©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Potential Role of WAF1/Cip1/p21 as a Mediator of TGF- Cytoinhibitory Effect (*)

(Received for publication, November 23, 1994 )

Chuan-Yuan Li(§)(¶) Laurent Suardet (§) John B. Little

From the Laboratory of Radiobiology, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression of many types of human cells by arresting them in the G(1) phase of the cell cycle. The arrest is mediated through interactions of various cyclin-dependent protein kinases (CDKs) and their inhibitors. We demonstrate that treatment with TGF-beta induces increased levels of WAF1/Cip1/p21, a potent inhibitor of various cyclin-CDK kinase activities, in two colon cancer cell lines (LS1034 and LS513), which are sensitive to TGF-beta-induced growth arrest. The induction in at least one of these cells lines (LS1034, p53-) is p53-independent. No WAF1 induction was observed after TGF-beta treatment in a third cell line (HT-29), which is completely insensitive to the cytoinhibitory effect of TGF-beta. In both LS513 and LS1034, WAF1 induction correlated with reduced cyclin E-associated kinase activity in vitro and suppression of the retinoblastoma susceptibility gene (Rb) protein phosphorylation in vivo. In addition, WAF1 was physically associated with cyclin E in the two sensitive cell lines. These results suggest that WAF1/Cip1/p21 is a mediator of cellular sensitivity to TGF-beta.


INTRODUCTION

In many mammalian cell types, TGF-beta (^1)treatment promotes growth arrest in G(1)(1, 2, 3) that is thought to be mediated through the many elements of the complex cell cycle machinery(4) . Among these elements, G(1) cyclin-CDK complexes, which include cyclin E-CDK2, cyclin D-CDK4, and cyclin D-CDK6, play important roles. It is now widely accepted that these complexes are needed during G(1) to phosphorylate and thus inactivate the retinoblastoma susceptibility gene (Rb), which inhibits cell cycle progression in its underphosphorylated state(4, 5) . It is thought that TGF-beta causes G(1) arrest by inhibiting G(1) cyclin-CDK kinase activities, thereby suppressing Rb phosphorylation. Consistent with this model, it has been shown that TGF-beta can inhibit cyclin E-CDK2 kinase activity (6) and cause the accumulation of Rb in the underphosphorylated form(7, 8) . In addition, it has been reported that TGF-beta treatment reduces the level of CDK4 and that overexpression of the CDK4 gene precludes the TGF-beta cytoinhibitory effect in a sensitive cell line(9) . The exact mechanism by which TGF-beta inhibits CDK-associated kinase activities has remained elusive, however, until the recent discoveries of several mammalian CDK kinase inhibitors, which include WAF1 (also called p21/Cip1/Sdi1)(10, 11, 12, 13) , p16(14, 15) , p27(16, 17) , and p15(18) . Two of these, p27 and p15, have been implicated as effectors of TGF-beta-induced cell cycle arrest (16, 18) .

We sought to examine whether WAF1 is involved in mediating TGF-beta-induced growth arrest in human cells, as it is a potent inhibitor of a wide range of cyclin-CDK complexes. To this end we employed three colon cancer cell lines previously characterized as having varying degrees of sensitivity to the cytoinhibitory effect of TGF-beta1(19) : HT-29, which is completely insensitive; LS513, which is moderately sensitive; and LS1034, which is very sensitive to TGF-beta1.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

The cell lines and their culturing conditions are described in detail elsewhere(19) . Antibodies against cyclin E (HE67), Rb, and WAF1 were purchased from Pharmingen. TGF-beta1 was purchased from R& Research Systems.

Measurement of TGF-beta Production and Sensitivity

The TGF-beta sensitivity of the cell lines was measured as described(19) . The production of TGF-beta by the cell lines was measured by sandwich enzyme-linked immunosorbent assay (SELISA)(20) .

Single Strand Conformational Polymorphism and Direct Sequencing Analysis of p53 Mutations

p53 status in these cell lines was determined by first screening P-labeled PCR products of exons 5-9 of the p53 gene by use of the single strand conformational polymorphism technique and then sequencing the PCR products with abnormal bandshifts with the direct sequencing kit from U. S. Biochemical Corp. The primers and PCR conditions were described in (26) .

Ionizing Radiation

Radiation exposure was carried out with a Co irradiator at a dose rate of 15.8 centi-gray/s.

Cell Cycle Analysis

Cell cycle analysis was performed by fluorescence-activated cell sorter analysis following the protocol of Kasten et al.(22) . Cells were labeled for 1 h with bromodeoxyuridine 17 h after irradiation and stained with propidium iodine and fluorescein-labeled anti-bromodeoxyuridine antibody prior to fluorescence-activated cell sorter analysis. TGF-beta1-treated cells were analyzed in the same fashion after 17 h of treatment.

Northern Analysis

Total RNA extraction was from cells grown in a 10-cm Petri dish to 80% confluency. RNA extraction was performed by first suspending the cells in a solution with 10 mM Tris, 10 mM vanadyl ribonucleoside complex, and 1% Nonidet P-40 detergent and then lysing them in 10 mM Tris, 150 mM NaCl, 1% SDS, followed by two extractions of phenol chloroform and one extraction of chloroform. The RNA was then separated by agarose gel electrophoresis in MOPS buffer and transferred to nitrocellulose membranes. Standard Northern analysis of WAF1 was performed by use of a WAF1 probe derived by PCR fragment amplification of cDNA from a human fibroblast cell line using the primers A (5` AGTTCCTTGTGGAGCCGGAGC 3`) and B (5` TGTAGAGCGGGCCTTTGAGGC 3`).

Western Analysis

Protein extraction was performed from cells grown to 80% confluency. After lysis of the cells directly in the Petri dish in a buffer consisting of 50 mM Tris-HCl at pH 7.4, 250 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na(3)VO(4), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 1 mM benzamide, 10 µg/ml trypsin inhibitor (all from Sigma), the lysed cells were centrifuged at 16,000 times g for 5 min, and the supernatants were kept and analyzed. About 60 µg of protein were loaded into each lane in a 15% polyacrylamide gel, electrophoresed, and blotted onto polyvinylidene difluoride (Millipore) membrane. A monoclonal antibody to WAF1 (Pharmingen) was used as the primary antibody. The signal was then developed with the ECL system from Amersham Corp.

Histone H1 Kinase Assay

The lysis buffer (21) consists of 40 mM Hepes, pH 7.5, 250 mM NaCl, 15 mM MgCl(2), 1% Triton X-100, 5 mM NaF, beta-glycerophosphate, 2 mM EGTA, and 2 mM EDTA. The lysate from two T-75 flasks were centrifuged at 16,000 times g for 5 min, and the supernatant was collected. A monoclonal antibody against cyclin E (clone HE67 from Pharmingen) was used to immunoprecipitate cyclin E-associated kinase. After washing the immunoprecipitate several times with the wash buffer (20 mM Hepes, 20 mM MgCl(2), and 15 mM EGTA), the immunoprecipitate was incubated with kinase buffer (20 mM Hepes, 20 mM MgCl(2), 15 mM EGTA, 1 mM dithiothreitol, 0.01 µg/µl protein kinase A inhibitor peptides, 0.5 µg/µl histone H1, 50 µM ATP, 0.1 µCi/µl [-P]ATP) for 20 min at 30 °C. The whole mixture was then combined with sample loading buffer, boiled, and loaded into 12% polyacrylamide gel and electrophoresed. The gel was then dried and exposed to a Kodak X-OMAT film for 48 h.

Metabolic Labeling and Immunoprecipitation

These were done according to procedures described in (12) .


RESULTS AND DISCUSSION

Dose-response curves for treatment of the three colon cancer cell lines with TGF-beta1 are shown in Fig. 1. As can be seen in Table 1, SELISA measurement of TGF-beta1 production revealed an inverse relationship between TGF-beta1 sensitivity and its production in these cells. However, the correlation may not be of functional significance as none of the TGF-beta1 produced was in its biologically active form(19) . Other characteristics of these cell lines, including their p53 status and cell cycle analysis after treatment with TGF-beta1 and ionizing radiation, are also shown in Table 1.


Figure 1: TGF-beta sensitivity of the three colon cancer cell lines. The methods employed to measure the cytoinhibitory effects of TGF-beta in such cell lines have been described in detail elsewhere(19) .





In order to determine sensitivity of these cell lines to the induction of a G(1) arrest, cell cycle analysis after exposure to either TGF-beta1 or ionizing radiation exposure was carried out by analysis of fluorescence-activated cell sorting (Table 1). HT-29 demonstrated no G(1) arrest after either TGF-beta1 or radiation exposure, whereas LS513 showed a significant G(1) arrest after -irradiation but only a moderate G(1) arrest after TGF-beta1 treatment. LS1034 showed no G(1) arrest after irradiation but a significant G(1) arrest in TGF-beta1-treated cells. The results obtained with ionizing radiation is consistent with previous reports that p53 status determines whether a G(1) arrest will occur(22) . The TGF-beta-induced G(1) arrest observed in LS1034 and its sensitivity to TGF-beta in the absence of p53 indicate that the TGF-beta cytoinhibitory effect in this cell line is not mediated by p53.

Cells exposed to either radiation or TGF-beta1 were assayed for WAF1 mRNA expression by Northern blot analysis (Fig. 2A). Almost no WAF1 mRNA was observed after either radiation or TGF-beta1 exposure in HT-29 cells, while in LS513 there was a clear induction of WAF1 mRNA in irradiated cells, consistent with the role of p53 as the positive transcriptional regulator of the WAF1 gene after irradiation. A small induction of WAF1 mRNA was observed in LS513 after exposure to TGF-beta1. In LS1034, on the other hand, a significant induction occurred after TGF-beta1 treatment, whereas no WAF1 expression was observed after irradiation (Fig. 2A). As p53 is mutated in LS1034, we conclude that the induction of WAF1 in this cell line is p53-independent. Western analysis of the three cell lines confirmed the Northern analysis (Fig. 2B). A time course study of the induction of WAF1 expression in LS1034 by TGF-beta1 treatment was also performed (Fig. 3). As can be seen in Fig. 3, induction is clearly visible beginning at 3 h after exposure to TGF-beta1, reaching its peak level by 6 h. Induction remained steady up to at least 48 h, dropping to near background level at 96 h.


Figure 2: A, Northern analysis of WAF1/p21 mRNA level in cell lines HT-29, LS513, and LS1034. For radiation exposure, the cells were given doses of 2 and 10 gray (Gy). For TGF-beta treatment, the cells were incubated with 60 ng/ml TGF-beta1. RNA were extracted 5 h after exposure to either radiation or TGF-beta. Twenty µg of the total RNA were loaded into each lane for analysis. Equal loading of the RNAs is indicated in the lowerpanel, in which the 18 S ribosomal RNA band is shown prior to capillary transfer to a nitrocellulose filter (Schleicher and Schuell). B, Western analysis of WAF1 protein levels. Proteins were isolated at the same time when RNA was isolated. About 50 µg of protein was loaded into each lane.




Figure 3: A Northern analysis of the time course of induction of WAF1 mRNA by TGF-beta1. RNA was extracted at various time points after incubation with TGF-beta (as indicated at the top of each lane). Twenty µg of the total RNA were loaded into each lane for analysis.



As WAF1 has been shown to be induced by radiation and implicated as an inhibitor of cyclin kinase activities in irradiated cells(23, 24) , the cyclin E-CDK2 complex was assayed for its kinase activity after immunoprecipitation against cyclin E by use of histone H1 as the substrate. As can be seen in Fig. 4, no reduction in the kinase activity of the complex was observed after irradiation in the HT-29 cell line. In LS513, the activity of the cyclin complex was reduced significantly by radiation and to a lesser extent by TGF-beta1 treatment. In LS1034, the kinase activity was almost completely abolished after TGF-beta1 treatment, while the change was much less significant in the irradiated cells.


Figure 4: Histone H1 kinase activity of the cyclin E complex after 16 h of either radiation or TGF-beta treatment.



Immunoprecipitation of [S]methionine-labeled cell lysates using a monoclonal antibody against cyclin E indicated that a protein of molecular mass 21 kDa (as determined by polyacrylamide gel electrophoresis) coprecipitated with cyclin E in both LS1034 and LS513 but not in HT-29 cells (data not shown).

In order to further examine cyclin-CDK kinase activity and to determine if TGF-beta1 sensitivity of these colon cancer cell lines is mediated through the Rb protein, Western analysis was performed using a monoclonal antibody that recognizes both the underphosphorylated and hyperphosphorylated forms of Rb. As can be seen in Fig. 5, the Rb protein was in both the hyper- and hypophosphorylated forms in the untreated control cultures of all three cell lines, with the majority in the hyperphosphorylated form. This is consistent with the fact that most of the cells are actively proliferating. In HT-29, which has a mutated form of p53 and is insensitive to TGF-beta1, Rb remained in the hyperphosphorylated form in the majority of the cells after both TGF-beta1 and radiation exposure, similar to control untreated cells (Fig. 5). In LS513, most of the cells showed an underphosphorylated form of the Rb gene product 16 h postirradiation, which is consistent with the wild type p53 status of the cell line. After TGF-beta1 treatment, a significant albeit smaller increase in the amount of the underphosphorylated form of Rb was observed, which correlates well with the moderate sensitivity of the cell line. In LS1034, however, the majority of the cellular Rb protein is converted into the underphosphorylated form after TGF-beta1 treatment, while no significant change in the distribution of Rb was observed after radiation (Fig. 5). Thus, the mechanisms of growth arrest induced by both ionizing radiation and TGF-beta1 are consistent with a model in which both WAF1 and Rb are essential players.


Figure 5: Western analysis of the retinoblastoma protein (Rb). In each cell line, there were three different treatment groups: control, TGF-beta-treated, or irradiated. The protein was extracted 16 h after radiation and TGF-beta exposure. A monoclonal antibody to Rb (G1-245) from Pharmingen was used as the primary antibody. The polyacrylamide gel was used at 8%.



Northern analysis of p16 mRNA expression revealed no correlation with either the p53 status or cellular sensitivity to TGF-beta1 (data not shown). In fact, the mRNA expression of the p16 gene was detected only in HT-29, which is completely insensitive to TGF-beta1 and has no normal p53 activity. Expression was not altered in either radiation or TGF-beta1-treated cells. Experiments are also under way to test whether the newly identified p27 and p15 kinase inhibitors are involved in mediating the TGF-beta sensitivity of these colon cancer cell lines. It is possible that all these factors participate in mediating the cellular sensitivity to TGF-beta1 by inhibiting different cyclin-CDK complexes.

In summary, these results provide evidence that TGF-beta1 can induce WAF1 in a manner that correlates with its cytoinhibitory effect and that cell cycle arrest induced by both radiation and TGF-beta1 appears to be mediated through WAF1, but the upstream signal transduction pathways are different as the former is mediated through p53 and the latter not. Further understanding of the various cell cycle regulators involved will greatly facilitate our understanding of the complex pathways constituting cellular sensitivity to TGF-beta.


FOOTNOTES

*
This work was supported by Research Grant CA-47542 and Center Grant ES0002 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

To whom correspondence should be addressed: Laboratory of Radiobiology, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Tel.: 617-432-1184; Fax: 617-432-0107.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; SELISA, sandwich enzyme-linked immunosorbent assay; CDK, cyclin-dependent protein kinase; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.