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Cell Growth & Differentiation Vol. 11, 395-408, July 2000
© 2000 American Association for Cancer Research


Articles

Transformation of Mortal Human Fibroblasts and Activation of a Growth Inhibitory Pathway by the Bovine Papillomavirus E5 Oncoprotein1

Lisa M. Petti2 and F. Andrew Ray

Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York 12208

Abstract

The 44-amino acid bovine papillomavirus E5 protein induces tumorigenic transformation of immortal rodent fibroblasts by binding to and activating the platelet-derived growth factor ß receptor (PDGFßR). Here E5 was expressed in mortal human diploid fibroblasts (HDFs), which lack the accumulated genetic changes that are present in immortal rodent cells. E5 induced focus formation and morphological transformation of HDFs without inducing anchorage independence or immortalization. Similar effects were observed with the v-sis and neu* oncogenes. E5-PDGFßR complexes were observed in the E5-expressing HDFs, as was constitutive PDGFßR activation, which was required for the transforming activity of E5. The E5 HDFs attained a higher saturation density than the control cells, expressing increased levels of hyperphosphorylated retinoblastoma protein at subconfluent densities. However, when these cells reached confluence, growth inhibition accompanied by dramatic down-regulation of the PDGFßR, and retinoblastoma protein was induced apparently by a factor secreted into the medium. This may represent a novel negative feedback mechanism controlling PDGFßR-induced proliferation and thereby protecting against complete transformation.

Introduction

The E5 protein of BPV3 rapidly induces tumorigenic transformation of certain immortalized rodent fibroblasts and is the primary transforming protein of this virus (1–4) . The hydrophobic 44-amino acid E5 protein forms a disulfide-linked homodimer with a subunit size of Mr 7000 and localizes primarily to intracytoplasmic membranes in BPV-transformed cells (5, 6) . An important cellular target for the BPV E5 protein in fibroblasts is the PDGFßR. In E5-transformed rodent fibroblasts, the E5 protein forms a stable complex with PDGFßR and induces constitutive activation of the PDGFßR (7, 8) , an event that is required for cell transformation (9, 10) . The E5 protein is thought to activate the PDGFßR by binding to the receptor as a homodimer and facilitating receptor dimerization, which then leads to receptor activation (11) .

Studies of E5-mediated transformation have been performed primarily in mouse C127 cells, a line of immortalized fibroblasts derived from a mouse mammary carcinoma (12) , and the effect of E5 expression alone in mortal and completely untransformed fibroblasts has not been investigated. Analysis of mortal cells has been limited to primary rodent or bovine fibroblasts expressing the entire BPV genome. For example, BPV induced morphological transformation of primary hamster embryo fibroblasts at a low frequency, with the transformed cells being anchorage independent and tumorigenic (13–15) . BPV infection of primary bovine dermal fibroblasts resulted in morphological transformation, with the transformed cells possessing activated PDGFßR associated with the E5 protein (16) . Transfection of primary mouse fibroblasts with BPV DNA resulted in immortalization after serial subcloning, but not in focus formation (17, 18) . Finally, transfection of rat embryo fibroblasts with BPV mutants resulted in immortalization that was dependent on the BPV E6 gene (19) . From these studies, it was not possible to determine the traits that were directly attributed to E5 expression because of the presence of the entire viral genome and/or the high spontaneous transformation rates of rodent cells (20) .

Here we assessed the effects of E5 expression in mortal human fibroblasts to determine the transforming characteristics that can be ascribed solely to this small but potent oncogene. Specifically, we used a retroviral vector containing the E5 gene to introduce E5 into HDFs. We chose human cells because rodent cells are susceptible to spontaneous immortalization and tumorigenic transformation and because young mortal HDFs lack a background of accumulated genetic changes that might modify the effects of E5. We found that the E5 gene can morphologically transform and induce focus formation of these fibroblasts, which are notoriously difficult to transform. In agreement with several earlier studies (21–24) , we also found that v-sis, the viral homologue of the PDGF B gene, can induce a similar transformed phenotype. Unlike these previous studies, however, here we show that the transformed phenotype of HDFs expressing E5 or v-sis is associated with and dependent on activation of the PDGFßR. We also found that neu* (the oncogenic form of neu which encodes the p185neu receptor tyrosine kinase, activated by a single point mutation resulting in a Val to Glu substitution at position 664 in transmembrane domain) could induce focus formation and morphological transformation of these cells. Although the E5-expressing HDFs lost their contact inhibition for growth and continued to proliferate beyond the saturation density of the normal cells, their growth was still impeded once they reached confluence. This growth inhibition at confluence was accompanied by down-regulation of the PDGF receptor, and both events appeared to be induced by a factor secreted into the medium. We propose that the E5-expressing HDFs activate a negative feedback mechanism to limit the proliferative effects of sustained PDGFßR activation.

Results

Phenotypic Effects of Stable E5 Expression in HDFs.
E5 was stably expressed in HSF4012 cells (NHDF4012; Clonetics, San Diego, CA), a human foreskin fibroblast cell strain with a normal karyotype4 that typically undergoes senescence after 55 PDs.5 E5 was introduced into these cells by infecting low-passage (approximately 15 PDs) HSF4012 cells with a high titer of an E5-expressing retrovirus containing a puromycin resistance marker. Cells stably expressing E5 were established after 1 week of selection in puromycin-containing medium and then verified for expression of the E5 protein by immunoblotting (Fig. 2ACitation ). Using the analogous retroviral construct without an insert and one containing the v-sis gene, control and v-sis-expressing HDFs, respectively, were established in parallel. The control HDFs were indistinguishable from normal HSF4012 cells (Fig. 1ACitation ). In contrast, the E5-expressing HDFs displayed an altered morphology similar to that of murine fibroblast cell lines transformed by this oncogene (25) , appearing more elongated, refractile, and dense than the control cells (compare Fig. 1BCitation with Fig. 1ACitation ). Virtually every cell that survived selection acquired the transformed morphology within just a few PDs (by 5–6 days after retroviral infection and just before confluence), suggesting that morphological transformation was a direct effect of E5 expression. Generally, the E5-expressing cells appeared the most refractile and elongated at higher cell densities, i.e., near and at confluence (Fig. 1ECitation ). The E5-expressing cells also were multilayered and growing on top of each other (Fig. 1BCitation ), whereas the control cells appeared as a flat monolayer (Fig. 1ACitation ). Moreover, the E5-expressing HDFs resembled HDFs stably expressing v-sis, as shown here (Fig. 1, C and F)Citation and in earlier studies (22, 24) . A similar morphology was observed previously for HDFs overexpressing c-sis (26) or HDFs treated with PDGF B (21) .6 Therefore, E5, like v-sis, can induce morphological transformation of HDFs.



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Fig. 2. The PDGFßR exists in a complex with the E5 protein and is constitutively activated in E5-expressing HDFs. Extracts of HDFs stably expressing E5, v-sis, neu*, or the empty retroviral vector (C) were prepared as described in "Materials and Methods." In A, approximately 1000 µg of protein were immunoprecipitated with an anti-E5 antiserum (E5 IP) and then subjected to anti-E5 immunoblotting (E5 blot). In B, approximately 400 or 40 µg of protein were immunoprecipitated with anti-E5 (E5 IP) or anti-PR (PR IP) antiserum, respectively, and then subjected to anti-PDGF receptor immunoblotting (PR blot). In C, subconfluent HDFs stably expressing E5, v-sis, or neu* were either left untreated (-) or treated (+) with AG1296 for 24 h and then lysed. Approximately 900 or 600 µg of extracted protein were immunoprecipitated with anti-PDGF receptor (PR IP) or anti-p185neu (Neu IP) antibodies, respectively. Ninety percent of each immunoprecipitation was subjected to immunoblotting with an antiphosphotyrosine antibody (PY blot). The remaining 10% of each immunoprecipitation was subjected to anti-PDGF receptor (PR blot) or anti-p185neu (Neu blot) immunoblotting. The arrows in A and B point to the E5 protein and the mature (m) and precursor (p) forms of the PDGF receptor (PR), respectively. Numbers to the right in C indicate the position of molecular weight markers in thousands.

 


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Fig. 1. HDFs stably expressing the BPV E5 and the v-sis oncogenes are morphologically transformed. Photomicrographs of normal HDFs harboring the retroviral vector alone (A) or HDFs stably expressing the BPV E5 (B and E) or v-sis (C and F) oncogenes are shown. E5-expressing HDFs treated with AG1296 before confluence are also shown (D). A-D, x47; E and F, x283.

 
Previous studies showed that in murine and bovine fibroblasts, the E5 protein could complex with and activate both mature and intracellular metabolic precursor forms of the PDGFßR (7, 9) . Therefore, we determined whether the PDGFßR was constitutively tyrosine phosphorylated and complexed with the E5 protein in HDFs. To detect E5-PDGFßR complexes in HDFs, coimmunoprecipitation analysis was performed by E5 immunoprecipitation followed by PDGF receptor immunoblotting. Both the slower migrating mature and faster migrating precursor forms of the PDGFßR were detected in E5 immunoprecipitates from cells expressing E5, whereas no coimmunoprecipitation of the receptor was detected in the control cells (Fig. 2BCitation ). This indicates that the E5 protein can form a stable complex with the PDGFßR in HDFs. Next, the tyrosine phosphorylation status of PDGFßRs in the E5-expressing HDFs was examined by antiphosphotyrosine immunoblotting of PDGF receptor immunoprecipitates from subconfluent cells. As expected, when compared with the control cells, tyrosine phosphorylation of both mature and precursor forms of the PDGFßR was dramatically increased in the E5-expressing HDFs (Fig. 2Citation C, top left panel), indicating that this receptor is constitutively activated in these cells. A similar increase in PDGF receptor tyrosine phosphorylation was observed in the v-sis-expressing HDFs. In both the E5- and v-sis expressing cells, the increase in tyrosine phosphorylation was not due to a proportionate increase in the total amount of receptor expressed (Fig. 2Citation C, bottom left panel). Thus, the E5 protein complexes with the PDGFßR and induces its constitutive activation in mortal human fibroblasts.

Focus Formation of HDFs by E5.
A focus-forming assay was performed by infecting low-passage (approximately 15 PDs) HSF4012 cells with a low concentration of retrovirus expressing E5 or v-sis. As a control, cells were also infected with the analogous retrovirus without an insert. After infection, cells were split and then maintained in the absence of biochemical selection and observed for the development of foci. Fig. 3Citation shows that no foci were induced after infection with the control retroviral vector (Fig. 3, Puro)Citation . In contrast, E5 readily induced the formation of many large, dense foci of piled up cells. The foci induced by E5 had a star-shaped morphology similar to that documented previously for foci induced by v-sis or overexpression of c-sis (the cellular gene encoding the PDGF B chain) in HDFs (21, 23, 24, 26) . Focus formation induced by E5 was typically observed by 14 days after infection, suggesting that it was a direct effect of E5 expression rather than a result of mutation during long-term culture. Corroborating the results of previous studies (21, 23, 24) , we showed that v-sis also could induce the formation of numerous star-like foci in HDFs (Fig. 3)Citation . In addition, the neu* oncogene also was assessed for focus-forming activity in HDFs. neu* encodes a mutated form of the epidermal growth factor receptor family member p185neu that is constitutively activated by a Val to Glu substitution at position 664 in the transmembrane domain (27, 28) . We found that retrovirus expressing the activated p185neu* receptor readily induced focus formation of HDFs, whereas the corresponding empty retroviral vector (LXSN) did not (Fig. 3)Citation . Thus, constitutive activation of a receptor tyrosine kinase other than the PDGF receptor can result in focus formation in HDFs.



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Fig. 3. The BPV E5, v-sis, and neu* oncogenes induce focus formation of HDFs. HSF4012 cells were infected with recombinant retroviral vectors expressing the BPV E5, v-sis, or neu* oncogenes. For controls, cells were infected with the retroviral vectors without an insert (Puro or LXSN). One day after infection, cells were split into new dishes, and 3 days later, the cells were either left untreated (-) or treated (+) with AG1246. Monolayers were maintained at confluence for 3 weeks thereafter. Crystal violet-stained cells are shown for visualization of foci.

 
Inhibition of Transformation by the Kinase Inhibitor AG1296.
We also examined the effect of treating the E5- and v-sis-expressing HDFs with AG1296, a kinase inhibitor reported to be specific for the PDGF receptor (29) . AG1296 treatment before confluence dramatically reduced tyrosine phosphorylation of the PDGF receptor in the E5- and v-sis-expressing cells, indicating that this agent inhibited the tyrosine kinase activity of the PDGF receptor as expected (Fig. 2Citation C, top panel). AG1296 treatment before confluence also reversed the morphology change of the E5- (Fig. 1DCitation ) and v-sis (data not shown)-expressing HDFs without preventing continued growth to confluence. Specifically, the AG1296-treated cells became flatter and less refractile, resembling the control cells. It is important to note that the effects of AG1296 in the E5-expressing cells were reversible; after its removal, tyrosine phosphorylation of the PDGF receptor was restored within an hour, and the characteristic transformed morphology of the cells was reestablished within 1–2 days (data not shown). This suggests that E5 expression was not lost in the AG1296-treated cells. AG1296 treatment also inhibited focus formation by E5 or v-sis (Fig. 3)Citation . This inhibition was not simply due to inefficient retroviral infection because cells treated with AG1296 were taken from the same infections that resulted in focus formation. Thus, these results indicate that constitutive activation of the PDGF receptor is required for E5- and v-sis-induced morphological transformation and focus formation of HDFs. To ensure that AG1296 inhibitor was specific for the PDGF receptor, we assessed whether it could inhibit morphological transformation and focus formation induced by constitutive activation of a different receptor tyrosine kinase, p185neu*. HDFs stably expressing the activated p185neu* were established by retroviral-mediated gene transfer as described in "Materials and Methods." These cells appeared to be morphologically transformed (data not shown) and expressed abundant amounts of tyrosine-phosphorylated p185neu* as assessed by p185neu immunoprecipitation followed by antiphosphotyrosine immunoblotting (Fig. 2Citation C, top panel). AG1296 treatment before confluence could not inhibit tyrosine phosphorylation of p185neu* (Fig. 2CCitation ), nor could it reverse morphological transformation by neu* (data not shown). AG1296 was also unable to inhibit focus formation induced by neu* (Fig. 3)Citation and therefore was not a general inhibitor of focus formation. Taken together, these data indicate that the AG1296 inhibitor is specific for the kinase activity of the PDGF receptor. A structurally similar inhibitor, AG1295, was shown to be specific for E5-activated PDGF receptors and not for activated p185neu* in mouse C127 cells (30) .

Growth Kinetics of E5-expressing HDFs.
HDFs stably expressing E5, v-sis, or the empty retroviral vector were seeded into multiple dishes at the same cell concentration and then counted at intervals thereafter. As illustrated by representative growth curves (Fig. 4Citation ) the E5- and v-sis-expressing HDFs consistently reached a 2–3-fold higher saturation density than the control cells. During the initial phase of exponential growth, the growth rate of the E5-and v-sis-expressing cells was not significantly different than that of the control cells. However, the E5- and v-sis-expressing cells continued to proliferate exponentially after growth of the control cells had slowed down. Notably, both control and transformed cells continued to grow, albeit slowly, for many days after exponential growth. Therefore, although the E5- and v-sis-expressing cells did not display an increased growth rate initially, the exponential growth phase of these cells was extended, allowing them to attain a higher saturation density than the control cells.



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Fig. 4. Stable expression of E5 and v-sis increases the saturation density of HDFs. HDFs expressing E5 ({blacksquare}), v-sis (•), or empty vector (control, *) were seeded at equal cell densities into multiple dishes and then counted 48 h later and every 24 h thereafter. For each time point, cells were counted in triplicate. Growth curves were generated after plotting the ln of the cell number versus the hours after plating. The mean value of the ln of the cell number with the SD is shown.

 
Assessment of E5-expressing HDFs for Other Transformed Traits.
The E5-expressing HDFs were also examined for other characteristics of transformed cells. First, these cells were assessed for their ability to grow under low serum conditions (Fig. 5Citation ). Control or E5-expressing HDFs were seeded into medium containing either 1% or 10% FBS and then counted at various times thereafter. Growth of both the control and E5-expressing cells in 1% FBS was severely restricted compared with growth in 10% FBS. This indicates that the E5-expressing cells, like the normal cells, required higher serum concentrations for optimal growth. However, even in 1% FBS, the E5-expressing HDFs were able to attain a higher final cell density than the control cells. Thus, although the E5 HDFs were serum dependent for growth, they still maintained a growth advantage under low serum conditions.



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Fig. 5. E5-expressing HDFs display limited growth in low serum. HDFs expressing E5 or empty vector were seeded at equal cell densities into media containing either 10% ({blacktriangleup}, control cells; {blacksquare}, E5 cells) or 1% (*, control cells; •, E5 cells) FBS and then counted at intervals thereafter. For the first four time points, the mean and SD of the ln of the cell number were calculated from triplicate plates. The fifth and sixth time points were derived by counting two plates and one plate, respectively. The SD for some points was too small for an error bar to be visualized.

 
The E5- and v-sis-expressing HDFs were also assessed for anchorage-independent growth. Equal numbers of normal, empty vector-, E5-, or v-sis-expressing HDFs were mixed with methylcellulose-containing media, plated, and examined 3 weeks later for the formation of colonies. As a positive control, the HT1080 human fibrosarcoma cell line was also tested. In three independent experiments, the E5- and v-sis-expressing cells, like the normal cells, formed either no or few colonies in this semisolid medium (data not shown). In contrast, a high percentage of the input HT1080 cells formed colonies in methylcellulose, as was expected (data not shown). Therefore, neither E5 nor v-sis was able to induce anchorage-independent growth of HDFs.

The E5-expressing cells were also tested for the ability to bypass senescence. We found that the E5-expressing cells, like the normal cells, senesced after serial passaging (data not shown), indicating that E5 was unable to directly induce immortalization of HDFs. Furthermore, E5 expression did not appear to induce premature senescence of HDFs, as was reported previously for ectopic expression of activated ras in these cells (31) .

Down-Regulation of the PDGF Receptor in E5-expressing HDFs.
For the PDGFßR and other receptor tyrosine kinases, ligand binding not only incites receptor activation but also triggers the clustering of receptors in coated pits, followed by endocytosis and lysosomal degradation of receptor-ligand complexes (32, 33) , a process referred to as receptor down-regulation. To determine the effect of E5 on PDGFßR down-regulation in HDFs, we examined PDGF receptor levels at different cell densities. An equal number of normal or E5-expressing cells was seeded into multiple dishes, and, at various times during expansion of the cultures, monolayers were lysed for immunoblot analysis or trypsinized in parallel for cell counts. To detect PDGF receptor levels in the cells, whole cell lysates were subjected to PDGF receptor immunoblotting. Fig. 6ACitation shows that at any given cell density, the level of PDGF receptors in the E5-expressing cells was significantly less than that in the control cells. This is likely due to receptor down-regulation in response to E5-induced receptor activation. Strikingly, when E5-expressing HDFs had just reached confluence (at approximately 2 x 106 cells/60-mm dish; Fig. 6BCitation ), PDGF receptor levels were nearly abolished (Fig. 6Citation A, upper panel, E5 Lane 3). A similar decrease in the amount of activated, tyrosine-phosphorylated receptor was observed when the cells reached confluence (data not shown). Thereafter, the growth rate of these cells was greatly reduced, and PDGF receptor levels remained diminished for 3 days (Fig. 6ACitation , upper panel, E5 Lanes 4 and 5). Dramatic receptor down-regulation also was observed when v-sis-expressing cells reached confluence (data not shown). In contrast, the levels of receptor in the control cells remained unchanged after the cells attained confluence and exhibited a reduced growth rate (Fig. 6Citation A, control Lane 3). Receptor down-regulation at confluence in the E5 HDFs was inhibited by AG1296 treatment (Fig. 9)Citation , suggesting that down-regulation required the kinase activity of the PDGF receptor. Therefore, constitutive activation of the PDGFßR by E5 in HDFs appears to lead to dramatic down-regulation of the receptor when the cells reach confluence. Others have demonstrated down-regulation of the PDGFßR in response to activation by E5 (34) , v-sis (35) , or v-fps (36) . However, none of these studies related PDGFßR down-regulation to cell density.



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Fig. 6. Rapid down-regulation of the PDGF receptor and Rb in E5-expressing cells occurs at confluence. An equal number of E5-expressing or control HDFs were seeded into dishes, and at various times thereafter, cells were trypsinized and counted or, in parallel, lysed directly in Laemmli sample buffer. A, whole cell extracts were electrophoresed on two 7% polyacrylamide-SDS gels. The upper portion of each gel was immunoblotted with either anti-PDGF receptor antibody or anti-Rb antibody. Anti-actin blotting of a lower portion of each gel indicated that an equal amount of protein was loaded in each lane. Arrows to the left indicate PDGF receptor (PR), Rb, or actin bands. Arrow on the right points to an apparent degradation product of Rb of approximately Mr 68,000–70,000. Numbers on the right indicate the position of molecular weight markers in thousands. B, counts of E5 ({blacksquare}) and control (•) cells were plotted as the ln of the cell number versus hours post seeding. The growth curve in B indicates the densities of the cultures examined by immunoblotting in A, with points 1–6 in B corresponding to Lanes 1–6 in A.

 


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Fig. 9. PDGF receptor down-regulation in the E5 HDFs requires serum factors as well as the kinase activity of the receptor. E5-expressing HDFs were plated in 60-mm dishes at 2.5 x 105 cells/dish. Cells were either left untreated or subjected to a media change or AG1296 treatment just before or at confluence (72 or 84 h after plating, respectively). The media replacement was performed with either serum-free medium or medium containing 10% FBS. AG1296 treatment was performed as described in "Materials and Methods" by adding the inhibitor (+) or DMSO (-) directly to the cells without replacing the media. Cell lysates were prepared at the times indicated after plating and subjected to SDS-PAGE followed by PDGF receptor or actin immunoblotting. Arrows on the left point to the mature (m) and precursor (p) forms of the PDGF receptor (PR) as well as to actin.

 
In Fig. 6ACitation (top panel), the conversion of distinct PDGF receptor bands to faint, smeary bands suggested that this receptor was being rapidly degraded. This degradation was specific for the PDGF receptor because the levels of actin remained constant at different cell densities, as assessed by an anti-actin immunoblot of a lower portion of the same gel. A pulse-chase experiment confirmed that the half-life of the PDGF receptor was markedly reduced in the E5-expressing HDFs when they reached confluence (Fig. 7Citation ). Twelve h before confluence (72 h after plating), a significant amount of receptor was still present after a 1-h chase, and complete degradation occurred after a 4.5-h chase. It is important to note that at this time, the PDGF receptor form with the longest half-life is one with an electrophoretic mobility between the mature (Fig. 7, m)Citation and precursor (Fig. 7, p)Citation forms. This may represent a more stable form of the receptor that is either a specific degradation product of the mature form or a different, incompletely processed precursor. In contrast to the subconfluent cells, in confluent cells (84 h after plating) metabolically labeled PDGF receptor is almost completely degraded after a 1-h chase. Thus, increased degradation accounts for down-regulation of the PDGF receptor in confluent E5 HDFs.



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Fig. 7. Degradation of the PDGF receptor in the E5 HDFs is increased at confluence. Control and E5-expressing HDFs were metabolically labeled with a mixture of [35S]methionine (71%) and [35S]cysteine (29%) 12 h before confluence (72 h after plating) or at confluence (84 h after plating). After 45 min, the label was removed, and the cells were either lysed immediately (0 h of chase) or incubated with media containing an excess of cold methionine and cysteine. After 1, 2.5, or 4.5 h of this cold chase, cells were lysed. PDGF receptor was immunoprecipitated from cell lysates and then subjected to SDS-PAGE followed by autoradiography. Arrows on the left point to the mature (m) and precursor (p) forms of the PDGF receptor (PR). The total amount of PDGF receptor present in the E5 HDFs at 72 and 84 h after plating is shown on the left of the PDGF receptor immunoblot in Fig. 9Citation .

 
We also examined the levels of Rb protein in the E5-expressing and control HDFs at different cell densities. Fig. 6ACitation (bottom panel) shows that in subconfluent control and E5 HDFs (Lanes 1 and 2) both hyper- and hypophosphorylated forms of Rb (corresponding to the upper and lower bands, respectively) were present. This is to be expected because the hyperphosphorylated form should be prevalent in dividing cells, allowing for cell cycle progression by releasing the transcription factor E2F. The amount of Rb in subconfluent E5-expressing cells appeared to be significantly increased compared with that in the control cells, particularly when the cells were approximately 50% confluent (Lanes 1). This was due primarily to an increase in hyperphosphorylated Rb (indicated by a lighter exposure of the Rb blot in Fig. 6ACitation ) and could therefore account for the extended exponential growth of the E5-expressing HDFs compared with the control cells. When both the control and E5 HDFs reached confluence (Lanes 3), a significant reduction in hyperphosphorylated Rb was observed. In addition, when the E5-expressing HDFs reached confluence, a faster migrating Rb form of approximately Mr 68,000–70,000 appeared that was not evident in the control cells. The presence of this smaller Rb form may indicate specific cleavage of Rb, implying that both Rb and the PDGF receptor are degraded in the E5 HDFs when confluence is reached.

We next determined whether the state of confluence was the direct cause of PDGF receptor down-regulation in the E5-expressing cells. For example, it is possible that the increased number of cell-cell contacts in a confluent culture might trigger a signal for receptor degradation. E5-expressing HDFs were plated at a high density (2.7 x 106 cells/dish, which is at or slightly higher than confluence) or at a low density (2 x 105 cells/dish). At various times after plating, cells were lysed and examined for PDGF receptor levels by immunoblotting. Fig. 8Citation shows that the PDGF receptor was not immediately degraded after plating at saturation density. Instead, receptor degradation began some time between 1 and 3 days after plating. Thus, in addition to confluence, there appears to be a time requirement for PDGF receptor down-regulation in the E5-expressing cells. In another experiment, we measured whether the E5 HDFs could continue to grow after being plated at confluence (approximately 2.3 x 106 cells/dish). Indeed, these cells continued to grow, although not at the exponential rate, reaching a final density of approximately 3 x 106 cells/plate 2 days after plating (data not shown). This corresponded to the time that a more transformed morphology and degradation of the PDGF receptor became evident (data not shown). Continued growth after plating at confluence may be due to the ability of these cells to grow on top of each other.



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Fig. 8. Forced confluence does not directly induce PDGF receptor down-regulation in the E5 HDFs. A culture of subconfluent E5-expressing HDFs was trypsinized and plated at subconfluent (2 x 105 cells/dish) or confluent (2.7 x 106 cells/dish) densities in 60-mm dishes. Cells were lysed at various times thereafter, and lysates were subjected to SDS-PAGE followed by PDGF receptor and actin immunoblotting of the same gel. PDGF receptor and actin levels were also examined in cells immediately before (Lane b) and after (Lane a) trypsinization. Arrows to the left indicate the position of the mature (m) and precursor (p) forms of the PDGF receptor (PR).

 
We also assessed the effect of fresh media replacement on receptor down-regulation. Briefly, the media of E5 HDFs that were nearing or had just reached confluence were replaced with either serum-free media or serum-containing media, and at various times thereafter, the cells were lysed and examined for PDGF receptor levels by immunoblotting. Fig. 9Citation shows that serum deprivation clearly inhibited PDGF receptor down-regulation, even when serum was removed just as the cells were starting to degrade their receptors (i.e., at 84 h). If the media on the cells were replaced with serum-containing media, receptor down-regulation still occurred, although the response was delayed by 1 day and reduced. These results indicate that serum factors are required for receptor down-regulation and argue against the possibility that receptor down-regulation is caused by a depletion of factors in the medium. If this were the case, down-regulation should not occur after replacement with fresh serum-containing medium. A more likely possibility is that the cells secreted a factor that induced receptor down-regulation and that media replacement removed such a factor. Media replacement with serum-containing medium would still remove such a factor, but the presence of serum might allow cells to secrete more of this factor and eventually induce receptor down-regulation.

To test the hypothesis that a secreted factor induces PDGF receptor degradation, we determined whether conditioned media from confluent E5 HDFs that were degrading their PDGF receptors could induce receptor down-regulation in subconfluent E5 cells that had not yet begun to degrade their receptors (Fig. 10ACitation ). Briefly, the media on subconfluent E5 cells was replaced with media from E5 cells that had just reached confluence and were down-regulating their PDGF receptors (S lane in Fig. 10ACitation ). Both treated and untreated cells were lysed 2.5 and 20 h later, and PDGF receptor levels were examined by immunoblotting. Strikingly, a 20-h treatment with conditioned media from confluent cells dramatically down-regulated the PDGF receptors in subconfluent cells (Fig. 10ACitation ). Even a short treatment of 2.5 h induced some receptor degradation. Media taken from confluent control cells not expressing E5 could not induce receptor down-regulation (Fig. 10BCitation ). Receptor down-regulation was not a result of the subconfluent cells becoming confluent and down-regulating their receptors independent of the treatment because receptor degradation was not evident in the untreated cells. Moreover, the fact that subconfluent cells could degrade their PDGF receptors in response to the conditioned media provides further evidence that the confluent state is not a direct cause of receptor degradation, although it may be required for secretion of a factor. Interestingly, a 20-h treatment with this conditioned medium also induced down-regulation of hyperphosphorylated Rb (Fig. 10, A and B)Citation . Thus, down-regulation of hyperphosphorylated Rb and degradation of the PDGF receptor are induced by the same stimulus, presumably a factor secreted into the medium at confluence.



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Fig. 10. Conditioned medium from confluent E5 HDFs induces down-regulation of the PDGF receptor and Rb in subconfluent cells. Subconfluent E5 HDFs in 60-mm dishes were either left untreated (-) or had their media replaced (+) with conditioned media from E5 HDFs that had just reached confluence and were degrading their receptors (Lane S in A). Cells were lysed at the time of treatment (time 0, untreated) or at the indicated times (h) after treatment. If unspecified, cells were lysed 20–24 h after treatment. In B, E5 HDFs were treated with conditioned media from confluent E5 HDFs (+E5) or confluent control HDFs (+C). In C, subconfluent E5 or control HDFs were treated with medium from E5 HDFs. In D, one dish of E5 HDFs was treated with conditioned E5 HDF medium that had been boiled for 5 minutes (Lane +b). In E, E5 HDFs were treated with whole E5 medium (+) or with medium that was fractionated by centrifugation through a filter device with a molecular weight cutoff of 3000. The retentate or concentrated unfiltered fraction containing high molecular weight solutes was added directly into the existing medium on the cells. The filtrate fraction containing the low molecular weight solutes was added to the cells by media replacement as described in A-D. Cell lysates were subjected to SDS-PAGE followed by PDGF receptor (PR), Rb, or actin immunoblotting as indicated. Arrows on the left point to the mature (m) and precursor (p) forms of the PDGF receptor (PR), hypophosphorylated Rb (pp), hyperphosphorylated Rb (ppp) and actin.

 
Treating control fibroblasts with conditioned medium from confluent E5 cells resulted in down-regulation of the PDGF receptor that was delayed with respect to the E5-expressing HDFs (Fig. 10CCitation ). After 20 h of treatment, there was minimal receptor down-regulation, but by 43 h, there was significant receptor down-regulation in these cells. Conditioned medium did not induce receptor activation in the control cells (data not shown). By 43 h, hyperphosphorylated forms of Rb were also down-regulated in the control cells (data not shown).

We next began to characterize the factor in the conditioned medium of confluent E5 HDFs. First, boiling the conditioned media before treatment did not inhibit the ability of the media to induce down-regulation of the PDGF receptor in the E5 HDFs (Fig. 10DCitation ), suggesting that the down-regulating activity in the medium was not enzymatic. To estimate the size of the factor, we treated subconfluent E5 HDFs with medium that was fractionated through a centrifugal filter device with a molecular weight cutoff of 3000. The majority of the down-regulating activity in the medium fractionated to the filtrate containing low molecular weight solutes (Mr less than 3000) and was depleted from the retentate containing the high molecular weight solutes (Fig. 10ECitation ). Taken together, these results suggest that when E5 HDFs reach confluence, they secrete a small, heat-stable soluble factor that induces down-regulation of the PDGF receptor and hyperphosphorylated Rb.

We also determined the effect of this conditioned medium on DNA synthesis and cell proliferation. Subconfluent E5 or control HDFs were either treated with conditioned medium from confluent E5 HDFs or left untreated. At various times after treatment, cells were examined for incorporation of [3H]thymidine and counted in parallel. Fig. 11Citation shows that by 43 h of treatment, DNA synthesis in the treated E5 HDFs was decreased 7-fold compared with the untreated cells. In addition, the treated E5 HDFs could only reach a peak density that was half that of the untreated cells, suggesting that cell growth also was inhibited. By 65 h after treatment, the E5 HDFs showed a 3-fold reduction in cell number compared with the untreated cells. Thus, conditioned medium from confluent E5 HDFs suppressed DNA synthesis and cell proliferation in subconfluent E5 HDFs. It is important to note that this effect occurred after the time (20 h) when down-regulation of the PDGF receptor and Rb was typically observed. We expected to see a decrease in DNA synthesis in the treated control cells 1 day after receptor down-regulation typically occurred (e.g., as shown in Fig. 10CCitation ). In this particular experiment, however, these cells probably initiated the normal mechanism of density-dependent growth arrest before factor-dependent growth arrest could occur.



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Fig. 11. Conditioned medium from confluent E5 HDFs suppresses DNA synthesis and cell proliferation. Subconfluent E5 or control HDFs in 24-well plates were either left untreated ({square}) or treated () with conditioned medium from E5 HDFs that had just reached confluence. At the time of treatment and various times thereafter, the cells were assayed for DNA synthesis by measuring [3H]thymidine incorporation as described in "Materials and Methods" or trypsinized and counted in parallel. cpm/100,000 cells (top panels) and cell number (bottom panels) were determined in triplicate. Mean values and SD are shown.

 
Discussion

In this report, we demonstrate that the 44-amino acid BPV E5 protein can induce morphological transformation and focus formation and increase the saturation density for growth of mortal HDFs. Similar effects were observed for expression of the v-sis oncogene, as shown previously (21–24) . Unlike these earlier studies, here we provide evidence that constitutive activation of the PDGF receptor mediates the transforming effects of E5 or v-sis in HDFs. We also found that constitutive activation of another receptor tyrosine kinase, p185neu*, imparts similar phenotypic changes in these cells. Therefore, two different receptor tyrosine kinases can apparently use common signaling pathways to elicit transforming phenotypes in human fibroblasts.

One pathway shared by receptor tyrosine kinases is the Ras/MAPK pathway (37) . However, instead of inducing transformation, exogenous expression of activated ras in HDFs has been shown to induce premature senescence associated with increased p16INK4a and p53 expression (31) . In this study, it was proposed that premature senescence occurred to protect against an initial mitogenic stimulus by ras that could lead to extensive proliferation and tumorigenesis. Similar results were observed after exogenous expression of constitutively activated forms of Raf or MAPK kinase in HDFs (38, 39) , suggesting that the Ras/MAPK pathway is involved in the induction of p16INK4a and p53 expression, which, in turn, promotes senescence. Therefore, activated ras has a very different effect in HDFs than constitutive activation of the PDGFßR or p185neu, although Ras is a downstream effector of these receptors. This may be because the phenotypic changes induced by sustained activation of a receptor tyrosine kinase represent the combined effect of several interconnected signaling pathways, which may be very different from the effect of activating only the Ras/MAPK pathway. In the E5-, v-sis-, and neu*-expressing HDFs, perhaps another receptor tyrosine kinase signaling pathway inhibits senescence by suppressing MAPK activation. For example, in certain cell systems, the Raf-MAPK kinase-MAPK pathway is inhibited by activation of the phosphatidylinositol 3'-kinase-protein kinase B pathway through inhibitory phosphorylation of Raf by protein kinase B (40, 41) . We have preliminary evidence suggesting that MAPK activity is only transiently increased in the E5- and v-sis-expressing cells; compared with the control cells, it is only increased at very low densities and is significantly reduced at higher densities (data not shown). A lack of sustained MAPK activity may explain why these cells do not undergo premature senescence. Nonetheless, a different growth inhibitory pathway, which leads to quiescence rather than senescence, is activated in the E5-expressing HDFs (see below). Importantly, HDFs appear to have multiple growth control pathways that may serve to protect against complete transformation.

Despite the focus-forming activity of E5, which indicates a loss of contact inhibition, the growth rate of the E5-expressing HDFs was still was greatly impeded once they reached confluence. We also found that the PDGF receptor was rapidly and dramatically down-regulated in the E5-expressing cells and not in the control cells when confluence was reached. We propose that the E5-expressing HDFs activate an alternative growth inhibitory pathway at confluence, resulting in density-dependent growth arrest in the absence of contact inhibition. Specifically, our data suggest that a soluble factor secreted by these cells induces degradation of both the PDGFßR and hyperphosphorylated Rb, leading to subsequent growth inhibition. Because of its insensitivity to heat and its small size, the factor secreted by E5 HDFs is not likely to be a protease that directly degrades the PDGFßR and Rb. Instead, such a factor is more likely to stimulate an intracellular degradation pathway. This is substantiated by the fact that only cycling cells and not quiescent cells can degrade their receptors in response to this factor (data not shown). Thus, we present the possibility that an intracellular proteolysis pathway may be part of a back up densitydependent growth control mechanism in HDFs. Additional experiments are required to identify the secreted factor, determine the basis for its expression, ascertain the degradation pathway it induces, and determine the role of this pathway in growth inhibition.

The confluent state is probably involved in secretion of the factor rather than the degradation pathway per se because degradation was activated in subconfluent cells in response to the factor. However, the confluent state is unlikely to directly cause secretion of the factor because plating cells at a confluent density did not immediately induce PDGF receptor down-regulation. Under these conditions, the ability of the cells to assume the typical elongated and refractile "transformed" morphology (data not shown) was also delayed and coincided with PDGF receptor down-regulation. Therefore, it may be the change in cell shape associated with the confluent state that stimulates the secretion of a factor.

The inability of the control cells to down-regulate the PDGF receptor at confluence may reflect their inability to secrete a factor rather than respond to one. Indeed, a soluble factor from the E5 HDFs could induce down-regulation of the PDGF receptor and Rb in control cells, albeit more slowly, despite the fact that the control cells have only a low basal level of activated PDGF receptor. Therefore, the down-regulation pathway probably does not require a high level of PDGF receptor activation, although the increased level of activated receptors in the E5 HDFs may expedite this process. This seems to be inconsistent with previous studies suggesting that internalization and degradation of the PDGFßR require the receptor to be active and able to phosphorylate one of its substrates, phosphatidylinositol 3'-kinase (42, 43) . It is possible that instead of receptor-mediated endocytosis, the E5 HDFs use another pathway such as ubiquitin-mediated proteolysis to degrade the PDGF receptor and Rb at confluence. Because conditioned medium from confluent control cells could not induce receptor down-regulation in the E5 cells (Fig. 10)Citation , receptor activation is probably required for secretion of the factor. This may explain why AG1296 inhibits receptor down-regulation at confluence in the E5 cells (Fig. 9)Citation . Thus, receptor activation appears to be required for secretion of the factor, but receptor activation over a basal level may not be required for the down-regulation process itself.

Like the PDGF receptor, Rb is probably down-regulated by proteolytic digestion when the E5 HDFs reach confluence, as evidenced by the disappearance of hyperphosphorylated Rb and the appearance of a smaller Mr 68,000–70,000 form. This raises the intriguing possibility that degradation of Rb and degradation of the PDGF receptor occur by the same mechanism. This is supported by the fact that down-regulation of Rb and the PDGF receptor in subconfluent control and E5 HDFs occurred in response to the same stimulus (i.e., a secreted factor from confluent E5 HDFs). A secreted factor could stimulate the activation of a specific protease that acts on the PDGF receptor and Rb as substrates, suggesting that the PDGF receptor and Rb share a common proteolytic recognition sequence. Similarly, the secreted factor could activate the specific targeting of these proteins for ubiquitinization. Alternatively, a proteolytic product of one protein could stimulate the degradation of the other. Whatever the mechanism, this represents the first report linking posttranslational processing of Rb to that of the PDGF receptor.

The Mr 68,000–70,000 Rb form is likely to be a specific COOH-terminal cleavage product because the antibody that detected it was raised against a COOH-terminal peptide. Others have detected a similar Mr 68,000 Rb form in human leukemia and lung cancer cell lines (44–46) . One group reported that this Rb form results from specific cleavage by a caspase-like activity during apoptosis (44, 45) . It is possible that degradation of Rb and the PDGF receptor in the E5 HDFs is also associated with apoptosis because a drop in cell number was observed after treatment of cells with conditioned medium from confluent E5 HDFs (Fig. 11)Citation . A different group also detected a COOH-terminal Mr 68,000 Rb form but found that it was not associated with apoptosis (46) . Additional experiments are required to characterize this Mr 68,000–70,000 Rb form and assess its role in growth control of the E5 HDFs.

We believe that that down-regulation of Rb and the PDGF receptor plays a role in growth inhibition at confluence because it stands to reason that a dramatic decrease in the amount of activated PDGFßR and hyperphosphorylated Rb might serve to limit cell growth. Furthermore, when subconfluent cells were treated with the secreted factor, down-regulation of both proteins appeared to precede growth inhibition. However, our data do not rule out the possibility that down-regulation of these proteins and growth inhibition result from parallel pathways induced by the same stimulus, and additional experiments are required to definitively demonstrate that the two events are causally related.

The E5-expressing cells attained a higher saturation density than the control cells under both high and low serum conditions, indicating that constitutive PDGFßR activation allowed a proliferative advantage. However, because the growth rate of the E5-expressing cells was significantly impaired under low serum conditions, constitutive activation of the PDGFßR is not sufficient for optimal growth of HDFs. This may explain why exponentially growing E5 HDFs had a growth rate similar to that of exponentially growing control cells. We believe that sustained PDGFßR activation provides a proliferative advantage by delaying the onset of a density-dependent growth control mechanism rather than by increasing the rate of cell cycle progression. In both the E5 and control HDFs, growth inhibitory mechanisms are activated once confluence is reached. However, the E5 HDFs attain confluence at a higher density than the control cells, thereby raising the threshold density for onset of growth inhibition. Two characteristics of the E5 HDFs may account for this: (a) reduced cell spreading at higher densities, which could cause each cell to occupy less space; and (b) the ability of the cells to grow on top of each other. Both factors may be related to alterations in cell attachment. The fact that a back up growth control mechanism is activated in the E5 HDFs at confluence despite a loss of contact inhibition suggests that confluence is an important growth-restricting state. Thus, sustained PDGFßR activation may effect changes in cell attachment, which allow the cells to attain a higher density before reaching a growth-restricting state.

Unlike previous studies that showed that v-sis, c-sis, or PDGF could induce anchorage-independent growth of HDFs (22, 26, 47) , we were unable to demonstrate this activity for either E5 or v-sis. One might argue that the level of E5 or v-sis expressed in our system was not sufficient to induce detectable anchorage-independent growth. By preselecting cells that expressed high levels of c-sis, Stevens et al. (26) showed that a threshold level of c-sis expression was required for efficient anchorage-independent growth of HDFs. In our system, there appeared to be sufficient E5 and v-sis expression to induce abundant tyrosine phosphorylation of the PDGFßR, yet anchorage independence was not induced. Alternatively, the discrepancy between previous studies and our study may be explained by our more rigorous assessment of anchorage independence. First, we used stricter criteria (>100 µ) for assigning anchorage-independent growth and were therefore less likely to consider false positives significant. Even when the E5- and v-sis-expressing cells displayed a low level of growth in semisolid medium (which was over 100-fold less than that of an anchorage-independent cell line), the same low level was also observed for the control cells. Second, by using cells that were derived from a transformed colony and expanded, the previous studies may have selected for additional mutations conferring anchorage independence. It is possible that during the expansion and passage of clonal populations, mutations occurred that led to increased numbers of anchorage-independent colonies. In our study, the cells were not cloned and were used soon after infection. In any event, our data suggest that E5 and v-sis are unable to directly induce anchorage-independent growth. This implies that constitutive activation of the PDGFßR in HDFs is not sufficient to induce anchorage independence. It is possible that the growth inhibitory pathway activated at confluence also may be activated when these cells are suspended in semisolid medium.

Because E5 was unable to induce anchorage-independent growth, immortalization, or complete growth in low serum of HDFs, we expect that it would be unable to fully transform these cells to tumorigenicity. These observations are consistent with those of several other studies that reported that v-sis could not impart an extended life span or make cells tumorigenic (22, 23, 24) . Thus, although E5 has a potent transforming effect in HDFs, it provides only some of the steps necessary for the multistage process of oncogenesis. Recent evidence suggests that one requirement for tumorigenic transformation of human cells is telomere maintenance and immortalization promoted by expression of the telomerase catalytic subunit hTERT (48) . Hahn et al. (48) showed that ectopic expression of hTERT, together with activated Ras and the SV40 large T antigen, was sufficient to allow human epithelial and fibroblast cells to form tumors when injected into nude mice. In this study, expression of hTERT with only activated Ras resulted in senescence, as shown previously for expression of activated Ras alone (31) . Because E5 did not appear to induce this premature senescence phenotype, it would be interesting to assess the effect of coexpressing E5 with hTERT and determine what additional oncogene functions, if any, would be necessary and sufficient to induce tumorigenic transformation. For example, if the Mr 68,000–70,000 form of Rb observed in the E5 HDFs plays a role in growth inhibition at confluence, human papillomavirus E7 might be able to contribute to complete transformation by inactivating this Rb form.

In conclusion, we provide evidence that constitutive activation of the PDGFßR can elicit transforming characteristics in mortal human fibroblasts. This stands to reason because the PDGFßR is capable of eliciting signals to induce cell proliferation and is abundantly expressed in fibroblasts. However, it appears that HDFs adopt a strategy to abate the proliferative effect of PDGFßR activation, perhaps as a protective mechanism against further transformation. We have evidence to suggest that part of this strategy may be to activate an intracellular proteolysis pathway. Additional experiments are required to identify this pathway and determine how it may function in normal cells. Thus, the E5-expressing HDFs may allow characterization of a novel growth regulatory mechanism. These cells should also be useful for examining the mechanistic relationship between loss of contact inhibition and morphological transformation apart from other transforming phenotypes.

Materials and Methods

Plasmids.
pRVH-E5 and pRVH-v-sis plasmids were obtained from Daniel DiMaio (Yale University, New Haven, CT). Standard subcloning procedures were used to subclone the BPV E5 and v-sis oncogenes from these plasmids into the pBabepuro retrovirus vector (a gift from Jeff Settleman; Harvard University, Cambridge, MA) to generate pBabepuro-E5 and pBabepuro-sis, respectively. The neu* cDNA was subcloned into the LXSN retrovirus vector from pSR{alpha}-NeuNT (a gift from David Stern; Yale University) using standard methods. High-titer amphotropic retrovirus was obtained after transient transfection of the retroviral plasmids into the Phoenix amphotropic producer cell line (American Type Culture Collection with permission from G. Nolan; Stanford University, Palo Alto, CA) as described previously (49) .

Stable Expression of E5, v-sis, and neu*.
Young HSF4012 (NHDF4012) human foreskin fibroblasts were purchased from Clonetics at passage 1 and maintained in MEM-{alpha}-10 [MEM-{alpha} (Life Technologies, Inc.) supplemented with 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin]. Cell strains stably expressing E5, v-sis, or neu* were established using high-titer recombinant retroviruses expressing these oncogenes to infect low-passage HSF4012 cells. Briefly, approximately 1 x 106 colony-forming units of retrovirus and 16 µg of Polybrene in 4 ml were added to HSF4012 cells at 80% confluence in a T75 flask. After 1 day, the infected cells were trypsinized and split into eight to ten 100-mm dishes. After allowing the cells to adhere to the dishes, puromycin (Sigma) or G418 (Life Technologies, Inc.) was added to the cells at a final concentration of 0.5 or 400 µg/ml, respectively. After 1 week of selection, stable cell strains were established. Control cells were established by infection with the corresponding retrovirus without an insert.

Focus-forming Assay.
Passage 7 HSF4012 cells, which were at 90% confluence in 60-mm dishes, were infected with approximately 1 x 103 colony-forming units of recombinant retrovirus. One day after infection, the cells in each dish were split 1:3. Three days later, when the cells were 90% confluent, 7 µl of DMSO or Tyrphostin AG1296 (Calbiochem; 17.5 µM, final concentration) were added to the cells. The cells were then maintained at confluence in MEM-{alpha}-10, with a weekly media change containing fresh AG1296 or DMSO. Foci were visible 10–14 days after infection, and 3 weeks after infection, monolayers were fixed and stained with 1% crystal violet (w/v) in 70% ethanol.

Growth Curves.
HDFs expressing E5, v-sis, or the empty retroviral vector were seeded into multiple 60-mm dishes at 1 x 105 cells/dish. At intervals thereafter, cells in triplicate dishes were trypsinized and counted using a Coulter counter. The number of cells per dish was converted to the ln, and the results from the triplicate dishes were used to determine the mean and SD at each time point. Cell doublings were calculated using the formula

where F = final cell number, and I = the initial cell number.

Growth in Low Serum.
Cells were diluted into media containing 1.0% FBS and counted. Cells were then added to media containing either 1% or 10% FBS and plated at a density of 105 cells/60-mm dish. Cells were trypsinized and counted in triplicate at intervals thereafter. Growth curves were plotted as described above.

Anchorage Independence Assay.
Control, E5- or v-sis-expressing cells (3 x 105) were suspended in 10 ml of 1.5% methylcellulose in complete media as described previously (50) . This suspension was mixed and distributed equally to three gridded 60-mm dishes that had been precoated with sterile 1.0% agarose in PBS. HT1080, a cell line derived from a human fibrosarcoma, was used as a positive control and found to be virtually 100% anchorage independent, based on daily observation of colony growth. Dishes were checked for clumps on the day after initiation of the assay, and no significant clumping problems were detected. After approximately 3 weeks, colonies that were greater than 100 µm were considered positive for anchorage-independent growth.

Immunoprecipitation and Immunoblotting.
For the experiment shown in Fig. 2CCitation , HSF4012 cells stably expressing E5, v-sis, or neu* in 60-mm dishes at 80% confluence were treated with 10 µl of DMSO or 10 µl of 10 mM AG1296 (final concentration, 25 µM). Twenty-four h later, these cells as well as the corresponding untreated control cells (expressing the empty retroviral vectors pBabepuro or LXSN) were lysed in cold EBC buffer as described previously (7, 8, 15) . For the experiment shown in Fig. 9Citation , cells were treated with AG1296 in the same manner but lysed 2 days after treatment in protein sample buffer as described below. For Fig. 2, A and BCitation , cells were lysed in cold 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid extraction buffer as described previously (29) . PDGF receptor was immunoprecipitated as described previously (8, 9, 15) by adding approximately 1 µl of anti-PR-C3a antibody (a gift from Daniel DiMaio; this antibody recognizes the COOH-terminal 13 amino acids of the human PDGFßR) per 100–200 µg of protein extract. p185neu* was immunoprecipitated from EBC cell extracts by adding 10 µl of anti-p185neu antibody Ab-4 (Calbiochem) to approximately 600 µg of protein extract. Immunoprecipitation of the E5 protein and its associated proteins was performed as described previously (8, 15) by adding approximately 1 µl of anti-E5 antibody (a gift from Daniel DiMaio; this antibody recognizes the COOH-terminal 16 amino acids of the E5 protein) per 100–200 µg of protein extract. Washed immunoprecipitates were resuspended in 2x Laemmli sample buffer. To prepare whole cell extracts for the experiments shown in Figs. 6–10Citation Citation Citation , the monolayers were washed twice with PBS and then lysed by adding 200–250 µl of hot 2x Laemmli sample buffer. Whole cell extracts or immunoprecipitates were boiled; electrophoresed on a SDS-7%, 7.5%, or 15% (for E5 immunoblotting) polyacrylamide gel; and transferred to nitrocellulose or polyvinylidene difluoride (for E5 immunoblotting) at 100 V for 2 hours. Immunoblotting was performed as described previously (8, 15) using a 1:1000 dilution of antiphosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc.), a 1:1000 dilution of anti-PDGF receptor antiserum (PR-C3a), a 1:100 dilution of anti-p185neu monoclonal antibody (Ab-3, Calbiochem), a 1:500 dilution of anti-E5 antiserum, a 1:750 dilution of anti-Rb antibody (C-15; Santa Cruz Biotechnology), or a 1: 500 dilution of anti-actin antiserum (Sigma). Proteins were detected by enhanced chemiluminescence using a protein A- or antimouse IgG (for p185neu immunoblot)-horseradish peroxidase conjugate (Pierce).

Pulse-Chase Experiment.
Monolayers of E5 or control HDFs that were either 80% (72 h after plating) or 100% (84 h after plating) confluent in 60-mm dishes were washed twice with PBS and preincubated for approximately 30 min in serum-free, methionine-free, and cysteine-free DMEM. Cells were then incubated in 1 ml of fresh serum-free, methionine-free, and cysteine-free medium containing approximately 418 µCi of Expre35S35S label (New England Nuclear; 71% [35S]methionine and 29% [35S]cysteine) for 45 min. Cells were then washed twice with PBS and either lysed immediately in EBC buffer or incubated in fresh medium containing 150 µg/ml L-methionine and 120 µg/ml L-cysteine for various times before lysis. After preclearing the extracts by incubation with protein A-Sepharose beads, PDGF receptor was immunoprecipitated and subjected to SDS-PAGE, followed by fluorography and autoradiography.

Fractionation of Conditioned Medium.
Conditioned medium from E5 HDFs that had just reached confluence was fractionated by centrifugation at 1600 x g for 3 h through a Centriplus (Millipore) centrifugal filter device with a molecular weight cutoff of 3000. The retentate, which was concentrated down to a smaller volume and contained solutes with molecular weights of >3000, was added directly to the existing medium of subconfluent E5 HDFs to restore its original concentration. The filtrate, which contained solutes with molecular weights of <3000, was added to subconfluent E5 HDFs after removal of the existing medium.

DNA Synthesis Assay.
For the experiment shown in Fig. 11, E5Citation Citation or control HDFs were seeded at 1–2 x 104 cells/well in 24-well plates, and 2 days later, when the cells were still subconfluent, they were either left untreated or treated with conditioned medium from E5 HDFs that had just reached confluence. At the time of treatment and various times thereafter, the cells were assayed for [3H]thymidine incorporation or trypsinized for cell counts. For [3H]thymidine incorporation, cells were incubated for 2 h with 100 µl/well of 1.5 µCi/ml [3H]thymidine (New England Nuclear). Triplicate wells were assayed for cold trichloroacetic acid-precipitable, hot perchloric acid-soluble radioactivity as described previously (30) . Cell counts were performed in parallel from triplicate wells using a hemocytometer.

Acknowledgments

We gratefully acknowledge Daniel DiMaio, David Stern, and Jeff Settleman for essential reagents and Julia Schaefer and Daniela Drummond-Barbosa for subcloning neu* and E5, respectively, into the appropriate retroviral vectors. We also thank Daniel DiMaio, Judith Laffin, Tom Friedrich, John Lehman, Ying Zhang, and Jeff Banas for invaluable discussions and advice.

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 Supported by National Cancer Institute Grant 1R29 CA73682-01A1 (to L. M. P.) and a Schaffer Fellowship from Albany Medical College (to F. A. R.). Back

2 To whom requests for reprints should be addressed, at Center for Immunology and Microbial Disease, MC-151, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. Phone: (518) 262-6285; Fax: (518) 262-5748; E-mail: pettil{at}mail.amc.edu Back

3 The abbreviations used are: BPV, bovine papillomavirus; PDGF, platelet-derived growth factor; PDGFßR; PDGF ß receptor; HDF, human diploid fibroblast; ln, natural logarithm; FBS, fetal bovine serum; Rb, retinoblastoma protein; MAPK, mitogen-activated protein kinase; PD, population doubling. Back

4 F. A. Ray, unpublished observations. Back

5 E. Okubo and F. A. Ray, unpublished observations. Back

6 Y. Zhang and L. M. Petti, unpublished observations. Back

Received for publication 3/17/00. Revision received 5/12/00. Accepted for publication 5/16/00.

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