Effect of sulphur mustard on human skin cell lines with differential agent sensitivity

SULPHUR MUSTARD EFFECTS ON HUMAN SKIN CELLS 115

© Crown copyright 2005. Reproduced with the permission of HMSO. J. Appl. Toxicol. 2005; 25: 115–128

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2005; 25: 115–128Published online 2 March 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1044

Effect of sulphur mustard on human skin cell lines withdifferential agent sensitivity

Rachel Simpson and Christopher D. Lindsay*

Biomedical Sciences Department, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK

Received 27 April 2004; Revised 6 September 2004; Accepted 13 September 2004

ABSTRACT: The ability of sulphur mustard (HD) to induce DNA damage places limits on the efficacy of approaches

aimed at protecting human cells from the cytotoxic effects of HD using a variety of protective agents such as thiol-

containing esters and protease inhibitors. In the present study, potential alternative strategies were investigated by examin-

ing the differential effects of HD on G361, SVK14, HaCaT and NCTC 2544 human skin cells.

The G361 cell line was more resistant to the cytotoxic effects of HD than the NCTC, HaCaT and SVK14 cell lines at

HD doses of >>>>>3 and <<<<<100 µµµµµM HD as determined by the MTT assay. At 72 h after exposure to 60 µµµµµM HD there was up

to an 8.8-fold difference (P <<<<< 0.0001) between G361 and SVK14 cell culture viability. Buthionine sulphoximine (BSO)

pretreatment increased the sensitivity of all four cell lines to HD. A substantial proportion of the resistance of G361 cells

to HD was attributable to BSO-mediated effects on antioxidant-mediated metabolism, although G361 cultures still retained

a high degree of viability at 30 µµµµµM HD following BSO pretreatment. Cell cycle analysis confirmed that SVK14 cells were

relatively more sensitive to HD, as shown by the 2.1-fold reduction (P <<<<< 0.0001) in the percentage of cells in G0/G1 phase

24 h after HD exposure compared with control cultures. This compared well with a 1.2-fold increase (P <<<<< 0.05) in the

percentage of G361 cells in G0/G1 phase following HD exposure, suggesting the existence of a more efficient G0/G1 check-

point control mechanism in this cell line. Manipulation of the cell cycle using various modulating agents did not increase

the resistance of cell lines to the cytotoxic effects of HD. © Crown copyright 2005. Reproduced with the permission of Her

Majesty’s Stationery Office Published in 2005 by John Wiley & Sons, Ltd.

KEY WORDS: sulphur mustard; human skin; viability; cell cycle; buthionine sulphoximine

* Correspondence to: C. D. Lindsay, Biomedical Sciences Department, Dstl

Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.

E-mail: [email protected]

© Crown Copyright 2004, Dstl

the alkylating effects of HD on the DNA of skin cells,

metabolic processes and connective tissue components

that are involved in skin regeneration and healing re-

sponses (Rice and Brown, 1999; Naghii, 2002).

The effect of HD on cells that are rapidly dividing is

particularly severe (Maynard, 1987). This may be due to

the formation of irreparable DNA cross-links (Murnane

and Byfield, 1981). According to Erickson et al. (1980),

cells that are highly sensitive to alkylating agents contain

a relatively higher proportion of DNA cross-links than

cells that are less sensitive, suggesting differences in

DNA repair efficiency. The position of a cell in the cell

cycle at the time of exposure to an alkylating agent

also may be important because mitotically active human

epidermal keratinocytes in culture are particularly sensi-

tive when they are in the S-phase of the cell cycle (Smith

et al., 1991).

Previous work suggests that increased resistance to HD

may be associated with elevated levels of proliferating

cell nuclear antigen (PCNA) (Smith et al., 2001), an ac-

cessory factor for DNA polymerase-δ and -ε in DNA

mismatch repair (Wood and Shivjii, 1997). The PCNA

also plays an essential role in nucleotide excision repair

(Miura et al., 1996; Tomkinson and Mackey, 1998).

Nucleotide excision repair is a DNA repair pathway that

utilizes DNA polymerase-δ or -ε to resynthesize bases

removed in the repair of pyrimidine dimers and other

Introduction

The chemical warfare agent sulphur mustard (HD) was

first used as a chemical weapon during World War I at

Ypres in 1917 (Haber, 1986). Sulphur mustard is a potent

vesicant and exposure to this agent may cause temporary

or permanent incapacitation. Within tissues, HD forms

irreversible covalent bonds with a variety of nucleophilic

groups, especially the genetic bases of DNA (Fox and

Scott, 1980; Klassen et al., 1986). The mono- and

bifunctional cross-links that HD forms with DNA results

in mitosis inhibition and death of the cell (Brooks and

Lawley, 1963). Sulphur mustard is bound rapidly by the

skin where it exerts cytotoxic effects predominantly upon

the mitotically active basal keratinocytes (Vogt et al.,

1984). However, the mechanism behind this and subse-

quent vesication is poorly understood. Recent work has

shown that, in vivo, epidermal melanocytes are the most

susceptible cell type in the dermo-epidermal region and

the severity of the lesion is related to skin type (Brown

and Rice, 1997). Sulphur mustard lesions typically take

many weeks or months to heal, and this may be due to

116 R. SIMPSON AND C. D. LINDSAY

Published in 2005 by John Wiley & Sons, Ltd. J. Appl. Toxicol. 2005; 25: 115–128

bulky DNA adducts (Wood and Shivjii, 1997) formed

by benzo[a]pyrene and nitrogen mustards (Smith and

Seo, 2002). According to Sparfel et al. (2002), nucleotide

excision repair is the major pathway for elimination

of DNA adducts formed by chemical carcinogens. This

indicates that it may be important also in the repair of

bulky lesions induced by HD. Previous reports have sug-

gested that the capacity of HD to induce DNA damage

places limits on the efficacy of approaches aimed at pro-

tecting human cells from the cytotoxic effects of HD

using scavenging agents such as hexamethylenetetramine

(Lindsay and Hambrook, 1997; Smith et al., 1997;

Andrew and Lindsay, 1998a), mono-isopropylglutathione

(Lindsay et al., 1997; Andrew and Lindsay, 1998b) and

di-isopropylglutathione (Lindsay and Hambrook, 1998).

In addition, the use of protease inhibitors such as E64

and mafenide hydrochloride (Lindsay et al., 1996) has

been shown to prevent dermo-epidermal separation in

human skin explants exposed to HD but did not prevent

the degradation of basal epidermal keratinocytes.

This report describes the differential effects of HD

on four human skin-derived cell lines: G361 malignant

melanoma cells, SVK14 cells (SV40 virus transformed

keratinocytes) and two spontaneously transformed kerat-

inocyte cell lines HaCaT and NCTC 2544; biochemical

assays were used to assess viability following HD

exposure and the consequences of inhibiting glutathione-

based antioxidant metabolism on sensitivity to HD.

Flow cytometry was used to determine the effect of HD

on cell cycle profiles of two cell lines with differential

sensitivity to HD. The effects of cell cycle inhibitors

on SVK14 and G361 cultures were determined further to

assess the therapeutic potential of modulating the cell

cycle on the cytotoxic effects of HD. The aim of the

present study was to determine if approaches based on

controlling cell cycle processes in human cells had thera-

peutic potential for enhancing the slow healing nature of

HD lesions.

Materials and Methods

Materials

Sulphur mustard was obtained from the Detection Depart-

ment, Dstl, Porton Down. The cell cycle profiles were

determined in cells treated with propidium iodide as

described in the DNA-Prep Coulter Reagents assay

kit (Beckman Coulter UK, High Wycombe). The cell

cycle inhibitors acyclovir, aphidicolin, olomoucine,

rapamycin, staurosporine and trichostatin were obtained

from Calbiochem (Merck Biosciences, Nottingham, UK).

Protein determinations were carried out using the Micro

BCA™ protein assay reagent kit (Pierce, Perbio Science

UK, Cheshire). All other chemicals were supplied by

Sigma-Aldrich (Poole, UK).

Cell Culture

The SVK14 human epidermal keratinocyte cell line (from

Dr Taylor-Papadimitriou, Imperial Cancer Research Fund,

London, UK) and the G361 human melanoma cell line

(European Collection of Cell Cultures, Health Protection

Agency, Porton Down, Salisbury, UK) were used for

these studies. The HaCaT and NCTC 2544 (hereafter

referred to as NCTC) cell lines were supplied by the

Biomedical Research Centre, Dundee, UK. The SVK14

and G361 cell lines were grown in a medium of 82%

(v/v) Dulbecco’s modified Eagle medium (DMEM) and

17% (v/v) foetal calf serum (FCS), containing 80 IU ml−1

penicillin, 80 µg ml−1 streptomycin, 1% non-essential

amino acids and 2 mM glutamine (hereafter referred to as

DMEM medium). The HaCaT cell line was grown in a

medium of DMEM, 10% (v/v) FCS, 100 IU ml−1 penicil-

lin, 100 µg ml−1 streptomycin and 2 mM glutamine (here-

after referred to as HaCaT medium). The NCTC cell

line was grown in a medium comprised of M199

medium and 10% (v/v) FCS, containing the same

antibiotics as for the HaCaT line and 2 mM glutamine

(hereafter referred to as NCTC medium). The cultures

were maintained at 37 °C in a humidified atmosphere

of 5% CO2–95% air.

The cells were grown in 150 cm2 flasks. Prior to

passaging or plating out, the monolayers (at 80–90%

confluency) were removed from the plastic substratum of

the flasks by incubation at 37 °C using a trypsin–EDTA

solution for 5–10 min. Following sedimentation of the

cell pellet by centrifugation at 300 g for 5 min at 4 °C

and resuspension of the pellet in DMEM, HaCaT or

NCTC medium, aliquots (100 µl) of each cell line were

pipetted into 96-well cell culture plates at a density of

50 000 cells per well. The cells were allowed to attach

and grow for 24 h at 37 °C in a humidified atmosphere of

5% CO2–95% air. The cell monolayers were subconfluent

at this time. The cell cycle analysis experiments em-

ployed 25 cm2 flasks containing cells seeded at 0.5 × 106

cells per flask. The SVK14 and G361 cells, once pipetted

into the flasks, were allowed to attach and begin growing

for 24 h at 37 °C in a humidified atmosphere of 5% CO2–

95% air prior to utilization, at which time the cultures

were subconfluent.

The MTT Assay

The cell culture viability assay used was the MTT assay.

The assay was carried out as described by Griffiths et al.

(1994) at various time points after HD exposure. Propan-

2-ol was used to extract the purple formazan product from

the cells. Absorbance of the solubilized formazan product

was measured at 570 nm against a solvent blank of

propan-2-ol using a Titertek Multiskan MCC plate-reader

(Flow Laboratories, Rickmansworth, Hertfordshire, UK).



Gentian Violet Assay

Cell culture density was determined by nuclear staining

with 0.1% (w/v) gentian violet (GV). The assay, origin-

ally described by Gillies et al. (1986), was carried out at

various time points after HD exposure. Briefly, cells in

the 96-well plates were stained with GV (100 µl well−1)

for 15 min. The stain was decanted and non-adherent

stain was removed by immersing the plates in running

tapwater for 10 min. The plates were then air-dried. The

dye was solubilized using 200 µl per well of a solvent

comprised of ethanol (50%, v/v) and acetic acid (1%, v/

v). Absorbance of the solubilized GV stain was measured

at 590 nm against the same solvent blank used to

solubilize the GV stain, using a Titertek Multiskan MCC

plate-reader. This assay permits quantification of cell

numbers in cultures because absorbance at 590 nm is

proportional to cell number (Gillies et al., 1986).

Dose–Response Study of the Effects of HD onSVK14, G361, HaCaT and NCTC Cell Viability

A stock solution of HD (50 mM) was prepared in

propan-2-ol. Working dilutions containing 0, 3, 10, 20, 30,

60, 100, 300 and 1000 µM were prepared in phosphate-

buffered saline (PBS, pH 7.4) and pipetted onto SVK14,

G361, HaCaT and NCTC cultures in 96-well plates

(100 µl per well). The HD-containing solutions were made

up and used immediately. The plates were incubated with

these solutions for 1 h at 37 °C in a humidified atmos-

phere of 5% CO2–95% air. The PBS solutions (contain-

ing 0–1000 µM HD) were removed and replaced with the

appropriate medium (100 µl per well) for each cell line.

Vehicle controls of up to 2% (v/v) were used. Cell cul-

ture viability was determined up to 72 h after HD expo-

sure using the MTT and GV assays as described above.

The data are shown as the mean absorbance ± SEM for

groups of eight cultures, expressed as a percentage of the

absorbance of control cultures (0 µM HD-treated).

Photography

Photographs of representative GV-stained cultures were

taken with a Nikon F-301 camera (using Kodak 64T colour

slide film) attached to a Nikon Diaphot phase-contrast

inverted microscope using a magnification of ×100.

Determination of SVK14, G361, HaCaT and NCTCCell Culture Growth Characteristics

To determine the growth rates of the four cell lines over

96 h, SVK14, G361, HaCaT and NCTC cultures were

sown into 96-well cell cultures plates at a seeding density

of 50 000 cells per well as described above. After an

incubation period of 24 h to allow the cells to adhere

(time t = 0 h), cell cultures in the 96-well plates were

fixed with 4% (w/v) formaldehyde in PBS at 4, 24, 48,

72 and 96 h after the initial period allowed for attachment

of cells. The cell density of the cultures was assessed

using the GV assay as described above. The data are

expressed as the mean absorbance (590 nm A) ± SEM for

groups of 24 cultures.

Effects of Depletion of Glutathione AntioxidantStatus on Cellular Sensitivity to HD UsingButhionine Sulphoximine

To determine if a significant element of the greater resist-

ance of G361 cells to HD was attributable to glutathione

(GSH) metabolism, SVK14, G361, HaCaT and NCTC

cells were sown into 96-well plates as noted above at a

density of 50 000 cells per well. After a period of attach-

ment, the cell cultures were exposed for 24 h to either

100 µM buthionine sulphoximine (BSO) (Armstrong

et al., 2002) in 100 µl of medium per well or to medium

only under the conditions noted above. The cultures were

subconfluent at this stage. Cells pretreated to ± BSO then

were exposed to 0, 20, 30 or 60 µM HD in PBS for 1 h

as described above. This was then replaced with medium

and the cultures incubated for a further period of 48 or

72 h. Cell viability was subsequently determined by the

MTT assay. The data are shown as the mean absorbance

(570 nm A) ± SEM for groups of eight cultures, ex-

pressed as a percentage of the absorbance of control cul-

tures (0 µM HD-treated). For comparison purposes, only

the data for the 72 h time point at 30 µM HD is shown.

Cell Cycle Analysis of HD-Exposed Cultures byFlow Cytometry

The effect of a mid-range dose of HD (30 µM) on cell

cycling activity in the most resistant (G361) and most

sensitive (SVK14) cell lines was determined by exposing

cultures to HD followed by flow cytometry analysis of

HD exposed and non-exposed cultures as described below.

A stock solution of HD (50 mM) was prepared in

propan-2-ol. The working dilution (30 µM) was prepared

in PBS. The medium overlying SVK14 and G361 cell

cultures, previously passaged into 25 cm2 flasks as de-

scribed above, was removed and immediately replaced by

5 ml per flask of either PBS or the 30 µM HD working

dilution. The flasks then were incubated for 1 h at 37 °C

in a humidified atmosphere of 5% CO2–95% air, after

which the PBS or HD solution was replaced with DMEM

growth medium (5 ml). The cultures then were incubated

at 37 °C in a humidified atmosphere of 5% CO2–95%

air. Cell cycle analysis was carried out at selected time



flasks at a density of 0.5 × 106 cells per flask in 5 ml of

DMEM medium. The SVK14 and G361 cells, once

pipetted into the flasks, were allowed to attach for 24 h at

37 °C in a humidified atmosphere of 5% CO2–95% air

prior to utilization. After the cells had adhered to the

plastic substrate of the flasks for 24 h, the medium was

removed and fresh DMEM medium (5 ml) was added.

The cell cycle synchronizing agents were made up in

the manufacturer’s recommended vehicle (refer to Table

1 for details) and 50-µl aliquots of inhibitor were added

to each flask. Vehicle controls were included and all

flasks were incubated at 37 °C in a humidified atmos-

phere of 5% CO2–95% air for 24 h to permit cell cycle

synchronization of the cell cultures. The cultures were

subconfluent prior to the addition of HD or control

buffer.

To determine the effect of 30 µM HD on partially

synchronized cultures of SVK14 and G361 cells after the

24 h incubation period with the cell cycle synchronizing

agent or vehicle control, cell cycle analysis was carried

out on control cultures (not treated with synchronizing

agent; n = 3) and on cultures treated with synchronizing

agent (n = 3). The remaining flasks were exposed to

30 µM HD or PBS for 1 h at 37 °C in a humidified

atmosphere of 5% CO2–95% air. The 30 µM HD or PBS

was removed from each flask and replaced with DMEM

medium; all flasks were returned to the 37 °C incubator

for further incubation periods of 24 or 48 h. Cell cycle

analysis was carried out at 24 and 48 h after HD expo-

sure as described above.

Statistical Analysis

The results were analysed using Welch’s approximate

two-tailed t-test on Instat™ Software (GraphPad Soft-

ware, San Diego, CA).

points (24, 48 and 72 h) after exposure to HD as

described below.

To prepare samples for flow cytometry, the contents

of each 25 cm2 flask was washed in PBS. The PBS was

aspirated and the cells removed from the flask by incuba-

tion at 37 °C with trypsin–EDTA for 5–10 min. The cells

were centrifuged (300 g, 4 °C, 5 min) and resuspended in

0.5 ml of DMEM medium. The DNA-Prep Reagent kit

(Beckman Coulter) was used to prepare SVK14 and

G361 cells for cell cycle analysis. Briefly, 0.1 ml of LPR

(a saponin and formaldehyde containing reagent used to

fix and permeabilize cells) was added to each sample in

DMEM medium. The samples were incubated at room

temperature for 10 min. A 1-ml aliquot of DP stain (the

propidium iodide component of the DNA-Prep kit) was

added to stain cellular DNA. The cell samples were

incubated at room temperature in the dark for 2 h.

Propidium iodide fluorescence (560–680 nm) was meas-

ured using a Beckman-Coulter EPICS® XL-MCL™ four-

colour flow cytometer, following excitation (at 488 nm)

with a 15 mW argon ion laser with the sample flow rate

set to high. EXPO™ 32 software (version 1.2; Beckman-

Coulter) was used to analyse the fluorescence output.

Fluorescence counts were collected over 300 s; the per-

centages of cells in each phase of the cell cycle were

determined by gating the G0/G1, S and G2/M phase cell

populations as described by Ormerod (1996), expressed as

the mean percentage of groups of eight cultures ± SEM.

Screening of Cell Cycle Synchronizing Agents

A series of cell cycle synchronizing agents were identi-

fied in the literature and screened at the concentrations

recommended in the literature (Table 1) for efficacy in

the cell lines used. The SVK14 and G361 cells were pre-

pared as described above by seeding cells into 25 cm2

Table 1. Cell cycle synchronising agent profiles for SVK14 and G361 cells

Inhibitor Classa Concb G361c SVK14c

Acyclovir (Cioe et al., 1992) G0/G1 500 µMD 0 G0/G1

Aphidicolin (Mutomba and Wang, 1996) Early S 0.1 µg ml−1D 0 G0/G1

Hydroxyurea (Lee et al., 1997) G1/S 375 µMW G1/Sd G1/Sd

Hydroxyurea G1/S 1 mMW S G2/M

Indole-3-carbinol (Cover et al., 1998) G0/G1 100 µME G0/G1d 0

Indole-3-carbinol G0/G1 225 µME Toxic Toxic

Indirubin-3-monoxime (Marko et al., 2001) G2/M 2 µMD S 0

Low Temperature (Doyle et al., 1996) G0/G1 4 °C 0 0

Mimosine (Hoffman et al., 1991) G1 300 µMW S/G2/M S

Olomoucine (Schutte et al., 1997) G1 200 µMD 0 G2/M

Rapamycin (Morice et al., 1993) G1 0.1 nmD 0 0

Staurosporine (Zong et al., 1999) G1 20 nmD G0/G1 G0/G1d

Trichostatin (Inkoshi et al., 1999) G1 17 nmD S/G2/M G0/G1

a The published activity for these cell cycle regulators.b The concentrations at which these compounds were used in the present study. The designations D, W and E refer to

the solvent vehicles DMSO, water and ethanol, respectively.c The predominant (although partial) activity found for the given regulators.d These agents were found to be the best at synchronizing the cell cycle at their respective positions (although only

partially) in SVK14, G361 or both cell lines.



Results

Dose–Response Study of the Effects of HD inSVK14, G361, HaCaT and NCTC Cells

The SVK14, G361, HaCaT and NCTC cells were

exposed to HD (up to 1000 µM) for 1 h, with viability

being measured at 4, 24, 48 and 72 h time points after the

exposure, using the MTT assay (Fig. 1) and GV assay

(for which similar results were obtained; data not shown).

Vehicle controls for all experiments were run, which

indicated that propan-2-ol had no significant effects on

cell culture viability (data not shown). Initially, at the 4 h

time point, the four cell lines exhibited similar responses

to HD, with viability only decreasing after exposure to

1000 µM HD. At 24 h after exposure, the HaCaT cell line

was found to be the most resistant to HD but only at

concentrations of <100 µM; the G361 cell line appeared

to be the more sensitive cell line at concentrations of

>30 µM HD. At 48 h after HD exposure, the dose–

response profile of G361 cells was largely maintained.

However, the relative viability of the remaining cell lines

was found to decline to a greater extent at HD concentra-

tions of >3 µM, so that at 72 h after HD exposure the

G361 cell line was found to be more resistant to the

cytotoxic effects of HD than the NCTC, HaCaT and

SVK14 cell lines at HD doses of >3 and <100 µM. More

specifically, at 72 h there were 1.3- (P < 0.001), 2.0- (P

< 0.0001), 3.4- (P < 0.0001), 4.7- (P < 0.0001) and 8.8-

fold (P < 0.0001) differences between G361 and SVK14

cell culture viability at 3, 10, 20, 30 and 60 µM HD,

respectively, although this pattern did not extend to

100 µM HD. An intermediate challenge concentration

of 30 µM HD was selected with which to investigate the

effects of HD on morphology, BSO and cell cycling

activity in G361 and SVK14 cells.

Figure 1. Effect of HD on SVK14 (�), G361 (�), HaCat (�) and NCTC (�) cells. Cells were exposed to HD (up to1000 µM) for 1 h. Cell culture viability was determined using the MTT assay at periods of 4 h (a), 24 h (b), 48 h (c)and 72 h (d) after exposure. Data are expressed as the mean percentage viability of control cultures (not exposedto HD) ± SEM (n = 8)



Growth of SVK14, G361, HaCaT and NCTC CellCultures

The growth characteristics of the four cell lines were

determined over 96 h using the GV assay as an index of

cell density (Fig. 3). It was found that the SVK14 cell

line grew more slowly compared with the other cell lines,

the difference being most apparent at the longer time

points so that at 72 h after t = 0 the order of cell culture

density was HaCaT>NCTC>G361>SVK14. The growth

curves for the cell lines shown were different to each

other, but overall the curves for the G361 cell line

most resembled those of the HaCaT and NCTC cell

lines. The differences in the growth profiles were particu-

larly marked at 72 and 96 h for G361 cultures, whose

density was found to be greater than that of SVK14 cul-

tures by 1.7-fold (P < 0.0001) and 1.4-fold (P < 0.0001),

respectively.

Figure 2. (a) Photomicrograph of a 72-h control culture of G361 cells stained with GV, demonstrating thedendritic morphology associated with this cell line. (b) Photomicrograph of a G361 culture 72 h after exposure to30 µM HD stained with GV, showing a reduction in culture density but maintaining the dendritic morphology seenin the control cultures. (c) Photomicrograph of a 72-h control culture of SVK14 cells stained with GV, demonstrat-ing the epithelial cobblestone morphology seen in this cell line. (d) Photomicrograph of an SVK14 culture 72 h af-ter exposure to 30 µM HD stained with GV, showing a necrotic population characterized by a reduction in cellculture density, loss of cell morphology, cellular fragmentation and nuclear condensation (magnification: ×100)

Photomicrography of HD-exposed G361 andSVK14 Cells

Photomicrographs taken of GV-stained cultures showed

the dendritic morphology of control (non-HD exposed)

G361 cells, with heavy GV staining of the nucleus

and the cytoplasm (Fig. 2a). At 72 h after exposure

to 30 µM HD the number of G361 cells in the culture

was reduced, although the cells had maintained their

morphology (Fig. 2b). The epithelial morphology of

SVK14 cells in control cultures is shown in Fig. 2c,

with dense staining of the nucleus and lighter staining

of the cytoplasm. At 72 h after exposure to 30 µM HD

there was a considerable reduction in the number of

cells, with cytoplasmic swelling and necrosis being

evident (Fig. 2d). This reduction of cell density in HD-

exposed SVK14 cells was greater than that found in

G361 cultures.



Figure 3. Determination of SVK14 (�), G361 (�),HaCat (�) and NCTC (�) cell culture growth rates. Thegrowth rates of the four cell lines are shown over peri-ods of up to 96 h. The cell density of the cultures wasassessed using the GV assay. The data are expressed asmean absorbance (A590 nm) ± SEM (n = 24)

Figure 4. The effect of BSO pretreatment on thecytotoxicity of HD in SVK14, G361, HaCat and NCTCcells 72 h after exposure to HD (30 µM). Cell cultureswere either exposed to medium only (black bars)or pre-exposed to BSO (100 µM) for 24 h prior to HDexposure (hatched bars). Cell viability was determinedby the MTT assay. The results show that BSO pretreat-ment significantly exacerbates the toxicity of HD. Dataare expressed as the mean percentage viability of con-trol cultures (not exposed to HD) ± SEM (n = 8). * P <0.05; ** P < 0.01; *** P < 0.0001

time point, show that BSO pretreatment in addition to

subsequent HD exposure (30 µM) resulted in a further

reduction in the viability of G361 (P < 0.0001), HaCaT

(P < 0.01), NCTC (P < 0.001) and SVK14 (P < 0.05)

cultures compared with cultures exposed to HD alone.

Vehicle controls showed that propan-2-ol did not contrib-

ute to the toxicity of HD (data not shown). The data

show that a substantial proportion of the resistance of

G361 cells to HD is attributable to BSO-mediated effects

on antioxidant-mediated metabolism, although G361 cul-

tures still retained a substantial degree of viability (43%)

at 30 µM HD following BSO pretreatment. Overall, the

magnitude of the BSO-mediated decrease in viability

in HD-exposed cells (expressed as a ratio relative to the

viability value of cultures exposed only to HD) was

of the order G361 (0.50)>SVK14 (0.58)>NCTC (0.63)>HaCaT (0.81), indicating that BSO pretreatment of G361

cells reduced viability by 50%. The relative similarity of

the level of BSO-induced viability reduction in SVK14

cell cultures (42%) further supported the selection of

G361 and SVK14 cells for further comparative analysis.

Cell Cycle Analysis of HD-exposed G361 andSVK14 Cultures by Flow Cytometry

Flow cytometry was carried out on G361 and SVK14

cells to determine the effect of HD on cell cycling activ-

ity. The flow cytometer output data set shown in Fig. 5

is a set of representative profiles for SVK14 and G361

cell control cultures (not exposed to HD; ‘−HD’ in the

figure), as well as cell cultures exposed to 30 µM HD

(‘+HD’ in the figure). Vehicle controls showed that

propan-2-ol did not affect the viability of the cultures

(data not shown). The data set shows that cell cycling

activity over 72 h in SVK14 cells was more sensitive to

disruption than cell cycling in G361 cells over this

period. This is exemplified by the 24-h data, which show

both a broadening of the G0/G1 peak and a shift to the

right indicating that DNA damage has occurred in

SVK14 cells. In contrast, although the cell cycle profile

for G361 cells retained much of its integrity over the 72-

h period, SVK14 cell counts had degenerated to back-

ground levels by this time. A complete data set (for 24

and 48 h only) is described in Table 2 for both cells lines,

in which the percentage values for the proportion of cells

in various phases of the cell cycle are shown. The table

contains data from cultures at 24 and 48 h after HD

exposure. Tabulated data are not shown for the 72-h time

point because only background counts were measured for

SVK14 cells at this time.

The results shown in Table 2 indicate that at 24 h the

control G361 cell cultures had a greater proportion of

their number in G0/G1 phase (by 1.3-fold; P < 0.001)

compared with control SVK14 cultures. In contrast, the

proportion of cells in S-phase was greater in SVK14

Buthionine Sulphoximine-mediated CellularSensitization to HD

The effects of BSO pretreatment on cellular sensitivity

to HD were determined in G361, SVK14, HaCaT and

NCTC cells. The use of 100 µM BSO in control cultures

showed that BSO did not affect significantly the viab-

ility of any of the four cell lines (data not shown). The

results in Fig. 4, depicting data collected at the 72 h



With respect to HD-induced effects on the cell cycle,

the data in Table 2 confirmed that at 24 h the SVK14

cells were relatively more sensitive to HD, as shown by

the 2.1-fold reduction (P < 0.0001) in the percentage of

cells in G0/G1 phase after HD exposure compared with

Figure 5. Cell cycle profiles of SVK14 and G361 cells to compare the effects of exposure to 30 µM HD. The cellcycle profiles of the SVK14 and G361 cells not exposed to HD (controls) are indicated by ‘−HD’ at 24, 48 and 72 h.Cell cycle profiles of cultures exposed to HD are denoted by ‘+HD’. The first peak denotes the G0/G1 phase cellpopulation; the smaller peak denotes the G2/M phase cells and S-phase cells are in the trough between the peaks.The x-axis of each graph denotes fluorescence intensity (arbitrary units); the y-axis denotes cell counts measured ateach fluorescence channel. The data are representative flow cytometer profiles for each cell line. The data fromthis experiment are described in Table 2 as relative percentages of cells in each phase of the cell cycle for ± HD-treated SVK14 and G361 cultures

Table 2. The relative proportion of SVK14 and G361 cells in each phase of thecell cycle 24 and 48 h after HD exposure (30 µM)a

Cell line Treatment G0/G1 S G2/M

Data at 24 h

SVK14 −HD 49.0 ± 1.5 11.2 ± 1.5 37.2 ± 2.8

+HD 23.7 ± 2.1 46.9 ± 2.0 26.6 ± 2.8

(P < 0.0001) (P < 0.0001) (P < 0.05)

G361 −HD 61.5 ± 1.8 7.2 ± 1.0 30.2 ± 2.3

+HD 72.8 ± 3.4 4.2 ± 2.6 22.6 ± 1.4

(P < 0.05) (NS) (P < 0.05)

Data at 48 h

SVK14 −HD 47.6 ± 1.1 14.6 ± 1.8 35.1 ± 2.2

+HD 14.9 ± 1.4 38.8 ± 3.1 36.1 ± 3.6

(P < 0.0001) (P < 0.0001) (NS)

G361 −HD 63.0 ± 1.4 9.7 ± 1.7 26.8 ± 1.8

+HD 43.9 ± 3.7 22.9 ± 2.7 32.0 ± 1.7

(P < 0.01) (P < 0.01) (NS)

a Data are presented as the mean percentage of cells in each phase of the cell cycle ± SEM (n = 8). The data

confirm that SVK14 cells are more sensitive to HD than G361 cells, as shown by the significant differences in

the percentage of cells in G0/G1 and S-phase between HD-exposed and control cells. There were differences in

the percentage of cells in each phase of the cell cycle when comparing exposed and control G361 cells, but these

differences were not as large as those measured for SVK14 cells. The HD-exposed SVK14 cells have a lower

percentage of cells in G0/G1 than control cells; the reduced proportion of cells is due to a shift in the G0/G1 peak

to the right, which is indicative of DNA damage.

cultures than in G361 cells (by 1.6-fold; P < 0.05). The

SVK14 cell cultures also had more cells in G2/M phase

than was the case for G361 cultures (by 1.2-fold),

although this was not statistically significant. A similar

overall pattern was found at 48 h.



control cultures. This compared well with a 1.2-fold

increase (P < 0.05) in the percentage of G361 cells in G0/

G1 phase following HD exposure. A 4.2-fold increase (P

< 0.0001) in the proportion of cells in S-phase was meas-

ured after SVK14 cells were exposed to HD, whereas the

proportion of G361 cells in S-phase fell by 1.7-fold fol-

lowing HD exposure, although this difference was not

significant. Sulphur mustard-induced perturbations in the

G2/M phase cell populations were found also in SVK14

and G361 cell cultures, resulting in 1.4- and 1.3-fold

reductions (P < 0.05 and P < 0.05, respectively) in the

proportion of cells in this phase of the cell cycle. At the

48-h time point, HDinduced disruption of G0/G1 phase in

SVK14 cells increased further, with a 3.2-fold reduction

(P < 0.0001) in the percentage of cells in this phase. This

contrasts with only a 1.4-fold reduction (P < 0.01) in

G361 cells. A 2.7-fold increase (P < 0.0001) in the

proportion of cells in S-phase was found in SVK14 cells

exposed to HD compared with control cultures, whereas

this proportion was lower (2.4-fold; P < 0.01) in G361

cells exposed to HD. No significant difference was found

in the relative proportions of SVK14 or G361 cells in G2/

M phase in HD-exposed and control cultures.

Screening of Cell Cycle Synchronizing Agentsand the Effect of HD (30 µµµµµM) on PartiallySynchronized Cultures of SVK14 and G361 Cells

A series of cell cycle synchronizing agents were screened

to determine if the cell cycle of SVK14 and G361

cultures could be halted reversibly at various stages of

the cell cycle (Table 1). Selected inhibitor treatments

were used to synchronize cultures of SVK14 and G361

cells prior to exposing them to HD, with cell cycle analy-

sis being carried out at 24 and 48 h after HD exposure

to determine if synchronization could modulate the

cytotoxicity of HD.

Some of the cell cycle inhibitor treatments tested

did not have any effect on SVK14 and G361 cells

(rapamycin, low temperature). Of the remainder,

acyclovir, aphidicolin, indole-3-carbinol, indirubin-3-

monoxime, mimosine, olomoucine and trichostatin pro-

duced different effects in SVK14 cells compared with

those obtained in G361 cell cultures; none of these

inhibitors produced completely synchronized cultures

(data not shown). In the case of indole-3-carbinol, a

lower concentration (100 µM) than the published one

(225 µM) had to be used because the higher dose was

found to be cytotoxic. Indole-3-carbinol was found to

have reversible but only partial G0/G1 modulatory effects

in G361 cells at 100 µM (data not shown). Figure 6

shows the effects of staurosporine on SVK14 cells, in

which only partial synchronization of cell cultures in

G0/G1 phase was obtained. At 48 h, it was found that

staurosporine increased the proportion of cells in G0/G1

phase from 51% in control cells to 54.5% for SVK14

cells. This partial synchronization was a pattern that was

found also for hydroxyurea in both cell lines (data not

shown).

It can also be seen from Fig. 6 that staurosporine

pretreatment of SVK14 cells exacerbated the effects of

Figure 6. The effect of 30 µM HD upon SVK14 cells partially synchronized in G0/G1 of the cell cycle with a 24-hstaurosporine pretreatment prior to HD exposure (30 µM). Cell cycle analysis was carried out 24 and 48 h after HDexposure. The cell cycle profiles of the SVK14 cells not exposed to HD (controls) are indicated by ‘−HD’ at 24 and48 h. Cell cycle profiles of cultures exposed to HD are denoted by ‘+HD’. The first peak denotes the G0/G1 phase cellpopulation; the smaller peak denotes the G2/M phase cells and S-phase cells are in the trough between the peaks.The x-axis of each graph denotes fluorescence intensity (arbitrary units); the y-axis denotes cell counts measured ateach fluorescence channel. The data are representative flow cytometer profiles for each cell line. The data from thisexperiment are described in Table 3 as total cell counts for ± staurosporine-pretreated and ± HD-exposed SVK14cultures. Partial synchronization of SVK14 cells with staurosporine was shown to exacerbate the effects of HD



Table 3. Effects of exposure to HD following pretreatment with/withoutstaurosporine on SVK14 cell cultures: total flow cytometer cell counts are shownat 24 and 48 h as an index of viability for SVK14 cultures given no pretreatmentand no exposure to HD, cultures pretreated with staurosporine only, culturesexposed to HD only and cultures both pretreated with staurosporine and thenexposed to HD as described in the Methods section (data are mean cell countvalues ± SEM; n = 3)

0 µM HD 30 µM HD

24 h 48 h 24 h 48 h

Control 17 100 ± 490 17 900 ± 352 8650 ± 620 1650 ± 150

(−staurosporine)

+Staurosporine 12 100 ± 600 15 000 ± 590 2720 ± 270 390 ± 30

(P < 0.0001) (P < 0.001) (P < 0.0001) (P < 0.0001)

subsequent HD exposure, so that at 48 h after HD expo-

sure the flow cytometer histogram of staurosporine-

pretreated cultures recorded only background counts.

Table 3 shows cell counts from an experiment in which

total cell numbers (counts) were compiled from flow

cytometry histograms from experiments described in

Fig. 6 as an index of culture viability for each treatment.

At the 48-h time point, it was found that cell counts

for cultures treated with staurosporine were 84% of that

of control cultures (where 17 900 counts = 100%, P <0.001; Table 3). At 48 h, the cell counts in cultures

exposed to HD only (30 µM) were 9.2% compared

with control cultures. However, cell counts in cultures

pretreated with staurosporine before HD exposure were

only 2.2% of control counts (P < 0.0001), indicating that

staurosporine pretreatment exacerbated the toxicity of

HD. The reversibility of the effects of staurosporine are

shown in Table 3: the cell counts for the staurosporine-

pretreated cultures at 48 h had increased to 15 000 from

12 100 at 24 h. In contrast, the histogram profile for HD-

treated SVK14 cells shows a characteristically broadened

G0/G1 peak with a lower number of recorded counts.

Hydroxyurea, reported to be a G1/S phase synchronizing

agent (Lee et al., 1997), partially synchronized cultures of

G361 and SVK-14 cells at G1/S phase of the cell cycle

but did not reduce the cytotoxicity of HD in either cell

line (data not shown).

Discussion

In this study, the human skin cell lines SVK14, HaCaT,

NCTC and G361 were used to model the cytotoxicity of

HD to obtain information on the biochemical basis for

differences in sensitivity to HD. Marked biochemical and

morphological differences in the responses of the cell

lines were found over a 72-h period following exposure

to HD. At the 4-h time point, relatively little response to

HD was seen except at the highest concentration of HD

(1000 µM). At the 24-h time point, the G361 cell line

was found to be more sensitive to HD than SVK14,

HaCaT and NCTC cells at 60, 100 and 300 µM HD,

whereas at 48 h the G361 cells became relatively more

resistant to HD, particularly at 10, 20 and 30 µM HD.

At 72 h this pattern became established further at 3–

60 µM HD.

The results confirm previous findings by Smith et al.

(2001), who found that cellular proliferation in G361 cul-

tures could recover following HD exposure, although in

the present study this was not evident at HD doses of

>60 µM. This is suggestive of a limited repair capability

and would explain the markedly greater resistance of

G361 cells at 72 h compared with SVK14 cells, signific-

ant differences in cell culture viability being found at

20, 30 and 60 µM HD. These observations were con-

firmed by photomicrography of control and HD-exposed

cell cultures, which indicated that, at 72 h after exposure

to 30 µM HD, although G361 cultures showed some

reduction in cell density the remaining cells were found

to maintain the dendritic morphology seen in control

cultures. In contrast, the normal epithelial cobblestone

morphology of control SVK14 cells was lost in HD-

treated cultures, with substantial losses in cell culture

density being found. The remaining SVK14 cells showed

a loss of normal cell morphology, as well as cellular frag-

mentation and nuclear condensation.

These findings are consistent with Smith et al. (2001),

who also showed that G361 cells were more resistant

than SVK14 cells to the effects of H2O2 to a similar ex-

tent, alluding to related resistance mechanisms involving

an upregulated DNA repair capacity. In the present study,

differences in cell population growth rates alone could

not account for the greater sensitivity of SVK14 cells to

HD, because despite the known deleterious effects of HD

on rapidly dividing cells (Maynard, 1987) the G361 cell

cultures were found to have a higher growth rate than

SVK14 cell cultures.

Intracellular antioxidant status, specifically the quantity

of intracellular glutathione (GSH) in cell lines, has been

reported to be important in the protection of cells against



HD toxicity (Gross et al., 1993). Thus, a series of experi-

ments were carried out using BSO to inhibit the activity

of γ-glutamyl cysteine synthetase (Armstrong et al.,

2002), with a view to determining if antioxidant status

was responsible for the relatively higher level of resist-

ance to HD found in G361 cultures. It was found that

although GSH metabolism played a significant part in the

mediation of HD toxicity in all four cell lines, the data

indicated that in G361 cell cultures the pretreatment with

BSO resulted in a relatively greater reduction in viability

compared with non-BSO-pretreated cultures at 30 µM HD

in relation to the other three cell lines investigated. This

implies that a large proportion of the resistance of G361

cultures to HD may be attributable to increased intra-

cellular antioxidant status. Previous studies have shown

that thiol agents such as mono-isopropylglutathione ester

and di-isopropylglutathione ester are capable of confer-

ring protection against HD in vitro (Lindsay et al., 1997;

Andrew and Lindsay, 1998b; Lindsay and Hambrook,

1998). However, other factors cannot be ruled out, be-

cause at 30 µM HD the G361 cultures contained a rela-

tively high proportion of cells that remained viable even

after BSO pretreatment. In order to probe this further, a

comparative analysis on cell cycling activity in SVK14

and G361 cultures was carried out because previous stud-

ies have suggested that G361 cells may have a more

efficient DNA repair capacity, as indicated by elevated

levels of PCNA in this cell line (Smith et al., 2001).

The DNA repair involved may be nucleotide excision

repair, because PCNA is an essential co-factor in this

type of repair (Bootsma, 2001). The SVK14 cells were

employed for comparative cell cycle studies because BSO

increased the toxicity of HD to a similar extent in both

cell lines.

In G361 cells the selected challenge dose of HD

(30 µM) was found to have relatively little effect on

the cell cycle profile at 24, 48 and 72 h post-exposure

compared with SVK14 cells. In particular, G361 cultures

maintained a high proportion of their population in G0/G1

phase even after HD exposure at 24 h. However, cell

cycling activity in the more sensitive SVK14 cell line

was found to be disrupted substantially, with most of the

propidium iodide-stained DNA accumulating in a broad-

ened G0/G1 peak that extended into the S-phase popula-

tion at 24 h after HD exposure. By 48 h the DNA signal

of the SVK14 cell cycle profile had degenerated sub-

stantially, and at 72 h only background noise could be

detected. This marked degeneration of the cell cycle pro-

file occurred in SVK14 cells only, and well before the

MTT assay in the HD titration experiments could detect

significant decreases in viability in this cell line compared

with the G361 cell line. Therefore, HD exposure caused

early damage to the DNA of SVK14 cells 24 h after HD

exposure, but this did not result in substantial loss of

viability (as determined by the MTT and GV assays)

until later time points. This indicates that early nuclear

events may be important in HD cytotoxicity, particularly

in relatively sensitive cell lines.

An increase in the width of the G0/G1 peak and a shift

in measured data counts to the right of the data collection

histogram are characteristic of increasing amounts of

genotoxic damage (Wagner et al., 1998). This can be in-

terpreted also as a reduction in G0/G1 peak counts if, as

was the case in the present study, the flow cytometry data

collection gates for HD-exposed cells are kept the same

as for control (unexposed) cultures. The results from the

cell cycle analysis therefore suggested that G361 cells

were more resistant to DNA damage than SVK14 cells,

even at later time points. One possible mechanism for

this difference in sensitivity is that in SVK14 cell cul-

tures a greater proportion of the cell population was in S-

phase; keratinocytes are known to be especially sensitive

to alkylating agents when in this phase of the cell cycle

(Smith et al., 1991). The finding that G361 cultures

tended to have a lower proportion of their cell population

in S-phase compared with SVK14 cultures may be

significant.

Attempts were made to modulate DNA damage and

concomitant cell cycle disruption by altering the progres-

sion of the cell cycle, because the efficiency of DNA

repair may be a function of the position of the cell in the

cell cycle (Gorczya et al., 1993). Relatively few studies

have assessed the effects of cell cycle regulation on DNA

repair (Smith and Seo, 2002). The G1 phase cell cycle

checkpoint effectively arrests the cell cycle if DNA dam-

age is detected and enables repair before progression to

S-phase (Smith and Seo, 2002). It should be noted that in

the present study the G361 cells were able to retain the

integrity of the cell cycle profile 24 h after HD exposure,

particularly with respect to G0/G1 phase counts, which

increased marginally from 61% of the total cell popula-

tion in control cultures to 73% of the cell population in

HD-exposed cultures. This is suggestive of the existence

of a more efficient G1 phase cell cycle checkpoint in

G361 cells compared with SVK14 cultures; in the latter

case, G0/G1 phase counts decreased substantially from

49% of the total cell population in control cultures to

24% of the cell population in HD-exposed cultures.

Effective repair in G1 phase is essential, because in S-

phase there may be no excision repair (Bell et al., 1999).

The expression of factors associated with nucleotide

excision repair, such as DNA polymerase-δ, has been

reported to be higher at the G1/S junction than elsewhere

in the cell cycle (Wang, 1996), therefore a variety of

cycle synchronizing agents were used to halt or increase

the relative proportion of cells in G0/G1 phase prior to

HD exposure. A major criterion for their use was that

their effects on cell cycle profiles should be reversible.

In the present study, indole-3-carbinol reversibly

but only partially held G361 cells at G0/G1 in the cell

cycle, and had no effect on SVK14 cells. Indole-3-

carbinol treatment has been reported to downregulate



cyclin-dependent kinase 6 (CDK6) transcription by selec-

tively disrupting the interactions of SP1 (transcription

factors) with a composite DNA-binding site within the

CDK6 promoter, arresting cells in G1 (Cram et al., 2001).

Inhibition of CDK6 correlates with a reduction in endo-

genous retinoblastoma protein (Cover et al., 1998). The

CD6-dependent phosphorylation of retinoblastoma protein

is required for the progression of cells through G0/G1

and into S-phase. Indole-3-carbinol had no beneficial

effect on G361 cells subsequently exposed to HD.

Staurosporine reversibly held SVK14 cells in G0/G1

phase of the cell cycle but the partial synchronization

obtained prior to HD exposure was found to exacerbate

the effects of HD. Staurosporine is reported to be a

broad-spectrum protein kinase inhibitor, examples of

whose targets include protein kinase C (Swe et al., 1996),

protein kinase A (Ruegg & Burgess, 1989), tyrosine pro-

tein kinase and CaM kinase (Yanagihara et al., 1991).

Staurosporine exploits the same potential hydrogen-bond

interactions as ATP (the cofactor for the protein kinases)

in its mechanism of binding (Furet et al., 1995). The

addition of staurosporine prior to HD exposure may

have resulted in the inhibition of these protein kinases

(which are involved in DNA double-strand break repair

mechanisms), possibly increasing the sensitivity of the

cells to the damaging effects of HD (Tuteja and Tuteja,

2001).

Hydroxyurea reversibly (but only partially) held

G361 and SVK14 cells at S-phase in the cell cycle. The

partial synchronization of G361 and SVK14 cells with

hydroxyurea prior to HD exposure was found to exacerb-

ate the effects of HD in both cell lines. Hydroxyurea

has been reported to destroy the tyrosyl free radical at the

active site of the M2 protein subunit of ribonucleotide

reductase, inactivating the enzyme (Yarbro, 1992). This

blocks the de novo synthesis of deoxyribonucleotides

(Hendricks and Matthews, 1998), synchronizing DNA

synthesis in S-phase. If no excision repair occurs in S-

phase (Bell et al., 1999), HD will cause greater damage

to cells synchronized in S-phase because potentially no

DNA repair may occur after the HD exposure until the

cells have completed the cell cycle. This may explain

why 30 µM HD caused cell cycle disruption in G361

cells partially synchronized in S-phase prior to HD expo-

sure, when previously this concentration of HD had pro-

duced only limited cell cycle disruption in non-pretreated

cells. Aphidicolin (an early S-phase inhibitor) also was

used in the present study, although both SVK14 and

G361cell cultures were found to be affected adversely by

treatment with aphidicolin. This may be because DNA

polymerase-δ and -ε associated with nucleotide excision

repair are specifically inhibited by this agent (Wood and

Shivjii, 1997).

In summary, the use of various cell cycle synchroniz-

ing agents was found to produce variable effects in

SVK14 and G361 cells. These agents had either no

protective action or exacerbated the cytotoxicty of

HD. More efficient methods for maintaining cells in

G0/G1 phase will be required to determine if maintaining

a relatively greater proportion of the cell population in

this part of the cell cycle at the time of HD challenge has

potential therapeutic benefit. The finding that, in G361

cells, a larger proportion of the cell population was in

G0/G1 phase at any time (before or after HD exposure)

compared with the more sensitive SVK14 cell line

may be significant in relation to the greater resistance to

HD-induced cell cycle disruption measured in G361 cell

cultures.

The greater resistance of G361 cells to HD may be

related to the role of PCNA, which not only is associated

with nucleotide excision repair but also may have a

role in cell cycle regulation. The PCNA interacts with

cyclin D, a molecular species that regulates cell cycle

progression from G1 phase to S phase (Xu et al., 2001).

Therefore, the basis for the greater resistance of the

G361 cell line to HD could be due to a combination of

greater DNA repair capacity (e.g. through an upregulated

nucleotide excision repair capability) and more efficient

cell cycle control processes that halt the cycle at the

appropriate checkpoints. The failure of mitotically active

cells to halt at the appropriate checkpoints would

result in the cell attempting to replicate codons with

HD-induced monofunctional DNA base alterations and

bifunctional inter- and intra-strand cross-links. This

would have several consequences, such as strand breaks,

mutations, disruption of DNA replication and transcrip-

tion processes and eventually cell death (Wang, 1998).

According to Smith and Seo (2002), p53 mediates the G1

phase checkpoint following DNA damage and it is likely

that it is this action that allows DNA repair to progress

by giving the cell a greater amount of time for DNA

repair to occur (in G1 phase) before the cell enters S-

phase. Further work will investigate the biochemical

basis of increased HD resistance in G361 cells, particu-

larly with respect to p53 activity following DNA damage

and the activation of its associated genes Gadd45 and

p48XPE (Smith and Seo, 2002) to determine if the differ-

ences in susceptibility to HD between SVK14 and G361

cells are attributable to differential expression of selected

components of the nucleotide excision repair pathway.

Acknowledgement—We would like to thank John Hayes for supplyingthe HaCaT and NCTC cells and for helpful technical discussions andadvice.

References

Andrew DJ, Lindsay CD. 1998a. Protection of human upperrespiratory tract cell lines against sulphur mustard toxicity byhexamethylenetetramine (HMT). Hum. Exp. Toxicol. 17: 373–379.

Andrew DJ, Lindsay CD. 1998b. Protection of upper respiratory tractcell lines against sulphur mustard by glutathione esters. Hum. Exp.

Toxicol. 17: 387–395.



Armstrong JS, Steinauer KK, Hornung B, Irish JM, Lecane P, BirrellGW, Peehl DM, Know SJ. 2002. Role of glutathione depletion andreactive oxygen species generation in apoptotic signalling in a humanB lymphoma cell line. Cell Death Differ. 9: 252–263.

Bell LM, Lannon CL, Yhap M, Pyesmany AF, Henry M, Laybolt K,Riddell DC, van Velzen D. 1999. Drug resistance in leukemia andlymphoma III. Adv. Exp. Med. Biol. 457: 289–296.

Bootsma D. 2001. The ‘Dutch DNA Repair group’ in retrospect. Mutat.

Res. 485: 37–41.Brooks P, Lawley PD. 1963. Effects of alkylating agents on T2 and T4

bacteriophage. Biochem. J. 89: 138–144.Brown RF, Rice P. 1997. Histopathological changes in yucatan minipig

skin following challenge with sulphur mustard. A sequential study ofthe first 24 h. Int. J. Exp. Pathol. 78: 9–20.

Cioe L, Mukhopadhyay S, Rovera G. 1992. Selective-inhibition ofproliferation in V-ABL-transformed and BCR-ABL-transformed cellsby a nucleoside analogue. J. Biol. Chem. 267: 22178–22182.

Cover CM, Hsieh SJ, Tran SH, Hallden G, Kim GS, Bjeldanes LF,Firestone GL. 1998. Indole-3-carbinol inhibits the expression ofcyclin-dependent kinase-6 and induces a G1 cell cycle arrest ofhuman breast cancer cells independent of estrogen receptor signal-ling. J. Biol. Chem. 273: 3838–3847.

Cram EJ, Liu BD, Bjeldanes LF, Firestone GL. 2001. Indole-3-carbinolinhibits CDK6 expression in human MCF-7 breast cancer cells bydisrupting sp1 transcription factor interactions with a compositeelement in the CDK6 promoter. J. Biol. Chem. 276: 22332–22340.

Doyle A, Griffiths JB, Newell DG. 1996. Cell + Tissue Culture: Labo-

ratory Procedures. John Wiley: Chichester; Section 4E: 3.3.Erickson LC, Laurent G, Sharkey NA, Kohn KW. 1980. DNA cross-

linking and monoadduct repair in nitrosourea-treated human tumourcells. Nature 288: 727–729.

Fox M, Scott M. 1980. The genetic toxicology of nitrogen and sulphurmustard. Mutat. Res. 75: 131–168.

Furet P, Caravatti G, Lydon N, Priestle JP, Sowadski JM, Trinks U,Traxler P. 1995. Modelling study of protein kinase inhibitors: bind-ing mode of staurosporine and origin of the selectivity of CGP52411. J. Comput. Aid. Mol. Des. 9: 465–472.

Gillies RJ, Didier N, Denton M. 1986. Determination of cell number inmonolayer cultures. Anal. Biochem. 159: 109–113

Gorczya W, Gong J, Ardelt B, Traganos F, Darzynkiewicz Z. 1993.The cell-cycle related differences in susceptibility of HL-60 cells toapoptosis induced by various anti-tumour agents. Cancer Res. 53:3186–3192.

Griffiths GD, Lindsay CD, Upshall DG. 1994. Examination of thetoxicity of several protein toxins of plant origin using bovine pulmon-ary endothelial cells. Toxicology 90: 11–27.

Gross CL, Innace JK, Hovatter RC, Meier HL, Smith WJ. 1993. Bio-chemical manipulation of intracellular glutathione levels influencescytotoxicity to isolated human lymphocytes by sulphur mustard. Cell

Biol. Toxicol. 9: 259–268.Haber LF. 1986. The Poisonous Cloud: Chemical Warfare in the First

World War. Oxford University Press: Oxford.Hendricks SP, Matthews CK. 1998. Differential effects of hydroxyurea

upon deoxyribonucleoside triphosphate pools, analyzed with vacciniavirus ribonucleotide reductase. J. Biol. Chem. 273: 29519–29523.

Hoffman BD, Hanauskeabel HM, Flint A, Lalande M. 1991. A newclass of reversible inhibitors. Cytometry 12: 26–32.

Inkoshi J, Katagiri M, Arima S. 1999. Neuronal differentiation of neuro2a cells by inhibitors of cell cycle progression trichostatin A andbutyrolactone. Biochem. Biophys. Res. Commun. 256: 372–376.

Klassen CD, Amdur MO, Doull J. 1986. Casarett and Doull’s Toxicol-

ogy: The Basic Science of Poisons. Macmillan Publishing: New York.Lee SE, Mitchell RA, Cheng A, Hendrickson EA. 1997. Evidence for

DNA-PK-dependent and -independent DNA double-strand breakrepair pathways in mammalian cells as a function of the cell cycle.Mol. Cell Biol. 17: 1425–1433.

Lindsay CD, Hambrook JL. 1997. Protection of A549 cells against thetoxic effects of sulphur mustard by hexamethylenetetramine. Hum.

Exp. Toxicol. 16: 106–114.Lindsay CD, Hambrook JL. 1998. Diisopropylglutathione ester protects

A549 cells from the cytotoxic effects of sulphur mustard. Hum. Exp.

Toxicol. 17: 606–612.Lindsay CD, Hambrook JL, Smith CN, Rice P. 1996. Histological as-

sessment of the effects of percutaneous exposure of sulphur mustardin an in vitro human skin system and the therapeutic properties of

protease inhibitors. Symposium paper, In Medical Defense

Biosciences Review, vol. II. US Army Medical Research and Devel-opment Command: Baltimore; 899–908.

Lindsay CD, Hambrook JL, Lailey AF. 1997. Monoisopropylglutathioneester protects A547 cells from the cytotoxic effects of sulphurmustard. Hum. Exp. Toxicol. 16: 636–644.

Marko D, Schatzle S, Friedel A, Genzlinger A, Zankl H, Meijer L,Eisenbrand G. 2001. Inhibition of cyclin-dependent kinase 1 (CDK1)by indirubin derivatives in human tumour cells. Br. J. Cancer 84:283–289.

Maynard RL. 1987. A medical review of chemical warfare agents.

Technical Paper No. 484. CBDE: Porton Down.Miura M, Sasaki T, Takasaki Y. 1996. Characterization of x-ray-

induced immunostaining of proliferating cell nuclear antigen inhuman diploid fibroblasts. Radiat. Res. 145: 75–80.

Morice WG, Brunn GJ, Wiederrecht G, Siekierka JJ, Abraham RT.1993. Rapamycin-induced inhibition of p34cdc2 kinase activation isassociated with G1/S-phase growth arrest in lymphocytes-T. J. Biol.

Chem. 268: 3734–3738.Murnane JC, Byfield JE. 1981. Irreparable DNA cross-links and

mammalian cell lethality with bifunctional alkylating agents. Chem.

Biol. Interact. 38: 75–86.Mutomba MC, Wang CC. 1996. Effects of aphidicolin and hydroxyurea

on the cell cycle and differentiation of trypanosoma brucei bloodstream forms. Parasitology 80: 89–102.

Naghii MR. 2002. Sulfur mustard intoxication, oxidative stress, andantioxidants. Mil. Med. 167: 573–575.

Ormerod MG. 1996. Analysis of DNA — general methods. In Flow

Cytometry: a Practical Approach, Ormerod MG (ed.). IRL Press:Oxford; 19–135.

Rice P, Brown RFR. 1999. The development of lewisite vapour inducedlesions in the domestic white pig. Int. J. Exp. Pathol. 80: 59–67.

Ruegg UT, Burgess GM. 1989. Staurosporine, K-252 and UCN-01:potent nonspecific inhibitors of protein kinases. Trends Pharmacol.

Sci. 10: 218–220.Schutte B, Nieland L, Engeland MV, Henfling MER, Meijer L,

Ramaekers FCS. 1997. The effect of the cyclin-dependent kinaseinhibitor olomoucine on cell cycle kinetics. Exp. Cell. Res. 236: 4–15.

Smith ML, Seo YR. 2002. p53 regulation of DNA excision repairpathways. Mutagenesis 17: 149–156.

Smith WJ, Sanders KM, Gales YA, Gross CL. 1991. Flow cytometricanalysis by vesicating agents in human cells in vitro. J. Toxicol. Cut.

Ocul. Toxicol. 10: 33–42.Smith CN, Lindsay CD, Upshall DG. 1997. Presence of methenamine/

glutathione mixtures reduces the cytotoxic effect of sulphur mustard.Hum. Exp. Toxicol. 16: 247–253.

Smith CN, Lindsay CD, Hambrook JL. 2001. An in vitro comparisonof the cytotoxicity of sulphur mustard in melanoma and keratinocytecell lines. Hum. Exp. Toxicol. 20: 483–490.

Sparfel L, Langouet S, Fautrel A, Salles B, Guillouzo A. 2002. Invest-igations on the effects of oltipraz on the nucleotide excision repair inthe liver. Biochem. Pharm. 63: 745–749.

Swe M, Bay BH, Sit K. 1996. Interphase and M-phase oral KBcarcinoma cells are targeted in staurosporine-induced apoptosis.Cancer Lett. 104: 145–152.

Tomkinson AE, Mackey ZB. 1998. Structure and function of mamma-lian DNA ligases. Mutat. Res. 407: 1–9.

Tuteja N, Tuteja R. 2001. Unraveling DNA repair in humans: molecularmechanisms and consequences of DNA repair defect. Crit. Rev.

Biochem. Mol. Biol. 36: 261–290.Vogt RF, Dannenberg Jr AM, Schofield BH, Hynes NA, Papirmeister

B. 1984. Pathogenesis of skin lesions caused by sulphur mustard.

Fundam. Appl. Toxicol. 4: S71–S83.Wagner ED, Rayburn AL, Anderson D, Plewa MJ. 1998. Analysis of

mutagens with single cell gel electrophoresis, flow cytometry andforward mutation assays in an isolated clone of chinese hamsterovary cells. Environ. Mol. Mutagen. 32: 360–368.

Wang JYJ. 1998. Cellular responses to DNA damage. Curr. Opin. Cell

Biol. 10: 240–247.Wang T. 1996. Cellular DNA Polymerases. In DNA Replication

in Eukaryotic Cells, DePamphilis, LM (ed.). Cold Spring HarbourLaboratory Press: Cold Spring Harbour; 461–493.

Wood RD, Shivjii MKK. 1997. Which DNA polymerases are used forDNA-repair in eukaryotes? Carcinogenesis 18: 605–610.



Xu H, Zhang P, Liu L, Lee MYWT. 2001. A novel PCNA-bindingmotif identified by the panning of a random peptide display library.Biochemistry 40: 4512–4520.

Yanagihara N, Tachikawa E, Izumi F, Yasugawa S, Tamamoto H,Miyamotot E. 1991. Staurosporine-An effective inhibitor for Ca2+/calmodulin-independent protein kinase II. J. Neurochem. 56: 294–298.

Yarbro JW. 1992. Mechanism of action of hydroxyurea. Semin. Oncol.

19: 1–10.Zong ZP, Fujikawa-Yamamoto K, Li AL, Yamaguchi N, Chang YG,

Murakami M, Odashima S, Ishikawa Y. 1999. Both low and highconcentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and CDK2 expression. Cell Struct. Funct. 24:457–463.

Effect of sulphur mustard on human skin cell lines with differential agent sensitivity

Documents

Transcript of Effect of sulphur mustard on human skin cell lines with differential agent sensitivity