Cancer Letters, 67 (1992) 199-206 Elwvier Scientific Publishers Ireland Ltd.

Aphidicolin

selectively

199

kills neuroblastoma

cells in vitro

Jindrich Cinatlb, Jaroslav Cinatl”, Marion Mainke”, Albrecht WeiDflog”, Gabriele Steigrnannb, Holger Rabenaub, Hans-Wilhelm Doerrb and Bernhard

Kornhuber”

Tentre of Pediatrics, Deportment of Haematology and Oncology and bCentre of Hygiene, Department of Medical Virology, 2. W. Goethe-Ckversity, Frankfurt a. G. (FRGJ (Received 6 October 1992) (Accepted 14 October 1992)

Summary

Introduction

Aphidicolin is a tetracyclic diterpene antibiotic which is known to inhibit the growth of eucaryotic cells by reversible binding to DNA polyrnerase Q without significant ejject on cell viability in most common human cell lines. We

Neuroblastoma (NB), a tumor of the sympathetic nervous system, is the most common solid malignancy of childhood outside the central nervous system [24]. The prognosis of this disease depends on its clinical presentation (local or metastatic spread and age of the children at diagnosis) and the cellular and molecular characterization of the tumor [2,7,8,19 - 211. Only recently, major progress has been achieved, both in antitumoral response to first line chemotherapy and in the consolidation with megatherapy [ 181. However, in typical stage 4 metastatic NB, the overall survival rate does not exceed 20% at 5 years. Therefore, new therapeutic agents which inhibit the growth of NB cells are still strongly desirable. Aphidicolin is a tetracyclic diterpene tetraol, obtained from Cephalosporium aphidicola and certain other fungi [3,11]. Aphidicolin inhibits the growth of eucaryotic cells by inhibiting the activity of DNA polymerase (Ywithout interfering with the activities of DNA polymerase fl and y [ 121. The effect of aphidicolin on DNA polymerase (Yis reversible without significant effect on cell viability in most common human cell lines [12,13,16,23]. In some leukemic cell lines aphidicolin was shown to influence cell differentiation [9,16,17].

observed that aphidicolin at a concentration of 5 x 10m7 M kills all cells of four human neuroblastoma cell lines. In contrast, viability of normal human embryonal cells and of human continuous cell lines including HeLa, H9, A549 and Caco-2 was influenced only moderately by aphidicolin. In addition, neuroblastoma cells were killed ajter treatment with 5 x 10e7 M aphidicolin in cocultures with normal embryonal cells which continued to proliferate after removal of aphidicolin. These results show that aphidicolin provides an agent which selectively kills neuroblastoma

cells in vitro.

Keywords: aphidicolin; neuroblastoma cells; cell killing

Correspondenceto: Jindrich Cinatl, Abteilung fiir Medizinische Virologie, UniversitBtsklinikum, J. W. Goethe-Universitit, PaulEhrlich-Str. 40, D-6000 Frankfurt a. M. 70, FRG.

0304-3835/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

200

In this study, we demonstrate that aphidicolin kills human NB cells in vitro. On the other hand, viability of several human continuous cell lines of different origin and of normal human embryonal cells is not significantly affected by aphidicolin. Materials and Methods Cell culture NB cell line IMR-32 was obtained from American Type Culture Collection (Rockeville, MD). NB cell lines designated UKF-NB-1, UKF-NB-2 and UKF-NB-3 were established from bone marrow metastasis harvested in relapse in three of our patients with Evans stage 4 NB as described previously in preliminary form [6]. In our hands, all NB cell lines showed neuroblastic morphology, i.e. small, rounded, loosely adherent cell bodies with neurite-like processes. The cell morphology in different NB cultures differed in length and number of neurite-like processes and degree of adhesiveness to the surface. Human T-lymphoid H9, colon adenocarcinema Caco-2, lung carcinoma A549 and cervix carcinoma HeLa cell lines were obtained from American Type Culture Collection. Primary cultures of human embryonal cells including lung fibroblasts (HEL) and amnion epithelial (HA) cells were established from lung tissue of human embryo or full-term placental material, respectively. The methods were performed as described previously [5]. All culture media and media supplements were purchased from Seromed (Berlin, FRG) . NB cell lines and U937 cells were grown in IMDM medium supplemented with 10% fetal bovine serum (FBS); HeLa, A549 and HEL cells were grown in MEM medium supplemented with 10% FBS; H9 cells were grown in RPM1 medium supplemented with 10% FBS; HA cells were grown in Ml99 medium supplemented with 20% FBS. The cells were routinely tested for mycoplasma by the HOECHST 33258 staining method [4] and found to be free of contamination.

Determination of the effect of aphidicolin on cell proliferation and suruiual Aphidicolin was purchased from Sigma (Deisenhofen, Germany) and dissolved in dimethyl sulfoxide. Stock solution of aphidicolin was stored at - 20°C. The percentage of dimethyl sulfoxide added to the growth medium did not exceed 0.01 ok;. To determine the effect of aphidicolin on cell proliferation, NB cell lines, HeLa, A549, H9 and Caco-2 were seeded at a density of 5 x lo4 cells, HEL at a density of 1 x lo5 cells and HA at a density of 3 x lo5 cells per 1 ml of a culture medium containing different concentrations of aphidicolin. To determine the effect of aphidicolin on cell survival, aphidicolin was added to cell cultures 3 days after seeding. In all tests, cells in 35 x 10 mm culture dishes (Nunc, Wiesbaden, FRG) were incubated at 37OC with 5% C02. Viable cells were counted using a hemocytometer. Viability of the cells was determined by the dye exclusion method after staining with 0.5% trypan blue solution.

Measurement of DNA synthesis DNA synthesis was measured by the incorporation of 5-bromo-2’-deoxyuridine (BrdU) into cellular DNA. Incorporated BrdU was detected using immunoenzymatical alkaline phosphatase anti-alkaline phosphatase method. Anti-BrdU monoclonal antibody (MoAb) and other chemicals were obtained as assay kits from Boehringer (Mannheim, FRG) . The procedure was performed according to the manufacturer’s instructions. Labelling indices were determined by counting 1000 cells.

Cocultures of NB cells with normal embryonal cells Confluent monolayers of primary cultures of HEL or HA cells in 25-cm2 culture flasks were incubated with different NB cells which were added at a density of 1 x lo6 cells per culture flask. Twenty-four hours after seeding of NB cells, different concentrations of aphidicolin were added. The presence of NB

201

100 60

60

60

60

40

40

2

-

UKPNB-1

-

UKF-NB2

-

UKPNB-1

-

UKP-NBZ

-

UKPNB-3

-

IMP-32

-

UKF-NE-3

-

IMP-32

0)

2; 0

5

100

A

C

D

100

100 60

60

40 60

40 60 t

20

20 1

-

EEL

+

AA

-

-

A549

-

A9

-cue2

Hek

-

EEL

-

HA

-

-

A549

-

a9

-cue-2

BcLa

Days of Treatment Fig. 1. aphidicolin

Time course experiment of cell survival in different cell types treated with aphidicolin. Three days after seeding, was added to a culture medium at a concentration of 5 x lo-’ M (A and C) or of 5 x 10m6M (B and D).

Table1. Effect of aphidicolin on the proliferation of different cell types. CeW

IC,, (MI

UKF-NB- 1 UKF-NB-2 UKF-NB-3 IMR-32 HEL HA HeLa A549 H9 Caco-2

8.1 8.5 6.5 7.9 2.9 3.8 2.1 2.5 1.8 3.0

l

1.33 f 0.91 + 0.42 f 1.12 f 0.33 +z 0.16 f 0.31 zt 0.19 ztz 0.09 ztz 0.24

nb x x x x x x x x x x

lo-* 1o-8 lO+s lo-* lo-’ lo-’ 1o-7 lo-’ lo-’ lo-’

3 3 4 4 3 3 2 2 2 2

aThe cells were seeded in triplicate in culture medium containing different concentrations of aphidicolin. Viable cells were counted 3 days after seeding. Values are means + S.E.M. bNumber of experiments.

cells was monitored by microscopic examination of cell morphology and immunoperoxidase staining with MoAb directed against neuron specific enolase (Dianova, Hamburg, FRG). Immunocytochemical analysis was performed as previously described by Hsu et al.

WI. Results Effectsof aphidicolin

on cell proliferation

and

suruiual

The proliferation of NB cells was inhibited by aphidicolin to a greater extent than the proliferation of other cell types as demonstrated by determining of I& (50% inhibitory concentration) values after 3 days of aphidicolin treatment (Table I). No viable cells in NB cultures were observed after 5 and 4 days of treatment

F&

2.

preparations,

of UKF-NBS

medium.

Note the small-round-cell

300 x

(C)

Native

M aphidicolin. The cells treated for 3 days showed signifi-

cells. The cells were grown 3 days in aphidicolin-free

(B) while the cells treated for 5 days were lysed (D). The untreated cell controls showed confluent cell layer within 8 days after seeding

phase contrast, magnification

cant cell enlargement

appearance

the cultures were grown for 3 days and 5 days in medium with 5 x 10 -’

Effect of aphidicolin on the morphological

bodies with processes (A). Thereafter,

Fig. 3. Effect of aphidicolin treatment on NB cells in cocultures with normal embryonal cells. HEL cells were mixed with IMR-32 cells and grown 5 days in medium without aphidicolin (A) or with 5 x 10 -’ M aphidicolin (B). Note in cocultures without aphidicolin IMR-32 cells attached to HEL monolayers. In cocuitures treated with aphidicolin IMR-32 cells were killed and only fibroblast cells are present. HA cells were mixed with UKF-NB-3 cells and grown 5 days in medium without aphidicolin (C) or with 5 x 10 -’ M aphidicolin (D) Note in cocultures without aphidicolin IMR-32 cells attached as compact aggregates to HA monolayers. In cocultures treated with aphidicolin UKF-NB-3 cells were killed and only epithelial cells are present. The cell layers were fixed in a mixture of methanol-acetone and stained with MoAb directed against neuronspecific enolase. Phase contrast, magnification 150 x

204 Table II. Effect of aphidicolin on DNA synthesis in different cell types. Cells

46 of BrdU labeled cells” Without aphidicolin

UKF-NB-1 UKF-NB-2 UKF-NBS IMR-32 HEL HA HeLa A549 H9 Caco-2

33.6 36.5 39.7 31.2 36.5 21.3 38.5 38.1 39.7 32.3

l

f zt f zt f f zt zt zt

Aphidicolin Aphidicolin (5 x 10e7 M) (5 X 10e6 M)

4.9 6.1 * 0.73 5.1 5.8 * 0.81 3.3 7.3 ztz0.57 4.2 4.9 l 0.62 2.6 3.9 zt 0.38 3.9 4.2 zt 0.87 3.6 5.3 zt 0.33 2.7 7.8 zt 0.65 3.4 3.5 zt 0.42 3.9 6.9 +z 0.93

0.5 0.6 0.9 0.7 0.6 0.3 0.2 0.9 0.7 0.5

f zt f f zt f * f f f

0.05 0.05 0.11 0.07 0.12 0.1 0.06 0.13 0.02 0.03

‘Aphidicolin was added to a culture medium 3 days after cell seeding. 12 h after aphidicolin addition, the cells were labelled for 30 min with BrdU. Values are means f S.E.M. from two independent experiments.

ment, they were washed with phosphatebuffered saline and incubated in aphidicolinfree medium after 5 days of treatment with 5 x low6 M aphidicolin. The results showed that non-neuroblastoma cell types reinitiated DNA synthesis within 4 h after removal of aphidicolin and incubation in aphidicolin-free medium (data not shown). Effect of aphidicolin on neuroblastoma cells in cocultures urith normal embryonal cells In cocultures of NB cells with normal embryonal cells, no morphologically discernable NB cells or cells positive for neuron specific enolase were found after 5 days of treatment with 5 x 10 -7 M aphidicolin. In Fig. 3 representative results are shown dealing with IMR-32 cells cocultured with HEL cells and UKF-NB-3 cells cocultured with HA cells. Both HEL and HA cells purged of NB cells proliferated after subculture in aphidicolin-free medium (data not shown). Discussion

with 5 x 10m7 M and 5 x 1O’6 M aphidicolin, respectively (Fig. 1A and B) . In contrast, the viability of normal human embryonal cells and of human continuous cell lines was influenced only moderately by aphidicolin treatment Fig. 1C and D). In all NB cultures aphidicolin treatment resulted in cellular enlargement and extension of cellular processes before cell lysis occured as shown in Fig. 2 for UKF-NB-3 cells. Other cell types treated with aphidicolin at concentrations up to 3 x 10 -5 M (maximum concentration tested) showed no change in cell morphology. Effect of aphidicolin on DNA synthesis The treatment with 5 x 10 -7 M aphidicolin for 12 h resulted in significant reduction of DNA synthesis in all cell types (Table II). DNA synthesis was inhibited almost completely after 12 h treatment with 5 x lob6 M aphidicolin both in NB and other cell types (Table II). To show whether non-neuroblastoma cell types reinitiate DNA synthesis after aphidicolin treat-

In the present study we showed that aphidicolin at a concentration as low as 5 x 10 -7 M kills human NB cells in vitro. Notably, aphidicolin has a little effect on cell viability at significantly greater concentrations and its inhibitory effect on cell growth is completely reversible in most common human cell lines [1,13,17,23]. In fact, we found that human cell lines of different origin treated with aphidicolin at concentrations up to 3 x 10 -5 M (maximum concentration tested) synthesized DNA and proliferated after removal of aphidicolin. Moreover, experiments dealing with cocultures of NB cells and normal embryonal cells showed that NB cells killed by aphidicolin did not liberate substances toxic for normal ceils. Among the inhibitors of DNA replication, aphidicolin is of a special interest because it specifically inhibits DNA polymerase a, which is the central element in the replication system. This substance is not incorporated into DNA, does not affect RNA synthesis and can be

washed out of the cell by removal from the culture [1,13,17]. The mechanism of cell killing by aphidicolin in human NB cells is not clear. In the Chinese hamster cell line (CHO strain AA@, cell killing by aphidicolin was found to require a period of inhibition-free growth following removal of the drug and was associated with characteristic aberrant mitotic processes [14]. These results supported a hypothesis that the cytotoxic effect of aphidicolin on CHO cells does not stem directly from its biochemical action per se, but that cell death results from processes that are evoked by the dissociation of normally integrated cell cycle events [ 141. Since we showed that NB cells are killed in the continuous presence of aphidicolin at concentrations which completely inhibit cell proliferation, its direct biochemical action in killing NB cells can not be excluded. Only recently, aphidicolin found attention as a substance with the capacity to suppress DNA repair and to partially restore the activity of cisplatin in cultured drug resistant human ovarian cancer cells [15]. In clinical trials a aphidicolinrelated water-soluble agent, glycinate, proved to be a relatively non-toxic agent with a maximum tolerated dose of 4500 mg/m2 in a 24-h continuous infusion study [22]. Therefore, aphidicolin and/or its chemical derivatives seem to be promising substances for further testing to show whether they could be used in patients with NB. In addition, studies are encouraged to investigate whether aphidicolin may be useful for chemopurification of NB cells contaminating the bone marrow for autologous marrow transplantation.

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3

4

5

6

References 1

Arabshahi,

L.,

Brown,

N.,

Khan,

N. and

Wright,

G.

Chen, T.R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent HOECHST 33258 stain. Exp. Cell Res., 104, 255-262. Cinatl, J. Jr., Cinatl, J., Rabenau, H., Ahn, J.-W. and Doerr, H.W. (1991) Quality control testing of surfaces for mammalian cell culture using cells propagated in a proteinfree medium. Biologicals, 19, 87 - 92. Cinatl, J, Gussetis, E.S., Cinatl, J. Jr., Ebener, U., Mainke, M., Schwabe, D., Doerr, H.W., Kornhuber, B. and Gerein, V. (1990) Differentiation arrest in neuroblastoma cell culture. J. Cancer Res. Clin Oncol., 116 (SuppI.), 9.

7

Combaret, V., Wang, Q., Favrot M.C., Thiesse, P., Philip, I., Bouffet, E., Bailly, C., Bouvier, R., Chauvin, F., Zucker, J.M., Bernard, J.L., Lenoir, G. and Philip, T. (1989) Clinical value of N-myc oncogene amplification in 52 patients with neuroblastoma included in recent therapeutic protocols. Eur. J. Cancer Clin. Oncol., 24, 1607- 1621.

8

Evans, A.E., D’Angio, G.J., Propert, K., Anderson, J. and Hann, H.W.L. (1987) Prognostlc factors in neuroblastoma. Cancer (Philadelphia), 59, 1853 - 1859. Griffin, J., Munroe, D., Major, P. and Kufe, D. (1982) Induction of differentiation of human myeloid leukemia cells by inhibitors of DNA synthesis. Exp. Hematol., 10,

9

10

11

Acknowledgments

This research was supported in part by organization ‘Verein fiir krebskranke Kinder, Frankfurt a. M. e. V.’ We are grateful to Alena Cinatlova for the microphotographs.

(1988) Inhibition of DNA polymerase alpha by aphidicolin derivatives. Nucl. Acids Res., 16, 5107- 5113. Brodeur, G.M., Seeger, R.C., Barrett, A., Berthold. F., Castelberry, R.P., D’Angio, J., De Bernardi, B., Evans, A.E., Favrot, M., Freeman, AI., Hasse, G., Hartmann, 0.. Hayes, F. A., Helson, L., Kemshead, J., Lampert, F., Ninane, J., Ohkawa, H., Philip, T., Pinkerton, C.R., Pritchard, J., Sawada, T., Siegel, S., Smith, E.I., Tsuchida, Y. and Voute, P.A. (1988) International criteria for diagnosis, staging and response to treatment in patients with neuroblastoma. J. Clin. Oncol., 6, 1874- 1881. Brundret, K.M., Dalziel, W., Hesp, B., Jarvis, J.A.J. and Neidle, S. (1972) X-ray crystallographic determination of the structure of the antibiotic aphidicolin: a tetracycline diterpenoid containing a new ring system. J. Chem. Sot. Chem. Comm., 1027 - 1028.

12

13

774-781. Hsu, S.M., Raine, L. and Fauger, H. (1981) Use of avidinbiotin-peroxidase complex in immunoperoxidase techniques. J. Histochem. Cytochem., 29, 577-580. Huberman, J.A. (1981) New views of the biochemistry of eucaryotic DNA replication revealed by Aphidicolin, an unusual inhibitor of DNA polymerase o. Cell, 23, 547-648. Ikegami, S., Taguchi, T., Ohashi. M., Oguro, M., Nagano, H. and Mano, Y. (1978) Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase CY.Nature (London). 275, 458 - 460. Iliakis, G., Nusse, M. and Bryant, P. (1982) Effects of aphidicolin on cell proliferation, repair of potentially lethal damage and repair of DNA strand breaks in Ehrlich ascites tumor cells exposed to X-rays. Int. J. Radiat. Biol., 42, 417-434.

206 14

15

16

17

18

Kung, A.L., Zetterberg, A., Sherwood, S.W. and Schimke, R.T. (1990) Cytotoxic effects of cell cycle phase specific agents: result of cell cycle pertubation. Cancer Res., 50, 7307 - 7317. Masuda, M., Tanaka, T., Matsuda, H. and Kusaba, I. (1990) Increased removal of DNA-bound platinum in a human ovarium cancer cell line resistant to cisdiamminedichlorplatinum (II). Cancer Res., 50, 1863 1866. Murate, T., Hotta, T., Tsushita, K., Suzuki, M., Yoshida, T., Saga, S., Saito, H. and Yoshida, S.A. (1991) Aphidicolin, an inhibitor of DNA replication, blocks the TPA-induced differentiation of a human megakaryoblastic cell line, MEG-01. Blood, 78, 3168-3177 Murate, T., Kagami, Y., Hotta, T., Yoshida, T., Saito, H. and Yoshida, S. (1990) Terminal differentiation of human erythroleukemia cell line K562 induced by aphidicolin. Exp. Cell Res., 191, 45-50. Philip, T., Zucker, J.M., Bernard, P., Lutz, P., Bordigoni, P., Plouvier, E., Robert, A., Roche, H., Souillet, G., Bouffet, E., Michon, J., Lopez, M., Vilcoq, J.M., Gentet, J.C., Philip, I., Ladenstein, R., Favrot, M. and Chauvin, F. (1991) Improved survival at 2 and 5 years in the LMCEl unselected group of 72 children with stage IV neuroblastoma older than one year of age at diagnosis: is cure possible in small subgroup? J. Clin. Oncol., 9, 1037 - 1044.

19

20

21

22

23

24

Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A., Siegel, S.E., Wong, K.Y. and Hammond, D. (1985) Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N. Engl. J. Med., 313, llll1116. Shimada, H., Chatten, J., Newton, W.A., Sachs, N., Hamoudi, A.B., Chiba, T., Marsden, H.R. and Misugi, K. (1984) Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastomas and an age-linked classification of neuroblastomas. J. Natl. Cancer Inst., 73, 405-441. Silber, J.H., Evans, A.E. and Fridman, M. (1991) Models to predict outcome from childhood neuroblastoma: the role of serum ferrkin and histology. Cancer Res., 51, 1426 - 1433. Swess, C., Zucchetti, M., Davoli, E., Califano, R., Cavalli, F., Frustaci, S., Gumbrell, L., Sulkes, A., Winograd, B. and D’Incalci, M. (1991) Phase I and clinical pharmacological evaluation of aphidicolin glycinate. J. Natl. Cancer Inst., 83, 1160- 1164. Tang, S., Poulin, L. and Levy, J.A. (1992) Lack of human immunodeficiency virus type 1 (HIV-l) replication and accumulation of viral DNA in HIV-l-infected T cells blocked in cell replication. J. Gen. Virol., 73, 933 - 939. Voute, P.A. (1984) Neuroblastoma. In: Clinical Pediatric Oncology, pp. 559 - 587. Editor: W.W. Sutow. Louis,

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