Paradoxical Increase in Retinoblastoma Protein in …...Paradoxical Increase in Retinoblastoma...

Paradoxical Increase in Retinoblastoma Protein in ColorectalCarcinomas May Protect Cells from Apoptosis1

Hirofumi Yamamoto, Jae-Won Soh,Takushi Monden, Micheal G. Klein,Li Ming Zhang, Haim Shirin, Nadir Arber,Naohiro Tomita, Ira Schieren, C. A. Stein, andI. Bernard Weinstein2

Herbert Irving Comprehensive Cancer Center [H. Y., J-W. S.,M. G. K., H. S., C. A. S., I. B. W.], Center for Neurobiology andBehavior [I. S.], Department of Medicine [L. M. Z., C. A. S.,I. B. W.], and Department of Pharmacology [C. A. S.], ColumbiaUniversity, College of Physicians and Surgeons, New York, NewYork 10032; Department of Surgery II, Osaka University MedicalSchool, Osaka, 565-0871, Japan [H. Y., T. M., N. T.]; andDepartment of Gastroenterology, Tel Aviv Sourasky Medical Center,Tel Aviv, 64239, Israel [N. A.]

ABSTRACTThe retinoblastoma (Rb) gene is inactivated in a variety

of human cancers, but in colorectal carcinomas there isfrequently increased expression of this gene. This is para-doxical in view of the known role of Rb as a tumor suppres-sor gene. In the present study, we compared the levels ofexpression of the Rb protein (pRb) in normal human colo-rectal mucosa, adenomatous polyps, and carcinomas by im-munohistochemistry. In vitro studies were also done to ex-amine the phenotypic effects of an antisense oligo-deoxynucleotide (AS-Rb) targeted to Rb mRNA in theHCT116 colon carcinoma cell line that expresses a relativelyhigh level of pRb. The incidence of pRb-positive cells wasincreased during multistage colorectal carcinogenesis.Invitro treatment of HCT116 cells with AS-Rb decreased thelevel of pRb by about 70% and also decreased the levels ofthe cyclin D1 protein and cyclin D1-associated kinase activ-ity. AS-Rb inhibited growth of HCT116 cells and inducedapoptosis. Reporter assays indicated about a 17-fold in-crease in E2F activity. These findings suggest that the in-creased expression of pRb in colorectal carcinoma cells mayprovide a homeostatic mechanism that protects them from

growth inhibition and apoptosis, perhaps by counterbalanc-ing potentially toxic effects of excessive E2F activity.

INTRODUCTIONStudies done about 10 years ago on cases of hereditary

Rbs3 led to the discovery of theRb gene, the first tumorsuppressor gene to be identified (1–3). Subsequent studies in-dicated that the function of this gene is also lost, at the somaticlevel, in a variety of nonhereditary human cancers, includingcancers of the breast, lung, soft tissue, bladder, and prostate. Onthe other hand, deletions and loss of expression of theRb geneare rare in human colorectal carcinomas (for review see Ref. 4).Indeed, in these cancers, and in cell lines derived from thesecancers, there is often increased expression of theRb gene(5–8), and in some tumors, there are additional copies of theRbgene due to amplification of the corresponding DNA region onchromosome 13 or nonrandom increases in chromosome 13 (4).This increase in the expression of a tumor suppressor gene incolorectal cancers seems to be paradoxical, and its biologicalsignificance is not known.

Studies done within the past few years indicate that theprotein encoded by theRbgene, pRb, normally plays a key roleas a negative regulator of the G1 to S transition in the cell cycleby binding the transcription factor E2F and preventing it fromactivating the transcription of genes required for the S phase.Phosphorylation of pRb by cyclin D1/CDK4 or cyclin D1/CDK6 complexes, and possibly by the cyclin E/CDK2 complex,during the latter part of G1 relieves this inhibitory effect, thusactivating E2F (for review see Ref. 9). Human colorectal car-cinomas frequently display abnormalities in the expression ofseveral genes that play either positive or negative roles in thispathway. Thus, there is frequently increased expression of thecyclin D1, cyclin E, CDK1, and CDK2 proteins (7, 10, 11), andin a subset of tumors and cell lines, there is amplification of thecyclin E and CDK2 genes (12, 13). Alterations in cyclin de-pendent kinase inhibitors are also seen in colorectal carcinomas.Thep16Ink4 gene is inactivated in about 40% of the cases due tode novomethylation of a 59CpG island (14), and expression ofthe p21Waf1 gene is frequently decreased (15, 16). Decreasedlevels of p27Kip1 due to proteolytic degradation have been seenin a subset of cases, and this is associated with a poor prognosisor high-grade tumors (17, 18).

To further explore the role of pRb in colon cancer, in thepresent study, we have examined the expression of pRb byimmunohistochemistry, and we found that there was an increasein the expression of pRb during the multistage process of

Received 12/23/98; revised 3/31/99; accepted 3/31/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by an award for cancer research from the FUSO Pharma-ceutical Company (Osaka, Japan; to H. Y.), a Deutshes Krebsforschun-gszentrum (DKFZ) award (to N. A.), and by National Cancer InstituteGrant CA 63467 and an award from the National Foundation for CancerResearch (to I. B. W.).2 To whom requests for reprints should be addressed, at Herbert IrvingComprehensive Cancer Center, Columbia University, College of Phy-sicians and Surgeons, 701 West 168th Street, New York, NY 10032.Phone (212) 305-6921; Fax: (212) 305-6889; E-mail: [email protected].

3 The abbreviations used are: Rb, retinoblastoma; pRb, Rb protein;oligo, oligodeoxynucleotide; TUNEL, terminal deoxynucleotide trans-ferase; DAPI, 49,6-diamidino-2-phenylindole; AS-Rb, antisense Rboligo; S-Rb, sense Rb oligo.

1805Vol. 5, 1805–1815, July 1999 Clinical Cancer Research

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colorectal carcinogenesis. To examine the phenotypic and bio-chemical effects of decreasing the level of expression of pRb incolon cancer cells, we carried outin vitro studies using theHCT116 human colon carcinoma cell line because it expressesa relatively high level of pRb. Our strategy was to transfect intothese cells an antisense phosphorothioate oligonucleotide tar-geted to the 59region of Rb mRNA. Our results provide evi-dence that the increased expression of pRb has a positive selec-tion value in HCT116 colon carcinoma cells because it protectsthem from growth inhibition and apoptosis. Furthermore, ourstudies provide evidence that cellular levels of pRb can mark-edly influence the expression of other proteins involved in cellcycle control.

MATERIALS AND METHODSCell Lines and Tissues. The HCT116 human colon car-

cinoma and the WI38 human lung fibroblast cell lines wereobtained from the American Type Culture Collection. TheHCT116 cell line was grown in DMEM, and the fibroblastswere grown in RPMI 1640 plus 10% fetal bovine serum, 100units/ml penicillin, and 100mg/ml streptomycin in 5% CO2 at37°C. Fifty colorectal carcinomas and paired adjacent normalmucosa samples, 50 samples of adenomatous colorectal polyps(37 tubular and 13 tubulo-villous adenomas), and 15 polypscontaining focal carcinomas were collected during surgery orduring endoscopic polypectomy in the Department of Surgery IIin Osaka University Medical School from 1994–1996. Thesamples of normal mucosa were cut in the longitudinal directionto examine the top, middle, and bottom parts of the glands, andthe polyps and carcinomas were cut across the maximum diam-eter. These samples were fixed in buffered formalin at 4°Covernight and embedded in paraffin. For Western blot analyses,a piece of tissue was immediately frozen in liquid nitrogen andstored at280°C.

Immunostaining. Immunostaining was performed by anavidin-biotin complex method, as described previously (7). Ananti-pRb antibody (G3-245; Pharmingen) was applied to thesections at a dilution of 1:50. A negative control section, towhich normal mouse serum had been applied rather than thespecific antibody, was included in each staining procedure. Theentire series of samples was stained at least twice, with sepa-rately prepared sections, and similar results were obtained. Witheach section, 10 fields were randomly picked under high powermagnification of the microscope, more than 700 cells werescored for positive or negative nuclear staining, and the percent-age of pRb-positive cells was determined. If a nucleus displayedbrown staining, it was considered positive.

Construction of Antisense and Sense Oligos to RbmRNA. An antisense phosphorothioate oligo to Rb mRNA(AS-Rb) was designed to target the translation start site. It wassynthesized by standard methods using a 380B DNA synthesizer(Applied Biosystems Inc., Foster City, CA; Ref. 19). As acontrol, the corresponding S-Rb was synthesized. The se-quences of these oligos and the corresponding region of thehuman Rb mRNA are shown below.Antisense oligo: 39-CAGTACGGCGGGTTTTGG-59;sense oligo: 59-GTCATGCCGCCCAAAACC-39;

Rb mRNA: 59-GUCAUGCCGCCCAAAACC (AUG is thetranslation start site).These 18 mer oligos are identical or similar to those used inprevious studies (20–22).

Transfection of Oligos. Cells were plated at the appro-priate numbers, according to the plate size, and grown for 48 hin the standard medium, at which time they were in the expo-nential growth phase and the monolayer was 40–50% confluent.They were then rinsed once with serum-free medium and trans-fected with a 1mM solution of the indicated oligo together withthe lipofectin reagent (Life Technologies, Inc., Gaithersburg,MD), which were premixed for 15 min after dilution, as recom-mended by the manufacturer, in the medium (DMEM forHCT116 cells and RPMI 1640 for WI38 cells). The amount oflipofectin (19mg/ml) and volume of medium (3–4 ml for a 6-cmdish) used were proportional to the plate area. Four hours later,fetal bovine serum was added at a final concentration of 10%.The toxicity of the lipofectin was monitored by cell viabilityusing trypan blue dye exclusion and found to be minimal be-cause the viability for HCT116 and WI38 cells at 48 h aftertransfection was over 96%.

Western Blot Analysis. Western blot analysis was per-formed, as described previously (23). Equal loading of theprotein samples was confirmed either by Coomassie blue stain-ing or by immunoreactivity with an antiactin antibody (Sigma).The intensities of the bands were quantitated with an imagescanner (Molecular Dynamics).

Antibodies. The following antibodies were used for de-tection of various human proteins, using the concentrationsrecommended by the manufacturer: (a) mouse monoclonal an-tibodies: Rb (G3-245) and p16Ink4 (Pharmingen), and p21Waf1

(Ab-1; Oncogene Science); (b) rabbit polyclonal antibodies:cyclin A, cyclin D1, CDK2, CDK4, and cyclin E (UBI); actin(Sigma); p27Kip1 (C-19) and CDK6 (Santa Cruz).

Flow Cytometric Analysis. Flow cytometric analysiswas performed, as described previously (23).

In Vitro Assay for Cyclin D1, Cyclin E, and CDK2-associated Kinase Activities. In vitro assays for cyclin D1-,cyclin E-, and CDK2-associated kinase activities were per-formed as described previously (23). For the cyclin D1-associ-ated kinase assay, 200mg of the cell extracts were immunopre-cipitated with 2mg of the cyclin D1 antibody. The GST-Rbfusion protein (1mg) was incubated with the immunoprecipitateplus 10mCi [g-32P]ATP for 30 min at 30°C. For the cyclin E-or CDK2-associated kinase assay, 100mg or 50 mg of total cellextracts, respectively, were immunoprecipitated with 2mg ofthe cyclin E antibody or 1mg of the CDK2 antibody. Theseimmunoprecipitates were incubated with 5mg of Histone H1(Sigma) as the substrate and 5mCi of [g-32P]ATP for 30 min at30°C. The reaction mixtures were then subjected to SDS-PAGE,and the intensities of phosphorylation of the substrates weredetermined by autoradiography.

In Situ Apoptosis Detection Using the TUNEL Assayand DAPI Staining. For the detection of apoptotic cells, aninsitu apoptosis detection kit ApopTag (Oncor, Gaithersburg,MD) was used, as recommended by the manufacturer (20). Forthe DAPI staining, the cells were incubated with DAPI solution(Boehringer Mannheim, Indianapolis, IN) at a concentration of1.5mg/ml PBS for 30 min and then washed in PBS for 2 h. For

1806Antisense to Rb in Colon Cancer Cells



quantitation of apoptotic cells, 10 microscopic fields were ran-domly chosen at325 magnification, and a series of pictureswere taken under light (for whole cells) or fluorescence (forTUNEL-positive cells or for DAPI-positive cells) microscopy.The total number of the cells and the number of apoptotic cells(those with a fluorescent signal by TUNEL staining and thosewith a nuclear fragmentation or a condensation by DAPI stain-ing) were counted in each field. Over 700 total cells werecounted in each sample, and the percentage of the apoptotic cellswas calculated.

Luciferase Reporter Assay for E2F Activity. E2F ac-tivity was determined by a luciferase reporter assay. The lucif-erase reporter plasmid E2F-Luc contains four repeats of thewild-type E2F site (TTTCGCGC) upstream of a minimal TATAbox, followed by the luciferase coding sequence (24). To nor-malize the transfection efficiency, the E2F-Luc plasmid wascotransfected with a CMV-bgal (galactosidase) plasmid at aratio of 2:1. At the indicated time, the cell extracts were pre-pared and assayed for luciferase andb-galactosidase, as de-scribed previously (25). The luciferase level in each sample wasnormalized by the corresponding value forb-gal activity.

Colony Efficiency Assay. Cells were seeded at a densityof 1 3 105/well in 6-cm dishes. Twenty four hours later, thevectors encoding wild-type E2F (CMV-Ap12-Stu) or mutanttype E2F (CMV-Ap12DDS) under CMV promoter and thecorresponding control vector (26) were cotransfected with thepBabe plasmid encoding puromycin resistance at a ratio of 5:1,using the lipofectin reagent. Forty-eight hours after transfection,the cells were subplated into 15-cm dishes and then selected inthe presence of 0.5mg/ml puromycin in complete medium for2 weeks. Individual drug-resistant clones were stained withGiemsa solution (Sigma).

Statistical Analyses. Statistical analyses were performedusing In Stat version 2.01. To determine correlations betweenthe expression of pRb and histological types, Fisher’s exact testwas used. The differences obtained between the S-Rb andAS-Rb treatment of the HCT116 cells was determined by theunpairedt test.

RESULTSpRb Expression during Multistage Colorectal Carcino-

genesis. The expression of pRb was examined by immunohis-tochemistry in the following set of colorectal samples: normalmucosa (n5 50), adenomatous polyps (n5 50), focal carcino-mas within adenomatous polyps (n 5 15), and various stages ofcarcinomas (n 5 50; Fig. 1 and Table 1). The extent of expres-sion of pRb was divided into three categories, based on thepercentage of cells that were positive for pRb, as follows:,10%, 10–50%, and.50%. In the normal mucosa samples,pRb-positive nuclei were seen only in the transitional zone ofthe crypt in the lower portion of the glands and not the upperportions of the crypts, and also in the germinal centers oflymphoid follicles (Fig. 1A). The percentage of pRb-positiveepithelial cells in all of the normal mucosa samples was,10%(Table 1). When compared with the normal mucosa samples, 14of 50 (28%) adenomatous polyps, 13 of 15 (87%) focal carci-nomas in polyps, and 39 of 50 (78%) carcinomas displayedincreased expression of pRb. When a cut off of.50% was usedto define very high expressors of pRb, none of the normalmucosa samples, 8% of the adenomatous polyps, 47% of thefocal carcinomas, and 34% of the carcinomas fit this criterion(Table 1). The differences between the normal mucosa andtumor samples and between the adenomatous polyps and focal

Fig. 1 Immunohistochemicalstaining for pRb expressionwith an antihuman pRb anti-body in normal colonic mucosa(A), an adenomatous colonicpolyp (B), a focal cancer in anadenomatous polyp (C), and acolon carcinoma (D). In thenormal mucosa, the positivestaining for nuclear pRb wasconfined to the lower portion ofthe crypts and the germinal cen-ter of lymphoid follicles (A).The upper regions of the nor-mal crypt (not shown here) didnot show pRb staining. In thepolyps and carcinomas, pRb-positive cells were scatteredthroughout the lesions. Magni-fication: A, 366; B, 366; C,350; D, 350.

1807Clinical Cancer Research



cancers were significant (P 5 ,0.0001 and 0.0018, respective-ly), but the differences between the focal carcinomas and car-cinomas was not significant (P 5 0.381). These findings indi-cate that during the transition from normal mucosa toadenomatous polyps to carcinomas there is a progressive in-crease in pRb expression. No significant correlations were foundbetween the extent of pRb expression and other clinicopatho-logic parameters (Table 1), but we should emphasize that onlya relatively small number of cases were available in each dataset. Phosphorylation status of pRb was also examined by West-ern blot analyses with the same antibody that was used forimmunostaining. Ten normal mucosa expressed solely under-phosphorylated pRb, whereas 9 of 10 cancer tissues exhibitedboth hyper- and underphosphorylated pRb. In the adenomatouspolyps, seven of eight samples displayed mainly the underphos-phorylated pRb (data not shown).

Effects of AS-Rb on pRb Expression and Cell Growth.An antisense phosphorothioate oligo to Rb mRNA (AS-Rb) wasdesigned to target the translation start site, and the correspond-ing S-Rb served as a control, as described previously (20–22).The lipofectin reagent (Life Technologies, Inc.) was used as thevehicle. To assess the self-association properties of these oligos,0.5 OD of the AS-Rb or S-Rb was subjected to PAGE in anondenaturing gel (12%), and the gels were stained withethidium bromide solution. A single band was seen, whichmigrated at the appropriate rate for a monomer. No higher-orderstructures were observed (data not shown). To determine theeffects of reducing the level of pRb expression in the HCT116colon cancer cell line, the cells were transfected with 1mM

AS-Rb, 1mM S-Rb, or only lipofectin, as described in “Mate-rials and Methods.” Cell extracts were harvested 46 h later whenthe cells were confluent or still growing and examined byWestern blot analysis with a pRb antibody. With extracts of theconfluent cultures only single Rb bands were detected that wereabout 110 kDa (underphosphorylated forms of pRb; Fig. 2A,top). The treatment with AS-Rb led to a marked reduction in thelevel of pRb. Densitometric analysis indicated that the residuallevel was only about 30% that of the control culture, which wastreated only with lipofectin. In contrast, treatment of the cellswith S-Rb, a sense Rb oligonucleotide, led to only a slight

(,10%) reduction in the level of pRb. In the growing condition,the control culture treated only with lipofectin displayed aprominent doublet, consisting of both the hyperphosphorylated(higher molecular weight) and underphosphorylated (lower mo-lecular weight) forms of pRb (Fig. 2A, bottom). Similar resultswere obtained with untreated HCT116 cells (data not shown).The treatment with AS-Rb reduced the total level of pRb byabout 70%, and the residual pRb protein was mainly the hyper-phosphorylated form. Equal loading of these lanes was con-firmed either by Coomassie blue staining or by parallel Westernblotting with an antiactin antibody (data not shown).

Table 1 pRb expression during multistage colorectal carcinogenesisIn the polyps group of samples, the percentage of pRb-positive

cells showed no significant correlation with the following clinicopath-ologic parameters: location in the colorectum (right side, transverse, leftside, or rectum), size (,10 mm, 10–20 mm, or.20 mm), shape(pedunculate, pseudosessile, or sessile), histology (tubular or tubulo-villous). In the carcinomas group of samples, the percentage of pRb-positive cells showed no significant correlation with tumor size (,3.9cm or .4.0 cm), depth of invasion, Dukes’ stage, site, or metastasis tolymph nodes or the liver.

pRb expression (% of cells positive)

,10% 10–50% .50% Total

Normal mucosa 50 (100%) 0 0 50Adenomatous polyps 36 (72%) 10 (20%) 4 (8%) 50Focal cancers in

adenomatous polyps2 (13%) 6 (40%) 7 (47%) 15

Carcinomas 11 (22%) 22 (44%) 17 (34%) 50

Fig. 2 Effects of AS-Rb and S-Rb on the expression of pRb (A) andon growth (B) in HCT116 cells.A, cells were plated at 1–23 105 in a6-cm diameter dish or at 3–53 105 in a 10-cm dish (for the confluentcultures 5 3 105 cells/6-cm diameter dish were seeded) and thentransfected with 1mM AS-Rb, S-Rb, or lipofectin alone. At 46 h aftertransfection, extracts were prepared and examined by Western blotanalysis for the level of expression of pRb.B, for the growth studies,2 3 104 cells were plated in 24-well plates (1.5-cm diameter) intriplicate for each treatment 2 days before transfection, and the cellnumber was about 83 104 at the time of transfection. At 24, 36, and48 h after transfection, the cells were trypsinized and counted in aCoulter counter to provide growth curves. The data indicate the meanvalues and SDs for triplicate assays.




Growth curves indicated that the culture treated withAS-Rb displayed marked growth inhibition when comparedwith the lipofectin-treated control culture; but, the growth of theculture treated with S-Rb was similar to that of the controlculture (Fig. 2B). These effects of AS-Rb, with respect tocausing a decrease in the level of pRb expression and markedlyinhibiting the growth of HCT116 cells, were reproduced in threeadditional experiments and with three independent preparationsof AS-Rb and S-Rb. In contrast to the effects obtained with theHCT116 cells, when cultures of WI38 human lung fibroblastswere treated under identical conditions with AS-Rb, a reproduc-ible 1.5–2.0 fold increase in growth was seen when comparedwith the lipofectin-treated control culture at 48 h after transfec-tion, but the S-Rb had no significant effect on growth (data notshown). The latter result is consistent with previous evidencethat AS-Rb stimulates the growth of HEL human lung fibro-blasts and keratinocytes (21, 22).

Effects of AS-Rb on Cell Morphology, Apoptosis, andCell Cycle Kinetics in HCT116 Cells. Treatment of mono-layer cultures of HCT116 cells with AS-Rb under the conditionsdescribed above not only inhibited growth but also increased thenumber of nonadherent cells. At 48 h, the number of floatingcells, expressed as percentage of the total cell culture, for thecultures treated with lipofectin alone, S-Rb or AS-Rb, were:4.8 6 0.01%, 7.36 0.36%, and 13.36 0.92%, respectively.The difference between AS-Rb- and S-Rb-treated cultures wassignificant (P5 0.014). In addition, by 48 h some adherent cellstreated with AS-Rb displayed triangular or irregular shaped cellmorphologies, and other adherent cells had an enlarged cyto-plasm (Fig. 3Aa). The S-Rb-treated adherent cells were roundand had a thin cytoplasm (Fig. 3Ab); their morphology wassimilar to that of cultures treated only with lipofectin or theuntreated HCT116 cultures (data not shown).

To investigate whether the above-described effects of

Fig. 3 A, morphologic changes in HCT116 cells.a, at 48 h after transfection with AS-Rb, the HCT116 cells displayed cytoplasmic enlargement andirregular shaped cell morphologies.b, comparable cultures transfected with S-Rb exhibited no morphological change. Magnification,3100.B, in situTUNEL assays for apoptosis. For these assays, 1–23 104 cells were plated in duplicate in 24-well plates (1.5-cm diameter) and were treated withthe oligos or only with lipofectin. Forty-eight hours later, floating cells and adherent cells were collected, pooled, and centrifuged. TUNEL assayswere performed using anin situapoptosis detection kit, ApopTag (Oncor, Gaithersburg, MD), as recommended by the manufacturer. A representativefluorescence microscopy photograph of the cells at 48 h after treatment with AS-Rb displays chromatin condensation and nuclear fragmentationcharacteristic of apoptosis. Magnification,380. C, cell kinetics in HCT116 cultures treated with the AS-Rb. Exponential cultures of HCT116 cellswere transfected with either AS-Rb or S-Rb and, 48 h later, analyzed by DNA flow cytometry, as described in “Materials and Methods.” The figurealso indicates the mean percentage of the cell population in each phase of the cell cycle from three independent experiments. Control cultures gaveresults similar to those seen with the S-Rb-treated culture (data not shown). The AS-Rb-treated cells displayed a subdiploid DNA fractioncharacteristic of apoptosis.




AS-Rb on growth inhibition and morphological changes inHCT116 cells were associated with apoptosis, we carried outinsitu TUNEL assays to assess the extent of DNA cleavage. Theculture treated with AS-Rb for 48 h displayed numerous positivecells. A representative photograph of the AS-Rb-treated cells isshown in Fig. 3B. The changes of the fluorescein-stained cellswere characteristic of apoptosis (i.e., nuclear condensation andfragmentation). The cultures treated with lipofectin or S-Rbdisplayed only an occasional positive staining cell, and controlsamples in which PBS was added instead of the TdT enzymegave negative results (data not shown). The percentage ofTUNEL-positive cells was calculated (see “Materials and Meth-ods”), and the values for the cultures treated with lipofectinalone, S-Rb or AS-Rb, were: 1.646 0.81%, 2.876 0.64%, and17.39 6 0.47%, respectively. Thus, when compared with thelipofectin-treated culture, S-Rb caused less than a 2-fold in-crease, but AS-Rb caused about a 10-fold increase in apoptosis.The difference between AS-Rb- and S-Rb-treated cultures wassignificant (P5 0.0015). These TUNEL assays were repeatedthree times, and similar results were obtained. DAPI stainingalso confirmed this difference in apoptosis (P 5 0.003), with thefollowing values of percentage of apoptotic cells: lipofectin,2.24 6 0.25%; S-Rb, 4.126 0.17%; and AS-Rb, 12.9460.61%.

To further assess the mechanism of growth inhibition ofHCT116 cells by AS-Rb, we analyzed cell cycle kinetics byDNA flow cytometry of cultures treated for 48 h with eitherS-Rb or AS-Rb. The results obtained with the S-Rb were similarto those obtained with cultures treated only with lipofectin (datanot shown). The mean values and SD for each cell cycle phasewere determined from three independent experiments and areshown in Fig. 3C. There was no significant difference in thefraction of cells in each phase of the cell cycle between AS-Rb-and S-Rb-treated cells. However, the AS-Rb-treated cells dis-played an increase in the early sub-G1 DNA fraction, providingfurther evidence for an increase in apoptotic cells (Fig. 3C).

Effects of AS-Rb on the Expression of Other Cell Cycle-Related Genes. It was of interest to examine the effects ofreducing the level of pRb in HCT116 cells on the expression ofvarious positive and negative regulators of the cell cycle.HCT116 cells were transfected with either S-Rb or AS-Rb, and24, 32, or 46 h later, extracts were prepared and examined byWestern blot analyses for levels of expression of several pro-teins (Fig. 4A). The intensities of the various bands were quan-titated by densitometry.

Treatment of the cells with AS-Rb led to about a 50%reduction in the levels of cyclin D1 and p21Waf1 and about a30% reduction in the levels of p27Kip1, cyclin E, and CDK4.Slight or no major changes were seen in the levels of cyclin A,CDK6, or CDK2. We did not detect the p16Ink4 protein in anyof the HCT116 cultures, presumably reflecting inactivation ofthis gene, although when used as a positive control, WI38 cellsgave a strong p16Ink4 band on a Western blot.

In vitro kinase assays were also performed with the 46-hprotein extracts (Fig. 4B). Assays for cyclin D1-associated ki-nase activity using a GST-Rb fusion protein as substrate indi-cated that there was an appreciable reduction of this activity inthe AS-Rb-treated cells. This activity was only 486 5% thatobtained with the extract from the S-Rb-treated cells. On the

other hand, there was no major change in cyclin-E-associated orCDK2-associated histone H1 kinase activity. The studies shownin Fig. 4 were repeated three times and gave similar results.

E2F Reporter Assays. Because it seems that the majorfunction of pRb in regulating the G1 to S transition of the cellcycle is to inhibit activity of the transcription factor E2F, it wasof interest to use an E2F-luciferase reporter to examine E2Factivity in HCT116 cells cotransfected with S-Rb or AS-Rb. Theluciferase activity was normalized by performing simultaneouscotransfection assays forb-galactosidase (see “Materials andMethods”). Treatment with S-Rb caused a slight (,3.5-fold)increase in E2F activity, but treatment with AS-Rb caused abouta 17-fold increase in E2F activity (P 5 0.003) (Fig. 5A). Similarresults were obtained in two additional studies (data not shown).

The finding of a marked increase in E2F activity in theAS-Rb-treated cells suggested that this might be the reason forthe induction of apoptosis because enhancement of E2F activitycan induce apoptosis in other types of carcinoma (27). There-fore, we examined the direct effect of E2F overexpression on thegrowth of HCT116 cells, using the following plasmids; CMVwild-type E2F, CMV mutant E2F, and a CMV vector controlplasmid. To determine the level of E2F activity that could beinduced with the wild-type E2F plasmid, we first carried out

Fig. 4 A, effects of AS-Rb on the expression of cyclins, CDKs, andCKIs. HCT116 cells were transfected with S-Rb or AS-Rb, and cellextracts were prepared 24, 32, and 46 h later. Protein (50mg) wasapplied to each lane and subjected to PAGE and Western blot analysiswith the appropriate antibodies. The relative intensities of the proteinbands were determined by densitometry. For additional details see“Materials and Methods.”B, in vitro kinase assays. Cells (3–53 105)were plated in a 10-cm dish and then treated with oligos. Forty-six hlater, the cells were harvested and lysed with kinase lysis buffer [50 mM

HEPES, 150 mM NaCl, 2.5 mM EGTA, 1.0 mM EDTA, 1.0 mM DTT,0.1% Tween 20, 10% Glycerol, 10 mM glycerophosphate, 1.0 mM NaF,0.1 mM Na3VO4, 10 mg/ml leupeptin, 10mg/ml aprotinin, and 1.0 mMPMSF (pH 7.5)]. After sonification, the extracts were clarified at15,000 3 g for 10 min at 4°C, and the supernatant fraction wascollected.In vitro assays for cyclin D1-, cyclin E-, and CDK2-associ-ated kinase activities were performed as described previously (23).




reporter assays. We found that cotransfection of the wild-typeE2F plasmid and the E2F reporter plasmid into HCT116 cellsyielded about a 25-fold increase in E2F activity when comparedwith the vector control plasmid, but this marked induction wasnot seen by cotransfection with the mutant E2F plasmid (Fig.5B). In an additional assay, the level of E2F activity using thewild-type E2F plasmid was again about 17-fold higher than thatwith vector control plasmid. This high level was similar to thatinduced with AS-Rb on growth (Fig. 5A). We then performedcolony efficiency assays to examine the effects of this markedincrease in E2F activity. The cells were transfected with each ofthe above plasmids, transfectants were selected with puromycinfor 2 weeks, and the puromycin-resistant colonies were stained.A clear inverse correlation was seen between the ability to formcolonies and the levels of E2F activity (Fig. 6). Furthermore,during selection, morphological changes similar to those seen inthe AS-Rb-treated cells, including cytoplasmic enlargement and

irregular-shaped cell morphologies, were frequently seen in thecultures transfected with the wild-type E2F plasmid, but not inthe cultures transfected with the mutant E2F or vector controlplasmids (data not shown). These assays on colony formationwere repeated three times, and similar results were obtained.

DISCUSSIONIn the present study, we performed immunostaining of the

pRb protein in normal human colonic mucosa, adenomatouspolyps, early carcinoma in polyps, and advanced colon cancers.To our knowledge, this is the first immunohistochemical anal-ysis for pRb expression using boundary lesions such as adenomaand carcinoma in adenoma. In the normal mucosa, pRb waslocated in the transitional zone of the crypt at the lower part ofthe glands where there is a homeostatic state of equilibriumbetween cell proliferation and cell differentiation (28). It was ofinterest that in the normal crypt, lack of expression of pRb wasobserved in the upper portion of crypt where growth arrest andapoptosis occurs (29, 30). This is consistent with the findings ofour in vitro study (Figs. 2–4), indicating that decreased expres-sion of pRb, induced by AS-Rb, leads to growth inhibition andapoptosis in HCT116 colon cancer cells. Thus, if loss of Rbexpression is mechanistically associated with growth inhibitionand apoptosis in the normal crypt, the maintenance of increasedRb in the colonic epithelial cell may prevent this negativegrowth process from occurring. Previous studies (5–8) havefound that there is often increased expression of pRb in humancolorectal carcinomas. The present study confirms this finding

Fig. 5 A, luciferase reporter assays for E2F activity. The HCT116cells were plated at 0.53 105/well (3.5-cm diameter) in triplicate 48 hbefore transfection. The E2F-Luc (2mg) and CMV-b gal (1 mg) plas-mids were cotransfected together with either AS-Rb or S-Rb oligos orwith only lipofectin. Twenty-four hours after transfection, the cells werewashed twice with cold PBS and lysed with 300ml of reporter lysisbuffer (Promega) for 15 min at room temperature. After centrifugation,30 ml of supernatant fraction was used for the luciferase assays, asdescribed previously (25).b-gal activity was determined using theb-Galactosidase Enzyme Assay System (Promega), as recommended bythe manufacturer. The luciferase level in each sample was normalizedby the corresponding value forb-gal activity. The increase in E2Factivity in the AS-Rb-treated cells when compared with the S-Rb-treatedcells was highly significant (P 5 0.0031).B, induction of E2F activityby forced expression of wild-type E2F. Onemg of wild type E2F,mutant E2F, or control vector was cotransfected with 1mg of E2Freporter plasmid and 0.5mg of the CMV-b gal plasmid. Forty-eight hlater, cells were harvested and then examined for the luciferase activity.Luciferase assays indicated that the E2F wild type construct yieldedincreased E2F activity by about 20-fold.

Fig. 6 Colony efficiency assay with overexpression of wild type, ormutant type E2F. Cells were seeded at a density of 13 105/well in 6-cmdishes. Twenty-four h later, the vectors encoding wild-type E2F, ormutant type E2F under CMV promoter, and the corresponding controlvector were cotransfected with the pBabe plasmid encoding puromycinresistance, at a ratio of 5:1 (2.5mg of each plasmid and 0.5mg of pBabeplasmid), using the lipofectin reagent. Forty-eight hours after transfec-tion, the cells were subplated into 15-cm dishes and then selected in thepresence of 0.5mg/ml of puromycin in complete medium, for 2 weeks.Individual drug-resistant clones were stained with Giemsa solution(Sigma).




and, furthermore, demonstrates that this increase is progressiveduring the multistage process of colorectal carcinogenesis be-cause we found a progressive increase in pRb expression duringthe transition from normal mucosa to adenomatous polyps tocarcinomas (Table 1 and Fig. 1). This is surprising because inprevious studies (10, 17) we found that during colorectal carci-nogenesis there is an increase in staining for the proliferationmarker Ki67, and also an increase in the expression of cyclinD1, which would be expected to enhance cell proliferation.Western blot analyses for pRb with the same antibody used forimmunostaining indicated that this antibody reacts with both thehyper- and underphosphorylated forms of pRb, as describedpreviously (7, 31). It is known that the percentage of phospho-rylation of pRb is higher in colon carcinoma tissues than innormal mucosa, thus suggesting a mechanism for inactivation ofpRb in the colon carcinomas. However, in a subset of coloncarcinomas, there is a significant increase in the absoluteamount of underphosphorylated pRb (6, 7), which could bind toand inactivate E2F. These findings seem paradoxical for a tumorsuppressor gene, which in its active form, inhibits the G1 to Stransition in the cell cycle (32).

In an attempt to understand this paradox, we carried outmechanistic studiesin vitro to examine the effects of decreasingthe level of pRb expression in the human colon cancer cell lineHCT116, which expresses a high level of this protein. For thispurpose, we used an antisense oligonucleotide sequence (AS-Rb) targeted to Rb mRNA because previous investigators hadshown the specificity of this AS-Rb in other cell systems (20–22). We found that introduction into the HCT116 cell line of theAS-Rb caused about a 70% inhibition in the level of expressionof pRb. This was associated with a marked inhibition of cellgrowth in vitro, changes in cell morphology, induction of apo-ptosis, altered levels of expression of several cell cycle relatedproteins, inhibition of cyclin D1 associated kinase activity, anda marked increase in E2F activity. These pleiotropic effectswere highly reproducible and seem to be secondary to thereduced levels of pRb because of their time course and the factthat they occurred with relatively low concentrations of AS-Rb(1 mM). A similar sense Rb oligonucleotide, S-Rb, had a slighteffect on some of these parameters, perhaps due to partialinhibition of translation of Rb mRNA, but the effects of theAS-Rb were always much more dramatic (Figs. 2–5). In addi-tion, a preliminary study on tumorigenicity in nude mice showedan apparent growth inhibition of HCT116 cells when treatedwith AS-Rb (data not shown).

As previously emphasized (33), studies using antisenseoligos must be interpreted with caution because of the possibil-ity of obtaining nonspecific effects with these materials. In thepresent study, we followed several guidelines (34, 35) to min-imize such effects. These included using a relatively short (18mer) oligomer that does not have apparent higher order struc-ture, using a low concentration of the oligomer (1mM), andusing the cationic lipid carrier lipofectin (which allows us to usea lower concentration of the oligomer and also reduces thepossibility of degradation of the oligomer to toxic derivatives).The use of lipofectin as a carrier (36, 37) also permits egress ofcharged oligos from the intracellular vesicular compartment.Thus, the extracellular concentration of phosphorothioate oligoscan be kept low, and nonsequence specificity can be avoided.

Furthermore, it is clear that our AS-Rb exerted the expectedbiochemical effects because the treated cells displayed a markeddecrease in the level of pRb and a marked increase in E2Factivity, and these effects were not seen with the S-Rb construct(Figs. 2 and 5). In addition, it is unlikely that the phenotypiceffects of the AS-Rb on HCT116 cells were due to nonspecifictoxicity because, as mentioned below, treatment of WI38 cellswith the same AS-Rb construct actually stimulated the growthof these cells.

Previous studies indicated that introduction of a full-lengthRb cDNA or micro injection of pRb into Rb-deficient osteosar-coma cells or melanoma cells inhibited growth (32, 38) and thatantisense oligos to Rb stimulated the growth of human fibroblastand keratinocytes (21, 22). We also confirmed that transfectionof our AS-Rb into WI38 human lung fibroblasts under theidentical conditions used with HCT116 cells stimulated growth.These findings are what would be expected with a tumor sup-pressor gene. The growth inhibition seen when the level of pRbwas reduced in HCT116 cells is, therefore, somewhat surprising.This situation is not, however, unique since a previous studyindicated that an antisense Rb oligonucleotide also inhibited thegrowth of rat hepatocytes (20). In addition, although introduc-tion of a wild-type Wilms’ tumor suppressor gene (WT1) cDNAinto NIH3T3 cells inhibited proliferation (39), treatment ofchronic leukemia cells with an WT1 antisense oligonucleotideinhibited growth (19, 40). Thus, the phenotype effects of de-creasing the expression of certain tumor suppressor genes ishighly dependent on cell context.

The reduction in the level of pRb in the HCT116 cells wasassociated with changes in several other cell cycle control genes.The most striking change was a decrease in the level of cyclinD1 (Figure 4A). This, plus a decrease in the level of CDK4,probably explains the marked decrease in cyclin D1-associatedkinase activity (Fig. 4B). The latter effect may be responsible, atleast in part, for the overall growth inhibition seen in theAS-Rb-treated HCT116 cells. Our finding that decreased ex-pression of pRb was associated with decreased expression ofcyclin D1 and p27Kip1 is consistent with previous evidence forthe existence of feedback control mechanisms that coordinatethe levels of these three proteins. Thus, pRb-deficient tumorsoften have decreased levels of cyclin D1 (41), and increasedexpression of cyclin D1 is often associated with increased levelsof pRb and p27Kip1 in human esophageal and colon carcinomacells (42, 43). It is of interest that the AS-Rb-treated cells alsodisplayed some reduction in the levels of p21Waf1 (Fig. 4A). Thedecreased levels of p21Waf1, and possibly CDK4, in the AS-Rb-treated cells might be secondary to the decrease in cyclin D1because there is evidence that cyclin D1 can induce p21Waf1

expression (44). Furthermore, in unpublished recent studies, wehave found that retrovirus-mediated transduction of a cyclin D1cDNA sequence into HCT116 cells results in a marked increasein the expression of pRb, p27Kip1, p21Waf1, and CDK4. It is alsoof interest that increased expression of pRb, cyclin D1, andp27Kip1 is often seen in primary colorectal carcinomas (5–8, 10,17–18), and concurrent overexpression of cyclin D1 and CDK4is seen in the intestinal adenomas of patients with familialadenomatous polyposis coli (45). Thus, the abrupt drop incellular levels of pRb in the HCT116 cells, which occurred inthe present study after transfection of AS-Rb, could disrupt a




complex network of feedback control mechanisms in coloncancer cells.

It is also of interest that treatment of the HCT116 cells withAS-Rb induced apoptosis, as assessed by the TUNEL assay,DAPI staining, and DNA flow cytometry (Fig. 3). These resultssuggests that the high level of expression of pRb in HCT116colorectal carcinoma cells might have a selective advantage byprotecting them from apoptosis. This finding is consistent withevidence that pRb can protect osteosarcoma cells, bladder car-cinoma, and hepatic carcinoma cells from apoptosis induced byceramide, INF-g, ionizing radiation and transforming growthfactor-b (20, 46–48). Using an E2F-luciferase reporter assay,we found that treatment of HCT116 cells with AS-Rb led to a17-fold increase in E2F activity as compared with control cul-tures treated with lipofectin (Fig. 5A). Although E2F can func-tion as either an oncogene or tumor suppressor gene, dependingon the cellular environment (49, 50), our findings of the toxicityand morphological changes with forced expression of wild-type-E2F (Figs. 5B and 6) suggest that dysregulation of E2F activitymay be responsible for the induction of apoptosis and alsocontribute to the growth inhibition seen in the AS-Rb-treatedHCT116 cells. Similar effects of E2F on apoptosis were seen inseveral cell lines, including breast and ovarian cancer cells (27,50, 51). However, we do not know the mechanism that mediatesthe apoptosis because the AS-Rb-treated HCT116 cells showedno change in the level of expression of p53, Bax, or Bcl-2,although they did show a decrease in Bcl-XL (data not shown).Nor can we rule out the possibility that other transcriptionfactors that bind to pRb (52, 53) may also be involved in thegrowth inhibition and apoptosis seen with AS-Rb.

Our strategy to transfect the AS-Rb into another coloncancer cell line, SW480, to reduce the level of pRb has thus farnot been successful, probably because the transfection effi-ciency using lipofectin was much higher in the HCT116 cells(data not shown). However, the present findings are not limitedto HCT116 cells because CMV plasmids encoding wild-typeE2F or Rb cDNA in the antisense orientation significantlyinhibited colony formation in the SW480 colon cancer cell line,as well as in HCT116 cells (data not shown). Because the statusof the p53 gene is wild type in HCT116 cells and mutant inSW480 cells (54, 55), these findings are consistent with aprevious study indicating that overexpression of E2F can induceapoptosis in breast cancer cell lines, irrespective of their p53status (27).

Regardless of the precise mechanisms, it is apparent fromthe present study that a decrease in the level of pRb in HCT116cells adversely affects their growth and viability, perhaps be-cause the increased level of pRb maintains a homeostatic bal-ance between positive and negative factors that control the cellcycle and cellular proliferation at least in certain colon carci-noma cell lines. Indeed, it seems that a majority of colon cancercases display overexpression of the E2F-1 protein (56). Homeo-static mechanisms may also explain why a subset of somehuman tumors express relatively high levels of two additionalnegative regulators of the cell cycle, p27Kip1 (17–18, 57–59) andp21Waf1 (60–62). Evidence is accumulating that increased ex-pression of pRb occurs in subsets of bladder, leukemia, andbreast cancer (31, 63–66). It is also likely that increased ex-pression of pRb in some tumors may be the results of loss of

upstream pRb-regulatory genes such as p16 (67). Taken to-gether, these findings suggest that the multistage process ofcarcinogenesis does not simply involve the step-wise activationof growth-promoting proto-oncogenes and inactivation ofgrowth inhibitory tumor suppressor genes. The evolving popu-lation of tumor cells may also have to upregulate the expressionof certain negative-acting genes to maintain a homeostatic bal-ance that favors optimal growth and viability. This conceptwould help to explain the complex and heterogeneous pheno-types of cancer cells and might also have implications withrespect to novel approaches to cancer chemoprevention andtherapy.

ACKNOWLEDGMENTSWe thank Dr. S. Chellappan (Columbia University, New York,

NY) for providing the plasmid used for the E2F luciferase reporterassay; Dr. W-H. Lee (The University of Texas Health Science Center atSan Antonio, San Antonio, TX) for providing the wild type and mutantE2F expression plasmids; Y. J. Zhang, E. Okin, and W-Q. Xing forexcellent assistance; and other members of the Weinstein Laboratory forhelpful advice and discussions. We are also grateful to B. Castro and P.Jean-Louis for valuable assistance in the preparation of this manuscript.

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1999;5:1805-1815. Clin Cancer Res Hirofumi Yamamoto, Jae-Won Soh, Takushi Monden, et al. Carcinomas May Protect Cells from ApoptosisParadoxical Increase in Retinoblastoma Protein in Colorectal

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