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THE EFFECTS OF PURINE NUCLEOSIDE
PHOSPHORYLASE (PNP) DEFICIENCY ON
THYMOCYTE DEVELOPMENT
by
Taniya Papinazath
A thesis submitted in conformity with the requirements
PNP is a crucial enzyme in purine metabolism, and its inherited defects result in severe
T-lineage immune deficiency in humans. I hypothesized that PNP deficiency disrupts the
development of late CD4-CD8- double negative (DN) thymocytes and induces mitochondrial-
mediated apoptosis of CD4+CD8+ double positive (DP) thymocytes. By using PNP-deficient
(PNP-/-) mice as well as an OP9-DL1 co-culture system simulating PNP-deficient conditions, I
demonstrated that PNP deficiency interferes with the maturation of DN thymocytes at the
transition from DN3 to DN4 stage. Although PNP deficiency does not affect the generation or
proliferation of DP thymocytes, PNP-/- DP thymocytes were observed to undergo apoptosis at a
higher rate. My results suggest that apoptosis is induced through a mitochondrial mediated
pathway. Additionally, re-introduction of PNP into PNP-/- thymocytes protected the cells from
the toxic effects of deoxyguanosine by preventing the formation of deoxyguanosine
triphosphate, indicating that the toxic metabolite in PNP deficiency is deoxyguanosine.
iii
Acknowledgements
First and foremost, I would like to thank my supervisor, Dr Eyal Grunebaum for his
wonderful supervision, guidance and mentoring. In the past two years under his supervision,
he has been encouraging and patient in both my scientific and artistic pursuits. Also, thank
you for allowing me to explore without inhibition and learn from my mistakes, I am truly
grateful for this experience.
I would like to extend my appreciation to the members of my program advisory
committee Dr.Yigal Dror, Dr. Cynthia Guidos and Dr. Chetankumar Tailor. I thank you all
for sitting through the numerous PAC meetings listening to me talk time and time again
about how PNP deficiency causes T-cell immune deficiency, and for giving me valuable
suggestions, without which the successful completion of this masters thesis would not have
been possible.
A special thank goes out to the entire Grunebaum lab. Weixian Min for helping with
all the animal handling, Yongmao Yu for giving timely input on flow data, Alka Arora for
making me feel at home when I first joined the lab, and Alireza Mansouri for bringing an
element of humour and joy into a sometimes frustrating scientific environment (and not to
forget all the entertaining coffee breaks).
I would also like to acknowledge the members of the Dror lab; for being patient in
answering all my western blot queries, for allowing me to borrow the cell separation magnets
God knows how many times, and for hearing me out in times of despair. Thank you all for
making me feel like an “honorary” member of your lab, inviting me for lab luncheons and
other lab celebrations.
My thanks is also extended to members of the PMH flow facility, Casey and Francis
for helping me get started in the world of flow cytometry.
My graduate life would be nothing without all the friends I made along the way. You
are far too many to list but thank you for your camaraderie and friendship. A special shout
out goes to Geneve Awong for clearing my OP9-DL1 co-culture queries.
Last but definitely not the least; I’d like to thank my loving family for all their
support and encouragement, giving me the strength to overcome all obstacles faced during
my graduate studies.
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Table of Contents
Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Table of Contents ............................................................................................................... iv List of Figures .................................................................................................................... vi List of Abbreviations ....................................................................................................... viii CHAPTER 1: INTRODUCTION ....................................................................................... 1
1.1. PNP Deficiency .................................................................................................... 2 Clinical Aspects .............................................................................................................. 4 Human PNP structure, chemistry, genome and mutations ............................................. 5 Purine Metabolism .......................................................................................................... 6 Pathophysiology .............................................................................................................. 7 Proposed mechanisms of the cellular immunodeficiency in PNP deficiency ................. 7 PNP as a target enzyme for chemotherapeutic applications ......................................... 10 Treatment Options ........................................................................................................ 11 TAT-PNP ...................................................................................................................... 12 Study Model .................................................................................................................. 13 1.2. Murine Thymocyte Development ...................................................................... 14 Introduction ................................................................................................................... 14 T-Cell Receptor (TCR) ................................................................................................. 15 Thymic Organization .................................................................................................... 16 Murine Thymocyte Development ................................................................................. 18 Notch Signaling ............................................................................................................ 22 OP9-DL1 ....................................................................................................................... 23 IL-7 and Flt3L ............................................................................................................... 24 Thymocyte abnormalities in PNP deficiency ............................................................... 25 Effect of PNP deficiency on T-lymphocytes ................................................................ 25 Selective dGuo toxicity to thymocytes and T-lymphocytes ......................................... 26 Thymocyte apoptosis associated with purine deficiencies ........................................... 27 • Insights gained from ADA deficiency ............................................................... 27 • Insights gained from PNP deficiency ................................................................ 29
CHAPTER 2: RATIONALE, HYPOTHESIS AND EXPERIMENTAL OBJECTIVES 33
4.1. Abnormal lymphocyte subpopulation in thymus of PNP-/- mice. ...................... 52
Aim 1: To determine whether PNP deficiency interferes with the transition of thymocytes from the DN3 to the DN4 stage ................................................................. 54 4.2. PNP deficiency disrupts thymocyte maturation at the DN3 to DN4 stage ........ 54 transition. ........................................................................................................... 54 Conclusion .................................................................................................................... 65
Aim 2: To determine whether the reduced number of DP thymocytes in PNP deficiency is due to increased apoptosis caused by dGuo ............................................ 66 4.3. Increased apoptosis in freshly isolated PNP-/- DP thymocytes. ......................... 66 4.4. PNP Deficiency does not affect proliferation or the maturation of DP thymocytes. ................................................................................................................... 68 4.5. dGuo causes increased thymocyte apoptosis. .................................................... 71 Direct effects of dGuo phosphorylation product dGMP on thymocyte apoptosis. ....... 72 Re-introduction of PNP into the cells increases the survival of PNP-/- thymocytes. .... 74 4.6. dGuo preferentially causes the apoptosis of PNP-/- DP thymocytes. ................. 76 Conclusion .................................................................................................................... 79
Aim 3: To examine whether the increased apoptosis of DP thymocytes in PNP deficiency is initiated in the mitochondria .................................................................... 80 4.7. Fas expression is not upregulated in PNP deficiency. ....................................... 80 4.8. Dissipation of mitochondrial membrane potential (MMP). ............................... 82 4.9. Changes in MMP occur before nuclear DNA fragmentation: ........................... 84 4.10. Caspase inhibition prevents dGuo induced DNA fragmentation but does not .. 87 prevent apoptosis of the cells. ............................................................................ 87 Conclusion .................................................................................................................... 89
Figure 1 Schematic representation of the role of PNP in purine metabolism, salvage of purines from ribo and deoxyribonucleosides.
3
Figure 2 Metabolic effects of PNP deficiency. 9
Figure 3 Structure of BCX-1777. 10
Figure 4 Traffic of thymocytes through the thymus. 17
Figure 5 Schematic representation of murine thymocyte development. 21
Figure 6 A schematic representation of postulated apoptotic mechanisms caused by ADA and PNP deficiencies. 32
Figure 7 Stages in murine thymoctye development. 35
Figure 8 Schematic representation of the PNP pathway. 37
Figure 9 Proposed mechanism of PNP deficiency. 38
Figure 10 Distribution of thymocyte populations in PNP-/- mice. 53
Figure 11 Characterization of maturation of DN thymocytes in PNP- deficient mice. 55
Figure 12 Characterization of maturation of DN thymocytes under PNP- deficient conditions using OP9-DL1 co-culture. 57,58
Figure 13 Characterization of maturation of DN thymocytes under PNP- deficient conditions using excess dGuo. 60,61
Figure 14 Characterization of DN3 and DN4 subsets by upregulation of CD71 expression. 63
vii
Figure 15 Characterization of the DN3 and DN4 subsets by intracellular TCRβ staining. 64
Figure 16 Increased apoptosis in freshly isolated DP thymocytes from PNP-deficient mice. 67
Figure 17 BrdU labeling profiles for PNP-/- mice show normal thymocyte proliferation but indicate a survival defect most strikingly obvious on day 5 after labeling.
69,70
Figure 18 Schematic representation of the role of PNP in purine degradation and salvage. 71
Figure 19 Demonstration of apoptosis of thymocytes after incubation in vitro with different purine metabolites. 73
Figure 20 Introduction of PNP into PNP-deficient cultures increases the rate of thymocyte survival. 75
4.8. Dissipation of mitochondrial membrane potential (MMP).
The intrinsic pathway (Figure 6, Pathway B) involves the release of pro-apoptotic proteins
that activate caspase enzymes from the mitochondria ultimately triggering apoptosis (Fulda
et al., 2006; Mayer et al., 2003). If the apoptotic signals are generated intramitochondrially,
the disruption of MMP occurs before nuclear DNA fragmentation. In order to determine
whether PNP deficiency disrupts the mitochondrial membrane, the integrity of the
mitochondrial membrane was analyzed by staining with MitoTracker® Red CMXRos.
Following 24 hours of dGuo treatment, a significantly higher percentage of PNP-/-
thymocytes loses MMP (M2), as indicated by a decrease in fluorescence intensity (Figure
23A). Results demonstrate a significantly higher percentage of dGuo treated PNP-/-
thymocytes losing MMP compared to the controls (Figure 23B).
83
A)
B)
0
5
10
15
20
25
PNP+/+ PNP-/-
Perc
enta
ge o
f cel
ls lo
sing
MM
P
PNP+/+PNP-/-
Figure 23: Disruption of MMP. Thymocytes from PNP-/- mice and age matched controls
were labeled with MitoTracker (a sensitive indicator of MMP) after 24 hours of dGuo
treatment. (A) In the histrograms, M1 indicates the percentage of cells maintaining MMP and
M2 indicates the percentage of cells losing MMP. (B) Graph represents the mean ±SD of the
percentage of PNP-/- (white bar) and PNP+/+ (black bar) thymocytes losing MMP from 3
separate experiments containing one or two mice in each (*p=0.003).
PNP-/-
*
M1= 95.48% M2= 4.34%
M1= 90.27% M2= 9.47%
M1
M1
M2M2
M1= 95.22% M2= 4.55%
M1= 78.95% M2= 20.95%
M1M1
M2M2
PNP+/+
Untreated dGuo treated
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4.9. Changes in MMP occur before nuclear DNA fragmentation:
When apoptotic signals are initiated within the mitochondria, the disruption of MMP
occurs prior to nuclear DNA fragmentation (Ravagnan et al., 1999). Thus, I was interested to
confirm that indeed change in MMP occurs prior to nuclear DNA fragmentation in PNP
deficiency. Therefore, I measured MMP using a mitochondria specific dye, and DNA
fragmentation by the TUNEL assay in dGuo treated PNP-/- thymocytes at several time points.
The time points that I examined included;
6-8 hours treatment, which I hypothesized is prior to initiation of apoptosis,
12 hours treatment, which is when I hypothesized apoptosis is initiated,
16-24 hours of treatment, when apoptosis is fully developed in both the mitochondria and
nucleus (summarized in Figure 24G).
As expected, 6-8 hours after treating PNP-/- thymocytes with dGuo, there was no
increase in TUNEL+ cells or increase in the percentage of cells with abnormal MMP (Figure
24G). In marked contrast, at 12 hours, a significant higher percentage of cells had disrupted
MMP (Figure 24A is a representative example), without a significant change in DNA
fragmentation (Figure 24B), and the results of 4 separate experiments are summarized in
Figure 24C. At 16-24 hours, the percentage of cells with changes in MMP (Figure 24D) was
similar to the percentage of cells undergoing DNA fragmentation (Figure 24E), with the
results of 4 separate experiments summarized in Figure 24F. In conclusion, these results
suggest that apoptosis in PNP-/- thymocytes exposed to dGuo, is initiated in the mitochondria.
85
A) B)
Mitotracker TUNELMitotracker TUNEL
C)
12 hour treatment
0
2
4
6
8
10
12
14
UT dGuo
Perc
enta
ge o
f cel
ls
DNA fragmentationLoss in MMP (M2)
D) E)
*
86
F)
16 hour treatment
0
5
10
15
20
25
UT dGuo
Perc
enta
ge o
f cel
ls
DNA fragmentationLoss in MMP
G)
0
5
10
15
20
25
6 hours 12 hours 16 hours
Thymocytes treated with 12.5μM dGuo
Perc
enta
ge o
f cel
ls
DNA fragmentationLoss in MMP
Figure 24: Effects of dGuo on MMP and DNA fragmentation after 12 hours and 16 hours of treatment. PNP-/- thymocytes were incubated in RPMI supplemented with 5% FBS in the presence of 12.5μM dGuo for the indicated time points. At the indicated time points the cells were stained with Mitotracker to investigate loss of MMP and nuclear DNA fragmentation was assayed by the TUNEL technique. (A) and (D) demonstrate changes in MMP at 12 h and 16 h respectively. Percentages of cells are indicated above the bars. (B) and (E) demonstrate nuclear DNA fragmentation measured by the TUNEL assay at 12 h and 16 h respectively. Percentages of apoptotic cells are indicated above the bars. (C) and (F) summarize DNA fragmentation (black) and changes in MMP (white) in untreated and 12.5μM dGuo treated samples at 12 h (*p=0.001) and 16 h respectively. The data represents mean ± SD for 4 independent experiments at each time point with one or two mice in each. (G) Graph summarizes the DNA fragmentation (black) and changes in MMP (white) profiles following 6 h, 12 h and 16 h dGuo treatment of PNP-/- thymocytes (*p=0.001) (UT, untreated).
*
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4.10. Caspase inhibition prevents dGuo induced DNA fragmentation but does not
prevent apoptosis of the cells.
To confirm that changes in MMP indeed occured before DNA fragmentation I used an
alternative strategy which determined the effect of caspase inhibition on MMP and DNA
fragmentation. When the apoptotic signals are initiated within the mitochondria, the
disruption of MMP does not involve the release and activation of caspases. Hence this event
is considered to be a caspase independent process. In contrast, when apoptotic signals
originate outside the mitochondria, then the subsequent disruption of MMP is a caspase
dependant process (Ravagnan et al., 1999). Consequently, if apoptosis originated within the
mitochondria then thymocyte apoptosis would not be affected by caspase inhibition.
However, if apoptosis signals originated due to nuclear DNA damage then the apoptosis
would be haulted by caspase inhibition. Therefore I examined the effect of caspase
inhibition (using the pan caspase inhibitor z-VADfmk) on dGuo induced apoptosis.
I first demonstrated that z-VADfmk prevented apoptosis (measured by the percentage
of Annexin+ cells) induced by dexamethasone (Dex) (Figure 25A), which is a well known
caspase-dependent apoptosis inducing agent. In contrast, z-VADfmk was unable to protect
thymocytes from dGuo induced apoptosis, as indicated by Annexin-V staining (Figure 25B).
Interestingly, z-VADfmk did prevent dGuo induced DNA fragmentation, which is
considered to be a late apoptotic event suggesting that in addition to the early mitochondria
induced apoptosis, dGuo also has a late, DNA fragmentation effect (Figure 25C).
88
A)
42.02
22.77
Annexin
Cel
l cou
nt
Dex Dex+ zVAD
42.02
22.77
Annexin
Cel
l cou
nt
Dex Dex+ zVAD
B)
Annexin
Cel
l cou
nt
dGuo dGuo + zVAD
17.48
25.61
Annexin
Cel
l cou
nt
dGuo dGuo + zVAD
17.48
25.61
C)
14.091.65
TUNEL
Cel
l cou
nt
dGuo dGuo + zVAD
14.091.65
TUNEL
Cel
l cou
nt
dGuo dGuo + zVAD
Figure 25: Effects of pan-caspase inhibition on dGuo induced apoptosis and nuclear DNA
fragmentation. PNP-/- thymocytes maintained in RPMI 1640 medium with 5% FBS, were
pre-treated for 30 minutes with 50μM z-VADfmk followed by the addition of (A) 3.75 μM
Dex or (B) and (C) 12.5μM dGuo. 20-24 hours later, apoptosis was measured by Annexin-V
staining (Figures A and B) and DNA fragmentation was measured by the TUNEL assay
(Figure C). Results are representative of 4 independent experiments with one or two mice in
each. The percentage of apoptotic cells are indicated above the bars.
89
CONCLUSION
In summary, these results demonstrate that a change in MMP occurs prior to nuclear DNA
fragmentation. Also, the loss in MMP induced apoptosis is resistant tocaspase inhibition
suggesting it is a caspase independent mechanism. Taken together, these findings suggest
that the accumulation of dGuo and its phosphorylated products in PNP-/- thymocytes
increased apoptosis in a mitochondrial mediated pathway.
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CHAPTER 5: DISCUSSION
91
Discussion
PNP deficiency disrupts thymocyte development. However, the lack of availability of
thymus tissue from affected patients has prevented detailed analysis of the precise effects of
abnormal purine metabolism on thymocytes. PNP-/- mice displaying metabolic and immune
phenotype similar to the human disease have helped understand some of the mechanisms
associated with toxic purine metabolites, and hence were extensively used in this study.
Although PNP is a ubiquitous enzyme, its preferential effect on thymocytes and T-cells is not
surprising, as these cells, unlike many other cell types, are unique in that they undergo
constant selection and proliferation during their lifetime. To maintain a constant homeostasis
in these cells requires a balance in the nucleotide pools to compensate for the DNA damage
and repair induced during the process of selection and subsequent proliferation. This may be
be the most likely explaination for the selective toxicity of thymocytes to the effects of PNP
deficiency and thus, has been the area of focus for most researchers. However, preliminary
data from our lab and previous observations by others suggests involvement of cells beyond
the thymus. Neurological dysfunction has been reported in more than half of the patients
(Markert, 1991). Similar to PNP-deficient patients PNP-/- mice manifest with neuroloigal
abnormalities as tested by their inability to sustain on a spinning rotorod and increased
apoptosis indicated by in situ TUNEL staining on purkinje cells from PNP-/- mice
cerebellums (Grunebaum, unpublished). Also, PNP-/- skin fibroblasts from mice have been
seen to proliferate at a slower rate compared to normal controls as tested by the thymidine
uptake assay (Grunebaum, unpublished). Few patients also developed bone marrow
abnormalities possibly due to hypersensitivity to irradiation (Dror et al., 2004). Reduced
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B- cell numbers and abnormal humoral function have been reported amongst a few patients
(Markert, 1987). These findings are indicative that PNP is essential in the maintainance and
functioning of many cells in the body.
We and others have found significant increases in the number of DN thymocytes in
the thymi of PNP-/- mice, which is particularly impressive when considering that the thymus
of these mice is atrophic with marked reduction in other thymocyte populations. This is
suggestive of abnormal maturation within the DN subset. Therefore our focus was initially
targeted towards studying the development of DN thymocytes. The significant increase in
the percentage and numbers of DN3 and reduction of DN4 and DP cells that I found in
freshly isolated thymocytes and ex-vivo cultures simulating PNP deficiency, clearly
demonstrate that maturation from the DN3 stage to the later stages are affected by PNP
deficiency. The limitations of these experiments as mentioned earlier, is that by the third
week of co-culture when most of the cells had developed into the later DN stages (DN3 and
DN4) and to the DP and SP stage, CD4+/CD8+ were not gated out to analyse the DN subsets.
This makes interpretation of results difficult as the “DN4” subset is really a heterogeneous
population consisiting of DN4, DP and SP cells. However, the significant increase in the
percentage of CD25+ cells at this stage was evidence enough for us to conclude that the delay
in thymocyte development in PNP deficiency begins at the DN3 stage. Another method to
study DN generation and expansion would be to carry out a dynamic analysis by a short term
BrdU pulse chase experiment. This would further show whether the early DNs are being
generated and proliferating at a proper rate. Moreover, investigating the exact stage of
developmental delay, i.e. whether between DN3 and DN4 or between DN4 and DP stage,
proved to be a challenge due to the absence of a distinct cell surface marker for the DN4
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subset. The transition between DN3 to DN4 is a crucial check-point in thymocyte
maturation, and it has been estimated that 4 out of 9 DN3 cells die at this stage as a result of
failure to rearrange their TCRβ locus (Gärtner et al., 1999) and hence my results are not
unexpected. I therefore also determined the percentage of PNP-/- DN3 and DN4 thymocytes
that express intracellular TCRβ chain. I found a decrease in the percentage of DN3 cells
transiting to the DN4 stage by down regulating CD25 and upregulating intracellular TCRβ
expression in PNP-/- mice compared to controls, further indicating a developmental delay
between the DN3 and DN4 stage. However, the similar percentages of late DN3 cells in
PNP-/- and PNP+/+ mice indicate that TCRβ rearrangement is taking place normally. My
results are also in concordance to those reported in ADA deficiency, wherein ADA
deficiency did not seem to compromise TCRβ chain rearrangement in developing
thymocytes (Thompson et al.,2000). Collectively, these results suggest that rearrangement of
the TCRβ locus and expression of the TCRβ chain is not affected by PNP deficiency. It will
be beneficial to study the TCR-vβ repertoire in the periphery of PNP-/- mice compared to
controls by using a panel of antibodies to different TCR-vβ chains, which is a quantitative
approach. A qualitative and more in-depth analysis of TCR-vβ repertoire is by a PCR
technique such as spectra typing.
Another possibility for the decreased DN4 population could be related to sensitivity
of these cells to factors released by dying thymocytes, such as toxic purine metabolites,
particularly dGuo. Alternatively, the increased death of DN4 thymocytes could be due to
abnormal signalling through the TCR complex, as has been seen in mice deficient in Lck
(Levin et al., 1993), pTα deficient mice (Fehling et al., 1995), absence of ZAP-70 (Sugawara
et al., 1998), RAG-/- mice (Shinkai et al., 1992; Mombaerts et al., 1992) and knock down
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models of pTα, CD3 ε, γ and ζ chains (Fehling et al., 1995; Haks et al., 1998; Dave et al.,
1997; DeJarnette et al., 1998). This explanation, which needs to be examined by future
experiments, is also supported by studies of peripheral PNP-deficient T-cells that showed
reduced responses and decreased IL-2 secretion, again implicating impaired intracellular
signalling (Arpaia et al., 2000; Dror et al., 2004; Toro et al., 2006).
In addition to the effect that PNP deficiency has on DN thymocytes described above,
striking reductions in the numbers and percentages of DP thymocytes in PNP-/- mice were
found. BrdU incorporation into the DNA of newly formed DP thymocytes in PNP-/- mice, as
well as in DP cells developed ex-vivo, demonstrated that proliferation of PNP-/- DP cells was
not impaired (refer Figures 16B, 16C). Moreover, the DP cells generated ex-vivo in PNP-
deficient conditions was reduced, although there is no selection ex-vivo. Thus I concluded
that proliferation and selection were not the causes for the decreased DP thymocytes. As an
alternative explanation, I studied apoptosis in PNP-/- DP thymocytes. An indication for
decreased cell survival was already provided by the rapid reduction in the percentage of
BrdU labelled DP thymocytes, which was not accounted for by reduced proliferation of DN
thymocytes or enhanced maturation into SP thymocytes. The increased apoptosis of PNP-/-
DP thymocytes was further confirmed by the significantly increased Annexin-V+ PI+ cells
among PNP-/- DP thymocytes. The presence of Annexin-V binding is also an indication that
the cells are truly undergoing apoptosis in contrast to other mechanisms of cell death such as
necrosis. Although the value of apoptosis in DP cells increased (on average) only from 1.5%
in normal control to approximately 3.5% among PNP-/- thymocytes, this increase was
statistically significant, was observed consistently among PNP-/- mice and was similar to that
reported previously (Arpaia et al., 2000). We suspect that the relatively low number of
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apoptotic cells observed at a steady state among fresh thymocytes reflects both the ongoing
gradual effect that toxic purine metabolites have throughout the 6 week period from birth to
the time thymocytes were collected, and also the quick removal of apoptotic cells by the
abundant scavenger macrophages found in the thymus of PNP-/- mice. This is in
concordance to reports by Thompson et al., wherein a moderate increase in thymocyte
apoptosis was not detectable by PI staining or in situ TUNEL staining in ADA-deficient
cultures (Thompson et al., 2000).
Importantly, the increased apoptosis of PNP-/- DP thymocytes cultured ex-vivo
indicated that the apoptosis was not caused by external events, such as surge of
corticosteroids in PNP-deficient mice, unidentified infections, or enhanced signalling through
the Fas-receptor. The latter hypothesis was also supported by demonstrating that PNP-/- DP
thymocytes did not upregulate the surface expression of Fas receptor, further indicating that
the extrinsic pathway mediated by Fas expression may not be the contributing mechanism for
apoptosis. Moreover, death receptor signaling via Fas was eliminated as the apoptotic
mechanism in ADA deficiency, as lpr mice (mice homozygous for lymphoproliferation
spontaneous mutation (Faslpr)) remained sensitive to ADA deficiency. However, the same
group also showed that the use of a caspase-8 inhibitor prevented the apoptosis of ADA
deficient thymocytes, indicating the possible role of extrinsic apoptotic mechanisms via other
death receptor pathways such as tumor necrosis factor receptor (TNFR) family (Thompson et
al., 2003), which was not tested in this study. It is also possible that p53 may be involved,
since Benveniste and Cohen showed that p53-/- mice were resistant to apoptosis when
induced with ADA deficient conditions using dCF and dAdo (Benveniste and Cohen, 1995).
p53 can cause apoptosis by inducing transcription of pro-apoptotic Bcl-2 family members
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such as Bax, PUMA and Noxa (Miyashita and Reed, 1995, Yu et al., 2001, Oda et al., 2000).
It was later shown by some preliminary studies that FTOCs from p53 knock-out mice were
susceptible to the consequences of ADA inhibition (Thompson et al., 2003). Of course, it is
still possible that a p53 independent mechanism may involve the pro-apoptotic Bcl-2 family
members. None of the above mentioned apoptotic pathways were addressed in my study
nevertheless, my preliminary experiments in attempt to study the extrinsic pathway, although
not conclusive, together with findings from ADA deficiency studies, lead us to narrow the
cause of apoptosis to an intrinsic mechanism.
We next asked whether the accelerated apoptosis of DP thymocytes in PNP
deficiency were caused by increased dGuo and its phosphorylation products, dGMP and
dGTP, or whether it was the depletion of PNP products (i.e. GTP). Both, increased dGuo
and dGTP concentrations, as well as decreased GTP concentrations were observed in PNP-
deficient thymocytes (Arpaia et al., 2000). Moreover, abnormal peripheral T-cell function
have been associated with increased dGuo and dGTP (Kicska et al., 2001; Bantia et al., 2003)
and reduced GTP. Here I demonstrated that apoptosis of PNP-/- thymocytes was induced by
dGuo at concentrations equivalent to those recorded in the plasma of PNP-deficient patients
(Hershfield and Mitchell, 2001) and mice (Arpaia et al., 2000). Moreover, apoptosis of
thymocytes could be prevented by restoring PNP activity within the cells, which provided
unequivocal evidence to the role of the abnormal purine homeostasis in induction of
apoptosis. Apoptosis was also induced in PNP-/- thymocytes by dGMP, an intermediate
metabolite between dGuo and dGTP, which further implicates the latter as the cause of
apoptosis. Thus our study shows that accumulation of dGuo in PNP deficiency, followed by
the conversion of dGuo to dGMP, and probably to dGTP, causes thymocyte apoptosis in
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PNP-/- mice. We suspect that dGTP is the cause of apoptosis, as intracellular dGTP pools are
normally kept under tight regulation (Bjursell et al., 1980; Reichard et al., 1985), and PNP
inhibition using BCX-1777 in T-ALL, led to accumulation of dGuo in the plasma and dGTP
in circulating leukemia cells that were induced to die rapidly (Bantia et al., 2003). The
suspected role of dGTP in mediating the apoptosis of PNP-deficient thymocytes, also
suggested that the mitochondria was the cellular compartment most affected by PNP
deficiency, as dGTP formation is dependent on the function of dGK, which is a
mitochondrial enzyme (Johansson et al., 1996). Indeed, I found that accumulation of dGuo
led initially to the dissipation of mitochondrial membrane potential and that inhibition of
caspases, prevented dGuo induced nuclear DNA damage, but not mitochondrial damage. In
the experiments conducted to study changes in MMP, apoptosis induced by dGuo was not
seen immediately but rather took 12 hours to observe the toxic effects, possibly
recapitulating the gradual toxicity of purine metabolites to exert its effects on thymocytes in
vivo. The mitochondrial abnormalities that I found in PNP-/- thymocytes were similar to
previous observations in normal thymocytes treated with a PNP inhibitor (Arpaia et al.,
2000), in ADA-deficient thymocytes (Thompson et al., 2000), and in inherited thymidine
phosphorylase deficiency, which causes mitochondrial neurogastrointestinal
encephalomyopathy (MNGIE) disease (Nishino et al., 1999, Hiarano et al., 2006). Thus, our
findings suggest particular sensitivity of mitochondria to abnormalities in purine
homeostasis, which could be due to interference of purine metabolites with the down
regulation of anti-apoptotic molecules such as Bcl-2, as introduction of a Bcl-2 transgene
into PNP-deficient thymocytes increased their survival (Arpaia et al., 2000). Alternatively,
the depletion of mtDNA due to particular sensitivity of mitochondria to abnormal purine
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metabolism is also suggested by the mitochondrial abnormalities reported in ADA deficiency
(Van De Wiele et al., 2002) and MNGIE (Lopez et al., 2009). Other observations also
provide additional evidence that thymocyte damage in PNP deficiency may originate in the
mitochondria. PNP-deficient cells were found to be sensitive to gamma irradiation but were
still able to replicate their nuclear DNA in response to mitogen in the presence of IL-2
(Arpaia et al., 2000). Also, over-expression of dGK in the mitochondria was found to
increase sensitivity to anti-cancer deoxyguanosine analogues in cancer cell lines (Zhu et
al.,1998).
In conclusion, by this study, I demonstrated that PNP deficiency affects murine
thymocyte development at two distinct stages, and my data also suggests the possibility of
different mechanisms affecting the two stages. The first is at the maturation of thymocytes
from the DN3 to DN4 stage. The precise mechanism leading to this defect is still not known
and requires further investigation. The second thymocyte population affected in PNP-
deficient mice is the DP population. PNP-/- DP thymocytes are undergoing apoptosis at an
increased rate. The apoptosis is induced by accumulating dGuo, and probably by the
secondary formation of dGTP, which interferes with mitochondrial integrity. Importantly,
restoration of PNP activity within the cells increased survival, suggesting that enzyme or
gene replacement therapy may help in correcting the thymocyte abnormalities in PNP-
deficient patients.
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Future Directions 1) Identify the cause of decreased DN4 thymocytes in PNP-/- mice. Specifically, determine
intracellular signalling in DN3 and DN4 thymocytes. Alternatively, measure cell cycling
in PNP-deficient DN3 and DN4 thymocytes in vivo and ex-vivo.
2) Determine the contribution of GTP depletion in thymocyte abnormalities by GTP
supplementation.
3) Distinguish between the effects of dGuo and those of dGTP by preventing
phosphorylation of dGuo into dGTP, for example by cross-breeding PNP-/- mice with
dGK-deficient mice. If dGTP is the cause of apoptosis, then the double knockout will
prevent the dGuo-induced thymocyte apoptosis.
4) Assess the importance of anti-apoptosis mechanisms in PNP-/- thymocytes. Specifically,
determine whether over-expression of Bcl-2 protects PNP-/- deficient thymocytes from
dGuo induced apoptosis, by cross breeding of PNP-/- mice with Bcl-2 transgenic mice.
Alternatively, measure pro-apoptotic molecules associated with the mitochondrial
pathway, such as BAX and BAK.
5) Determine the sequence of events triggered by the mitochondrial damage that leads to
cell death. Specifically, release of cytochrome c from the intermembrane space of the
mitochondria to the cytoplasm, and activation of specific caspases, such as caspase 3 and
caspase 9.
6) Improve the ability to follow development of PNP-deficient thymocyte ex-vivo by
100
producing bone marrow stroma cells that do not express PNP, thereby more closely
simulating in vivo conditions in PNP-deficient patients. This can be done by inhibiting
PNP (using siRNA to PNP) in the OP9-DL1 cells.
7) Confirm that dGuo is indeed the metabolite responsible for thymocyte toxicity by
preventing its uptake into thymocytes. This can be achieved by the use of a nucleoside
transport inhibitor.
8) Explore the interactions (cross-talk) between thymocytes and the thymic epithelial cells,
which are also affected by the abnormal purine metabolism and possibly contribute to the
immune dysregulation observed in PNP-deficient patients and mice.
101
References Aifantis, I., Buer, J., von Boehmer, H., Azogui, O. (1997). Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor beta locus. Immunity. 7:601-7. Akashi, K., Kondo, M., von Freeden-Jeffry, U., Murray, R., Weissman, I.L. (1997). Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell. 89:1033-41. Allen, P.M. (1994). Peptides in positive and negative selection: A delicate balance. Cell 76:593–96. Allman, D., Sambandam, A., Kim, S., Miller, J.P., Pagan, A., Well, D., Meraz, A., Bhandoola, A. Thymopoiesis independent of common lymphoid progenitors. (2003). Nat Immunol. 4:168-74. Alonso, R., López-Guerra, M., Upshaw, R., Bantia, S., Smal, C., Bontemps, F., Manz, C., Mehrling, T., Villamor, N., Campo, E., Montserrat, E., Colomer, D. (2009). Forodesine has high antitumor activity in chronic lymphocytic leukemia and activates p53-independent mitochondrial apoptosis by induction of p73 and BIM. Blood. 114:1563-75. Anderson, G., Owen, J.J., Moore, N.C., Jenkinson, E.J. (1994). Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro. J Exp Med. 179:2027-31. Apasov, S.G., Blackburn, M.R., Kellems, R.E., Smith, PT., Sitkovsky, M.V. (2001). Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling. J Clin Invest. 108:131-41. Arpaia, E., Benveniste, P., Di Cristofano, A., Gu, Y., Dalal, I., Kelly, S., Hershfield, M., Pandolfi, P.P., Roifman, C.M.,and Cohen, A. (2000). Mitochondrial basis for immune deficiency: evidence from purine nucleoside phosphorylase–deficient mice. J Exp Med. 191:2197–07. Ashley, N., Adams, S., Slama, A., Zeviani, M., Suomalainen, A., Andreu, A.L., Naviaux, R.K., Poulton, J. (2007). Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances. Hum. Mol. Genet. 16:1400–11. Ashton-Rickardt, P.G., Van Kaer, L., Schumacher, T.N., Ploegh, H.L., and Tonegawa, S. (1993). Peptide contributes to the specificity of positive selection of CD8 þositive T cells in the thymus. Cell. 73:1041–49. Awong, G., La Motte-Mohs, R.N., Zúñiga-Pflücker, J.C. (2008). In vitro human T cell development directed by notch-ligand interactions. Methods Mol Biol. 430:135-42.
102
Baguette, C., Vermylen, C., Brichard, B., Louis, J., Dahan, K., Vincent, M.F., Cornu, G. (2002). Persistent developmental delay despite successful bone marrow transplantation for purine nucleoside phosphorylase deficiency. J Pediatr Hematol Oncol. 24:69-71. Balakrishnan, K., Nimmanapalli, R., Ravandi, F., Keating, M.J., Gandhi, V. (2006). Forodesine, an inhibitor of purine nucleoside phosphorylase, induces apoptosis in chronic lymphocytic leukemia cells. Blood. 108:2392-98. Bantia, S., Ananth, S.L., Parker, C.D., Horn, L.L., Upshaw, R. (2003). Mechanism of inhibition of T-acute lymphoblastic leukemia cells by PNP inhibitor--BCX-1777. Int Immunopharmacol. 3:879-87. Barankiewicz, J., Gelfand, E.W., Issekutz, A., Cohen, A. (1982). Evidence for active purine nucleoside cycles in human mononuclear cells and cultured fibroblasts. J Biol Chem. 257:11597-600. Benveniste, P., Cohen, A. (1995). p53 expression is required for thymocyte apoptosis induced by adenosine deaminase deficiency. Proc Natl Acad Sci U S A. 92:8373-77. Bell, J.J., Bhandoola, A. (2008). The earliest thymic progenitors for T cells possess myeloid lineage potential. Nature. 452:764-7. Bjursell, G., Skoog, L. (1980). Control of nucleotide pools in mammalian cells. Antibiot Chemother. 28:78-85. Broome, C.B., Graham, M.L., Saulsbury, F.T., Hershfield, M.S., Buckley, R.H. (1996). Correction of purine nucleoside phosphorylase deficiency by transplantation of allogeneic bone marrow from a sibling. J Pediatr. 128:373-76. Bzowska, A., Kulikowska, E., Shugara, D. (2000). Purine nucleoside phosphorylases: properties, functions, and clinical aspects. Pharmacology & Therapeutics. 88:349-425. Carlyle, J.R., Michie, A.M., Furlonger, C., Nakano, T., Lenardo, M.J., Paige, C.J., Zúñiga-Pflücker, J.C. (1997). Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes. J Exp Med. 186:173-82. Carson, D.A., Kaye, J., and Seegmiller, J.E. (1977). Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: Possible role of nucleoside kinase(s). Proc Natl Acad Sci U S A. 12:5677–81. Carson, D.A., Kaye, J., Matsumoto, S., Seegmiller, J.E., Thompson, L. (1979). Biochemical basis for the enhanced toxicity of deoxyribonucleosides toward malignant human T cell lines. Proc Natl Acad Sci U S A. 76:2430-3. Chan, T.S. (1978). Deoxyguanosine toxicity on lymphoid cells as a cause for immunosuppression in purine nucleoside phosphorylase deficiency. Cell. 14:523-30.
103
Chan, B., Wara, D., Bastian, J., Hershfield, M.S., Bohnsack, J., Azen, C.G., Parkman, R., Weinberg, K., Kohn, D.B. (2005). Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). Clin Immunol. 117:133-43. Ciofani, M., Knowles, G.C., Wiest, D.L., von Boehmer, H., and Zuniga-Pflucker, J.C. (2006). Stage-specific and differential notch dependency at the alphabeta and gammadelta T lineage bifurcation. Immunity. 25:105–116. Cohen. A., Gudas, L.J., Ammann, A.J., Staal, G.E., Martin, D.W. Jr. (1978). Deoxyguanosine triphosphate as a possible toxic metabolite in the immunodeficiency associated with purine nucleoside phosphorylase deficiency. J Clin Invest. 61:1405-9. Cohen, A., Lee, J.W., Dosch, H.M., Gelfand, E.W. (1980). The expression of deoxyguanosine toxicity in T lymphocytes at different stages of maturation. J Immunol. 125:1578-82. Cohen, A. (1986). Role of intracellular deoxynucleoside triphosphate levels in DNA repair in human lymphocytes. Adv Exp Med Biol. 195:201-5. Cohen, A., Grunebaum, E., Arpaia, E., Roifman, C.M. (2000). Immunodeficiency caused by purine nucleoside phosphorylase deficiency. Immunology and Allergy Clinics of North America. 20:143-159. Conry, R.M., Bantia, S., Turner, H.S., Barlow, D.L., Allen, K.O., LoBuglio, A.F., Montgomery, J.A., Walsh, G.M. (1998). Effects of a novel purine nucleoside phosphorylase inhibitor, BCX-34, on activation and proliferation of normal human lymphoid cells. Immunopharmacology. 40:1-9. Dalal, I., Grunebaum, E., Cohen, A., Roifman, C.M. (2001). Two novel mutations in a purine nucleoside phosphorylase (PNP)-deficient patient. Clin Genet. 59:430-37. Dave, V.P., Cao, Z., Browne, C., Alarcon, B., Fernandez-Miguel, G., Lafaille, J., de la Hera, A., Tonegawa, S., Kappes, D.J. (1997). CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage. EMBO J. 16:1360-70. DeJarnette, J.B., Sommers, C.L., Huang, K., Woodside, K.J., Emmons, R., Katz, K., Shores, E.W., Love, P.E. (1998). Specific requirement for CD3epsilon in T cell development. Proc Natl Acad Sci USA. 95:14909-14. de Pooter, R., Zúñiga-Pflücker, J.C. (2007). T-cell potential and development in vitro: the OP9-DL1 approach. Curr Opin Immunol. 19:163-8. Dietz, G.P., and Bahr, M. (2004). Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol Cell Neurosci. 27:85–131.
104
Di Santo, J.P., Rodewald, H.R. (1998). In vivo roles of receptor tyrosine kinases and cytokine receptors in early thymocyte development. Curr Opin Immunol. 10:196-207. Dror, Y., Grunebaum, E., Hitzler, J., Narendran, A., Ye, C., Tellier, R., Edwards, V., Freedman, M.H., Roifman, C.M. (2004). Purine nucleoside phosphorylase deficiency associated with a dysplastic marrow morphology. Pediatr Res. 55:472-7. Ealick, S.E., Rule, S.A., Carter, D.C., Greenhough, T.J., Babu, Y.S., Cook, W.J., Habash, J., Helliwell, J.R., Stoeckler, J.D., Parks, R.E., Jr, et al. (1990). Three-dimensional structure of human erythrocytic purine nucleoside phosphorylase at 3.2 A resolution. J Biol Chem. 3:1812-20 Fairbanks, L.D., Taddeo, A., Duley, J.A., Simmonds, H.A. (1990). Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency. J Immunol. 144:485-91. Fehling, H.J., Krotkova, A., Saint-Ruf, C., von Boehmer, H. (1995). Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells. Nature. 375:795-8. Frank, S.J., Samelson, L.E., Klausner, R.D. (1990). The structure and signalling functions of the invariant T cell receptor components. Semin Immunol. 2:89-97. Fugmann, S.D., Lee, A.I., Shockett, P.E., Villey, I.J., Schatz, D.G. (2000). The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu Rev Immunol. 18:495-527 Fulda, S., Debatin, K.M. (2006). Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 25:4798-811. Gandhi, V., Kilpatrick, J.M., Plunkett, W., Ayres, M., Harman, L., Du, M., Bantia, S., Davisson, J., Wierda, W.G., Faderl, S., Kantarjian, H., Thomas, D. (2005). A proof-of-principle pharmacokinetic, pharmacodynamic, and clinical study with purine nucleoside phosphorylase inhibitor immucillin-H (BCX-1777, forodesine). Blood. 106:4253-60. Gärtner, F., Alt, F.W., Monroe, R., Chu, M., Sleckman, B.P., Davidson, L., Swat, W. (1999). Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion. Immunity. 10:537-46. Gelfand, E.W., Dosch, H.M., Biggar, W.D., Fox, I.H. (1978). Partial purine nucleoside phosphorylase deficiency. Studies of lymphocyte function. J Clin Invest. 61:1071-80. Gelfand, E.W., Lee, J.J., Dosch, H.M. (1979). Selective toxicity of purine deoxynucleosides for human lymphocyte growth and function. Proc Natl Acad Sci U S A. 76:1998-2002.
105
Germain, R.N. (2002). T-cell development and the CD4-CD8 lineage decision. Nat. Rev.Immunol. 2:309–22. Giblett, E.R., Ammann, A.J., Wara, D.W., Sandman, R., Diamond, L.K. (1975). Nucleoside-phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet. 1:1010-3. Giblett, E.R. (1985). ADA and PNP deficiencies: how it all began. Ann N Y Acad Sci. 451:1-8. Gilbertsen, R.B., Posmantur, R., Nath, R., Wang, K.K. (1997). Apoptotic death induced in MOLT-4 T-lymphoblasts by purine nucleoside phosphorylase inhibition. Inflamm Res. 46:S151-2. Goddard, J.M., Caput, D., Williams, S.R., Martin, D.W., Jr. (1983). Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli. Proc Natl Acad Sci USA. 14:4281-5. Godfrey, D.I., Kennedy, J., Suda, T., Zlotnik, A. (1993). A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol. 150:4244-52. Gudas, L.J., Zannis, V.I., Clift, S.M., Ammann, A.J., Staal, G.E., Martin, D.W. Jr, (1978). Characterization of mutant subunits of human purine nucleoside phosphorylase. J Biol Chem. 253:8916-24. Guidos, C.J. (2002). Notch signaling in lymphocyte development. Semin Immunol. 14: 395-404. Gump, J.M., Dowdy, S.F. (2007). TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 13:443-8. Haks, M.C., Krimpenfort, P., Borst, J., Kruisbeek, A.M. (1998). The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages. EMBO J. 17:1871-82. Hasserjian, R.P., Aster, J.C., Davi, F., Weinberg, D.S., Sklar, J. (1996). Modulated expression of notch1 during thymocyte development. Blood. 88:970-6. Hayday, A.C., Barber, D.F., Douglas, N., Hoffman, E.S. (1999). Signals involved in gamma/delta T cell versus alpha/beta T cell lineage commitment. Semin Immunol. 11:239-49.
106
Haynes, B.F., and Heinly, C.S. (1995). Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment. J Exp Med. 181:1445-58. Hershfield, M.S., Chaffee, S., Koro-Johnson, L., Mary, A., Smith, A.A., Short, S.A. (1991). Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol. Proc Natl Acad Sci USA. 88:7185-9. Hershfield, M.S. and Mitchell, B.S. (2001) Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds.), The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp. 2585-2625. Hirano, M., Nishino, I., Nishigaki, Y., Martí, R. (2006). Thymidine phosphorylase gene mutations cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Intern Med. 45:1103. Hirota, Y., Yoshioka, A., Tanaka, S., Watanabe, K., Otani, T., Minowada, J., Matsuda, A., Ueda, T., Wataya, Y. (1989). Imbalance of deoxyribonucleoside triphosphates, DNA double-strand breaks, and cell death caused by 2-chlorodeoxyadenosine in mouse FM3A cells. Cancer Res. 49:915– 9. Hirschhorn, R. (1986). Inherited enzyme deficiencies and immunodeficiency: adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies. Clin Immunol Immunopathol. 40:157-65. Hoffmann, M.W., Allison, J., Miller, J.F. (1992). Tolerance induction by thymic medullary epithelium. Proc Natl Acad Sci U S A. 89:2526-30. Husain, M., Grunebaum, E., Naqvi, A., Atkinson, A., Ngan, B.Y., Aiuti, A., Roifman, C.M. (2007). Burkitt's lymphoma in a patient with adenosine deaminase deficiency-severe combined immunodeficiency treated with polyethylene glycol-adenosine deaminase. J Pediatr. 151:93-5. Janeway, C.A. Jr. (1992). The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol. 10:645-74. Jenkinson, E.J and Owen, J.J. (1990). T-cell differentiation in thymus organ cultures. Semin Immunol. 2:51-8. Jenuth, J.P., Mably, E.R., Snyder, F.F. (1996). Modelling of purine nucleoside metabolism during mouse embryonic development: relative routes of adenosine, deoxyadenosine, and deoxyguanosine metabolism. Biochem Cell Biol. 74:219-25.
107
Johansson, M., Karlsson, A. (1996). Cloning and expression of human deoxyguanosine kinase cDNA. Proc Natl Acad Sci U S A. 93:7258-62. Kane, L.P., Lin, J., Weiss, A. (2000). Signal transduction by the TCR for antigen. Curr Opin Immunol. 12:242-9. Kazmers, I.S., Mitchell, B.S., Dadonna, P.E., Wotring, L.L., Townsend, L.B., Kelley, W.N. (1981). Inhibition of purine nucleoside phosphorylase by 8-aminoguanosine: selective toxicity for T lymphoblasts. Science. 214:1137-9. Kelly, A.P., Finlay, D.K., Hinton, H.J., Clarke, R.G., Fiorini, E., Radtke, F., Cantrell, D.A. (2007). Notch-induced T cell development requires phosphoinositide-dependent kinase 1. EMBO J. 26:3441-50. Kim, K., C. K. Lee, T. J. Sayers, K. Muegge, and S. K. Durum. (1998). The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways. J. Immunol. 160: 5735–41 Kruisbeek, A.M., Haks, M.C., Carleton, M., Michie, A.M., Zúñiga-Pflücker, J.C., Wiest, D.L. (2000). Branching out to gain control: how the pre-TCR is linked to multiple functions. Immunol Today. 21:637-44. Kwon, Y.D., Oh, S.K., Kim, H.S., Ku, S.Y., Kim, S.H., Choi, Y.M., Moon, S.Y. (2005). Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction. Mol Ther. 12:28–32. Lehar, S.M., Dooley, J., Farr, A.G., Bevan, M.J. (2005). Notch ligands Delta 1 and Jagged1 transmit distinct signals to T-cell precursors. Blood. 105:1440-7. Levin, S.D., Anderson, S.J., Forbush, K.A., Perlmutter, R.M. (1993). A dominant-negative transgene defines a role for p56lck in thymopoiesis. EMBO J. 12:1671-80. Liao, P., Toro, A., Min, W., Lee, S., Roifman, C.M., Grunebaum, E. (2008). Lentivirus gene therapy for purine nucleoside phosphorylase deficiency. J Gene Med. 10:1282-93. Lind, E.F., Prockop, S.E., Porritt, H.E., Petrie, H.T. (2001). Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development. J. Exp. Med. 194:127–34. López, L.C., Akman, H.O., García-Cazorla, A., Dorado, B., Martí, R., Nishino, I., Tadesse, S., Pizzorno, G., Shungu, D., Bonilla, E., Tanji, K., Hirano, M. (2009). Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice. Hum Mol Genet. 18:714-22.
108
Lucas, B., Vasseur, F., Penit, C. (1993). Normal sequence of phenotypic transitions in one cohort of 5-bromo-2’-deoxyuridine-pulse-labeled thymocytes. Correlation with T cell receptor expression. J Immunol. 151:4574-82. Mackarehtschian, K., Hardin, J.D., Moore, K.A., Boast, S., Goff, S.P., Lemischka, I.R. (1995). Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity. 3:147-61. Maki, K., Sunaga, S., Ikuta, K. (1996). The V-J recombination of T cell receptor-gamma genes is blocked in interleukin-7 receptor-deficient mice. J Exp Med. 184:2423-7. Maraskovsky, E., O'Reilly, L.A., Teepe, M., Corcoran, L.M., Peschon, J.J., Strasser, A. (1997). Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice. Cell. 89:1011-9. Markert, M.L., Hershfield, M.S., Schiff, R.I., Buckley, R.H. (1987). Adenosine deaminase and purine nucleoside phosphorylase deficiencies: evaluation of therapeutic interventions in eight patients. J Clin Immunol. 7:389-99. Markert, M.L., (1991). Purine nucleoside phosphorylase deficiency. Immunodefic Rev. 3: 45-81. Markert, M.L., Finkel, B.D., McLaughlin, T.M., Watson, T.J., Collard, H.R., McMahon, C.P., Andrews, L.G., Barrett, M.J., Ward, F.E. (1997). Mutations in purine nucleoside phosphorylase deficiency. Hum Mutat. 9:118-21. Martin. O.W., JR and Gelfand, E. W. (1981). Biochemistry of diseases of immunodevelopment. Ann. Rev. Biochem. 50:845-77. Matei, I.R., Gladdy, R.A., Nutter, L.M., Canty, A., Guidos, C.J., Danska, J.S. (2007). ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes. Blood. 109:1887-96. Mayer, B., Oberbauer, R. (2003). Mitochondrial regulation of apoptosis. News Physiol Sci. 18:89-94. Mazza, G., Housset, D., Piras, C., Gregoire, C., Lin, S.Y., Fontecilla-Camps, J.C., Malissen, B. (1998). Glimpses at the recognition of peptide/MHC complexes by T-cell antigen receptors. Immunol Rev. 163:187-96. Meuth, M. (1989). The molecular basis of mutation induced by dNTP pool imbalances in mammalian cells. Exp Cell Res. 181:305– 16. Michie, A.M., Zúñiga-Pflücker, J.C. (2002). Regulation of thymocyte differentiation: pre-TCR signals and beta-selection. Semin Immunol. 14:311-23.
109
Michie, A.M., Chan, A.C., Ciofani, M., Carleton, M., Lefebvre, J.M., He, Y., Allman, D.M., Wiest, D.L., Zúñiga-Pflücker, J.C., Izon, D.J. (2007). Constitutive Notch signalling promotes CD4 CD8 thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals. Int Immunol. 12:1421-30. Miller, J.F. (1961). Immunological Function of the Thymus. Lancet, 2:748-49. Mitchell, B.S., Mejias, E., Daddona, P.E., Kelley, W.N. (1978). Purinogenic immunodeficiency diseases: selective toxicity of deoxyribonucleosides for T cells. Proc Natl Acad Sci USA. 75:5011-4. Miyashita T, Reed JC. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 80:293-9. Molina, T.J., Bachmann, M.F., Kündig, T.M., Zinkernagel, R.M., Mak, T.W. (1993). Peripheral T cells in mice lacking p56lck do not express significant antiviral effector functions. J Immunol. 151:699-706. Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S., Papaioannou, V.E. (1992). RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 68:869-77. Morris, P.E. Jr., Omura, G.A. (2000). Inhibitors of the enzyme purine nucleoside phosphorylase as potential therapy for psoriasis. Curr Pharm Des. 6:943-59. Morrissey PJ, McKenna H, Widmer MB, Braddy S, Voice R, Charrier K, Williams DE, Watson JD. (1994). Steel factor (c-kit ligand) stimulates the in vitro growth of immature CD3-/CD4-/CD8- thymocytes: synergy with IL-7. Cell Immunol. 157:118-31. Nagahara, H., Vocero-Akbani, A.M., Snyder, E.L., Ho, A., Latham, D.G., Lissy, N.A., Becker-Hapak, M., Ezhevsky, S.A., Dowdy, S.F. (1998). Transduction of full length TAT fusion proteins into mammalian cells:TAT-p27Kip1 induces cell migration. Nat Med. 4:1449–52. Nishino, I., Spinazzola, A., Hirano, M. (1999). Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science. 283:689-92. Nitta, T., Murata, S., Ueno, T., Tanaka, K., Takahama, Y. (2008). Thymic microenvironments for T-cell repertoire formation. Adv Immunol. 99:59-94. Nyhan WL. (2005). Disorders of purine and pyrimidine metabolism. Mol Genet Metab. 86:25-33. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N. (2000). Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science. 288:1053-8.
110
Ogasawara, J., Suda, T., Nagata, S. (1995). Selective apoptosis of CD4+CD8+ thymocytes by the anti-Fas antibody. J Exp Med. 181:485-91. Ohashi, P.S. Negative selection and autoimmunity. (2003). Curr Opin Immunol. 15:668-76. Osborne, W.R. (1980). Human red cell purine nucleoside phosphorylase. Purification by biospecific affinity chromatography and physical properties. J Biol Chem. 255:7089-92. Owen, J.J., and Ritter, M.A. (1969). Tissue interaction in the development of thymus lymphocytes. J Exp Med. 129:431-42. Penit, C. (1988). Localization and phenotype of cycling and postcycling murine thymocytes studied by simultaneous detection of bromodeoxyuridine and surface antigens. J.Histochem. Cytochem. 36:473–78. Peschon, J.J., Morrissey, P.J., Grabstein, K.H., Ramsdell, F.J., Maraskovsky, E., Gliniak, B.C., Park, L.S., Ziegler, S.F., Williams, D.E., Ware, C.B., Meyer, J.D., Davison, B.L. (1994). Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med. 180:1955-60. Petrie, H.T., Livak, F., Schatz, D.G., Strasser, A., Crispe, I.N., Shortman, K. (1993). Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes. J Exp Med. 178:615-22. Petrie, H.T. (2002). Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol Rev. 189:8-19. Petrie, H.T., Zúñiga-Pflücker, J.C. (2007). Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu Rev Immunol. 25:649-79. Porritt, H.E., Gordon, K., Petrie, H.T. (2003). Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice. J Exp Med. 198:957-62. Pugmire, M.J., Ealick, S.E. (2002). Structural analyses reveal two distinct families of nucleoside phosphorylases. Biochem J. 361:1-25. Pui, J.C., Allman, D., Xu, L., DeRocco, S., Karnell, F.G., Bakkour, S., Lee, J.Y., Kadesch, T., Hardy,R.R., Aster, J.C., and Pear, W.S. (1999). Notch1 Expression in Early Lymphopoiesis Influences B versus T Lineage Determination. Immunity. 11:299–308. Radtke, F., Wilson, A., and MacDonald, H. R. (2004). Notch signaling in T- and B-cell development. Curr. Opin. Immunol. 16:174-9. Ravagnan, L., Marzo, I., Costantini, P., Susin, S.A., Zamzami, N., Petit, P.X., Hirsch, F., Goulbern, M., Poupon, M.F., Miccoli, L., Xie, Z., Reed, J.C., Kroemer, G. (1999).
111
Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene. 18:2537-46. Reichard, P. (1978). From deoxynucleotides to DNA synthesis. Fedn Proc. Fedn Am. Socs exp. Biol. 37:9- 14. Reichard P. (1985). Ribonucleotide reductase and deoxyribonucleotide pools. Basic Life Sci. 31:33-45. Ricciuti, F., Ruddle, F.H. (1973). Assignment of nucleoside phosphorylase to D-14 and localization of X-linked loci in man by somatic cell genetics. Nat New Biol. 241:180-2. Rich, K.C., Majias, E., Fox, I.H. (1980). Purine nucleoside phosphorylase deficiency: improved metabolic and immunologic function with erythrocyte transfusions. N Engl J Med. 303:973-7. Saada, A. (2008). Mitochondrial deoxyribonucleotide pools in deoxyguanosine kinase deficiency. Mol Genet Metab. 95:169-73. Saint-Ruf, C., Ungewiss, K., Groettrup, M., Bruno, L., Fehling, H.J., von Boehmer, H. (1994). Analysis and expression of a cloned pre-T cell receptor gene. Science. 266: 1208-12. Sakiyama, T., Iwase, M., Horinouchi, K., Akatsuka, A., Yoshida, Y., Kikuchi, T., Shimatake, H., Kitagawa, T. (1989). Clinico-biochemical and molecular studies of purine nucleoside phosphorylase deficiency. Adv Exp Med Biol. 253A:73-9. Sasaki, Y., Iseki, M., Yamaguchi, S., Kurosawa, Y., Yamamoto, T., Moriwaki, Y., Kenri, T., Sasaki, T., Yamashita, R. (1998). Direct evidence of autosomal recessive inheritance of Arg24 to termination codon in purine nucleoside phosphorylase gene in a family with a severe combined immunodeficiency patient. Hum Genet. 103:81-5. Schmitt, T.M., Zúñiga-Pflücker, J.C. (2002). Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity. 17:749-56. Schmitt, T.M., Ciofani, M., Petrie, H.T., Zúñiga-Pflücker, J.C. (2004). Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions. J Exp Med. 200:469-79. Schneider, Y.J., Limet, J.N., Octave, J.N., Otte-Slachmuylder, C., Crichton, R.R., Trouet, A. (1982) The role of receptor-mediated endocytosis in iron metabolism. Prog Clin Biol Res. 91:495–521. Schwarze, S.R., Ho, A., Vocero-Akbani, A., Dowdy, S.F. (1999). In vivo protein transduction:delivery of a biologically active protein into the mouse. Science. 285:1569-72.
112
Scollay, R.G., Butcher, E.C., Weissman, I.L. (1980). Thymus cell migration. Quantitative aspects of cellular traffic from the thymus to the periphery in mice. Eur J Immunol. 10:210-8. Scollay, R., Bartlett, P., and Shortman, K. (1984). T cell development in the adult murine thymus: Changes in the expression of the surface antigens Ly2, L3T4 and B2A2 during development from early precursor cells to emigrants. Immunol. Rev. 82:79–103. Scollay, R., Wilson, A., D’Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L., and Shortman, K. (1988). Developmental status and reconstitution potential of subpopulationsof murine thymocytes. Immunol. Rev. 104:81–120. Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A.M., et al. (1992). RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 68:855-67. Sidi, Y., Gelvan, I., Brosh, S., Pinkhas, J., Sperling, O. (1989). Guanine nucleotide metabolism in red blood cells: the metabolic basis for GTP depletion in HGPRT and PNP deficiency. Adv Exp Med Biol. 253A:67-71. Simmonds H.A., Fairbanks, L.D., Morris, G.S., Morgan, G., Watson, A.R., Timms, P., Singh, B. (1987). Central nervous system dysfunction and erythrocyte guanosine triphosphate depletion in purine nucleoside phosphorylase deficiency. Arch Dis Child. 62:385-91. Snyder, F.F., Jenuth, J.P., Mably, E.R., and Mangat, R.K. (1997). Point mutations at the purine nucleoside phosphorylase locus impair thymocyte differentiation in the mouse. Proc. Natl. Acad. Sci. USA. 94:2522–27. Sprent, J., Kishimoto, H. (2002). The thymus and negative selection. Immunol Rev. 185:126-35. Staal, G.E., Stoop, J.W., Zegers, B.J., Siegenbeek van Heukelom, L.H., van der Vlist, M.J., Wadman, S.K., Martin, D.W. (1980). Erythrocyte metabolism in purine nucleoside phosphorylase deficiency after enzyme replacement therapy by infusion of erythrocytes. J Clin Invest. 65:103-8. Sitnicka, E., Buza-Vidas, N., Ahlenius, H., Cilio, C.M., Gekas, C., Nygren, J.M., Månsson, R., Cheng, M., Jensen, C.T., Svensson, M., Leandersson, K., Agace, W.W., Sigvardsson, M., Jacobsen, S.E. (2007). Critical role of FLT3 ligand in IL-7 receptor independent T lymphopoiesis and regulation of lymphoid-primed multipotent progenitors. Blood. 110:2955-64. Stoeckler, J.D., Agarwal, R.P., Agarwal, K.C., Parks, R.E. Jr. (1978). Purine nucleoside phosphorylase from human erythrocytes. Methods Enzymol. 51:530-8.
113
Stoeckler, J. D. (1984). Purine nucleoside phosphorylase: a target for chemotherapy. In R. J. Glazer (Ed.), Developments in Cancer Chemotherapy (pp. 35± 60). Boca Raton. CRC Press. Sugawara, T., Di Bartolo, V., Miyazaki, T., Nakauchi, H., Acuto, O., Takahama, Y. (1998). An improved retroviral gene transfer technique demonstrates inhibition of CD4-CD8- thymocyte development by kinase-inactive ZAP-70. J Immunol. 161:2888-94. Surh, C.D., Sprent, J. (1994). T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature. 372:100-3. Takahama, Y., Suzuki, H., Katz, K. S., Grusby, M. J., and Singer, A. (1994). Positive selection of CD4þ T cells by TCR ligation without aggregation even in the absence of MHC. Nature. 371:67–70. Takahama, Y. (2006). Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol. 6:127-35. Thompson, L.F., Van de Wiele, C.J., Laurent, A.B., Hooker, S.W., Vaughn, J.G., Jiang, H., Khare, K., Kellems, R.E., Blackburn, M.R., Hershfield, M.S., Resta, R. (2000). Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase-deficient fetal thymic organ cultures. J Clin Invest. 106:1149-57. Thompson, L.F., Vaughn, J.G., Laurent, A.B., Blackburn, M.R., Van De Wiele, C.J. (2003). Mechanisms of apoptosis in developing thymocytes as revealed by adenosine deaminase-deficient fetal thymic organ cultures. Biochem Pharmacol. 66:1595-9. Toro, A., Paiva, M., Ackerley, C., Grunebaum, E. (2006). Intracellular delivery of purine nucleoside phosphorylase (PNP) fused to protein transduction domain corrects PNP deficiency in vitro. Cell Immunol. 240:107-15. Toro, A., Grunebaum, E. (2006). TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice. J Clin Invest. 116:2717-26. Tourigny, M.R., Mazel, S., Burtrum, D.B., Petrie, H.T. (1997). T cell receptor (TCR)-beta gene recombination: dissociation from cell cycle regulation and developmental progression during T cell ontogeny. J Exp Med. 185:1549-56. Trowbridge, I.S., Shackelford, D.A. (1986). Structure and function of transferrin receptors and their relationship to cell growth. Biochem Soc Symp. 51: 117–129. Ullman, B., Gudas, L.J., Clift, S.M., Martin, D.W. Jr. (1979). Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: genetic model for immunodeficiency disease. Proc Natl Acad Sci USA. 76:1074-8.
114
Van De Wiele, C.J., Vaughn, J.G., Blackburn, M.R., Ledent, C.A., Jacobson, M., Jiang H., Thompson, L.F. (2002). Adenosine kinase inhibition promotes survival of fetal adenosine deaminase-deficient thymocytes by blocking dATP accumulation. J Clin Invest. 110:395-02. Van De Wiele, C.J., Joachims, M.L., Fesler, A.M., Vaughn, J.G., Blackburn, M.R., McGee, S.T., Thompson, L.F. ( 2006). Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture. J Immunol. 176:5925-33. Van de Wiele CJ, Marino JH, Tan C, Kneale HA, Weber J, Morelli JN, Davis BK, Taylor AA, Teague TK. (2007). Impaired thymopoiesis in interleukin-7 receptor transgenic mice is not corrected by Bcl-2. Cell Immunol. 250:31-9. Visan, I., Yuan, J.S., Tan, J.B., Cretegny, K., Guidos, C.J. (2006). Regulation of intrathymic T-cell development by Lunatic Fringe- Notch1 interactions. Immunol Rev. 209:76-94. von Boehmer, H. (1994). Positive selection of lymphocytes. Cell. 76:219–228. von Boehmer, H., Fehling, H.J. (1997). Structure and function of the pre-T cell receptor. Annu Rev Immunol.15:433-52. von Freeden-Jeffry, U., Vieira, P., Lucian, L.A., McNeil, T., Burdach, S.E.G., Murray, R. (1995). Lymphopenia in interleukin IL-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med. 181:1519-26. von Freeden-Jeffry, U., Solvason, N., Howard, M., Murray, R. (1997). The earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression. Immunity. 7:147-54. Wadia, J.S., and Dowdy, S.F. (2003). Modulation of cellular function by TAT mediated transduction of full-length proteins. Curr Protein Pept Sci. 4:97–104. Wang, H., Pierce, L.J., Spangrude, G.J. (2006). Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system. Exp Hematol. 34:1730-40. Wang, L., Hellman, U., Eriksson, S. (1996). Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA. FEBS Lett. 390:39-43. Watson, A.R., Evans, D.I., Marsden, H.B., Miller, V., Rogers, P.A. (1981). Purine nucleoside phosphorylase deficiency associated with a fatal lymphoproliferative disorder. Arch Dis Child. 56:563-5.
115
Williams, S.R., Goddard, J.M., Martin, D.W. Jr. (1984). Human purine nucleoside phosphorylase cDNA sequence and genomic clone characterization. Nucleic Acids Res. 12:5779-87. Wilson, A., MacDonald, H.R., and Radtke, F. (2001). Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194:1003-12. Witt, C. M., Raychaudhuri, S., Schaefer, B., Chakraborty, A. K., and Robey, E. A. (2005). Directed migration of positively selected thymocytes visualized in real time. PLoS Biol. 3:e160. Wu, L., Li, C. L. and Shortman, K. (1996). Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J. Exp. Med. 184:903-11. Xiong, N., Raulet, D.H. (2007). Development and selection of gammadelta T cells. Immunol Rev. 215:15-31. Yang, Y., Ashwell, J.D. (1999). Thymocyte apoptosis. J Clin Immunol. 19:337-49. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. (2001). PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell. 7:673-82. Yu, Y., Arora, A., Min, W., Roifman, C.M., Grunebaum, E. (2009). EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations. J Immunol Methods. 350:29-35 Zannis, V., Doyle, D., Martin, D.W. Jr. (1978). Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits. J Biol Chem. 253:504-10. Zhu, C., Johansson, M., Permert, J., Karlsson, A. (1998). Enhanced cytotoxicity of nucleoside analogs by overexpression of mitochondrial deoxyguanosine kinase in cancer cell lines. J Biol Chem. 273:14707-11.