Clinical Cancer Research 13, 3174-3181, June 1, 2007. doi:
10.1158/1078-0432.CCR-06-1720

Methylation and Silencing of Protein Tyrosine Phosphatase Receptor Type O in
Chronic Lymphocytic Leukemia
Tasneem Motiwala1, Sarmila Majumder1, Huban Kutay1, David Spencer Smith1, Donna
S. Neuberg4, David M. Lucas2, John C. Byrd2,3, Michael Grever2,3 and Samson T.
Jacob1,2,3
Authors' Affiliations: 1 Department of Molecular and Cellular Biochemistry, 2
Division of Hematology-Oncology, Department of Internal Medicine, and 3
Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, and 4
Department of Biostatistics and Computational Biology, Dana-Farber Cancer
Institute, Boston, Massachusetts

Requests for reprints: Samson T. Jacob, Department of Molecular and Cellular
Biochemistry, 333 Hamilton Hall, Ohio State University, 1645 Neil Avenue,
Columbus, OH 43210. Phone: 614-688-5494; E-mail: Jacob.42@... .

Purpose: Previous studies in our laboratory have shown the progressive
methylation and suppression of the gene encoding protein tyrosine phosphatase,
PTPRO, in the livers of rats fed a methyl-deficient diet that induces
hepatocarcinogenesis. Subsequently, we observed the methylation of PTPRO in
primary human lung tumors and also showed its potential tumor suppressor
characteristics. The present study was undertaken to investigate whether the
truncated form of PTPRO (PTPROt), specifically expressed in naïve B lymphocytes,
was also methylated and suppressed in chronic lymphocytic leukemia (CLL), a
disease generally affecting B lymphocytes.

Experimental Design and Results: Initial screening showed that 60% of the 52 CLL
samples analyzed using methylation-specific PCR assay were methylated compared
with B lymphocytes from normal individuals, which were not methylated. The
expression of PTPROt, as measured by semiquantitative reverse transcription-PCR,
inversely correlated with methylation in the few samples tested. Analysis of
additional samples (n = 50) by combined bisulfite restriction analysis showed
that the PTPRO CpG island was methylated in 82% of patients with CLL compared
with B lymphocytes from normal individuals. Furthermore, overall expression of
PTPRO was reduced in CLL relative to normal lymphocytes. The PTPRO gene was also
suppressed by methylation in the CLL cell line WaC3CD5, where it could be
reactivated upon treatment with the DNA hypomethylating agent 5-AzaC. Ectopic
expression of PTPROt in a nonexpressing cell line increased growth inhibition
with fludarabine treatment, a therapy commonly used for CLL.

Conclusion: This study reveals the potential role of PTPRO methylation and
silencing in CLL tumorigenesis and also provides a novel molecular target in the
epigenetic therapy.


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