Kidney International, Vol. 61, Symposium 1 (2002), pp. S125–S131

c-myc antisense oligonucleotide treatment amelioratesmurine ARPKD

JUSTIN L. RICKER, JOHN E. MATA, PATRICK L. IVERSEN, and VINCENT H. GATTONE II

Department of Anatomy and Cell Biology, The University of Kansas Medical Center, Kansas City, Kansas 66160-7400,USA;AVI BioPharma, 4575 SW Research Way, Suite 200, Corvallis, Oregon 9733, USA; and Department of Anatomy and CellBiology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120, USA

c-myc antisense oligonucleotide treatment ameliorates murine slower progression than ARPKD, which usually affectsARPKD. newborns or young children. ARPKD can cause massive

Background. Overexpression of c-myc is postulated to play bilateral enlargement of the kidneys. Most individualsa role in the pathogenesis of polycystic kidney disease (PKD).with ARPKD who survive the neonatal period developc-myc expression is increased in all rodent models of PKDrenal failure.that have been examined as well as in human ADPKD. To

determine whether overexpression of renal c-myc contributes The human ARPKD gene is located on chromosometo renal cyst formation, C57BL/6J-cpk litters (an animal model 6 [1], but has not yet been cloned. Nonetheless, a numberof ARPKD) were treated with an antisense oligomer (ASO) of mouse models of ARPKD exist to study this conditionto c-myc mRNA.

[2]. The C57BL/6J mouse homozygous for the congenitalMethods. Injections of 30 �g of a c-myc ASO were givenpolycystic kidney (cpk) gene mutation is one of the mostto C57BL/6J-cpk litters on postnatal days 7–20. Control mice

received either sham injections or injections of an equal amount widely utilized models of ARPKD [3, 4]. While the cpkof a scrambled ASO. At 20 days, kidney weight, body weight, gene is not analogous to the human ARPKD gene, theserum urea nitrogen (SUN), hematocrit, and renal concentra- PKD in this well characterized mouse model exhibitstion of ASO were determined. In kidney, c-Myc and PCNA

severe bilateral renal cysts in a pattern very similar toprotein were assessed by immunoblotting and steady state lev-human ARPKD. The collecting duct cysts are the pre-els of renal RNA for c-myc, EGF, SGP-2, and histone H4

were assessed by northern blot hybridization. c-Myc and PCNA dominant pathology and replace the normal renal paren-proteins were localized by immunohistochemistry. chyma leading to morbid uremia by approximately four

Results. Cystic mice treated with the c-myc ASO had a de- weeks of age.creased relative kidney weight, improved renal function, andC57BL/6J-cpk/cpk mice have previously been showna reduced amount of cystic change compared with sham and

to overexpress renal c-myc mRNA [5, 6]. The c-mycscrambled ASO controls. The abnormal expression of severalPKD related proteins and mRNAs were partially reversed by mRNA is overexpressed in epithelia from cystic as wellc-myc antisense treatment. c-myc staining appeared to be re- as normal sized tubules [7]. Renal c-myc mRNA is alsoduced in the noncystic tubules. Treatment with the c-myc ASO overexpressed in other rodent models of PKD such asdid not cause a reduction in hematocrit or total body weight

Han:SPRD-cy/cy rats [8], CD-1-pcy/pcy mice [9], andindicating that the beneficial effects were not due to a general-BALB/c-cpk/cpk mice [10]. Interestingly, mice overex-ized inhibition of cell proliferation in rapidly growing tissue.

Conclusions. c-Myc appears to play a role in the cystogenesis pressing a c-myc transgene develop PKD and die of renalof cpk-induced murine PKD and antisense targeting the over- failure by six weeks to three months of age [11, 12].expression of c-myc partially ameliorated the renal changes. Human ADPKD kidney also has been shown to overex-

press c-myc mRNA [13]. Therefore, renal c-myc overex-pression must play an important role in renal cystogen-INTRODUCTIONesis since it is always associated with PKD. To determinePolycystic kidney disease (PKD) is a major cause ofif increased c-myc mRNA expression contributes to PKDend stage renal disease in humans. PKD is characterizeddevelopment, C57BL/6J-cpk/cpk mice were treated withby severe dilation of renal tubules and can be inheriteda c-myc antisense oligomer (ASO) (AVI BioPharma, Cor-as an autosomal dominant (AD) or autosomal recessivevallis, OR).(AR) trait. In humans, ADPKD has a later onset and

MATERIALS AND METHODSKey words: polycystic kidney disease, C57BL/6J-cpk/cpk mice, c-myc,antisense oligonucleotide, EGF, clusterin (SGP-2), PCNA. C57BL/6J-cpk/� mice (colony established at KU Med-

ical Center, stock originally obtained from Jackson Lab- 2002 by the International Society of Nephrology

S-125

Ricker et al: c-Myc ASO therapy in PKDS-126

oratory, Bar Harbor, ME) were bred to produce the tors (catalog number 1836170, Boehringer Mannheim,Mannheim, Germany). Protein was isolated and pro-normal (�/� or cpk/�) and cystic (cpk/cpk) mice used

in this study. Litters of C57BL/6J- cpk mice received i.p. cessed for western blot analysis by standard techniques.Protein samples were separated on a 12.5% SDS/acryl-injections of 4.3 nmoles (30 �g) per day of the c-myc

ASO (AVI-4126, AVI BioPharma, Corvallis, OR) on amide gel with a 6% SDS/acrylamide stacking gel at 33mA for 14 h. The gels were soaked in transfer bufferpostnatal days 7 through 20. A few litters were treated

with higher (14.3 nmoles; 100 �g) or lower (1.4 nmoles; (192 mM glycine, 25 mM Tris base, and 10% methanol,pH 8.3) for 20 minutes and transferred to Immobilon-P10 �g) dosages of the c-myc ASO. The c-myc ASO had

a phosphorodiamidate Morpholine backbone with a se- transfer membranes (Millipore Corporation, Bedford,MA, at 80 volts for 80 min). The membranes were soakedquence of 5�-ACG TTG AGG GGC ATC GTC GC-3�.

As controls, mice were either sham injected or given an briefly in methanol and dried at 40�C. Membranes wererehydrated briefly in methanol, rinsed in wash bufferequal amount of a scrambled ASO (ACT GTG AGG

GCG ATC GCT GC). These mice were sacrificed four (PBS with 0.3% Tween 20) and incubated in blockingbuffer (PBS, 0.3% Tween 20 and 3% powdered milk)hours after their last injection. Control, scrambled and

c-myc ASO (AVI BioPharma, Corvallis, OR) treated for 30 minutes. Membranes were incubated for one hourwith primary antibodies to: c-Myc (catalog number sc-C57BL/6J-cpk litters were anesthetized (65 mg/kg so-

dium pentobarbital, i.p.) and sacrificed at 20 days of age. 764, Santa Cruz Biotechnology, Inc., Santa Cruz, CA),glucocorticoid receptor (catalog number sc-1004, SantaTotal body weight, total kidney weight and kidney weight

as a percent of body weight were determined. Blood was Cruz Biotechnology, Inc., Santa Cruz, CA) and PCNA(catalog number sc-7907, Santa Cruz Biotechnology,collected from the heart, hematocrit was measured, and

serum was used to assess renal function. SUN was deter- Inc., Santa Cruz, CA). The membranes were washedthree times for 5 minutes with wash buffer and incubatedmined utilizing a colorimetric assay (catalog number

640-A, Sigma Diagnostics, St. Louis, MO). The left kid- for 30 minutes with the appropriate horseradish peroxi-dase (HRP) conjugated secondary antibody. Membranesney was removed, weighed, snap frozen in liquid nitro-

gen, and stored at –80�C until processed for RNA isola- were rinsed again with wash buffer and incubated withECL detection reagent (Promega, Madison, WI) fol-tion. The animals were then perfusion fixed with 4%

paraformaldehyde in 0.1 M phosphate buffer through a lowed by autoradiography using Kodak Bio Max film.catheter placed in the left ventricle. The right kidney

Extraction and analysis of renal c-myc ASOwas processed for paraffin embedment and sections werestained with periodic acid Schiffs reaction or immuno- 500 ng of an internal standard (AVI-0-1-22-242, AVI

BioPharma, Corvallis, OR; a 15 mer phosphorodiami-stained for c-Myc.date Morpholine oligomer whose sequence is derived

Immunohistology from a 5� truncation of the c-myc ASO) was mixed witha 500 �L aliquot of kidney lysate prepared in RIPAParaffin sections of kidneys from c-myc ASO treated

and untreated 20 day-old normal and cystic C57BL/6J- buffer. An equal volume of methanol was added, andthe resulting precipitate was centrifuged for 10 minutes.cpk mice were processed for c-Myc immunohistochemis-

try. After endogenous peroxidase activity was quenched The supernatant was collected, the pellet was washedwith 100 �L of 0.025 M Tris buffer (pH 9.0) and recentri-with methanolic peroxide, the sections were treated with

normal goat blocking serum. Kidney sections were incu- fuged. The resulting supernatants were collected, heatedat 70�C for 10 minutes and recentrifuged. The superna-bated with a primary antibody to c-Myc (1:25, catalog

number sc-764, a rabbit polyclonal antibody to human tant was lyophilized following the addition of 100 �L of0.025 M Tris buffer (pH 9.0). The resulting residue wasc-Myc, Santa Cruz Biotechnology, Inc., Santa Cruz, CA,

18 hours at 4�C). The bound antibody was visualized reconstituted with 100 �L of a 5�-fluorescein labeledDNA (FDNA, Hybridon, Inc., MA) solution (0.2 OD’susing an avidin biotin complex method (Vector Labora-

tories, Inc., Burlingame, CA) with nickel-enhanced DAB in 100 �L of 0.025 M Tris buffer). The FDNA has acomplementary sequence to that of the c-myc ASO andas the chromagen. Negative control sections were pro-

cessed in an identical manner, except normal rabbit se- forms a stable duplex. The entire sample was injectedand analyzed by HPLC (Varian 9010 HPLC pump, Palorum replaced the primary antibody.Alto, CA, attached to a Rainin AI-200 autosampler and

Western blot analysis a Varian 9075 Fluorescence detector connected to aDNAPac PA-100 anion exchange column, Dionex Corp.,Fresh kidneys from c-myc ASO treated and untreated

C57BL/6J-cpk/cpk mice were removed at 21 days and Sunnyvale, CA). A binary gradient (10% B at 0 min to45% B at 20 min) consisting of 0.025 M Tris buffer (pHimmediately homogenized in Radio Immuno Precipita-

tion Assay (RIPA) buffer (1X PBS, 1% Nonidet P-40, 9.0) as mobile phase A and 0.025 M Tris buffer (pH 9.0)/1 M NaCl as mobile phase B was used to achieve the0.5% deoxycholic acid, 0.2% SDS) with protease inhibi-

Ricker et al: c-Myc ASO therapy in PKD S-127

chromatographic separation. The fluorescence excitation renal function (Table 1). Total body weight and hemato-crit were not affected by the c-myc ASO treatment (Ta-and emission wavelengths were set at 494 nm and 518ble 1), suggesting that ASO treatment did not signifi-nm, respectively. The amount of the c-myc ASO presentcantly affect normal highly proliferative, extrarenalin the kidney lysate samples was determined using aprocesses. While ASO treatment slowed renal enlarge-standard curve generated by running a series of c-mycment, the C57BL/6J-cpk/cpk kidneys from treated miceASO solutions containing the internal standard.still exhibited significant cystic pathology (Fig. 1). Norenal changes were seen in normal C57BL/6J mice fol-RNA isolation and northern hybridizationlowing c-myc ASO treatment (data not shown).RNA was extracted and purified from kidneys of 20

c-myc mRNA was clearly over-expressed in the cysticday-old normal and cystic mice as well as 20 day-oldkidney. This over-expression was unaltered by c-mycc-myc ASO and scrambled oligomer treated normal andASO treatment (Fig. 2). However, treatment inhibitedcystic mice using the acid guanidinium thiocyanate-phe-translation of the c-myc mRNA into c-Myc protein asnol-chloroform extraction method as previously de-evinced by lower c-Myc protein (a 59% reduction byscribed [14]. The frozen kidneys were homogenized inwestern blot analysis, Fig. 3). EGF mRNA is highly ex-

Tri-Reagent (Molecular Research Center, Inc., Cincin-pressed in normal renal distal tubules, but severely di-

nati, OH) and RNA was isolated according to the manu- minished in the cystic kidney [17]. Following c-myc ASOfacturer’s instructions. The RNA was separated on a 1% treatment, EGF mRNA expression was increased nearlyagarose/formaldehyde gel via electrophoresis, with each twelve fold in the cystic kidney (Fig. 2). Messenger RNAlane containing 10 �g of total renal RNA. The RNA for SGP-2, or clusterin, a marker of immature collectingwas then transferred overnight to a Zetabind membrane duct, was over-expressed in cystic versus normal kidney,(Cuno, Inc., Meriden, CT), and stained with methylene but was decreased 40% after c-myc ASO treatment (Fig.blue to ensure equivalent amounts of RNA in each lane. 2). Increased EGF mRNA and decreased SGP-2 mRNAThe RNA was baked (2 hours at 80�C) and hybridized expression indicate that c-myc ASO treatment led toovernight at 65�C in Church’s buffer [15] (1% BSA and an increased maturation of both the distal tubules and7% SDS in 0.5 M sodium phosphate with 1 mM EDTA) collecting ducts, respectively. mRNA for Histone H4, awith 32P-labeled cDNA probes (random prime labeling marker associated with increased cell proliferation, wasmethod, Rediprime II, Amersham Pharmacia Biotech, over-expressed in cystic compared to normal kidney.Arlington Heights, IL). The probes identified c-myc [7], c-myc ASO treatment did not significantly reduce theclusterin (SGP-2) [16], prepro epidermal growth factor Histone H4 message level in cystic kidney (Fig. 3). How-

ever, ASO treatment was associated with a 30% decrease[17], and Histone H4 [4] mRNAs. Hybridized northernin PCNA, a protein marker of cell proliferation (Fig. 3).blots were washed for 10 minutes each at 65�C; twice in

c-Myc protein was elevated in the cystic compared toWash Buffer A (0.5% BSA and 5% SDS in 40 mMnormal kidneys [5] (Figs. 3 and 4). By immunohistochem-sodium phosphate with 1 mM EDTA), and four timesistry, the c-Myc was localized to the nuclei of epithelialin Wash Buffer B (1% SDS in 40 mM sodium phosphatecells lining cystic and noncystic tubules. After c-mycwith 1 mM EDTA). Autoradiography was used to visual-ASO treatment, the lower c-Myc protein levels appearize the relative expression of mRNA. Specific mRNAto be due largely to a reduction in c-Myc protein in thewas quantitated using densitometry of the autoradio-noncystic tubules (Fig. 4A and C). Therefore, abnormali-graph. All of the northern blot hybridization studies wereties typically seen in noncystic tubules of the cystic kidneyperformed in duplicate with different sets of RNAs.were reduced by c-myc ASO treatment.

Statistical analysisQuantification of renal c-myc ASO

Statistical analysis of SUN, total body weight, kidneyThe c-myc ASO was detected in kidney lysate at a

weight as a percent of body weight, and hematocrit were concentration of 0.47 �M one hour after the final injec-performed using analysis of variance (ANOVA) (P � tion in animals treated for 14 days with 4.3 nmoles (30 �g)0.05) with the aid of MINITAB software (State College, per day. There was no phosphorodiamidate morpholinePA). Data are presented as mean � SEM. oligomer detected in kidney lysate from 20 day-old un-

treated C57BL/6J-cpk mice which served as a negativecontrol.RESULTS

Effects of c-myc morpholine antisense Dose responseoligomer treatment Higher (14.3 nmoles; 100 �g) and lower (1.4 nmoles;

The c-myc ASO treatment significantly reduced the 10 �g) dosages of the c-myc ASO also ameliorated thePKD. Relative kidney weight as well as SUN levels werecystic renal enlargement and inhibited the decline in

Ricker et al: c-Myc ASO therapy in PKDS-128

Table 1. Effect of treatment of C57BL/6J-cpk mice with c-myc antisense oligonucleotide (Mean � SEM, N)

Control c-myc antisense Scrambled antisense

Group phenotype Normal Cystic Normal Cystic Normal Cystic

Kidney weight 1.48�0.03 21.68�1.88 1.64�0.02 11.91�0.45a 1.54 �0.02 17.93�0.65(as a percent of (14) (11) (15) (5) (12) (8)total body weight)

Serum urea nitrogen 16.3�1.0 62.4�6.8 17.5�0.7 38.4�3.2a 20.3 �0.7 59.8�4.8mg/dL (15) (11) (15) (5) (11) (8)

Total body weight 10.5�0.35 10.6�0.65 10.1�0.42 9.0�0.40 9.53�0.34 10.0�0.30grams (15) (11) (15) (5) (12) (8)

Hematocrit %b 30.9�1.12 23.8�2.35 31.7�0.45 22.0�1.68 30.5�1.01 25.6�0.98(15) (11) (11) (5) (10) (8)

a P � 0.05 for difference of treated from control/scrambled value within the phenotypeb P � 0.05 for difference between cystic and normal, but not with treatment

Fig. 1. Light micrographs of 20 day-old control and c-myc ASO treatedC57BL/6J-cpk cystic kidneys. Compared with (a, b ) control cystic kid-ney, (c, d ) c-myc ASO treated cystic kidney exhibits an increasedamount of normal renal parenchyma. a, b: 8X; c, d: 30X.

reduced to a similar extent to that observed after treat-ment with 4.3 nmoles (30 �g) of c-myc ASO. Total bodyweight and hematocrit levels were also unaffected bythese dosages of c-myc ASO (data not shown).

Fig. 2. Renal mRNA expression in 20 day-old control, scrambled oligo-nucleotide and c-myc ASO treated normal and cystic C57BL/6J-cpkDISCUSSIONmice. The c-myc, histone H4, and SGP-2 messages were overexpressedin cystic compared to normal kidney. c-myc ASO treatment did notThe c-myc gene, located on mouse chromosome 15,alter c-myc or histone H4 mRNA expression, but did decrease SGP-2encodes 64 to 67 kD proteins containing domains thatmRNA expression nearly 40% in cystic kidney. EGF mRNA was se-

function in nuclear localization, non-specific and se- verely diminished in cystic compared to normal kidney, but was in-creased 12-fold in cystic kidney following c-myc ASO treatment. Allquence-specific DNA binding, and oligomerization [18].of the northern blot hybridization studies were performed in duplicatec-Myc functions as a transcription factor that dimerizesusing different sets of RNAs. Representative autoradiographs as well

with other proteins and binds DNA [18, 19]. These heter- as methylene blue staining of 28S and 18S ribosomal RNA to ensureequal loading and transfer of RNA are shown.odimeric DNA-binding complexes both activate and re-

Ricker et al: c-Myc ASO therapy in PKD S-129

Fig. 3. Western blot analysis in 20 day-old control and c-myc ASOtreated C57BL/6J-cpk cystic kidney. The c-Myc and PCNA proteinlevels were reduced 59% and 30%, respectively, in cystic kidney follow-ing c-myc ASO treatment. All of the western blot hybridization studieswere performed in duplicate using different sets of total renal proteinsamples. Glucocorticoid receptor was used as a control for loading(based on unpublished data from R. Maser, personal communication).

press transcription of a number of genes [19]. Increasedexpression of c-myc is seen in proliferating cells andregulates cell growth, transformation, inhibition of dif-ferentiation, and apoptosis [18, 19].

Fig. 4. Localization of c-Myc protein in 20 day-old control and c-mycRenal c-myc mRNA is overexpressed in all PKD mod-ASO treated C57BL/6J-cpk cystic kidney. (a, b ) c-Myc protein is local-els examined [5, 6, 9, 11, 12]. In the kidney, c-Myc playsized to the nuclei of epithelial cells lining cystic (arrowheads) and

an important role in both apoptosis [12, 13, 20–23], and noncystic (*) tubules in control cystic kidney. (c ) After c-myc ASOtreatment, the c-Myc protein is reduced in the nuclei of epithelial cellsproliferation [12, 13, 22–24]. In vitro, overexpression oflining the noncystic tubules, but unchanged in cystic tubules. a: 100X;c-myc promotes proliferation of cells in the presence ofb, c: 400X.

normal serum [18], but leads to apoptosis of cells in theabsence of growth factors [18, 25]. We hypothesized thatc-myc ASO treatment would result in a decrease in cellu-lar proliferation and amelioration of the PKD. Renal c-myc may contribute to the abnormal behavior of bothepithelial cell hyperplasia appears to play an important cystic and noncystic tubules since some of the detrimen-role in PKD initiation and progression [26, 27]. However, tal effects of this overexpression of c-myc were reducedevidence of the effects of our ASO treatment on cell by ASO therapy.proliferation has been equivocal. Western blot data sug- The use of phosphorodiamidate Morpholino oligomergests that the c-myc ASO treatment reduced prolifera- (PMO) chemistry to inhibit c-Myc in the cystic kidneytion (reduced renal PCNA), however, renal histone H4 avoids possible adverse effects associated with the moremRNA was not reduced by treatment. c-myc ASO treat- prevalent phosphorothioate oligonucleotides (PSOs). Thement appears to reduce c-Myc protein, but primarily in internucleoside charges of the phosphorothioate analogsthe noncystic renal tubules. Since both cystic and noncys- are responsible for nonspecific effects through bindingtic tubules overexpress c-myc, both are exhibiting abnor- to cellular and extracellular proteins [28–30]. Nonspecificmalities. The effect of ASO treatment on cystic tubules interactions can lead to detrimental physiological effectsis suggested by the reduced renal size and less prominent in vivo including inhibition of the clotting cascade, activa-cystic change. This c-myc ASO treatment also acts in tion of the complement cascade and severe hypotensionpart by inhibiting the detrimental effects of c-myc over- [31–36]. Further, bolus injection of PSOs can lead toexpression on the noncystic tubules so they retain a more blockade of sympathetic tone due to antagonism ofnormal function. This effect of c-myc ASO treatment on alpha-1 adrenergic receptors [37]. A second disadvan-noncystic tubules is suggested by an increase in matura- tage of the PSO chemistry involves the RNase H mecha-tion of the distal tubule and collecting duct as evinced nism of action. RNase H is a ubiquitous enzyme presentby increased EGF, and decreased SGP-2 mRNA concen- in the cell nucleus and cytoplasm in concentrations suffi-

cient to mediate the degradation of the RNA in an RNA/trations after treatment, respectively. Overexpression of

Ricker et al: c-Myc ASO therapy in PKDS-130

Correspondence to Vincent H. Gattone II, Ph.D., Department ofDNA heteroduplex [38]. Hence, RNase H competentAnatomy and Cell Biology, Indiana University School of Medicine, 635

oligonucleotide chemistries such as PSO lack specificity, Barnhill Drive Indianapolis, Indiana 46202, USA.E-mail: gattone@anatomy.iupui.eduin part, because duplexes as short as five base pairs in

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