JOURNAL OF PATHOLOGY, VOL. 178: 71-77 (1996)

THE ASSOCIATION BETWEEN CELL PROLIFERATION AND APOPTOSIS: STUDIES USING THE CELL

CYCLE-ASSOCIATED PROTEINS Ki67 AND DNA POLYMERASE ALPHA

PHILIP J . COATES, SALLY A. HALES AND PETER A. HALL

Division of Pathological Sciences, University of Dundee, Ninewells Hospital and Medical School, Dundee, DDI 9SY, U. K.

SUMMARY The process of apoptosis is associated with the inappropriate expression of cell cycle regulatory proteins, which has led to the proposal

that the apoptotic pathway represents an abortive attempt to pass through the cell proliferation cycle. To investigate this hypothesis, we examined the expression of two proliferation-associated antigens in apoptotic cells. Apoptotic bodies seen in a range of normal and pathological tissues are often positive for the Ki67 antigen, indicating that these cells were in the cell cycle during the period that they died. In contrast, spontaneous apoptosis of human polymorphonuclear leukocytes maintained in culture was not associated with the expression of either Ki67 or DNA polymerase a. In addition, apoptotic bodies in the pre-menstrual endometrium did not express the Ki67 antigen. These results indicate that, contrary to previous suggestions, apoptosis does not always depend on cell cycle entry. The use of antibodies to Ki67 should be valuable in defining the association of apoptosis with proliferation in a wide range of cells and tissues.

KEY WORDS-apOptOSiS; Ki67; cell cycle

INTRODUCTION

Apoptosis, or programmed cell death, is a form of cell death which is characterized by an ordered series of morphological and biochemical changes. Apoptosis is an active process, involving the co-ordinated expression of a variety of genes. It is a basic biological process, critical for metamorphosis in insects and amphibia, for tissue modelling during development of mammals, in the deletion of T- and B-lymphocytes during negative selec- tion and the immune response, in tissue atrophy after hormonal stimuli, and in the regulation of cell number in adult tissues. 1-6 In addition, abnormalities of apoptosis appear to play a vital role in tumorigenesis,' and apop- tosis is a key mechanism by which chemotherapeutic agents kill

Although many diverse stimuli can cause apoptosis in a range of different cell types, there is good evidence to suggest that the final pathway of cell death is common to all cells. Recent studies have highlighted the close associ- ation between cell death and cell replication and have suggested that apoptosis involves entry of cells into the cell Thus, it has been suggested that even non-proliferating cells must re-enter the cell cycle before they can undergo apoptosis. This proposition is based on a wealth of circumstantial data obtained from a variety of experimental models of apoptosis, including apoptosis occurring in terminally differentiated cells, or in non-proliferating cells in culture. In particular, many studies have shown that apoptosis of non-proliferating cells is accompanied by an induction of proto-oncogenes

Addressee for correspondence: Dr P. J. Coates, Department of Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DDl 9SY, U.K.

CCC 0022-341 7/96/01007 1-07 0 1996 by John Wiley & Sons, Ltd

and other molecules involved in cell cycle progression. For instance, in the rat prostate, fully differentiated epithelial cells undergo apoptosis after removal of androgens. Even though these cells are normally non- proliferative, this phenomenon is accompanied by a transient induction of c-fos, followed by an induction of c-myc," a sequence of events which is generally associ- ated with cellular proliferation. In apoptosis of lym- phoid cells deprived of growth factors, there is a similar induction of c-fos and c-jun, even though the absence of growth factors would be expected to cause a down- regulation of these proteins. The induction of c-fos and c-jun appears to be a necessary part of the apoptotic pathway in these cells.I2 It has also been shown that expression of c-myc accompanies the apoptosis of T-cells following activation and is a requirement for this pro- cess.13 In non-proliferating fibroblasts, overexpression of c-myc leads to a rapid induction of a p o p t ~ s i s . ' ~ * ' ~ Similarly, continuous expression of c-fos precedes cell death in vivo, and expression of c-fos in serum-deprived cells in vitro induces apoptosis. l6 Finally, inappropriate activation of p34cdc2, which regulates entry into mitosis, is required for apoptosis induced by a lymphocyte p r~ tease . ' ~

These data indicate that apoptosis of non- proliferating cells is associated with the expression of proteins involved in cell cycle entry, but do not show that the apoptotic cells have themselves entered the cell cycle. Evidence that non-proliferating cells do indeed enter the cell cycle before undergoing apoptotis has come from studies of the rat prostate. Here, the cell cycle-associated antigen, PCNA, is up-regulated in pros- tate epithelial cells after androgen ablation. In addition, there is an increase in the number of cells which incor- porate bromodeoxyuridine (BrdU), used as a marker of

Received 7 June I994 Accepted 7 July 1995

72 P. J. COATES ET AL.

S-phase. Most notably, the nuclei of apoptotic cells contain BrdU, suggesting that these normally quiescent cells had entered into S-phase prior to undergoing apoptosis.

In addition to the above, there is substantial evidence to indicate that abnormal expression of negative regula- tors of the cell cycle is also associated with apoptosis. In particular, overexpression of the tumour suppressor proteins 53 or Rb causes apoptosis in certain situation^!^-^' It is clear that expression of p53 is essential for apoptosis of a variety of cell types after genotoxic insults, but is not required for apoptosis following other

In an attempt to explain these data, many authors have suggested that apoptosis represents an abort- ive attem t b cells to pass through the cell cycle. 2.4- 7 7 ps524~2rIn this model, the conflicting expres- sion of negative and positive regulators of cell prolifera- tion in the same cell will cause apoptosis, as will the inappropriate expression of positive regulators of prolif- eration in cells which are unable successfully to complete the cell cycle, It has also been suggested that apoptosis specifically represents an abortive attempt to pass through An integral part of this proposal is that it is impossible for any cell to undergo a o tosis

As a direct test of this hypothesis, we studied the expression of the replication-associated antigens Ki67 and DNA polymerase a in apoptotic cells in vivo, and in an in vitro model of apoptosis involving a non- proliferating cell population. The model that we chose to use is the apoptosis of aged neutrophils.26 In this system, apoptosis occurs in neutrophils after short-term culture, mimicking the process seen during the resolution of an acute inflammatory response, where the apoptotic cells are specifically recognized and engulfed by macro- phages.,’ Apoptosis of neutrophils in vitro shows the classic features of apoptosis seen elsewhere, including the characteristic morphological changes and chromatin fragmentation;26 unlike many other commonly used models of apoptosis, cell death occurs without the addition or removal of any growth factors or other compounds.

without first entering into a proliferative state. ?4,l?,24,25

MATERIALS AND METHODS

Apoptosis in human polymovphonucleav leukocytes Neutrophil granulocytes were isolated from the

peripheral blood of healthy human volunteers by a modification of the method previously described.26 It was noticed in initial experiments that standard isola- tion procedures using Ficoll-Hypaque (Pharmacia Biotech Ltd., Milton Keynes, U.K.) resulted in sub- stantial clumping of cells, and an inhibition of apoptosis, as noted.26 Therefore, neutrophils were iso- lated using Percoll (Pharmacia) gradients as follows. Solutions were sterilized by ultrafiltration or auto- claving and sterile pipettes and containers were used throughout. Blood (3040 ml) was collected into citrated tubes (Vaccutainer, Beckton Dickinson, Oxford, U.K.) and centrifuged at 300g for 20 rnin at

room temperature. The serum was drawn off and 5 ml was calcified by the addition of CaCI, to 2 0 m ~ final concentration, to produce platelet-rich, plasma-derived serum (PPRDS). The remainder of the serum was centrifuged at 1500 g for 15 min to produce platelet- poor plasma (PPP). Meanwhile, the cell pellet was resuspended in 27 ml of 0.9 per cent saline, to which was added 3 ml of 6 per cent dextran sulphate (500 000 molecular weight; Pharmacia). Cells were left to stand at 37°C for 30 min to allow erythrocytes to sediment. The top fraction was removed and centrifuged at 275 g for 6 rnin at room temperature. The cell pellet was then resuspended in 2 ml of PPP in a 15 ml conical centrifuge tube. This was underlayered by 2ml of 60 per cent stock Percoll (9:l vol/vol Percoll-9 per cent saline) in 0.9 per cent saline, which was in turn underlayered with 2 ml of 95 per cent Percoll in 0.9 per cent saline. The gradients were centrifuged at 700 g for 20 min, after which time the neutrophils form a tight band at the junction of the 60 and 95 per cent Percoll. The cells were removed carefully, washed three times in RPMI medium (Life Technologies Ltd., Paisley, U.K.) and finally resuspended in 3040ml of RPMI medium containing 100 unitdl of penicillin and streptomycin and supplemented with 10 per cent autologous PRPDS. Cells were transferred to individual Petri dishes and maintained at 37°C in a tissue culture incubator with a 5 per cent CO, atmosphere.

Cells were removed after 2, 6, and 20 h of culture. Cytospin preparations were made using 20Opl of cell suspension, centrifuged at 700rpm for 3 rnin in a cytospin 2 (Shandon Scientific Ltd., Runcorn, Cheshire, U.K.). Cells were fixed in a 50:50 mixture of acetone and methanol for 10 rnin at - 20°C and air-dried for 1 h. Slides were wrapped in aluminium foil and stored at - 70°C until use.

Slides were allowed to warm to room temperature and air-dried for 1 h before immunostaining with MIBl or the SJKl32 monoclonal antibody, which reconizes DNA polymerase For these preparations, an in- direct immunofluorescent technique was used, to avoid confusion with the high levels of endogenous peroxidase activity in the granulocytes. In addition, slides were stained with haematoxylin and eosin to estimate the purity of the isolated cells and the amount of apoptosis which had occurred.

Induction of apoptosis in pvoh$evating human cells

Hep2 cells, which are of human epithelium origin, were cultured in Dulbecco’s modified Eagles medium (DMEM; Life Technologies) containing 10 per cent fetal calf serum at 37°C with an atmosphere of 10 per cent CO,., Cells were plated at approximately 10 per cent confluency and allowed to adhere and grow for 24 h. Some cells were then treated with etoposide (Vepesid; Bristol Myers Pharmaceuticals, Hounslow, U.K.) at a concentration of 1 0 0 , ~ ~ in DMEM containing 10 per cent fetal calf serum. Replica cytospin preparations were made from treated and untreated cells after detachment of the cells by trypsinization. The cytospins were fixed,

APOPTOSIS AND CELL PROLlFERATION 73

Fig. 1 .-Apoptosis in Hep2 cells treated with 1 0 0 ~ ~ eptoposide for 16 h. (A) shows propidium iodide counterstain of an apoptotic cell. (B) shows localization of the Ki67 antigen in this apoptotic cell

stored, and immunostained as for polymorphonuclear cell preparations.

Tissues

A range of human tissue samples were retrieved from the files of the Department of Pathology at Ninewells Hospital. Samples were selected to contain examples of apoptosis seen in the steady-state kinetics of adult tissues, in normal involution of the endometrium, and as a consequence of cell-mediated immunity.29 Sections were collected onto silanated slides and used for immunocytochemistry with the monoclonal antibody MIB 1, which recognizes the Ki67 antigen.30 Immuno- staining was achieved using the avidin-biotin complex peroxidase technique, after microwave treatment of the sections, as previously de~cribed.~' Immunoreactive sites were identified by diaminobenzidine/H,O, and the sections were lightly counterstained with haematoxylin.

In situ end labelling In situ end labelling (ISEL) to identify apoptotic cells

was carried out on polymorphonuclear leukocytes essen- tially as described p rev i~us ly .~~ Dried cytospins were incubated directly in the labelling mixture, containing the Klenow fragment of DNA polymerase at a con- centration of 10 unitslml, biotinylated dATP (Life Technologies Ltd.). Slides were incubated for 1 h at 37°C

and reactive sites were identified with FITC-avidin DCS (Vector Laboratories, Peterborough, U.K.), used for 30 min at a concentration of 40puglml.

RESULTS

Ki67 antigen is expressed when cycling human cells undergo apoptosis in vitro

Cells of the human epithelial cell line, Hep2, grow rapidly in the presence of 10 per cent fetal calf serum. In the starting population of cells, over 95 per cent showed staining with the monoclonal antibody MIB1, and the majority were similarly positive for DNA polymerase a. Treatment of these cells with etoposide resulted in the accumulation of many apoptotic cells, which can be seen with propidium iodide staining (Fig. IA), and were demonstrated by the ISEL technique. The majority of the nuclei (over 75 per cent) of these cells were seen to be stained by MIB1, indicating expression of the Ki67 antigen (Fig. 1B). In most cells, Ki67 was associated with nuclear fragments, but occasional cells seemed to show Ki67 reactivity excluded from regions of con- densed DNA. In addition, apoptotic Hep2 cells showed strong reactivity for DNA polymerase a. This result demonstrates that neither the Ki67 antigen nor DNA polymerase is degraded during the process of apoptosis and these antigens can therefore be used to assess the proliferative state of apoptotic cells in culture.

74 P. J. COATES ET AL.

Fig. 2. -Apoptosis in polymorphonuclear leukocytes. In (A) and (B), cytospin preparations have been stained with haematoxylin and eosin. Cells in (A) were taken 2 h after isolation from peripheral blood, whilst cells in (B) were stained 20 h after isolation. Notice the characteristic features of apoptosis in many cells in (B). In (C), cells prepared 20 hours after isolation have been stained using the ISEL technique, demonstrated with fluorescein. The strong reactivity indicates the presence of fragmented DNA

Ki67 antigen is not expressed during apoptosis of ageing neutrophils in vitro

Using Percoll gradients to isolate neutrophils, the nucleated cell population was seen to contain over 90 per cent granulocytes, although there were substantial numbers of erythrocytes. The cells showed little evidence of clumping during the course of the experiment, but showed a gradual accumulation of apoptotic cells, reaching approximately 40 per cent after 20 h (Figs 2A and 2B). These apoptotic cells were labelled using the ISEL technique, showing that they contained frag- mented DNA (Fig. 2C). Staining of cells taken at 2, 6, or 20 h after isolation with the monoclonal antibody MIBl showed that no cells expressed the Ki67 antigen. In addition, no cells were seen to contain detectable levels of DNA polymerase a.

Ki67 antigen can be identijied in naturally occurring apoptosis in vivo

A range of epithelial and lymphoid tissues which contained easily identifiable apoptotic bodies were studied. MIB 1 -positive immunostaining was seen in apoptotic bodies present amongst the proliferating cells of lymphoid germinal centres of tonsil (Fig. 3A). Apoptotic nuclei in the crypts of the large and small intestinal mucosa were also MIB1-positive. In an example of cell-mediated immunity (piecemeal necrosis in chronic active hepatitis), positive immunostaining with MIBl was again noted in apparently apoptotic lymphocytes. However, apoptotic bodies in pre- menstrual phase endometrium were unstained with MIBl (Fig. 3B).

DISCUSSION

The mechanisms by which cells undergo apoptosis are currently receiving much attention, due largely to the realization that programmed cell death has important roles in normal development, in neoplasia, and in the chemotherapy of turnours.’-’ Much information has accumulated on the expression of various genes during apoptosis and it has been found that in many cases these genes are involved in the regulation of the cell cycle. In addition, the expression of these genes has been shown to be necessary for apoptosis to occur. This has led to the idea that apoptosis represents an abortive attempt by a cell to proceed through the cell cycle, and in particular to pass through mitosis. This notion is supported by changes in the levels and distribution of various cell cycle regulatory proteins,’’ and the activation of cyclin- dependent kinases during apopto~is,’~,’~ which are key regulators of the cell cycle and which initiate both chromatin condensation and the disruption of the nuclear membrane, two features of apoptosis. In ad- dition, the characteristic nuclear morphology of apopto- sis has been suggested to be similar to the ‘mitotic catastrophe’ seen in cells which inappropriately express high levels of p34cdc2.’7

These data, therefore, have been used to suggest that cells must be in the cell cycle if they are to undergo apoptosis. Conversely, non-proliferating (Go) cells will be unable to die through apoptosis, unless they Jirst enter the cell cycle. This suggestion fits also with the observed efficiencies of various chemo- therapeutic drugs, which kill cells through the induc- tion of apopt~s is**~ and which are less effective on slowly growing turnours.”

APOPTOSIS AND CELL PROLIFERATION 75

Fig. 3.-Ki67 antigen detection in formalin-fixed, paraffin-embedded tissues. In (A), an apoptotic nucleus in a germinal centre in normal human tonsil is seen to stain for Ki67 (arrow), as are many proliferating cells. In (B), apoptotic cells in pre-menstrual endometrium are seen to be negative for Ki67 (arrows), although proliferating cells are positive

To study the association of apoptosis with prolifer- ation, we used the Ki67 antigen as a marker of prolif- eration. The association of Ki67 with proliferating cells is well established and several aspects of the molecule make it suitable for the purpose of assessing whether apoptotic cells must of necessity have entered the cell cycle. Firstly, it is clear that the Ki67 antigen is expressed throughout the cell cycle and is not restricted to a small phase of the cycle.39 Secondly, expression is strictly limited to proliferating cells and the protein is rapidly degraded, so that non-cycling cells do not express the antigen, nor is expression seen during DNA repair processes.3941 Thirdly, it appears that expression of the Ki67 antigen is required for cells to pass through the cell cycle, since abrogation of expression inhibits cell p r~ l i fe ra t ion .~~ For these reasons, cells which do not contain detectable levels of the antigen are highly unlikely to be in the cell cycle. One potential pitfall with this approach was that this highly labile antigen4' may be degraded during the process of apoptosis, leading to false-negative results. To validate the use of Ki67 in our studies, we showed that Ki67 staining is retained by proliferating cells grown in culture and induced to undergo apoptosis with etoposide.

Using Ki67 to investigate the proliferative state of apoptotic cells, we found that many apoptotic nuclei seen in tissue sections are Ki67-positive. In particular, apoptosis in lymphoid germinal centres and in crypts of the intestinal epithelium is associated with Ki67 expres- sion. These data indicate that these cells were actively proliferating at the time that they underwent apoptotic cell death. The data do not, however, indicate that entry into the cell cycle is a prerequisite for apoptosis. To investigate this, we used a well-characterized model of

apoptosis which occurs in a non-proliferating popu- lation of cells. We chose to use freshly isolated primary cultures of human polymorphonuclear leukocytes, since mature, circulating granulocytes do not proliferate under normal condition^.^^ We found that the apoptosis occurring in aged neutrophils was not associated with Ki67 positivity, which suggests that these cells had not entered the cell cycle during the time that they under- went apoptosis. To confirm our proposition, we also used an antibody to DNA polymerase a, and showed no expression of this molecule in apoptotic neutrophils. Again, DNA polymerase a is an essential requirement for cell division, since it is used for DNA replication, but is not involved in the cellular response to DNA damage.41 These results clearly show that apoptosis of polymorphonuclear cells occurs independently of cell cycle entry and therefore argue that not all forms of apoptosis are tied to cell cycle entry.

In addition to this in vitro model of apoptosis, we also observed that apoptosis in the endometrium is not associated with Ki67 expression. This tissue was used as an example of apoptosis occurring naturally as a consequence of variations in levels of trophic hormones. Our results may therefore suggest that apoptosis of hormone-dependent cells is also independent of cell cycle entry. This suggestion contradicts certain previous observations in androgen-sensitive prostate epithelium, where apoptosis after androgen ablation has been linked to expression of PCNA and incorporation of BrdU." However, recent studies using this model have shown that the majority of apoptotic cell death occurs in Go cells? The discrepancies between these two conflicting sets of results can be explained by the induction of DNA repair systems in cells undergoing apoptosis, leading to

76 P. J. COATES ET AL.

the expression of PCNA and incorporation of BrdU into damaged DNA as a response to the apoptotic pathway, rather than as part of the pathway.44

In conclusion, this study is the first to use the analysis of Ki67 expression as a marker of proliferation in apoptosis and we have validated the use of this approach both in tissue sections and in cells grown in culture. The analysis of Ki67 expression in apoptotic cells provides a convenient measure of the cell cycle association of apoptosis in tissues. This may be useful in the future for determining the importance of the relative proportion of apoptotic deaths occurring within the cycle in normal tissue development and in pathological conditions, including neoplasia. From our current studies, whilst it is clear that apoptosis in many cell types is dependent on cell cycle-associated gene expression, we have shown that non-proliferating cells in vitro and in vivo can undergo apoptosis without first entering the cell cycle. It is tempting to speculate that different cell types, under- going apoptosis in response to different stimuli and for different biological reasons, will show differences in their relationship to cell cycle events. In other words, it appears likely that some forms of apoptosis are depend- ent on cell cycle entry, whilst others are not, in the same way that induction of apoptosis in a given cell type may depend on the expression of p53 or may be independent of p53 expression, depending on the particular stimu- lUS,22.2' Support for our proposal comes from recent observations indicating that activation-induced apop- tosis of T-cells is cell cycle-dependent and requires c-myc and cyclin B expression, whilst apoptosis induced in these cells by corticosteroids does not require expression of these genes.".45

ACKNOWLEDGEMENTS

This work was supported by the Cancer Research Campaign (Grant SP2148). We would like to thank M. Greer for help with polymorphonuclear leukocyte isolation and we are grateful to S. McPherson for photography and for technical assistance.

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