Cancer Res Treat.  2013 Jun;45(2):126-133.

Src Family Kinase Inhibitor PP2 Has Different Effects on All-Trans-Retinoic Acid or Arsenic Trioxide-Induced Differentiation of an Acute Promyelocytic Leukemia Cell Line

• Yoon SG
• Cheong HJ
• Kim SJ
• Kim KH
• Lee SC
• Lee N
• Park HS
• Won JH

Abstract

PURPOSE
Leukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (ATO). Recent studies have shown that inhibition of Src family kinases (SFKs) resulted in enhancement of retinoic acid-induced myeloid differentiation.
MATERIALS AND METHODS
In this study, we investigated the question of whether the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well as when combined with all-trans-retinoic acid (ATRA). In addition, we attempted to determine the difference in retinoic acid-induced gene expression between cells treated with PP2 in combination with ATRA and in combination with ATO.
RESULTS
SFK inhibitor PP2 induced significant enhancement of ATRA- or ATO-induced differentiation of NB4 cells. A significantly stronger synergistic effect was observed when PP2 was combined with ATRA than when combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by nitroblue tetrazolium staining. These effects were not related to apoptosis. Results of Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. Intercellular adhesion molecule-1 expression showed a significant increase in cells treated with PP2 in combination with ATRA, whereas cathepsin D expression showed a significant increase in cells treated with PP2 in combination with ATO.
CONCLUSION
Our data showed that SFK inhibitor PP2 enhanced acute promyelocytic leukemia cell differentiation when combined with either ATRA or ATO with difference in activation of retinoic acid-induced genes.

Keyword

Acute promyelocytic leukemia; All-trans-retinoic acid; Arsenic trioxide; Src kinase; Cell differentiation

MeSH Terms

Apoptosis
Arsenic
Arsenicals
Cathepsin D
Cell Differentiation
Cell Line
Gene Expression
Granulocyte Precursor Cells
Granulocytes
Humans
Intercellular Adhesion Molecule-1
Leukemia, Promyelocytic, Acute
Nitroblue Tetrazolium
Oxides
Phosphotransferases
Pyrimidines
src-Family Kinases
Tretinoin
Arsenic
Arsenicals
Cathepsin D
Intercellular Adhesion Molecule-1
Nitroblue Tetrazolium
Oxides
Phosphotransferases
Pyrimidines
Tretinoin
src-Family Kinases

Figure

• 

  Fig. 1 Effect of PP2 on all-trans-retinoic acid (ATRA)- or arsenic trioxide (ATO)-induced differentiation of NB4 cells. NB4 cells were treated with 10 µM of PP2 alone, 0.5 µM of ATO alone, 0.001 µM of ATRA alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2 for 72 hours. Cells were harvested, incubated with anti-CD11b-PE antibody or isotype-matched antibody, and analyzed by flow cytometry. A total of 10,000 cells per sample were analyzed for expression of CD11b. (A) Open histograms, cells stained with isotype control antibody; shaded histograms, staining with CD11b antibody. Values in the upper right corner illustrate the percentage of CD11b-positive cells. (B) Columns represent the averages for five independent experiments with similar results. Control cells that were not treated with any reagents. a)p<0.05 significantly higher than ATO alone, b)p<0.05 significantly higher than ATRA alone, c)p<0.05 significantly higher than PP2 combined with ATO.

• 

  Fig. 2 Effect of PP2 on all-trans-retinoic acid (ATRA)- or arsenic trioxide (ATO)-induced granulocytic differentiation of NB4 cells. NB4 cells were treated with 10 µM of PP2 alone, 0.5 µM of ATO alone, 0.001 µM of ATRA alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2 for 72 hours. After treatment for 72 hours, cells were evaluated for granulocytic differentiation on the basis of nitroblue tetrazolium reduction assay. Using a light microscope, a minimum of 200 cells were counted, in order to determine the percentage of differentiated cells. (A) Differentiated cells were identified by their intracellular blue formazan deposits. (B) Columns represent the averages for four independent experiments with similar results. C indicates control that cells were not treated with any reagents. a)p<0.05 significantly higher than ATO alone, b)p<0.05 significantly higher than ATRA alone, c)p<0.05 significantly higher than PP2 combined with ATO.

• 

  Fig. 3 Effect of PP2 on all-trans-retinoic acid (ATRA)- or arsenic trioxide (ATO)-induced apoptosis of NB4 cells. NB4 cells were treated with 10 µM of PP2 alone, 0.5 µM of ATO alone, 0.001 µM of ATRA alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2 for 72 hours. After treatment for 72 hours, cells were stained with Annexin V-FITC and analyzed by flow cytometry (results representative of three independent experiments). Values in the center present the percentage of apoptotic cells.

• 

  Fig. 4 Retinoic acid-induced gene expression. NB4 cells were treated for 72 hours with 10 µM of PP2 alone, 0.5 µM of arsenic trioxide (ATO) alone, 0.001 µM of all-transretinoic acid (ATRA) alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2. Fifty micrograms of whole cell lysates were resolved by sodium dedecyl sulfate polyacrylamide gel electrophoresis and subjected to western blotting with antibodies against intercellular adhesion molecule-1 (ICAM-1) or cathepsin D. Equal loading was determined by actin. Similar results were obtained in three independent experiments.

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