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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

RhoA: A therapeutic target for chronic myeloid leukemia

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Poonam R Molli, Madhura B Pradhan, Suresh H Advani, Nishigandha R Naik
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-16) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

N.R.N. conceived and designed the experiments. P.R.M. did experiments with clinical samples. S.H.A. was a clinical collaborator who identified the patients and provided clinical samples. M.B.P. did growth inhibition assay using cell lines. P.R.M and M.B.P. analyzed the respective data under guidance of N.R.N. P.R.M. and N.R.N. wrote the paper.

Abstract

Background

Chronic Myeloid Leukemia (CML) is a malignant pluripotent stem cells disorder of myeloid cells. In CML patients, polymorphonuclear leukocytes (PMNL) the terminally differentiated cells of myeloid series exhibit defects in several actin dependent functions such as adhesion, motility, chemotaxis, agglutination, phagocytosis and microbicidal activities. A definite and global abnormality was observed in stimulation of actin polymerization in CML PMNL. Signalling molecules ras and rhoGTPases regulate spatial and temporal polymerization of actin and thus, a broad range of physiological processes. Therefore, status of these GTPases as well as actin was studied in resting and fMLP stimulated normal and CML PMNL.

Methods

To study expression of GTPases and actin, Western blotting and flow cytometry analysis were done, while spatial expression and colocalization of these proteins were studied by using laser confocal microscopy. To study effect of inhibitors on cell proliferation CCK-8 assay was done. Significance of differences in expression of proteins within the samples and between normal and CML was tested by using Wilcoxon signed rank test and Mann-Whitney test, respectively. Bivariate and partial correlation analyses were done to study relationship between all the parameters.

Results

In CML PMNL, actin expression and its architecture were altered and stimulation of actin polymerization was absent. Differences were also observed in expression, organization or stimulation of all the three GTPases in normal and CML PMNL. In normal PMNL, ras was the critical GTPase regulating expression of rhoGTPases and actin and actin polymerization. But in CML PMNL, rhoA took a central place. In accordance with these, treatment with rho/ROCK pathway inhibitors resulted in specific growth inhibition of CML cell lines.

Conclusions

RhoA has emerged as the key molecule responsible for functional defects in CML PMNL and therefore can be used as a therapeutic target in CML.
Zusatzmaterial
Additional file 1: Figure S1. H-ras localization in normal PMNL: Middle Z planes and 3D LCM images showing distribution of H-ras in unstimulated and fMLP stimulated (0.5, 5, 10, 30, 45 min) normal PMNL. The central panel shows the corresponding transmission images of the fluorescent images. For studying distribution of GTPases, 'thermo1' LUT based on the fluorescence intensity was applied to all the fluorescence images in this figure. Fluorescence intensity reflects the expression level of the protein of interest. The pseudocolour code used was as follows: Dark blue: extremely low level of expression; Light blue: low level of expression; Yellow: moderate level of expression; Orange: high level of expression; Red: extremely high level of expression. (JPEG 71 KB)
12943_2011_1005_MOESM1_ESM.JPEG
Additional file 2: Figure S2. H-ras localization in CML PMNL: Middle Z planes and 3D LCM images showing distribution of H-ras in unstimulated and fMLP stimulated (0.5, 5, 10, 30, 45 min) CML PMNL. The central panel shows the corresponding transmission images of the fluorescent images. For studying distribution of GTPases, 'thermo1' LUT based on the fluorescence intensity was applied to all the fluorescence images in this figure. Fluorescence intensity reflects the expression level of the protein of interest. The pseudocolour code used was as follows: Dark blue: extremely low level of expression; Light blue: low level of expression; Yellow: moderate level of expression; Orange: high level of expression; Red: extremely high level of expression. (JPEG 58 KB)
12943_2011_1005_MOESM2_ESM.JPEG
Additional file 3: Figure S3. Rac1 localization in normal PMNL: Middle Z planes and 3D LCM images showing distribution of rac1 in unstimulated and fMLP stimulated (0.5, 5, 10, 30, 45 min) normal PMNL. The central panel shows the corresponding transmission images of the fluorescent images. For studying distribution of GTPases, 'thermo1' LUT based on the fluorescence intensity was applied to all the fluorescence images in this figure. Fluorescence intensity reflects the expression level of the protein of interest. The pseudocolour code used was as follows: Dark blue: extremely low level of expression; Light blue: low level of expression; Yellow: moderate level of expression; Orange: high level of expression; Red: extremely high level of expression. (JPEG 87 KB)
12943_2011_1005_MOESM3_ESM.JPEG
Additional file 4: Figure S4. Rac1 localization in CML PMNL: Middle Z planes and 3D LCM images showing distribution of rac1 in unstimulated and fMLP stimulated (0.5, 5, 10, 30, 45 min) CML PMNL. The central panel shows the corresponding transmission images of the fluorescent images. For studying distribution of GTPases, 'thermo1' LUT based on the fluorescence intensity was applied to all the fluorescence images in this figure. Fluorescence intensity reflects the expression level of the protein of interest. The pseudocolour code used was as follows: Dark blue: extremely low level of expression; Light blue: low level of expression; Yellow: moderate level of expression; Orange: high level of expression; Red: extremely high level of expression. (JPEG 94 KB)
12943_2011_1005_MOESM4_ESM.JPEG
Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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