Name: focus formation: a cell-based assay to determine the oncogenic potential of a gene
Uploaded: 2014-12-31T15:00:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

この作品は、NIHのディレクターの新イノベーター賞プログラム（ED）からの助成金によってサポートされていました。 AAは国立がん研究所、国立科学財団からの学部賞によって部分的にサポートされていました。

1.ウイルスベクターを作る目的の遺伝子、ならびに陽性および陰性対照のためのコード配列は、従来のクローニング法（PCR増幅、制限消化およびライゲーション）によってpBABEpuroに挿入される。 BamHIでSnaBIで、EcoRIおよびSalIで：DNAを挿入することができるベクター上の4つの制限部位がある。 <ol><li> QIAGENプラスミドミディキットを使用してコンピテント細胞からトランスフェクショングレードのプラスミドDNAを準備します。 </li><li> NanoDrop2000分光光度計を用いてDNA濃度を測定します。 </li></ol> 2.レトロウイルス生産のPlat-Eパッケージング細胞株は、NIH 3T3細胞に目的のcDNAを送達するエコトロピックレトロウイルスを産生するために使用される。 <ol><li>凍結細胞からのPlat-Eの文化の確立<ol><li>すぐに37℃の水浴中でのPlat-E細胞を解凍し、15mlのコニカルチューブに移す。ゆっくりとのPlat-E培地9ml（デュを追加lbecco改変イーグル培地（DMEM）+ 10％ウシ胎児血清（FBS）+ペニシリン及びストレプトマイシン）。 </li><li> 180 xでのチューブを遠心 gで5分間、上清を捨てる。 </li><li>再懸濁のPlat-E培地及び10cmの培養皿に移し、10 mlの細胞。 </li><li>それらは80〜90％コンフルエント（約2日）になるまで37℃、5％CO 2インキュベーター中で細胞をインキュベートする。 </li></ol></li><li>セル分割<ol><li>メディアを吸引除去し、PBSで細胞を1回洗浄します。 </li><li> 0.05％トリプシンEDTAの2ミリリットルを加え、室温で1分間インキュベートする。 </li><li>指タッピングによって細胞を剥離、ぷらっと-E培地10mlを追加し、15ミリリットルチューブに細胞懸濁液を移す。 </li><li> 180 xでのチューブを遠心 gで5分間、上清を捨てる。 </li><li>ぷらっと-E培地10mlで細胞を再懸濁し、1から新しい料理でそれらをシード：4-1：6の希釈。 </li></ol></li><li> PLAT-Eシードおよびトランスフェクション<ol><li>シード2×10 6任意の抗生物質を含まないのPlat-E培地を用いて10cmの培養皿当たり細胞。 </li><li> 37℃、5％CO 2インキュベーター中で細胞をO / Nでインキュベートする。 </li><li>翌日、1.5mlマイクロチューブにのOpti-MEM300μlのを転送する。 </li><li>のOpti-MEMでプリペアチューブに、ポリエチレンイミンの27μL（PEI）、費用対効果の高いトランスフェクション試薬12を追加します。指タッピングにより穏やかに混合し、室温で5分間インキュベートする。 </li><li> 、のOpti-MEM / PEIチューブにトランスフェクショングレードのプラスミドDNAの9μgのを追加し、ボルテックスにより穏やかに混合し、室温で15分間インキュベートする。 </li><li>追加DNA / PEIのPlat-E皿に、37℃でO / Nインキュベート複雑なワイズドロップ、5％のCO 2。 </li><li>翌日、トランスフェクション試薬を含む培地を吸引し、（抗生物質を含まない）新鮮なのPlat-E培地10mlを追加します。 </li><li>インキュベーターにセルを返します。 </li></ol></li></ol> 3. NIH 3T3細胞と感染<ol><li> NIHの確立3T3文化や播種<ol><li>すぐに37℃の水浴中でNIH 3T3細胞を解凍し、15mlチューブに移す。ゆっくりとNIH 3T3培地（DMEM + 10％FBS）の9ミリリットルを追加します。 </li><li> 180 xでのチューブを遠心 gで5分間、上清を捨てる。 </li><li> 10cmの培養皿にNIH 3T3媒体と転送10mlで細胞を再懸濁。 </li><li> 37℃、5％CO 2インキュベーター中で細胞をインキュベートする。これは、自発的な変換（および病巣形成の、したがって基礎レベル）の周波数として文化がコンフルエントに達すると増加、NIH 3T3細胞は常にunderconfluent（百分の50から60）を保持していることが非常に重要です。 </li><li>シード3×10 10 cmディッシュあたり5個の細胞と、翌日感染のためにO / Nインキュベートする。 </li></ol></li><li>感染<ol><li>孔径0.45μmのナイロン膜フィルターを介して濾過する10mlの使い捨てシリンジを使用し、そして15mlにそれを転送することによってのPlat-E皿からレトロウイルス上清を収穫チューブ。 </li><li> （抗生物質を含まない）を細胞に新鮮なのPlat-E培地10mlを追加し、インキュベーターに戻す。 </li><li>感染していることがNIH 3T3細胞ディッシュから培地を吸引し、ウイルス含有濾過した上清の5の正規NIH 3T3培地1mlと5ミリリットルを追加します。 </li><li> 610μg/ mlの濃度で、皿にポリブレンを追加します。 </li><li> 37℃、5％CO 2で細胞をO / Nでインキュベートする。 </li><li>感染の第二ラウンドのために3.2.5 - 次の日、繰り返して3.2.1を繰り返します。 </li><li>定期的なNIH 3T3培地でウイルス含有培地を交換してください。 </li><li> NIH 3T3細胞は、必要に応じて培地を交換、2-3週間成長できるようにします。レプリカプレートから全細胞溶解物に従来のタンパク質電気泳動およびイムノブロットにより、目的のタンパク質の発現を確認してください。 </li></ol></li></ol> 4.染色および定量<ol><li>クリスタルバイオレット染色<ol><li> NIH 3T3培地を吸引し、食器を置く氷の上で。 </li><li>氷冷PBSで2回皿を洗う。 </li><li> 10分間氷冷メタノールで細胞を固定します。 </li><li>氷から皿を外し、メタノールを吸引し、25％のメタノール（RT）で行われた、0.5％クリスタルバイオレット溶液の3ミリリットルを追加します。 </li><li> RTで5分間皿をインキュベートする。 </li><li>クリスタルバイオレット溶液を吸引し、色がリンスに外れなくなるまで注意深くのMilli-Q H 2 Oとの皿をすすぐ。 </li><li>料理はベンチトップ（発見）にO / Nを乾燥することができます。 </li></ol></li><li>病巣の定量<ol><li>料理が乾燥したら、プレート上の細胞の暗色コレクションを測定するために定規を使用しています。として、直径5ミリメートルよりも大きくされているもののみをカウント &quot;巣&quot;を病巣の数を記録し、対照と比較して平均値と有意性を計算。 </li></ol></li></ol>

<ol>
	<li>Clark, G. J., Cox, A. D., Graham, S. M., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+assays+for+Ras+transformation.">Biological assays for Ras transformation.</a> Methods in enzymology. , 255-395 (1995).</li><li>Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell biology : a laboratory handbook. , 345-352 (2006).</li><li>Raptis, L., Vultur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+assays.">Neoplastic transformation assays.</a> Methods in molecular biology. 165, 151-164 (2001).</li><li>Johnson, P. J., Coussens, P. M., Danko, A. V., Shalloway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressed+pp60c-src+can+induce+focus+formation+without+complete+transformation+of+NIH+3T3+cells.">Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.</a> Molecular and cellular biology. 5, 1073-1083 (1985).</li><li>Bonner, T. I., Kerby, S. B., Sutrave, P., Gunnell, M. A., Mark, G., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+biological+activity+of+human+homologs+of+the+raf/mil+oncogene.">Structure and biological activity of human homologs of the raf/mil oncogene.</a> IMolecular and cellular biology. 5, 71400-71407 (1985).</li><li>Yancopoulos, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+can+cooperate+with+ras+to+transform+normal+cells+in+culture.">N-myc can cooperate with ras to transform normal cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 82, 5455-5459 (1985).</li><li>Fujiwara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+activity+of+purinergic+receptor+P2Y,+G+protein+coupled,+8+revealed+by+retroviral+expression+screening.">Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.</a> Leukemia &amp; lymphoma. 48, 978-986 (2007).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene. 7, 1063-1066 (2000).</li><li>Morgenstern, J. P., Land, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+mammalian+gene+transfer:+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line.">Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.</a> Nucleic acids research. 18, 3587-3596 (1990).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397, 3173-3178 (2010).</li><li>Klaver, B., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+5'+and+3'+long+terminal+repeat+promoter+function+in+human+immunodeficiency+virus.">Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus.</a> Journal of virology. 68, 3830-3840 (1994).</li><li>Fukumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+gene+transfection+by+DNA+compaction+at+pH+4.0+using+acidified,+long+shelf-life+polyethylenimine.">Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine.</a> Cytotechnology. 62, 73-82 (2010).</li><li>Fox, E. J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells.">S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells.</a> The Biochemical journal. 359, 361-367 (2001).</li><li>Yun, J. S., Rust, J. M., Ishimaru, T., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+Mad+family+member+Mad3+in+cerebellar+granule+neuron+precursor+proliferation.">A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation.</a> Molecular and cellular biology. 27, 8178-8189 (2007).</li><li>Gore, Y., Lantner, F., Hart, G., Shachar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad3+negatively+regulates+B+cell+differentiation+in+the+spleen+by+inducing+Id2+expression.">Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression.</a> Molecular biology of the cell. 21, 1864-1871 (2010).</li><li>Barisone, G. A., Yun, J. S., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cerebellar+proliferation+to+tumorigenesis:+new+insights+into+the+role+of+Mad3.">From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3.</a> Cell cycle. 7, 423-427 (2008).</li><li>Barisone, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+MXD3+in+proliferation+of+DAOY+human+medulloblastoma+cells.">Role of MXD3 in proliferation of DAOY human medulloblastoma cells.</a> PloS One. 7, e38508 (2012).</li><li>Nair, S. K., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structures+of+Myc-Max+and+Mad-Max+recognizing+DNA.+Molecular+bases+of+regulation+by+proto-oncogenic+transcription+factors.">X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.</a> Cell. 112, 193-205 (2003).</li></ol>

著者らは、開示することは何もない。

FFAは、in vitroでの悪性形質転換を評価するための迅速かつ容易な方法を提供する。これは、候補遺伝子の比較的多数のスクリーニングに適用され、その控えめな技術的要件は、費用対効果を高める。さらに、2つ以上の遺伝子を共発現させることができる組み合わせの腫瘍形成能を評価するために（時には「協力」アッセイと呼ばれる）。このアッセイの利点は、その単純な手法、定量...

腫瘍細胞は、遺伝子発現のパターンの形態学的および増殖性の変化にエピゲノミクスに、変化の広い範囲によってそれらの正常な対応物と区別することができる。後者の中では、、、接触の損失（密度）阻害を血清への依存を軽減足場非依存性増殖の取得と、最終的には、動物に注射した場合に腫瘍を形成する能力は、悪性形質転換3の有効、測定可能な指標である。 in vitroおよびin vivoアッセイにおけるいくつかの細胞の形質転換のために開発されてきた。 インビトロアッセイは、（成長速度、飽和密度）及び増殖因子（低血清成長）、培養形態（フォーカス形成アッセイ）の変化を識別し、測定時に培養ダイナミクスを目指すまたは要件アンカレッジ（ソフトアガーでの成長）。細胞型の悪性の性質を決定するためのゴールドスタンダードは、実験動物における腫瘍形成（異種移​​植片）のままである。しかし、T彼は、コストとのインビボ研究の長さは、常に候補癌遺伝子の最初の検証ステップまたはスクリーニング、それらに正当ことはありません。何のin vitroアッセイは、遺伝子の発癌性の明確な評価を提供していないが、これらは、生体内の研究で未来を絞り込むことが発癌性への洞察を提供します。 インビトロでの発癌性を評価するための最も広く使用されているシステムの一つはフォーカス形成アッセイ2である。このアプローチは、NIH 3T3マウス線維芽細胞、強く接触阻害を示し、非形質転換細胞株の使用に依存している。密度依存性増殖の損失の癌遺伝子の過剰発現。形質転換された細胞は、次に簡単に非形質転換細胞のバックグラウンド単層に対して可視化「病巣」を形成する、複数の層で成長することができる。フォーカス形成アッセイは、その後、コンタクトInhibが喪失によって証明されるように、悪性形質転換を誘導する候補癌遺伝子の能力を測定する測定可能な表現型としてition。 FFAは、プロテインキナーゼ（ 例えば 、Srcの4、BRAF 5）の過剰発現により形質転換を評価するために使用されている、転写因子（ 例えば 、N-mycの6）、Gタンパク質共役受容体（ 例えば 、P2RY8 7）およびGTPアーゼ（ 例えばとりわけラス1）。このアッセイの相対的な容易さは、遺伝子の過剰発現は、インビトロで NIH 3T3マウス線維芽細胞を形質転換するのに十分であるか否かを迅速かつ視覚的に明確な答えを提供する適切な選択肢となる。 FFAは、このプロトコルに記載のレトロウイルスを産生するウイルスパッケージングタンパク質を提供するのPlat-Eパッケージング細胞株8、およびレトロウイルスベクターpBABEpuro 9（Addgeneプラスミド1764）を使用する。目的の遺伝子を含むpBABEpuro構築物でトランスフェクションした後、のPlat-E細胞株は、NIH 3T3細胞を感染させるために使用することができるエコトロピックレトロウイルスを産生する。 T遺伝子送達の彼のウイルスの方法は、従来の化学的なトランスフェクション法よりも効率的であり、それは持続的に遺伝子10を表現する方法を提供しています。 NIH 3T3細胞のゲノムに組み込まれると、目的の遺伝子の過剰発現は、ウイルスの長い末端反復（LTR）プロモーター11によって駆動される。この定数式は、NIH 3T3細胞に、病巣の形成によって測定されるように、目的の遺伝子は、発癌活性を有するかどうかを決定するために使用することができる。

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3は、MYC / MAX / MADネットワークのメンバーである塩基性ヘリックス - ループ - ヘリックスロイシンジッパー（bHLHZ）転写因子である。それは、MADファミリー13-15の異型メンバーであり、発癌16,17に関与することが報告されている。 pBABEpuro（陰性対照）およびMYC（陽性対照）と比較した場合、MXD3を過剰発現させたNIH 3T3料理が著しく少ない病巣（ 図1A）を有してい?...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

フォーカス形成アッセイは、候補癌遺伝子の形質転換能を評価するための簡単​​な方法を提供する。

フォーカス形成：細胞ベースのアッセイ遺伝子の発癌性を決定するために、

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.8K ビュー.  University of California, Davis. フォーカス形成アッセイは、候補癌遺伝子の形質転換能を評価するための簡単​​な方法を提供する。 

ビデオ: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

癌細胞スフェロイド培養における潜在的に新規薬剤標的を同定するためのスクリーニング機能ゲノミクスを利用

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

がん研究

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

ヒト造血細胞のためのコロニー形成細胞（CFC）アッセイ

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

免疫・感染学

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

C-fos の役割と Dusp1 の遺伝子依存性の評価方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

ヒトP53を研究する機能アッセイを開発するシャーシとしての酵母

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

アンカー多重ポリメラーゼ連鎖反応を用いた腫瘍性遺伝子融合検出、次世代シーケンシング

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

トランスクリプトーム解析を用いた無秩序発がん性転写因子の構造機能関係のマッピング

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

培養細胞における癌関連シグナル伝達を問い合わせるための多重ルシフェラーゼベースのスクリーニング·プラットフォーム

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

がん遺伝子由来の転写-複製コンフリクトを研究するための近接ライゲーションアッセイ

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

膵管細胞株におけるpH変化を伴うがん遺伝子発現解析

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

軟寒天コロニー形成アッセイ

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...