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Subject: Potent Bystander Effect in Suicide Gene Therapy Using Neural Stem Cells Transduced with Herpes Simplex Virus Thymidine Kinase Gene
Date: Wed, 24 Jan 2007 15:31:16 +0100
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                <TD width=3D500><!-- Hidden ToCLink <p><a =
href=3D/produkteDB/produkte.asp?Aktion=3DAusgabe&ArtikelNr=3D91032&Produk=
tNr=3D223857&Ausgabe=3D231596&Anfang=3DP&ContentOnly=3Dfalse>Vol. 69, =
No. 6, 2005</a></p> --><A=20
                  name=3DOLN>
                  <HR SIZE=3D1>

                  <P><I>Laboratory/Clinical Translational =
Research</I></P>
                  <P><B class=3Darticletitel>Potent Bystander Effect in =
Suicide=20
                  Gene Therapy Using Neural Stem Cells Transduced with =
Herpes=20
                  Simplex Virus Thymidine Kinase Gene</B><BR>Shaoyi Li, =
Tsutomu=20
                  Tokuyama, Junkoh Yamamoto, Masayo Koide, Naoki Yokota, =
Hiroki=20
                  Namba<BR><BR>Department of Neurosurgery, Hamamatsu =
University=20
                  School of Medicine, Hamamatsu, Japan<BR></P>
                  <P><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#AC">Address=20
                  of Corresponding Author</A></P>
                  <P><I>Oncology</I> 2005;69:503-508 (DOI:=20
10.1159/000091032)</P>
                  <HR SIZE=3D1>
                  </A>
                  <P>&nbsp;<B class=3Dsection1>Outline</B></P>
                  <UL>
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#kw">Key=20
                    Words</A>
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#abstract">Abstract</A>=20

                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SA1">Introduction</A>
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SA2">Materials=20
                    and Methods</A>
                    <UL>
                      <LI><A=20
                      =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SC1">Isolation=20
                      of NSCs and Establishment of the NSCtk Cells</A>
                      <LI><A=20
                      =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SC2">In=20
                      vitro Bystander Effect between NSCtk and C6 =
Cells</A>
                      <LI><A=20
                      =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SC3">In=20
                      vivo Bystander Effect between NSCtk and C6 Cells =
in=20
                      Sprague-Dawley Rats</A></LI></UL>
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SA3">Results</A>
                    <UL>
                      <LI><A=20
                      =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SC4">In=20
                      vitro Bystander Effect between NSCtk and C6 =
Cells</A>
                      <LI><A=20
                      =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SC5">In=20
                      vivo Bystander Effect between NSCtk and C6 Cells =
in=20
                      Sprague-Dawley Rats</A></LI></UL>
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#SA4">Discussion</A>=20

                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#REFS">References</A>=20
                    </LI></UL>
                  <UL>
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#AC">Author=20
                    Contacts</A>=20
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#AI">Article=20
                    Information</A>=20
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#PD">Publication=20
                    Details</A>=20
                    <LI><A=20
                    =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#Dosage">Drug=20
                    Dosage / Copyright</A> </LI></UL><A name=3Dkw>
                  <HR SIZE=3D1>
                  </A>
                  <P>&nbsp;<A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<B class=3Dsection1>Key =
Words</B></P>
                  <UL>
                    <LI>Herpes simplex virus thymidine kinase
                    <LI>Ganciclovir
                    <LI>Bystander effect
                    <LI>Neural stem cell
                    <LI>Suicide gene therapy</LI></UL><A =
name=3Dabstract>
                  <HR SIZE=3D1>
                  </A>
                  <P>&nbsp;<A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<B =
class=3Dsection1>Abstract</B></P>
                  <P><I>Objective:</I> The herpes simplex virus =
thymidine kinase=20
                  (HSVtk)/ganciclovir suicide gene therapy system has =
been=20
                  considered as one of the most promising therapeutic =
strategies=20
                  for malignant gliomas. We have been using <I>HSVtk</I> =

                  gene-transduced neural stem cells (NSCtk) that possess =
an=20
                  ability to migrate toward a tumor mass for the =
treatment of=20
                  experimental brain tumors. In the present study, we =
evaluated=20
                  the potency of anti-tumor effect mediated by the =
bystander=20
                  effect between NSCtk and C6 glioma cells in the=20
                  HSVtk/ganciclovir suicide gene therapy system. =
<I>Methods:</I>=20
                  NSCtk and C6 glioma cells were mixed at various ratios =

                  (NSCtk:C6 cell ratios of 1:1 to 1:64) and the =
bystander effect=20
                  was evaluated both under in vitro and in vivo =
conditions.=20
                  <I>Results:</I> In vitro co-culture experiment showed =
a=20
                  complete tumor growth inhibition at the NSCtk:C6 =
ratios as low=20
                  as 1:16. In vivo co-implantation study in the rat =
brain showed=20
                  no visible tumors at the NSCtk:C6 ratios as low as =
1:16 and=20
                  all those rats survived more than 100 days. =
<I>Conclusion:</I>=20
                  The results clearly demonstrated an extremely potent =
bystander=20
                  effect between NSCtk and C6 cells, and the minimum =
number of=20
                  NSCtk cells needed for the treatment of tumors was =
roughly=20
                  estimated.</P>
                  <P>Copyright =A9 2005 S. Karger AG, Basel</P>
                  <HR SIZE=3D1>

                  <P><B class=3Dsection1><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A =
name=3DSA1>Introduction</A></B><BR>
                  <P>The infiltrative and invasive nature of glioma, as =
well as=20
                  the proximity to critical intracranial structures, =
presents a=20
                  major hurdle to effective treatment by conventional =
means of=20
                  surgery and radiotherapy [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r001')">1</A>]. In fact,=20
                  despite the continuous refinement of the techniques of =

                  diagnosis, surgical intervention, radiotherapy and/or=20
                  chemotherapy, the prognosis for patients with =
malignant=20
                  gliomas remains poor [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r002')">2</A>].=20
                  Fortunately, the encouraging advances in molecular =
biology=20
                  have been contributing to the development of gene =
therapies.=20
                  To date, a number of target genes have been used or =
are=20
                  expected to be applied. Among them, the most feasible =
and=20
                  well-established approach is the herpes simplex virus=20
                  thymidine kinase/ganciclovir (HSVtk/GCV) system, one =
of the=20
                  suicide gene therapies. This strategy is also called =
the=20
                  enzyme/prodrug system because the suicide gene encodes =
an=20
                  enzyme that modifies a nontoxic prodrug into a toxic =
molecule=20
                  in the cells. The HSVtk converts the nontoxic =
nucleoside=20
                  analogue GCV into GCV monophosphate, which is then =
further=20
                  phosphorylated to GCV triphosphate by the cellular =
kinases.=20
                  Incorporation of GCV triphosphate into elongating DNA =
during=20
                  cell proliferation results in premature chain =
termination and=20
                  eventually selectively kills dividing cells [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r003')">3</A>]. One of=20
                  the most attractive elements in the HSVtk/GCV system =
is the=20
                  so-called 'bystander effect', where brain tumor cells =
that=20
                  were not transduced with the <I>HSVtk</I> gene were =
also=20
                  eliminated along with <I>HSVtk</I> gene-transduced =
tumor cells=20
                  [<A =
href=3D"javascript:NewWindow('000091032.r004')">4</A>, <A=20
                  =
href=3D"javascript:NewWindow('000091032.r005')">5</A>]. Though=20
                  its mechanism is still not clear, it is considered =
that=20
                  phosphorylated GCV is capable of passing through =
cellular gap=20
                  junctions to confer cytotoxic effects on nontransduced =

                  neighboring cells. The biosafety and clinical safety =
of the=20
                  strategy have already been tested in clinical trials =
by direct=20
                  injection of the HSVtk retroviral vector-producing =
cells into=20
                  the tumor or the brain walls around the excised tumor, =

                  although only limited therapeutic benefit was achieved =
[<A=20
                  href=3D"javascript:NewWindow('000091032.r006')">6</A>, =
<A=20
                  =
href=3D"javascript:NewWindow('000091032.r007')">7</A>]. Since=20
                  the extracellular virus moves by passive diffusion or =
fluid=20
                  convection, it is not possible for the virus to spread =
far=20
                  from the site of injection. On the other hand, glioma =
cells=20
                  are known to migrate a long way along the white matter =
beyond=20
                  the therapeutic radius.</P>
                  <P>In order to increase the ability of targeting the=20
                  infiltrating tumor cells, we tried to treat an =
experimental=20
                  brain tumor through the bystander effect by injecting=20
                  <I>HSVtk</I> gene-transduced tumor cells (TK tumor =
cells) in=20
                  the vicinity of the tumor followed by GCV =
administration [<A=20
                  href=3D"javascript:NewWindow('000091032.r008')">8</A>, =
<A=20
                  =
href=3D"javascript:NewWindow('000091032.r009')">9</A>]. Tumor=20
                  growth was drastically suppressed through the potent =
bystander=20
                  effect achieved between the pre-existing tumor cells =
and the=20
                  injected TK tumor cells. Though, theoretically, all =
the=20
                  injected TK tumor cells were to be completely killed =
by GCV,=20
                  ethical problems still exist with the use of viable =
tumor=20
                  cells even in fatal gliomas. Therefore, we tested the =
use of=20
                  neural stem cells (NSCs) transduced with <I>HSVtk</I> =
gene=20
                  (NSCtk) instead of TK tumor cells, because NSCs =
exhibit an=20
                  innate tumor-homing capability in experimental brain =
tumor=20
                  models. NSCs implanted at distant sites from the =
tumor, even=20
                  in the opposite side of the brain, migrate through =
normal=20
                  brain tissue targeting the tumor cells [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r010')">10</A>,<A=20
                  =
href=3D"javascript:NewWindow('000091032.r011')">11</A>,<A=20
                  =
href=3D"javascript:NewWindow('000091032.r012')">12</A>,<A=20
                  =
href=3D"javascript:NewWindow('000091032.r013')">13</A>]. In our=20
                  previous study, we showed a potent bystander effect =
between=20
                  NSCtk and C6 cells, and complete tumor eradication was =

                  obtained by the 'NSCtk therapy' when the same number =
of NSCtk=20
                  cells as the tumor cells were intracranially implanted =
[<A=20
                  =
href=3D"javascript:NewWindow('000091032.r014')">14</A>]. The=20
                  bystander effect observed between NSCtk and C6 cells =
was much=20
                  more potent than that observed between tumor cells [<A =

                  href=3D"javascript:NewWindow('000091032.r008')">8</A>, =
<A=20
                  =
href=3D"javascript:NewWindow('000091032.r009')">9</A>], and=20
                  therefore, this strategy seemed appropriate for =
clinical use=20
                  not only from the aspect of clinical safety, but also =
for its=20
                  anti-tumor potency. In the present study, we precisely =

                  evaluated the potency of anti-tumor effect mediated by =
the=20
                  bystander effect between NSCtk and C6 cells by a =
stepwise=20
                  decrease in the ratios of the NSCtk cells against C6 =
cells=20
                  both under in vitro and in vivo conditions in order to =
provide=20
                  basic experimental data for clinical application of =
the 'NSCtk=20
                  therapy'.</P>
                  <P><B class=3Dsection1>
                  <P>&nbsp;</P><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSA2>Materials and=20
                  Methods</A></B><BR>
                  <P><B class=3Dsection4><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSC1>Isolation of NSCs =
and=20
                  Establishment of the NSCtk Cells</A></B></P>
                  <P>All of the following experiments were performed =
according=20
                  to the Rules of Animal Experimentation and the Guide =
for the=20
                  Care and Use of Laboratory Animals of the Hamamatsu =
University=20
                  School of Medicine. As we described previously, fetal =
cerebral=20
                  hemisphere tissue was obtained from 14-day =
Sprague-Dawley rat=20
                  embryos and mechanically dissociated in NSC growth =
medium=20
                  using the Neural Stem Cell Expansion Kit/Neurosphere =
system=20
                  (R&amp;D Systems, Inc., Minneapolis, Minn., USA) [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r014')">14</A>]. The=20
                  cells were plated at a concentration of 4 =D7 =
10<SUP>5</SUP> per=20
                  milliliter in an ultra-low attachment plate and =
incubated at=20
                  37=B0C under 5% CO<SUB>2</SUB>. The medium was =
supplemented with=20
                  20 ng/ml human epidermal growth factor and human =
fibroblast=20
                  growth factor basic each day. Thereafter, bulk =
cultures were=20
                  generated by passaging the cells repeatedly.</P>
                  <P>The PA317 cells (HSVtk retrovirus-producing mouse=20
                  fibroblast cell line, provided by Genetic Therapy =
Inc.,=20
                  Gaithersburg, Md., USA) were cultured in NSC growth =
medium=20
                  with 20 ng/ml of human fibroblast growth factor basic =
to=20
                  prepare the HSVtk supernatant. After the supernatant =
of HSVtk=20
                  retrovirus and 8 =B5g/ml polybrene (Aldrich Chemical =
Company=20
                  Inc., Milwaukee, Wisc., USA) were added, the NSCs were =

                  incubated for 3 h. Then the cells were washed and =
cultured for=20
                  an additional 2 days. The drug-resistant neurospheres =
were=20
                  collected after drug selection with 150 =B5g/ml G418=20
                  (Sigma-Aldrich Japan K.K., Tokyo, Japan) for 1 week. =
The=20
                  clone, having a high GCV sensitivity and the same in =
vitro=20
                  proliferative activity as wild-type NSCs, was selected =
(NSCtk=20
                  cells). All the NSCtk cells were used as a single cell =

                  suspension for the following experiments by =
mechanically=20
                  dissociating them with a fire-polished Pasteur =
pipette.</P>
                  <P><B class=3Dsection4><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSC2>In vitro Bystander =
Effect=20
                  between NSCtk and C6 Cells</A></B></P>
                  <P>The C6 rat glioma cell line was purchased from ATCC =

                  (Manassas, Va., USA). To determine the lowest =
necessary number=20
                  of NSCtk cells which could provide effective =
anti-tumor effect=20
                  in our strategy, we co-cultured NSCtk cells with 5 =D7 =

                  10<SUP>3</SUP> C6 cells at NSCtk:C6 cell ratios of =
1:1, 1:4,=20
                  1:16, 1:32 and 1:64 in DMEM medium (including 10% =
fetal bovine=20
                  serum) containing 1 =B5g/ml GCV in a 96-well tissue =
culture=20
                  plate. C6 cells alone (5 =D7 10<SUP>3</SUP>, with or =
without=20
                  GCV) or mixed with the same number of NSCtk cells =
(NSCtk:C6=20
                  cell ratio of 1:1, without GCV) were also cultured in =
the DMEM=20
                  medium. The conditional medium was changed every 2 =
days and=20
                  the number of living cells was determined by =
tetrozolium-based=20
                  colorimetric assay (MTT assay) on day 10 [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r015')">15</A>].=20
                  Sensitivity to GCV was expressed as the percent =
absorbance of=20
                  the C6 cells alone, cultured without GCV.</P>
                  <P><B class=3Dsection4><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSC3>In vivo Bystander =
Effect=20
                  between NSCtk and C6 Cells in Sprague-Dawley =
Rats</A></B></P>
                  <P>Seventy-two male Sprague-Dawley rats (280-320 g, 9 =
weeks=20
                  old; Nippon SLC, Hamamatsu, Japan) were anesthetized =
with 0.4=20
                  ml/100 g equithesin and placed in a stereotaxic =
apparatus=20
                  (Narishige Scientific Instrument Lab., Tokyo, Japan). =
NSCtk=20
                  cells mixed with 1 =D7 10<SUP>5</SUP> C6 cells at =
ratios of 1:1,=20
                  1:4, 1:16 and 1:32 (n =3D 12 for every group) in 10 =
=B5l DMEM=20
                  medium were infused with a 50-=B5l microsyringe to the =
point of=20
                  5 mm ventral from the dura through the burr hole =
(coordinates=20
                  with respect to bregma: 2 mm posterior, 3 mm right). =
The=20
                  animals were administered intraperitoneally with 15 =
mg/kg GCV=20
                  twice daily from day 0 for 10 days. Twelve rats, =
implanted=20
                  similarly with 1 =D7 10<SUP>5</SUP> NSCtk cells mixed =
with 1 =D7=20
                  10<SUP>5</SUP> C6 cells in 10 =B5l DMEM medium, were =
given=20
                  physiological saline (0.9% NaCl solution) =
intraperitoneally=20
                  for 10 days. Another 12 rats were implanted with 1 =D7 =

                  10<SUP>5</SUP> C6 alone and treated with GCV as above. =
The=20
                  animals were kept under the same laboratory =
conditions. No=20
                  steroids or antibiotics were used. Half of the animals =
(n =3D 6)=20
                  for each group were sacrificed and histologically =
examined on=20
                  day 14, the other half (n =3D 6) served for the =
survival study=20
                  group. When the rats developed symptoms such as severe =
paresis=20
                  and/or ataxia, or when their body weight decreased to =
less=20
                  than 80%, they were sacrificed. The total volume of =
the tumor=20
                  (in cubic millimeters) was calculated by summing up =
the=20
                  cross-sectional areas. Survival was analyzed by a =
log-rank=20
                  test based on the Kaplan-Meier survival analysis using =

                  Statview 5.0 software.</P>
                  <P><B class=3Dsection1>
                  <P>&nbsp;</P><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSA3>Results</A></B><BR>
                  <P><B class=3Dsection4><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSC4>In vitro Bystander =
Effect=20
                  between NSCtk and C6 Cells</A></B></P>
                  <P>No proliferation inhibitions of C6 cells were =
detected when=20
                  they were cultured with GCV (fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig01">1</A>,=20
                  black column). When C6 cells were co-cultured with the =
same=20
                  number of NSCtk cells in the medium without GCV (fig. =
<A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig01">1</A>,=20
                  gray column), we did not observe proliferation =
inhibition=20
                  either, as compared with the condition of C6 only =
without GCV,=20
                  which, therefore, was used as the control for other=20
                  experimental conditions. The anti-tumor effect =
mediated by the=20
                  bystander effect was quite evident in the co-cultured =
NSCtk/C6=20
                  cells at ratios from 1:1 to 1:16 when cultured in the =
medium=20
                  containing 1 =B5g/ml GCV, where we could hardly detect =
any=20
                  viable cells on day 10. Even at the NSCtk:C6 cell =
ratios of=20
                  1:32 and 1:64, over 80 and 20% growth inhibition could =
be=20
                  achieved (fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig01">1</A>,=20
                  white columns).</P>
                  <P><A name=3DFIG01></A>
                  <TABLE width=3D"100%" border=3Don justify=3D"center">
                    <TBODY>
                    <TR>
                      <TD align=3Dmiddle><IMG alt=3DFIG01=20
                        =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"00009=
1032_f01.GIF"'></TD></TR>
                    <TR>
                      <TD><FONT size=3D-1><B>Fig. 1.</B> In vitro =
bystander=20
                        effect between NSCtk and C6 cells. No =
proliferation=20
                        inhibitions were observed in the plates of C6 =
alone with=20
                        GCV (black column) and at the NSCtk:C6 ratio of =
1:1 with=20
                        physiological saline (PS) (gray column), while =
hardly=20
                        any viable cells could be detected at the =
NSCtk:C6=20
                        ratios from 1:1 to 1:16 when cultured with GCV =
on day 10=20
                        (MTT assay, triplicate, mean =B1 standard error; =
error=20
                        bars smaller than icons do not appear). Over 80 =
and 20%=20
                        growth inhibition could also be achieved even at =
the=20
                        NSCtk:C6 ratios of 1:32 and 1:64 when cultured =
with GCV=20
                        (white columns; p &lt; 0.01 compared with C6 =
alone with=20
                        GCV).</FONT></TD></TR></TBODY></TABLE></P>
                  <P>Representative phase-contrast photomicrographs of =
the=20
                  bystander anti-tumor effect between NSCtk and C6 cells =
at the=20
                  ratio of 1:1 co-cultured in the medium with and =
without 1=20
                  =B5g/ml GCV on day 1 through day 10 are shown (fig. <A =

                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig02">2</A>,=20
                  bottom two rows). The C6 cells showed no obvious =
change on day=20
                  1 even after GCV exposure. However, the proliferation =
of C6=20
                  cells began to be held down from day 2 in the =
existence of=20
                  GCV, and dramatic cell death could be observed from =
day 4. The=20
                  C6 cells, together with NSCtk cells, degenerated and =
gradually=20
                  detached from the bottom of the plate, and finally, no =
viable=20
                  cells could be seen in the plate on day 10 (fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig02">2</A>,=20
                  bottom row). But the NSCtk cells did not exert any =
inhibiting=20
                  effect on the surrounding C6 cells when they were not =
exposed=20
                  to GCV (fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig02">2</A>,=20
                  third row). C6 cells alone were also cultured with or =
without=20
                  GCV to examine the direct effect of GCV on C6 cells =
(fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig02">2</A>,=20
                  top two rows).</P>
                  <P><A name=3DFIG02></A>
                  <TABLE width=3D"100%" border=3Don justify=3D"center">
                    <TBODY>
                    <TR>
                      <TD align=3Dmiddle><IMG alt=3DFIG02=20
                        =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"00009=
1032_f02.JPG"'></TD></TR>
                    <TR>
                      <TD><FONT size=3D-1><B>Fig. 2.</B> Phase-contrast=20
                        photomicrographs of the bystander anti-tumor =
effect=20
                        between NSCtk and C6 cells at the ratio of 1:1 =
on day 1=20
                        through day 10. When C6 cells were cultured =
alone, GCV=20
                        exerted no effect on tumor cell proliferation =
(top two=20
                        rows). Even when C6 cells were cultured with =
NSCtk=20
                        cells, no inhibitory effect on C6 cells was =
exerted when=20
                        not cultured with GCV (third row). When cultured =
with=20
                        GCV, the cells degenerated and gradually =
detached from=20
                        the bottom of the plate, and finally, no viable =
cells=20
                        could be seen in the plate on day 10 (bottom =
row).=20
                        =D7100.</FONT></TD></TR></TBODY></TABLE></P>
                  <P><B class=3Dsection4><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A name=3DSC5>In vivo Bystander =
Effect=20
                  between NSCtk and C6 Cells in Sprague-Dawley =
Rats</A></B></P>
                  <P>On histological examination on day 14, no visible =
brain=20
                  tumors were found in the groups implanted with an =
NSCtk/C6=20
                  mixture (1:1, 1:4 and 1:16) and treated with GCV (fig. =
<A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig03">3</A>c-e),=20
                  while all the rats in the group implanted with C6 =
alone (fig.=20
                  <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig03">3</A>a)=20
                  and in the group implanted with NSCtk/C6 cells (1:1) =
but not=20
                  treated with GCV (fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig03">3</A>b)=20
                  developed a large tumor. All 6 rats in the group =
implanted=20
                  with an NSCtk/C6 mixture at the ratio of 1:32 and =
treated with=20
                  GCV showed a small tumor along the needle path (fig. =
<A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig03">3</A>f).=20
                  The measured tumor volume is shown in figure <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig03">3</A>g.</P>
                  <P><A name=3DFIG03></A>
                  <TABLE width=3D"100%" border=3Don justify=3D"center">
                    <TBODY>
                    <TR>
                      <TD align=3Dmiddle><IMG alt=3DFIG03=20
                        =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"00009=
1032_f03.JPG"'></TD></TR>
                    <TR>
                      <TD><FONT size=3D-1><B>Fig. 3.</B> In vivo =
bystander=20
                        effect between NSCtk and C6 cells in =
Sprague-Dawley=20
                        rats. <B>a-f</B> Representative photomicrographs =
of a=20
                        coronal section from the rat brain. A large =
tumor was=20
                        observed in the rats implanted with C6 alone =
treated=20
                        with GCV (<B>a</B>) and with NSCtk/C6 (1:1) =
treated with=20
                        physiological saline alone (<B>b</B>). No =
visible tumors=20
                        were observed in all the rats co-implanted with =
NSCtk/C6=20
                        at the ratios of 1:1 (<B>c</B>), 1:4 (<B>d</B>) =
and 1:16=20
                        (<B>e</B>) and treated with GCV. The rats =
co-implanted=20
                        with NSCtk/C6 at the ratio of 1:32 showed a =
small tumor=20
                        along the needle path (<B>f</B>). <B>g</B> =
Measured=20
                        tumor volume in the six groups (n =3D 6, mean =
=B1 standard=20
                        error; error bars smaller than icons do not =
appear). All=20
                        the rats in the groups implanted with an =
NSCtk/C6=20
                        mixture at ratios of 1:1 to 1:32 and treated =
with GCV=20
                        showed potent growth inhibition of tumor (white =
column,=20
                        p &lt; 0.01 compared with C6 alone treated with =
GCV).=20
                        <B>h </B>Kaplan-Meier survival curve. The rats =
in the=20
                        group of C6 alone treated with GCV and in the =
group of=20
                        NSCtk/C6 (1:1) treated with physiological saline =
(PS)=20
                        died 2-3 weeks after tumor implantation, while =
all the=20
                        rats in the groups of NSCtk/C6 (1:1, 1:4, 1:16) =
treated=20
                        with GCV survived more than 100 days. The mean =
survival=20
                        time in the group of NSCtk/C6 (1:32) treated =
with GCV=20
                        prolonged to 28 days (p &lt; 0.01 compared with =
any=20
                        group of C6 alone treated with GCV or NSCtk/C6, =
1:1,=20
                        treated with physiological=20
                  saline).</FONT></TD></TR></TBODY></TABLE></P>
                  <P>On survival study, all the rats in the groups =
implanted=20
                  with an NSCtk/C6 mixture (1:1, 1:4 and 1:16) and =
treated with=20
                  GCV survived more than 100 days. All the rats in the =
group=20
                  implanted with C6 alone and in the group implanted =
with=20
                  NSCtk/C6 cells (1:1) but not treated with GCV died 2-3 =
weeks=20
                  after tumor implantation due to tumor growth, and =
there was no=20
                  statistical significance between them (p =3D 0.89, =
log-rank=20
                  test). The mean survival time of the 6 rats in the =
group=20
                  implanted with an NSCtk/C6 mixture at the ratio of =
1:32 and=20
                  treated with GCV was 28 days and was significantly =
longer than=20
                  in the previous two control groups (p &lt; 0.01, =
log-rank=20
                  test) (fig. <A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#fig03">3</A>h).</P>
                  <P><B class=3Dsection1>
                  <P>&nbsp;</P><A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<A =
name=3DSA4>Discussion</A></B><BR>
                  <P>In the present study, we have demonstrated the =
feasibility=20
                  of the 'NSCtk therapy' for the treatment of =
experimental=20
                  gliomas and observed an extremely potent bystander =
effect=20
                  between NSCtk and C6 glioma cells. We have shown in =
the=20
                  co-implanted study that an NSCtk:C6 ratio of at least =
1:16 is=20
                  required for complete eradication of the tumor. This=20
                  information would be of importance when preparing a =
clinical=20
                  protocol of this strategy.</P>
                  <P>There have been several intracranial =
co-implantation=20
                  studies using neural stem/progenitor cells and tumor =
cells=20
                  similar to the present study. Barresi et al. [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r016')">16</A>]=20
                  implanted 8 =D7 10<SUP>4</SUP> immortalized neural =
progenitor=20
                  cells expressing cytosine deaminase and 2 =D7 =
10<SUP>4</SUP> C6=20
                  glioma cells in Sprague-Dawley rats and administered=20
                  5-fluorocytosine. Though four times more effector =
cells were=20
                  used, the cytosine deaminase/5-fluorocytosine system, =
which=20
                  was another suicide gene therapy and also generated =
the=20
                  bystander effect, only produced 50% reduction in tumor =
size 10=20
                  days after implantation. Shah et al. [<A=20
                  =
href=3D"javascript:NewWindow('000091032.r012')">12</A>]=20
                  implanted 0.75 =D7 10<SUP>6</SUP> mouse neural =
precursor cells=20
                  expressing tumor necrosis factor-related =
apoptosis-inducing=20
                  ligand and 0.25 =D7 10<SUP>6</SUP> glioma cells in =
nude mice=20
                  (three times more effector cells) and obtained =
significant=20
                  tumor reduction 2 weeks after implantation but could =
not=20
                  obtain complete eradication. Although the used =
effector cells=20
                  and tumor cells are not the same among studies, and =
therefore,=20
                  the results cannot be simply compared, the results of =
the=20
                  present study strongly indicate that the 'NSCtk =
therapy' is=20
                  extremely potent as compared with other strategies =
using=20
                  neural stem/progenitor cells.</P>
                  <P>In the present study, we obtained NSCs from the =
embryos of=20
                  the Sprague-Dawley rat, from which C6 glioma was also =
derived.=20
                  A strong bystander effect was achieved by GCV =
treatment in the=20
                  co-implantation experiments of NSCtk and C6 cells =
derived from=20
                  a syngeneic animal. This bystander effect was much =
stronger=20
                  than that observed between the <I>HSVtk</I> =
gene-transduced=20
                  and <I>HSVtk </I>gene-nontransduced C6 rat glioma =
cells and=20
                  even more potent than that observed between the =
<I>HSVtk=20
                  </I>gene-transduced and <I>HSVtk</I> =
gene-nontransduced 9L rat=20
                  glioma cells that had a higher expression of =
connexin43 than=20
                  C6 cells [<A=20
                  href=3D"javascript:NewWindow('000091032.r008')">8</A>, =
<A=20
                  =
href=3D"javascript:NewWindow('000091032.r017')">17</A>]. The=20
                  reasons for this extremely potent bystander effect by =
NSCtk=20
                  cells in the present study are not fully understood, =
and=20
                  further studies are needed to elucidate the mechanism. =
A=20
                  higher in vivo resistance of differentiated NSCs to =
GCV might=20
                  partly explain why the bystander effect observed =
between NSCtk=20
                  and C6 cells is much more potent than that observed =
between=20
                  tumor cells. Whatever the mechanism, this surprisingly =

                  stronger bystander effect, as well as the higher =
biological=20
                  safety, suggests a broad prospect of the 'NSCtk =
therapy' in=20
                  clinical applications.</P><A name=3DREFS>
                  <HR SIZE=3D1>
                  </A>
                  <P>&nbsp;<A=20
                  =
href=3D"http://content.karger.com/produktedb/produkte.asp?typ=3Dfulltext&=
amp;file=3DOCL2005069006503#OLN"><IMG=20
                  alt=3D"goto top of outline"=20
                  =
src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
es/global/odstop.gif"'=20
                  border=3D0></A>&nbsp;<B =
class=3Dsection1>References</B><BR>
                  <DL compact><BR><BR><!-- 1-->
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                    <DD>DeAngelis LM: Brain tumors. N Engl J Med=20
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multiforme.=20
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src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
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                    width=3D20 border=3D0></A> <BR><BR><!-- 9-->
                    <DT>9
                    <DD>Namba H, Tagawa M, Miyagawa T, Iwadate Y, =
Sakiyama S:=20
                    Treatment of rat experimental brain tumors by herpes =
simplex=20
                    virus thymidine kinase gene-transduced allogeneic =
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 10-->
                    <DT>10
                    <DD>Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, =
Yang W,=20
                    Small JE, Herrlinger U, Ourednik V, Black PM, =
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 11-->
                    <DT>11
                    <DD>Ehtesham M, Kabos P, Kabosova A, Neuman T, Black =
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                    JS: The use of interleukin 12-secreting neural stem =
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                    2002;62:5657-5663.<A=20
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 12-->
                    <DT>12
                    <DD>Shah K, Bureau E, Kim DE, Yang K, Tang Y, =
Weissleder R,=20
                    Breakefield XO: Glioma therapy and real-time imaging =
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 13-->
                    <DT>13
                    <DD>Zhang Z, Jiang Q, Jiang F, Ding G, Zhang R, Wang =
L,=20
                    Zhang L, Robin AM, Katakowski M, Chopp M: In vivo =
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                    resonance imaging tracks adult neural progenitor =
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 14-->
                    <DT>14
                    <DD>Li S, Tokuyama T, Yamamoto J, Koide M, Yokota N, =
Namba=20
                    H: Bystander effect-mediated gene therapy of gliomas =
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                    genetically engineered neural stem cells. Cancer =
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 15-->
                    <DT>15
                    <DD>Mosmann T: Rapid colorimetric assay for cellular =
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                    and survival: application to proliferation and =
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                    assays. J Immunol Methods 1983;65:55-63.<A=20
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 16-->
                    <DT>16
                    <DD>Barresi V, Belluardo N, Sipione S, Mudo G, =
Cattaneo E,=20
                    Condorelli DF: Transplantation of prodrug-converting =
neural=20
                    progenitor cells for brain tumor therapy. Cancer =
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es/note.gif"'=20
                    width=3D20 border=3D0></A> <BR><BR><!-- 17-->
                    <DT>17
                    <DD>Namba H, Iwadate Y, Kawamura K, Sakiyama S, =
Tagawa M:=20
                    Efficacy of the bystander effect in the herpes =
simplex virus=20
                    thymidine kinase-mediated gene therapy is influenced =
by the=20
                    expression of connexin43 in the target cells. Cancer =
Gene=20
                    Ther 2001;8:414-420.<A=20
                    href=3D"javascript:NewWindow('000091032.r017')"><IMG =
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src=3D'http://content.karger.com/produktedb/Showpic.asp?filename=3D"/imag=
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                    width=3D20 border=3D0></A> </DD></DL>
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                  <P>&nbsp;<A=20
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                  border=3D0></A>&nbsp;<B class=3Dsection1>Author =
Contacts</B></P>
                  <P>Hiroki Namba, MD<BR>Department of Neurosurgery, =
Hamamatsu=20
                  University School of Medicine<BR>1-20-1=20
                  Handayama<BR>Hamamatsu, 431-3192 (Japan)<BR>Tel. +81 =
53 435=20
                  2281, Fax +81 53 435 2282, E-Mail <A=20
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                  Information</B></P>
                  <P>Received: June 21, 2005<BR>Accepted after revision: =
October=20
                  15, 2005<BR>Published online: January 16, =
2006<BR>Number of=20
                  Print Pages : <B>6</B><BR>Number of Figures : =
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                  <P>Oncology (<I>International Journal of Cancer =
Research and=20
                  Treatment</I>) </P>
                  <P>Vol. 69, No. 6, Year 2005 (Cover Date: January =
2006)</P>
                  <P>Journal Editor: Trump, D.L. (Buffalo, =
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                  <P>Drug Dosage: The authors and the publisher have =
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