PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 99/44067
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`GOlN 33/68, 33/53, 33/543
`
`(11) International Publication Number:
`
`Al
`
`(43) International Publication Date:
`
`2 September 1999 (02.09.99)
`
`(21) International Application Number:
`
`PCT/US99/04015
`
`(22) International Filing Date:
`
`24 February 1999 (24.02.99)
`
`(30) Priority Data:
`60/075,908
`
`25 February 1998 (25.02.98)
`
`us
`
`(71) Applicants: NEW YORK MEDICAL COLLEGE [US/US];
`Valhalla, NY 10595 (US). BECTON DICKINSON AND
`COMPANY [US/US]; I Becton Drive, Franklin Lakes, NJ
`07417-1880 (US).
`
`(72) Inventors: DARZYNKIEWICZ, Zbigniew; 37 Meadow Lane,
`Chappaqua, NY 10514 (US). TRAGANOS, Frank; Apart(cid:173)
`ment 17D, 301 East 66th Street, New York, NY 10021 (US).
`JUAN, Gloria; Apartment 2W, 20 Maple Street, Sleepy Hol(cid:173)
`low, NY 10591 (US). GRUENWALD, Stefan; 450 Jolina
`Way, Encinitas, CA 92024 (US).
`
`(74) Agents: RODRICK, Richard, J. et a!.; Becton Dickinson and
`Company, I Becton Drive, Fmnklin Lakes, NJ 07417-1880
`(US).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD,
`GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
`KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF,
`BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN,
`TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: FLOW CYTOMETRIC DETECTION OF CONFORMATIONS OF pRB IN SINGLE CELLS
`
`(57) Abstract
`
`and kits are
`reagents,
`Methods,
`provided
`that permit
`flow
`cytometric
`determination
`of
`the
`phosphorylation
`of
`retinoblastoma
`susceptibility
`status
`gene protein
`(pRB)
`in
`individual cells.
`Methods are described
`that permit
`the
`hypophosphorylated, active, form of pRB to
`be measured either as an absolute quantity
`or as a proportion of total cellular pRB.
`Further described are methods that permit
`pRB phosphorylation status to be correlated
`with cell cycle phase and with protein
`components of the cell cycle. Screening
`of chemical compounds for antiproliferative
`and antineoplastic activity using the flow
`cytometric assays is demonstrated. Reagent
`kits that facilitate the subject methods are
`also provided.
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`--;fl. -·.!l
`
`a;
`()
`CJ) > ::;
`Cii
`E.
`
`pRbT
`
`pRbP.
`
`0
`
`10
`15
`20
`5
`Tune of stimulation {h)
`
`25
`
`30
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`Cl
`CM
`CN
`cu
`cz
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`Ll
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Narn
`Yugoslavia
`Zimbabwe
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`FLOW CYTOMETRIC DETECTION OF CONFORMATIONS OF pRB IN SINGLE CELLS
`
`5
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
`
`10
`
`This work was supported in part by Grant
`CA R01 28704 from the National Cancer Institute,
`National Institutes of Health. The government has
`certain rights in this invention.
`
`FIELD OF THE INVENTION
`
`15
`
`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.
`In
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`particular, 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
`5 using multiparameter flow cytometry.
`
`BACKGROUND OF THE INVENTION
`
`The recent revolution in genomics technology,
`and in particular, the development of high density
`nucleic acid microarrays, has focused extraordinary
`10 attention on the differential expression of genes as
`markers of cellular differentiation, prognosticators of
`disease, and potential targets for interventional
`therapy. Brown et al., Nature Genet. 21(Suppl.) :33-37
`
`(1999); Duggan et al., Nature Genet. 21(Suppl.) :10-14
`
`15
`
`(1999); Cole et al., Nature Genet. 21(Suppl.) :38-41
`
`20
`
`(1999); Debouck et al., Nature Genet. 21(Suppl.) :48-50
`(1999). Although an understanding of the cell's
`transcriptional program is indeed important to all of
`these goals, the function of many critical proteins is
`regulated, at least in part, at the posttranslational
`level, a level to which transcription-based approaches
`are perforce indifferent.
`One such critical protein is that encoded by
`the retinoblastoma susceptibility gene (pRB; pRb),
`25 which plays a pivotal role in the regulation of the
`cell cycle.
`pRB restrains cell cycle progression by
`maintaining a checkpoint in late G1 that controls
`commitment of cells to enter S phase. The critical
`role that pRB plays in cell cycle regulation explains
`its status as archetypal tumor suppressor: loss of pRB
`
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`function results in an inability to maintain control of
`the G1 checkpoint; unchecked progression through the
`cell cycle is, in turn, a hallmark of neoplasia.
`pRB activity is controlled by changes in
`5 phosphorylation.
`pRB is hypophosphorylated in normal
`quiescent cells (in G0 phase) and in cells that are in
`early G1 • With continued progression through G1 ,
`cyclin-dependent kinases (Cdk; Cdk), in association
`with their respective cyclins, phosphorylate pRB at a
`10 number of serine and threonine residues.
`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
`15 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.
`25 Mice homozygously deleted for the RB gene show
`defective differentiation of various tissues.
`Given the critical role that pRB
`phosphorylation plays in controlling progression of
`cells through the cell cycle and in mediating terminal
`30 differentiation, there exists a need for assays that
`permit the ready determination of pRB phosphorylation
`
`20
`
`status.
`
`Typically, the phosphorylation status of pRB
`is assayed in vitro, measuring 32P-labeling and/or
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`electrophoretic mobility of the protein after isolation
`and identification by Western blotting. The procedure
`is cumbersome, and more importantly risks artifactual
`activation of phosphatases that may dephosphorylate the
`5 protein during or after cell lysis. There thus exists
`a need for methods of measuring pRB phosphorylation in
`intact cells.
`Furthermore, the existing methods measure
`phosphorylation of pRB in bulk culture. Several
`10 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
`15 pRB molecules at varying proportions throughout the
`cycle? What proportion of pRB molecules is
`phosphorylated within the cell during G1 , 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?
`Is it the
`ratio of hypo- to hyperphosphorylated pRB or, rather,
`the total level of the latter that is critical for cell
`commitment to enter s phase?
`Study of the average behavior of cells in
`bulk culture also precludes evaluation of heterogeneity
`in the cycling of individual cells in the population.
`Such heterogeneity in cell cycle kinetics in tumor cell
`populations is recognized as a major impediment to
`successful therapy of cancer. There thus exists a need
`for methods that permit the heterogeneity of cell cycle
`kinetics to be assayed, and a particular need for
`methods that would permit cell cycle heterogeneity to
`be assessed in populations of tumor cells.
`
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`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
`5 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
`
`10 need for methods that permit the phosphorylation status
`of pRB to be measured in individual cells without
`exogenous intervention in the cell cycle.
`Methods permitting the phosphorylation status
`of pRB to be measured in individual cells would prove
`15 useful additionally in identifying and characterizing
`antiproliferative agents that act by halting
`progression through the cell cycle.
`Onconase®, initially named protein P30, is a
`basic protein of 12,000 MW isolated from oocytes or
`20 early embryos of Rana pipiens. Onconase® shows
`
`antiproliferative activity in vitro, 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
`
`25 vivo in mice.
`Although 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
`30 understood. The protein is known to have both
`cytostatic and cytotoxic effects, the former
`manifesting as an increase in the proportion of cells
`in G1 phase of the cell cycle; but the mechanism by
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`which the drug effects such cell cycle arrest is
`unknown.
`It would be advantageous to have an assay
`that would permit such a drug's effects on the cell
`cycle, and in particular, its effect, if any, on pRB
`5 phosphorylation, readily to be assayed on a single-cell
`basis.
`
`Reliable measures of pRB phosphorylation
`status in individual cells would permit pRB to serve as
`a marker for distinguishing quiescent from cycling
`10 cells. Although certain cell features, such as
`cellular RNA content, nucleolar mass, chromatin
`structure (degree of chromatin condensation),
`expression of the Ki-67 antigen, or expression other
`proliferation-associated proteins have been proposed as
`15 markers distinguishing cycling from noncycling cells,
`there is as yet no generally accepted, easily
`measurable marker which discriminates G0 from G1 cells.
`There thus exists a need in the art for a marker that
`reliably discriminates quiescent cells from cycling
`20 cells.
`
`Recently, two mAbs recognizing human pRB have
`been described, one of which specifically detects the
`underphosphorylated form of this protein (pRBP-), the
`other of which reacts with total pRB, regardless of
`25 phosphorylation state (pRBT). Dunaief et al., Cell
`79:119-130 (1994); Wang et al., Oncogene 8:279-288
`(1993); Terada et al., J. Immunol. 147:698-704 (1991);
`Zarkowska et al., Oncogene 14:249-266 (1997). These
`antibodies have been used to study several aspects of
`pRB metabolism in bulk culture. There exists a need to
`adapt these antibodies to methods permitting detection
`of pRB phosphorylation states in intact cells on a
`single-cell basis.
`
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`SUMMARY OF THE INVENTION
`
`10
`
`The present invention solves these and other
`problems in the art by presenting methods and reagents
`that permit the concurrent and discriminable detection
`5 of discrete functional conformations of proteins within
`a single cell.
`In particular, the invention provides
`methods and reagents for the flow cytometric
`determination of multiple pRB phosphorylation states in
`individual cells.
`We have demonstrated, for the first time,
`that 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
`25 single cells.
`Using these fluorophore-conjugated antibodies
`with dyes that bind stoichiometrically to DNA, we have
`found that the phosphorylation status of pRB may now be
`correlated with the cell's position in the cell cycle,
`30 without the need for artificial synchronization of the
`cycle. When further used with antibodies specific for
`other protein components of the cell cycle machinery -
`
`15
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`20
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`5
`
`10
`
`such as cyclins, cyclin dependent kinases, and Cdk
`inhibitors -
`the methods permit complex intracellular
`interactions of pRB to be assessed.
`Thus, in a first aspect, 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.
`Where the conformational state of the cell
`may change rapidly, the method may further comprise the
`antecedent step of fixing the cell. Where the protein
`is internal to the cell - either cytoplasmic or
`nuclear -
`the method further comprises the step, before
`flow cytometric detection, of permeabilizing the cell.
`In preferred embodiments, the antibodies are
`labeled with fluorophores and the fluorophores are
`distinguishable by a laser cytometer. Where the laser
`cytometer is a flow 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. Although any fluorophore that permits laser
`30 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.
`
`15
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`20
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`In another aspect, 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
`5 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;
`
`10
`
`20
`
`A method for determining the relative
`1.
`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
`15 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.
`In preferred embodiments of this aspect of
`the invention, each of the pRB conformations is
`correlated with a discrete phosphorylation state of the
`protein.
`In one such embodiment, the first antibody is
`specific for a conformation assumed by the
`25 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.
`
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`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
`5 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,
`10 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.
`In preferred
`embodiments, the second protein may be a cyclin, a
`15 cyclin dependent kinase, or a cyclin dependent kinase
`inhibitor.
`In another aspect, the invention provides
`methods of assaying, or screening, compounds for
`antiproliferative activity, comprising: contacting a
`
`20
`
`sample of proliferating cells with said compound in
`
`vitro; 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
`25 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.
`In a further aspect, the invention provides
`methods of assessing the in vivo antiproliferative
`effect of a compound, comprising: contacting a sample
`
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`5
`
`15
`
`of cells obtained from a subject - after the in vivo
`administration of said compound to said subject - with
`a first antibody, said first antibody specific for a
`conformation assumed by the hypophosphorylated form of
`pRB and a secon~ antibody, said second antibody
`specific for at least one other conformation of pRB and
`flow cytometrically distinguishable from said first
`antibody; and flow cytometrically detecting the binding
`of each of said antibodies concurrently by said cell;
`10 wherein an increased ratio of hypophosphorylated pRB to
`total pRB, as compared to the ratio obtained from cells
`identically assayed that were obtained prior to
`administration of said agent, indicative of in vivo
`antiproliferative effect.
`In yet a further aspect, 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
`20 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
`25 antibodies concurrently by said cell; wherein cells
`with a decreased ratio of hypophosphorylated pRB to
`total pRB are determined to have increased
`proliferative potential.
`In another aspect, the invention provides
`reagent kits that facilitate the practice of the
`subject methods. Thus, in one embodiment, the
`invention provides a kit for detecting the
`phosphorylation status of pRB in individual cells,
`comprising: a first antibody, said first antibody
`
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`specific for a first phosphorylation state of pRB; and
`a second antibody, said second antibody specific for at
`least one other phosphorylation state of pRB; wherein
`said first and second antibodies are flow
`In
`5 cytometrically distinguishable from one another.
`preferred embodiments, the first antibody is specific
`for the hypophosphorylated form of pRB and the second
`antibody is specific for total pRB.
`In embodiments
`particularly suited to flow cytometric analysis, each
`10 of said antibodies is conjugated to a fluorophore, and
`said fluorophores are flow cytometrically
`distinguishable from one another.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`15
`
`The above and other objects and advantages of
`the present invention will be apparent upon
`consideration of the following detailed description
`taken in conjunction with the accompanying drawings, in
`
`which:
`
`FIG. 1 presents frequency distribution
`
`20 histograms representing the intensity of fluorescence
`of stimulated lymphocytes reacting with pRBP- mAb (left
`panels) or pRBT mAbs (right panels) at different times
`(0 to 24 hours) after administration of PHA. The
`dashed lines represent the maximal level of background
`
`25
`
`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
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`experiments, each providing essentially identical
`results;
`
`FIG. 2 shows changes in percentage of
`lymphocytes reacting with anti-pRBP- or anti-pRBT mAb,
`as well as the cells expressing cyclin D3, as a
`function of the duration of incubation with PHA;
`
`5
`
`10
`
`FIG. 3 shows bivariate distribution of DNA
`content vs. reactivity with anti-pRBP- 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
`15 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-pRBP- mAb: panel A, unstimulated lymphocytes;
`
`lymphocytes stimulated with PHA for 24 h;
`20 panel B,
`lymphocytes stimulated for 24 hours but pre-
`panel C,
`incubated with alkaline phosphatase prior to incubation
`with anti-pRBP- 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-pRBP- after treatments with STP (panel D) or
`phosphatase (panel C) . Because the cell frequency
`
`25
`
`coordinate scale varied between the samples
`(automatically adjusted by the software of the flow
`30 cytometer to present major peaks at similar heights)
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`the apparent loss of the subpopulation unreactive with
`anti pRBP- mAb is not seen to be compensated by the
`increase in size of the peak representing the reactive
`population;
`
`5
`
`FIG. 5 presents bivariate distributions of
`
`HL-60 cells from the control culture showing expression
`of pRBT
`(CyChrome®, A panels), the pRB~/pRBT ratio
`(FITC/CyChrome®, B panels), and pRBP-
`(FITC, C panels)
`
`15
`
`versus cellular DNA content (DAPI). The three top
`10 panels show gating analysis of pRBP- positive cells, and
`the bottom three panels show gating analysis of G1 cells
`expressing high (suprathreshold) levels of pRBT.
`Variability of G1 cell population with respect to pRBT
`is evident in panels A (note that the CyChrome® scale
`is exponential). Arrows indicate a threshold
`representing minimal pRBT level of S phase cells; G1
`cells with a subthreshold pRBT level do not enter S
`phase. Panel C, top, shows the gate used to select the
`cells which have hypophosphorylated pRB
`(pRBP- positive
`20 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 pREP(cid:173)
`
`positive cells, color labeled (green), show variable
`levels of pRBT when revealed in panel A and have a pRBP-
`
`25
`
`/pRBT ratio above the threshold value, greater than that
`of S phase cells (B). The G1 cells with high pRBT
`values, above the threshold, gated as shown in panel A,
`bottom (marked in red), show variable pRBP-/pRBT (panel
`B) and pRBP-
`(panel C); some of these cells are pRBP-
`30 positive, others negative (C);
`
`FIG. 6 shows changes in pRBT during
`
`differentiation of HL-60 cells. The bars represent
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`mean pRBT fluorescence of G1 , S, and G2 /M cells from the
`control cultures and from the cultures treated with RA
`or vitamin D3 , calculated as described under Material
`and Methods for Example 2. G1 , S, and G2 /M cells were
`5 distinguished and gated based on differences in their
`DNA content, their mean pRBT fluorescence is expressed
`per unit of DNA and normalized to the mean fluorescence
`of G1 cells from the control culture, which was
`expressed as 100;
`
`10
`
`FIG. 7 shows changes in pRBP- during
`
`differentiation of HL-60 cells. The bars represent
`mean values of pRBP- fluorescence of the control cells
`and the cells from vitamin D3 and RA-treated cultures
`estimated for G1 , S, and G2 /M cells, as described in the
`legend to Fig. 6 and in Example 2, below;
`
`15
`
`FIG. 8 demonstrates changes in pRBP-/pRBT during
`
`differentiation of HL-60 cells. The bars represent
`mean values of pRB~/pRBT ratio estimated for G1 , S, and
`G2 /M cells from control and vitamin D3 and RA-treated
`20 cultures;
`
`FIG. 9 shows bivariate distributions of pRBT and
`pRBP-/pRBT 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
`25 vitamin D3 (panels C and F), obtained as described under
`Material and Methods for Example 2. The mean pRBT
`
`fluorescence of cells from the differentiating cultures
`is increased (note that the pRBT scale is exponential).
`Also increased is their pRBP-/pRBT ratio. The
`arrowheads indicate the threshold levels of pRBT or
`
`30
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`pRBP- /pRBT representing minimal pRBT and maximal pRBP(cid:173)
`/pRBT of early S phase cells, respectively;
`
`FIG. 10 presents a Western blot showing
`
`reactivity of anti -pRBP- mAb (A) and anti -pRBT mAb with
`
`5 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);
`
`10
`
`FIG. 11 presents growth and viability curves
`
`15
`
`(a) as well as cell cycle distribution (b) of U937
`cells maintained in the absence (CTRL) and presence of
`170 nM (2.0 pg/ml) Onconase® (ONC) for up to 72 h. The
`percentage of dead cells was estimated based on the
`trypan blue exclusion test. The percentage of cells in
`the respective phases was estimated flow cytometrically
`based on their DNA content. Apoptotic cells (Ap) were
`recognized as the cells with fractional DNA content
`(sub-G 1 ), as previously described;
`
`20
`
`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,
`25 exponential growth) or in the presence of 170 nM
`
`Onconase® for 48 hours (One) . There were 48%, 38% and
`14% of cells in G1 , S, and G2 /M in Exp culture compared
`with 64%, 24% and 13% cells in G1 , S, and G2 /M,
`respectively, in One culture. The scattergrams
`represent bivariate distributions of the cells with
`
`30
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`5
`
`10
`
`respect to their cyclin D3 vs. DNA content associated
`fluorescence intensities. The upper level of the
`isotype-matched control (mean fluorescence + 3 standard
`deviations) is marked in the Exp panel with a dashed
`line. The bar plots represent the mean FITC (anti(cid:173)
`cyclin D3 mAb) fluorescence values (arbitrary units) of
`the cells in different phases of the cycle (gated based
`on differences in DNA content as shown in the Exp
`panel), after subtraction of the background
`fluorescence, i.e., mean fluorescence of the same cells
`but stained with isotype-matched control IgG antibody.
`Western blots (b) show expression of cyclin D3 in
`control culture and in the culture treated with
`Onconase 48 h;
`
`15
`
`FIG. 13 shows expression of p16 1NK4A 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-p16 1
`4A fluorescence estimated
`for cells in G1 S and G2 /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;
`
`NK
`
`20
`
`FIG. 14 shows expression of p21 WAFl/crPl in U937
`
`cells growing exponentially (Exp) and in the culture
`
`treated with 170 nM Onconase® for 72 hours (One). The
`25 bar plot shows mean anti-p21wAn/crPl mAb fluorescence
`estimated for cells in G1 , S and G2 /M as described in
`the legend to Figure 12. Western blot (B) shows the
`p21 WAFl/CIPl band only in the Onconase®-treated cells;
`
`FIG. 15 shows expression of p27nPl in U937 cells
`
`30 growing exponentially (Exp) and in the culture treated
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`with 170 nM Onconase® for 72 hours (One). The bar plot
`shows mean anti-p27KrP 1-mAb fluorescence estimated for
`cell in G1