この研究助成金に支えられないです。国立がん研究所から CA 81301。我々 は貴重な議論のため博士アンナ, ミシェル博士アダム t. Zarth とフリーメーソンのがんセンターの分析化学共有リソースの質量分析支援のため迅明博士ピーター Villalta ボブ カールソン編集者の支援のために感謝します。入力します。分析化学共有リソースを部分的にサポートによって国立がん研究所がんセンター サポート助成金 CA-77598

1。 材料と一般的な手順 <ol><li>合成 NNN 前述した 15。Norcotinine、P450 2A6 Baculosomes、NADPH 再生システム、0.5 x 反応バッファー、および他のすべての化学物質を取得または試薬グレードの商業ソースから溶剤</li>。
	<li>500 MHz 分光器の記録の NMR スペクトル。レポート化学シフトごとの部分として百万 (ppm)。(7.26 ppm CDCl 3) 1 H NMR の内部参照と 13 C NMR (77.2 ppm CDCl 3) として残留溶媒のピークを使用します 。
	注: は、次の略語を使用ピーク分裂: s 一重項、d = ダブレット、dd = ダブレット、dt のダブレットを = = dq、三つ子のダブレット四重奏曲、ddd のダブレットを = ダブレットと m のダブレットのダブレットを = = マルチプレット</li>。
	<li>焼入れ Orbitrap ベロスと m/z としてレポート データの選択した化合物の高分解能質量分析法 (HRMS) を実行します</li>。
	<li>使用ポリグラム シリカゲル TLC 板 (40 mm x 80 mm、厚さ 0.2 mm) 薄層クロマトグラフィー (TLC) の 254 nm の蛍光指示薬がプレコート。紫外線ランプ照射による TLC の版を視覚化します</li>。
	<li>、60 Å、70 150 メッシュによるシリカゲル 1.5 cm × 15 cm のガラスカラム フラッシュ クロマトグラフィーを実行します</li>。
</ol> 2。Nitrosamide 肯定的な制御の準備 (N &#8242;-nitrosonorcotinine、NNC) 電磁攪拌棒を含む <ol><li>ドライ、一晩オーブンで (〜 140 ° C 16 h) 25 mL 丸底フラスコ。朝は、クールなストリームの N 2 N 2 の流れを維持するオイルのバブラーを使用しながら下のフラスコ レート 1 秒あたり 1 つのバブルします 。
	注: 冷却した後、N 2 の下でフラスコを保つために注意する必要はありません</li>。
	<li>ヒューム フードで冷却のフラスコに norcotinine (31.8 mg、0.196 モル)、(5 mL)、無水酢酸、酢酸 (1 mL) を追加。水氷浴で 0 の ° C に冷却しながら連続的にこの混合物をかき混ぜる 。
	注意:無水酢酸と酢酸はある腐食性</li>。
	<li>追加ナノ 2 (33.3 mg、0.483 モル) 1 つの部分し、連続的に 2.5 h. TLC によって反応の進行状況を監視の混合物をかき混ぜる (100% エチル、R f = 0.19、1.4 の手順を参照してください). 
	注: この時間をかけて混合物黄色になりますますバブルで時折</li>。
	<li>(18 mL) 冷たい H 2 O に混合物を注ぐことによって反応を抑制します。18 ml の CH 2 Cl 2 スズの水溶液を即座に抽出します。CH 2 Cl 2 (9 mL) 少なくとも 2 の追加回水の層を抽出します</li>。
	<li>は、MgSO 4 2 分フィルターのための ~ 100 mg 以上のプールされた有機物を乾燥、粗、黄色の油を生成する回転蒸発によってソリューションを集中し、なさい。残留の酢酸を削除する蒸発熱 30 ° C の水浴 。
	注: プロトコルを一時停止できるここで CH 2 Cl 2 および 2-8 ° c. で暗い保存されてソリューションに化合物を溶解している場合</li>
	<li>カラムクロマトグラフィーにより原油の化合物を浄化する (手順 1.5 参照) 100% 固定相としてシリカゲルを用いる溶離液 16 としてエチル 。
	注意:彼らは人間の発癌物質と見なされますよう、NNC と関連 nitrosamides を注意して処理する必要があります</li>。
	<li>CDCl 3 で純粋な化合物を溶解し、NMR を使用 (手順 1.2 参照) して構造を確認し、このソリューションの 17 のモル濃度を決定します。2-8 ° c まで必要な暗闇の中で、このソリューションを保存します 。
	注: このソリューションは、(ステップ 3.5) の in vitro アッセイのポジティブ コントロールとして使用されます。サポート情報では NMR スペクトルや化学シフトします</li>。
	<li>純粋な NNC ソリューションを確認正確な質量を親し、1.3 と 4.3 の手順で説明するパラメーターの下で高分解能質量分析計 (HRMS) に直接注入による製品イオン質量を決定します 。
	注: 製品の質量は、体外 代謝アッセイ (ステップ 4.3) でこの化合物を検出するため使用されます</li>。
</ol> 3。インキュベーション の in vitro 例ニトロソアミン P450 <ol><li>-80 ° C のフリーザーから P450 2A6 Baculosomes、鮮やかな NADPH 再生システム、および 10 mM NADP + 在庫ソリューションを削除し、聞かせ融解は氷の上。解凍後、希釈 Baculosomes と NADPH 再生システム 1:10 と 1:50、0.5 X 反応バッファーを単一の 1 mL の管に、それぞれ。同様に、反応バッファー X nadp + 在庫ソリューション 0.5 97 μ L に 3 μ L を追加します 。
	注: NADP + は、光に敏感です。このソリューション コンテナーをアルミ箔で包むことによって保護をしてください。酵素のソリューションは、凍結融解サイクルに敏感以上 2 サイクルに制限します。今後の実験に必要な因数を準備します</li>。
	<li>50 μ L の酵素液 (5 pmol P450 を含む) を 1 mL 管含む 40 μ L 反応バッファーは、4 μ M NNN ソリューションの各インキュベーションを追加。中古水槽を併用した 37 ° C で 2 分間この新しい混合物をインキュベートし、NADP + の希釈液の 10 μ L を追加します。37 分の 1 30 分の完全なシステムをインキュベート ° C 
	注: を用いたインキュベーション時間 5 分間隔 (例えば 5、10、15 分など) 十分な nitrosamide 形成時間コースを提供する</li>。
	<li>3.0 N ZnSO 4 の最初の追加 10 μ l と 3.0 N Ba(OH) の 10 μ L を癒す目的の培養時間後 2.このソリューションと白い沈殿物を形成する渦。沈殿物をペレットに 4 分間、8000 x g でサンプルを遠心します 。
	注: 手順する必要があります一時停止この段階で形成された nitrosamides 水溶液中での安定性が限られていると。偽陰性に長い一時停止があります</li>。
	<li>すぐに上澄みをピペットし、2 μ L を高分解能液体クロマトグラフィー肯定的な nanoelectrospray をイオン化タンデム質量分析法による分析 (LC NSI +-人事管理システム/MS 手順 4.1 4.3).</li>
	<li>肯定的な制御の孵化のストリームの N 2 の下で 1 mL 管に 100 fmol の合成 NNC ソリューションを含む、因数を蒸発させます。このバイアルによる酵素システムを 3.2 から追加 3.3 3.4 の手順の説明に従ってし、</li>。
	<li>陰性対照の孵化の実験を説明されている (手順 3.2 3.4) として反応バッファー X 0.5 交換、50 μ L の酵素液を除いて実行します</li>。
</ol> 4。LC NSI + の nitrosamide 検出パラメーターの例-人事管理システム/MS <ol><li>、LC のコンポーネント、商業、手パック 18 C18 を使用して高精度システムに 4.2 の手順で次の多段グラデーションを適用 (5 μ m)100 mm x 75 μ m, 15 μ m オリフィス キャピラリーカラム</li>。
	<li>を使用して 5 mM NH 4 OAc 溶剤、溶剤 B として MeCN としては 1 μ L/分で 5 %b で実行することによって列にサンプルをロードします。0 ~ 5 分と 0.3 μ L/分の流量をその後減速します。ランプ 55% B 10 分以上にし、5% に再平衡化に続いて 4 分以上にグラデーションを 5 から 20% の B を次に、実行 B.</li>
	<li>実行 LC NSI +-焼入れ Orbitrap の人事管理システム/MS。NNC フルスキャンして MS 2 断片化を監視します。60,000 の解像度でフル スキャンを実行し、5 ppm の質量公差で正確な親質量 (192.07670) を抽出します。衝突誘起解離 (CID) 衝突エネルギーと 25 eV、15,000、解像度によって親イオン (2.0 amu) とフラグメントを分離し、30 ミリ秒のスキャン時間使用 MS 2 断片化 NNC (m/z のプロダクト イオンの正確な質量を抽出します。192 m/z 134.04739 と 162.07874) 5 ppm の質量公差で</li>。
</ol>

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著者が明らかに何もありません。

ニトロソアミン代謝の解明は、その発がん性を理解するための重要なコンポーネントです。関与するチトクローム p450 および他の代謝酵素はポリモーフィックなので、さらにこの知識の応用では危険度が高い個人1,4が識別潜在的でした。新しいデータ α-hydroxynitrosamines、DNA 結合に関与するニトロソアミンの推定の主要な代謝産物のさらに酸化反応?...

N-ニトロソアミンが食事、タバコ製品、および一般的な環境は、発ガン性物質の大規模なクラス人間の体の1に内生的に形成されることも。以上 300 のn-ニトロソ化合物をテストされていると &#62; 90% で、発がん性評価動物モデル2,3。その発がん性を展示するには、これらの化合物はシトクロム p4501,2,3でまずアクティブする必要があります。研究を示していますそれチトクローム p450 は容易に酸化する α-hydroxynitrosamines (図 1)、半減期 ~ 5 で反応性の高い化合物であるニトロソアミン s alkyldiazohydroxides に自発的に分解する前に。後者は、H2O および N2の損失の後アルキレートの DNA することができます。結果として得られる DNA 付加体、修復されていない場合はがん開発1につながる重要な新規または腫瘍のサプレッサー遺伝子の場合、突然変異を起こすことができます。このため、代謝経路の完全に理解を取得する多くの努力が注がれてきました、DNA 付加体のおよび発がん性ニトロソアミンのチトクローム P450 酸化の下流の代謝産物。この知識は、個々 のがんリスク評価4の潜在的なアプリケーションです。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg"> 
図 1: 一般的なニトロソアミンの提案の代謝。 ニトロソアミン (1) α-hydroxynitrosamines (2) alkyldiazohydroxides (3) に自発的に分解する p450 で酸化します。これらの化合物は DNA 付加体を形成する DNA にバインドできます。それは、仮定されるその2はさらに nitrosamides 4に p450 によって酸化されます。これらは、DNA 付加体または3知られている DNA 付加体を形成するために加水分解を形成する DNA に直接バインドできます。R1 R2は、任意のアルキル基を表します。<a href="//ecsource.jove.com/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
Α hydroxynitrosamine 仮説は、広範なデータでしっかりとサポートされて、いくつかの矛盾があります。主要な 1 つは α hydroxynitrosamines5,6の半減期が短いです。これらの化合物を小胞体膜で生成される核 DNA を後アルキレートと知られています。数秒の一生を考えると、これらの中間体が生き残る方法必要な旅行しかし細胞質不可解です。1 つの仮説は、α hydroxynitrosamines の一部 processively nitrosamides7、8比較9ではかなり安定である酸化されてことです。これがおそらく、チトクローム P450 活性部位の α hydroxynitrosamines の保持によって発生します。このタイプの酸化のための先例は、ニコチン10、アルコール11、単純な alkylnitrosamines12,13と見られています。さらに、nitrosamides は、直動発ガン性物質2,3です。その反応性9に基づいて、これらの化合物は、DNA を作り出すため付加体の α-hydroxynitrosamines、新から生じるものと同じ、未踏の DNA 付加体の (図 1) を考えられています。したがって、この仮説だけではなく、細胞質を通じてトランスポートをについて説明します、また DNA の形成は、製品を損傷します。
本稿で体外シトクロム P450 を介する変換ニトロソアミンの nitrosamides を評価するための一般的なプロトコルが記載されています。例14 n ′- nitrosonorcotinine (NNC) チトクローム P450 2A6 によってn ′- nitrosonornicotine (NNN) の以前に報告された変換は展示されています。このプロトコルの幅広い基質酵素システムへの応用は、全体的なニトロソアミン代謝における nitrosamides の重要性を判断に役立ちます。

<table>
	<tbody>
		<tr>
			<td>Norcotinine</td>
			<td>AKoS GmbH (Steinen, Germany)</td>
			<td>CAS 17708-87-1, AKoS AK0S006278969</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>242845</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>431311</td>
			<td></td>
		</tr>
		<tr>
			<td>Barium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>433373</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>151823</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>ViVid CYP2A6 Blue Screening Kit</td>
			<td>Life Technologies</td>
			<td>PV6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>221376</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL tubes</td>
			<td>Fisher</td>
			<td>AB0533</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z510424</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Erlenmeyer flask</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4980125</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Separatory Funnel</td>
			<td>Sigma-Aldrich</td>
			<td>Z261017</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z278262</td>
			<td></td>
		</tr>
		<tr>
			<td>500 MHz NMR Spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-22R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LC vials</td>
			<td>ChromTech</td>
			<td>CTC&#8211;0957&#8211;BOND</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir bar</td>
			<td>Sigma-Aldrich</td>
			<td>Z127035</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR tube</td>
			<td>Sigma-Aldrich</td>
			<td>Z274682</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000, P200, and P10 pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Sigma-Aldrich</td>
			<td>Z691410</td>
			<td></td>
		</tr>
		<tr>
			<td>RSLCnano UPLC system</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Water Bath</td>
			<td>Fisher</td>
			<td>FSSWB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6795420</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoFrit Column</td>
			<td>New Objective</td>
			<td>PF3607515N5</td>
			<td></td>
		</tr>
		<tr>
			<td>Luna C18, 5 um</td>
			<td>Phenomenex</td>
			<td>535913-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

N-ニトロソアミンは、チトクローム P450 酸化活性を示すことを必要とする環境発がん物質の老舗グループ。代謝活性化の利用可能なメカニズムを含む DNA アルキル化剤に自発的に分解する α-hydroxynitrosamines の形成。DNA 損傷と結果として得られる突然変異の蓄積は、癌にもつながります。新しい証拠は、α hydroxynitrosamines することができますさらに酸化する nitrosamides に processively シトクロム p450 を示します。Nitrosamides α-hydroxynitrosamines よりも一般的より安定、DNA をアルキレートもすることができますので、nitrosamides は発がんにおける役割を果たす可能性があります。本報告では、ニトロソアミンの体外チトクローム p450 の新陳代謝からの nitrosamide 生産を評価するための一般的なプロトコルについて述べる。このプロトコルは、関連する nitrosamides と体外チトクローム P450 代謝アッセイ検出液体クロマトグラフィー nanospray イオン化・高解像度タンデム質量分析法を用いた合成への一般的なアプローチを採用しています。このメソッドは、例におけるn ′nitrosonornicotine のマイナーな代謝産物としてn ′- nitrosonorcotinine を検出しました。高感度・選択的に正確のための質量計測手法がある.ニトロソアミン チトクローム P450 システムの多種多様なこのメソッドのアプリケーションは、この変換の一般性を決定するのに役立ちます。シトクロム p450 はポリモーフィックし、活動が異なる、ためには、個々 のがんのリスク評価に役立つ可能性が nitrosamide 形成の理解を深める。

白らの作業に基づいています。19、norcotinine され、NNC にニトロソ化きれいに高収率 (80-92%)の in vitro実験のための標準。1H nmr 法、 13C NMR、居心地の良い、HRMS 親質量 [M + H] を確認すると共に HSQC (サポート情報) などの分光学的解析から得られた正常反応の構造証拠+の 5 ppm 以内、理論値 (図 2)?...

Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

Α-水酸化チトクローム p450 によって発がん性ニトロソアミンが受け入れられる代謝経路突然変異を引き起こす DNA 損傷中間体を生成します。ただし、新しいデータは、nitrosamides への酸化がさらに発生することができますを示します。一般について述べるニトロソアミンのチトクローム p450 代謝体外産 nitrosamides を検出する手法。

チトクローム p450 によるニトロソアミンのIn Vitro代謝 Nitrosamide 形成を検出するための一般的な方法

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

a-general-method-for-detecting-nitrosamide-formation-vitro-metabolism

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Cancer Research

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

9.9K ビュー.  University of Minnesota. Α-水酸化チトクローム p450 によって発がん性ニトロソアミンが受け入れられる代謝経路突然変異を引き起こす DNA 損傷中間体を生成します。ただし、新しいデータは、nitrosamides への酸化がさらに発生することができますを示します。一般について述べるニトロソアミンのチトクローム p450 代謝体外産 nitrosamides を検出する手法。

ビデオ: A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

ザ<em&gt;ショウジョウバエ</em&gt;成虫盤腫瘍モデル：遺伝的モザイクを用いた遺伝子発現と腫瘍侵襲性の可視化と定量化

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

がん研究

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

蒸発低減文化条件は、マイクロタイタープレートに多細胞スフェロイド形成の再現性を高め

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

A Unified Methodological Framework for Vestibular Schwannoma Research

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.

The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

前庭シュワン細胞腫研究のための統一方法論的枠組み

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

前立腺癌におけるリン酸化プロテオームを調べる定量的ラベル無料質量と相まってリン酸化ペプチド濃縮

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超音波画像によるマウスにおける肺腫瘍の進行の検出

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

生物発光を用いたマウスにおける乳がん増殖および転移性コロニー形成のモニタリング

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

核磁気共鳴分光法によるアルギニンメチロームの探索

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

治療誘発性老化癌細胞における複数の蛍光老化マーカーの同時イメージングおよびフローサイトメトリーベースの検出

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...