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[A1CR]

CR may counter the decrease in stems cells, may be a message
from (1). Although CR seems to prevent cancers, a popular
press report (2) seems to say that a new study (3) reports
that stem cells are the cause for colon cancer
re-occurrence. May this be why CR also
does not seem to help for previously initiated cancers?

1. Bauer JH, Poon PC, Glatt-Deeley H, Abrams JM, Helfand SL.
Neuronal expression of p53 dominant-negative proteins in
adult Drosophila
melanogaster extends life span.
Curr Biol. 2005 Nov 22;15(22):2063-8.
PMID: 16303568

Hyperactivation of p53 leads to a reduction in tumor
formation and an
unexpected shortening of life span in two different model
systems . The
decreased life span occurs with signs of accelerated aging,
such as
osteoporosis, reduction in body weight, atrophy of organs,
decreased stress
resistance, and depletion of hematopoietic stem cells. These
observations
suggest a role for p53 in the determination of life span and
the speculation
that decreasing p53 activity may result in positive effects
on some aging
phenotypes . In this report, we show that expression of
dominant-negative
versions of Drosophila melanogaster p53 in adult neurons
extends life span
and increases genotoxic stress resistance in the fly.
Consistent with this,
a naturally occurring allele with decreased p53 activity has
been associated
with extended survival in humans . Expression of the
dominant-negative
Drosophila melanogaster p53 constructs does not further
increase the
extended life span of flies that are calorie restricted,
suggesting that a
decrease in p53 activity may mediate a component of the
calorie-restriction
life span-extending pathway in flies.

2. Recurrence of colon cancer linked to stem cells
Last Updated: Monday, November 20, 2006
CBC News

Findings that suggest stem cells are at the root of colon
cancer may lead to
more effective treatments for the disease, and explain its
tendency to
recur.

Canadian and Italian researchers discovered the key role
played by stem
cells by implanting cells from human colorectal tumours in
specially bred
mice lacking an immune system.

(CBC) The researchers asked if every cell in a tumour has
the same ability
to keep cancer going. The answer they got was no.

It turns out only one in 60,000 cells - the stem cell - in
the tumour
actually has the ability to generate the cancer again ...

http://tinyurl.com/y6yqgm

3. A human colon cancer cell capable of initiating tumour
growth in
immunodeficient mice
Catherine A. O'Brien, Aaron Pollett, Steven Gallinger and
John E. Dick
Nature advance online publication 19 November 2006

Colon cancer is one of the best-understood neoplasms from a
genetic
perspective1, 2, 3, yet it remains the second most common
cause of
cancer-related death, indicating that some of its cancer
cells are not
eradicated by current therapies4, 5. What has yet to be
established is
whether every colon cancer cell possesses the potential to
initiate and
sustain tumour growth, or whether the tumour is
hierarchically organized so
that only a subset of cells-cancer stem cells-possess such
potential6, 7.
Here we use renal capsule transplantation in immunodeficient
NOD/SCID mice
to identify a human colon cancer-initiating cell (CC-IC).
Purification
experiments established that all CC-ICs were CD133+; the
CD133- cells that
comprised the majority of the tumour were unable to initiate
tumour growth.
We calculated by limiting dilution analysis that there was
one CC-IC in 5.7
104 unfractionated tumour cells, whereas there was one CC-IC
in 262 CD133+
cells, representing >200-fold enrichment. CC-ICs within the
CD133+
population were able to maintain themselves as well as
differentiate and
re-establish tumour heterogeneity upon serial
transplantation. The
identification of colon cancer stem cells that are distinct
from the bulk
tumour cells provides strong support for the hierarchical
organization of
human colon cancer, and their existence suggests that for
therapeutic
strategies to be effective, they must target the cancer stem
cells.

Human tumour biology has long been studied in experimental
xenogeneic colon
cancer models, typically generated by injecting cell lines
or implanting
pieces of primary tumours into immunodeficient mice8, 9, 10.
However, cell
lines do not recapitulate all aspects of primary tumours and
a quantitative
assay for single cells is required to determine whether
CC-ICs exist in
colon cancer. Therefore, we developed a reliable xenograft
model through
subrenal capsule implantation of human colon cancer cell
suspensions into
pre-irradiated non-obese diabetic (NOD)/severe-combined
immunodeficient
(SCID) mice. Tumour formation occurred in 17 out of 17
samples tested,
comprising six primary colon cancers, ten liver metastases,
and one
retroperitoneal metastasis (Table 1 and Supplementary Fig.
1a and b). The
histology and degree of differentiation of all xenografts
resembled the
original tumours from which they were derived (Fig. 1). The
tumours were
positive for cytokeratin-20 (CK-20) and negative for
cytokeratin-7 (CK-7), a
pattern seen almost exclusively in colonic adenocarcinoma11.
Xenografts and
parent tumours exhibited similar patterns of expression for
multiple mucin
antigens and for markers highly associated with colon
cancers including
carcinoembryonic antigen (CEA)12 and p53 (ref. 13). The
degree of tumour
cell proliferation, as revealed by MIB-1 staining12, was
similar in
xenografts and parent tumours (Fig. 1). Thus, the xenografts
generated in
this model matched the phenotypes of the original tumours.

To determine whether this xenotransplant system was
quantitative and able to
detect single CC-ICs, we performed limiting dilution
experiments. Groups of
NOD/SCID mice were transplanted with replicate doses of
human colon cancer
cells over a range from doses unable to initiate tumour
growth to doses that
always initiated tumour formation (Table 2). The
tumour-forming capacity and
phenotypic appearance were the same for primary xenografts
and tumours
passaged into secondary and tertiary recipients. The similar
behaviour of
the primary and passaged tumours made it possible to combine
data to
calculate the average frequency of CC-ICs in these tumours
using the
maximum-likelihood estimation method of limiting dilution
assay14, 15. We
calculated that on average there was one CC-IC per 5.7 104
(95% confidence
interval: one per 3.4 104 to one per 9.3 104)
unfractionated colon cancer
cells, although the limited number of recipients used for
any single sample
prevented precise estimation of the patient-to-patient
variation. Thus, as
has been shown for breast16 and brain17 cancer, only a small
subset of colon
cancer cells are able to initiate tumour growth.

By combining the quantitative assay with cell fractionation,
we were able to
test whether human colon cancer adheres to the stochastic
model, in which
every tumour cell has equal tumour initiation potential6, 7,
18, or to the
cancer stem cell (CSC) model, in which some cell fractions
are enriched for
CC-IC activity while others are completely devoid of
CC-ICs6, 7, 18 .We
focused on fractionation based on CD133 expression. The
phylogenetically
conserved protein CD133 was recently identified19, 20 as a
potential CSC
marker in brain17 and prostate19 cancer. CD133 expression
ranged from 1.8 to
24.5% in the colon cancer samples described in Table 1 (Fig.
2a). We used
immunohistochemistry to show that CD133 was expressed in
clusters amid
negative cells (Fig. 2b). Normal colon tissue also expressed
CD133 but at
much lower levels than primary colonic tumours (0.4-2.1%
normal versus
8.9-15.9%) (Table 1; Fig. 2a). To determine whether CD133
expression
enriches for CC-ICs, colon cancer cells were separated into
CD133- and
CD133+ fractions and injected into NOD/SCID mice. Of 47 mice
(dose range: 2
103 to 2.5 105) injected with CD133- cells, only one mouse
transplanted
with the highest cell dose (Table 2) generated a tumour.
Because the CD133-
fraction was contaminated with 5-15% of cells expressing
only low levels of
CD133, we conclude that neither CD133- nor CD133low cells
possess CC-IC
activity. In contrast, tumours were consistently generated
after injection
of 1 103 colon cancer cells expressing the highest levels
of CD133
(CD133+), and injection of 100 CD133+ cells resulted in
tumour growth in one
of four mice. Thus, while significantly enriched, not every
CD133+ cell
represents a CC-IC. In total, 45 of 49 mice injected with
CD133+ cells
developed tumours (Table 2). All tumours generated from
CD133+ cells were
phenotypically similar to the original tumours (Fig. 1).
Moreover, CD133
expression ranged from 1.7% to 22.4% in the xenografts,
similar to the range
seen in the original tumours. The isolation of tumorigenic and
non-tumorigenic fractions, based on CD133 expression,
provides strong
support for the cellular organization of human colon cancer
according to the
CSC model.

Another prediction of the CSC model is that CC-ICs should
self-renew to
generate new CSCs and differentiate to generate
non-tumorigenic progeny.
Serial transplantation experiments from ten primary
xenografts demonstrated
that only CD133+ and not CD133- cells were able to initiate
tumour growth in
serially transplanted secondary and tertiary mice. Tumours,
either primary
or passaged, could have been infiltrated with non-malignant
cell types, but
the high proportion (>98%) of CD133- cells that co-expressed
the
human-specific protein epithelial specific antigen from both
primary and
passaged tumours confirmed they were human colon cancer
cells and not
infiltrating murine cells or non-epithelial human cells that
had somehow
been co-passaged (Supplementary Fig. 2). Additionally, we
showed that CD133-
cells remained viable and stained positive for epithelial
specific antigen
under the renal capsule but were unable to regenerate
tumours for as long as
15 to 21 weeks post-injection (Supplementary Fig. 3a, b and
c). Furthermore,
cells positive for epithelial specific antigen were also
malignant, staining
positive for p53, in cases where the parent tumours were
p53+ (Supplementary
Fig. 3d). These studies were performed on passaged tumours,
so it is highly
unlikely the CD133- cells are pre-malignant cells, rather
than malignant
cells. Therefore, the CD133- cells are generated from the
CD133+ cells. Thus
we can conclude that only CD133+ CC-ICs can be serially
passaged, forming
xenografts that re-establish tumour heterogeneity,
generating both CD133+
and CD133- progeny in a ratio similar to that in the patient
tumour (Fig.
2c).

To determine the frequency of CC-ICs within the CD133+
subset we carried out
a limiting dilution assay, using the same principles as
described for
unfractionated tumour cells14, 15. The passaged xenografts
matched the
phenotype and tumour-forming capacity of the parent tumours,
enabling us to
combine data from passaged and primary cells. The frequency
was calculated
to average one CC-IC in 262 CD133+ colon cancer cells (95%
confidence
interval: one in 129 to one in 534), representing a 216-fold
enrichment of
CC-ICs compared to unfractionated colon cancer cells.

Interestingly, the estimate of CC-IC frequency when
back-calculated to take
into account the proportion of CD133+ cells within the
unfractionated tumour
is 20-fold higher than when unfractionated cell suspensions
were assayed.
For example, multiplication of the CC-IC frequency (one in
262) by the mean
level of CD133 expression for all samples (12%) yields an
estimate of 20
CC-ICs per 57,000 unfractionated tumour cells, instead of
the one in 57,000
measured in the initial limiting dilution assay. One
possible explanation
for this finding is that the CD133- progeny are negatively
regulating the
growth of the CD133+ CC-IC fraction, thereby requiring a
greater overall
number of CD133+ cells to give rise to a tumour, as has been
observed in
human haematopoietic stem cells21.

Here we have identified and characterized CC-ICs from human
colon tumour
samples on the basis of their ability to initiate human
colon cancer after
transplantation into NOD/SCID mice. CC-ICs possessed two key
criteria that
define stem cells: after transplantation at limit dilution,
single CC-ICs
proliferated extensively and differentiated to produce
tumours that were
phenotypically similar to the original patient tumours, and
as a population
they self-renewed, enabling re-establishment of colon cancer
in secondary
and tertiary recipient mice. CC-ICs were almost exclusively
CD133+, while
the CD133- fraction that comprised 81-98% of the tumour mass
had no CC-IC
activity. Thus colon cancer, like acute myelogenous
leukaemia22, breast16
and brain17 cancer is organized as a hierarchy in which a
small population
of CSCs sustain the tumour. The calculated frequency of
CC-ICs was, on
average, one in 262 CD133+ cells, so clearly the majority of
CD133+ cells
are not CC-ICs. As described for CD34 expression on acute
myelogenous
leukaemic stem cells, this result suggests there may be a
hierarchy of
CC-ICs and progenitors23. Thus, future studies using
additional cell surface
markers in combination with CD133 are necessary to purify
the CC-IC fraction
further. Finally, clonal tracking studies need to be carried
out to
establish self-renewal at the single cell level and
determine whether
different subclasses of CC-ICs exist23.

Although we found CD133+ CC-ICs in primary and metastatic
tumours, most
primary colon cancers tested were derived from right-sided
tumours and may
not be representative of all forms of colon cancer.
Nevertheless, our
findings should stimulate future studies directed towards
increasing the
range of colon cancer samples tested and addressing whether
qualitative or
quantitative CC-IC differences have prognostic value.
Analysis of CC-ICs
using molecular genetic techniques should further our
understanding of the
genetic abnormalities commonly associated with colon cancer,
such as
microsatellite status. Furthermore, as our understanding of
normal colon
stem and progenitor cell biology improves, it should be
possible to gain
insight into the cells that are the origin of colon cancer
and the cellular
context within which the well-characterized sequence of
genetic events
occurs24, 25.

The existence of tumorigenic and non-tumorigenic cells
within colon cancers
implies that not all the cells within a tumour are able to
initiate and
sustain neoplastic growth. This concept has important
therapeutic
implications, and may explain the observation that small
numbers of
disseminated cancer cells can be detected in the circulation
of patients
that never develop metastatic disease18. The identification
of CC-ICs
provides a powerful tool with which to develop a better
understanding of
tumour progression and the metastatic process, given that
the CSC model
predicts that the unit of selection in tumour progression
would be the CSC
itself. Moreover, because CC-ICs are the driving force
sustaining tumour
growth, developing adjuvant therapies directed at
specifically eliminating
the CC-IC fraction may prove to be a more effective strategy
for reducing
both local and distant recurrence6, 26. The model described
here will
provide the means of further purifying and functionally
characterizing the
biological properties of the CC-IC fraction, with the goal
of developing new
therapeutic strategies directed specifically against CC-ICs.