[CANCER RESEARCH57. 2961-2965, July 15, 1997]

ABSTRACT

A novel gene encoding a protein containing Sm motif-like domains wasfound to have elevated expression in pancreatic cancer and in several

cancer-derived cell lines. CaSm (for Cancer-associated Sm-like) mRNA isup-regulated in 87.5% (seven of eight) of pancreatic tumor/normal pairs.Similarly, cell lines from cancers originating in liver, ovary, lung, andkidney show increased CaSm expression compared to their normal tissuecognates. CaSm encodes a 133-amino acid open reading frame that contalus the two Sm motifs found in the common snRNP proteins, with the

greatest homology to the Sm G protein (60% similarity). Two hypotheticalproteins from Caenorhabditis elegans and Saccharomyces ceres'isiae shareeven greater similarity (72.8 and 67.7%, respectively), suggesting a broadfamily of proteIns containing Sm motifs. Antisense CaSm RNA is able toalter the transformed phenotype of pancreatic cancer cells by reducingtheir ability to form large colonies in soft agar when compared to untransfected cells. Therefore, CaSm expression appears to be necessary formaintenance of the transformed state.

INTRODUCTION

Ductal adenocarcinoma of the exocrine pancreas is the most common pancreatic tumor type and is the fourth leading cause of cancerdeaths in the United States (1). Because it is usually diagnosed at anadvanced stage (not resectabbe), pancreatic cancer has an extremelypoor prognosis with the 5-year survival of less than 3% (2). Mediansurvival after diagnosis is 6 months. Improved survival is anticipatedif pancreatic cancer can be identified and resected at an early stage.Recent surgical literature reports a higher 5-year survival (up to 20%),primarily in patients with small (<2 cm) tumors (3). Staging of

pancreatic cancer is based upon the degree of metastasis, and patientspresenting with early-stage disease have a much better prognosis thanthose presenting at a bate stage. Successful treatment, therefore, isdependent upon very early diagnosis; thus, it is important to findadditional pancreatic cancer markers that may facilitate this earlydetection.

Aberrant regulation of the mechanisms that control cell growth anddifferentiation results in cellular transformation leading to cancer.

Molecular analysis has demonstrated that multiple mutations in oncogenes and tumor suppressor genes are required to manifest themalignant phenotype. Although the molecular etiology of pancreaticcancer is not defined, several genetic alterations have been detected.For example, the most common changes yet recognized are mutationsin the K-ras oncogene (4) and mutations or homozygous deletions inseveral tumor suppressor genes, including TPS3 (5), p16/MTS-1 (6,

Received 1/27/97; accepted 5/15/97.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by Department of Energy Grant DE-TGO5-94ER61893.

M. W. G. and P. L. B. were supported, in part, by a career development award from theAmerican Cancer Society.

2To whom requests for reprints should be addressed, at Center for Molecular andStructural Biology, Hollings Cancer Center, Medical University of South Carolina, 171Ashley Avenue, Charleston, SC 29425-2213.

3 Present address: Department ofPathology and Laboratory Medicine, Albany Medical

Center Hospital, A-81, Albany. NY 12208.

7), and DPC4 (8). In addition, gene amplification plays a role in some

pancreatic cancers (9).To understand further the mechanisms that bead to pancreatic can

cer, other genetic events need to be identified. Toward this goal, wehave used subtractive hybridization cloning to identify and isolategenes the expression pattern of which is associated with pancreaticcancer (10, 11). This technique was used because it allows theisolation of genes based only upon their preferential expression pattern and is not dependent upon any previous knowledge of the gene.We describe here the isolation and initial characterization of a genecalled CaSm4 that was isolated because of its elevated expression inpancreatic cancer.5 Sm motifs are regions of protein-protein interaction identified in the common core Sm proteins, a subset of snRNPproteins (12). The CaSm gene product was previously unknown andhas limited homology to the Sm motifs found in the common snRNPproteins (12, 13). Most significantly, its expression appears to berequired for at least one characteristic of transformed cells: the abilityof pancreatic cancer cells to grow in an anchorage-independent manner. Its expression is not limited to pancreatic cancer, but it is alsodetected in a variety of normal tissues. Elevated CaSm expression inseveral additional cancer-derived cell lines suggests that it may beimportant in the development of other malignancies.

MATERIALS AND METHODS

Tissues, Cell Lines, and Tissue Culture. Human tissues were procuredfrom The Cooperative Human Tissue Network, Mt. Sinai Medical Center(Miami Beach, FL; from Dr. Saul Suster) and from The Medical University ofSouth Carolina. The cell lines HS680.PAN, Capan-l, and PANC-l wereobtained from American Type Culture Collection (Rockville, MD). They were

maintained in DMEM/10% FBS, RPMI 1640/15% FBS, and DMEM/l0%

FBS, respectively. Transfection of PANC-l cells was performed in 35-mmwells using 2 @.tgof DNA and 10 pi of LipofectAmine (Life Technologies,

Inc., Bethesda, MD) per well (60—80%confluent). Selection for stable transfectants was in 500 @tg/mlG41 8. Soft agar growth assays were performed insix-well plates (35-mm wells). Duplicate assays initiated with 1000, 5000, and

25,000 cells were scored after 3 weeks. The soft agar assay was performedtwice, independently.

eDNA Cloning. Subtractive hybridization was performed betweenHS680.PAN and CAPAN-l as described previously (14, 15). Clone CA3-30was isolated from among the subtractive library clones. Its elevated expressionin CAPAN-l compared to HS680.PAN was verified by Northern blot hybridization. The full-length sequence partially contained in CA3-30 is henceforthknown as CaSm. The insert from CaSm was subcloned in the antisenseorientation into the eukaryotic expression vector pSGneoSK [a modification ofPS05 (Stratagene, La Jolla, CA) containing a neomycin/G4l8-resistance cassette and the multiple cloning site from pBluescript II SKI. This construct wasused for the transfection of PANC-l cells.

RNA Isolation and Analysis. RNA from cultured cells and from humantissues was purified using RNAzol B (Tel-Test, Inc., Friendswood, TX)

according to the manufacturer's protocol. Total RNA was fractionated on 1.2

or 1.5%agarose gels containing 0.66 Mformaldehyde (2.2 Min the sample) by

4 The abbreviations used are: CaSm, cancer-associated Sm-like; FBS, fetal bovineserum.

5 GenBank data base accession no. AF000I77.

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CaSm: An Sm-like Protein That Contributes to the Transformed State in

Cancer Cells'

Clifford W. Schweinfest,2 Michael W. Graber,3 Jeannie M. Chapman, Takis S. Papas, Paul L. Baron, and

Dennis K. Watson2Centerfor Molecular and Structural Biology [C. W. S.. M. W. G.. J. M. C.. 7'. 5. P., D. K. W.J and Department ofSurgerv (P. L BJ, Hollings Cancer Center. Medical University ofSouth Carolina, Charleston, South Carolina 29425

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CaSm PROTEIN CONTRIBUTED TO TRANSFORMATION

nucleotides including a polyadenylation signal at nucleotides 878—883.The translational start signal is contained within the sequenceTCAAAAT@1A (nucleotides 160—168;underline identifies the startingmethionine codon), which contains the requisite purines at positions —3and +4 (17). The largest open reading frame can encode a 133-aminoacid pobypeptide (nucleotides 165—563)ofpredicted molecular weight Mr15,179 and isoelectric point of 4.97. The 133-amino acid polypeptideshares significant homology with the snRNP Sm 0 protein (Fig. 3A). Acomputerized BESTFIT of CaSm and human Sm 0 protein is 32%identical and 60% similar (allowing for conservative amino acid substi

tations). This similarity is nearly completely confined to the NH2 te@@mi@@alhalf of CaSm (amino acids 4—78).Interestingly, this homology localizesto the two Sm motifs that characterize the Sm protein family (12). Smmotif 1 and Sm motif 2, 32, and 14 amino acids, respectively, areresponsible for protein-protein interactions, presumably necessary for the

assembly of snRNP complexes (12). The level of identity between CaSmand Sm G protein is low (32%) compared to the bevel of identity betweenthe Sm G proteins of very distantly related species such as plants andyeast (>50% identity). Other Sm proteins from snRNPs are even lesssimilar to CaSm than Sm 0. Also, at 133 amino acids, CaSm protein isnearby twice the size of human Sm G protein (76 amino acids). Finally,with the exception of Sm F protein (p1, 4.6), all of the Sm proteins havebasic isoelectric points (18). Therefore, it seems unlikely that CaSm is atrue member of the Sm protein family. Nonetheless, most key featuresthat constitute the Sm motifs are retained in CaSm. Specifically, the100% conserved glycine and asparagine residues at positions 13 and 23,respectively, of Sm motif 1 are also found in CaSm. Overall, 12 ofthe 15defined positions in the consensus for Sm motif 1 are conserved in CaSm.Furthermore, 10 of the 11 defmed positions in the Sm motif 2 consensusarealsoconservedinCaSm(Fig.3A).Interestingly,twohypotheticalproteins from Caenorhabditis elegans and Saccharomyces cerevisiae

share even greater similarity to CaSm (72.8 and 67.7%, respectively; Fig.3, B and C@.These two proteins also contain Sm motifs and are mostsimilar to CaSm in those regions.

The predicted open reading frame of CaSm was confirmed by itsexpression in a coupled transcription and translation reaction (data notshown). The putative coding strand translates an Mr 18,000 pobypeptide, whereas the putative noncoding strand produces a much smallerproduct. The Mr 18,000 product observed is somewhat larger than theMr 15,200 predicted from its deduced amino acid sequence. Further

more, only antisense probe to the putative coding strand hybridizes tomRNA from pancreatic cancer cells (data not shown).

No significant similarities were found to any motifs listed in thePROSITE database.

the method of Lehrach et a!. (16). Gels were transferred to Duralon filters(Stratagene) in 0.1 M sodium phosphate (pH 6.8), UV cross-linked, andhybridized in Quik-Hyb (Stratagene) according to the manufacturer's instruc

tions. RNA quantity and quality were monitored by ethidium bromide visualization of the 285 and 185 ribosomal bands.

RESULTS

CaSm Is Up-Regulated in Pancreatic Cancer Tissue. Differentially expressed mRNAs in pancreatic cancer were first identified byperforming subtractive hybridization between the pancreatic cancercell line CAPAN-l and the diploid, more normal pancreatic epithebiabcell line H5680.PAN. cDNA clones obtained by subtractive hybridization were confirmed to be differentially expressed by two assays:(a) DNA from 600 subtractive cDNA clones was dot blotted ontonylon membranes followed by differential hybridization with totalcDNA to CAPAN-l and HS680.PAN mRNA. CaSm was amongthose clones that had a much stronger hybridization signal withCAPAN-l cDNA compared to HS680.PAN cDNA; and (b) CaSmcDNA insert (along with other tentatively identified differentiallyexpressed cDNA clones) was labeled and used to probe a Northernblot of tumor and normal pancreatic tissue RNAs. Fig. 1 shows arepresentative Northern blot for CaSm that includes both matchedpairs of samples (tumor and normal tissues from the same patient) aswell as individual tumor and normal specimens. Eight of nine matchedpairs show significantly higher levels of a 1.2-kb CaSm mRNA intumor/pancreatitis compared to normal. The absolute level of CaSmmRNA is somewhat variable among the samples such that sometumor samples express less mRNA than nonmatched normal samples(Fig. I, compare Lane 1 7T to Lane 18W). However, the matched

samples show a consistent pattern of overexpression in tumor tissue.Nine of nine individual tumor/pancreatitis specimens show high bevelsof CaSm mRNA, comparable to the levels in the matched tumorspecimens.

In addition to pancreatic cancer, CaSm mRNA is expressed innormal thymus, breast, colon, spleen, and esophagus tissues; lowbevels of expression are seen in normal pancreas, lung, brain, placenta,kidney, ovary, testis, and heart (Fig. 14). Several pancreatic cancercell lines express high levels of CaSm mRNA. These include AsPC- 1,CAPAN-2, C0LO357, PANC-l, CAPAN-l, and HPAC (Fig. 2B).Other cancer-derived cell lines that express high levels of CaSmmRNA include those from prostate, liver, ovary, lung, rectum, bladder, kidney, and non-erythroid hematopoietic cells (Fig. 2B).

CaSm Encodes a Small Protein with Homology to snRNP SmProteins. The original CA3-30 cDNA clone was used to isolate afull-length clone. The clone was sequenced and found to consist of 894

2962

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Fig. I. Expression of CaSm mRNA in pancreatic tissues. Total RNA (5 @sgflane)from surgically obtained pancreas samples was electrophoresed on 1.2% agarose containingformaldehyde. transferred to a nylon membrane, and hybridized with 32P-labeled CaSm probe. T, tumor (or suspect mass); N, normal; P. pancreatitis. Specimens isolated from the samepatient are shown with an os'erbar, these pairs constitute a laneset. Otherwise, single specimens from separate individuals are shown in unpaired lanes. Laneset 1, benign mass; Laneset2. adenocarcinoma; Laneset 3, adenocarcinoma; Lane 4. insulinoma; Lane 5, adenocarcinoma metastasis to colon; Lane 6, adenocarcinoma; Lane 7, pancreatitis; Lane 8, neoplasm withlow to moderate malignant potential; Lane 9, normal pancreas; Lane 10. adenocarcinoma; Lane I 1. adenocarcinoma; Lane 12. adenocarcinoma; Laneset 13, adenocarcinoma; Laneset14, adenocarcinoma; Laneset 15. pancreatitis; Laneset 16. adenocarcinoma; Laneset I 7. adenocarcinoma; Laneset 18. adenocarcinoma; Lane 19, adenocarcinoma. Lower panels,ethidium bromide-stained RNA.

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CaSm PROTEIN CONTRIBUTED TO TRANSFORMATION

0

Fig. 2. CaSm mRNA expression in human tissues andcancer cell lines. Total RNA (10 @sg/lane)was electrophoresed on 1.2% agarose containing formaldehyde,transferred to a nylon membrane, and hybridized with32P-labeledCaSm probe. A, Northern blot analysis usingRNA from the indicated human tissues. B, Northern blotanalysis using RNA from the indicated cancer cell lines.The cell lineswerederivedfromtumorsoriginatinginhuman pancreas (AsPC-l, Capan-2, Colo357, PANC-l,Capan-l. and HPAC), prostate (PC-3 and LNCaP), breast(BT2O and MCF-7), liver (Hep G2 and SK-HEP-l), cervix (HeLa), ovary (OVCAR-3), lung (A-427), rectum(SW1463), bladder (T24), non-erythroid hematopoieticcells (MOLT-4, NC-37, Raji, H9, KG-l, and HL-60), andkidney (Caki-l). The exposure for the first four pancreaticcell lines (AsPC-I, Capan-2, Co1o357,and PANC-1) wasapproximately 3-fold more than for the remaining celllines. Lawer panel, ethidium bromide-stained RNA.

.,-,-,

Inhibition of CaSm Expression Reduces the Transformed Phenotype in Pancreatic Cancer Cells. To assess whether up-regulationof CaSm in pancreatic cancer cells is related to the transformed stateof these cells, we performed an mRNA “knock-down―experiment. Aplasmid that constitutively expresses an antisense RNA of CaSm was

stably transfected into PANC-1 cells. After selection in G41 8 forstable uptake of the plasmid, individual clones were screened byNorthern blot hybridization for a decrease in the expression of theendogenous 1.2-kb CaSm mRNA. Most of the clones screened did notshow any decrease in the level of the endogenous 1.2-kb CaSmmRNA. Rather, antisense RNA is expected to interfere primarily withmRNA translation. However, to assure that CaSm expression wasreduced, clones that showed “knock-down―of the endogenous mRNA

were preferentially selected for further study. Fig. 4 shows that severalclones were obtained in which the endogenous CaSm mRNA transcript was significantly reduced in the presence of the antisensetranscript (0.8 kb). Note that not all clones show a decrease in theamount of the endogenous transcript. Three clones, along with theparental cells, were chosen for analysis of anchorage-independentgrowth. A significant decrease in the ability of the antisense transfectants to grow in soft agar was observed (Fig. 5). After 3 weeks in softagar, only the parental cell line, PANC-l, retained the ability toproduce large colonies in the agar (Fig. SA). All three antisensetransfectants failed to produce barge colonies. Specific quantitation ofanchorage-independent colony formation for clone K shows that thereduction oflarge (>280 @m)and medium (140—280Mm) colonies is

significantly higher than for small colonies (< 140 @m;Fig. SB). Thereduction of colony formation in soft agar does not appear to be dueto growth rate because clone K and the parental cell line PANC-lhave very similar growth rates when grown on plastic (data notshown). These results were reproduced in another pancreatic cancercell line. Four independent, stable antisense transfectants of AsPC-lcells produced significantly fewer large and medium colonies in softagar compared to the untransfected cell line (data not shown).

DISCUSSION

We have described the isolation and characterization of a newcDNA clone, CaSm, corresponding to a gene that is up-regulated inpancreatic cancer tissues and cell lines. Importantly, antisense RNAinduced inhibition of expression of CaSm in pancreatic cancer celllines dramatically reduces the ability of these cells to form anchorageindependent colonies in soft agar. This supports the idea that CaSmexpression in pancreatic epithelia contributes to the transformed statein pancreatic cancer. The mechanism by which CaSm contributes to

neoplastic transformation is unknown. Stable transfectants expressingCaSm antisense RNA grow at essentially the same rate as untransfected cells; therefore, a direct role in growth regulation seems unlikely. Furthermore, CaSm expression is not induced by serum stimubation in PANC-l cells or in NIH3T3 cells. Finally, the integrationsite of the antisense construct does not seem to play a role because

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CaSm may be genuinely elevated in pancreatitis, perhaps as a predisposing condition to pancreatic cancer (19). Although there is no strong

consensus among published reports, K-ras mutations have been observed in patients 18 and 40 months prior to clinical diagnosis ofpancreatic cancer (20). Similarly, 24% of hyperplastic foci examinedhad a K-ras mutation (21); (b) the samples tested may contain occultpancreatic cancer; (c) because we have also observed high levels ofexpression of CaSm in lymphocytic cell lines (Fig. 2B), it could bethat the apparent up-regulation observed in pancreatitis is due to thelarge number of activated lymphocytes that are part of the inflammatory response. In fact, both pancreatitis specimens were from patientswith chronic pancreatitis with evidence of acute inflammation. Todistinguish among these possibilities, it will be necessary to observe

these tissue specimens by immunohistochemical methods.We observe that cancer cell lines from liver, ovary, lung, and

kidney appear to have increased CaSm expression compared to theirnormal cognates (Fig. 2, compare A and B). To assess this possibilitydirectly, it will be necessary to compare matched tumor/normal spec

imens from these organs. In addition, immunohistochemical methodswill be needed to discern whether the low level of CaSm expressionobserved in normal liver, ovary, lung, and kidney is due to low overallexpression or to relatively high expression in a small subset of celltypes found in normal tissue.

Among known proteins, the predicted CaSm protein is most similarto the human Sm 0 protein, a “commonprotein―component of thesnRNP (12). Interestingly, the region of greatest homology is in theso-cabled Sm motifs 1 and 2 that characterize the Sm proteins. Thesemotifs are required for protein-protein interaction among members ofthe Sm protein family; however, they are also found in proteins thatdo not belong to the major Sm protein family (12). All eight snRNP

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Sm motif 1 Sm motif 2CaSio 3 OYMPGTASLIEDIDKK@ILVLLRDGRTLIGFLRSIDQFANLVLHQTVEP1IHVGKKYGDIPP@GIFVVRGENWLL1

I:II.:IJ:I::IIIIII:IIIII.IIIJIIIIJII!II:I:.III.l:I'C .elegans 7 OYLPGAISLFEQLDKK@LLWLRDGRKLIGFLRSIDQFANLILEDWE FVEKYFCET GFMLIRGENVELA

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liii ..:I.IIIIIII .1:1::.I@.I.IIII I.:C .elegans GElD. .DTIETGLTQVSPEEF . .RRLEDEYIAKNPPKFLKRQAEKTEEO 122

C Smmotif1 Smmotif2CaSm 4 OMPGTASLIEDIDKKLVLLRDGRTLIGFLRSIDQFANLVLHQTVER@IHVG ..KKYGDIPP@GIFVVRGENVVLL

:..II.::..:I:I:IIIjlIII:J.JI.:IJ:III:I::.IIII..:Yeast 40 @FTTTAAIVSSVDR FVLLRDGRNLFGVLRTFDQYANLILQDCVE YFSEENKYAEE GIFMIRGENVVML

GEIDLEKESDT. .PLQQVSIEEILEEQRVEQQTKLEAEKLKVQALKDRGL. .SIPRADTLO 130f:I::II.:_:::.:.:.I. :::.::..I.I..::1:

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Fig. 3. Homology of CaSm protein to Sm G protein and to hypothetical proteins. O@, the sequence is not shown in its entirety. A. alignment of CaSm to human Sm G protein. Boxedareas, Sm motifs I and 2, as indicated. The core consensus for the Sm motifs is that deduced by Hermann et a!. (12). U. uncharged, hydrophobic amino acids (L, I, V. A, F, W, Y,C. M); Z an uncharged. hydrophobic amino acid plus T or S. B. alignment of CaSm protein to C. elegans hypothetical protein deduced from cosmid F40F8 (GenBank accession numberZ69302). C. alignment of CaSm protein with the S. cerevisiae hypothetical protein open reading frame YJL124c (GenBank accession number Z49399).

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four independent clones in each of two cell lines all demonstrate thesame phenotype.

A large majority of the pancreatic cancer samples examined showelevated expression of CaSm mRNA. However, we also note that twoof the samples tested that show up-regulation compared to matchednormal tissue are not neoplastic tissues; rather, they are pancreatitissamples (Fig. 1). Several possible explanations may be considered: (a)

28S

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Fig. 4. Reduction of endogenous CaSm expression in stable antisense transfectants.RNA (5 @sg/lane)from individual stable transfectants containing the CaSm antisenseconstruct was electrophoresed on I .5% agarose containing formaldehyde, transferred to anylon membrane, and hybridized with 32P-labeled CaSm probe. Sizes of the endogenousCaSm mRNA (1.2 kb) and the transfected antisense RNA (0.8 kb) are indicated. Lowerpanels. ethidium bromide-stained RNA.

2964

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CaSm PROTEIN CONTRIBUTED TO TRANSFORMATION

remains to be determined whether CaSm mediates transformation directlyand if so, whether the Sm motifs present in CaSm (and absent in its splicevariant) mediate protein interactions that facilitate cellular transformation.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of Mary Jane McWilhams, Jana Veseley, and Amy Smialowicz. Dr. Saul Suster is acknowledged

for providing some of the specimens used along with a pathological description. Dr. Shengmei Qi and Dr. Richard Ascione are acknowledged for contri

butions in the early phase of this project. Dr. Mark C. Willingham is thanked

for critical review of the manuscript.

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16. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. RNA molecular weightdeterminations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 16: 4743—4751,1977.

17. Kozak, M. An analysis of vertebrate mRNA sequences: intimations of translationalcontrol. J. Cell Biol., 115: 887—903,1991.

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19. Bansal, P.. and Sonnenberg. A. Pancreatitis is a risk factor for pancreatic cancer.Gastroenterology, 109: 247—251, 1995.

20. Berthélemy,P., Bouisson, M., Escourrou, J., Vaysse, N., Rumeau, J. L., and Pradayrol, L. Identification of K-nix mutations in pancreatic juice in the early diagnosis ofpancreatic cancer. Ann. Intern. Med., 123: 188—191,1995.

21. Tada. M., Ohashi, M., Shiratori, Y., Okudaira, T., Komatsu, Y., Kawabe, T., Yoshida,H., Machinami, R., Kishi, K., and Omata, M. Analysis of K-ras gene mutation inhyperplastic duct cells of the pancreas without pancreatic disease. Gastroenterology,110: 227—231,1996.

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Clone 2

COLONYSIZE(pm)

Fig. 5. Reduction of anchorage-independent growth in antisense transfectants. A. softagar colonies formed after 3 weeks from parental pancreatic cancer cell line PANC-I andfrom three antisense transfectant clones (Clone K, Clone L, and Clone 2). B, quantitationof the soft agar colonies, by size, from PANC-1 and from clone K.

common core proteins have been cloned and sequenced, yet CaSmshares only limited homology with this group. Therefore, CaSm is notlikely to be a member of this common core group. A search of proteinsequence databases revealed two hypothetical proteins with higherlevels of similarity than Sm G protein. These proteins derive from theopen reading frames of a C. elegans cosmid clone (accession numberZ69302) and a S. cerevisiae clone (accession number Z40399). The C.elegans sequence is 54.4% identical and 72.8% similar over aminoacids 3—121,whereas the S. cerevisiae clone is 37.8% identical and67.7% similar over amino acids 4—130.Both of the Sm motifs areincluded in these regions. Furthermore, the important amino acids thatform the Sm consensus are conserved here as well. This is consistentwith the previous model that the Sm motifs define a broader family ofinteractive proteins that extends beyond the spliceosomal Sm proteins(12). Recently, we have found a possible splice variant of CaSm thateliminates amino acids 22—32of Sm motif 1 and all of Sm motif 2.6 It

6 M. W. Graber, C.W. Schweinfest, and D. K. Watson, unpublished results.

140-280

2965

Clone L

El PANC-l

.

>280

on July 6, 2018. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1997;57:2961-2965. Cancer Res   Clifford W. Schweinfest, Michael W. Graber, Jeannie M. Chapman, et al.   State in Cancer CellsCaSm: An Sm-like Protein That Contributes to the Transformed

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on July 6, 2018. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from