Prothymosin a Functions as a Cellular Oncoprotein by Inducing Transformation of Rodent Fibroblasts in Vitro *

Prothymosin a (ProT a ), a cellular molecule known to be associated with cell proliferation, is transcriptionally up-regulated on expression of c- myc and interacts with histones in vitro and associates with histone H1 in cells. Previous studies have also shown that ProT a is involved in chromatin remodeling. Recent studies have shown that ProT a interacts with the acetyl transferase p300 and an essential Epstein-Barr virus protein, EBNA3C, involved in regulation of viral and cellular transcription. These studies suggest a potential involvement in regulation of histone acetylation through the association with these cellular and viral factors. In the current studies, we show that heterologous expression of ProT a in the Rat-1 rodent fibroblast cell line results in increased proliferation, loss of contact inhibition, anchor-age-independent growth, and decreased serum dependence. These phenotypic changes seen in transfected Rat-1 cells are similar to those observed with a known oncoprotein, Ras, expressed under the control of a heterologous promoter and are characteristic oncogenic growth properties. These results demonstrate that the ProT a gene may function as an oncogene when stably expressed in Rat-1 cells and may be an important downstream cellular target for inducers of cellular transformation, which may include Epstein-Barr virus and c-myc. gel, followed by transfer to 0.22- m m nitrocellulose. Immune detection of Myc-tagged ProT a from pA3M-ProT a was carried out by incubation with monoclonal anti-Myc antibody, followed by incubation with anti- mouse horseradish peroxidase secondary antibody and detection by standard chemiluminescence. Western blot analysis reveals the pres- ence of a small ( ; 22 kDa) protein in all four Rat-1 pA3M-ProT a clones, but not in either pA3M- or pA3M-Ras-transfected Rat-1 cells (compare lanes 2–5 with lane 1 ).

Prothymosin ␣ (ProT␣) 1 is implicated in the growth of normal cells as well as in the proliferation of mammalian cells undergoing malignant transformation. However, the physiological role of this protein in cell proliferation remains to be elucidated. Nonetheless, expression of the gene is generally correlated with cellular proliferation and is low in quiescent cells (1)(2)(3)(4). This prompted us to determine whether ProT␣ has any oncogenic potential for cellular transformation in a rodent fibroblast assay. The present study indicates that overexpres-sion of ProT␣ in Rat-1 cells is sufficient to induce a transformed phenotype of these cells in vitro that is similar to the phenotype produced by the ras oncogene when overexpressed under similar conditions.
ProT␣ is a small (12.5 kDa), highly acidic nuclear protein, which contains a putative nuclear localization signal at the carboxyl terminus and a small basic domain at the amino terminus (1,(5)(6)(7)(8)(9). Additionally, ProT␣ is highly conserved and ubiquitously expressed in a wide variety of cells, tissues, and organisms (1)(2)(3)(4)8), which suggests that it is required for an essential function of the cell. Subsequently, numerous research findings have indicated that ProT␣ may be involved in cell proliferation. This relationship has been supported by the direct correlation of both ProT␣ mRNA and protein levels with levels of proliferation. The transcript is induced upon growth stimulation of resting lymphocytes (4), thymocytes (10), NIH 3T3 fibroblasts (4,11), and hepatocytes during liver regeneration (10,12). Moreover, ProT␣ concentrations were reported to be higher in tumor samples than in normal breast tissue (13), and were phosphorylated in stimulated proliferating cells (14). ProT␣ gene expression is elevated in normal proliferating tissue (15), but repressed in quiescent cells (13). Expression of ProT␣ correlates with proliferation (16), whereas ProT␣ antisense oligonucleotides induce apoptosis in HL-60 cells (17). Additionally, ProT␣ expression is also known to occur in proliferating B and T lymphocytes (18). Overexpression of ProT␣ has been shown to accelerate proliferation, and to retard HL-60 promyelocyte differentiation (19). Antisense oligonucleotides of ProT␣ mRNA have also been shown to inhibit cell proliferation in myeloma cells (20).
c-myc is a proto-oncogene that has been implicated in normal proliferation and diverse forms of tumorigenesis (21). Overexpression of c-myc stimulates cell cycle progression, and induces cell transformation and apoptosis (22). The transcriptional activation of c-myc leads to an increase in the level of transcription of ProT␣, and ProT␣ mRNA levels vary with c-myc expression during tissue proliferation, viral infection, and heat shock (23). Overexpression of ProT␣ is concomitant with that of c-myc during rat hepatic carcinogenesis (24). An E-box element localized in the first intron mediates transcriptional regulation of the gene for ProT␣ by c-myc (25). Furthermore, the human papilloma virus type 16 E6 oncogene can transactivate the ProT␣ and c-myc promoters (26,27), indicating that ProT␣ is a transcriptional target of at least two known oncogenes. Conditional expression of c-myc in human neuroblastoma cells increases ProT␣ and accelerates early progression into S-phase after mitogenic stimulation of quiescent cells (28). Recent studies have shown that ProT␣ binds to histones in vitro (29), thereby suggesting a possible role for this molecule in chromatin remodeling in mammalian cells (7,30,31). Despite the documented evidence for a role of ProT␣ in cell proliferation, its potential role in induction and maintenance of cell transformation has not been investigated. Since ProT␣ is closely associated with cell proliferation and is induced upon expression of the known oncogene c-myc and the human papillomavirus E6 protein, we decided to investigate the potential role of ProT␣ when overexpressed in Rat-1 cells by phenotypic transformation assays. Here we show that overexpression of ProT␣ induces the typical transformed phenotype in vitro as shown by increased proliferation, anchorage-independent growth, loss of contact inhibition, and decreased serum dependence of the transfected Rat-1 cells, which suggest that ProT␣ may function as a cellular oncogene and is likely one of the important downstream targets for inducers of transformation like c-myc.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfections-Rat-1 cells, obtained from George Mosialos and Elliott Kieff, were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Gemini Bioproducts, Inc.), penicillin (25 units/ml), streptomycin (25 g/ml), and gentamicin (10 g/ml) and maintained in a humidified atmosphere at 37°C with 5% CO 2. After two passages, Rat-1 cells were seeded at 1 ϫ 10 5 cells/35-mm diameter six-well plate, and transfected when ϳ60% confluent with 5 g of pA3M vector, pA3M-ProT␣, or pA3M-Ras. 10 l of Superfect reagent (Qiagen) and 5 g of each plasmid were resuspended in 140 l of DMEM without serum and incubated for 10 min at room temperature, followed by addition of 1.5 ml of DMEM (with 10% serum). The cells were placed under selection 24 h after transfection with DMEM containing 800 g/ml G418. The cells were trypsinized after growing for 48 h under selection and plated in DMEM containing 10% fetal bovine serum, penicillin (25 units/ml), streptomycin (25 g/ ml), and gentamicin (10 g/ml). Photographs were taken at the beginning of the fourth week, and colonies were counted.
For studies of serum dependence, 1 ϫ 10 5 cells were plated in 35-mm six-well plates containing DMEM with 10% serum. The cultures were washed twice with serum-free medium and then resuspended in DMEM with 1.0%, 2.5%, 5.0%, and 10% fetal bovine serum. Cells were trypsinized on the fourth day and were counted using a hemocytometer with trypan blue staining. Photographs were taken at 10ϫ magnification using a BK40 Olympus phase contrast microscope.
Soft Agar Assay-Soft agar plates were prepared as described previously (32). Briefly, the bottom layer was made by melting 1.4% DNA grade agarose (American Bioanalytical) in sterile water, which was cooled to 40°C in a water bath and added to an equal quantity of 1ϫ DMEM enriched with 20% glutamine, and 1.6 mg/ml G418, yielding a final concentration of 0.7% agar in DMEM. The top agar layer was prepared by melting 0.7% agar, followed by equilibration at 40°C in a water bath. Trypsinized cells (1.8 ϫ 10 5 ) grown under selection for 48 h were added to 5 ml of the 0.7% top agar and poured onto the prepoured bottom layer in a 100-mm plate. The soft agar was covered with 1 ml of DMEM containing 10% serum and 800 g/ml G418 every 5 days and incubated at 37°C with 5% CO 2 . Colonies were observed and quantified 4 weeks after transfection.
Cell Growth Experiment-To measure the growth of cells, 1 ϫ 10 5 cells were plated into 35-mm plates and allowed to grow for 12 days. The cells were trypsinized at 2-day intervals up to 12 days and counted on a hemocytometer with trypan blue staining. A total of six time points were taken for each transfection. This assay was repeated three times, and data points were plotted as mean Ϯ S.D.
Plasmid Constructs-ProT␣ was cloned by ligation of the entire coding sequence amplified from pAV1 (33) in frame with the Myc epitope into the pA3M mammalian expression vector (34). Primers flanking the coding sequence of ProT␣ contained EcoRI and EcoRV restriction sites in the forward and reverse primers, respectively (forward primer, 5Ј-GGAATTCCATGTCAGACGCAGCCGTAGACA-3Ј; reverse primer, 5Ј-GGATATCGGGTCATCCTCGTCGGTCTTCTG-3Ј) and were used to amplify ProT␣ cDNA with Vent polymerase (New England Biolabs). The ras cDNA was cloned in a similar fashion using forward and reverse primers (5Ј-TTAGAATTCATGACAGAATACAAGCTT-GTG-3Ј and 5Ј-TTAGATATCTAGGACAGCACACACTTGCA-3Ј, respectively). The polymerase chain reaction product obtained was ligated into the prepared EcoRI and EcoRV sites of pA3M in frame with the Myc epitope tag at the carboxyl terminus.

Induction of Cell Proliferation by
ProT␣-Rat-1 cells were transfected with ProT␣, Ras, or pA3M vector alone. Transfected cells were grown in DMEM complete medium with 10% fetal bovine serum and 800 g/ml neomycin. The cells were trypsinized and counted at 2-day intervals up to 12 days when cell transfected with Ras, and ProT␣ reached confluence. Little difference was noted up to 6 days, during which time the Rasand ProT␣-transfected cultures proliferated at rates similar to that observed for the control vector transfectants. After 6 days, however, the rate of proliferation in cells transfected with ProT␣ and Ras was substantially greater than that observed in cells transfected with empty pA3M vector. This trend continued until day 12, at which time the Ras and ProT␣ cultures reached confluency ( Fig. 1). Interestingly, ProT␣ demonstrated an enhanced ability to induce proliferation when compared with the Rastransfected cells (Fig. 1). This suggests that ProT␣ may have more potent effects on cell cycle by driving the cells through the G 1 phase more rapidly than those effects that are due to Ras expression. Each data point is based on counts from multiple transfections done in triplicate. These results clearly show that ProT␣ induces proliferation of the rodent fibroblast Rat-1 cell line in vitro in a similar manner to the known oncoprotein Ras, which indicates that these closely related phenotypes are induced in vitro by these two cellular proteins.
ProT␣ Induces Foci Formation in Monolayer Cultures-Rat-1 cells were transfected with pA3M-ProT␣ or pA3M-Ras expression vector as well as the pA3M vector alone, followed by selection with neomycin after 24 h. Phenotypic changes were FIG. 1. Growth rate in Rat-1 cell clones. Rat-1 cells were passaged twice and plated in 35-mm six-well plates. The cells were transfected at 60% confluence with pA3M vector, pA3M-Ras, and pA3M-ProT␣. Cells were selected with 800 g/ml G418 after 24 h. Cells were trypsinized every 2 days from a new well, and counted on hemocytometer. Cells were counted in duplicate, with three independent readings. Data points represent mean values. Standard deviations ranged from 3% to 22%. Triangles indicate ProT␣, squares indicate Ras, and diamonds indicate vector control. Starting at day 6 and continuing through the completion of the experiment at day 12, ProT␣ and Ras Rat-1 clones displayed a significantly increased growth rate compared with vector control. observed 20 days after transfection for the Ras and ProT␣ transfections but not in vector alone control. Little or no foci formation was observed in cultures that were transfected with vector alone (Fig. 2, panel A and D) in which the cells under selection continued to grow in a monolayer until they reached confluency. However, in the Ras-and ProT␣-transfected cultures, many foci were readily apparent by 20 days after transfection. This phenotypic change was macroscopically visible on average between 18 -21 days after transfection but could be seen within 10 days microscopically. The foci in these cultures were typically 1-2 mm in size and continued to grow in size as they remained in culture. Foci that developed from Ras-transfected cells were similar to that of foci developing from ProT␣transfected Rat-1 cells (Fig. 2, compare panels B and E with panels C and D). The cells in these foci continued to grow upon one another with an apparent absence of contact inhibition until they would break off from the dish and again continue to stack on each other as the foci grew larger. The number of foci obtained on an average from the Ras-transfected cells was slightly higher than that seen with ProT␣ transfected cells (Table I). However, this was not obvious by visual inspection when comparing plates containing the transfected cultures (see Fig. 2, panels B and C). A small number of foci less than 0.5 mm in size were seen in the vector alone control; however, only foci greater than 1 mm in size were counted. Additionally, the foci formed in the vector alone control were observed only after the cultures reached confluence and did not grow in size over time (Fig. 2, panels A and D), whereas foci were formed in ProT␣ and Ras cultures at an earlier time, before the respective monolayer eventually became confluent. Foci were counted in each of three cultures 25 days after selection. The data shown in Table  I represent three independent experiments and show that ProT␣ induces a loss of contact inhibition phenotype and, like Ras, allows Rat-1 cells to grow upon one another and form multicellular foci.
ProT␣ Promotes Growth of Rat-1 Cells in Low Serum-Typically, cells that are transformed have a decreased dependence on serum for growth. To determine if ProT␣ is capable of inducing serum-independent growth, cells transfected with Ras, ProT␣, and vector alone were grown in media containing 1.0%, 2.5%, 5.0%, and 10.0% serum for 4 days. The cells were trypsinized, and viable cells were counted on a hemocytometer with trypan blue staining. At low serum concentration (1%), growth was not pronounced for any of the samples (Fig. 3a, A-C), whereas at concentrations of 2.5% to 5% vigorous growth was observed with Rat-1 cells transfected with both Ras or ProT␣, but not in the vector alone control (Fig. 3a, D-F and G-I). At concentrations of 10% serum, cell cultures grew at a relatively similar rate. As expected, vector control cells did not grow significantly in media that contained 1% serum and showed a clear dose response to serum as the concentration was increased (Fig. 3b). Both ProT␣-and Ras-transfected cultures, however, rapidly reached confluence (Table II) and similarly achieved much higher cell density (Fig. 3b) in 2.5%, 5%, and 10% serum concentrations. In all dilutions of serum, 1-10% of the Ras-and ProT␣-transfected cells had similar growth properties in terms of number of cells (Fig. 3b) and morphological properties (Fig. 3a) and levels of confluence (Table II). These results show that ProT␣ promotes growth of Rat-1 cells in low serum concentrations with striking similarity to that of the known oncoprotein Ras. This indicates that cells transfected with ProT␣ can stimulate cellular processes required for cell proliferation even under low serum conditions, again indicating a transformed phenotype.
ProT␣ Induces Colony Formation of Rat-1 Cells in Soft Agar-Based on the previous results, we became interested in whether ProT␣ could induce anchorage-independent growth of Rat-1 cells as determined by growth in soft agar. To this end we used anchorage-independent growth as another in vitro parameter to monitor the expression of a fully transformed phenotype (32,35). Cells were transfected as described above, with equivalent amounts of empty pA3M vector, pA3M-ProT␣, and pA3M-Ras. 24 h after transfection, the cells were placed under selection with 800 g/ml neomycin in DMEM. After 3 more days of selection, 1.8 ϫ 10 5 cells were trypsinized, counted and seeded on soft agar. The cells in soft agar were fed with 1 ml of selection medium once a week, and plates were observed every 3 days. 22 days after transfection, colonies were readily apparent in the case of Ras, but not in control cultures. In the ProT␣ culture, colonies were apparent but were initially smaller than those observed in the Ras-transfected culture. After 5-6 weeks of selection, ProT␣ colonies had grown to the size of the Rasinduced colonies (Fig. 4, panels B and E compared with panels C and F). Photographs were taken at the end of week 7 for all plates. Overall, ProT␣ colonies initially grew slower than Rasexpressing colonies in soft agar but eventually attained similar size. Moreover, on average the number of colonies obtained with ProT␣ was about 50% lower than that seen with Ras but much greater than vector alone control (Table III). Additionally, the approximate number of ProT␣ colonies after 12 weeks

FIG. 2. ProT␣ induces Rat-1 cells to form foci similar to Ras.
Rat-1 cells were passaged twice and seeded in DMEM containing 10% serum. Cells were transfected with pA3M vector alone or with vector containing Ras or ProT␣ cDNA. Cells were maintained in DMEM containing 10% serum and 800 g/ml G418. Representative photomicrographs were taken after 4 weeks of selection with a phase contrast microscope (magnifications, ϫ1.5 and ϫ10) and show the ability of ProT␣ to induce cellular foci in a fashion similar to that observed with the known Ras oncoprotein (compare panels A-C and D-F). was similar to that of the Ras-transfected cells. Once the ProT␣ colonies reached a size similar to that of Ras, no other obvious morphological differences were detected in this assay. Typically, the vector alone culture showed no sign of colony growth even after extended incubations in 12 weeks of selection. These results indicate that ProT␣ can induce anchorage-independent growth when stably expressed in Rat-1 cells.
The Transformed Rat-1 Foci Express ProT␣ from the Transfected Heterologous Promoter-To demonstrate that the transformed phenotype seen in Rat-1 cells transfected with ProT␣ was due to the overexpression of ProT␣ from the heterologous cytomegalovirus immediate-early promoter, we did Western blots on cells obtained from individual colonies or foci from the experiments above. Signals of ProT␣ were detected using monoclonal antibodies against the Myc tag fused to ProT␣. A specific band was detected in all the ProT␣-transfected cells and was not detected in the cells that were transfected with vector alone (Fig. 5, compare lane 1 with lanes 2-5). A smaller band was seen below the specific band in lanes 2-5, suggesting proteolytic degradation or varying levels of modification of the Myc-tagged ProT␣ expressed from the heterologous cytomegalovirus immediate-early promoter. DISCUSSION Although it has been implicated in cell proliferation (1)(2)(3)(4)8) and chromatin remodeling (29,30), the physiological function of the ProT␣ protein remains somewhat obscure. Recent work in our laboratory has shown that ProT␣ is involved in transcriptional regulation at the level of histone acetylation through interaction with p300 and one of the essential EBV antigen, EBNA3C (36). EBNA3C has been shown to be required for the in vitro transformation of primary human lymphocytes by EBV (37) and has been suggested to have oncogenic potential and deregulatory effects on the cell cycle (38). Moreover, the in vitro interaction as well as the in vivo association of ProT␣ and EBNA3C suggested that ProT␣ might be a cellular target of EBNA3C, which results in regulation of cell cycle events. The additional studies demonstrating that ProT␣ can interact with histone H1 in vitro and in cells (30,31,36) strengthened the previous findings that ProT␣ may have a role FIG. 3. a, ProT␣ induces serum-independent growth of Rat-1 cells. After transfection, Rat-1 cells were grown in 1.0%, 2.5%, 5.0%, and 10.0% serum for 6 days and photographed with phase contrast microscopy (magnification, ϫ10) to show fields that reflect typical cell morphology and density. Rat-1 cells expressing ProT␣ and Ras display significantly enhanced ability to grow in low serum concentrations (compare panels B, E, H, and K and panels C, F, I, and L with panels A, D, G, and J). b, effect of serum on cell growth. Transfected Rat-1 cells were grown in the presence of 1.0%, 2.5%, 5.0%, and 10.0% serum concentrations for 4 days, at which time cells were trypsinized and viable cells were counted on a hemocytometer with trypan blue staining. Data points in Fig. 3b represent mean values Ϯ S.D.
in regulating the assembly of nucleosomes. These studies prompted us to investigate a possible oncogenic role for ProT␣ in triggering cellular growth transformation.
The experiments presented here show that ProT␣ is capable of inducing not only significant cell proliferation, but also every assayed feature of transformed cells. Specifically, ProT␣ expression resulted in the marked proliferation of transfected Rat-1 cells, elimination of contact inhibition, and both anchorage-and serum-independent growth of these cells in vitro. This was shown by the observation and quantification of multicellular foci, growth in soft agar, and growth in the presence of low serum. These results were strikingly similar to those observed upon expression of the known oncoprotein, Ras, whereas these phenotypes were not observed in Rat-1 clones containing only the control pA3M vector. Taken together, this indicates that ProT␣ retains the capacity to function as a transforming oncoprotein.
The mechanism by which ProT␣ induces transformation, however, remains unclear. Although ProT␣ does not bear sequence homology to any other known oncogenes, the similarity between the phenotypes induced by ProT␣ and Ras is striking. Since ProT␣ is almost completely localized to the nucleus (39), it is unlikely that it functions by stimulating a signal transduction cascade from the plasma membrane, as does the Ras oncoprotein (40,41). As suggested by recent work in our laboratory, it is more likely that ProT␣ functions at the level of gene transcription by modulating histone acetyltransferase activity in producing its oncogenic effects (36). Overexpression of ProT␣ may thereby result in the transcriptional dysregulation of promoters that drive expression of cell cycle regulatory proteins. ProT␣ has structural similarities to other cellular proteins with acidic domains and includes the high mobility group proteins, nucleolin, and proliferating cell nuclear antigen (7,39,42). The functions of these molecules all relate to the modification of chromatin structure during activation/repression of transcription and replication of DNA templates (43). It is possible that ProT␣ plays similar roles as an ancillary factor for the basal transcriptional or replicative machinery in a global sense in lieu of the fact that ProT␣ is ubiquitously expressed and up-regulated in proliferating cells (23,44,45). These putative roles correlate with known studies demonstrating that ProT␣ is required for cell division and is up-regulated in proliferating, transcriptionally active cells increasing the transition of the cells through G 1 (23,44,45).
As already stated, ProT␣ is a transcriptional target of c-Myc (44). Transcription of the ProT␣ gene rapidly increases upon Myc activation probably due to Myc transcriptional activity on an E-box DNA element located within the first intron of the ProT␣ gene (25). c-myc is a well characterized oncogene known to play an important role in the transformation of primary cells into immortal cell lines, which can be continually grown in culture (22). The present work suggests that activation of the ProT␣ promoter may be central to the cellular proliferation induced by c-Myc activation. Although at this time, the exact role of ProT␣ FIG. 4. Colony formation in soft agar. Rat-1 cells were transfected with pA3M vector alone, pA3M-ProT␣, and pA3M-Ras, followed by selection in 800 g/ml G418 after 24 h. 48 h later, cells were trypsinized and transferred to soft agar covered with DMEM. Representative photomicrographs were taken with a phase contrast microscope (magnification, ϫ10), after 7 weeks of G418 selection. Photographs of whole plates were taken with a camera (magnification, ϫ1.5).   downstream of c-Myc is not clear, it is still intriguing to note that ProT␣ may be a critical target for c-Myc-mediated induction of cell proliferation and transformation. ProT␣ is certainly not the only target for c-Myc activation, but it is possible that this ubiquitous cellular protein may be one of the critical targets required to induce the transformed phenotype. Current studies also suggest that the proliferation and transformation induced by the DNA tumor virus EBV in various human malignancies may in part involve targeting of ProT␣ similar to c-Myc. It would therefore be interesting to note whether or not ProT␣ could be activated by the EBVencoded nuclear proteins required for B cell immortalization. As stated before, ProT␣ has been shown to interact with the EBV EBNA3C, a viral protein required for transformation of primary human B lymphocytes, and the p300 coactivator/histone acetyltransferase (36). By interacting with p300, ProT␣ may regulate transcriptional activity through modulation of the histone acetylase function of p300. This would then result in a change in transcriptional activity of promoters that drive expression of genes regulating proliferation and cell cycle progression. These studies, which include the identification of the specific promoters that are affected, are currently under study. It is possible that ProT␣ may have a role on specific promoters through its interaction with the basal transcription factors or that it is part of specific complexes required for activation of specific gene promoters. The latter is less likely to be true in lieu of the fact that ProT␣ interacts with coactivators, which include p300 and possibly others and is generally up-regulated in proliferating cells (36). The fact that the ProT␣/p300 complex is also bound by EBNA3C in EBV-infected cells does provide some clues. Further studies will also be necessary to determine what role EBNA3C may play in cell transformation in the context of ProT␣, documented in this study. We are currently targeting specific cellular pathways utilized by ProT␣ in an attempt to determine the mechanism by which ProT␣ can induce transformation of mammalian cells in vitro.
Studies have also demonstrated that ProT␣ expression is induced by the E2F transcription factor known to regulate cell cycle-related genes or genes involved in cell proliferation (45). These genes are typically required for DNA synthesis and cell proliferation (45)(46)(47). The disruption of the E2F⅐pRb complex due to hyperphosphorylation of the pRb molecules occurs in late G 1 allowing the cells to exit the G 1 restriction point (48 -51). Moreover, pRb is a known tumor suppressor or suppressor of cell proliferation and can be considered to be a regulator of ProT␣ expression through its association with E2F, as E2F can activate the ProT␣ promoter (45). ProT␣ expression also correlates with the expression of cyclin B with an increase in S/G 2 and reducing as the cell traverses or enters the G 1 phase of a new cycle (45,52). This similarity in expression patterns is not fully understood, as studies are still left to be done that may explain the similarities in terms of transcription regulation or a functional relationship that is intimately tied with the cell cycle. Overall, these data indicate that ProT␣ is a cell cycleregulated molecule intricately intertwined in the processes of DNA synthesis and cell proliferation.
It is noteworthy that ProT␣ appears to be targeted by at least two diverse oncogenic stimuli. Specifically, the cellular c-myc oncogene as well as the DNA tumor virus, EBV, both seem to interface directly with ProT␣ or through its transcriptional regulatory function in the process of cellular transformation (36). The current study, in which transfected ProT␣ alone is shown to induce many features of malignancy in rodent fibroblasts, serves to corroborate the importance of this molecule in having a critical role in studies of basic cancer biology.