Camptothecin causes cell cycle perturbations within T-lymphoblastoid cells followed by dose...

Pergamon

PII: SO1452126(97)00077-S

Leukemia Research Vol. 21, No. IO, 961-972, 1997. pp. 0 1997 Elsevier Science Ltd. All rights reserved

Printed in Great Britain 0145-2126/97 $17.00 + 0.00

CAMPTOTHECIN CAUSES CELL CYCLE PERTURBATIONS WITHIN T-LYMPHOBLASTOID CELLS FOLLOWED BY DOSE DEPENDENT

INDUCTION OF APOPTOSIS

Nicholas Johnson, Tony T. C. Ng and Jacqueline M. Parkin Department of Immunology, St. Bartholomew’s and the Royal London College of Medicine and Dentistry,

38 Little Britain, West Smithfield, London EClA 7BE, U.K.

(Received 13 January 1997. Accepted 16 May 1997)

Abstract-We have investigated the effect of the anticancer compound, camptothecin on Jurkat T-cells, a lymphoblastoid leukemic cell-line. Exposure to low concentrations led to rapid cessation of DNA (more than 95%) and RNA (more than 75%) synthesis. Perturbations to the cell cycle were observed following exposure which caused a significant accumulation of cells within Gl (P= 0.03) with a concomitant decrease in G2/M (P= 0.025). Concentrations below 0.1 PM could inhibit DNA synthesis but not induce apoptosis. Induction of apoptosis was dose dependent and could be detected as early as 3 h post exposure. The apoptotic population appeared to be derived from Gl and S-phase cells but not G2/M, coinciding with the cell cycle compartments in which DNA and RNA polymerases function. However, direct inhibition of DNA polymerase alone was not shown to be associated the induction of apoptosis or with a decrease in susceptibility to camptothecin-induced cell death. The effects of camptothecin on Jurkat T-cells and the potential mechanisms involved are discussed in the context of observations made in other transformed cell lines. 0 1997 Elsevier Science Ltd

Key words: Camptothecin, apoptosis, Jurkat T-cell, cell cycle.

Introduction

Apoptosis plays a fundamental role in the development and functioning of the immune system [ 1,2]. However, the mechanisms that mediate this process within haemopoetic cells remain unresolved. The induction of apoptosis by chemical agents has provided a useful model for the elucidation of this process [3]. Camp- tothecin and related compounds are highly cytotoxic, and provide potent therapeutics against cancer [4-71. Its mode of action is through the inhibition of DNA topoisomerase I by reversible binding to the enzyme- DNA complex [8]. Camptothecin could theoretically affect many aspects of DNA metabolism including transcription, recombination and chromosome segrega- tion at mitosis [9, lo] and a direct result of exposure to this and related compounds including the topoisomerase II inhibitor, etoposide, is a complete cessation of DNA synthesis.

Abbreviations: CPT, camptothecin; APH, aphidicolin; NOC, nocodazole; TdT, terminal transferase.

Correspondence too: Dr. N. Johnson, Department of Biochemistry and Molecular Biology, University College London, Gower Street, London EClE 6BY, UK.

Early studies investigating the action of camptothecin, which were based on the myeloid cell line HL-60, suggested that camptothecin-induced cell differentiation to a more mature phenotype [ 11,121. However, it was soon recognised that camptothecin caused a rapid dose- dependent stimulation of an apoptotic pathway [ 13-151. The differentiation status of HL-60 cells appeared to influence the susceptibility to apoptosis. Differentiation following treatment with phorbol esters inhibited the induction of DNA fragmentation by a range of topoisomerase [16]. This could be interpreted as evidence that DNA replication is necessary for the induction of apoptosis, with phorbol ester treatment causing a cessation of cell division [17]. Original investigations indicated susceptibility to cell death was dependent upon entry into S phase. Recent studies suggest that HL-60 cells which undergo apoptosis are derived exclusively from the S-phase of the cell cycle [18] although other investigators, using different tech- niques, cell lines and exposure times, have argued that camptothecin induces apoptosis within cells from all stages of the cell cycle [19]. The mechanisms which stimulate apoptosis following camptothecin treatment are unresolved but could provide important information

961

962 N. Johnson et al.

towards the understanding of the anticancer properties of this class of compounds and the pathways that activate the apoptotic process.

The data presented demonstrate the susceptibility of Jurkat cells, a human T-cell line derived from lymphoblastoid leukemia, to camptothecin-induced apoptosis. This study also examines the kinetics of both the inhibition of RNA/DNA synthesis and subsequent induction of apoptosis. Important differences were found between Jurkat and other cell lines in terms of the effects of camptotbecin on cell-cycle responses as well as the relationship between cell-cycle stage and susceptibility to camptotbecin-induced apoptosis.

Materials and Methods

Cell culture Jurkat cells were obtained from the MRC AIDS

directed program (Potters Bar, UK). Cells were cultured at a concentration of 1 x lo6 per ml in RPM1 1640 media supplemented with 10% foetal calf serum (TCS Biologicals Ltd., Buckingham, UK), 2 mm01 1-l L-

glutamine, 100 U ml-’ penicillin and 100 pg ml-’ streptomycin. Camptothecin (CPT), aphidicolin (APH) and nocodazole (NOC) (all Sigma Chemicals Ltd., Dorset, UK) were used at the concentration indicated.

Cell cycle synchronisation Synchronisation within Gl/S was achieved using a

double aphidicolin block [20]. Jurkat cells were washed and resuspended in 1 ug ml-’ aphidicolin and cultured for 16 h. Cells were then washed in fresh media and cultured for a further 10 h in medium alone. Treated cells were then exposed to a further 16 h with 1 ug ml-’ aphidicolin. Synchronisation was checked by propidium iodide staining and flow cytometry (see below). Cell viability was checked immediately following release from aphidicolin and was routinely greater than 90%. Synchronised cells were used for experimentation immediately to avoid cells re-entering the cell cycle.

Jurkat cells were blocked in S/G2/M by a 16 h incubation with 10 ug ml-’ nocodazole. Cells were thoroughly washed and resuspended in fresh media prior to exposure to camptothecin. Viability was checked and found to be greater than 85%.

Proliferation assays Jurkat cells were cultured in 96-well plates with the

indicated concentrations of camptothecin or aphidicolin for the indicated time at 37°C. Each well was pulsed with 0.5 pCi 3H-labelled thymidine or uridine (both ICN, Thame, UK), 30 min prior to harvesting onto glass fibre filters (Whatman, Maidstone UK). Nucleotide incorporation was measured by liquid scintillation in a B counter (Wallac, Milton Keynes, UK). Triplicate wells

for each time point were set up which gave a mean value for counts per minute (CPM). Results are shown as inhibition index, calculated by dividing the mean CPM of each treated sample by the mean CPM obtained for the untreated control. Control values were 3270 CPM (k379) for uridine incorporation and 1283.5 CPM (+ 84) for thymidine incorporation. Data are representative of two separate experiments.

Cell viability Cell samples were diluted l/10 with 0.1% trypan blue

in phosphate buffered saline (PBS). Samples were counted in disposable glasstic slides (HYCOR Bio- medicals Inc., California, USA) and the mean percentage of viable cells in nine microscopic fields was presented (each containing a minimum of 50 cells).

Staining with propidium iodide The percentage of hypodiploid cells was derived

using an established method [21]. Briefly, cells were washed with PBS and fixed in 50% ethanol at 4°C for 1 h. After further washing, the cells were resuspended in 50 pg ml-’ propidium iodide (Cambridge Bioscience, Cambridge, UK) in buffer containing 0.1% sodium citrate, 0.1% Triton X-100 and RNAse A 100 ug ml-’ (Sigma). Samples were allowed to equilibrate at room temperature for 1 h prior to analysis using a FACScan flow cytometer (Becton-Dickinson Ltd, Oxford, UK). Cell cycle analysis was performed using Cellfit software (Becton-Dickinson) and the percentage of hypodiploid cells analysed using the Lysis II program (Becton- Dickinson).

DNA fragmentation Confirmation of apoptosis was made by visualisation

of DNA fragmentation using agarose gel electrophoresis. Treated cells (approximately 106), were pelleted and resuspended in 15 ul of hypotonic lysis buffer containing 5 mM Tris pH 7.4, 5 mM EDTA, 0.5% Triton X-100 and 1 mM PMSF. 5 ~1 of 100 ug ml-’ RNAse A was added and the sample incubated at 37°C for 1 h. After addition of 5 u15 x loading buffer (0.25% bromophenol blue, 40% sucrose), the sample was loaded onto a 1.5% Tris-acetate agarose gel. Following electrophoresis at 50 V for 3 h DNA fragmentation was visualised by staining with 1 ug ml-’ ethidium bromide and UV transillumination.

Measurement of apoptosis using the terminal transferase (TDT) assay

Evidence of early apoptotic endonuclease activity was assessed by the incorporation of labelled nucleotides [ 151 and adapted for use with propidium iodide [22]. Samples were fixed with 40% formaldehyde for 15 min on ice, washed and resuspended in 70% ethanol and

Cell cycle perturbations within T-lymphoblastoid cells 963

IMABCD~

Fig. 1. DNA fragmentation induced by camptothecin treatment of Jurkat T-cells. 123 base pair DNA marker (M), untreated cells (A) and camptothecin treated cells at 0.01 FM (B),

0.1 j.tM (C) and 1 PM. (D).

stored at 4°C for up to 5 days. Prior to labelling, cells were rehydrated with PBS for 15 min. After centrifuga- tion each sample was resuspended in a labelling solution made up of 5 units TdT and 0.5 nmol biotinylated dUTP (both from Boehringer Mannheim, East Sussex, UK) prepared in a cacodylate buffer containing 0.2 M

potassium cacodylate, 2.5 mM Tris-HCl pH 6.6, 2.5 mM cobalt chloride and 0.25 ml ml-’ BSA. The negative control was left to incubate with cacodylate buffer only. Samples were incubated at 37°C for 30 min and then washed and resuspended in 5 pg ml-’ fluor- esceinated avidin (Boehringer Mannheim), prepared in 4 x SSC, 0.1% Triton X-100 and 5% (w/v) non-fat dry milk. After a final wash, samples were resuspended in PBS containing 50 pg ml-’ propidium iodide, 100 pg ml-’ RNAse A and analysed by flow cytometry.

Statistical analysis Non-parametric Wilcoxon signed rank analysis using

the SPSS/PC + Statistics 4.0 package was employed to analyse the difference between treated and untreated samples. A P value of less than 0.05 was considered significant.

Results

Kinetics of camptothecin-induced apoptosis within jurkat T-cells

When exposed to camptothecin, Jurkat cells appear highly susceptible to apoptosis. Following exposure to camptothecin at concentrations as low as 0.1 PM, Jurkat cells show the distinctive morphology of apoptotic cells (data not shown). This includes the appearance of nuclear shrinkage and disaggregation [23]. To confirm that these morphological changes were caused by the induction of apoptosis, similarly treated cells were analysed by gel electrophoresis and demonstrated the “ladder” pattern associated with activation of an endogenous endonuclease (Fig. 1) [24]. The dose dependency of camptothecin action is also illustrated with concentrations of camptothecin greater than 1 PM following 24 h exposure providing the strongest stimulus for the induction of apoptosis. Table 1 shows the dose-dependent inhibition of DNA synthesis in Jurkat cells by camptothecin as measured by 3H-thymidine uptake. Inhibition was observed at camptothecin concentrations as low as 0.01 PM, a concentration of 1 pM was required to totally inhibit DNA synthesis after 16 h.

Table 1. The effect of camptothecin on Jurkat T-cells

Camptothecin (pM) Thymidine incorporation Viability (%)

0 20148 f 414* 91.0 &- 2 0.001 21176 + 574 85.0 f 4 0.01 9384 f 2912 64.5 f 18.5 0.1 1022 f 345 46.5 f 3.5 1 398 f 200 47 f 0.8

* Data represents CPM ( f S.D.) measured 16 hrs following addition of camptothecin. Viability measured by trypan blue dye exclusion. Apoptosis measured by increase in hypodipliod cells.

Apoptosis (%)

12.8 f 6.5 15.5 + 9.5 31.7 f 12.2 51.1 + 11.6 57.2 f 3.7


Fig.

Hypodiploid Cells (S)

Dl

[Cl

[Bl

MI

Time (hrs)

Fig. 2. Induction of cell death by camptothecin over a 24 hr timecourse measured by an increase in hypodiploid cells.

- Nick Formation (%) 40-

Cells T Incorporating

dUTF’ (%) 30- 1

20-

0; , , , , , , , , { 0 12 3 4 5 6 7 8 9

Tiie (hrs)

3. Induction of apoptosis by camptothecin (1 PM) measured by incorporation of biotin labeled dUTP into DNA strand breaks (TdT assay). Error bars represent S.E.M.

The cessation of DNA synthesis was accompanied by a concomitant increase in apoptosis as determined by propidium iodide staining and loss of cell viability.

Kinetic analysis of camptothecin-induced cell death shows the rapid induction of apoptosis within 4 h at drug concentrations greater than 1 pM (Fig. 2). Measurement of cell viability by trypan blue exclusion and DNA fragmentation, by agarose gel electrophoresis, could only be detected at 6 h following exposure to camptothecin at concentrations greater than 1 pM (data not shown). To confirm these observations, an in vitro nick translation technique was used to identify the earliest evidence of apoptosis. Figure 3 shows the time course of nick formation. An increase in the incorporation of biotin-labelled dUTP can be observed as early as 3 h and reached a plateau at 6 h of camptothecin treatment. Comparison of the cell cycle profile by simultaneous cell cycleRdT analysis (Fig. 4), suggests that the source of these apoptotic cells were the Gl and S phases but not G2/M. Only these two phases of the cell cycle appeared to incorporate labelled dUTP between 3 and 5 h.

In addition to these observations, camptothecin also

showed rapid inhibition of nucleotide synthesis. Figure 5 and Fig. 6 compare the kinetics of DNA and RNA synthesis inhibition, respectively, between camptothecin and aphidicolin, a known inhibitor of DNA polymerase. A camptothecin concentration of 1 PM was used to allow interpretation of results with respect to the induction of apoptosis. Within 1 h, aphidicolin caused over 90% inhibition of DNA synthesis. Camptothecin required 2 h to reach similar levels of inhibition. In contrast, aphidicolin is a poor inhibitor of RNA synthesis reaching a maximum 45% inhibition after 2 h, confirming its specificity for DNA polymerases. Camptothecin caused rapid inhibition of RNA synthesis (77% reduction within 1 h compared to no treatment control), although the level of inhibition did not reach that for DNA synthesis within the time course of the investigation.

Cell cycle changes associated with exposure to camptothecin

Investigation of the cell cycle during the early stages of camptothecin exposure revealed distinct perturbations


Rl R2 R3

FL2-‘N=Lz-W-.~ -->

FL2-WFL2-Rma --->

Fig. 4. Measurement of cell cycle profile by propidium iodide staining (X-axis) and apoptosis by the TdT assay (Y-axis) between 3- 6 hrs following exposure to camptothecin (1 PM). Due to non-specific uptake of label by cells in Gl the majority of negatively staining cells have been pushed into the X-axis. The boxes have been arbitrarily placed to indicate the possible source of apoptotic

events in relation to the cell cycle and represent Gl (Rl), S (R2) and G2N (R3).

to the distribution ‘of asynchronously dividing Jurkat cells. Under optimal conditions of culture, the ratio of cells within the three principal phases of the cell cycle remained stable at 53.8% k 1.34 (Gl), 29.1% f 2.4 (S) and 17.2 f 1.1 (G2N), as shown by propidium iodide- staining alone (data not presented). Figure 7 shows a representative experiment of cell-cycle changes at various concentrations of camptothecin during 24 h of exposure. By 24 h, the influence of camptothecin at greater than 0.1 pM caused such a distortion to the DNA content profile that it proved impossible to model. The cause of this was the loss of the G2A4 population. At a concentration of 0.01 PM, cells became blocked in S phase, elevating the percentage within this stage to over 59% ( f 3%). There appears to be partial inhibition of thymidine uptake at this concentration (Table 1) suggesting that cells cycle into S phase but undergo partial inhibition of DNA synthesis and are delayed in completing this stage. No significant increase in

apoptosis was observed over unstimulated cells at this or lower concentrations. Figure 8 shows the gradual increase of cells within the Gl compartment over the first 8 h of culture with 1 pM camptothecin. This increase reached statistical significance at 3 h (P= 0.03). Over this time period, the percentage of cells within S phase appears variable but essentially stable until the 7 h time point where the percentage of cells begins to decrease. Within G2/M there is a decline in the number of cells within this phase over the time course, reaching significance at 2 h (P= 0.025). The kinetics of this decline and the observations presented above (Fig. 4) suggest that a loss of GUM cells cannot be explained by apoptotic cell death and may be related to the inhibitory action of camptothecin on S-phase transit. These observations are consistent with the known function of camptothecin causing arrest of DNA synthesis, preventing cells from entering S phase and blocking those cells that are in this phase from


..w-

- Aphidicdin

0 1 2 3 4

Time (Hrs)

Fig. 5. Inhibition of 3H labeled thymidine uptake by aphidicolin (1 &ml) and carnptothecin (1 PM).

completing it. Presumably G2/M is relatively unaf- fected, allowing cells to complete mitosis and return to Gl, resulting in an accumulation witbin this compartment.

This data is in partial agreement with previous investigations of the cell cycle consequences of camptothecin who have shown an accumulation of cells in all phases [2.5]. However, when compared to results obtained witbin HL-60 myeloid cells [15] there appears to be quite a significant difference. At 3 h following exposure to camptothecin (0.1 PM), I-K-60 cells showed an increase in the number of cells within Gl by almost two-fold, whilst showing a four-fold decrease in S-phase cells. This lead to the deduction that only S phase cells were susceptible to apoptosis within this cell line. No changes were observed within the G2/M phase of the cell cycle. This suggests that different transformed cell

lines, and, by analogy, leukaemias show variable responses to this compound.

Cell cycle synchronisation does not influence the susceptibility of camptothecin-induced apoptosis in Jurkat T-cells

To assess the influence of the cell cycle on camptothecin-induced apoptosis, Jurkat cells were blocked in GUS with aphidicolin, and G2A4 with nocodazole 0. Blocked and untreated cells were washed and exposed to 10 pM camptothecin for up to 6 h. This concentration was used to induce apoptosis rapidly before cells could begin cycling (see Fig. 2). Figure 9 and Fig. 10 show the induction of DNA fragmentation within asynchronous, aphidicolin blocked and nocodazole blocked Jurkat cells. Aphidicolin alone did not induce apoptosis and resuspension in fresh media allowed the release of cells back into the cell cycle

- Aphidicdin

Inhibition Index

Fig. 6. Inhibition of 3H labeled uridine uptake by aphidicolin (1 &ml) and camptothecin (1 PM).


SAMPLE

Fig. 7. Cell cycle changes induced by exposure to carnptothecin. Histograms are representative of three separate experiments.

with a distinctive peak of cells traversing S phase within 3 h (data not shown). Observation of both DNA fragmentation and the development of a hypodiploid population suggest that aphidicolin increased the sensitivity of Jurkat cells to camptothecin-induced cell death (Fig. 11) confirming the observation that apoptotic cells could potentially be derived from both Gl and S. In a similar manner, nocodozole treatment did not inhibit camptothecin-induced cell death. Figure 9 indicates that treatment with this compound can induce apoptosis itself after exposure for greater than 16 h with the development of DNA fragmentation in all samples treated with nocodazole. Although this complicates the role of camptotbecin within G2A4 blocked cells, it does indicate that cells trapped in this compartment of the cell cycle remain susceptible to apoptosis.

Discussion and Conclusion

The data presented within this study demonstrate the susceptibility of Jurkat T-cells to undergo apoptosis when exposed to camptothecin. A comparison of the time courses of different methods of measuring apoptosis shows a distinct sequence of events. Camp- tothecin acts very rapidly to inhibit both DNA and RNA synthesis. Evidence of DNA strand breaks is measurable following 3 h of exposure and is followed by an increase in hypodiploid cells at 4 h indicating active digestion of DNA. Morphological signs of nuclear changes can be observed between 4 and 5 h of exposure (data not

shown). No change in membrane integrity was observed over this time period. This agrees with a hypothetical model of apoptosis which divides the process into four stages; detection of the apoptotic stimulus, signal transduction pathways to the effecters of apoptosis, effecters of apoptosis including proteases act on the cell, and finally the post-mortem stage in which the DNA is degraded [26]. It also demonstrates the rapidity of the process with the post-mortem stage being reached within 4 h among susceptible cells. Comparison of these data with published observations suggest that the changes observed within this lymphoblastoid cell-line are very different from those seen in the lymphocytic cell-line Molt-4, which show a marked accumulation of cells within the G2/M compartment upon 24 h exposure to camptothecin [27]. Jurkat cells also appear less sensitive than the myeloid cell-line HL-60, Independent results have shown that HL-60 cells demonstrate advanced signs of apoptosis at lower concentrations [15] and following shorter treatment periods [ 161. Although some of these differences may be explained by inter- laboratory variation, the widely varying effects on the cell cycle suggest distinct mechanisms. Differences between the two cell-lines could be related to the ability of Jurkat cells to tolerate blocks within the cell cycle WI.

The relationship between cell cycle distribution and susceptibility to camptothecin-induced apoptosis remains a controversial issue. Data presented suggest that apoptotic cells could arise from both Gl and S phases of

968 N. Johnson er al.

Gl 80

* - Media Only l

Cells in Cl 70 - -m-o--- (%I CFT (lpg/ml:

40 I I I I I I I I 0 12 3 4 5 6 7 8 9

Time (Hrs)

S 40

1 Cells in S

(So)

25 -

20 I I I I 1 I I 0 1 2 3 4 5 6 7 8 9

Time (Hrs)

GUM

Cells in G2iM @)

T/

I lo- ‘rl. * *

b---Z- * I - ---;---,-

J. * A. 5- l . *

“o __-w- -- -z l _. -.--

P

0 I I I I I I I I

0 12 3 4 5 6 7 8 5

- Media Only

---O--- CFT(lpg/ml)

- Media Only

---o--- CR(lpg/ml)

Time (Hrs)

Fig. 8. Changes in the percentage of cells within each phase of the cell cycle within untreated and camptothecin treated Jurkat T- cells. Error bars represent S.E.M. from three (untreated) and six (treated) independent measurements for each time point. * indicates

p < 0.05.

the cell cycle, which coincide with the phases in which camptothecin inhibits RNA- and DNA-polymerase activities (Figs 5 and 6). The rapid appearance of apoptotic cells, apparently from Gl, within 3 h of addition of CPT and before apoptotic events from S- phase cells arise, suggests that degradation and loss of DNA probably does not account for this population. However, during the repeated washing steps of the TUNEL procedure, some extraction of low molecular

weight DNA from apoptotic cells may occur, resulting in a shift of their DNA content towards lower values. The relative proportion of Gl cells undergoing apoptosis as detected by this assay may therefore be a slight overestimate. Aphidicolin alone did not induce a high level of apoptosis in Jurkat T-cells within 24 h. This indicates that inhibition of DNA synthesis alone in the absence of DNA damage (in the form of camptothecin- trapped topoisomerase-DNA cleavable complex, for


M 123 456 7 8 9 101112 131415 161718

Fig. 9. DNA fragmentation within samples pretreated with aphidicolin and nocodazole, and subsequently exposed to camptothecin (10 PM). Track M represents DNA markers. Each group of three represents samples taken at 0, 2 and 4 hrs. Tracks l-3 are asynchronous cells; tracks 4-6 are asynchronous cells plus camptothecin; tracks 7-9 are aphidicolin blocked cells; tracks 10-12 are aphidicolin blocked cells plus camptothecin; tracks 13-15 are nocodazole blocked cells; tracks 1618 are nocodazole blocked cells

plus camptothecin.

instance) does not provide a sufficient signal for the induced apoptosis (Fig. 11). These data reinforce the initiation of apoptosis. Resuspension of cells in fresh notion that S-phase cells may have an enhanced media following exposure to aphidicolin allowed the susceptibility to apoptosis induced by DNA-damaging resumption of cell cycling with a distinctive peak of agents. cells transversing S phase within 3 h (data not Apoptosis induced by vincristine [29] and anti-CD3 presented), an event which was coincident with the treatment [28] can be reversed by DNA synthesis aphidocolin-associated enhancement of camptothecin- inhibition with aphidicolin. This, combined with the

35

SAMPLE

Fig. 10. Hypodiploid cell formation within Jurkat cells treated as in Fig. 9.

970 N. Johnson er al.

Fig. 11.

,,..-- ,.--ii .A “““” “““” ---- A ---- A ApH+/epT+ ApH+/epT+

------- -------

/.-- ----o---- ----0”-.- ApH+/cme ApH+/cW-

,,p _._..“- ,,p _._..“-

“I... . . . . . . . “I... . . . . . . .

- 0 - 0

APH-/CPT+ APH-/CPT+

APH-/Cm- APH-/Cm-

,/-• ,/-• T T

0 0 1 1 2 3 4 5 6 2 3 4 5 6

Tiie (lxs) Tiie (lxs)

Kinetics of camptothecin-induced apoptosis in untreated and aphidicolin-blocked Jurkat cells. Kinetics of camptothecin-induced apoptosis in untreated and aphidicolin-blocked Jurkat

observation that low concentrations of camptothecin can inhibit DNA synthesis resulting in an apparent block in S phase (Fig. 7) without causing apoptosis, suggests that the proapoptotic effect of camptothecin could operate through a number of different pathways. Inhibition of RNA synthesis could provide a possible alternative. Bruno et al. [30], have shown that inhibition of RNA synthesis and DNA synthesis in lymphocytes entering the cell cycle following PHA stimulation occurs at concentrations greater than 1.5 PM. In addition, the recent observation that topoisomerase I is a kinase that both binds ATP and phosphorylates the SR-family of RNA-splicing enzymes [31] links this enzyme to both RNA synthesis and functions unrelated to DNA synthesis. The inhibition of both RNA and DNA synthesis in Jurkat T-cells by camptothecin treatment also implies that the signal(s) that triggers the onset of apoptosis (evident within a few hours of exposure to camptothe- tin) may not require de ~OVO macromolecular synthesis. The evidence for G2/M cells entering an apoptotic pathway is less clear between 3 and 5 h exposure and the decline in cell percentage in this compartment during this period could be related instead to the camptothecin- induced slow-down in S-phase transition. However, the observation that nocodazole blocked cells, i.e. cells trapped in G2/M, remain highly susceptible to both spontaneous and camptothecin-induced apoptosis, indicate that cells within this phase of the cell cycle are not resistant to apoptosis.

The TdT data suggest that initiation of DNA strand cleavage starts 3 h following exposure to camptothecin. Similar data were obtained by using similar methodol- ogy and interpreted as clear evidence of apoptosis [ 151.

The terminal transferase used in the assay recognises both single- and double-strand breaks as a template for dUTP incorporation. The appearance of single DNA strand-breaks could be due to the action of camptothecin itself and may not necessarily reflect the degree of apoptosis. However, a comparison of the time courses of DNA synthesis inhibition (Fig. 5) and nick formation (Fig. 3) demonstrates that camptothecin has caused more than 95% DNA synthesis inhibition by 2 h post- exposure but with no concomitant increase in the number of DNA strand breaks. It is possible that the transferase does not recognise strand breaks generated by camptothecin, perhaps owing to the close association of camptothecin with the DNA-topoisomerase covalent complex at the site of cleavage [S]. Alternatively the level of single-stranded breaks required to inhibit DNA synthesis is lower than that detectable by this assay. These data indicate that active signs of apoptosis as measured by the TdT assay are demonstrable at 3 h following exposure to camptothecin, or 1 h following the total cessation of DNA synthesis.

The exact mechanism by which the signal for the activation of apoptotic mediators is delivered following camptothecin treatment remains unclear. The significant accumulation of cells within Gl implicates ~53, a known inducer of apoptosis in response to DNA damaging agents [32-341. Jurkat cells constitutively express functional p53 which has been shown to associate with the SPl transcription factor following TNF receptor engagement [35]. We are currently investigating changes in the nuclear/cytoplasmic ratio of ~53 expression in Jurkat cells following exposure to camptothecin. Another intriguing possibility is that


cyclin dependent kinases (cdk), which control the cell cycle, may also be related to the onset of apoptosis. Activation of cyclin BUcdc2 kinase has been observed within HL-60 cells following exposure to camptothecin within the first 30 min, followed by a rapid loss of activity implicating that this could form the start of an apoptotic pathway [36]. However, our studies have not shown a change in the levels or the phosphorylation status of either cyclin B or cdk2 during the 24 h window in which apoptosis is triggered within Jurkat cells (unpublished observations). Recent studies have demonstrated the influence of a range of cyclin- dependent kinases on the susceptibility of HELA cells to chemical induced apoptosis [37]. However, the authors suggest that great caution should be used in interpreting these links until intermediates between cdks and the effecters of apoptosis can be identified.

Camptothecin is a potent inducer of apoptosis in Jurkat T-cells. Targeting either the topoisomerase-DNA complex or other unspecified targets may, therefore, offer an effective therapeutic approach against T lymphoblastoid leukemias. Characteristic features of the cytotoxic action of camptothecin against these leukaemic cells such as cell cycle specificity and dose response differ significantly from that shown in various myeloid leukaemic models. Further studies are required to elucidate the molecular basis underlying these differences which may improve our understanding of the pathways that activate the apoptotic process within the haemopoietic system.

Acknowledgements-The authors would like to thank Dr R. G. Wickremasinghe for his constructive comments on this research. NJ was supported by the Joint Research Board of St. Bartholomew’s Hospital, London. TN was supported by a Medical Research Council Clinical Fellowship (G106/620).

References

1. Lynch, D. H., Ramsdell, F. and Alderson, M. R., Fas and FasL in the homeostatic regulation of immune responses. Immunology Today, 1995, 16, 569.

2. Akbar, A. N., Borthwick, N., Salmon, M., Gombert, W., Bofill, M., Shamsadeen, N., Pilling, D., Pett, S., Grundy, J. E. and Janossy, G., The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory. Journal of Experimental Medicine, 1993, 178, 427.

3. Dive, C. and Hickman, J. A., Drug-target interactions: only the first step in a commitment to a programmed cell death? British Journal of Cancer, 1991, 64, 192.

4. Slichenmeyer, W. J., Rowinsky, E. K., Donehower, R. C. and Kaufmann, S. H., The current status of camptothecin analogs as anticancer agents. Journal of the National Cancer Institute, 1993, 85, 271.

5. Lui, L. F., DNA topoisomerase poisons as antitumour drugs. Annual Review of Biochemistry, 1989, 58, 351.

6. Schellens, J. H. M., Creemers, G. J., Beijnen, J. H., Rosing,

7.

8.

9.

10.

11.

12.

13.

14.

H., De Boer-Dennert, M., McDonald, M., Davies, B. and Verwij, J., Bioavailability and pharmacokinetics of oral topecan: a new topoisomerase I inhibitor. British Journal of Cancer, 1996, 73, 1268. Dancey, J. and Eisenhauer, E. A., Current perspectives on camptothecin in cancer treatment. British Journal of Cancer, 1996, 74, 327. Hertzberg, R. P., Caranfa, M. J. and Hecht, S. M., On the mechanism of topoisomerase inhibition by camptothecin: Evidence for binding to an enzyme-DNA complex. Biochemistry, 1989, 28,4629. Wang, J. C., DNA topoisomerases. Annual Review of Biochemistry, 1985, 54, 665. Hsiang, Y.-H., Lihou, M. G. and Liu, L. F., Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Research, 1989, 49, 5077. Chou, S., Kaneko, M., Nakaya, K. and Nakamura, Y., Induction of differentiation of human and mouse myeloid leukemia cells by camptothecin. Biochemical and Biophy- sical Research Communications, 1990, 166, 160. Aller, P., Ruis, C., Mata, F., Zorilla, A., Cabanas, C., Bellon, T. and Bemabeu, C., Camptothecin induces differentiation-related genes in U937 human promonocytic leukemia cells. Cancer Research, 1992, 52, 1245. Kaufmann, S. H., Induction of endonucleolytic DNA cleavage in human myelogenous leukemia ceils by etoposide, camptothecin and other cytotoxic anticancer drugs: a cautionary note. Cancer Research, 1989, 49, 5870. Lennon, S. V., Martin, S. J. and Cotter, T. G., Dose- dependent induction of apoptosis in human tumor cell lines by widely divergent stimuli. Cell Proliferation, 1991, 24, 203.

15. Gorczyca, W., Gong, J., Ardelt, B., Traganos, F. and Darzynkiewicz, Z., The cell Cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumour agents. Cancer Research, 1993, 53, 3186.

16. Solary, E., Bertrand, R., Kohn, K. W. and Pommier, Y., Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase I and II inhibitors. Blood, 1993, 81, 1359.

17. Porliri, E., Hoffhrand, A. V. and Wickremasinghe, R. G., Granulocytic differentiation of HL-60 human promyelocytic leukemia cells is preceded by a reduction in levels of inositol lipid-derived second messengers. Experimental Hematology, 1988, 16, 641.

18. Gorzyca, W., Melamed, M. R. and Darzynkiewicz, Z., Apoptosis of S-phase HL-60 cells induced by DNA topoisomerases: Detection of DNA strand breaks by flow cytometry using the in situ nick translation assay. Toxicology Letters, 1993, 67, 249.

19. Cotter, T. G., Glynn, J. M., Echeverri, F. and Green, D. G., The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle. Anticancer Research, 1992, 12, 773.

20. O’Conner, P. M., Ferris, D. K., Pagano, M., Draetta, G., Pines, J., Hunter, T., Longo, D. L. and Kohn, D. W., G2 delay induced by nitrogen mustard in human cells affects cyclin Bl/cdc2kinase complexes differently. Journal of Biological Chemistry, 1993, 268, 8298.

21. Nicolleti, I., Migliorati, G., Pagliacci, M. C., Grignani, F. and Riccardi, C., A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. Journal of Immunological Methods, 1991, 139, 271.


22. Chapman, R. S., Whetton, A. D., Chresta, C. M. and Dive, C., Characterisation of drug resistance mediated via the suppression of apoptosis by abelson protein tyrosine kinase. Molecular Pharmacology, 1995, 48, 334.

23. Wyllie, A. H., Kerr, J. F. R. and Currie, A. R., Cell death: the significance of apoptosis. International Review of Cytology, 1980, 68, 25 1.

24. Arends, M. J., Morris, R. G. and Wyllie, A. H., Apoptosis: the role of the endonuclease. American Journal of Pathology, 1990, 136, 593.

25. Poot, M., Hiller, K. H., Heimpel, S. and Hoehn, H., Distinct patterns of cell cycle disturbance elicited by compounds interfering with DNA topoisomerse I and II activity. Experimental Cell Research, 1995, 218, 326.

26. Vaux, D. L. and Strasser, A., The molecular biology of apoptosis. Proceedings of the National Academy of Sciences of the USA, 1996, 93, 2239.

27. Del Bino, G. and Darzynkiewicz, Z., Camptothecin, teniposide, or 04’-(9-acridylamine)-3-methanesulfon-m- aniside, but not mitoxantrone or doxorubicin, induces degradation of nuclear DNA in the S phase of HL-60 cells. Cancer Research, 1991, 51, 1165.

28. Zhu, L. and Anasetti, C., Cell cycle control of apoptosis in human leukaemia T cells. Journal of Immunology, 1995, 154, 192.

29. Bomer, M. M., Myers, C. E., Sartor, P., Sei, Y., Toku, T., Trepel, J. B. and Schneider, E., Drug induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3. Cancer Research, 1995, 55, 2122.

30. Bruno, S., Giaretti, W. and Darzynkiewicz, Z., Effect of

camptothecin on mitogenic stimulation of human lymphocytes: Involvement of DNA topoisomerase I in cell transition from GO to Gl phase of the cell cycle and in DNA replication. Journal of Cellular Physiology, 1992, 151, 478.

31. Rossi, F., Labourier, E., Fome, T., Divita, G., Riou, J. F., Antoine, E., Cathata, G., Brunel, C. and Tazi, J., Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature, 1996, 381, 80.

32. Lane, A. P., A death in the life of ~53. Nature, 1993, 362, 786.

33. Lowe, S. W., Schmitt, E. W., Smith, S. W., Osborne, B. A. and Jacks, T., ~53 is required for radiation induced apoptosis in mouse thymocytes. Nature, 1993, 362, 847.

34. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L. and Wyllie, A. H., Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature, 1993, 362, 849.

35. Gualberto, A. and Baldwin, A. S. Jr., ~53 and Spl interact and cooperate in the tumour necrosis factor-induced transcriptional activation of the HIV-l long terminal repeat. Journal of Biological Chemistry, 1995,270, 19680.

36. Shimuzu, T., O’Connor, P. M., Kohn, K. W. and Pommier, Y., Unscheduled activation of cyclin Bl/Cdc2 kinase in human promyelocytic leukaemia cell line HL60 cells undergoing apoptosis induced by DNA damage. Cancer Research, 1995, 55, 228.

37. Meikrantz, W. and Schegel, R., Suppression of apoptosis by dominant negative mutants of cyclin-dependent protein kinases. Journal of Biological Chemistry, 1996, 271, 10205.

Camptothecin causes cell cycle perturbations within T-lymphoblastoid cells followed by dose...

Documents

Transcript of Camptothecin causes cell cycle perturbations within T-lymphoblastoid cells followed by dose...