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Cell Growth & Differentiation Vol. 10, 601-610, September 1999
© 1999 American Association for Cancer Research

Loss of Cell Cycle Control by Deregulation of Cyclin-dependent Kinase 2 Kinase Activity in Evi-1 Transformed Fibroblasts1

Anna Kilbey2, Victoria Stephens and Chris Bartholomew2,, 3

Cancer Research Campaign Beatson Laboratories, Beatson Institute for Cancer Research, Glasgow, Scotland


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Evi-1 transcriptional repressor protein has two distinct zinc finger DNA binding domains designated ZF1 and ZF2 and is implicated in the progression of human and murine leukemias, in which it is abnormally expressed. In this report, we show that Evi-1-expressing Rat1 fibroblasts are anchorage independent, have an abbreviated G1 phase of the cell cycle, and have a reduced requirement for serum mitogens for S-phase entry. These biological changes are accompanied by a moderately increased production of cell cycle-regulatory proteins cyclin A and cyclin-dependent kinase (Cdk) 2, a dramatic deregulation of Cdk2 kinase activity, and a corresponding increase in the levels of hyperphosphorylated retinoblastoma protein (pRb). We show that the elevated cyclin A-Cdk2 activity is due to the combination of increased accumulation and stabilization of cyclin A bound to a faster-migrating species of Cdk2 believed to be the active threonine 160 phosphorylated form and a substantial reduction in complexed p27. Cyclin E kinase activity is also elevated due to a reduction in p27. A significant reduction in total cellular p27 protein levels and a moderate reduction in p27 mRNA are observed, but no changes in Cdk regulatory kinases and phosphatases occur. The Evi-1 transcriptional repressor domain and the ZF1 DNA binding domain are required for both cell transformation and induction of Cdk2 catalytic activity. We propose that one consequence of Evi-1 expression is to repress the transcription of target genes, which may include p27, that deregulate the normal control of the G1 phase of the cell cycle, providing a cellular proliferative advantage that contributes to transformation in vitro and leukemogenesis in vivo.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The Evi-1 proto-oncogene was originally identified as a common site of retroviral integration in interleukin 3-dependent murine myeloid cell lines (1) . Elevated expression of EVI-1 has subsequently been observed in cases of human AML4 (2) , chronic myelogenous leukemia in blast crisis (3) , acute promyelocytic leukemia, and myelodysplastic syndrome (4) . Overexpression has largely been attributed to translocations and inversions of chromosome 3q26, which is where EVI-1 resides (2) , but many cases have emerged with no gross cytogenetic abnormalities of this region, implying that additional mechanisms can deregulate EVI-1 expression in certain hematopoietic cells (4) .

The Evi-1 gene encodes a Mr 145,000 nuclear zinc finger protein (5) with NH2-(ZF1) and COOH (ZF2)-terminal domains containing seven and three Cys2His2 zinc finger motifs, respectively (1) , that have distinct DNA binding specificities (6 , 7) . In addition, we have identified two distinct transcriptional repressor domains in the Evi-1 protein designated Rp (8) and IR (9) . Direct trans-repression (8) or the antagonism of existing trans-acting cellular transcription factors GATA-1 (10) and AML1 (11) by EVI-1 or an AML1/EVI-1 fusion protein, respectively, have emerged as potential mechanisms of Evi-1 activity in vivo.

Although the mode of action of Evi-1 in cellular transformation is poorly understood, several lines of evidence implicate a role in cell proliferation. Constitutive ectopic expression transforms Rat1 fibroblasts (12) . This expression is absolutely dependent on the ZF1, ZF2, and Rp domains (8 , 9 , 12) and is partially dependent on the IR domain (9) . Intervention experiments using antisense oligonucleotides specifically inhibit the proliferation of leukemic cells that express the AML1/EVI-1 fusion protein (13) . The embryonic lethal phenotype observed in gene targeting experiments is accompanied by a widespread hypocellularity consistent with a function in general cell proliferation (14) .

Because Evi-1 encodes a transcriptional repressor protein that appears to allow cell proliferation but not differentiation (15) , it has been proposed to repress target genes that are negative regulators of cell growth, survival, or tumor suppression (8) . Molecular components of the cell cycle machinery are frequently targeted in cancer, which, in some cases, results in activation of gene expression (cyclin D1 and cyclin A; Refs. 16 and 17) . However, in many cancers, inhibition of cell cycle-regulatory proteins is seen. Several mechanisms are invoked including: (a) deletion and subsequent loss of heterozygosity by mutation of tumor suppressor proteins pRb, p53 (18) , or p16 (19) ; (b) transcriptional repression by hypermethylation of the p16 gene (20) ; and (c) direct interactions of viral oncoproteins E1A, E7, and SV40-Tag with pRb and other pocket proteins, EIB, E6, and SV40-Tag with p53 (18) , and E1A and E7 with p27 (21) and p21 (22) , respectively. Similar cell cycle-regulatory proteins might be targets for Evi-1 transcriptional repression.

The altered growth properties of Evi-1-expressing cells suggest that it mediates changes in cell cycle control. The major transitions of the eukaryotic cell cycle are controlled by the Cdks. Their kinase activities are dependent on the interaction of constitutively expressed catalytic subunits (Cdks) with periodically expressed cyclins (23) . The activity of these complexes, in turn, is dependent on the phosphorylation status of the kinase (24) and the interaction with Cdk inhibitors [p15, p16, p18, and p19 (Inks) and p21, p27 and p57 (Kips); Ref. 25 ].

In fibroblasts, the decision to progress beyond the restriction point (26) through the G1 and G1/S phases of the cell cycle is determined by mitogenic and anchorage-dependent signals controlled by complexes of D-type cyclins with Cdk4 (or Cdk6) and cyclins A or E with Cdk2 (27) . One of the principal targets of cyclin D- and E-dependent kinases is pRb, which becomes hyperphosphorylated on multiple sites during G1 (28) , relieving its growth-inhibitory effects by releasing bound E2F/DP family transcription factors that subsequently activate target genes with key roles in cellular proliferation (29) .

In this report, we describe the consequences of Evi-1 expression on cell cycle progression in Rat1 fibroblasts. We show that overexpression of Evi-1 induces anchorage independence, accelerated G1 progression, reduced serum requirements, and elevated Cdk2 activity. These observations suggest that Evi-1 induces cell proliferation that is mediated through a perturbation of the G1 phase of the cell cycle.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Isolation of Evi-1-expressing Rat1 Single Cell Clones.
Several independent single cell clones designated RatFL and RatMX were derived from the infection of Rat1 cells with recombinant retroviral vectors containing an Evi-1 full-length cDNA or an empty vector control, respectively (9) , and Western blots confirmed that the RatFL cells expressed Evi-1 (data not shown). We have previously shown that Evi-1 induces an anchorage-independent phenotype in Rat1 fibroblasts (8) , and the RatFL cell lines share this property (data not shown). RatFL clones are morphologically indistinguishable from Rat1 and RatMX cells at low cell densities but adopt a more rounded refractile morphology as cells approach confluence (data not shown). The studies described below have been performed with similar results with two independent RatFL clones, clones 6 and 7; therefore, cell lines are referred to as RatFL cells only.

Evi-1 Reduces Serum Requirements in Rat1 Fibroblasts.
Mitogenic stimuli are required to traverse the G1 restriction point of the cell cycle in dividing cells (26) . To investigate the effect of constitutive Evi-1 expression upon both the growth rate and serum growth factor dependence of Rat1 fibroblasts, RatFL, Rat1, and RatMX clones were cultured in normal (5%) and reduced (0.2%) serum, and the cell numbers were monitored over several days. Under normal growth conditions, RatFL cells typically grow faster and reach higher saturation densities than their normal counterparts, with culture doubling times of approximately 21 and 24 h, respectively (Fig. 1 A)Citation . Under low-serum culture conditions, RatFL clones consistently fail to quiesce and continue to proliferate slowly, at least in the short term (Fig. 1A)Citation . This contrasts with the Rat1 and RatMX cells, which quiesce within a single doubling (Fig. 1B)Citation .



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  		Fig. 1. Comparison of the growth rate of Rat1 ({diamond}), RatMX ({blacksquare}), and RatFL ({square}) cell lines in the presence of 5% (A) or 0.2% (B) NBCS. A representation of three independent experiments is shown. C, the proportion of cells incorporating BrdUrd (24 h continuous labeling) into newly synthesized DNA in RatMX ({blacksquare}) and RatFL ({square}) cells grown for 48 h in the indicated levels of serum.

 
These results suggest that Evi-1 reduces the doubling time and partially alleviates the serum requirement of Rat1 cells. To confirm the reduced serum requirement, we determined the proportion of cells entering the S-phase of the cell cycle by monitoring the incorporation of BrdUrd into newly synthesized DNA by flow cytometric analysis of cultures grown in different amounts of serum. As the serum concentration was reduced, the proportion of BrdUrd-labeled RatMX cells diminished rapidly, indicating a reduction in the number of cells entering S phase (Fig. 1C)Citation . In contrast, optimal stimulation of RatFL cells is still achieved in 1% serum, and even at serum concentrations as low as 0.2%, a significantly higher proportion of RatFL cells enter S phase as compared with RatMX cells (Fig. 1 C)Citation . This analysis shows that constitutive Evi-1 expression is sufficient to stimulate S-phase entry in reduced serum.

Evi-1 Accelerates the G1 Phase of the Cell Cycle.
These studies suggest that Evi-1 deregulates cell cycle control. To determine more precisely which phase is affected, asynchronous cultures of both RatFL and RatMX cells growing in 5% serum were pulse-labeled for 1 h with BrdUrd, and the labeled cohort was subsequently analyzed at 4-h intervals for cell cycle distribution by flow cytometric analysis of propidium iodide-stained cells (Fig. 2 a)Citation . In 12 h, the labeled cells exit S phase and traverse G2/M phase with similar kinetics as judged by their synchronous entry into G1 (Fig. 2, b and c)Citation . However, the RatFL cells exit G1 approximately 12 h after BrdUrd labeling, whereas the RatMX cells begin to exit this phase after 15 h (Fig. 2 d)Citation . This difference shows that cells expressing Evi-1 traverse G1 more rapidly than their normal counterparts.



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  		Fig. 2. Flow cytometric analysis of DNA content. Fluorescence-activated cell-sorting dot plots show (a) a population of RatMX cells and RatFL cells that have been pulse-labeled for 1 h with BrdUrd and monitored at 4-h intervals from S-phase through the cell cycle. Graphs summarizing the proportion of RatMX ({diamondsuit}) and RatFL ({blacksquare}) cells in S phase (b), G2/M phase (c), and G1/G0 (d) phase are also shown. Similar results were obtained in two independent experiments.

 
Evi-1 Induces Hyperphosphorylation of pRb and Persistence of Cyclin A and Cdk2.
Because Evi-1 accelerates progression through the G1 phase of the cell cycle in Rat1 fibroblasts, we performed Western blots to examine the expression of G1-regulatory proteins in both growing and serum-deprived cells. This analysis revealed that components of the G1 and G1/S-phase-regulatory kinases, cyclin A and Cdk2, persist upon serum deprivation for 24 and 48 h, respectively, in RatFL cells as compared with only 8 and 24 h in RatMX cells (Fig. 3)Citation . The levels of cyclin D1, cyclin E, Cdk4, and Cdk6 are all similar in growing and serum-deprived RatFL and RatMX cells (data not shown). Under the same conditions, flow cytometric analysis of propidium iodide-labeled cells revealed a delay in the kinetics of cell cycle exit in RatFL cells (Fig. 3)Citation .



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  		Fig. 3. Histogram summarizing flow cytometric analysis of propidium iodide-labeled RatMX ({blacksquare}) and RatFL ({square}) cells in the G1/G0 phase of the cell cycle of cells growing in 5% NBCS (O) or in 0.2% NBCS for the indicated times. Bottom panel, an analysis of the corresponding levels of cell cycle regulators in Evi-1-expressing and control cells. Western blots show equivalent amounts of RatMX and RatFL protein cell extracts derived from either growing cultures (O) or serum-deprived cells for the times shown and probed with the indicated antibodies. Even loading of Western blot filters for cyclin A was confirmed by reprobing for Cdk4 as a reference protein that remains invariant (data not shown).

 
The phosphorylation status of pRb is indicative of G1 cyclin-dependent kinase activity. Growing RatFL cells produce 5–10-fold elevated levels of pRb compared with RatMX cells (Fig. 3Citation time 0), which, based on the retarded electrophoretic mobility, is hyperphosphorylated in both cases. Upon serum deprivation of RatFL cells, the abundance of pRb is gradually diminished, but a high proportion of hyperphosphorylated protein was maintained even after 24 h (Fig. 3)Citation , in contrast to RatMX cells, in which only the hypophosphorylated form was detected after 16 h without serum (Fig. 3)Citation .

Evi-1 Enhances Cyclin-Cdk2 Activity in Growing and Serum-deprived Rat1 Fibroblasts.
The persistence of Cdk2 and cyclin A in serum-deprived cells and the elevated levels of the hyperphosphorylated substrate molecule pRb suggested that cyclin-dependent catalytic activity might be altered in Evi-1-expressing Rat1 cells. A comparison of Cdk2, Cdk4, and Cdk6 activities in growing and serum-deprived RatMX and RatFL cells revealed major changes in Cdk2 catalytic activity only (Fig. 4, a and b)Citation . Cdk2 kinase activity is elevated in growing RatFL cells (Fig. 4, bCitation , time 0), although comparable amounts of Cdk2-containing complexes were coimmunoprecipitated from both cell lines with {alpha}Cdk2-specific antisera (Fig. 4bCitation ; WB-{alpha}Cdk2). This activity remained high in serum-deprived RatFL cells (Fig. 4 b)Citation but was rapidly lost in RatMX cells (Fig. 4 b)Citation . Therefore, one of the functional consequences of Evi-1 expression in Rat1 fibroblasts is to deregulate cyclin/Cdk2 activity. Interestingly, Cdk2 activity increases moderately in control cells after serum deprivation for 4 h (Fig. 4, b, and h)Citation , but the significance of this observation has not been pursued further.



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  		Fig. 4. In vitro kinase assays of Cdk4 (a), Cdk6 (a), and Cdk2 (b) immunoprecipitated with the corresponding Cdk-specific antibody (IP-{alpha}Cdk) from equivalent amounts of cell extracts derived from cells growing in 5% NBCS (O) or cells deprived of serum for the indicated times, using either glutathione S-transferase/pRb fusion protein (KA-GST-pRb) or histone H1 (KA-Histone H1) as substrates. Control lanes show nonspecific kinase activity associated with any proteins binding to protein A-Sepharose alone. Also shown are Western blots with anti-Cdk2 (b) indicating similar levels of Cdk2 from individual immunoprecipitations (WB-{alpha}Cdk2).

 
To determine whether cyclin A-Cdk2 or cyclin E-Cdk2 activity is deregulated, kinase assays were performed after coimmunoprecipitations with the corresponding antibodies. Elevated Cdk2 activity was observed in RatFL cells independently of the antisera used in either growing or serum-deprived cells (Fig. 5, a–c)Citation , showing that both cyclin A-Cdk2 and cyclin E-Cdk2 complexes are deregulated in Evi-1-expressing cells. The magnitude of cyclin A-associated kinase activity was significantly greater, which might reflect the relative activities of the two complexes and the efficiencies of the antisera used, or some activity might also be due to cyclin A-Cdc2 complexes, which are immunoprecipitated by {alpha}cyclin A.



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  		Fig. 5. In vitro kinase assay of Cdk2 activity of equivalent amounts of cell extracts after immunoprecipitation with Cdk2 (IP-{alpha}Cdk2; a), cyclin E (IP-{alpha}cyclin E; b), or cyclin A (IP-{alpha}cyclin A; c). RatMX and RatFL cells were compared, both while growing and after a 16-h serum deprivation. Exposure times of kinase assays are 30 min and 8 h for Cdk2/cyclin A and cyclin E, respectively. Also shown are Western blots of immunoprecipitated protein cell extracts with Cdk2 (WB-{alpha}Cdk2) and p27 antibodies (WB-{alpha}p27). d, {alpha}Cdk2 Western blot (WB) analysis of equivalent amounts of the indicated growing (5% NBCS) cell extracts that have been immunoprecipitated (IP) with either {alpha}cyclin A or {alpha}Cdk2. Migration of inactive nonphosphorylated (Cdk2) and active (Cdk2-P) Cdk2 are indicated. Also shown are control immunoprecipitations with protein A-Sepharose beads only (PAS).

 
Cdk2 activity is regulated primarily by the abundance of cyclins A and E, the inhibitor protein p27, and phosphorylation. The moderate increase in Cdk2 immunoprecipitated by either {alpha}Cdk2 or {alpha}cyclin A in RatFL cells, which is consistent with their increased cellular levels, might contribute in part to the elevated kinase activity (Fig. 5, a and cCitation , WB-{alpha}Cdk2). Furthermore, the p27 contents of the Cdk2, cyclin E, and cyclin A immunoprecipitated complexes were determined by Western blot analysis showing that the ratio of cyclin-Cdk2-associated p27:Cdk2 is significantly lower in RatFL cells than in RatMX cells in each case (Fig. 5, a–cCitation , WB-{alpha}p27). In addition, {alpha}Cdk2 immunoprecipitates two Cdk2 species (Fig. 5aCitation , WB-{alpha}Cdk2), and the faster-migrating species probably corresponds to the active threonine 160 phosphorylated form (Cdk2-P; Refs. 30 and (31) . Only one Cdk2 species coimmunoprecipitates with {alpha}cyclin A and {alpha}cyclin E, and this corresponds with Cdk2-P (Fig. 5d)Citation . The abundance of cyclin A-complexed Cdk2-P is elevated in RatFL cells, which could be due to increased levels of both cyclin A and Cdk2 or to an increase in Cdk2 phosphorylation.

The Evi-1 Repressor and ZF1 DNA Binding Domains Are Both Required for Deregulated Cyclin/Cdk2 Activity.
We have previously shown that both Evi-1 ZF1 and Rp domains are required for the transformation of Rat1 fibroblasts (8 , 9) . To determine whether the induction of Cdk2 activity is also dependent on the DNA binding and transcriptional repressor properties of Evi-1, Rat1 cell clones expressing mutant proteins (designated Rat{Delta}ZF1 and Rat{Delta}Rp) were isolated (data not shown).

Cdk2 activity was examined in growing cells and in serum-deprived cells, and the results show that the enzyme activity in both mutant cell lines is the same as that observed in Rat1 and RatMX cells under both conditions (Fig. 6, a and b)Citation . In particular, deregulated catalytic activity that persists in the absence of serum is only observed in cells expressing the full-length protein (Fig. 6b)Citation . This has been verified with other clones expressing the mutant proteins (data not shown). Therefore, the induction of cyclin-Cdk2 activity by Evi-1 in Rat1 fibroblasts is dependent on the ZF1 DNA binding and transcriptional repressor domains of the protein, and this correlates with transforming activity.



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  		Fig. 6. In vitro kinase assays of Cdk2 activity derived from equivalent amounts of the indicated immunoprecipitated protein cell extracts using anti-Cdk2 (IP-{alpha}Cdk2), growing cells (a, 5% NBCS), and cells deprived of serum for 12 h (b) as described in the Fig. 5 legend. Also shown are Western blots with anti-Cdk2, indicating the Cdk2 and Cdk2-P forms from individual immunoprecipitations (WB-{alpha}Cdk2). Control immunoprecipitations with protein A-Sepharose only are also shown (PAS).

 
Western blots show a moderate increase in Cdk2 immunoprecipitated from RatFL cells (Fig. 6, a and bCitation , WB-{alpha}Cdk2). Furthermore, the relative abundance of total cellular Cdk2-P appears to increase in the RatFL cells (Fig. 6, a and bCitation , WB-{alpha}Cdk2). This correlates with increased Cdk2 activity, elevated Cdk2-P complexed with cyclin A (Fig. 7Citation , IP-{alpha}Cyclin A and WB-{alpha}Cdk2), and a significant reduction in bound p27 (Fig. 7Citation , WB-{alpha}p27), which are all dependent on the transcriptional repressor and ZF1 DNA binding activities of the Evi-1 protein. The bands below Cdk2 and Cdk2-P are nonspecific (Fig. 7)Citation . Taken together, these data suggest that Evi-1 transcriptional activity either directly or indirectly modulates intracellular levels of cyclin A, Cdk2, Cdk2 phosphorylation, and p27, which all contribute to the deregulation of cyclin-Cdk2 activity and cell transformation.



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  		Fig. 7. Anti-cyclin A (IP-{alpha}CyclinA) and anti-Cdk2 (IP-{alpha}Cdk2) immunoprecipitated protein cell extracts of the indicated growing (5% NBCS) cell lines were Western blotted with either anti-Cdk2 (WB-{alpha}Cdk2; showing the two species Cdk2 and Cdk2-P described in the Fig. 6 legend) and anti-p27 (WB-{alpha}p27).

 
Evi-1 Reduces Total Cellular Levels of the Cell Cycle Inhibitor Protein p27, but Components of CAK and cdc25a Remain Unchanged.
To investigate whether Evi-1 might affect the activity of Cdk2 by influencing its phosphorylation status, the production of the CAK complex components Cdk7,cyclin H, and Mat1, the putative Cdk-inactivating tyrosine kinase Wee1, and the corresponding activating phosphatase Cdc25a was determined. Western blots with specific antibodies revealed no significant changes in the relative abundance of these proteins in the cell lines examined (Fig. 8 a)Citation . Wee1 was undetectable in these cells (data not shown).



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  		Fig. 8. a and b, Western blots of equivalent amounts of Rat1, RatMX, and RatFL protein cell extracts derived from either growing cultures (O, 5% NBCS) or cells deprived of serum for 12 h, with the indicated antibodies. c, Northern blot analysis of 20 µg of total RNA derived from growing (5% NBCS) Rat1, RatMX, RatFL, and Rat{Delta}Rp cells with the indicated probes.

 
Reduced levels of the inhibitor protein p27 could also explain the increased Cdk2 catalytic activity in RatFL cells. Because less p27 is associated with cyclin-Cdk2 in RatFL cells, we determined the total p27 levels in both cell lines. The abundance of p27 is significantly reduced in growing and serum-deprived RatFL cells (Fig. 8b)Citation . Furthermore, the relative abundance of the p27 protein in the RatFL cells remained consistently lower even after 48 h of serum deprivation (data not shown). p16 and p21 levels were also examined in this study, but they were not detectable (data not shown).

Because p27 protein levels are reduced in RatFL cells, we investigated whether the gene is a potential target for Evi-1-mediated transcriptional repression by examining endogenous mRNA levels in various Rat1 cell lines grown in 5% serum using Northern blot analysis. These data show a moderate reduction in p27 mRNA levels in RatFL cells that are restored to normal in Rat{Delta}Rp cells (Fig. 8 c)Citation . The mRNA levels of cyclin A2 were also examined, and these were slightly elevated in RatFL cells (Fig. 8c)Citation . Glyceraldehyde-3-phosphate dehydrogenase mRNA levels confirmed that similar amounts of total RNA were loaded in each case. These data show that a reduction in the abundance of p27 mRNA and an increased accumulation of cyclin A2 mRNA coincide with elevated Evi-1 expression.


   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The importance of mitogenic signals during G1 has long been recognized, but it has recently become increasingly apparent that G1 progression is also regulated by cell anchorage to the extracellular matrix. We suggest that Evi-1 accelerates the transit time through the G1 phase of the cell cycle by reducing the requirements for both anchorage and serum growth factors. Phenotypic consequences of Evi-1 expression include an increased proliferation and saturation density, anchorage-independent growth, and a distinct cellular morphology at confluence. These changes are dependent on both the DNA binding and transcriptional repressor activities of the Evi-1 protein. This is the first demonstration that Evi-1 expression induces cell proliferation and provides significant new insights into the mechanism by which ectopic expression of this transcriptional repressor protein may contribute to leukemia progression.

These Evi-1-mediated phenotypic changes are accompanied by a number of molecular changes that include: (a) the deregulated expression of cyclin A and Cdk2 proteins that persist in serum-deprived cells; (b) a reduction in the abundance of the p27 protein that also has delayed induction kinetics in response to serum deprivation; and (c) elevated levels of pRb that persist in the hyperphosphorylated form in the absence of serum. These alterations are consistent with deregulated catalytic activity of the G1-G1/S-phase-dependent cyclin-Cdk2 kinases, and both cyclin A-Cdk2 and cyclin E-Cdk2 complex activities are significantly elevated and persist upon serum deprivation in Evi-1-expressing Rat1 fibroblasts.

Cyclins D1, A, and E, their associated kinase activities, pRb phosphorylation, and reduced p27 have all been shown to have a role in anchorage-dependent growth (32, 33, 34, 35) , G1 progression (36) , and the G1-to-S-phase transition (37, 38, 39, 40) of fibroblasts. Unscheduled expression or antisense studies with these genes partially mimic the features observed with Evi-1. Therefore, any one or a combination of these molecular changes could be responsible for the Evi-1-induced phenotype in Rat1 fibroblasts. However, it is difficult to determine the primary targets of Evi-1 that result in Cdk2 deregulation because of the interdependence of proteins regulating cell proliferation.

The pRb protein is a key substrate molecule for phosphorylation by Cdk2, Cdk4, and Cdk6. Our studies show that Cdk2 catalytic activity, but not Cdk4 and Cdk6 catalytic activity, is significantly elevated in RatFL cells relative to RatMX cells. The persistence of hyperphosphorylated pRb in RatFL cells after serum deprivation demonstrates that the pRb kinase is deregulated in vivo, which is consistent with our in vitro observations with Cdk2 catalytic activity.

The significance of the elevated levels of pRb in RatFL, a portion of which remains hyperphosphorylated after serum deprivation, and the molecular mechanism responsible are unclear. Increased pRb levels are also observed in cells transformed by other nuclear oncogenes (41) . Persistent hyperphosphorylated pRb might explain the elevation in pRb protein levels in RatFL cells. One of the consequences of pRb phosphorylation is to relieve its growth-inhibitory effects by releasing and derepressing the activity of bound E2F/DP-1 family transcription factors (29) . This would result in concomitant E2F-dependent trans-activation of the pRb gene promoter (42 , 43) and, consequently, increased pRb production. A similar mechanism could explain the persistent expression of Cdk2 and cyclin A observed because both genes also have E2F-responsive promoters (44) . The increased abundance of cyclin A2 mRNA levels observed are also consistent with this.

However, despite the persistence of hyperphosphorylated pRb, the abundance of hypophosphorylated pRb in RatFL cells after just 8 h of serum deprivation is similar to that observed in RatMX cells after 16 h of serum deprivation. Presumably, this complexes with and inactivates E2F/DP-1. Hence, the presence of free E2F/DP-1 family transcription factors might depend on their abundance in RatFL cells. Alternatively, the elevated levels of Cdk2 activity might induce unscheduled entry of RatFL cells into the S phase of the cell cycle by bypassing the activation of the pRb/E2F pathway (45) . pRb is not the only target substrate for Cdk2 catalytic activity. A number of other Cdk2 substrates that can modulate cell proliferation have recently been identified including NPAT (46) , Id2 (47) , Id3 (48) , p130 (49) , p107 (50) , and p27 (51) .

A reduction in the abundance of the stoichiometric Cdk2 inhibitor protein p27 (52 , 53) could account for elevated Cdk2 catalytic activity in RatFL cells. This is a key regulatory molecule that inhibits cell proliferation in response to environmental stimuli including either the absence of growth factors, contact inhibition, or growth inhibitors such as TGF-ß. p27 binds cyclin-Cdk2 complexes, masking the catalytic cleft (54) and inhibiting the activation of Cdk2 by steric hindrance of CAK (55) . p27 complexes with and inhibits both cyclin A- and cyclin E-associated Cdk2 activities (51) .

Our results show that both cyclins A and E associate with active Cdk2-P in preference to the unphosphorylated species. In the case of cyclinA-Cdk2 complexes, the level of the active species is significantly elevated in RatFL cells and is dependent upon the ZF1 DNA binding and repressor domains of the Evi-1 protein and inversely proportional to p27. The moderate increase in cyclin A and Cdk2 in RatFL cells and the reduced level of p27 probably all contribute to the increase in both the abundance and activity of these active complexes in growing and serum-deprived cells. Cyclin E-Cdk2 catalytic activity is likely to be more dependent on reduced p27 because cyclin E levels are similar in all cells examined. Reduced p27 would facilitate the access of CAK to cyclin-Cdk2 complexes, enabling phosphorylation/activation of Cdk2 and more stable interactions with cyclins A and E. Alternatively, CAK activity itself may be elevated in these cells. Although this cannot be formally ruled out, we see no changes in the abundance of the components cyclin H, Cdk7, or Mat1 of the CAK complex. Furthermore, we see no changes in expression of the Cdk2-activating tyrosine phosphatase Cdc25a.

The target genes of the Evi-1 transcription factor are unknown, but this study shows changes in the production of four cell cycle-regulatory proteins, cyclin A, Cdk2, pRb, and p27 in Evi-1-expressing fibroblasts. One mechanism by which the Evi-1 transcriptional repressor protein transforms cells could involve a reduction in p27 mRNA production. We show here that p27 mRNA levels are moderately decreased in Evi-1-expressing cells. Interestingly, the murine p27 promoter (56) contains four potential Evi-1 binding sites, two each for the ZF1 and ZF2 DNA binding domains, but it is currently unclear whether Evi-1 directly or indirectly represses p27 mRNA levels. The potential functional significance of p27 mRNA down-regulation in cell transformation has been demonstrated with the v-Src oncoprotein (57) . The moderate change observed in our study may be significant because it has recently been demonstrated that a 50% reduction in p27 gene dosage is sufficient to predispose mice to tumorigenesis (58) . A small reduction in the production of p27 might lead to an increase in Cdk2 activity, which, in turn, would result in a further decrease in cellular p27 levels through phosphorylation and subsequent degradation (51) .

In addition to explaining the increased catalytic activity of Cdk2, Evi-1-mediated p27 reduction could also contribute to blocking TGF-ß signaling. The p27 protein is known to be one of the target genes for TGF-ß (54) , and the strong growth-inhibitory effect of TGF-ß is inhibited by the expression of Evi-1 in mink Mv1lu cells, which coincides with persistent pRb hyperphosphorylation (59) . The effect is partly mediated by targeting signaling molecule Smad3 and by an unknown mechanism that requires the Evi-1 repressor domain we have shown to be necessary for the induction of Cdk2 activity and a reduction in p27 protein and mRNA levels.

Constitutive ectopic expression of Evi-1, like that of cyclins D1, D2, or E, shortens G1 in mammalian fibroblasts and reduces their serum requirement for S-phase entry. This property is likely to be very important in leukemia progression. Ectopic expression of D-type cyclins prevents hemopoietic cell differentiation in 32D cells (60) . Ectopic expression of Evi-1 in 32D cells produces similar results (15) . The growth factor stem cell factor, granulocyte macrophage colony-stimulating factor and interleukin 2 induce cell proliferation that coincides with a decrease in p27 in M07e (61) and T cells (62) , respectively. Conversely, inhibition of cyclin-Cdk activity by ectopic expression of p27 induces U937 cell differentiation (63) . This suggests that cells with reduced p27 levels and increased cyclin-Cdk activity are more sensitive to growth factors and that increased p27 and reduced cyclin/Cdk activity favor cell differentiation. Increased growth factor sensitivity would impart a selective advantage on cells in the hemopoietic microenvironment, where cells compete for the limited availability of cytokines. Therefore, the role of Evi-1 in leukemia could be to influence the decision to proliferate or differentiate by deregulation of cyclin-Cdk activity.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cells and Cell Culture.
Adherent human BOSC 23 cells were selected and maintained in a modification of Eagle’s MEM with double concentrations of amino acids and vitamins (SLM; Life Technologies, Inc.) and 10% FCS (Advanced Protein Products Ltd.) supplemented with hypoxanthine-aminopterin-thymidine medium (Life Technologies, Inc.), 250 µg/ml xanthine, 25 µg/ml adenine, and 50 µg/ml mycophenolic acid (Sigma). In transfection experiments, cells were supplemented with hypoxanthine-thymidine (Life Technologies, Inc.), 250 µg/ml xanthine, 25 µg/ml adenine, 100 µg/ml streptomycin, and 37.5 µg/ml penicillin. Rat1 cells were maintained in DMEM with 5% newborn calf serum (Life Technologies, Inc.).

Virus production by transient transfection of BOSC 23 cells, Rat1 cell infections, and G418 selection (800 µg/ml, Life Technologies, Inc.) were all performed as described previously (8) . Single cell clones were derived by limiting dilution in 96-well microtiter plates.

Retroviral Vectors.
The construction of the retroviral vectors has been described previously (8 , 9) .

Growth Curves and Soft Agar Colony Assays.
Exponentially growing cell cultures were trypsinized, washed in DMEM, and seeded at 1 x 105 or 5 x 105 cells/90-cm dish in DMEM containing 5% or 0.2% newborn calf serum, respectively. Every 24 h (5% serum) or 48 h (0.2% serum), cells were trypsinized, and the viable cell counts were determined with a hemocytometer. Experiments were performed in triplicate for each time point. Soft agar colony assays were performed as described previously (8) .

Northern Blot Analysis.
Total RNA was prepared from various cell lines using RNAzol B (Biogenesis Ltd). Northern blots were performed with 20 µg of total RNA/lane, essentially as described previously (64) .

Western Blot Analysis.
Preparation of whole cell protein extracts, Western blots, and detection were performed as described previously (8) . The antibodies used were {alpha}Evi-1 (1806; Ref. (9) , {alpha}Cdk2 (rabbit polyclonal antibody, Santa Cruz sc-163), {alpha}Cdk4 (rabbit polyclonal antibody, Santa Cruz sc-260), {alpha}Cdk6 (rabbit polyclonal antibody, Santa Cruz sc-177), {alpha}Cyclin A (rabbit polyclonal antibody, Santa Cruz sc-596; mouse monoclonal Serotec E67), {alpha}Cyclin D1 (mouse monoclonal antibody, Santa Cruz sc-450), {alpha}Cyclin E (rabbit polyclonal antibody, Santa Cruz sc-481), {alpha}p16 (murine antibody, a gift from Dr. C. J. Sherr, St. Jude Children’s Research Hospital, Memphis, TN), {alpha}p21 (rabbit polyclonal antibody, Santa Cruz sc-756), {alpha}p27 (rabbit polyclonal antibody, a gift from Dr. S. Coats, AMGEN Inc., Thousand Oaks, CA), {alpha}pRb (mouse monoclonal antibody, PharMingen 14001A), {alpha}Cdk7 (rabbit polyclonal antibody, Santa Cruz sc-529), {alpha}Cyclin H (rabbit polyclonal antibody, Santa Cruz sc-609), {alpha}Mat1 (rabbit polyclonal antibody, Santa Cruz FL-309), {alpha}cdc25a (rabbit polyclonal antibody, Santa Cruz), and {alpha}Wee1 (rabbit polyclonal antibody, Santa Cruz sc-325).

Immunoprecipitations and in Vitro Kinase Assays.
Rat 1 fibroblasts (5.0 x 106) were extracted in 0.5 ml of Cdk2 lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM sodium fluoride, 1% NP40, 1 mM phenylmethylsulfonyl fluoride, 10 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin] or 0.5 ml of Cdk4/Cdk6 lysis buffer [50 mM HEPES (pH 7.5), 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1 mM DTT, 0.1% Tween 20, 10 mM ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 2 µg/ml aprotinin, 5 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride]. Protein concentrations of Cdk2 and Cdk4/6 extracts were determined by bicinchoninic acid/copper sulphate binding using the Sigma protein assay and Bradford assays, respectively. Precleared cell extracts (500 µg) were incubated (1 h at 4°C with rocking) in lysis buffer with 0.5 µg of antiserum, and the immune complexes were collected by incubation (1 h at 4°C with rocking) with 50 µl of protein A-Sepharose (Sigma). For subsequent analysis, the immunoprecipitates were washed four times in ice-cold lysis buffer and two times in 50 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT (Cdk2 assays) or 50 mM HEPES (pH 7.5), 1 mM DTT, 5 mM MnCl2, 5 mM EGTA, and 5 mM ß-glycerophosphate (Cdk4/Cdk6 assays) and either fractionated on reducing SDS gels or used for kinase assays.

For Cdk2 the kinase reactions were started by adding 1 µg of histone H1, 50 µM ATP, 0.1 mM protein kinase A inhibitor (Sigma p0300), and 10 µCi of [{gamma}-32P]ATP (3000 Ci/mmol) in a final volume of 20 µl and incubated for 30 min at 30°C. Cdk4 and Cdk6 kinase reactions were performed by the addition 50 µM ATP, 10 µCi of [{gamma}-32P]ATP (6000 Ci/mmol), and 2.5 µg of pRb substrate (Santa Cruz sc-4112) in 30 µl, 30°C, 30 min. Reactions were stopped by the addition of Laemmli sample and histone H1, and pRb phosphorylation was determined by SDS gel electrophoresis (10% acrylamide), transfer to nitrocellulose membrane, and autoradiography. Blots were subsequently examined by Western blot analysis to determine complex composition.

Flow Cytometric Analysis.
To quantitate cells engaged in DNA synthesis, BrdUrd (Sigma) was added to the growth medium to a final concentration of 10 µM for a 1-h pulse or added continuously for 24 h. When chase periods were required, these were performed using conditioned medium from replicate cultures. After labeling, adherent cells were detached by trypsinization, washed once in PBS, and fixed in 70% ethanol. For antibody staining, the cells were resuspended in PBS, and the DNA was partially denatured by the addition of an equal volume of 4 N HCl for 15 min. Cells were then washed twice with PBS and once with PBT and incubated in PBT containing a 1:40 dilution of anti-BrdUrd monoclonal antibody (DAKO) for 30 min at room temperature. Cells were then washed once with PBT and incubated in PBT containing a 1:128 dilution of FITC-conjugated goat antimouse IgG (Sigma) for 30 min at room temperature. After two additional washes in PBT, cells were resuspended in PBS containing 10 µg/ml propidium iodide and 250 µg/ml RNase A and incubated for an additional hour at room temperature. The samples were then analyzed by two-color flow cytometry using a Becton Dickinson FACScan flow cytometer.


   Acknowledgments
 
We thank Profs. J. A. Wyke and P. R. Harrison and Drs. D. A. F. Gillespie and W. Kolch for critical reading of the manuscript and J. McColl and P. McHardy for technical assistance.


   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 Leukaemia Research Fund Grant 9408 (A. K.), Wellcome Trust vacation scholarship (to V. S.), and by the Cancer Research Campaign. Back

2 Present address: Glasgow Caledonian University, School of Biological & Biomedical Sciences, City Campus, Cowcaddens Road, Glasgow G4 OBA, Scotland. Back

3 To whom requests for reprints should be addressed. Phone: 0141-331-3213; E-mail: c.bartholomew{at}gcal.ac.uk Back

4 The abbreviations used are: AML, acute myelogenous leukemia; Cdk, cyclin-dependent kinase; BrdUrd, bromodeoxyuridine; CAK, Cdk-activating kinase; TGF, transforming growth factor; pRb, retinoblastoma protein; Cdk2-P, phosphorylated Cdk2; PBT, PBS containing 0.5% BSA and 0.1% Tween 20; NBCS, newborn calf serum. Back

Received for publication 3/ 4/99. Revision received 6/17/99. Accepted for publication 7/19/99.


   References
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 Results
 Discussion
 Materials and Methods
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