Multiple Forms of Dihydrofolate Reductase

Abstract

Dihydrofolate reductase (DHFR) is an enzyme which is essential for DNA synthesis. Because of its critical role in cell multiplication, it has been a target enzyme in cancer chemotherapy. Methotrexate (MTX) is the drug which is commonly used in various cancers and its therapeutic action is based on its ability to inactivate DHFR. However, it has been frequently reported that certain cancer cells do not respond to the conventional doses of this drug. To circumvent their resistance, sometimes high or even mega doses ( 1 gm 12 gm/m2) of MTX have been used which unfortunately are toxic to the normal cells as well. Scientific community has been striving hard to find out various causes of this resistance to MTX therapy. One of the mechanisms of resistance that has been proposed just few years ago is the presence (in these cells) of a form of DHFR which binds MTX very weakly and, therefore, enzyme action will not be inhibited at conventional doses of the drug. r he present study was undertaken xp ore th· s phenome o to identify those forms of DHFR, which have low affinity for this drug, in various cancer cells. Such an identification would have been of great help in correctly assessing the optimal therapeutic doses of MTX in a particular type of cancer so that the tumor cells could be killed with minimal toxic effects to the normal cells of the body. It would have also indicated whether a particular person is likely to benefit from MTX or not. With that objective in mind, the plan of work proposed in the grant request was as follows : i. Identification of low and high affinity forms of enzyme in various human leukemia cells and in cancer tissues using MTXbinding assay system. ii. Development of Enzyme-linked immunosorbent assay (ELISA) for 2 DHFR. iii. Using ELISA for id?ntification and distribution of inactive forms of enzyme in cancer cells as well as in serum and urine of cancer patients. iv. Characterization of these multiple forms of enzyme. We are happy to report !hat all of the aims and objectives have been successfully achieved during this period of time. 'l;i, f .. I' re·;)., Nineteen samples of human leukemia cells and 27 samples of normal and tumor tissues from gastrointestinal cancer patients were analyzed and the low-affinity form of enzyme was found to be present in most of them. The results pertaining to human leukemia cells were presented in the 633rd meeting of The Biochemical Society, London (Abstract enclosed) and later published in the j urnal, Biochemical Society Transactions, 18:633-634 (1990). As far as the second objective is concerned, using calf liver DHFR, an ELISA has been developed which in conjunction with [3H]MTX binding assay was used to quantitate functional and non-functional forms of DHFR in various leukemia cells. T?ese results were presented in ___, ., __ u , Dow Medical College and Civil Hospital, Karachi (Abstract enclosed), and in the 11th Paki tan Co gress f zoology, Khanuspur, Murree Hills (Abstract enclosed) The results pertaining to the development of ELISA have been published· e Italian J. Biochem., 40: 207-215 ( 1991). The data concerning heterogeneity of the enzyme in colon t mor cells have recently n published in the Journal of Pakistan Medica 139 ( 1991). The results presented at the Khanuspur meeting on immunoreactive forms of DHFR are being put together in the form of a manuscript which will be submitted for publication in a journal o international standing. The publication of the data ·n refe eed lUld ndexed journals is .indica. i e...__,__,?-?U,,J::??ri.-·i results. These findings, we believe, would be of value to both medical and scientific communities