Interphase and M-phase oral KB carcinoma cells are targetted in staurosporine-induced apoptosis

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CANCER LETTERS

Cancer Letters IO4 (1996) 145-l 52

Interphase and M-phase oral KB carcinoma cells are targetted in staurosporine-induced apoptosis Myint Swe, Boon-Huat Bay*, Kwok-Hung Sit Department

qf‘Anatomy,

National

University

c~f’Sin,~upore, Kent Ridge S119620,

Sinprpore

Received 20 December 1995;revision received 2 I March 1996;accepted2 1 March I996

Abstract

The effect of staurosporine, an antimicrobial agent and inhibitor of protein kinase C (PKC) on programmed cell death/apoptosis (PCD) was investigated in the human oral cavity epidermoid carcinoma KB cell line. Staurosporine-treated oral KB carcinoma cells exhibited morphological features characteristic of apoptosis such as (a) cell shrinkage and increased nuclear fluorescence (quantitated by image analysis with laser scanning confocal microscopy), (b) nuclear condensation and fragmentation observedunder fluorescencemicroscopy with propidium-iodide-DNA staining and (c) chromatin condensation seen under transmission electron microscopy, Specific terminal deoxynucleotidyl transferase (TdT)-mediated labeling of 3’OH ends of DNA breaks in staurosporine-treated cells confirmed DNA fragmentation. In addition, we show the concomitant existence of M-phase PCD with interphase PCD in staurosporine-treated KB cells. It would appear that staurosporine induces apoptosis regardless of the cell cycle phase and that mitosis and apoptosis are not necessarily mutually exclusive events. Keywords: Staurosporine; Cell cycle; M-phase PCD; Fluorescence microscopy; Ultrastructure; In situ nick end labeling ---

1. Introduction Programmed cell depth/apoptosis (PCD), an event often associated with characteristic morphological and biochemical changes, is believed to be a critical component of tumorigenesis [9,24]. The tumorigenic process which is characterized by the accumulation of cells (either as a result of increased proliferation or failure of the cells to undergo apoptosis) has long been attributed to dysregulation of cell growth [27]. Activation of mechanisms that protect the cells from undergoing apoptosis would result in cancer progression ] 151. * Corresponding author. Tel.: +65 772 6139; fax: +65 778 7643.

There appears to be a compelling relationship between apoptosis and inappropriate cell cycle regulation as evidenced by the association of c-myc overexpression with high rates of cell proliferation and apoptosis [29]. In fact, it has been proposed that arrest of GUS transition in the cell cycle can lead to apoptosis [5]. Mitosis and apoptosis have been suggested as mutually exclusive events. Cells which have entered M-phase do not perish by PCD but by necrosis only unless they could complete cytokinesis and return to interphase [ 10,281. Although it was previously thought that M-phase specific cells do not undergo apoptosis, recent cell cycle DNA phase studies of PCD from glucocorticoid treatment, serum growth factor deprivation and

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hydroxyl radical damage with vanadyl(4) ions, have indicated that 2N DNA from Gl and S and 4N DNA from G2A4 cell cycle phases were degradable to sub2N fragmented DNA [21,22,26]. In the present study, we provide evidence to show the existence of Mphase PCD in apoptosis induced by staurosporine, a potent microbial inhibitor of protein kinase C [25]. Staurosporine is a known inducer of apoptosis in many tumorigenic and non-tumorigenic cell lines [3,1 I].

spun down onto clean slides in the Cytospin-2 (Shandon, UK). (b) For quantitation of cell profile areas, cells were fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer and stained with 1% methylene blue. Image analyses were performed with the Carl Zeiss LSM 410 Inverted Confocal Microscope equipped with Windows-based LSM 4 software version 3.5. The propidium iodide dye was excited using a 488 nm laser line with a laser power of 10%.

2. Materials and methods 2.4. Transmission electron microscopy (TEM) 2.1. Cell cultures and chemicals The human oral cavity epidermoid carcinoma cell line ( ATCC, CCL 17; obtained from the American Type Culture collection) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel). Cells were grown at 37°C in a 10% CO2 incubator in 25 cm2 flasks (Costar Corp., Cambridge, MA). Staurosporine (Cat. No. S-4400), propidium iodide (Cat. No. P-4170) and ribonuclease A (RNase A, Type IIA; Cat. No. R5000) were obtained from Sigma.

For TEM, cells were fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2). Osmication was with 2% osmium tetroxide followed by dehydration in an ascending series of methanol and infiltration with araldite. Subsequently, the araldite was allowed to polymerize for 18 h at 60°C. Ultrathin sections were doubly stained with uranyl acetate and lead citrate before viewing in a Philips CM 120 electron microscope. 2.5. Evaluation of DNA fragmentation by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end labeling

2.2. Induction of apoptosis Staurosporine was added to 25 cm2 culture flasks containing 4 ml DMEM with 10% serum to give concentrations of 0, 1, 20 and 200 PM strength and incubated for 20 h under the same conditions as above. 2.3. Confocal laser microscopy image analysis of (a) nuclear area andfluorescence and (6) cell profile area (a) For nuclear area and fluorescence estimations, cells incubated in staurosporine for 20 h, were detached and processed as follows. Briefly, cells were fixed in 1% paraformaldehyde, permeabilized in 70% ethanol and incubated in 1 ml of freshly prepared propidium iodide (PI) solution (Tris 1.21 g/l, NaCl 05’89 @l, m’OYOXJ1 a) which contained 6mg~&onuclease A per ml PI solution, added just before incubation. Incubation time was 15 min over an ice bath. Cells were washed with PBS and an aliquot was

The manufacturer’s protocol (ApoTag, Oncor, USA) was followed. Briefly, template-independent addition of dUTP-digoxigenin to 3’-OH ends of the fragmented DNA was catalyzed by exogenous TdT [8]. Subsequent detection of fluorescence in positive cells with fluorescein-labeled antidigoxigenin antibody was by flow cytometry and fluorescence microscopy. Microccocal nuclease (Worthington Diagnostics, Flow General, USA) digestion of fixed cell samples were used as positive controls. Eighty units of the DNase were added to 1 ml of the cell suspension in complete Dulbecco’s phosphate buffered saline as previously described [23]. 2.6.

Flow cytometry

For flow cytometric evaluation of fluorescence intensity, the COntter EPICS ELITE.ESP flow cytometer equipped with a 15 mW argon laser at 488 nm was used. Fluorescence intensity was detected at emission wavelength of 525 nm.

M. Swe et (11.I Cancer Letters 104 (1996) 145-152

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Reichert-Jung Univar fluorescence microscopy with an attached xenon lamp source. Fluorescence micrographs were taken using Kodak Gold III film IS0 400. 0

0

1

20

2.8, Statistical analysis

200

CONCENTRATtON (EM)

Result were analyzed using Student’s two-tailed t-

Fig. I. Effect of staucosporine(dose response)on the nuclear area of orat KB cells as measured by image analysis with the Carl Zeiss LSM 410 confocal microscope. Bar = standarderror, n = 20.

test.

2.7. Fluorescence microscopy

Treatment of KB oral staurosporine resulted in a nuclear area (P c 0.05) with 20pM concentration (Fig. 20pM staurosporine-treated

Ceils processed as above (PI stained and fluorescein labeled) were observed under incident light using excitation/emission 488/630 nm filters in the 06

3. Results carcinoma cells with signifiiant reduction in maximal effect seen at 1) The mean area ot nuclei was 41 it 2.5 /Lrn’

T

05

04

5‘ 5 5 ii

03

s 'Z 5 al L 02

0.1

0 80-1cKJ

100120

120-140

140-t60

160-180

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fluorescence of nuctei

/ n 20pM aControl Fig. 2. Fluorescenceintensity of 20@4 staurosporine-treatedoral KB nuclei compared to untreatedcells. Nuclei were stained with propidium iodide dye and fluorescencequantitated by confocal microscopy.

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M. Swe et al. I Cancer Letters 104 (1996) 145-152

Fig. 3. Fluorescence micrographs of propidium iodide stained oral KB cells. (A) Untreated cells. Note the uniform staining of the ce:Ils. A telo] phase cell (indicated by arrowhead) and a metaphase cell with the characteristic equatorial plate (indicated by arrow) is seen. (W) 20/JrM staurosporine-treated cells showing the presence of nuclear condensation and fragmentation. Apoptotic bodies am indicated by thin x630. XI-0 ws and brightly fluorescent M-phase nuclei by thick arrows. Magnification

hf. Sweet al. I CancerL.etters 104 (1996) 145-152

CONCENTRATION

(PM)

Fig. 4. Histogram of cell profile area of 20,~4M treated oral KB cells and controls quantitated by image analysis with the confocal microscope Bar = standard error, n = 20.

(SEM) compared to untreated nuclei which was 100 2 2.5 pm2, showing that nuclear areas in the staurosporine-treated cells have been reduced to 40% that of controls. As shown in Fig. 2, staurosporinetreated cells exhibited increased nuclear fluorescence compared to untreated cells as demonstrated by the distribution of these cells at the high end of the fluorescence scale. Corroborative evidence of increased fluorescence is shown in Fig. 3, where the nuclei of staurosporinetreated celIs were much brighter than the nuclei of control cells. ‘Ibe nuclei of the staurosporine-treated cells showed characteristics morphological features of apoptosis such as condensation and fragmentation [ 14,301. Increased nuclear fluorescence with PI-DNA staining has also been cited as a feature of apoptosis [ 121. Brightly fluorescent mitotic cells seen in Fig. 3B,C (compared to untreated cells in Fig. 3A) are therefore suggestive of mitotic programmed cell death. A significant reduction in the cell profile areas of the staurosporine-treated cells compared to controls (P < 0.05) is seen in Fig. 4. Cell shrinkage was evident in staurosporine-treated cells as shown by the reduction of the ceI1 profile areas to only 40% that of untreated cells. Chromatin condensation typical of apoptosis were observed in 201(.M staurosporinetreated cells (Fig. 5B,C compared to Fig. 5A). TdT-mediated labeling of 3’OH ends of frag-

Fig. 5. Transmission electron micrograph of oral KB cells. (A) Untreated cell with well defined nucleus. (BC) 20,~M staurosporine-treated cells showing chromatin condensation (arrowheads) and intact plasma membranes. Treated KB cell in (C) also has a convoluted nuclear membrane (arrow). Bar = 2,um.

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M. SW et al. I Cancer Letter.? 104 (I 996) 145-152

150

B

staurosporine 20pMnuclease 80 u/ml .......

,

1

mented DNA in staurosporine-treated cells are shown in Figs. 6 and 7. Staurosporine-treated cells are bright in fluorescence confirming the presence of DNA fragmentation. Micrococcal nuclease treated cells were used as positive controls as this DNase is known to fragment DNA [2]. 4. Discussion

.l

1000

DNA nicks’(TdT-medikcl

3’OH :z-labeling)

Fig. 6. Flow cytometric evaluation of TdT-mediated 3’OH ends of DNA breaks. There is a right shift of cence intensity of cells treated with DNase digestion An even more pronounced right shift is seen staurosporine-treated cells (93%).

labeling of the fluoresby 57.7%. in 20pM

The antimicrobial agent, staurosporine, a potent inhibitor of protein kinases and positive regulator of apoptosis, is believed to induce apoptosis by its actions on protein kinase C (PKC). PKC is known to affect a wide variety of cellular processes including cell proliferation and differentiation, in addition to its role as a negative regulator of apoptosis [7,16]. Staurosporine appears to interact directly with the catalytic domain of PKC to induce apoptosis [ 181. High concentrations of staurosporine are believed to

Fig. 7. Fluorescence micrograph of TdT-mediated dUTP nick end labeling of DNA breaks seen in 20pM staurosporine-treated KB cells of the same sample as in Fig. 6. Positive cells are fluorescent. Magnification x240.

M. SW et (11.I Cancer Letters 104 (19%) 145-152

interfere with intracellular signaling activated by extracellular survival factors [ 13,171. Another suggested pathway for staurosporine induced PCD is its ability to block the cell cycle at Cl (l-10 rig/ml) and G2 (100 rig/ml) phases [l]. Recently, staurosporine dose dependent increase in DNA fragmentation typical of apoptosis has been demonstrated in S, G2IM and Cl cell cycle phases by filter binding assays and DNA electrophoresis in CA46 cells [3j. The discovery of the involvement of the bcl-2 gene as both apoptosis protectors and at the same time apoptosis inducers has potentiated research into the relationship between apoptosis and the cell cycle [ 5 1. Programmed cell death has even been viewed as abortive mitosis [6] due to premature activation of mitosis specific phosphorylation via cyclin dependent cdc2 kinase [ZO]. In line with the observation that staurosporine could induce apoptosis at all cell cycle phases, we show in this study that staurosporine induces programmed cell death in both interphase and M-phase KB oral carcinoma cells. The mechanistic pathway for staurosporine-induced M-phase PCD could be similar to autophagic sequestration of mitotic chromosomes induced by vanadyl(4) ions observed in Chang liver cells recently [22]. Since staurosporine is able to induce apoptosis regardless of the cell cycle phase, inhibit tumor invasion and mitosis specific cdc2 kinase [4,19], it would make a very efficacious chemotherapeutic agent against cancer, the scourge of mankind from time immemorial. Acknowledgements This research was supported by research grant RP950332 from the National University of Singapore. The authors thank Mr. L.S Liau and Mr. Myint Sein for technical assistance rendered. References /I ] Abe. K., Yoshidn, M., Usui, T., Horinouchi, S. and Beppu, T. (1991) Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein klnases. Exp. Cell Res., 192, 122-127. 121 Arcnds, M.J., Morris, R.J. and Wyllie, A.H. (1990) Apoptosis: the role of endonuclease. Am. J. Pathol., 136, 593-609. 1i] Bertrand. R , Salary, E., O’Connor, P., Kohm, K.W. and

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