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Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability.

Chen M, Huang JD, Deng HK, Dong S, Deng W, Tsang SL, Huen MS, Chen L, Zan T, Zhu GX, Guan XY - BMC Cancer (2011)

Bottom Line: Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region.This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification.This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Oncology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, China.

ABSTRACT

Background: Amplification of 3q26 is one of the most frequent genetic alterations in many human malignancies. Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region. Functional study has demonstrated the oncogenic role of eIF-5A2 in the initiation and progression of human cancers. In the present study, we aim to investigate the physiological and pathological effect of eIF-5A2 in an eIF-5A2 transgenic mouse model.

Methods: An eIF-5A2 transgenic mouse model was generated using human eIF-5A2 cDNA. The eIF-5A2 transgenic mice were characterized by histological and immunohistochemistry analyses. The aging phenotypes were further characterized by wound healing, bone X-ray imaging and calcification analysis. Mouse embryo fibroblasts (MEF) were isolated to further investigate molecular mechanism of eIF-5A2 in aging.

Results: Instead of resulting in spontaneous tumor formation, overexpression of eIF-5A2 accelerated the aging process in adult transgenic mice. This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification. Investigation of the correlation between cellular senescence and aging showed that cellular senescence is not required for the aging phenotypes in eIF-5A2 mice. Interestingly, we found that activation of eIF-5A2 repressed p19 level and therefore destabilized p53 in transgenic mouse embryo fibroblast (MEF) cells. This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase. Additionally, a significantly increase in number of aneuploidy cells (p < 0.05) resulted from an increase in the incidences of misaligned and lagging chromosomal materials, anaphase bridges, and micronuclei in the transgenic mice.

Conclusion: These observations suggest that eIF-5A2 mouse models could accelerate organismal aging by increasing chromosome instability.

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Generation of eIF-5A2 transgenic mice. (A) Three eIF-5A2 transgenic mouse founders (mouse No.: 10, 11, and 89) and their offspring (166-169) were determined by PCR. One mouse without eIF-5A2 transgene (No. 9) was used as negative control. Mouse fibroblast cell line NIH 3T3 and eIF-5A2 transgenic construct was used as negative (-) and positive (+) controls. (B) The transgene eIF-5A2 was mapped to one mouse chromosome site in line 11 by FISH. The metaphase spread was prepared from bone marrow lymphocyte. Arrow indicates the hybridization signals of eIF-5A2. (C) Expression of eIF-5A2 in transgenic mice was confirmed by Northern blot analysis. A human eIF-5A2 cDNA probe was used and it did not detect the endogenous eIF-5A2 mRNA in both wild-type and transgenic mice. (D) Western blot showed the overexpression of human eIF-5A2 in transgenic mice using liver lysates. The lower bands (17dD) were eIF-5A2 and the upper bands were shifted eIF-5A2 bands caused by posttranslational hypusination. (E) Expression of transgene eIF-5A2 in various tissues of transgenic mouse was detected by RT-PCR using a pair of human-specific primers.
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Figure 1: Generation of eIF-5A2 transgenic mice. (A) Three eIF-5A2 transgenic mouse founders (mouse No.: 10, 11, and 89) and their offspring (166-169) were determined by PCR. One mouse without eIF-5A2 transgene (No. 9) was used as negative control. Mouse fibroblast cell line NIH 3T3 and eIF-5A2 transgenic construct was used as negative (-) and positive (+) controls. (B) The transgene eIF-5A2 was mapped to one mouse chromosome site in line 11 by FISH. The metaphase spread was prepared from bone marrow lymphocyte. Arrow indicates the hybridization signals of eIF-5A2. (C) Expression of eIF-5A2 in transgenic mice was confirmed by Northern blot analysis. A human eIF-5A2 cDNA probe was used and it did not detect the endogenous eIF-5A2 mRNA in both wild-type and transgenic mice. (D) Western blot showed the overexpression of human eIF-5A2 in transgenic mice using liver lysates. The lower bands (17dD) were eIF-5A2 and the upper bands were shifted eIF-5A2 bands caused by posttranslational hypusination. (E) Expression of transgene eIF-5A2 in various tissues of transgenic mouse was detected by RT-PCR using a pair of human-specific primers.

Mentions: To characterize the role of eIF-5A2 in vivo, we generated transgenic mouse lines overexpressing human eIF-5A2 ubiquitously. Pronuclear injection of the pCAGGS-eIF-5A2 construct resulted in the generation of three founder mice, as identified by genomic Southern blot analysis and PCR (Figure 1A). To confirm the single integration event, an eIF-5A2 probe was used to hybridize to the metaphase spreads from bone marrow lymphocytes by fluorescence in situ hybridization (FISH). The transgene was mapped to one chromosome site in line 11 (Figure 1B). All the founders were able to transmit the transgene to their offspring in the expected Mendelian ratio. Three lines were expanded, and the expression of the transgene was confirmed in two out of three founder lines (10 and 11). Expression of eIF-5A2 at the RNA level was detected by RT-PCR and Northern blot hybridization using liver and testis tissues (Figure 1C). Also, EIF-5A2 protein expression was demonstrated by immunohistochemistry (IHC) and Western blot analysis (Figure 1D) in multiple organs. Of note, no difference was observed in the expression of murine endogenous eIF-5A2 among the founders and wild-type controls. To ensure that the observed phenotypes were not a result of genomic insertion positional effects, we performed all experiments with both transgenic lines and obtained similar results for both lines. Expression of transgene eIF-5A2 in trnasgenic mice could be detected in all tested tissues by RT-PCR (Figure 1E).


Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability.

Chen M, Huang JD, Deng HK, Dong S, Deng W, Tsang SL, Huen MS, Chen L, Zan T, Zhu GX, Guan XY - BMC Cancer (2011)

Generation of eIF-5A2 transgenic mice. (A) Three eIF-5A2 transgenic mouse founders (mouse No.: 10, 11, and 89) and their offspring (166-169) were determined by PCR. One mouse without eIF-5A2 transgene (No. 9) was used as negative control. Mouse fibroblast cell line NIH 3T3 and eIF-5A2 transgenic construct was used as negative (-) and positive (+) controls. (B) The transgene eIF-5A2 was mapped to one mouse chromosome site in line 11 by FISH. The metaphase spread was prepared from bone marrow lymphocyte. Arrow indicates the hybridization signals of eIF-5A2. (C) Expression of eIF-5A2 in transgenic mice was confirmed by Northern blot analysis. A human eIF-5A2 cDNA probe was used and it did not detect the endogenous eIF-5A2 mRNA in both wild-type and transgenic mice. (D) Western blot showed the overexpression of human eIF-5A2 in transgenic mice using liver lysates. The lower bands (17dD) were eIF-5A2 and the upper bands were shifted eIF-5A2 bands caused by posttranslational hypusination. (E) Expression of transgene eIF-5A2 in various tissues of transgenic mouse was detected by RT-PCR using a pair of human-specific primers.
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Related In: Results  -  Collection

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Figure 1: Generation of eIF-5A2 transgenic mice. (A) Three eIF-5A2 transgenic mouse founders (mouse No.: 10, 11, and 89) and their offspring (166-169) were determined by PCR. One mouse without eIF-5A2 transgene (No. 9) was used as negative control. Mouse fibroblast cell line NIH 3T3 and eIF-5A2 transgenic construct was used as negative (-) and positive (+) controls. (B) The transgene eIF-5A2 was mapped to one mouse chromosome site in line 11 by FISH. The metaphase spread was prepared from bone marrow lymphocyte. Arrow indicates the hybridization signals of eIF-5A2. (C) Expression of eIF-5A2 in transgenic mice was confirmed by Northern blot analysis. A human eIF-5A2 cDNA probe was used and it did not detect the endogenous eIF-5A2 mRNA in both wild-type and transgenic mice. (D) Western blot showed the overexpression of human eIF-5A2 in transgenic mice using liver lysates. The lower bands (17dD) were eIF-5A2 and the upper bands were shifted eIF-5A2 bands caused by posttranslational hypusination. (E) Expression of transgene eIF-5A2 in various tissues of transgenic mouse was detected by RT-PCR using a pair of human-specific primers.
Mentions: To characterize the role of eIF-5A2 in vivo, we generated transgenic mouse lines overexpressing human eIF-5A2 ubiquitously. Pronuclear injection of the pCAGGS-eIF-5A2 construct resulted in the generation of three founder mice, as identified by genomic Southern blot analysis and PCR (Figure 1A). To confirm the single integration event, an eIF-5A2 probe was used to hybridize to the metaphase spreads from bone marrow lymphocytes by fluorescence in situ hybridization (FISH). The transgene was mapped to one chromosome site in line 11 (Figure 1B). All the founders were able to transmit the transgene to their offspring in the expected Mendelian ratio. Three lines were expanded, and the expression of the transgene was confirmed in two out of three founder lines (10 and 11). Expression of eIF-5A2 at the RNA level was detected by RT-PCR and Northern blot hybridization using liver and testis tissues (Figure 1C). Also, EIF-5A2 protein expression was demonstrated by immunohistochemistry (IHC) and Western blot analysis (Figure 1D) in multiple organs. Of note, no difference was observed in the expression of murine endogenous eIF-5A2 among the founders and wild-type controls. To ensure that the observed phenotypes were not a result of genomic insertion positional effects, we performed all experiments with both transgenic lines and obtained similar results for both lines. Expression of transgene eIF-5A2 in trnasgenic mice could be detected in all tested tissues by RT-PCR (Figure 1E).

Bottom Line: Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region.This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification.This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Oncology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, China.

ABSTRACT

Background: Amplification of 3q26 is one of the most frequent genetic alterations in many human malignancies. Recently, we isolated a novel oncogene eIF-5A2 within the 3q26 region. Functional study has demonstrated the oncogenic role of eIF-5A2 in the initiation and progression of human cancers. In the present study, we aim to investigate the physiological and pathological effect of eIF-5A2 in an eIF-5A2 transgenic mouse model.

Methods: An eIF-5A2 transgenic mouse model was generated using human eIF-5A2 cDNA. The eIF-5A2 transgenic mice were characterized by histological and immunohistochemistry analyses. The aging phenotypes were further characterized by wound healing, bone X-ray imaging and calcification analysis. Mouse embryo fibroblasts (MEF) were isolated to further investigate molecular mechanism of eIF-5A2 in aging.

Results: Instead of resulting in spontaneous tumor formation, overexpression of eIF-5A2 accelerated the aging process in adult transgenic mice. This included decreased growth rate and body weight, shortened life span, kyphosis, osteoporosis, delay of wound healing and ossification. Investigation of the correlation between cellular senescence and aging showed that cellular senescence is not required for the aging phenotypes in eIF-5A2 mice. Interestingly, we found that activation of eIF-5A2 repressed p19 level and therefore destabilized p53 in transgenic mouse embryo fibroblast (MEF) cells. This subsequently allowed for the accumulation of chromosomal instability, such as errors in cell dividing during metaphase and anaphase. Additionally, a significantly increase in number of aneuploidy cells (p < 0.05) resulted from an increase in the incidences of misaligned and lagging chromosomal materials, anaphase bridges, and micronuclei in the transgenic mice.

Conclusion: These observations suggest that eIF-5A2 mouse models could accelerate organismal aging by increasing chromosome instability.

Show MeSH
Related in: MedlinePlus