Open Access

Identification of genes involved in ceramide-dependent neuronal apoptosis using cDNA arrays

  • Charles Decraene1, 4,
  • Bernard Brugg2,
  • Merle Ruberg3,
  • Eric Eveno1,
  • Christiane Matingou1,
  • Fariza Tahi1, 5,
  • Jean Mariani2,
  • Charles Auffray1 and
  • Geneviève Pietu1, 4Email author
Genome Biology20023:research0042.1

https://doi.org/10.1186/gb-2002-3-8-research0042

Received: 23 November 2001

Accepted: 8 May 2002

Published: 31 July 2002

Abstract

Background

Ceramide is important in many cell responses, such as proliferation, differentiation, growth arrest and apoptosis. Elevated ceramide levels have been shown to induce apoptosis in primary neuronal cultures and neuronally differentiated PC 12 cells.

Results

To investigate gene expression during ceramide-dependent apoptosis, we carried out a global study of gene expression in neuronally differentiated PC 12 cells treated with C2-ceramide using an array of 9,120 cDNA clones. Although the criteria adopted for differential hybridization were stringent, modulation of expression of 239 genes was identified during the effector phase of C2-ceramide-induced cell death. We have made an attempt at classifying these genes on the basis of their putative functions, first with respect to known effects of ceramide or ceramide-mediated transduction systems, and then with respect to regulation of cell growth and apoptosis.

Conclusions

Our cell-culture model has enabled us to establish a profile of gene expression during the effector phase of ceramide-mediated cell death. Of the 239 genes that met the criteria for differential hybridization, 10 correspond to genes previously involved in C2-ceramide or TNF-α signaling pathways and 20 in neuronal disorders, oncogenesis or more broadly in the regulation of proliferation. The remaining 209 genes, with or without known functions, constitute a pool of genes potentially implicated in the regulation of neuronal cell death.

Background

Ceramide is an intracellular lipid second messenger generated in response to a large number of extracellular signals [1,2]. These include tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), ionizing and ultraviolet radiation, anti-cancer drugs, growth-factor withdrawal, infection by human immunodeficiency virus (HIV) or bacteria. It is reported to participate in cell differentiation [3], senescence [4], growth arrest or programmed cell death [1,2], depending on the cell type.

The role of ceramide in programmed cell death or apoptosis has been described in lymphocytes [5], macrophages [6], neurons in primary culture [7,8] and neuronally differentiated PC12 cells [9,10,11]. A number of downstream targets of ceramide have been identified. The best documented are the ceramide-activated protein phosphatases (CAPP) and the ceramide-activated protein kinase (CAPK). The former, represented by the PP1 and PP2A families, mediate the effect of ceramide on the transcription factors c-Myc [12] and c-Jun [13]. CAPK is involved in the mitogen-activated protein (MAP) kinase (MAPK) cascades that include the extracellular-signal regulated kinases (ERK), the c-Jun N-terminal kinases or stress-activated kinases (JNK/ SNK/SAPK) and the p38 family [14].

Recently, it has been shown that C2-ceramide rapidly decreases phosphorylation of ERKs, but increases p38 and JNK phosphorylation, activating the transcription factors c-Fos, c-Jun and p53, during the effector phase of apoptosis in primary cortical neurons [15]. It also regulates the protein kinase B (Akt/PKB)-dependent survival pathways, inactivating Akt by dephosphorylation and activating the Bcl-2-related protein BAD by phosphorylation [16,17,18]. Ceramide-induced apoptosis in neurons or in neuronally differentiated PC12 cells has been associated with mitochondrially produced reactive oxygen species (ROS) as well as activation and nuclear translocation of the transcription factor NFκB [10,11,19]. All these molecular events are observed during the effector phase of ceramide-induced apoptosis which also includes gene expression and new protein synthesis required for ceramide-mediated cell death, as it has been shown that neuronal cell death can be inhibited by cycloheximide [7].

The genes that are transcriptionally regulated during ceramide-mediated cell death are still poorly documented. To study gene expression during neuronal cell death, we carried out a differential screen of an array of 9,120 cDNA clones from a human infant brain library (library 1NIB [20]) with complex cDNA targets derived from neuronally differentiated rat pheocytochroma PC12 cells treated with C2-ceramide compared to control PC12 cells. This model is particularly suitable for establishing a gene-expression profile during ceramide-mediated neuronal death because first, the neuronal cell population is synchronized and homogeneous, unlike brain tissue or primary neuronal cultures, and second, because the use of exogenous C2-ceramide eliminates the risk of interference by transcripts activated by signal transducers upstream of ceramide in the cell-death pathway or in pathways activated in parallel.

Results

Cell death induced in neuronally differentiated PC12 cells by C2-ceramide

The morphological characteristics of differentiated PC12 cells after 24 hours in the presence of 25 μM C2-ceramide were compatible with cell death by apoptosis. Compared with control cultures, as viewed by phase-contrast microscopy (Figure 1a), C2-ceramide-treated cells lost their neurites and became rounded and shrunken after 24 hours of treatment (Figure 1b). The cells that remained viable in the C2-ceramide-treated cultures were refringent (Figure 1b), like those in the control cultures (Figure 1a), and excluded the vital marker propidium iodide (Figure 1c), whereas the dead cells took up propidium iodide that intercalated into their DNA (Figure 1d), revealing condensed and fragmented nuclei. As previously described, when neuronally differentiated PC12 cells or primary cultures of mesencephalic neurons were treated with cell-permeant C2-ceramide (10-50 μM), they died in a dose-dependent manner [7,10]. At 25 μM no significant cell death was observed until 12 hours after the initiation of treatment (Figure 2a). After 24 hours, 50% of the cells had died. By 48 hours, no viable cells remained. Furthermore, we observed activation of caspase-3/CPP32, a member of the cysteine-activated aspartate family of cell-death proteases [21], that started 8 hours after the beginning of ceramide treatment and was five times the control value by 18 hours (Figure 2b). No significant cell death and caspase-3/CPP32 activity were observed using the inactive C2 analog of ceramide, C2-dihydroceramide (Figure 2).
Figure 1

Morphological characteristics of nerve growth factor (NGF)-differentiated PC12 cells during C2-ceramide-induced apoptosis. (a) Control cultures of PC12 cells after 6 days in the presence of NGF viewed by phase-contrast microscopy; (b) NGF-differentiated PC12 cells after 24 h treatment with 25 μM C2-ceramide. Open arrows, viable cells; white arrows, dead cells. (c,d) Condensed and fragmented nuclei of dead cells in (c) control and (d) NGF-differentiated PC12 cells visualized by intercalation of propidium iodide into DNA were viewed under epifluorescence illumination.

Figure 2

Characterization of C2-ceramide-induced apoptosis. (a) Time course of cell death induced by 25 μM C2-ceramide (circles) or by 25 μM C2-dihydro-ceramide (triangles). Cells were counted in at least 10 randomly chosen fields with a 20x objective. The percentage of cells excluding the vital dye propidium iodide was calculated at each time point after the beginning of C2-ceramide treatment with respect to the corresponding control. (b) Time course of caspase-3-like activity after 25 μM C2-ceramide (circles) or 25 μM C2-dihydroceramide (triangles) treatment. Data are mean ± SEM (bars) values of at least three experiments, performed in triplicate. The black arrows indicate the time of C2-ceramide treatment of the cell cultures used in the expression study.

Validation of hybridization signals

Hybridization of 9,120 cDNA clones with complex cDNA targets from poly(A)+ RNA extracted from C2-ceramide-treated or control cells produced signals of varying intensities (Figure 3a). In order to eliminate clones for which no reproducible hybridization signals were obtained, the signal-intensity values were validated as described in Materials and methods. Thus, 7% of the clones hybridized with the control cDNA target (634) and 14% of clones hybridized with the C2-ceramide-treated cDNA target (1,297) were excluded from further analysis. The remaining 6,494 clones were analyzed for differential hybridization.
Figure 3

Hybridization signal analysis. (a) Macroarray of 9,120 cDNA clones hybridized with complex cDNA targets derived from mRNA of neuronally differentiated PC12 cells without C2-ceramide treatment (control) or treated with C2-ceramide (stimulated). (b) Distribution of the hybridization signal intensities between control and stimulated cells. Some genes identified in the present study are indicated.

Differential gene expression in neuronally differentiated PC12 cells treated with C2-ceramide compared to controls

Changes in gene expression were analyzed during the effector phase of neuronal death, 7 hours after the beginning of C2-ceramide treatment. This time point was chosen because on the one hand it is preceded by the activation of the transcription factor NFκB and c-Jun observed 4 to 6 hours after C2-ceramide treatment in PC12 cells [10,22], and on the other, the apoptotic process is still not induced by caspase-3 activation, which occurs 8 hours after the beginning of C2-ceramide treatment.

Hybridization between the rat PC12 cell-derived targets and the human cDNA macroarray was carried out as described in Materials and methods. Modulation of gene expression was quantitated by calculating the ratio of the intensity of the normalized hybridization signal obtained with the C2-ceramide cDNA target to that obtained with the control target. Clones were considered to be differentially hybridized in C2-ceramide-treated cells compared to control cells if the ratio between the corresponding hybridization intensity values was ≥ 2 (up-hybridized clones) or ≤ 0.5 (down-hybridized clones) which are the limits of confidence for the method. To decrease the risk of false-positive results, clones with hybridization signals that were less than twofold above background were also excluded, resulting in the elimination of 538 clones. In addition, the remaining clones were hybridized with complex cDNA targets from poly(A)+ RNA extracted from C2-dihydroceramide-treated cells used as negative control and compared to untreated cells. No modulation of expression was observed (except for one clone excluded from the analysis) in the presence of this inactive analog of C2-ceramide (data not shown). Among the 239 clones that met the criteria for differential hybridization, 132 were up-hybridized in C2-ceramide-treated cells and 107 were down-hybridized. The distribution of the hybridization-intensity values between the control and the C2-ceramide complex cDNA targets is presented in Figure 3b. Approximately 55% (72/132) of the up-hybridized clones were hybridized 3-6-fold more in C2-ceramide-treated cells than in the control and 40% (41/107) of the down-hybridized clones were hybridized 3-9-fold less.

Partial 5' and 3' sequences of the 239 clones were compared with all the sequences in the database developed in our laboratory (the Genexpress Index [23]) and in public databases. Of the 239 clones, 179 clones corresponded to already identified human genes, 113 of which have defined functions. The remaining 60 clones corresponded to genes with limited characterization. Under the hypothesis that differential hybridization of the clones reflects linear modulation of expression of the corresponding genes, we assume that we have detected differential gene expression using cDNA array technology that can be interpreted according to the information available.

Ten differentially expressed genes encode proteins with a role in ceramide or TNF-α signaling pathways (Figure 4, Table 1; see [24] for links to database entries for each gene). Two of these genes, PLA2G4C [25] and CLN3 [26,27] seem to have a role in ceramide-mediated cell death or survival. Two upregulated genes (ETV5 [28], NPTX2 [29,30]) and two downregulated genes (COL1SA1 [31,32], TNFAIP1 [33]) encode proteins that are modulated by TNF-α. Four genes, three upregulated (AXL [34], BIRC1 [35], RSU1 [36]) and one downregulated (MAPK10 [37]) encode proteins with a role in the TNF-α signaling pathway.
Figure 4

Differentially expressed genes that encode proteins with functions involved in ceramide-dependent apoptosis. Black boxes, Genes involved in the ceramide signaling pathway; gray boxes, genes transcriptionally stimulated by TNF-α; white boxes, genes involved in the TNF-α signaling pathway.

Table 1

Genes differentially expressed in ceramide-dependent apoptosis and involved in the ceramide and TNF-α pathways

Clone ID

GENX

GenBank accession number

Unigene

C. int.

C. SD

S. int.

S. SD

Ratio

Similarity

Gene symbol

Genes involved in the C2-ceramide signaling pathway

   yf59e08

5705

R13531

Hs.18858

3.56

0.54

12.01

0.86

3.37

Phospholipase A2, group IVC (cytosolic, calcium-independent)

PLA2G4C

   yf71a08

115123

R12998; R40387

Hs.194660

1.14

0.23

2.53

0.15

2.21

Ceroid-lipofuscinosis, neuronal 3, juvenile (Batten, Spielmeyer-Vogt disease)

CLN3

Genes transcriptionally stimulated by TNF-α

   yg86b08

4272

R53048; R53135

Hs.43697

1.72

0.18

8.77

0.58

5.09

Ets variant gene 5 (ets-related molecule)

ETV5

   yc86d06

5838

F12910; T75064

Hs.3281

3.61

0.48

11.54

0.61

3.19

Neuronal pentraxin II

NPTX2

   yc88f11

197

F10424; F12821

Hs.78409

1.18

0.21

0.55

0.11

0.47

Collagen, type XVIII, alpha 1

COL18A1

   yf78g01

200888

R14176; R40470

Hs.76090

1.19

0.28

0.54

0.06

0.46

TNF-α-induced protein 1 (endothelial)

TNFAIP1

Genes involved in the TNF-α signaling pathway

   yf76d09

1017

R13424; R40936

Hs.83341

1.49

0.23

5.45

0.54

3.65

AXL receptor tyrosine kinase

AXL

   yg49f10

116415

R20716

Hs.79019

4.54

0.13

12.69

1.38

2.80

Baculoviral IAP repeat-containing 1

BIRC1

   c-26g10

1350

F07467

Hs.75551

1.37

0.27

2.81

0.56

2.05

Ras suppressor protein 1

RSU1

   c-08d10

4997

F05370; Z38358

Hs.151051

4.94

1.06

2.41

0.51

0.49

Mitogen-activated protein kinase 10

MAPK10

Clone ID, clone name according to the public databases. GENX, cluster name including the corresponding cDNA sequence in the Genexpress Index 2 ([23] and R. Mariage-Samson et al., unpublished data). UniGene, cluster name in the UniGene database [85]; C. int., mean of the normalized and validated intensity values obtained after filter hybridization with complex cDNA target derived from control mRNA. C. SD, standard deviation derived from the C.int. S. int., mean of the normalized and validated intensity values obtained after filter hybridization with complex cDNA target derived from ceramide-stimulated cultured cell mRNA. S. SD, standard deviation derived from the S. int. Ratio, ratio of S. int. to C. int. Similarity, gene similarity.

Twenty clones correspond to genes encoding proteins that have been involved in the regulation of apoptosis and/or cell growth (Figure 5, Table 2, see [24]). Fourteen are up-hybridized and six are down-hybridized by C2-ceramide. Ten of the upregulated and two of the downregulated genes encode proteins stimulating apoptosis and/or growth arrest. The other genes (four upregulated and four downregulated) encode proteins downregulating apoptosis and/or stimulating growth.
Figure 5

Differentially expressed genes that encode proteins involved in the regulation of apoptosis and/or cell growth. Gray boxes, genes stimulating apoptosis and/or growth arrest; white boxes, genes downregulating apoptosis and/or stimulating growth.

Table 2

Differentially expressed genes that encode proteins involved in the regulation of apoptosis and/or cell growth

Clone ID

GENX

GenBank accession number

Unigene

C. int.

C. SD

S. int.

S. SD

Ratio

Similarity

Gene symbol

Proteins stimulating apoptosis and/or growth arrest

   yg01b10

2112

R18353; R42557

Hs.286

1.68

0.17

7.95

0.55

4.74

Ribosomal protein L4

RPL4

   yl73h11

567

H06473

Hs.9663

1.60

0.15

7.42

0.54

4.64

Programmed cell death 6-interacting protein

PDCD6IP

   yc92h11

673

F13260; T77039

Hs.75709

2.12

0.33

9.83

0.95

4.64

Mannose-6-phosphate receptor (cation dependent)

M6PR

   yg94h08

6030

R56149

Hs.78776

1.96

0.34

7.09

1.25

3.61

Putative transmembrane protein

NMA

   yf90d04

25970

R15366

Hs.20912

1.34

0.22

4.71

0.58

3.51

Adenomatous polyposis coli like

APCL

   yg76b02

3804

R51346; R51453

Hs.78935

0.95

0.18

2.89

0.40

3.03

Methionine aminopeptidase; eIF-2-associated p67

METAP2

   yd01h06

9451

R39334; T78769

Hs.274348

1.98

0.16

5.89

0.66

2.97

HLA-B associated transcript-3

BAT3

   c-22F12

2915

F08770

Hs.75323

1.44

0.23

3.26

0.29

2.26

Prohibitin

PHB

   yf69g07

115124

R14126

Hs.132955

1.82

0.30

3.99

0.96

2.19

BCL2/adenovirus E1B 19 kD-interacting protein 3-like

BNIP3L

   yd02b11

115910

T79985

Hs.63984

0.88

0.19

1.88

0.30

2.14

Cadherin 13, H-cadherin (heart)

CDH13

   c-3ke04

781

F10823; F13223

Hs.12409

1.17

0.10

ND

ND

0.43

Somatostatin

SST

   yg64g08

115205

R35542; R51110

Hs.288986

3.01

0.41

0.90

0.15

0.30

Survival of motor neuron 1 1, telomeric

SMN1

Proteins downregulating apoptosis and/or stimulating growth

   yg44d03

408

R25503

Hs.155212

1.74

0.40

7.67

0.40

4.40

Methylmalonyl coenzyme A mutase

MUT

   yg68d10

2957

R36284; R49571

Hs.89582

1.80

0.25

7.26

0.57

4.04

Glutamate receptor, ionotropic, AMPA 2

GRIA2

   yl81d04

9379

H05457; H07007

Hs.150423

2.64

0.36

8.72

1.06

3.31

Cyclin-dependent kinase 9 (CDC2-related kinase)

CDK9

   yd02a11

78693

T79973

Hs.107911

2.52

0.42

5.38

0.44

2.14

ATP-binding cassette, sub-family B (MDR/TAP), member 6

ABCB6

   yg51a11

17820

R21694; R46587

Hs.223014

1.09

0.24

ND

ND

0.46

Antizyme inhibitor

OAZIN

   yh10g09

4858

R61276; R61277

Hs.8073

1.59

0.23

0.66

0.16

0.41

Septin 3

SEP3

   yf53a12

3165

R12025; R37093

Hs.356245

1.14

0.21

0.29

0.02

0.25

Apoptosis regulator

LOC51283

   yg67b12

115951

R35827;

Hs.285754

2.38

0.48

0.50

0.12

0.21

Met proto-oncogene

MET

Abbreviations and column headings are as in Table 1.

The remaining 83 clones corresponding to 82 genes with known or putative functions have no obvious relation to the apoptosis process (Table 3, see [24]). Of the total number of differentially hybridized clones, 66 correspond to mRNA sequences (Table 4, see [24]) and 60 to poorly characterized genes (Table 5, see [24]) that encode proteins without known function.
Table 3

Known genes differentially expressed in ceramide-dependent apoptosis with no identified direct interaction with the ceramide-dependent apoptosis process

Clone ID

GENX

GenBank accession number

Unigene

C. int.

C. SD

S. int.

S. SD

Ratio

Similarity

Gene symbol

Signal transduction

   yl85b10

1842

H05211

Hs.22003

1.62

0.40

7.99

0.37

4.94

Solute carrier family 6 (neurotransmitter transporter, GABA), member 1

SLC6A1

   yf77g11

3900

R14207; R37490

Hs.75819

1.12

0.12

5.02

0.43

4.46

Glycoprotein M6A

GPM6A

   yg63f10

1552

R26636; R49665

Hs.24212

1.01

0.15

4.02

0.61

3.98

Latrophilin

KIAA0786

   c-2ee07

116218

Z45003

Hs.107979

1.75

0.35

6.17

0.85

3.52

Small membrane protein 1

SMP1

   yf60h11

12653

R13771

Hs.61628

1.43

0.18

4.68

0.61

3.28

Calcium binding atopy-related autoantigen 1

CBARA1

   yf88a09

9668

R15201

Hs.181326

4.01

0.50

11.65

2.01

2.90

Myotubularin-related protein 2

MTMR2

   yg11b08

107475

R17181; R41731

Hs.5462

0.72

0.12

1.54

0.33

2.14

Solute carrier family 4, sodium bicarbonate cotransporter, member 4

SLC4A4

   c-2mh12

1997

Z41050; Z45338

Hs.108787

1.08

0.21

0.52

0.04

0.47

Phosphatidylinositol glycan, class N

PIGN

   yc87e10

115203

F10343; F12737

Hs.173717

1.24

0.17

0.50

0.06

0.40

Phosphatidic acid phosphatase type 2B

PPAP2B

   yf48c10

9043

R12286; R12797

Hs.10842

1.10

0.24

0.43

0.03

0.39

RAN, member RAS oncogene family

RAN

   yd09f12

2991

R39085

Hs.306359

2.39

0.46

0.90

0.22

0.38

Hect domain and RCC1 (CHC1)-like domain (RLD) 1

HERC1

   c-3ie05

5307

F10685; F13091

Hs.9347

1.48

0.24

0.53

0.02

0.36

Regulator of G-protein signaling 14

RGS14

   yg16c08

5294

R17962; R43452

Hs.1440

1.05

0.23

0.29

0.04

0.27

Gamma-aminobutyric acid (GABA) A receptor, beta 3

GABRB3

   yf50c04

1366

R11777; R37698

Hs.5985

1.13

0.12

0.17

0.01

0.15

Non-kinase Cdc42 effector protein SPEC2

LOC56990

Transcription/translation

   yf71g02

5232

R40420

Hs.16313

0.90

0.13

2.30

0.15

2.55

Kruppel-like zinc-finger protein GLIS2

GLIS2

   c-26a02

451

F07446

Hs.13993

1.64

0.38

3.39

0.73

2.07

TBP-like 1

TBPL1

   c-05c07

4917

Z38284; Z41997

Hs.26973

1.21

0.20

2.45

0.52

2.02

Bromodomain adjacent to zinc-finger domain, 2B

BAZ2B

   c-24a11

114423

F07382

Hs.75678

1.38

0.23

0.66

0.16

0.47

FBJ murine osteosarcoma viral oncogene homolog B

FOSB

   yg90e12

10904

R56427; R56428

Hs.239

1.28

0.23

0.59

0.03

0.46

Forkhead box M1

FOXM1

   yf61e03

4401

R13803; R37662

Hs.182447

7.20

1.01

2.75

0.60

0.38

Heterogeneous nuclear ribonucleoprotein C (C1/C2)

HNRPC

   yf64g02

993

R37803

Hs.6151

4.87

0.77

1.87

0.46

0.38

Pumilio homolog 2 (Drosophila)

PUM2

   yg53f10

1678

R62465; R25720

Hs.520

1.41

0.20

ND

ND

0.35

Nuclear receptor subfamily 2, group C, member 2

NR2C2

   yg47e10

1548

R21283; R45373

Hs.14520

1.55

0.26

0.53

0.13

0.34

Eukaryotic translation initiation factor 2C, 1

EIF2C1

   yg36d06

1872

R24568; R44373

Hs.76177

10.91

1.23

3.67

0.10

0.34

Transcription factor CP2

TFCP2

   yg60b12

303

R35123; R49511

Hs.2186

3.12

0.63

0.94

0.07

0.30

Eukaryotic translation elongation factor 1 gamma

EEF1G

   yg27a08

4127

R43968

Hs.278589

9.43

1.24

2.76

0.40

0.29

General transcription factor II, i, pseudogene 1

GTF2IP1

Cellular traffic or structure proteins

   yg19f05

200119

R20424; R43544

Hs.169793

1.51

0.36

7.37

1.32

4.86

Ribosomal protein L32

RPL32

   yc86h03

2760

F12918; T75229

Hs.182625

2.38

0.26

7.78

1.17

3.27

Vamp (vesicle-associated membrane protein)-associated protein B and C

VAPB

   yc87f04

5084

R38549; T75126

Hs.22826

1.83

0.11

5.59

0.64

3.06

Tropomodulin 3 (ubiquitous)

TMOD3

   yf98g01

8512

R18713

Hs.75196

2.96

0.63

9.29

0.80

3.14

Ankyrin repeat-containing protein

G9A

   yh17e09

1304

R59488; R59489

Hs.30991

0.78

0.19

2.32

0.11

2.97

Ankyrin repeat domain 6

ANKRD6

   yf76d11

424

R13426; R40938

Hs.119324

0.84

0.08

2.07

0.35

2.48

Kinesin-like 4

KNSL4

   c-27f03

1382

F07488

Hs.89497

2.32

0.31

5.60

0.64

2.42

Lamin B1

LMNB1

   yc96a12

11155

F13331; T77651

Hs.159613

4.50

0.32

10.84

2.13

2.41

Thyroid hormone receptor binding protein

AIB3

   yf57c11

1225

R12822; r20734

Hs.1501

0.94

0.22

2.25

0.22

2.39

Syndecan 2

SDC2

   yl71a06

10804

H05894

Hs.6682

1.33

0.11

2.94

0.20

2.21

Solute carrier family 7, cationic amino acid transporter, y+ system, member 11

SLC7A11

   yc99f07

11082

T78361

Hs.103042

2.21

0.07

0.98

0.19

0.44

Microtubule-associated protein 1B

MAP1B

   yf72e08

2558

R13080; R40510

Hs.7979

2.05

0.34

0.80

0.15

0.39

Likely ortholog of mouse synaptic vesicle glycoprotein 2a

SV2

   yc87h12

2952

F10545; F12946

Hs.21611

5.68

0.59

1.93

0.17

0.34

Kinesin family member 3C

KIF3C

   yg54d05

604

R25813; R46810

Hs.117977

1.62

0.33

0.50

0.11

0.31

Kinesin 2 (60-70 kD)

KNS2

   yf91b02

1980

R16352; R42300

Hs.103042

3.50

0.41

1.01

0.24

0.29

Microtubule-associated protein 1B

MAP1B

   yf72a03

115963

R13048; R40479

Hs.187958

1.46

0.34

0.40

0.06

0.28

Solute carrier family 6, member 8, accessory proteins BAP31/BAP29

SLC6A8, DXS1357E

Immunity/inflammatory response

   yg75d06

25621

R54423

Hs.179661

1.88

0.18

8.01

1.02

4.26

FK506-binding protein 1A (12 kD)

FKBP1A

   yg65b03

2453

R35324

Hs.9688

0.86

0.13

3.67

0.60

4.26

Leukocyte membrane antigen

IRC1

   yg57f05

190007

R34428

Hs.181244

3.83

0.24

9.76

1.22

2.55

MHC class I gene family

 

   yf51e08

2563

R12005; R39844

Hs.75682

0.89

0.04

2.05

0.21

2.31

Autoantigen

RCD-8

   c-2bh04

190137

F03851; F07604

Hs.284394

1.13

0.07

0.56

0.07

0.50

Complement component 3

C3

   yf59h02

5580

R13549; R20669

Hs.82689

1.05

0.21

0.47

0.10

0.44

Tumor rejection antigen (gp96) 1

TRA1

   yc86g03

8628

F10456; F12856

Hs.302749

1.51

0.35

0.58

0.09

0.39

FK506-binding protein 9 (63 kD)

FKBP9

Protein processing

   yf68a10

1071

R40190;

Hs.75890

0.55

0.13

2.09

0.23

3.80

Site-1 protease (subtilisin-like, sterol-regulated, cleaves sterol regulatory element binding proteins)

S1P

   c-2na07

2001

F04230; F07978

Hs.102

1.01

0.16

0.46

0.10

0.45

Aminomethyltransferase (glycine cleavage system protein T)

AMT

   yc85d05

6301

F10498; F12892

Hs.170197

1.45

0.22

0.59

0.10

0.41

Glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2)

GOT2

   c-2ge12

2793

Z40826; Z46090

Hs.183212

1.14

0.19

0.45

0.09

0.39

Isoprenylcysteine carboxyl methyltransferase

ICMT

   yg52f04

202164

R21082; R46258

Hs.235887

1.55

0.31

0.54

0.08

0.34

HMT1 (hnRNP methyltransferase, Saccharomyces cerevisiae)-like 1

HRMT1L1

Proteases

   yf64f07

2813

R13707; R37801

Hs.171501

1.27

0.24

ND

ND

0.39

Ubiquitin specific protease 11

USP11

Metabolism

   yc97f08

1805

R39698; T78043

Hs.2838

2.73

0.24

9.35

0.86

3.42

Malic enzyme 3, NADP(+)-dependent, mitochondrial

ME3

   yg97d06

3929

R59198; R59256

Hs.78989

0.68

0.07

1.99

0.18

2.94

Alcohol dehydrogenase 5 (class III), chi polypeptide

ADH5

   c-2ca07

1549

F03858; F07608

Hs.180616

1.04

0.12

0.50

ND

0.48

CD36 antigen (collagen type I receptor, thrombospondin receptor)-like 1

CD36L1

   yc95g06

3164

R39463; T77281

Hs.155247

1.01

0.20

0.31

0.05

0.30

Aldolase C, fructose-bisphosphate

ALDOC

Miscellaneous

   yg24g06

3097

R19249; R44514

Hs.22654

0.48

0.08

2.93

0.67

6.12

Sodium channel, voltage-gated, type I, alpha polypeptide

SCNIA

   yc89d05

10816

F10796; F13191

Hs.12365

1.26

0.20

5.46

0.36

4.34

Synaptotagmin XIII

SYT13

   c-28e05

4334

F07514

Hs.6126

2.04

0.50

7.19

1.11

3.52

Mannosidase, beta A, lysosomal-like

MANBAL

   yg35g09

4463

R20330

Hs.88764

3.30

0.19

9.88

2.40

2.99

Male-specific lethal-3 (Drosophila)-like 1

MSL3L1

   yg36c01

292

R24560; R44360

Hs.6430

1.50

0.22

4.40

0.78

2.93

Protein with polyglutamine repeat; calcium (Ca2+) homeostasis endoplasmic reticulum protein

ERPROT213-21

   yc98a06

1475

R37847; T78111

Hs.301789

1.55

0.27

4.52

0.35

2.92

Capping protein (actin filament) muscle Z-line, alpha 1

CAPZA

   yf54h02

924

R11969

Hs.4865

1.41

0.17

3.05

0.18

2.17

Voltage-gated sodium channel beta-3 subunit (scn3b gene)

HSA243396

   yf91a04

434

R16348; R42296

Hs.12152

1.00

0.24

2.03

0.16

2.04

APMCF1 protein

APMCF1

   c-1ia09

4199

Z39718; Z43661

Hs.8834

0.64

0.15

1.30

0.19

2.02

Ring finger protein 3

RNF3

   yg83b04

4211

R53332; R53937

Hs.7022

1.19

0.28

0.59

0.09

0.50

Dedicator of cytokinesis 3

DOCK3

   yf57d02

4610

R12627; R20528

Hs.334688

1.51

0.25

0.75

0.13

0.50

Phytanoyl-CoA hydroxylase interacting protein

PHYHIP

   yg36h06

3087

R24595; R44400

Hs.7122

1.08

0.13

ND

ND

0.46

Scrapie responsive protein 1

SCRG1

   yf99b05

2822

R18211; R42149

Hs.79284

1.07

0.21

0.48

0.07

0.45

Mesoderm specific transcript (mouse) homolog

MEST

   c-24h06

92359

Z40467; Z44591

Hs.171545

1.02

0.20

0.45

0.11

0.44

HIV-1 Rev binding protein

HRB

   yd05d01

2346

R38832; T80384

Hs.13493

1.06

0.24

0.44

0.06

0.42

Like mouse brain protein E46

E46L

   yf74e11

2106

R13277; R40723

Hs.334851

1.95

0.41

0.80

0.17

0.41

LIM and SH3 protein 1

LASP1

   yd01e11

3181

T78746

Hs.168640

1.17

0.27

0.47

0.10

0.40

Homolog of mouse Ank

ANK

   yf48e09

414

R12292; R12804

Hs.21050

1.22

0.26

0.47

0.07

0.38

g20 protein

LOC51161

   yg16d07

1087

R43459; R17969

Hs.87125

9.91

2.06

3.35

0.51

0.34

EH-domain containing 3

EHD3

   yf57d07

12763

R12632; R20533

Hs.109706

1.78

0.23

0.58

0.09

0.33

Hematological and neurological expressed I

HN1

   c-2Ia12

200991

F04056; F07796

Hs.74376

1.00

0.07

0.33

0.01

0.33

Olfactomedin related ER localized protein

NOE1

   yf61b05

1882

R13783; R37641

Hs.297743

2.04

0.16

0.63

0.12

0.31

Carbonic anhydrase X

CA10

   yf61a10

3960

R39112; R13989

Hs.2288

12.49

1.82

3.38

0.84

0.27

Visinin-like 1

VSNL1

   yg53b12

8924

R25707; R62451

Hs.169047

2.34

0.46

0.54

0.12

0.23

Chondroitin sulfate proteoglycan 3 (neurocan)

CSPG3

Abbreviations and column headings are as in Table 1.

Table 4

Messenger RNA or protein sequences differentially expressed in ceramide-dependent apoptosis

Clone ID

GENX

GenBank accession number

Unigene

C. int.

C. SD

S. int.

S. SD

Ratio

Similarity

Upregulated clones

   yg51f11

229

R21710

Hs.64691

1.32

0.16

6.34

0.43

4.80

KIAA0483 protein

   yg30b04

5093

R44721

Hs.12896

1.31

0.12

5.99

0.29

4.57

KIAA1034 protein

   yf53g09

13

R12046

Hs.90424

1.56

0.10

7.09

0.67

4.55

Homo sapiens cDNA: FLJ23285 fis, clone HEP09071

   yc94b11

223

F13362; T77404

Hs.101375

1.40

0.15

6.38

1.10

4.55

cDNA DKFZp434H205 (from clone DKFZp434H205)

   yg37d06

2008

R19640

Hs.264636

1.70

0.39

7.39

1.60

4.34

KIAA0781 protein

   yf75c06

425

R13300; R40783

Hs.26409

0.83

0.18

3.55

0.67

4.27

cDNA DKFZp547K204 (from clone DKFZp547K204)

   yf79e07

2479

R14269; R40562

Hs.19150

0.93

0.15

3.91

0.25

4.22

cDNA DKFZp564A2164 (from clone DKFZp564A2164)

   yf49e01

6657

R11708

Hs.21710

1.64

0.20

6.61

1.00

4.04

Hypothetical protein DKFZp761G0313

   yg07h12

200578

R22668

Hs.7734

1.00

0.09

3.71

0.56

3.70

H. sapiens cDNA: FLJ21380 fis, clone COL03329

   yg32e10

5469

R23681

Hs.106825

1.38

0.15

5.09

0.55

3.68

Hypothetical protein FLJ20300

   yf90d07

6203

R15369; R42110

Hs.323396

1.28

0.20

4.66

0.41

3.63

Hypothetical protein RP1-317E23 (LOC56181)

   yg91g03

1624

R56083; R56195

Hs272814

0.83

0.15

2.89

0.30

3.48

Chromosome 20 open reading frame 67

   yg11e11

2086

R17284

Hs.106210

4.25

0.56

14.04

2.67

3.30

Hypothetical protein FLJ10813

   ym11b06

6715

H11788

Hs.125034

1.98

0.16

6.52

0.85

3.29

H. sapiens cDNA FLJ10733 fis, clone NT2RP3001392

   yf80c08

772

R14304; R40254

Hs.59236

1.21

0.25

3.94

0.63

3.24

Hypothetical protein DKFZp434L0718

   yg18e11

4391

R20224

Hs.41185

3.49

0.86

11.27

1.23

3.23

cDNA DKFZp564O1262 (from clone DKFZp564O1262)

   yc90h10

1343

F13218; T75433

Hs.141003

1.32

0.24

4.19

1.05

3.18

H. sapiens cDNA: FLJ21691 fis, clone COL09555

   yg42a11

200671

R24764; R45496

Hs.288368

0.45

0.10

1.41

0.11

3.14

H. sapiens cDNA: FLJ21314 fis, clone COL02248

   yc85f03

255

F12760; T74722

Hs.318401

3.03

0.42

9.46

0.80

3.12

HSPC039 protein (LOC51124)

   yc86g12

32

F12859; T75226

Hs.180948

4.61

0.28

14.27

1.89

3.10

KIAA0729 protein

   c-2lb03

1917

Z45263

Hs.155182

6.48

1.55

19.98

3.34

3.08

KIAA1036 protein

   yf94d09

2836

R16328; R41404

Hs.6343; HS.306400

3.16

0.48

9.58

0.97

3.03

KIAA1464 protein

   yf49g10

2696

R11887

Hs.40094

4.42

0.68

13.38

1.70

3.03

Human DNA sequence from clone 167A19 on chromosome 1p32.1-33

   yg67h02

1136

R35733; R49366

Hs. 325825

3.76

0.34

11.27

0.80

3.00

H. sapiens cDNA: FLJ20848 fis, clone ADKA01732

   yc89d09

2388

F13194; T75317

Hs.22109

3.52

0.07

10.11

1.40

2.87

KIAA0945 protein

   yf72d11

4469

R13137; R40616

Hs.6311

2.38

0.54

6.80

0.92

2.86

H. sapiens cDNA: FLJ20859 fis, clone ADKA01617

   yg73c09

51540

R51740

Hs.288959

1.31

0.18

3.70

0.65

2.83

H. sapiens cDNA: FLJ20920 fis, clone ADSE00877

   yf50h09

9583

R11919;

Hs.11637

3.87

0.33

10.71

1.31

2.77

H. sapiens mRNA; cDNA DKFZp547J125 (from clone DKFZp547J125)

   c-2ba02

4345

Z41723; Z44845

Hs.15921

1.93

0.37

5.31

1.02

2.75

Hypothetical protein FLJ10759

   yg36f12

11000

R25011; R45019

Hs.118983

1.13

0.27

3.00

0.46

2.65

H. sapiens cDNA FLJ12150 fis, clone MAMMA1000422

   c-24b10

1689

Z44563

Hs.154919

2.67

0.43

6.60

1.44

2.47

KIAA0625 protein

   yf76a11

1849

R13420; R40930

Hs.7822

1.12

0.09

2.73

0.33

2.43

cDNA DKFZp564C1216 (from clone DKFZp564C1216)

   yc91c07

162

F10758; F13156

Hs.140833

0.61

0.14

1.46

0.32

2.41

H. sapiens mRNA full length insert cDNA clone EUROIMAGE 29222

   yc94c08

4310

R38361; T77413

Hs.119004

0.53

0.05

1.25

0.22

2.36

KIAA0665 gene product

   yg57f04

3072

R34427; R48960

Hs.326416

0.90

0.16

1.98

0.35

2.20

cDNA DKFZp564H1916 (from clone DKFZp564H1916)

   yg15g12

5559

R18075; R42970

Hs.22370

0.66

0.14

1.45

0.21

2.19

cDNA DKFZp564O0122 (from clone DKFZp564O0122)

   yg97d02

1018

R59194; R59252

Hs.5324

0.64

0.07

1.32

0.16

2.06

Hypothetical protein (CL25022)

   yc95f04

3851

F13386; R39459

Hs.7888

0.58

0.07

1.16

0.25

2.02

H. sapiens clone 23736 mRNA sequence

Downregulated clones

   yg42e05

4312

R45416; R25077

Hs.169330

1.05

0.23

0.52

0.05

0.49

Neuronal protein (NP25)

   yg89f11

2081

R55970; R55969

Hs.16443

1.16

0.27

0.56

0.06

0.49

H. sapiens cDNA: FLJ21721 fis, clone COLF0381

   yg33e09

5446

R20455; R44158

Hs.333389

1.39

0.19

0.67

0.17

0.48

Hypothetical protein MGC13090

   c-2aa11

1485

Z40609; Z44824

Hs.13485

1.44

0.23

0.70

0.16

0.48

KIAA1918 protein

   yf65e06

5690

R13865; R37007

Hs.301685

1.03

0.21

ND

ND

0.48

KIAA0620 protein

   yl76d07

37588

H05960; H06010

Hs.92418; Hs.63510

3.95

0.73

1.85

0.18

0.47

KIAA0141

   c-2cg09

201091

F03885; F07635

Hs.288361

1.06

0.10

0.49

0.02

0.47

H. sapiens cDNA: FLJ22696 fis, clone HSI11696

   yg64h02

2829

R35543; R51112

Hs.12239

2.94

0.61

1.35

0.30

0.46

CGI-10 protein (LOC51004)

   yf49c08

23982

R11699; R17677

Hs.322844

1.29

0.32

0.58

0.12

0.45

Hypothetical protein DKFZp564A176

   yg33g08

636

R20203; R44989

Hs.7750

8.39

1.43

3.60

0.64

0.43

Novel human gene mapping to chromosome 1

   yf53d08

532

R11837; R36955

Hs.246885

1.07

0.05

0.44

0.08

0.42

Hypothetical protein FLJ20783

   yg65h10

10701

R35431; R49229

Hs.222746

1.04

0.25

0.42

0.09

0.40

KIAA1610 protein

   yg69e11

1257

R36317; R49249

Hs.216958

1.16

0.28

0.44

0.10

0.38

KIAA0194 protein

   yf79f12

5599

R14349; R40677

Hs.179946

2.55

0.37

0.86

0.20

0.34

KIAA1100 protein

   yf86c11

1909

R15181; R41632

Hs.286013

1.06

0.14

0.34

0.08

0.32

Short coiled-coil protein

   yf78c09

1664

R14217; R40635

Hs.351029

10.44

1.29

3.36

0.75

0.32

H. sapiens cDNA FLJ31803 fis, clone NT2RI2009101

   yf61c10

1067

R13997; R39120

Hs.5008; Hs.21515

1.11

0.09

0.35

0.08

0.32

CG-87 protein

   yd06g01

2455

R38891; T81283

Hs.165570

1.41

0.07

0.45

0.10

0.32

H. sapiens clone 25052 mRNA sequence

   yf64f10

111134

R36936

Hs.80285

8.72

0.83

2.76

0.35

0.32

mRNA cDNA DKFZp586C1723 (from clone DKFZp586C1723)

   yc91e03

11140

F13018; T77597

Hs.337629

5.27

1.08

1.67

0.22

0.32

cDNA DKFZp434D115 (from clone DKFZp434D115)

   yf65b02

8918

R13839; R36985

Hs.227913

1.09

0.24

0.32

0.05

0.30

API5-like 1

   yf60a03

1485

R13618; R38474

Hs.13485

2.13

0.36

0.65

0.09

0.30

KIAA1918 protein

   yf56a05

5140

R12419

Hs.7132

1.97

0.33

0.57

0.08

0.29

KIAA0574 protein

   yg78h08

884

R51917; R54309

Hs.6449

1.20

0.28

0.34

0.08

0.28

CGI-87 protein (LOC51112)

   yf69g12

713

R40161

Hs.288776

1.21

0.23

0.29

0.04

0.24

H. sapiens cDNA: FLJ21304 fis, clone COL02111

   yf93b11

2192

R16295; R40219

Hs.108504

1.25

0.16

0.24

0.05

0.20

Hypothetical protein FLJ20113

   yf51g08

2572

R12017; R39856

Hs.20977

1.11

0.20

0.19

0.04

0.17

Human DNA sequence from clone RP5-881L22 on chromosome 20

   yg26c11

904

R19006; R44076

Hs.226396

3.13

0.50

0.35

0.08

0.11

Hypothetical protein FLJ11126

Abbreviations and column headings are as in Table 1.

Table 5

Unknown genes differentially expressed in ceramide-dependent apoptosis

Clone ID

GENX

GenBank accession number

Unigene

C. int.

C. SD

S. int.

S. SD

Ratio

Similarity

Upregulated clones

   yf66a04

17755

R18781

 

1.04

0.24

5.19

0.59

4.98

ESTs

   yf88d07

3024

R15141; R41563

Hs.12381

1.21

0.09

5.83

0.85

4.80

ESTs

   yc85h07

11132

F12902; T74741

 

1.38

0.16

5.75

0.53

4.16

ESTs

   yg38a10

2564

R19870; R45098

Hs.182503

1.32

0.23

5.46

0.63

4.15

ESTs

   c-25h01

1301

Z44625

Hs.29672

4.51

0.68

18.31

3.99

4.06

ESTs

   yg53c11

5862

R25710; R62454

 

1.83

0.19

7.36

0.59

4.02

ESTs

   yg02a02

5691

R18381; R42444

Hs.240816

1.52

0.23

6.08

0.94

4.00

ESTs

   yh09g12

4411

R61781; R61782

 

1.20

0.30

4.55

0.24

3.78

ESTs

   yf80c09

943

R14362

 

2.24

0.32

8.34

0.16

3.73

ESTs

   yd02e05

761

R39357; T80134

Hs.306425; Hs.327350

1.20

0.19

4.47

0.73

3.72

ESTs

   yg17c05

5291

R18746; R43067

Hs.238956

1.09

0.11

3.79

0.39

3.48

ESTs

   c-2ef12

1659

F07687

 

3.08

0.02

10.53

2.42

3.42

ESTs

   yf58e03

1072

R12737; R39789

Hs.119714

3.06

0.60

10.30

0.39

3.36

ESTs

   yl69a01

160

H00104

Hs.21417

3.24

0.54

10.72

1.23

3.31

ESTs

   yc93d09

438

T77119

Hs.21417

2.08

0.45

6.77

1.60

3.25

ESTs

   c-28f03

1425

F07517; Z40576

 

2.49

0.35

7.77

0.52

3.12

ESTs

   yg60e11

2509

R35134

 

4.12

0.92

12.87

1.02

3.12

ESTs

   yl96g09

11047

H09060

 

2.97

0.51

9.23

1.00

3.11

ESTs

   yg02f03

2758

R18419

HS.18585

3.51

0.62

10.83

1.03

3.09

ESTs

   yf94d10

11844

R16329; R41405

Hs.197143

2.73

0.46

8.41

1.14

3.08

ESTs

   yf63f02

201117

R13594

Hs.155639

1.92

0.25

5.75

0.77

3.00

ESTs

   yf98b09

16058

R18177; R42241

Hs.106359

1.07

0.24

3.17

0.27

2.95

ESTs

   yf80c07

1885

R14303

Hs.32565

0.76

0.04

2.18

0.33

2.85

ESTs

   yc92a01

11141

F13028; T76925

 

4.87

0.12

13.72

2.61

2.82

ESTs

   yf76a02

711

R13339

Hs.7913

5.21

0.78

14.52

3.06

2.79

ESTs

   yf55h04

664

R12357

 

3.64

0.18

9.99

1.24

2.75

ESTs

   yc85h06

11131

F12901; T74740

 

5.20

0.54

14.15

2.43

2.72

ESTs

   yc88c03

10642

F12878; R38624

Hs.106313

1.50

0.30

4.04

0.70

2.70

ESTs

   yg39a10

10317

R19899; R45120

Hs.89388

4.91

0.67

12.91

2.40

2.63

ESTs

   yh15d09

6818

R61465

 

4.60

0.37

11.88

1.21

2.58

ESTs

   yg02g01

1987

R18425; R42486

Hs.4983

1.11

0.27

2.75

0.51

2.47

ESTs

   yg08h03

201114

R22721; R43427

Hs.244482

0.70

0.17

1.60

0.08

2.28

ESTs, moderately similar to alternatively spliced product using exon 13A (H. sapiens)

   yg33b02

4208

R20161; R44947

Hs.22905

0.91

0.19

2.05

0.17

2.26

ESTs

   yg44c04

3106

R25497; R45563

None

1.11

0.27

2.60

0.50

2.33

ESTs

   yg46g12

5388

R20696; R45358

Hs.311444; Hs.6591

0.90

0.17

1.95

0.48

2.16

ESTs

   yg42a06

2573

R25050; R45389

Hs.23558

0.57

0.14

1.22

0.22

2.13

ESTs

   yf63f11

5521

R36919

Hs.25205

0.99

0.14

2.11

0.19

2.13

ESTs

Downregulated clones

   c-2eg10

1662

F03955; F07692

 

1.04

0.19

0.51

0.08

0.49

ESTs

   c-29f04

201571

Z40598; Z44804

Hs.184780

1.06

0.15

0.52

0.11

0.49

ESTs

   c-2la08

1913

Z40977; Z45261

Hs.125266

1.03

0.22

ND

ND

0.49

ESTs

   c-2ch10

3050

F03889; F07637

Hs.27278

2.42

0.49

1.12

0.24

0.46

ESTs, weakly similar to chain A, cyclophilin A complexed with cyclosporin A (H. sapiens)

   yg83b05

20476

R53938; R53333

 

1.27

0.10

0.56

0.13

0.44

ESTs

   yg36f04

5214

R24580

Hs.27104

2.18

0.53

0.95

0.17

0.43

ESTs

   yf60a12

3065

R38592; R13746

 

6.52

1.15

2.61

0.42

0.40

ESTs

   yc86e07

2935

F10326; F12716

Hs.227993

7.76

0.94

3.02

0.59

0.39

ESTs

   yc90f10

10752

F10679; F13085

Hs.12395

1.12

0.20

0.43

0.08

0.38

ESTs

   yc97e12

4395

T78036

Hs.23213

1.11

0.20

0.41

0.05

0.37

ESTs

   yf50h10

477

R11920; R39108

Hs.6777

2.39

0.56

0.82

0.16

0.34

ESTs

   yf74a06

16024

R13206; R40294

 

1.32

0.28

0.45

0.10

0.34

ESTs

   yg96d11

3143

R59141; R59142

 

1.30

0.19

0.43

0.08

0.33

ESTs

   yf51a04

958

R11976; R39818

Hs.4241

1.25

0.23

0.40

0.09

0.32

ESTs

   yg51e05

5025

R46483; R21387

Hs.23187

6.26

0.92

1.98

0.19

0.32

ESTs

   yg02h09

2969

R17514; R42608

Hs.139270

10.09

0.93

3.19

0.46

0.32

ESTs

   yf66f03

978

R37086

Hs.23210

1.72

0.18

ND

ND

0.29

ESTs

   yf67b06

115094

R18860

Hs.203213

1.72

0.28

ND

ND

0.29

ESTs

   yl91f12

4185

H08130; H08131

Hs.19515

2.86

0.33

0.70

0.17

0.25

ESTs

   yg14a03

2782

R17432; R42778

Hs.22217

1.57

0.27

0.34

0.06

0.22

ESTs

   yf52e12

4147

R12228; R39947

Hs.7237

1.57

0.30

0.34

0.06

0.22

ESTs

   yf50g11

1829

R11917; R39107

Hs.352354; Hs.244624

2.37

0.24

0.48

0.10

0.20

ESTs

   yf84f08

2237

R14545; R41206

Hs.349648

1.06

0.17

0.19

0.03

0.18

ESTs, weakly similar to KIAA1157 protein (H. sapiens)

Abbreviations and column headings are as in Table 1.

To confirm the results obtained by macroarray analysis, differentially expressed transcripts representing upregulated or downregulated genes were analyzed for differential expression by reverse transcription PCR (RT-PCR) or northern blots. As shown in Figure 6, the upregulation of ETV5, M6PR and APCL was confirmed by RT-PCR, and the downregulation of two genes with unknown function (mRNA DKFZp586C1723 and GENX 2969) was confirmed by northern blotting.
Figure 6

Confirmation of macroarray results by RT-PCR and northern blotting. The percentage of signal modulation (PCR amplification signal or hybridization signal) in relation to control cells (without C2-ceramide treatment) has been calculated in each condition to compare the expression of each gene in neuronally differentiated PC12 cells with (black boxes) or without (white boxes) C2-ceramide treatment. The PCR amplification signal and the hybridization signal for the positive controls (HPRT and 18S rRNA genes, respectively) are indicated.

Discussion

Extracellular signaling molecules such as cytokines, growth-factor deprivation and DNA damage caused by chemotherapeutic agents or irradiation activate ceramide-mediated signal transduction pathways leading to cell death. These pathways have been investigated in the immune system, where they are known to have an important role, and in neurons, as they are suspected to play a part in neurodegenerative disorders [1]. A number of steps in the signaling cascades have been elucidated. However, although the translation inhibitor cycloheximide inhibits the ceramide-mediated death of mesencephalic neurons [7], the expression patterns of genes modulated during ceramide-mediated cell death remain unknown. In a global approach to this question, we have used cDNA macroarray technology to determine the profile of gene expression in a neuronal model of cell death, neuronally differentiated and C2-ceramide-treated PC12 cells, in which ceramide-dependent changes in gene expression could be isolated from the effects of other transcription modulators.

Identification of genes closely implicated in the ceramide and/or TNF-α signaling pathway

We were able to detect differential expression of 10 genes known to be involved in the ceramide or TNF-α signaling pathways (see Figure 4, Table 1) thus validating our study. A summary illustration of the putative role of these genes is presented in Figure 7. Briefly, two genes, encoding phospholipase A2 group IVC (PLA2G4C) and ceroid-lipofuscinosis, neuronal 3, juvenile (CLN3) are already known to be involved in ceramide-mediated signal transduction. The first, PLA2G4C, belongs to the cytosolic phospholipase A2 gene family that encodes two different proteins: calcium-independent and calcium-dependent cytosolic phospholipases [38]. TNF-α regulates the expression of PLA2G4A mRNA in HeLa cells [39] and in human bronchial epithelial cells [40], which is indirect evidence of modulation by ceramide, but the role of ceramide was not demonstrated directly in these studies. However, ceramide was shown directly to upregulate the expression of the gene encoding cytosolic phospholipase A2 in the fibroblast cell line L929 [41]. Conversely, the activation of this gene was reported to be necessary for ceramide accumulation and cell death in the same cells [25]. We show for the first time that this gene is involved in neuronal apoptosis.
Figure 7

Schematic illustrating the putative roles of the proteins encoded by the genes noted in Figures 4 and 5.

The second gene, CLN3, is expressed in a variety of human tissues including the brain, where the product is necessary for neuronal survival [26,27]. Interestingly, CLN3 does not inhibit C2-ceramide-induced apoptosis but modulates endogenous ceramide synthesis and suppresses apoptosis by preventing generation of ceramide [42]. Thus, C2-ceramide can activate a negative feedback mechanism regulating endogenous ceramide generation as well as activate the downstream targets of the endogenous lipid.

Four other genes or families of genes known to be transcriptionally regulated by TNF-α were also modulated by C2-ceramide in our model (Table 1). Of these, ETS variant 5 (ETV5) belongs to the family of ETS transcription factor genes. Increased expression of both ETS1 mRNA and the protein has been observed in human fibroblasts after TNF-α or IL-1β stimulation [28]. PEA3 (a mouse protein corresponding to ETV5) inhibits tumorigenesis in vivo [43]. Moreover, ETV5 and ETS1 can cooperate with c-Jun/c-Fos [44,45], potential regulators of apoptosis in many cell types and specially in the mammalian nervous system [46]. The second gene regulated by TNF-α is NPTX2, encoding neuronal pentraxin II. Pentraxins are a family of proteins that include C-reactive protein and serum amyloid P. They have been found in the brain plaques characteristic of Alzheimer's disease and are toxic to neuronal cell cultures [47,48]. Furthermore, the expression of NPTX3 is increased in response to TNF-α or IL-1β stimulation via activation of NFκB [29,30]. The regulation of the pentraxin gene family by C2-ceramide treatment is consistent with our previous studies showing NFκB activation by C2-ceramide in PC12 cells and in primary cultures of neurons [10,19]. The last two genes known to be regulated by TNF-α and identified in our model are COL18A1, encoding type XVIII collagen alpha 1, and TNFAIP1, encoding TNF-α-induced protein 1. These proteins, downregulated by C2-ceramide, are modulated by TNF-α in various cell types [31,32,33].

We also identified four genes encoding proteins known to participate in TNF-α-activated signal transduction pathways. Thus AXL, upregulated by a factor of 3.65 (Table 1), encodes a tyrosine kinase receptor. Signaling through this receptor is reported to protect against TNF-α-induced apoptosis in fibroblasts and its absence increases apoptosis after serum deprivation [34]. Interestingly, ARK, the mouse protein corresponding to AXL, activates the survival pathway mediated by the serine-threonine kinase Akt [49], which is negatively regulated by ceramide [16,17,50], and is also reported to modulate ceramide synthesis [51]. The second gene we identified is BIRC1, encoding baculoviral IAP repeat-containing 1 protein. This protein, putatively involved in spinal muscular atrophy [52], is an inhibitor of cell death induced by various apoptotic stimuli, including TNF-α [35]. The third identified gene, RSU1, encodes Ras suppressor protein 1, which is involved in TNF-α signaling by blocking the Ras-dependent response. Levels of both RSU1 mRNA and protein have been correlated with a decrease in growth rate and tumorigenic potential in U251 glioblastoma cells [53] and it induces growth arrest in PC12 cells [36]. This is consistent with the report that ceramide regulates apoptosis via modulation of the Ras signaling pathway [18]. In addition, RSU1 has been identified as an inhibitor of Jun kinase activation [37]. This point is interesting, as the fourth gene presenting in this group, MAPK10/J.NK3, encoding the JNK family member mitogen-activated protein kinase 10, is downregulated by C2-ceramide in our model.

The identification of these eight genes, which are involved in the TNF-α signaling pathway, in C2-ceramide treated PC12 cells, suggests that their modulation of expression by TNF-α could be the result of a ceramide-dependent mechanism.

Commitment to apoptosis: upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes by the ceramide pathway

Twenty genes regulated by C2-ceramide correspond to genes known to be involved in regulation of apoptosis and/or cell growth (Figure 5, Table 2). Twelve of these genes are known to be associated with oncogenesis and four with neuronal disorders. Of the upregulated genes, 10 out of 14 are known to be associated with a pro-apoptotic or anti-proliferation process and 3 out of 14 are mainly implicated in protection of the cell against cytotoxicity or damage. Of the downregulated genes, 4 out of 6 are associated with an anti-apoptotic or a proliferation process. This highlights the fact that the cells are engaged in programmed cell death. The putative roles of these genes are illustrated in Figure 7, which focuses on the pro-apoptotic or anti-proliferation process versus anti-apoptotic or proliferation processes.

Briefly, of the known pro-apoptotic or anti-proliferative genes that are upregulated in our model, RPL4 encodes the ribosomal protein L4 that has been shown to be transcriptionally stimulated prior to apoptosis induced by the 5-azacytidine in the PC12 cells [54]. PDCD6IP, upregulated by C2-ceramide in our model, encodes a protein that interacts with ALG2, a Ca2+-binding protein that is required for apoptosis induced by diverse stimuli, including ceramide treatment [55,56,57]. M6PR encodes the cation-dependent mannose-6-phosphate receptor, which has been implicated in retinoid-induced apoptosis [58]. NMA, encoding a putative transmembrane protein, is expressed at low levels in metastatic human melanoma cell lines and xenografts, and is completely absent in highly metastatic human melanoma cell lines [59]. APCL, encoding adenomatous polyposis coli like protein, is a tumor-suppressor gene [60]. METAP2 encodes methionine aminopeptidase eIF-2-associated p67, which interacts with eukaryotic translation initiation factor eIF-2 [61] and could regulate p53 signaling [62]. BAT3, downregulated in some transformed cells, encodes HLA-B associated transcript-3, which interacts with the tumor-suppressor protein DAN that contains growth or tumor suppressive activity in vitro [63]. PHB encodes the protein prohibitin, a potential tumor-suppressor protein that binds to the retinoblastoma (Rb) protein and represses E2F transcriptional activity [64,65]. BNIP3L, encoding BCL2/adenovirus E1B 19 kD-interacting protein 3-like, is a pro-apoptotic gene which has a growth-inhibitory effect on cancer cells [66]. CDH13, encoding cadherin 13, is significantly downregulated in human breast carcinoma cell lines and breast cancer, whereas its overexpression decreases tumor-cell growth [67,68].

Of the known anti-apoptotic or proliferative genes that are downregulated in our model, LOC51582 encodes an antizyme inhibitor, which regulates the antizyme activity proposed to be involved in the polyamine biosynthesis pathway [69,70]. Interestingly, overexpression of antizyme inhibits cell growth [71,72], whereas LOC51582 is downregulated in our model. This observation is consistent with a role of antizyme in the apoptotic process and suggests that ceramide can regulate its activity. LOC51283, a regulator of the activity of the Bcl-2 family proteins, encodes a novel apoptosis regulator, which has been identified as an inhibitor of Bax-induced cell death [73]. Its downregulation by C2-ceramide confirms its involvement in the ceramide-dependent regulation of cell death. The last gene presented here, MET, encodes the MET proto-oncogene, known to be a receptor of the hepatocyte growth factor that has been described to protect neuronal cells from apoptosis via the phosphatidylinositol-3 kinase/Akt pathway [74].

Four genes out of the other genes presented in Table 2 have already been implicated in neuronal disorders, suggesting that ceramide may be a key second messenger in these pathologies. The upregulation of the glutamate receptor gene (GRIA2) seems to be an indicator of tolerance to ischemia [75]. The absence of somatostatin, encoded by SST (downregulated in our model), is associated with apoptotic neurons in patients with Alzheimer's disease [76]. SMN, encoding Survival of motor neuron 2, downregulated by C2-ceramide, strongly contributes to the severity of the spinal muscular atrophy [77]. MUT mRNA is upregulated in ischemia, in relation to a decrease in the accumulation of its neurotoxic metabolite [78].

In conclusion, our cell culture model has enabled us to establish a profile of gene expression during the effector phase of ceramide-mediated cell death. In spite of the stringency of the criteria adopted for differential hybridization, a large number of cDNA clones, 239 of the 9,120 in our cDNA array derived from a normalized infant brain library, correspond to genes up- or downregulated by C2-ceramide treatment. Already-known genes account for 179 of the transcripts, 113 of which have a putative function.

On the basis of their putative functions, we have made an attempt at classifying these transcripts, first with respect to known effects of ceramide or ceramide-mediated transduction systems, then with respect to regulation of cell growth and apoptosis. The 30 genes in Tables 1 and 2 met these criteria, validating the approach and suggesting that the other modulated genes may also be relevant with regard to the progression of the cell-death mechanisms. These genes were classified as having no obvious relation to cell death or survival (Table 3), no known function (Table 4) or as poorly characterized (Table 5). As a result of our study, these genes now have tentative functions. The full list can be consulted with the relevant data on the dedicated website [24].

Interestingly, given the large number of genes known to be modulated by NFκB in the immune system [79], it was surprising that only pentraxin was detected in our model. This suggests either that NFκB is less important in neurons than in lymphocytes, or that its targets are different. Conversely, the transcriptional regulators responsible for the differential expression of the genes detected in our study remain to be discovered. In any case, our results show that transcriptional regulation plays an important role in ceramide-mediated cell death and that some of the modulated transcripts, in agreement with published studies, are involved in other cell-death mechanisms as well.

Materials and methods

Cell culture

Rat PC12 cells [80], which acquire a neuronal phenotype in the presence of nerve growth factor (NGF), were plated at a density of 2,000-3,000 cells/cm2 in 75 cm2 culture flasks coated with polyethylenimine (1 mg/ml) in Leibovitz modified L15 medium (Gibco BRL) supplemented with 2% horse serum and 150 ng/ml NGF (grade II; Alomone Labs, Jerusalem, Israel) as previously described [81]. Apoptosis was induced, after 6 days in the presence of NGF, with the cell-permeant C2 analog of ceramide (C2-ceramide), N-acetylsphingosine (Biomol Research Laboratories, Plymouth Meeting, PA), at a concentration of 25 μM. As negative control, an inactive C2 analog of ceramide (C2-dihydroceramide), N-acetylsphinganine (Biomol Research Laboratories), was used in the same condition as C2-ceramide.

Morphological characterization of apoptosis and cell counts

Neurite retraction and cell shrinkage were visualized by phase-contrast microscopy. Condensed and fragmented nuclei were made visible in situ as described in [7], by intercalation into nuclear DNA of the fluorescent probe propidium iodide. Propidium iodide, which only enters dead cells that have become permeable, was visualized by epifluorescence with a rhodamine filter (excitation, 548-580 nm; emission, 580-610 nm). Viability was quantified by counting cells in at least 10 randomly chosen fields with a 20x objective. The percentage of cells excluding the vital dye propidium iodide was calculated at each time point after the beginning of C2-ceramide or C2-dihydroceramide treatment with respect to the corresponding control.

Measurement of caspase-3-like activity

Caspase-3-like activity was measured using the CaspACE Assay system (Promega, Madison, WI). Cell extracts containing equivalent amounts of protein were used to measure DEVDase (caspase-3-like) activity: the chromophore p-nitroaniline (pNA), released from the colorimetric substrate (Ac-DEVD-pNA) upon cleavage by DEVDase produces a yellow color that is monitored by a photometer at 405 nm.

Preparation of the cDNA macroarray

cDNA clones from a normalized infant brain library (library 1NIB; [20]) were randomly selected to provide a set of 9,120 cDNA clones. The 3' and/or 5' ends of these clones had been previously sequenced [25]. The sequences, registered in GenBank [82], were compared to those in public data bases, permitting tentative identification of the corresponding gene transcripts. The cDNA clones were used to prepare PCR products using oligonucleotide primers complementary to sequences in the vector. They were spotted by robot (Flexis; Perkin Elmer, Shelton, CT) at medium density (25 PCR products/cm2) on nylon membranes (Hybond-N+; Amersham Biosciences, Uppsala, Sweden) as previously described [83]. The entire collection of 9,120 cDNA clones was spotted on a set of four filters.

Purification of poly(A)+mRNA

Total RNA was extracted from control PC12 cultures and from PC12 cultures treated with C2-ceramide or C2-dihydroceramide (approximately 106 cells) with the RNeasy midi kit (Qiagen, Courtaboeuf, France), according to the manufacturer's instructions. The integrity of the RNA was confirmed by agarose gel electrophoresis. Poly(A)+ mRNA was extracted from total RNA with oligo(dT)-conjugated magnetic beads (Dynabeads; Dynal, Oslo, Norway), as described in the manufacturer's protocols.

Complex cDNA target synthesis

Complex cDNA targets were synthesised by reverse transcription of 500 ng poly(A)+ mRNA extracted from control, C2-dihydroceramide- or C2-ceramide-treated PC12 cells. The reaction was performed with the Superscript™ Preamplification System (Invitrogen) as previously described [84]. The reaction mixture contained random-oligonucleotide primers (500 ng), 50 μCi [α-33P]dATP, 3,000 Ci/mmol (Amersham), 500 μM d(T, C, G)TP (Amersham) and 50 μM dideoxyGTP (Invitrogen).

Filter hybridization

The filters were prehybridized at 68°C for 30 min in ExpressHyb hybridization solution (Clontech, Palo Alto, CA), hybridized for 2 h in the same solution to which the radiolabeled complex cDNA target was added, then washed twice for 30 min at 25°C in standard saline citrate (SSC) 1×/0.1% sodium dodecyl sulfate (SDS) and twice for 30 min at 25°C in SSC 0.1×/0.1% SDS. The washed filters were exposed to phosphorus screens (Molecular Dynamics, Sunnyvlae, CA) for 16 h.

Hybridization signal quantitation

Image acquisition was carried out with the Phosphorlmager (Molecular Dynamics). The hybridization signal corresponding to each cDNA clone was quantitated with a specifically designed software (XdotsReader; Cose, Dugny, France) and the local background signal was subtracted. The intensity of the hybridization signal for each clone was then divided by the average intensity of all the clones on each filter to obtain normalized values. Hybridization was done in quadruplicate so that, for each clone/target combination, four values were obtained, compared and validated if at least three out of the four values were similar (SD ± 25%). The final value assigned to each clone was the average of the validated values.

Northern blotting

Total RNA (20 μg) were fractionated under denaturing conditions in a 1.2% agarose gel and transferred onto a Hybond-N+ membrane (Amersham). Specific probes were generated from cDNA clones of interest by PCR using vector-specific primers. The PCR products were purified using the microcon kit (Amicon, Wageningen, The Netherlands) and radiolabeled by random priming (Gibco BRL). Oligonucleotides corresponding to 18S rRNA (control probe) were 32P-labeled using [γ-33P]ATP and T4 RNA kinase. For northern blot analysis, the blots were prehybridized 2 h in ULTRAhyb hybridization buffer (Ambion, Austin, TX), hybridized with the labelled probe (1-2 × 106 cpm/ml) for 16 h at 42°C in the same solution, and washed as for the high-density filters. The washed filters were exposed to phosphorus screens (Molecular Dynamics) for 48 h. The hybridization signal of the specific probes was analyzed with the ImageQuant software (Molecular Dynamics) and compared to the signal obtained with the control probe.

RT-PCR

Total RNA of PC12 cells cultured with or without C2-ceramide was purified according to the protocol described above. Total RNA (2 μg) were reverse transcribed using the Superscript™ Preamplification System (Invitrogen) according to the manufacturer's protocol. An aliquot of the reaction was then used for PCR amplification with the Advantage PCR kit (Clontech) and primers specific to the gene of interest. The amplification products were visualized after electrophoresis in a 1.5% agarose gel with ethidium bromide. The signals were analyzed with ImageQuant software and compared to HPRT (hypoxanthine phosphoribosyl transferase) as control gene.

Declarations

Acknowledgements

This work was supported by the CNRS and grants from European Union to J.M. (TMR projet Neuril) and BIOMED2 programs (EURO-IMAGE Consortium, BMH4-CT-97-2284) to C.A. C.M. was supported by Genome Express and C.D. acknowledges fellowships from the Fédération Française des Groupements Parkinsoniens (FFGP) and the Association pour la Recherche sur le Cancer (ARC).

Authors’ Affiliations

(1)
Genexpress, CNRS FRE 2376
(2)
Neurobiologie des Processus Adaptatifs, UMR 7102 CNRS-UPMC, Université Pierre et Marie Curie
(3)
INSERM U289, Hôpital de la Salpêtrière
(4)
CEA Service de Génomique Fonctionnelle, 2 rue Gaston Crémieux
(5)
Université d'Evry-Val d'Essonne

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