DiscussionThe C13orf19 mRNA inhibition by D5 has no effects on cellular growth properties. We suppose

that the inhibition leads to reduced apoptosis in PCa cells. Therefore, studies on the potential chemoprotective effects of siRNA D5 are underway. Surprisingly, relatively high dosis of etoposide does not induce apoptosis in PC-3 cells. Therefore, experiments using a different chemotherapeutic agent are underway.

Material & MethodsWe compared the downregulation of the C13orf19 mRNA

expression by five different siRNA duplexes (D1: nt 2245‑2263; D2: nt 2215‑2233; D3: nt 1024‑1042; D4: nt 440‑458; D5: nt 1288‑1306) by DOTAP-mediated transfection of the PCa cell line PC-3 (FIG.7 A). The PC-3 cells showed higher C13orf19 mRNA expression compared to other PCa cell lines (Du145, LNCap, 22RV1).

The transfection conditions were optimized and selected the most efficient siRNA. The mRNA expression of C13orf19 and the PBGD reference gene were measured by quantitative PCR. The relative expression values were normalized to the non-silencing (ns) siRNA-control.

Cellular viability was analyzed by the WST‑1 viability assay (Roche) and apoptosis by annexin V-propidium iodide staining using FACS (BD Biosciences). Also, cell cycle distribution and clonogenic survival were examined.

In addition, the effects of C13orf19 down-regulation in combination with chemotherapy on overall cell survival were examined. Therefore, cells were exposed to etoposide (20 µM) for 24 h one day after transfection (FIG.1 B).

Results All five siRNAs duplexes inhibit the C13orf19 mRNA expression in

PC-3 cells already 12 h after transfection (FIG.1). The most efficient siRNA is duplex D5. After 12 h siRNA D5 inhibits the C13orf19 mRNA expression down to 12%. After 72 h there is still an inhibition to 31%. However, the application of these siRNAs showed no obvious effects on doubling time and cellular morphology.

Moreover, cell viability (FIG.2), cell cycle distribution (FIG.3), apoptosis (FIG.6) and clonogenic survival (data not shown), showed no alterations as compared to the controls.

Administering etoposide to PC-3 cells leads to a cell cycle arrest in the G2-M-phase (FIG.4) but does not induce apoptosis (FIG.5). As seen in FIG.7, cells treated with etoposide enlarge as a consequence of the G2-M arrest. There were no significant differences in cell cycle distribution and apoptosis of siRNA D5 transfected PC-3 cells treated with etoposide compared to the ns-siRNA+ etoposide and etoposide controls.

WST-1 cell viability assay showed a higher viability of PC-3 cells treated with siRNA D5 and etoposide compared to ns-siRNA+ etoposide (data not shown). But this result is difficult to interpret due to problems with the weak adhesion of the PC-3 cells in the 96well format.

http://urologie.uniklinikum-dresden.de, *e-mail: doreen.kunze@gmx.de

Inhibition of C13orf19 mRNA expression by siRNA in Inhibition of C13orf19 mRNA expression by siRNA in prostate cancer cellsprostate cancer cells

Doreen Kunze*, Uta Schmidt, Susanne Fuessel, Axel Meye, Manfred P. WirthDoreen Kunze*, Uta Schmidt, Susanne Fuessel, Axel Meye, Manfred P. Wirth Department of Urology, Technical University Dresden, GermanyDepartment of Urology, Technical University Dresden, Germany

Introduction Chromosome 13q is often affected by genetic aberrations in prostate cancer

(PCa), above all loss of heterozygosity [1]. These changes have led to the hypothesis that at least one prostate-specific tumor suppressor gene (TSG) resides on chromosome 13q.

C13orf19 (NM 017569) was previously identified to be down-regulated in PCa. It ist localized on chromosome 13q13 between the TSGs BRCA-2 and RB-1. The mRNA was expressed exclusively in the epithelia as shown by in situ hybridization [2]. The entire chromosomal region is often affected by loss of heterozygoty [3]. The presumed corresponding protein of C13orf19 with 733 amino acids resembles a transcription factor because of its putative nuclear localization signal and the glutamine clusters at the C terminus.

Until now C13orf19 function is unknown. We therefore inhibited its mRNA expression by lipid-mediated siRNA transfection (tf) in order to study the effects of this inhibition in both BPH and Pca cell lines.

References[1] R. von Knobloch, L. Konrad, P.J. Barth, H. Brandt, S.Wille, A. Heidenreich, et al., Genetic pathways and new progression markers for prostate cancer suggested by microsatellite allelotyping. Clin. Cancer Res. 10 (2004) 1064-1073 [2] U. Schmidt, U. Fiedler, C.P. Pilarsky, W. Ehlers, S. Fuessel, M. Haase, G. Faller, G. Sauter, and M.P. Wirth. Identification of a novel gene on chromosome 13 between BRCA-2 and RB-1. Prostate 47 (2001) 91-101

[3] U. Fiedler, W. Ehlers, A. Meye, S. Fussel, G. Faller, U. Schmidt and M.P. Wirth. LOH analyses in the region of the putative tumour suppressor gene C13 on chromosome 13q13. Anticancer Res. 21 (2001) 2341-2350

Table1. Inhibition of C13orf19 mRNA expression in comparison to ns-siRNA

FIG.6 clonogenic survival , PC-3, 125 nM siRNA

siRNA-D5

ns-siRNA

untreated

3.9

1.6

3.6

1.3

3.5

0.8

FIG.6 apoptosis rate in PC-3 cells after transfection with 125 nM siRNA. The percentages of early apoptotic cells (annexin V-positive, PI-negative; lower right) and cells died by apoptosis (annexin V-positive, PI-positive; upper right) are shown.

FIG.3 Cell cycle distribution of PC-3 cells after transfection with 125 nM siRNA

FIG.2 Results of WST‑1 viability assay for PC-3 cells 48 h after transfection

0%

20%

40%

60%

80%

100%

siR

NA

-D5

ns-s

iRN

A

untr

eate

d

siR

NA

-D5

ns-s

iRN

A

untr

eate

d

siR

NA

-D5

ns-s

iRN

A

untr

eate

d

G2-M

S-phase

G0-G1

24 h 48 h 72 h

0,0

0,4

0,8

1,2

1,6

ns-s

iRN

A

siR

NA

-D3

siR

NA

-D5

ns-s

iRN

A

siR

NA

-D3

siR

NA

-D5

untr

eate

d

OD

450

nm

125 nM 250 nM

0%

20%

40%

60%

80%

100%

ns-s

iRN

A

siR

NA

-D5

eto

po

sid

e

ns-s

iRN

A +

eto

po

sid

e

siR

NA

-D5 +

eto

po

sid

e

untr

eate

d

G0-G1 S-phase G2-M

FIG.4 Cell cycle distribution of PC-3 cells after treatment with 250 nM siRNA + 20 µM etoposide at 72 h after transfection

0

4

8

12

16

ns-s

iRN

A

siR

NA

-D5

etop

osid

e

ns-s

iRN

A+

etop

osid

e

siR

NA

-D5

+et

opos

ide

untr

eate

d

apo

pto

sis

in %

FIG.5 Apoptosis rate in PC-3 cells after treatment with 250 nM siRNA + 20 µM etoposide at 72 h after transfection

FIG.1 Treatment scheme of PCa cells in vitro (A) Dotap-mediated transfection with 250 nM siRNA(B) combination of siRNA transfection and chemotherapy

seeding transfection(4 h)

24 – 72 h CT(24 h)

20 h WST-1 assay 

cell countingapoptosis detection

cell cycle distribution

24 – 72 h(B)

seeding transfection(4 h)

24 – 72 h WST-1 assay 

cell countingRNA isolation

apoptosis detectioncell cycle distributionclonogenic survival

20 – 68 h(A)

A

B

FIG.7 PC-3 cells (125x)(A) 250 nM siRNA-D5

(B) 250 nM siRNA-D5 + 20 µM etoposide