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Vol. 6, 673-680, june 1995 Cell Growth & Differentiation 673

Recombinant Human Retinoblastoma ProteinInhibits Cancer Cell Growth

Lance C. Pagliaro, Douglas Antelman, Duane E. Johnson,Todd Machemer, Ernest A. McCulloch,2 Emil J. Freireich,Sanford A. Stass,3 H. Michael Shepard, Dan Maneval,and Jordan U. Gutterman4

Divisions of Medicine IL. C. P., E. J. F., J. U. G.J and Laboratory MedicineIE. A. M., S. A. SI, The University of Texas M. D. Anderson CancerCenter, Houston, Texas 77030; and Canji, Inc., San Diego, California

92121 ID. A., D. E. J., T. M., H. M. S., D. M.I

Abstrad

Aberrant expression of the tumor suppressor gene RB1 isassociated with a variety of solid tumors andhematopoietic neoplasms. Certain cancer cell lines inwhich the protein encoded by RB1 (p1 1 0RB) is absenthave been reported to show decreased growth rate,clonogenicity, or tumorigenicity following insertion of atranscriptionally adive RB1 gene. We asked whetherthese RB-deficient cells could be growth inhibited bydired exposure to purified p1 I 0R0#{149}We report adecrease in uptake of tritiated thymidine by 5637bladder carcinoma cells (RB-negative) when purifiedrecombinant p1 1 0RB is added to culture media.Internalization of the protein by cells and translocationto the nucleus are demonstrated byimmunohistochemistry, FACS, and detedion ofradiolabeled protein in subcellular fradions. Next, wechose a well-described leukemia cell culture model toinvestigate the potential effect of recombinant p1 1 0R8 inclinical disease. We observed dose-related decreases incell number or colony formation in vitro in 8 of 20acute myelogenous leukemia samples, 7 of which didshow endogenous p1 1 0RB detectable byimmunohistochemistry. Histological appearancefollowing exposure to p1 1 0RB shows cytoplasmicvacuolization and nuclear lobulation of degeneratingcells. We conclude that purified p1 1 0RB added toculture media is internalized by cells, translocated to thenucleus, and exerts a growth-inhibitory effed on certaincancer cell types.

IntrodudionTumor suppressor genes have been shown to play impor-tant roles in the pathogenesis of cancer in humans (1 , 2).Their attraction as potential therapeutic targets lies in thepossibility that restoration of their function in tumor cells

Received 2/1/95; revised 3/21/95; accepted 3/29/95.1 This research was supported in part by a grant from the Biomedical

Research Foundation.2 Present address: Ontario Cancer Institute/Princess Margaret Hospital,Toronto, Ontario, Canada M4X 1 K9.

3 Present address: Department of Pathology, The University of Maryland,Baltimore, MD 21201.4 To whom requests for reprints should be addressed, at The University of

Texas M. D. Anderson Cancer Center, Box 41 , 1 51 5 Holcombe Blvd.,Houston, TX 77030.

will interfere with cell proliferation. This idea is supportedby experimental evidence in the case ofthe RB!5 gene: thein vitro proliferation and in vivo tumorigenicity of RB-negative tumor cell lines is inhibited by insertion of a tran-scriptionally active copy of the gene (3-5). The RB! geneprotein, p1 1 0RB has been shown to block progressionthrough the G1 phase of the cell cycle when a truncatedmolecule containing the binding domain is microinjecteddirectly into cultured RB-negative tumor cells (6). The Mr1 1 0,000 nuclear protein binds DNA and interacts withother nuclear proteins including the transcription factorE2F, the nuclear tyrosine kinase c-abl, and D cychins (7-10).Phosphorylation of multiple serine and threonine residuesinactivates p1 10RB and allows DNA synthesis and celldivision to occur (1).

The p1 10RB protein plays a crucial role in regulating theproliferation of cells (1). Patients who inherit an abnormalRB! allele develop retinal tumors, osteosarcomas, and softtissue sarcomas associated with loss of the remaining wild-type allele (1 1-1 3). Mice that are heterozygous for mutantRB! develop pituitary tumors, whereas homozygous mutantembryos die by the sixteenth day ofgestation with abnormaldevelopment of the hematopoietic and central nervous sys-tern tissues (1 4, 1 5). The mechanism through which p1 1 0RBcontrols gene expression is not known, but the presence ofbinding to DNA and to transcription factors such as E2Findicates participation in a complex that binds to regulatoryelements in the genome. In experimental models and in thenatural history of diseases such as hereditary retinoblas-toma, loss of p1 10RB is associated with uncontrolled celldivision and progression toward malignancy.

We have produced recombinant human p1 1 0RB from E.co/i at the laboratories of Canji, Inc., making it possible totest the protein at concentrations up to 1 M for its effect oncells in culture. We asked whether direct exposure topllO#{176}could regulate cell growth. There are several pre-cedents for extracellular proteins affecting gene expressionthrough cellular uptake and nuclear translocation. Theseinclude the tat protein of HIV, a 60-amino acid syntheticpeptide (pAntp) derived from the Drosophila antennapediahomeobox gene, bFGF, and insulin (16-20).

The bladder cancer cell line 5637 (ATCC HTB-9) ex-presses no functional pi 1 0RB and has been reported previ-ously to exhibit decreased growth in vitro, decreased col-ony formation in soft agar, and reduced tumorigenicity invivofollowing transfection with the wild-type RB! gene (1,5). First, we asked whether exposure of the 5637 parent cellline to recombinant p1 1 0RB would result in growth inhibi-tion. Second, to further investigate the effect of recombinantp1 10RB on self-renewal in a neoplastic cell population, welooked for an effect on clonogenicity of blast cells frompatients with AML. Previous reports have shown decreased

5 The abbreviations used are: RB, retinoblastoma; bFGF, basic fibroblastgrowth factor; AML, acute myelogenous leukemia; cdk, cyclin-dependentkinase; FBS, fetal bovine serum; CSF, colony-stimulating factor.

3H-Thy Uptake(% of Medium

Control)

120

i:

10 100

p11ORB (nM)

1000

Patient number

A

C80I

B

=

C0

(3

Fig. 2. A, dose-dependent reduction in AML blast clonogenic cell recovery.

Prior to plating, cells were incubated 48 h in liquid media with singleaddition of buffer (LI), p1 1 0 1 00 nM (a), or p1 1 0R11 200 na (U). Significantinhibition is seen at 200 nM (P< 0.03 compared to buffer). The means of fourreplicate wells are shown; bars, SEM. Patients nos. 3 and 10 are not shown

(see text). B, a p1 10-binding peptide, but not a mutant peptide, neutralizesgrowth inhibition by recombinant p1 10R#{149}T peptide has the amino acidsequence NLFCSEEMPSSDDE, which is derived from SV4O T-antigen andbinds the p110#{176}A/B pocket. K peptide has a single amino acid substitution

(NLFCSKEMPSSDDE( and does not bind p1 Prior to plating, blast cellsfrom patient no. 14 were incubated with mixtures as shown for 24 h

(concentrations: 200 ma pllO#{176}#{176};250 nsa T peptide; and 250 nsi K peptide).Reduction in colony formation is significant with p1 l0 alone and with

p1 1 0R0 K peptide (P = 0.03). The means offour replicate wells are shown;bars, SEM.

674 Recombinant RB Protein Inhibits Cancer Cell Growth

Fig. 1. Dose-response relationship of p1 1 53 added to culture niedia andnwasured uptake of I Hithymidine by 5637 cells. Concentrations of p1 10R

at the beginning of the experiment are shown. One-half of the cell medium

was removed and replaced with p1 10Rcontaining medium twice each dayfor 3 days. I HiThymidine incorporation assayed by liquid scintillation

counting; mean of tour replicate samples shown. Bars. SEM.

or absent levels of p1 1 0Rtt in AML blasts from 25-30% ofpatients studied, despite only rare occurrence of detectableRB! gene alterations (21-25). We chose the AML blast cellculture model to study the effect of pi i 0RB on clonogenic-ity because: (a) the genotype of freshly obtained blastsreflects that of cells in vivo, unlike cell lines that bearmutations accumulated during serial passage; (b) largenumbers of blast cells can be obtained from patients for cellculture; and (C) previous studies have demonstrated thateffects on clonogenic cell recovery in vitro are predictive ofevents in vivo (26-28).

In this report, we show that recombinant p1 1 0 inhibitsthe growth rate of 5637 cells, and we describe 8 cases inwhich self-renewal of cultured AML blasts, measured asclonogenic cell recovery, is inhibited by pi 10RB Data arealso presented to show that pi 10RB is internalized by cellsin vitro and translocated to the nucleus.

Results

5637 Cells Exposed to p1 10R8 Are Growth Inhibited. Di-rect exposure to pi i 0RU is associated with a dose-relateddecrease in uptake of l#{176}Hlthymidine by 5637 cells, mdi-cating decreased DNA synthesis (Fig. i ). Continuous expo-sure to pi 10Rt3 concentrations greater than 100 nM weregrowth inhibitory. Cells incubated with medium aloneyielded counts of i 26,000 to i 32,000 cpm. At the highestconcentration of pi 1 0 (1 pM), incorporation wasapproximately 50% of control (P < 0.Oi).

Decreased Growth Rate and Clonogenicity of Blast Cellsfrom Patients with AML. Blasts were obtained from periph-eral blood or bone marrow specimens from 20 patients withAML. Inhibition of growth rate or clonogenic cell recoveryfollowing exposure to pi i 0RB was observed in eight casesand was dose dependent (Fig. 2A). Table i shows the resultsof immunostaining as a measurement of endogenouspi i 0R and the clinical characteristics of these eight cases.The sample obtained from patient no. 3 did not yield suf-ficient cells for the clonogenic assay but did show a markeddecrease in cell number relative to buffer control after 72 hin suspension culture with pi 1 0R0 adde