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Keywords:

  • apoptosis;
  • cell cycle;
  • cyclin-dependent kinase 1;
  • tyrosine phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

Summary. The K562 leukaemic cell line expresses an inherent survival signal due to the antiapoptotic properties of Bcr-abl, which is, in part, mediated by prolonging the G2M checkpoint and allowing DNA repair mechanisms to operate post genotoxic insult. Arrest of the cell cycle is mediated by retaining an inactivating state of phosphorylation of cyclin-dependent kinase 1 (Cdk1) on tyrosine 15. Our data confirmed that cell survival in K562 was promoted by cell cycle arrest at G2M in response to the genotoxin etoposide. There was no predicted cell cycle arrest in Bcr-abl-positive derivative cell lines of K562 that did not survive the same genotoxic insult but, paradoxically, Cdk1 tyrosine phosphorylation was enhanced to a higher extent compared with the parental cell line where arrest of the cell cycle was observed. To ascertain that this was not an anomaly of the derivative lines, HL60 cells were treated with concentrations of etoposide that induced arrest of the cell cycle or apoptosis. Only HL60 cells that subsequently underwent apoptosis elicited the same effect of increased Cdk1 tyrosine phosphorylation. It is proposed that the augmented tyrosine phosphorylation status of Cdk1 is associated with the abolition of cell survival, in addition to the previously reported induction of cell cycle arrest in myeloid cell lines.

The K562 cell line is a p53-negative Bcr-abl-positive cell line derived from a patient with chronic myeloid leukaemia (CML) and has been used as an in vitro model for this disease (Lozzio & Lozzio, 1975; Koeffler et al, 1986; Bi et al, 1992). It is resistant to cell death from a range of anticancer agents. This resistance is, in part, due to Bcr-abl inhibiting apoptosis and prolonging arrest of the cell cycle at the G2M cell cycle checkpoint, following treatment with ionizing radiation or chemotherapeutic agents (McGahon et al, 1994; Bedi et al, 1995; Nishii et al, 1996). Moreover, using a temperature sensitive mutant of the Bcr-abl gene introduced into the Baf-3 cell line, it was shown that protection, mediated by Bcr-abl at permissive temperatures, was due to phosphorylation of cyclin-dependent kinase 1 (Cdk1); a protection that could be reversed by the ability of caffeine to induce dephosphorylation of Cdk1 even in the presence of Bcr-abl (Nishii et al, 1996).

The cell cycle proceeds via induction of a series of cyclins that are expressed at prerequisite points of the cell cycle (Nurse, 1990; Morgan, 1995). For each cyclin, there is a cyclin-dependent kinase (Cdk) partner, which, on binding to the cyclin, forms an active complex that drives transition from one phase of the cell cycle to the next (Sherr, 1996). Checkpoints, or pauses in the cell cycle, can be induced by the cell in response to its environment or by DNA-damaging chemotherapeutic agents. These checkpoints occur at progression from G1 into S-phase, during S-phase, where replicon initiation is retarded, and at progression from G2 into mitosis to circumvent an aberrant mitosis (Murray, 1992; Elledge, 1996; O'Connor & Fan, 1996; Kaufman, 1998).

The G2M cell cycle checkpoint prevents cells from entering mitosis with unreplicated or damaged DNA. Under normal circumstances, cells enter mitosis as a result of activation of the mitotic phase promoting factor (MPF), which is a complex of cyclin B and Cdk1 (Nurse, 1990). Cyclin B/Cdk1 induces changes in cell morphology associated with mitosis, including nuclear membrane breakdown, and microtubule and actin filament rearrangements (Jackman & Pines, 1997). Cyclin B is synthesized during late S-phase and G2, and binds to Cdk1. This complex then requires an activating phosphorylation of a threonine residue (T161) on Cdk1 by Cdk1-activating kinase (CAK). The complex is kept inactive by phosphorylation of specific threonine (T14) and tyrosine (Y15) residues in the ATP binding site of Cdk1. Y15 and T14 phosphorylation is maintained by the nuclear Wee1 kinase (Parker & Piwnica-Worms, 1992; Morgan, 1995) and the cytosolic membrane-associated kinase Myt1 respectively. Myt1 is reported to phosphorylate both T14 and Y15 but preferentially phosphorylates T14 (Mueller et al, 1995; Liu et al, 1997). Activation of the cyclin B/Cdk1 complex is brought about by dephosphorylation of T14 and Y15 by the cell division cycle (Cdc)25 family of phosphatases (Dunphy & Kumagai, 1991; Gautier et al, 1991).

In response to DNA damage, the G2M checkpoint is governed by the activation of checkpoint kinase (Chk)1, and Chk2 via ATR (Liu et al, 2000) and ATM (Matsuoka et al, 1998), resulting in phosphorylation of Cdc25c on serine residue 216 (Peng et al, 1997; Sanchez et al, 1997; Zeng et al, 1998). This allows the binding of 14-3-3 proteins, leading to the cytoplasmic sequestration of Cdc25c, which is then unable to dephosphorylate and activate nuclear cyclin B/Cdk1 (Peng et al, 1997; Sanchez et al, 1997; Lopez-Girona et al, 1999). Cytoplasmic sequestration of the cyclin B/Cdk1 complex also regulates the G2M DNA damage checkpoint. Cyclin B and Cdk1 are found primarily in the cytoplasm during interphase and only relocate to the nucleus late in G2 (Hagting et al, 1998; Toyoshima et al, 1998) but, following DNA damage, the 14–3-3 σ protein is reported to sequester the cyclin B/Cdk1 complex, thus promoting a prolonged G2M cell cycle arrest. This does not occur in cells lacking both alleles of the 14–3-3 σ gene, where cyclin B and Cdk1 translocated to the nucleus despite induced DNA damage. The result was mitotic catastrophe, a form of cell death that has many characteristics in common with apoptosis (Chan et al, 1999).

As many therapeutic agents used for the management of leukaemia ultimately target DNA, the G2M checkpoint can be seen as a form of cellular resistance to chemotherapeutic regimens. This is because the checkpoint has evolved to allow DNA repair mechanisms to be activated, and successful DNA repair promotes continued cell cycling and likely disease progression. Also, arrest of the cell cycle prevents continued cycling on a compromised DNA template and reduces the potential for cell death as a result of an aberrant mitosis.

This study has used the K562 cell line, two derivative cell lines of K562 that have a weak G2M checkpoint and the HL60 cell line to investigate the relationship between cell cycle progression, cell cycle arrest and cell death. Our data show that post-genotoxic insult, continued cell cycling and an ensuing cellular demize can occur despite augmented Cdk1 tyrosine phosphorylation, if the degree of phosphorylation is in excess of that required for arrest of the cell cycle at G2M.

Reagents and antibodies.  Antibodies to Cdk1 and antiphospho-tyrosine (mouse monoclonal antibodies) were purchased from Santa Cruz (Insight Biotechnology, Wembley, UK). Secondary antibody to mouse monoclonal antibodies (Dako, Ely, UK) was horseradish peroxidase (HRP) conjugated. The antiphospho-tyrosine antibody was directly conjugated to HRP. Buffer reagents and 30% acrylamide/bis-acrylamide were purchased from Sigma UK (Gillingham, UK). Antibody class-specific rat anti-mouse IgG2A-coated Dyna-Beads were obtained from Dynal, Bromborough, UK. Phosphate-buffered saline (PBS) tablets were obtained from Oxoid, Basingstoke, UK. Drug preparations used were Vepesid (Bristol Myers, Swindon, UK) for etoposide.

Cell lines. The acute myeloid leukaemic cell line HL60 and the erythro–myeloid leukaemic cell line K562 subclone 6 were originally obtained from the European Collection of Animal Cell Cultures (ECACC) Porton Down, UK. The K/Dau300 and K/Dau600 cells lines were developed by prolonged culture of the parental K562 cell line in 300 and 600 ng/ml daunorubicin, as previously described (Jiang et al, 1994). These cell lines remain p53 negative, express Bcr-abl protein and express the multidrug resistance (MDR-1) gene product Pgp170. Continued culture under these conditions has selected cells that proliferate and divide in the presence of drug, and hence exhibit a poor ability to undergo cell cycle arrest at the G2M checkpoint. There are no known links between expression of the drug efflux pump, Pgp170, and induction of apoptosis or cell cycle regulation. All cell lines were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal calf serum, 2 mmol/l l-glutamine (Sigma), 100 units penicillin and 100 μg/ml streptomycin in a humidified incubator containing 5% CO2 at 37°C.

Cell cycle analysis and assessment of cell death. Exponentially growing cells were harvested, washed in medium and resuspended in supplemented RPMI medium at 0·5 × 106/ml in 2 ml volumes in 24 well plates (Costar, Corning Inc., NY, USA). Drugs were added at this stage at the concentrations stated in the text. At the relevant time points, aliquots of 500 000 cells were removed, washed twice in PBS and permeabilized in ice cold 70% ethanol for 60 min. Cells were washed twice in PBS and resuspended in 500 μl of PBS, containing 50 μg/ml propidium iodide and 200 μg/ml RNAse, and incubated for 60 min at 37°C prior to analysis. Cell cycle analysis was achieved by flow cytometry on either an Epics Elite (Coulter Electronics, High Wycombe, UK) or a FACSVantage (Becton Dickinson, Cowley, Oxford, UK) cytometer using pulse processing for aggregate discrimination. Dead cells were defined as the sub G0/G1 peak; a feature used to detect apoptotic cells (Darzynkiewicz et al, 1992).

Cell lysate and polyacrylamide gel electrophoresis (PAGE). Cell lysates were generated by lysing 1 × 107 cells in 500 μl radio immunoprecipitation assay buffer (PBS containing 1% non-idet P40, 0·5% sodium deoxycholate, 0·1% sodium dodecyl sulphate, 4% aprotinin, 1% sodium orthovanadate and 0·5% phenylmethylsulphonyl fluoride) on ice and sheared by repeated passage through a 19 gauge Microlance needle. Protein concentration was assessed using the direct Lowry method (Sigma). Precisely 50 μg protein/lane of total cell lysate was analysed on a 12% polyacrylamide gel for the resolution of Cdk1. Electrophoresis was performed using 100 V constant voltage for 1·5 h on a Mini Protean II (Bio-Rad) electrophoresis rig.

Immunoprecipitation was carried out by taking 250 μl of appropriate species anticlass-specific antibody bound to Dyna-Beads, which were then coated with 15 μg of primary antibody (200 μg/ml). These beads (75 μl) were added to 500 μg of cell lysate and precipitated by magnetic separation. Beads were washed four times in PBS + 0·1% bovine serum albumin (BSA) and eluted directly into 30 μl denaturing sample buffer, containing 2-mercaptoethanol and boiled for 5 min prior to loading on the gel. Gels were run as above.

Western blots.  Western blots were performed using a Bio-Rad Immersion Electroblotter overnight at 30 V on to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Sigma) in Tris-base/glycine/methanol transfer buffer. Proteins were visualized by blocking membranes with 5% BSA for the phosphotyrosine studies and in 5% dried milk solution for all other blots, in Tris-buffered saline (TBS)10 mmol/l Tris HCL, 150 mmol/l sodium chloride, pH 8·0 containing 0·1% Tween 20 (Sigma). Membranes were then incubated with primary antibody at predetermined optimized concentrations in 1% dried milk/TBS solution for 1 h at room temperature or in 0·1% TBS/Tween 20 for the phosphotyrosine studies. Membranes were washed six times in TBS/Tween 20 and secondary antibody was then added at a dilution of 1:2000 for 1 h. Excess secondary antibody was removed by a further four washes of TBS/Tween 20 and two washes of TBS only. Any protein bands were identified using Pearce and Warriner (Chester, UK)-enhanced chemiluminescence reagents and exposing membranes to Hyper-film (Amersham International, Little Chalfont, UK). All Western blot data are representative of three or more independent experiments.

Protein expression was quantified as integrated density values (IDVs) and were obtained from an Alpha Imager 2000 (Alpha Innotech, Staffs., UK).

G2M cell cycle arrest studies and mitotic index.  K562 and the derivative cell lines were cultured at 0·5 × 106/ml in 2 ml volumes in 24 well plates for 24 h to which etoposide at 10 μg/ml, nocodazole at 1 μg/ml or both had been added. Cytopsins were made from 100 μl aliquots of each culture and stained using the May–Grunwald Giemsa stain (Sigma). Three fields of 100 cells each were counted per cytospin and the number of mitotic cells was counted. Mitotic index was calculated as:

  • image

Results are a mean of three independent experiments.

G2M cell cycle synchronization.  HL60 cells were cultured at a concentration of 0·5 × 106/ml in the presence of 0·25 mmol/l thymidine for 24 h. The cells were then washed twice in PBS and placed back into thymidine-free supplemented RPMI medium for 8 h. Cells were returned to medium containing 0·25 mmol/l thymidine for a further 16 h. Cells were washed twice in PBS and restored to thymidine-free RPMI culture medium. Cells were found to be in the G2M phases of the cell cycle 6 h post release from the second thymidine block.

Statistics. Data were analysed using a paired t-test with P < 0·05 being considered significant.

Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

The parental K562 cell line and the derivative cell lines, K/Dau300 and K/Dau600, were incubated continuously with the concentrations of etoposide shown in Fig 1 and analysed for cell cycle distribution flow cytometrically at the time points shown. As etoposide, a topoisomerase II inhibitor, is a potent inducer of apoptosis (Kaufmann et al, 1993; McGahon et al, 1994; Bedi et al, 1995; Nishii et al, 1996; Martins et al, 1997), a characteristic marker of apoptosis was used to assess cell death. In this study, the sub G0/G1 population was used to this effect. Incubation of K562 cells with lower concentrations of etoposide induced cells to undergo G2M cell cycle arrest. For example, 1 μg/ml and 5 μg/ml of etoposide induced cells to undergo cell cycle arrests of 37·63% (P < 0·05) and 33·58% (P < 0·05), respectively, at 24 h, compared with a control of 16·20%. This resulted in excess of a 300% increase in G2M arrest at 48 h. Failure to induce arrest of the cell cycle was mirrored by an ability to induce an apparent apoptotic cell death. There was no significant induction of apoptosis for as long as 36 h at etoposide concentrations inducing cell cycle arrest in the K562 cell line. This resistance was maintained for a further 12 h at doses of 1 μg/ml and 5 μg/ml and was only broken by high-dose etoposide for 48 h, where 20 μg/ml and 80 μg/ml induced the modest increases in apoptosis to 15·13% (P = 0·003) and 27·73% (P = 0·006), respectively, compared with a control of 6·63% (P < 0·05). However, significant arrest of the cell cycle was not observed under these conditions. In fact, 80 μg/ml etoposide failed to induce any additional increase in the percentage of cells in G2M when compared with the control for the 48 h time period.

image

Figure 1. Etoposide-induced cell cycle arrest and cell death in K562, K/Dau300 and K/Dau600 cell lines. Flow cytometric analysis of parental K562 cL.6 cells, and the K/Dau300 and K/Dau600 derivative cell lines, showing the percentage cells in G2M (top panel) and the percentage of sub G0/G1 cells (bottom panel) observed in cultures treated with etoposide at the concentrations shown after incubation times of 4, 8, 24, 36 and 48 h. Results are the mean of four experiments and error bars show standard error of the mean which did not exceed 9·87 for the cell death data or 7·03 for the G2M arrest data.

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In contrast, there was no increase of the percentage of cells in the G2M phases of the cell cycle in the K/Dau300 cell line at concentrations of etoposide that subsequently induced cell death. Etoposide (1 μg/ml) induced an increase in G2M cell cycle arrest from a control of 20·92% to 28·72% at 24 h (P < 0·05) but this drug concentration was a poor inducer of apoptosis over the time period studied. Higher drug concentrations of 5 μg/ml, 20 μg/ml and 80 μg/ml did not induce cell cycle arrest at 24 h yet, compared with a control of 6·72%, induced apoptosis of 12·65%, 23·02% and 31·43% (P < 0·05) respectively. This was augmented considerably in the treatment groups of 48 h. A similar pattern of results was observed in the second derivative cell line, K/Dau600, where a propensity for failure to induce the G2M checkpoint resulted in cell death.

Thus, etoposide-induced cell cycle arrest is drug concentration and time dependent, and imparts resistance to apoptotic cell death.

Constitutive levels of expression of Cdk1 are less in the derivative cell lines

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

One reason for the lack of arrest of the cell cycle and a greater propensity for etoposide-induced apoptosis in the derivative cell lines could have been a loss of functional Bcr-abl. However, fluorescent in situ hybridization (FISH) showed that multiple copies of the Bcr-Abl translocation were present in all K562 cell lines. Western blots showed that the chimaeric 210 kDa protein was expressed at a similar level in the three cell lines. Also, immunoprecipitation showed that it was phosphorylated on tyrosine residues in all three cell lines: an indicator of probable autophosphorylation of the 210 kDa protein. This lack of variation in Bcr-abl expression across the cell lines suggests that Bcr-abl expression is not responsible for the differential response of these cell lines to etoposide (data not shown). As arrest of the cell cycle at the G2M checkpoint was associated with a poor cell-death response, the level of expression of Cdk1, the pivotal kinase for transition from G2 into mitosis, was measured. Western blots showed that Cdk1 was expressed at lower levels in the derivative cell lines than in the parental cell lines (Fig 2A), suggesting the possibility that constitutive expression of Cdk1 above a certain threshold may be required to promote cell survival following genotoxic insult.

image

Figure 2. Effects of etoposide on Cdk1 expression and Cdk1 tyrosine phosphorylation. (A) Western blot for constitutive Cdk1 expression in K562 cL.6 (lane 1), K/Dau300 (lane 2) and K/Dau600 (lane 3) cell lines. Integrated density values (IDVs) are 83520, 72384 and 41760 respectively. β-actin expression is shown as a lane loading control. (B) Western blot for Cdk1 expression in K562 cL.6 (lanes 1 and 2, IDVs = 89088 and 93264), K/Dau300 (lanes 3 and 4, IDVs = 70992 and 75168) and K/Dau600 (lanes 5 and 6, IDVs = 44544 and 51504) cell lines treated with 10 μg/ml etoposide (lanes 2, 4 and 6) compared with non-treated controls (lanes 1, 3 and 5). β-actin expression is shown as a lane loading control. (C) Western blot showing tyrosine phosphorylation (pY) of immunoprecipitates of Cdk1. Tyrosine phosphorylation of Cdk1 in K562 cL.6 (lanes 1 and 2, IDVs = 37125 and 48375), K/Dau300 (lanes 3 and 4, IDVs = 25875 and 87750) and K/Dau600 (lanes 5 and 6, IDVs = 9000 and 69750) cell lines treated with 10 μg/ml etoposide for 24 h (lanes 2, 4 and 6) compared with non-treated controls (lanes 1, 3 and 5).

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Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

To determine whether the observed depression of constitutive Cdk1 expression in K/Dau300 and K/Dau600 cells was in any way influential on the outcome of genotoxic insult on these cells, the expression of Cdk1 was measured post treatment with etoposide. A concentration of 10 μg/ml etoposide was chosen because optimization studies showed that it doubled the percentage of K562 cells arrested at G2M compared with control cultures at 24 h. Also, there was no accumulation of cells in G2M in the derivative cell lines (Fig 3). These data were corroborated using a sub G0/G1 assay, where 10 μg/ml etoposide induced minimal cell death in the K562 cell line (control, 4·7%vs 12·5%, n = 6, P > 0·005), and cell death levels of 26·1% in K/Dau300 cells (n = 6, P < 0·005) and 26·2% in K/Dau600 cells (n = 6, P < 0·005) compared with controls of 7·6% and 12·2%, respectively, after 48 h continuous incubation. The effect of etoposide on Cdk1 expression was examined in each cell line. There was little change in Cdk1 expression, as a result of etoposide treatment at 24 h, in any of the K562 cell lines (Fig 2B). The percentage increase of Cdk1 expression due to etoposide, compared with that of the control cultures (as measured by densitometry), is shown in Table I and indicates that etoposide had little effect on Cdk1 expression.

image

Figure 3. The effect of 10 μg/ml etoposide for 24 h on cell cycle distribution (right-hand panel) compared with control cultures (left-hand panel). Percentage of cells in G2M after etoposide treatment are 37% parental cells (top row), 11% K/Dau300 cells (middle row) and 20% K/Dau600 cells compared with controls of 20%, 22% and 22% respectively.

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Table I.  The percentage increase from control cultures of: Cdk1 expression in etoposide-treated cells (from Fig 2B), tyrosine-phosphorylated (YP) Cdk1 in etoposide-treated cells (from Fig 2C) and the ratio of relative abundance of YP–Cdk1 in etoposide-treated cells to control cells, as measured by integrated density values (IDV).
 K562K/Dau300K/Dau600
  • *

    Relative abundance = IDV YP–Cdk1/IDV Cdk1.

  • Change in relative abundance = relative abundance in etoposide-treated cells/relative abundance in control cells.

Percentage increase in Cdk1 expression post etoposide treatment 4·7%  5·9% 15·6%
Percentage increase in YP-Cdk1 post etoposide treatment30%239%675%
Change in relative abundance* of YP-Cdk1 post etoposide treatment 1·245  3·203 6·703

Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

The function of Cdk1 as a kinase is regulated by the state of phosphorylation of the T14 and Y15 residues within the ATP binding site of Cdk1. Therefore, Cdk1 was immunoprecipitated from the lysates generated above and assessed for phosphotyrosine status.

Figure 2C shows that augmented tyrosine phosphorylation occurred in K/Dau300 and K/Dau600 cell lines after exposure to 10 μg/ml etoposide for 24 h (lanes 4 & 6), i.e. the cell lines that exhibited a failure to induce cell cycle arrest and subsequently went on to die. There was modest augmentation of tyrosine phosphorylation on Cdk1 in K562 cells treated with 10 μg/ml etoposide for 24 h. Densitometry readings of these data are summarized in Table I, where the percentage change in tyrosine phosphorylation of Cdk1 is shown, compared with that of control cultures. The change in relative abundance of tyrosine-phosphorylated Cdk1 in Table I also demonstrated that, in instances where genotoxic insult resulted in cell cycle checkpoint failure and cell death, the increased tyrosine phosphorylation of Cdk1 was in excess of that required to produce a G2M cell cycle arrest.

Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

To determine the effects of Cdk1 tyrosine phosphorylation on the G2 to M phase transition, the effects of etoposide-mediated Cdk1 tyrosine phosphorylation with respect to cell cycle regulation were investigated. Cdk1 inactivation due to Y15 phosphorylation stops cells progressing from G2 into mitosis. Nocodazole is a microtubule assembly inhibitor and blocks cells in mitosis at metaphase. Thus it can be used to trap cells that have passed from G2 into mitosis.

K562 parental and derivative cell lines were treated for 24 h with 10 μg/ml etoposide as previously described, and a combination of 10 μg/ml etoposide and 1 μg/ml nocodazole for 24 h to trap any cells progressing from G2 into mitosis. Cells were morphologically analysed for the presence of mitotic cells. The parental K562 cell line did not show any increase in the amount of mitosis in cells treated with etoposide and trapped by nocodazole, when compared with etoposide alone, confirming the presence of an intact G2M checkpoint. However, in a similar comparison, the derivative cell lines showed an increase in the number of cells undergoing mitosis when treated with etoposide and trapped in mitosis with nocodazole. Calculation of a mitotic index (Table II) showed that etoposide-treated parental K562 cells arrested at the G2M checkpoint and did not enter mitosis whereas the derivative cell lines, post genotoxic insult, entered mitosis and were arrested only by the nocodazole trap in the metaphase stage of mitosis.

Table II.  Mitotic index for cell lines treated with etoposide, nocodazole and a combination of both agents.
 K562K/Dau300K/Dau600
Control0·0480·0470·068
Nocodazole0·2470·2380·291
Etoposide0·0120·0350·026
Nocodazole + Etoposide0·0090·1070·137

These data show that the G2M checkpoint was not intact in derivative cell lines despite the increased Cdk1 tyrosine phosphorylation.

Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

To ascertain that the increased tyrosine phosphorylation of Cdk1 seen in response to etoposide was not unique to the K562 derivative cell lines, studies were also performed using a different myeloid cell line HL60. HL60 cells were treated with three concentrations of etoposide that induce either G2M cell cycle arrest (0·1 μg/ml) or a concentration-dependent loss of cell survival (1 and 10 μg/ml) (data not shown). Timed experiments showed that by 4 h incubation with etoposide, there was no difference in the expression of Cdk1 per se in cells treated with 0·1 μg/ml compared with 1·0 μg/ml (Fig 4A) but loss of cell survival induced by 1 μg/ml was associated with an increased level of Cdk1 tyrosine phosphorylation (Fig 4B). Cell death induced by 10 μg/ml etoposide at 4 h gave protein yields that were too low to analyse.

image

Figure 4. Effects of etoposide on HL60 cells. (A) Effect of etoposide treatment on Cdk1 levels in HL60 cells. Western blot showing Cdk1 levels in HL60 cells treated for 4 h with 0 μg/ml (lane 1), 0·1 μg/ml (lane 2) and 1 μg/ml (lane 3) of etoposide. β-actin expression is shown as a lane loading control. (B) Tyrosine phosphorylation (pY) of immunoprecipitates of Cdk1. Tyrosine phosphorylation of Cdk1 in HL60 cells treated with 0 μg/ml (lane 1), 0·1 μg/ml (lane 2) and 1 μg/ml (lane 3) of etoposide for 4 h. IDVs = 14175 (lane 1), 17955 (lane 2) and 33075 (lane 3).

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HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

Hyperphosphorylation of Cdk1 on tyrosine residues in response to etoposide treatment occurs in cells that fail to undergo cell cycle arrest at the G2M checkpoint. To ascertain if increased tyrosine phosphorylation of Cdk1 occurred in cells in the G2M phase of the cell cycle, HL60 cells were synchronized by a double thymidine block and allowed to proceed to G2M. G2M-synchronized cells treated with either 1 μg/ml or 10 μg/ml of etoposide exhibited an augmented level of Cdk1 tyrosine phosphorylation. This was seen within 1 h with 10 μg/ml of etoposide (Fig 5B, lane 3). The same treatment of etoposide in asynchronous HL60 cells did not show the same increase in Cdk1 tyrosine phosphorylation (Fig 5A, lane 2). This indicated that cells in G2M were more sensitive to changes in Cdk1 tyrosine phosphorylation associated with loss of cell survival than cells distributed throughout the cell cycle.

image

Figure 5. Western blot showing tyrosine phosphorylation (pY) of immunoprecipitated Cdk1 from asynchronous and G2M synchronized HL60 cells. (A) Tyrosine phosphorylation of Cdk1 immunoprecipitated from asynchronous control cells (lane 1) and cells incubated with 10 μg/ml etoposide for 1 h (lane 2). (B) Tyrosine phosphorylation of Cdk1 immunoprecipitated from asynchronous control cells (lane 1), G2M-synchronized cells (lane 2) and G2M-synchronized cells plus 10 μg/ml etoposide for 1 h (lane 3).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References

The response of leukaemic cells to genotoxic insult dictates the efficacy of many antileukaemic regimens. The response can be varied but includes induction of cell cycle arrest and/or cell death. While both of these responses are desirable in the prevention of disease progression, only the latter results in a potential state of remission. This is because an arrest of the cell cycle can be seen as a form of cellular resistance to genotoxins because checkpoints have evolved to enable DNA repair to take place and allow cells to re-enter the cell cycle, resulting in disease progression.

This study tackles the early response of Bcr-abl-positive and -negative leukaemic cell lines to genotoxic insult from etoposide. The data presented in Fig 1 is in accordance with previous findings, concerning the resistance to cell death of the K562 cell line to genotoxins (Kaufmann et al, 1993; McGahon et al, 1994; Martins et al, 1997). The lower concentrations of etoposide caused a pronounced arrest at G2M while the higher concentrations induced relatively low levels of cell death that manifest after 48 h continuous exposure to the drug. In contrast, the derivative cell lines were shown to die far earlier (24 h) and this was associated with very low or non-existent G2M arrest.

In response to genotoxic stress, the G2M checkpoint is activated by maintaining a state of Cdk1 inactivation due mainly to phosphorylation of Cdk1 on Y15 (Nurse, 1990; Parker & Piwnica-Worms, 1992; Morgan, 1995). However, in the K/Dau300 and K/Dau600 cell lines, tyrosine phosphorylation of Cdk1 was greatly enhanced as a result of etoposide treatment, yet paradoxically there was no arrest of the cell cycle. Table I summarizes the data, in that where genotoxic stress failed to activate cell cycle arrest with a subsequent failure of cells to survive, the change in relative abundance of tyrosine-phosphorylated Cdk1 to total Cdk1 in etoposide-treated cells, compared with control cells, was in excess of that required to induce a cell cycle arrest.

Nocodazole trap experiments were performed to determine the relationship between Cdk1 phosphorylation and cell cycle control. Etoposide reduced the mitotic index of asynchronous proliferating K562 cells, which is commensurate with an arrest of the cell cycle at the G2M checkpoint. This was confirmed by the observation that the addition of nocodazole did not trap etoposide-treated cells in mitosis (Table II), i.e. the cells were arrested at the interface between G2 and mitosis. In contrast, K/Dau300 and K/Dau600 cell lines treated with etoposide did not arrest at this checkpoint. This was confirmed by the observation that these cells could be trapped in mitosis by nocodazole. This was indicated by an increased mitotic index in cells treated with both etoposide and nocodazole when compared with etoposide alone. As tyrosine phosphorylation was augmented in the derivative cell lines treated with etoposide, these findings were surprising, because they infer that tyrosine phosphorylation is not associated with induction of cell cycle arrest as would be predicted by phosphorylation of Y15. In summary, K562 cells' response to etoposide was a block in the transition of cells from G2 into mitosis coupled to cell survival for at least 24 h. In the derivative cell lines, the same genotoxic insult greatly augmented Cdk1 phosphorylation, which did not impart cell cycle arrest, neither was a state of cell survival maintained.

To show that this phenomenon was not restricted to the K562 derivative cell lines the HL60 cell line was also studied. The response of the HL60 cell line to etoposide also showed an increased level of tyrosine-phosphorylated Cdk1 in cells that subsequently underwent apoptosis above the level required to induce cell cycle arrest at the G2M checkpoint. Also, cells synchronized in the G2M phases of the cell cycle showed an increased propensity for the induction of tyrosine phosphorylation of Cdk1 compared with asynchronous cells, when treated with etoposide, suggesting that events at G2M are critical to cell survival.

The tyrosine phosphorylation of Cdk1 observed in this study did not induce G2M cell cycle arrest as predicted. The fact that the phosphorylation was lower in cells that undergo cell cycle arrest than in those that do not was an unexpected finding. To induce cell cycle arrest,phosphorylated Cdk1 must be nuclear. In the Hela cell line, the block in cells at the G2M transition, following either radiation or etoposide treatment, has been shown to be associated with an accumulation of inactive, cyclin B-associated, tyrosine-phosphorylated, Cdk1 in the nucleus. Cdk1 was found in the cytoplasm but it was not phosphorylated. Moreover, caffeine treatment, which abrogated the G2M checkpoint, also abrogated nuclear accumulation of tyrosine-phosphorylated Cdk1 (Kao et al, 1999). Also, this checkpoint can be abrogated by enforced expression of a non-inhibitable Cdk1 mutant (cdc2AF) plus a mutated cyclin B incapable of nuclear export (Jin et al, 1998). The nuclear localization of these kinase-active mutants in DNA damage-arrested cells promoted cell cycle progression that resulted in abnormal chromosomal morphology, and apoptosis was subsequently observed to be a result of premature mitotic events (Jin et al, 1998). Thus, the heightened phosphorylation seen in our study may reflect the cellular location of Cdk1. In cells that fail to undergo cell cycle arrest, the phosphorylated Cdk1 may have remained cytoplasmic. The corollary of this is that active Cdk1 is nuclear. As cytoplasmic Cdk1 activity is required for a normal mitosis, the imbalance of active Cdk1 in the nucleus and inactive Cdk1 in the cytoplasm would promote an aberrant mitosis and subsequent cell death. Why cytoplasmic Cdk1 should have enhanced phosphorylation levels is not known.

Another possibility is that the Cdk1 phosphorylation observed is associated with activation of the cyclosome- or anaphase-promoting complex (APC/C). Jin et al (1998) showed that cells expressing a non-degradable form of cyclin B were capable of inducing an enforced exit from mitosis and that this was associated with increased tyrosine phosphorylation of Cdk1. It was also reported that Cdk1 phosphorylation could activate the APC/C, thus driving cells through anaphase, again enforcing an exit from mitosis (Listovski et al, 2000). Therefore, the Cdk1 phosphorylation observed in our studies might be that relating to APC/C activation and an enforced mitosis rather than that associated with a G2M cell cycle arrest. This is supported by the observation that taxol induces apoptosis in cycling cells via a Cdk1-dependent upregulation of p55cdc, an apoptosis-promoting gene product found in proliferating cells. Cell death via p55cdc is also due to APC/C activation (Makino et al, 2001), which is simultaneous to, or followed by, an inactivation of Cdk1 by tyrosine phosphorylation.

Therefore, the etoposide-induced tyrosine phosphorylation of Cdk1 observed in our studies could be either a result of, or a mediator of, premature exit from mitosis rather than an inducer of cell cycle arrest. Enforced cell division in a cell with etoposide-damaged DNA would promote mitotic catastrophe and a subsequent apoptotic death similar to that detected in both the HL60 and derivative K562 cell lines.

Cdk1 phosphorylation allied to poor cell cycle checkpoint instigation has been shown post etoposide treatment in HL60 and derivative K562 cell lines. Thus, the phosphorylation status of Cdk1 appears to dictate the response of leukaemic cells to etoposide independently, or downstream of, Bcr-abl.

Our data indicates that enhanced tyrosine phosphorylation of Cdk1 is paradoxically related to a conversion from a cell cycle arrest scenario into that of cell death, probably as a result of events at mitosis that are incompatible with cell survival. What dictates the decision between arrest and death in this context should be important in determining the outcome of therapeutic intervention in leukaemia.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Etoposide treatment induced either arrest of the cell cycle at G2M or cell death that is time and dose dependent
  6. Constitutive levels of expression of Cdk1 are less in the derivative cell lines
  7. Cdk1 expression does not change on the induction of etoposide-induced cell cycle arrest or cell death
  8. Augmented tyrosine phosphorylation of Cdk1 is indicative of a failure to induce cell cycle arrest
  9. Augmented Cdk1 tyrosine phosphorylation does not prevent cells entering mitosis
  10. Verification that augmented Cdk1 tyrosine phosphorylation is not unique to the derivative cell lines
  11. HL60 cells synchronized in G2M show augmented Cdk1 tyrosine phosphorylation in response to etoposide
  12. Discussion
  13. Acknowledgment
  14. References
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