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[Cancer Research 65, 1352-1360, February 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Angiogenin Is Translocated to the Nucleus of HeLa Cells and Is Involved in Ribosomal RNA Transcription and Cell Proliferation

Takanori Tsuji, Yeqing Sun, Koji Kishimoto, Karen A. Olson, Shumei Liu, Saori Hirukawa and Guo-fu Hu

Center for Biochemical and Biophysical Sciences and Medicine, Department of Pathology, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Guo-fu Hu, Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, MA 02115. Phone: 617-432-6582; Fax: 617-432-6580; E-mail: guofu_hu{at}hms.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiogenin is an angiogenic protein known to play a role in rRNA transcription in endothelial cells. Nuclear translocation of angiogenin in endothelial cells decreases as cell density increases and ceases when cells are confluent. Here we report that angiogenin is constantly translocated to the nucleus of HeLa cells in a cell density–independent manner. Down-regulation of angiogenin expression by antisense and RNA interference results in a decrease in rRNA transcription, ribosome biogenesis, proliferation, and tumorigenesis both in vitro and in vivo. Exogenous angiogenin rescues the cells from antisense and RNA interference inhibition. The results showed that angiogenin is constitutively translocated into the nucleus of HeLa cells where it stimulates rRNA transcription. Thus, besides its angiogenic activity, angiogenin also plays a role in cancer cell proliferation.

Key Words: rRNA transcription • cell proliferation • cancer progression • angiogenin • nuclear translocation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiogenin is a 14-kDa angiogenic protein originally isolated from the conditioned medium of HT-29 human colon adenocarcinoma cells based on its angiogenic activity (1). Angiogenin has been shown to play a role in tumor angiogenesis (2, 3). Its expression is up-regulated in many types of cancers including breast (4), cervical (5), colon (6), colorectal (7), endometrial (8), gastric (9), kidney (10), ovarian (11), pancreatic (12), prostate (13), and urothelial (14) cancers, as well as astrocytoma (15), leukemia (16), lymphangioma (17), melanoma (18), Wilms tumor (19), and others.

Angiogenin undergoes nuclear translocation in endothelial cells, which has been shown to be necessary for angiogenesis. Inhibition of nuclear translocation of angiogenin (20) or mutagenesis at its nuclear localization sequence (21) both abolished the angiogenic activity. Nuclear translocation of angiogenin in endothelial cells is rapid (22) and independent of microtubules and lysosomes (23), but is strictly dependent on cell density (22). It decreases as cell density increases and ceases when cells are confluent. No nuclear angiogenin can be detected in normal, non–blood vessel cells regardless of the cell density. Consistently, angiogenin does not induce any detectable cellular response in epithelial cells and fibroblasts. These results suggested that the nuclear function of angiogenin in normal cells is specific for endothelial cells and occurs only when cells are not confluent.

Angiogenin interacts with endothelial cells to induce a wide range of cellular response including migration (24), proliferation (25), and tube formation (26). All these are necessary steps in the process of angiogenesis. The activity of angiogenin is relatively low when compared with that of classic endothelial cell mitogens such as vascular endothelial growth factor and basic fibroblast growth factor. However, angiogenin has a comparable angiogenic activity in various in vivo angiogenesis assays (1, 27) . Moreover, angiogenin antagonists potently inhibited the establishment, progression, and metastasis of human cancer cells inoculated in athymic mice (2, 3). These results suggested that angiogenin has a distinct, yet to be uncovered role in angiogenesis and tumorigenesis. Recently, we discovered that angiogenin is able to bind to the rRNA gene and stimulate rRNA transcription (28). An angiogenin-binding element has been identified from rDNA and we have shown that this DNA sequence has angiogenin-dependent promoter activity (29), suggesting that the function of nuclear angiogenin is related to rRNA transcription.

Cancer is characterized by sustained cell growth requiring continuous protein synthesis that depends on a constant supply of ribosomes (30). Ribosome biogenesis is a multistep process involving assembly of ribosomal proteins and rRNA in an equal molar ratio. During tumorigenesis, the transcription of ribosomal proteins is known to be up-regulated through the Akt-PI3K-mTOR-S6K pathway. However, it is less clear how rRNA transcription is up-regulated. Here we report that angiogenin is continuously translocated into the nucleus of HeLa cells in a cell density–independent manner. Down-regulation of angiogenin expression inhibited rRNA transcription, ribosome biogenesis, cell proliferation, and tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture. Human umbilical vein endothelial cells (HUVEC) were cultured in human endothelial serum-free basal growth medium + 5% fetal bovine serum (FBS) and 5 ng/mL basic fibroblast growth factor. HeLa cells were cultured in DMEM + 10% FBS. Cell viability was determined by trypan blue exclusion method. Cell number was determined with a Coulter counter.

Nuclear Translocation of Angiogenin. HUVEC and HeLa cells were seeded at various densities on a coverslip for 24 hours, washed with serum-free medium and incubated with 1 µg/mL angiogenin at 37°C for 1 hour. Cells were washed with PBS, fixed with methanol at –20°C for 10 minutes, and washed again with PBS containing 30 mg/mL bovine serum albumin. The fixed cells were then incubated with 50 µg/mL of anti-angiogenin monoclonal antibody 26-2F for 1 hour, washed, and incubated with Alexa 488–labeled goat F(ab')2 anti-mouse IgG at 1:100 dilution for 1 hour. The cells were then washed, mounted in 50% glycerol, and monitored with a Nikon Labphot epifluorescent microscope.

[3H]Thymidine and [3H]Uridine Incorporation. Cells were cultured for 24 hours, serum starved for another 24 hours, stimulated with 10% FBS, and continuously labeled with 1 µCi/mL [3H]thymidine or [3H]uridine for 24 hours. The cells were washed with PBS, precipitated with 10% trichloroacetic acid, and solubilized with 0.2 N NaOH plus 0.2% SDS. After neutralization with 0.2 volume of 1 N HCl, the radioactivity was determined by liquid scintillation counting.

Northern Hybridization. The probe used for 45S rRNA (5'-GGTCGCCAGAGGACAGCGTGTCAG-3') hybridizes with the first 25 nucleotides of the 45S rRNA (31). The probe used for actin (5'-CTCTGTGCTCGCGGGGCGGAC-3') hybridizes with the 5' end of cytoplasmic ß-actin mRNA. The probe used for angiogenin was a 569-bp DNA fragment containing the complete 441-bp angiogenin coding sequence and 44 bp from the 5' and 15 bp from the 3' noncoding regions.

ELISA Detection of Angiogenin. ELISA plates were coated with 1 µg 26-2F and blocked with 5 mg/mL bovine serum albumin in PBS. Samples were added in triplicates and the plates were incubated at 4°C overnight, washed with PBS, and incubated with 100 µL/well anti-angiogenin polyclonal antibody R112 (1:4,000) at room temperature for 2 hours. After washing with PBS, an alkaline phosphatase–labeled goat anti-rabbit antibody (1.25 µg/mL) was added and incubated at room temperature for 1 hour. The plate was washed with PBS and p-nitrophenyl phosphate (5 mg/mL, 100 µL/well) dissolved in 0.1 mol/L diethanolamine containing 10 mmol/L MgCl2 (pH 9.8) was added. After 1-hour incubation at room temperature, the absorbance was measured at 410 nm with a turbidity reference at 630 nm. A standard curve (50-1,000 pg angiogenin per well) was done each time on every plate.

Silver Staining of Nucleolar Organizer Region. Cells cultured on a coverslip were fixed with methanol/acetic acid (9:1, v/v) at room temperature for 10 minutes and incubated in 60 mmol/L NaAc (pH 4.1) containing 0.8 g/mL AgNO3, 15% formaldehyde, and 3% methanol at 37°C in the dark for 30 minutes. The slip was washed with water, mounted on a glass slide, and observed at 1,000x magnification. nucleolar organizer region (NOR) dots were counted in 30 randomly selected nuclei.

[32P] Labeling of Newly Synthesized RNA. Cells were pulse-labeled with 25 µCi/mL [32P]Pi for 2 hours and washed with PBS. Total cellular RNA was extracted with Trizol. Equal amounts of RNA were applied for agarose/formaldehyde gel electrophoresis and transferred onto a nylon membrane. Radiolabeled RNA was visualized by autoradiography.

Stable Transfection of HeLa Cells with Angiogenin Antisense Vector. The entire coding region of the human angiogenin cDNA was amplified from pAngC plasmid and cloned into the pCI-neo vector in the antisense orientation. The pCI-Ang(–) plasmid was transfected into HeLa cells using Lipofectin and stable transfectants were selected with 2 mg/mL G418. Integration of the transfected gene into chromosome was confirmed by genomic DNA PCR with the forward primer (5'-AGTACTTAATACGACTCACTATAGGC-3') from the T7 sequence of the pCI vector and the reverse primer (5'-ATGCAGGATAACTCCAGGTACAC-3') from the inserted angiogenin sequence. Transcription of the antisense human angiogenin mRNA was confirmed by reverse transcription–PCR using the same set of primers.

Stable Transfection of HeLa Cells with a Plasmid Containing Angiogenin RNA Interference Cassette. Three regions corresponding to the nucleotides 106 to 126, 122 to 142, 381 to 401, respectively, of the angiogenin mRNA were originally selected for small interfering RNA targeting. Double-stranded, 21-nucleotide-long RNA with 2-nucleotide overhang at the 3' end were synthesized and transfected into HeLa cells to test the efficiency of these small interfering RNA in inhibiting angiogenin expression. ELISA showed that the third region (381-401) with the sequence of 5'-GGTTCAGAAACGTTGTTGTTA-3' was most effective in reducing angiogenin expression. This sequence was therefore used to construct an angiogenin RNA interference (RNAi) plasmid in the pBS/U6 vector according to the method of Sui et al. (32). This plasmid (pAng-RNAi) and pBabe-puro were cotransfected into HeLa cells in the presence of Lipofectin and the stable transfectants were selected with 0.5 µg/mL puromycin for 2 weeks.

Soft Agar Assay. Cells were seeded at a density of 4 x 103 cells per 35-mm cell culture dishes in 0.33% agar and cultured for 7 days. Dishes were stained with crystal violet solution (0.05%) overnight at 4°C. Colonies were counted in 10 fields at x25 magnification. Two-tailed Student's t test was used to verify the differences between the groups.

Xenografic Growth of HeLa Cell Tumors in Athymic Mice. pCI or pCI-Ang(–) transfectants, 8 x 105 cells per mouse, were injected s.c. into the left shoulders of male athymic mice (eight mice per group). Tumor sizes were measured with a microcaliper and recorded in cubic millimeters (length x width2). Mice were sacrificed on day 13 and the wet weight of the tumor was recorded.

Immunohistochemistry. Dako's (Carpinteria, CA) Envision system was used. Neovesssels were stained with an anti–von Willebrand's factor IgG at a 1:200 dilution. von Willebrand's factor–possitive vessels in each tumor were counted in five most vascularized areas at 200x magnification. Proliferating cells were stained with an anti–proliferating cell nuclear antigen (PCNA) IgG. Angiogenin was stained with 26-2F as the primary antibody at the concentration of 10 µg/mL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Angiogenin Is Constitutively Translocated to the Nucleus of HeLa Cells. Immunofluorescent staining showed that nuclear translocation of exogenous angiogenin in HUVECs occurred only when cells were not confluent. Bright nuclear/nucleolar staining was detected when cells were cultured at 5 x 103 cells/cm2. Virtually no nuclear angiogenin was observed when cells were cultured at a density exceeding 1 x 105 cells/cm2 (Fig. 1A). However, angiogenin was detected in the nuclei of HeLa cells cultured under the densities ranging from 5 x 103 to 2 x 105 cells/cm2 (Fig. 1A).



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  		Figure 1. Nuclear translocation and nuclear function of angiogenin in HeLa cells is independent of cell density. A, HUVEC and HeLa cells, seeded at the densities indicated, were incubated with 1 µg/mL angiogenin at 37°C for 1 hour. The cells were washed with PBS and fixed with methanol at –20°C for 10 minutes. Angiogenin was visualized with 10 µg/mL 26-2F and Alexa 488–labeled goat anti-mouse IgG. B, nuclear proteins were extracted from HUVEC and HeLa cells cultured under various densities and analyzed by Western blotting (150 µg per lane) with an anti-angiogenin polyclonal antibody (R112). C, HeLa cells were cultured for 24 hours and nuclei were isolated by subcellular fractionation. Nuclear run-on transcription was carried out with 5 x 104 nuclei isolated from HeLa cells cultured under various densities in the presence or absence of 1 µg/mL angiogenin. D, nuclei were isolated from HeLa cells transfected with an angiogenin RNAi plasmid (pAng-RNAi) and the vector control (pBS/U6) and nuclear run-on assay was done.

 
The primary antibody (26-2F) used in this experiment is specific for human angiogenin (33). It does not recognize angiogenin from other species including bovine, porcine, rabbit, and mouse. X-ray structural analysis of angiogenin-antibody complex has shown that 26-2F interacts with two segments consisting of residues 34 to 41 and 85 to 91, respectively (34). These two regions are apart in the primary but close in the 3-dimentional structures. No fluorescence was observed when 26-2F was replaced by a nonimmune IgG (data not shown). Western blotting analysis confirmed that comparable amounts of angiogenin protein were detected when equal amounts of nuclear proteins, extracted from HeLa cells cultured under various densities, were applied. However, in HUVECs, angiogenin was detectable only in the nuclear proteins extracted from nonconfluent cells (Fig. 1B). Fig. 1C shows that the nuclei isolated from HeLa cells cultured under various densities responded to angiogenin in transcribing 45S rRNA in a nuclear run-on assay. We have also found that angiogenin is translocated to the nucleus in other types of cancer cells including MB231 breast, PC-3 prostate, and HT-29 colon cancer cells (data not shown).

These results suggested that nuclear translocation of angiogenin occurs in HeLa cells regardless of the cell confluence status and that angiogenin stimulates rRNA synthesis in HeLa nuclei. They also indicated that HeLa cell nuclei are not yet saturated with endogenous angiogenin because they can still be stimulated to synthesize rRNA by exogenous angiogenin. However, down-regulation of endogenous angiogenin expression did reduce the transcription potential of HeLa nuclei. As shown in Fig. 1D, the rRNA transcription activity of the nuclei isolated from angiogenin-underexpressing HeLa cells after angiogenin RNAi transfection was lower than that from vector control transfectants. These results showed that the rRNA transcription activity of the nuclei is correlated with angiogenin level.

Transient Transfection of an Angiogenin Antisense Compound Inhibited Cell Proliferation. To confirm that endogenous angiogenin is indeed involved in rRNA transcription, we transiently transfected HeLa cells with an angiogenin antisense oligo to inhibit angiogenin expression and examined the resultant change in rRNA transcription. Fig. 2A shows that transfection of HeLa cells with CT-1, a second-generation antisense oligo for angiogenin (28), decreased the level of 45S rRNA but had no effect on that of glyceraldehyde-3-phosphate dehydrogenase. Transfection with CT-2, a scrambled control oligo, had no effect. A decrease in angiogenin mRNA and protein levels was confirmed by Northern blotting and ELISA, respectively (Fig. 2A and B).



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  		Figure 2. The effect of angiogenin antisense oligonucleotide CT-1 on HeLa cell growth. A, HeLa cells were treated with 1 µmol/L CT-1 or CT-2 in the presence of 5 µL/mL Lipofectin. Total RNA from 2 x 106 cells was isolated and analyzed by Northern blotting for 45S rRNA, glyceraldehyde-3-phosphate dehydrogenase, and angiogenin. B, HeLa cells, 5 x 104 cells per 35-mm dish, were cultured in DMEM + 10% FBS for 24 hours. The cells were washed by OptiMEM and transfected with 1 µmol/L CT-1 or CT-2 in the presence of 5 µL/mL Lipofectin. The culture media were collected at 72 hours and angiogenin levels were determined by ELISA and normalized to cell numbers. C, cell numbers were determined at 48, 72, and 96 hours. Points, means of three experiments; bars, SD.

 
HeLa cell proliferation was inhibited by CT-1 transfection (Fig. 2C). CT-2 had no effect on cell proliferation indicating that the inhibitory activity of CT-1 was not due to a nonspecific cytotoxic effect. These results showed a positive correlation between angiogenin level, rRNA transcription, and cell proliferation. CT-1 also significantly inhibited the proliferation of MB231, PC-3, and HT-29 cells, whereas CT-2 had no effect (data not shown).

Stable Transfection of an Angiogenin Antisense Plasmid Inhibited rRNA Transcription. To further show that endogenous angiogenin is required for rRNA transcription and cancer cell growth, we transfected HeLa cells with pCI-Ang(–), a plasmid containing the antisense sequence of angiogenin cDNA, and selected stable transfectants with G418. Angiogenin level in the antisense transfectants was reduced as determined by ELISA and Western blotting analysis (Fig. 3A).



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  		Figure 3. Down-regulation of angiogenin inhibits HeLa cell growth. HeLa cells were transfected with pCI-Ang(–) or pAng-RNAi and their corresponding control vectors, pCI and pBS/U6. Stable transfectants were selected with 2 mg/mL G418 and 0.5 µg/mL puromycin for pCI-Ang(–) and pAng-RNAi, respectively. Similar results were obtained from all the individual clones and from the pooled populations. A-C, pCI-Ang(–) and pCI transfectants. D-F, pAng-RNAi and pBS/U6 transfectants. A and D, angiogenin secretion levels determined by ELISA (left), and cellular angiogenin levels detected by Western blotting with actin as the loading control (right). B and E, cell morphology changes in angiogenin-underexpressing cells. C and F, the steady-state level of 45S rRNA detected by Northern blotting with 18S rRNA as the loading control (left) and the numbers of AgNOR (right).

 
The morphology of the cells changed dramatically in the antisense transfectants (Fig. 3B). The steady-state level of the 45S rRNA was significantly lower in the antisense transfectants as determined by Northern blotting (Fig. 3C, left). Ribosome biogenesis was measured by silver staining of NOR (Fig. 3C, right). NORs are actively transcribing rDNA loops and reflect the ribosome biogenesis status (35). They are associated with argyrophilic proteins and can be visualized by silver staining. The size and number of NORs reflect the capacities of the cell to transcribe rRNA and are therefore indices for cell proliferation, transformation, and even malignancy (36). The decrease in 45S rRNA level and in NOR numbers in pCI-Ang(–) transfectants indicated that rRNA transcription and ribosome biogenesis were decreased when angiogenin expression was inhibited.

RNAi was also used to down-regulate angiogenin expression in HeLa cells. Very similar results were obtained as in antisense transfection except that they were more significant due to a greater degree of inhibition in angiogenin expression (Fig. 3E). We used a DNA vector-based RNAi method (32) to construct a pAng-RNAi plasmid, with the insertion DNA template corresponding to the nucleotides 381 to 401 of angiogenin mRNA. It was transfected into HeLa cells together with pBabe-puro (37) and those cells that stably express angiogenin RNAi were selected by puromycin resistance. Stable transfection of pAng-RNAi resulted in an 80% reduction of angiogenin expression, as shown by ELISA and Western Blotting (Fig. 3D). The 45S rRNA level and NOR numbers were also decreased more significantly (Fig. 3F).

The effect of down-regulating angiogenin expression on the de novo synthesis of rRNA was determined by metabolic labeling with 32P. Cells were pulse-labeled with [32P]Pi for 2 hours and the total cellular RNA was isolated with Trizol. Fig. 4A shows that the newly synthesized 45S rRNA was significantly decreased in pAng-RNAi transfectants than that in pBS/U6 control transfectants when equal amounts of total RNA was loaded (Fig. 4B). In the 2-hour period, some of the newly synthesized 45S rRNA has been processed to 32S rRNA whose level was significantly lower in pAng-RNAi transfectants (Fig. 4A). Because of the large cytoplasmic pool of stable ribosomes, the cellular levels of 28S and 18S rRNA were relatively stable (38). Thus, 28S and 18S rRNA still serve as the best loading control. However, it should be noted that the RNAi transfectants do have a reduced steady-state level of 45S rRNA, which results in a decrease in the cellular level of 28S and 18S rRNA. Therefore, use of 28S rRNA as a loading control actually underestimated the difference in newly synthesized 45S rRNA per cell after antisense and RNAi transfection. In any event, these results indicated that the de novo synthesis of 45S rRNA decreased when angiogenin expression was inhibited, confirming that the nuclear function of angiogenin is related to rRNA transcription.



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  		Figure 4. Angiogenin RNAi transfection inhibits the de novo synthesis of 45S rRNA. pBS/U6 or pAng-RNAi transfectants were cultured in DMEM + 10% FBS. [32P]Pi (25 µCi/mL) was added to each dish and incubated for 2 hours. Total cellular RNA was isolated with Trizol and the amount determined by absorbance at 260 nm. Equal amounts of RNA were loaded to each lane and subjected to agarose/formaldehyde electrophoresis. The newly synthesized RNA were visualized by autoradiography (A). 28S and 18S rRNA were stained by ethidium bromide (B). Representative experiment of three repeats.

 
Cell Proliferation Was Inhibited by Down-regulating Angiogenin Expression. Because of the central importance of rRNA transcription in cell growth, decreased rRNA transcription should slow cell proliferation. This is confirmed by three different cell proliferation assays (Fig. 5). Direct cell number counting (Fig. 5A and B) showed that both antisense and RNAi transfectants have reduced cell proliferation rates than that of their corresponding vector control transfectants. However, the extent of inhibition was greater in RNAi (Fig. 5B) than in antisense transfectants (Fig. 5A), probably reflecting the fact that RNAi is more effective in inhibiting angiogenin expression.



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  		Figure 5. Angiogenin-underexpressing HeLa cells have reduced growth rate. A, C, and E, antisense transfectants. B, D, and F, RNAi transfectants. A and B, cell proliferation was determined by direct cell number counting. C and D, DNA synthesis determined by [3H]thymidine incorporation. E and F, RNA synthesis determined by [3H]uridine incorporation. Cells were cultured for 24 hours, serum-starved for another 24 hours, stimulated with 10% FBS, and continuously incubated with 1 µCi/mL [3H]thymidine (C and D) or 1 µCi/mL [3H]uridine (E and F) for 24 hours.

 
The rate of DNA and RNA synthesis was determined by [3H]thymidine and [3H]uridine incorporation, respectively. Cells were continuously labeled for 24 hours and the incorporated radioactivity was normalized to cell numbers. pCI-Ang(–) and pAng-RNAi transfectants had slower DNA (Fig. 5C and D) and RNA (Fig. 5E and F) synthesis rates than do the respective vector control transfectants.

These results showed that endogenous angiogenin in HeLa cells plays an important role in rRNA transcription, ribosome biogenesis, and cell proliferation.

Colony Formation in Soft Agar Was Inhibited by Angiogenin RNAi. The anchorage-independent growth of the pBS/U6 and pAng-RNAi transfectants was analyzed by colony formation assay in soft agar (Fig. 6A-C). RNAi transfection decreased both colony number and size significantly. Colony number per 35-mm dish was 1,631 ± 52 and 908 ± 42, and the average colony size was 115 ± 8 and 65 ± 8 µm in vector control and in pAng-RNAi transfectants, respectively (Fig. 6A and B). A complete recovery was obtained when exogenous angiogenin (0.1 µg/mL) was added (Fig. 6C), indicating that the decrease in colony formation in pAng-RNAi transfectants was caused by reduced angiogenin expression. These results showed that the tumorigenicity of HeLa cells is decreased by down-regulating angiogenin expression.



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  		Figure 6. Angiogenin RNAi transfectants have reduced tumorigenicity in soft agar. Anchorage-independent growth of pBS/U6 control (A) and pAng-RNAi transfectants in the absence (B) or presence of 0.1 µg/mL angiogenin (C) was determined in soft agar. The numbers of colonies in the entire 35-mm dish were counted. Average colony size was determined by measuring the diameters of colonies in 10 microscope fields with a microcaliber. Pictures from representative areas.

 
Angiogenin Down-regulation Inhibited Ecotopic Growth of HeLa Cell Tumors in Athymic Mice. The effect of angiogenin antisense transfection on tumor growth was examined in athymic mice. Figure 7 shows that the appearance, establishment, and growth of HeLa cells inoculated in athymic mice were all significantly inhibited after angiogenin antisense transfection. There was a delay in tumor occurrence (Fig. 7A) and the progression was slower (Fig. 7B) in angiogenin antisense-transfected HeLa cells. When tumors did develop, their average size was about half of those in the control group (Fig. 7B). These data confirmed the results obtained in soft agar assays that the tumorigenicity of HeLa cells decreased when angiogenin expression was down-regulated.



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  		Figure 7. Angiogenin antisense HeLa transfectants have reduced tumorigenicity in athymic mice. pCI vector control and pCI-Ang(–) antisense transfectants, 8 x 105 cells per mouse, were injected s.c. into the left shoulder of the mice. Eight mice were used per group. A, tumor appearance. Mice were examined for tumor growth thrice per week. B, tumor volume. Tumor sizes were measured with a caliper and recorded in cubic millimeters (length x width2). C, neovessels were stained with an anti–von Willebrand's factor IgG. Vessel density (vessels/field) was shown as mean ± SD for each group. D, proliferating cells were stained by an anti-PCNA monoclonal antibody. PCNA-positive and total numbers of cells were counted in 10 randomly selected areas at 200x magnification. E, 26-2F was used to reveal nuclear angiogenin in HeLa tumors derived from pCI and pCI-Ang(–) transfectants. No staining was observed when a nonimmune IgG was used to replace 26-2F. Pictures from a representative animal of each group.

 
Immunohistochemical staining with an anti–von Willebrand's factor antibody (Fig. 7C) showed that the HeLa tumor tissue grown from pCI-Ang(–) transfectants had about half of the blood vessel density (11 versus 21 vessels per field at 200x, peripheral regions of the tumors) as compared with those grown from vector control transfectants, indicating that angiogenesis was inhibited in angiogenin antisense transfectants, in agreement with the known role that angiogenin plays in tumor angiogenesis. More importantly, PCNA staining showed that cell proliferation in the pCI-Ang(–) tumor was significantly lower than that in the control (Fig. 7D), indicating that not only tumor angiogenesis but also tumor cell proliferation per se was inhibited by angiogenin down-regulation. Staining with 26-2F confirmed that angiogenin in the nuclei of tumor cells decreased after antisense transfection (Fig. 7E). These results further showed that angiogenin plays a dual role in tumor growth by contributing both in angiogenesis and in cancer cell proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Angiogenin was isolated as a tumor angiogenic factor based solely on its angiogenic activity (1). Therefore, subsequent studies had mainly focused on how it induces angiogenesis and how its angiogenic activity can be intervened. Angiogenin has been considered to interact only with endothelial cells and vascular smooth muscle cells. Results presented in this article indicated that cancer cells are also targets for angiogenin.

We have showed in this article that angiogenin is constitutively translocated to the nucleus of HeLa cells where it plays a role in rRNA transcription. Because rRNA transcription regulates ribosome production and, consequently, the translation potential of a cell, it is conceivable that deregulation of rRNA transcription may be an important determinant in neoplastic transformation. Continuous nuclear translocation of angiogenin in cancer cells can certainly be one of the contributing factors. Indeed, inhibiting angiogenin expression reduced tumorigenicity and reversed the malignant phenotype of HeLa cells.

Several animal models have been used to examine the role angiogenin plays in tumor growth (2, 39). Whereas the antitumor activity of angiogenin antagonists was obvious, the mechanism by which they prevented or delayed tumor appearance was not fully understood. Based on the long-held assumption that angiogenin is a tumor angiogenic protein, these data seemed to support the proposition that the observed antitumor effects were due to inhibition of angiogenin-induced tumor angiogenesis. However, the tumors that eventually developed in angiogenin antagonist–treated mice were substantially smaller than those in the control group. It is therefore possible that other effects such as repression of rRNA transcription of cancer cells also contributed to the marked anticancer activity of angiogenin antagonists. Our results shown in Fig. 7 showed that this is truly the case. Down-regulating angiogenin expression in HeLa cells not only reduced tumor angiogenesis (Fig. 7D) but also inhibited tumor cell proliferation (Fig. 7E).

The finding that angiogenin is involved in rRNA transcription in cancer cells is significant for our understanding of cancer biology and neoplastic transformation. One of the hallmarks of cancer is sustained cell growth and this can only be achieved by increased protein synthesis. To accommodate this need, there must be an increase in ribosome biogenesis. Up-regulation of ribosomal proteins and rRNA transcription is an important factor in cancer transformation (40). It has been reported that the proliferation effects of estrogen in the rat induces pituitary tumors (41). However, the susceptibility to formation of such tumors is highly strain dependent. The particularly susceptible Fisher 344 strain develops tumors after 30 to 55 days of estrogen treatment. In contrast, the Sprague-Dawley strain is resistant to such tumors. The major difference between the two strains is the marked increase (250%) of rRNA in the pituitaries of Fisher 344 rats (42). It is notable that the increase in rRNA accumulation occurs within 3 days of estrogen treatment when the increase in the number of lactotrophs is minimal or nonexistent. These results indicated that estrogen induces rRNA synthesis in the early stages of tumorigenesis before any tumor growth is detectable.

Androgen-dependent growth of the prostate has been well documented in prostatic hyperplasia and prostatic carcinoma (43). It has been shown that androgens regulate the accumulation of rRNA during androgen-dependent cell growth (44) and that androgen-stimulated rRNA synthesis is the mechanism by which androgens affect growth (45). Recently, it has been reported that angiogenin is the most up-regulated gene in prostate intraepithelial neoplasia in the murine prostate restricted Akt kinase transgenic mice (13). In these mice, expression of Akt1 in the prostate results in activation of the p70S6K pathway that up-regulates ribosomal proteins through the Akt-p70S6K-mTOR pathway. Because rRNA production has to increase in an equimolar ratio to that of ribosomal proteins for ribosome biogenesis, we are currently investigating whether rRNA transcription in the prostate is stimulated by angiogenin and whether inhibition of angiogenin expression will inhibit prostate intraepithelial neoplasia development.

The role angiogenin plays in rRNA transcription in cancer cells suggests that up-regulation of angiogenin expression in various cancer cells not only induces tumor angiogenesis but also directly contributes to cell proliferation. Thus, inhibitors targeting angiogenin will be more effective than those that inhibit either angiogenesis or cancer cell proliferation alone. We have found that neomycin, an aminoglycoside antibiotic that blocks nuclear translocation of angiogenin (20), significantly inhibits both tumor angiogenesis and cancer cell proliferation in an ecotopic human tumor model in athymic mice.1

The mechanism by which angiogenin stimulates rRNA transcription is unclear at present. We believe that angiogenin acts in cancer cell nucleus in a similar manner as it does in endothelial cell nucleus. Angiogenin has been shown to undergo nuclear translocation in endothelial cells through endocytosis and classic nuclear pore import (27). The detailed transportation steps across the cytoplasm are unknown, but it seems to be lysosome and microtubule independent (29). More extensive work is needed to understand how angiogenin is translocated to the nucleus, how it interacts with the RNA polymerase I machinery, and whether it also affects transcription catalyzed by RNA polymerase II. We have carried out a DNA array analysis (Atlas Human 1.2 Array I, II, III, Clontech, Palo Alto, CA) and found that the ribosomal proteins are universally down-regulated (19-80% of the control) in pAng-RNAi transfectants.2 All these results point to an important role angiogenin plays in ribosome biogenesis of cancer cells.


   Acknowledgments
 
Grant support: NIH grant CA91086 (G-F. Hu) and the Endowment for Research in Human Biology, Inc.

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.


   Footnotes
 
Note: T. Tsuji and Y. Sun contributed equally. Y. Sun is currently in the Institute of Environmental Science and Systems Biology, Dalian Maritime University, Dalian 116026, China. K. Kishimoto is currently in the Department of Oral and Maxillofacial Surgery, Okayama University Graduate Schools, Okayama 700-8525, Japan. K.A. Olson is currently in Archemix Corp., 1 Hampshire Street, Cambridge, MA 02139.

1 Hirukawa et al., unpublished data. Back

2 Tsuji et al., unpublished data. Back

Received 6/ 7/04. Revised 11/ 9/04. Accepted 11/22/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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