• No results found

On the cytotoxicity of HCR NTPase in the neuroblastoma cell line SH SY5Y

N/A
N/A
Protected

Academic year: 2020

Share "On the cytotoxicity of HCR NTPase in the neuroblastoma cell line SH SY5Y"

Copied!
8
0
0

Loading.... (view fulltext now)

Full text

(1)

Open Access

Short Report

On the cytotoxicity of HCR-NTPase in the neuroblastoma cell line

SH-SY5Y

Markus Pasdziernik

1

, Barbara Kaltschmidt

2

, Christian Kaltschmidt

3

,

Claudia Klinger

1

and Michael Kaufmann*

1

Address: 1The Protein Chemistry Group, Witten/Herdecke University, Stockumer Str 10, 58453 Witten, Germany, 2Institute for

Neurobiochemistry, Witten/Herdecke University, Stockumer Str 10, 58453 Witten, Germany and 3Dept. of Cell Biology, Bielefeld University, Universitätsstr 25, 33501 Bielefeld, Germany

Email: Markus Pasdziernik - markus.pasdziernik@uni-due.de; Barbara Kaltschmidt - b.kaltschmidt@uni-wh.de;

Christian Kaltschmidt - c.kaltschmidt@uni-bielefeld.de; Claudia Klinger - cklinger@uni-wh.de; Michael Kaufmann* - mika@uni-wh.de * Corresponding author

Abstract

Background: The human cancer-related nucleoside triphosphatase (HCR-NTPase) is

overexpressed in several tumour tissues including neuroblastoma. HCR-NTPase is an enzyme exhibiting a slow in vitro activity in hydrolysing nucleosidetriphosphates. However, its in vivo

function is still unknown. To learn more about the physiological role of HCR-NTPase, we both overexpressed and silenced it in the neuroblastoma cell line SH-SY5Y.

Findings: No effect was observed when the expression of endogenously expressed HCR-NTPase in the cells was silenced by RNA interference. On the other hand, overexpression of HCR-NTPase led to cytotoxicity of the protein in SH-SY5Y cells. Even if the catalytic essential amino acid glutamate 114 was replaced by alanine (E114A-HCR-NTPase), the protein remained cytotoxic. The results could be confirmed by successfully rescuing the cells via RNA interference.

Conclusion: Although expressed in several tumours, at least in SH-SY5Y, HCR-NTPase is not essential for the cells to survive. Increased levels of the protein lead to cytotoxicity due to physical intracellular interactions rather than hydrolysis of nucleosidetriphosphates by its intrinsic residual enzymatic activity.

Reseach hypothesis

A screen of the cancer genome anatomy project (CGAP) database [1,2] for expressed sequence tags that compared to normal tissue are highly expressed in human tumours revealed the human cancer-related nucleoside triphos-phatase (HCR-NTPase) [3]. HCR-NTPase, the gene prod-uct of the mRNA NM_032324 (synonyms: MGC13186, LOC84284, C1orf57, GI:14150100) is described to exhibit an increased expression profile in liver cholangi-ocarcinoma when compared to normal tissue [3]. In

addi-tion, as retrieved from SOURCE [4,5] when this project was initiated, HCR-NTPase was stated to be expressed in many human tumours, several of them located in the brain such as medullablastoma, glioblastoma, and neu-roblastoma.

A homology search using BLAST [6,7] retrieved significant sequence homologies between HCR-NTPase and proteins assigned to COG1618 of the COG database [8]. As a rep-resentative of COG1618 proteins, aaTHEP1 from the Published: 11 June 2009

BMC Research Notes 2009, 2:102 doi:10.1186/1756-0500-2-102

Received: 20 August 2008 Accepted: 11 June 2009

This article is available from: http://www.biomedcentral.com/1756-0500/2/102 © 2009 Kaufmann et al; licensee BioMed Central Ltd.

(2)

hyperthermophilic bacterium Aquifex aeolicus was bio-chemically characterised [9] and its crystal structure was resolved [10]. Both HCR-NTPase and aaTHEP1 hydrolyse ATP and GTP in vitro with Km in the micromolar range and kcat in the range between 5 and 9 × 10-3 s-1 corresponding to approximately 20–30 molecules hydrolysed per hour and enzyme molecule. In addition, the proteins resemble each other in terms of structure. They belong to the class of P-loop NTPases, and their regular secondary structures can be superimposed with a backbone RMSD of 2.8 Å [3]. Thus, aaTHEP1 and consequently COG1618 proteins may serve as model systems for HCR-NTPase.

Analysing the phylogenetic distribution of COG1618 pro-teins by phylogenetic COG ranking [11,12] reveals that they are absent from almost all mesophiles and present in all thermophiles, most of them archeae. In addition, HCR-ATPase is present in many eucarya analysed thus far. Therefore, HCR-ATPase and COG1618 proteins are at least very similar to PACE proteins i. e. proteins from archaea without assigned function that are conserved in eukarya as described by Matte-Tailliez et al. [13]. The authors argue that most of the PACE proteins are informa-tional proteins and could be of strong biomedical interest.

The aim of this study was to obtain further insight into the role of HCR-NTPase in tumour cells. For that purpose we investigated the phenotype of the neuroblastoma cell line SH-SY5Y [14] as a model system for brain tumours under both conditions overexpressing and silencing HCR-NTPase.

Methods

Eucaryotic cell culture and transfections

SH-SY5Y neuroblastoma cells [14] were obtained from ATCC, Manassas, USA and grown at 37°C and 5% CO2 in Dulbecco's modified Eagle's Medium containing 1% pen-icillin/streptomycin and 15% fetal calf serum (growth medium; GIBCO, Eggenstein, Germany) in a humidified incubator. To keep the cells in logarithmic growth, conflu-ent cells were washed with phosphate buffered saline (PBS), detached by 0.05% Trypsin and 0.5 mM EDTA in PBS, followed by washing and diluting them in fresh growth medium. 106 cells were transfected with 2 μg Plas-mid by electroporation in 50 μl of Cell Line Nucleofector V solution (Amaxa, Cologne, Germany). After transfec-tion, cells were washed in growth medium, transferred to 6-well plates and judged microscopically after 24 h of incubation at 37°C and 5% CO2 using an Axiovert100 microscope (Zeiss, Oberkochen, Germany). Red fluores-cent cells were analysed at 584 nm and green fluoresfluores-cent cells at 482 nm excitation. Three different fields of view were evaluated for each experiment and all experiments were performed independently in triplicate.

Silencing HCR-NTPase in SH-SY5Y by RNA interference

The expression of HCR-NTPase in SH-SY5Y cells was blocked via RNA interference [15] using pSilencer1.0-U6 (Ambion, Darmstadt, Germany). shRNA coding inserts flanked by ApaI and EcoRI restriction sites were con-structed by phosphorylating 25 pmol of oligonucleotides (Operon, Cologne, Germany) using T4 poynucleotide kinase, followed by assembling complementary pairs by incubation for 5 min at 100°C and 60 min at 37°C. pSilencer1.0-U6 sequentially was cut by ApaI (Fermentas, St. Rot, Germany) and EcoRI (Fermentas, St. Leon-Rot, Germany), the inserts were inserted and the plasmids were amplified in E. coli SURE (Stratagene, San Diego, USA). Expression of HCR-NTPase was blocked by trans-fecting SH-SY5Y cells with purified plasmids. The follow-ing pairs of oligonucleotides were used:

siRNA1:

5'-ATC CAT AAA GCC AGT GAT TCT CAA GAG AAA TCA CTG GCT TTA TGG ATC ATT TTT T-3' and

5'-AAT TAA AAA ATG ATC CAT AAA GCC AGT GAT TTC TCT TGA GAA TCA CTG GCT TTA TGG ATG GCC-3'

siRNA2:

5'-AGA GCC TCC ACC TGG AAT TCT CAA GAG AAA TTC CAG GTG GAG GCT CTA ATT TTT T-3' and

5'-AAT TAA AAA ATT AGA GCC TCC ACC TGG AAT TTC TCT TGA GAA TTC CAG GTG GAG GCT CTG GCC-3'

siRNA3:

5'-GAA TGC CGA CTG CAG CAT TCT CAA GAG AAA TGC TGC AGT CGG CAT TCC TTT TTT T-3' and

5'-AAT TAA AAA AAG GAA TGC CGA CTG CAG CAT TTC TCT TGA GAA TGC TGC AGT CGG CAT TCG GCC-3'

siRNA4:

5'-TTC CTA AAG GAA AGC CAT TCT CAA GAG AAA TGG CTT TCC TTT AGG AAC TTT TTT T-3' and

5'-AAT TAA AAA AAG TTC CTA AAG GAA AGC CAT TTC TCT TGA GAA TGG CTT TCC TTT AGG AAG GCC-3'

Overexpressing HCR-NTPase in SH-SY5Y cells

(3)

overexpressed using the plasmid pRc/CMV (Invitrogen, Karlsruhe, Germany). The gene was amplified from pET101/D-TOPO/HCR-NTPase using the primers

5'-AAA AGC TTA TGG CCC GGC ACG TGT TCC-3' and

5'-AAA ATC TAG ATC ACT TCC TGC TGC TCT G-3'.

The PCR-fragment was digested sequentially by XbaI (Fer-mentas, St. Leon-Rot, Germany) and HindIII (Fer(Fer-mentas, St. Leon-Rot, Germany), inserted into pRc/CMV and amplified in E. coli TOP10 (Invitrogen, Karlsruhe, Ger-many).

HCR-NTPase as a fusion with red fluorescent protein [16] (HCR-NTPase-RFP) was overexpressed using the plasmid pmaxFP-Red-C (Amaxa, Cologne, Germany). The gene was amplified from pET101/D-TOPO/HCR-NTPase using the primers

5'-AAA AGA TCT GGA GGA GGA GGA ATG GCC CGG CAC GTG TTC-3' and

5'-AAG GTA CCT CAC TTC CTG CTG CTC TG-3'.

Before digesting with BglII (Fermentas, St. Leon-Rot, Ger-many) and KpnI (Fermentas, St. Leon-Rot, GerGer-many), the PCR-fragment was inserted into pCR2.1-TOPO (Invitro-gen, Karlsruhe, Germany). The resulting fragment was inserted into pmaxFP-Red-C and amplified in E. coli

TOP10. As a control for the efficiency of transfection, pmaxGFP (QIAGEN) was used. HCR-NTPase was overex-pressed by transfecting SH-SY5Y cells with purified plas-mids.

Construction of a E114A-HCR-NTPase mutant in pRc/ CMV

An enzymatically inactive HCR-NTPase mutant was con-structed by mutating a conserved catalytic glutamate [17]. For that purpose, E114 of HCR-NTPase in pRc/CMV was replaced by an alanin using the Phusion™ Site-Directed Mutagenesis Kit (Finnzymes, Espoo, Finland) according to the instructions of the manufacturer. The utilised phos-phorylated primers were

5'-P-CGT CAT CGA TGC GAT TGG GAA GA-3' and

5'-P-CAC ACT CTT TGC CCT GGG CCA-3'.

Inhibition of apoptosis

Apoptosis was inhibited by adding the caspase inhibitor Z-VAD-FMK (Promega, Mannheim) to the culture medium directly after transfection at a final concentration of 20 μM.

Bioinformatics

ClustalW-alignments were performed as previously described [18,19] and for the graphical representation, Genedoc was used [20].

All investigations have been performed in accordance to German regulatory affairs.

Results

Loss-of-function: silencing HCR-NTPase by RNA interference

Since HCR-NTPase is overexpressed in neuroblastoma we were interested in the phenotype of SH-SY5Y if the intrin-sic expression of HCR-NTPase was suppressed. For that purpose we constructed plasmids containing siRNAs directed to the regions within HCR-NTPase that are shown in figure 1. Untreated cells and those transfected with plasmids containing siRNAs were analysed by their cell counts as judged via phase contrast microscopy. To show the effect caused by the transfection procedure, cells transfected with constructs encoding GFP and RFP were included as controls. As can be seen in figure 2, the cell counts are reduced by about one fourth by the transfec-tion procedure alone. However, compared to the GFP-and RFP-controls the cells remain mainly unaffected regardless of which siRNA constructs were introduced.

Gain-of-function: overexpressing HCR-NTPase in SH-SY5Y

(4)

Rescue of SH-SY5Y cells by RNA interference

To confirm that the expression of HCR-NTPase was indeed the cause of the observed cell death after transfec-tion with HCR-NTPase containing plasmids, we tried to rescue the cells by means of RNA interference. Whereas siRNA1 and siRNA4 showed no effect, siRNA2 signifi-cantly increased the survival rate and siRNA3 was even able to completely rescue the cells yielding the same cell counts observed when the cells were transfected with plas-mids encoding solely RFP or GFP (figure 4).

The E114A-HCR-NTPase mutant

HCR-NTPase belongs to the ASCE (additional strand, cat-alytic E) class of NTPases [3]. In ASCE NTPases, NTP-hydrolysis typically depends on a conserved catalytic glutamate (E114 in HCR-NTPase and E107 in aaTHEP1). This essential glutamate is described to prime a water mol-ecule for a nucleophilic attack on the γ-phosphate [17,21].

Consequently, a mutant protein lacking this essential glutamate is expected to be catalytically inactive. Such a mutant was then suitable to decide whether unspecific intracellular hydrolysis of nucleotides or the physical interaction with other intracellular compounds is respon-sible for the observed cytotoxicity. Hence, we constructed the E114A-HCR-NTPase mutant and expressed it in SH-SY5Y cells. As shown in figure 5, the effect of E114A-HCR-NTPase on SH-SY5Y cells resembles that one observed with the wild type protein. Although to a slightly lesser degree, the cells are killed by E114A-HCR-NTPase and can be rescued by siRNA3 mediated RNA interference exclud-ing unspecific NTP-hydrolysis as the cause of cytotoxicity. The cells died due to apoptosis as could be seen morpho-logically. In addition, as judged by green fluorescence, we successfully rescued them by the caspase inhibitor Z-VAD-FMK (table 2).

Positions of siRNAs used to silence HCR-NTPase in SH-SY5Y Figure 1

Positions of siRNAs used to silence HCR-NTPase in SH-SY5Y. ClustalW-alignment of the eight most diverse sequences out of a group of 29 species that have been found to contain Walker A and B motifs similar to those of HCR-NTPase [3]. The sequences were from Homo sapiens, Mus musculus, Drosophila melanogaster, Aquifex aeolicus (aaTHEP1), Ther-mophilum pendens, Methanopyrus kandleri, an uncultured archaeon, Sulfolobus tokodaii, and Aeropyrum pernix, respectively.

HCR-NTPase : - - - M A R - - - - H V F L T G P P G V G K T T L I H K A S E V L K S S G V P V D G F Y T E E V R - - Q G G R R I G F D V V T L Q 9 C Q A 9 : - - - M S R - - - - H V F L T G P P G V G K T T L I Q K A I E V L Q S S G L P V D G F Y T Q E V R - - Q E G K R I G F D V V T L Q 4 V 5 T 6 : - - - M K N Q K N I T I I L T G P P G V G K T T L V H K I C S A L Q D R G R I L Q G F Y T E E M R - - G A S Q R I G F D V V T L aaTHEP1 : - - - M K I I I T G E P G V G K T T L V K K I V E R L G K R - - - A I G F W T E E V R D P E T K K R T G F R I I T T A 1 R Y X 5 : - - - M A A K N F L L T G R P G I G K T T C V V K T A E L L V S R G V K V G G M V T H E V R - - E G G S R V G F K V R D L Q 8 T X 4 9 : - - - M P H N V V L T G R P G I G K T T V C L K V R N V L E E E G Y T V G G I Y C P E I R - - E G G R R I G F E I V D L Q 6 4 D Z 8 : - - - M G I R I G I T G K P R I G K S T I I K A V I R R L K A E G I A V G G M L T A D I Q - - E G G V R V G F S L E D I Q 9 6 Y L 7 : - - - M Q T R L R V Y I T G E P G V G K T T I F L K V I D K L K S Q G Y S I S G F Y C P E V R - - E K G Q R I G F K I K S L Q 9 Y D Y 8 : M Q S R E L R E Q L L G C V K S R K S L H V T G P P G S G K S T F V S R L A E A L R V K G C R L G G F M A P E V R - - R G G R R V A F K I V D I

HCR-NTPase : S - G T R G P L S R V G L E P P P - G K R E C R V G Q Y V V D L T S F E Q L A L P V L R N A D C S S G - P G Q R V C V I D E I G K M E L F S Q L Q 9 C Q A 9 : S - G A Q G P L S R V G S Q P L P - G K P E C R V G Q Y V V N L D S F E Q L A L P V L R N A G S S C G - P K H R V C I I D E I G K M E L F S Q P Q 4 V 5 T 6 : A - G K R A I L S R K N P G D Q L - R R P - - K V G E Y S V F V Q D F D S L A L P V L G T Q D S Q - - - P E P D L L V V D E V G K M E L L S K R aaTHEP1 : E - G K K K I F S S K F F T S K - - - K L V G S Y G V N V Q Y F E E L A I P I L E R A Y R E A K K D R R K V I I I D E I G K M E L F S K K A 1 R Y X 5 : L T G R E G F L A K V G A G - - - A G P R V G K Y V V H V E E L E A V G V G A I L R - - - A V S E A Q - - V V V I D E I G P M E L Y S P S Q 8 T X 4 9 : T E G D R Y L L A R E G A - - - S G P R V G R Y G V F V D N L E R A - A E S I E R - - - A V K R T D - - V V I V D E V G P M E L K S N A Q 6 4 D Z 8 : N T G E K G I L A H V H H R Q T G - - - P K V K V G K Y T V N L A D L D S I G A N S I K N - - - A R A Q P D P I I I I V D E I G P M E L K S K R Q 9 6 Y L 7 : D N E V E D W L A S I Y A K - - - S S I K I G K Y Y I T I N - - - E D I I N K I K E - - - K I S K S E - - I I G I D E I G P M E L S V P K Q 9 Y D Y 8 : A S G E E G Y L A V A D E S L A A P G G R R A R H G R Y L V L V D E A W R V M S H A I R N - - - A F E H A D - - I I I V D E I G P M E L A V P G

HCR-NTPase : F I Q A V R Q T L S T P G T I I L G T I P V P K G K P L A L V E E I R N R K D V K V F N V T K E N R N H L L P D I V T C V Q S S R K Q 9 C Q A 9 : F I Q A V R Q M L S T P G I I V V G T I P V P K G K P L A L V E E I R K R R D V K V F N V T R D N R N S L L P D I V A V V Q S S R T Q 4 V 5 T 6 : F E S A M A D L L K K K R A L L V T I P E K S T L A L V E Q L R K S A G S K I Y Q V T K F N R N A L A G E I T E E I T K A L L aaTHEP1 : F R D L V R Q I M H D P N V N V V A T I P I R D V H P L V K E I R R L P G A V L I E L T P E N R D V I L E D I L S L L E R A 1 R Y X 5 : F L P A V L K A L D S D K P V L A T I H E R E S S S G R L R G I L E R G D V K L Y T V T L Q N R D L L P P Q L A R E I A S L V A R Q 8 T X 4 9 : F V D A V R R A A D A H T P A I F V V H E R S R H P V V V D L R E E R P D V V R F R V T L S N R D E L S D R I L E H V L E W L E E R Q 6 4 D Z 8 : F I E A V E E S I E S E K S M L A S V H Q R S E H E L V K R V K K E F E M F E V T Q E N R D E M A N R I I Q R F S G L E V Q 9 6 Y L 7 : L K E I I D Y V L N E K P I V V A V V H R K I S F K D G K T F V V T Y E N R N R L D N E I F N Y I I S S I Q -Q 9 Y D Y 8 : F R E A L T E I L D S - G K P L V T V F H R R L R T L D P G L Y K L L E R G C I V W L D E - - R N R G P L I R L V S E I A S A L S N E A C G N S

siRNA1

siRNA2 siRNA3

(5)

Silencing HCR-NTPase in SH-SY5Y has no effect Figure 2

Silencing HCR-NTPase in SH-SY5Y has no effect. Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids containing GFP, RFP, siRNA1, siRNA2, siRNA3, and siRNA4, respectively. The data for the GFP- and RFP-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independ-ently performed experiments.

0

10

20

30

40

50

60

70

80

90

100

co

nt

rol

GF

P

RF

P

si

R

N

A1

si

R

N

A

2

siR

N

A

3

siR

N

A

4

%

of

vit

a

l c

[image:5.612.69.544.93.392.2]

ells

Table 1: Quantitative analysis of the cytotoxicity of HCR-NTPase expressed in SH-SY5Y

Total red green

Control 100 -

-GFP6 76 (2) - 61 (2)

RFP6 67 (10) 43 (8)

-HCR-NTPase9 31 (5) -

-HCR-NTPase-RFP3 24 (3) 1 (1)

-GFP + HCR-NTPase6 30 (9) - 7 (2)

GFP + HCR-NTPase-RFP3 30 (4) 1 (1) 6 (2)

(6)

Cytotoxicity of HCR-NTPase expressed in SH-SY5Y Figure 3

Cytotoxicity of HCR-NTPase expressed in SH-SY5Y. Phase contrast and fluorescence images of SH-SY5Y cells are shown. The cells were either untreated (A) or transfected with Plasmids encoding NTPase (B), GFP (C), GFP + HCR-NTPase (D), RFP (E), and an HCR-HCR-NTPase-RFP fusion product (F).

A

C

E

B

D

F

Rescue of SH-SY5Y cells by RNA-interference Figure 4

Rescue of SH-SY5Y cells by RNA-interference. Normalised cell counts (in % of untransfected control) of SH-SY5Y transfected with plasmids encoding a fusion protein between HCR-NTPase and RFP alone (HCR-NTPase-RFP) or cotrans-fected with siRNA1, siRNA2, siRNA3, and siRNA4, respectively. Compared to the control, less than 1% of cells containing the HCR-NTPase-RFP fusion protein could be detected by fluorescence microscopy (data not shown). The data for the GFP-, RFP- and HCR-NTPase-RFP-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

0 10 20 30 40 50 60 70 80 90 100

contr ol

GF P

RF P

HC R-N

TPa se-R

FP siR

NA1 siR

NA 2

siR NA3

siR NA

4

%

of

vit

a

l c

(7)
[image:7.612.88.518.90.376.2]

Cytotoxicity of the E114A-HCR-NTPase mutant and rescue of SH-SY5Y cells by siRNA3 Figure 5

Cytotoxicity of the E114A-HCR-NTPase mutant and rescue of SH-SY5Y cells by siRNA3. Normalised cell counts

(in % of untransfected control) of SH-SY5Y transfected with plasmids encoding the E114A-HCR-NTPase mutant (E114A) alone or cotransfected with either GFP or siRNA3. Whereas fGFP + E114A were analysed by fluorescence microscopy, all other cells were counted via phase contrast microscopy. The data for the GFP-, RFP- and HCR-NTPase-controls are the same as those shown in table 1. The error bars indicate the standard deviations of at least 3 independently performed experiments.

0

10

20

30

40

50

60

70

80

90

100

co

ntr

ol

GF

P

RF

P

HC

R

-NT

Pa

se

E

114

A

GF

P

+

E

114

A

fGF

P +

E

114

A

E

114

A +

si

R

N

A3

%

of

vit

a

l c

ells

Table 2: Effect of Z-VAD-FMK on the cytotoxicity induced by HCR-NTPase and E114A-HCR-NTPase in SH-SY5Y

Treatment Fluorescent cells

GFP (control) 100 (20)

GFP + Z-VAD-FMK 93 (15)

GFP + HCR-NTPase 4 (4)

GFP + HCR-NTPase + Z-VAD-FMK 91 (21)

GFP + E114A-HCR-NTPase 79 (12)

GFP + E114A-HCR-NTPase + Z-VAD-FMK 95 (17)

[image:7.612.61.560.520.674.2]
(8)

Publish with BioMed Central and every scientist can read your work free of charge

"BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime."

Sir Paul Nurse, Cancer Research UK

Your research papers will be:

available free of charge to the entire biomedical community

peer reviewed and published immediately upon acceptance

cited in PubMed and archived on PubMed Central

yours — you keep the copyright

Submit your manuscript here: BioMedcentral

Competing interests

All authors disclose any financial and personal relation-ships with other people or organisations that could inap-propriately influence (bias) their work.

Authors' contributions

MP carried out the cell culture studies and participated in the cloning experiments. BK designed the siRNA con-structs and participated in the design of the study. CKA and MK conceived of the study and participated in its design. CKL carried out the molecular biology experi-ments and participated in the cloning steps. MK drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Daniela Kaufmann for her help in preparing the manuscript and Thomas Dittmar for affording the opportunity to use the AMAXA nucleofector device.

References

1. Strausberg RL, Buetow KH, Greenhut SF, Grouse LH, Schaefer CF:

The cancer genome anatomy project: online resources to reveal the molecular signatures of cancer. Cancer Invest 2002,

20:1038-1050.

2. The Cancer Genome Anatomy Project [http://cgap.nci.nih.gov/ ]

3. Placzek WJ, Almeida MS, Wuthrich K: NMR structure and func-tional characterization of a human cancer-related nucleo-side triphosphatase. J Mol Biol 2007, 367:788-801.

4. Diehn M, Sherlock G, Binkley G, Jin H, Matese JC, Hernandez-Bous-sard T, Rees CA, Cherry JM, Botstein D, Brown PO, Alizadeh AA:

SOURCE: a unified genomic resource of functional annota-tions, ontologies, and gene expression data. Nucleic Acids Res

2003, 31:219-223.

5. SOURCE search [http://smd.stanford.edu/cgi-bin/source/source Search]

6. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.

7. NCBI BLAST [http://www.ncbi.nlm.nih.gov/BLAST/]

8. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, et al.:

The COG database: an updated version includes eukaryotes.

BMC Bioinformatics 2003, 4:41.

9. Klinger C, Rossbach M, Howe R, Kaufmann M: Thermophile-spe-cific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase. BMC Biochem 2003, 4:12.

10. Rossbach M, Daumke O, Klinger C, Wittinghofer A, Kaufmann M:

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold. BMC Struct Biol

2005, 5:7.

11. Meereis F, Kaufmann M: PCOGR: phylogenetic COG ranking as an online tool to judge the specificity of COGs with respect to freely definable groups of organisms. BMC Bioinformatics

2004, 5:150.

12. The Protein Chemistry Group·PCOGR online [http:// www.uni-wh.de/pcogr]

13. Matte-Tailliez O, Zivanovic Y, Forterre P: Mining archaeal pro-teomes for eukaryotic proteins with novel functions: the PACE case. Trends Genet 2000, 16:533-536.

14. Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS: Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res 1978, 38:3751-3757.

15. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC:

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391:806-811. 16. Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA,

Labas YA, Semenova TN, Ugalde JA, Meyers A, Nunez JM, et al.:

GFP-of functional features and structural complexity. Mol Biol Evol

2004, 21:841-850.

17. Aravind L, Iyer LM, Leipe DD, Koonin EV: A novel family of P-loop NTPases with an unusual phyletic distribution and trans-membrane segments inserted within the NTPase domain.

Genome Biol 2004, 5:R30.

18. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD: Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 2003, 31:3497-3500. 19. EBI Tools: ClustalW2 [http://www.ebi.ac.uk/Tools/clustalw2/

index.html]

20. Nicholas KB, Nicholas HBJ, Deerfield DWI: Analysis and Visuali-zation of Genetic Variation. EMBNEWNEWS 1997, 4:14. 21. Leipe DD, Koonin EV, Aravind L: Evolution and classification of

P-loop kinases and related proteins. J Mol Biol 2003,

Figure

Table 1: Quantitative analysis of the cytotoxicity of HCR-NTPase expressed in SH-SY5Y
Table 2: Effect of Z-VAD-FMK on the cytotoxicity induced by HCR-NTPase and E114A-HCR-NTPase in SH-SY5Y

References

Related documents

This study is aimed at assessing the knowledge, attitude, and practice of women in Butajra Town towards cervical cancer and screening so as to develop ways to improve the

In this study LPS was used as a positive control and have shown the same multiplication index of the parasite with dexamethasone treated macrophages, this study suggests that

Reported exposures included male-to-male sexual contact (with or without a history of injecting drug use) in over 60 per cent of cases, injecting drug use for three per cent of

In the first experiment, the mouse cDNA expression array was hybridized using total probes derived from the antropyloric tumor of 8-week-old TFF1 null mice (total probe

Whereas flyash based geopolymer blended with blast furnace slag gives a compressive strength equal to 13.63 MPa at a curing regime of 35 o C for 24 hours.. The dissolution of

Numerical investigations, based on the mechanical properties of two comparative rock samples, Tennenbach und Pfinztal Sandstone, have been performed to investigate their

Due to the close relationship between clinical prognosis and HVPG, 9,28 a study reports that portal pressure-guided therapy significantly improves survival in cirrhosis not only

replacements from 3% to 6% of mS or nS increased concretes compressive strength levels compared to the control. The enhancement in strength level was a function of