8iochimica et Biophysica Acta, 1088(1991) 285-291 © 1991 ElsevierSciencePublishersB.V.0167-4781/91/$03.50 ADONIS 016747819100088P

285

BBAEXP92214

Evidence that a protein kinase enhances amsacrine mediated formation of topoisomerase II-DNA complexes in murine mastocytoma cell nuclei Sandra J. Darkin-Rattray

*

and Raymond K. Ralph

Department of Cellular and Molecular Biology, University of Auckland Auckland (New Zealand)

(Received5 May 1990) (Revisedmanuscriptreceived9 October 1990) Key words: Amsacrine;TopoisomeraseII-DNAcomplexformationenhancingfactor; Caseinkinase11:Nucleus:(Murine mastocytomacell)

Cytoplasmic extracts of K21 murine mastoc~ytoma cells contain a protein factor, distinct from topoisomerases I and 11, that facilitates formation of amsacrine-induced topoisomerase ii-DNA complexes 0PDC) in isolated 1(21 cell nuclei (Darkin, S.J. and Ralph, R.K. (1988) Biochim. Biophys. Acta 1007, 295-300). The PDC enhancing activity was shown to reside in a protein kinase with specificit) for a casein kinase !! substrate and sensitive to heparin and anti-casein kinase I! antiserum. This appears to be the first direct evidence of a protein factor that modulates ~ c r i n e - i n d u c e d topoisomerase ii action. Introduction DNA topoisomerase II is the nuclear target of several anticancer drugs, including amsacrine [2]. The drugs interfere with DNA breakage-rejoining by stabilizing a cleavable complex between DNA and topoisomerase II [3,4]. The amount of drug-induced complex serves as a measure for drug activity [5] and the 'frozen" topoisomerase II-DNA complex is thought to be a key factor in drug-mediated cytotoxicity. However, the mechanism by which amsacrine-induced topoisomerase II-DNA complexes translate into a lethal event is not understood although stabilization of the cleavable complex appears to be an important step in a chain of events involving additional factors which eventually causes cell death [5]. Accordingly, inhibition of protein synthesis with cycloheximide protects Balb/c 3T3, CCRF-CEM, * Present address: Department of Pharmacologyand Therapeutics. CoBege of Medicine,JHMHC, Universityof Florida, Gainesville. FL, U.S.A. Abbreviations:SDS, sodium dodecyl sulphate: PDC, topoisomerase ll-DNAcomplex;Mops,4-morpholinepropanesulphonicacid; PMSF, phenyimethylsulfonylfluoride; DFP, diisopropylfluorophosphate; mAMSA, amsacrine (4'-(9-acridinylamino)methanesulfon-m-anisidide; BSA,bovineserum albumin:CKIL caseinkinase II. Correspondence: R.K. Ralph, Departmentof Cellularand Molecular Biology, Universityof Auckland,Private Bag. Auckland,New Zealand.

L1210 and K21 mouse mastocytoma cells against the cytotoxic action of anticancer drugs such as etoposide or amsacrine without decreasing topoisomerase II activity [6,7]. Furthermore, we previously demonstrated and partially characterized a factor that enhanced the formation of amsacrine-induced topoisomerase II-DNA complexes in isolated nuclei of K21 murine mastocytoma cel~s [1] This factor appeared to be a labile protein, distinct from topoisomerases I and II, that was rapidly lost from cells grown with protein and RNA synthesis inhibitors. Because the resistance of some cancer cells, e.g., non-cycling tumour cells, to drugs such as amsacrine could be related to the absence or availability of the protein factor that enhances cleavable complex formation or stabilizes preformed~ complexes, the nature of the protein from K21 cells that increases amsacrine-induced protein-DNA complex formation was further investigated. We now report that the enhancing activity resides in a 70 kDa protein kinase, with properties reminiscent of casein kinase II. Materials and Methods Materials

Digitonin, Mops and dextran grade B 150-200 were from BDH. High and low molecular weight markers, EGTA and proteinase inhibitors aprotinin, leupeptin, a2-macroglobulin, PMSF and DFP were from Sigma.

286 DEAE-cellulose (Cellex-D) and Aquacide were from Bio-Rad and Calbiochem, respectively. Bisacrylamide and acrylamide were from Scrva. RPMI 1640 and horse serum were from Gibco, New Zealand. [methyl3H]Thymidine (70-90 Ci/mmol) was from Amersham. [y-32p]Adenosine 5'-triphosphate was prepared as previously descEbed [8]. Amsacrine, provided as the isethionate salt by Dr. B.C. Baguley, University of Auckland Medical School, was stored as a 1 mM stock solution in water at - 2 0 °C from which working dilutions were made immediately prior to use.

Cell culture A clonal subline (K21, wild type) ~,i P815-X2 mouse mastocytoma cells was kindly provided by Dr. R. Schindler, University of Berne, Switzerland. The cells were grown in RPMI 1640 medium, supplemented with 10% horse serurra in a 5% C O j 9 5 % air atmosphere and subcultu:ed as recommended [9].

Preparation of cytoplasmic extracts Cytoplasmic extracts were prepared by digitonin extraction of log-phase K21 ceils as previously described [1] and stored in 100 #1 aliquots at - 9 0 ° C .

Quantitation of amsacrine-stimulated covalent proteinDN,J complex formation Drug-induced factor-enhanced formation of proteinDNA complexes (PDC) in isolated nuclei was measured by S D S / K + precipitation as previously described [1]. Radioactive labelling of the DNA of proliferating K21 cells, conditions for cell lysis and preparation oi nuclei were as described [1]. Essentially, to induce drug-stimulated factor-enhanced PDC formation, 105 nuclei were added to the following ieaction mixture in 96-well plates in a total volume of 50 #l/well: 50 mM Tris-HC! (pH 7.5); 10 mM MgCI:; 120 mM KCI; 5 mM EGTA; 2.25 mM EDTA; 0.5 mM dithiothreitol; 30 # g / m l fraction V bovine serum albumin; 2 mM ATP; with or without 10 #M amsacrine; 0-100 #g cytoplasmic extract or column fractions, followed by incubation for 10 rain at 37°C. The nuclei were then collected by centrifugation, protein-DNA complexes precipitated with SDS and K + and the samples processed as described [1]. The total acid-precipitable radioactivity per assay (i.e., 105 nuclei) was routinely approx. ~ • 104 c p m It was necessary to determine optimal amounts of each cytoplasmic extract (#g protein), therefore, a concentration range (O-100 #g protein) was tested for the ability to enhance amsacrine-stimulated PDC formation and the amount of cytoplasmic extract (#g protein) that produced maximal protein-DNA complex formation was defined as 1 unit. In each experiment, the stimulation of PDC formation in whole cells by 10 ttM amsacrine was determined

as an internal control [1]. A 10-12-fold stimulation of PDC in whole cells was achieved with 10 #M amsacrine. The data presented are means of triplicate determinations from two or three separate experiments. Standard errors were less than 5%. In the absence of drug a maximum of 1% of total DNA was precipitated.

Determination of protein concentration Protein concentrations were determined according to Bradford [10].

Ammonium sulphate precipitation of cytoplasmic extracts Four broad ammemam sulphate fractions (0-20%, 20-60%, 60-80%, > 80% sl,.pernatant) were recovered at 0 ° C according to Scopes [11]. The fractions were centrifuged at 10000 × g for 30 rain at 4 ° C and the precipitates were redissolved in 1-2 pellet voi. of 'isolation buffer' (20 mM Tris-HCI (pH 7.5), 1% (v/v) aprotinin, 10 mM Na2S205, 0.1 m g / m l DFP, 1 mM PMSF). To remove (NH4)2SO 4, the precipitates and the final supernatant were dialysed against 2000 vol. of the above solution for 6 h at 4°C. After dialysis, the protein concentration in each sample was determined and an aliquot of each fraction was assayed for PDC enhancing activity. The remainder of the fractions were stored at - 90 ° C.

Purification of the PDC enhancing factor by DEAE-ceilulose anion.exchange column chromatography DEAE-ceUulose (Cellex-D, Bio-Rad) columns (1 × 2.5 cm) were prepared in plastic disposable syringes Columns were equilibrated with isolation buffer before samples (200 #!) of the dialys~,~ 20-60% (NH4)2SO 4 cytoplasmic extract fraction, containing approx. 2-3 mg protein, were loaded. The cohinms were eluted with a linear 0.01-0.2 M KCI gradient (50 ml) in isolated buffer followed by a 0.5-1 M KCI wash (10 ml). The flow rate was 30 m l / h and 1 ml fractions were collected after the emergence of the void volume (2 rnl). The salt concentration and protein content of each fraction was measured by refractive index and absorbance at A2so nm, respectively. After chromatography, selected fractions were pooled, concentrated, dialysed against isolation buffer including 2~% glycerc,i and then assayed for PDC enhanci~.g activity. Dilution and low proteie content necessitated that pooled column fractions were concentrated prior to assays for ~ctivity. The pooled fractions were transferre:~l to dialysis bags and dehydrated with ~quacide tCalbiochem) until the desired concentration factor was achieved. Routinely, fractions were 10-fold concentrated with Aquacide for 2 h at 4°C. To hasten the concentration process, at 30 min intervals rehydrated Aquacide gel was removed and the dialysis bags were covered with fresh, dry Aquacide powder.

287

SDS-polyacrylamide gel electrophoresis

TABLE I

Linear 7-15% gradient SDS-polyacrylamide gels were prepared according to Laemmli [12] and electrophoresed in a Bio-Rad Protean II gel apparatus for 2.5 h at 50 V/gel. Proteins were visualised by staining with Coomassie blue or silver [13].

Purificationof the PDC enhancingfactor

Elution and renaturation of proteins from SDS-polyacrylamide gels Enzymatically active proteins were recovered from SDS-PAGE gels essentially as described by Hager and Burgess [14]. Protein elution from gels and renaturation were performed at room temperature for 16 h and 12 h, respectively.

Protein kinase activity assays The acidic peptide Ser-Glu-Glu-Glu-Glu-Glu was synthesized by Dr. D. Harding (Massey University, Paimerston North, New Zealand). This synthetic peptide is a specific substrate for the protein kinase referred to as casein kinase II [15]. Phosphorylation of the peptide was assayed for 30 rain at 37°C in 50/~1 containing 100 mM Tris-HCl (pH 7.5), 10 mM MgCI 2, 100 mM NaCI, 30 /~M [y-32P]ATP (6951 cpm/pmol), 0.5 mg substrate/ml and an aliquot (2-5/~!) of crude cytoplasmic extract, column fraction or renatured protein. Reactions were terminated with glacial acetic acid added to a final concentration of 30% then the volume was adjusted to 500 pl with 30% acetic acid. To recover the radioactive peptide, approx. 0.4 g of Dowex 1-8 anion-exchanger (equilibrated in 30% acetic acid then filtered) was added and mixed with the reaction mixture for 5+-10 min. The tubes were then centrifuged for 20 s in a microfuge to remove free ATP and Pi tightly bound to the Dowex gel leaving protonated phosphopeptide in the supernatant. The supernatants were transferred directly to glass scintillation vials and the excess acetic acid was removed by evaporation before 5 ml of aqueous scintillant (ACS II) was added and radioactivity associated with peptide int,asured.

Fraction

Total T o t a l Specific Purification protein activity activity (-fold) (rag) (units) (units/rag) 6 83.3 14 1

Crude extract (NH4)2SO4, 20-607o 2.7 DEAE 0.17 Renatured 70 kDa protein 0.0002

540 340

200 2000

10

50000

The PDC enhancing factor was purified by DEAEcellulose ion-exchange chromatography of ammonium sulphate-fractionated cytoplasmic extracts. The elution profile of the 20-60% ammonium sulphate fraction from a DEAE cellulose column is shown in Fig. 1. The PDC enhancing activity eluted with 120-160 mM KCI in a single peak with 143-fold purification (Table I).

Gel electrophoresis of columns fractions Fig. 2A shows a representative Coomassie blue stained SDS-PAGE gel of protein samples taken from various stages of the purification process. Although a 14-fold purification of PDC enhancing activity was achieved on fractionation of cytoplasmic extracts with 20-60% ammonium sulphate, there was little change in total proteins (cf. lanes 2 and 3). Lane 4 merely highlights the loss of proteins on filtration through 0,22 #m filters, during preparation for FPLC chromatography. With Coomassie blue staining, no protein bands were detected in the DEAE-cellulose column active fraction (lane 5). However, Fig. 2B shows the same gel (lanes 5 and 6 only) after silver staining when a predominant band at approx. 68-70 kDa was apparent, together with A

0.2

" i J,++ ,l 'ii+

0.1

I0

Ammonium sulphate precipitation of cytoplasmic extracts As an initial step in the purification of PDC enhancing activity, four broad ammonium sulphate fractions were recovered from mastocytoma cytoplasmic extracts. The 20-60% fraction contained the bulk of the PDC enhancing activity, with 1 unit of activity contributed by 5 /~g of protein. After taking dilution factors into consideration, this corresponded to a 14-fold purification (Table I). Typically, 40-45% of the total protein was recovered in the 20-60% fraction which was taken for subsequent chromatographic purification.

3571

Purification of the PDC enhancing factor by column chromatography

E

Results

14.2 143

w

o

o

0.0 10

20

30

40

SO

60

7o

8

Fraction Number

Fig. I. Purificationof PDC enhancingactivity by DEAE-cellulose chromatography. Dialysed 20-60t~ (NH4)2SO4 precipitated cytoplasmic extract (2-3 mg protein) from K21 cells was applied to a I x 2.5 cm DEAE-cellulosecolumn.Proteinswere eluted with a linear 10-200 mM KCI gradient in isolationbuffer (50 ml), followed by a 0.5 M KC1 wash (10 ml). The flow rate was 30 ml/h and 1 ml fractions werecollected,Selectedfractionswere pooled, concentrated. dialysed then tested for PD(' enhancingactivity(O).

288 minor bands of lower molecular weight. When active fractions from other D E A E columns were fractionated by gel electrophoresis and silver stained, a similar band at approx• 70 kDa predominated.

10~ ,< Z Cl

!i' ~s

Elution and renaturation of the 70 kDa protein from SDS-polyacrylamide gels Samples of the bulked DEAE-cellulose column fractions containing PDC enhancing activity were electrophoresed on a 7-15% polyacrylamide gel with bovine

/--..

o

~4 O

u.

2 0.0

0.04

o.oe 0.12 0.16

0.20

-0.24

O

Concentration of Renatured 70 kDa

A,

LANE

1

2

3

4

5

6

Protein (pg/ml)

kDa

it ~k

m97 .~ P . - 6 6 n45

m29

jj~

m24 m20.1 m14

B,

Fig. 3. The PDC enhancing activity of the renatured 70 kDa protein. The 70 kDa protein region was excised from an SDS-polyacrylamide gel and the protein eluted and renatured (see Materials and Methods). The enhancement of l0 #M amsacrine-induced topoisomeraseII-DNA complexes in K21 nuclei by the 70 kDa protein (0-0.2 ttg) was determined by SDS/K* precipitation. K21 nuclei with: 70 kDa protein, ($). Fold increase in PDC formation in controls; ×, intact cells; o, crude extract (1 unit; 30 ~g protein); 20-60% (NH4)2SO4 fraction (l unit: 5 lag protein).

LANE ;Do 116 97 66 45 36 29 24 201 14

Fig. 2. SDS-PAGEelectrophoresis of cytoplasmic extracts at various stages of fraetionation. Cytoplasmic extracts were fractionated by procedures described in Materials and Methods and proteins in afiquots from successivestages were separated by SDS-PAGE (7-15% acrylamide gradient). (A) Coomassie blue-stained gel: lane 1, molecular weight standards; lane 2, crude cytoplasmic extract from K21 cells; lane 3, 20-60% (NH402SO,t cytoplasmic extract fraction; lane 4, sample as in lane 3 after filtration through 0.22 ~am filters; lane 5, DEAE-cellulosepooled column fractions containing maximum PDC enhancing activity; lane 6, molecular weight standards. (B? Gel (A), after silver staining. Lanes 5 and 6 only.

serum albumin (BSA) as a 66 kDa marker and the protein region at 70 kDa was excised and renatured. The renatured 70 kDa protein was then tested for P D C er.hancing activity. As shown in Fig. 3, the 70 kDa protein reconstituted PDC forming activity in nuclei to levels seen with intact cells, crude cytoplasmic extracts and the salt fractionated extract. Moreover, the very sharp decline in P D C enhancing activity previously observed with crude extracts at higher concentrations [1] was not seen. This may indicate that the renatured protein was relatively free from contaminating proteins that negate its action. To estimate the concentration and purity of the renatured protein, an aliquot was electrophoresed alongside known amounts of BSA on a 7-15% SDS gel and then stained with silver. The resulting gel contained 0.2 #g of a single 70 kDa protein with an activity corresponding to a 3571-fold purification (Table I).

Prefiminary identification of the 70 kDa protein with PCD enhancing activity Topoisomerase II activity can be modulated by phosphorylation in Drosophila cell free systems and in oivo with HeLa or Drosophila cell lysates [15-17]. However, although topoisomerase II was phosphorylated in Drosophila cell homogenates under conditions which specifically stimulated a variety of protein kinases, Ackerman et al. [17] found that modification of the enzyme was always sensitive to inhibitors specific for casein kinase II (CKII), namely heparin and anti-casein kinase II antiserum. Furthermore, the modification of topoisomerase 11 by C K I I is known to stimulate topoisolaerase II activity in vitro [15]. Therefore, we investigated whether the properties of the 70 kDa PDC en-

289

6

0 0.0 Heparin

0.1

0.2

0.3

Concentration Wmtl

Fig. 4. The effect of heparin on PDC enhancing activity. The enhancement of amsacrine-induced PDC formation by crude K21 cytoplasmic extracts in the presence of O-O.3 cg hepatin/ml was determined with isolated K21 nuclei by SDS/K+ precipitation. K21 nuclei with cytoplasmic extracts and O-O.3 &ml heparin, (0); K2I nuclei only (0).

hancing factor isolated from K21 cell extracts (previously shown to stimulate drug induced topoisomerase II activity) were similar to the casein kinase family of enzymes.

Fig. 5. The sensitivity of PDC enhancing activity at successivestages of purification to heparin and anti-casein kinase If antiserum. PDC enhancing activity (1 unit) from crude cytoplasmic extracts, the DEAE-cellulose column active fraction and the renatured 70 kDa protein were incubated with or without SO pg of anti-CKII antibody (Ab) or 0.2 pg/ml heparin under standard conditions. PDCs induced by 10 PM amsacrine in isolated K21 nuclei were measured by SDS/K+ precipitation. Treatments are as indicated.

body to CKII reduced activity approx. 9-lo-fold three fractions tested.

in all

Protein kinase activity of the 70 kDa protein Heparin-sensitivity

of the PDC

enhancing factor

The sensitivity to heparin of the PDC enhancing activity in crude cytoplasmic extracts was tested over a range of heparin concentrations known to specifically inhibit CKII (O-O.3 kg heparin/ml). As shown in Fig. 4, heparin inhibited the PDC enhancing activity with 0.12 pg heparin/ml reducing PDC formation approx. &fold. It was noticeable that heparin reduced complex forming activity in nuclei in the presence of cytoplasmic extract to levels lower than those achieved with nuclei alone. Therefore, the slight stimulation of complex formation usually observed in isolated nuclei may be due to residual heparin-sensitive PDC enhancing activity. The PDC e.nhancing activity in the active DEAE-cellulose column fractions and the 70 kDa renatured protein were also sensittve to heparin (0.2 pg/ml) which reduced PDC formation approx. 4.5fold (Fig. 5). Sensitivity of the PDC enhancing activity to anti-rat casein kinme II antiserum

The ability of the renatured 70 kDa protein to phosphorylate the synthetic peptide substrate, Ser-Glu-GluGlu-Glu-Glu, specific for casein kinase II [19] was also tested. As shown in Fig. 6, the 70 kDa, protein phosphorylated the synthetic substrate for CKII which incorporated approx. 10 pmol “‘P/n& per mg protein. Moreover, phosphorylation of the substrate was sensitive to 0.2 pg/ml heparin and SO gg of anti-CKII antiserum which reduced incorporation of phosphate to 4.6 pmol/min per mg protein and 0.7 pmol/min per mg protein, respectively. The polyamine spermine (1 mM), is known to stimu-

liver

The sensitivity of the PDC enhancing activity to rat liver casein kinase II antiserum was tested. The antibody used was highly specific for cytosolic CKII and nuclear kinase (NKII), but not reactive against CAMPdependent protein kinase, casein kinase I and nuclear protein kinase-NI [XI]. PDC enhancing factor (1 unit), in crude extracts, active column fractions and the renatured 70 kDa protein, was incubated with 50 cg of CKII antibody in standard reaction conditions and PDC quantitated by SDS/K+ precipitation. As shown in Fig. 5, the anti-

Treatment Fig. 6. Protein kinase activity of the renalured 70 kDa protein. Phosphorylation of a synthetic peptide substrate for casein kinase 11 by the renatured 70 kDa protein was tested as described in Materials and Methods. Reactions contained 2 yl aliquots of 70 kDa protein. Sensitivity to 0.2 pg/ml heparin, 50 cg anti-CKII antibody of 1 mM spermine was also tested as indicated.

290 late pnosphorylation by CK II 2-3-fo!d [20]. However, under our conditions only a 1.3-fold stimulation of kinase activity was observed (Fig. 6). Similarly, spermine had little detectable stimulatory effect on the PDC enhancing activity of the 70 kDa protein as measured by S D S / K ÷ precipitation (data not shown). The precise reason(s) for the reduced effect of spermine under our experimental conditions is unclear since spermine is known to affect DNA structure and topoisomerase II activity. Furthermore, the salt concentration in our assays may not be optimal for stimulation by spermine. In a preliminary study, we found that heparin-sensitive CKII purified from calf thymus according to Zandomeni et al. [21] caused an ll-fold increase in PDC formation when added to factor-depleted K21 mastocytoma cell nuclei. This effect was comparable to the increase of PDC formation observed with the 70 kDa protein. Moreover, the enhancement of amsacrineinduced PDC formation by calf thymus CKII was totally abolished when the purified CKII was incubated with antiserum to rat liver CKII prior to the assays. Taken together, these results suggest that the 70 kDa PDC enhancing factor is a het'arin-sensitive protein kinase with properties similar to casein kinase II or NKII. Discussion

Previously, we have shown that a factor present in tumour cell extracts, and distinct from topoisomerases I and I1, is required for topoisomerase II-DNA complex formation in amsacrine-treated isolated mastocytoma cell nuclei [1]. When this protein was recovered from gels and renatured, it showed heparin-sensitive proteinkinase activity with a specific CKII substrate, it stimulated formation of topoisomerase II-DNA complexes and was inhibited by antibody to rat liver CKII. These observations suggest that the 70 kDa PDC enhancing protein is related to casein kinase II or nuclear kinase II. In addition, CKII, purified from calf thymus, restored drug-induced PDC formation when added to factor depleted K21 mouse mastocytoma cell nuclei. Casein kinase II belongs to a family of casein kinases comprising CKI, CKII, CKIII and NKII (reviewed in Ref. 20), CKII appears to be a growth related enzyme [22,23] which preferentially phosphorylates acidic protein substrates, such as casein and phosvitin on serine or threonine residues [24,251. CKII is found in the cytoplasm and nucleus of cells consistent with its action on a variety of substrates, including topoisom*rases I and II. Various workers have suggested that there are two pools of topoisomerase II, representing functional states or isoforms [26-28] which may have different sensitivity to amsacrine. Our results indicate that amsacrine-induced topoisomerase II-DNA complex formation in

nuclei is dependent on the presence of a 70 kDa protein kinase with CKII-like properties. Furthermore, it is known that phosphorylation of topoisomerase II by CKII in vitro stimulates enzyme activity [15,29], therefore, it is conceivable that modifcation of topoisomerase II by phosphorylation may be responsible for the different isoforms of the enzyme. It is possible that phosphorylation of topoisomerase II (TII) by casein kinase II (CKII) or a related enzyme converts TII to a drug-sensitive PDC forming state, possibly by affecting its action on, or association with, the nuclear matrix or scaffold, since it has been proposed [28] that one form of topoisomerase II, the matrix or scaffold associated enzyme bound tightly to DNA loses its DNA strand passing catalytic function, whereas a second non-matrix bound or 'soluble' form (which may be phorphorylated), produces topoisomerization and forms readily reversible complexes with DNA. This model is supported by the release of PDC forming activity when normally inactive K21 nuclei are extracted with high concentrations of salt [30]. As a consequence of factor depletion and dephosphorylation durin~ nuclei isolation only a fraction of the potential drug-induced PDCs in whole cells would be expected in isolated nuclei, as seen with K21 cell nuclei [1] and in L1210 cell nuclei when measured by alkaline elution [31-33]. Therefore, the fraction of PDCs produced in isolated nuclei may reflect the residual amount of factor that activates matrix associated topoisomerase II and the different proportions of PDC formation obtained with various nuclei may reflect the amount of factor retained using different nuclei isolation methods [34-36, cf. 30-33] and the state of phosphorylation of topoisomerase Il. From such a model one can envisage a role for the PDC enhancing factor in drug resistance. For example, reduced or modified kinase activity, increased phosphatase activity, or an altered phosphorylation site on topoisomerase II could directly affect the action or association of topoisomerase II with DNA or the affinity of topoisomerase II-targeted drugs for the enzyme shifting the equilibrium between cleavable and noncleavable-complexes to the latter. It has been reported that dephosphorylation of topoisomerase I prevents the formation of camptothecin-induced topoisomerase IDNA complexes [37]. Therefore, the possibility that topoisomerase 11 must be phosphorylated to facilitate drug binding or protein-DNA complex formation is important since the role of topoisomerase II in growth or drug resistance is not well understood, and qualitative alterations of topoisomerase |I, either by covalent modification or specific inhibitors of topoisomerase II activity may be involved. This is the first demonstration of a protein factor that directly influences drug-induced topoisomerase II action.

291 Acknowledgements O u r r e s e a r c h w a s s u p p o r t e d b y t h e C a n c e r Society o f N e w Z e a l a n d a n d a s e n i o r s c h o l a r s h i p to S . J . D . - R . f r o m t h e A u c k l a n d M e d i c a l R e s e a r c h F o u n d a t i o n . W e gratefully t h a n k D r s . K . A h m e d a n d S.A. G o u e l i , V e t e r a n s Administration Medical Center, Minneapolis, U.S.A., f o r their g e n e r o u s gift o f t h e a n t i - C K I I a n t i b o d y .

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