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[Cancer Research 61, 462-468, January 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

RB2/p130 Gene-enhanced Expression Down-Regulates Vascular Endothelial Growth Factor Expression and Inhibits Angiogenesis in Vivo1

Pier Paolo Claudio, Peter Stiegler, Candace M. Howard, Cristiana Bellan, Corrado Minimo, Gian Marco Tosi, Janusz Rak, Al Kovatich, Paola De Fazio, Pietro Micheli, Mario Caputi, Lorenzo Leoncini, Robert Kerbel, Giovan Giacomo Giordano and Antonio Giordano2

Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 [P. P. C., P. S., C. M. H., C. M., A. K., A. G.]; Dipartimento di Scienze Odontostomatologiche e Maxillo-Facciali, Universita di Napoli "Federico II," Napoli, Italy [P. P. C.]; Istituto di Anatomia Patologica e Histology [C. B., L. L.] e Dipartimento di Scienze Oftalmologiche e Neurochirurgiche [G. M. T.], Universita di Siena, Siena, Italy; Division of Cancer Biology Research, Sunnybrook Health Science, Toronto, Ontario, M4N 3M5 Canada [J. R., R. K.]; Servizio di Anatomia ed Istologia Patologica e Citologia Diagnostica, Azienda Ospedaliera "Cotugno," Napoli, Italy [P. D. F., P. M.]; Istituto di Malattie dell’Apparato Respiratorio, II Universita’ degli Studi di Napoli and Istituto di Ricerca Cardio-Pneumologico A. O. "Monaldi," Napoli, Italy [M. C.]; and Istituto di Anatomia Patologica, Facolta’ di Medicina, II Universita’ degli studi di Napoli, Napoli, Italy [G. G. G.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Angiogenesis is an essential step in the progression of tumor formation and development. The switch to an angiogenetic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to genetic changes such as mutations in key cell cycle regulatory genes. The pathogenesis of the angiogenetic phenotype may involve the inactivation of tumor suppressor genes such as the "guardian of the genome," p53, and the cyclin-dependent kinase inhibitor p16. Retinoblastoma family member RB2/p130 encodes a cell cycle regulatory protein and has been found mutated in different tumor types. Overexpression of RB2/p130 not only suppresses tumor formation in nude mice but also causes regression of established tumor grafts, suggesting that RB2/p130 may modulate the angiogenetic balance. We found that induction of RB2/p130 expression using a tetracycline-regulated gene expression system as well as retroviral and adenoviral-mediated gene delivery inhibited angiogenesis in vivo. This correlated with pRb2/p130-mediated down-regulation of vascular endothelial growth factor protein expression both in vitro and in vivo.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Tumorigenesis is a multistep process that involves several genetic changes resulting in uncontrolled cellular proliferation and inhibition of apoptosis (1) . Tumor growth and cellular proliferation are linked by the ability of the tumor to foster proper vascularization from the host to the alien tumor graft. Recent evidence shows that tumors do not grow larger than a few millimeters in size unless vascularized by the host (2) . Tumor progression and growth require an appropriate rate of blood vessel formation related to the rate of neoplastic cellular proliferation; otherwise, tumor necrosis and eventual calcification result.

Angiogenesis is driven by a balance between different positive and negative effector molecules influencing the growth rate of capillaries. Various angiogenetic and antiangiogenetic factors have been cloned to date (3, 4, 5, 6, 7) . VEGF3 and TSP-1 are two of the most well studied. VEGF is a potent tumor-secreted angiogenic factor as opposed to TSP-1, which functions as an antiangiogenic molecule (8 , 9) . Normal growth results by balanced and coordinated expression of these opposing factors. A switch from normal to uncontrolled vessel growth can occur by up-regulating angiogenesis stimulators or down-regulating angiogenesis inhibitors, suggesting that the angiogenetic process is tightly regulated by the oscillation between these opposing forces (10) . The switch to an angiogenic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to epigenetic or genetic changes (11) . In support of this theory, mRNA expression of VEGF is up-regulated in aggressive tumor cell lines expressing an activated ras oncogene (12) . Conversely, transcription of VEGF is down-regulated in these same tumor cell lines after disruption of the mutant ras allele, thus eliminating VEGF expression and rendering the cells incapable of tumor formation in vivo (13) . The switch to an angiogenic phenotype has also been associated with the inactivation of the tumor suppressor gene p53 (14) . Conversely, cell lines that are p16 deleted revert to an antiangiogenic phenotype upon the restoration of wild-type cyclin-dependent kinase inhibitor p16 (15) .

Different gene therapy approaches using tumor suppressor genes have been tested in vivo to date with varying results (16) . Recent studies have indicated that angiogenesis may be regulated to some extent via the p53 tumor suppressor function, but no reports have been published regarding the RB family. The RB gene family includes three members: the Rb tumor suppressor RB/p105, p107, and RB2/p130. These proteins are highly homologous in the "pocket" region, composed of subdomains A and B separated by a spacer region that is highly conserved among each of the proteins (17, 18, 19, 20, 21) . This functional domain is targeted by viral oncoproteins and is responsible for many functional interactions (22) . Functionally, all of the RB family members show cell type-specific, growth-suppressive properties unique to each member. They each bind and temporally modulate in a distinct manner the activity of specific members of the E2F family of transcription factors and are regulated by phosphorylation in a cell cycle-dependent manner (23) . The structural identities of these proteins underlie similar but distinct functional properties. In fact, all three family members inhibit cell cycle progression in the G1 phase of the cell cycle (24, 25, 26) . Interestingly, the RB family of proteins exhibits unique growth-suppressive properties that are cell type specific, suggesting that although they may complement each other, their functions are not fully redundant (27) .

In several tumor cell lines, pRb2/p130 mediates a G0-G1 phase cell cycle arrest, including the human T98G glioblastoma cell line, which is resistant to the suppressive effects of both pRb/p105 and p107 (24 , 25 , 27) . Additionally, we have demonstrated that RB2/p130 expression suppresses tumor growth in vivo by inhibiting tumor formation in nude mice as well as causing regression of established tumor grafts using a tetracycline-regulated expression system and retroviral-mediated gene delivery (28 , 29) . Our results mentioned previously led us to investigate retrospectively the possibility that RB2/p130 may modulate tumor progression by affecting the fine-tuned angiogenetic balance, because only tumors of 1–2 mm of diameter can receive all sufficient nutrients by diffusion; therefore, additional growth depends on the development of an adequate blood supply through angiogenesis (30) . We decided to test whether a link between RB2/p130 expression and inhibition of angiogenesis may be involved in RB2/p130-mediated tumor suppression/regression.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Culture.
H23 cells (human lung adenocarcinoma) have been described previously (28) . HJC {Delta}5 cells and their clone HJC 12 (JC-T antigen-transformed hamster glioblastoma) expressing pRb2/p130 under an inducible tetracycline promoter have been described previously (29) . Briefly, we used a modified tetracycline-regulated method to create an autoregulatory-inducible RB2/p130 gene expression system created in the HJC 15c cell line, originating from a human polyomavirus-induced (JC virus) hamster brain tumor (29) . The parental cell line HJC 15c was used to create the control cell line HJC {Delta}5 that contains the tetracycline transactivator under the control of the Tetp promoter. HJC {Delta}5 cells were used to form the HJC 12 cell line, which contains, in addition to tetracycline transactivator, the full length cDNA of the human RB2/p130 gene downstream of the Tetp promoter. In this system, pRb2/p130 expression is repressed in the presence of the antibiotic tetracycline (+) and induced in its absence (-) to 100-fold at the protein level (29) . The 293T/17 cell line (human renal carcinoma; Ref. 22 ) was purchased from the American Type Culture Collection upon authorization of the Rockefeller University. H23 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine. The 293T/17 cell line was maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine. HJC {Delta}5 and HJC 12 cells were grown in DMEM supplemented with 5% FCS (Sigma Chemical Co., St. Louis, MO) and the antibiotics streptomycin (10 mg/ml) and penicillin (100 units/ml) and in the presence or not (+/-) of 2 µg/ml tetracycline (Sigma).

Adenovirus and Retrovirus Vectors.
Retroviral and adenoviral vectors expressing RB2/p130 or controls expressing the bacterial ß-Gal (Lac-Z) or the puromycin resistance (Pac) gene alone have been described previously (28 , 31) . Briefly, retrovirus-mediated gene transfer studies were carried out with a MLV-based system (28) . Transient and DNA cotransfection of the 293T/17 cells using PHIT60 (CMV-MLV-gag-pol-SV40 ori) and PHIT456 (CMV-MLV-amphotropic env-SV40 ori) vectors, along with MSCV-based transfer vectors MSCV-Pac, MSCV-Pac-LacZ, and MSCV-Pac-RB2/p130, were performed by calcium phosphate precipitation (28) . The retroviral supernatant was collected 48 h after transfection, filtered through 0.45 µm filters, and titered as described previously (28) to produce retroviruses carrying the puromycin resistance gene alone or in combination with the Lac-Z gene or the RB2/p130 ORF, respectively. Viral titers of 1 x 107 infectious units/ml were obtained (28) .

Adenoviruses were generated by subcloning the full-length ORF of the RB2/p130 gene into the pAd.CMV-Link1 vector to form the Ad.CMV-RB2/p130 virus, as described previously (31) . The pAd.CMV-Link1 vector alone (to produce the Ad-CMV virus) was used as a negative control to assay the effects of viral infection alone without delivering a transgene. The above-mentioned viruses were generated by cotransfection of the constructs mentioned previously with an adenoviral backbone into the packaging cell line 293 primary embryonal human kidney cells, transformed by sheared human adenovirus type 5. The adenoviruses were recovered, screened, and expanded as described previously (31) . After purification by sequential equilibrium, density gradients using CsCl viral stocks were made at 5 x 1012 particles/ml and stored at -80°C in a solution containing 10% glycerol. A viral titer of 22 x 109 pfu/ml was determined by plaque assay for the Ad-CMV and Ad-CMV-RB2/p130 viruses. Infection of nonpermissive cells confirmed that the viruses were replication defective.

Northern Blot Analysis.
H23 cells were grown to 70% confluency then infected with 50 multiplicity of infection of adenoviruses carrying RB2/p130 ORF or with the control Ad-CMV. After 14 h, the medium was changed, and the cells were harvested after a total of 48 h after infection.

VEGF Northern blot analysis was performed essentially as described previously (32) . Briefly, RNA was extracted using the RNAzol kit (Tel-Test, Inc., Friendswood, TX), following the manufacturer’s instructions. The RNA was resolved on a 1% agarose gel containing 6.6 M formaldehyde, transferred to a Zeta Probe (Bio-Rad, Hercules, CA) membrane, and hybridized at 65°C with a 32P-labeled cDNA probe containing either the 200-bp fragment of the human VEGF sequence common to all four known isoforms of VPF/VEGF protein or TSP-1 (32 , 33) . The amount of RNA loaded in each lane was evaluated by ethidium bromide gel staining of the gel before the transfer. The TSP-1 probe was purchased from the American Type Culture Collection.

Antibodies, Immunohistochemical Analysis, and IMD Assessment.
Rabbit polyclonal anti-VEGF was the kind gift of Genentech, Inc. (San Francisco, CA). Purified antimouse CD31 (PECAM-1), clone MEC 13.3 was purchased from (PharMingen, San Diego, CA). Anti-VEGF was used at a dilution 1:500, and anti-CD31 was used at a dilution of 1:50, following the manufacturer’s instructions for immunohistochemical analysis.

VEGF staining intensity was graded on a scale of 0 to 3: 0, no detectable staining; 1, traces of staining; 2, moderate amount of diffuse staining; and 3, a large amount of diffuse staining. This grading scale is a modification of that of Takahashi et al. (34) .

Intratumoral microvessels were highlighted by immunostaining different serial, formalin-fixed, paraffin-embedded sections of the same tumor graft with anti-CD31. IMD was determined as described previously (35) . Briefly, CD31-stained sections underwent an individual microvessel count on a x400 magnification in the areas of most intense neovascularization (hot spots). IMD was expressed as microvessels/mm2.

ELISA.
The VEGF ELISA was performed as described previously using the anti-VEGF from Genentech, Inc. (1:2000 dilution) and an antirabbit horseradish peroxidase-conjugated antibody (Amersham, Arlington Heights, IL; 1:5000 dilution) as secondary antibody and the 3,3',5'5-tetramethylbenzidine liquid substrate system following the manufacturer’s recommendations (Sigma; Ref. 36 ).

Luciferase Assay.
Luciferase assay was performed by transfecting a total of 3 µg of either mouse VEGF promoter (37) or an artificial E2F promoter containing three consecutive E2F consensus binding sites (38) linked to the luciferase reporter gene for each point in the HJC 12 cells, in the presence of the antibiotic tetracycline (+; uninduced status) and in its absence (-; induced pRb2/p130 protein status). HJC 12 cells were plated at 60% confluency in six-well dishes the day before the experiment, and transfections were performed by the standard calcium-phosphate method as reported previously (29) . Normalization was performed by cotransfecting a total of 1 µg of CMV Lac-Z (Promega, CA) for each experimental point. The experiment was performed in triplicates and repeated twice. Luciferase activity was assayed using the luciferase kit assay according to the manufacturer’s instructions (Promega Corp., Madison, WI) and measured using a luminometer (Corning Costar Corp., Cambridge, MA).

Western Blot.
Protein concentration was assayed by Bradford analysis (Bio-Rad Laboratories, Inc., Melville, New York) and confirmed by running 10 µg of protein on a 12% SDS-polyacrylamide gel (SDS-PAGE); staining was with Coomassie blue. For Western blotting purposes, an equal amount of 100 µg of protein extract for each sample was electrophoresed into 12% SDS-polyacrylamide gels (SDS-PAGE) and transferred to 0.2 µm nitrocellulose membranes (Schleicher & Schuell, Germany). The loading and transfer of equal amounts of protein were confirmed by staining the membranes with Red Ponceau (Sigma). Membranes were quenched at 4°C overnight in a solution of TBS-T (Tris-buffered saline + 0.5% Tween 20) and 5% dry milk for blocking nonspecific binding. Either primary rabbit polyclonal anti-VEGF (Genentech) or anti-VEGF (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:200 in a solution of TBS-T and 3% dry milk was used independently to incubate the blots. After several washes in a solution of TBS-T, the blots were incubated with a solution of TBS-T containing an antirabbit secondary antibody (horseradish peroxidase conjugated; Amersham, Life Science), diluted 1:20,000 for 1 h at room temperature. The blots were then washed several times in TBS-T, reacted with a ECL (Enhanced Chemiluminescence kit; DuPont NEN, Boston, MA), and exposed to X-ray films.

Animal Studies.
Animal care and humane use and treatment of mice were in strict compliance with the following: (a) institutional guidelines; (b) the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 1996); and (c) the Association for Assessment and Accreditation of Laboratory Animal Care International. Tumors were generated by the s.c. injection of 2.5 x 106 H23 or of 5 x 106 HJC {Delta}5 or HJC 12 cells into nude mice (female nu/nu-nuBR outbred, isolator-maintained mice, 4–5 weeks of age, from Charles Rivers Wilmington, MA), as described previously (28 , 29) .

For H23 injected cells, when the tumors reached a volume of ~20 mm3 after 15 days, each tumor was transduced with 5 x 106 retroviruses carrying the Pac gene alone or the Pac gene and the Escherichia coli ß-Gal (Lac-Z) gene as control or the Pac gene and RB2/p130 ORF with three animals/group by direct injection of 20 µl of retroviral supernatant directly into each of the tumors.

For the HJC nude mice group, the mice were treated with tetracycline for 4 days prior to injection. The mice were injected s.c. along their left and right flanks at two sites/mouse with 5 x 106 cells/flank while under anesthesia with isopropane gas. There were four groups of animals with three animals/group. Two groups were injected with HJC 12 cells, and treatment with tetracycline continued after injection in one group (12+), whereas another group (12-) ceased to be administered tetracycline after injection of the cells. The two control groups were injected with HJC {Delta}5 cells, and one ({Delta}5+) continued to receive tetracycline while the other control group ({Delta}5-) did not.

Animals were sacrificed by CO2 asphyxiation when Pac and Lac-Z retrovirus-transduced tumors or HJC {Delta}5 (± tetracycline) and HJC 12 tumors (+ tetracycline) reached a size of 300–350 mm3 . Tissues to be sectioned were placed in OTC (Sakura Finetek USA, Inc., Torrance, CA), frozen in liquid nitrogen, and stored at -80°C or preserved in neutral-buffered formalin at 4°C before embedding in paraffin.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
RB2/p130 Enhanced Expression Down-Regulates VEGF Expression.
We have shown previously that RB2/p130 enhanced expression inhibits tumor formation (28 , 29) and induces tumor regression (28 , 29) . Interestingly, we found that some of the tumors treated with retroviruses delivering RB2/p130 underwent central necrosis and subsequent calcification. Following these observations, we tested the hypothesis that RB2/p130 overexpression could inhibit angiogenesis. We chose to study the expression levels of two well-studied proteins involved in angiogenesis, VEGF and TSP-1.

H23 cells were infected with adenoviruses carrying RB2/p130 or with the control Adeno-CMV and harvested after 48 h. Interestingly, Northern blot analysis with a probe against VEGF showed a down-regulation of 2- fold of the vascular endothelial growth factor upon overexpression of RB2/p130 (Fig. 1)Citation . On the other hand, no modification of TSP-1 was observed in the same experimental conditions (Fig. 1)Citation . Because low abundance of gene expression can result from either enhanced mRNA degradation or promoter regulation, we thought to analyze the effects of forced RB2/p130 gene expression on the VEGF promoter. HJC 12 cells transfected with the VEGF promoter and cultured in the absence of the antibiotic tetracycline (RB2/p130 induced) showed 2–3-fold down-regulation with respect to the uninduced HJC 12 (+ Tet) cells and to the vector control-transfected cells in either the induced (-Tet) or uninduced status (+ Tet; Fig. 2Citation ). A promoter containing E2F consensus binding sites linked to the luciferase reporter gene was used as a positive control for pRb2/p130 transcriptional repression activity. These results led us to explore the VEGF protein abundance in vitro and in vivo after enhanced RB2/p130 expression.



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  		Fig. 1. Northern blot analysis of H23 cells transduced with either Ad-CMV or Ad-RB2/p130. Left, the probes used. Lower panel, ethidium bromide staining for equal gel loading. A 2-fold reduction of VEGF abundance was observed upon enhanced RB2/p130 expression.

 


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  		Fig. 2. Graphic representation of VEGF luciferase activity in HJC 12 cells VEGF and E2F promoter luciferase constructs were transiently transfected into HJC 12 cells, and subsequently, pRb2/p130 expression was induced (- Tet). Top, the promoters used. A 2–3-fold reduction of VEGF promoter activity was observed upon enhanced RB2/p130 expression. Bars, SD.

 
RB2/p130 Modulates VEGF Protein Expression in Vitro.
To test the hypothesis that RB2/p130 may modulate VEGF protein expression in vitro, we studied (39) the conditioned culture medium of H23 cells transiently transduced with either adenovirus carrying RB2/p130 or with the CMV control adenovirus, by means of an ELISA. Additionally, we tested the conditioned medium of the HJC 12 cells in which the RB2/p130 expression is regulated by a tetracycline-inducible promoter (29) . In both systems, overexpression of RB2/p130 resulted in a down-regulation of the VEGF protein abundance by 3-fold with respect to the controls (Fig. 3)Citation . To further our analysis, we also tested VEGF abundance in the intracellular compartment. H23 cells were transiently transduced with either adenovirus carrying RB2/p130 or with the CMV control adenovirus. We also tested protein extracts from HJC 12 cells in which the RB2/p130 expression is regulated by a tetracycline-inducible promoter. Western Blot analysis using rabbit polyclonal antibodies against VEGF showed a 2–3-fold reduction of intracellular protein abundance upon enhanced RB2/p130 expression (Fig. 4)Citation .



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  		Fig. 3. Graphic representation of a single experiment of a VEGF ELISA in the conditioned medium of H23 and HJC 12 cells after RB2/p130 overexpression. Columns 1–4, control medium; Columns 5–9, conditioned medium. Columns 1–3, negative controls. Column 4, background. The graph is the representation of a single experiment that was repeated three times with the same result.

 


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  		Fig. 4. Western blot analysis of VEGF protein abundance upon pRb2/p130 enhanced expression in H23 and HJC 12 cells. Top, cell lines. H23 cells were transduced with either adenoviral vector carrying RB2/p130 (pRb2) or empty adenoviral vector (CMV). HJC 12 cells were grown under an induced (- Tet) or uninduced (+ Tet) condition. A 3-fold reduction of VEGF protein abundance was observed upon enhanced RB2/p130 expression. Coomassie blue staining of 10 µg of protein of total lysate is shown to verify protein concentration and equal loading. Contr, control.

 
RB2/p130 Inhibits Angiogenesis in Vivo.
Using samples from two previous studies in which we showed that RB2/p130 inhibits tumor formation and induces tumor regression in nude mice (28 , 29) , we were able to identify a novel mechanism of tumor inhibition for RB2/p130. Serial sections of tumors grown in nude mice and treated or untreated with RB2/p130 were immunostained for VEGF and CD31. CD31 is a specific marker for endothelial cells (40) . We chose to grade the VEGF staining on a scale from 0 to 3, following previous work by Takahashi et al. (34) , with some modification. We considered a score of 0 equal to no detectable staining; 1, traces of staining; 2, a moderate amount of diffuse staining; and 3, a large amount of diffuse staining. Interestingly, RB2/p130 overexpression caused VEGF immunostaining to drop from a large amount of diffuse staining (score, 3), characteristic of the control samples (Fig. 5, A–CCitation , and Fig. 6, A–DCitation ), to traces of staining (score, 0; Fig. 5DCitation and Fig. 6, E and FCitation ) in both the two tumor graft groups examined (see also Table 1Citation ).



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  		Fig. 5. Immunohistochemical analysis of VEGF and CD31 of HJC {Delta}5 and HJC 12 tumor grafts grown in nude mice. A, high VEGF expression in HJC {Delta}5 (+ Tet) tumor (control; x100). B, high VEGF expression in HJC {Delta}5 (- Tet) tumor (control; x100). C, high VEGF expression in HJC 12 (+ Tet, pRb2/p130 not induced) tumor (control; x100). D, low VEGF expression in HJC 12 (- Tet, pRb2/p130 induced) tumor (x100). E, VEGF expression in a human colon cancer. Lower left corner, high VEGF expression in the tumor; upper right corner, low VEGF expression in the normal colon tissue (x100). F, CD31 immunostaining of HJC {Delta}5 (+ Tet) tumor (control). G, CD31 immunostaining of HJC {Delta}5 (- Tet) tumor (control; x400). H, CD31 immunostaining of HJC 12 (+ Tet, pRb2/p130 not induced) tumor (control; x400). I, CD31 immunostaining of HJC 12 (- Tet, pRb2/p130 induced) tumor (x400). J, CD31 immunostaining of normal mouse lung (x400).

 


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  		Fig. 6. Immunohistochemical analysis of VEGF and CD31 of H23 tumor grafts grown in nude mice. A, high VEGF expression in H23 tumor transduced with control retrovirus (Pac; x100). B, high power field (x400) of A. C, high VEGF expression in H23 tumor transduced with retrovirus carrying Lac-Z (x100). D, high power field (x400) of B. E, low VEGF expression in H23 tumors transduced with retrovirus carrying RB2/p130. Upper side of the panel, normal mouse neurovascular formation (x100). F, high power field (x400) of E showing the lack of VEGF immunostaining. G, CD31 immunostaining of H23 tumor transduced with control retrovirus (Pac; x400). H, CD31 immunostaining of H23 tumor transduced with retrovirus carrying Lac-Z (x400). I, CD31 immunostaining of H23 tumors transduced with retrovirus carrying RB2/p130 (x100). Upper side of the panel, normal mouse neurovascular formation stained for CD31. J, high power field (x400) of I showing the only vessels found in the slide. K, CD31 immunostaining of normal mouse lung (x400).

 

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  		Table 1 VEGF staining intensity and IMD in nude mice tumors

 
IMD assessment showed at least an 81% (confidence interval, 1.95–10.5) reduction of microvessels count after CD31 immunostaining in all tumor grafts (H23 and HJC) in which RB2/p130 was overexpressed (Table 1)Citation . Fig. 5, F–HCitation , shows a few representative examples of microvessel density in the HJC {Delta}5 (+ Tet), HJC {Delta}5 (- Tet) and HJC 12 (+ Tet) control tumor grafts, respectively. Fig. 6, G and HCitation , instead show samples of H23 tumor grafts treated with the control retroviruses carrying Pac or ß-Gal, respectively. Fig. 5ICitation , however, demonstrates very poor microvessel density upon induction of RB2/p130 expression in HJC 12 (- Tet) tumor grafts as evidenced by CD31 immunostaining. Fig. 6ICitation at low magnification power (x100) shows poor microvessel density in a H23 tumor graft treated with retroviruses carrying RB2/p130. Additionally, Fig. 6ICitation contains on its upper side a portion of normal nude mouse tissue demonstrating a normal neurovascular formation that was stained by the CD31 antibody, proving that the lack of CD31 staining is indeed specific to enhanced pRb2/p130 expression in tumor tissues. Fig. 6JCitation is a higher magnification field (x400) of Fig. 6ICitation , showing the only vascular formation present on this particular slide. Finally, Figs. 5JCitation and 6KCitation show the specificity of the CD31 staining to neurovascular bundles in normal embryonal mouse lung endothelium in the conditions used. The effects upon VEGF staining intensity and IMD were specific to pRb2/p130 expression because withdrawal of tetracycline from the HJC {Delta}5 tumors did not alter VEGF intensity and actually enhanced IMD.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
We have shown previously that RB2/p130 potently inhibits tumor formation in nude mice and causes tumor regression of fully established tumor grafts (28 , 29) . In the present retrospective study, RB2/p130 significantly decreased VEGF RNA and protein expression in vitro and in vivo in two different cell types, glioblastoma and lung adenocarcinoma, in both rodent and human tumor cell lines, using either retrovirus/adenovirus-mediated gene transfer or a tetracycline-regulated gene expression system. Additionally, enhanced RB2/p130 gene expression down-regulated the activity of the VEGF promoter in a tetracycline-regulated pRb2/p130 system. We analyzed the VEGF promoter for putative E2F regulatory binding sites and found that it does not contain any known responsive site of regulation of the RB pathway. Therefore, the down-regulation of the VEGF promoter that we have observed could be attributable to an indirect mechanism that needs to be further investigated. The fact that these data were reproduced using different expression systems indicate also that the VEGF down-regulation observed at the RNA, promoter, and protein levels was not attributable to a mere viral bystander effect but was contingent upon the enhanced expression of the RB2/p130 gene within the tumor itself. Previous reports have indicated that the tumor suppressor gene p53 and the cyclin-dependent kinase inhibitor p16 control tumor angiogenesis by regulating TSP-1 or VEGF expression (9 , 15) . However, the exact mechanism by which p53 and p16 operate the VEGF down-regulation is still unknown. VEGF and TSP-1 are two important factors regulating angiogenesis and antiangiogenesis, respectively. High tumorigenic potential is associated with elevated levels of VEGF (32) . On the other side, TSP-1 overexpression has been reported to suppress tumor growth and metastasis potential in some cell types (41) . Additionally, another group showed instead that wild-type p53 suppresses VEGF expression in an anaplastic thyroid carcinoma cell line (42) . Our data that forced expression of RB2/p130 significantly determined tumor regression and inhibited VEGF RNA abundance by 2-fold and protein expression as well as VEGF promoter activity by 3-fold, causing at least 81% (confidence interval, 1.95–10.5) reduction in vessel formation, indicate a novel tumor-regulatory property for the RB-related gene RB2/p130. This new growth regulatory feature seems to involve the control of the basic nutritional supplies that the tumor extracts from the host. Moreover, the knowledge that p53, p16, and now also pRb2/p130 regulate angiogenesis via inhibition of the vascular endothelial growth factor, suggests that cell cycle regulatory proteins generally acting in the G1 phase of the cell cycle could have similar effects, or that these proteins control distinct pathways with a common end point. This similarity, along with the fact that DNA tumor viruses simultaneously evolved the ability to repress both p53 and RB family function to accomplish cellular transformation, suggests a cooperation between the proteins in their strategies to regulate proliferation and tumor progression.

The roles of these effectors in tumorigenesis need to be studied more closely to define the mechanisms behind their inhibition of angiogenesis. This would aid in designing more targeted and effective combined gene therapy strategies.


   ACKNOWLEDGMENTS
 
We thank Dr. K. Hubner (Thomas Jefferson University, Philadelphia, PA) for providing the H23 cell line and the normal mouse tissues used to test the CD31 and the VEGF antibodies and Dr. P. B. Fisher (Columbia University, College of Physicians and Surgeons, New York, NY) for providing the mouse VEGF promoter.


   FOOTNOTES
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grants RO1 CA 60999–01A1 and PO1 NS 36466 (to A. G.) and by the Sbarro Institute for Cancer Research and Molecular Medicine (to G. G. G.). P. P. C. is the recipient of a fellowship from the "Associazione Leonardo di Capua," Napoli, Italy. Back

2 To whom requests for reprints should be addressed, at Pathology, Anatomy and Cell Biology, Kimmel Cancer Institute, Thomas Jefferson University, 1020 Locust Street, Room 226, Philadelphia, PA 19107. Phone: (215) 503-0781; Fax: (215) 923-9626; E-mail: agiordan{at}lac.jci.tju.edu Back

3 The abbreviations used are: VEGF, vascular endothelial growth factor; TSP, thrombospondin; IMD, intratumoral microvessel density; RB, retinoblastoma; MLV, murine leukemia virus; ORF, open reading frame; Ad-CMV, adenovirus-cytomegalovirus; ß-Gal, ß-galactosidase; MSCV, murine stem cell virus. Back

Received 5/ 8/00. Accepted 11/ 7/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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