Aldehyde dehydrogenase 3A1 confers oxidative stress resistance accompanied by altered DNA damage response in human corneal epithelial cells

Journal Pre-proof Aldehyde dehydrogenase 3A1 confers oxidative stress resistance accompanied by altered DNA damage response in human corneal epithelial cells Georgia-Persephoni Voulgaridou, Ilias Tsochantaridis, Christos Tolkas, Rodrigo Franco, Alexandra Giatromanolaki, Mihalis I. Panayiotidis, Aglaia Pappa PII:

S0891-5849(19)32380-9

DOI:

https://doi.org/10.1016/j.freeradbiomed.2020.01.183

Reference:

FRB 14588

To appear in:

Free Radical Biology and Medicine

Received Date: 13 December 2019 Revised Date:

27 January 2020

Accepted Date: 27 January 2020

Please cite this article as: G.-P. Voulgaridou, I. Tsochantaridis, C. Tolkas, R. Franco, A. Giatromanolaki, M.I. Panayiotidis, A. Pappa, Aldehyde dehydrogenase 3A1 confers oxidative stress resistance accompanied by altered DNA damage response in human corneal epithelial cells, Free Radical Biology and Medicine (2020), doi: https://doi.org/10.1016/j.freeradbiomed.2020.01.183. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

ROS

HCE-2 isogenic cell line pair H2DCFDA

Environmental stressors

O2

UVR

DCF cel

l

ROS

cellular oxidative status Comet Assay

NAD(P) +

GSSG

NAD(P)H

Lipid Peroxidation

Aldehydes (4-HNE, M DA)

Mock/HCE-2

GSH

ALDH3A1

ALDH3A1/HCE-2 O HO

H

O

HO

H

DNA damage

O

nucle

H

RT2 Profiler Assay

O

O

O H

us

O

H 2 O2

Tert-butyl peroxide

OH

O O

O

Etoposide

TREATMENTS

DDR genes

DDR gene regulation

DNA Damage

Apoptosis

?

Cell cycle

?

1

Aldehyde dehydrogenase 3A1 confers oxidative stress resistance accompanied by

2

altered DNA damage response in human corneal epithelial cells

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Georgia-Persephoni Voulgaridou1, Ilias Tsochantaridis1, Christos Tolkas1, Rodrigo

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Franco2,3, Alexandra Giatromanolaki4, Mihalis I. Panayiotidis5 and Aglaia Pappa1*

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1

Department of Molecular Biology & Genetics, and 2Redox Biology Center, 114 VBS

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0905, University of Nebraska-Lincoln, Lincoln, NE 68588, USA;

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Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln,

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Lincoln, NE 68583, USA; 4Department of Pathology, Democritus University of

9

Thrace, University General Hospital of Alexandroupolis, Alexandroupolis, Greece,

10

5

11

Neurology & Genetics, Nicosia, 2371, Cyprus

3

School of

Department of Electron Microscopy & Molecular Pathology, The Cyprus Institute of

12 13

*Corresponding author:

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Aglaia Pappa, Ph.D.

15

Department of Molecular Biology & Genetics

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Democritus University of Thrace

17

University Campus, Dragana,

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68100 Alexandroupolis, GREECE

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Phone: +30-25510-30625

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Fax: +30-25510-30625

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E-mail: [email protected]

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1

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Abstract

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Aldehyde dehydrogenase 3A1 is constitutively expressed in a taxon-specific

26

manner in the cornea, where, due to its high abundance, it has been characterized as a

27

corneal crystallin. ALDH3A1 has been proposed to be a multifaceted protein that

28

protects cellular homeostasis through several modes of action. The present study

29

examines the mechanisms by which ALDH3A1 exerts its cytoprotective role under

30

conditions of oxidative stress. To this end, we have utilized an isogenic HCE-2

31

(human corneal epithelium) cell line pair differing in the expression of ALDH3A1.

32

Single cell gel electrophoresis assay and H2DCFDA analysis revealed that the

33

expression of ALDH3A1 protected HCE-2 cells from H2O2-, tert-butyl peroxide- and

34

etoposide-induced oxidative and genotoxic effects. Furthermore, comparative qPCR

35

analysis revealed that a panel of cell cycle (Cyclin A, B1, B2, D, E), apoptosis (p53,

36

p21, BAX, BCL-2, BCL-XL) and DNA damage response (DNA-PK, NBS1) proteins

37

were up-regulated in the ALDH3A1 expressing HCE-2 cells. Moreover, the

38

expression profile of a variety of DNA damage signaling (DDS)-related genes, was

39

investigated (under normal and oxidative stress conditions) by utilizing the RT2

40

profilerTM PCR array in both isogenic HCE-2 cell lines. Our results demonstrated that

41

several genes associated with ATM/ATR signaling, cell cycle regulation, apoptosis

42

and DNA damage repair were differentially expressed under all conditions tested. In

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conclusion, this study suggests that ALDH3A1 significantly contributes to the

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antioxidant defense of corneal homeostasis by maintaining DNA integrity possibly

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through altering the expression of specific DDS-related genes. Further studies will

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shed light on the precise role(s) of this multifunctional protein.

47 48

Keywords

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ALDH3A1, aldehyde dehydrogenase 3A1, ALDHs, corneal homeostasis, DNA

50

damage response, DDR, DNA damage signalling, DDS, oxidative stress, DNA

51

damage, antioxidant

52 53

3

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1. Introduction

55

The aldehyde

dehydrogenase (ALDH) superfamily comprises a group of

56

NAD(P)+-dependent cytoprotective proteins which oxidize and thus detoxify

57

endogenous and exogenous aldehydes with cytotoxic effects1. Apart from their

58

essential metabolic roles though, ALDHs emerge as multifaceted proteins implicated

59

in various cellular homeostatic mechanisms like differentiation, embryogenesis and

60

development2.

61

Aldehyde dehydrogenase 3A1 (ALDH3A1), a member of the ALDHs family,

62

is a homodimeric protein that detoxifies medium chain saturated and unsaturated

63

aldehydes like hexanal, octanal, benzaldehyde and 4-hydroxy-nonenal (4-HNE)3. Its

64

expression is inducible in liver, following exposure to certain xenobiotics, or

65

constitutive in the epithelial layers of lung, stomach, urinary bladder, skin and

66

cornea4–6. Specifically, in the cornea of most mammals, ALDH3A1 accumulates in

67

high abundance (5-50% of total soluble proteins) and thus has been characterized as a

68

corneal crystallin

69

products of lipid peroxidation, ALDH3A1 possess strong cytoprotective properties.

70

Consequently, its constitutive expression in tissues that serve as first line of defense

71

(e.g. cornea, lung, skin, stomach) is not surprising. Indeed, ALDH3A1 appeared to

72

protect corneal epithelial cells against apoptosis caused by UV and 4-HNE by

73

inhibiting caspase-3 activation and 4-HNE-protein adduct formation8,10.

7–9

. Due to its ability to oxidize toxic aldehydes, generated as by-

74

The detoxification function of ALDH3A1 could be indispensable for its

75

cytoprotective actions. However, its high abundance in cornea (at levels exceeding

76

those needed for metabolism alone), in combination with novel experimental findings,

77

suggest a multifunctional role for ALDH3A1, involving non-catalytic properties.

78

Briefly, it has been proposed that the enzyme contributes to the maintenance of the 4

79

optical properties of cornea through a chaperone-like activity and participates in cell

80

cycle regulation9,11.

81

The multifaceted function of ALDH3A1 is supported by several experimental

82

data. For instance, ectopic expression of ALDH3A1 in corneal epithelial cells (at

83

levels similar to those found in vivo) was associated with elongation of cell cycle,

84

decreased plating efficiency, and reduced DNA synthesis accompanied with

85

significant alteration of major cell cycle regulators12,13. ALDH3A1 expression was

86

also associated with increased resistance to apoptosis caused by a variety of DNA

87

damaging agents12. In addition, although the enzyme is normally considered being in

88

the cytosol, it has also been found in the nucleus of corneal epithelial cells, thus

89

supporting for novel nuclear role(s) of this protein associating its proliferation-

90

suppressive function with protection against oxidative DNA damage13,14.

91

In a previous study, an isogenic cell line pair of human epithelial cells (HCE-

92

2) differing only in ALDH3A1 expression was established by stable transfection and

93

showed that the ALDH3A1 expressing HCE-2 cells were resistant to the cytotoxic

94

effects of both hydrogen peroxide (H2O2) and tert-butyl peroxide11. In this study, we

95

utilized this isogenic HCE-2 cell line pair to further investigate the cytoprotective role

96

of ALDH3A1 in the corneal epithelium homeostasis in order to elucidate the

97

molecular mechanisms by which ALDH3A1 confers a cell survival advantage under

98

conditions of oxidative stress.

99

100 101

2. Materials and methods 2.1 Materials

5

102

Human corneal epithelium HCE-2 cell line was obtained from ATCC

103

(Manassas, VA, USA) while the culture media along with the various additives, fetal

104

bovine serum (FBS), antibiotics and trypsin were either from Biosera (East Sussex,

105

UK), Gibco (Life Technologies, Carlsbad, CA, USA), Sigma-Aldrich Co.

106

(Taufkirchen, Germany) or Biochrome (Berlin, Germany). Etoposide was purchased

107

from Sigma-Aldrich Co. (Taufkirchen, Germany). Primers, dNTPs, Trizol and

108

Platinum SYBR Green were purchased from Invitrogen (Life Technologies Carlsbad,

109

CA, USA) while the random hexamers and PrimeScript Reverse Transcriptase were

110

from Takara (Shiga, Japan). RT2 Profiler PCR array for DNA damage signalling

111

pathway was purchased from Sabioscience (Qiagen, Venlo, Netherlands). The

112

oxidants used in the study were either obtained from Sigma-Aldrich Co. (Taufkirchen,

113

Germany), Cayman chemicals (Michigan, USA) or Carl Roth GmbH (Karlsruhe,

114

Germany). 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased

115

from Invitrogen while black 96-well plates with glass clear bottom were from Perkin

116

Elmer,Waltham, MA, USA.

117

2.2 Cell culture

118

Human corneal epithelium cell line HCE-2 was maintained in 1:1 mixture of

119

Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture (DMEM/F12)

120

supplemented with 15% FBS, 0.5% (v/v) dimethyl sulfoxide (DMSO), 0.1 µg/ml

121

cholera toxin, 10 ng/ml epidermal growth factor, 5 µg/ml insulin, 40 µg/ml

122

gentamycin, 100 µg/ml streptomycin and 100 units/ml penicillin. Mock- and

123

ALDH3A1-HCE-2 stably transfected cell lines11 were cultured in the same medium

124

with the extra addition of 0.2 mg/ml hygromycin. Cells were cultivated at 37°C with

125

5% CO2 in a humidified incubator.

6

126

2.3 H2DCFDA assay

127

In brief, 1 x 104 HCE-2 cells per well were seeded in black 96-well plates with

128

glass clear bottom, cultured for 24h and then treated with different concentrations of

129

H2O2 (0 – 500µΜ) for 16h, tert-butyl peroxide (0 – 50µΜ) for 16h and etoposide (0 –

130

50µM) for 16 h Then, the PBS-washed cells were incubated with 10µM H2DCFDA

131

(diluted in fresh culture medium) in dark at 37oC for 40 minutes. The cell-permeant

132

2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used as an indicator for

133

reactive oxygen species (ROS) in cells. After the incubation period, medium was

134

removed, cells were returned to fresh culture medium and fluorescence was measured

135

at 495nm excitation and 520nm emission using a multi-plate reader (Perkin

136

Elmer,Waltham, MA, USA). Unstained cells were used as negative controls.

137

2.4 Single cell gel electrophoresis assay (Comet assay)

138

In brief, 3 x 105 HCE-2 cells per well were seeded in 60mm cell culture plates

139

a day prior to the experiment. Then, cells were treated with various concentrations of

140

H2O2 (200µΜ & 500µΜ), tert-butyl peroxide (25µΜ, 50µΜ) and etoposide (25µΜ,

141

50µM) for 16h. Comet assay was conducted as described previously15. In brief,

142

approximately, 8 x 103 HCE-2 cells (2x104 cells/ml of PBS w/o Ca2+/Mg2+) were

143

suspended in 1 mL low-melting-point agarose (1% w/v) and placed onto super-frosted

144

glass microscope slides. The agarose was allowed to set at room temperature and

145

subsequently slides were immersed in lysis solution (1.2M NaCl, 100mM Na2EDTA,

146

0.1% sodium lauryl sarcosinate, 0.26M NaOH, pH∼13) for 1 h at 4°C in the dark to

147

induce DNA denaturation (alkaline unwinding). Then, slides were washed with rinse

148

solution (0.03M NaOH, 2mM Na2EDTA, pH ~12.3) twice for 20 min at room

149

temperature and subjected to electrophoresis at 13V for 25 min (in the presence of

7

150

rinse solution). Following electrophoresis slides were neutralized in dH20 and stained

151

with 10 µg/ml propidium iodide for 20 min. A Nikon ECLIPSE E200 fluorescence

152

microscope was used for their observation. Scoring of DNA damage and image

153

analysis was performed as described previously (Figure 2D) 16.

154 155

2.5 Real time PCR

156

Real time PCR was conducted as described earlier17. Primers for real time

157

PCR were designed with the Primer Express 3.0 software (Applied Biosystems) and

158

are presented in Table 1. Total RNA from HCE-2 cells was extracted using Trizol

159

reagent according to the manufacturer's instructions. For cDNA synthesis, 4.5 µg of

160

total RNA with 1 mM dNTPs and 50 pmol of random hexamers were used. For real-

161

time PCR analysis, Platinum SYBR Green was used according to the manufacturer’s

162

instructions. Reactions were carried out on an Applied Biosystems Step One

163

Instrument. Reactions were run in triplicate in three independent experiments.

164

Expression data were normalized to beta-actin using the 2-∆∆CT method18.

165

Table 1. Primers used for the real time PCR comparative quantification studies Gene

Forward Primer

Reverse Primer

β-actin

GCGCGGCTACAGCTTCA

CTTAATGTCACGCACGATTTCC

Cyclin A

ACGGGTTGCACCCCTTAAG

CCAAGGAGGAACGGTGACA

Cyclin B1

GGCCTCTACCTTTGCACTTCCT

GCTCGACATCAACCTCTCCAA

Cyclin B2

AAGCTTTTTCTGATGCCTTGCT

AGGGTTCTCCCAATCTTCGTTAT

Cyclin D

AGACCTTCGTTGCCTCTTGTG

ATGGAGGGCGGATTGGAA

Cyclin E

GGCCTTGTATCATTTCTCGTCAT

GCGACCACTGATACCCTGAA

DNA-PK

TGCGTTTGGATCCGCTACA

AGTTAGCCGAAAAGGCATCAAC

NBS1

GCAGGAGGAGAACCATACAGACTT

TGGCACAGTTTTTCCTTCCAA

p53

TCTGTCCCTTCCCAGAAAACC

CAAGAAGCCCAGACGGAAAC

8

P21

GCGGGGCTGCATCCA

AGTGGTGTCTCGGTGACAAAGTC

BAX

CCAAGGTGCCGGAACTGA

CCCGGAGGAAGTCCAATGT

BCL-2

TGCGGCCTCTGTTTGATTTC

GGGCCAAACTGAGCAGAGTCT

BCL-XL

AACGGCGGCTGGGATAC

GCTCTCGGCTGCTGCATT

166 167

2.6 RT2 profilerTM PCR array for DNA damage signaling pathway

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The gene expression analysis of pathway-focused genes was done using the

169

96-well RT2 ProfilerTM PCR Array for Human DNA Damage Signaling Pathway

170

(PAHS-029Z) (Qiagen, USA).

171

Total RNA from HCE-2 cells was extracted using Trizol reagent and 500 ng

172

of total RNA were reverse transcribed via the RT2 First Strand Kit. The amplified

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cDNA was subsequently mixed with RT2 SYBR Green Mastermix and the mixture

174

was aliquoted into the 96-well PCR array, according to the manufacturer's

175

instructions. Reactions were performed in a Roche LightCycler 480 with the

176

following program: 10 min at 95oC (heat activation), 15 sec at 95oC, 1 min at 60oC

177

(for 45 cycles), 15 sec at 60oC and continuous at 95oC (melt curve analysis). Cycle

178

thresholds (Cts) were calculated for each gene with the absolute quantification/second

179

derivative max selection of the Roche LightCycler. Basic result analysis was

180

conducted with the web-based Sabiosciences RT2 Profiler PCR array data Analysis

181

version 3.5 (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The

182

experimental data were analyzed through the comparative ∆∆Ct method, while a two-

183

tailed unpaired Student's t-test analysis was performed for statistics. The web based

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Sabiosciences RT2 Profiler PCR array data Analysis version 3.5 was used for the

185

generation of clustergrams.

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2.7 Statistical analysis 9

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At least three independent experiments were conducted per sample for each

188

condition tested. All values are expressed as the mean ± S.D. GraphPad Prism

189

software (version 8.3.0) was used for all statistical analyses. Comparison of results

190

between two groups was performed by Student’s t test. Analysis of two variables

191

among multiple groups was performed with a two-way ANOVA, followed by

192

Tukey’s multiple comparison test. A value of p<0.05 was considered significant.

193

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3. Results 3.1 ALDH3A1 exhibits enhanced antioxidant properties

195

In a previous report, an isogenic HCE-2 cell line stably expressing human

196

ALDH3A1 was established and ALDH3A1 was shown to protect against thermal and

197

oxidative stress conditions11. In this study, we sought to investigate the antioxidant

198

capacity of ALDH3A1 under the influence of various oxidants with different modes

199

of action19–22. ALDH3A1/ and mock/HCE-2 cells (transfected with the empty

200

expression vector) were treated with H2O2 (0-500µM, 16h) (Figure 1A), tert-butyl

201

peroxide (0-50 µΜ, 16h) (Figure 1B) or etoposide (0-50 µΜ, 16 h) (Figure 1C). The

202

intracellular levels of reactive oxygen species (ROS) in HCE-2 cells were evaluated

203

using the H2DCFDA assay. ALDH3A1-expressing cells exhibited lower ROS levels

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compared to the mock-transfected HCE-2 cells in all conditions tested (Figure 1),

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with the protection effect being more prominent in the cases of treatment with H2O2

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and tert-butyl peroxide (Figure 1A, 1B). Nevertheless, ALH3A1 also reduced ROS

207

levels in HCE-2 cells treated with 25 µM and 50 µΜ of etoposide in a statistically

208

significant manner (Figure 1C).

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Figure 1. Protective effects of ALH3A1 on H2O2-, tert-butyl peroxide- and

211

etoposide-induced increase in intracellular ROS levels in HCE-2 cells. Cells were

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treated with A. H2O2 (0-500µM, 16h), B. tert-butyl peroxide (0-50µΜ, 16h) or C.

213

etoposide (0-50µΜ, 16h) and the intracellular ROS levels were assessed by the

214

H2DCFDA assay. ROS levels are expressed as fold change relative to control

215

(untreated) cells. Results are shown as mean ± S.E. At least three independent

216

experiments were performed for each condition. *P ≤0.05, ** P≤0.01, *** P≤0.001,

217

**** P≤0.0001.

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3.2 ALDH3A1 expression protects HCE-2 cells from DNA damage

220

Next, we examined the extent of the DNA damage induced by these oxidants

221

in the isogenic HCE-2 cell line pair. ALDH3A1/ and mock/HCE-2 cells were 11

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incubated for 16h with H2O2 (200 µM & 500 µM) (Figure 2A), tert-butyl peroxide (25

223

µM & 50 µM) (Figure 2B) or etoposide (25 µM & 50 µM) (Figure 2C). DNA damage

224

was assessed by single cell gel electrophoresis assay (comet assay) conducted under

225

alkaline conditions in order to detect both single and double strand DNA breaks.

226

ALDH3A1 expression was associated with lower DNA damage levels, in a

227

statistically significant manner, for all oxidants tested (Figure 2).

228

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Figure 2. Protective effects of ALDH3A1 on H2O2-, tert-butyl peroxide- and

230

etoposide-induced DNA damage in HCE-2 cells. Cells were treated with A. H2O2

231

(200µM & 500µM), B. tert-butyl peroxide (25µM & 50µM) or C. etoposide (25µM &

232

50µM) for 16 h and subjected to cell gel electrophoresis (comet) assay. D

233

Representative comets of classes 0 (i), 1 (ii), 2 (iii), 3 (iv) and 4 (v) are shown. The

234

parameters used for classifying DNA damage in individual cells are: the size and

235

integrity of comet’s head, the intensity of the tail and the head/tail analogy. The

12

236

scored arbitrary units (AU) represent the extent of DNA damage under each examined

237

condition. Results are shown as mean ± S.E. At least three independent experiments

238

were performed for each condition. * p≤0.05, **** p≤0.0001.

239

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3.3 ALDH3A1 expression leads to differential expression of a panel of cell cycle,

242

apoptosis and DNA damage response-related genes in HCE-2 cells

243

We showed that ALDH3A1 protects from the genotoxic effects of H2O2, tert-

244

butyl peroxide and etoposide, all of which induce DNA damage through different

245

modes of action. This prompted us to investigate the gene expression profile of

246

various proteins involved in cell cycle (Cyclin A, B1, B2, D, E) (Figure 3A), DNA

247

damage response (DDR) (DNA-PK, NBS1) (Figure 3B) and apoptosis (p53, p21,

248

BAX, BCL-2, BCL-XL) (Figure 3C). Real time (RT)- PCR experiments revealed that

249

Cyclin B1, Cyclin B2, Cyclin D, Cyclin E, DNA-PK, NBS1, p53, BAX, BCL-2 and

250

BCL-XL were significantly up-regulated in the ALDH3A1/HCE-2 compared to

251

mock/HCE-2 cells (Figure 3).

13

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Figure 3. ALDH3A1 up-regulates the mRNA levels of a set of cell cycle, DNA

254

damage response and apoptosis-related genes in HCE-2 cells. Total RNA from the

255

ALDH3A1/ and mock/HCE-2 cells was extracted, and the mRNA levels of the target

256

genes was determined by quantitative real-time PCR (comparative quantification

257

∆∆Ct method). A-C. Fold regulation of the examined genes, functionally grouped, in

258

the isogenic HCE-2 cell lines. A. Cell cycle, B. DDR and C. Apoptosis. The

259

expression levels of the examined genes were normalized to those of β-actin, while

260

the control (untreated) cells were used as reference sample. Results are shown as

261

mean ± S.E. At least three independent experiments were performed for each

262

condition. * p≤0.05, ** p≤0.01, *** p≤0.001

263

264

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3.4 ALDH3A1 affects the expression of a set of DNA damage signaling-related

266

genes in HCE-2 cells

267

We showed that ALDH3A1 expressing cells demonstrated remarkable

268

resistance against the H2O2, tert-butyl peroxide and etoposide -induced DNA damage.

269

Moreover, several cell cycle, apoptosis and DDR-related genes were shown to be up-

270

regulated in the ALDH3A1 expressing HCE-2 cells. Next, we analysed, in a wide-

271

scale, the gene expression profile of DDS-related proteins in the isogenic HCE-2 cell

272

line pair, by utilizing the RT2 profiler RT-PCR array. We simultaneously quantified,

273

through a comparative ∆∆Ct method, the expression profile of 84 different genes

274

implicated in DDS. PCR array results demonstrated that ALDH3A1 expressing HCE-

275

2 cells exhibit a significant differentiation in the expression pattern of the examined

276

genes compared to the non-expressing (mock) HCE-2 cells (Figure 4A). For further

277

analysis, we set a threshold of fold regulation ≥1.5 or ≥0.5 and p-value <0.01 (for the

278

samples with Ct <30) and selected a set of 18 genes (Figure 4B/C). The clustergram

279

of the selected genes (Figure 4B) demonstrated the existence of two distinctive

280

clusters with reverse expression patterns in the HCE-2 isogenic cell lines: cluster A.

281

representing the down-regulated and cluster B. representing the up-regulated genes in

282

the ALDH3A1/HCE-2 compared to mock/HCE-2 cells (Figure 4B). Most of the

283

selected genes appeared to be up-regulated in the ALDH3A1/HCE-2 cell line

284

(BRCA1, FANCD2, RBBP8, RAD1, RNF8, CDC25C, CDK7, CDC25A, MAPK12,

285

TP53, SIRT1, MRE11A, FEN1, APEX1, ERCC2, DDB1), while only CDKN1A and

286

GADD45G (Figure 4B/C) were found to be significantly down-regulated.

15

287

288

16

289

290

291

292

Figure 4. ALDH3A1 alters the gene expression profile of a panel of DNA damage

293

signaling genes in HCE-2 cells. Total RNA was extracted from the ALDH3A1/ and

294

mock/HCE-2 cells and RT2 ProfilerTM PCR Array for Human DNA Damage 17

295

Signaling Pathway (PAHS-029Z) was utilized to determine the expression profile of

296

84 different DDS-related genes. Five different housekeeping genes were used for

297

normalization (ACTB, B2M, GAPDH, HPRT1 and RPLP10) of gene expression. The

298

web based Sabiosciences RT2 Profiler PCR array data Analysis (version 3.5) tool was

299

used for analyzing the results and generating clustergrams. Hierarchical clustering of

300

A. all the examined genes and B. the selected genes (fold regulation ≥1.5 or ≥0.5, p-

301

value <0.01) in the isogenic HCE-2 cell line pair. Clustergrams display a heat map

302

indicating the magnitude of gene expression along with a dendrogram which

303

organizes genes in proportion to their expression pattern. The color saturation

304

represents magnitude of gene expression with red squares indicating higher gene

305

expression, black squares no change and green squares lower gene expression. C.

306

Fold change of gene expression of the selected genes in ALDH3A1/ vs mock/HCE-2

307

cells. Genes are grouped based on their function. a. ATM-ATR signaling, b. cell

308

cycle, c. apoptosis and d. DNA damage repair. Results are shown as mean ± S.E.

309

Three independent experiments were performed for each condition Results are shown

310

as mean ± S.E. *p≤0.05, **p≤0.01, *** p≤.001, **** p≤.0001.

311

312

313

3.5 The isogenic HCE-2 cell line pair exhibits altered expression profile of DNA

314

damage signalling -related genes under oxidative stress conditions

315

Next, we examined the expression profile of the DDS-related genes in the

316

isogenic HCE-2 cell line pair under oxidative conditions. Specifically, we treated

317

HCE-2 cells for 16 h with 200 µM H2O2 and investigated the expression of DDS

318

genes through the RT2 profiler PCR array kit. Again, we comparatively quantified

18

319

(through the ∆∆Ct method) the expression of 84 genes included in the PCR array

320

among the ALDH3A1/ and mock/HCE-2 cells under conditions generating oxidative

321

stress. As expected, ALDH3A1 altered the expression profile of the examined genes

322

(Figure 5A). A set of 20 genes was selected based on the fold change of gene

323

expression (≥1.5 or ≥0.5) and statistical significance (p-value <0.01) (only genes with

324

Ct <30 were selected) (Figure 5B/C). The heat-map of the selected genes revealed two

325

main clusters. Genes in cluster A were negatively regulated, while genes in cluster B

326

were positively regulated in the ALDH3A1 expressing cells compared to non-

327

expressing (mock) HCE-2 cells (Figure 5B). Specifically, genes BRCA1, FANCD2,

328

RBBP8, RAD1, CDC25A, H2AFX, CDK7, CDC25C, MAPK12, SIRT1, MRE11A,

329

FEN1, ERCC2, DDB1 and NTHL1 were up-regulated in the ALDH3A1/HCE-2 cell

330

line and genes PPM1D, CDKN1A, BBC3, DDB2 were down-regulated in

331

ALDH3A1/HCE-2 in comparison to mock/HCE-2 cells (Figure 5B/C).

332

19

333

334

20

335 336

337

338

Figure 5. DNA damage signaling genes exhibit different expression profile

339

between ALDH3A1 expressing and non-expressing HCE-2 cells. ALDH3A1/ and 21

340

mock/HCE-2 cells were treated with 200 µM H2O2 for 16 h and their total RNA was

341

extracted. The expression profile of 84 DDS-related genes in the isogenic HCE-2 cell

342

lines was then analysed with the RT2 ProfilerTM PCR Array for Human DNA Damage

343

Signaling Pathway (PAHS-029Z). Gene expression of the target genes was

344

normalized to five different housekeeping genes (ACTB, B2M, GAPDH, HPRT1 and

345

RPLP10). For the analysis of the results and the generation of clustergrams, the web

346

based Sabiosciences RT2 Profiler PCR array data Analysis (version 3.5) tool was

347

utilized. Hierarchical clustering of A. all the examined genes and B. the selected

348

genes (fold regulation ≥1.5 or ≥0.5, p-value <0.01) in the isogenic HCE-2 cell line

349

pair under oxidative stress generating conditions. Clustergrams display a heat map

350

indicating the magnitude of gene expression along with a dendrogram which

351

organizes genes in proportion to their expression pattern. The color saturation

352

represents magnitude of gene expression with red squares indicating higher gene

353

expression, black squares no change and green squares lower gene expression. C.

354

Fold change of gene expression of the, functionally grouped, selected genes in

355

ALDH3A1/ vs mock/HCE-2 cells under oxidative stress conditions. a. ATM-ATR

356

signaling, b. cell cycle, c. apoptosis and d. DNA damage repair. Results are shown as

357

mean ± S.E. Three independent experiments were performed for each condition

358

Results are shown as mean ± S.E. *p≤0.05, **p≤0.01, *** p≤.001, **** p≤.0001.

359

360

4. Discussion

361

Aldehyde dehydrogenase 3A1 (ALDH3A1) has been characterized as corneal

362

crystallin due to its constitutive-abundant expression in cornea (up to 50% of the

363

water-soluble proteins). Its ability to detoxify by-products of lipid peroxidation is

22

364

considered crucial for the survival of corneal epithelium, which is constantly exposed

365

to oxidative environmental stressors (light, UV exposure, molecular oxygen). Beyond

366

its apparent role in detoxification though, ALDH3A1 abundant expression is

367

suggested to be associated with a variety of additional functions contributing to

368

enhancing the cellular defenses against oxidative stress.

369

The main aim of our study was to explore the protective effect of ALDH3A1

370

under conditions of oxidative stress. Therefore, we utilized an HCE-2 cell line, stably

371

transfected with human ALDH3A1 cDNA, established previously11. The expression

372

of ALDH3A1 in HCE-2 cells was associated with enhanced resistance against the

373

oxidative and genototoxic effects of H2O2, tert-butyl peroxide and etoposide.

374

Furthermore, a set of cell cycle (Cyclin A, B1, B2, D, E), apoptosis (p53, p21, BAX,

375

BCL-2, BCL-XL) and DDR (DNA-PK, NBS1) proteins were up-regulated in the

376

ALDH3A1 expressing HCE-2 cells. Moreover, by utilizing the RT2 profilerTM PCR

377

array, we demonstrated in a wide-scale, that ALDH3A1/HCE-2 cells exhibited

378

differentiated expression of genes implicated in ATM/ATR signaling, cell cycle

379

regulation, apoptosis and DNA damage repair compared to the control (mock) HCE-2

380

cells under normal and as well as oxidative stress conditions.

381

The antioxidant properties of ALDH3A1 against H2O2-, tert-butyl peroxide-

382

and etoposide-induced oxidative stress confirmed in this study by the detection of

383

H2DCFDA-derived fluorescence (Fig. 1) are in line with previous reports supporting

384

the antioxidant capacity of ALDH3A1. Specifically, it was demonstrated previously

385

that ALDH3A1 expressing HCE (human corneal epithelium) cells exhibited higher

386

tolerance to the cytotoxic effects 4-HNE and UVC radiation in comparison to the

387

mock transfected HCE cells8. Moreover, in another report, it was shown that

388

ALDH3A1 was able to protect rabbit corneal fibroblastic cells from apoptosis and 23

389

cellular oxidative damage caused by 4-HNE, H2O2, etoposide, mitomycin C and

390

UVR12. Similarly, in another study utilizing stably transfected rabbit corneal

391

keratocytes with ALDH3A1 cDNA it was shown that ALDH3A1 expression was

392

associated with resistance against 4-HNE-induced apoptosis, by preventing 4-HNE

393

protein-adduct formation, maintaining GSH homeostasis and preserving 20S

394

proteasome function10. Along these lines, ALDH3A1-expressing HCE-2 cells

395

exhibited increased viability under treatment with H2O2 and tert-butyl peroxide in

396

comparison to non-expressing (mock) HCE-2 cells.

397

Next, we showed that ALDH3A1 reduced the levels of H2O2-, tert-butyl

398

peroxide- and etoposide-induced DNA damage in HCE-2 cells. To this end, only a

399

few studies have documented the potential of ALDH3A1 to attenuate DNA damage.

400

For instance, Jang et al., demonstrated that overexpression of ALDH3A1 alleviated

401

DNA damage caused by cigarette smoking extract in primary human bronchial

402

epithelial cells, as indicated by H2AX phosphorylation23.

403

Interestingly, ALDH3A1 expression appeared to associate with attenuation of

404

DNA damage caused by agents that exert DNA damaging effects via different modes

405

of action. Etoposide forms a complex with the free ends of DNA and with the nuclear

406

enzyme topoisomerase II which inhibits topoisomerase II binding thus resulting in

407

DNA double strand break formation19,20. On the other hand, H2O2 and tert-butyl

408

peroxide mainly induce oxidative damage in cells, causing a spectrum of DNA

409

lesions, including single and double strand breaks21,22. Therefore, we assessed the

410

effect of ALDH3A1 on the expression of genes implicated in DDR, apoptosis and cell

411

cycle progression in HCE-2 cells by qPCR. Our results indicated that ALDH3A1 up-

412

regulated Cyclin B1, Cyclin B2, Cyclin D, Cyclin E, DNA-PK, NBS1, p53, BAX,

413

BCL-2 and BCL-XL in HCE-2 cells. Our findings agree with previous experimental 24

414

data indicating that ALDH3A1 can modulate key cellular mechanisms, such as cell

415

cycle progression and apoptosis. Specifically, Pappa et al., reported that the

416

expression of ALDH3A1 was reduced in proliferating human primary corneal

417

epithelium cells, while ALDH3A1-expressing HCE cells appeared to have reduced

418

plating efficiency, decreased DNA synthesis and elongated cell cycle in comparison

419

to the non-expressing HCE cells. ALDH3A1 expression was also associated with

420

reduced phosphorylation of the retinoblastoma protein and decreased cyclin A- and

421

cyclin B-dependent kinase activities13. Accordingly, Koppaka et al. showed that

422

ALDH3A1 expression attenuated cell proliferation and induced the accumulation of

423

p53 in the nucleus of hTCEpi cells24. Finally, similar results were also demonstrated

424

in a recent study in which ectopic expression of ALDH3A1 resulted in slower

425

proliferation rate and differentiated gene expression of cyclins A, B1, B2, D1 and p21

426

in human breast adenocarcinoma cells (MCF-7)17.

427

The results of RT-PCR analysis prompted us to further investigate the

428

expression of DDS-related genes, by the RT2 profilerTM PCR array (84 different target

429

genes), in the isogenic HCE-2 cell lines both under normal and oxidative stress

430

generating conditions (i.e., 200 µM H2O2). Our results demonstrated that ALDH3A1

431

altered the expression profile of a panel of DDS-related genes in both conditions

432

tested. Specifically, 18 and 20 genes for normal and oxidative stress conditions

433

respectively were selected based on statistical significance (p-value≤0.01) and

434

up/down- regulation (fold regulation ≥1.5 and ≤0.5 in the ALDH3A1/ vs. mock/HCE-

435

2 cells). In general, ALDH3A1 expression induced similar alterations in the gene

436

expression profile of the examined genes in both conditions tested, considering that

437

14 of the selected genes (BRCA1, FANCD2, RBBP8, RAD1, CDC15A, CDK7,

438

CDC25C, MAPK12, CDKN1A, SIRT1, MRE11A, FEN1, ERCC2 and DDB1) were 25

439

common both in normal and oxidative stress generating conditions (Table 2). Among

440

the selected genes, BRCA1, FEN1 and MAPK12 exhibited the highest up-regulation

441

and CDK1A (p21) the highest down-regulation in ALDH3A1/HCE-2 compared to

442

mock/HCE-2 cells.

443 444

Table 2. Differential expression of DNA damage signaling genes in ALDH3A1/HCE-

445

2 vs mock/HCE-2 cells both under normal and oxidative stress generating conditions

Untreated

Function

Symbol BRCA1

ATM/ATR signaling

Cell cycle

Apoptosis

DNA damage repair

Gene name BRCAI/BRCC1/BROVCA1/IRIS

200 µM H2O2

Fold change

p-value

Fold change

p-value

2,44

0,000157

2,19

0,00058 0,00074

FANCD2

DKFZp762A223/FA-D2/FA4/FACD/FAD/FAD2/FANCD

1,67

0,000020

1,49

RBBP8

CTIP/RIM/SAE2

1,83

0,000024

1,79

8,7E-05

RAD1

HRAD1/REC1

1,68

0,003100

1,84

0,01189

CDC25A

CDC25A2

1,53

0,014738

1,45

0,00098

CDK7

CAK1/CDKN7/MO15/STK1/p39MO15

1,83

0,000513

1,67

0,0004

CDC25C

CDC25

1,49

0,006656

1,51

0,00304

MAPK12

ERK3/ERK6/P38GAMMA/PRKM12/SAPK-3/SAPK3

2,13

0,001781

1,96

0,00106

CDKN1A

CAP20/CDKN1/CIP1/MDA-6/P21/SDI1/WAF1/p21CIP1

0,50

0,001284

0,41

2,8E-05

SIRT1

SIR2L1

1,72

0,001638

1,76

0,00179 0,00132

MRE11A

ATLD/HNGS1/MRE11/MRE11B

1,75

0,001021

1,69

FEN1

FEN-1/MF1/RAD2

2,59

0,000149

2,44

2,6E-05

ERCC2

COFS2/EM9/MGC102762/MGC126218/MGC126219/TTD/XPD

1,47

0,002823

1,57

0,00151

DDB1

DDBA/UV-DDB1/XAP1/XPCE/XPE/XPE-BF

1,84

0,007247

1,82

4,2E-05

446

26

447

448

BRCA1 gene (breast cancer, early onset) encodes for a nuclear protein

449

associated with DDR signaling cascade and tumor suppression. Mutations of BRCA1

450

are related with high risk of breast and ovarian cancer while its deficiency is

451

associated with genetic instability and apoptosis. When DNA damage occurs, BRCA1

452

protein facilitates the phosphorylation of p53 by ATM and consequently induces cell

453

cycle arrest and DNA repair 25,26. FEN1 encodes for a flap endonuclease participating

454

in laggind strand replication and base excision repair Specifically, in laggind strand

455

replication FEN1 removes the 5' end of Okazaki fragments, while in DNA repair, it

456

removes 5' overhanging flaps. Inhibition of FEN1 activation has been associated with

457

cell cycle abnormalities as well as with genomic instability27–30. MAPK12 (p38

458

gamma or ERK6) gene encodes for a serine/threonine kinase. MAPK12 is activated

459

through phosphorylation in response to DNA damage and translocates in the nucleus,

460

where it induces DNA repair and G2/M cell cycle checkpoint31. CDK1A gene encodes

461

for the cyclin-dependent kinase inhibitor p21, a protein involved in cell cycle

462

regulation, apoptosis and DNA damage response. p21 inhibits the activity of certain

463

cyclin-dependent kinases as a response to various stimuli thus negatively regulates

464

cell cycle progression. Its role in DNA repair is considered controversial, with some

465

studies suggesting its direct involvement and others its inhibitory role in DNA

466

repair.32–34.

467

The effect of ALDH3A1 on the expression of these genes, is of pivotal

468

importance as it could explain the protective effect of ALDH3A1 in tissues like

469

cornea, that constitutes a tissue of first line of defense, under conditions of oxidative

470

stress. Furthermore, it will unfold new perspectives regarding the role of ALDH3A1,

471

and ALDHs in general, in cancer stem cell (CSC) phenotype35–37. We propose that the 27

472

positive regulation of DDS signaling even under non-oxidative conditions could keep

473

corneal cells to a state of cellular alertness, leading into a more dynamic and quick

474

response when DNA damage occurs, thus conferring a distinct survival advantage in

475

cells expressing ALDH3A1. Additionally, the reported nuclear localization of

476

ALDH3A1 in HCE and hTCEpi cells could be related with our findings as ALDH3A1

477

presence in the nucleus further supports the assumption that it could participate in the

478

regulation of gene expression24. Nevertheless, ALDH3A1 could additionally alter the

479

regulation of certain genes through its metabolic role. ALDH3A1 contributes to the

480

anti-oxidant defense either by the direct scavenging of ROS or indirectly through re-

481

cycling NADPH14 Therefore, ALDH3A1 could participate in gene expression

482

regulation by controlling cellular ROS levels and redox signaling. Further studies will

483

shed light on the precise roles of ALH3A1 in DDS cascade as well as on the

484

molecular mechanism underlying through which ALDH3A1 exert its cytoprotective

485

properties.

486

487

ACKNOWLEDGMENTS

488

This research has been co-financed by the European Union (European Social Fund-

489

ESF) and Greek national funds through the Operational Program “Education and

490

Lifelong Learning” of the National Strategic Reference Framework (NSRF) −

491

Research Funding Program: Heracleitus II. Investing in knowledge society through

492

the European Social Fund.

493

494

28

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•

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a multi-faceted protein with important implications in the corneal epithelial homeostasis.

•

Ectopic expression of ALDH3A1 protected human corneal epithelial (HCE-2) cells from H2O2-, tert-butyl peroxide- and etoposide-induced oxidative and genotoxic effects.

•

ALDH3A1 expression affected the regulation of certain cell cycle-, apoptosisand DNA damage signaling (DDS)-related genes in HCE-2 cells.

•

Pathway-focused gene expression profiling of ALDH3A1-expressing and nonexpressing HCE-2 cells demonstrated that several genes associated with ATM/ATR signaling, cell cycle regulation, apoptosis and DNA damage repair were differentially expressed under normal and oxidative stress conditions.

•

ALDH3A1 may contribute to the antioxidant defenses of corneal epithelial homeostasis by maintaining DNA integrity through altering the expression of specific DDS-related genes.