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Cell Growth & Differentiation Vol. 10, 93-100, February 1999
© 1999 American Association for Cancer Research

Retardation of Cell Proliferation after Expression of p202 Accompanies an Increase in p21WAF1/CIP11

Jordan U. Gutterman and Divaker Choubey2

Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [J. U. G., D. C.], and Department of Radiotherapy, Loyola University Chicago-Hines VAMC, Hines, Illinois 60141 [D. C.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
p202 is an IFN-inducible, primarily nuclear, phosphoprotein (52-kDa) whose constitutive overexpression in transfected cells inhibits colony formation. To investigate the molecular mechanism(s) by which expression of p202 protein impairs colony formation, we established stable cell lines that inducibly express p202. Using this cell model, we demonstrate that the induced expression of p202 in asynchronous cultures of these cells was accompanied by: (a) an increase in steady-state levels of p21WAF1/CIP1/SDI1 (p21) mRNA and protein; (b) a decrease in Cdk2 protein kinase activity; and (c) an increase in the functional form of retinoblastoma protein (pRb). Transient transfection of a p202-encoding plasmid in Saos-2 cells, which do not harbor a wild-type p53 protein, resulted in an increase in p21 protein, which indicated that p202 could regulate expression of p21 protein independent of p53 protein. Moreover, we demonstrate that expression of p202 in these cells increased cell doubling time without accumulation of cells in a particular phase of the cell cycle. Taken together, these results are consistent with the possibility that p202 protein contributes to the cell growth retardation activity of the IFNs, at least in part, by modulating p21 protein levels.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IFNs exert antiproliferative effects on a variety of normal and tumor cell lines in culture and in vivo (1, 2, 3, 4, 5) , and IFN-inducible proteins are thought to mediate this activity (2 , 3) . p202 is an IFN-inducible, primarily nuclear, phosphoprotein (52-kDa) whose levels are increased in cultured cells in response to treatment with IFN-{alpha} (6, 7, 8) . In IFN-treated murine AKR-2B fibroblasts, the induced p202 is first detected in the cytoplasm; after a delay of about 30–36 h, it is detected in the nucleus (7) . In vitro p202 binds to double- and single-stranded DNA nonspecifically (9) , and a sequence conserved among the members of the 200-family proteins is sufficient for self-association of p202 (10) . Moreover, constitutive overexpression of p202 in transfected cell lines inhibits colony formation (7 , 11, 12, 13) , 3 and decreased levels of p202 in murine fibroblasts correlate with increased susceptibility to apoptosis (14) .

p21CIP1/WAF1/SDI1 (15, 16, 17, 18) is a member of the CIP/KIP family of CDK inhibitors (19) and is a transcriptional target of p53. p21 is a critical determinant of the G1 arrest in response to DNA damage (20, 21, 22) and may play an important role at the G2-M-phase transition (23 , 24) . However, p21 also responds to other signals independently of p53 (25 , 26) and has been implicated in terminal differentiation and senescence (18 , 27 , 28) . Furthermore, increased levels of p21 were reported to retard S phase progression primarily by inhibiting Cdks (29) .

IFN-treatment of cultured cells is shown to increase p21 mRNA and protein levels independent of p53 protein levels (30, 31, 32) and inhibit pRb phosphorylation (33, 34, 35) . However, it is not known which IFN-inducible protein(s) participates in the regulation of p21 protein levels. Furthermore, in the case of hematopoietic cells, including Daudi Burkitt’s lymphoma and M1 myeloid cells, IFN-treatment is known to arrest them at G0-G1 phase of the cell cycle (2) . However, IFN-{alpha} or -ß treatment of other cells is known to retard cell proliferation by prolonging all of the phases of the cell cycle (2 , 5 , 36) .

To investigate the molecular mechanism(s) by which p202 expression in transfected cells inhibits colony formation, we used a tetracycline-repressible promoter to control expression of p202 in murine AKR-2B fibroblasts. We found that the induced expression of p202 in asynchronously growing cells, which correlated with retardation of cell proliferation without a significant change in cell-cycle distribution, accompanied by an increase in p21 mRNA and protein levels, a decrease in Cdk2 kinase activity in extracts, and an increase in the functional form of pRb.4 Moreover, transient transfection of a p202-encoding plasmid in Saos-2 cells also resulted in an increase in p21 protein levels. Taken together, these observations are consistent with the possibility that p202 contributes to the cell growth retardation activity of the IFNs, at least in part, by modulating p21 protein levels.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Inducible Expression of p202.
Cell lines constitutively overexpressing p202 have been very difficult to establish because of the growth-inhibitory effects of p202 (7 , 11) .3 Consequently, we established stable murine AKR-2B cell lines (these cells express very low levels of endogenous p202) in which we could conditionally overexpress p202 under the control of a tetracycline-repressible promoter (37) . Three independent clones expressed increased levels of p202 in the absence of tetracycline, and I-33 clonal cell line was chosen for additional studies on the basis of its lower basal levels of p202. In the presence of tetracycline (2 µg per ml), expression of p202 in I-33 cells was comparable with endogenous levels in the parental AKR-2B cells; removal of tetracycline resulted in increased (3- to 4-fold) levels of p202 within 24 h (Fig. 1ACitation ; compare Lane 2 with Lane 3). As reported previously (7) , elevated levels of p202 were detected in both the cytoplasm and the nucleus, and the induced levels of p202 accumulated in the nucleus similar to IFN-treated AKR-2B cells (Fig. 1, B and C)Citation . Thus, these results suggested that nuclear accumulation of p202 was not altered in these cells.



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Fig. 1. Inducible expression of p202 and its subcellular localization. A, inducible expression of p202 in I-33 cells. I-33 cells were grown in the presence (Lane 2) or absence (Lanes 3-5) of tetracycline (2 µg/ml) for 24 h (Lane 3), 48 h (Lane 4), or 72 h (Lane 5). Total cell extracts were analyzed by immunoblotting using polyclonal anti-p202 antiserum (upper panel) or antiactin antibody (lower panel) for equal amounts of protein loading. As a control, extracts from AKR-2B cells were loaded in Lane 1. The p202 protein band is indicated by a white arrow. B, subcellular localization of inducible p202 in both nuclear and cytoplasmic fractions. I-33 cells were grown in the absence of tetracycline (Lanes 3 and 4) for 48 h and fractionated into the nuclear (Lane 3) and cytoplasmic (Lane 4) fractions. As a control, AKR-2B parental cells were treated with IFN (1000 units/ml) for 48 h and fractionated into the nuclear (Lane 1) and cytoplasmic (Lane 2) fractions. The fractions containing approximately equal amounts of proteins were analyzed by immunoblotting using polyclonal anti-p202 antiserum. The p202 protein band is indicated by the arrow. C, localization of inducible p202 in both nucleus and cytoplasm by indirect immunofluorescence microscopy. I-33 cells were grown (c and d) on glass coverslips in the presence (c) or absence (d) of tetracycline for 48 h. As a control, parental AKR-2B cells were grown (a and b) and left untreated (a) or treated with IFN (1000 units/ml) for 48 (b). Cells were processed for indirect immunofluorescence microscopy using immunoaffinity-purified anti-p202 antibodies. Anti-p202 antibodies were detected by rhodamine-conjugated antirabbit sheep antibodies.

 
Induced Expression of p202 Retards Cell Proliferation.
To study the effects of p202 expression on cell proliferation, we seeded equal number of cells in the presence or absence of tetracycline in culture medium and counted cells (the average number of cells from at least two plates were used for proliferation rate determinations) at the indicated times. As shown in Fig. 2Citation , uninduced cells continued to proliferate in the presence of tetracycline, whereas proliferation of cells in which p202 expression was induced slowed down considerably. The decrease in proliferation rate was apparent around 48 h after removal of tetracycline and correlated with the levels of p202 in these cells. Induction of p202 in subconfluent cultures of I-33 cells under the growth conditions tested did not result in cell death (as measured by the number of dead floating cells). Thus, these observations, which are consistent with our previous findings (14) , provide support to the conclusion that a decrease in cell count of I-33 cells after p202 induction (see Fig. 2Citation ) was due primarily to retardation in the proliferation rate and not to cell death.



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Fig. 2. Retardation of cell proliferation after p202 induction. Growth rates of I-33 cells in the presence or absence of tetracycline were measured at the indicated times as described in "Materials and Methods." Two independent samples were counted for each time point, and the average of two experiments was plotted against the number of days after the removal of tetracycline.

 
To test whether induced expression of p202 accompanied any alterations in the cell cycle distribution of I-33 cells, we grew cells in the absence of tetracycline for the indicated times and analyzed them by flow cytometry. As shown in Table 1Citation , induced expression of p202 in asynchronous cultures of I-33 cells in two experiments did not appear to result in accumulation of cells in any particular phase of the cell cycle.


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Table 1 Induced expression of p202 in I-33 cells and cell-cycle distribution

 
p202 Induction Is Accompanied by an Increase in p21.
IFN-treatment of cultured cells has been reported to increase p21 mRNA and protein levels (30, 31, 32) . Therefore, we explored whether induced expression of p202 in I-33 cells correlated with the levels of p21 mRNA. As seen in Fig. 3ACitation , the removal of tetracycline from the medium of I-33 cells was followed by about a 3-fold increase in steady-state levels of p21 mRNA (compare Lane 2 with Lanes 3 and 4). Moreover, as shown in Fig. 3BCitation , induced expression of p202 was accompanied by a marked increase in p21 protein levels (compare Lane 2 with Lanes 3–5), which accumulated in the nucleus (Fig. 3C)Citation . Similar results were obtained when p202 expression was induced in cells of two stable L929 cell lines.3



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Fig. 3. Increase in steady-state levels of p21 mRNA and protein after p202 induction. A, upper panel, I-33 cells were grown in the presence (Lane 2) or absence (Lanes 3–5) of tetracycline for 24 h (Lane 3), 48 h (Lane 4), or 72 h (Lane 5). Total cytoplasmic RNA was analyzed by Northern blot analysis using human p21 cDNA probe as described in "Materials and Methods." As a control, total cytoplasmic RNA from the parental AKR-2B cells was also included in Lane 1. Arrow, the p21 mRNA band. Lower panel, the cytoplasmic RNA applied was visualized with ethidium bromide stain for equal amounts of RNA. B, as shown in Fig. 1ACitation , I-33 cells were grown in the presence (Lane 2) or absence (Lanes 3–5) of tetracycline for 24 h (Lane 3), 48 h (Lane 4), or 72 h (Lane 5). Total protein extracts were analyzed by immunoblotting using anti-p21 antibodies (upper panel) or antiactin antibody (lower panel) for equal amounts of protein loadings. As a control, extracts from the parental AKR-2B cells were also included in Lane 1. The p21 protein band is indicated by the white arrow. C, I-33 cells were grown on glass coverslips in the presence (Tet+) or absence (Tet-) of tetracycline for 48 h. As a control, AKR-2B cells (upper panel) were left untreated (Cont.) or treated with IFN (IFN) for 48 h. Cell were processed for indirect immunofluorescence microscopy using anti-p21 antibodies as described in "Materials and Methods."

 
Because elevated levels of p21 protein are shown to retard cell proliferation by inhibiting the activity of cdks (29 , 38 , 39) and IFN-{alpha} treatment of cells was reported to inhibit Cdk2 protein kinase activity by increasing p21 protein levels (30 , 31) , we tested whether induced expression of p202 correlated with a decrease in Cdk2 protein kinase activity. As shown in Fig. 4Citation (upper panel), induced expression of p202 followed a marked (50–70%) decrease in Cdk2 histone protein kinase activity. Taken together, these observations are consistent with the possibility that increased levels of p21 protein, which accompanied induction of p202 in these cells, contribute to a decrease in Cdk2 protein kinase activity.



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Fig. 4. Increase in p21 protein levels after p202 induction in I-33 cells correlates with a decrease in Cdk2-associated histone protein kinase activity. I-33 cells were grown in the presence (Lane 2) or absence (Lanes 3–6) of tetracycline for 12 h (Lane 3), 24 h (Lane 4), 36 h (Lane 5), or 48 h (Lane 6). Total protein extracts containing equal amounts of proteins were subjected to immunoprecipitation using polyclonal anti-Cdk2 antibodies followed by protein kinase assays using histone H1 as a substrate as described in "Materials and Methods." The upper panel indicates autoradiogram resulting from phosphorylation of histone H1, and the lower panel indicates Coomassie blue staining of the same gel for equal amount of histone H1 in the reaction.

 
To rule out the possibility that the decrease in Cdk2 protein kinase activity in extracts of p202-expressing I-33 cells was due to a decrease in Cdk2 or associated cyclin protein levels, we examined their protein levels in extracts. As shown in Fig. 5Citation , induced expression of p202 did not significantly alter detectable protein levels of Cdk2, cyclin A, or cyclin E. These observations indicated that induced expression of p202 did not affect protein levels of Cdk2/cyclin A or E complexes. Furthermore, in these cells protein levels of cyclin D1 (Fig. 5)Citation , Cdk4, and p16 were also not altered (data not shown).



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Fig. 5. Analyses of cell cycle-regulatory proteins after p202 induction by Western blot analysis. I-33 cells were grown in the presence (Lane 2) or absence (Lanes 3 and 4) of tetracycline for 24 h (Lane 3) or 48 h (Lane 4). Total protein extracts containing equal amounts of proteins (50 µg) were analyzed by Western blot analysis using anti-Cdk2, anticyclin A, anticyclin E, or anticyclin D1 as described in "Materials and Methods." As a control, extracts from the parental AKR-2B cells were also included in Lane 1. The Cdk2 (cdk2), cyclin A, cyclin E, and cyclin D1 protein bands are indicated.

 
Increase in p21 Protein Levels Is Independent of p53.
Because constitutive low-level expression of p202 in murine 10T1/2 cells resulted in 2.4-fold increase in p53 protein levels (40) , we tested whether in I-33 cells, which possess a wild-type p53 protein,3 induced expression of p202 accompanied an increase in p53 protein levels. We found that induced levels of p202 in I-33 cells correlated with a moderate decrease in p53 protein levels (data not shown). This observation is not unexpected because p202 expression in transfected cells inhibits E2F (E2F-1/DP-1 and E2F-4/DP-1) activity (12 , 41) , and inhibition of E2F activity was shown to decrease p53 levels (42) . Moreover, overexpression of E2F-1 was shown to induce accumulation of p53 (43) . Taken together, these results indicated that in I-33 cells, increase in p21 protein levels was independent of an increase in p53 protein levels.

To test further whether p202 expression could alter p21 protein levels independent of p53 protein levels, we transiently transfected human Saos-2 cells (these cells harbor a mutant p53) with a p202-encoding plasmid (pCMV-202) and analyzed cell extracts for p202 and p21 protein expression. As shown in Fig. 6Citation (upper panel), transfection of Saos-2 cells with pCMV-202 but not with pCMV resulted in an increase in p202 levels. Moreover, increased levels of p202 correlated with increased levels of p21 protein (lower panel), which suggested that overexpression of p202 in these cells could increase p21 protein levels independent of a wild-type p53 protein.



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Fig. 6. Transient transfection of a p202-encoding plasmid in human Saos-2 cells increases p21 protein levels. Cells were grown in 150-mm plates and transfected with plasmid pCMV-202 DNA (10 µg/ml; Lane 2) or equal amount of plasmid pCMV DNA (Lane 1). Cells were lysed after 48 h of transfections, and the extracts containing equal amounts of proteins (40 µg) were analyzed by immunoblotting using anti-p202 (upper panel) or anti-p21 (lower panel). Arrows, p202 and p21 protein bands.

 
Induction of p202 Is Followed by an Increase in the Functional Form of pRb.
IFN-treatment of several cell lineages has been reported to alter levels of cell-cycle regulatory proteins, including the pRb protein (44) . Therefore, we examined whether induced levels of p202 in I-33 cells correlated with an alteration in the functional form (the hypophosphorylated form) of pRb. As seen in Fig. 7Citation , removal of tetracycline from the medium of I-33 cells accompanied a moderate increase in pRb protein levels (compare Lane 2 with Lanes 4 or 5). Moreover, a significant fraction of pRb migrated as the hypophosphorylated form [as noted previously (45) , in extracts prepared from murine cells, pRb migrated as only two protein bands]. These observations indicated that induction of p202 in I-33 cells was accompanied by an increase in the functional form of pRb.



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Fig. 7. Inducible expression of p202 correlates with an increase in the functional form of pRb. I-33 cells were grown in the presence (Lane 2) or absence (Lanes 3–5) of tetracycline for 24 h (Lane 3), 48 h (Lane 4), or 72 h (Lane 5) as described in Fig. 1ACitation . Total cell extracts containing equal amounts of proteins (as shown in Fig. 1A and 3BCitation ) were analyzed by immunoblotting using anti-pRb antibodies. As a control, total cell extracts from the parental AKR-2B cells were also included in Lane 1. White arrow, the hypophosphorylated form of pRb protein; black arrow, the hyperphosphorylated form of pRb protein.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Constitutive overexpression of p202 in transfected cells inhibits colony formation (7 , 11 , 13) , and the resulting clonal cell lines that constitutively express moderate levels (2 to 3-fold above the endogenous levels) of p202 are very difficult to maintain in culture (7 , 11, 12, 13) .3 Moreover, these observations made it conceivable that the inhibition of colony formation by p202 overexpression in these transfected cells may have been a consequence of cell death and/or retarded cell proliferation. Therefore, to study the effects of p202 expression on cell proliferation, we used a tetracycline-repressible promoter to control expression of p202. Using these cell models, we were able to demonstrate that inducible expression of p202 in murine AKR-2B cells (I-33 cells) resulted in retardation of cell proliferation without significantly altering the cell-cycle distribution and accompanied by: (a) an increase in steady-state levels of p21 mRNA and protein; (b) a decrease in Cdk2 protein kinase activity; and (c) a moderate increase in the functional form of pRb. Moreover, our observations reported herein shed light on a molecular mechanism by which p202 may contribute to IFN’s ability to retard cell proliferation by modulating p21 protein levels.

Because increased levels of p21 protein are known to inhibit activity of Cdks by interacting with Cdk complexes (19) , it is likely that increased levels of p21 protein in I-33 cells, which accompanied induced levels of p202, contribute to the retardation of cell proliferation cycle by decreasing the activity of Cdks. Because AKR-2B cells possess a normal pRb (11) , a decrease in phosphorylation of pRb was predictable (Fig. 7)Citation . It may be worthwhile to note that IFN- treatment of the parental AKR-2B cells also resulted in retardation of cell proliferation without a significant accumulation of cells in a particular phase of the cell cycle (data not shown) and was accompanied by an increase in p21 protein levels (Fig. 3CCitation , upper panel).

Inducible expression of p202 in I-33 cells accompanied a moderate increase in the steady-state levels of p21 mRNA (Fig. 3A)Citation and a large increase in p21 protein levels (Fig. 3B)Citation . Because p21 mRNA and protein expression are shown to be uncoupled in response to various stimuli (46) [including in response to IFN-treatment (30) ] in a variety of cells and p21 protein levels are found to be substantially modulated on the posttranscriptional level (46) [including the stabilization of p21 protein (47 , 48) ], it is possible that similar mechanisms may also contribute to increased p21 protein levels in these cells. Consistent with this notion, we found that induced levels of p202 protein in I-33 cells seem to stabilize p21 protein (data not shown). Taken together, it is conceivable that p202 protein regulates p21 protein levels by both transcriptional and posttranscriptional mechanisms.

In our previous study (40) , involving transient transfection of a p202-encoding plasmid (pCMV-202) in human C-33A cells, overexpression of p202 inhibited the p53-stimulated transcription of a reporter gene whose transcription was driven by a 2.4-kb DNA fragment from the 5'-flanking region of p21 gene. However, constitutive low-level expression of p202 in stably transfected pools of 10T1/2 cells, which accompanied increased levels (2.4-fold) of p53 protein, did not significantly alter the endogenous levels of p21 protein (40) . However, overexpression of p202 in I-33 cells did not accompany an increase in p53 protein levels. Furthermore, inducible overexpression of p202 in murine AKR-2B (I-33 cells) and L929 cells but not in human K562 or 293 cells3 was found to accompany increased p21 protein levels. Thus, taken together, these observations make it conceivable that the ability of p202 to modulate p21 protein levels depends on the cellular context. Additional work will be needed to examine this possibility.

E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription by p21 (49 , 50) . Our earlier observations that: (a) IFN-treatment or p202 expression in transfected AKR-2B cells inhibits the sequence-specific DNA-binding of E2F complexes in extracts (12 , 41) ; and (b) overexpression of E2F-4 in AKR-2B cells overcomes p202-mediated inhibition of colony formation (41) support the hypothesis that p202 expression slows down cell proliferation, at least in part, by inhibiting the pRb-E2F pathway. In addition, the model predicts that increased levels of p202 may be tolerated in cells because of overexpression of E2Fs (E2F-1 to E2F-5). Consistent with this prediction, we found that I-33 cells that inducibly overexpressed p202 had higher levels of E2F-1, E2F-3, and E2F-4.3

Although the molecular basis for our observation that the increase in induced levels of p202 in I-33 cells accompanied by an increase in steady-state levels of p21 protein did not result in the accumulation of cells in the G1 phase of the cell cycle remains to be explored, it may be worthwhile to note that higher levels of p21 protein were reported in human glioma cells and may account for their slow proliferation rate without an arrest in the G1 phase of the cell cycle (51) . Moreover, regulated ectopic expression of cyclin D1 was reported to induce transcriptional activation of the p21 gene without altering cell cycle progression (52) . Therefore, taken together, our observations reported here make it conceivable that in I-33 cells, increased p21 protein levels were not sufficient to arrest cells at the G1 phase of the cell cycle. This is consistent with a recent report in which the increase in cellular p21 levels did not result in growth arrest of cells in a particular phase of the cell cycle (53) . Because overexpression of E2F-1 can bypass a G1 arrest caused by the inhibition of G1-specific Cdk activity and by {gamma}-irradiation (54 , 55) and E2F family members can up-regulate expression of p21 protein (56) , higher levels of E2Fs (E2F-1, E2F-3, and E2F-4) detected in extracts prepared from I-33 cells after p202 induction may also, in part, account for the increased accumulation of p21 protein without arrest of cells in the G1 phase.

IFN-{alpha} treatment of cells is known to increase levels of p21 (30, 31, 32) , p15 (31) and p19 (57) Cdk inhibitors. Therefore, it is likely that an increase in p21 protein levels is not the only mechanism by which IFNs retard cell proliferation. Moreover, p202 expression in transfected cells modulates the transcriptional activity of E2Fs (E2F-1 and E2F-4; Refs. 12 , 41 ), p53 (40) , AP-1 (c-Fos and c-Jun), and NF-{kappa}B (p50 and p65; Ref. 13 ), making it conceivable that p202 retards cell proliferation by regulating levels of numerous gene products. Further work is in progress to delineate the contributions of molecular changes that occur after induced expression of p202 in the retardation of cell proliferation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Plasmids.
The pCMV-202 and pCMV plasmids have been described previously (12) . The pUHD15–1-neo plasmid contains the tTa transactivator gene and the neomycin gene as described in (58) . p202 cDNA was cloned into the BamHI site of plasmid pUHD-3. The cloning sites of the resulting plasmid (Tet-202) were sequenced to rule out any cloning artifacts. pUHD15–1-neo and Tet-202 allowed conditional expression of p202 as described previously (37) .

Cell Lines and Transfections.
The AKR-2B cells (originally a gift from Dr. H. L. Moses, Vanderbilt University, Nashville, TN) or human Saos-2 cells (from American Type Culture Collection) were grown in DMEM supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO2. If so indicated, recombinant IFN (1000 units/ml; Universal Type-1, Research Diagnostic, Inc., Flanders, NJ) was added to subconfluent cultures as described previously (7) . To generate stable cell lines expressing p202 inducibly, a one-step procedure was used. AKR-2B cells were transfected with pUHD15–1-neo and plasmid Tet-202 (in 50:1 ratio), and clones were selected and maintained in 500 and 2 µg/ml G418 (Geneticin, Life Technologies) and tetracycline, respectively. To test for cloned cell lines capable of inducing expression from the tetracycline promoter, the cells were grown in the presence or absence of tetracycline in the growth medium supplemented with serum tested for the absence of Tet (Tet System Approved, Clontech Lab., Inc.). Cells were extracted at the indicated times, and expression of the p202 protein was determined by immunoblotting as described previously (7) . Of 38 clonal AKR-2B cell lines screened for the inducible expression of p202, 3 cell lines were found to overexpress p202 upon withdrawal of tetracycline. The I-33 cell line was chosen for additional studies on the basis of its lower basal levels of p202.

Transfections were performed using the calcium chloride method (59) . Subconfluent cultures of Saos-2 cells were transfected with pCMV-202 plasmid (10 µg/150-mm cell culture plate) or pCMV. After 48 h of transfections, cells were lysed and extracts containing equal amounts of proteins were used to detect p202 and p21 protein levels by immunoblotting.

Proliferation Rate and Cell-Cycle Analyses.
To determine the rate of cell proliferation, 1 x 105 cells were seeded per 100-mm plate with or without tetracycline. The medium was replaced with fresh medium with or without tetracycline every 48 h. At indicated times, cells were trypsinized and collected individually from at least two plates. Cells from each plate were counted with Coulter counter at least twice. The average number of cells from at least two plates were used for proliferation rate determinations.

For cell-cycle analysis, 2.5 x 105 cells were seeded per 100-mm plate with or without tetracycline. The medium was replaced with fresh medium with or without tetracycline every 48 h. At indicated times, cells were trypsinized and fixed with 2 ml of 70% ethanol for at least 30 min. For flow cytometry, the fixed cells were centrifuged at 1500 x g for 5 min and resuspended in 1 ml of PBS solution containing 50 µg/ml RNase A (Sigma) and 10 µg/ml propidium iodide (Sigma). The stained cells were analyzed by flow cytometry (FACSCaliber, Becton Dickinson) within 4 h. The percentage of cells in various cell cycle phases was determined by using the CellFit program.

Northern Blot Analysis.
Cells of the I-33 cell line were grown in the presence or absence of tetracycline (2 µg/ml), and cytoplasmic RNA was prepared as described previously (12) . RNA samples were subjected to Northern blot analysis as described previously (60) . A labeled human cDNA probe for p21 (kindly provided by Dr. J. R. Smith, Baylor College of Medicine, Houston, TX) was hybridized to RNA at the desired temperature for 1–1.5 h using rapid hybridization buffer (Clontech Inc.) as suggested by the supplier.

Western Blot Analysis.
Cells were collected from plates in PBS, resuspended in modified radioimmunoprecipitation assay lysis buffer (11) supplemented with protease inhibitors (leupeptin, 50 µg per ml; Pepstatin A, 50 µg per ml; PMSF, 1 mM), and incubated at 4°C for 30 min. Lysates were sonicated briefly before centrifugation at 14,000 x g in a microfuge for 10 min. Supernatants were collected, and equal amounts of proteins were processed for immunoblotting as described previously (7) . Polyclonal anti-p202 antiserum and the immunoaffinity-purified anti-p202 antibodies have been described previously (7) . Monoclonal (F-5) or rabbit polyclonal (C-19) anti-21 antibodies, anti-Cdk2 (M2), anti-pRb (C-15), and anticyclin E (M-20) were from Santa Cruz Inc. Anticyclin A was from PharMingen.

Protein Kinase Assays.
Cell extracts containing equal amounts of proteins were subjected to immunoprecipitations using polyclonal antibodies against Cdk2 protein kinase (M2; Santa Cruz Biotech., Inc.). The immunoprecipitates bound to Protein A/G Sepharose beads (Pierce) were first washed 5 times with radioimmunoprecipitation assay buffer and finally with kinase buffer. Beads (10 µl) were incubated with histone H1 (1 µg) in kinase reaction (25 µl) supplemented with 10 µCi [{gamma}-32P]ATP. The reaction products were analyzed by SDS-PAGE followed by autoradiography.

Indirect Immunofluorescence Microscopy.
Immunofluorescence microscopy was performed essentially as described previously (61) . Cells were incubated with immuno-affinity purified anti-p202 antibodies (7) , and p202 was detected with affinity-purified antirabbit sheep IgG antibodies conjugated to rhodamine (Sigma). After washing, cells were mounted in 3% propyl gallate in 70% glycerol and viewed under the microscope.


    Acknowledgments
 
We thank Manfred Gossen and Hermann Bujard for providing plasmids and suggestions, Peter Lengyel for suggestions and discussions, and Gordon B. Mills for discussions and a critical reading of this article.


    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 NIH Grant CA69031 (to D. C.) and, in part, by funds from the Clayton Foundation for Research (to J. U. G.). Back

2 To whom requests for reprints should be addressed, at Department of Radiotherapy, Loyola University Chicago-Hines VAMC, 5th Avenue and Roosevelt Road (114-B), Hines, IL 60141. Phone: (708) 343-7200, Ext. 3407; Fax: (708) 216-2647; E-mail:dchoube{at}rdth2.rdth.luc.edu Back

3 D. Choubey, unpublished data. Back

4 The abbreviations used are: pRb, retinoblastoma protein; Cdk, cyclin-dependent kinase. Back

Received for publication 5/15/98. Revision received 12/ 3/98. Accepted for publication 12/ 3/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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