The Effect of Sclareol on Growth and Cell Cycle Progression of Humain Leucemic Cell Line!!!_2

7/27/2019 The Effect of Sclareol on Growth and Cell Cycle Progression of Humain Leucemic Cell Line!!!_2

1/18

Leukemia Research 23 (1999) 217234

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

a Department of Immunology , Hellenic Anticancer Institute , Athens GR -115 22 , Greeceb Laboratory of Pharmacognosy , Department of Pharmacy , Uni 6ersity of Athens , GR 157 -71, Athens , Greece

c Clinical Laboratory , Kesarias 2 , Nikea , Greeced Department of Pharmacology , Uni 6ersity of Ioannina , Athens GR 451 -10 , Greece

e Department of Biology , Uni 6ersity 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. Thecompound showed an IC 50 lower than 20 mg/ml in most cell lines tested, while it was higher for resting peripheral bloodmononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic celllines used. At a concentration of 20 mg/ml the compound showed a signicant cytostatic effect as soon as 4 h after continuousincubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareolin 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 apoptoticsigns is time and dose dependent. From the ow cytometry experiments, a delay of the cell population on G 0 / 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 labdanediterpene which is easily obtained from Clary sage(Sal 6ia sclarea Linn.). Sclareol has high antimicrobial

activity [1] and is used as avoring agent and as asynthon for preparation of Ambra odorants in per-fumery [2].

Abbre 6 iations : 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 dihy-drochloride.

* Corresponding author. Present address: G. Papandreou str. 110,Zografou 157 73, Athens, Greece. Tel.: + 1-7487233; fax: + 1-7487233; e-mail: [email protected].

Labdane diterpenes are known for their cytotoxic prop-erties [3 8]. Sclareol, has been reported to exhibitstrong cytotoxic activity against P-388, KB, and

0145-2126 /99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved.PII: S0145-2 126(98)00 134-9


2/18

K . Dimas et al . / Leukemia Research 23 (1999) 217234 218

NSCLC-N6 cell lines [3]. In this study, the effect of sclareol, which had previously been isolated from theleaves of Cistus incanus subsp. creticus (L.) [9], wasobserved on established human leukemic cell lines. Thecytostatic and apoptotic effect of sclareol on some of thecell lines tested, was also studied along with a owcytometric 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 Creteand was veried by Dr A. Yiannitsaros. A voucherspecimen (No 170 Athens Pharmacognosy Herbarium) isdeposited in the herbarium of the division of Pharmacog-nosy University of Athens.

2 .2 . Isolation of sclareol

Sclareol had been isolated from the leaves C . incanussubsp. creticus (L.) Heywood and its structure wasdetermined by spectroscopic methods [9].

2 .3 . Cytotoxic acti 6ity

Sclareol was tested for cytotoxic activity on severalhuman leukemic cell lines. The following cell lines wereused: CCRF-CEM [11,12], MOLT3 [13], H33AJ-JA13[14], HUT78 [15], H9 [16] (T cells), KM3 [17], NA-MALWA [18], DAUDI [19,20], SDK [21], JIYOYE [22],CCRF-SB [23] (B cell lines), HL60 [24] (promyelocyticcell line), K562 [25] (proerythrocytes), U937 [26] (mono-cytes). All cell lines were maintained as exponentiallyproliferating suspension cultures in RPMI-1640 medium(Gibco Europe, Scotland UK), supplemented with 10%heat inactivated fetal calf serum (Myoclone Gibco), 2mM L -glutamine (Gibco) and 50 mg/ml gentamycin. Allincubations were carried out at 37C, in a humidiedatmosphere with 5% CO 2 . Sclareol was also tested

against peripheral blood mononuclear leukocytes(PBML), obtained from the blood of normal volunteersafter FicollHypaque centrifugation [27]. Sclareol wasdissolved in DMSO at specify concentrations and storedin small aliquots at 40C. To determine the cytotoxicactivity, the diterpene was added at the same time to eachcell line or PBML (1 106 cells/ml nal cell density) in16 well at-bottomed microplates. Viability of the cellswas assessed by trypan blue dye exclusion, at thebegining of incubation time and was always greater than98%. Cultures used as controls contained an equivalentamount of DMSO (negative) or vincristine (vincristinesulphate, Pharmachemie, Haarlem, Netherlands) (posi-

tive). After the addition of the diterpene, cells werecultured for 48 h in a moist 5% CO 2 atmosphere. TheIC 50 for each cell line was determined by the MTTmethod [28,29]. Briey, 4 h before the end of the 48 hincubation period, MTT dissolved in PBS, was added tothe cell cultures to give a nal concentration of 50 mg/ml.At the end of the 48 h incubation period, acid iso-propanol was added to the wells and the optical densitywas measured with an ANTHOS HT II Microelisareader, using a test wavelength of 550 nm.

Cell death, due to the drug was determined by trypanblue dye exclusion. Cells were incubated with threedifferent concentrations of the compound (20, 10, and 2mg/ml) under the same conditions as for the MTT assay.Doses higher than 20 mg/ml were found to be rathercytotoxic for resting PBML. Therefore 20 mg/ml was thehighest concentration examined in the following experi-ments. Trypan blue-excluding cells were counted with theaid of a hematocytometer using aliquots removed fromcultures at the designated times (1, 4, 24, and 48 h after

the addition of sclareol). Viability of the controls (cellsincubated either with DMSO or only in culture medium)was also assessed by this method and was always greaterthan 95%. Vincristine at two concentrations, 5 and 1mg/ml, was used as a positive control.

Data representing the mean of experiments done intriplicate 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 afteraddition of the compound to be tested. The cells wereincubated with 10 mCi of [3 H] thymidine (Amersham,UK), added 1 h before the end of each interval, underthe same conditions as for the MTT assay. At the endof each incubation period the cells were harvested withan automatic cell harvester and the amount of radioac-tivity incorporated into the macromolecules was mea-sured in a liquid scintillation counter (Packard IL) andexpressed as CPM. Sclareol was tested at three concen-trations 2, 10, and 20 mg/ml. DNA synthesis measure-ments were done at the cell lines used for the

proliferation assay. Control cell cultures were incubatedwith DMSO (negative) or vincristine at two concentra-tions, 5 and 1 mg/ml (positive).

The data representing the mean of experiments wasdone in triplicate and was analyzed by a two-tailedStudents t -test. P B 0.05 was considered signicant.

2 .5 . Apoptosis and morphological assessment

Exponentially growing cells (5 105 cells/ml) fromMOLT3, H33AJ-JA13 (T cell lines) and HL 60 (promye-olcytic cell line) cells were incubated for 8 and 24 h withtwo concentrations of sclareol, 20 and 10 mg/ml. Control


3/18


cultures with DMSO (negative control) or withetoposide (Vepesid, Bristol-Myers Squibb, Germany)(positive control [3032]) at a concentration of 20 mg/ml,were also tested in parallel. At the dened times aliquotsfrom each culture were removed, xed with cytospinonto microscopic slides, followed by the addition of 70%methanol, stained with WrightGiemsa dye, and ob-served under a light microscope (1000 magnication).

DNA from these cell lines was also analysed forstudying endonucleolytic DNA damage. At the designedtime cell aliquots (2 106 cells) were collected, washedand the cells were lysed with TNE buffer. DNA wasextracted and puried. DNA from each sample wasanalyzed on 1.2% horizontal agarose gels in TBE buffer.Electrophoresis was performed at 2.5 V /cm and theDNA was visualized under UV light after staining withethidium 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) wasstudied. Cells were incubated with 20 and 10 mg/ml of sclareol for 4, 8, 24, and 32 h extended to 48 and 56 hfor the concentration of 10 mg/ml. DMSO or 10 mg/mletoposide, were used as controls. At the given timesaliquots were removed and the cells harvested by cen-trifugation. The cells (1 106 cells) were then resus-pended in PBS, washed and resuspended in ice-cold 70%ethanol. DAPI was then added at a nal concentrationof 1.0 mg/ml. Cells were analyzed for DNA content byquantitation of green uorescence in a Partec PAS III iow cytometry system (Partec GmbH, Munster, Ger-many). At least 10 000 events for H33AJ-JA13 and16 000 for MOLT3 were counted. One parameter his-tograms were analyzed using the programme for cellcycle analysis supplied from the manufacturer.

3. Results

3 .1. Cytotoxic acti 6ity on human leukemic cell lines

After 48 h of incubation, sclareol exhibited an IC 50below 20 mg/ml at the most cell lines tested (results aresummarized in Table 1). Only the B-cell Namalwa(Burkitt lymphoma, immature B-cell) was not affected atdoses 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 notaffect signicantly the viability of resting human PBMLat doses up to 25 mg/ml.

3 .2 . Effect on cell growth and DNA synthesis

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

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

Table 1In vitro cytotoxicity of sclareol on leukemic cell lines. Vincristine wasused as control and exhibited an IC 50 B 1 mg/ml in cell lines tested

IC 50 (mg/ml)Cell lines

T cell linesCCRF-CEM 17.8MOLT3 14.2H33AJ-JA13 13.2

9.5HUT 786.0H9

B cell linesKM3 13.5NAMALWA a N.A.JIYOYE 11.3DAUDI 12.9CCRF-SB 13.0SDK 18.0

GRANULOCYTIC K562 24.2

PROMYELOCYTIC 12.0HL60

MONOCYTIC U937 12.7

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


4/18


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 incubatedfor 1, 4, 24, and 48 h, in the presence of sclareol. Viability and DNA synthesis were assayed as described in Section 2. The values representmeans 9 SD.

3 .3 . Morphological changes and assessment of DNAclea 6age

Sclareol at a concentration of 20 mg/ml inducedsevere morphological changes in all cell lines examined.Eight hours after the addition of the compound, treatedcells (Fig. 3D, E; Fig. 4 D; Fig. 5D, E), compared withnegative control (Fig. 3B; Fig. 4B; Fig. 5B), showed areduction in cell volume, condensation of nuclear chro-

matin and nuclear fragmentation in many cells. Thischaracteristic morphology, which also appeared inetoposide (Fig. 3C; Fig. 4C; Fig. 5C) treated cells isconsistent with apoptosis. Necrotic cells were alsopresent. These predominated 16 h later, after 24 h of incubation. At 10 mg/ml some morphological changesconsistent with apoptosis exist only in H33AJ-JA13after 24 h of incubation (Fig. 3E). The most obviouschange in the other two cell lines is the appearance of


5/18


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 for1, 4, 24, and 48 h, in the presence of sclareol. Viability and DNA synthesis were assayed also as described in Section 2. The values representmeans 9 SD.

bubbles in almost the entirety of the HL60 popula-tion, at 8 h of incubation with a concentration of 10mg/ml (Fig. 5F, G).

Furthermore sclareol did not induce endonucleolyticDNA cleavage either at MOLT3 or at H33AJ-JA13 inany of the concentrations at any time intervals tested(Fig. 6A, B). In MOLT3 etoposide did not induce anendonucleolytic DNA cleavage (Fig. 6A). The only cellline that sclareol induced DNA cleavage was the HL60cell line (Fig. 6C). In this cell line the DNA cleavageseems to be dose and time dependent: it appears after24 h of incubation with 10 mg/ml and after 8 h with 20mg/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 owcytometer for H33AJ-JA13, while Fig. 8 shows thesame data for MOLT3. In H33AJ-JA13, with 20 mg/mlof sclareol, dead cells appeared after 4 h of incubation(Fig. 7C). Additionally the cells of the G 0 / 1 showed aperturbance of the DNA content and there are fewercells in the G 2 / M phase (7.1% against 13.2% of thecontrol). After 8 h apoptotic cells appeared along with


6/18


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 after24 h.

necrotic. The whole histogram of DNA distribution isquite different compared to that of the controls. Latter16 h after, only dead cells (necrotic and apoptotic) weretracked. After the addition of 10 mg/ml of sclareol (Fig.7D), we observed that the relative distribution of thecells remained the same after 4 h of incubation 47.6% inG 0 / 1 , 39.8% in S, and 12.6% in G 2 / M against 49.4, 37.3,and 13.3%, respectively of the control) but the numberof the cells in late G 1 /early S seems to increase. At 8 hthe histogram was similar to that of the controls, while

after 24 h of incubation a small number of apoptoticcells had appeared near to the early S phase. At thistime the percentage of cells to G 0 / 1 is 60.1% while the Sphase falls to 31.2% and the G 2 / M to 8.7%. The apop-totic population continues to be present until the end of the incubation time, after 56 h. An accumulation inG 0 / 1 is also observed after 48 h of incubation (57.6%).

In MOLT3 the results at 20 mg/ml, are more or lessthe same with that of H33AJ-JA13, at the same concen-tration. At 10 mg/ml we have the appearance of small


7/18


Fig. 4. Light microscopy examination of MOLT3 exposed to sclareol and etoposide (see Section 2) (1000 ): (A) Untreated cells; (B) DMSOtreated 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 increasealong with the increase of the incubation time. At 32 hof incubation an accumulation of the cell population isobserved in G 0 / 1 which rises to 67.0% against 52.2% of the control while S is 29.2% and G 2 / M 3.8% (against38.8 and 9.0% of the controls).

4. Discussion

We report the effect of labdane type diterpenesclareol, which was isolated from the leaves of C .incanus subsp. creticus (L.) on a panel of humanleukemic cell lines. Sclareol showed signicant cytotoxicactivity in all cell lines tested, with the exception of NAMALWA (Burkitt lymphoma, immature B-cell). Itexhibited IC 50 s below 20 mg/ml in most of the remain-ing cell lines, while it was not cytotoxic for restingPBML up to this concentration (IC 50 for resting PBML: 25 mg/ml). The effect of sclareol in T lineage, seemsto have a correlation with the maturity phase. No sucha correlation occurs in B lineage. Sclareol also has acytostatic 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 phenomenontakes place very early after the addition of the com-pound, while at lower concentrations (10 mg/ml) seemsto be dependent upon the sensitivity of the cell line andthe time. A rst indication for the synchronization of the cell population, as far as the DNA synthesis isconcerned, also exists (peaks above the level of thecontrols). The similarities between the DNA synthesisrate curves of MOLT3 and DAUDI may be underlyinga specic mechanism of action in a pathway, which ismore or less common to the two lineages.

The morphological assessment on three cell lines (twoof the T and one of the promyelocytic lineage) revealedthe appearance of morphological signs consistent withapoptosis in all the cell lines tested, at a concentrationof 20 mg/ml and as soon as 8 h after the addition of thecompound. However the DNA cleavage assessmentshowed that low molecular weight DNA fragments(DNA laddering) occurred only in the promyelocyticcell line HL60. It would appear that in HL60 theintensity of the electrophoretic pattern is dose and timedependent. For MOLT3 and H33AJ-JA13, as the ne-crotic cells seems to predominate, the absence of

these fragments can be due to a high grade insult from


8/18


Fig. 5. Light microscopy examination of HL60 exposed to sclareol and etoposide (see Section 2) (1000 ): (A) Untreated cells; (B) DMSO treatedcells; (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.


9/18


Fig. 6. (A) Agarose gel analysis of sclareol induced DNA cleavage in MOLT3: (Lanes 1, 2) DNA extracted from untreated and DMSO treatedcells 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 20mg/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, respectivelyand; Lane 8: Hin dIII standards. (B) Agarose gel analysis of sclareol induced DNA cleavage in H33JA-AJ13: (Lanes 1, 2) DNA extracted fromuntreated 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 treamentwith 10 mg/ml sclareol for 8 and 24 h, respectively. (C) Agarose gel analysis of sclareol induced DNA cleavage in HL60: (Lanes 1, 2) DNAextracted 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 treamentwith 10 mg/ml sclareol for 8 and 24 h respectively and; (Lane 8) Hin dIII standards.


10/18


F i g

. 7 . D N A h i s t o g r a m s o f H 3 3 J A - A

J 1 3 c e l l s : ( A ) H i s t o g r a m s o f u n t r e a t e d c e l l s a f t e r d i f f e r e n t t i m e i n t e r v a l s ; ( B ) : D M S O t r e a t e d c e l l s ; ( C ) h i s t o g r a m s o f c e l l s t r e a t e d w i t h 2 0

m g / m l o f s c l a r e o l ;

( D

) h i s t o g r a m s o f c e l l s t r e a t e d w i t h 1 0

m g / m l . a n d ; ( E ) c e l l s t r e a t e d w i t h 1 0

m g / m l o f e t o p o s i d e ( s e e S e c t i o n 2 )

. A r r o w s s h o w t h e a p o p t o t i c c e l l p o p u l a t i o n .


11/18


F i g

. 7 . ( C o n t i n u e d )


12/18


F i g

. 7 . ( C o n t i n u e d )


13/18


F i g

. 7 . ( C o n t i n u e d )


14/18


F i g

. 8 . D N A h i s t o g r a m s o f M O L T 3 c e l l s : ( A ) H i s t o g r a m s o f u n t r e a t e d c e l l s a f t e r d i f f e r e n t t i m e i n t e r v a l s ; ( B ) D M S O t r e a t e d c e l l s ; ( C ) h i s t o g r a n s o f c e l l s t r e a t e d w i t h 2 0

m g / m l o f s c l a r e o l ; ( D )

c e l l s t r e a t e d w i t h 1 0

m g / m l s c l a r e o l a n d ; ( E ) c e l l s t r e a t e d w i t h 1 0

m g / m l o f e t o p o s i d e ( s e e S e c t i o n 2 )

. A r r o w s s h o w t h e a p o p t o t i c c e l l p o p u l a t i o n .


15/18


F i g

. 8 . ( C o n t i n u e d )


16/18


F i g

. 8 . ( C o n t i n u e d )


17/18


F i g

. 8 . ( C o n t i n u e d )


18/18


sclareol at the concentration of 20 mg/ml that occurs,which does not allow to the majority of the cells toactivate the apoptotic machinery [33]. The ow cyto-metric analysis of the two T cell lines revealed thatthere is in fact a large number of dead cells (necroticand apoptotic) which begin to occur as early as 4 hafter commencing incubation.

Finally the cell cycle analysis indicates that the actionof sclareol may be phase specic, as a G 0 / 1 accumula-tion of cells may occur in both the two cell lines tested,although in different times. This can be a result of thelargest cell cycle that MOLT3 cells have, compared tothat of H33AJ-JA13. The accumulation of the cellpopulation in one phase of the cell cycle, may cause, asynchronization of the population, a phenomenonwhich is also present in DNA synthesis curves.

It could be concluded that sclareol seems to killleukemic cells activating the apoptotic machinery. Thisactivation probably takes place through a specicmechanism of action, activated by sclareol, while this

action of sclareol may also be phase specic. Moreexperiments to clarify the mechanism of action of sclareol are now in progress in our laboratories.

References

[1] Ulubelen A, Miski M, Johansson C, Lee E, Mabry TJ, MatuSA. Terpenoids from Sal 6 ia palaestina . Phytochemistry1985;24:1386.

[2] Decorzant R, Vial C. A short synthesis of Ambrox from sclareol.Tetrahaedron 1987;43:1871.

[3] Chinou I, Demetzos C, Harvala C, Roussakis C, Verbist JF.Cytotoxic and antibacterial Labdane-type diterpenes from theaerial parts of Cistus incanus subsp. creticus . Planta Med1994;60:34.

[4] Darias V, Bravo L, Rabanal R, Sandez-Mateo CC, Martin-Her-rera DA. Cytostatic and antibacterial activity of some com-pounds isolated from several lamiaceae species from CanaryIslands. Planta Med 1990;56:70.

[5] Malochet Grivois C, Cotelle P, Biard JF, et al. Dichlorolisso-climide a new cytotoxic labdane derivative from Lissoclinumvoeltzkowi Michaelson (Urochordata). Tet Lett 1991;32:6701.

[6] Itokawa H, Morita H, Katou I, et al. Cytotoxic diterpenes fromthe rhizomes of Hedychium . Planta Med 1988;3:11.

[7] Demetzos C, Mitaku S, Couladis M, Harvala C, Kokkinopoulos

D. Natural metabolites of ent-13-epi-manoyl oxide from theresin Ladano. Planta Med 1994;60:590.[8] Ik-Soo L, Xianjian M, Hee-Byung C, Domingo A, Madulid R,

Brian L. Novel cytotoxic labdane diterpenoids from Neou 6ariaacuminatissima . Tetrahedron 1995;51:21.

[9] Demetzos C, Harvala C, Philianos SM, Skaltsounis AL. A newlabdane type diterpene and other compounds from the leaves of Cistus incanus subsp. creticus (L.) Heywood. J Nat Prod1990;53:1315.

[10] Greuter W, Raus T. Med-checklist Notule 4. Willdenowia1991;11:275.

[11] McCarthy RE, Junius V, Farber S, Lazarus H, Foley GE.Cytogenetic analysis of human lymphoblasts in continuous cul-ture. Exp Cell Res 1965;40:197.

[12] Uzman BG, Foley GE, Farber S, Lazarus H. Morphologicvariations in human leukemic lymphoblasts (ccrf-cem cells) afterlong term culture and exposure to chemotherapeytic agents.Cancer 1966;19:1725.

[13] Minowada M, Ohnuma T, Moore GE. Rosette-forming humanlyphoid cells I: establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer Inst 1990;49:891.

[14] Weiss A, Stobo JD. Requirement for the coexpression of T3 andT cel antigen receptor on a malignant human T-cell line. J ExpMed 1994;160:1284.

[15] Gootenberg JE, Ruscetti FW, Mier JW, Gardar A, Gallo RC.Human cutaneous T cell lymphoma and leukemia cell linesproduce and respond to T cell growth factor. J Exp Med1981;154:1403.

[16] Popovic M, Read-Connole E, Gallo RC. T4 positive humanneoplastic 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 fromchildren with acute lymphoblastic leukemia and leukemic trans-formed non-Hodgkin lymphoma. Int J Cancer 1977;19:521.

[18] Klein G, Dombos L, Gothoskar B. Sensitivity of EBV producerand non producer human lymphoblastoid cell lines to superinfec-tion with EB-virus. Int J Cancer 1972;10:44.

[19] Klein E, Klein G, Nadkarni JS, Nadkarni JJ, Wigzell H, CliffordP. Surface IgM specicity on cells derived from a Burkittslymphoma. Lancet 1967;2:1068.

[20] Nisson K, Giovanella BC, Stehlin JS, Klein G. Tumorigenicityof human hematopoietic cell lines an athymic nude mice. Int JCancer 1977;19:337.

[21] Kottaridis S, Perez S, Kokkinopoulos D, et al. Establishmentand characterization of a B-cell line from a patient with acutelymphoblastic 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 theexperimental 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 promyelocyticleukemia cells by tumor promoters. Science 1981;204:868.

[25] Alder S, Ciampi A, McCullosh EA. A kinetic and clonal analysisof 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 mar-row. Scand J Clin Lab Invest 1968;21(97):1.

[28] Mossman T. Rapid colorimetric assay for cellular growth andsurvival: application to proliferation and cytotoxicity assays. JImmunol Methods 1983;65:55.

[29] Denizot F, Lang R. Rapid colorimetric assay for cell growth andsurvival: modications to the tetrazolium dye procedure giving

improved sensitivity and reliability. J Immunol Method1986;89:271.[30] Marks ID, Fox MR. DNA damage, poly(ADP-ribosyl)ation and

apoptotic cell death as a potential common pathway of cytotoxicdrug 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 -glycopyra-noside) (etoposide) cytotoxicity. Cancer Res 1983;43:120.

[32] Kaufmann SH. Iduction of endonucleolytic DNA cleavage inHuman acute myelogenous leukemia cells by etoposide, camp-tothecin, and other cytotoxic anticancer drugs: a cautionarynote. Cancer Res 1989;49:5870.

[33] Gotter GT. Induction of apoptosis in cells of the immune systemby cytotoxic stimuli. Sem Immunol 1991;4:399.

.

Documents

The Effect of Sclareol on Growth and Cell Cycle Progression of Humain Leucemic Cell Line!!!_2