cyb5d2 enhances hela cells survival of etoposide-induced cytotoxicity

CYB5D2 enhances HeLa cells survival ofetoposide-induced cytotoxicity

Yanyun Xie, Anthony Bruce, Lizhi He, Fengxiang Wei, Lijian Tao, and Damu Tang

Abstract: Cytochrome b5 domain containing 2 (CYB5D2) (neuferricin) belongs to the family of membrane-associated pro-gesterone receptors (MAPRs). MAPRs affect multiple cellular processes, including proliferation, differentiation, and survival.Consistent with these observations, we report here that CYB5D2 enhances HeLa cells survival of etoposide (ETOP)-mediatedcytotoxicity. Overexpression of CYB5D2 enhanced the survival of HeLa cells compared with HeLa cells transfected withempty vector (EV) upon ETOP treatment. As ETOP initiates ATM-dependent DNA damage response (DDR), we were ableto show that CYB5D2 did not affect ETOP-induced DDR. In line with these observations, CYB5D2 did not protect HeLacells from UV-induced cytotoxicity. Additionally, CYB5D2 had no effects on TNFa-induced apoptosis. Collectively,CYB5D2 enhances HeLa cell survival of ETOP-induced cytotoxicity with some specificity. CYB5D2 contains a cytochromeb5 (cyt-b5) domain and a transmembrane (TM) motif. Both domains are required for CYB5D2-mediated protection of HeLacells from ETOP-induced cytotoxicity. In an effort to search for the underlying mechanisms, we have profiled gene expressionbetween HeLa–CYB5D2 and HeLa–EV cells. Although ectopic CYB5D2 does not massively alter gene expression, the ex-pression of several transcripts was affected more than 2-fold, suggesting that they may contribute to CYB5D2-mediated HeLacell survival of ETOP treatment.

Key words: CYB5D2/neuferricin, cytotoxicity, apoptosis, cell cycle and DNA damage response.

Résumé : La neuferricine (CYB5D2, acronyme de Cytochrome b5 domain containing 2) appartient à la famille des récep-teurs de la progestérone associés à la membrane (MAPR). Les MAPR affectent plusieurs processus cellulaires, notammentla prolifération, la différenciation et la survie. De manière cohérente avec ces observations, nous rapportons ici queCYB5D2 augmente la survie des cellules HeLa soumises aux effets cytotoxiques de l’étoposide (ETOP). La surexpressionde CYB5D2 augmentait la survie des cellules HeLa comparativement aux cellules HeLa transfectées avec un vecteur vide(VV) à la suite du traitement à l’ETOP. Puisque le l’ETOP initie une réponse aux dommages à l’ADN dépendante del’ATM, nous avons pu démontrer que CYB5D2 n’affectait pas cette réponse induite par l’ETOP. Dans la foulée de ces ob-servations, CYB5D2 ne protégeait pas les cellules HeLa de la cytotoxicité induite par les UV. De plus, CYB5D2 n’avait pasd’effet sur l’apoptose induite par le TNF-a. En somme, CYB5D2 augmente la survie des cellules HeLa traitées à l’ETOPavec une certaine spécificité. CYB5D2 contient un domaine cytochrome b5 (cyt-b5) et un motif transmembranaire. Lesdeux domaines sont requis à la protection conférée par CYB5D2 vis-à-vis la cytotoxicité induite par l’ETOP chez les cellu-les HeLa. Afin de découvrir les mécanismes sous-jacents, nous avons dressé un profil de l’expression génique chez les cel-lules HeLa-CYB5D2 et HeLa-VV. Même si CYB5D2 ne modifie pas massivement l’expression génique, les niveauxd’expression de plusieurs transcrits étaient affectés de plus de deux fois, ce qui suggère que ces derniers peuvent contribuerà la survie des cellules HeLa conférée par CYB5D2 à la suite d’un traitement à l’ETOP.

Mots‐clés : CYB5D2/neuferricine, cytotoxicité, apoptose, cycle cellulaire, réponse aux dommages à l’ADN.

[Traduit par la Rédaction]

IntroductionNongenomic actions of progesterone are mediated by the

membrane progesterone receptors (mPRs), which include the

7 transmembrane PRs and the single transmembrane PRs(Bramley 2003; Brinton et al. 2008; Fernandes et al. 2008;Thomas 2008). The latter belongs to the family of membrane

Received 12 November 2010. Revision received 2 February 2011. Accepted 17 February 2011. Published atwww.nrcresearchpress.com/bcb on 2011.

Y. Xie, A. Bruce, L. He, and D. Tang. Division of Nephrology, Department of Medicine, McMaster University, St. Joseph’s Hospital, 50Charlton Avenue East, Hamilton, ON L8N 4A6, Canada; Division of Urology, Department of Surgery, McMaster University, St. Joseph'sHospital, 50 Charlton Avenue East, Hamilton, ON L8N 4A6, Canada; Father Sean O’Sullivan Research Institute, McMaster University, St.Joseph’s Hospital, 50 Charlton Avenue East, Hamilton, ON L8N 4A6, Canada.; Hamilton Center for Kidney Research, St. Joseph’sHospital, 50 Charlton Avenue East, Hamilton, ON L8N 4A6, Canada.F. Wei. Department of Cell Biology, Guangdong Pharmaceutical University, 280 Waihuan East Road, Guangzhou Higher Education MegaCenter, Guangzhou, Guangdong 510006, P.R. China.L. Tao. Division of Nephrology, Department of Medicine, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha,Hunan 410008, P.R. China.

Corresponding author: Damu Tang (e-mail: [email protected]).

341

Biochem. Cell Biol. 89: 341–350 (2011) doi:10.1139/O11-004 Published by NRC Research Press

6 June Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

associated progesterone receptors (MAPRs). The conservedstructural features of MAPRs consist of an N-terminal singletransmembrane domain and a C-terminal cytochrome b5 (cyt-b5) domain (Thomas 2008), which binds to heme (Rohe etal. 2009). The MAPR family includes progesterone receptormembrane component 1 (PGRMC1), Neudesin, cytochromeb5 domain containing 2 (CYB5D2), and others (Cahill 2007).While research activity related to MAPR is increasing, func-tions of MAPRs remain unclear.The most intensively investigated MAPR is PGRMC1, with

62 publications listed in PubMed by September 2010. Besidescontributing to progesterone signalling, PGRMC1 also acti-vates Akt (Hand and Craven 2003), increases membrane-bound epidermal growth factor receptor (EGFR) (Ahmed etal. 2010a), potentially enhances cholesterol biosynthesis byactivating lanosterol demethylase (the P450 protein CYP51)(DeBose-Boyd 2007; Hughes et al. 2007; Rohe et al. 2009),promotes the tumorigenesis of breast and other cancers (Cra-ven 2008; Neubauer et al. 2008, 2009; Rohe et al. 2009;Ahmed et al. 2010b), and protects cancerous cells from DNAdamage-associated cytotoxicity induced by the inhibitors oftopoisomerase II (doxorubicin) (Crudden et al. 2006) and top-oisomerase I (cisplatin) (Peluso et al. 2008, 2009).As a member of the MAPR family, CYB5D2 (also known

as neuferricin) shares a similar structural topology as PGRMC1with an N-terminal transmembrane domain and a conservedcyt-b5 domain (Kimura et al. 2010). Consistent with thesestructural similarities with PGRMC1, we report here thatCYB5D2 also offers certain protections from etoposide (a wellknown topoisomerase II poison)-induced cytotoxicity. By com-parison with empty vector-transfected HeLa cells, ectopicCYB5D2-expressing HeLa cells survive significantly better fol-lowing etoposide treatment. This protection requires the pres-ence of the transmembrane and cyt-b5 domains.

Materials and methods

Cell lines and plasmidsHeLa cells were purchased from the American Type Culture

Collection (ATCC) and cultured in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal bovine serum.CYB5D2 was amplified by PCR from a normal human

prostate cDNA library (Clontech, Palo Alto, Calif.) andsubcloned into pCDNA3/N-2xHA (2 HA tags) andpLHCX. The transmembrane deletion mutant of CYB5D2(CYB5D2-DTM) was produced by PCR using primers(5′-CCCAAGCTTATGGGTCCCCGCGCTGGCT-3′, and5′-GCTCTAGATTAGAGTGGAAAGGAGCATGTGATGG-3′).The following PCR conditions were used: 5 cycles at 94 °Cfor 45 s, 61 °C for 45 s, and 72 °C for 2 min, followed by30 cycles at 94 °C for 45 s, 66 °C for 45 s, and 72 °C for2 min. Deletion of the cyt-b5 (CYB5D2-Dcyt-b5) wasachieved by PCR amplification of the 5′fragment (residues1–34 AA) and the 3′ fragment (residues 135–264 AA), fol-lowed by ligation of the 2 fragments by using BamH1.Both CYB5D2-DTM and CYB5D2-Dcyt-b5 mutants wereconfirmed by DNA sequencing.

Generation of anti-CYB5D2 antibodyTo exclude the possibility that the anti-CYB5D2 antibody

raised may recognize other members of MAPRs, we generated

a GST-fusion protein that contained the CYB5D2 region C-terminal to the conserved cyt-b5 domain (GST-C-CYB5D2).Recombinant GST-C-CYB5D2 was purified from E. coli BL-21 cells and used to immunize 2 rabbits (8–10 weeks old). Af-finity purification was performed using a 2-step procedure. Asthe GST-C-CYB5D2 was produced at a low level, it was im-practical to use it for affinity purification of the anti-CYB5D2antibody. To overcome this difficulty, we generated GST-CYB5D2-DTM, which was produced at a much higher level.Isolated GST or GST-CYB5D2-DTM was coupled to cyano-gen bromide (CNBr)-activated-sepharose 4B (Sigma) to a finalconcentration of 9 mg recombinant protein/ml CNBr-sepharose4B beads. Fifteen millilitres of rabbit serum was first passedthrough a GST-coupled CNBR activated sepharose 4B columnto exclude IgG against GST. The flow-through was then ap-plied to a GST-CYB5D2-DTM-coupled sepharose 4B columnto purify the anti-CYB5D2 antibody. The antibody specificallyrecognizes CYB5D2, as signals could be specifically com-peted by addition of GST-CYB5D2-DTM (1 mg/mL) but notGST (1 mg/mL) during Western blot (data not shown).

Cell cycle analysisCell cycle distribution was determined by dispersing cells

using PBS containing 0.02% EDTA, followed by examinationof cell cycle profile according to our published procedure(Wei et al. 2010).

Western blot analysisCells lysates were prepared in a buffer containing 20 mmol/L

Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/LEGTA, 1% Triton X-100, 25 mmol/L sodium pyrophosphate,1 mmol/L NaF, 1 mmol/L b-glycerophosphate, 0.1 mmol/L so-dium orthovanadate, 1 mmol/L PMSF, 2 µg/mL leupeptin, and10 µg/mL aprotinin. Fifty micrograms of total cell lysate wasseparated by SDS–PAGE and transferred onto Immobilon-Pmembranes (Millipore, Billerica, Massachussetts, USA).Membranes were blocked with 5% skim milk and then incu-bated with the indicated antibodies at 4 °C overnight. Signalswere detected using an ECL Western Blotting Kit (Amer-sham, Burlington, Ontario, Canada). Primary antibodies andconcentrations used were: anti-H2AX (Santa Cruz, 1:1000);anti-gH2AX (Upstate, 1:1000), anti-phospho-ATM (S1981)(Rockland, 1:500), anti-ATM (Rockland, 1:500), anti-HA(Covance, 1:1000), and anti-actin (Santa Cruz, 1:1000), anti-caspase3 (Transduction Laboratory, 1:1000), anti-caspase9(Cell signalling, 1:1000), anti-Bcl-XL (Transduction Labora-tory, 1:1000), anti-Bcl-2 (Santa Cruz, 1:500), and anti-Bax(Santa Cruz, 1:1000).

Immunofluorescence stainingCells were treated as outlined in the figure captions. Im-

munofluorescent staining was carried out by fixing cellswith 4% paraformaldehyde for 20 min. Anti-HA (Covance,1:1000) was then added to the slides at 4 °C overnight. Afterwashing, secondary antibodies, FITC-donkey anti-mouse IgG(1:200, Jackson Immuno Research Lab), were applied for 1 hat room temperature. The slide was subsequently coveredwith Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame,Calif.). Images were taken with a fluorescent microscope(Carl Zeiss, Axiovert 200).

Xie et al.342

Published by NRC Research Press

Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

Assay of apoptosisDetermination of apoptosis was performed following our

published procedure (Tang et al. 2000, 2002). Apoptotic celldeath was observed under a light microscope for typical mem-brane blubbing. Apoptosis was quantified by terminal deoxy-nucleotidyl transferase (TdT)-mediated dUTP nick endlabeling (TUNEL) assay. Briefly, cells were seeded in a cham-ber slide containing 8 chambers 1 day before treatment. Upona specific treatment, apoptotic cell death was detected using a

TUNEL kit (GenScript USA, Inc, Cat No: L00290) followingthe manufacturer’s instruction. At least 200 cells were countedin 4 randomly selected fields under a light microscope andpercentage of apoptotic cells was then calculated.

Clonogenic survival assayCells were seeded at 6 × 105 cells per well in 6-well plates

for 24 h. Cells were then treated with etoposide (ETOP) at thedesignated doses for 8 h, followed by culture for 2–3 weeks

Fig. 1. Alignment of the cytochrome b5 (cyt-b5) domain of CYB5D2 with that of PGRMC1. Identical and similar residues are indicated byvertical lines and dots, respectively. The cyt-b5 domain of CYB5D2 shares 40.4% identity and 55.8% similarity with the counterpart ofPGRMC1. Alignment was performed using the EMBOSS Align Program.

Fig. 2. CYB5D2 enhances HeLa cell survival of ETOP treatment. (A) HeLa cells were stably infected with empty vector (EV) or HA-taggedCYB5D2 retrovirus. The expression of ectopic CYB5D2 and actin was examined by Western blot using anti-HA and anti-actin antibodies,respectively. (B) Typical profiles of cell cycle distribution for HeLa EV and HeLa CYB5D2 cell lines. (C) HeLa EV and HeLa CYB5D2 cellswere seeded in 6-well plates at 6 × 105 cells per well overnight, followed by etoposide treatment at the indicated doses for 8 h. Cells werethen cultured in normal medium for 2–3 weeks until surviving cell colonies formed, which were stained with crystal violet. Experiments wererepeated 3 times. Typical images from 1 experiment are shown. (D) Quantification of surviving cells. Data were graphed as means ± SE.Asterisk (*), p < 0.05 by comparison with EV cells.

343Biochem. Cell Biol. Vol. 89, 2011


Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

until colonies were formed. Surviving cells were then stainedwith crystal violet (0.5%). Colonies numbers were countedusing ImageJ 1.44 (National Institutes of Health, BethedaMaryland, USA).

DNA microarray analysisTotal RNA was purified using TRIzol (Invitrogen, Burling-

ton, Ont.). Affymetrix analysis was performed by the UHNMicroarray Centre using Human 1.0ST Gene Array Chip.The cut-off value was 2-fold.

Statistical analysisStatistical analysis was performed using Student’s t-test,

and p < 0.05 was considered statistically significant.

Results

CYB5D2 enhances HeLa cells survival of etoposide(ETOP), but not UV treatmentWhile CYB5D2 shares 19.8% identity and 26.2% similar-

ity with PGRMC1 (data not shown), both proteins have40.4% identity and 55.8% similarity between their cyt-b5 do-mains (Fig. 1). As cyt-b5 domain-mediated heme-binding isrequired for Dap1 (the yeast homologue of PGRMC1) andPGRMC1 to protect cells from DNA damage-induced toxic-ity (Mallory et al. 2005; Crudden et al. 2006), this suggests

that CYB5D2 may also play a role in resistance to DNAdamage. To investigate this possibility, we have constructedHeLa cells stably transfected with empty vector (EV) orCYB5D2 (Fig. 2A). Ectopic CYB5D2 does not apparentlyaffect cell cycle distribution (Fig. 2B) and cell proliferation(data not shown). EV and CYB5D2 cells were subsequentlytreated with different doses of ETOP for 8 h, and the cell’sability to survive these conditions was then examined by clo-nogenic assay. By comparison with HeLa EV cells, HeLaCYB5D2 cells formed significantly more colonies (Figs. 2Cand 2D). We then examined whether CYB5D2 also offers re-sistance to DNA damage induced by other reagents. Our pre-liminary data indicate that hydroxyurea used at 1 mmol/Ldoes not produce significant cytotoxic effects, and that UVtreatment is potently toxic to HeLa cells. We thus treatedHeLa cells with a variety of doses (5, 7.5, or 10 mJ/cm2)and cultured cells for 2–3 weeks to allow surviving cells toform colonies. By comparison with EV, CYB5D2 has no ef-fect on UV-mediated cytotoxicity (Fig. 3).We further determined whether CYB5D2 generally reduces

apoptosis. When treated with TNFa in combination with theprotein synthesis inhibitor cycloheximide, comparable levelsof apoptosis were induced in both HeLa EV and HeLaCYB5D2 cells (data not shown). Additionally, UV-inducedapoptosis is also not affected by CYB5D2 (data not shown).As TNFa and UV initiate apoptosis through the death recep-

Fig. 3. CYB5D2 has no effect on UV-induced cytotoxicity. (A) HeLa empty vector (EV) and HeLa CYB5D2 cells were treated with UV atthe indicated doses, followed by culture of the treated cells for 2–3 weeks for the formation of surviving colonies. (B) Quantification of sur-viving cell colonies derived from UV 5 mJ/cm2 was performed by solubilization of stained crystal violet with 0.33% acetic acid, and thenmeasured with a spectrophotometer at A550 (left panel) following our published methodology (Tang and Kidd, 1998). Quantification of sur-viving cell colonies derived from UV 7.5 mJ/cm2 and UV 10 mJ/cm2 was performed using ImageJ (right panel)..

Xie et al.344


Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

tor and the mitochondrial pathway, respectively (Kulms andSchwarz 2000), we concluded that CYB5D2 does not impactthe core processes of apoptosis. This is consistent with thecomparable levels of caspases 9, Bax, and Bcl-2, major com-ponents of the apoptotic core machinery (Cryns and Yuan1998), in both EV and CYB5D2 cells (Fig. 4). Although aslightly higher level of caspase 3 is expressed in CYB5D2cells (Fig. 4), this does not apparently affect DNA damage-in-duced apoptosis in HeLa CYB5D2 cells, as Bcl-xL is alsoslightly elevated in HeLa CYB5D2 cells (Fig. 4). Taken to-gether, the above observations demonstrate that CYB5D2 me-diates some specific resistance to ETOP-induced cytotoxicity.As ETOP is a well established topoisomerase II poison (Bur-den and Osheroff 1998), our observations therefore are con-sistent with the reported protection by PGRMC1 againstdoxorubicin toxicity, also a topoisomerase II poison (Pommieret al. 2010).

Both the transmembrane and cyt-b5 domains arerequired for CYB5D2-mediated resistance to ETOPSince the MAPR family has 2 structure features, a single

transmembrane (TM) motif and a cyt-b5 domain (Thomas2008), we have investigated their involvement in resistanceto ETOP. For this purpose, CYB5D2 mutants with either theTM (CYB5D2-DTM) or cyt-b5 (CYB5D2-Dcyt-b5) domaindeleted were constructed (Fig. 5A, left panel). WhileCYB5D2, as expected, enhanced the survival of HeLa cellstreated with ETOP (Figs. 5B and 5C), deletion of either do-main significantly compromised the resistance (Figs. 5B and5C). To fully appreciate the impact of these ectopic proteins

on the survival of HeLa cells treated with ETOP, we have ex-amined the expression of both endogenous and ectopicCYB5D2 proteins using the anti-CYB5D2 antibody. Thisantibody was affinity purified, and specifically recognizesCYB5D2 (see Materials and methods for details). AlthoughCYB5D2-Dcyt-b5 was expressed at a reduced level, com-pared with CYB5D2 and CYB5D2-DTM (Fig. 5A, leftpanel), ectopic CYB5D2-Dcyt-b5 was expressed at a sub-stantially increased level compared with the endogenousCYB5D2 (Fig. 5A, right panel). Because of the high levelsof ectopic CYB5D2 and DTM expressed, as well as theirmolecular weights being very close to that of endogenousCYB5D2, we were unable to distinguish between the ectopicand endogenous proteins (data not shown). These observa-tions suggest that by comparison with ectopic CYB5D2, theinability of CYB5D2-Dcyt-b5 to enhance the survival ofHeLa cells to treatment with ETOP (Figs. 5B and 5C) maynot be attributable to its low level of expression (Fig. 5A,left panel). This concept is consistent with the observationthat while CYB5D2-DTM was expressed at a level compara-ble with that of CYB5D2 (Fig. 5A, left panel), CYB5D2-DTM is also incapable of offering HeLa cell survival advant-age against ETOP treatment (Fig. 5B and 5C). Inside the cell,CYB5D2 displays perinucleus staining, suggesting its associ-ation with the ER membrane (Fig. 5D). This pattern of stain-ing is consistent with the staining of PGRMC1 in MCF7 andHeLa cells (Min et al. 2004; Neubauer et al. 2009).CYB5D2-DTM does not apparently associate with cellularplasma membranes (Fig. 5D). Collectively, our observationsreveal the requirement of the TM and cyt-b5 domains forCYB5D2 to protect cells from ETOP-induced cytotoxicity.

CYB5D2 mediates resistance to ETOP via pathwaysindependently of DNA damage response (DDR)ETOP induces DDR by activating the ATM pathway,

which results in G2/M arrest (Wei et al. 2010). To determinethe mechanisms responsible for CYB5D2-mediated resistanceto ETOP, we have examined whether CYB5D2 affects ETOP-induced DDR. In response to ETOP, both EV and CYB5D2cell lines displayed comparable levels of phosphorylation ofATM Serine 1981 (S1981) (Fig. 6A), an event that convertsinactive ATM dimmers into active ATM monomers (Bakken-ist and Kastan 2003; Cann and Hicks 2007). ETOP also indu-ces comparable levels of gH2AX in both cell lines (Fig. 6A).While ETOP treatments were associated with more CYB5D2cells in G2/M and fewer cells in S phase than EV cells(Fig. 6B; Table 1), the same trend was also observed in thecontrol cells (DMSO treated) (Table 1). Additionally, most ofthese differences were not statistically significant, except fordifferences in S phase in 3 treatments including the controltreatment (Table 1). Furthermore, these differences did notcorrelate with ETOP doses used (Table 1). Taken together,we concluded that ectopic expression of CYB5D2 may notactivate ETOP-induced G2/M arrest. This notion is consistentwith the biochemical evidence that ectopic CYB5D2 did notobviously change ETOP-induced ATM S1981 phosphoryla-tion and the production of gH2AX (Fig. 6A).

CYB5D2 affects the expression of specific genesTo further investigate the underlying mechanisms, we have

examined whether CYB5D2 may regulate, either directly or

Fig. 4. HeLa empty vector (EV) and HeLa CYB5D2 cells expresscomparable levels of apoptotic proteins, except caspase 3, caspase 9,and Bcl-xL that were expressed at slightly higher levels in CYB5D2cells than in EV cells. Both cell lines were examined for the indi-cated apoptotic proteins by Western blot.



Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

Fig. 5. Both the transmembrane and cyt-b5 domains are required for CYB5D2 to enhance HeLa cell survival of ETOP-induced toxicity. (A)CYB5D2 mutants were generated with either the TM (residues 1–29) or the cyt-b5 (residues 35–134) deleted. HeLa cells were stably infectedwith the indicated retrovirus. The expression of ectopic CYB5D2 mutants (HA-tagged) was detected by Western blot with anti-HA antibody(left panel). Ectopic CYB5D2-Dcyt-b5 was also examined by anti-CYB5D2 antibody (right panel). The CYB5D2 bands in the right panelrepresent the endogenous protein in both empty vector (EV) and DCyt-b5 lines. (B and C) HeLa EV, CYB5D2, DTM, or DCyt-b5 cells wereassayed for the survival of ETOP treatment by clonogenic assay as described in Fig. 1 legend. Experiments were repeated 3 times. Represen-tative images from a single experiment were shown (B). Quantification of surviving cell colonies (means ± SE) was also presented (C). As-terisk (*), p < 0.05 by comparison with EV cells. (D) The HeLa stable lines of HA-tagged CYB5D2, CYB5D2-DTM, and CYB5D2-DCyt-b5 were immunofluorescently stained with an anti-HA antibody. Nucleoli were counter stained with DAPI. Merge images are also shown.Scale bar represents 10 µm.

Xie et al.346


Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

Fig.

6.CYB5D

2does

notaffect

etoposide-inducedDNA

damageresponse.(A

)HeL

aEVandCYB5D

2cells

weretreatedwith

theindicateddosesof

ETOPfor8h.

S1981phosphory-

latedATM

(p-ATM),ATM,gH2A

X,a

ndH2A

Xweredeterm

ined

byWestern

blot,u

sing

specific

antib

odies.Experim

ents

wererepeated

3tim

es.Representativeim

ages

from

asingle

experimentwereshow

n.(B)HeL

aEVandCYB5D

2cells

weretreatedwith

theindicateddosesof

ETOPfor24

h,follo

wed

bydeterm

inationof

cellcycleprogression.



Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

indirectly, the expression of candidate genes, which may con-tribute to CYB5D2-mediated resistance to etoposide. TotalRNA was isolated from both HeLa EV and HeLa CYB5D2cells and analyzed using the Affymetrix platform. Surpris-ingly, CYB5D2 does not massively affect gene expression.Among 28 869 genes (Human 1.0 ST Gene Chip, Affyme-trix), 2-fold or greater changes in gene expression were foundin 12 genes (Table 2), while ectopic CYB5D2 was overex-pressed more than 28-fold (Table 2). However, it is not obvi-ously clear how changes in any one of them may contributeto CYB5D2-mediated resistance to ETOP.

DiscussionCYB5D2 is also known as neuferricin, based on its expres-

sion in the central nervous system and its involvement in pro-moting neurogenesis (Kimura et al. 2010). However, it ismost likely that CYB5D2 has additional functions, as theprotein is expressed at much higher levels in the heart, adre-nal gland, and kidney than in the brain (Kimura et al. 2010).This is consistent with the suggested neuroprotection activityof PGRMC1 (Labombarda et al. 2003; Meffre et al. 2005), a

member of the MAPR family, in addition to its other func-tions (Rohe et al. 2009).In line with the involvement of PGRMC1 in resistance to

topoisomerase II poison-induced cytotoxicity, we demonstratehere that CYB5D2/neuferricin also enhances the survival ofcells exposed to topoisomerase II poison etoposide-inducedcytotoxicity. This activity requires the TM and cyt-b5 do-mains of CYB5D2. The requirement of the TM domain mayindicate that CYB5D2 protects against ETOP toxicity via reg-ulation of specific organelle’s (i.e., the endoplamic reticulum)function. As interaction with heme is required for PGRMC1-mediated resistance to doxorubicin, the requirement of thecyt-b5 domain suggests that association with heme may alsocontribute to CYB5D2-mediated resistance to etoposide. Con-sistent with this possibility, CYB5D2/neuferricin was reportedto bind to heme (Kimura et al. 2010). However, it is also pos-sible that deletion of the cyt-b5 domain may cause CYB5D2conformation changes, which may attenuate its protectionagainst ETOP-derived cytotoxicity. Evidence that may supportthis possibility was the observation that deletion of the cyt-b5domains significantly reduced its expression, when comparedwith CYB5D2 and CYB5D2-DTM (Fig. 5A, left panel).Therefore, further research will be required to properly exam-ine the contribution of heme-binding to CYB5D2’s ability toprotect cells from ETOP-induced toxicity.It is unlikely that CYB5D2 protects against etoposide-

induced cytotoxicity by modulation of DDR, as CYB5D2has no observed effects on etoposide-induced ATM activa-tion, gH2AX production, and G2/M arrest (Fig. 6; Table 1).Additional support for this concept is the observation thatCYB5D2 does not affect UV-induced apoptosis. UV indu-ces DDR primarily through the ATR pathway (Batista etal. 2009). Additionally, CYB5D2 also does not affect thecore process of apoptosis. Taken together, CYB5D2 maymodulate etoposide-induced cytotoxicity via novel or un-identified pathways. One of these pathways may protectheme from chemical-induced damage, as this mechanismhas been proposed for PGRMC1-mediated resistance toDNA damage (Rohe et al. 2009). PGRMC1 has beenshown to interact with and activate the P450 proteinCYP51A (Hughes et al. 2007). P450 proteins are knownto detoxify toxic compounds, which may contribute toPGRMC1-mediated resistance to DNA damage reagents. Itwill be an intriguing possibility that P450 proteins mayplay a role in CYB5D2-initiated resistance to etoposide.

Table 1. Ectopic CYB5D2 does not affect ETOP-induced G2/M arrest in HeLa cells.

ETOP (µmol/L)

DMSO 0.2 0.4 0.6 0.8 1.0G1 phase

EV 48.2±4.1 44.0±4.3 37.5±6.0 33.4±5.9 21.3±3.6 10.8±1.0CYB5D2 54.5±1.3 49.7±4.2 42.5±5.2 35.8±5.6 24.0±2.9 11.7±0.6

S phaseEV 26.4±4.2 28.8±5.9 29.3±4.1 24.4±1.2 19.6±5.9 21.5±6.1CYB5D2 15.9±1.5a 18.6±2.2 15.1±0.6a 12.4±1.0a 12.5±1.8 13.8±2.7

G2/M phaseEV 25.4±5.8 27.2±6.0 33.2±9.5 42.2±6.0 59.2±4.2 67.7±6.0CYB5D2 29.6±1.7 31.7±5.8 42.4±5.1 51.8±4.8 63.5±1.7 74.5±2.2

Note: Cell cycle distributions were derived from 3 independent experiments and were presented as mean ± SE.aStatistically significant at p < 0.05 (by comparison with DMSO treatment).

Table 2. Ectopic CYB5D2 affects gene expression.

Gene symbol Fold change Regulationa ChromosomeCYB5D2 28.8 Up 175S_rRNA 2.80 Up 11Y_RNA 2.12 Up XU6 –3.28 Down 5UBE2L7P –2.57 Down 14SNORD77b –2.51 Down 1Y_RNA –2.43 Down 13SNORD50 –2.37 Down 128043502c –2.25 Down 2RBMS1d –2.20 Down 12SNORA73Ae –2.16 Down 17896748c –2.02 Down 18165692c –2.0 Down Mf

aHeLa CYB5D2 vs. HeLa EV (empty vector). CYB5D2 in HeLa CYB5D2cells is up-regulated at 28.8 fold.

bSmall nucleolar RNA, C/D box 77.cTranscripts cluster Id.dRNA binding motif, single stranded interacting protein 1.eSmall nucleolar RNA, H/ACA box 73A.fMitochondrial chromosome.

Xie et al.348


Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

Potential candidates that affect ETOP-induced resistance toetoposide may include those genes that their expression isaffected by CYB5D2 (Table 2).

AcknowledgmentsThis work was supported by a Heart and Stroke Founda-

tion grant to D. Tang. We also like to acknowledge the finan-cial support from St. Joseph's Health Care at St. Joseph’sHospital, Hamilton, Ontario, Canada, to the Hamilton Centrefor Kidney Research (HCKR).

ReferencesAhmed, I.S., Rohe, H.J., Twist, K.E., and Craven, R.J. 2010a.

Pgrmc1 (progesterone receptor membrane component 1) associ-ates with epidermal growth factor receptor and regulates erlotinibsensitivity. J. Biol. Chem. 285(32): 24775–24782. doi:10.1074/jbc.M110.134585. PMID:20538600.

Ahmed, I.S., Rohe, H.J., Twist, K.E., Mattingly, M.N., and Craven, R.J.2010b. Progesterone receptor membrane component 1 (Pgrmc1): aheme-1 domain protein that promotes tumorigenesis and is inhibitedby a small molecule. J. Pharmacol. Exp. Ther. 333(2): 564–573.doi:10.1124/jpet.109.164210. PMID:20164297.

Bakkenist, C.J., and Kastan, M.B. 2003. DNA damage activates ATMthrough intermolecular autophosphorylation and dimer dissocia-tion. Nature, 421(6922): 499–506. doi:10.1038/nature01368.PMID:12556884.

Batista, L.F., Kaina, B., Meneghini, R., and Menck, C.F. 2009. HowDNA lesions are turned into powerful killing structures: insightsfrom UV-induced apoptosis. Mutat. Res. 681(2–3): 197–208.PMID:18845270.

Bramley, T. 2003. Non-genomic progesterone receptors in themammalian ovary: some unresolved issues. Reproduction, 125(1):3–15. doi:10.1530/rep.0.1250003. PMID:12622691.

Brinton, R.D., Thompson, R.F., Foy, M.R., Baudry, M., Wang, J.,and Finch, C.E. 2008. Progesterone receptors: form and function inbrain. Front. Neuroendocrinol. 29(2): 313–339. PMID:18374402.

Burden, D.A., and Osheroff, N. 1998. Mechanism of action ofeukaryotic topoisomerase II and drugs targeted to the enzyme.Biochim. Biophys. Acta, 1400(1–3): 139–154. PMID:9748545.

Cahill, M.A. 2007. Progesterone receptor membrane component 1: anintegrative review. J. Steroid Biochem. Mol. Biol. 105(1–5): 16–36. doi:10.1139/O07-135. PMID:18059525.

Cann, K.L., and Hicks, G.G. 2007. Regulation of the cellular DNAdouble-strand break response. Biochem. Cell Biol. 85(6): 663–674. doi:10.1139/O07-135. PMID:18059525.

Craven, R.J. 2008. PGRMC1: a new biomarker for the estrogenreceptor in breast cancer. Breast Cancer Res. 10(6): 113. doi:10.1186/bcr2191. PMID:19090968.

Crudden, G., Chitti, R.E., and Craven, R.J. 2006. Hpr6 (heme-1domain protein) regulates the susceptibility of cancer cells tochemotherapeutic drugs. J. Pharmacol. Exp. Ther. 316(1): 448–455. doi:10.1124/jpet.105.094631. PMID:16234411.

Cryns, V., and Yuan, J. 1998. Proteases to die for. Genes Dev. 12(11):1551–1570. doi:10.1101/gad.12.11.1551. PMID:9620844.

DeBose-Boyd, R.A. 2007. A helping hand for cytochrome p450enzymes. Cell Metab. 5(2): 81–83. doi:10.1016/j.cmet.2007.01.007. PMID:17276348.

Fernandes, M.S., Brosens, J.J., and Gellersen, B. 2008. Honey, weneed to talk about the membrane progestin receptors. Steroids,73(9–10): 942–952. doi:10.1016/j.steroids.2007.12.004. PMID:18221973.

Hand, R.A., and Craven, R.J. 2003. Hpr6.6 protein mediates celldeath from oxidative damage in MCF-7 human breast cancer cells.

J. Cell. Biochem. 90(3): 534–547. doi:10.1002/jcb.10648. PMID:14523988.

Hughes, A.L., Powell, D.W., Bard, M., Eckstein, J., Barbuch, R.,Link, A.J., and Espenshade, P.J. 2007. Dap1/PGRMC1 binds andregulates cytochrome P450 enzymes. Cell Metab. 5(2): 143–149.doi:10.1016/j.cmet.2006.12.009. PMID:17276356.

Kimura, I., Nakayama, Y., Konishi, M., Kobayashi, T., Mori, M.,and Ito, M. 2010. Neuferricin, a novel extracellular heme-binding protein, promotes neurogenesis. J. Neurochem. 112(5):1156–1167. doi:10.1111/j.1471-4159.2009.06522.x. PMID:19968755.

Kingma, P.S., and Osheroff, N. 1998. The response of eukaryotictopoisomerases to DNA damage. Biochim. Biophys. Acta,1400(1–3): 223–232. PMID:9748592.

Kulms, D., and Schwarz, T. 2000. Molecular mechanisms of UV-induced apoptosis. Photodermatol. Photoimmunol. Photomed. 16(5):195–201. doi:10.1034/j.1600-0781.2000.160501.x. PMID:11068857.

Labombarda, F., Gonzalez, S.L., Deniselle, M.C., Vinson, G.P.,Schumacher, M., De Nicola, A.F., and Guennoun, R. 2003. Effectsof injury and progesterone treatment on progesterone receptor andprogesterone binding protein 25-Dx expression in the rat spinalcord. J. Neurochem. 87(4): 902–913. doi:10.1046/j.1471-4159.2003.02055.x. PMID:14622121.

Mallory, J.C., Crudden, G., Johnson, B.L., Mo, C., Pierson, C.A.,Bard, M., and Craven, R.J. 2005. Dap1p, a heme-binding proteinthat regulates the cytochrome P450 protein Erg11p/Cyp51p inSaccharomyces cerevisiae. Mol. Cell. Biol. 25(5): 1669–1679.doi:10.1128/MCB.25.5.1669-1679.2005. PMID:15713626.

Meffre, D., Delespierre, B., Gouézou, M., Leclerc, P., Vinson, G.P.,and Schumacher, M. 2005. The membrane-associated progester-one-binding protein 25-Dx is expressed in brain regions involvedin water homeostasis and is up-regulated after traumatic braininjury. J. Neurochem. 93(5): 1314–1326. doi:10.1111/j.1471-4159.2005.03127.x. PMID:15934950.

Min, L., Takemori, H., Nonaka, Y., Katoh, Y., Doi, J., and Horike, N.2004. Characterization of the adrenal-specific antigen IZA (innerzone antigen) and its role in the steroidogenesis. Mol. Cell.Endocrinol. 215(1–2): 143–148. doi:10.1016/j.mce.2003.11.025.PMID:15026187.

Neubauer, H., Clare, S.E., Wozny, W., Schwall, G.P., Poznanovic, S.,and Stegmann, W. 2008. Breast cancer proteomics revealscorrelation between estrogen receptor status and differentialphosphorylation of PGRMC1. Breast Cancer Res. 10(5): R85.doi:10.1186/bcr2155. PMID:18922159.

Neubauer, H., Adam, G., Seeger, H., Mueck, A.O., Solomayer, E.,and Wallwiener, D. 2009. Membrane-initiated effects of proges-terone on proliferation and activation of VEGF in breast cancercells. Climacteric, 12(3): 230–239. doi:10.1080/13697130802635637. PMID:19340614.

Peluso, J.J., Liu, X., Saunders, M.M., Claffey, K.P., and Phoenix, K.2008. Regulation of ovarian cancer cell viability and sensitivity tocisplatin by progesterone receptor membrane component-1. J.Clin. Endocrinol. Metab. 93(5): 1592–1599. doi:10.1210/jc.2007-2771. PMID:18319313.

Peluso, J.J., Gawkowska, A., Liu, X., Shioda, T., and Pru, J.K. 2009.Progesterone receptor membrane component-1 regulates thedevelopment and Cisplatin sensitivity of human ovarian tumorsin athymic nude mice. Endocrinology, 150(11): 4846–4854.doi:10.1210/en.2009-0730. PMID:19797399.

Pommier, Y., Leo, E., Zhang, H., and Marchand, C. 2010. DNAtopoisomerases and their poisoning by anticancer and antibacterialdrugs. Chem. Biol. 17(5): 421–433. doi:10.1016/j.chembiol.2010.04.012. PMID:20534341.

Rohe, H.J., Ahmed, I.S., Twist, K.E., and Craven, R.J. 2009. PGRMC1



Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

(progesterone receptor membrane component 1): a targetable proteinwith multiple functions in steroid signaling, P450 activation anddrug binding. Pharmacol. Ther. 121(1): 14–19. doi:10.1016/j.pharmthera.2008.09.006. PMID:18992768.

Tang, D., and Kidd, V.J. 1998. Cleavage of DFF-45/ICAD bymultiple caspases is essential for its function during apoptosis. J.Biol. Chem. 273(44): 28549–28552. doi:10.1074/jbc.273.44.28549. PMID:9786842.

Tang, D., Lahti, J.M., and Kidd, V.J. 2000. Caspase-8 activation andbid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. J.Biol. Chem. 275(13): 9303–9307. doi:10.1074/jbc.275.13.9303.PMID:10734071.

Tang, D., Wu, D., Hirao, A., Lahti, J.M., Liu, L., and Mazza, B. 2002.ERK activation mediates cell cycle arrest and apoptosis after DNAdamage independently of p53. J. Biol. Chem. 277(15): 12710–12717. doi:10.1074/jbc.M111598200. PMID:11821415.

Thomas, P. 2008. Characteristics of membrane progestin receptoralpha (mPRalpha) and progesterone membrane receptor compo-nent 1 (PGMRC1) and their roles in mediating rapid progestinactions. Front. Neuroendocrinol. 29(2): 292–312. doi:10.1016/j.yfrne.2008.01.001. PMID:18343488.

Wei, F., Xie, Y., Tao, L., and Tang, D. 2010. Both ERK1 and ERK2kinases promote G2/M arrest in etoposide-treated MCF7 cells byfacilitating ATM activation. Cell. Signal. 22(11): 1783–1789.doi:10.1016/j.cellsig.2010.07.007. PMID:20637859.

Xie et al.35


0

Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y SA

VA

NN

AH

RIV

NA

TL

AB

BF

on 1

1/22

/14

For

pers

onal

use

onl

y.

cyb5d2 enhances hela cells survival of etoposide-induced cytotoxicity

Documents