Cell Cycle Regulation. Applied Reagents and Technologies

Transcription

1 Cell Cycle Regulation Applied Reagents and Technologies

2 Highlighting Reagents for: Flow Cytometry Immunohistochemistry Western Blot Analysis Immunoprecipitation Ribonuclease Protection Assays Table of Contents Introduction to the Cell Cycle Cyclins Cyclin Product Listing Cyclin-Dependent Kinases Cyclin-Dependent Kinase Product Listing Cell Cycle Regulators Retinoblastoma Protein (Rb) p Cell Cycle Regulator Product Listing Apps.= Applications F= FITC FC= Flow Cytometry F Set= FITC Set Hu= Human IH= Immunohistochemistry IP= Immunoprecipitation Ms= Mouse PU= Purified R= Rat Rb= Rabbit RPA= Ribonuclease Protection Assay Rxns= Reactions SE= Antiserum W= Western Blot Reacts with mouse and rat. * Sennes Drug Innovations, Inc. (Houston, TX) has granted PharMingen exclusive rights to commercialize the p21 monoclonal and polyclonal antibody technology for non-clinical research purposes. Patent has been issued. All products are for research use only and not for use in diagnostic or therapeutic procedures. 1 Orders MABS (6227) Phone

3 Introduction to the Cell Cycle Cyclins and cyclin-dependent kinases (cdks) are essential for cell-cycle control in eukaryotes. Cyclins (regulatory subunits) bind to cdks(catalytic subunits) to form active cyclin-cdk complexes. Cdk subunits by themselves are inactive and binding to a cyclin is required for their activity. Cyclins A, B1, D, and E undergo periodic synthesis and degradation, thereby providing a mechanism to regulate cdk activity throughout the cell cycle. Cdk activity is further regulated by activating or inhibitory phosphorylation, and by small proteins (p15, p18, p19, p21, and p27), called inhibitors of cdk activity, that bind to cyclins, Cdks, or cyclincdk complexes. Active cyclin-cdk complexes drive cells through particular cell cycle phases, called checkpoints, by phosphorylating the unique sets of protein substrates that are essential to achieve transition to the next phase. PharMingen offers an expanding line of antibodies and reagents for the analysis of cyclins, cdks, and cell cycle regulators. Technical Assistance TALK-TEC ( ) Fax

4 Cyclin D1 DCS-6 IgG2a 1 2 kd Figure 1. Western blot analysis of cyclin D1(clone DCS-6, Cat. No A) in WS1 human fibroblast cells. Lane 1, DCS-6. Lane 2, mouse IgG2a isotype (negative) control. Cyclins Nine different cyclins have been identified to date and are designated A through I. As denoted by their name, cyclins are synthesized and degraded in a precisely timed sequence within the cell. The levels of the particular cyclins are regulated at the level of transcription as well as by targeted degradation via the ubiquitin pathway. D- and E-type cyclins are expressed during G 0 /G 1 and are referred to as start cyclins. Start, also known as the restriction checkpoint in mammalian cells, is the point in late G 1 at which the cell commits itself to another round of DNA replication. The D-type cyclins (D1, D2, and D3) are expressed in response to growth factors or mitogens, and rapidly degrade when mitogens are withdrawn. In cells that are proliferating continuously, their levels are less variable during the cell cycle than cyclins E, A or B1. Expression of a particular D-type cyclin is tissue specific. For example, T lymphocytes express more cyclin D3 than D2, and are cyclin D1- negative. D-type cyclins appear to promote G 0 to G 1 transitions and the rate of G 1 progression. The absence of D-type cyclins in specific cell types may signal a switch between proliferation and differentiation. In general, cyclin E is induced later in G1 than the D-type cyclins. Cyclins A and B1 are mitotic cyclins. Cyclin A is synthesized during S phase and degrades during anaphase. Cyclin B1, the first identified cyclin, is synthesized during late S, maximally expressed during the transition from G 2 to M, and degraded during anaphase Cyclin B1 FITC DNA Content (PI) 1000 Mouse IgG2a FITC DNA Content (PI) Figure 2. Profile of MOLT-4 human leukemia cells analyzed by FACScan (BDIS, San Jose, CA). Cells were stained using the cyclin B1 isotype set (Cat. No. 1371KK). DNA was stained with propidium iodide (PI). 3 Orders MABS (6227) Phone

5 Description Clone(s) Specificity Apps. Format Size Cat. No. Cyclin A BF683 Hu W, IP PU 0.1 mg 14531A 0.25 mg 14531C FC F 100 test 13824X Cyclin A BF683, Hu FC F Set 100 test 1370KK Isotype Set IgE-3 Cyclin B1 GNS-11 Hu W, IP PU 0.1 mg 14551A 0.25 mg 14551C Cyclin B1 GNS-1 Hu W PU 0.1 mg 14541A 0.25 mg 14541C FC F 100 test 13844X Cyclin B1 GNS-1, Hu FC F Set 100 test 1371KK Isotype Set MOPC-21 Cyclin D1 DCS-6 Hu, Ms, R W, IP PU 0.1 mg 66271A Cyclin D1 G Hu W PU 0.1 mg 14561A 0.25 mg 14561C FC F 100 tests 13864X Cyclin D1 G , FC F Set 100 test 1372KK Isotype Set MOPC-21 Cyclin D2 G Hu W, IP PU 0.1 mg 14821A 0.25 mg 14821C FC F 100 tests 13874X Cyclin D2 G132-43, Hu FC F Set 100 test 1373KK Isotype Set MOPC-173 Cyclin D3 G Hu W, IP PU 0.1 mg 14781A 0.25 mg 14781C Cyclin D3 G , Hu FC F Set 100 test 1374KK Isotype Set MOPC-21 Cyclins D1/D2/D3 G Hu W, IP PU 0.1 mg 14841A 0.25 mg 14841C FC F 100 tests 13904X Cyclins D2/D3 G Hu W, IP PU 0.1 mg 14711A 0.25 mg 14711C Cyclins D1/D2/D3 G , Hu FC F Set 100 test 1375KK Isotype Set MOPC-21 Cyclin E HE12 Hu W PU 0.1 mg 14591A 0.25 mg 14591C Cyclin E HE67 Hu W, IP PU 0.1 mg 14761A 0.25 mg 14761C Cyclin H G301-1 Hu W PU 0.1 mg 13971A V-Cyclin G319-1 Hu W PU 0.1 mg 65931A Western Blot Controls Recombinant Cyclin A W Lysate 0.5ml 16116Y Recombinant Cyclin B1 W Lysate 0.5ml 16126Y Recombinant Cyclin D1 W Lysate 0.5ml 16136Y Recombinant Cyclin D2 W Lysate 0.5ml 16146Y Recombinant Cyclin D3 W Lysate 0.5ml 16156Y Recombinant Cyclin E W Lysate 0.5ml 16166Y RiboQuant Ribonuclease Protection Assay (RPA) System RiboQuant In Vitro Transcription Kit RPA 25 Rxns 45004K RiboQuant RNase Protection Assay Kit RPA 200 Rxns 45014K RiboQuant RPA Starter Package RPA 45024K (In Vitro Transcription, RPA, one Multi-Probe Template Set) hcyc-1 RiboQuant Human Cyclin RPA 10 Rxns 45352P Multi-Probe Template Set hcyc-2 RiboQuant Human Cyclin RPA 10 Rxns 45353P Multi-Probe Template Set HeLa Control RNA RPA 10 Rxns 45201Z Cyclin A Cyclin B Cyclin C Cyclin D1 Cyclin D2 Cyclin D3 Cyclin A1 L32 GAPDH Cyclin E Cyclin F Cyclin G1 Cyclin G2 Cyclin I Cyclin H L32 GAPDH hcyc Probe Yeast trna HeLa hcyc Probe Yeast trna HeLa Figure 3. Autoradiograms of RiboQuant RPA Assays utilizing Human Multi-Probe Template Sets for cyclin genes: hcyc-1, Cat. No P hcyc-2, Cat. No P Technical Assistance TALK-TEC ( ) Fax

6 Cyclin-Dependent Kinases Cyclin-dependent kinases (cdks) are catalytic proteins whose activity requires interaction with their regulatory subunits, the cyclins, as well as specific phosphorylation events. The precise timing of cyclin-cdk activity during the cell cycle determines whether the cell cycle continues or becomes blocked. Cdks 2,4,5 and 6 associate with D-type cyclins. When complexed to cyclin D, cdk4 and cdk6 phosphorylate the retinoblastoma protein Rb, removing the G 1 block caused by underphosphorylated Rb. Interaction between cdk2 and cyclin E during G 1 /S transition creates a complex with histone H1 kinase activity. This complex is thought to be required for the initiation and progression of DNA replication during S phase. Cdk2-cyclin A complexes appear during late S phase and also play a role in progression of DNA replication. Cdk1 forms a complex with cyclin B1. This complex is activated by the protein phoshatase cdc25 and subsequently drives the cell to enter mitosis. Cdk7 (CAK, for cdk-activating kinase) has been shown to phosphorylate and activate many different cdks in vitro. 5 Orders MABS (6227) Phone

7 IgG heavy chain Cdk4 kd CdK1 CdK2 CdK3 CdK4 p27 p21 PISSLRE hcc Figure 4. Immunoprecipitation (IP)/Western blot (W) analysis of cdk4 (clone ACD1, Cat. No.13961A) in 3T3 cells. Lane 1, ACD1 W. Lane 2, ACD1 IP followed by W with ACD1. Lane 3, IP with rat IgGs, followed by W with ACD1. kd Cdk T3 293 HeLa Saos-2 Negative Figure 5. Western blot analysis of cdk4 (clone ACD1, Cat. No.13961A) in cell lysates. Lane 1, mouse (3T3), lane 2, human 293, lane 3, human HeLa, and lane 4, human Saos-2. Rat IgGs were used as a negative (isotype) control. P16 L32 GAPDH Probe Yeast trna HeLa Figure 6. Autoradiogram of RiboQuant RPA Assays utilizing a Human Multi- Probe Template Set for cdk genes: hcc-1, Cat. No P Description Clone Specificity Apps. Format Size Cat. No. Cdc25B Polyclonal Hu W SE 0.1 ml 14746E Cdk1 A-17 Ms W, IP PU 0.1 mg 14391A Cdk2 Polyclonal Hu W, IP SE 0.1 ml 15536E Cdk2 G Hu W PU 0.1 mg 14771A Cdk3 G Hu W PU 0.1 mg 13221A Cdk4 ACD1 Ms W, IP PU 0.1 mg 13961A Cdk5 Polyclonal Hu W, IP SE 0.1 ml 15166E Cdk5 G162-9 Hu W PU 0.1 mg 13491A Cdk6 Polyclonal Ms W, IP SE 0.1 ml 13446E Cdk7 Polyclonal Hu W, IP SE 0.1 ml 13776E Cdk7 MO-1.1 Hu W, IP PU 0.1 mg 65011A Western Blot Controls Recombinant Cdk1 Lysate Hu W 0.5ml 16216Y Recombinant Cdk2 Lysate Hu W 0.5ml 16256Y RiboQuant Ribonuclease Protection Assay (RPA) System hcc-1 RiboQuant Human Cell Cycle Multi-Probe Template Set RPA 10 Rxns 45352P HeLa Control RNA RPA 10 Rxns 45201Z Technical Assistance TALK-TEC ( ) Fax

8 MW (kd) 97 p Figure 7. Western blot analysis of human p16 INK4a (clone G , Cat. #13381A) in Saos-2 cells. Lane 1, G Lane 2, IgG1 isotype (negative) control. Cell Cycle Regulators Cyclin-cdk complexes form holoenzymes that phosphorylate Retinoblastoma-family proteins (prb, p107 and p130) through specific stages of the cell cycle. Cell cycle regulators such as p15, p16 (see Figure 7), p18 and p21 block the activity of cyclin-cdk complexes, preventing phosphorylation of their targets. The transcription factors E2F (1-4) and DPI (1 and 2) which drive the DNA replication machinery are negatively regulated by direct interaction with prb, p107 and p130. A common feature of prb-related proteins and tumor suppressors such as p53 and p33 ING1 is their ability to inhibit cell proliferation. p33 ING1 facilitates the activity of p53 and leads to increased expression of p21 (see Figure 8), a major target of p53 transcription regulation. c-myc is a positive regulator of cyclin G1-cdk complexes and of cyclin E-cdk2 complexes. c-myc can enhance cdk activity via functional inactivation of the cdk inhibitors as well as by inducing the cdk-activating phosphatase Cdc25. Figure 8. Immunohistochemical analysis of human p21 (clone SX118, Cat. No A). Formalin-fixed, paraffin-embedded tissue section of human small intestine stained with SX118 using a DAB chromogen and Hematoxylin counterstain. 7 Orders MABS (6227) Phone

9 Retinoblastoma Protein (Rb) Rb appears to play a key role in the control of cell division and differentiation. Rb is a nuclear phosphoprotein that undergoes cell cycle-dependent phosphorylation. During G 0 /G 1, terminal differentiation, senescence and quiescence, Rb is underphosphorylated. Before the onset of DNA synthesis in late G 1, Rb becomes phosphorylated on serine and threonine residues. It undergoes additional phosphorylation as cells progress into S and G 2 /M. Most of the phosphate groups are removed as the cell re-enters into G 0 /G 1. Rb migrates as a single band or as multiple, closely-spaced bands between ~ kd when sized on denaturing polyacrylamide gels (SDS-PAGE). The different bands represent different Rb phosphorylation states which may be referred to as underphosphorylated, phosphorylated, and highly phosphorylated (see Figure 10). The higher molecular weight bands are more highly phosphorylated than the lower molecular weight bands. The level of phosphorylation is cell cycle dependent. Underphosphorylated forms predominate in G 0 and early G 1, whereas more highly phosphorylated forms are present in S, G 2 and M. Additionally, the level of phosphorylation may also be cell type dependent (not all forms are seen in all cell types that express Rb). G G3-245 G3-349 G4-340 C G XZ XZ XZ kd Q G 1 S M pprb prb Figure 10. Western blot analysis of Rb (clone G3-245, Cat. No A) during the cell cycle in MOLT-4 human leukemia cell cultures. Rb migrates as multiple bands due to varying degrees of phosphorylation. Cell cycle stages are denoted as Q (quiescent), G 1 (late G 1 ), S, and M. prb, underphosphorylated Rb. pprb, phosphorylated and highly phosphorylated Rb species. Rb G XZ55 Figure 9. Epitopes recognized by different Rb monoclonal antibodies. p130 Rb p107 p53 hcc p130 Rb p107 DP1 hts Figure 11. Immunohistochemical analysis of human Rb (clone G3-245, Cat. No.14001A). Formalin-fixed, paraffin-embedded tissue section of human osteosarcoma stained with G3-245 using a DAB chromogen and Hematoxylin counterstain. p57 DP2 p27 E2F1 p21 E2F2 p19 p18 E2F4 p16 p14/15 L32 GAPDH L32 GAPDH Probe Yeast trna HeLa Probe Yeast trna HeLa Figure 12. Autoradiograms of RiboQuant RPA Assays utilizing Human Multi-Probe Template Sets for cell cycle regulator genes: hcc-2, Cat No.45091P hts-1, Cat. No.45091P Technical Assistance TALK-TEC ( ) Fax

10 Relative Cell Number Log Fluorescence Intensity Figure 13. Anti-human p53 (clone DO-7, Cat. No.15801A). Profile of permeabilized CEM human leukemia cells analyzed on a FACScan (BDIS, San Jose, CA). Cells were stained with DO-7 or a mouse IgG2b isotype (negative) control followed by FITC-conjugated second step). p53 kd DO-7 migg2b p53 p53 is a 53 kd nuclear phosphoprotein that acts as a tumor suppressor protein, by inhibiting cell proliferation when DNA damage occurs. Wild type p53 protein has a very short half-life and is usually not detectable with monoclonal antibodies (mabs) in normal tissues. Mutant p53 proteins typically have an increased half-life, accumulate to high levels, and are detectable by mabs. Mutant p53 can complex with wild type p53, increase its half-life, and lead to an accumulation detectable by mabs. Other stabilizing factors may also lead to an accumulation of wild type p53. PharMingen offers a panel of p53 specific antibodies. Many of the antibodies are suitable for multiple applications. We find that the DO-1 (Cat. No A) and DO-7 (Cat. No A; see Figure 15) antibodies are the best choices for immunohistochemical staining of acetone-fixed, frozen and formalin-fixed, paraffin-embedded human tissue sections. DO-1 and DO-7 (see Figure 14) are also excellent choices for western blot analysis and immunoprecipitation for most experimental systems. However, they do not cross-react with mouse or rat p53. The DO-7 antibody has also been developed for flow cytometric analysis (see Figure 13). G59-12 (Cat. No A) is reactive with mouse, rat and human p53 and can be used for immunoprecipitation as well as for flow cytometric analysis. PAb 240 (Cat Nos A and 14461C) exclusively recognizes mutant forms of p53 in immunoprecipitation. Both PAb 122 (Cat. No A) and PAb 240 have been documented for wide species reactivity. p53 antibodies should be selected based on application, species recognition, and epitope recognition. PAb PAb PAb G p DO-1 DO PAb Figure 14. Western blot analysis of human p53 (clone DO-7, Cat. No.15801A) in human leukemia cell lysates. Lane 1, DO-7. Lane 2, IgG2b isotype (negative) control. Figure 16. Epitopes recognized by different p53 monoclonal antibodies. Figure 15. Anti-human p53 (clone DO-7, Cat. No.15801A). Formalinfixed, paraffin-embedded tissue section of human breast carcinoma stained with DO-7 using a DAB chromogen and Hematoxylin counterstain. 9 Orders MABS (6227) Phone

11 Description Clone Specificity Apps. Format Size Cat. No. APC 5B2 Hu W PU 0.1 mg 66651A BRCA-1 Polyclonal Hu W, IP PU 50 µg 66036N BRCA-1 Polyclonal Hu W, IP PU 50 µg 66046N BRCA-1 Polyclonal Hu W, IP PU 50 µg 66056N BRCA-2 Polyclonal Hu W, IP SE 0.1 ml 66066E BRCA-2 Polyclonal Hu W, IP SE 0.1 ml 66076E DCC G92-13 Hu FC PU 0.1 mg 15031A DCC G Hu FC, IH PU 0.1 mg 15041A DP-1 TFD10 Hu W PU 0.1 mg 66201A E2F-1 KH95/E2F Hu, Ms, R W, IP PU 0.1 mg 14971A E2F-2 TFE22 Hu W PU 0.1 mg 66711A E2F-3 TFE31 Hu W PU 0.1 mg 66721A Maspin Polyclonal Hu W SE 0.1 ml 15716E Maspin G Hu W, IH PU 0.1 mg 15781A p16 INK4a Polyclonal Hu W, IP, IH SE 0.1 ml 15126E p16 Kip1 G Hu W, IP, IH PU 0.1 mg 13251A p16 Kip1 G Hu W, IP PU 0.1 mg 13381A p18 A33-1 Ms W, IP PU 0.1 mg 65561A p19 INK4c 3B6 Hu, Ms W, IP PU 0.1 mg 65911A p19 INK4c Polyclonal Ms W, IP SE 0.1 ml 13636E p21* SX118 Hu, Ms, R W, IP, IH PU 0.1 mg 65951A p21* SXM30 Hu, Ms, R W, IP, IH PU 0.1 mg 65961A p21* Polyclonal Hu W, IP+ SE 0.1 ml 15431E p21* 6B6 Hu W, IP, IH PU 0.1 mg 15091A p21* 18A10 Hu W, IP PU 0.1 mg 15101A p21* 2G12 Hu W, IP, IH PU 0.1 mg 15441A p21* Polyclonal Ms W, IP SE 0.1 ml 13436E p27 Kip1 G Hu, Ms W, IP PU 0.1 mg 13231A p27 Kip1 Polyclonal Ms W, IP SE 0.1 ml 15596E p27 Kip1 Polyclonal Ms W, IP SE 0.1 ml 15606E p33 ING1 Polyclonal Hu, Ms W SE 0.1 ml 66156E p53 G59-12 Hu, Ms, R W, IP, IH PU 0.1 mg 14211A p53 PAb 122 Hu, Ms, R W, IP PU 0.1 mg 14091A p53 PAb 240 Hu, Ms, R W, IH PU 0.1 mg 14461A 0.25 mg 14461C p53 DO-1 Hu W, IP, IH PU 0.1 mg 15791A p53 DO-7 Hu W, IP, IH, FC PU 0.1 mg 15801A p53 Isotype Set DO-7,27-35 Hu FC F Set 100 tests 1580KK p53 PAb 1801 Hu W, IP, IH PU 0.1 mg 14471A 0.25 mg 14471C p53 PAb 246 Ms IP PU 0.1 mg 14451A p57 KIP2 A120-1 Hu W PU 0.1 mg 65021A p107 SD9 Hu W, IP PU 0.1 mg 14911A p300 NM11 Hu W, IP PU 0.1 mg 14991A PRK B37-2 Ms W PU 0.1 mg 66641A Rb G3-245 Hu, Ms, R W, IP, IH, FC PU 0.1 mg 14001A Rb C36 Hu W, IP PU 0.1 mg 14031A Rb G Hu W, IP PU 0.1 mg 14441A Rb XZ55 Hu W, IP PU 0.1 mg 14051A Rb XZ91 Hu W, IP PU 0.1 mg 14061A Rb XZ133 Hu W PU 0.1 mg 14071A Rb, G3-245 mab W, IH, FC Peptide 0.1 mg 66671K Blocking Peptide Set Set each Von Hippel-Landau Ig32 Hu W, IP, IH PU 0.1 mg 65031A Protein Wilms' Tumor 2C12 Hu IH PU 0.1 mg 15921A Western Blot Controls Recombinant mutant p53 Lysate W 0.5ml 16236Y Recombinant wildtype p53 Lysate W 0.5ml 16226Y MOLT-4 Lysate W 0.5ml 16246Y COS-7 Lysate W 0.5ml 16266Y HeLa Carcinoma Lysate W 0.5ml 16286Y RiboQuant Ribonuclease Protection Assay (RPA) System hcc-2 RiboQuant Human Cell Cycle RPA 10 Rxns 45091P Multi-Probe Template Set hts-1 RiboQuant Human Tumor Suppressor RPA 10 Rxns 45101P Multi-Probe Template Set HeLa Control RNA RPA 10 Rxns 45201Z Technical Assistance TALK-TEC ( ) Fax

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