MOLECULAR AND CELLULAR BIOLOGY, Jan. 1994, p. 851-8580270-7306/94/$04.00+0

Loss of Thrombospondin Transcriptional Activity inNickel-Transformed Cells

K. SALNIKOW, S. COSENTINO,t C. KLEIN, AND M. COSTA*

Department ofEnvironmental Medicine and Kaplan Comprehensive Cancer Center,New York University Medical Center, New York, New York 10016

Received 18 June 1993/Returned for modification 5 August 1993/Accepted 30 September 1993

mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an

excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report therecovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtainedby this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels ofthrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin.To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies tohuman thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatlyreduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts innormal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNAexpression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewerthrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations ofrestriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggestingthat there were no major deletions or rearrangements of the gene in the nickel-transformed cells. Theinactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of thisgene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normaland Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformedcells, suggesting that the promoter region ofthrombospondin was less efficiently transcribed in Ni-transformedcells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumorsuppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of anexpression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondinpromoter such that the expression was higher in normal cells than in transformed cells.

Nickel and its compounds are among the few well-docu-mented human carcinogens, and only recently have themolecular mechanisms of Ni carcinogenesis been addressed(6). Ni ions have a much higher affinity for proteins than forDNA, and thus Ni compounds have been found to selec-tively damage protein-rich heterochromatic chromosomalregions (3-6). In Chinese hamster (CH) cells the longestcontiguous region of heterochromatin is found on the Xchromosome. This is thought to explain the disproportion-ately high incidence of transformation among male Chinesehamster embryo (CHE) cells compared with female cells,since over half of the male Ni-transformed cells had a

complete deletion of the long arm of the X chromosome astheir primary chromosomal aberration (3). The striking de-letion of protein-rich heterochromatin may simply modelother protein-DNA interactions occurring in genetically ac-tive promoter regions. CHE cells are used in cell transfor-mation studies because, unlike mouse cells, they can betransformed by nickel compounds with a low spontaneousbackground. Human cells are not generally transformed bychemical carcinogens, and other mammalian cell types (e.g.,mouse cells) exhibit such high transformation backgroundsthat it is difficult to attribute the transformation to thecarcinogen treatment.

* Corresponding author. Mailing address: Department of Envi-ronmental Medicine, New York University Medical Center, 550First Avenue, New York, NY 10016. Phone: (914) 351-2368 or (212)263-5280. Fax: (914) 351-2118 or (212) 263-5019.

t Present address: University of Cagliari, Cagliari, Italy.

In further studies, the introduction of the normal Chinesehamster X chromosome into Ni-transformed male CHE cellsresulted in senescence of these cells, suggesting that one ofthe genes inactivated during Ni-induced transformation wasapparently X linked and related to senescence (13). Addi-tional studies revealed that this X chromosome-senescingactivity was under methylation control, and the resultssuggested that Ni might influence carcinogenesis and cellularsenescence by an epigenetic mechanism involving changesin DNA methylation (13, 32). However, these studies alsodemonstrated that the striking deletion of the Xq was prob-ably not the site of the senescence gene, since Ni-trans-formed cells without any microscopic X chromosome dele-tion also senesced when a normal X chromosome wastransferred (32).

Since a senescence gene or genes were inactivated duringNi carcinogenesis, we searched for candidate genes thatwere transcribed in normal CHE cells but not in the Ni-transformed cells. A new method was devised to subtractcDNA libraries for analysis of genes that were inactivatedduring Ni-induced transformation. mRNA obtained fromlow-passage CHE cells was converted into single-strandcDNA and was subtracted with mRNA from Ni-transformedcells by using a protocol that allowed paramagnetic beads tocapture nonhybridized single-strand CHE-specific cDNA(5). A number of clones were obtained, and one of the genesequences was found to be highly homologous to the mousethrombospondin I gene (17).Thrombospondin is a 420-kDa protein composed of three

identical subunits of 140 kDa that are linked by disulfide

851

Vol. 14, No. 1

852 SALNIKOW ET AL.

bonds (see reference 24 for a review). It is produced andsecreted by endothelial cells, muscle cells, and fibroblasts(11, 19, 23, 25). Thrombospondin biosynthesis is inverselyproportional to cell density and can be stimulated by serum,

platelet-derived growth factor, basic fibroblast growth fac-tor, and transforming growth factor ,B (7, 8, 20). Thrombo-spondin mRNA is superinduced by cycloheximide in a

manner similar to the induction of c-fos and other immedi-ate-early growth-regulatory genes (20). One of the mostinteresting features of the thrombospondin protein functionis its ability to inhibit angiogenesis in vivo and inhibit themigration of endothelial cells in vitro (9, 27). This wouldsuggest that the protein levels should be lower in tumor cellswhere neovascularization is important for tumor growth (9,27). Therefore, cells producing high levels of thrombospon-din cannot form tumors in vivo, illustrating the obviousimportance of this gene in cancer development. However,high levels of thrombospondin conferred serum- and anchor-age-independent growth (2).

In the present study, we found that the expression ofthrombospondin was very low in tumorigenic Ni-trans-formed Ni-2 and Ni-6 cells as measured by both mRNAanalysis and immunostaining with a monoclonal antibody tothrombospondin. The tumorigenic properties of these celllines have been well characterized (3). These and otherstudies established that thrombospondin transcription can besilenced by Ni.

MATERIALS AND METHODS

Cell cultures. Primary male CHE cells were used atpassages 4 to 27 and were always maintained at a subcon-fluent state by passage every third day at a dilution ratio of1:2. The Ni-2 and Ni-6 transformed cell lines were obtainedby treatment of male CHE cells with NiCl2 or crystallineNiS, respectively (3). All cell lines were routinely main-tained in a minimal essential medium supplemented with10% fetal bovine serum (Sigma, St. Louis, Mo.), 100 U ofpenicillin, 100 jig of streptomycin (GIBCO-BRL, Gaithers-burg, Md.) per ml, and 2 mM glutamine. Wild-type andnickel-resistant BALB/3T3 cells were derived earlier andmaintained as described previously (31). Cells resistant to

200 ,uM nickel (B-200) were used in the present study.Preparation of cDNA libraries and subtractive hybridiza-

tion. The mRNA for the cDNA library preparation was

obtained using a Fast Track mRNA isolation kit tInvitrogen,San Diego, Calif.). Typically 6 x 107 to 8 x 10 cells were

used for the mRNA isolation. The mRNA quality was

checked on denaturing 1.2% agarose gels with formalde-hyde. cDNA libraries were constructed from the mRNA ofCHE and Ni-2 cells by using XZAP bacteriophage vectors

and commercial kits for cDNA synthesis (GIBCO-BRL) andcloning (Stratagene, La Jolla, Calif.). For screening the CHEcDNA library, probes were obtained by subtractive hybrid-ization of single-stranded cDNA from CHE cells and mRNAfrom Ni-2 cells. Single-stranded radiolabeled cDNA was

synthesized by oligo(dT)-primed reverse transcription ofpoly(A) RNA (5 ,ug) from CHE cells and coprecipitated witha 10-fold excess of poly(A) RNA from Ni-2 cells. Theprecipitate was resuspended in a final volume of 8.2 ,ulcontaining 500 mM sodium phosphate buffer (pH 6.8), 6 mMEDTA, and 0.2% sodium dodecyl sulfate (SDS). The hybrid-ization was performed at 68°C for 24 h in a siliconized 20-,uglass capillary tube. The reaction mixture was then trans-

ferred into 1 ml of SS buffer (0.12 M sodium phosphate [pH6.8], 0.1% SDS), and the unhybridized CHE-specific cDNA

was recovered by using a 3'-biotinylated oligo(dA) primer.After the hybridization mixture had been heated at 65°C for10 min, 10 ,ul of the biotinylated oligo(dA) (50 pmol/Vl) wasadded, and the poly(T) region of the cDNA molecules wasannealed to this primer by slow cooling at room temperaturefor 30 min. These cDNA-oligo(dA)-biotin complexes werecaptured by using streptavidin-coupled paramagnetic parti-cles and a magnetic stand (Promega, Madison, Wis.). Theparticles were washed four times with 0.1 x SSC (lx SSC is0.15 M NaCl plus 0.015 M sodium citrate), and the single-stranded cDNA was finally eluted from the biotin-magnetic-particle complexes by addition of double-distilled water. Theelution step was repeated once to ensure complete removalof the cDNA.

After ethanol precipitation the cDNA was resuspended inwater, and 32P-labeled probes were generated by randompriming (U.S. Biochemical Corp., Cleveland, Ohio). Theseprobes were then used to screen the CHE cDNA library. Atotal of 25,000 clones were plated onto five 150-mm LB-agardishes (5,000 clones per dish), and 19 positive signals weredetected after hybridization. The clones were isolated andreplated at low density on 60-mm LB-agar dishes. Replicafilters were screened with CHE and Ni-2 cDNA probes, and10 plaques which gave stronger signals with the CHE probewere purified and converted to plasmid pBluescript SK. Forslot blot hybridization of the selected cDNA clones, mRNAfrom CHE and Ni-2 cells was labeled with [32P]dCTP byusing reverse transcriptase.

Sequencing. Double-stranded plasmid clones were se-quenced with Sequenase 2.0 (U.S. Biochemical Corp.) asrecommended by the manufacturer. [a-35S]dATP was usedas the label. The T-3 and T-7 primers which correspond tothe flanking region of the cDNA inserts were used to initiatesequencing. Subsequent sequence analyses were performedwith synthetic oligonucleotide primers (National Bio-sciences, Plymouth, Minn.) whose sequences were deter-mined by the previous sequencing experiments. The over-lapping DNA sequence information was assembled bycombining all the data. For comparison of the acquired datawith known sequences, the GCG program was used.

Northern (RNA) blot. mRNA (5 ,ug) was separated on a1.2% formaldehyde agarose gel and then transferred to anylon Nytran membrane (Schleicher & Schuell, Keene,N.H.) in 20x SSC buffer. Hybridization was carried out asrecommended by the manufacturer. The filter was hybrid-ized with the thrombospondin 32P-labeled probe. Thrombo-spondin-specific mRNA was detected by autoradiography.The filter was rehybridized with a P-actin probe to controlfor equal gel loading. For detection of thrombospondin RNAin Ni-resistant mouse cells, 20 ,ug of total RNA was sepa-rated in a 1% formaldehyde agarose gel. For detection ofretinoblastoma (Rb) expression, 20 ,ug of total RNA from therespective cell lines was used.

Southern blot. Digested genomic DNA (15 ,ug) was sepa-rated in a 0.8% agarose gel and then transferred to a nylonNytran membrane (Schleicher & Schuell) in 20x SSC bufferovernight. The blot was hybridized as recommended by themanufacturer. The thrombospondin hybridization probe wasobtained by separation of digested plasmid DNA in a 1%agarose gel, and the derived insert was extracted withGeneclean (Bio 101, Inc., La Jolla, Calif.). The probe waslabeled with [a-32P]dCTP by using a random primer kit (U.S.Biochemical).

Transient transfection. Two expression vectors,pTSPCAT3A and pTSPCAT7A, containing portions of thehuman thrombospondin promoter region, from -1659 to

MOL. CELL. BIOL.

NICKEL HAS INHERITED EFFECTS ON TRANSCRIPTION 853

+749 and -234 to +749, respectively, were kindly providedby V. M. Dixit, University of Michigan (15). The plasmidDNA was introduced into CHE and Ni-2 cells by calciumphosphate coprecipitation procedures, using a MammalianTransfection kit (Stratagene). For cotransfection, 15 ,ug ofpTSPCAT7A plasmid DNA was mixed with 15 ,ug of eitherthe mouse Rb expression plasmid (provided by R. Bernards,Harvard Medical School) or plasmid vector that does notcontain the Rb insert. Transfection was carried out bycalcium phosphate coprecipitation. The efficiency of DNAuptake was monitored by transfection with plasmid pCH-110containing the 1-galactosidase gene driven by the simianvirus 40 (SV-40) promoter (Pharmacia, Piscataway, N.J.).For each transient-transfection experiment, 1-galactosidase(3-Gal) activity was measured (see below) and the transfec-tion efficiency was standardized against the activity of thisgene. Additionally, to ensure that the SV-40 promoter wasnot differentially regulated in normal and Ni-transformedcells, cells were transfected with a pSV2-CAT plasmid andthe activity of chloramphenicol acetyltransferase (CAT) wasfound to be equivalent in all cell types examined.

Analysis of the quantity of CAT and 13-Gal. The CAT and1-Gal activity in cell lysates was measured by use of theCAT and 13-Gal enzyme-linked immunosorbent assay(ELISA) kit (5 Prime -> 3 Prime, Inc., Boulder, Colo.). Allreactions were performed in duplicate, and the extinctioncoefficients of the samples were measured at 405 nm in amicroplate reader. Calibration curves were obtained asrecommended by the manufacturer with the standards sup-plied with the ELISA kits.

Immunofluorescence. For immunofluorescence micros-copy, cells were grown on 22-mm2 glass coverslips (ComingGlass, Coming, N.Y.) in six-well plates. Glass coverslipswere coated overnight with 1.5% gelatin (Difco, Detroit,Mich.), rinsed with serum-free a minimal essential medium,and seeded with 5 x 10' cells per coverslip. The cells weregrown for 42 h, washed with phosphate-buffered saline(PBS), fixed with freshly prepared 3% paraformaldehyde for3 min, and treated with 0.5% Triton X-100 for 15 min. Thecells were washed again with PBS and overlaid with primarymonoclonal antibody against human thrombospondin(Biodesign, Kennebunkport, Maine). Coverslips were incu-bated in a humid chamber for 40 min at room temperature.After being washed in PBS, the coverslips were incubated inan appropriate diluted fluorescein isothiocyanate-tagged sec-ond antibody (Sigma) for 30 min. The coverslips weremounted in 70% glycerol diluted with PBS. Fluorescentimages were recorded on Kodak T-Max 3200 film with aZeiss photomicroscope and were developed in a KodakT-Max developer.

RESULTS

Cloning strategy. We have developed a strategy to isolategene sequences that were expressed in normal CHE cells butnot in Ni-transformed (Ni-2) cells. The protocol used avidin-coupled magnetic particles for separation of single-strandedCHE-specific cDNA from a mixture of cDNA-mRNA hy-brids. However, unlike the protocol provided by Promegafor isolation of mRNA, we used biotinylated poly(A) thatpermitted the capture of single-stranded poly(T)-tailedcDNA from CHE cells. Such separation is efficient andoccurred without significant loss of the probe. Figure 1describes the scheme that was used to obtain the CHE-specific cDNA probes. The probe was highly enriched forCHE-specific sequences since only 2 to 3% of the starting

CHEcellsmRNA X+ reverse transcriptase

CHE cellssscDNA

Hi

+ biotinylated poly A

Ni-2 cellsmRNA

lybridization1:10 ratio

Separation of ss CHE cells specificcDNA with avidin magnetic particles

+ 32P-dCTP, random primer, DNA-polymerase

ds 32P-labelled subtracted probe

screening CHE cell cDNA library 4

CHE cell specific clones

FIG. 1. Scheme for obtaining CHE-specific cDNA subtractiveprobes. Abbreviations: ss, single-stranded DNA; ds, double-stranded DNA.

material was recovered as single-stranded cDNA after sep-aration from cDNA-mRNA hybrids.The cDNA libraries were constructed from the mRNA of

CHE and Ni-2 cells. The CHE library contained 1.3 x 106recombinants with an average insert length of 1.0 to 1.5 kbon the basis of T7 and T3 primer-directed PCR analysis ofthe phage-derived DNA. Individual cDNA clones wereisolated as the result of screening the CHE cDNA librarywith probes obtained following subtractive hybridization(see Materials and Methods). At least 25,000 clones werescreened, resulting in 19 positive clones, which were re-screened by using CHE and Ni-2 cDNA probes. Ten cloneswere selected for further analysis. The results of the hybrid-izations confirmed highly specific expression in normal CHEcells for 5 of the 10 cloned sequences. Cross-hybridizationrevealed that these five clones were unique.The CHE-specific clones were sequenced by using the T-3

and T-7 flanking primers. Comparisons of the identifiedsequences with the gene bank data demonstrated that oneclone with an insert length of 2.6 kb showed striking homol-ogy with the mouse thrombospondin I. It matched exons 19to 22 of the mouse thrombospondin I gene (Fig. 2A) with96% homology. Only 22 base substitutions per 523 nucle-otides were found in this portion of the coding region of thehamster sequence. A complete sequence of this clonedfragment (GenBank accession number L22614) will be pub-lished elsewhere (5). Figure 2B shows the insert size and theportion of the hamster thrombospondin gene coding regionthat was obtained. These results clearly illustrate that thethrombospondin gene was one of the genes that appeared tobe present in normal cells but may be less abundant inNi-transformed cells.

Analysis of thrombospondin mRNA and protein expressionin Ni-transformed cells. RNA blot analysis with the clonedDNA fragment of the thrombospondin gene as a proberevealed that an approximately 5.3-kb mRNA was present inCHE cells at different passages (Fig. 3A). In the Ni-trans-formed Ni-2 and Ni-6 cell lines this mRNA was not detect-able. A 13-actin probe was used to control for gel loading,

VOL. 14, 1994

854 SALNIKOW ET AL.

EXON XIX0 .0

gtaGGTTATGATGAGTTTAATGCTGTGGACTTCAGCGGTACCTTCTTCG Y D E F N A V D F S G T F F

gtaGGTTATGATGAGTTTAATGCCGTGGACTTTAGTGGTACCTTCTTCG Y D E F N A V D F S G T F F

0 0 @0ATCAACACCGAGAGAGATGATGACTACGCTGGCTTTGTATTCGGCTACCAGTCCI N T E R D D D Y A G F V F G Y Q S

ATCAACACTGAGAGAGATGATGACTATGCTGGCTTTGTGTTTGGCTACCAGTCCI N T E R D D D Y A G F V F G Y Q S

0AGCAGCCGCTTCTACGTTGTGATGTGGAAACAAGTCACCCAGTCCTACTGGGACS S R F Y V VM W K Q V T Q S Y W DAGCAGCCGCTTCTACGTTGTGATGTGGAAACAAGTTACCCAGTCCTACTGGGACS S R F Y V V M W K Q V T Q S Y W D

ACCAACCCCACAAGGGCTCAGGGATACTCAGGCCTGTCTGTAAAGGTTGTGAACT N P T R A Q G Y S G L S V K V V NACCAACCCCACAAGGGCTCAGGGATACTCAGGCCTGTCTGTAAAGGTTGTGAACT N P T R A Q G Y S G L S V K V V N

TCCACCACCGGCCCTGGCGAGCACCTGCGGAATGCACTGTGGCACACAGGAAACS T T G P G E H L R N A L W H T G NTCCACCACCGGCCCTGGCGAGCACCTGCGGAATGCTCTGTGGCACACAGGAAACS T T G P G E H L R N A L W H T G N

Akb4.8 -

1.9 -

B

CHE

a-aL aC Ni2N6

IIMiINIU.Em .m

FIG. 3. (A) Northern blot analysis of the mRNA expressed innormal CHE cells at different passages compared with the expres-sion of thrombospondin mRNA in Ni-2 and Ni-6 transformed cells.(B) Hybridization with P-actin confirms equal loading of mRNA inall lanes.

ACCCCTGGCCAGgtaagaT P G QACCCCTGGCCAGgtT P G Q

EXON XX. *agGTGCGCACCCTGTGGCATGACCCTCGCCACATCGGCTGGAJ

V R T L W H D P R H I G W IGTGCGCACCCTGTGGCATGACCCTCGTCACATAGGCTGGAJV R T L W H D P R H I G W I

0TACAGATGGCGTCTCAGCCACAGGCCAAAGACCGGTTATiY R W R L S H R P K T G Y

TACAGATGGCGTCTCAGCCACAGGCCAAAGACAGGTTACJY R W R L S H R P K T G Y

EXON XXI 0agaGTGGTGATGTATGAAGGAAAGAAAATCATGGCTGACT4

V V M Y E G K K I M A D 'gaGTGGTGATGTATGAAGGAAAGAAAATCATGGCCGACTC

V V M Y E G KK I M A D '0 ~~0

GACAAAACCTACGCCGGCGGTAGACTAGGCCTGTTCGTCTTCD K T Y A G G R L G L F V F

GATAAAACCTACGCCGGTGGTAGACTAGGCCTGTTTGTCTTCD K T Y A G G R L G L F V F

GTGTTCTTCTCAGACATGAAATACGAGTGTCGAV F F S D M K Y E C R

GTGTTCTTCTCAGACATGAAATACGAATGTCGAV F F S D M K Y E C R

MOUSE aGATTCCTAAamino a. D SHAMSTER GATTCCTAAamino a. D S

B

EXON XXII

Untranslated Region

p Biuescript

2

FIG. 2. (A) Comparison of the DNA sequence ai

composition of the previously reported (17) mouse thiand the fragment of CH gene cloned in this work. Th4the coding region of the mouse and CH thromboswhich corresponds to exons 19 to 22. Differences in Ition between mouse and CH thrombospondin are shdots. (B) Structure of the cloned fragment of the CHdin DNA. The figure shows a 2.6-kb pBluescript ins527 bp of the thrombospondin gene, which corresponto 22, and approximately 2.0 kb of the 3' untranslate

which was equivalent for all lanes (Fig. 3B). Figure 4 showsthe monoclonal antibody staining of thrombospondin protein

*A *TTCACTGC in normal and transformed cells. By using cells fixed withK D F T A paraformaldehyde and then treated with Triton X-100, it wasAGGA=CFTCAC possible to observe both cell surface-bound and internal

0 thrombospondin. In CHE cells (Fig. 4A), thrombospondinICARGtaa was evenly distributed in the cytoplasm of the cell, with

ATCAGA beadlike inclusions. Little thrombospondin was observed inthe nucleus. Ni-transformed cells (Fig. 4B and C) showed a

lower level of thrombospondin, with the protein concen-c0GG^cccATcTAT trated primarily in the perinuclear area. In some transformedCGGGACCCATCTATS G P I Y cells, the thrombospondin staining was minimal and theCAGGACCCATCrAT thrombospondin was localized in lysosome-like vesicles.

Thrombospondin seems to be a highly conserved proteinS Q E M since monoclonal antibodies prepared against human platelet

TCTCAAGAAATG

thrombospondin reacted with both hamster and calf serumQ E

thrombospondin. Although the amount of calf serum-derivedthrombospondin in the cell culture media was 3 to 4 ,ug/ml,little thrombospondin was detected in Ni-transformed cells.Our results from the Northern blots clearly show that theexpression of cellular thrombospondin was high in normalCHE cells but almost undetectable in Ni-transformed cells.In Ni-transformed cells, the low level of stained materialcould represent either calf serum thrombospondin taken upby cells and degraded in the lysosomes or cellular thrombo-

T-7 spondin that was accumulated in the granules, reflectingJ7_T7] either poor cellular synthesis or reduced uptake or both.

Is thrombospondin transcription depressed as a result ofp Bluescript transformation or nickel treatment? We examined the ex-

pression of thrombospondin mRNA in wild-type and Ni-kb resistant mouse BALB/3T3 cells (31). Wild-type cells ex-

3kb pressed thrombospondin at abundant levels, whereas B-200

Ni-resistant cells exhibited less thrombospondin mRNAnd amino acid (Fig. 5). These results indicate that the loss of thrombospon-rombospondin din transcription is related to Ni exposure, since the B-200

e figure shows cells had acquired resistance to 200 ,uM NiCl2 (31).base composi- Mechanism for decreased expression of thrombospondin in

iown by black Ni-transformed cells compared with normal cells. There werethrombospon- several possible reasons for the reduced expression of,ert containing thrombospondin in Ni-transformed cells compared with nor-ds to exons 19 mal cells. One possibility was inactivation of the gene byd region. rearrangement or deletion. Figure 6 is a Southern blot

showing restriction enzyme polymorphisms of the thrombo-spondin gene in normal and Ni-transformed cells, as well asin the immortalized cell lines CHO and MCA-1. There were

AMOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOUSEamino a.HAMSTERamino a.

MOL. CELL. BIOL.

1

NICKEL HAS INHERITED EFFECTS ON TRANSCRIPTION 855

B-200Wild Type 3T3 (Nickel Resistant clone)

Thrombospondin

O- Actin

FIG. 5. Expression of thrombospondin in wild-type BALB/3T3and nickel-resistant B-200 cells. The level of thrombospondinmRNA was examined by hybridization as described in Materials andMethods. Actin mRNA is shown in the lower portion as a standardfor RNA loading. Nickel-resistant cells were obtained as previouslydescribed (31).

other genes such as thrombospondin. To investigate whetherthrombospondin expression was lower in Ni-transformedcells than in normal cells, we obtained two CAT reporterplasmids that contained portions of the human thrombospon-din promoter and exon 1 of the gene (pTSPCAT3A andpTSPCAT7A, respectively) (15). The pTSPCAT3A plasmidcontained a larger part of the thrombospondin promoter,region from -1659 to +749. This promoter had most of theknown regulatory sequences including Spl and AP-2 sites, a

c-fos direct repeat, a serum-responsive element, and a cyclicAMP (cAMP) response element. Figure 7A shows the higherlevel of expression of thrombospondin in normal cells com-pared with transformed cells as judged by the amount ofCAT activity following transfection of the pTSPCAT3Aplasmid. Figure 7B shows the results of transfection of thepTSPCAT7A plasmid, which contained a smaller portion ofthe thrombospondin promoter region from -234 to +749. Itdid not contain the c-fos-like SRE element, the cAMPresponse element, or the AP-2 or c-fos direct repeats, but itdid contain two binding sites for the nuclear transcriptional

FIG. 4. Monoclonal antibody staining of thrombospondin in nor-mal cells (A) and in Ni-2 (B) or Ni-6 (C) transformed cells. Cellswere stained with a primary monoclonal antibody against humanthrombospondin and then incubated with a fluorescein isothiocy-anate-tagged second antibody as described in Materials and Meth-ods.

no apparent differences in the restriction enzyme digestionpatterns with EcoRI and BamHI, PvuII and NheI, or XhoIand Xba .

Since Ni-transformed cells were previously described tohave sustained an apparent inactivation of a senescence-related gene(s) (13), it was possible that the senescencegenes act as a regulatory protein driving the transcription of

kb

23.1 -

9.46.6 -

4.3 -

2.3 o2.0 >

CHEABC

Ni2A B C

..#

MCAA B C

I..

WV.

CHOA B C

E --

A - EcoR 1 + BamHIB - Pvu 11 + NheC - Xba + Xho

FIG. 6. A Southern blot examining restriction enzyme polymor-phisms of the thrombospondin gene in normal (CHE) cells, Ni-transformed (Ni-2) cells, and two other immortalized cell lines(MCA-1 and CHO). Enzyme digestions were carried out with EcoRIand BamHI (A), PvuII and NheI (B), or XbaI and XhoI (C), asdescribed in Materials and Methods.

VOL. 14, 1994

856 SALNIKOW ET AL.

0-wx0

-

0Eco

150

120

A1

-1 669

90 r

s0

0

150

I, .

0 +749 CAT 12o

120

0

gz 90

cso

0

Ei

NI-2

pTSPCAT7A

l-234 0 +749

I T~~~~

B

o0CHE

MOL. CELL. BIOL.

CAT

NI-2

FIG. 7. Activity of the thrombospondin promoter linked to a CAT reporter gene in normal and Ni-2 transformed cells. (A) ThepTSPCAT3A construct contained region -1659 to +749 of the human thrombospondin promoter and the first untranslated exon of thethrombospondin gene. (B) The pTSPCAT7A construct contained region -234 to +749 of the thrombospondin promoter and exon 1 of thethrombospondin gene. For examination of CAT reporter gene activity in normal and Ni-transformed cells, plasmid DNA was transfected intothe cells. CAT and P-Gal expression was measured by ELISA following transient transfection of these vectors into the cells. CAT activitywas measured and standardized as described in Materials and Methods. The values are the means and standard deviations of duplicatedeterminations from three experiments.

factor Spl. Again, the lowered CAT activity in Ni-trans-formed cells than in normal cells suggested that the throm-bospondin promoter was less efficiently transcribed in Ni-transformed cells. These experiments were standardized fortransfection efficiency and promoter activity by using ,B-Galand the SV40 promoter (see Materials and Methods).

Since senescence genes and possible transcription factorswere inactivated in Ni-transformed cells, we investigated theeffects of overexpression of the Rb gene on the transcrip-tional activity of the heterologous human thrombospondinpromoter (Fig. 8). Whereas the cotransfection of the plasmidwithout the Rb coding region had no effect on the transcrip-tion of the thrombospondin gene, cotransfection of theplasmid with the Rb coding region stimulated the thrombo-spondin promoter in both normal and Ni-transformed cells(Fig. 8). This effect was clearly greater in normal cells thanin Ni-transformed cells. Figure 9 examines the expression ofthe Rb gene in normal and Ni-transformed CHE cells. TheRb gene was expressed at equivalent levels in normal andNi-transformed CHE cells (Fig. 9), when the gel loading is

15

0L-C

0a00

-o0

-

0

12

taken into consideration (see ,B-actin lanes). As a control forRb expression, human osteosarcoma cells were examined.

DISCUSSION

Changes in gene expression are clearly very important inthe multistage process of carcinogenesis, and it is likely thatmany genes are increased or decreased in their expression,through either amplification or alterations in their transcrip-tion. However, there have been few studies demonstratingthat genes are specifically altered in their transcriptionalregulation during the carcinogenic process. In general, rear-

wzU0

04, (D c0

Rb (4.7kb)I I9 ,

61-

31

oCHE Ni-2

FIG. 8. Analysis of the activity of the thrombospondin promoterin the presence of a mouse retinoblastoma expression vector. CATexpression in CHE and Ni-2 cells was determined as described inthe legend to Fig. 7. The values are the means and standarddeviations from three independent experiments. Symbols: M,pTSPCAT7A; M, +Rb; ED, -Rb.

FIG. 9. Expression of the Rb gene in normal and nickel-trans-formed cells CHE cells. A mouse probe for the Rb gene was used toassess the expression of Rb mRNA in normal and nickel-trans-formed cells by standard Northern blot analysis as described inMaterials and Methods. Also shown is the expression of the Rb genein human osteosarcoma cells (HOS) as a control.

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NICKEL HAS INHERITED EFFECTS ON TRANSCRIPTION 857

rangements, amplification, or mutations of genes have beenimplicated as the causative factors yielding altered pheno-types during carcinogenesis (1).

In previous studies, we have shown that the selectivity ofNi interaction with the heterochromatin of CH chromo-somes yielded striking heterochromatic damage and, in somecases, deletions of the entire heterochromatic long arm ofthe X chromosome (3). Ni has approximately 5 to 7 orders ofmagnitude greater affinity for amino acids and proteinscompared with its affinity for DNA or its bases, and thehigher protein content of heterochromatin may account forthe selectivity of Ni binding and effects in this region (18,21). Ni ions have also been shown to affect specific proteinbinding to DNA. For example, in a mouse system in whichheterochromatin is found primarily in the centromere, Niinhibited specific protein binding to mouse satellite DNA(10). Therefore it was proposed that Ni, by affecting protein-DNA interactions, may affect transcription factors in cells.Ni-induced transformation could be inhibited by elevatingthe magnesium ion concentration in the extracellular me-

dium (4). The ability of Ni ions to damage heterochromatinwas probably not only due to the Ni binding to chromatinproteins, which are more concentrated in the heterochroma-tin, but also may be related to the ability of Ni to undergooxidation reactions following its binding to certain ligands,such as the imidazole nitrogen of histidine, where theoxidation potential of Ni can be substantially lowered (14).This could generate oxygen radicals in a locally cagedfashion, further contributing to the heterochromatin-specificdamage induced by Ni compounds. However, the hetero-chromatin damage and chromosomal deletion is clearly notthe site of the inactivated senescence gene that we havepreviously described (13, 32) but may model how Ni couldaffect other proteins present in gene-regulatory promoterregions. Ni has been reported to catalyze the oxidation ofboth DNA and protein, leading to DNA-protein cross-links(14).We have found that one of the genes that was transcrip-

tionally affected during Ni carcinogenesis was the thrombo-spondin gene. The thrombospondin gene has previouslybeen cloned in mouse, human, and chicken cells (16, 17, 35)but not in hamster cells. We examined the structural integ-rity of the thrombospondin gene in Ni-transformed cells andfound no alterations in the restriction endonuclease digestionpattern; however, the transcription of the gene was down-regulated in the Ni-transformed cells. The down-regulationof the transcription of the thrombospondin gene could be dueeither to the presence of an inhibitor or to the absence of a

positive transcription factor(s). It is interesting to speculatethat since Ni-induced transformation in this system alsoinvolved the loss of a senescence gene, it was possible thatthis senescence gene could interact with the promoter regionof the thrombospondin gene and regulate its transcription.Thrombospondin has a number of regulatory elements in itscomplex promoter region, including AP-1, AP-2, Spl, SRE,and many others. It has already been reported that throm-bospondin gene expression is dependent on the expressionof an unknown tumor suppressor gene, which is presumablylocated on human chromosome 1 (30). Inactivation of thischromosome 1-related gene following transformation re-

sulted in the inactivation of thrombospondin and the loss ofangiogenesis-suppressing activity (9, 27). Cells containinghigh levels of thrombospondin cannot forms tumors in vivobecause the thrombospondin inhibits capillary growth (9,27).The identification of the factor(s) missing in Ni-trans-

formed or -resistant cells may led to clues about the identityof a senescence gene. What has been clearly established inthis study, however, is that the thrombospondin protein isnot expressed in Ni-transformed cells to the same level as itis in normal cells and that this seems to be a direct result ofthe nickel effects on transcription. The thrombospondinprotein itself has many important regulatory functions, in-cluding its known inhibition of capillary angiogenesis andregulation of growth and differentiation in many systems (19,27, 28). In fact, a number of studies have also linked thethrombospondin protein to senescence in a premature agingsyndrome (Werner's syndrome), in which the thrombospon-din gene expression was increased (26). In addition, there isconsiderable linkage of thrombospondin protein functionand cell growth properties that lead to carcinogenesis.Thrombospondin levels were not decreased in SV40-immor-talized cells but were found to be lower in transformedhuman cells (24, 25).

In understanding the multistage nature of carcinogenesis,it is important to identify control proteins that are bothincreased and decreased in expression. The study of mech-anisms involved in gene expression will identify importantregulatory processes that may be disrupted in cancer cells. Anumber of proteins whose inactivation of function by muta-tion, have been shown to be important in the carcinogenicprocess, including the Rb gene and the p53 gene (29, 33).

It was interesting to find that the Rb gene apparently can

affect the transcription of thrombospondin and does so to a

greater extent in normal than in Ni-transformed cells. How-ever, the basic mRNA levels of the Rb gene are quite similarin CHE and Ni-2. In general, the Rb gene is thoughtto interact with other protein transcription factors, and inthis manner, it regulates transcription (33, 34). BothpTSPCAT3A and pTSPCAT7A plasmids were expressed incells at approximately similar levels as judged by CATactivity. Despite the shorter thrombospondin promoter inplasmid pTSPCAT7A, both plasmids harbored two Splregulatory elements. Rb has been shown to positively regu-late Spl-mediated transcription of insulin-like growth factorII, and we therefore suggest that Rb functions in this mannerto activate thrombospondin gene expression (12).The inactivation of the thrombospondin gene could also

involve mutations or changes in methylation in its promoter.We have also shown that Ni-resistant mouse cells exhibitdecreased transcription of thrombospondin. Preliminarydata indicate that the thrombospondin promoter is hyper-methylated in nickel-resistant cells compared with wild-typecells on the basis of HpaII and MspI restriction enzymedigestion patterns (6a). Future studies will examine hownickel induces hypermethylation of the thrombospondinpromoter as well as the promoter of other genes.

ACKNOWLEDGMENTSThe original cloning procedure and initial identification of the

thrombospondin gene were carried out by S. Cosentino. We thankTomasz Kluz for technical assistance and Jane Galvin for secretarialassistance.

This work was supported by grants ES 00260, ES 04715, ES05512, and ES 04895 from the National Institute of EnvironmentalHealth Sciences and by National Cancer Institute grant CA 13343.

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