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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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Effect of mutant N-RAS expression on cell cycle distribution. Representative histograms are shown from one of four experiments.  Filled histograms represent infected cells (β-galactosidase positive); open  histograms show uninfected cells (β-galactosidase negative) within the  same culture. Values indicate the proportion of cells in S + G2M ± SD.  **P <0.001.
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Figure 4: Effect of mutant N-RAS expression on cell cycle distribution. Representative histograms are shown from one of four experiments. Filled histograms represent infected cells (β-galactosidase positive); open histograms show uninfected cells (β-galactosidase negative) within the same culture. Values indicate the proportion of cells in S + G2M ± SD. **P <0.001.

Mentions: Fig. 2 illustrates β-galactosidase expression in control cells (expressing lacZ alone) and in RAS-infected cells (coexpressing mutant N-RAS and lacZ). By applying a threshold of positivity defined by an identically treated mock-infected culture, we were able to assess the relative proportion of infected cells in the culture. This enabled us to determine the relative expansion of the infected cells in relation to the uninfected cells throughout the subsequent culture period. Fig. 3 demonstrates that the relative expansion of control-infected erythroblasts was close to that of uninfected cells, demonstrating that there was stable expression of β-galactosidase in these cells and that expression of this selectable marker did not overtly affect their proliferative capacity. On the other hand, the proportion of RAS-infected erythroblasts rapidly declined, indicating that the effect of activated RAS on these cells was to reduce their overall proliferative rate in comparison with the nonexpressing cells within the same culture. This rate of decline appeared to be constant, since logarithmic transformation of these data yielded a linear plot (r = −0.9982). This allowed us to calculate that the effective doubling time of these cells was increased from 14.6 to 18.9 h, a 29% average increase over the time frame indicated. The cumulative effects of this increase resulted in the gradual disappearance of RAS-expressing cells from these cultures. A number of explanations could account for this observation: (a) cell cycle time (tc) is increased, (b) cell cycle is unaffected, but there is an increased rate of premature cell death, or (c) a combination of the above. To determine whether expression of mutant N-RAS caused perturbation of the normal cell cycle distribution, we supravitally stained these cultures for DNA content in combination with staining for β-galactosidase activity. Fig. 4 shows that the proportion of cells in cycle was indeed reduced in the RAS-expressing cells (43%) when compared with the control-infected cells (59%); P <0.001. Since these cultures contained mixed populations of infected and uninfected cells (as defined by β-galactosidase positivity), we were also able to analyze the cells not expressing mutant RAS within the same culture; this analysis effectively acts as an internal control. The proportion of cells in cycle in this population (57%) was effectively identical to the controls and demonstrates that the changes in cell cycle distribution were specific to the mutant RASexpressing cells within the culture. The most likely explanation for the observed decrease in the proportion of cells in S + G2M is that, on average, the RAS-expressing cells spent more time in the G1 phase of the cell cycle. If this is the case, the increased proportion in G1 could be accounted for by an overall increase in tc of 30% to 19 h. This value is close to the actual increased doubling time observed above and suggests that increase in cell cycle time alone could account for the decreased proliferative rate of these cells.


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)

Effect of mutant N-RAS expression on cell cycle distribution. Representative histograms are shown from one of four experiments.  Filled histograms represent infected cells (β-galactosidase positive); open  histograms show uninfected cells (β-galactosidase negative) within the  same culture. Values indicate the proportion of cells in S + G2M ± SD.  **P <0.001.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Effect of mutant N-RAS expression on cell cycle distribution. Representative histograms are shown from one of four experiments. Filled histograms represent infected cells (β-galactosidase positive); open histograms show uninfected cells (β-galactosidase negative) within the same culture. Values indicate the proportion of cells in S + G2M ± SD. **P <0.001.
Mentions: Fig. 2 illustrates β-galactosidase expression in control cells (expressing lacZ alone) and in RAS-infected cells (coexpressing mutant N-RAS and lacZ). By applying a threshold of positivity defined by an identically treated mock-infected culture, we were able to assess the relative proportion of infected cells in the culture. This enabled us to determine the relative expansion of the infected cells in relation to the uninfected cells throughout the subsequent culture period. Fig. 3 demonstrates that the relative expansion of control-infected erythroblasts was close to that of uninfected cells, demonstrating that there was stable expression of β-galactosidase in these cells and that expression of this selectable marker did not overtly affect their proliferative capacity. On the other hand, the proportion of RAS-infected erythroblasts rapidly declined, indicating that the effect of activated RAS on these cells was to reduce their overall proliferative rate in comparison with the nonexpressing cells within the same culture. This rate of decline appeared to be constant, since logarithmic transformation of these data yielded a linear plot (r = −0.9982). This allowed us to calculate that the effective doubling time of these cells was increased from 14.6 to 18.9 h, a 29% average increase over the time frame indicated. The cumulative effects of this increase resulted in the gradual disappearance of RAS-expressing cells from these cultures. A number of explanations could account for this observation: (a) cell cycle time (tc) is increased, (b) cell cycle is unaffected, but there is an increased rate of premature cell death, or (c) a combination of the above. To determine whether expression of mutant N-RAS caused perturbation of the normal cell cycle distribution, we supravitally stained these cultures for DNA content in combination with staining for β-galactosidase activity. Fig. 4 shows that the proportion of cells in cycle was indeed reduced in the RAS-expressing cells (43%) when compared with the control-infected cells (59%); P <0.001. Since these cultures contained mixed populations of infected and uninfected cells (as defined by β-galactosidase positivity), we were also able to analyze the cells not expressing mutant RAS within the same culture; this analysis effectively acts as an internal control. The proportion of cells in cycle in this population (57%) was effectively identical to the controls and demonstrates that the changes in cell cycle distribution were specific to the mutant RASexpressing cells within the culture. The most likely explanation for the observed decrease in the proportion of cells in S + G2M is that, on average, the RAS-expressing cells spent more time in the G1 phase of the cell cycle. If this is the case, the increased proportion in G1 could be accounted for by an overall increase in tc of 30% to 19 h. This value is close to the actual increased doubling time observed above and suggests that increase in cell cycle time alone could account for the decreased proliferative rate of these cells.

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