[CANCER RESEARCH 52. 3067-3072, June I, 1992]

Increased Expression of Human Ribosomal Phosphoprotein PO Messenger RNA inHepatocellular Carcinoma and Colon Carcinoma1

Graham F. Barnard, Raymond J. Staniunas, Shideng Bao, Ken-ichi Manine. Glenn D. Steele, Jr., John L. Gollan,and Lan Bo Chen3

Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute ¡G.F. B., S. B„M. L., L. B. C.J; Department of Surgery, New England Deaconess Hospital[R. S., K. M., G. D. SJ; and Division of Gastroenterology, Brigham <*Women's Hospital [J. L. G., G. F. B./, Harvard Medical School, Boston, MA 02115

ABSTRACT

To search for differentially expressed gene products in selected cancersof endoderma! origin, cDNA libraries derived from mRNA in humanhepatocellular carcinoma and adjacent grossly normal tissue were generated. From these parent libraries, subtracted cDNA libraries of tumorminus normal and normal minus tumor tissues were constructed. Afterscreening these subtracted libraries by ±hybridization, a cDNA clonethat is overexpressed in hepatocellular carcinoma and encodes the humanacidic ribosomal phosphoprotein PO (PO) was identified. We then evaluated the expression of this phosphoprotein PO in human colon carcinomasamples. Surgical specimens of primary tumors and liver métastaseswereexamined by Northern hybridization of total RNA with one of 2 'I'

labeled PO probes. The mRNA level of the PO was greater in primarycolon carcinoma than in paired adjacent normal colonie epithelium in 36of 38 cases; the mean tumor/normal ratio was 2.7 (range, up to 13). Thetumor/normal ratio, when plotted against the Dukes' stage of disease,

gave evidence for increasing PO expression with increasing stage of coloncarcinoma (P = 0.02). In all 8 cases of paired colon carcinoma metastaticto liver and 2 cases of paired primary hepatocellular carcinoma, the POmRNA level was greater in tumor than in adjacent normal liver tissue.The mean tumor/normal ratio was 4.0 (range, up to 11) for the coloncancers metastatic to liver and 4.2 for the primary hepatocellular carcinoma samples. These findings support a common increased expressionof selected gene products in different tumors of endodermal origin andsuggest that increased POexpression, in line with certain other ribosomalproteins, may be associated with human colorectal cancer progressionand biological aggressiveness.

INTRODUCTION

The identification of markers for colorectal carcinoma withincreased expression in poorly differentiated, advanced, or metastatic lesions could prove extremely useful in tumor detectionor in the estimation of prognosis. Potential new markers fromour laboratories include the laminin-binding protein and ubi-quitin hybrid protein, whose increased mRNA expression appears to correlate with Dukes' classification of colorectal car

cinoma (1, 2). The technique of subtractive cDNA cloning hasbeen used to determine tissue-specific mRNA expression; forexample, the isolation of T-cell versus B-cell receptors (3) andthe finding of a 10-fold reduced expression of low-abundancepreprosomatostatin I mRNA in patients with Alzheimer's dis

ease (4).The common expression of carcinoma-related antigens in

different tissues of endodermal origin, such as liver and colon.

Received 7/23/91; accepted 3/24/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by N1H Grants CA44704 and DK07533. G. F. B.is the recipient of National Research Service Award Grant l F32 OA-0900 fromthe National Cancer Institute. Presented in part at the annual meeting of theAmerican Gastroenterological Association. New Orleans. LA, 1991 (34).

2 Present address: Second Department of Surgery, University of Tokyo 7-3-1Hongo, Bunkyo-ku, Tokyo 113, Japan.

' To whom requests for reprints should be addressed, at Division of Cellularand Molecular Biology, Mayer 840, Dana-Farber Cancer Institute. 44 BinneyStreet. Boston, MA 02115.

has been reported previously (5). Thus, to screen for other suchcommon gene products we elected to generate subtractivecDNA libraries from HCC1 tissue and adjacent normal tissue

from the same patient and to screen the differentially expressedclones against mRNA from a series of gastrointestinal tumorsand their respective adjacent normal tissues. Recent interest inhuman ribosomal phosphoproteins has developed for severalreasons: (a) their increased expression in specific primary andmetastatic tumors (6, 7); (b) the presence of autoantibodiesagainst these proteins in systemic lupus erythematosus (8); and(c) the possibility that these same proteins may function in adual capacity as DNA repair proteins (9). We report our findings with a cDNA clone isolated with preferential overexpres-sion in HCC tissue, which codes for PO, and which was used toprobe samples of primary colon adenocarcinoma, colon ade-nocarcinoma metastatic to liver, and primary hepatocellularcarcinoma.

MATERIALS AND METHODS

Tissue Specimens. Surgical specimens were obtained from the NewEngland Deaconess Hospital Department of Surgery. Thirty-eight pairs

of primary human colon carcinomas and adjacent normal colon tissue,8 pairs of metastatic colon carcinoma to liver and adjacent normalliver, and 3 pairs of primary hepatocellular carcinoma and adjacentgrossly normal liver tissue were obtained fresh from the operatingroom. Necrotic and ulcerated parts of the tumors were removed andnormal colonie epithelium was dissociated from muscle and connectivetissue as applicable. All tissue samples were then frozen rapidly inliquid nitrogen.

Reverse Transcription-Polymerase Chain Reaction Amplification. Fivejig of RNA from each of the HCC and adjacent normal liver samplesused to prepare the cDNA libraries were separately converted to cDNAin the presence of HBV S-gene (10) and HCV 5'-nontranslated or NS3

sequence (11) downstream oligonucleotide primers. PCR amplification(nested PCR for HCV sequences) was completed in the presence of therelevant upstream primers as detailed (10, 11). Controls utilized RNAfrom hepatoma cell line CRL 8024, which contains integrated HBV,and from a donor human liver rejected for liver transplantation becauseof serological markers for HCV. Negative controls omitted the reversetranscriptase enzyme from the incubation. PCR products were separated on agarose gel electrophoresis, transferred to GeneScreenAYuinylon filters (DuPont, Boston, MA) (12), and probed by DNA hybridization with relevant 12P-labeled HBV or HCV probes internal to the

PCR primers (10, 11).Extraction of Total RNA and Northern Blot Hybridization. Total

cellular RNA was extracted from surgical specimens or cell linesaccording to the methods described previously (13), with some modifications and as detailed (1). Equal amounts (IS ¿ig)of total cellularRNA were electrophoresed in 1% agarose-formaldehyde gels. RNA wastransferred to GeneScreen/Yus nylon filters (DuPont) (14). Only those

4The abbreviations used are: HCC, hepatoccllular carcinoma; PO. human

acidic ribosomal phosphoprotein PO: ds, double stranded; ss, single stranded; IBLuria-Bertani medium; HBV, hepatitis B virus; HCV, hepatitis C virus; T, tumor;N, normal; TL, metastatic lesion to liver; NL. adjacent normal liver; TC, primarycolon cancer: NC. adjacent normal colon mucosa; PCR. polymerase chainreaction.

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RIBOSOMAL PO mRNA IN HEPATIC AND COLONIC CANCERS

filters or samples with visually similar intensities of non-degraded T orN 28S and 18S rRNA under UV illumination were used to calculateT/N ratios. Filters were prehybridized, hybridized, washed, and exposedas outlined (1).

For generation of probes: (a) cDNA inserts were cut from clones ofinterest using Xba\/Hind\\\ or Xhol/Sacl endonuclease digestion; (b)independently, a full length PO cDNA clone was isolated from an HT-29 human colon carcinoma cell line cDNA library and was used as asecond PO probe; and (c) a cDNA clone coding a partial sequence of ß-actin (15) was used as an internal control as indicated. The inserts werepurified by agarose gel electrophoresis, recovered using Geneclean (Bio101, La Jolla, CA), and labeled with «-[32P]dCTP(20 MCi, 3000 Ci/

mmolj using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Specific activities of the probes were ~2-5 x10" cpm/^ig DNA. Hybridization signals were quantitated by an LKB

Ultroscanner XL enhanced laser densitometer (LKB Produkter AB,Bromma, Sweden) and analyzed by computer using the LKB 2400GelScan XL software package. The integrated area under the densitycurves produced the tumor/normal ratios as an estimate of mRNAexpression, although the signal density may not be fully linearly relatedto mRNA expression.

cDNA Library Construction. Selection of polyadenylated mRNAfrom total cellular RNA was based on methods described previously( 16) or utilized the Fast-track mRNA selection kit (Invitrogen, La Jolla,CA). A plasmid cDNA library construction system (Librarian II; Invitrogen) was used, with modifications, to generate cDNA libraries fromhepatocellular carcinoma and adjacent grossly normal tissues fromthe same patient. Double stranded cDNAs were synthesized by published methods (17) from 2-5 n% poly(A)* RNA with Oligo(dT)-

priming. Nonpalindromic BST XI cut linkers were (¡gatedto the blunt-ended ds cDNA. Unligated linkers and ds cDNA of size <400 nucleo-tides were removed by size selection through a Sephacryl S-400 exclusion column (Pharmacia, Piscataway, NJ). The sized ds DNA wasligated to pre-prepared BST XI cut vector pcDNA II. Ligation mixtureswere used to transform freshly thawed competent Escherichia coliINVaF'. The cDNA library was stored in 15% glycerol at -70°C, and

aliquots were plated onto LB medium with 50 ¿ig/mlampicillin, 1.5%agar, containing l mM isopropyl l-thio-0-D-galactoside (BRL, Gaith-ersberg, MD) and 0.1% 5-bromo-4-chloro-3-indoyl-|8-D-galactoside(BRL).

Subtraction Libraries. Plasmid cDNA libraries were prepared asoutlined above from: (a) HCC tissue; and (b) adjacent grossly normalliver from the same patient. Two subtraction libraries, HCC minusnormal and normal minus HCC, were prepared using a subtractioncDNA library construction system (Invitrogen) with modifications. Theparent plasmid cDNA libraries were amplified on solid medium (LBwith 50 Mg/ml ampicillin) and then converted to ss DNA. Twenty Mgss DNA from either the HCC or normal liver source were irradiatedfor 15 min under a sun lamp (GE#RSM, 275 W) in the presence ofphotobiotin acetate. The photobiotinylation process was repeated toincrease the efficiency of cross-linking; 2.5 Mg of ss DNA from thealternate HCC or normal liver tissue source were ethanol-precipitatedin the presence of an approximately 8-fold M excess of the photobioti-nylated DNA and, after heating to 100°Cfor 1 min, hybridization wascontinued at 68°Cfor 16-20 h. Photobiotinylated DNA and hybridized

fragments were selectively precipitated with streptavidin (1 mg/ml) and5 M ammonium acetate. The subtractive hybridization process wasrepeated with an 8-fold M excess of the photobiotinylated ss DNA. Thesubtracted ss DNA (about 200 ng) was converted to ds DNA and thentransformed into competent E. coli INVaF' to generate the doubly-subtracted libraries that were stored in 15% glycerol at —¿�70°C.Thesizes of the generated subtracted libraries varied from 1.2 x IO4to 1.3x 10s colonies.

Synthesis of 32P-labeled cDNA from Poly (A)* mRNA. About 1 ^gpoly (A)+ mRNA was converted to ss cDNA using an oligo dT primer,and including 5 n\ (-10 nCi) a-["P]dCTP and avian myeloblastosis

virus reverse transcriptase as detailed in the cDNA probe system(Invitrogen). Labeled cDNA was recovered by exclusion from a Select-D G-50 spin column (5 prime 3 prime, Boulder, CO) and ~1 x IO6cpm with specific activity of ~2-8 x IO7cpm/Vg DNA added to 10 ml

prehybridization medium. Alternatively, aliquots of mRNA sampleswere partially degraded in the presence of carrier tRNA in NaHCO3,pH 9.2, at 85°Cfor 5 min, and then end-labeled with 7-["P]ATP and

T4 polynucleotide kinase according to details outlined in the subtraction cDNA probe kit (Invitrogen p 18-19) to yield probes of similarspecific activity.

Library Screening: ±Hybridization. Aliquots of the subtracted librarywere grown on LB medium with 50 fig/ml ampicillin, 1 mM isopropyll-thio-/J-D-galactoside, and 0.1% 5-bromo-4-chloro-3-indoyl-/3-D-gal-actoside. Selected white colonies with inserts were transferred in triplicate to Duralose-UV membranes (Stratagene) in a grid pattern for ±hybridization using 32P-labeled cDNA probes prepared from the origi

nal tumor and normal sources of mRNA. Probed ±pairs of filters werecompared and differentially expressed clones replated from the masterand reprobed to verify selective expression.

Clinicopathological Data. Age, sex, tumor location, histology, tumorsize, carcinoembryonic antigen level, pathological data relating to depthof invasion, lymph node metastasis, liver metastasis, and follow-up datawere obtained from the hospital records of each patient. The Dukes'staging (18, 19) of the primary tumors was: Dukes' A (tumor invadinginto, but not through the bowel wall); Dukes' B (tumor invading throughthe bowel wall, without lymph node involvement); Dukes' C (withinvolvement of regional lymph nodes); and Dukes' D (with distant

metastasis).Statistical Analysis. For data with n > 30, a standard normal distri

bution was used. For n< 12, Wilcoxon's signed rank statistic was used

to determine significance levels. To examine for a trend towards higherexpression in different Dukes' stage of disease, an analysis of covariance

was used. A P value of 0.025 was required to perform separate tests fortrend for stages A-D and stages A-D and liver metastasis.

RESULTS

cDNA Library Generation and Isolation of Clones EncodingPO. Both HCC minus normal and the complementary normalminus HCC subtraction cDNA libraries representing overex-pressed and underexpressed gene products were prepared, witha view to the inclusion of potential oncogene products andtumor suppressor gene products, respectively. Parent cDNAplasmid libraries in E. coli INVaF' were prepared from mRNA

isolated from samples of human hepatocellular carcinoma andadjacent grossly normal tissue of the same patient. The unam-plified libraries contained about 4 x 104-2 x 10s colonies. After

amplification, subtraction cDNA libraries were prepared usingss DNA and 2 cycles of subtractive hybridization with an 8-foldM excess of the photobiotinylated "undesired" source of DNA.

Initial screening of about 2200 colonies with inserts by ±hybridization yielded 33 clones coding for 17 distinct sequences,which had consistent T > N or N > T expression upon repeatedplating and hybridization. One of these clones had 100% ho-mology over an 873-nucleotide overlap at the 3'-end of the

mRNA of human acidic ribosomal phosphoprotein PO (20),and lacked the 5'-terminal 151 nucleotides from the initiationcodon and the 77 nucleotide 5'-untranslated region. Independ

ently, a full-length PO cDNA clone was isolated from an HT-

29 colon carcinoma cell line cDNA library. Inserts from these2 PO cDNA clones were used as probes for the Northern RNAhybridizations. There were no mutational changes in the cDNAsequence from the clones isolated from either the HT-29 cellline or from the hepatoma library that might have contributedto the altered PO expression.

HBV and HCV may be important in the etiology of HCC(21, 22); we therefore checked the samples of HCC and adjacentnormal liver used to prepare the cDNA libraries for any evidence of HBV or HCV sequences by reverse transcription-polymerase chain reaction and Southern hybridization and

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RlBOSOMAl, PO mtNA IN HEPATIC AND COLONIC CANCERS

found no evidence for these hepatitis viruses in these samples.Furthermore, no hepatitis-related sequences were isolated from

the subtraction and screening process.Northern Blot Analysis of Primary Colon Carcinoma. Fig. 1

illustrates a representative autoradiogram from a NorthernRNA blot, containing 8 paired total RNA samples from patientswith primary lesions of colon carcinoma and adjacent normalmucosa probed first with 12P-labeled PO derived from the HT-29 colon carcinoma cell line and then with "P-labeled cDNAencoding a portion of the /3-actin gene. Each individual pair ofT/N tissues was derived from the same patient. No differencewas noted whether the PO probe was derived from the HCCsubtractive cDNA library (873 nucleotide overlap) or from theHT-29 colon carcinoma cell line (full length). The hydridizationsignals were quantitated by a densitometer scanner. Fig. 2includes the mean tumor/normal hybridization signal densityfor the paired primary colon carcinoma samples (n = 38, mean2.73 significantly different from a ratio of 1.0, P < 0.001). Ahistogram of the distribution of T/N ratios for the individualpaired samples of primary colorectal carcinoma is shown inFig. 3, with a maximum of 13 and median 2.2. To investigatewhether the increased expression of PO mRNA in colon carcinoma correlated with the extent of disease, the T/N ratio of POexpression for paired samples of primary colon cancer wasplotted against the Dukes' stage of disease for the 38 samples

(Fig. 4). A trend of increasing PO expression with increasingstage of colon cancer was observed. Analysis of covariance gaveevidence for a significant effect for stage (P = 0.02 analyzingstages A-D and P = 0.001 analyzing stages A-D and livermétastases).

Clinicopathological Data. The study cohort for the coloncarcinoma patients presented here consisted of 46 patients, 21males, and 25 females. The mean age was 69 years (range, 41-87 years). The distribution of primary colon tumors was asfollows: cecum, 11; "right" colon, 4; transverse colon, 4; sig-

moid colon, 15; rectosigmoid, 3, rectal, 1. Histologically, 40tumors were classified as moderately differentiated adenocar-cinomas; there were one well differentiated and 5 poorly differentiated adenocarcinomas. Samples were classified as Dukes'A, n = 5; Dukes' B, n = 13; Dukes' C, n = 9; and Dukes' D, n

= 11. Liver metastasis was present in 8 patients. Mediancarcinoembryonic antigen level, available for n = 5 Dukes' Apatients was 2 (range, 1-27); n = 8 Dukes' B patients was 3(range, 1-122); n = 7 Dukes' C patients was 7 (range, 1-98); n= 9 Dukes' D patients was 108 (range, 3-4944). The mean (and

median) follow-up for n = 44 patients with more than 1 month

follow-up was 18 months (range, 2-47 months). The diseasestatus at last follow-up was alive and disease-free, 25; alive with

disease, 10; and expired or expired with disease, 11. There wasno correlation of PO mRNA expression with age, sex, tumorlocation, tumor size, or carcinoembryonic antigen level. Noconclusion could be made with respect to the differentiationbecause the majority of specimens was moderately differentiated. There was no change in the quoted levels of significancewhen the extent of disease was staged by the tumor-node-metastasis system.

Northern Blot Analysis of Hepatic Tumors. Hepatic tumorsaccounted for a total of 10 samples. Eight were paired met-astatic colon carcinomas and 2 were primary HCC; all of these10 had T/N ratios >1.0 for the PO hybridization signals. TwoHCC cell lines (HB 8065 and CRL 8024) had signals greaterthan any normal (or tumor) liver tissue values for the sameamount of applied total RNA (data not shown). Fig. 5 illustratesan autoradiogram from a Northern blot of RNA from patientswith primary hepatocellular carcinoma (Fig. 5A, n = 2) andcolon carcinoma metastatic to liver (Fig. 5Ä,n = 3). A singlepatient had 4 simultaneous tissues resected (Fig. 6/4): TL, NL,TC, and NC. This provided the opportunity to compare thelevel of PO mRNA expression, between liver and colon, withoutany confounding interpatient variability. For this patient, theTC/NC ratio of the primary colon carcinoma was 2.2, the TL/NL ratio for the metastatic lesion was 5.0, the metastatic andprimary lesions had similar intensities with a TL/TC of 1.1,and the normal colon had a somewhat higher signal than thenormal liver, NC/NL = 1.7. Two other patients had simultaneous resections of hepatic métastasesand primary colon cancer lesions yielding 3 samples (TL, TC, and NC). Both primaryand metastatic colon tumors showed higher levels of PO mRNAthan did normal colon mucosa in these 2 patients (Fig. 6B). Noconsistent difference was noted in the PO mRNA levels betweenthe primary tumor and the respective liver métastases:in onecase, the signal from the metastatic lesion was stronger (patient2, TL > TC); in the other, the primary was stronger (patient 3,TC > TL) and, as mentioned, the patient of Fig. 6A had TL =TC. To facilitate further comparison between T/N hybridization signals from primary colonie lesions and metastatic lesions,a comparison was made between unpaired normal colon mucosa(n = 4) and unpaired normal liver tissue (n = 5,). Comparingranked or mean signal intensities gave a NC/NL ratio of 1.27(data not shown).

Fig. 2 includes the T/N hybridization signal density of theautoradiogram for the liver tumors probed with PO. The mean

Fig. 1. Northern blot analysis of RNAfrom paired samples of primary colon carcinoma and adjacent normal colonie epitheliumprobed with "P-labeled cDNA encoding hu

man ribosomal phosphoprotein PO(toppanel).and then with "P-labeled cDNA encoding ft-

actin (middlepanel). Bottom panel, agarose gelunder UV illumination with positions of 28SrRNA and I8S rRNA indicated, n = 8 pairs;patient 7 has degraded RNA.

TNTNTNTNTNTNTNTN

PO •¿�-• -

ACTIN

-18S

-18S

-28S

-18S

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RIBOSOMAL PO mRNA IN HEPATIC AND COLONIC CANCERS

0.0*Ho PRIMARY HCC L-MET TOTAL

N = 3B N = 2

Fig. 2. Results of Northern blot analysis of RNA from carcinoma and pairednormal samples probed with 32P-labeled cDNA encoding human ribosomal phos-phoprotein PO. Tumor/normal ratio of hybridization signal density is expressedas mean ±SE (except for HCC ±range). Ho, null hypothesis that tumor =normal; PRIMARY COLON CA, paired samples of primary colon carcinomaand adjacent normal mucosa (n = 38); HCC, paired samples of HCC and adjacentnormal liver (n = 2); L-MET, paired samples of colon carcinoma metastatic tothe liver and adjacent normal liver (n = 8); TOTAL combines the T/N ratios forall the tumors (n = 48).

16-1

<1 1-2 2-3 3-4 4-5 5-1010-15

TUMOR / NORMAL RATIO

Fig. 3. Results of Northern blot analysis of RNA from primary colon carcinomaand paired normal colon samples probed with 32P-labeled cDNA encoding human

ribosomal phosphoprotein PO. The number of tumor pairs is plotted against therange of tumor/normal ratios of hybridization signal density for paired samples(n = 38), each pair from the same patient.

T/N ratio for the metastatic colon carcinoma samples (n = 8)was 4.03, and for all of the hepatic tumors it was 4.06, significantly different from a ratio of 1.0 (P = 0.001). The 2 HCCsamples had a mean T/N ratio of 4.2. The distribution of T/Nratios for the hepatic tumors had a maximum of 10.8 andmedian of 2.1.

To summarize, a total of 48 carcinoma tissue samples andcorresponding paired normal samples were analyzed. ProbingNorthern blots with 32P-labeled PO yielded an autoradiography

signal from T tissue greater than or equal to N in 46 of 48 cases(96%). The signals were consistent with a transcript size of 1.2-1.6 kilobases. Of the paired primary colon carcinomas, 87%had a T/N ratio >1.2, 10.5% had an approximately equal ratio(defined as T/N 0.8-1.2), and 2.5% had a signal less dense thanthe tumor (defined as T/N < 0.8). All of the hepatic tumorshad a T/N ratio >1.2.

DISCUSSION

There is evidence for the dual expression of carcinoma-relatedantigens in tissues of endodermal origin, such as liver andcolon. For example, a monoclonal antibody (SF-25) raisedagainst a human hepatoma cell line (FOCUS) detected a M,125,000 cell surface antigen in 23 of 23 colon adenocarcinomatissues, but not in the adjacent normal mucosa (5). We thus

screened differentially expressed clones, isolated by subtractivecDNA hybridization of hepatocellular carcinoma versus normaltissue, against a series of colorectal carcinoma tissues. One ofthe selectively overexpressed clones in HCC was found toencode the ribosomal phosphoprotein PO, which shares a highlyconserved C-terminal sequence with P1/P2, which are believedto interact with eukaryotic initiation, elongation, and releasingfactors (23-25). PO may be analogous to the prokaryotic LIOprotein (20). The level of PO mRNA expression was increasedin the majority of tumors tested. To reduce the effect of theinherent variability in expression between patients, only T/Nratios (calculated with both T and N samples from the samepatient) were utilized in the analysis.

P-proteins may be readily able to exchange on/off the ribo-some (26). This may contribute to the formation of autoanti-bodies to P-proteins in some patients with systemic lupuserythematosus (8, 27). This raises the possibility that autoanti-bodies may be generated against overexpressed P-proteins inpatients with colorectal cancers. However, assays for IgM/IgGP-protein antibodies in the serum of 11 patients with Dukes'

stage D colon carcinoma were negative. The use of antibodiesagainst P-proteins may permit evaluation of the expression ofthis gene product, at the protein level, on tumor cells fromarchival specimens and an attempt to correlate the data withsurvival statistics. The ready removal of the P-proteins fromribosomes also may be relevant in their proposed role as dualfunction proteins. There is significant homology (65% in 317amino acid overlap) between human PO and Drosophila repairgene AP3 (9). Thus, it is possible that PO has a function bothas an integral ribosomal protein, and additionally as a DNArepair protein free of ribosomal attachment. Phosphorylationof the P-proteins may increase their affinity for the ribosome(28). The proposed role as dual function protein/enzyme isattractive for a protein with the capacity to be phosphorylated,since there are many examples of phosphorylation of tyrosine,serine, and threonine residues affecting regulatory processes(29). Ribosomal protein S6 is phosphorylated during development, tissue regeneration, growth, and transformation, in partby tyrosine-specific kinases (for review, see Ref. 30). Therefore,it will be of considerable interest to further define the natureand role of the phosphorylation status and corresponding struc-tural/enzymic activities of the P-proteins, particularly PO.

With respect to ribosomal protein expression in neoplastictissues, increased expression of L31 mRNA in 23 of 23 colo-

A B C D L-METN r, N = 13 N-.-9 N=11 NI!

DUKES' STAGE

Fig. 4. Results of Northern blot analysis of RNA from primary colon carcinomaand paired normal colon samples, and colon cancer samples metastatic to liverprobed with "P-labeled cDNA encoding human ribosomal phosphoprotein PO.

Tumor/normal ratio of hybridization signal density is expressed as mean ±SE.A, B, C, and D, stage of colon carcinoma by Dukes' classification: A, n = 5; B, n= 13; C, «= 9; D, n = 11. L-MET, colon carcinoma samples metastatic to liver,

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Fig. 5. Northern blot of RNA from primary HCC and colon cancer metastatic to liverprobed with 3!P-labelcd cDNA encoding human ribosomal phosphoprotein PO.A. primaryHCC and adjacent normal liver (n = 2); B,metastatic colon cancer and adjacent normalliver (n = 3).

A.Patient 1

T N

1.2 kb-

2T N

B.123

T N T N T N

A.Patient 1111

TL NL Te Ne

B.222Ne Te TL

333Te Ne TL

1.2kb--

Fig. 6. Northern blot analysis of RNAfrom primary colon carcinoma and metastaticcolon carcinoma samples probed with 32P-la-belcd cDNA encoding human ribosomal phosphoprotein PO. .4, 4 samples from a singlepatient (patient I). TL, NL, TC, and NC; B, 2other patients (patients 2 and 3) with simultaneous primary, metastatic. and normal colonsamples.

rectal tumors was noted (31). After submission of this paper,ribosomal protein S3 mRNA was reported to be increased incolon cancer (32). Additionally, P2 had enhanced mRNAexpression in liver métastasesand in primary colon carcinomacompared to normal colonie mucosa (n = 3) (6, 7). Contrary toexpectations, it also was increased ~5-fold in breast fibroaden-

omas compared to breast carcinomas (7). Thus, for P2 at least,the increased P-protein mRNA expression was not specific forcancer. We did not assess the level of PO mRNA in othernonmalignant colonie diseases (such as diverticulitis, inflammatory bowel disease, or adenomas), and therefore cannotexclude the possibility that increased PO expression may occurin benign conditions. However, we have noted no increased POmRNA expression in gastric carcinoma samples,5 thus the

elevation is not a uniform finding in all cancers, nor even allgastrointestinal cancers. In our laboratory, we have noted increased expression in human colorectal cancer of some otherribosomal proteins including S6 (33) and the ubiquitin-S27ahybrid protein that correlated with Dukes' stage of disease (2).Not all studied ribosomal proteins were increased.'' The basis

for the increased expression of PO observed in this study is notreadily apparent. It is possible that protein synthesis is highlyactivated during progression and métastasesof colon tumors,and that these selected ribosomal proteins, particularly thosesuch as PO, whose expression correlates with Dukes' stage of

disease, may prove useful as markers of biological aggressiveness in colorectal carcinoma.

ACKNOWLEDGMENTS

We thank Dr. B. Spiegelman for providing the actin clone. Dr. P.Lavin for his assistance in statistical analysis of the data. Cathy Grayfor accrual of the clinicopathological data, and Drs. A. Schneebaumand L. Cook at the Lahey Clinic, Burlington, MA, for performing theserum assays for P-protein antibodies.

5G. F. Barnard, unpublished observations.6 R. J. Staniunas. unpublished observations.

REFERENCES

1. Mafune. K., Ravikumar. T.. Wong. J., Yow, H., Chen. L., and Steele, G., Jr.Expression of a M, 32.000 laminin-binding protein mRNA in human coloncarcinoma correlates with disease progression. Cancer Res.. SO: 3888-3891.1990.

2. Mafune, K., Wong, J., Staniunas. R., Lu, M.. Ravikumar, T., Chen, L., andSteele. G., Jr. Ubiquitin hybrid protein gene expression during human coloncancer progression. Arch. Surg.. 126: 462-466, 1991.

3. Hedrick, S.. Cohen, D.. Nielson, E., and Davis, M. Isolation of cDNA clonesencoding T cell specific membrane-associated protein. Nature (Lond.), 308:149-153, 1984.

4. Travis, G., and Sutcliffe. J. Phenol emulsion enhanced DNA driven subtrac-tive cDNA cloning: isolation of low abundance monkey cortex specificmRNAs. Proc. Nati. Acad. Sci. USA. 85: 1696-1700. 1988.

5. Takahashi. H.. Carlson, R., Ozturk. M.. Sun, S.. Motte. P., Strauss. W..Isselbacher, K., Wands. J., and Shouval. D. Radioimmunolocation of métastases from adenocarcinoma of the colon: using a hepatoma antibody. Gastro-enterology, 96: 1317-1329. 1989.

6. Elvin, P., Kerr. I.. McArdle. ( .. and Birnie. G. Isolation and preliminarycharacterization of cDNA clones representing mRNAs associated with tumour progression and metastasis in colorectal cancer. Br. J. Cancer, 57: 36-42, 1988.

7. Sharp. M.. Adams. S.. Elvin, P., Walker, R.. Brammar. W., and Varley, J.A sequence previously identified as metastasis-related encodes an acidicribosomal phosphoprotein P2. Br. J. Cancer, 61: 83-88, 1990.

8. Schneebaum, A., Singleton. J.. West, S., Blodgett. J., Allen, L., Cheronis, J.,and Kotzin, B. Association of psychiatric manifestations with antibodies toribosomal P proteins in systemic lupus crythematosus. Am. J. Mcd., 90: 54-62, 1991.

9. Kelley, M., Venugopal. S., Harless. J., and Deutsch, W. Antibody to humanDNA repair protein allows for cloning of a Drosophila cDNA that encodesan apurinic cndonuclease. Mol. Cell. Biol., 9: 965-973, 1989.

10. Paterlini, P.. Gerken. G., Nakajima, E.. Terre. S., D'Edrico, A., Griglioni.

W., Nalphas, B., Franco, D., Wands, J., Kew. M.. Pisi, E., Tiollais, P., andBrechot, C. Polymerase chain reaction to detect hepatitis B virus DNA andRNA sequences in primary liver cancers from patients negative for hepatitisB surface antigen. N. Engl. J. Med., 323: 80-85, 1990.

11. Shieh, Y., Shim, K-S., Lampertico, P., Balart, L., Jeffers, L., Thung, S.,Regerstein, F., Reddy, K.. Farr, G.. Schiff, E., and Gerber. M. Detection ofhepatitis C virus sequences in liver tissue by the polymerase chain reaction.Lab. Invest.. 65:408-411. 1991.

12. Maniatis. T.. Fritsch, E., and Sambrook. J. Molecular Cloning: a Laboratory'Manual, pp. 393-389. Cold Spring Harbor. NY: Cold Spring Harbor Laboratory Press, 1983.

13. Chirgwin, J., Przybyla, A., MacDonald, R., and Rutter, W. Isolation ofbiologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry. 18: 5294-5299, 1979.

14. Sambrook, J., Fritsch, E., and Maniatis. T. Molecular Cloning: A LaboratoryManual (2nd ed.). Chap. 7.46-7.50. Cold Spring Harbor, NY: Cold SpringHarbor Laboratory Press, 1989.

3071

Research. on February 20, 2021. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

RIBOSOMAL PO mRNA IN HEPATIC AND COLONIC CANCERS

15. Spiegelman, B., Frank, G., and Green, H. Molecular cloning of mRNA from3T3 adipocytes: regulation of mRNA content for glycerophosphate dehy-drogenase and other differentiation-dependent proteins during adipocytedevelopment. J. Biol. Chem., 258: 10083-10089, 1983.

16. Maniatis, T., Fritsch, E., and Sambrook, J. Molecular Cloning: a LaboratoryManual, pp. 197-198. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1983.

17. Gubler, U., and Hoffman, B. A simple and very1effective method for generating cDNA libraries. Gene, 25: 263-269. 1983.

18. Dukes, E. The classification of cancer of the rectum. J. Pathol. Bacterio!.,35: 323-332, 1932.

19. Astler, U., and Collar, F. The prognostic significance of direct extension ofcarcinoma of the colon and rectum. Ann. Surg., 139: 846-851, 1954.

20. Rich. B., and Steitz. J. Human acidic ribosomal phosphoprotein PO. PI, P2:analysis of cDNA clones, in vitro synthesis, and assembly. Mol. Cell. Biol..7:4065-4074. 1987.

21. Beasley. R., and Hwang, L. Viral Hepatitis and Liver Disease, pp. 209-224.New York: Gruñe& Stratton, 1984.

22. Kiyosawa, S., Sodeyama, T., Tanaka, E., Gibo, Y., Yoshizawa, K., Nakano,Y.. Furuta, S.. Akahane, Y., Nishioka, K., Purcell, R., and Alter, H. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellularcarcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology.12: 671-675, 1990.

23. Elkon, K., Skelly, A., Parnassa, A., Moller, W., Dahno, W., Weissbach, H..and Brot, N. Identification and chemical synthesis of a ribosomal proteinantigenic determinant in systemic lupus erythematosus. Proc. Nati. Acad.Sci. USA, 83: 7419-7423, 1985.

24. Elkon, K., Parnassa. A., and Foster, C. Lupus autoantibodies target ribosomalproteins. J. Exp. Med.. 162:459-471, 1985.

25. Towbin, H., Ramjoue, H., Küster,H., Livertani, D., and Gordon, J. Molecular antibodies against eukaryotic ribosomes: use to characterize a ribosomal

protein not previously identified and antigenically related to the acidicphosphoproteins P1/P2. J. Biol. Chem., 257: 12709-12715, 1982.

26. Tsurugi, K.. and Ogata, K. Evidence for the exchange ability of acidicribosomal proteins on cytoplasmic ribosomes in regenerating rat liver. J.Biochem.. 98: 1427-1431. 1978.

27. Bonfa, E., Golombek, S., Kaufman, L., Kelly, S., Weissbach, H., Brot, N.,and Elkon, K. Association between lupus psychosis and anti-ribosomal Pprotein antibodies. N. Engl. J. Med.. 317: 265-271, 1987.

28. Sanchez-Madrid, F., Vidales, F., and Ballesta, J. Effect of phosphorylationon the affinity of acidic proteins from Saccfiaromyces cerevisiae for theribosomes. Eur. J. Biochem., 114: 609-613, 1981.

29. Edelman, A., Blumenthal, D., and Krebs, E. Protein serine/threonine kinases.In: C. Richardson, P. Boyer, I. Dawid, and A. Meister (eds.), Annual Reviewsof Biochemistry, pp. 567-613. Palo Alto, CA: Annual Reviews, Inc., 1987.

30. Erikson, R. Structure, expression, and regulation of protein kinases involvedin the phosphorylation of ribosomal protein S6. J. Biol. Chem., 266: 6007-6010, 1991.

31. Chester, K., Robson, L., Begent, R., Talbot, I., Pringie, J., Primrose, L.,McPherson, A., Boxer, G., Sont hai I, P., and Malcolm, A. Identification of ahuman ribosomal protein mRNA with increased expression in colorectaltumors. Biochim. Biophys. Acta, 1009: 297-300, 1989.

32. Pogue-Geile, K., Geiser, J., Shu, M., Miller, C, Wool, L, Meisler, A., andPipas, J. Ribosomal protein genes are overexpressed in colorectal cancer:isolation of a cDNA clone encoding the human S3 ribosomal protein. Mol.Cell. Biol., //: 3842-3849, 1991.

33. Staniunas, R., Mafune, K., Lu, M., Chen. L., and Steele, G., Jr. Increasedexpression of ribosomal protein S6 in human colon cancer. Surg. Forum, 41:457-459, 1990.

34. Barnard. G., Bao, S., Staniunos, R.. Lu, M., Matune, K., Steele, G., Jr,(rollan. J. and Chen L. Increased expression of human acidic ribosomalphosphoprotein PD mRNA in carcinomas of colonie and hepatocellularorigin (Abstract). Gastroenterology, 100: 348A, 1991.

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1992;52:3067-3072. Cancer Res   Graham F. Barnard, Raymond J. Staniunas, Shideng Bao, et al.   CarcinomaMessenger RNA in Hepatocellular Carcinoma and Colon Increased Expression of Human Ribosomal Phosphoprotein P0

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