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WO1999044067A1 - DETECTION PAR CYTOMETRIE DE FLUX DE CONFORMATIONS DE pRB DANS DES CELLULES INDIVIDUELLES - Google Patents

DETECTION PAR CYTOMETRIE DE FLUX DE CONFORMATIONS DE pRB DANS DES CELLULES INDIVIDUELLES Download PDF

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Publication number
WO1999044067A1
WO1999044067A1 PCT/US1999/004015 US9904015W WO9944067A1 WO 1999044067 A1 WO1999044067 A1 WO 1999044067A1 US 9904015 W US9904015 W US 9904015W WO 9944067 A1 WO9944067 A1 WO 9944067A1
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Prior art keywords
prb
cells
cell
cyclin
antibody
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PCT/US1999/004015
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English (en)
Inventor
Zbigniew Darzynkiewicz
Frank Traganos
Gloria Juan
Stefan Gruenwald
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New York Medical College
Becton Dickinson And Company
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Application filed by New York Medical College, Becton Dickinson And Company filed Critical New York Medical College
Priority to AU33100/99A priority Critical patent/AU3310099A/en
Priority to JP2000533764A priority patent/JP2002505433A/ja
Priority to EP99936123A priority patent/EP1057036A1/fr
Publication of WO1999044067A1 publication Critical patent/WO1999044067A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4736—Retinoblastoma protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the present invention relates to methods, reagents, and reagent kits for detecting discrete functional conformations of proteins concurrently in individual cells, particularly by flow cytometry.
  • the invention relates to methods, reagents, and reagent kits for the determination of the phosphorylation status of the retinoblastoma susceptibility gene protein (pRB) in individual cells using multiparameter flow cytometry.
  • pRB retinoblastoma susceptibility gene protein
  • pRB retinoblastoma susceptibility gene
  • pRB activity is controlled by changes in phosphorylation.
  • pRB is hypophosphorylated in normal quiescent cells (in G 0 phase) and in cells that are in early G ⁇ .
  • cyclin-dependent kinases Cdk; Cdk
  • Cdk cyclin-dependent kinases
  • pRB Unphosphorylated, pRB binds to and sequesters transcription factors of the E2F family. Phosphorylated, pRB discharges these factors, the factors in turn activating transcription of genes coding for proteins regulating DNA replication and cell proliferation. These events commit the cell to entry into S phase. Later, in late mitosis, type 1 protein phosphatases dephosphorylate pRB, restoring the active, E2F-sequestering form, thus resetting the cycle. pRB is also essential in the terminal differentiation of cells of various lineages. During terminal differentiation, when cells exit the cycle, pRB expression is upregulated and the protein remains in the active — that is, hypophosphorylated — state. Mice homozygously deleted for the RB gene show defective differentiation of various tissues.
  • the phosphorylation status of pRB is assayed in vi tro, measuring 32 P-labeling and/or - 4
  • the existing methods measure phosphorylation of pRB in bulk culture.
  • Several questions regarding the mechanism by which pRB controls cell cycle progression cannot be answered using such assays. For example, is phosphorylation of pRB within the cell an all-or-none phenomenon, or is there instead a mixture of hypophosphorylated and hyperphosphorylated pRB molecules at varying proportions throughout the cycle? What proportion of pRB molecules is phosphorylated within the cell during G l f prior to entrance to S phase? Is there a critical threshold in the ratio of hypophosphorylated to hyperphosphorylated pRB molecules that determines the transition of cells to quiescence or to commitment to enter S?
  • the heterogeneity in the cycling kinetics and timing of cells in culture typically obligates artificial synchronization of the cells in culture to permit meaningful results to be obtained using the existing bulk assays; and yet this cell cycle synchronization, when induced by inhibitors of DNA polymerase, is associated with growth imbalance and unscheduled expression of cyclins. Gong et al . , Cell Growth Differ. 6:1485-1493 (1995). There thus exists a need for methods that permit the phosphorylation status of pRB to be measured in individual cells without exogenous intervention in the cell cycle.
  • Onconase ® is a basic protein of 12,000 MW isolated from oocytes or early embryos of Rana pipiens . Onconase ® shows antiproliferative activity in vi tro, suppressing proliferation of tumor cell lines of various lineages, including those of hematological origin. Onconase ® has also been shown to inhibit growth of certain tumors in vivo in mice.
  • Onconase ® is currently in clinical trials for treatment of patients with advanced pancreatic adenocarcinoma and malignant mesothelioma, the mechanism of its antitumor activity is still poorly understood.
  • the protein is known to have both cytostatic and cytotoxic effects, the former manifesting as an increase in the proportion of cells in G x phase of the cell cycle; but the mechanism by - 6 -
  • the present invention solves these and other problems in the art by presenting methods and reagents that permit the concurrent and discriminable detection of discrete functional conformations of proteins within a single cell.
  • the invention provides methods and reagents for the flow cytometric determination of multiple pRB phosphorylation states in individual cells.
  • anti-pRB antibodies that distinguish the phosphorylation state of pRB may successfully be conjugated to fluorophores (fluorochromes) without loss of specificity; that when conjugated to such fluorophores, these antibodies provide sufficient signal to permit detection of pRB in individual cells; that when simultaneously applied to cells that have been fixed and permeabilized, these antibodies bind to their respective functional conformations of pRB within the cell without mutual interference; and that when conjugated to flow cytometrically distinguishable fluorophores, these antibodies permit the concurrent detection of discrete functional conformations (phosphorylation states) of pRB to be detected in single cells.
  • fluorophores fluorochromes
  • the methods permit complex intracellular interactions of pRB to be assessed.
  • the present invention provides a method for determining the relative intracellular conformational states of a protein, comprising: contacting a cell with a first antibody, said first antibody specific for a first conformation of said protein, and a second antibody, said second antibody specific for at least one other conformation of said protein, said first and second antibodies being distinguishably labeled; detecting the binding of each of said antibodies concurrently by said cell; and determining the relative binding thereof.
  • the method may further comprise the antecedent step of fixing the cell.
  • the method further comprises the step, before flow cytometric detection, of permeabilizing the cell.
  • the antibodies are labeled with fluorophores and the fluorophores are distinguishable by a laser cytometer.
  • the fluorophores are flow cytometrically distinguishable.
  • the fluorophores may conjugated directly or indirectly to the antibodies, with the direct conjugation of at least one, preferably at least two, antibodies presently preferred.
  • any fluorophore that permits laser cytometric detection may usefully be employed, those presently preferred are selected from the group consisting of: FITC, PE, PerCP, APC, PE-CY5 tandem fluorophore and PerCP-CY5.5 tandem fluorophore. - 9 -
  • the present invention provides a method for determining the relative intracellular conformational states of pRB, comprising: contacting a cell with a first antibody, said first antibody specific for a first conformation of pRB, and a second antibody, said second antibody specific for at least one other conformation of pRB, said first and second antibodies being distinguishably labeled;
  • a method for determining the relative intracellular conformational states of a protein comprising: contacting a cell with a first antibody, said first antibody specific for a first conformation of said protein, and a second antibody, said second antibody specific for at least one other conformation of said protein, said first and second antibodies being distinguishably labeled; detecting the binding of each of said antibodies concurrently by said cell; and then determining the relative binding thereof.
  • each of the pRB conformations is correlated with a discrete phosphorylation state of the protein.
  • the first antibody is specific for a conformation assumed by the hypophosphorylated form of pRB, and the second antibody is specific for at least one other conformation of pRB; this may include specificity for all functional conformations of pRB. In another embodiment, the first antibody is specific for all conformations of pRB, and the second antibody is specific for a subset thereof. - 10 -
  • the methods of this second aspect of the invention may further comprise the step of contacting said cell with a fluorescent nucleic acid stain, and then detecting, preferably by laser cytometry, most preferably by flow cytometry, the binding to DNA of said stain concurrently with detecting the binding to pRB of said first and second antibody.
  • the methods of this aspect may further comprise contacting said cell with a third antibody, said third antibody being specific for a second protein and distinguishable from each of said first and second antibodies, and then detecting the concurrent binding of each of said antibodies to said cell.
  • the second protein may be a cyclin, a cyclin dependent kinase, or a cyclin dependent kinase inhibitor.
  • the invention provides methods of assaying, or screening, compounds for antiproliferative activity, comprising: contacting a sample of proliferating cells with said compound in vi tro; contacting said cells with a first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and being flow cytometrically distinguishable from said first antibody; flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein an increased ratio of hypophosphorylated pRB to total pRB indicates antiproliferative activity of said compound.
  • the invention provides methods of assessing the in vivo antiproliferative effect of a compound, comprising: contacting a sample - 11 -
  • the invention also provides methods of assessing the proliferative potential of a heterogeneous population of cells, comprising: contacting said cells with a first antibody specific for a conformation assumed by the hypophosphorylated form of pRB and a second antibody, said second antibody specific for at least one other conformation of pRB and flow cytometrically distinguishable from said first antibody; and then flow cytometrically detecting the binding of each of said antibodies concurrently by said cell; wherein cells with a decreased ratio of hypophosphorylated pRB to total pRB are determined to have increased proliferative potential.
  • the invention provides reagent kits that facilitate the practice of the subject methods.
  • the invention provides a kit for detecting the phosphorylation status of pRB in individual cells, comprising: a first antibody, said first antibody - 12 -
  • first and second antibodies are flow cytometrically distinguishable from one another.
  • first antibody is specific for the hypophosphorylated form of pRB and the second antibody is specific for total pRB.
  • each of said antibodies is conjugated to a fluorophore, and said fluorophores are flow cytometrically distinguishable from one another.
  • FIG. 1 presents frequency distribution histograms representing the intensity of fluorescence of stimulated lymphocytes reacting with pRB p" mAb (left panels) or pRB ⁇ n-iAbs (right panels) at different times (0 to 24 hours) after administration of PHA.
  • the dashed lines represent the maximal level of background fluorescence (i.e., the maximal level of fluorescence measured from cells incubated with isotype-matched control IgGl antibody) .
  • Fluorescence intensity is plotted on a three-log exponential scale. The results are representative of three repetitions of the - 13 -
  • FIG. 2 shows changes in percentage of lymphocytes reacting with anti-pRB p" or anti-pRB ⁇ Ab, as well as the cells expressing cyclin D3, as a function of the duration of incubation with PHA;
  • FIG. 3 shows bivariate distribution of DNA content vs . reactivity with anti-pRB p ⁇ mAb on the third day of lymphocyte mitogenic stimulation with PHA.
  • the threshold (dashed line) represents the background fluorescence of the same cells stained with FITC labeled isotype-matched IgGl. Similar distribution was seen in cultures of PHA-stimulated lymphocytes maintained in exponential growth for 6 days in the presence of interleukin 2;
  • FIG. 4 shows the effect of pretreatment with alkaline phosphatase and effect of staurosporine (STP) added at time 0, together with PHA, on cell reactivity with anti-pRB p" mAb: panel A, unstimulated lymphocytes; panel B, lymphocytes stimulated with PHA for 24 h; panel C, lymphocytes stimulated for 24 hours but pre- incubated with alkaline phosphatase prior to incubation with anti-pRB p" mAb; panel D, lymphocytes stimulated with PHA for 24 hours in the presence of 20 nM STP. Note the loss of cell subpopulation unreactive with anti-pRB p" after treatments with STP (panel D) or phosphatase (panel C) . Because the cell frequency coordinate scale varied between the samples (automatically adjusted by the software of the flow cytometer to present major peaks at similar heights) 14 -
  • FIG. 5 presents bivariate distributions of
  • HL-60 cells from the control culture showing expression of pRB ⁇ (CyChrome ® , A panels), the pRB p" /pRB ⁇ ratio (FITC/CyChro e ® , B panels), and pRB p" (FITC, C panels) versus cellular DNA content (DAPI) .
  • the three top panels show gating analysis of pRB p" positive cells, and the bottom three panels show gating analysis of G x cells expressing high (suprathreshold) levels of pRB ⁇ .
  • Variability of G 1 cell population with respect to pRB ⁇ is evident in panels A (note that the CyChrome ® scale is exponential) .
  • Panel C top, shows the gate used to select the cells which have hypophosphorylated pRB (pRB p ⁇ positive cells; the dashed line indicates mean FITC fluorescence + 3 SD of the cells stained with the same mAb but after treatment with alkaline phosphatase) .
  • the gated pRB p" positive cells, color labeled (green) show variable levels of pRB ⁇ when revealed in panel A and have a pRB p" /pRB ⁇ ratio above the threshold value, greater than that of S phase cells (B) .
  • FIG. 6 shows changes in pRB ⁇ during differentiation of HL-60 cells. The bars represent - 15 -
  • G x , S, and G 2 /M cells mean pRB ⁇ fluorescence of G x , S, and G 2 /M cells from the control cultures and from the cultures treated with RA or vitamin D 3 , calculated as described under Material and Methods for Example 2.
  • G x , S, and G 2 /M cells were distinguished and gated based on differences in their DNA content, their mean pRB ⁇ fluorescence is expressed per unit of DNA and normalized to the mean fluorescence of G x cells from the control culture, which was expressed as 100;
  • FIG. 7 shows changes in pRB p" during differentiation of HL-60 cells.
  • the bars represent mean values of pRB p ⁇ fluorescence of the control cells and the cells from vitamin D 3 and RA-treated cultures estimated for G x , S, and G 2 /M cells, as described in the legend to Fig. 6 and in Example 2, below;
  • FIG. 8 demonstrates changes in pRB p" /pRB ⁇ during differentiation of HL-60 cells.
  • the bars represent mean values of pRB p ⁇ /pRB ⁇ ratio estimated for G x , S, and G 2 /M cells from control and vitamin D 3 and RA-treated cultures;
  • FIG. 9 shows bivariate distributions of pRB ⁇ and pRB p" /pRB ⁇ versus cellular DNA content of exponentially growing cells from untreated cultures (panels A and D) and cultures treated with RA (panels B and E) and with vitamin D 3 (panels C and F) , obtained as described under Material and Methods for Example 2.
  • the mean pRB ⁇ fluorescence of cells from the differentiating cultures is increased (note that the pRB ⁇ scale is exponential) . Also increased is their pRB p" /pRB ⁇ ratio.
  • the arrowheads indicate the threshold levels of pRB ⁇ or - 1 6 -
  • FIG. 10 presents a Western blot showing reactivity of anti-pRB p" mAb (A) and anti-pRB ⁇ mAb with proteins extracted from control (CON) and PMA-treated cells. Note the increased intensity bands representing hypophosphorylated pRB (panel A, also bottom band in B) , hyperphosphorylated pRB (top band in B) , and total pRB (both bands in B) ;
  • FIG. 11 presents growth and viability curves
  • FIG. 12 demonstrates the effect of Onconase ® on expression of cyclin D3 by U937 cells.
  • Anti-cyclin D3 mAb immunofluorescence in combination with DNA content was measured by multiparameter flow cytometry (a) .
  • the cells were growing either in the absence ⁇ Exp, exponential growth) or in the presence of 170 nM
  • Onconase ® for 48 hours ⁇ One) There were 48%, 38% and 14% of cells in G x , S, and G 2 /M in Exp culture compared with 64%, 24% and 13% cells in G x , S, and G 2 /M, respectively, in One culture.
  • the scattergrams represent bivariate distributions of the cells with - 17 -
  • FIG. 13 shows expression of pl ⁇ INK4A in U937 cells growing exponentially ⁇ Exp) and in the culture treated with 170 nM Onconase ® for 72 hours ⁇ One) .
  • the bar plot shows mean anti-pl6 INMA fluorescence estimated for cells in G x S and G 2 /M, as described in the legend to Figure 12. Proportions of cells in different phases of the cycle were the same as in Figure 12;
  • FIG. 14 shows expression of p 21 AF1CIP1 in U937 cells growing exponentially ⁇ Exp) and in the culture treated with 170 nM Onconase ® for 72 hours ⁇ One) .
  • the bar plot shows mean anti-p21 WAF1CIP1 mAb fluorescence estimated for cells in G x , S and G 2 /M as described in the legend to Figure 12.
  • Western blot (B) shows the P21 AFI/CIPI band only in the onconase®-treated cells;
  • FIG. 15 shows expression of p27 KIP1 in U937 cells growing exponentially ⁇ Exp) and in the culture treated - 18 -
  • FIG. 16 shows detection of U937 cells having underphosphorylated pRB in exponentially growing control culture (panel a) and in the cultures treated with 170 nM Onconase ® for 48 hours (panel b) and 72 hours (panel c) . Also shown are cells from the culture at higher cell density (>10 6 cells/ml) where proliferation declined because of cell crowding (d) . The cells reacting with anti-pRB p" were in G 0 x phase (open arrows) . The dashed line indicates the upper level of background fluorescence, i . e . of the cells pretreated with alkaline phosphatase.
  • the percentage of cells reacting with anti-pRB p" mAb was increased in the Onconase ® -treated and crowded cultures compared to the control. No significant changes in expression of pRB detected by the antibody that reacts with this protein regardless of its phosphorylation status (anti- pRB ⁇ ) were observed between Onconase ® -treated and control cells (not shown) .
  • the phosphorylation status of pRB is typically measured by assessing 32 P-labeling and/or electrophoretic mobility on a Western blot. Not only is the procedure cumbersome, but it also risks the artifactual activation of phosphatases that may dephosphorylate the protein during or after cell lysis, confounding results.
  • Hybridoma clone G99-549 was derived originally from a fusion in which underphosphorylated human pRB, produced in sf9 insect cells, was used as immunogen.
  • the hybridoma clones that produced antibody that recognized pRB in Western blots only a single clone, G99-549, produced antibody that identified the faster- migrating, and presumably underphosphorylated, forms of pRB.
  • Several control experiments have since demonstrated that the faster migrating forms of pRB recognized by this mAb on Western blot indeed lack phosphorylation. - 22 -
  • the epitope recognized by G99-549 lies between pRB amino acids 514-610, within an "A box" of a T-antigen binding pocket domain of human pRB.
  • the location of this epitope suggests that G99-549 mAb recognizes a structural conformation that pRB assumes in the hypophosphorylated state, rather than directly recognizing the unphosphorylated form of a phosphorylation consensus sequence.
  • clone G3-245 anti-pRB ⁇ ; pRB ⁇ mAb; also termed Mh-RB-02
  • Mh-RB-02 anti-pRB ⁇ ; pRB ⁇ mAb; also termed Mh-RB-02
  • conjugation to fluorophore is required; yet not all antibodies can successfully be conjugated to fluorophore with retention of antigen specificity.
  • the antibodies once conjugated, the antibodies must provide sufficient signal to permit detection in individual cells.
  • the inhibitor permits the signal to be amplified before assay through accumulation of protein in the cytoplasm.
  • the assays have not been directed to detection of unamplified nuclear proteins.
  • the reagents that distinguish these conformations must not interfere with or abrogate the binding of one another. Where the conformations are mutually exclusive, this requirement is easily met, since the antibodies must bind to separate molecules; where one antibody recognizes a subset of the conformations recognized by the other, however, the antibodies must both bind to a single molecule in one or another conformation, implicating steric hindrance as a potential problem.
  • each antibody must permit conjugation to a fluorophore, the two fluorophores being flow cytometrically distinguishable, without loss of specificity and without fluorescence quenching.
  • the flow cytometric methods of the present invention have been applied to normal peripheral blood lymphocytes after stimulation with polyclonal mitogen (Example 1), to the human promyelocytic leukemic cell line HL-60 (Example 2), and to human histiocytic U937 cells (Example 3) .
  • the procedure is similar for each of these cell types.
  • cells are fixed, labeled with antibodies conjugated to flow cytometrically detectable fluorophores ( fluorochromes ) , and then detected using single- or dual-laser multiparametric flow cytometry.
  • fluorophores fluorochromes
  • fixation The first step in the flow cytometric detection of pRB functional conformations is fixation of the cells; fixation both arrests metabolic processes and, simultaneously, renders the cytoplasm and nucleus accessible to the labeling antibodies.
  • fixation was accomplished either by: (1) suspending in 1% formaldehyde in PBS for 15 min on ice, followed after washing by resuspension in ice- cold 80% ethanol, further followed after rinsing by resuspension in 0.25% Triton X-100 (Sigma); or by
  • Cells may, for example, be simultaneously fixed and permeabilized in HBSS (HEPES buffered saline) with 10 mmol/L HEPES buffer, 4% paraformaldehyde, and 0.1% saponin for 10 to 15 minutes at 4°C.
  • HBSS HEPES buffered saline
  • cells may be permeabilized using commercially available reagents, such as FACS TM Permeabilization Solution (Becton Dickinson Immunocytometry Systems cat. no. 340457) .
  • FACS TM Permeabilizing Solution is designed to overcome the limitations of saponin-based permeabilizing reagents; saponin, a compound derived from plants, is a common source of variability in intracellular immunophenotypic staining because of its heterogeneous composition.
  • the cells are labeled with antibodies conjugated to flow cytometrically detectable fluorophores (fluorochromes) .
  • fluorophores flow cytometrically detectable fluorophores
  • the antibodies may be directly conjugated to fluorophores or may be labeled indirectly, using a fluorophore-conjugated secondary antibody that - 26 -
  • Fluorophores that may readily be used in the practice of the present invention include FITC (fluorescein isothiocyanate), PE (phycoerythrin), PerCP (peridinium chlorophyll protein) , APC (allophycocyanin) , PE-CY5 tandem fluorophore (phycoerythrin-cyanine 5 tandem resonance energy transfer fluorophore; CyChrome ® ) and PerCP-CY5.5 tandem fluorophore.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • PerCP peridinium chlorophyll protein
  • APC allophycocyanin
  • PE-CY5 tandem fluorophore phycoerythrin-cyanine 5 tandem resonance energy transfer fluorophore
  • CyChrome ® PerCP-CY5.5 tandem fluorophore
  • Other fluorophores known in the art may also be used, as may molecules such as biotin, for which specific binding cognates (such as streptavidin) may then
  • CyChrome ® and FITC to be a particularly useful combination (anti-pRB ⁇ conjugated to CyChrome ® , and anti-pRB p" mAb conjugated with FITC) .
  • PE may also be used in combination with FITC, and presents certain advantages in quantitation. PE is particularly well-suited to quantitative analysis: it is bright, and it does not self-quench. Fluorophores such as fluorescein isothiocyanate (FITC), CY3 and CY5 can self-quench due to overlap of their excitation and 27 -
  • PE for antigen density quantitation
  • a further advantage of PE as a fluorophore is the commercial availability of pelletized bead standards (QuantiBRITETM, Becton Dickinson Immunocytometry Systems, San Jose, CA) that provide defined levels of PE fluorescence, permitting standard curves to be constructed that then permit the derivation of the number of antibodies bound per cell.
  • antibodies may be used that bind to cyclins, including cyclin A, cyclin Bl, cyclin C, cyclin DI, cyclin D2, cyclin D3, cyclin E, cyclin F, cyclin G, cyclin H, cyclin I, and cyclin K.
  • Antibodies may also be used that have specificity for cyclin dependent kinases, such as Cdkl, Cdk2, Cdk3, Cdk4, Cdk5, Cdk6, Cdk7 and Cdk8, or for cyclin dependent kinase inhibitors, including pl5 INK4b , pl6 INK4a , pl8 INK4 , pl9 INK4c , P 21 WAF1CIP1 , and p27 Kie1 .
  • Such antibodies are commercially available (PharMingen, San Diego, CA;
  • multiparametric analysis permits the concurrent measurement of DNA content. Measurement of DNA content enables the pRB phosphorylation state to be correlated in each cell with the cell's position in the cell cycle; this, in turn, permits direct analysis of pRB phosphorylation status in asynchronous, exponentially growing cell populations.
  • the DNA content of the cell may simultaneously be determined using a fluorescent nucleic acid stain.
  • cells are counterstained with 1 ⁇ g/ml of 4, 6-diamidino-2-phenyl indole (DAPI; Molecular Probes Inc., Eugene, OR) in PBS, or with 5 ⁇ g/ml of propidium iodide (PI; Molecular Probes) in PBS with 0.1% RNase (Sigma) .
  • DAPI 4, 6-diamidino-2-phenyl indole
  • PI propidium iodide
  • nucleic acid stains may also be used, including both cell-permeant nucleic acid stains and, with concomitant use of fixatives that permeabilize the cell, with cell-impermeant nucleic acid stains as well.
  • Hoechst 33258 and Hoechst 33342 which, like DAPI, are blue fluorescent dyes that bind to the minor groove of DNA at AT-rich sequences.
  • Hoechst 33342 more rapidly permeates cells than Hoechst 33258, and is thus commonly used for determining the DNA content of viable cells without detergent treatment or fixation; when cells are fixed and/or permeabilized for flow cytometric detection of other intracellular components (such as pRB and cyclins), Hoechst 33258 also proves useful.
  • the Hoechst dyes can be excited by a mercury-arc lamp, the UV spectral lines of the argon- ion laser, or the 325 nm spectral line of the He-Cd laser.
  • the Hoechst dyes preferentially bind to AT-rich sequences and exhibit higher quantum yields when bound to AT-rich nucleic acids, thus introducing a strong bias into the measurements of nuclear DNA content.
  • data obtained with Hoechst 33342, 33358 and DAPI correlate very well with each other but less well with data obtained with propidium iodide, a red fluorescent, cell-impermeant nucleic acid stain.
  • Ethidium bromide, ethidium homodimer-1 (Molecular Probes, Inc., Eugene, OR, cat. no. E-1169) , and hexidium iodide (Molecular Probes cat. no. H-7593) are phenanthridinium derivatives that intercalate between the bases of DNA in fixed cells, yielding red fluorescent signal. Unlike DAPI and the Hoechst dyes, these dyes bind DNA stoichiometrically with little or no sequence preference. Because these base-intercalators are not specific for DNA, cell-cycle analysis requires treatment of fixed samples with RNase to eliminate fluorescence resulting from dye binding to RNA. - 30 -
  • SYTOX Green nucleic acid stain (cat. no. S-7020, Molecular Probes Inc., Eugene, OR; excitation maximum 504 nm, emission maximum 523 nm) is particularly useful for cell-cycle analysis on RNase- treated fixed cells.
  • SYTOX Green is a high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes, such as those that have been permeabilized and/or fixed.
  • SYTOX green produces lower coefficients of variation than propidium iodide, and unlike DAPI or Hoechst dyes, shows little base selectivity.
  • nucleic acid fluorescent dyes include acridine orange, which emits green fluorescence when bound to double-stranded nucleic acids and red fluorescence when bound to single-stranded nucleic acids. This spectral property has been exploited in methods for simultaneously analyzing the DNA and RNA content of a cell culture. Also useful for measurement of DNA content, and thus determination of cell-cycle status, is 7-aminoactinomycin D (7-AAD, cat. no. A- 1310, Molecular Probes, Inc., Eugene, OR). 7-AAD is a fluorescent intercalator that undergoes a spectral shift upon association with DNA; 7-AAD/DNA complexes can be excited by the argon-ion laser and emit beyond 610 nm. This visible light-excitable nucleic acid stain is suitable for cell-cycle analysis, although it exhibits selectivity for binding to GC regions of DNA.
  • E ⁇ gene, OR E ⁇ gene, OR
  • cells may be grown in media that permits incorporation into DNA of halogenated pyrimidine analogues that may then be detected flow cytometrically.
  • cells may be pulse- labeled with 5-bromodeoxyuridine (5-BrdU), and then subsequently contacted with an anti-5-BrdU antibody.
  • 5-bromodeoxyuridine 5-BrdU
  • Wilson "Analysis of DNA- measurement of cell kinetics by the bromodeoxyuridine/anti-bromodeoxyuridine method," in Flow Cytometry: A Practical Approach pp. 137 - 156, Ormerod (ed.), IRL Press (Oxford, UK) 1994, incorporated herein by reference.
  • Example 1 we demonstrate that two anti-pRB monoclonal antibodies — anti-pRB p" and anti- pRB ⁇ — are able to recognize and to bind their cognate epitopes simultaneously, a prerequisite to the concurrent measurement of multiple functional conformations of the pRB protein.
  • Example 1 further demonstrates that these antibodies may, after conjugation to flow cytometrically distinguishable fluorophores, be used in multiparameter flow cytometric assays to report the phosphorylation status of pRB in individual cells.
  • Example 1 further demonstrates that phosphorylation of pRB within a cell is rapid and complete, since reactivity of individual lymphocytes with anti-pRB p" mAb was lost abruptly, rather than step-wise, during stimulation.
  • the data show that phosphorylation affects nearly all pRB molecules within each individual cell.
  • pRB is phosphorylated by Cdk4 and in some cell types, also by Cdk6.
  • Cdk4 is activated by cyclins D2 and D3.
  • Adding a third antibody to the multiparametric flow cytometry assay described herein permits pRB phosphorylation to be correlated, on a cell-by-cell basis, with the presence of these other proteins.
  • experiments reported in Example 1 demonstrate that the initiation of pRB phosphorylation during lymphocyte stimulation, seen 3 hours after addition of mitogen, coincides with the appearance of cells expressing cyclin D3 (FIG. 2) .
  • the flow cytometric assay may be performed using reagents that permit the concurrent measurement of DNA content, allowing pRB status to be correlated with the cell's position in the cell cycle, even in asynchronous, exponentially growing cell populations, without necessity for culture synchronization.
  • Cell synchronization by inhibitors of DNA replication introduces experimental bias due to induction of a severe imbalance of cyclin expression.
  • the present approach therefore, may be uniquely suitable to assay the status of pRB phosphorylation in individual cells without perturbing their progression through the cycle.
  • Example 1 further demonstrates the utility of the methods of the present invention in elaborating the mechanism of action of antiproliferative agents. It - 34 -
  • staurosporine a nonspecific protein kinase inhibitor, arrests lymphocytes and fibroblasts early in G x , past the point of induction of cyclins D3 and D2 but prior to induction of cyclin E.
  • Fibroblasts and cells of human lung or breast cancer lines that have been arrested in Gl by STP have underphosphorylated pRB.
  • the possible target of STP in all these cells may be Cdk4, but evidence is mixed: other data suggest that the STP- induced arrest in G x may be associated with the induction and accumulation of pl8 and p27 ⁇ p Cdk inhibitors.
  • Example 3 further demonstrates the utility of the present methods for analyzing, and screening for, antiproliferative activity of chemical agents. Because the assay utilizes intact cells rather than cell extracts, the permeability of the studied agents through the plasma membrane may be determined at the same time. Of course, permeability-enhancing adjuncts, such as are known in the art, may be employed to study the effects of otherwise impermeable agents.
  • the methods of the present invention make it possible to estimate the intercellular variability in cell cycle kinetics, and to detect rare cells or cell subpopulations having different properties. This is of particular importance in light of the evidence that heterogeneity of tumor cell populations contributes to therapeutic failure; it would be clinically useful to identify the few cells that survive and are responsible for the relapse. - 35
  • pRB phosphorylation was further analyzed in HL- 60 leukemic cells during proliferation in culture and during induction of differentiation of these cells by various agents .
  • pRB and its state of phosphorylation were flow cytometrically assayed in HL-60 cells during their proliferation and after induction of differentiation.
  • Correlated measurements of pRB p_ , pRB ⁇ , pRB p" :pRB ⁇ , and cellular DNA content revealed that following mitosis (during the exponential phase of cell growth) , a mixture of hypo- and hyperphosphorylated pRB was present within the cell for less than 2 hours, i . e . , early in G x ; no hypophosphorylated pRB was detected throughout remainder of the cycle.
  • the present data indicate that regardless whether differentiation of HL-60 cells was induced along a myelogenous pathway, as in the presence of RA, or along a monocytic pathway, as by vitamin D3 or PMA, the hypophosphorylated pRB predominated over its hyperphosphorylated form within individual cells. This observation is in agreement with the vast amount of data in the literature that indicates that pRB becomes hypophosphorylated during differentiation of cells of various lineages. Likewise, the observed rise in total pRB per cell as reflected by the increased binding of anti-pRB ⁇ is concordant with the published data showing that expression of this protein increases during cell differentiation regardless of the cell system.
  • Example 3 further demonstrates that the methods of the present invention may be used to investigate the mechanism of action of antiproliferative (including antineoplastic) agents, and may be used to screen compounds for such effects.
  • the mechanism of antitumor activity of antiproliferative (including antineoplastic) agents may be used to investigate the mechanism of action of antiproliferative (including antineoplastic) agents, and may be used to screen compounds for such effects.
  • Onconase ® a 12 kDa amphibian protein homologous to pancreatic RNase A, is still poorly understood. The protein shows cytostatic and cytotoxic activity in - 37 -
  • Onconase ® suppresses cell proliferation
  • Human histiocytic lymphoma U937 cells were treated with Onconase ® and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) pl6 INK ⁇ p21 WAE,1/CIP1 and p27 KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRB) .
  • CKIs cyclin-dependent kinase inhibitors
  • Onconase ® -arrested G x cells but was phosphorylated in the cells that were still progressing through S and G 2 /M in the presence of Onconase ® .
  • the cytostatic effect of Onconase ® thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of pl6 INK4A , P21 AFI/ C IPI and p27 ⁇ p ⁇ , the events which may prevent phosphorylation of pRB during G 0 x and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.
  • lymphocytes to HL-60 leukemic cells, to human histiocytic lymphoma U937 cells.
  • flow cytometric methods may also be applied to any adherent cell type that permits of dispersal, before or after fixation, as a unicellular suspension in fluid media.
  • the present reagents and methods may readily be adapted for detection by LSC ® devices. Furthermore, the methods of the present invention are not limited to fluorescence-based detectors.
  • the antibodies of the present invention may equally be conjugated, directly or indirectly, to moieties that generate light-absorptive reporters.
  • the enzymes may be conjugated, directly or indirectly, to enzymes such as alkaline phosphatase that generate dyes, the light absorption of which may be detected using colorimeters, including appropriately-filtered scanning confocal microscopes. By whatever method detected, however, the antibodies must be mutually distinguishable.
  • the methods of the present invention find use both in advance of, and following, therapy.
  • survey of the cell cycle kinetics of a tumor sample acquired by biopsy, excision, or venipuncture, will provide an indication of the percentage of cells that may be targeted by such therapy.
  • a similar assessment may identify cells that have escaped treatment but that retain abnormal proliferative potential. Proliferative states short of neoplasia, such as those found, e . g. , in psoriasis, adenomas, benign prostatic hypertrophy, uterine fibroids, and the like, may similarly be analyzed.
  • the present invention provides - 40 -
  • kits that permit the assay readily to be performed on an as-needed basis.
  • a typical embodiment of such a kit includes two antibodies, discriminably labeled, each of which recognizes a different functional conformation of the pRB protein.
  • one antibody recognizes the hypophosphorylated form of human pRB, and the other recognizes total pRB.
  • the present methods readily permit pRB phosphorylation to be assessed concurrently with cytokine production, CD69 expression, and the like.
  • the pRB p ⁇ mAb appears to bind a conformational epitope in the SV40 large T binding region on pRB. Abrogation of the binding of the antibody may thus usefully permit screening of antibodies or anti-tumor drugs that target those processes that are altered by tumor antigens.
  • the pRB p may, of course, also be used to monitor, direct, and effect purification of the hypophosphorylated form of pRB.
  • antigen-binding domain may further be used as a model for drug screening, evaluation, or design; as a model for an inhibitor of Rb; and as an agent directly useful in gene therapy.
  • Fluorophore abbreviations are as follows: phycoerythrin (PE) , peridinium chlorophyll protein (PerCP) , allophycocyanin (APC) , fluorescein isothiocyanate (FITC), cyanine 5 (CY5) , cyanine 5.5 - 42 -
  • CY5.5 CyChrome ®
  • PerCP/CY5.5 PerCP/CY5.5 tandem fluorophore
  • Human peripheral blood lymphocytes obtained from healthy volunteers by venipuncture, were isolated by density gradient centrifugation. The cells were washed twice with phosphate-buffered saline (PBS) , resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 2 mM L-glutamine at a density of 10 6 cells/ml, treated with 10 ⁇ g/ml of phytohemagglutinin (PHA) and incubated at 37.5°C in the mixture of 95% air and 5% C0 2 for the periods of time shown in the respective figure legends.
  • PHA phytohemagglutinin
  • Staurosporine an alkaloid (MW 466 Dalton) isolated from the culture broth of Streptomyces staurospores is a nonspecific protein kinase inhibitor. This reagent was used in cultures at a final concentration of 20nM, which arrests lymphocytes in Gl - 43
  • the cells were washed with PBS and fixed in suspension in 1% formaldehyde in PBS for 15 min on ice. The cells were then washed with PBS and resuspended in ice-cold 80% ethanol for up to 24 h. After fixation the cells were rinsed twice with PBS and resuspended in 100 ⁇ l of PBS containing 0.25% Triton X-100 (Sigma), 1% bovine serum albumin (BSA; Sigma) and 1.2 ⁇ g/ml of the anti-pRB ⁇ mAb (clone G3-245; PharMingen cat. no.
  • the cells were counterstained with 1 ⁇ g/ml of 4, 6-diamidino-2-phenyl indole (DAPI;
  • the cells were washed twice with PBS, suspended in 1 ml of 0.25% Triton X-100 in PBS, kept on ice for 5 min, centrifuged (300g; 5 min) and the cell pellet resuspended in 100 ⁇ l of PBS containing 0.5 ⁇ g of the anti-cyclin D3 mAb (clone G107-565, mouse IgG x ; PharMingen cat. no. 14781) and 1% BSA and incubated for 2 hours at room temperature.
  • the anti-cyclin D3 mAb clone G107-565, mouse IgG x ; PharMingen cat. no. 14781
  • BSA 1% BSA
  • the cells were then rinsed with PBS containing 1% BSA and incubated with the FITC-conjugated goat anti-mouse IgG antibody (Molecular Probes) diluted 1:30 in PBS containing 1% BSA for 30 min at room temperature in the dark.
  • the cells were rinsed again, resuspended in 5 ⁇ g/ml of PI and 0.1% RNase in PBS and incubated at room temperature for 20 min prior to fluorescence measurements.
  • Isotype-matched IgG (Sigma) was used as a control to define the background fluorescence. Detection of cyclins by flow cytometry was performed essentially as described in Juan et al . , Cell . Prolif. 29:259-266 (1996); Gong et al . , Leukemia 9:893-899 (1995); Darzynkiewicz et al . , Methods Cell Biol . 41:421-435 (1994) .
  • ELITE ESP flow cytometer/cell sorter (Coulter Inc., Miami, FL) using either an argon ion laser (emission at 488 nm) alone or combined with a helium-cadmium laser (emitting UV light) .
  • fluorescence signals were collected using the standard configuration of the flow cytometer (green fluorescence, representing FITC labeled anti-pRB p" mAb; red fluorescence - 45
  • lymphocytes a large fraction of lymphocytes (-80%) did not react with anti-pRB p" mAb and at 24 hours proportions of the reacting and nonreacting cells were approximately similar.
  • negative cells however, varied markedly, decreasing from 98 to nearly 20% between 3 and 4 hours of stimulation, remaining at about 20% level up to 14 h, and then increasing, to reach 45% at 24 h.
  • the decrease in percentage of cells reacting with anti-pRB p" .MAB coincided in time with the appearance of cells expressing cyclin D3 (FIG. 2) .
  • Over 98% of the nonstimulated lymphocytes did not react with anti-cyclin D3 mAb.
  • the cyclin D3-positive cells however, became apparent as early as 3 hours after addition of PHA, and their proportion was on steady rise afterwards, reaching nearly 70% at 12 h, and then declining to 40% at 24.
  • FIG. 3 shows the bivariate distribution of pRB p" vs. DNA content (cell cycle position) in a population of PHA-stimulated lymphocytes, 3 days after addition of the mitogen, i.e., when the cells were actively proliferating, distributed in all phases of the cycle.
  • mitogen i.e., when the cells were actively proliferating, distributed in all phases of the cycle.
  • relatively few pRB p" -positive cells were present in this culture.
  • Nearly all cells reacting with anti-pRB p" mAb had a G 0 /G x DNA content; the cells in S, G 2 M, as well as most cells in G 0 /G x phase were pRB p ⁇ - negative.
  • the preincubation did not lead to the increase in the degree of stainability of either nonstimulated lymphocytes or of the subpopulation of cells from PHA cultures which were already stainable with anti-pRB p" prior to the preincubation (FIGS. 4B and 4C) .
  • the presence of 20 nM of STP during culture with PHA precluded the appearance of the cell population unreactive with anti- pRBP " mAb.
  • the hybridoma clone G99-549 which secretes mouse mAb of IgGl isotype against pRB p" , was derived from a fusion for which underphosphorylated human pRB produced in Sf9 insect cells had been used as an immunogen. From the hybridoma clones positive for pRB in Western blots, only a single clone identified the underphosphorylated forms of plOS 1 ⁇ . The epitope recognized by clone G99-549 was mapped to be between amino acids 514 and 610, which is located within "A - 49 -
  • anti- pRb p ⁇ mAb is a specific marker of underphosphorylated pRb and that anti-pRb ⁇ detects both phosphorylated and - 50 -
  • the present data show that pRB phosphorylation during lymphocyte stimulation occurs as early as 3 hours after addition of PHA., is rapid and total, and affects nearly all pRB molecules within each individual cell.
  • the latter conclusion is drawn from the observation of discontinuous, quantum-like loss of the cells' stainability with the anti-pRb p " mAb, manifested by the absence of cells characterized by intermediate reactivity with this antibody at any time during the stimulation.
  • the observation that preincubation with phosphatase did not additionally increase reactivity of the already pRb p" positive subpopulation of lymphocytes in PHA cultures with the antibody (FIG. 2) gives further support to this conclusion.
  • pRB phosphorylation within cells having different levels of total pRB must be compared.
  • pRB is phosphorylated by Cdk4 and in some cell types also by Cdk6.
  • Cdk4 is activated by cyclins D2 and D3.
  • STP Phosphorylation of pRB in PHA-stimulated lymphocytes was prevented by STP (FIG. 4) . It has previously been shown that STP arrests lymphocytes and fibroblasts early in G x , past the point of induction of - 52 -
  • Multiparameter analysis of pRb p" vs. DNA content (FIG. 3) or pRb p" /pRb ⁇ ratio enables one to analyze the status of pRB phosphorylation in relation to the position of the cell in the cycle in asynchronous, exponentially growing cell populations, obviating their synchronization.
  • Cell synchronization by inhibitors of DNA replication which is necessary in the case of tumor cells (which unlike normal cells cannot be synchronized by withdrawal of growth factors or serum) or in the case suspension cultures which cannot be synchronized by mitotic detachment, introduces experimental bias due to induction of a severe imbalance of cyclin expression.
  • the present approach therefore, may be uniquely suitable to assay status of pRB phosphorylation in individual cells but without perturbing their progression through the cycle.
  • the cells with a G 0 x DNA content having hyperphosphorylated pRB can be classified as G x , while the cells with underphosphorylated pRB, as G 0 .
  • Antitumor drug development is now focused on agents which target Cdk4 or other Cdks to stop their pRB phosphorylating activity.
  • the simple assay of pRB phosphorylation as shown here to demonstrate the effect of STP (FIG. 4), can be conveniently used to rapidly screen activity of such agents. Because the assay utilizes intact cells rather than cell extracts, the permeability of the studied agents through the plasma membrane is screened at the same time. Most important, however, because individual cells are analyzed, it makes it possible to estimate the intercellular variability and detect rare cells or cell subpopulations with different properties.
  • the human promyelocytic leukemic cell line HL- 60 was kindly provided by Dr. Harry A. Crissman of the Los Alamos National Laboratory (Los Alamos, NM) .
  • the cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin,
  • Example 1 Methods were essentially as described in Example 1. Briefly, the cells were fixed in suspension in 1% formaldehyde in PBS for 15 min on ice, then washed with PBS, and resuspended in ice-cold 80% ethanol for up to 24 h. After fixation, the cells were washed twice with PBS and then suspended in 1 ml of 0.25% Triton X-100 in PBS on ice for 5 min.
  • the cell pellet was suspended in 100 ⁇ l PBS containing 1% bovine serum albumin (BSA; Sigma) and 0.5 ⁇ g of the anti-pRB ⁇ mAb (PharMingen, clone G3-245) conjugated with CyChrome ® and/or with 1 ⁇ g of anti-pRB p" mAb (PharMingen, clone G99-549) conjugated with FITC and incubated for 2 h at room temperature. The cells were then rinsed with PBS containing 1% BSA, counterstained with 4, 6-diamidino-2-phenyl indole (DAPI) (Molecular Probes Inc., Eugene, OR), and their fluorescence was measured by flow cytometry.
  • BSA bovine serum albumin
  • Tris buffer (Sigma) at pH 9.4 for 30 min. Other controls consisted fluorochrome-conjugated isotype matched tagged mAb to irrelevant antigen (IgGl; clone MOPC-21, PharMingen) .
  • the cells were gated based on differences in their DNA content to distinguish cells in G x , S, and G 2 /M, and from the mean values of the FITC fluorescence the mean value of the respective control cells (FITC-conjugated isotype-matched IgGl) in the same phase of the cycle was subtracted.
  • the mean values of S phase cells were multiplied by 0.75 and of G 2 /M cells by 0.5 to express the pRB-associated cell fluorescence per unit of DNA.
  • the data were normalized to the mean fluorescence value of G x cells from the untreated, control culture (100), as shown in FIGS. 7- 10.
  • the experiments were repeated, inducing cell differentiation by each of RA, vitamin D 3 , and PMA at least three times, with essentially identical results.
  • Figure 5 presents the bivariate distribution of pRB ⁇ , pRB p" , and pRB p" /pRB ⁇ versus the cellular DNA content of proliferating HL-60 cells. Because the measurement of DNA content reveals the cell cycle position, the bivariate analysis as shown in this figure allowed us to correlate expression of total pRB as well as its phosphorylation state (pRB p J pRB p" /pRB ⁇ ) with the cell cycle phase of individual cells. As is evident, the G x cell population was characterized by great intercellular variability in expression of pRB ⁇ .
  • a threshold in pRB ⁇ during G x was apparent, namely, nearly all cells entering S, as well as the cells in S and G 2 /M, expressed pRB ⁇ above the threshold level (Fig. 5A, arrow) .
  • the absence of cells with the subthreshold pRB ⁇ that would be in S or G 2 /M was a very characteristic and consistent feature of all the pRB ⁇ versus DNA content distributions, whether representing cells from the untreated or differentiating cultures.
  • the mean pRB ⁇ per cell was increasing during S and G 2 in the control cultures: the cells in G 2 /M bound about 30% more pRB ⁇ mAb than the G x cells. However, when pRB ⁇ expression was calculated per unit of DNA, a reduction by about 40% was observed for G 2 /M cells compared to cells in G x (Fig. 6) . Unlike G x cells, the cells in S and G 2 /M were much more uniform in terms of pRB ⁇ expression (Fig. 5) .
  • the pRB p" /pRB ⁇ of the pRB p" -positive cells was above the threshold level of the S phase cells (Fig. 5B, top) , and, as the pattern of cell distribution on the bivariate scattergrams indicates, these cells were not directly entering S.
  • Figures 6-9 illustrate changes in pRB ⁇ and pRB p" in HL-60 cells undergoing differentiation following treatment with RA or vitamin D 3 . Similar changes were also observed in the case of cells induced to differentiation by PMA (not shown) .
  • the treatment led to an increase in the proportion of cells in G 0/X coinciding with a decrease of S and G 2 /M cells (Fig. 9) .
  • the differentiating cells showed, on average, increased pRB ⁇ compared to the cells from the control cultures.
  • the increase was greater for S and G 2 /M cells than for G o i cells (Fig. 6) .
  • the increase was also apparent from the Western blots (Fig. 10) .
  • the S and G 2 /M cells in the RA- or vitamin D 3 -treated cultures had nearly twofold higher pRB ⁇ than their respective counterparts from the untreated cultures.
  • the G 0/x cells from differentiating cultures showed only a 20 (for vitamin D 3 ) or a 60% (for RA) increase (Fig. 6) .
  • the threshold in pRB ⁇ during G 0/X was more pronounced in populations of - 60 -
  • the prominence of the threshold was due to the fact that the G 01 cell population with the subthreshold level of pRB ⁇ was more numerous and more heterogenous, in terms of pRB ⁇ expression, than that of the G x cells from the untreated cultures.
  • the aim of this study was to analyze expression of pRB and reveal the state of its phosphorylation in individual cells during the cell cycle as well as after induction of differentiation. This was accomplished by - 61 -
  • t P _ is length of time when pRb remains underphosphorylated
  • T duration of the cell cycle
  • This rate is determined by the complex machinery regulating activation of Cdk4, which involves its phosphorylation, association with D type cyclins, and interaction with a plethora of Cdk inhibitors.
  • New pRb is being synthesized as well, and it also undergoes phosphorylation at that time.
  • Such a sequence of events provides an explanation for the apparent lack of correlation between the degree of pRb phosphorylation and the total level of this protein, as observed. It also is consistent with the observed heterogeneity of G x cells with respect to degree of pRb phosphorylation.
  • Phosphorylation of pRb during G x is gradual and sequential, initially carried out by cyclin D/Cdk4 and subsequently, affecting other pRb sites, by cyclin E/Cdk2. It is possible that conformation of pRb following phosphorylation by cyclin D/Cdk4 is different than conformation of this protein after phosphorylation of the additional sites by cyclin E/Cdk2.
  • the anti- pRb p" mAb used in the present study detects pRb for only a short period of time (1.8 h) during G x , well before entrance of the cells to S, i.e., prior to the expected activation of cyclin E/Cdk2. In our previous study we observed that this mAb stops to recognize pRb 4 hours - 64 -
  • pRb p" mAb recognizes hypophosphorylated pRb prior to its phosphorylation by cyclin D/Cdk4 and is nonreactive with pRb which is phosphorylated either by cyclin D/Cdk4 or by cyclin D/Cdk4 followed by cyclin E/Cdk2.
  • the present data indicate that regardless whether differentiation of HL-60 cells was induced along a myelogenous pathway, as in the presence of RA, or along a monocytic pathway, as by vitamin D3 or PMA, the hypophosphorylated pRb predominated over its hyperphosphorylated form within individual cells. This observation is in agreement with the vast amount of data in the literature that indicates that pRb becomes hypophosphorylated during differentiation of cells of various lineages. Likewise, the observed rise in total pRb per cell as reflected by the increased binding of anti-pRb ⁇ is concordant with the published data showing that expression of this protein increases during cell differentiation regardless of the cell system.
  • Inhibition of pRb phosphorylation observed during differentiation is attributed to suppression of Cdk4/6 activity through downregulation of D type cyclins, unregulation of Cdk inhibitors, as well as the upstream changes in growth factor receptors or signal transduction, affecting the pRb pathway.
  • the S and G 2 /M cells from differentiating cultures contained a significant proportion of pRb in a hypophosphorylated state.
  • the cells induced to differentiation initiate DNA replication and progress through S having a significant proportion of pRb molecules in a hypophosphorylated state.
  • phosphorylation of only a fraction of pRb molecules may be sufficient to release enough E2F to activate transcription of the genes committing the cell to enter S phase. If this is the case then the overall pRb level within the cell as well as its state of phosphorylation both are critical for cell commitment to enter S phase.
  • the pRb level may be regulated at the stage of its transcription, translation, and/or degradation, while pRb phosphorylation in G x involves activation of Cdk4/6.
  • vitamin D 3 also are characterized by the increased cyclin E to p27 ⁇ ipl ratio, which would indicate that activation of Cdk2 by cyclin E contributes to abrogation of the vitamin D 3 -induced arrest in G x .
  • the vitamin D 3 -induced increase in level of pRb in this case, appears to be compensated by the increased activity of Cdk2. .Analysis of total cellular pRb and its state of phosphorylation at particular phases of the cycle in the cell systems that have cell cycle arrest uncoupled from differentiation may provide additional information about a function of p.Rb in these processes .
  • Example 1 mitogenic stimulation of G 0 lymphocytes results in rapid phosphorylation of pRb, without a change in cellular level of this protein: proliferating lymphocytes during G x have total pRB contents similar to that of their counterparts in G 0 .
  • the cell transition to G 0 which is a transient state, thus, unlike the irreversible cell arrest during terminal differentiation, appears to involve only dephosphorylation of pRb, with no change in its cellular content.
  • Human histiocytic lymphoma U-937 cells were purchased from .American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 ⁇ g/ml streptomycin and 2mM - 68 -
  • the suspension cultures were passaged by re- seeding the cells at a density of 1 x 10 s cells/ml.
  • the cells were studied during their exponential and asynchronous growth, generally within 72 hours of their re-seeding in medium with fresh serum, before reaching density of 8 x 10 s cells/ml.
  • the cultures were periodically tested for Mycoplasma infection by staining of cytocentrifuge preparations with the DNA fluorochrome 4, 6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA) .
  • DAPI DNA fluorochrome 4, 6-diamidino-2-phenylindole
  • Onconase ® provided by Alfacell (Bloomfield, NJ, USA) , was diluted in RPMI to the final concentrations as shown in the Results below and figure legends above.
  • the cells were washed with phosphate-buffered saline (PBS) and fixed in suspension in either ice-cold 80% ethanol or absolute methanol for up to 24 h. After fixation, the cells were washed twice with PBS and then suspended in 1 ml of 0.25% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS on ice for 5 min.
  • PBS phosphate-buffered saline
  • the cells were then centrifuged (300 g, 5 min) , the cell pellet suspended 100 ⁇ l PBS containing 0.5 ⁇ g of the anti-cyclin (or anti-CKI) mAb and 1% bovine serum albumin (BSA; Sigma) , and incubated for 2 h at room temperature.
  • Anti-cyclin D3 (clone G107- 565); anti-cyclin E (clone HE12) ; anti-pl6 INK4A (clone G175-405); and p2 WAF1/CIP1 (clone SX118) and anti-p27 KIP1 (clone G173-524) mAbs, all were obtained from
  • FITC- conjugated goat anti-mouse IgG antibody diluted 1:30 in PBS containing 1% BSA for 30 min at room temperature in the dark.
  • the cells were washed again, resuspended in 5 ⁇ g/ml of propidium iodide (PI; Molecular Probes) and 0.1% RNase A (Sigma) in PBS, and incubated at room temperature for 20 min prior to measurement.
  • PI propidium iodide
  • RNase A Sigma
  • Immunocvtochemical detection of pRB phosphoirylation The status of phosphorylation of pRB in individual cells was monitored using a combination of anti-pRB antibodies, one specifically detecting underphosphorylated pRB (anti-pRB p_ mAb) and another which reacts with total pRB, regardless of its phosphorylation (anti-pRB ⁇ mAb) . Briefly, following incubations in the presence or absence of Onconase ® , the cells were washed with PBS and fixed in suspension in 1% methanol-free formaldehyde in PBS for 15 min on ice. The cells were then washed with PBS and resuspended in ice-cold 80% ethanol for up to 24 h.
  • DNA content was analyzed based on DAPI fluorescence (blue emission) excited by UV light, while the anti-pRB p_ mAb (FITC) and anti-pRB ⁇ (Cy-Chrome ® ) related emission was excited with blue light laser.
  • FITC anti-pRB p_ mAb
  • Cy-Chrome ® anti-pRB ⁇
  • E expression in the untreated U937 cells was similar to that seen in the nontumor cells such as proliferating lymphocytes or fibroblasts, i.e. its maximal expression was on schedule, timely correlated with the cell entrance to S phase.
  • expression of cyclin E was not significantly changed in the cells growing in the presence of Onconase ® (not shown) . - 73 -
  • cyclin D3 or CKIs may be held accountable for the suppression of pRb phosphorylation leading to cell arrest in G 0 ⁇ .
  • the D-type cyclins are activators of Cdk4/6, the protein kinases which specifically target pRb for phosphorylation during G x .
  • Cyclins of the D type are tissue-specific and cyclins D2 and D3 - 76 -
  • p2i WAF1 CIP1 binds stoichiometrically to multiple Cdks blocking activation of the Cdk/cyclin complexes and suppressing their ability to phosphorylate substrates, notably pRb.
  • This inhibitor can be induced either via activation of the tumor suppressor gene p53, or independently of p53, by two growth suppressing factors, transforming growth factor ⁇ (TGF- ⁇ ) and interferon regulatory factor 1 (RF-1) .
  • TGF- ⁇ transforming growth factor ⁇
  • RF-1 interferon regulatory factor 1
  • the interaction between interferons and p2i WAF1CIP also involves signal transducing activators of transcription (STAT) proteins since one of these proteins (STAT1) induces expression of this CKI .
  • p27 KIP1 binds to the Cdk/cyclin complexes and targets multiple Cdks. Also, as p21 WAF1/CIP1 , it mediates TGF- ⁇ activity via affecting pRb phosphorylation and arresting cells in G x and S. It is not surprising, therefore, that p27 KIP1 deficient mice show similarity to retinoblastoma gene-deficient mice, both characterized by hyperplasia of many organs having pituitary dysfunction.
  • the INK family member pl6 INK4A specifically binds to Cdk4 (rather than to the Cdk/cyclin complex) and inhibits activity of Cdk4/cyclin D.
  • pl6 INK4A specifically binds to Cdk4 (rather than to the Cdk/cyclin complex) and inhibits activity of Cdk4/cyclin D.
  • a vast body of literature indicates that overexpression or ectopic expression of pl6 INK4A prevents pRb phosphorylation.
  • Onconase ® by downregulating expression of cyclin D3 as well as enhancing expression of each of the three CKIs, has multiple intermediate pathways to prevent pRb phosphorylation in G x .
  • a common feature of tumor cells is the abrogation of cell cycle checkpoints, in particular the - 77 -
  • Gl checkpoint either by aberrant expression of positive regulators (cyclins and Cdks), loss of negative regulators (CKIs) , or both. All components of the cell cycle regulatory machinery whose expression, as shown in the present study, was modulated by
  • Onconase ® are frequently abnormal or deleted in cancer.
  • MTS1 multiple tumor suppressor-1
  • chromosome 9p21 the so-called multiple tumor suppressor-1
  • the gene encoding p27 KIP1 is located on chromosome 12pl3 and this chromosome fragment is aberrant in several hematological tumors and myeloproliferative disorders.
  • the gene of p2l AF1/CIP1 present on chromosome 6p21 appears to be involved in various tumors by virtue of its downstream of p53 functional role.
  • the antitumor potency of Onconase may thus be related to its ability to substitute for loss of a function of each of the above gene products in terms of prevention of pRb phosphorylation.
  • Onconase ® is a novel drug and its mechanism of action is still an enigma.
  • the ribonuclease activity of Onconase ® appears to be a prerequisite for its cytostatic and cytotoxic properties. This protein is present in amphibian eggs and early embryos where its physiological role is unknown. Since fertilized eggs do divide one may expect that some inhibitor of enzymatic activity of Onconase is present within the eggs. It is difficult to visualize the mechanism by which this ribonuclease may selectively affect the cell cycle regulatory molecules as presently observed, namely downregulate expression of cyclin D3, have no effect on cyclin E, and augment expression of all three - 78 -
  • RNA or ribonuclein complex which has regulatory function may be present in the cell and that this RNA may be a target of Onconase. Its degradation may trigger a specific response in terms of modulation of the cell cycle progression, as presently seen.
  • An interesting possibility is that activity of the double-stranded (ds) RNA-dependent protein kinase (PKR) , the enzyme which phosphorylates I ⁇ B and thereby activates the ubiquitous transcription fact NFKB, may be affected by Onconase via degradation of the dsRNA regulatory of this kinase. .
  • ds double-stranded
  • PTR RNA-dependent protein kinase
  • dsRNA is one of the Onconase targets and its degradation results in inhibition of PKR which may lead to suppression of cell proliferation.
  • Onconase induces cell apoptosis.
  • the apoptotic pathway can also be linked with Onconase via NFKB, since interference with activation of this transcription factor is known to prevent expression of the cell survival factors and enhances sensitivity to apoptosis.

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Abstract

L'invention concerne des méthodes, des réactifs et des trousses permettant de procéder à la détermination, par cytométrie de flux, de l'état de phosphorylation de la protéine du gène de sensibilité au rétinoblastome (pRB) dans des cellules individuelles. Les méthodes selon l'invention permettent de mesurer la forme hypophosphorylée, active du pRB soit comme une quantité absolue soit comme une proportion du pRB cellulaire total. L'invention concerne en outre des méthodes permettant de mettre en corrélation l'état de phosphorylation du pRB avec une phase du cycle cellulaire et avec des composants protéiques du cycle cellulaire. L'invention concerne aussi le criblage de composés chimiques pour en étudier l'activité antiproliférative et antinéoplasique au moyen de dosages par cytométrie de flux. L'invention concerne enfin des trousses de réactifs facilitant les méthodes selon l'invention.
PCT/US1999/004015 1998-02-25 1999-02-24 DETECTION PAR CYTOMETRIE DE FLUX DE CONFORMATIONS DE pRB DANS DES CELLULES INDIVIDUELLES WO1999044067A1 (fr)

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