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Real-time in vivo imaging of p16gene expression: a new approach to study senescence stress signaling in living animals.

Ohtani N, Yamakoshi K, Takahashi A, Hara E - Cell Div (2010)

Bottom Line: To date, much of our current knowledge of how human p16(INK4a )gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells.However, since human p16(INK4a )gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16(INK4a )gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress.Here, we discuss the molecular mechanisms that direct p16(INK4a )gene expression in vivo and its potential for tumor suppression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology, Cancer Institute, Japanese Foundation for Cancer Research (JFCR), 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan.

ABSTRACT
Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16(INK4a). To date, much of our current knowledge of how human p16(INK4a )gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16(INK4a )gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16(INK4a )gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16(INK4a )gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16(INK4a )gene expression in vivo and its potential for tumor suppression.

No MeSH data available.


Related in: MedlinePlus

Strategy for in vivo imaging of p16INK4a gene expression. A large genomic DNA segment (195.4 kb) of human chromosome that contains the entire INK4a/ARF gene locus and surrounding sequences was engineered to express luciferase-tagged p16Ink4a. FISH technique reveals that the transgenic mice line (p16-luc) contanins a single copy of the human chromosome segment. The arrow shows the transgene. The p16-luc mouse was anesthetized and subjected to in vivo bioluminescence imaging after injection of luciferin.
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Figure 1: Strategy for in vivo imaging of p16INK4a gene expression. A large genomic DNA segment (195.4 kb) of human chromosome that contains the entire INK4a/ARF gene locus and surrounding sequences was engineered to express luciferase-tagged p16Ink4a. FISH technique reveals that the transgenic mice line (p16-luc) contanins a single copy of the human chromosome segment. The arrow shows the transgene. The p16-luc mouse was anesthetized and subjected to in vivo bioluminescence imaging after injection of luciferin.

Mentions: In order to monitor human p16INK4a gene expression as accurately as possible, we used a large genomic DNA segment of the human chromosome that contains the entire INK4a/ARF gene locus(Figure 1). Furthermore, this human chromosomal segment was engineered to express a fusion protein of human p16INK4a and firefly luciferase without deleting any genomic DNA sequences of the INK4a/ARF gene locus (Figure 1). This is crucial, because BMI-1, which is a negative regulator of p16INK4a gene expression[38], has been shown to bind not only to the promoter region, but also to the intron region of the p16INK4a gene locus[39]. Moreover, the expression of the p16-luc fusion protein enables us to specify p16INK4a gene expression, but not ARF gene expression, from this overlapping gene locus.


Real-time in vivo imaging of p16gene expression: a new approach to study senescence stress signaling in living animals.

Ohtani N, Yamakoshi K, Takahashi A, Hara E - Cell Div (2010)

Strategy for in vivo imaging of p16INK4a gene expression. A large genomic DNA segment (195.4 kb) of human chromosome that contains the entire INK4a/ARF gene locus and surrounding sequences was engineered to express luciferase-tagged p16Ink4a. FISH technique reveals that the transgenic mice line (p16-luc) contanins a single copy of the human chromosome segment. The arrow shows the transgene. The p16-luc mouse was anesthetized and subjected to in vivo bioluminescence imaging after injection of luciferin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821322&req=5

Figure 1: Strategy for in vivo imaging of p16INK4a gene expression. A large genomic DNA segment (195.4 kb) of human chromosome that contains the entire INK4a/ARF gene locus and surrounding sequences was engineered to express luciferase-tagged p16Ink4a. FISH technique reveals that the transgenic mice line (p16-luc) contanins a single copy of the human chromosome segment. The arrow shows the transgene. The p16-luc mouse was anesthetized and subjected to in vivo bioluminescence imaging after injection of luciferin.
Mentions: In order to monitor human p16INK4a gene expression as accurately as possible, we used a large genomic DNA segment of the human chromosome that contains the entire INK4a/ARF gene locus(Figure 1). Furthermore, this human chromosomal segment was engineered to express a fusion protein of human p16INK4a and firefly luciferase without deleting any genomic DNA sequences of the INK4a/ARF gene locus (Figure 1). This is crucial, because BMI-1, which is a negative regulator of p16INK4a gene expression[38], has been shown to bind not only to the promoter region, but also to the intron region of the p16INK4a gene locus[39]. Moreover, the expression of the p16-luc fusion protein enables us to specify p16INK4a gene expression, but not ARF gene expression, from this overlapping gene locus.

Bottom Line: To date, much of our current knowledge of how human p16(INK4a )gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells.However, since human p16(INK4a )gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16(INK4a )gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress.Here, we discuss the molecular mechanisms that direct p16(INK4a )gene expression in vivo and its potential for tumor suppression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology, Cancer Institute, Japanese Foundation for Cancer Research (JFCR), 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan.

ABSTRACT
Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16(INK4a). To date, much of our current knowledge of how human p16(INK4a )gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16(INK4a )gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16(INK4a )gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16(INK4a )gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16(INK4a )gene expression in vivo and its potential for tumor suppression.

No MeSH data available.


Related in: MedlinePlus