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IkappaBalpha overexpression delays tumor formation in v-rel transgenic mice.

Carrasco D, Perez P, Lewin A, Bravo R - J. Exp. Med. (1997)

Bottom Line: Overexpression of IkappaBalpha in v-rel transgenic mice resulted in an extended survival, and the development of cutaneous T cell lymphomas of CD8(+)CD4(-) phenotype.These phenotypic alterations were associated with a dramatic reduction of p50/v-Rel, but not v-Rel/v-Rel nuclear DNA binding activity and an increased expression of the intercellular adhesion molecule 1.Our results indicate that v-Rel homodimers are active in transformation and that the capacity of v-Rel-containing complexes to escape the inhibitory effect of IkappaBalpha may be a key element in its transforming capability.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.

ABSTRACT
We have previously shown that transgenic mice expressing the oncoprotein v-Rel under the control of a T cell-specific promoter develop T cell lymphomas. Tumor formation was correlated with the presence of p50/v-Rel and v-Rel/v-Rel nuclear kappaB-binding activity. Since experimental evidence has led to the suggestion of a potential tumor suppressor activity for IkappaBalpha, we have studied the role of IkappaBalpha in the transforming activity of v-Rel by overexpressing IkappaBalpha in v-rel transgenic mice. Overexpression of IkappaBalpha in v-rel transgenic mice resulted in an extended survival, and the development of cutaneous T cell lymphomas of CD8(+)CD4(-) phenotype. These phenotypic alterations were associated with a dramatic reduction of p50/v-Rel, but not v-Rel/v-Rel nuclear DNA binding activity and an increased expression of the intercellular adhesion molecule 1. Our results indicate that v-Rel homodimers are active in transformation and that the capacity of v-Rel-containing complexes to escape the inhibitory effect of IkappaBalpha may be a key element in its transforming capability.

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Reduced IκBα inhibition of v-Rel DNA binding  activity (A) and increased levels  of ICAM-1 in v-rel/ikba double  transgenic T cells (B and C). (A)  Equal amounts of nuclear protein extracts from Δc-rel (a) or  v-rel (b) transgenic thymocytes  were incubated in the presence  of increased amounts of IκBα  protein and analyzed by EMSA  using a palindromic κB-binding  site. (B) Total RNA was prepared from ikba transgenic, v-rel  transgenic or v-rel/ikba double  transgenic lymph node–derived  cells. After cDNA synthesis,  one-tenth of the reaction mixture was amplified in the presence of 0.1 μCi of [32P]dCTP using Taq polymerase and sequence-specific primers. One-fifth  of the amplified products were separated on 6% nondenaturing polyacrilamide gels. Gels were dried and exposed to x-ray film. (C) Purified T cells from lymph  nodes of v-rel transgenic (a) and v-rel/ikba (b) double transgenic mice were spun onto slides and incubated with anti–ICAM-1 FITC-labeled antibody.
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Figure 6: Reduced IκBα inhibition of v-Rel DNA binding activity (A) and increased levels of ICAM-1 in v-rel/ikba double transgenic T cells (B and C). (A) Equal amounts of nuclear protein extracts from Δc-rel (a) or v-rel (b) transgenic thymocytes were incubated in the presence of increased amounts of IκBα protein and analyzed by EMSA using a palindromic κB-binding site. (B) Total RNA was prepared from ikba transgenic, v-rel transgenic or v-rel/ikba double transgenic lymph node–derived cells. After cDNA synthesis, one-tenth of the reaction mixture was amplified in the presence of 0.1 μCi of [32P]dCTP using Taq polymerase and sequence-specific primers. One-fifth of the amplified products were separated on 6% nondenaturing polyacrilamide gels. Gels were dried and exposed to x-ray film. (C) Purified T cells from lymph nodes of v-rel transgenic (a) and v-rel/ikba (b) double transgenic mice were spun onto slides and incubated with anti–ICAM-1 FITC-labeled antibody.

Mentions: To understand why higher levels of IκBα are required to reduce nuclear translocation of v-Rel and p50/v-Rel κB-binding activity in transgenic thymocytes, we compared the inhibitory effect of IκBα on κB binding activity in nuclear protein extract derived from v-rel transgenic (Fig. 6 A) and Δc-rel transgenic thymocytes. Δc-rel transgenic animals express a truncated version of the mouse c-Rel protein (Δc-Rel) under the control of the lck promoter, and, like the v-Rel oncogenic protein, lacks part of the COOH-terminal transcriptional activation domain keeping the RHD intact. The generation of Δc-rel transgenic mice has been previously described (17). Increasing levels of purified baculovirus expressed IκBα protein were added to identical amounts of nuclear protein extracts from Δc-rel transgenic thymocytes (Fig. 6 A a) and v-rel transgenic thymocytes (Fig. 6 A b) before starting the κB-binding reaction. The total amount of protein extract used in the reaction was tested by Oct 1 DNA-binding activity and the identification of the complexes by antibody supershifts (data not shown). In the absence of added IκBα, similar amounts of total κB binding activities were detected in Δc-rel transgenic (Fig. 6 A a, lane 1) and in v-rel transgenic thymocytes (Fig. 6 A b, lane 1). However, when increased amounts of IκBα protein were added to the binding reaction, the binding of p50/Δc-Rel was dramatically reduced (Fig. 6 A a, lanes 2 and 3), whereas the binding of v-Rel containing complexes was slightly affected (Fig. 6 A b, lanes 2 and 3). At 100 ng of IκBα, the binding of v-Rel–containing complexes is significantly reduced (Fig. 6 A b, lane 4), and only when the highest amounts of IκBα were used (1 μg) and when endogenous p50/p50 κB binding also began to be affected (Fig. 6 A a, lane 5), was the binding of v-Rel–containing complexes completely reduced (Fig. 6 A b, lane 5). These results demonstrate that the binding of v-Rel–containing complexes is more resistant than the binding of complexes containing the cellular homologue Δc-Rel to the inhibitory effect of IκBα, and offer an attractive explanation for the molecular mechanisms involved in the transforming activity of v-Rel. This is in agreement with previous in vitro studies that demonstrated a reduced IκBα inhibition of DNA binding by the oncogenic v-Rel protein when compared to the nononcogenic c-Rel protein (28).


IkappaBalpha overexpression delays tumor formation in v-rel transgenic mice.

Carrasco D, Perez P, Lewin A, Bravo R - J. Exp. Med. (1997)

Reduced IκBα inhibition of v-Rel DNA binding  activity (A) and increased levels  of ICAM-1 in v-rel/ikba double  transgenic T cells (B and C). (A)  Equal amounts of nuclear protein extracts from Δc-rel (a) or  v-rel (b) transgenic thymocytes  were incubated in the presence  of increased amounts of IκBα  protein and analyzed by EMSA  using a palindromic κB-binding  site. (B) Total RNA was prepared from ikba transgenic, v-rel  transgenic or v-rel/ikba double  transgenic lymph node–derived  cells. After cDNA synthesis,  one-tenth of the reaction mixture was amplified in the presence of 0.1 μCi of [32P]dCTP using Taq polymerase and sequence-specific primers. One-fifth  of the amplified products were separated on 6% nondenaturing polyacrilamide gels. Gels were dried and exposed to x-ray film. (C) Purified T cells from lymph  nodes of v-rel transgenic (a) and v-rel/ikba (b) double transgenic mice were spun onto slides and incubated with anti–ICAM-1 FITC-labeled antibody.
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Figure 6: Reduced IκBα inhibition of v-Rel DNA binding activity (A) and increased levels of ICAM-1 in v-rel/ikba double transgenic T cells (B and C). (A) Equal amounts of nuclear protein extracts from Δc-rel (a) or v-rel (b) transgenic thymocytes were incubated in the presence of increased amounts of IκBα protein and analyzed by EMSA using a palindromic κB-binding site. (B) Total RNA was prepared from ikba transgenic, v-rel transgenic or v-rel/ikba double transgenic lymph node–derived cells. After cDNA synthesis, one-tenth of the reaction mixture was amplified in the presence of 0.1 μCi of [32P]dCTP using Taq polymerase and sequence-specific primers. One-fifth of the amplified products were separated on 6% nondenaturing polyacrilamide gels. Gels were dried and exposed to x-ray film. (C) Purified T cells from lymph nodes of v-rel transgenic (a) and v-rel/ikba (b) double transgenic mice were spun onto slides and incubated with anti–ICAM-1 FITC-labeled antibody.
Mentions: To understand why higher levels of IκBα are required to reduce nuclear translocation of v-Rel and p50/v-Rel κB-binding activity in transgenic thymocytes, we compared the inhibitory effect of IκBα on κB binding activity in nuclear protein extract derived from v-rel transgenic (Fig. 6 A) and Δc-rel transgenic thymocytes. Δc-rel transgenic animals express a truncated version of the mouse c-Rel protein (Δc-Rel) under the control of the lck promoter, and, like the v-Rel oncogenic protein, lacks part of the COOH-terminal transcriptional activation domain keeping the RHD intact. The generation of Δc-rel transgenic mice has been previously described (17). Increasing levels of purified baculovirus expressed IκBα protein were added to identical amounts of nuclear protein extracts from Δc-rel transgenic thymocytes (Fig. 6 A a) and v-rel transgenic thymocytes (Fig. 6 A b) before starting the κB-binding reaction. The total amount of protein extract used in the reaction was tested by Oct 1 DNA-binding activity and the identification of the complexes by antibody supershifts (data not shown). In the absence of added IκBα, similar amounts of total κB binding activities were detected in Δc-rel transgenic (Fig. 6 A a, lane 1) and in v-rel transgenic thymocytes (Fig. 6 A b, lane 1). However, when increased amounts of IκBα protein were added to the binding reaction, the binding of p50/Δc-Rel was dramatically reduced (Fig. 6 A a, lanes 2 and 3), whereas the binding of v-Rel containing complexes was slightly affected (Fig. 6 A b, lanes 2 and 3). At 100 ng of IκBα, the binding of v-Rel–containing complexes is significantly reduced (Fig. 6 A b, lane 4), and only when the highest amounts of IκBα were used (1 μg) and when endogenous p50/p50 κB binding also began to be affected (Fig. 6 A a, lane 5), was the binding of v-Rel–containing complexes completely reduced (Fig. 6 A b, lane 5). These results demonstrate that the binding of v-Rel–containing complexes is more resistant than the binding of complexes containing the cellular homologue Δc-Rel to the inhibitory effect of IκBα, and offer an attractive explanation for the molecular mechanisms involved in the transforming activity of v-Rel. This is in agreement with previous in vitro studies that demonstrated a reduced IκBα inhibition of DNA binding by the oncogenic v-Rel protein when compared to the nononcogenic c-Rel protein (28).

Bottom Line: Overexpression of IkappaBalpha in v-rel transgenic mice resulted in an extended survival, and the development of cutaneous T cell lymphomas of CD8(+)CD4(-) phenotype.These phenotypic alterations were associated with a dramatic reduction of p50/v-Rel, but not v-Rel/v-Rel nuclear DNA binding activity and an increased expression of the intercellular adhesion molecule 1.Our results indicate that v-Rel homodimers are active in transformation and that the capacity of v-Rel-containing complexes to escape the inhibitory effect of IkappaBalpha may be a key element in its transforming capability.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.

ABSTRACT
We have previously shown that transgenic mice expressing the oncoprotein v-Rel under the control of a T cell-specific promoter develop T cell lymphomas. Tumor formation was correlated with the presence of p50/v-Rel and v-Rel/v-Rel nuclear kappaB-binding activity. Since experimental evidence has led to the suggestion of a potential tumor suppressor activity for IkappaBalpha, we have studied the role of IkappaBalpha in the transforming activity of v-Rel by overexpressing IkappaBalpha in v-rel transgenic mice. Overexpression of IkappaBalpha in v-rel transgenic mice resulted in an extended survival, and the development of cutaneous T cell lymphomas of CD8(+)CD4(-) phenotype. These phenotypic alterations were associated with a dramatic reduction of p50/v-Rel, but not v-Rel/v-Rel nuclear DNA binding activity and an increased expression of the intercellular adhesion molecule 1. Our results indicate that v-Rel homodimers are active in transformation and that the capacity of v-Rel-containing complexes to escape the inhibitory effect of IkappaBalpha may be a key element in its transforming capability.

Show MeSH
Related in: MedlinePlus