tumor suppressor proteins as regulators of cell differentiation1

[CANCER RESEARCH 58. 4282-4287. October I, 1998]

Advances in Brief

Tumor Suppressor Proteins as Regulators of Cell Differentiation1

Zhan-Rong Li, Rosemary Hromchak, Anuradha Mudipalli, and Alexander Bloch2

Rnxm'll Park Cancer Institute. Buffalo, New York 14263

Abstract

The products of the tumor suppressor genes are considered to functionas specific inhibitors of tumor cell growth. In this communication, wepresent evidence to show that these proteins inhibit tumor cell proliferation by participating in the activation of tumor cell differentiation. TheMI -I human myeloblastic leukemia cells used in this study proliferatewhen treated with insulin-like growth factor I and transferrin but differ

entiate to monocytes when exposed to tumor necrosis factor a or transforming growth factor /Â¡I,or to macrophage-like cells when treated with

both these cytokines. Initiation of proliferation but not of differentiationwas followed by a 20- to 25-fold increase in the nuclear level of the DNApolymerase-associated processivity factor PCNA and of the proliferation-

specific transcription factor E2F1. In contrast, induction of differentiationbut not of proliferation was followed by a 25- to 30-fold increase in the

nuclear level of the tumor suppressor proteins p53 (wild type), pRb, andpl30/Rb2 and of the p53-dependent cyclin kinase inhibitor p21/Cipl. p53

and p21/Cipl, respectively, inhibit the expression and activation of PCNA,whereas pI30 and pRb, respectively, inhibit the expression and activationof E2F1. As a result, Gt-S-associated DNA and mRNA synthesis is inhib

ited, growth uncoupled from differentiation, and maturation enabled toproceed. Where this function of the tumor suppressor proteins is impaired, the capacity for differentiation is lost, which leads to the sustainedproliferation that is characteristic of the cancer cell.

Introduction

Functionally mature cells that are formed in the tissues of anorganism derive from primitive stem cells that, along discrete stagesof development, differentiate to their fully mature state. The proportion of proliferating and differentiating cells that are present at intermediate stages of the maturation path is tightly regulated to assure abalanced supply of functionally competent cells. Regulation of thissupply is mediated by cytokines produced by cells that are part of thematuration sequence itself or that belong to functionally related developmental paths. If maturation is permanently arrested at an intermediate stage of the developmental sequence without concomitantinhibition of cell proliferation, a population of morphologically andfunctionally immature "cancer" cells accumulates, which interferes

with the normal functioning of the affected organism.Numerous mechanisms that underlie such oncogenic transforma

tion have been identified. They predominantly involve alterations inproliferation-related signal transduction, which lead to sustained cell

growth without cell differentiation (1). Tumor suppressor proteinshave been shown to be capable of abolishing such sustained growth byinterfering with cell cycle progression, such interference having beenconsidered to form a specific surveillance mechanism (2). However,in this study, we provide evidence to show that the inhibition of tumor

Received 5/20/98; accepted 8/13/98.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.

1This study was aided by Grant CA-36241 from the National Cancer Institute.

Department of Health and Human Services.: To whom requests for reprints should be addressed, at Roswell Park Cancer Institute.

Elm and Carllon Streets. Buffalo. NY 14263. Fax: (716) 845-8857.

cell proliferation by the tumor suppressor proteins relates to their roleas natural mediators of cell differentiation.

Materials and Methods

Antibodies and Reagents. TGF-/311 was kindly provided by Bristol-

Myers Squibb (Seattle. WA). and TNF-a by Asahi Chemical Industry (Tokyo,Japan). IGF-I was purchased from Austral Biologicals (San Ramon. CA).Antibodies to PCNA (PC10), p53 (DO-1). p21/WAFl/Cipl (C-19), pRb(C-15), E2F1 (C-20), pl30 (C-20), as well as goat antimouse and goat

antirabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz,CA.) Immobilized protein A was purchased from Pierce (Rockford. IL), andthe molecular standards used in SDS-PAGE were obtained from Bio-Rad

Laboratories (Hercules. CA). Western blotting reagents were supplied byAmersham International (Arlington Heights, IL). The phosphorothioate-mod-ified antisense oligonucleotides to pRb, 5'-GGG GOT TTT GGG CGG CATGAC-3' (3); to p53. 5'-CGG CTC CTC CAT GGC AGT-3' (4); and to asegment of the pl30 promoter. 5'-CGA CGG CAT AGC OCA CCC CT-3' (5);

and their sense oligonucleotides were synthesized by the Roswell Park CancerInstitute Biopolymer Facility. SuperFect transfection reagent was supplied byQiagen. Inc. (Chatsworth, CA).

PCNA-prornoter and E2F1-promoter DNA was provided to us. respectively,

by Dr. Swati P. Deb (University of Texas Health Science Center. San Antonio.TX: Ref. 6). and Dr. David G. Johnson (The University of Texas M. D.Anderson Cancer Center. Smithville. TX; Ref. 7).

Cell Cultures. ML-1 cells were maintained in RPMI 1640 containing 10%FBS. Cultures containing 5 X IO5 cells/ml were grown in this medium for 3days at 37Â°Cin a humidified atmosphere. Fresh medium was then added to

reduce the cell number to the starting concentration. To perform the assaysdescribed, the cells were synchronized in G0 by incubation for 3 days in RPMI1640 containing 0.05% FBS. After washing with serum-free RPMI 1640,5 X IO5 cells/ml were suspended in RPMI 1640 containing 0.5% FBS, which

was added to preserve their viability during the prolonged (70 h) assay period.Proliferation of the cells was initiated by treatment with 40 ng/ml IGF-I plus0.5 /xg/ml Tf; monocytic differentiation was induced with 60 ng/ml TGF-/31 orwith 20 units/ml TNF-a; and macrophage-like cells were obtained by adding10 ng/ml TGF-/31 together with 10 units/ml TNF-a. The number of cells

obtained under these conditions was counted by hemocytometer and theirviability assayed by trypan blue exclusion; the extent of differentiation wasassessed by determining the mature phenotype.

Nuclear and Cytoplasmic Extracts. To obtain nuclei, 2-5 x 1()7 cells

were washed with serum-free, ice-cold RPMI 1640, followed by suspension in

0.3 ml of nuclear buffer consisting of 2 mM MgCl,. 5 mM K2HPO4, 0.1 mMEDTA, 1 mM PMSF. 20 fj.g/ml aprotinin, 20 (Â¿g/mlleupeptin. and 0.1 mMNa,VO4. An additional 0.3 ml of nuclear buffer containing 0.7% of TritonX-100 was then added; after standing on ice for 8 min. the cells were

homogenized, and the homogenate centrifuged at 800 rpm. the supernatantntbeing designated the cytoplasmic fraction. The pellet was washed once withnuclear buffer, and the nuclear extract was prepared as described previously(8). Briefly, the pellet was resuspended in butter containing 20 mM HEPES(pH 7.9). 25% glycerol, 0.42 M NaCl. 1.5 mM MgCl,, 0.2 mM EDTA, 0.5 mMPMSF. 0.5 mM DTT, 0.1 mM Na,VO4, 50 mM NaF. 30 mM PP,. 20 ng/mlleupeptin. and 20 /j.g/ml aprotinin; homogenization was carried out using aKontes Duali tissue grinder. After centrifugation of the homogenate, the

*The abbreviations used are: TGF-ÃŸl. transforming growth factor 01; TNF-a. tumor

necrosis factor a; IGF-I. insulin-like growth factor I; Tf. transferrin: PCNA. proliferating

cell nuclear antigen: FBS. fetal bovine serum: EMSA. electrophoretic mobility shift assay:PMSF. phenylmethylsulfonylfluoride; pRB. retinoblastoma protein: DF. differentiationfactor.

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DIFFERENTIATION REGULATORS

supernatantâ€”designated the nuclear extractâ€”was frozen and stored at-70Â°C.

Western Blotting and Immunoprecipitation. The levels of p130, p53,pRB. p21/Cip, PCNA, and E2F1 in the nuclear and cytoplasmic fractions wereassayed by Western blotting, and immune complexes were detected by immu-

noprecipitation followed by Western blotting (8). Briefly, nuclear or cytoplasmic extract (300 ^ig of protein) was added to 300 Â¡j.\of immunoprecipitationbuffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 20 mM

EDTA, 0.1 mM Na,VO4, 50 mM NaF, 1 mM PMSF, 1 mM benzamidine, 10fig/ml trypsin inhibitor, 20 /xg/ml leupeptin. and 20 /Â¿g/mlaprotinin. To eachsample, 1.5 fig of the desired affinity-purified antibody was added. Afterrotating at 4Â°Cfor 1 h, 40 fil of immobilized protein A was added to each

sample, and rotation continued overnight. Immunoprecipitates were collectedby centrifugation, and after washing, the immunecomplexes were analyzed byWestern blotting as described above.

Measurement of DNA Synthesis and Rapidly Turning-over mRNA.DNA synthesis was determined by methyl-[3H]thymidine incorporation (8).

Rapidly turning over mRNA was measured as described previously (9).Briefly, cells were labeled for l h with 5-[3H]uridine (20 Â¿iCi/ml, 23.6

Ci/mmol),and RNA was extracted with a concentrated solution of Nal, mRNAbeing selectively bound to nitrocellulose paper. 3H-labeled mRNA was then

counted by liquid scintillation spectrometer.EMSA. For determining p53 binding to the PCNA promoter, PCNA pro

moter DNA was cut with Hindlll. and the segment â€”¿�272to â€”¿�46was isolatedas described (6). The fragment was end-labeled with 32P using T4 polynucle-

otide kinase. Binding reactions were carried out using 10 /xg of nuclear proteinand the labeled PCNA promoter DNA fragment (50,000 cpm) in binding bufferconsisting of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA,0.5% (v/v) glycerol, and 1.5 fig poly(dl-dC) in a final volume of 25 /il. The

reactions were carried out at room temparature for 30 min. The samples wereelectrophoresed at 175 V on 5% nondenaturing polyacrylamide gel with 0.5 MTris-borate/EDTA buffer. Specificity of binding was confirmed by the addition

of 50 x unlabeled probe. p53 in the complex was identified by incubating thereaction mixtures with 1.5 fig of anti-p53 antibody for 30 min (supershiftreaction). EMSA was also used to determine pl30-E2Fl promoter binding.The pGL3 E2F1 promoter DNA (7) was digested with Eagl, and the 101-bpsegmentâ€”purified by agarose gelâ€”was end-labeled with 12P. Specificity of

binding was confirmed by adding 75 x unlabeled probe to the reaction mixture,and antibody to p 130 was used to detect the presence of p 130 in the complex.

Effect of Antisense Oligonucleotides of p53, p 130, and pRb on MI,-ICell Differentiation. Ten, 5, and 20 /xg, respectively, of the antisense oligo-nucleotides to p53, p 130, and pRb were incubated with 2 /il superFect at 37Â°C

for 15 min. diluted with 100 /xl of serum-free medium, and added to I ml ofcell suspension (5 X IO5 cells/ml) containing 10 ng/ml TGF-ÃŸl and 10

units/ml TNF-a. Corresponding sense Oligonucleotides were used as controls.

After 2 days of incubation, the cell number was counted and the extent ofdifferentiation determined.

Results and Discussion

ML-1 human myeloblastic leukemia cells (10), suspended in serum-depleted RPMI 1640, proliferate when treated with IGF-I plus Tf(Fig. la) but differentiate to monocytes when exposed to TGF-ÃŸl(Fig. \b) or to TNF-a (not shown). Macrophage-like cells are formed

when these two cytokines are applied jointly (Fig. le). By adding0.5% FBS to these cultures, their long-term (>48 h) viability is

enhanced without their maturation status being affected. When treatedwith 60 ng/ml of TGF-/31 or with 20 units/ml of TNF-a, -85-90% of

the cells convert to monocytes (Fig. \b). When both these cytokinesare applied (10 ng/ml TGF-ÃŸland 10 units/ml TNFa), 90-95% of thecells mature to macrophage-like cells (Fig. le). Stimulation of prolif

eration leads the cell population to double after approximately 70 h ofincubation (Fig. Id), whereas induction of differentiation results incessation of cell growth. Because the 0.5% amount of FBS that isadded to the RPMI 1640 to enhance viability contains a small amountof IGF-I and Tf, ~15%-20% of the cells in controls are able to

proliferate (Fig. \d). This amount of FBS does not affect the numberof differentiation-induced cells.

Because induction of ML-1 cell differentiation is accompanied by

proliferation arrest at the GrS border (8), we began an examination ofthe mechanisms by which this proliferation-arrest is regulated. We,

first, focused our attention on the expression of PCNA (11) and E2F1(12), two key mediators of the proliferation-associated G,-S transit.

Because the level of these mediators, like that of other regulatoryproteins, varies as the cells progress along the cell cycle, all cellpopulations used in these assays were synchronized in G0 by serumdepletion (13). This modification assured that the results obtainedderived from an essentially homogeneous cell population, rather thanfrom mixed populations containing cells present at multiple stages ofcell growth or differentiation. Because such regulatory proteins can bepresent at different levels and in different molecular forms in nuclei ascompared to cytoplasm, separation of the two compartments was alsocarried out routinely to allow an accurate assessment of the site(s) atwhich these proteins exert their primary activity unimpeded by artifacts that may result from intermixing the components of different cellcompartments.

Employing these assay conditions, we found that, as shown in Fig.2a, the level of the PCNA was low in the cytoplasm of control as wellas of proliferation- and differentiation-induced ML-1 cells. In contrast, as shown in Fig. 2b, an approximately 20-fold increase in thelevel of PCNA occurred in the nuclei of proliferation-stimulated cells.

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O 20 40 60 80 (h)Fig. I. Morphology of ML-1 human myeloblastic leukemia cells stimulated to proliferate with IGF-I and Tf (Â«)or to differentiate to monocytes with TGF-ÃŸl(/;) or macrophage-like

cells with TGF-/31 + TNF-a (<â€¢).The lime-dependent change in ihe number of unstimulated (O). proliferation-stimulalcd (â€¢).or differenliation-induced cells (D) is shown in (</).The

data points shown in Fig.\d represent the mean Â±SD derived from five separate experiments.

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DIFFERENTIATION REGULATORS

Fig. 2. Time-dependent change in the levels of PCNA(n, h) and E2FI (d, r) proteins present in the cytoplasmand nuclei of untreated (O), proliferation-stimulated (â€¢).or differentiation-induced (D) ML-I cells, as related to

the extent of DNA (r) and mRNA synthesis (/) occurringin these cells. All values shown in the panels a, h, J, ande were determined by Western blots normalized to 0-lime

control. The data given in the panels are representative ofthree separate experiments each. The insets given in thepanels show peak levels of the respective proteins ingrowth (G) and differentiation (/J).

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The level of this processivity factor remained low in the nuclei ofcontrol cells and of differentiation-induced cells. In keeping with the

determinant role PCNA plays in DNA synthesis (11), the pronounceddifference in the level at which this protein was expressed in growthversus differentiation (Fig. 20) was reflected in the extent to whichDNA synthesis took place in proliferation-stimulated as compared tocontrol cells or to differentiation-induced cells (Fig. 2c). ExtensiveDNA synthesis occurred only in growth-induced cells but not incontrol or in differentiation-induced cells.

In parallel with PCNA, the transcription factor E2F1 was present atonly a very low level in the cytoplasm of control as well as of growth-and differentiation-induced cells (Fig. Id). In contrast, this transcription factor was expressed at a high level in the nuclei of proliferation-

induced cells (Fig. 2e), its concentration being low in the nuclei ofcontrol and of differentiation-stimulated cells. E2F1 regulates thetranscription of various genes required for the proliferation-associatedG,-S transit (12), and the difference in level at which E2F1 is present

in growth as compared to differentiation is, thus, expected to affect theextent of transcription that ensues. As shown in Fig. 2/, E2F1-

mediated mRNA synthesis was, indeed, high in growth and very lowin differentiation. These data were in agreement with previous findings demonstrating that PCNA and E2F1 serve, respectively, as determinants of DNA and mRNA synthesis required for the proliferation-associated cell cycle transit from G, to S-phase. Their lack of

expression in differentiation, thus, provided a mechanistic basis forthe absence of the G,-S transit in the maturation process.

This recognition raised questions, concerning the mechanisms bywhich the expression and activation of PCNA and E2F1 was regulated. As summarized in Fig. 3, various reports had shown that thelevel and activity of PCNA and E2F1 is controlled by diverse tumorsuppressor proteins. Thus, wild-type p53, present in ML-1 cells (14),

was reported able to interfere with PCNA synthesis by repressing thePCNA promoter (15-17). p53 was also shown capable of regulating

the expression of the cyclin kinase inhibitor p21/Cipl/Waf/l via a

binding site in the p21 promoter (18). p21/Cipl, in turn, was observedable to inhibit DNA replication by direct binding to PCNA (19-21),and by interacting with cyclin-dependent kinases (22-25). In parallel,

the tumor suppressor protein pl30/Rb2 was found capable of inhibiting transcription from the E2F1 promoter (7), whereas the pRB wasobserved able to inhibit the E2F1-dependent transactivation of S-phase genes by direct binding to E2F1 (26-28).

The in vitro demonstration that these tumor suppressor proteins candown-regulate the expression and activation of PCNA and E2F1 in aconcentration-dependent manner suggested that, in intact cells, they

may act in a parallel manner. That expectation was borne out by thedata shown in Fig. 4. The tumor suppressor protein p53 (Fig. 4, a andb), its auxiliary factor p21/Cipl (Fig. 4, c and d), and the tumorsuppressor protein pRb (Fig. 4, g and h), were all expressed at 20- to30-fold elevated levels in the nuclei of differentiation-induced cells,

remaining at very low levels in the nuclei of control and of proliferation-stimulated cells. p21 deviated from this pattern by the fact that

its level was also increased to a significant extent in the cytoplasm of

Fig. 3. Schematic outline of metabolic sites at which the synthesis of proliferation-associated DNA and mRNA is inhibited by tumor-suppressor proteins expressed indifferentiation-induced cells.

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DIHLRI'.MÃ•ATION REGULAIORS

Fig. 4. Time-dependent change in the levels of

the tumor suppressor proteins p53 (a, b}. pl30/Rb2(e. /), and pRb (g, H) and of the auxiliary proteinp21/Cipl (c, i/l in cytoplasm and nuclei of untreated(O), proliferation-stimulated (â€¢).or differentiation-induced (D) ML-1 cells. The values shown weredetermined by Western blots normalized to 0-time.The data presented in the panels are representativeof at least three separate experiments each.

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the differentiation-induced cells (Fig. 4c). A deviation from the gen

eral pattern was also provided by pl30/Rb2 (Fig. 4/), its initialnuclear concentration being elevated in control as well as in growth-and differentiation-induced cells. That level increased further withtime in differentiation but decreased in control and in growth-stimulated cells (Fig. 4/). Isolated differentiation-associated increases in the

level of one or the other of these tumor suppressor proteins were alsoobserved in diverse other cell lines (for examples, see Refs. 29-31).

These results clearly demonstrated that expression of these tumorsuppressor proteins and of the auxiliary protein p21/Cipl is uniquely

associated with the process of differentiation. This conclusion isfurther supported by the data shown in Fig. 5. The EMSA analysispresented in Fig. 5a demonstrated that p53 binds to the PCNApromoter only in extracts from differentiation-induced (D) but notfrom proliferation-induced (G) ML-1 cells. That selective complex-

ation correlates with the fact that p53 is expressed at markedlyelevated levels only in the nuclear extracts from differentiation-

stimulated cells (Fig. 4b).Similarly, as shown in Fig. 5b-2, significant complexation of pi 30

with the E2F1 promoter occurred at the 40-50 h time point in extracts

Fig. 5. Interaction of tumor suppressor proteinswith the processivity faclor PCNA and the transcription factor E2FI. Fig. 5Â«shows the binding ofwild-type p53 to the human PCNA promoter. Segment -272 to -46 (6) was labeled with "P and

incubated for 30 min with nuclear extract fromML-1 cells untreated (O. treated with IGF-I (G), orwith TNF-a + TGF-01 (O). Specificity of bindingwas confirmed by competition with 50X unlabeledprobe (D + cold 50X). The PCNA/p53 complexformed in differentiation was identified using anti-p53 antibody (D + ap53). Fig. 5. b-l and b-2.

shows pl30/Rb2 binding to the E2F1 promotersequence in nuclear extracts from ML-1 cells untreated (O or treated with IGF-I (C) orTNFa + TGF-/31 (D) for 5 h (/>-/> or for 46 h(b-2). A 101-bp Eag\ fragment, derived from thehuman E2FI promoter, was labeled with 33P and

used as the probe. A 75 X unlabeled probe was usedto demonstrate the specificity of binding. The antibody to pl30/Rb2 was used to demonstrate thepresence of p 130 in the complex. Fig. 5, c and il,shows the protein-protein interactions betweenp21/Cipl-PCNA and pRb-E2Fl in nuclear extracts(300 u.g protein/sample I from differentiation-stimulated cells (Lane D). The extracts were immuno-precipitated with either anli-PCNA or anti-p21/Cipl (5c) and with anti-pRb or anti-E2FI <5</>.Theimmune complexes were collected using immobilized protein A beads, and were probed with antibodies for the presence of p21/Cipl. PCNA. E2F1.and pRb.

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4285

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Fig. 6. The effect of antisense (AS} and sense (Sioligonuclcolides of pRb. p53. and pl.30 on Ihenumber and the differentiation status of ML-1 cells.The antisense (n and r) or sense (b and </) oligonu-cleolides were added to the ML-1 cell culturesdevoid of (control) or containing the DFs TGF-ÃŸland TNF-a. Cell numbers were determined by he-mocylometer. and maturation was determined by-

assessing the differentiated morphology.

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from only differentiation-stimulated (D) but not from proliferation-

stimulated (G) cells. That complexation correlates with the high levelof p 130 that, at this time interval, is found present in the nuclei ofdifferentiation-induced cells (Fig. 4/). The elevated levels of p 130that were present in control cells and in proliferation-stimulated cellsat early time points (0-15 h; Fig. 4/), likely account for the significant

extent of complexation between p 130 and E2F1 that occurred at thesetime points (Fig. 5b-l) in growth-induced as well as in differentiation-

induced cells. It seems that early transcription from the E2F1 promoter is suppressed in both growth and differentiation with growth-

associated E2F1 expression being allowed only at later time points,when the pl30 concentration declines to a low level (Fig. 4f versusFig. 2e).

Analogous to the differentiation-associated inhibition of the PCNA

promoter by p53 and of the E2F1 promoter by p 130, direct binding ofp21/Cipl to PCNA (Fig. 5c) and of pRb to E2F1 (Fig. 5d) occurredonly in differentiation-induced cells, such binding contributing to theinhibition of G,-S-associated DNA synthesis and transcription (Fig. 2,rand/; Refs. 6, 7, 24-26).

To gain additional experimental support for the regulatory role thattumor suppressor proteins are indicated to play in the process of celldifferentiation, sense and antisense oligonucleotides of p53, p 130, andpRb were added to ML-1 cells untreated (control) or treated with theDFs TGF-ÃŸland TNF-a. As shown in Fig. 6, a significant reduction

in the number of differentiated cells was obtained only in the presenceof the antisense oligonucleotides of p53, pi30. and pRb, with theirnumber being decreased by 44, 68, and 63%, respectively. No significant changes in the number of differentiated cells occurred in culturestreated with the coresponding sense oligonucleotides. In the absenceof DF, the cells did not respond to treatment with antisense oligonucleotides, which supports the indication that their action pertains to thedifferentiation process.

By participating in the inhibition of the PCNA-dependent andE2F1-dependent G,-S transit, the tumor suppressor proteins are indi

cated to uncouple growth from differentiation (32), allowing entryinto the maturation path. Where this function of the tumor suppressorproteins is impaired, sustained proliferation at a stage of incompletematuration is expected to ensue, that condition being a prime characteristic of the cancer cell.

4286

Acknowledgments

We are grateful to Bristol-Myers Squibb (Seattle. WA) for providingTGF-/31 and to Asahi Chemical Industry (Tokyo, Japan) for TNF-a. We also

thank Swati P. Deb (University of Texas Health Science Center at SanAntonio. TX) for providing PCNA-promoter DNA and David G. Johnson(University of Texas M. D. Anderson Cancer Center. Science Park-ResearchDivision, Smithville. TX) for donating E2F-1-promoter DNA.

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[>IH-Ã•-:RI;NTIATK>NRW;II.AIOKS

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1998;58:4282-4287. Cancer Res Zhan-Rong Li, Rosemary Hromchak, Anuradha Mudipalli, et al. Tumor Suppressor Proteins as Regulators of Cell Differentiation

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