JOURNBl OF MillTBRV PHBRMBCO-MCDICINC N°7-2ai4
INVESTIGATION OF NOVEL GENE REGULATION CAUSED BY LOW DOSE EXPOSURE OF BENZO[A]PYRENE IN JURKAT CELLS USING SUPPRESSIVE SUBTRACTIVE HYBRIDIZATION
Nguyen Minh Phuong'; William K Chan"; Pham Thi Thanh Huong' SUMMARY
Aims of this study are to investigate the differential gene expression caused by a carcinogen Our hypothesis Is that by employing suppressive subtractlve hybridization PCR (SSH PCR), we may Identify hovel rare up- and down-regulated transcripts that are missed in conventional mlcroarray and proteomic analyses We exposed a non-cytotoxic dose of benzofajpyrene (BaP) to AhR-deflclent Jurkat cells and investigated the cellular response In genome-wide gene expression. After treating the cells with 2.5 pM BaP for 48h, we Isolated mRNA for SSH PCR analysis. We screened approximately 15,000 clones to obtain a final of 41 positive clones (20 up- and 21 down-regulated clones). The use of this approach to Identify the regulated transcnpts which was validated by real-time qPCR. Among these 41 transcripts, we observed that 27 of them are related to cell growth, DNA repair and gene regulation; 3 of them (TCF7, D0CK2, and ZAP70) are related to immune response. Two of the down-regulated transcnpts BCL2 and TCF7 have been reported to be also down-regulated by cigarette smoke and the rest are novel BaP-regutated transcripts. Our data suggested that Jurkat cells are sensitive to DNA damage at a non-cytotoxlc dose of BaP because of Jurkat cells down-regulate genes (SART1.
SNRK, HIRA, and BCL2) that promote cell growth and up-regulate DNA repair related genes (GTF2H2 and RNASEH1). In addition. Immune response Is suppressed even though no apparent change in morphology has been observed when comparing the BaP-treated cells with the untreated cells.
' Key words: Benzo[a]pyrene: Gene regulation: Suppressive subtractlve hybridization;
Jurkat cells.
INTRODUCTION individuals are exposed to a low level of BaP over an extended period of time.
Benzo[a]pyrene (BaP) is a polycydic The food chain is considered the dominant aromatic hydrocarbon (PAH) that is formed pathway for routine human exposure through incomplete combustion of organic and accounts for about 97% of the total materials and is found in air, tobacco daily intake of BaP [3], Inhalation and smoke, automobile exhaust, and food [1]. consumption of contaminated water are Depending on the sources, BaP may enter considered minor pathways of BaP exposure our body by inhalation, dermal absorption, for the general population, except for or ingestion [2], Human exposure to BaP tobacco users or workers in the coal may be have an acute event; but generally, industry [4].
* Vietnam Military Medical University
" University of the Pacific, Stockton, California, USA
Corresponding author: Nguyen Minh Phuong ([email protected]) 32
JOURNBl OF MIUTRRV PHRRMRCO-MCDICINC N ° 7 - 2 a i 4 It is widely accepted that BaP is
carcinogenic, mutagenic, cytotoxic, immunotoxic, and teratogenic in various species and tissues [4], Similar to many PAHs, BaP is an aryl hydrocarbon receptor (AhR) agonist. Several studies have demonstrated that the etiology of toxicology and cancer caused by BaP is mainly due to the fact that upon binding of BaP to AhR, numerous enzymes that are involved in activation and detoxification of BaP are induced [5). The BaP-bound AhR translocates into the nucleus where it dimerizes wiith Amt.
The AhR/Arnt complex binds to the ORE, resulting in the activation of gene transchptbn [6], ORE is present in promoters of proto- oncogenes, cytokine genes and genes encoding PAH-metabolizing enzymes (e.g.
CYP1 Al) [7]. Many studies have demonstrated that CYP1A1 is involved in the metabolic activation of BaP into a reactive intermediate which is capable of binding to the DNA and proteins. Hundreds of reports have shown that the reactive intermediates, rather than the parent compound, are responsible for the BaP-mediated toxicity, cancer, and mutations [8], However, it has been reported that another AhR ligand TCDD causes apoptosis in T cell lines in the absence of AhR [9] and the mechanism is largely unknown. To examine whether BaP affects the cellular immune response and other responses that are not AhR- dependent, we used suppressive subtractlve hybridization PCR (SSH PCR) to investigate genes that are regulated by BaP in Jurkat cells.
SSH PCR is a powerful and yet simplistic technique to identify genes that are either up-or down-regulated [10]. This PCR-based method selectively suppresses ttie amplification
of unregulated sequences and equalizes for the abundance of cDNAs within a target population by the incorporation of a normalization step [11]. Thus, this approach should enhance the probability of identifying regulation of rare (low abundance) transcripts. In this study, we exposed Jurkat cells to a non-cytotoxic dose of BaP for 48 hours and examined the cellular response with regard to the gene regulation of the rare transcripts.
MATERIAL AND METHODS 1. Reagents.
BaP and cell culture media were purchased from Sigma. Other cell culture reagents were purchased from Invitrogen.
Fetal calf serum was purchased from Tissue Culture Biologjcals. Jurkat cells were grown in RPMI-1640 supplemented with 10% fetal calf serum, 10 U/ml of ampicillin, and 10 pg/ml of streptomycin.
The cell popufation was maintained at densities between 1 x 1 0 ^ and 1 x 1 0 ^ cells/ml. MCF~7 cells was grown in DMEM supplemented with 10% fetal calf serum, 10 U/ml of ampicillin, and 10 pg/ml of streptomycin. Ail cells were maintained at 37°C and 5% CO2.
2. BaP exposure to Jurkat cells.
Jurkat cells were prepared in a 75 cm^
flask containing 40 ml of complete media at a concentration of 2 x 10* cells/ml. On the following day, when the cell density reached at 4 X 10* ceils/ml, BaP in DMSO was added to the cells to a final concentration of 2.5 pM. Control cells were treated with the vehicle DMSO. After 48h at 37°C, cells were han/ested to initiate SSH PCR.
JOURNRl OF MIUTRRV PHBRMBCO-MCDICINC N°7-2014 3. SSH and cDNA library construction.
Total RNA was extracted from the vehicle control or the BaP-treated cells using the TRI reagent and then poly(A)RNA was isolated from the total RNA using the Oligotex mRNA kits (Qiagen, Valencia, CA).
Synthesis of cDNA and SSH PCR (scheme 1) were performed using the PCR-Select cDNA subtraction kit. 8 microgram of poly(A)RNA from the control or the BaP- treated cells was used for the first strand cDNA synthesis. The whole reverse transcriptase reaction was subjected to the second strand synthesis. 10 pg of the cDNA library was used to start the
SSH PCR method, according to the manufacturer's recommendation. For the forward subtraction to identify the up- regulated genes, cDI^A from the control cells was used as the driver whereas cDNA from the BaP-treated ceils was used as the tester. The opposite would apply for the reverse subtraction to identify the down-regulated genes. The PCR products obtained from the forward and reverse subtractions were cloned into the pGEM-TA plasmid. The ligated products were transformed into XL10- Gold ultracompetent cells for blue/white screening.
Dnv8t(i
Linear amplification No ampimcatjon } EXfwnentlal amplltlcatloiri
Scheme 1: Flow chart showing how regulated genes are determined by SSH PCR. Bold lines/an-ows represent the Rsal digested tester or driver. Clear boxes represent the outer part of adapter 1 and adapter 2 where PCR primer 1 is located. Solid boxes represent the inner part of adaptor 1 where nested PCR primer 1 is located. Shaded boxes represent the inner part of adaptor 2 where nested PCR primer 2 is located. Two sequential PCR reactions were perfonned to amplify the regulated transcripts: first with PCR primer 1 and then with nested primers 1 and 2. ds, double stranded; ss, single stranded.
JOURNAL OF MIUTRRV PHRRMRCO-MCDICiNC N°7-2014 4. Secondaiy screen using DNA dot
blot hybridization.
Each of the pGEM cloned plasmids was extracted from 3 ml overnight bacterial culture of a single white colony. To an overnight culture, the routine miniprep solutions I - III were used to release the plasmid into the solution.
After centrifugation at 16,000 g for 30 min at 4°C, the supematant was directly used for dot blot hybridization. Two microliter of the supernatant was spotted onto 82 mm GE nylon membrane. DNA dot blot hybridization was performed using BrightStar psoralen- biotin labeled subtracted cDNA probe generated from cDNA of either the fora/ard orthe reverse subtraction. The blot membranes were pre-hybridized at 42°C for 30 min in the ultrasensitive hybridization buffer and then the denatured probe was added to the buffer, followed by hybridization at 42°C overnight. After hybridization, the membranes were washed twice in 2X SSC with 0.1X SDS at 42°C for 5 min, twice in 0.1X SSC with 0.1X SDS at 42°C for 15 min. After the final wash, the membrane was blocked with 5% BSA in IX TBST for I h and then incubated for an additional I h in 1X TBST containing streptavidin-peroxidase and 5% BSA.
Aftenflrards, the membrane was washed six times with IX TBST for 5 min. The signal was visualized using Pierce SuperSignal West Pico chemlluminescent substrate.
The signal intensities were analyzed using the Un-Scan-lt software. Clones with a five-fold difference in signal intensity between the forward and the reverse probe hybridization were sequenced and identified using the BLAST database.
5. Western blot analysis.
Whole cell extracts (WCE) from BaP or DMSO-treated Jurkat cells were used to
determine whether Jurkat cells express AhR. 75 cm^ f ask, 300 pi of lysis buffer (25 mM HEPES, pH 7.4, 0 4 M KCI, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1 mM PMSF, and 2 pg/ml of leupeptin) were used to harvest the cells After 3 cycles of freeze/thaw, lysates were kept on ice for 30 min and then centrifuged at 14,000g at 4°C for 10 mm. The supematants were WCE.
The transferred nitrocellulose membrane was blocked with 5% nonfat dry milk (Bio- Rad) in 1XTBST. Polyclonal against AhR (H-211) (Santa Cruz Biotechnology, Santa Cruz, CA) was used (1:100) in an overnight incubation containing 5% milk and 1XTBST to detect AhR expression Anti-rabbit IgG-HRP (Sigma) was used (1.10,000) as the secondary antibody. AhR was visualized using Pierce SuperSignal West Pico chemlluminescent substrate (Thermo Scientific, Rockford, IL).
6. Statistical analysis.
We perfomied unpaired one-tailed t.test to detemiine the statistical significance using Prism 5 software.
RESULTS
1. AhR expression and CYP1A1 activity in BaP- and DMSO-treated Jurkat cells.
40ua 601X1 20ua 40un 60ua 20|jq BaP-treated Juikat DMSO-treated Jurkat MCF-7
Fig. 1: Western blot analysis showing that AhR is not detected in BaP- and DMSO- treated Jurkat cells. Jurkat cells were treated with 2.5 pM BaP or DMSO for 48h.
Different amounts of WCE from BaP- or DMSO-treated Jurkat cells (20 pg, 40 pg, and 60 pg) and MCF-7 cells (20 pg) were used to detect AhR protein using AhR- specific (H-211) antibody.
JOURNAL OF MIUTRRV PHflRMIICO-MCDICINC N ° 7 - 2 0 1 4 To determine whether our Jurkat cells
expressed AhR, WCE fronn BaP-or DMSO- treated Jurkat cells were prepared for Western blot analysis. WCE from MCF-7 cells was used as a positive control. No detectable AhR protein was observed in
both BaP- and DMSO-treated Jurkat cells up to 60 |jg of protein whereas 20 pg of MCF-7 WCE showed a strong signal for the AhR protein (Fig. 1), indicating that there was minimal, if any, AhR in Jurkat cells.
2. Determination of the up- and down-regulated transcripts in Jurl<at cells after BaP exposure.
BaP- treated
cDNA DMSO- treated cDNA subtraction
bp
_ Control
500 — 300 —
1 2 3 4 5
Fig. 2: Agarose gel analysis of PCR products obtained from SSH PCR. The PCR products using different templates as follows: (1) cDNA from forward subtraction;
(2) unsubtracted BaP-treated cDNA: (3) cDNA from reverse subtraction;
(4) unsubtracted DMSO-treated cDNA; (5) unsubtracted control cDNA.
The PCR products obtained from the forward and the reverse subtractions were cloned into the pGEM-TA plasmid.
Expectedly, we observed less cDNA in the subtracted samples when compared with the con-esponding unsubtracted samples (Flg.2), indicating that the subtraction process was effective in eliminating the unregulated transcripts.
From a total of about 15,000 colonies, we selected 150 and 200 white colonies from the fora/ard and the reverse subtraction, respectively, which corresponded to 150 up-and 200 down-regulated genes after BaP
treatment. In an effort to minimize false positive clones, DNA dot blot hybridization was performed using biotinylated probes synthesized from the subtracted cDNA to screen all clones from the fora/ard and the reverse subtractions (Fig. SjTOnly the clones that had at least a five-fold difference in intensity behveen the fonward and the reverse probes were selected to proceed with further characterization. Forty-one clones ranging from 350 to 1,100 base pairs were then selected, identified, and characterized (tables 1 and 2 and Flg.4).
36
JOURNfll OF MILITARV PHRRMRCO-MCDICINC N°}-2014
Forward probe
Reverse Subtraction clones (1-50)
^ :
Fig.3: DNA dot blot hybridization screening of clones obtained after fonward and reverse cDNA subtractions from Jurkat cells treated with BaP or DMSO (vehicle). Each membrane contained 50 clones. They were hybridized with a BrightStar psoralen-biotin labeled subtracted cDNA probe from either fonward or reverse subtractions (forward and reverse probes). Fonward probe contains cDNA of up-regulated transcripts whereas reverse probe contains cDNA of down-regulated transcripts Arrows indicate the selected clones representing either the up- or down regulated transcripts that are at least 5-fold difference in intensity between the up- and down-regulation cDNA populations.
b p f 5 F13 F15 F19 F23 F26F2BF30 F34 F35 b p ^ F 1 F 9 Faa F3G F10BF115 F125 Ft29 F-13B 8 0 0 - _ 1100-™.
P14 R30 R46 R5SH63 R6fl R70Rr2 R85R101 R1H ( , • * * ' " ^''^^ R14B P14'i R151 R162 R163 R173 R133 R18S
5 0 0 - J ^
Fig.4: Agarose gel showing the PCR products using cloned pGEM-T plasmids as templates. Sp6 and T7 primers are used to amplify the selected positive clones. The size of the positive clones ranges from 350 to 1,100 bp. The 20 up-regulated (forward subtraction) clones are prefixed with T " (above) whereas the 21 down-regulated (reverse subtraction) clones are prefixed with "R" (below).
JOURNAL OF MIUTHRV PHRRMnCO-MCDICINC N'>7-2014
Table 1: Up-regulated transcripts by BaP in Jurkat cells obtained from fonward subtraction (tester is BaP-treated cDNA whereas driver is DMSO-treated cDNA).
Cfltegartea RfflBlated g e B o RcgnlaCton/Effe*! g r i d
cetlgrourth cellgniuiih
DNArepmi gene ragulacion gene i^ulMion sencr>B|!iilation
rSIOMAl}
^mall nibtm^ ^HOcemiDiiie CGiofKiDeiil (UTP IS) GTPose activalioa [irol^a-bindii^ pnXciu 2 Ki3BF2) sfalidKie3CNE(i3)
protcm ^roa-iBia ])liras[)kiiiiti« 4A2 (PTP4A2>
gi!»enl traas(^i|itian haai iSHl (C)TF2H2) EaxMUicleBS« III (RNas>:H I)
<ly«ket^Hci£ts ccmgcnka t (DKCI) ca»SOinal gKOlem L7 (OPLT) nbosomal protein S11 <SPS 11)
jumllnuetesrifliDEtticltiopmieltiDI ( S N B F O I } ,' r!itibunilt{PSMEl) tMomodOfiinln conraninig 3 (BRD3;
ziBC finger{KOKin I 0 7 ( Z N F I « 7 ) ELOVL &fflityinert^iei-5 f E L O V U ) p^rophospfa^ate 2 (F'PA2) dnomoflMtK I of 151 ( C l o t f l S i )
» ^ 7 0 A S { H S P A 8 >
$eac n^nlaf ion piotEotMrnc at.-tivaiaf !it
apfSA u p / N A 1^1/t !(«"*•*
i q t / T g r o w A u p / r gtt>w*
u p / j repair t i p / f n^Mir
•V up up T up up Up up up u p up up
2 6 - U ie.ti 16.94 5-37 5.06 8.74 5-98 3 * « 2635 19.51 IZ-JS UJl 10 59 S.I5 23.1 5.36 9 3 4 7.02 12.07
J a i 147157 N M 1600!
NM 203315 NM 006656 KM 0S0391 USA 001515 NM_002Si36 NM 001363 NM 000971 NM_00IO(5 NM_006938 N M _ 0 0 6 a 3 NM 007371 N M 0(HfllJ746
N M 021814 }^£J16»S9 NM_001032363
N M _ 0 0 2 ^ 4
!»M_«J6537
Table 2: Down-regulated transcripts by BaP in Juri^at cells obtained from reverse subtraction (tester is DMSO-treated cDNA and driver is BaP-treated cDNA)
mtnnftocid ITBlMpOlt ccllfrowth
cellgiDirlli L-eHgnnvih Ctllgtowtb
cictljjfowU)
RiegitlMWlgciiCT Bqt»l«aBw!Hft« F B M Nnmhw
gene riigubtioD gme icgulalkm
^ene teptUdoa NA S!A
MliHo canto- fiimily 7, nwrnbet I <CAT5 MjuanvHQ iKil ajromasaaBQiigeii tSASTl) SNF relMeil kisate (HfllK)
betone ceUc^^-lc r c E ^ i i o n d e f ^ l v e hoflmlog A (HERA) B-celi rLL/Eympfa>niia2 ( B C L ^
WD Kpeai douuiiii 6 CWDRfi) a ^ a i C ffl«f golgi 3 !f£Rl3tC3>
ting fio^ j m u u i ) « t R N F t 4 « ) ling finger |m»ein 139|1ENF13e}
Uwwcripioo &CIM 7 <TCF7) dedfcBiQTJircyuiknKsiES (DOCK2>
juta-cbam ^sjclotod ptnein ijaase 70 (ZAP70) XPA b m d ^ praieiD 2 (XAIH)
etdaiTyoEicirBnjla£on.dl)i^tH>nEii;u)r I dduCEKFtD)
^Kcitig &dior am/ser 4 (SPlt54) kelcb-like ECtl-awoeuiud pmtUB I <)CGAPt>
clitoo»os<w«elOarf4(C10or«) iiypodieiicalpnJWiiHl.OC100i29034) Bardet-Biedl liyncbonH- 9 (BBS9) sQsiu domain comaiaiiia 4 (Sli^D4)
down / 1 ttanspan d o w n / l ^ o w t h dowiVlgrowtli dcnwn* i grontli davaf X etmrth dOKii/1 gtowOi d o w n / N A d o u n / K A d>^TO/^fA iloWn/NA dowD/iiespontE tliMi'n/|ceapDn«e d o w n / J response
ik>»n down down
<to*ti down down
<to»n dovwi
59.66
> « t
>6Ii UJ 10.05 7.42
>68 5 1 J 3 7-43 S.38 26.R3 5.55 5.1»
21.68
<;7 57 65.1 7A9 20.91 9.56 11.13 20.15
NM 003045 NM O0SI46 fiM 00(100594
NM 003325 NM 000657 NM OlSCBI NM 01S96S
!<JM^»0963 NM 01B4J4 NM 199250 NM 003202 NM 00494*
N'M 207519 NM 020196 NM 001968 NM O0S626 NM D I 2 2 » NM 145246 XM 001726040 NM OOI0336ffi5 NMJtM&a 38
JOURNfll OF MIUTflRV PHRRMflCO.mCDICINE N°7.20)4 DISCUSSION
BaP is a well known AhR Ilgand which activates the AhR signaling pathway. The BaP-hound receptor modulates the expression of target genes at the transcriptional level, which should explain in part the BaP-mediated toxicities [6], In addition, the upregulation of the AhR target enzyme CYP1A1 is responsible for the bioactivation of BaP, leading to DNA damage [5], The AhR is therefore an important component of the BaP-induced gene expression and the loss of AhR has been shown to inhibit BaP-induced carcinogenicity in mice [12], However, AhR-independent promotion of apoptosis has been reported for the AhR ligand TCDD in L-MAT cells [13]. In addition, TCDD-mediated immunotoxicity appears to be AhR-independent in Jurkat and L-MAT cells [9], With regard to BaP, it causes DNA damage in Jurkat cells which do not have AhR; this BaP-mediated DNA damage would likely change the gene expression pattern in these cells. We are interested in the cellular response to a non-cytotoxic dose of BaP in AhR deficient Jurkat cells. We performed Western blot analysis on our Jurkat cells and found that there was no detectable amount of AhR in them. This finding allowed us to use our Jurkat cells to Investigate how AhR- independent gene regulation is affected by BaP. We have used low dose of BaP for study because we are interested in human response to BaP exposure in environment which Is often a low level exposure. Other investigators had shown
that 2.5 pIM BaP was a non-cytotoxic dose for Juri<at cells up to 48h exposure [14].
We also found that Jurkat ceils that were treated with 2.5 pM BaP for 48h appeared normal and showed no sign of cell death.
We investigated genome-wide changes in gene expression in BaP-treated Juri<at cells using SSH PCR. One of the intrinsic characteristics of SSH PCR is that this method normalizes the abundant and the rare transcripts through two hybridization steps, so that the rare transcripts are preferentially enriched during the process [10]. Therefore, we expected to identify novel transcripts that had not been reported for BaP treatment. We screened approximately 7,500 clones for each of the up-and down-reguiated populations by performing blue/white screening and DNA dot blot hybridization to minimize false positive clones. Arbitrarily we selected clones with at least a 5-fold difference in our dot blot screening to be considered positive. Other groups had used the same cutoff for their SSH screenings [15]. The 41 identified genes were categorized according to their function-cell growth, DNA repair, gene regulation, immune response, stress response, metatrolism, amino acid transport, and protein interaction. It appears that in response to BaP exposure, Jurkat cells alter the machinery for transcription, RNA processing, translation, and protein degradation to suppress cell proliferation and to promote DNA repair. Specifically, 11 regulated transcripts are involved in transcription, RNA splicing, translation, and protein degradation processes, in which 7 39
JOURNRl OF MIUTRRV PHARMRCO-MCDICINC N ° 7 - 2 0 1 4 transcripts (DKC1, RPL7, RPS11, SNRPD1, PSME1, BRD3, and ZNF107) are up- regulated and 4 transcripts (XAB2, EEF1D, SFRS4, and KEAP1) are down-regulated.
Four of them had a direct effect on nbosome assembly and protein synthesis (RPL7, SNRPD1, RPS11, and EEF1D), suggesting that modulation of active protein synthesis is a response to low dose exposure of BaP. Genes involving in cell growth, DNA repair, and gene regulation contribute to 66% (27 out of 41 transcripts) of the identified transcripts. Among them, there are 14 up-regulated genes (SIGMA1, PTP4A2, G3BP2, UTP18, NEU3, GTF2H2, RNASEH1, DKC1, RPL7, RPS11, SNRPD1, PSME1, BRD3, and ZNF107) and 13 down- regulated genes (HIRA, SART1, WDR6, BCL2, ERGIC3, RNF130, SNRK, RNF146, C19orf48, XAB2, EEF1D, SFRS4, and KEAP1). Although there are similar number of transcripts to either promote or suppress growrth, the 2 transcripts SART1 and SNRK which have the highest fold increase (> 68-fold) suppress growth. In addition, two DNA repair-implicated transcripts GTP2H2 and RNASEH1 are up-regulated, suggesting that a low BaP dose of triggers DNA repair and slows down cell grovrth in Jurkat cells.
Down-regulation of the histone regulator HIRA in our BaP-treated cells suppresses cell growth in part by suppressing the bci2 gene transcription. We observed that the bcl2 transcript is down-regulated by BaP and this decrease has been reported in cells that have been exposed to cigarette smoke and TCDD [9]. In addition, down-
regulation of BCL2 promotes apoptosis in lymphocyte whereas Jurkat cells stably transfected with BCL2 are resistant to apoptosis.
Down-regulation of the KEAP1 transcript is interesting. KEAP1 promotes the degradation of Nrf2 which is i key transcription factor for the up-regulatlon of phase II enzymes upon oxidative stress Upon exposure to reactive oxygen species, modification of KEAP1 occurs which acts as a switch to allow Nrf2 protein accumulation to occur. Interestingly, low dose exposure of BaP down-regulates KEAP1, which may trigger a general cellular defense against chemical insult by increasing the Nrf2- mediated production of redox enzymes.
Regarding the effect of BaP on the immune response, 3 genes (ZAP70, TCF7, and D0CK2) are found to be down- regulated. Both ZAP70 and D0CK2 are known as players in TCR-mediated T-cell activation. ZAP70 is a protein tyrosine kinase which plays an important role in T cell development and lymphocyte activation.
This enzyme, which is phosphoryiated on tyrosine residues upon TCR stimulation, functions in the initial step of TCR-mediated signal transduction in combination with the Src family kinases Lok and Fyn. The D0CK2 gene encodes a hematopoietic cell-specific CDM family protein that is indispensable for lymphocyte chemotaxis.
DOCK2 mediates TCR-dependent activation of Rac2, leading to the regulation of IL-2 promoter activity in T cells. Both ZAP70 and DOCK2 are down-regulated to a small extent (5-fold) in our screening, suggesting that they are rare transcripts
40
JOURNDL OF MIUTflRV PHRRMflCO-MCDICINC H''^•20^^
that are preferentially enrictied by SSH PCR mettiod. These 2 genes miglit be essential in the eariy T cell response to low dose exposure of BaP. In addition, TCF7 is also down-regulated in our screening by more than 26-fold. TCF7 is an HMG box transcription factor which plays a role in WNT signaling pathway in T cell development. Expression of TCF7 is abundant in CD34+ hematopoietic cells, recomended that this expression may contribute to hematopoiesis. It would be interesting to explore how down-regulation of TCF7 plays a role in BaP-mediated immunotoxicity
A low dose of BaP appears to elicit stress response in Jurkat cells. Among the 3 stress-related transcripts that are up-regulated, HSPA8 has the highest fold increase. This heat shock protein has been reported to be elevated in response to stress. The observed increase of the HSPA8 transcript upon BaP treatment Indicated that Jurkat cells are sensitive to a 2 5 |JM concentration of BaP and consider it a stress condition. It is noteworthy that proteomic analysis of Jurkat cells treated with the same dose of BaP did not show any change of heat shock protein expression, reinforcing the added benefit of using SSH to reveal regulated transcripts that are missed by proteomic screening.
CONCLUSIONS
We have reported novel transcripts in Jurkat cells that are regulated by a non- cytotoxic dose of BaP using SSH which preferentially enriched rare transcripts.
Thirty-nine of the 41 regulated clones reported here have not been identified as BaP or cigarette smoke regulated transcnpts.
Collectively, our data reveals how Jurkat cells respond to a non-cytotoxic dose of BaP by altering gene expression independent of the AhR action.
ABBREVIATIONS AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; BaP, benzo[a]pyrene;
CYP, cytochrome P450; DRE, dioxin response element; PAH, polycydic aromatic hydrocarbon, SSH, suppressive subtractlve hybridization; TCDD, 2,3,7,8- tetrachlorodibenzo-p-dioxin.
REFERENCES
1. DH Phillips. Polycydic aromatic hydrocarbons in diet. Mutation research, 1999, 443. (1-2).
pp.139-147.
2. EM Faustman, GS Omenn. Risk assessment, In- C D Klaassen (Ed ). Casaret and DouH's toxicology: tlie basic science of poisons, IVlcGraw-Hill, New York, 2008, pp.107-128,
3 HA Hattemer-Frey, CCTravis. Benzo-a- pyrene: Environmental partitioning and human exposure, Toxicol, ind. Health. 1991, 7 (3), pp 141-157.
4 KP Miller, KS Ramos. Impact of cellular metabolism on the biological effects of benzo(a}pyrene and related hydrocarbons, Drug metabolism reviews. 2001, 33 (1), pp.1-35.
5. T Shimada, A Sugie, T Yamada, H Kavmzoe, M Hashimoto, E Azuma, T Nakajima, K Inoue, Y Oda Dose-response studies on the induction of liver cytochromes P450 1A1 and 1B1 by
JOURNAL Of MILITflRV PHflRMflCO-MCDICINC N°7-2014 polycydic aromatic hydrocarbons in arylhydrocarbon-respnsive C57BL/6J mice, Xenobiotica. 2003, 33 (9), pp.957-971.
6. DWNebert. A Puga, V Vasiliou. Role of the Ah receptor and the dioxin-inducible [Ah]
gene battery in toxicity, cancer, and signal transduction, Ann N Y Acad Sci. 1993, 685 (Jun 23), pp.624-640,
7 ZWLai, T Pineau, C Esser Identification of dioxin-responsive elements (DREs) in the 5' regions of putative dioxin-inducible genes Chemico-Biological Interaction 1996, 100 (2), pp.97-112.
ft AH Conney. RL Chang. DMJerina, SJ Wei.
Studies on the Metabolism of Benzo[a]pyrene and dose-dependent differences in the mutagenic profile of Its ultimate carcinogenic metabolite.
Drug Metabolism Reviews. 1994, 26 (1) pp.125-163,
9. A Hossain, S Tsuchiya. M Minegishi. k Osada, S Ikawa, FA Tezuka, M Kaji. T Konrio, M Watanabe. H Kikuchi. The Ah receptor is not involved in 2,3,7,8-tetrachlorodibenzo-p- dioxin-medlated apoptosis in human leukemic T cell lines. The Joumal of Biological Chemistfy 1998, 273 (31), pp, 19853-19858.
10. L Diatchenko. YF Lau, AP Campbell.
A Chenchik, F Moqadam. B Huang, S Lukyano/, K Lukyanov, N Gurskaya, ED Sverdlov, PB Siebert. Suppression subtractlve hybridizatior, a method for generating differentially regulated oi tissue-specific cDNA probes and libraries, Proceeding of National Academy of Sciences USA. 1996, 93 (12), pp 6025-6030.
