DMA FRAGMEfSsT ATlON ASSAY

BACKGROUND OF THE INVENTION

5

Sr normally ^f unctioning Dioiogscal systems cell number ss oy the Ssiance Detween ce s crouferatson and apopiosss An inappropriate balance betweer ce ! o^feratci and apoptoSiS πas Deeπ implicated *n the etiology of many d seases -o instance, ar exacerbation o ^f apoptot c mechanisms ,s thought to contnbute ^í o neurodegenerative I') d.seases sjch as A zhe ner's disease, autoimmune diseases exerrp.ήeα by fviosipie Sclerosis and iscrema-assoaated njunes sυcn as sroke Conversely, a rπt.gatø~ of appropriate asoptctc pathways ss thought to be an jnderly.πg mechanism ct diseases s«cr as cancer

i5 AooDtosis is chasactenzeα by severa, haiimark features ncluαing ceS snnnkage ano cytoplasmic merrorane oiebbing, chromatin condensation, nuclear DNA fragne"tavon a^o p ^r otein degraαation Tnere are at least two d stsnguishab ⁱ e apoptotsc pathways - t ^H e ex^s.c and intrinsic oamways (fo- ^r eview, see Oncogene 23 2881-2874, 2C04, ^nctochem PhotoDio Sa 3 ⁷ 21~729, 2004; The extnnsic or receotor-medfatea pathway s snαtcec Dy

2^ deatn recepjo" ,gards such as tumor recrosis factor Tie death receptor isgands s gna througn the caspase cascade, uk nately resuiting n nuclear DMA d.gestiOn by caspase ac: _; vated nucseases The ntπnsic painway, signaling th"o _^ .gh mstocrondrsai mecran STC, is sensitive to envuonmentai st ^r essors like ultraviolet nght or dr _^ gs Tηese stresses cause pe-mea!ization of the riitochonαriai membrane leading to tne release of cytocnroiie c

25 endonuclease G apopϊos,s nducing factor (AlF) as well as τ?any Qtnsr o-MαenOf ed moiecuies ^" " ^"h e rstative cortπbutton ot the extnnssc o- sntr nsic patnway to apoptos.s is deterπired by ;he oa'ance setween proapoptotsc and cell sun/ val facfo's (tor review see J intern Med 258 47S-517, 2C05)

10 Apoptos s can be >ndαced tπrough death receptor hganα Dsndsng, acttvatio ^r o ^* apoptosis i^αucers, acrsvation of caspases, down regUat.on of cell su^<va! πoiecules, o" tnrough other <nowr aπα unknown rove! mechanisms These a!, leaα to DNA f-agmemat on, th _^ s, a phenotyp.c DNA fragmentation assay wsSS identify ail anoptos s- rκLc.ng corrDounds irrespec ^t ive of rrode o ^f act.oi and potertiafiy identify compounds witn noves mecran STSS A

^5 gel-Daseα DNA sadder assay is the go Q standard assay for DNA fragτentatc ^r However v~e labor-inie^sive and πjiti-step nature of the gel-based DNA Sadoer assay ,s rot amerab e tc

high-throughput screening efforts. Thus, there is a need for a screening assay that would identify agents acting through the classical apoptosis pathways and novel mechanisms as well. Cytotoxic assays could potentially be employed but these assays are non-selective in that they identify compounds involved in both apoptosis- and nonapoptosis-mediated cell death and can lead to significant false positives. A non-radioactive and robust assay that is amenable to high-throughput screening wouid be preferred.

Currently, three different formats have been utilized for screening of compounds involved in apopiosis. The first one is based on a radiometric filtration method, where ceils are grown in 3H-thymidine and then intact DNA is separated from fragmented DNA using a glass-fiber filter plate (Anal. Siochem. 242:187-196, 1996). The throughput of the radiometric assay is limited by the hazard associated with large amounts of radioactivity and the laborious nature of the assay. The second assay format is a TUNEL assay which is based on labeling of 3' double-stranded DNA (dsDNA) with fiuorescent-dUTP by a transferase enzyme and then detection by flow cytometry or imaging methods. There are many TUNEL assay kits available but ail of them are labor intensive and only a few samples can be tested per assay. The third assay format is a sandwich ELISA assay using anti-DNA and ant ^' t-histone antibodies. This assay is aiso iabor intensive and the need for two antibodies makes it relatively costly for high-throughput compound screening.

An efficient and nonradioactive assay format would be to employ a ONA intercaiator such as PicoGreen or propidium iodide to detect fragmented DNA. For example, PicoGreen is a small organic molecuie that intercalates into the major groove of dsDNA. PicoGreen has been a useful tool to study DNA ieveis in blood samples (Scan. J. Immunol. 57:525 - 533, 2003; Clin. Immunol. 106:139 -147, 2003; Blood 102(6):2243 - 2250, 2003). Using DNA intercalators, the present invention provides methods for the detection of agents that modify formation of DNA fragments in cells and is amendable to high throughput screening applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the change in PicoGreen fluorescence in relative fluorescence units (RFU) in HL-80 iysates following treatment of ceils with camptothecin (campto) or DMSO (control). (RFU, relative fluorescence units; DMSO, dimethyl sulfoxide)

^P igure 2 snows the PiCoGreen iuorescence signal (RFU, relative ^uorescence _^ n S) s dependent or tie levei of DNA ,n tne cei ^! iysates follow ng treatTβrt of H-.-6Q ce.ss wt ^r camptothec.n (carroto) (RFU, -eϊative fluorescence units)

5 Figure 3 shows tre effects of seiected compounds such as camotoinecπ (earπpfo) staurospoπn and oteorryon on DNA fragme ⁿ tation in HL-60 cells as detecteσ Dy (RFU, "eiat've fl jorescence units)

Figure 4 shows the effects of camptot^eciπ (campto) on DNA *ragmentat,cn in ht-60 cei s as I ^{1 )} αetecteα by propidiurr ioaide (RFU, relative fiuorescence uπts)

Figure 5 snows 'he effects of camptothe&n (campto) staurosponn ano bleomycin o^ DKA fragmentation >n riL-δC cells as detected Dy ELISA (Abs, absorbance)

15 F.gure 6 snows "ne effect o* an aooptosis inhibitor, ZnCi ₂ , on camptotnec ^!r - nαuced DKA fragmentation aε αefecteo oy PicoGreen (RFl. relative florescence unsts*

Fsgtre 7 shovv'S that RNase ?eatme"t improves the DHA fragrnerta:,on s!g ⁿ a> to Dackgroυ".α ratio r HL-60 ceil iysates as detected by PicoGreen (RFU, relative *iuoresceice un.ts; 0

Figure 8 shows the time course of camptothecin (camoto) effects on DKA fragrreπtation detected by PscoG'βer n riL-60 iysates (RFU, relative fiuorescence unαs, nr, hou' DWSC d.methy ¹ su.foxsde)

5 Figure 9 shows ne e ^f fect of HL-60 cell density on DMA fragme ⁿ tation Detected by PjcoG-ee" \r HL-QQ Sysates ^RFJ, resatsve fiuorescence units, DMSO, d.methyi suifox.αe)

Figure " 0 snows ,he fotd-inαuctfon <n PicoGreen DNA fragmentatior ssgna in relsttc to HL- 60 cesi density 0

Figu-e 11 shows tne effect of DMSO concentrations on HL-60 cells (RFb, ~eiat,ve fiuo'-escerce ^ "its, DMSO, cϋmethyi sulfoxide)

Figure 12 shows the .nαuctran of DNA fragmentation detected αy fo low ^g 5 of HL-δC ce!>s with vainomycin, vinblastine or vincristine

Figure 13 shows the induction of DMA fragmentation detected by PicoGreeπ following incubation of HL-60 cells with etoposide, genistein, purornycin or rapamydn.

Figure 14 shows the data distribution of screening a random chemical library using PicoGreen detection of DNA fragmentation in HL-60 lysates. (RFU, relative fluorescence units)

DETAILED DESCRIPTION OF THE INVENTION

AIi publications cited herein are hereby incorporated by reference, ϋniess defined otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

The terminology used in this specification and the appended claims is for the purpose of describing particular embodiments only and use in the specification is not intended to be limiting of the invention. The singular forms of a word are intended to include the plural forms unless the context clearly indicates otherwise. For example, the singular forms of "a", "an" and "the" are intended to include the plural forms as well. Further, reference to an agent may include a mixture of two or more agents. Thus, the term "an agent" includes a plurality of agents, including mixtures and/or enantiomers thereof, it should aiso be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, elements, components, and/or groups thereof.

Furthermore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (herein "Sambrook et a!., 1989"}: DNA Cloning: A Practical Approach, Volumes i and Il (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J, Gait ed. 1984); Nucleic Acid Hybridization [B. D. Names & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds.

(1984 ^N !], An.πai Celt Culture [R I Freshney, eel (1986)], Irnmobilizeσ Cells Anσ Erzynes ^r JRL Press, (1986)1, B Perba', A Practical Guide To Molecular CsO-mg ("984^, ^ρ ,Vi AuSJDe. et al {βas ) Current Protocols in Mo ecular Biology John Wi ey & So~s, me ("994}

5 Thus, a' ^v embodime ⁿ t of :he invention is a method of identifying an agent that moαi^es ihe *ormatιor o* DNA fragments, the method compr s ^! ng (a) pOvsαing ceiiS in ar array of receptacles, (D) aααsπg an agent to at leas; one receptacle, ^c; ncubafrg tie agent λ'ith the cells ^* or a predetermned penod of time, (c) lysing ihe cei.s, {&) ado.ng a cetectaole compound capao e of intercalating into DNA fragments to sasc at least one receptace, _v f) H) measuring the amount of detectaoie compound intercalated, and (g) oompa ^r rg the amo^n, o ^f intercalated detectaole compound io a control to detem.ne a d.ffererce thereo/ denisfyng saiα agen: as a modifying agent when the di ^f ference exceeαs a psedetemmeα t^resio.α

The assay detects DNA fragments The DNA fragments may Dθ srraL do _^ ,D e- 15 strayed DNA (dsDNA) fragments in the cytoplasmic fraction of ce i iysates and csDKA fragments released ^rom apopiotic cells mto the mecium Further, the DKA f'agmer ts may ^e ssngse-stranded DMA (ssDiMA) fragments in the cytoplasmic f-actioπ of eel ,/sates &M ssDNA fragments -eleased *rom apoptotic ceils into ;he τιedi _^ .m m ar embααsrren: of tne snvemson, the assay ss utilized to measure spontaneous apootosss s^cr as b~t no; ^τ,,teα to 20 apoptosis during co-culture in tne presence or aosence of different eel types o- αsffereπt Cultur ng corαitions THUS DNA fragment formation may be induced Dy removing ar ingred'ent fro τi tie culture meaium such as feta> bovine serum Anot ^h er em&σcs ment of ;ne invention j;.!izes the assay to measure the absence of apoptos.s m anot ^h er etjaod men* the presence or πcease of apootosis or the aosence or decrease o^ apopiosfs can De rreasu^ed 25 during treatment o* ce ^! ls wth an agent In a farther embocirpent of trie nvertion the assay s utilized to measure ceh surv vai or c&\, proliferation

A *urtre' embodiment of tne invention §s a methoα of ιdent,fyιng an ageni that modif.es the formation o ^f DNA fragments, the method composing (a) proviαmg ceLs n a"

30 array of receptacles, (b) aααing to at >east one receptacie a componert selected from tre group cons sting of an induce', ao rhibitor, a modulator, a nodulato" o ^" the .nd _^ cer ana a modulator of the tnn.Ditor, (c) incubating the component with tne ceils fora predeternsred oeπod of t me, (α) add'ng an agent to said at least one -eceptacle, (e) sncjoat rg the ageii with ihe cells lor a predetermined penoα of time, (f) lysing fne ceiis, (g) adα ng a oetecfab e

>5 compound capaole o* intercalating into DNA fragments to said at ieast o^e "eceotsc.e, (h) measυr.ng the aπo jit of detectable compound intercalated and {>) conoanng the arrount o*

sntercasaied detectable compound to a control to determine a difference thereby idenifying said agent as a modifying agent when the difference exceeds a predetermined threshold

Refinements such as adding the inducer, the inhibitor or the modulator with the agent sn a single step are well within the Knowledge and capability of the sailed arcsan and are considered embodiments of the invention

An emoodiment of the invention is a component that modifies tne forma: on of DMA fragments by affecting apoptosss, ceii survival or ce ! proi.feration. The component is selectee from the group consisting of an inαucer an inhibitor, a modulator, a modulator of tne .nducer and a modulator of the inhibitor.

Ceil survival is the aoiϋty of a ceil to stay alive sn favoraDie or unfavorable conditio ⁿ s. Unfavorable corctitions include but are not limited to the presence <f one or more toxsc compounds, nutrient deprivation, or lack of oxygen As a non-hmmng example, some cancer ceils nave increased expression of survival proteins, for exampie Bci2, wnicn make the cete resistant to apoptos.s Otners cancer cells nave developed mechanisms which make tne cells survive better or &e iess prone to apoptosss under conditions of tow oxygen level

Cell proliferation is an increase in cell number Non-limiting examples o* ^~ ceii proliferation are an increase in ceil number due to norma* ceii division, an ϋiduct.on of ceil division or an 'nhibstion of cell death

An embodiment of the invention is a method of identifying an agent that mocifies ire formation of DNA fragments by ceil undergoing apoptosss. However, the snventon is not limited to any particular form of cell death The invention can be appiieα to any mechanism ct cell death where DNA fragmentation is a terminal event.

The term "inhibitor" encompasses any drug, chemrcai, protein or protein fragment capable of blocking, >nterruptιng or preventing a cellular response, activity or pathway involved in apopfosss, cell survival or ceil proliferation. Further, an inhibitor may De, e.g , a molecular chape'one. antibody or inhibitory RNA (RNAi) that blocks expression of cellar protesns inere&y inhioiting pathways directly or indirectly. An "inhibitor" may be tne manipulation of cuituπng conditions such as oxygen augmentation of deprivation or changing media components in such a manner as to blocκ, interrupt or prevert a ceiiUar response.

π

The term "inducer" encompasses any drug, chemical, protein or protein fragment capable of initiating or stimulating a cellular response, activity or pathway involved in apoptosis, eel! survival or cef! proliferation. Further, an inducer may be a molecular cnaperone, antibody or inhibitory RNA (RNAi) that blocks expression of cellular proteins thereby removing inhibition or directly initiating or stimulating a ceiluiar response, activity or pathway. An "inducer" may be the manipulation of culturing conditions such as oxygen augmentation or deprivation or changing media components in such a manner as to block, interrupt or prevent a ceiluiar response.

The term "modulator" encompasses any drug, chemical, protein or protein fragment capable of adjusting the intensity, proportion or the characteristics of a ceiiuiar response, activity or pathway involved in apoptosis, ceil survival or cell proliferation. Further, a modulator may be a moiecuiar cnaperone, antibody or Inhibitory RNA (RNAI) that blocks expression of ceiluiar proteins thereby removing inhibition or inducing a ceiluiar response, activity or pathway. A "modulator" may be the manipulation of culturing conditions such as oxygen augmentation or deprivation or changing media components in such a manner as to block, interrupt or prevent a ceiluiar response.

In a further embodiment of the invention, a modulator may be used in conjunction with an inducer such that a modulator of an inducer makes the inducer more potent (e.g., resulting in an enhanced ceiluiar response) or the inducer iess potent (e.g., resulting in a reduced cellular response). A modulator of an inhibitor makes the inhibitor less potent (e.g., enhanced cellular response) or the inhibitor more potent (e.g., reduced ceiluiar response).

Inhibitors, inducers or modulators can be utilized to mimic pathways or aspects of disease states. As a non-limiting illustrative example, an embodiment of the invention would be to induce apoptosis with fl-amyioid fragments to mimic aspects of Alzheimer's diseasein the presence or absence of potential modulators such as inflammatory cytokines. A further non-limiting illustrative example, an embodiment of the invention is to identify agents that promote apoptosis in one or multiple aspects of cancer. Using such a paradigm, a contemplated embodiment of the invention is to quantify the ability of a test agent to induce or enhance apcpfosis In cancer ceils, tissues or organs. An inducer of apoptosis can be broad- acting encompassing many pathways leading to ceil death. Alternatively, an inducer of apoptosis can be very specific to a single apoptotic pathway or limited to treating a specific disease or pathological condition. Thus, an embodiment of the invention encompasses a screening method for identifying a test agent that may ameliorate a disease state where

apoptosis is thought to be inhibited, for example, cancer. Such cancers include, but are not I limited to, a \ - ^ -->- ^ o , _v ^« c _o, multiple myeloma, non-Hodgkin iympnoma, cnronic lymphocytic leukemia and solid tumors.

Another embodiment of the invention is the use of -v.ore than one inducer, inhibitor or modulator. Using more than one inducer, inhibitor or modulator could, but is not iirrted to, having additive effects, counter effects, synergistic effects or affecting multiple pathways.

An embodiment of the invention utilizes an intercalating detectable compound. A non- limiting example is an intercalating fluorescent aye. lntercalators commonly are heteroaromafic poiycyclic molecules that insert between two base pairs in a DNA duplex. However, the invention is not limited to hβterαarornatic polycyciic molecules. Any intercalating molecule that shows a significant fiuorescent enhancement or shift in emission or excitation ρararneter(s) in the presence of DNA fragments with little or no nonselective binσing to RNA or proteins is contemplated by the present invention. Such intercalating ayes are known to those skilled in the art and include, but are not limited to, the bssbenzlmsde dye Hoecnst 33258. Another useful intercalating detectable compound is propidiυm iodide. An embodiment of tne invention utilizes PicoGreen. PicoGreen belongs to the family of unsymmetric monomethine cyanine dyes, it exhibits high binding constants with DNA and is highly fiuorescent wnen bound to DNA, while virtually non-fluorescent when free in solution. A further embodiment of the invention uses propidiurn iodide. Further, some intercalators are capable of binding to ssDNA such as TOTO (Nucleic Aαds Res. 23 1215-1222, 1995) and OiiGreen (Molecular Prooes, Cat # 07582, Cat. #011492). An embodiment of the invention utilizes cell-permeant DNA probes such as BENA435 (Nudeic Acids Res. 34:} and thus may eliminate the neeά to lyse the ceils in order to iabei the DNA. A further embodiment of the invention utilizes YOPRO, Hoechst 33342, DAPS and DRAQ5.

The intercalating detectable molecule is not limited to a fluorescent dye. The amount of DNA fragment can be quantified using any methodology known to those skilled in the art The amount of intercalating molecules incorporated into DNA can be quantified by laoeis such as, but not limited to, radioisotopes or scintillant-activating compounds. Detection methods include but are not limited to, a peptide tag, enzymatic activity, absorbance, fluorescence, time-resolved fluorescence, poiarizeα: fluorescence, fluorescence resonance energy transfer, luminescence, bioiuminescence resonance energy transfer, radioactive labeling and scintillation proximity or other methods commonly used in the field, in another embodiment, indirect labeling methods may be used including, but not limited to. ussng

J),

labeled antibodies, using streptavidirvbiotin interactions, metai chelating affinity reagents or GST-glutathione affinity reagents. Any direct or indirect labeling method known to those skilled in the art is contemplated as part of this invention. In a further embodiment, the amount of DMA can be quantified by the determination of absorbance at 280nm (A260).

A further embodiment of the invention is to separate chromosomal DNA from DNA fragments before measuring the amount of detectabie compound that has intercalated. Separation of chromosomal DNA from DMA fragments may be performed by methods known in the art. Non-limiting examples include centrif ligation, filtration, sedimentation. electrophoresis, size-exclusion, affinity purification and precipitation. Any method of separation may be employed by one skilled in the art to separate or remove chromosomal DNA from the DNA fragments.

An embodiment of the invention involves iysing the ceils. Cells can be iysed by the addition of a detergent containing iysis buffer. However, the invention is not limited to the use of detergent in the lysis buffer but may include any method that is appropriate for iysing ceils. For example, celis may be iysed by exposure to hypotonic buffer, sonicatioπ or freeze/thaw. Other methods of iysing ceils are weii known to those skilled in the art.

A further embodiment of the invention utilizes DNase free RNase to remove RNA in the cell lysates for the purpose of increasing signal to background ratio by reducing background fluorescent signal due to endogenous cellular RNA.

An embodiment of the invention comprises an array of receptacles that can receive ceils and other materials such as culture media. An array of receptacles can be any number of receptacles from at least one or more than one receptacle suitable for holding ceils within the scope of the invention. Examples include but are not limited to fiasks, culture dishes, tubes such as 1.5 mi tubes, 12 weii plates, 96 weii plates, 384 well plates and miniaturized microtiter plates with perhaps 4000 receptacles (U.S. Patent Application 20050255580). The array of receptacles may be amendable to the addition of a protective covering thus preventing against entry of contaminants or evaporation of contents.

A further characteristic of the receptacles is that the receptacle may allow for analysis, non-limiting examples inciude, specfrophotometric analysis, scintillation counting and fluorescence measurements. However, this is not a limitation to receptacles that can be used within the scope of the invention given that samples can be transferred to a suitable container

aτiendao<e for f jrthe" ana.ysis A ion limst.ng examp e is :o ^" nod'fy the rnet ^r oα SvXh tnai ϊne methoα furtner comprises orovidmg a second array of receptac es wheren iti& step of ,ys>πg the celis furtne" corroπses separating supernatant from eel ¹ denrs and the "ext step fuτhe ^r comprises aαα.ng a detectable compound capabiβ of intercalating .ito DMA fragments to at 5 least one receptacle o* said seco^α aray o ^r receptacles containing a sarrp.e of sa α separated supenatani

An enbcαsment of the invention uses a control A control is a tβ'r ^* of a*t wei understood by skϋleα artisans An appropriate control may De dependent cv fre assay

10 patarrete's utι!s∑ed or :he experimental queston under investigatioi A cortro! πiay be a particular set o* assay conditions or the addition or elimination of a Daracusar corπpoυns to tne culture medium A conbol may be consiαereα a posit.ve con; o s ^f i :hat :he assay conditions or contro compounα aααed brings about the antiαpateα response ^or exampte ^fi' the agent urcer rvestsgatior is expected to nduce apoptosis, a positive co ⁿ tro' would be a

^ compound Known tc nduce apoptosis A ncn-imitng examp.e of a positive control .s tne addition of vjn&iastme sulfate A contro! may also be a negative co ⁿ tro A negat^e confo may be a particular set oτ assay condit ons or tne addition a- on of a paiicu.a- corrpoisnd to tne custαre medium tnat would bπng aooot the arstcpateα "espoose For example, if the agent under .nvestigatson is exoected to mααce apootos ^! s, i^en a negat ve 0 control woulα be expected to not .nαuce apoptosis A control may oe a "veiicie' confo Fc example, ,f the test agent ss αsssoiveα \r DMSO then the vehsce control WOJ c αe DMSC without test agent A contro! may Simply be the use of historical αata

An emDoα n&nt of the .nventioi uses ceil lines that are commerciaJy ava .aoie ^p o- "> exsniple, ceils trax can De used a-e available from the Ameπcar Tissue Cuitu e Conpa^y i one enDodime^ HL-60 ceS s are used Ceils may be prokaryot c o' ejkaryotc ^" ' ^" he nvention ss not iinrteα by tne type o ^f ceils used Pr.mary cultures n~ay asso be utshzed Non- dϊferentsated cehs rray be subjected to various agents to cause i ⁿ e cehs to afferent ate ,nto a particular pherotyoe Fo- examp.e progen.tor ce.is induced to differentiate nto (; o igoαeiαrocyies wouiα oe an emoodiment of t ⁿ e nvention ^he particular ce'l type useα may be selected by -narkers specifically expresseα by the desired cell tyoe, or aiter^atveiy, Dy tne loss of a oart'cjiar marker(s) Ce is can &e seoarateo o- sorted Dy netnocs such as flow cytomet-y that are commonly used by skilled artsans

-H-

An βmboαsmenf of tne snventen uses a homogeneous cei popu.ation An alte^at ve embodiment of .he inventors uses a heterogeneous ce.i population The ceils can be of a"\ type and in any proportion to complete the assay o* the invention

^ Ceiss rray De obtained from a biG'og'cal sample A bsoJogsca ^! sample may ircsuαe, D«ϊ

«s not ijfϊi ieα to, tissue or fluids, sections of tssues such as biopsy and autopsy samples, aro f ^r ozen sections ,akeπ for h stoiogic purposes Such sarrples biood, spjturr, tissue, c j lured ceils e g , pπmary cultures, expiants, and _^ ra reformed cehs, stoo , trπe, etc A biOiogica. sample can be obtained from a eukaryotic orgamsrr, molding ^f rom Tsarma's Such Iv) as a p-imate, e g chimpanzee macaque or human, cow, dog, cat, a 'odeni, e g guinea pα -at, rπθu.se, rabDit, or a bsrc, reotile, or fish

Anotner embodiment of the invention ss to use cells trans.enny or staoty irarsfoirsec io overexoress or not express at least one protein ana deteim ne if such express or> or *acκ

,5 thereof affects DXA fragmentation Expression can De induced o" const t Jt ve Agents car De tested for their ability to modulate DNA fragmentation sn the trarsforned ce is Fυther rest agents can be tested for tnesr ability to πoαuSate DNA *ragmentat'oπ >n transfected eels v t ⁿ e presence or aosence of nαucers or inhiottors of apoptosis Such an βmboosment ma> constitute a control

20

A recombinant expression vector of tne 'nvention compr.ses s HuCiesc adc mo ecλe sr a foπτs su faoiβ for exoressson of the nuciesc acid n a ⁿ ost ceά Thus, a "ecomo'iart expression vector of the present invention can snc u'ce one or more "egusaiory sequences, selected on the basss of the host cei'S to oe used fo ^r expression thai is ope'aoiy n<eα to ire

25 nu&eic acid to 3β exp ^r essed W'thsr a recombinant express.on vectoi , "ope ^r aD,y ϋrkeσ" 8 intended :o cieai that .he nucleotide sequence of interest ss linκed to tie -eg^atory sequencers", in a manner that allows for exoress.on of the nυoeot de seq jence (e g , n a^ T vitro transcr ptϊon/transiation systerr or \r a nost cei ¹ when the vector ts introduced ,nto ifte host cei.) Trse term "regulatory sequence" is snfenαed to ,nciude promoters enhancers anc

30 other expression control elements (e g , polyaαenyla: on signals) Such regu atory sequences a ^r e described, for example r Goeddes, Gene Exp'essior " ^" echnoiogy Methods n Enzymoiogy Vo. 185 Acaαernic Press, San Diego, Cai'f (19&0) Regulatory sequences include those tnat αirect constitut ve exp'ess.on o ^v ' the nuceotide sequence in rran^ tyoes cf host cells (e g t sstβ specific regulatory sequences) it wsli be appre&ateo oy those SKi.iec ~

35 the art that the αesign of t ⁿ e expression vector can depend on such factors as f ^κ e crosce o" nost CBu to be ransformed, the leve of expression cf protein αesred, etc The exp'βss.O"

vectors of the invention can be introduced into πost cells to proαuce profe rs or pept^es encodes oy nucleic aααs as described herein

The term "overexpression" as used herein, refers to tre expressior o* a poSypeotiee, 5 e g , a motecuse that may be involved π apoptosis or cell survival Tecraπ.sns by a ce I a a level that ,s greater than the norma! sevel of expression of the po ypept de n a cei tnat normally expresses Ire poiypeohde or m a eel, that does not normally express the polypeptide Fos exarroie expression of the polypeptide may Dy "0%, 20%, 30%, ^0%, 50%, 60%, 70, 80%, 90°/c 100%, or more as compared to expression of the polypeptide in a w 'ά- 10 type ceii that normally expresses the poiypeptiαe fvLtants, variants, or analogs of ne posypeptiαe of rterest may oe overexpressed

As tseα herein, the tern "transient" expression refers to expression o* exogenous n J&eic acα rnoιecjie(s) wnsch are separate from the chromosomes of the ce.! Trars er: 15 express.on generally reaches its maximum 2-3 days a ^f ter introGucrson of the exoge ⁿ ous nuciesc add ana suoseq jentiy declines

As used nere, tπe terrr "stable" expresssor refers to express.on of exogenous "ude c acic rno!ecυle(s, that are part of tie chromosomes of the cell In genera!, vectoss fcr stable 20 expresssor of genes inαude one or more se.ection markers

CeJ cuitunng techniques for transformed, non-transformed, ormary culture anc bsoiogica.' samples are well Known m the art Bsoiog.cai samples or cultured cei'S can be stored unti' required ^or use The media used for cJtuπng can be specsfscaay αes gned o" 25 αurcnased frorr commercia sources

The present rwentson provides methods for .dertifysng (e Q , screening, αetectsng, characterizing, analyzing and quantifying) agents that modu.ate the ^f ormation o ^f αsDNA or ssDMA tragrrents The ^í erm "agent", "test agent", "test cσrnpounc", "drug candsαa'e" or

3 ¹⁾ "modulato ¹ "" or giammatscai equivalents as used he-em descnoes any molecule eithe" iaturaiiy occurr ng or synthetic, e g , protein, oligopeptide (e g from about 5 to about 25 amino aαds n length, prefe'aoly from about 10 to 20 or 12 to 18 amino acsds ^! - iβngf- preferabiy "2, 15 or 18 amno aαds m length), small organic rrøiecu.e, po ysaccnaπce, >iθid (e g , a spn.ngolsp o), fatty acid, polynucleotide Oligonucleotide, etc , which s emptoyed ¹ P the

35 assays o* the nvent on and assayed for its ab lity to modulate DMA -ragmeitat'or o ^* apootosis There are no particular restrictions as to the comoounα tnat car be assa/eo

- .3-

Exarroies >ncluce s.ngle agents or libraries o* sma! , med,uτ or h gh ^■ no.ecJar weigh, chemical molecules, pursued proteins, expression p-oducts of gene .soranes sy-tπetfc peo.αe iioranes, ce 1 extracts ano culture supematants An agent ercoπpasses ary coπb ration o* dif ereni agents

An agent may include at leas* one or more soiuDle and factors, eel matpx comoonents, conditioned media, cei extracts tissue extracts, explarts ρ*ή τiod>t e ^r s gasses, osmotic pressurs moαsfsers, ionic strength modif.β's, viruses, DNA RNA or gene fragments Ar agent can oe >n the form of a lib ^r ary of test agents s _^ cn as a comb nator ai Q- r&ndorr zee i.orary tnat provides a suffioent range of dsvers.ty o" conversely are limited to similar structures or features Agents can be ootionaliy !snked to a fusion partre ¹ ", e g , target.rg compounds ,escue coTipouπds, dimeπzatson compounds, staoiiizirg compo _^ nos add ^r essaDle comoounds aid other functional moieties Conve^ttoπa.iy, πew chemsca entrues with useíυ properties are generated by identifying a test agert (caiteα a ",eao compound" o" a ",ead") with some desirable property or act v>ty e g .nh biting activity o ^r rroύdiatsng activity The iead compoi_nd is then used as a scaffoiα to cieste vaianis o ^c rhe lead compound and further evaluate tne property ana activity' of those vanant comoouπds

An agent Tsay iπciude treatment conditio ⁿ s ana manipalation o^ externa anc snte ^r na cond.tions or envirorment A non-1, mitmg example of such ar agent incsudes jstrav.oiet lsgnt

An embodiment of the invention is use in nsgh througnput sc ee ⁿ irg (ήTS) methods HTS is tne autø-iated simultaneous testing of thousands of csssf.nct chemtcai corrpounds ir assays dessgnec to model b ologics! mechanisms o' aspeαs of disease patho.ogies More than ore compounα e g , a plura ity of compojnds, can be tested sitiuitaieously, e g ψ one batch In one embodiment, the term HTS screening metnod refers to assays which .esi the abihty o ^f ore compounα or a plurality o* compounds to influence tne ^eaaout o^ choice

ϋqu.d handling systems, analytsca! equipment sucn as fluorescence eaαers or scirtiϋation counters and robot cs for cell culture and sample man puiat»on are wen k^own r the art Mechaπcai systems such as robotic arms o- "cherry-oicking" αev ces are avaaacle to tne skϋ ed artisan Commercial plate readers are available to ana yze corventiora> 96- wea or 3S4-weii plaLes Sing e sample, multiple sample or plate samp e reaαers a ^r e avasabe tnat analyze predetermneα welis and generate raw αata reports Tne raw data can be tra.nsfo ^p Tieα and presented in a variety of ways

AP embodiment of the nvention ,s an assay systerr for centifying an agent that rnodu ates the ^' orration of do«bie-sfanded DNA fragments, t^e assay s/sfeτ coπp'S rg (a) ar array of -eceptades, (b) lysis buffer, (c) a detectab'e compounα capaole o* intercalating into dcuDte-strarded DNA, and (d) at .east one compo ⁿ ent wherein the component ss selected í ^' rom the group consisting of the agent(s), 'rdϋcer(s) of apostos.s snhsb.toφ) or apoptosss, controlfs) and cei'S

A further emoodiment of the nventson ss a kit composing at ieasi ore e>errent of re assay system a^d instructions for use Thus, the components of re assay system may De provided separately or nay be provided together suci as .π a Kit Components of the assav systerr may Dθ prepareα ana included n a KA accorαing to methods rhar rnaxsTize tne stability of the ird!vsα_al componerts Sucn methods are farrlia' to tπose persons SK (sec v fne art For example, cells ot the assay system may oe orov άeά as a s ^soenssor c iyophiiized Aodit ona ¹ compone ⁿ ts of :~e system may also oe "cLαed sucr. as outers, containers for rrsxing re assay components sucn as mscrotife" plates or test tJDes ^" "*"e assay systen can be orovided in the form of a kit that includes ins: uctsons fc performing t' ^λ e assay and nstructions for date nandSing and interpretation

An embodiment of tie srventson is a pharmaceutical compos tion for tne moausatson o" DNA f ^r agment formation comprising a tnerapeuticaily effective amount of an age-i αeπtif ec by tne methods of the invention a^d a pharmaceutically acceotaole caτιe '

The terr "tneiaoeuticaiiy e ^f fective arount" refers to an anoun, of an ager" e ^f fective to feat a disease or disorder ,n a subject or mammal Ir the case o* ca cer the tnerapejficady elective anourt of the σrug may reduce the number of cancer ce! s, reαuce tne tumor Size .rh.bit (J e slow to some extent ana p'βfera&iy stop' carce' ces irf..tration into penpne.a! o ^r gans, .nhsbst (i e , slow to some extent a"d preferao.y stop) tjrsor metastasis, innicϋ, to some extent tumor growth, anα/or rei.eve to some extent one or mo'β of the symptoms associated with the cancer To the exte ⁿ t the d'ug may jDreven: growtn anα/or kill existing cancer ceils «. may be cytostatic ard/or cytotox.c The example to cancels non-i.mitng s nee an agent the modulates the formation c ^f csDNA or ssDNA *ragme ⁿ ts wodα nave app. cations to many varied diseases

A pnaιrπaceufιca ^! composition ^f or the modulation of DNA fragment formatsor may corroπse a tneraDeutical.y effective amount of a i agent wherein tne agent moαύates DNA f ^r agment formation v,a a receptor orote n Thus, the pharmaceutical compos t on may

compπse a test age"t tha; is an agonist, a partial agonist, ar antagonist or an πve^se agon.st Fuαtsβi, the composition may comoπse a test agent trat s a oepi.ce, peptsde f ^r agments thereof, cognates, congeners, m.rnics, analogs, or secreting ceife ana soiϋbiβ moiecuiβs tnereof A further emoodiment of the invention ,s a pharmaceutical 5 composition for the modulation of DNA ^f ragmert formation composing a tne-apeufica >y efective arrount of the identified agent and a pnaTiaceuticaϋy acceptable earner, wrere," tne phamnacett cal composit.on effectively modulates an apoptotic pathway o ^r "ieenan>sm

As used herein the teττi "agonist" re ^f ers to moieties (e g but rot 1 mitβo to sgarcs 10 and agents) Iiai activate tne intracei.uiar response when Dounα :o the receoto ^* , of erπance GTP among to rsembranes

As used herein, tre term "partial agonist" refers to moieties ^e g , Diit not nrr tec to ligaids and agents; τ.ai acf.vate the intracellular response when DOund to trie ' ^■ eceotor to a i5 lesser αegree-'extert than do agonists, or enhance GTP binding to memDranes to a esser deg-ee'extem ;han αo agoissts

As usec nerein, the term "antagonist" refe-s to moieties (e g , but not ivc tec to, iigancis ano agenis) that competstiveiy bind to the receptor at the same srte as αoes an 0 agonist However, ar antagonist does not activate tie rtracePu.ar resoonse ,nst ated b*f tne active ^f orm of tne receotor ana thereby can snhib t tne sntracei υ!ar responses Dy agonssts o" pa ^f tsai agonists In a related aspect, antagonists do not ciminssh the basei.ne .ntracβiLiar response ⁽ n tne absence of an agonist or partial agontst

25 As usec iβresn, the term "inverse agonist" refers to moieties ^e g , oj* ro, ,im>teα tc l.gand ard agent; that osnd to a constitutively active recepto' and nhibit tne base.ene )"itracei ular response The basei.ne response is initiated by tne active ^orm o* tne receotc^ oeiow the norma oase level of activity tnat is observeα n the absence o* agonists or part'a agonists c decrease of GTP bsndirg to membranes

30

As useα nereι ^« - _t> the term "hgand" refers to a rnosβty thai DSPOS to anorer rrosecu e, wherein the moiety '"dudes, but certasniy is not ϋrrnted to a normone or a reurotra ^r smitte- and fuπher refers to iganαs wnere.r the moiety sfereoseiect,veiy binds to a -eceoto-

^ 5 T^e pnarTsaceutical compositions of the present invention can be ^sees ,~ como.natio- wsth other tnerapeut c agents For example, in the treatment of career, the pha"-naceuoca,

coπoositioπ may be given in combiration with cytokines or va-ioυs chemo;he"apβvitsc compounds

A furthei evroodnvert of the invention \s a method of diagnos. ig or monster ^g a treatment c* a disease wherein a biomarker for the disease comprises the formation o* DNA ^f ragrnents, the method comprising (a; p-ovsαmg a biological sample in an aray of receptaoes, {&) adding a αeteαaoie conpojnd capable of intercalating into ONA fragments to at Seast one "eceptacie, (c) measuring the amount of αetectab e coτpoιrc ⁱ ntercaiatec and (o) coπoaπrg the amojni o* intercalated detectable eompo _^ nα to a reference to determine a dif ^f erence inereby diagnosing or monrtonng the treatment of the disease w ^κ e~ tne difference exceeds a predetermined threshold

A Dioma kei ss a term wβii KPOWπ to one sκil!ed .n the a*t A nor-i'm tirg exaπp e s tne use of tne rerm bioτjar><er to encompass any phys'o og.ca! response, ohenotype or characteristic that can be used to qua~titate or quaiitat.veiy snαicate a soecifc state o ^f tne cell, organism aid rnannmai

A DsomatKer is useful for aidπg .π the d agnosss monitoring a^c orecϊcton of α sease o ^r in monstonng the treatment of a Disease when it ss sigrificanfy α ^! fferert oβtween Vr.e sjbsets of oioiogical samples tested Levels of a bιoπarκer a'e

"siguf'cantiy different" when the probability that the pa't.chia' ^" biOTsarker has oeen idertfieo by chance is less fnan a Dredeterrnsned vaiue The metnod of ca!cu.aung Such probaDi's?/ w i αepend on the exact method u: lizeα to compare the leveis berweer tne subsets stcn as t test or similar statisiica! analysis As wsH oe understood by those r the ar _s tne ^reoeier^ nee inreshoid w \ va~y αeoendsng on the number of samples utilized

A bso.ogscat samp e may be organ sampies αeπveα ^rom o-gar.s of nor.-rumaπ animals or humans, όssue samples denved from tissues of non-hjman ansnais o- lαmans as wel! as ce.i samoies, froτi cells of non-human anima.s or humans cr fion cβi cultures For ansτ>as experimentation, Dioiogicas sampies comppse larger organ tissues oDtasneα afte- necroosy or osopsy and body fluids, sacn as biooα For ciinsca use of tne b.omarkers, part cu'ar preferred saπp ^s es comprise bcdy fluids, iκe D»ood sera, p.asma urne, synovial ^τ 1j,α spnai fluid, cerebrospiPa ^! fiMsα, semer o- lymph, as we I as boαy tss _^ es obtained Dy D opsy

A reference is understood by one skilled in the an A reference can include, but is not hmited to, a biological sample from a non-diseased subject wherein ϊhe subject is a ion- human ammai or human. Further, a reference can be a biological sample from a non-treated subject Alternatively, a reference can be from the same suoject before, duπig ano afte ^1* treatment A reference can De from the same subject but can be a different ce^s, bssue cr organ sample tnan ceil, tissue or organ source used to measure the otona-ker A reference does not have to De a biological sample but can be a sample with a known amount of DMA fragments.

The invention is further described sn the following examples which αo not (<mit the scope of the invention described in the claims. While the indention has been descr.bed and exemplified in sufficient deiail for ihose skilled in this art to produce and use it, various alternatives, modifications, and improvements shoulc be apparent without departing from ϊhe spirit and scope of the invention One skilled in the art read Jy appreciates that the presem invention ss wed aαapfed to carry out the objective and obtain the ends aid advantages mentioned, as well as those inherent there<n. The examples that foi'ow are descriptions of embodiments and are not intended as limitations on the scope of the invention Modificat.ons therein and other uses wii: occur to tnose skilled in the art These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims. Varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the indention.

The invention illustratively described herein may be practiced in the absence o ^f any element or elements, limitation or limitations, which are not speαficasiy discosed herein The terms and expressions which have been employed are used as terns of description ana no: of limitation, and there is no .ntention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it >s recognized that various modifications are possible within the scope of the irvemon claimed Thus, if should be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed may be made by those skilled in the art, and that such rnodif.catiors and variations are considered to be within the scope of this invention as defmec by the appenoed claims.

Example 1

Detection of DNA Fragments by PicoGreen:

General assay conditions and considerations are described. However, for subsequent examples provided beiow, assay conditions were modified to test variables and to accommodate the experimental purpose and do not necessarily limit the invention to specific embodiments.

HL-βQ is a human AML eel! line commercially available from ATCC (ATCC Cat # CCL- 240 ™). Complete cell culture medium was prepared as follows: 100 mi heat-inactivated fetal bovine serum, 20 mL 1-M HEPES (pH 7.5), 10 m!_ Penicillin/Streptomycin stock solution (see Table 1 ) was added to a 1 -liter RPMi-1640 medium. After mixing thoroughly, the complete medium is filtered through a 0.22-μrn sterilized filtration apparatus (Naigene). incubation medium was prepared as follows: 5 ml Penicillin/Streptomycin stock solution and 25-mi heat- inactivated fetal bovine serum were mixed with 500-mi RPMi-1640 (w/o phenol-red and L- glufamine).

The genera; experimental procedure was performed with the following protocol. Cells were cultured four to five days before compound treatment. Ceil viability should be approximately greater than 92 %. Cell density was counted and viability confirmed using the GUAVA PCA with Via count 2.12 program. Cells were aliquoted and centrifuged at 300χg for 6 minutes. The supernatant was discarded and the cell peiiet was resuspended to 0.15 million ceiis/mL with RPMl (phenol Red-Free) with 5% FBS and 1% Penicillin/Streptomycin. An aliquot of 40 uL of ceil suspension was dispensed to each well of a 384-weil. Cells were incubated for the appropriate time under particular experimental conditions. Then, 45 uL of ceil lysis buffer added to the ceii samples.

Lysis buffer was prepared as follows: to make 1 -liter of lysis buffer, 20 mL of 1-M Tris- HCi (pH 8.0) solution, 40 mL of 0.5-M EDTA (pH 8.0), 10 mL of 20 % Tween-20 solution, 10 mL of 20 % Triton X-100 solution are mixed with 920 mL deionized water. Just before use, 5ml of an RNase A stock solution (10 mg/mL) was added into the lysis buffer to a final concentration of 0.05 mg/mL. After the addition of lysis buffer, the plates are allowed to stand at room temperature for 60 minutes. The ceil culture plates were centrifuged at 2000 x g for 20 min and 10 uL of the supernatant of the cell iysates containing the DMA fragments was transferred with a CyBio-weii 384 info the detection plates (Corning Costar 384-weϊl Polystyrene assay plate, black, non-binding surface). An aliquot of 10 uL of PicoGreen

K)

detection solution was added to each sample we!! in the detection plates. The PicoGreen detection solution is made fresh before use by diluting the DMSO stock solution 1 :200 into the detection buffer. The detection buffer is prepared by mixing 50 ml 10χ Tris-HCL buffered saline (TBS, pH 8.0) and 2 mi_ 0.5-M EDTA (pH 8.0) stock solutions with delonized water to final volume of 500 mL. Fluorescence intensity was analyzed with PerkinEimer Envision.

Table 1 lists non-limiting exemplary reagents anci materials, concentrations, (unctions and supplier source.

Table 2 lists non-iimiting examples of equipment, how such equipment can be used arsα a supplier.

TABLE 2:

Measurement parameters on PerkinElmer Envision were as follows: for excitation, tne 5 mirror is FiTC; excitation filter is FlTC 485; emission filter is FlTC 535; number of flashes equals 25; excitation light is 1%: detection gain is equai to 1 and measurement height is 8 mm.

Each assay contains positive, negative and biank controis. The appropriate controls 10 used were determined by the experimental purposes to be achieved. Typically, the positive control was HL-60 cells treated with 5 uM vinblastine sulfate in 1 % DMSO. The negative control was HL-80 cells treated with 1% DMSO. The signal blank was incubation medium with 1% DMSO (no HL-60 ceϋs).

Data analysis can be adjusted to the experimental parameters or the paradigm under investigation. Typically, the relative amount of fragmented DNA formed was represented by the fluorescence intensity (Fl) of a sample. The effect of an agent treatment on DMA fragmentation in HL-60 cells was calculated based on the change in fluorescence intensity relative to the DMSO controi samoies. Percent effect was determined as:

20

% Effect = 100" [{FUgβn _t - mean(Fi _{Ilegatlve contro} ,))/ mean(Fi _; ve contro .l ^; )]

-?s

Example 2

PicoGreen Specifically Detects DNA Fragments Released in HL-60 Ceils:

Figcre 1 snows PscoGreen iuorescence intensity increased n HL-60 treated wit" camptotheciD -iL-δO ceϋs ,n mid-'og ohase (O 4 rwi.oπ ceι ^! s/m>_) were t-βatecs wstn e,rer G " % DMSO earner solvent or 3 2 uM camptothean for 5 5 hours An eq _^ as volume o* tie .ysts Duf ^f er i20 rhf, Tπs-HC! (ph 8 O) ₁ 20 rnfvl EDTA, 0 2 % 7ween-20i was aαdec into tie tcta cell culture After standing at room temperature for 45 mirutes, xhe cei iysafe was sub _^ ecfeo to centπfugaiior at 2000 x g for 20 mm and the top portion o ^f the supernatart was w.tncravvn arc DMA content was qua ⁿ titated using florescence irte^s ty readout oy msxsrg win PicoGreen dye Medium Diank ss the equal mixture of cen custure anc lysis fcuffe" ¹

\0

Example 3

PicoGreen F.uo ^{^} escence S.gna ts Dependent on the Level of DMA n the Ce.i ι_ysates

15

After treatment witn either 0 1 % DlvϊSO earner solvent or 8 2 LM camptotheαr fc 5 5 hours, an equal volume of the lysss buffer without EDTA (20 mM Tπs-hC ^s \ph 8 O^ 0 2 % Tween-20 and 3 jg/mL RNase) was aoded into tr>e HL-60 total cei, cufLres After εtand ng at room temperatu'e *o ^r 45 minutes, :he cei ^! iysates were centπfugec a^c tne top portion of trs

20 supernatant was w thdrawn and sncϋoated with RNase-free DNase at 3? ⁰ C ^f or tne .ndscateα tiTie (Figure ?) After 2 hours of treatment, DNase ! was aote to reduce the iuo-esceπce ϊnterssty of DMSO contros saπp ^! e oy about 50% The signal fo" carnototneci" treateα ce,!s had nigher fluo ^r escence .niensity before DNase ! treatment ana was reduceo effect've y to a .eves sim lar to inat of DMSO control with DNase I treatment These ^r esu!ts mα cate tηat t^e ^ PicoGreen fluorescent signal is due to the presence o* frag'renrec DNA ,n the cei iysates

Example 4 30

Compar.son of PscoG^een to Propsd urn iodide or ELISA for Detect rg αsDKA

HL-60 ce is in mid-log phase (0 3 msStor cells/m,} were treated wfth d,fte ^r e"t αoses cf camptotnecn, stauOspoπne or bleomycin for 20 hours An equa, volume of ;-e ιys ^r s oufer 35 (20 mM Tαs-hG (ph 8 0), 20 mM EDTA, 0 2 % Tween-20 and 5 jg,m._ RNase; was addee sπto tne iotas cell culture The cell lysate was centπf jged at 2000 x g for 20 m n ard top

portion of the supernatant was withdrawn and DNA content was quantltated using either an ELiSA kit (Roche Appiied Sciences) or fluorescence intensify readout using PicoGreen or propidiυm iodide. The dose response curve of camptotheciπ using PicoGreen detection (Figure 3) was compared to propidiυm iodide detection (Figure 4). Propidium iodide was 5 diluted from a 0.5 mg/mi stock to 0.00125 mg/mL working solution in 10 mM Tris-HCL (pH 7.5) with 1 mlVI EDTA. 20 uL of the propidium iodide working solution was mixed with 20 ul of sample solution before measurement of fluorescence intensity on PerkinEimer Envision with excitation wavelength: 531 nm; emission wavelength 835 nm.

10 The dose response curves for camptoihecin, staurosporine or bleomycin detected with PicoGreen are shown in Figure 3. The EC5Q value for camptothecin was 1.48 ufvi, staurosporine was 0.41 uM and bleomycin was greater than 100 uM. The EC50 values as determined by PicoGreen were in good agreement with the ELiSA detection kit (Figure 5). The EC50 values determined by ELiSA were 1.11 uM for camptothecin, 0.19 uM for

15 staurosporine and greater than 10OuM for bleomycin.

Example 5

20 Effect of ZnCl ₂ , an Apoptosis inhibitor, on Camptothβcin-induceσ DNA Fragmentation in HL- 60 Cells:

Zinc has been known to inhibit apoptosis induced by both chemical and death- receptor agonists. To further demonstrate the feasibility of applying the PicoGreen assay to

25 detect and quantify dsDNA as a measure of DMA fragmentation in apoptosis, HL-60 celis in mid-log phase (0.3 million ceiis/mL) were treated with 3.2 μM camptothecin in the presence of different doses of zinc chloride for 20 hours (Figure 6). DNA fragments released from celis were quantified with PicoGreen reagent as described above. For sampies with camptothecin, without zinc chloride or at low concentrations, fluorescence signal was more than 3 fold of

30 that from sampies with DMSO treatment. When zinc chloride concentrations were increased to 100 uM or higher, the magnitude of DNA fragmentation, which was reflected by the level fluorescent signal, decreased to the same level as sampies with DMSO treatment only.

-? <; Example δ

Effects of RNase T eatmert on DNA Fragmentation Signal to Background Rat o r ^_-6C Lysates

SL-60 cei s sn msd-log phase (0 4 million ceils/rnL; were treateα w.th either 0 1 % 5 DMSO earner sosveni or 3 2 uM camptoinecm for 5 5 hojrs An equa voL πe of ire _; ysis bufre>- (20 TiM Tns-HCi (pH 8 0), 20 nriM EDTA 0 2 % Tweeπ-20) w in afferent corcentratons of CNase-free RNase (Roche Applied Sciences ' 579681 ) were aααec <o tr.e total ceil culture After standing at room temperature for 45 minutes, the ce ^l! iysaie wss sυbjecteα to centrifuge at 2000 x g for 20 rr.sn The top Dortscr o ^f the suoernaant was 10 withdrawr ana DMA content was quantstated oy mtxipg witn PicoGreer aye ^reament of tie ce!i lysate Vλ:I rngh conce ⁿ trations of RNase decreaseα DacKgrouπd fluoresce ⁿ ce αue to celLlsr RNA ana mproved tne signal window (Figure 7}

15 Exampse ⁷

Time Course of Camptoihecm Effects on DNA Fragmentation n HL-δO Ceils

HL-60 ce Is .n mid-log pnase (0 4 m'iSion cesis/mL) were t'eateα \.vst ^λ either C 1 % 2(J DMSO Ca-TIe ^1- solvent or 3 2 JM camptothecn (Figure 8} At each are po>ri inαica.'eα ^x 0C ul of tne celi suspension was withdrawn to mix with equai volume c* the sys.s buffer (20 mM Tns-HC! (pH 8 05, 20 mM EDTA, 0 2 % Tween-20) Camptctnecn s a fast act ~g apootosts- induαng ager; Ai 4 hours of treatment, camptotheαn already causeα a Sign.f.cant increase in DNA fragmentation 25

Examp'e 8

Effects o^ Ceil Density on DNA Fragmentation in hL-80 Cells

M) hL-60 cei s »π ceil Culture medum were spun down at 300 ».g for β n ^ After d.scarding the med'um eel s were re-suspended into compound snejoation neot jm to sncicated concentration The cell suspens ons were tr.en incubateα w fh 1 '"0 vo,uτ>e of e tne ^* 0 1 % DMSO earner solvent or 3 2 tM camptothecir for 20 hours oefore lysis ana detectso"

^5 oroceaure Meo.um blank is tne equas mixture o ^f eel culLre rrecium anc >ys,s buffer Figure

9 slows the effect of ceil density on ϊne signal window and F gu e 10 graph ca! y represents tne fdd-snducton in signal -eiative to ceil density

S Exarrpie 9

DMSO Toierarce i HL-δC Cells

HL-60 ceiis sr, mid-log phase (O 3 mill on ceiis/mL) we'e treateα with afferent αoses of 0 DMSO fc 20 nours An ecual voiume of ire iysis buffer (20 rrM Tπs-^Ci (pβ 8 0} 2C n.V EDTA, 0 2 % Tween-20 and 5 ug/mL RKase) was adαed srtσ t~e totat ceπ culture The ce ' iysate was cenir.fugeα at 2000 x g for 20 mm The top portion of the Supernatant was withdrawn arc DIsA content was quan: fated using the PscoGreen fiuorescer i assa> FsgLre 11 s^ows rial JO to 1% DMSO could be tolerates Dy hL-60 cei.s for 20 nr ncubatson 5

Example 10

Dose Response Curves of a Pane ¹ of Cytotoxic Agents with Differed Mechanisms o ^* Act o^ 0

H,_~8C eel s in mid-iog pnase (C 3 million ceiis/mL) were treateα with d.ffetert αoses o* knowr apootosss inducing compojnαs for 20 hours Figure 12 shows the c>iotox c actsv Iy c* vaiinornyon, vinDiasαne and Figure 13 snows tne cytotox c act vity o ^* eiopos oe gen ste>n, aiα rapamyαn N

Examote 11

Data DisfnDjtior of Screening a Ranαom Chemical Library (>

The co TipOLnd .lorary was dispensed to a 384 well into weiis frorr coiumn ' io 22 Tne posiLve ccn.roi, vinbiastin (5 uM) was added to weils in column 24 We is r co αmπ 23 were αsβd for the negative contro. (without compound) HL-60 cei.s were aLq _^ cteo to each well ana were jncuoaied fo- 40 hours (Figure 14} DNA fragmentation was measured US^Q ^? the p-ocedure deserved <n Example 1