Authors:
Alok Bhushan,
Christopher Daniels and James C.K. Lai
Affiliation:
Title:
Antifolate Resistance: Role of a novel protein
Abstract: Cross
resistant to chemotherapeutic drugs is one of the major problems in treating
cancer patients. While studying the cross resistance patterns of a murine
leukemia cell line L1210/DDP selected for resistance to cisplatin, we observed
that the cell line was >25,000 fold resistant to methotrexate. The cell line was
tested for mechanisms of resistance to methotrexate. There was no amplification
of dihydrofolate reductase or its enzyme activity in the resistant cell line as
compared to the sensitive cell line. Activitiess of folypolyglutamate synthase,
the enzyme that catalyzes the polyglutamation of methotrexate, were not
different between the two cell lines. Although the efflux of methotrexate was
similar in resistance and sensitive cell lines, there was a decreased
association of methotrexate observed in the resistant cell line. To further
understand this association of methotrexate, photoaffinity analog of
methotrexate was synthesized and its covalent binding with various proteins was
assessed with time. The analog bound to a 66kD protein in the sensitive cell
line and was transported it to the 21kD dihydrofolate reductase. Although there
was binding at the 68kD protein in the resistant cell line, there was no
transport of the photoaffinity analogy of methotrexate to dihydrofolate
reductase. Due to the small change in mobility of the 66kD protein in the
resistance cell line, we determined post-translational modification,
specifically tyrosine phosphorylation of the proteins in the two cell lines. It
was observed that there was a membrane protein at 66kD that was tyrosine
phosphorylated in the sensitive cell line and was not tyrosine phosphorylated in
the resistant cell line. The protein was purified from the membranes of the
L1210 cells and the peptide fragments sequenced. The sequence was found to be
homologous to that of the family of heat shock proteins. Further investigation
of the protein and its function showed that the tyrosine phosphorylation of this
protein may have a role in methotrexate transport inside the cell and in
sensitivity for cells to methotrexate. Understanding the implications of the
presence or absence of the transport protein in tumors may be important in
deciding the chemotherapeutic regimen for cancer patients and will reduce
unnecessary toxicity due to methotrexate by not administering it to patients
that lack the activity of the protein to transport methotrexate inside the
cancer cell.
(Supported by American Cancer Society Grant DHP-170, University Research Grant
and Faculty Affairs Committee grant from