Leukemia Research 23 (1999) 217 234

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines
Kostas Dimas a, Dimitrios Kokkinopoulos a,*, Costas Demetzos b, Basilios Vaos c, Marios Marselos d, Mixalis Malamas d, Theodoros Tzavaras e
b

Department of Immunology, Hellenic Anticancer Institute, Athens GR -115 22, Greece Laboratory of Pharmacognosy, Department of Pharmacy, Uni6ersity of Athens, GR 157 -71, Athens, Greece c Clinical Laboratory, Kesarias 2, Nikea, Greece d Department of Pharmacology, Uni6ersity of Ioannina, Athens GR 451 -10, Greece e Department of Biology, Uni6ersity of Ioannina, GR 451 -10, Greece Received 30 March 1998; accepted 4 July 1998

Abstract Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 mg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 mg/ml the compound showed a signicant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using ow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the ow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the rst report, that a labdane type diterpene kills tumor cells via a phase specic mechanism which induces apoptosis. 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Apoptosis; Sclareol; Human leukemic cell lines; Cytotoxic/cytostatic effect; Cell cycle

1. Introduction Sclareol [(13R)-labd-14-ene-8,13-diol], is a labdane diterpene which is easily obtained from Clary sage (Sal6ia sclarea Linn.). Sclareol has high antimicrobial activity [1] and is used as avoring agent and as a synthon for preparation of Ambra odorants in perfumery [2].

Abbre6iations: PBML, peripheral blood mononuclear leukocytes; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; CPM, counts per minute; DAPI, 4%,6%-diamidin-2-phenylindol dihydrochloride. * Corresponding author. Present address: G. Papandreou str. 110, Zografou 157 73, Athens, Greece. Tel.: + 1-7487233; fax: + 17487233; e-mail: cdemetzo@atlas.uoa.gr.

Labdane diterpenes are known for their cytotoxic properties [38]. Sclareol, has been reported to exhibit strong cytotoxic activity against P-388, KB, and

0145-2126/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 9 8 ) 0 0 1 3 4 - 9

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NSCLC-N6 cell lines [3]. In this study, the effect of sclareol, which had previously been isolated from the leaves of Cistus incanus subsp. creticus (L.) [9], was observed on established human leukemic cell lines. The cytostatic and apoptotic effect of sclareol on some of the cell lines tested, was also studied along with a ow cytometric study of the sclareol action.

2. Materials and methods

2.1. Plant material

The plant C. incanus subsp. creticus (L.) Heywood (syn. C. creticus subsp. creticus (L.) Greuter et Burdet; C. creticus L.) [10], was collected on the island of Crete and was veried by Dr A. Yiannitsaros. A voucher specimen (No 170 Athens Pharmacognosy Herbarium) is deposited in the herbarium of the division of Pharmacognosy University of Athens.

2.2. Isolation of sclareol

Sclareol had been isolated from the leaves C. incanus subsp. creticus (L.) Heywood and its structure was determined by spectroscopic methods [9].

2.3. Cytotoxic acti6ity

Sclareol was tested for cytotoxic activity on several human leukemic cell lines. The following cell lines were used: CCRF-CEM [11,12], MOLT3 [13], H33AJ-JA13 [14], HUT78 [15], H9 [16] (T cells), KM3 [17], NAMALWA [18], DAUDI [19,20], SDK [21], JIYOYE [22], CCRF-SB [23] (B cell lines), HL60 [24] (promyelocytic cell line), K562 [25] (proerythrocytes), U937 [26] (monocytes). All cell lines were maintained as exponentially proliferating suspension cultures in RPMI-1640 medium (Gibco Europe, Scotland UK), supplemented with 10% heat inactivated fetal calf serum (Myoclone Gibco), 2 mM L-glutamine (Gibco) and 50 mg/ml gentamycin. All incubations were carried out at 37C, in a humidied atmosphere with 5% CO2. Sclareol was also tested against peripheral blood mononuclear leukocytes (PBML), obtained from the blood of normal volunteers after FicollHypaque centrifugation [27]. Sclareol was dissolved in DMSO at specify concentrations and stored in small aliquots at 40C. To determine the cytotoxic activity, the diterpene was added at the same time to each cell line or PBML (1 106 cells/ml nal cell density) in 16 well at-bottomed microplates. Viability of the cells was assessed by trypan blue dye exclusion, at the begining of incubation time and was always greater than 98%. Cultures used as controls contained an equivalent amount of DMSO (negative) or vincristine (vincristine sulphate, Pharmachemie, Haarlem, Netherlands) (posi-

tive). After the addition of the diterpene, cells were cultured for 48 h in a moist 5% CO2 atmosphere. The IC50 for each cell line was determined by the MTT method [28,29]. Briey, 4 h before the end of the 48 h incubation period, MTT dissolved in PBS, was added to the cell cultures to give a nal concentration of 50 mg/ml. At the end of the 48 h incubation period, acidisopropanol was added to the wells and the optical density was measured with an ANTHOS HT II Microelisa reader, using a test wavelength of 550 nm. Cell death, due to the drug was determined by trypan blue dye exclusion. Cells were incubated with three different concentrations of the compound (20, 10, and 2 mg/ml) under the same conditions as for the MTT assay. Doses higher than 20 mg/ml were found to be rather cytotoxic for resting PBML. Therefore 20 mg/ml was the highest concentration examined in the following experiments. Trypan blue-excluding cells were counted with the aid of a hematocytometer using aliquots removed from cultures at the designated times (1, 4, 24, and 48 h after the addition of sclareol). Viability of the controls (cells incubated either with DMSO or only in culture medium) was also assessed by this method and was always greater than 95%. Vincristine at two concentrations, 5 and 1 mg/ml, was used as a positive control. Data representing the mean of experiments done in triplicate was analyzed by a two-tailed Students t -test. P B 0.05 was considered signicant.

2.4. Measurement of DNA synthesis

DNA synthesis was assayed at 1, 4, 24, and 48 h after addition of the compound to be tested. The cells were incubated with 10 mCi of [3H] thymidine (Amersham, UK), added 1 h before the end of each interval, under the same conditions as for the MTT assay. At the end of each incubation period the cells were harvested with an automatic cell harvester and the amount of radioactivity incorporated into the macromolecules was measured in a liquid scintillation counter (Packard IL) and expressed as CPM. Sclareol was tested at three concentrations 2, 10, and 20 mg/ml. DNA synthesis measurements were done at the cell lines used for the proliferation assay. Control cell cultures were incubated with DMSO (negative) or vincristine at two concentrations, 5 and 1 mg/ml (positive). The data representing the mean of experiments was done in triplicate and was analyzed by a two-tailed Students t -test. P B 0.05 was considered signicant.

2.5. Apoptosis and morphological assessment

Exponentially growing cells (5 105 cells/ml) from MOLT3, H33AJ-JA13 (T cell lines) and HL 60 (promyeolcytic cell line) cells were incubated for 8 and 24 h with two concentrations of sclareol, 20 and 10 mg/ml. Control

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cultures with DMSO (negative control) or with etoposide (Vepesid, Bristol-Myers Squibb, Germany) (positive control [30 32]) at a concentration of 20 mg/ml, were also tested in parallel. At the dened times aliquots from each culture were removed, xed with cytospin onto microscopic slides, followed by the addition of 70% methanol, stained with Wright Giemsa dye, and observed under a light microscope (1000 magnication). DNA from these cell lines was also analysed for studying endonucleolytic DNA damage. At the designed time cell aliquots (2 106 cells) were collected, washed and the cells were lysed with TNE buffer. DNA was extracted and puried. DNA from each sample was analyzed on 1.2% horizontal agarose gels in TBE buffer. Electrophoresis was performed at 2.5 V/cm and the DNA was visualized under UV light after staining with ethidium bromide (Sigma, MO).

2.6. Cell cycle analysis

The effect of sclareol on the cell cycle progression of MOLT3 and H33AJ-JA13 (both from T lineage) was studied. Cells were incubated with 20 and 10 mg/ml of sclareol for 4, 8, 24, and 32 h extended to 48 and 56 h for the concentration of 10 mg/ml. DMSO or 10 mg/ml etoposide, were used as controls. At the given times aliquots were removed and the cells harvested by centrifugation. The cells (1 106 cells) were then resuspended in PBS, washed and resuspended in ice-cold 70% ethanol. DAPI was then added at a nal concentration of 1.0 mg/ml. Cells were analyzed for DNA content by quantitation of green uorescence in a Partec PAS III i ow cytometry system (Partec GmbH, Munster, Germany). At least 10 000 events for H33AJ-JA13 and 16 000 for MOLT3 were counted. One parameter histograms were analyzed using the programme for cell cycle analysis supplied from the manufacturer.

OYE and DAUDI) cell lines were used to study the effect of sclareol on growth and DNA synthesis of leukemic cells. Figs. 1 and 2 show the viability and DNA synthesis curves of these cell lines, in the presence of various concentrations of sclareol. It is obvious that 24 h after the addition of 20 mg/ml of sclareol, the viability in all cases decreased to very low levels compared to the control, while after 48 h the death rate is almost 100% in all cell lines (Fig. 1A, C and; Fig. 2A, C). Additionally DNA synthesis (Fig. 1B, D and; Fig. 2B, D) falls as early as 4 h after the addition of the compound and there is practically no cellular activity, as far as the DNA synthesis is concerned, after 24 h of continuous incubation. At 10 mg/ml the viability is less affected. The two B cell lines (Fig. 2A, C), showed a decreased growth : 60% for JIYOYE and to 80% for DAUDI compared to the control level after 48 h of incubation. At this concentration the DNA synthesis rate is higher than the control after 1 h of incubation, but after 48 h falls to a level of 80% for MOLT3 (Fig. 1B), H33AJ-JA13 (Fig. 1D) and DAUDI (Fig. 2D) and to a level of 20% for JIYOYE(Fig. 2B). At 2 mg/ml all cell lines tested were found to be resistant to the loss of viability even after 48 h of incubation. Also completely unaffected was the DNA synthesis in H33AJ-JA13. On the other three cell lines the curve has a peak at a level higher than that of the control, but falls again to normal levels at the end of the incubation period (Fig. 1B, D and Fig. 2B, D).

Table 1 In vitro cytotoxicity of sclareol on leukemic cell lines. Vincristine was used as control and exhibited an IC50B1 mg/ml in cell lines tested Cell lines T cell lines CCRF-CEM MOLT3 H33AJ-JA13 HUT 78 H9 B cell lines KM3 NAMALWA JIYOYE DAUDI CCRF-SB SDK GRANULOCYTIC K562 PROMYELOCYTIC HL60 MONOCYTIC U937
a

IC50 (mg/ml)

3. Results

3.1. Cytotoxic acti6ity on human leukemic cell lines

After 48 h of incubation, sclareol exhibited an IC50 below 20 mg/ml at the most cell lines tested (results are summarized in Table 1). Only the B-cell Namalwa (Burkitt lymphoma, immature B-cell) was not affected at doses up to 50 mg/ml. The most sensitive cell line was H9, which was the most mature T-cell line used (T-ALL, single positive, CD4 + ). Furthermore sclareol did not affect signicantly the viability of resting human PBML at doses up to 25 mg/ml.

17.8 14.2 13.2 9.5 6.0 13.5 a N.A. 11.3 12.9 13.0 18.0 24.2 12.0 12.7

3.2. Effect on cell growth and DNA synthesis

Two T (MOLT3 and H33AJ-JA13) and two B (JIY-

N.A., not active up to 50 mg/ml.

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Fig. 1. Effect of sclareol on viability (A and C) and DNA synthesis (B and D) of MOLT3 and H33AJ-JA13 cell lines. The cells were incubated for 1, 4, 24, and 48 h, in the presence of sclareol. Viability and DNA synthesis were assayed as described in Section 2. The values represent means 9 SD.

3.3. Morphological changes and assessment of DNA clea6age

Sclareol at a concentration of 20 mg/ml induced severe morphological changes in all cell lines examined. Eight hours after the addition of the compound, treated cells (Fig. 3D, E; Fig. 4 D; Fig. 5D, E), compared with negative control (Fig. 3B; Fig. 4B; Fig. 5B), showed a reduction in cell volume, condensation of nuclear chro-

matin and nuclear fragmentation in many cells. This characteristic morphology, which also appeared in etoposide (Fig. 3C; Fig. 4C; Fig. 5C) treated cells is consistent with apoptosis. Necrotic cells were also present. These predominated 16 h later, after 24 h of incubation. At 10 mg/ml some morphological changes consistent with apoptosis exist only in H33AJ-JA13 after 24 h of incubation (Fig. 3E). The most obvious change in the other two cell lines is the appearance of

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Fig. 2. Effect of sclareol on viability (A and C) and DNA synthesis (B and D) of JIYOYE and DAUDI cell lines. The cells were incubated for 1, 4, 24, and 48 h, in the presence of sclareol. Viability and DNA synthesis were assayed also as described in Section 2. The values represent means 9 SD.

bubbles in almost the entirety of the HL60 population, at 8 h of incubation with a concentration of 10 mg/ml (Fig. 5F, G). Furthermore sclareol did not induce endonucleolytic DNA cleavage either at MOLT3 or at H33AJ-JA13 in any of the concentrations at any time intervals tested (Fig. 6A, B). In MOLT3 etoposide did not induce an endonucleolytic DNA cleavage (Fig. 6A). The only cell line that sclareol induced DNA cleavage was the HL60 cell line (Fig. 6C). In this cell line the DNA cleavage seems to be dose and time dependent: it appears after 24 h of incubation with 10 mg/ml and after 8 h with 20 mg/ml. Additionally the DNA laddering is more intense

after 24 h of incubation with the later concentration, mentioned above.

3.4. Effect on cell cycle progression

Fig. 7 represents the data obtained from the ow cytometer for H33AJ-JA13, while Fig. 8 shows the same data for MOLT3. In H33AJ-JA13, with 20 mg/ml of sclareol, dead cells appeared after 4 h of incubation (Fig. 7C). Additionally the cells of the G0/1 showed a perturbance of the DNA content and there are fewer cells in the G2/M phase (7.1% against 13.2% of the control). After 8 h apoptotic cells appeared along with

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Fig. 3. Light microscopy examination of H33JA-AJ13 exposed to sclareol and etoposide (see Section 2) (1000 ): (A) Untreated cells; (B) DMSO treated cells; (C) etoposide treated cells after 8 h; (D) cells treated with 20 mg/ml sclareol after 8 h; (E) cells treated with 10 mg/ml sclareol after 24 h.

necrotic. The whole histogram of DNA distribution is quite different compared to that of the controls. Latter 16 h after, only dead cells (necrotic and apoptotic) were tracked. After the addition of 10 mg/ml of sclareol (Fig. 7D), we observed that the relative distribution of the cells remained the same after 4 h of incubation 47.6% in G0/1, 39.8% in S, and 12.6% in G2/M against 49.4, 37.3, and 13.3%, respectively of the control) but the number of the cells in late G1/early S seems to increase. At 8 h the histogram was similar to that of the controls, while

after 24 h of incubation a small number of apoptotic cells had appeared near to the early S phase. At this time the percentage of cells to G0/1 is 60.1% while the S phase falls to 31.2% and the G2/M to 8.7%. The apoptotic population continues to be present until the end of the incubation time, after 56 h. An accumulation in G0/1 is also observed after 48 h of incubation (57.6%). In MOLT3 the results at 20 mg/ml, are more or less the same with that of H33AJ-JA13, at the same concentration. At 10 mg/ml we have the appearance of small

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Fig. 4. Light microscopy examination of MOLT3 exposed to sclareol and etoposide (see Section 2) (1000 ): (A) Untreated cells; (B) DMSO treated cells; (C) etoposide treated cells after 8 h; (D) cells treated with 20 mg/ml sclareol after 8 h.

percentage of apoptotic cells after 24 h of incubation (as also for H33AJ-JA13), which appears to increase along with the increase of the incubation time. At 32 h of incubation an accumulation of the cell population is observed in G0/1 which rises to 67.0% against 52.2% of the control while S is 29.2% and G2/M 3.8% (against 38.8 and 9.0% of the controls).

4. Discussion We report the effect of labdane type diterpene sclareol, which was isolated from the leaves of C. incanus subsp. creticus (L.) on a panel of human leukemic cell lines. Sclareol showed signicant cytotoxic activity in all cell lines tested, with the exception of NAMALWA (Burkitt lymphoma, immature B-cell). It exhibited IC50s below 20 mg/ml in most of the remaining cell lines, while it was not cytotoxic for resting PBML up to this concentration (IC50 for resting PBML : 25 mg/ml). The effect of sclareol in T lineage, seems to have a correlation with the maturity phase. No such a correlation occurs in B lineage. Sclareol also has a cytostatic effect, inhibiting DNA synthesis, as it arises from the four cell lines tested. The effect of sclareol on

DNA synthesis seems to be dose and time dependent. At high concentrations (20 mg/ml) the phenomenon takes place very early after the addition of the compound, while at lower concentrations (10 mg/ml) seems to be dependent upon the sensitivity of the cell line and the time. A rst indication for the synchronization of the cell population, as far as the DNA synthesis is concerned, also exists (peaks above the level of the controls). The similarities between the DNA synthesis rate curves of MOLT3 and DAUDI may be underlying a specic mechanism of action in a pathway, which is more or less common to the two lineages. The morphological assessment on three cell lines (two of the T and one of the promyelocytic lineage) revealed the appearance of morphological signs consistent with apoptosis in all the cell lines tested, at a concentration of 20 mg/ml and as soon as 8 h after the addition of the compound. However the DNA cleavage assessment showed that low molecular weight DNA fragments (DNA laddering) occurred only in the promyelocytic cell line HL60. It would appear that in HL60 the intensity of the electrophoretic pattern is dose and time dependent. For MOLT3 and H33AJ-JA13, as the necrotic cells seems to predominate, the absence of these fragments can be due to a high grade insult from

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Fig. 5. Light microscopy examination of HL60 exposed to sclareol and etoposide (see Section 2) (1000 ): (A) Untreated cells; (B) DMSO treated cells; (C) etoposide treated cells after 8 h; (D) cells treated with 20 mg/ml sclareol after 8 h; (E) cells treated with 10 mg/ml sclareol after 8 h; (F) cells treated with 20 mg/ml sclareol after 24 h; (G) cells treated with 10 mg/ml sclareol after 24 h.

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Fig. 6. (A) Agarose gel analysis of sclareol induced DNA cleavage in MOLT3: (Lanes 1, 2) DNA extracted from untreated and DMSO treated cells respectively; (Lane 3) DNA extracted from cells treated with 20 mg/ml of etoposide after 8 h; (Lane 4, 6) DNA from cells treated with 20 mg/ml sclareol after 8 and 24 h of incubation, respectively; (Lane 5, 7) DNA after treament with 10 mg/ml of sclareol for 8 and 24 h, respectively and; Lane 8: Hin dIII standards. (B) Agarose gel analysis of sclareol induced DNA cleavage in H33JA-AJ13: (Lanes 1, 2) DNA extracted from untreated and DMSO treated cells, respectively; (Lane 3) DNA extracted from cells treated with 20 mg/ml of etoposide after 8 h; (Lanes 4, 5, 7) DNA from cells treated with 20 mg/ml sclareol after 3, 8, and 24 h of incubation, respectively and; (Lanes 6, 7) DNA extracted after treament with 10 mg/ml sclareol for 8 and 24 h, respectively. (C) Agarose gel analysis of sclareol induced DNA cleavage in HL60: (Lanes 1, 2) DNA extracted from untreated and DMSO treated cells, respectively; (Lane 3) DNA extracted from cells treated with 20 mg/ml of etoposide after 8 h; (Lanes 4, 6) DNA from cells treated with 20 mg/ml sclareol after 8 and 24 h of incubation, respectively; (Lanes 5, 7) DNA extracted after treament with 10 mg/ml sclareol for 8 and 24 h respectively and; (Lane 8) Hin dIII standards.

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Fig. 7. DNA histograms of H33JA-AJ13 cells: (A) Histograms of untreated cells after different time intervals; (B): DMSO treated cells; (C) histograms of cells treated with 20 mg/ml of sclareol; (D) histograms of cells treated with 10 mg/ml. and; (E) cells treated with 10 mg/ml of etoposide (see Section 2). Arrows show the apoptotic cell population.

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Fig. 7. (Continued )

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Fig. 7. (Continued )

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Fig. 7. (Continued )

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Fig. 8. DNA histograms of MOLT3 cells: (A) Histograms of untreated cells after different time intervals; (B) DMSO treated cells; (C) histograns of cells treated with 20 mg/ml of sclareol; (D) cells treated with 10 mg/ml sclareol and; (E) cells treated with 10 mg/ml of etoposide (see Section 2). Arrows show the apoptotic cell population.

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Fig. 8. (Continued )

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Fig. 8. (Continued )

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Fig. 8. (Continued )

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K. Dimas et al. / Leukemia Research 23 (1999) 217234 [12] Uzman BG, Foley GE, Farber S, Lazarus H. Morphologic variations in human leukemic lymphoblasts (ccrf-cem cells) after long term culture and exposure to chemotherapeytic agents. Cancer 1966;19:1725. [13] Minowada M, Ohnuma T, Moore GE. Rosette-forming human lyphoid cells I: establishment and evidence for origin of thymusderived lymphocytes. J Natl Cancer Inst 1990;49:891. [14] Weiss A, Stobo JD. Requirement for the coexpression of T3 and T cel antigen receptor on a malignant human T-cell line. J Exp Med 1994;160:1284. [15] Gootenberg JE, Ruscetti FW, Mier JW, Gardar A, Gallo RC. Human cutaneous T cell lymphoma and leukemia cell lines produce and respond to T cell growth factor. J Exp Med 1981;154:1403. [16] Popovic M, Read-Connole E, Gallo RC. T4 positive human neoplastic cell lines susceptible to and permissive for HTLV-III. Lancet 1984;29:1472. [17] Schneider U, Shwenk HU, Bernkamm G. Characterization of EBV-genome negative null and T cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer 1977;19:521. [18] Klein G, Dombos L, Gothoskar B. Sensitivity of EBV producer and non producer human lymphoblastoid cell lines to superinfection with EB-virus. Int J Cancer 1972;10:44. [19] Klein E, Klein G, Nadkarni JS, Nadkarni JJ, Wigzell H, Clifford P. Surface IgM specicity on cells derived from a Burkitts lymphoma. Lancet 1967;2:1068. [20] Nisson K, Giovanella BC, Stehlin JS, Klein G. Tumorigenicity of human hematopoietic cell lines an athymic nude mice. Int J Cancer 1977;19:337. [21] Kottaridis S, Perez S, Kokkinopoulos D, et al. Establishment and characterization of a B-cell line from a patient with acute lymphoblastic leucemia. Leuk Res 1985;9:113. [22] Pulvertaft RJ. Cytology of Burkitts tumour (African lymhoma). Lancet 1964;1:238. [23] Adams R. Formal discussion: The role of transplantation in the experimental investigation of human leukemia and lymphoma. Cancer Res 1967;27:2479. [24] Rovera C, OBrian TG, Schneider C, Newmann R, Kemslead J, Greaves M. Induction of differentiation in human promyelocytic leukemia cells by tumor promoters. Science 1981;204:868. [25] Alder S, Ciampi A, McCullosh EA. A kinetic and clonal analysis of heterogeneity in k562 cells. J Cell Physiol 1984;118:186. [26] Koren HS, Anderson SJ, Larrick JW. In vitro activation of human macrophage-like cell line. Nature 1979;279:328. [27] Boyum A. Separaton of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 1968;21(97):1. [28] Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55. [29] Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival: modications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Method 1986;89:271. [30] Marks ID, Fox MR. DNA damage, poly(ADP-ribosyl)ation and apoptotic cell death as a potential common pathway of cytotoxic drug action. Biochem Pharmacol 1991;42:1859. [31] Wozniac AJ, Ross WE. DNA damage as a basis for 4%dimethylepipodo-phylotoxin-9-(4,6-O -ethylidene-B-D-glycopyranoside) (etoposide) cytotoxicity. Cancer Res 1983;43:120. [32] Kaufmann SH. Iduction of endonucleolytic DNA cleavage in Human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res 1989;49:5870. [33] Gotter GT. Induction of apoptosis in cells of the immune system by cytotoxic stimuli. Sem Immunol 1991;4:399.

sclareol at the concentration of 20 mg/ml that occurs, which does not allow to the majority of the cells to activate the apoptotic machinery [33]. The ow cytometric analysis of the two T cell lines revealed that there is in fact a large number of dead cells (necrotic and apoptotic) which begin to occur as early as 4 h after commencing incubation. Finally the cell cycle analysis indicates that the action of sclareol may be phase specic, as a G0/1 accumulation of cells may occur in both the two cell lines tested, although in different times. This can be a result of the largest cell cycle that MOLT3 cells have, compared to that of H33AJ-JA13. The accumulation of the cell population in one phase of the cell cycle, may cause, a synchronization of the population, a phenomenon which is also present in DNA synthesis curves. It could be concluded that sclareol seems to kill leukemic cells activating the apoptotic machinery. This activation probably takes place through a specic mechanism of action, activated by sclareol, while this action of sclareol may also be phase specic. More experiments to clarify the mechanism of action of sclareol are now in progress in our laboratories.

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