitated using antibody Y13-259 • This antibody blocks the function of p21ras and reduces the ability of GAP to hydrolyze GTP, but doesn’t block the interaction of bound p21ras with GTPase-activating proteins Then recombined with GAP or NF1 catalytic domains See if p21ras behaves normally or shows evidence of undergoing activating mutation

15. Ras gene mutation

16. Ras gene mutation results • Transformed p21ras not affected by treatment – activated mutants are insensitive to GTPase-activating activity of GAP or NF1 • Normal p21ras cells and the neurofibrosarcoma cells were sensitive to GAP and NF1 • P21ras behaves normally and doesn’t undergo activating mutation

17. Ras regulatory mechanism defectiveness • Measure activity of the GTPase-activating protein p120GAP and NF1 GAP-related protein (GRP) from cells • See if functioning p120GAP or functional NF1 GRP • Tested for ability to stimulate the hydrolysis of GTP bound to added p21ras

18. Ras regulatory mechanism

19. Ras regulatory mechanism defect results • Lysates and immunoprecipitates of p120GAP have high GAP-like activity • Immunoprecipitates of NF1 GRP have high levels of activity when from sarcoma cells • IP’s of p120GAP have activity similar to other cells • Functional p120GAP but no functional NF1 GRP

20. Inhibited growth • Look at the inhibiting effects of the p21ras antibody, Y13-259 • Using neurofibrosarcoma cells (ST 88-14)

21. Antibody inhibition

22. Conclusions • Since p21ras antibody inhibits growth of ST 88-14 cells, p21 has a positive effect on proliferation of cells • Production of non functional NF1 GRP causes an activation of p21ras, even in the presence of functional p120GAP • NF1 GRP is a negative regulator of p21ras in neural crest cells, therefore affirming the negative control of ras proteins in cells and the inhibition of cell proliferation.

23. NF1 is a tumor suppressor gene normally essential for negative regulation of ras proteins in the cell

24. As for the position of the gene in the pathway… • Frequent mutations of p53 require an upstream position for the malignancies to form • If growth-inhibitory p21 was upstream of the NF1 GAP-related protein there would not be malignant tumors common to NF

25. NF1 acts upstream of ras

26. Future Implications • Therapeutic approaches aimed at the reduction of the Ras-GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms

27. References • Cappione A., French B., and Skuse G. 1997. A Potential role for NF1 mRNA editing in the pathogenesis of NF1 tumors. American Journal of Human Genetics 60 (2) : 305-12. • Donegan, K. 2000. National Neurofibromatosis Foundation. • http://www.neurofibromatosis.org • Kleinsmith, Becker, and Hardin. 2000. The World of the Cell. Benjamin/Cummings Publishing Co. New York.