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Mutant N-RAS induces erythroid lineage dysplasia in human CD34+ cells.

Darley RL, Hoy TG, Baines P, Padua RA, Burnett AK - J. Exp. Med. (1997)

Bottom Line: By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle.This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development.The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology, University of Wales College of Medicine, Cardiff, United Kingdom.

ABSTRACT
RAS mutations arise at high frequency (20-40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, beta-galactosidase, allows direct identification and study of N-RAS-expressing cells by incubating infected cultures with a fluorogenic substrate for beta-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.

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Expression of β-galactosidase after infection of CD34+ cells.  Mock-infected cells (open histograms) indicate the threshold for positivity. These data represent β-galactosidase expression after a 3 d induction  with EPO.
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Figure 2: Expression of β-galactosidase after infection of CD34+ cells. Mock-infected cells (open histograms) indicate the threshold for positivity. These data represent β-galactosidase expression after a 3 d induction with EPO.

Mentions: Acquired data were analyzed using Lysys II (Becton Dickinson) and WinMDI (gift of Joe Trotter, Scripps Institute, La Jolla, CA). Cell cycle analysis was carried out using the pragmatic approach of Watson et al. (21). Copies can be obtained from CytonetUK http://www.cf.ac.uk/uwcm/hg/hoy/. Infected cultures contained mixed populations of infected and uninfected cells. Infected cells were identified as those exhibiting greater fluorescence than the equivalent mock-infected culture (see Fig. 2). The threshold of positivity was set such that >90% of the selected cells had fluorescence greater than the mock-infected control. Membrane-damaged cells were excluded from the analysis on the basis of strong staining with 7AAD or PI as appropriate. In this way, both infected and uninfected populations were analyzed by gating on β-galactosidase positive and negative cells, respectively.


Mutant N-RAS induces erythroid lineage dysplasia in human CD34+ cells.

Darley RL, Hoy TG, Baines P, Padua RA, Burnett AK - J. Exp. Med. (1997)

Expression of β-galactosidase after infection of CD34+ cells.  Mock-infected cells (open histograms) indicate the threshold for positivity. These data represent β-galactosidase expression after a 3 d induction  with EPO.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196261&req=5

Figure 2: Expression of β-galactosidase after infection of CD34+ cells. Mock-infected cells (open histograms) indicate the threshold for positivity. These data represent β-galactosidase expression after a 3 d induction with EPO.
Mentions: Acquired data were analyzed using Lysys II (Becton Dickinson) and WinMDI (gift of Joe Trotter, Scripps Institute, La Jolla, CA). Cell cycle analysis was carried out using the pragmatic approach of Watson et al. (21). Copies can be obtained from CytonetUK http://www.cf.ac.uk/uwcm/hg/hoy/. Infected cultures contained mixed populations of infected and uninfected cells. Infected cells were identified as those exhibiting greater fluorescence than the equivalent mock-infected culture (see Fig. 2). The threshold of positivity was set such that >90% of the selected cells had fluorescence greater than the mock-infected control. Membrane-damaged cells were excluded from the analysis on the basis of strong staining with 7AAD or PI as appropriate. In this way, both infected and uninfected populations were analyzed by gating on β-galactosidase positive and negative cells, respectively.

Bottom Line: By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle.This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development.The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology, University of Wales College of Medicine, Cardiff, United Kingdom.

ABSTRACT
RAS mutations arise at high frequency (20-40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, beta-galactosidase, allows direct identification and study of N-RAS-expressing cells by incubating infected cultures with a fluorogenic substrate for beta-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.

Show MeSH
Related in: MedlinePlus