Elucidation of the Structure and Function of a new form of Dihydrofolate Reductase

Abstract

Dihydrofolate reductase (DHFR) is an enzyme required for DNA synthesis and hence, necessary for cell multiplication. Since there is uncontrolled multiplication of certain cells in cancer, this enzyme has been used as target enzyme for cancer chemotherapy. Methotrexate (MTX) is the most commonly used anticancer drug and it controls cancer by binding very tightly to this enzyme and, hereby, inhibiting its action. The therapy with MTX fails in certain cancer patients perhaps because of presence of certain forms of DHFR having low or little affinity for MTX. The primary objective of this project was to study the nature of these multiple forms of DHFR. By using MTX binding assay and an enzyme-linked immunosorbent assay we have screened scores of leukemia cells cytoplasm samples and leukocytes cytoplasm samples from patients with different types of cancer for active DHFR and the immunoreactive non-functional (IRE) form of DHFR. In majority of these samples the concentration of IRE was hundreds of fold higher than the active DHFR. Our initial observation that cancer patients in neutropenia (very low count of neutrophils in blood) have low quantities of multiple forms of DHFR in their leucocytes compared to their levels in non-neutropenic state provided impetus to our hypothesis that there appears to be an association between white blood cells proliferation and concentration of multiple forms of DHFR. This was further supported by our finding that colony-stimulating factors (G-CSF and GM-CSF), which are often used in cancer patients to resolve their neutropenia induce white cell proliferation by increasing the concentration of multiple form of DHFR. Similar results were obtained in non-neutropenic cancer patients who received these haematopoietic growth factors for their stem cells mobilization. These data have had a significant impact in this field and may lead to our understanding of the molecular mechanism by which colony-stimulating factors induce white cell proliferation. In addition, we have succeeded in raising monoclonal antibodies to DHFR. We also partially purified immunoreactive but non-functional form of DHFR (IRE) and determined its apparent molecular weight and isoelectric points. The IRE could not be converted into active enzyme by treatment with chaotropic agents and thiols suggesting that additional factors must be required for conversion of non-functional form into active DHFR. We are happy to report that we have achieved most of the objectives of this project. Some of the data pertaining to this study has already been presented in two international meetings, such as 27th World Congress, of the International Society of Hematology held in Amsterdam and 3rd International Cancer Congress, Allama Iqbal Medical College and European Society of Pediatric Hematology and Immunology, Lahore. Some of it is going to be presented in the 28th World Congress of International Society of Hematology at Toronto. The fact that our data have been published (or are being published) in the peer reviewed and an indexed journal of International standing it is indicative of the validity of these results. Five full papers and four abstracts resulting from this project indicate reasonable contribution to the scientific literature. In addition, two full papers are in preparation. The findings reported in these publications, we believe, would be of value to both medical and scientific communities. Another noteworthy point is our success in developing monoclonal antibodies to DHFR. Our laboratory perhaps is one of the few places in Pakistan where monoclonal antibodies have been produced. Ms. Fakhra Sultana, the Research Associate on this project is in the process of completing her M. Phil. from the Karachi University. This is another satisfying aspect of this project.