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Oncogenesis of T-ALL and nonmalignant consequences of overexpressing intracellular NOTCH1.

Li X, Gounari F, Protopopov A, Khazaie K, von Boehmer H - J. Exp. Med. (2008)

Bottom Line: Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL).We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells.In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level.

View Article: PubMed Central - PubMed

Affiliation: Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL). We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells. Early consequences are the generation of polyclonal nontumorigenic CD4(+)8(+) T cell receptor (TCR)-alphabeta(+) cells that do not qualify as tumor precursors despite the observation that they overexpress Notch 1 and c-Myc and degrade the tumor suppressor E2A by posttranslational modification. The first tumorigenic cells are detected among more immature CD4(-)8(+)TCR-alphabeta(-) cells that give rise to monoclonal tumors with a single, unique TCR-beta chain and diverse TCR-alpha chains, pinpointing malignant transformation to a stage after pre-TCR signaling and before completion of TCR-alpha rearrangement. In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level. Even though the tumors consist of phenotypically heterogeneous cells, no evidence for tumor stem cells was found. As judged by array-based comparative genomic hybridization (array CGH) and spectral karyotype (SKY) analysis, none of the tumors arise because of genomic instability.

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TCR expression by malignant and nonmalignant cells. (a) TCR-β rearrangement in various cell types. Analysis of TCR-β rearrangement by Southern blot analysis in GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) thymocytes derived from retroviral GFP-empty vector–transplanted mice, as well as GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) splenocytes derived from retroviral GFP-ICN1–transplanted mice at 2 and 8 wk after BMT. Molecular markers are shown on the left. (b) TCR-β and TCRV-α2 expression by various lymphocyte subsets. (left) Staining with the pan TCR-β antibody H57. (right) Staining with Vα2 TCR antibody.
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fig2: TCR expression by malignant and nonmalignant cells. (a) TCR-β rearrangement in various cell types. Analysis of TCR-β rearrangement by Southern blot analysis in GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) thymocytes derived from retroviral GFP-empty vector–transplanted mice, as well as GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) splenocytes derived from retroviral GFP-ICN1–transplanted mice at 2 and 8 wk after BMT. Molecular markers are shown on the left. (b) TCR-β and TCRV-α2 expression by various lymphocyte subsets. (left) Staining with the pan TCR-β antibody H57. (right) Staining with Vα2 TCR antibody.

Mentions: At 2 wk, a minor subset of CD4−8+ EGFP+ cells could be observed in peripheral lymphoid tissue, which, unlike the abnormal CD4+8+ cells, did not express an αβ TCR, and thus corresponded in surface phenotype to CD4−8+ immature thymocytes that represent precursors of CD4+8+ DP cells (Fig. 1, d and e). When these cells were transplanted into nu/nu mice, they generated tumors comprised of CD4+8+ and CD4−8+ cells with αβ TCRs on the cell surface. These data suggested that cells with malignant potential are generated after pre-TCR signaling–dependent TCR-β selection (14) and before TCR-α rearrangement. This was supported by the notion that the resulting monoclonal T-ALL carry a single TCR-β rearrangement (Fig. 2 a) but express diverse TCR-α chains, as indicated by the partial staining of αβ TCRs with antibodies specific for one particular Vα subgroup only (Fig. 2 b). Thus, malignant transformation of ICN1-overexpressing cells is an event that occurs after pre-TCR signaling, but before the acquisition of the CD4+8+ TCR-αβ+ phenotype. In fact, some of the monoclonal tumor cells do not express an αβ TCR on the cell surface, which is likely caused by the observation that they did not succeed in generating productive TCR-α rearrangements. The malignant potential of the phenotypically distinct tumor cells is reflected in their DNA labeling kinetics that are faster than those of their normal counterparts with the same surface phenotype. This is true for the “early” ICN1-overexpressing CD4−8+ TCR-αβ− cells that cause malignancy after transfer into nu/nu mice and also for the CD4+8+ and CD4−8+ TCR-αβ+ tumor cells that each can cause malignant tumors on their own after transfer into nu/nu recipients even when injected in low cell numbers. The analysis showing that each phenotypically distinct subset causes tumors with identical kinetics when injected in different doses refutes the possible notion that the subset with the most immature phenotype, i.e., the CD4−8+ TCR-αβ− subset features tumor stem cells, whereas the more mature subsets feature tumor mass but not necessarily highly tumorigenic cells (Fig. 1 d and Fig. 3). On the basis of these data, it appears useful to distinguish between normal CD4+8+ TCR-αβ+ thymocytes, abnormal nonmalignant and nontumorigenic ICN1-overexpressing CD4+8+ TCR-αβ+ cells, and tumorigenic CD4−8+ TCR-αβ−, as well as CD4+8+ TCR-αβ+ and CD4−8+ TCR-αβ+ tumor cells when further analyzing tumor development at the subcellular level. We assume that the nontumorigenic but abnormal CD4+8+ cells are derived from ICN1-overexpressing CD4−8+ TCR-αβ− precursors that have failed to undergo malignant transformation because of a genetic status not suited to cooperate with Notch1 overexpression to generate malignant transformation (see Discussion). Because this sequence of events after ICN1 overexpression has not been elaborated previously, it seems important to remember the contribution of the different subsets to malignancy when further analyzing gene expression.


Oncogenesis of T-ALL and nonmalignant consequences of overexpressing intracellular NOTCH1.

Li X, Gounari F, Protopopov A, Khazaie K, von Boehmer H - J. Exp. Med. (2008)

TCR expression by malignant and nonmalignant cells. (a) TCR-β rearrangement in various cell types. Analysis of TCR-β rearrangement by Southern blot analysis in GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) thymocytes derived from retroviral GFP-empty vector–transplanted mice, as well as GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) splenocytes derived from retroviral GFP-ICN1–transplanted mice at 2 and 8 wk after BMT. Molecular markers are shown on the left. (b) TCR-β and TCRV-α2 expression by various lymphocyte subsets. (left) Staining with the pan TCR-β antibody H57. (right) Staining with Vα2 TCR antibody.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2585834&req=5

fig2: TCR expression by malignant and nonmalignant cells. (a) TCR-β rearrangement in various cell types. Analysis of TCR-β rearrangement by Southern blot analysis in GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) thymocytes derived from retroviral GFP-empty vector–transplanted mice, as well as GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) splenocytes derived from retroviral GFP-ICN1–transplanted mice at 2 and 8 wk after BMT. Molecular markers are shown on the left. (b) TCR-β and TCRV-α2 expression by various lymphocyte subsets. (left) Staining with the pan TCR-β antibody H57. (right) Staining with Vα2 TCR antibody.
Mentions: At 2 wk, a minor subset of CD4−8+ EGFP+ cells could be observed in peripheral lymphoid tissue, which, unlike the abnormal CD4+8+ cells, did not express an αβ TCR, and thus corresponded in surface phenotype to CD4−8+ immature thymocytes that represent precursors of CD4+8+ DP cells (Fig. 1, d and e). When these cells were transplanted into nu/nu mice, they generated tumors comprised of CD4+8+ and CD4−8+ cells with αβ TCRs on the cell surface. These data suggested that cells with malignant potential are generated after pre-TCR signaling–dependent TCR-β selection (14) and before TCR-α rearrangement. This was supported by the notion that the resulting monoclonal T-ALL carry a single TCR-β rearrangement (Fig. 2 a) but express diverse TCR-α chains, as indicated by the partial staining of αβ TCRs with antibodies specific for one particular Vα subgroup only (Fig. 2 b). Thus, malignant transformation of ICN1-overexpressing cells is an event that occurs after pre-TCR signaling, but before the acquisition of the CD4+8+ TCR-αβ+ phenotype. In fact, some of the monoclonal tumor cells do not express an αβ TCR on the cell surface, which is likely caused by the observation that they did not succeed in generating productive TCR-α rearrangements. The malignant potential of the phenotypically distinct tumor cells is reflected in their DNA labeling kinetics that are faster than those of their normal counterparts with the same surface phenotype. This is true for the “early” ICN1-overexpressing CD4−8+ TCR-αβ− cells that cause malignancy after transfer into nu/nu mice and also for the CD4+8+ and CD4−8+ TCR-αβ+ tumor cells that each can cause malignant tumors on their own after transfer into nu/nu recipients even when injected in low cell numbers. The analysis showing that each phenotypically distinct subset causes tumors with identical kinetics when injected in different doses refutes the possible notion that the subset with the most immature phenotype, i.e., the CD4−8+ TCR-αβ− subset features tumor stem cells, whereas the more mature subsets feature tumor mass but not necessarily highly tumorigenic cells (Fig. 1 d and Fig. 3). On the basis of these data, it appears useful to distinguish between normal CD4+8+ TCR-αβ+ thymocytes, abnormal nonmalignant and nontumorigenic ICN1-overexpressing CD4+8+ TCR-αβ+ cells, and tumorigenic CD4−8+ TCR-αβ−, as well as CD4+8+ TCR-αβ+ and CD4−8+ TCR-αβ+ tumor cells when further analyzing tumor development at the subcellular level. We assume that the nontumorigenic but abnormal CD4+8+ cells are derived from ICN1-overexpressing CD4−8+ TCR-αβ− precursors that have failed to undergo malignant transformation because of a genetic status not suited to cooperate with Notch1 overexpression to generate malignant transformation (see Discussion). Because this sequence of events after ICN1 overexpression has not been elaborated previously, it seems important to remember the contribution of the different subsets to malignancy when further analyzing gene expression.

Bottom Line: Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL).We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells.In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level.

View Article: PubMed Central - PubMed

Affiliation: Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL). We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells. Early consequences are the generation of polyclonal nontumorigenic CD4(+)8(+) T cell receptor (TCR)-alphabeta(+) cells that do not qualify as tumor precursors despite the observation that they overexpress Notch 1 and c-Myc and degrade the tumor suppressor E2A by posttranslational modification. The first tumorigenic cells are detected among more immature CD4(-)8(+)TCR-alphabeta(-) cells that give rise to monoclonal tumors with a single, unique TCR-beta chain and diverse TCR-alpha chains, pinpointing malignant transformation to a stage after pre-TCR signaling and before completion of TCR-alpha rearrangement. In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level. Even though the tumors consist of phenotypically heterogeneous cells, no evidence for tumor stem cells was found. As judged by array-based comparative genomic hybridization (array CGH) and spectral karyotype (SKY) analysis, none of the tumors arise because of genomic instability.

Show MeSH
Related in: MedlinePlus