Scientific life

Senescence in microenvionment

The fundamental difference between senescence and apoptosis is alive or dead.
Both phenotypes prevent cellular proliferation, but one is dead and the other is alive.

Senescent cells are alive, though cell cycle is arrested.
This raises the possibility that senescence has other functions except cell cycle arrest.
They will talk with other cells.
I thought if senescent cells may suppress the tumor progression by inducing immune responses.

Two elegant studies from Scott Lowe's laboratory showed senescence triggers immune responses, which contribute to clearance of tumor and fibrosis.
These are very impressive studies for me.
However, the relation between senescence and immune responses is almost unknown. It is not simple but complex.

Senescent cells express inflammatory cytokines which attract immune cells. And immune infiltration is supposed to contribute to senescent tumor regression.
It was shown, for example, senescent cells are targeted by NK cells.
However, senescent cells also promote tumor progression through a paracrine mechanism.
The group of Judith Campisi reported senescence-associated secretory phenotype.
They modified antibody arrays to quantify proteins secreted by senescent cells and revealed common inflammatory functions of senescence.
They also showed senescent cells induced EMT in non-aggressive tumor cells and stimulated premalignant tumor cells to invade a basement membrane in a paracrine fashion.

Senescence can be both beneficial and deleterious.
The second stage of senescence study has been already started.

References
PLoS Biology 6, 2853-2868 (2008)
Nature 445, 656-659 (2007)
Cell 134, 657-667 (2008)

What is tumorigenic in vivo?

Efficient tumour formation by single human melanoma cells
Nature 456, 593-599 (2008)

Since tumor is heterogeneous, it consists of some different kinds of populations.
Different populations may have different tumorigenic potential.

If you inject tumor cells into immunocompromised mice, the tumorigenic population will form tumors.
If you culture tumor cells in suspension, the tumorigenic population will be allowed to grow.

Some people argue tumorigenic potential in vivo by using immunocompromised mice and insist cancer stem cell theory by providing evidences that only small subpopulations are responsible for cancer initiation.
But the detectable frequency of tumorigenic cells depends on the immunodeficiency of mice.

In this paper they demonstrated that tumorigenic cells are common in some cancer by using NOD/SCID/Il2rγ mice, high immunocompromised mice.
Modifications of xenotransplantation assay conditions dramatically increase the detectable frequencies of tumorigenic cells.
Even unselected single melanoma cell formed tumors in this mice.

Neither this study nor previous cancer stem cell studies have addressed which cells actually contribute to tumor growth or progression in patients.

Cre virus

Considering a wide resource of Cre-mediated conditional mice, it must be significant to create Cre-mediated shRNA virus.

Cre-loxP system is able to apply for the generation of inducible knockdown animals by crossing to transgenic mice expressing Cre in tissue specific fashion.

Cre-regulated viral shRNA vector system has been already reported from some laboratories.
They generated retroviral or lentiviral shRNA vector which express shRNA after addition of Cre recombinase. This technique relies on the insertion of loxP-flanked stuffer sequence between Pol?鶚 promoter and shRNA coding sequences.
Adeno, retro or lenti viral vector are used to deliver Cre recombinase in vitro.
Conditional knockdown mice are generated by infection of ES cells with Cre-regulated shRNA vector. The chimera mice are crossed to Cre transgenic mice, in which Cre is under the control of tissue specific promoters, to activate shRNA in tissue specific fashion.
Moreover, use of Cre-ER mice, where Cre remains inactive until administration of the estrogen analogue tamoxifen, allows temporal regulation of RNAi in vivo.

References
Proc. Natl. Acad. Sci. USA, 105, 13895–13900 (2008)
Proc. Natl. Acad. Sci. USA, 101, 10380 –10385 (2004)
Proc. Natl. Acad. Sci. USA, 101, 7347–7351 (2004)
Nature Methods, 3, 682-688 (2006)

Cell Cycle Imaging

Cell 132, 487-498, February 8, 2008
Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression

細胞周期を制御するユビキチンリガーゼであるSCFとAPCの直接の基質であるCdt1とGemininに着目し、細胞周期のG1期とS-G2-M期を明瞭に可視化することを可能にした論文です。
Figureを見るだけでもその技術に感嘆します。理研からの報告ですが、この論文を見ると日本の技術が健在であることを認識します。

G1期特異的に高発現するCdt1をRFPの下流に組み込んだベクターとS-G2-M期特異的に高発現するGemininをGFPの下流に組み込んだベクターをレンチウイルスでHela細胞に導入することで、G1期とS-G2-M期を明瞭に可視化することを可能にした。

培養細胞の細胞周期をリアルタイムで可視化することができるだけでなく、マウスに移植した細胞株の細胞周期や、Cdt1とGemininのトランスジェニックマウスを作製して組織発生過程の細胞周期を可視化することも可能にしている。

個体レベルで内在性の遺伝子変異を可視化することはできないかと考えたことがありましたが、それが無理にしても外来性のマーカーによって遺伝子特異的変化を可視化する技術が進めば、生理的変化でしか評価するできなかった非特異的な臨床検査に革命を起こすことができるかもしれない。

ヒトiPS細胞

Cell 131, 1-12, November 30, 2007
Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

今日の読売新聞の表紙を飾ったのは上記論文についての記事でした。
最新号のCellのImmediate Early Publicationに掲載された上記論文は京大の山中先生の研究グループによるヒトiPS細胞樹立の報告です。以前僕はiPS細胞という記事を書いたことがありましたが、今回彼らはヒトの皮膚線維芽細胞からiPS細胞を樹立することに成功しました。

ヒト線維芽細胞(HDF)はMEFに比べてレトロウイルスの感染効率が低いことは経験的事実です。彼らはGFP導入効率20％以下というHDFの低い感染効率を向上させるため、マウス受容体であるSlc7a1をレンチウイルスによってHDFに導入することでヒト細胞にエコトロピック受容体を発現させた。ウイルス受容体の宿主域を変えることで感染効率を向上させた点ではVSV-Gと同じですが、彼らはこの方法によりHDFの感染効率をMEF並みに上昇させることに成功したと述べています。

36歳の白人女性の顔面皮膚から得られた皮膚線維芽細胞にOct3/4、Sox2、Klf4、c-Mycの４種類の遺伝子をPlatEで産生させたレトロウイルスにより導入し、6日間10%FBS添加DMEMで培養した後にフィーダー細胞上に捲き、その後bFBS添加ES培地で24日間培養することでヒトiPS細胞樹立に成功した。