Distinct Mechanisms for Regulating the Tumor Suppressor and Antiapoptotic Functions of Rb*

Duanduan Ma {ddagger}, Ping Zhou {ddagger} and J. William Harbour §

From the Departments of Ophthalmology and Visual Sciences and of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, February 19, 2003 , and in revised form, March 19, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The retinoblastoma protein, Rb, suppresses tumorigenesis by inhibiting cell proliferation and promoting senescence and differentiation. Paradoxically, Rb also inhibits apoptosis, which would seem to oppose its tumor suppressor function. Further, most human cancer cells inactivate Rb by hyperphosphorylation and demonstrate increased proliferative capacity but not high levels of apoptosis. As a potential explanation for these findings, we show here that the tumor suppressor and antiapoptotic functions of Rb are regulated by distinct phosphorylation events. Phosphorylation of sites in the C terminus occurs efficiently every cell cycle and regulates proliferation. Phosphorylation of Ser567 is inefficient and does not occur during the normal cell cycle. However, high cyclin-dependent kinase activity promotes phosphorylation of Ser567 by inducing an intramolecular interaction that leads to release of E2F, degradation of Rb, and susceptibility to apoptosis. Thus, phosphorylation of Ser567 may limit excessive proliferation by triggering cell death under hyperproliferative conditions. These findings suggest that the antiproliferative and antiapoptotic activities of Rb may represent complementary functions that work in concert to maintain the proliferation rate of cells within certain limits. As a survival strategy, some cancer cells may exploit this dual role of Rb by phosphorylating sites that regulate tumor suppression but avoiding phosphorylation of Ser567 and consequent apoptotic stimulus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The retinoblastoma tumor suppressor protein (Rb) is involved in regulating a variety of cellular functions, including cell division, differentiation, senescence, and apoptosis (1). Rb in turn is regulated by phosphorylation; it is active when hypophosphorylated and inactive when hyperphosphorylated (2). Rb is phosphorylated by cyclin D- then cyclin E-associated kinases as cells progress from early G1 into S phase, and this sequential phosphorylation is necessary for complete hyperphosphorylation of Rb (3, 4, 5). Rb has 16 potential CDK1 phosphorylation sites, most of which have been shown to be phosphorylated in vivo (6). Some sites appear to regulate distinct biochemical activities. For example, Ser807 and Ser811 regulate binding of Rb to c-Abl (7), whereas Thr821 and Thr826 regulate binding to LXCXE proteins (7). Binding to E2F is more complex and appears to be regulated by multiple phosphorylation sites (8, 9). Phosphorylation of sites in the C terminus triggers a conformational change that allows phosphorylation of Ser567, which then leads to disruption of the A-B pocket and release of E2F (5). Despite these findings, it remains unclear whether specific phosphorylation sites govern distinct physiologic functions (such as cell proliferation and apoptosis) and whether specific sites are preferentially phosphorylated in cancer cells.

The Rb pathway is thought to be disrupted in virtually all cancers (10), and the tumor suppressor activity of Rb derives at least in part from its ability to inhibit cell transit from G1 into S phase (11). However, Rb also inhibits apoptosis, which would seem to oppose its tumor suppressor activity. The distinct phenotypes of Rb(+) cancer cells versus Rb(-) cancer cells may provide insight into this apparent paradox of Rb function. Rb(+) cancers in which the Rb protein is inhibited by hyperphosphorylation (including most adult solid tumors) are generally slow growing, resistant to apoptosis, and insensitive to chemotherapy and radiation (12). In contrast, Rb(-) cancers in which Rb is genetically disrupted (such as retinoblastoma and small cell lung cancer) usually have a high rate of both proliferation and apoptosis and are generally sensitive to chemotherapy and radiation (12). Similarly, in cultured cells and mouse models, genetic inactivation of Rb leads to increased cell proliferation and apoptosis (13, 14, 15, 16, 17, 18, 19, 20), whereas phosphorylation of Rb (e.g. as a result of p16ink4a inactivation) generally does not have this effect (21). A possible explanation for these findings is that the tumor suppressor functions of Rb (such as cell cycle inhibition) may be regulated by different phosphorylation events than is the antiapoptotic activity. In Rb(-) cancers, both proliferation and apoptosis may proceed unchecked, whereas in Rb(+) cancers, the tumor suppressor activity may be inhibited while keeping the antiapoptotic function intact.

To test this hypothesis, we analyzed Rb phosphorylation in U2OS osteosarcoma cells. These Rb(+) cells are deficient for p16ink4a, which leads to deregulation of cyclin D-CDK4 and constitutive phosphorylation of endogenous and exogenous Rb (3, 22, 23). Further, reintroduction of p16ink4a into these cells leads to accumulation of hypophosphorylated Rb and G1 arrest. This model allowed Rb phosphorylation sites to be studied in vivo without overexpressing cyclins or CDKs. We found that cell cycle regulation by Rb was controlled by multiple phosphorylation sites in the C terminus in a cumulative manner. In contrast, a distinct phosphorylation site (Ser567) that regulates binding to E2F was not phosphorylated every cell cycle but was only phosphorylated in the presence of high CDK2 activity. Phosphorylation of Ser567 led to inactivation and degradation of Rb and release of E2F. An Rb mutant that prevented phosphorylation of Ser567 had no effect on cell cycle progression or senescence in U2OS cells, but it inhibited proliferation and apoptosis in cells with high E2F1 levels and high CDK2 activity. These results suggest that the tumor suppressor and antiapoptotic functions of Rb may be regulated by distinct mechanisms and that Ser567 may be a sentinel for hyperproliferative conditions.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Expression vectors for most Rb constructs were previously described (5), with nomenclature as follows: G, a Gal4 tag; L, LexA tag; GST, GST tag; FL, full-length Rb; LP, large pocket (amino acids 379–928); A, domain A (amino acids 379–602); B, domain B (amino acids 612–792); AB, domains A and B plus the spacer region, amino acids 379–792); C, (the C terminus, amino acids 792–928); {Delta}567, Ser-to Ala substitution; {Delta}568, Pro-to-Ala substitution; {Delta}2S, Ser-to-Ala substitutions at Ser807 and Ser811; {Delta}2T, Thr-to-Ala substitutions at Thr821 and Thr826; {Delta}4, a combination of {Delta}2S and {Delta}2T; AB{Delta}4, elimination of Ser602 and Ser608 by deletion of the spacer and elimination of Ser780 and Ser788 by truncation at position 778. Rb{Delta}10 is a hemagglutinin-tagged full-length Rb mutant with the substitution of 10 phosphorylation sites (gift from J. Bartek) (24). Rb{Delta}9 is a full-length Rb mutant with the substitution of nine phosphorylation sites (gift of E. Knudsen) (8).

G-Rb{Delta}780 is a Ser-to-Ala substitution at Ser780 generated from the corresponding wild type construct by site-directed mutagenesis using the Mutagene in vitro mutagenesis kit (Bio-Rad). G-A{Delta}567 and G-A{Delta}568 were generated by digesting G-Rb{Delta}567 and G-Rb{Delta}568, respectively, with BamHI and BglII, and the domain A fragments were cloned into pM2 at the BamHI site. G-BC was created by digesting G-Rb-LP with BamHI and BglII and cloning the fragment corresponding to domain B and the C terminus (amino acids 612–928) into pM2 at the BamHI site. Similarly, G-BC{Delta}4 and G-BC7 were generated from G-RbLP{Delta}4 and PSM-7LP. Constructs were generated for co-expression of GFP by digesting the Rb fragments from their pM2 vector with EcoRI and XbaI and inserting the fragment into the pCMS-EGFP vector (Clontech). GST-A was generated by digesting G-Rb with BamHI and BglII and cloning the fragment into pGEX-2T (Amersham Biosciences). Similarly, GST-A{Delta}567 and GST-A{Delta}568 were generated from G-Rb{Delta}567 and G-Rb{Delta}568. Plasmids were checked for correct DNA sequence.

Cell Culture—U2OS cells, Rat2/E2F1-DP1-inducible cells (gift from W. Lee), and murine embryo fibroblasts lacking all three pocket proteins (gift from T. Jacks) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For Rat2/E2F1-DP1 cells, G418 (250 µg/ml) and tetracyclin (1 µg/ml) were added to the media. E2F and DP1 were induced by the removal of tetracycline for 2 days. Apoptosis was enhanced by reducing the fetal bovine serum to 0.5%.

Western Blot and Immunoprecipitations—Western blots were performed as previously described (5) using the indicated primary antibodies: anti-Rb (sc102; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Rb-phospho-Ser780 and anti-Rb-phospho-Ser807/Ser811 (Cell Signaling), and anti-Gal4 and anti-LexA (Santa Cruz Biotechnology). An antibody that recognizes Rb phosphorylated at Ser567 was created for our laboratory by Bethyl Laboratories, Inc. by immunizing rabbits with a peptide derived from the Rb sequence spanning Ser567 (CAWLSD-SPLFDL) with or without phosphorylation at Ser567. Phosphospecific antibodies were purified using an affinity column containing the unphosphorylated peptide. An enzyme-linked immunosorbent assay confirmed high affinity antibody against the phosphorylated peptide (dilution titer 1:56,870 versus 1:100). Secondary anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase (Santa Cruz) were applied, and immunoreactivity was detected by ECL chemiluminescence reaction (PerkinElmer Life Sciences). Immunoprecipitations were performed as previously described (5). Transfections were performed in C33A cells using the indicated expression vectors (10 µg for Rb vectors and 3 µg for cyclin E). 10% of the lysate was used for direct Western blot, and the remainder was immunoprecipitated with agarose beads conjugated to monoclonal anti-Gal4 or anti-LexA antibody (Santa Cruz Biotechnology). Immunoprecipitates were analyzed by Western blot as above using anti-LexA or anti-Gal4 primary antibodies.

Transcriptional Repression Assays—U2OS cells (106) were transfected with 4 µg of DNA, including the indicated expression vectors and a cyclin E promoter-luciferase reporter construct (gift from R. Weinberg). Empty expression vectors were included in control assays. Lysates were collected after 48 h in lysis buffer (50 mM Tris-MES, pH 7.8, 1 mM dithiothreitol, 1% Triton X-100), and the protein concentrations were normalized. A reaction mixture was generated containing a final concentration of 10 mM Mg(Oac)2, 50 mM Tris-MES, pH 7.8, 2 mM ATP. Luciferin (100 µl of 1 mM) was added, and the luciferase activity was measured by the luminometer.

BrdUrd Incorporation Assay—U20S cells (105) were transfected with 1 µg of the indicated pCMV-GFP expression vectors on coverslips. At the indicated time points, the cells were labeled with BrdUrd (Amersham Biosciences) at 1:1000 dilution. After 11 h, cells were rinsed with 1x PBS, fixed in 3.7% formaldehyde for 15 min, permeabilized in 0.3% Triton X-100 for 15 min, and then probed with an anti-BrdUrd antibody (PharMingen) at a 1:200 dilution in immunofluorescence buffer (PBS, 5 mg/ml bovine serum albumin, 0.5% Nonidet P-40, 10 mM MgCl2, 300 µg/ml DNase) for 1 h at 37 °C in a humidified chamber. After washing the coverslips twice with 1 ml/well PBS, the cells were incubated with AlexaFluor-conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR) at a 1:1000 dilution for 1 h. Cells were stained with Hoechst reagent and mounted onto slides using Vectashield mounting medium (Vector Laboratories) and analyzed by fluorescence microscopy.

Flow Cytometry—U2OS cells (2x 106) were transfected with 2 µg of the indicated expression vectors. GFP was co-expressed by using the pCMV-EGFP construct or by co-transfecting expression vectors for GFP and the target protein at a 1:5 ratio. At the indicated time points, cells were collected and fixed in 0.5% paraformaldehyde for 5 min, fixed in 70% ethanol for 1 h, and then stained with a propidium iodide solution (0.1% Triton X-100, 0.5 mM EDTA pH 7.4, 0.05 mg/ml RNase A, and 50 µg/ml propidium iodide in PBS, pH 7.4). Flow cytometry was performed on GFP-positive cells using the FACSCalibur machine, and data analysis was performed with Cell Quest software. Experiments with Rat2/E2F1-DP1 cells were performed in a similar fashion, except that the expression vectors were co-transfected with a puromycin resistance plasmid and selected for 3 days in media containing puromycin (0.8 µg/ml). To trigger apoptosis in some cells, tetracycline was removed from the media to induce expression of E2F1 and DP1, and serum was reduced to 0.5%. Cells were collected 48 h later, stained with propidium iodide, and analyzed by flow cytometry.

Colony Formation and Flat Cell Assays—U2OS cells (2x 106) were grown on 100-mm plates and transfected with the indicated expression vectors (10–16 µg of target DNA, 1–2 µg of puromycin resistance plasmid and carrier DNA). Puromycin (0.8 µg/ml) was added after 24 h, and medium was changed every 3 days until colonies grew out (usually within 2–3 weeks). Cells were fixed in 10% acetic acid and 10% methanol and stained with 0.4% crystal violet and 20% ethanol, and colonies were counted in a blinded fashion. Flat cell assays were performed in the same manner, except that flat cells (defined as at least 10 times greater diameter than a normal U2OS cell) were counted in 10–20 fields by a masked examiner under a 10x objective using the inverted microscope. To analyze senescence-associated {beta}-galactosidase, cells were washed with PBS, fixed with 2% formaldehyde and 0.2% glutaraldehyde for 3–5 min, washed with PBS, placed in solution containing 1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, and 2 mM magnesium chloride in 40 mM citric/sodium phosphate buffer, pH 6.0, and incubated overnight at 37 °C without CO2. Positive cells demonstrated blue staining.

In Vitro Phosphorylation—In vitro phosphorylation experiments were performed as previously described (5). GST-tagged cyclin E-CDK2 was obtained from baculovirus-infected SF9 insect cells. In vitro phosphorylation reactions were performed at room temperature for the indicated time periods in 40 µl of kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol), 150 µM ATP, 100 µCi of [{gamma}-32P]ATP, and 500 ng of the indicated Rb fragments. Phosphorylation products were resolved by 10% SDS-PAGE, detected by autoradiography, and quantitated by densitometry. Phosphorylation of Ser567 was also analyzed by expressing the indicated Rb fragments in BL21 cells (Stratagene). Proteins were purified using glutathione beads (Amersham Biosciences), phosphorylated using recombinant cyclin E-CDK2 as above, resolved by SDS-PAGE, and detected by Western blot using the anti-Rb-phospho-Ser567 antibody (1:500 dilution).

Viability Assays—Viability assays were performed essentially as previously described (25) except that Rat2/E2F1-DP1 cells were transfected with the indicated expression vectors along with a puromycin resistance plasmid, selected for 72 h in media containing puromycin, trypsinized, and plated into 96-well plates at 3 x 105 cells/well. After 24 h, tetracyclin was removed from the media, and serum was reduced to 0.5% in some cells for 48 h to induce E2F/DP1 expression and promote apoptosis. A methanethiosulfonate assay was performed according to the manufacturer's recommendations (Promega). Briefly, 20 µl of methanethiosulfonate and phenazine methosulfate solution was added to each well, cells were incubated at 37 °C for 3–4 h, and absorbance was measured at 496 nm using a plate reader.

Immunofluorescence Microscopy—U2OS cells (5 x 104) were plated on coverslips and transfected with 1 µg each of expression vectors for cyclin D and cyclin E, as indicated. After 48 h, cells were washed with PBS, fixed in methanol for 5 min at -20 °C, and then treated with 2% fish gelatin for 1 h. Anti-Rb-phospho-Ser567, anti-Rb-phospho-Ser780, or anti-Rb-phospho-Ser807/Ser811 (1:50 dilution) was applied for 1 h. As a control, some cells were analyzed with anti-Rb-phospho-Ser567 pretreated with the Rb Ser567 peptide described above (1 µg of antibody to 3 µg of peptide). Cells were then washed with PBS, treated with fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology) at a 1:100 dilution for 1 h, and then washed with PBS. Coverslips were mounted onto slides using the Vectorshield mounting medium, stained with 4',6-diamidino-2-phenylindole (Vector Laboratories) and analyzed with a fluorescence microscope.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ectopic Rb Is Hyperphosphorylated and Inactivated in U2OS Cells—Since we were interested in determining whether specific phosphorylation sites may regulate distinct Rb functions, we wished to identify a model in which ectopic Rb constructs could be expressed in vivo without overexpressing a cyclin or CDK. Consistent with previous reports (3, 22, 23), we found that ectopic Rb was hyperphosphorylated when expressed in U2OS cells (Fig. 1A). Since Rb represses expression of cyclin E and other genes involved in S phase entry (26), we tested the ability of ectopic Rb to repress a cyclin E promoter linked to a luciferase reporter. Expression of p16ink4a, which causes an accumulation of hypophosphorylated endogenous Rb, resulted in strong repression of the cyclin E promoter (Fig. 1B). In contrast, ectopic Rb demonstrated minimal repressor activity. By flow cytometry, expression of p16ink4a caused cells to accumulate with 2N DNA content, consistent with G1 arrest, whereas the DNA content of cells expressing Rb did not differ from untransfected cells or cells expressing an inactive fragment of Rb (Fig. 2A). BrdUrd incorporation assays were performed to further analyze the ability of ectopic Rb to prevent S phase entry. By 72 h after transfection, only 21% of cells expressing p16ink4a had incorporated BrdUrd, compared with 72% of cells expressing ectopic Rb (Fig. 2B). In colony formation assays, expression of p16ink4a reduced the number of colonies by about 90%, whereas Rb reduced colonies by only about 35% (Fig. 2C). These results suggest that ectopic Rb is phosphorylated sufficiently in U2OS cells to block its tumor suppressor activity. Hence, we were able to use these cells to analyze the physiologic role of individual CDK phosphorylation sites by expression of Rb phosphorylation site mutants without overexpressing a cyclin (Fig. 1C).



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FIG. 1.
Ectopic Rb is phosphorylated and inactivated in U2OS cells. A, ectopic Rb was expressed in U2OS cells with or without co-expression of cyclin E to facilitate full hyperphosphorylation of Rb and analyzed with antibodies that recognize all phosphorylated forms of Rb ({Delta}Rb), or Rb phosphorylated at Ser780 or Ser807/Ser811. B, the cyclin E promoter linked to a luciferase reporter was transfected into U2OS cells along with either Rb, Rb{Delta}10, or p16ink4a, and repressor activity was analyzed by luciferase activity. Control cells were transfected with an inactive fragment of Rb containing domain A. C, summary of Rb phosphorylation site mutants used in this study. Unbroken lines indicate the length of the expressed protein (either full-length or large pocket). Broken lines indicate that both full-length and large pocket proteins were used. Black boxes indicate amino acid substitutions at the corresponding serine or threonine residues. Note that Rb{Delta}568 results in loss of phosphorylation at Ser567.

 


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FIG. 2.
Tumor suppressor activity of Rb is regulated by C-terminal phosphorylation sites. A, flow cytometry experiments in U2OS cells expressing the indicated constructs at 24, 48, and 60 h after transfection. Cells were stained with propidium iodide and analyzed for DNA content. B, BrdUrd incorporation assays were performed in U2OS cells expressing the indicated constructs at 24, 48, or 72 h after transfection. For clarity, the Rb mutants with substitution of up to four C-terminal phosphorylation sites are shown separately in the right panel. C, colony formation assays were performed in U2OS cells expressing the indicated constructs. Cells were selected in 0.8 µg/ml puromycin, and colonies were counted after 2–3 weeks. D, flat cell assays were performed in U2OS cells expressing the indicated constructs. Flat cells were counted with an inverted microscope. Inset, flat cells (large arrow) were at least 10 times larger in diameter than regular U2OS cells (small arrow) and stained for senescence-associated {beta}-galactosidase (blue). Control cells were transfected with an inactive fragment of Rb containing domain A.

 

In colony formation assays (Fig. 2C), BrdUrd incorporation assays, and flow cytometry (data not shown), the large pocket fragment of Rb (amino acids 379–928) demonstrated no greater activity than full-length Rb. Thus, the six potential phosphorylation sites in the N-terminal region did not appear to be required for regulation of Rb activity. Other investigators have also found that the N terminus was not essential for Rb regulation (8, 27). In contrast, phosphorylation sites in the C terminus (amino acids 792–928) have been strongly implicated in regulating Rb activity. Ser807 and Ser811 regulate binding to c-Abl, Thr821 and Thr826 regulate binding to LXCXE proteins such as HDAC, and all four of these sites cooperate to regulate active transcriptional repression by Rb (5, 7). Ser780 is a cyclin D-specific site that also has been implicated in regulating Rb activity early in G1 (8, 28, 29). Thus, we initially focused our attention on these C-terminal phosphorylation sites.

CDK Sites in the Rb Large Pocket Cooperate to Regulate Cell Cycle Inhibition—Phosphorylation sites were first examined for their role in cell cycle regulation. Flow cytometry was performed at 24, 48, and 60 h after transfection of the phosphorylation site mutants (Fig. 2A). As mentioned above, expression of wild-type Rb did not alter the cell profile compared with controls. Rb{Delta}780 and Rb{Delta}2S (containing mutations at Ser807 and Ser811) had no significant effect on cell cycle profile at 24 h, but by 48 h, these mutants caused an increase in cells with 4N DNA content, consistent with a delay in G2/M phase. The effect was stronger with the double site mutant Rb{Delta}2S than the single site mutant Rb{Delta}780, suggesting a possible cumulative effect. Rb{Delta}9 and Rb{Delta}10, which contain mutations of the C-terminal phosphorylation sites as well as additional sites within the large pocket (e.g. Ser608 and Ser12 in the spacer region) (8, 24), both induced an accumulation of cells with 2N DNA content through 48 h, consistent with G1 arrest. Cells expressing these constructs later began to enter S phase, which was previously shown to be caused by persistent CDK2 activity in the absence of p16ink4a (24). Rb mutants were further analyzed for their ability to inhibit S phase entry by measuring BrdUrd incorporation at 24, 48, and 72 h (Fig. 2B). As mentioned above, wild-type Rb did not inhibit BrdUrd incorporation compared with an inactive Rb fragment. In contrast, Rb{Delta}9 strongly inhibited BrdUrd incorporation with activity comparable with p16ink4a. Single and double phosphorylation site mutants, including Rb{Delta}780, Rb{Delta}2S, and Rb{Delta}2T (containing mutations at Thr821 and Thr826), demonstrated inhibitory activity intermediate between Rb and Rb{Delta}9. Rb{Delta}4, which contains the C-terminal mutations present in both Rb{Delta}2S and Rb{Delta}2T, was more efficient than the single and double site mutants, but it was not as efficient as Rb{Delta}9. Taken together, these results suggest that the C-terminal phosphorylation sites cooperate with other sites in the large pocket (e.g. Ser608 and Ser612) to regulate G1 arrest in a cumulative manner. Residual G2/M-inhibitory activity may persist when Rb is in a more phosphorylated state.

CDK Sites in the Large Pocket Cooperate to Regulate Tumor Suppression—Because hypophosphorylated Rb can induce flat cells as a manifestation of a senescent or differentiated phenotype (30, 31), we examined the ability of our Rb mutants to induce flat cells. Rb{Delta}9, Rb{Delta}10, and p16ink4a efficiently induced flat cells that stained positively for senescence-associated {beta}-galactosidase (Fig. 2D). In contrast, Rb{Delta}4 and all of the single and double site mutants failed to induce flat cells. As another measure of tumor suppressor activity, the Rb phosphorylation site mutants were then tested in colony formation assays. Rb{Delta}9, Rb{Delta}10, and p16ink4a demonstrated strong inhibitory activity. The C-terminal mutants Rb{Delta}780, Rb{Delta}2S, and Rb{Delta}2T demonstrated inhibitory activity intermediate between wild-type Rb and p16ink4a (Fig. 2C). Interestingly, Rb{Delta}4 inhibited colonies as effectively as Rb{Delta}9, Rb{Delta}10, and p16ink4a. Therefore, senescence appeared to be regulated by phosphorylation sites in the C terminus as well as the spacer region, whereas inhibition of colony formation, possibly a more general measure of tumor suppression, could be regulated by as few as four sites in the C terminus. This colony-inhibitory activity may be due, at least in part, to the residual G2/M-inhibitory activity that Rb retains as it becomes more phosphorylated (see above).

CDK Sites in the Large Pocket Cooperate to Regulate the A-B Interaction—The above data are most consistent with a model in which phosphorylation sites on Rb regulate its cell cycle and senescence functions in a cumulative manner. What might be the molecular basis for this cumulative effect? We previously showed that phosphorylation of the C terminus induced an intramolecular interaction with the A-B pocket (5), possibly through interactions between the negatively charged C terminus and a positively charged lysine patch in domain B (32). This interaction led to disruption of the domain A-domain B complex and consequently to inactivation of Rb. Therefore, phosphorylating more CDK sites and thereby creating a more negatively charged C terminus may generate a stronger intramolecular interaction that more efficiently disrupts the A-B complex. To examine whether the C-terminal phosphorylation sites cumulatively regulate the A-B interaction, we expressed the large pocket as separate proteins, domain A (amino acids 379–602) and domain B linked to the C terminus (amino acids 612–928), in C33A cells, which do not efficiently phosphorylate Rb (Fig. 3A). The domain A fragment was tagged with LexA, the BC fragment was tagged with Gal4, and the complex was detected by immunoprecipitation with an anti-Gal4 antibody. Coexpression of cyclin E to activate endogenous CDK2 disrupted the complex. Mutation of four C-terminal phosphorylation sites in the BC fragment (corresponding to the sites mutated in Rb{Delta}4) did not inhibit the A-B interaction. However, when cyclin E was co-expressed, disruption of the A-B complex was reduced in comparison with the wild-type construct. Further, mutation of all seven phosphorylation sites in the C terminus further minimized disruption of the A-B complex when cyclin E was co-expressed. These results are consistent with a model in which phosphorylation sites in the C terminus cooperate in a cumulative manner to regulate the A-B pocket.



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FIG. 3.
Phosphorylation of C-terminal sites permits phosphorylation of Ser567 and disruption of the A-B pocket. A, immunoprecipitation experiments were performed to determine how phosphorylating the C-terminal CDK sites affects the interaction between Rb domains A and B. The indicated Gal4- and LexA-tagged proteins were expressed in C33A cells (which do not hyperphosphorylate ectopic Rb). L-A, indicates domain A (amino acids 379–602) tagged with LexA. G-BC, domain B and the C terminus (amino acids 612–928) tagged with Gal4. G-BC{Delta}4 and G-BC{Delta}7, the G-BC construct with mutation of 4 and 7 phosphorylation sites, respectively, in the C terminus. Cyclin E (cycE) was co-expressed as indicated. Lysates were immunoprecipitated with an anti-LexA antibody and analyzed by Western blot with an anti-Gal4 antibody (top panel). 10% of each lysate was directly analyzed by Western blot with the anti-Gal4 antibody (middle panel) and the anti-LexA antibody (bottom panel). B, in vitro phosphorylation assays were performed using the indicated GST-tagged proteins incubated with cyclin E-Cdk2 (purified from baculovirus) in the presence of [32P]ATP and analyzed at the indicated time points. Proteins were separated by SDS gel electrophoresis, and 32P intensities of individual Rb fragments were quantified using densitometric analysis of autoradiographs. AB, the A-B pocket (amino acids 379–792, containing phosphorylation sites at Ser567, Ser602, Ser608, Ser780, and Ser788). AB{Delta}4, the A-B pocket with Ser-to-Ala substitutions at 602, 608, 780, and 788, leaving Ser567 as the only available phosphorylation site. C, the C terminus (amino acids 792–928, containing phosphorylation sites at Ser795, Ser807, Ser811, Thr821, and Thr826). C, immunoprecipitation experiments were performed to examine how mutating Ser567 or Pro568 affects the domain A-domain B interaction. The indicated Gal4- and LexA-tagged proteins were expressed in C33A cells. Lysates were immunoprecipitated with an anti-Gal4 antibody and analyzed by Western blot with an anti-LexA antibody (top panel). 10% of each lysate was directly analyzed by Western blot with the anti-LexA antibody (middle panel) and the anti-Gal4 antibody (bottom panel). D, to determine how mutating Ser567 or Pro568 affected phosphorylation of Ser567, GST-tagged proteins containing domain A or the mutant A{Delta}567 or A{Delta}568 were expressed in BL21 cells. Proteins were purified using glutathione beads, phosphorylated in vitro using recombinant cyclin E-CDK2 as above, and analyzed by Western blot using an anti-Rb antibody that recognizes phospho-Ser567.

 

CDK Sites in the Large Pocket Are Phosphorylated in U2OS Cells—Since phosphorylation sites in the C terminus appear to be critical for regulating Rb activity, and since ectopic Rb is inactivated in U2OS cells, we expected the C-terminal sites to be phosphorylated in ectopic Rb. To examine the phosphorylation status of individual C-terminal sites, we expressed the large pocket fragment in U2OS cells and performed Western blot analysis using a pan-Rb antibody ({Delta}Rb) or phosphospecific antibodies that detect Rb phosphorylated at Ser780, Ser807, and Ser811. The pan-Rb antibody demonstrated a smear of bands representing the multiple phosphorylated forms of Rb. Coexpression of cyclin E to activate CDK2 reduced the smear to a single, slower migrating band representing the hyperphosphorylated form of Rb (Fig. 1A), suggesting that not all phosphorylation sites are maximally phosphorylated in U2OS cells without coexpression of cyclin E. Both {Delta}Rb-phospho-Ser780 and {Delta}Rb-phospho-Ser807/Ser811 detected bands corresponding largely to the slower migrating form of Rb, and co-expression of cyclin E did not increase the intensity of these bands, suggesting that these C-terminal sites are maximally phosphorylated in U2OS cells. We previously found that phosphorylation of Ser567, a CDK site in domain A, may regulate binding to E2F and was dependent on prior phosphorylation of the C terminus (5). To test whether Ser567 may be phosphorylated in U2OS cells, we generated an antibody directed against Rb phosphorylated at Ser567 ({Delta}Rb-phospho-Ser567). Using this antibody, we initially found no evidence that Ser567 was phosphorylated in these cells (see below).

Ser567 Is Phosphorylated Less Efficiently than the C-terminal CDK Sites—Ser567 is buried within the interface between domains A and B, where it is normally inaccessible to cyclin-CDK complexes. We previously showed that phosphorylation of the C terminus in the presence of high CDK activity triggers an intramolecular interaction that allows cyclin E-CDK2 access to phosphorylate Ser567, which disrupts the A-B complex and inhibits binding to E2F (5). In support of this model, the crystallographic solution of the Rb-E2F complex showed that Ser567 makes a key contact with the E2F binding site and that phosphorylation of this site would disrupt the integrity of the E2F binding site (33). Since we initially found no evidence that Ser567 was phosphorylated in U2OS cells, we wondered whether phosphorylation of this site was less efficient than phosphorylation of the C-terminal sites. To examine the rate of phosphorylation of the C-terminal sites and Ser567, we performed in vitro phosphorylation experiments in which the A-B pocket (amino acids 379–792) and the C terminus were incubated as separate recombinant proteins in the presence of cyclin E-CDK2 (Fig. 3B). The AB and C fragments both contained five CDK phosphorylation sites, allowing an estimate of the number of sites phosphorylated at various time points. When incubated separately, the C-terminal fragment was phosphorylated more rapidly than the A-B fragment. By 60 min, all five sites on the C-terminal fragment were phosphorylated, whereas only about four sites on the A-B fragment were phosphorylated. However, when the A-B fragment was phosphorylated in the presence of the C-terminal fragment, it was phosphorylated more rapidly, and all five sites were phosphorylated. To directly study Ser567, we mutated the other four phosphorylation sites in the A-B fragment (AB{Delta}4). As expected, the maximal intensity of phosphorylation of AB{Delta}4 was equivalent to approximately one phosphorylation site. The rate of phosphorylation of AB{Delta}4 was much slower than the C-terminal or AB fragments, but the rate of phosphorylation of Ser567 was greatly increased in the presence of the C-terminal fragment. These findings suggest that phosphorylation of Ser567 is inefficient, which could explain why Ser567 is minimally phosphorylated in U2OS cells, but this site can be phosphorylated in the presence of high CDK2 activity and prior phosphorylation of the C terminus.

Ser567 Regulates the Rb-E2F Interaction by Stabilizing the A-B Pocket—To determine how alteration of Ser567 may affect the interaction between domains A and B, we generated a domain A fragment in which Ser567 was converted to alanine. In immunoprecipitation assays, mutation of Ser567 prevented binding between domains A and B (Fig. 3C). This finding may explain why naturally occurring point mutations at Ser567 inactivate Rb in retinoblastoma patients (34, 35), and it supports the idea that altering Ser567 by phosphorylation may also disrupt the A-B pocket (33). To study the effect of blocking phosphorylation of Ser567 without inactivating Rb, we previously created a mutant (Rb{Delta}568) in which the adjacent Pro568, which is part of the CDK recognition motif, was converted to alanine (5). The P568A substitution reduced but did not eliminate interaction between domains A and B (Fig. 3C), and this reduced interaction did not inhibit transcriptional repressor activity or E2F binding (5). As expected, Rb{Delta}568 inhibited phosphorylation of Ser567 (Fig. 3D). Rb{Delta}568 had no inhibitory activity in BrdUrd incorporation assays or flow cytometry compared with wild-type Rb (Fig. 2, A and B), and it did not induce flat cells (Fig. 2D). These findings suggest that Ser567 does not play a role in regulation of tumor suppression functions of Rb, such as cell cycle inhibition and senescence. However, in colony formation assays in U2OS cells, Rb{Delta}568 demonstrated some inhibitory activity (Fig. 2C). Since Ser567 regulates interaction of Rb with E2F (5), we suspected that the activity observed with Rb{Delta}568 may stem from its resistance to release of E2F. Therefore, we tested Rb{Delta}568 in Rat2/E2F1-DP1 cells, which have a rapid growth rate, high CDK2 activity, and increased capacity to hyperphosphorylate Rb as a result of E2F1 and DP1 overexpression (36). In colony formation assays, wild-type Rb had no inhibitory activity in these cells, whereas Rb{Delta}568 strongly inhibited colony formation comparable with Rb{Delta}9 (Fig. 4A).



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FIG. 4.
Phosphorylation of Ser567 promotes apoptosis in Rat2/E2F1-DP1 cells with high CDK2 activity. A, colony formation assays were performed in Rat2/E2F1-DP1 cells expressing the indicated Rb constructs. B, cell viability was determined by methanethiosulfonate assays in Rat2/E2F1-DP1 cells expressing the indicated Rb constructs to determine their ability to block apoptosis. E2F1 and DP1 were induced 24 h after transfection of the indicated expression vectors by removing tetracycline from the media. Control vector expressed an inactive fragment of Rb containing domain A. C, flow cytometry was performed in Rat2/E2F1-DP1 cells expressing the indicated Rb constructs. To enhance apoptosis in the indicated cells, E2F1 and DP1 were induced by removal of tetracycline, and serum was reduced to 0.5%. After 48 h, cells were stained with propidium iodide and analyzed by flow cytometry for DNA content. Apoptosis was inferred by an increase in the fraction of cells with <2N DNA and a decrease in cells with 2N DNA.

 

Phosphorylation of Ser567 Renders Cells Susceptible to Apoptosis—Rb is thought to block apoptosis, at least in part, by binding and inhibiting free E2F, which can trigger both p53-dependent and -independent apoptosis (17, 37). Since Ser567 can regulate binding of Rb to E2F, we wondered whether this site may regulate the antiapoptotic activity of Rb. To address this question, we turned again to the Rat2/E2F1-DP1 cells, which have a high rate of apoptosis resulting from high levels of free E2F1. In viability assays, Rb{Delta}568 suppressed apoptosis more efficiently than Rb or even Rb{Delta}9 (Fig. 4B). Similarly, flow cytometry was performed to assess apoptotic cells with <2N DNA content. In cells expressing wild-type Rb or Rb{Delta}9, induction of E2F1/DP1 resulted in ~50% increase in cells with <2N DNA and ~50% decrease in cells with 2N DNA content, consistent with increased apoptosis (Fig. 4C). In contrast, in cells expressing Rb{Delta}568, there was virtually no change in the fraction of 2N or <2N cells following induction of E2F1/DP1. Thus, phosphorylation of Ser567 may render cells susceptible to apoptosis. In contrast, the nine CDK sites that are mutated in Rb{Delta}9 (including all seven sites in the C terminus) efficiently regulate cell cycle and senescence functions of Rb, but they do not regulate apoptosis as efficiently as the single CDK site at Ser567.

Phosphorylation of Ser567 Is Associated with Degradation of Rb—These findings suggest that the C-terminal CDK sites are efficiently phosphorylated and are critical for regulation of cell cycle and senescence functions of Rb, whereas Ser567 is inefficiently phosphorylated and appears to regulate the antiapoptotic activity of Rb. Thus, Ser567 may only be phosphorylated in vivo under extreme proliferative conditions where CDK activity is abnormally high. As stated above, we initially found no evidence that Ser567 was phosphorylated in U2OS cells by Western blot. Specifically, there was no detectable band where the intact large pocket protein should migrate (around 97 kDa). However, upon closer inspection, we noticed that the {alpha}-Rb-phospho-Ser567 antibody detected several discrete, small weak bands ranging in size from about 55 to 65 kDa (Fig. 5A). These bands were more intense when cyclins D and E were co-expressed. In contrast, none of the bands were detected in cells expressing Rb{Delta}567, suggesting that the antibody recognized specific Rb fragments that were only present when Ser567 was phosphorylated. These bands are reminiscent of Rb degradation products that associated with apoptosis (38) and suggest that Rb that is phosphorylated on Ser567 is mostly in a degraded form. The A-B pocket is normally resistant to proteases (39), but it may become susceptible to proteolytic cleavage after it is disrupted by phosphorylation of Ser567. This protein degradation could explain why phosphorylation of Ser567 was not identified in previous phosphopeptide mapping experiments (6).



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FIG. 5.
Phosphorylation of Ser567 leads to degradation and exclusion of Rb from the nucleus. A, Western blot analysis of ectopic Rb or Rb{Delta}567 expressed in U2OS cells with or without co-expression of cyclins D and E. The blot was analyzed with an anti-Rb antibody that recognizes phosphorylation at Ser567. The 97-kDa mark indicates the expected location of the Gal4-Rb fusion protein. Faint bands were detected at about 55 and 65 kDa (arrows). B, phosphospecific Rb antibodies recognizing phospho-Ser567, -Ser780, and -Ser807/Ser811 were used as indicated to analyze the cellular localization of phospho-Rb by immunofluorescence microscopy in U2OS cells or mouse embryo fibroblasts lacking Rb, p107, and p130 (TKO MEFs). 4',6-Diamidino-2-phenylindole staining (DAPI; blue) indicates location of nuclei. Fluorescein isothiocyanate staining (FITC; green) indicates phospho-Rb.

 

Degraded or inactive Rb has been shown to localize poorly to the nucleus (34). To examine whether Rb phosphorylated on Ser567 may have a different subcellular localization than active forms of Rb, we performed immunofluorescence experiments using {Delta}Rb-phospho-Ser780, {Delta}Rb-phospho-Ser807/Ser811,or {Delta}Rb-phospho-Ser567 (Fig. 5B). Rb phosphorylated at Ser780 and Ser807/Ser811 was localized almost entirely to the nucleus. In contrast, Rb phosphorylated at Ser567 was located mostly in the cytoplasm. Preincubation of {Delta}Rb-phospho-Ser567 with its peptide epitope virtually extinguished the signal, suggesting that the immunoreactivity was specific. Immunoreactivity with the anti-Ser567 antibody was weak, suggesting that a small fraction of Rb is normally phosphorylated at this residue. However, ectopic expression of cyclins D and E substantially increased the cytoplasmic staining, consistent with the idea that high CDK activity facilitates phosphorylation of Ser567 and degradation of Rb.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CDK Phosphorylation Sites Cumulatively Regulate Rb Tumor Suppressor Activity—How the 16 potential CDK phosphorylation sites regulate Rb activity is an area of active research and controversy. Our findings suggest that the phosphorylation sites in the C terminus are critical for regulating the cell cycle, senescence, and overall tumor suppressor activity of Rb. Our data are most consistent with a model in which these sites cooperate in a cumulative manner to regulate Rb activity. Interestingly, Rb mutants in which one or two phosphorylation sites in the C terminus were mutated (Rb{Delta}780 and Rb{Delta}2S) failed to arrest cells in G1 or to induce senescence, yet they could suppress colony formation. As a possible explanation for this inhibitory activity, both of these mutants inhibited transit of cells through G2/M. This finding is consistent with previous studies, which have shown that Rb can arrest cells in S phase and G2/M (40, 41, 42, 43, 44, 45, 46). This G2/M inhibitory activity was stronger in the double site mutant (Rb{Delta}2S) than the single site mutant (Rb{Delta}780), suggesting a cumulative effect. Thus, Rb may retain inhibitory activity in G2/M when it is in a more phosphorylated state than is required for inhibitory activity in G1, which would be consistent with a model in which Rb is progressively associated with different sets of chromatin remodeling enzymes that regulate different sets of genes as Rb is progressively phosphorylated through the cell cycle (5, 47).

Phosphorylation of Ser567 Requires Prior Phosphorylation of the C Terminus—Whereas the C-terminal sites were rapidly phosphorylated in vitro and in vivo, Ser567 was very inefficiently phosphorylated. The phosphorylated C terminus and high CDK2 activity significantly enhanced phosphorylation of Ser567. Ser567 is normally in an inaccessible location buried within the interface of domains A and B (32). However, the phosphorylated C terminus interacts with a positively charged patch of lysine residues in domain B, causing a conformational change in the pocket that exposes Ser567 (5). The C terminus also has docking sites for cyclin E-CDK2, and the aforementioned conformational change places this kinase complex into the proximity of Ser567 to further enhance phosphorylation of this site. These findings suggest a multistep process for phosphorylation of Rb in which the C-terminal sites are phosphorylated efficiently every cell cycle, whereas Ser567 is phosphorylated inefficiently and only under extreme conditions of high CDK activity. The inaccessible location of Ser567 and requirement for prior phosphorylation of the C terminus to phosphorylate Ser567 would ensure the proper ordering of phosphorylation events and prevent Ser567 from being inappropriately phosphorylated during the normal cell cycle.

Ser567 May Regulate the Antiapoptotic Activity of Rb—We previously showed that phosphorylation of Ser567 disrupts the A-B pocket, leading to release of E2F. In support of this model, the crystallographic solution of the Rb-E2F2 complex showed that Ser567 interacts through a critical hydrogen bond with a residue within the E2F binding site, and phosphorylation of Ser567 would perturb this binding site and release E2F (33). As further evidence of the critical role that Ser567 may play in Rb function, the Ser567-Pro568 CDK recognition motif is highly conserved in vertebrate Rb and p130 (Fig. 6A), and Ser567 missense mutants from human retinoblastomas are lacking in tumor suppressor activity and protein binding (34, 35). Loss of Rb leads to apoptosis largely by liberation of free E2F (48), which can trigger apoptosis by p53-dependent and -independent mechanisms (17, 37). Taken together with the present results, we propose a model in which phosphorylation of Ser567 may regulate the antiapoptotic activity of Rb through modulation of E2F binding (Fig. 6B). This would suggest that at least a portion of the cellular pool of E2F may remain bound to Rb throughout the cell cycle to maintain free E2F levels below an apoptotic threshold. Further, free E2F may only accumulate to sufficient levels to trigger apoptosis when Rb function is lost, either by genetic deletion or as a result of high CDK2 activity sufficient to phosphorylate Ser567. In this model, one would expect to find Rb-E2F complexes in normal cells beyond the G1/S transition. Indeed, chromatin immunoprecipitation assays demonstrated that promoters of endogenous genes containing E2F sites had no free E2F bound to their promoters during S phase, but rather, most promoters were occupied by E2F bound to Rb or one of the other pocket proteins (49).



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FIG. 6.
Ser567 is a conserved CDK phosphorylation site that may regulate the antiapoptotic function of Rb. A, amino acid sequence alignment of the region spanning the CDK recognition motif Ser567-Pro568 in Rb and p130 in vertebrate species. The serine-proline CDK recognition motif is indicated in boldface type. B, model illustrating how distinct phosphorylation sites may regulate the cell cycle and apoptotic functions of Rb. Early in G1, Rb is minimally phosphorylated and represses transcription of genes involved in S phase entry at least in part by recruiting chromatin remodeling enzymes such as histone deacetylase (HDAC). Initial phosphorylation of Rb on sites regulating the cell cycle (e.g. C-terminal sites) prevents Rb from binding HDAC and allows cells to traverse the G1/S boundary and proceed through the cell cycle. Rb is dephosphorylated after mitosis in preparation for reentry into G1. Some E2F may remain bound to a partially phosphorylated Rb throughout the normal cell cycle. However, under hyperproliferative conditions in which CDK2 levels are abnormally high, Ser567 may become phosphorylated, leading to irreversible degradation of Rb and release of E2F. The high levels of free E2F may then trigger an apoptotic response by p53-dependent and -independent mechanisms.

 

Ser567 Phosphorylation May Serve as a Sentinel for Excessive Proliferation—Phosphorylation of Ser567 leads not only to disruption of the A-B pocket and release of E2F but also to degradation of Rb. Thus, phosphorylation of Ser567 is likely to be an irreversible event, whereas phosphorylation of the C-terminal sites is likely to occur every cell cycle with subsequent dephosphorylation before re-entry into G1. Thus, Ser567 may serve as a sentinel for extreme hyperproliferative conditions, and phosphorylation of this site may initiate an irreversible signal that leads to elimination of the cell. Consequently, Rb(+) cancer cells may undergo selective pressure to keep CDK2 activity below the threshold for phosphorylating Ser567. Using phosphospecific antibodies to examine 32 primary Rb(+) melanoma specimens, we found that Ser807 and Ser811 were frequently phosphorylated in tumor cells (50), whereas phosphorylation of Ser567 was detected rarely and was largely confined to the cytoplasm.2 These melanomas were clinically slow growing and had minimal spontaneous apoptosis by histopathologic examination. Thus, some tumor cells may exploit the dual role of Rb by phosphorylating sites that regulate tumor suppression but avoiding phosphorylation of Ser567 and consequent apoptotic stimulus, which could explain why Rb(+) tumors like melanoma tend to proliferate slowly and to be resistant to therapy-induced apoptosis. This mechanism potentially could be exploited therapeutically to sensitize cancer cells to radiation and chemotherapy.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants K08 EY00382 and R01 EY13169 (to J. W. H.). 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. Back

{ddagger} These authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Campus Box 8069, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8989; Fax: 314-747-2797; E-mail: harbour{at}vision.wustl.edu.

1 The abbreviations used are: CDK, cyclin-dependent kinase; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline. Back

2 D. Ma, P. Zhou, and J. W. Harbour, unpublished observations. Back



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