Authors: Christopher K. Daniels1,3,
Ashwini Saini1, Tuoen Liu1, Peter R. Sheridan2,3,
James C.K. Lai1,3 and Alok Bhushan1,3.
Affiliation: (1) Department of
Biomedical and Pharmaceutical Sciences, College of Pharmacy, (2) Department of
Biological Sciences, and (3) the ISU Biomedical Research Institute, Idaho State
University, Pocatello, Idaho 83209.
Title: Characterization of the Novel Methotrexate Resistance Protein
Abstract: Bhushan and co-workers
have previously described an alteration in an unidentified
66-68 kDa membrane-associated, tyrosine-phosphorylated, methotrexate
(MTX)-binding protein in the L1210 murine leukemia cell line.
This protein was absent, at least in the tyrosine-phosphorylated form, in
cisplatin-resistant, methotrexate cross-resistant L1210/DDP cells.
Indeed, this 66-68 kDa protein was also demonstrated to be absent, again
at least in the tyrosine-phosphorylated form, in cells that were concomitantly
rendered MTX-resistant by treatment with the tyrosine kinase inhibitor,
genistein. This recently identified 66-68 kDa protein, thus, appeared to be a
new, novel player in the mechanisms that have been described accounting for
resistance to MTX. We have further
characterized this 66-68 kDa protein by affinity chromatography purification
using its two identified properties, MTX-binding and tyrosine phosphorylation,
yielding a single band of 66-68 kDa on phosphotyrosine western blot.
This purified protein was subjected to trypsin digestion and peptide
fragments isolated and sequenced yielding two partial peptide sequences:
TVTNAVVTVPAYFNDSQRQA and VEIIANDQ.
The peptide sequences were used to search the mouse genome at the NCBI database
for ORFs containing theses peptides using the TBLASTN function.
A single gene was identified containing both sequences, the HSPa8 gene,
which codes for the heat shock family protein, HSC70.
HSPa8, determined by reverse transcription PCR fo mRNA, was found to be
transcribed in both cell lines.
Western blot analysis showed that HSC70 protein is present in both cell lines.
Finally, using a binding assay with MTX-agarose, followed by Western
blotting for HSC70, we show HSC70 was present and was found in the MTX-binding
fraction. Interestingly, HSC70 is
present in the resistant L1210/DDP cells as well, but it shows very little
MTX-binding. Thus, we have
identified a new, novel role for the heat shock protein, HSC70, where an
apparent disruption of the normal function of HSC70 can lead to the development
of resistance to MTX-induced cell death.
This undocumented mechanism may yield an understanding into why some
tumor types are unresponsive to MTX treatment, and may provide a new clinical
screening tool to determine potential tumor sensitivity to MTX.
This work was supported in part by the Idaho INBRE program and NIH grant
#P20RR016454 of the