Proc. Natl. Acad. Sci. USAVol. 89, pp. 6599-6603, July 1992Medical Sciences

Interferon a induces the expression of retinoblastoma gene productin human Burkitt lymphoma Daudi cells: Role in growth regulation

(retinoblastoma protein/growth suppression)

RAKESH KUMAR* AND IAN ATLASLaboratory of Receptor Biology, Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021

Communicated by Lloyd J. Old, April 14, 1992 (received for review November 14, 1991)

ABSTRACT Interferon a (IFN-a) is a regulatory secretoryprotein with distinctive biological effects such as antiprolifer-ative actions against many tumor cell lines, including humanBurkitt lymphoma Daudi cells. The mechanism underlyinggrowth inhibition by IFN-a is not well established. The growthof many mammalian cell types is also regulated by tumorsuppressor retinoblastoma (RB) gene product, the RB protein.In the studies presented here, we explored the possible involve-ment ofRB protein in the growth inhibitory action of IFN-a inthe Daudi cell model system. We observed that IFN-a inducesa 3- to 10-fold increased expression of RB protein in growth-sensitive Daudi cells but not in the growth-resistant variant ofDaudi cells. IFN-a-mediated induction of RB protein was anearly event that preceded the period of growth inhibition ofDaudi cells. IFN-a-induced RB protein predominantly exists asthe underphosphorylated form. Addition of antibody againstIFN-a to Daudi cells resulted in the inhibition of constitutiveexpression of RB protein and stimulation of [3H]thymidineincorporation. These results demonstrate that the induction ofRB protein expression in IFN-a-treated Daudi cells couldconstitute an important mechanism ofIFN-a-mediated growthregulation.

Regulation of cell proliferation appears to be a complexprocess involving the regulated expression and/or modifica-tion of discrete gene products, including that of inhibitorygrowth regulators such as secretory proteins like interferons(IFNs) and nuclear phosphoproteins like retinoblastoma(RB) gene product. An obvious question that arises iswhether the negative growth regulatory actions of IFN aremediated through the induced expression and/or modifica-tion of the RB protein.IFNs are a family of hormone-like secretory proteins that

interact with neighboring cells and bring about many pheno-typical changes in these cells. These effects include regula-tion of cell differentiation and inhibition of cell growth andproliferation (1). IFNs consists of three antigenically distinctsubclasses: a, P3, y. Each type of IFN has been shown toinhibit growth of various untransformed and transformedcells in culture (2, 3). Recently IFNs have been considered toalso have a negative regulatory role in the autocrine regula-tion of cell proliferation, as several growth factors induce theformation ofIFN in the cells they stimulate (4-6). In addition,IFN-a and IFN-/3 are also constitutively expressed in cellsderived from the hematopoietic system: in lymphoblastoidcells, in leukemic and normal peripheral leukocytes, and inmacrophage cultures (7, 8).The mechanisms underlying growth inhibition by IFNs are

not well established. In sensitive cells, all growth regulatoryactions of IFNs are thought to be mediated by the productsof specific sets of genes, the IFN-induced genes, the expres-

sion of which is increased by IFN (9). The 2'-5'-oligoadenylate [2-5(A)] synthetase system constitutes onesuch IFN-inducible pathway, which has been implicated as amediator of many cellular responses of IFNs (1-3). Inducedexpression of 2-5(A) synthetase by IFNs appears to play animportant role in some cell types (3) but not in Burkittlymphoma Daudi cells. 2-5(A) synthetase and other IFN-induced genes, such as 561, MTII, and 2A, are induced wellby IFN-a in growth-sensitive and growth-resistant Daudicells (10-13), whereas some other genes, such as 1-8, 9-27,and 6-16, are induced only in growth-sensitive Daudi cells(13). Another suggested mechanism of phenotypical rever-sion of transformed cells by IFNs could involve modulationof expression of the oncogenes in target cells (14). IFNs havebeen shown to down-regulate the expression of c-myc inDaudi cells (10, 14) and of c-Ha-ras and c-src in bladdercarcinoma RT4 cells (15). In brief, the mechanism of IFN-a-mediated growth regulation is poorly understood.The RB gene is the prototype of the class of tumor

suppressor genes. The RB gene encodes a 105-kDa nuclearphosphoprotein with characteristics of growth-suppressorand cell cycle regulatory factor (16-19). The RB proteinexists in phosphorylated (pRBF) and underphosphorylated orunphosphorylated (pRB) forms and is ubiquitously present innormal mammalian cells. The state of phosphorylation ofRBprotein varies in a cell-cycle-dependent manner (17, 18). Invarious normal and tumor cells, pRB could be detected inGo/G1 phase and growth-arrested cells, and pRBP could bedetected in S and G2/M phases of the cell cycle (17, 19, 20).The growth-suppressive activity of RB protein has beendemonstrated by introduction of cloned RB gene into RBtumor cells (21) and into RB-deficient (RB minus) prostatecarcinoma and osteosarcoma cell lines (21, 22). Additionalsupport for a role of RB protein in growth regulation isprovided by studies involving inactivation of functional RBprotein (pRB) by DNA tumor viruses, which leads to loss ofgrowth suppression and activation ofthe mitogenic responsesusually associated with transformation (16, 17, 23). Takentogether, these observations raised the possibility that un-derphosphorylated RB protein may act as the active RBprotein with growth-suppressive activity (21-23).

In the present study, we have investigated the possible roleof RB protein in the growth regulatory action of IFN-a inDaudi cells. Here, we report that IFN-a induces the expres-sion of underphosphorylated RB protein in growth-sensitiveDaudi cells, but not in an IFN-a-responsive but growth-resistant variant of Daudi cells, and discuss the implicationsof this observation in understanding the IFN-mediatedgrowth regulation.

Abbreviations: IFN, interferon; RB, retinoblastoma; pRB, RB pro-tein; pRBP, slowly migrating form of RB protein; mAb, monoclonalantibody; TGF-,B1, transforming growth factor P1.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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MATERIALS AND METHODSCell Cultures and IFN. Human Burkitt lymphoma Daudi

wild-type and Daudi growth-resistant cell lines (11) weregrown in RPMI-1640 medium supplemented with 10% fetalbovine medium. Human colon carcinoma SW-480 cell line(24) was grown in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum. Recombinanthuman IFN-a 2a (no. R022-9859; specific activity, 5 x 107international units/mg of protein) was from Hoffman-LaRoche.Growth Assays. Cell proliferation was measured by count-

ing cell numbers in a Coulter Counter. Cell density was alsomeasured by the thiazolyl blue dye (Sigma) uptake method asdescribed (25). Briefly, dye taken up by live cells wasextracted in isopropyl alcohol/1 M HCl (96:4) for determi-nation of optical density at a wavelength of 570 nm. Formeasuring the DNA synthesis, triplicate cultures were la-beled with [3H]thymidine at 10,uCi/ml (5 Ci/mmol; 1 Ci = 37GBq) for 3 hr. Cells were collected in glass fiber filters(Whatman), washed twice with phosphate-buffered saline,twice with 5% trichloroacetic acid, dried, and assayed forradioactivity. Cell cycle distribution was determined bystaining DNA with propidium iodide as described (3).Immunoblot Analysis of RB Protein. Daudi cells were

treated with the IFN-a at a cell density of 2-4 x 105 cells perml. Cell lysates were made as described (26). Briefly, cellswere washed twice with TBS (25 mM Tris-HCl/150 mMNaCl, pH 8.0) and lysed in buffer (50 mM Tris-HCl, pH8.0/120 mM NaCl/0.5% Nonidet P-40/100 mM NaF/200,uMNaVO5/1 mM phenylmethylsulfonyl fluoride/10 Ag of leu-peptin per ml) for 15 min on ice. The lysates were centrifugedin an Eppendorf centrifuge at 4°C for 15 min. Cell lysatescontaining 50 ,ug of total protein were resolved on 7%SDS/polyacrylamide gel electrophoresis and transferredonto nitrocellulose, this was followed by immunoblottingwith anti-RB monoclonal antibody (mAb) RB-PMG3-245(PharMingen, San Diego) and detection of immune com-plexes using 1251-labeled protein A (25).Labeling of RB Protein with [35S]Methionine and

[32P]Orthophosphate and Immunoprecipitation. An equalnumber of cells (1 x 107 cells) from each experimentalcondition was incubated in methionine-free medium for 30min and labeled for 2 hr with 300 ,Ci of [35S]methionine perml in methionine-free medium containing 1% fetal calf serum.Cell lysates were prepared and aliquots containing 400 ,g oftotal cellular protein were immunoprecipitated with 10 ,ug ofanti-RB mAb per ml at 4°C for 3 hr. Immune complexes werecollected by adsorption to rabbit anti-mouse immunoglobulinconjugated to protein A-Sepharose beads as described (27,28). Beads were washed three times with buffer (20 mMTris'HCl, pH 8.0/100 mM NaCl/1 mM EDTA/0.5% NonidetP-40/100 mM NaF/250 ,uM NaVO5/1 mM phenylmethylsul-fonyl fluoride/10,g of leupeptin per ml) and boiled in samplebuffer for resolution on 7% SDS/PAGE. For labeling the RBprotein with 32p, cells cultured with or without IFN-a werelabeled with 700 ,uCi of [32P]orthophosphate per ml, inphosphate-free medium containing 1% fetal calf serum for 2hr.RNA Analysis. Total cytoplasmic RNA was isolated as

described (29). Northern hybridization was done with RNAsthat had been denatured in the presence of formaldehyde(30). The cDNA probe for RB mRNA was kindly provided byW.-H. Lee (31). As a negative control, the filter was rehy-bridized with actin cDNA probe.

RESULTSEffect of IFN-a on RB Protein Expression in Daudi Cells. To

explore the mechanism of IFN-a-mediated negative growth

regulation, we analyzed the effect ofIFN-a on the expressionof RB gene product in human Burkitt lymphoma Daudi cells[sensitive to growth inhibition by IFN-a (11)], by Westernimmunoblotting using anti-RB mAb (Fig. 1A), and correlatedthis with the kinetics of growth inhibition responses in Fig.1B. Fig. 1 shows that treatment of Daudi cells with IFN-aenhanced the steady-state levels ofRB protein expression by2.6-fold after 4 hr and by 10-fold after 16 hr of treatmentcompared to control cells, followed by a decline to near basallevels. RB protein exists in multiple forms comprised ofelectrophoretically slower migrating phosphorylated (pRBP)and faster migrating underphosphorylated (pRB) forms (16).As shown in Fig. 1, the RB protein in Daudi cells appearedas one broad band of -105-115 kDa, the induction of whichby IFN-a was associated with appearance of the fastermoving form (lanes 2-5; asterisks indicate differences in theprotein front migration). Since expression of phosphorylatedand underphosphorylated forms ofRB is induced early afterIFN-a treatment (see below), they cannot be adequatelyresolved in the immunoblot shown here. However, in otherexperiments with low levels of induction we can detect theappearance of a faster migrating band (underphosphorylatedform) of RB protein in IFN-a-treated Daudi cells with nodifference in the expression of slower migrating bands ofRBprotein between control and IFN-a-treated cells (see Fig. 6).Quantitation of the autoradiographs from 10 different immu-noblot experiments indicated a variable extent ofinduction ofRB protein levels by IFN-a in Daudi cells. This was depen-dent on the basal levels ofRB protein in untreated culture. Ingeneral, Daudi cells with lower basal RB protein exhibitedbetter enhancement by IFN-a. Other experiments demon-strated that the increased expression of RB protein was alsoIFN-a dose-dependent. The onset of increased expression ofRB protein in IFN-a-treated Daudi cells preceded the periodwhen growth inhibition occurred. During 16 hr of IFN-atreatment of Daudi cells, there was no significant change inthe growth rate of Daudi cells or in the rate of [3H]thymidine

A

B

pRB _

pRB Levels% 'd of contro

Cell Growth% of controlDNA Synthesis%/. of controL

100 lOG 9 7

1GG 9 84

FIG. 1. Analysis of regulation ofRB protein expression by IFN-ain Daudi cells. Cells were treated with 500 international units ofIFN-a per ml for the indicated times. Cell lysates were made and 50,ug of protein was analyzed by immunoblotting using anti-RB mAb.A and B are two independent experiments. The asterisks in B (*)indicate electrophoretic differences in the migration of RB proteinfront. (B) Quantitation of the data was done by densitometricscanning of the autoradiogram and is presented as fold induction ofcontrol. Cell proliferation was determined by counting cell numberin a Coulter counter and is presented as % of control. The effect onDNA synthesis was determined by measuring the [3H]thymidineincorporation into DNA of Daudi cells, and results are presented as% of control.

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-mRNA-RB

IFN-a - + ± -CHX - - + +

FIG. 2. Northern blot analysis of RB mRNA. Cells were treatedwith 1000 international units of IFN-a per ml with or without 20 Agof cycloheximide (CHX) per ml for 16 hr. Total cellular RNA wasprepared and 20 jig of RNA was electrophoresed for Northernblotting using RB1 cDNA probe (31).

incorporation. In contrast, the level ofRB protein expressionwas induced up to 10-fold during the same period of treat-ment, starting from 4 hr. In addition, there was only a 24%increase in the population of cells present in the Go/G1 phasein Daudi cells treated with IFN-a for 18 hr compared tocontrol cells (data not shown).To determine whether the increase in the levels of RB

protein expression in IFN-a-treated Daudi cells is associatedwith increased expression of RB mRNA, Northern blotanalysis was performed. Fig. 2 shows that 16 hr of IFN-atreatment increased the steady-state levels of 4.6-kilobasemRNA of RB1 by 2.6-fold, cotreatment of cultures withcycloheximide superinduced the RB mRNA by 5.6-fold (lane3), and cycloheximide-mediated stabilization of RB1 mRNAresulted in an increase of 3.4-fold (lane 4).

Analysis of the State of Phosphorylation of IFN-a-InducedRB Protein. Since RB protein appeared as one broad band inDaudi cells, and also to confirm that the observed fasterelectrophoretic migration of RB protein in IFN-a-treatedDaudi cells (Fig. 1) was due to differences in the state ofphosphorylation, IFN-a-treated Daudi cells were labeled inparallel with [32P]orthophosphate or [35S]methionine for an-alyzing labeled RB protein. Fig. 3A shows the kinetics ofexpression of32P-labeled RB protein (pRBP, upper panel) and35S-labeled RB protein (pRB, lower panel) in Daudi cells thathad been pretreated with IFN-a for different length of time.To quantitate the data, RB protein bands in Fig. 3A werescanned by a densitometer and plotted on an arbitrary scaleafter subtracting the background in each lane (Fig. 3B).Pretreatment of Daudi cells with IFN-a induced the levels ofpRBP and pRB differentially. The levels of pRBP wereinduced maximally by 3-fold during 4 hr and were below basallevels between 16 and 48 hr of IFN-a treatment. In contrast,

A32P Autorad

pRB XE*

35S Autorad

pRB { '

Hr: 0 4 16 24 4832p 35S

pRBP- pRB- -

IFN-a -+ -+

the levels of pRB were induced continually up to 5-foldbetween 4 and 24 hr and declined to 1.5-fold by 48 hr ofIFN-atreatment. Similar results were obtained when Daudi cellswere treated with IFN-a for 16 hr (Fig. 3A, lower autoradi-ograms). The observed increase in the levels of 35S-labeledRB protein in IFN-a-treated cells was not due to associatedphosphorylated form, as treatment of 35S-labeled RB proteinwith acid phosphatase (which converts the phosphorylatedform into underphosphorylated form) did not change theelectrophoretic mobility of RB band compared to nonphos-phatase treated (data not shown). Because IFN-a may alsoenhance the expression of RB protein by affecting the sta-bility ofRB protein, we have also performed experiments toexamine the effect of IFN-a treatment for 4 hr and 24 hr onthe turnover/degradation ofnewly synthesized (pulsed for 90min) 35S-labeled RB protein. There was no change in thedegradation rate of 35S-labeled RB protein with or withoutIFN-a (data not shown). Taken together these results suggestthat IFN-a-induced, newly synthesized RB protein predom-inantly exists in underphosphorylated form in addition tosome appearance of phosphorylated form, which may haveresulted from in vivo modification of induced RB protein inDaudi cells.

Specificity of Induction ofRB Protein Expression by IFN-a.Results in Fig. 4 show that the effect of IFN-a on theinduction ofRB protein in Daudi cells was specific in nature,as preincubation of IFN-a with neutralizing anti-IFN-a poly-clonal antibody (Ab) blocked the induction of RB proteinelicited by IFN-a (lanes 3 and 2). Other experiments indi-cated that the addition of four times excess of IFN-a couldcompletely abolish the inhibitory effect of Ab on the IFN-a-mediated RB protein expression, and there was also addi-tional enhancement of RB protein expression (data notshown). In experiments using anti-IFN-a Ab (in the absenceof IFN-a), there was a significant reduction in the levels ofbasal RB protein (lanes 1 and 4), and this was accompaniedby appearance of a slow migrating band of RB protein (lane5). Incubation of Daudi cells with anti-IFN-a Ab for 24 hrresulted in a 45% increase in the [3H]thymidine incorporationduring the 3-hr pulse period (data not shown). These resultssuggest that the constitutive expression of RB protein andgrowth of Daudi cells can also be regulated by endogenousIFN-a or related IFN. Since another growth inhibitor, TGF-P1, has been shown to modify the status of the phosphory-lation of RB protein in mink cells (26), we investigated the

B U)c05 -

Lf4Lo--2 4-

co3m.0aL 3 -

00

A2

010

DCD

to

LcoJ0cL ONco

12 24IFN-a Pretreatment, hr

FIG. 3. IFN-a regulates RB protein expression. (A) Effect of IFN-a on the levels of radiolabeled RB protein as a function of time. Daudicells were treated with 1000 international units of IFN-a per ml for indicated times, and 1 x 107 cells from each condition were labeled witheither [32P]orthophosphoric acid (upper panel) or [35S]methionine for 2 hr. Cell lysates were made and then immunoprecipitated with anti-RBmAb. Quantitation of the RB band was obtained by densitometric scanning of the autoradiogram. Background in each lane was also determinedand was subtracted from RB bands for plotting the data on an arbitrary scale (B). In an independent experiment Daudi cells were treated withIFN-a for 16 hr and labeled with [32P]orthophosphoric acid and [35S]methionine; immunoprecipitated RB protein was analyzed on 7%SDS/PAGE (A, lower autoradiograms).

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IFN-a - + +--Ab-IFN-a -- - + + +Ab-TGF -}TGF- 31

-4 ±-+

+

_pRBPhos4 j -pRBE I

Lanes 1 2 3 4 5 6 7 8 9

FIG. 4. Specificity of IFN-a-mediated regulation of RB protein.Daudi cells were treated for 16 hr under indicated conditions. Dosesof agents used were as follows: IFN-a, 500 international units/ml;polyclonal sheep Ab against human IFN-a (Advanced BiotherapyConcepts, Columbia), 2000 neutralizing units/ml; polyclonal rabbitAb against human TGF-.31 (R & D Systems, Minneapolis), 100,ug/ml; transforming growth factor #1 (TGF-#1), 5 ng/ml. IFN-a andanti-IFN-a Ab were preincubated together at 37C for 30 min beforeaddition to the cell culture (lane 3). Cell lysates were made, and RBprotein was analyzed by immunoblotting. Lane 5 has resulted fromeight times longer exposure of lane 4 and shows the distinct appear-ance of the slower migrating RB form, which was not seen in anyother overexposed lanes.

possibility of involvement of TGF-f31 in the effect of IFN-aon RB protein. Lanes 7 and 8 show that the inclusion ofneutralizing polyclonal rabbit Ab against TGF-,31 resulted inthe inhibition of IFN-a-induced RB protein expression. Ex-ogenous recombinant TGF-81 did not have any effect on RBprotein expression (lane 9). These results suggest that theIFN-a-induced enhanced expression of RB protein may bemediated by a TGF-f-related polypeptide recognized by theAb used and/or may also require presence of IFN-a.RB Protein Expressio and Growth Inhibiin by OEN. To

analyze the possible relationship between RB protein expres-sion and growth inhibition by IFN-a, we compared the IFN-agrowth-sensitive Daudi wild-type cells with IFN-a growth-resistant cells (which respond to IFN-a by inducing IFN-induced genes but are not growth-inhibited). Fig. 5 showsthat IFN-a induced RB protein expression in Daudi wild-typecells but not in Daudi resistant cells, which were also notgrowth inhibited by 500 international units of IFN-a per ml.We have also examined the effect of IFN-a on modulation

of RB protein expression in other carcinoma cells. Coloncarcinoma SW-480 cells were growth inhibited by 72 hr ofIFN-a treatment, and there was also 4-fold enhanced expres-sion of RB protein with faster electrophoretic migrationduring 16 hr of treatment with IFN-a (Fig. 6). IFN-a alsoinduces RB protein in IFN-a growth-responsive myeloidlymphoma K-562 cells. IFN-y did not have any effect on thegrowth of SW-480 and K-562 cells. In epidermoid carcinomaA-431 cells, which were growth inhibited by IFN-y and notby IFN-a (3), there was no effect of IFNs on RB proteinexpression. Daudi cells are shown in Fig. 6 as a control.

DISCUSSIONCellular growth and division are influenced by IFNs, whichhave been shown to exert growth inhibitory effects on

Daud Wildr%~~~~~~~~~~~~~~~~~~

Hr. 0 12 24 48

DO dRes_

0 12 48

FIG. 5. Regulation of RB protein by IFN-a in Daudi wild-type(Daudiid) cells and Daudi growth-resistant (DaudiRes) cells. Daudiidcells and DaudiRes cells, which are IEN-a sensitive but are not growthinhibited by IFN-a, were treated with IFN-a for the indicated times.Cell lysates were made and expression ofRB protein was determinedby immunoblot analysis as described in the legend to Fig. 1.

SW-48C.( K-562

pRP. --

3rowth Inhitfior.% of Contr-

FiG. 6. Effect of IFNs on the levels of RB protein in tumor celllines. SW-480 cells, K-562 cells, A-431 cells, and Daudi cells weretreated with 1000 units of IFNs per ml for 16 hr. Cell lysates weremade for immunoblot analysis of RB protein as described in thelegend to Fig. 1. Growth inhibition of cells was determined after 72hroftreatment with 1000 units ofIFN-a per ml. Results are presentedas % of control. Arrows indicate positions of two bands of RBprotein.

various cells in culture (2). Growth inhibitory actions ofIFN-a are thought to be dependent on the induced changesin expression and/or modification of specific cellular regu-latory gene products. In addition to IFN-a, the growth ofvarious mammalian cells has also been shown to be nega-tively regulated by the RB protein (16). The present studywas undertaken to explore the possible involvement of RBprotein in the growth regulatory actions of IFN-a usingIFN-a growth-sensitive human Burkitt lymphoma Daudicells as a model system.

Results presented here demonstrated that treatment ofDaudi cells with IFN-a significantly induced RB proteinexpression by 3- to 10-fold. Since RB protein has been shownto regulate the cell growth in a cell-cycle-dependent manner,induction ofRB protein by IFN (which does not regulate thecell growth in a cell-cycle-dependent manner) opens a wholenew area of investigation regarding the additional modes ofactions of these regulatory proteins. The finding that IFN-amediates induction of RB protein as an early event is inter-esting, as this induction precedes the appearance of growthinhibitory responses, such as inhibition of [3H]thymidineincorporation, and growth rate and GO/GI phase arrest ofDaudi cells. This implies that induction ofRB protein may actas a signal for growth inhibition of Daudi cells by IFN-a.Moreover, the evidence that IFN-a-induced RB proteinpredominantly exists as underphosphorylated RB proteinbefore and during the onset of GO/GI growth arrest maysuggest that absolute induction and state of phosphorylationofRB protein are important in IFN-a-treated Daudi cells andindicate a close relationship between growth inhibition ofDaudi cells and IFN-a-mediated induction of RB proteinexpression that was only related, in part, to Go/Gj cell arrestof Daudi cells. The.mechanism of observed IFN-a-mediatedinduction of RB protein remains to be delineated. This mayoccur at the transcriptional level and/or posttranscriptionallevel and may involve enhanced RB mRNA stability, leadingto an increase in RB protein expression. Earlier studies havesuggested that the RB protein is regulated at the protein levelby phosphorylation (16, 26). In addition, our present resultsindicate that the IFN-a may also regulate expression ofunderphosphorylated RB protein at the pretranslational levelof the gene. In this respect it is important to note that themitogen-mediated stimulation of hematopoietic cells hasbeen shown to be accompanied by increased expression ofphosphorylated RB protein due to reduced half-life of RBtranscript (32). Moreover, recent data (33, 34) on the RB geneexpression in lymphocytes in patients with germ-line deletionof one allele and differentiation-associated increased expres-sion ofRB protein in human testicular tumor also suggestedpossibilities of additional transcriptional regulation of RBgene.The observation that the constitutive expression of RB

protein was regulated by endogenous IFN-a or related IFNs,

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which also regulate the Daudi cell growth, raises a possibilitythat the reported variations of basal RB protein expression incontrol Daudi cells among different experiments could bedue, in part, to variable degrees of interaction with endoge-nous IFN. It is possible that the endogenous IFN could actas one of the cellular factor(s) that controls the action of theRB protein. This is important, as certain types of leukemiccancer cells with rearranged or deleted IFN genes (35) mayhave functional breakdown of this IFN-RB novel negativeregulatory pathway. Furthermore, our preliminary data withhuman colon carcinoma SW-480 cells, which do not havedetectable endogenous IFN-a expression, and with SW-480cells transfected with IFN-a-2a gene, indicated that theconstitutive expression of RB protein in transfected SW-480cells was 60%o higher than in parental SW-480 cells, and thegrowth rate of transfected SW-480 cells was 40% slower thanthat of the parental SW-480 cells-thereby raising the pos-sibility of interactions between endogenously derived IFN-aand RB protein for the benefit of cellular growth regulation(R.K. and N. B. K. Raj, unpublished observations).Data from the literature suggest that the growth inhibition

of Daudi cells by IFN-a could also involve down-regulationof c-myc mRNA (10). In this context, it is important to notethat TGF-f1-induced growth inhibition of human kerati-nocytes is accompanied by down-regulation ofc-myc mRNA,which is thought to be mediated by RB protein interactionwith c-myc promoter (36). Therefore, it is possible thatIFN-a-induced RB protein may play a role in the earlierreported c-myc mRNA repression (10) in IFN-a-treatedDaudi cells. Further investigations are needed to determinewhether induction of underphosphorylated RB protein byitself, or in conjunction with other mechanisms such asreduced expression of c-myc mRNA, is sufficient to accountfor IFN-a-induced growth inhibition of Daudi cells.The observed IFN-a-mediated induction of RB protein

appears to have a close relationship with the growth inhibi-tory action of IFN-a in Daudi cells, as IFN-a did not induceRB protein expression in Daudi growth-resistant cells andalso in RB minus prostate carcinoma DU-145 cells, whichwere not growth inhibited by IFN-a (data not shown).Induction of RB protein expression by IFN-a is not aphenomenon restricted to Daudi cells, as IFN-a also inducesRB protein expression in other human tumor cell lines, suchas colon carcinoma SW-480 cells, myeloid carcinoma K-562cells, and bladder carcinoma T-24 cells, which are growthinhibited by IFN-a. Taken together, the data presentedprovide evidence suggesting that induction of RB proteinexpression could constitute an important mechanism of IFN-a-mediated growth regulation of Daudi cells.

We thank Lawrence Pfeffer for generously providing Daudi celllines, N. Babu Raj for SW-480 cell lines, Joan Massague for theDU-145 cell line, Wen-Hwa Lee for RB cDNA probe, John Men-delsohn for encouragement and many helpful discussions, and LloydOld for helpful suggestions. The secretarial assistance of FrantziePaul is gratefully acknowledged. This work was supported by fundsto the Laboratory of Receptor Biology.

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