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Identify Werner protein molecular partners in S phase alt cell

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Identify Werner protein molecular partners in S phase alt cell

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IDENTIFY WERNER PROTEIN MOLECULAR PARTNERS
IN S PHASE ALT CELL

by

Fangjin Huang

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2012

Copyright 2012 Fangjin Huang

ii

Table of Contents
List of Tables ............................................................................................................... iii
List of Figures ............................................................................................................. iv
Abstract ......................................................................................................................... v
Introduction ................................................................................................................... 1
Werner Syndrome Protein (WRN) ......................................................................... 1
Alternative lengthening of telomeres (ALT) .......................................................... 2
The relationship between WRN and ALT .............................................................. 4
Chapter 1: Materials and Methods ................................................................................ 8
Chapter 2: Results ....................................................................................................... 13
Optimize conditions for S phase CCL75.1 cell synchronization using double
thymidine block ................................................................................................... 13
WRN associated proteins in S phase ALT cell ..................................................... 20
The function of WRN complex in telomerase activity and processivity ............. 22
Chapter 3: Discussion ................................................................................................. 24
Werner protein and its molecular partners in ALT cell ........................................ 24
The function of WRN complex in telomerase activity and processivity ............. 30
References ................................................................................................................... 33

iii

List of Tables
Table 1: Profile of FACS analysis results of Hela and CCL75.1 cells from
Figure 1, showing percentage of cells in different stages of cell cycle. ........ 15
Table 2: The experimental design and results of L
9
(3
4
) orthogonal test ..................... 17
Table 3: One way ANOV A result of the influence of 3 factors on the percentage
of S phase cells at 3 h point ........................................................................... 17
Table 4: One way ANOV A result of the influence of 3 factors on the percentage
of cells moving from G1 phase to S phase between 0 h and 3 h ................... 18

iv

List of Figures
Figure 1: The different outcome of cell cycle synchronization of Hela cell and
CCL75.1 cell after double thymidine block. .................................................. 15
Figure 2: T-test results of orthogonal test. ................................................................... 18
Figure 3: Histogram of CCL75.1 cells synchronized to S phase with different
duration of 2
nd
blocking. ................................................................................ 19
Figure 4: WRN complex in asynchronous and S phase Hela and CCL75.1 cells.
........................................................................................................................ 21
Figure 5: Telomerase activity of Hela cells transfected with hTERT and hTR. .......... 23

v

Abstract
The Werner protein (WRN) is encoded by the WRN gene. Mutations in this gene are the
causal factor for the outset of an autosomal recessive progerid disorder known as Werner
Syndrome. WRN has been proposed to function in ALT, a pathway that maintains
telomere length independent of telomerase and involves high level of recombination
processes observed in about 15% of cancer cells. These functions are probably
accomplished by the interactions between WRN and many other proteins. Thus I studied
the WRN complex formation in ALT cells to further investigate the potential role of
WRN in ALT pathway. In this study, I used CCL75.1 cell as a representative for ALT
cell lines and synchronized CCL75.1 cells to S phase using optimized double thymidine
block. I found out that the optimal condition for harvesting S phase CCL75.1 cells was as
follows: cells were blocked with media containing 4 mM thymidine for 18 hours,
released in regular media for 9 hours, blocked again in 4 mM thymidine media for 19
hours and harvested after regular media incubation for 3 hours. Protein extracts from
asynchronous and S phase CCL75.1 and Hela cells were subjected to
immunoprecipitation of WRN protein followed by Western Blot analysis to determine the
interaction between WRN and Ku70, PARP1 and TRF2. In both Hela and CCL75.1 cell
lines, the interactions of WRN with Ku70 and PARP1 can be detected in asynchronous
cells, but the association with TRF2 can only be detected during S phase. My results
vi

suggest that WRN complex in CCL75.1 cell is similar to that of Hela cell during S phase,
indicating that the cell cycle related regulation of WRN complex is not different in ALT
cells and that the difference between telomerase positive cell and ALT cell is probably
not due to the variation of WRN complex composition in S phase. It is likely that WRN
influences the activity of ALT cells in other ways.
1

Introduction
Werner Syndrome Protein (WRN)
Werner syndrome protein (WRN), an 180kDa nuclear protein that belongs to the family
of RecQ helicases, is encoded by the WRN gene. Mutations in the gene lead to an
autosomal recessive progerid disorder known as Werner Syndrome (WS). Werner
syndrome patients suffer from premature aging and genomic instability and the symptoms
usually begin around the third decade of life (Huang et al., 2006). The defect of
functional WRN also increases the incidence of rare cancers in these patients, especially
sarcomas (Futami et al., 2008). WRN is unique among the family of RecQ helicases in
that it possesses 3’-5’ exonuclease activity in addition to the 3’-5’ helicase activity of all
the family members (Shen et al., 1998). WRN is thought to be involved in DNA
replication and repair processes. Studies have indicated that WRN is capable of
unwinding many DNA structures including linear duplex DNA, synthetic replication
forks, Holliday junctions and G-quadruplexes (Mohaghegh et al., 2001). A delayed
S-phase progression is observed from WRN depletion cells because a decreased rate of
replication fork elongation, suggesting that WRN is involved in replication fork stalling
recovery, base excision repair and double strand break repair (Rossi et al., 2010).
Consistent with these studies, it is not surprising to find that cells derived from WS
2

patients exhibit elevated genomic instability including increased level of DNA deletions,
translocations and chromosomal breaks, when compared to normal cells (Fukuchi et al.,
1989). In addition to WRN’s role in guarding the genome integrity, WRN participates in
telomere maintenance as well. Cells from WS patients have accelerated telomere
shortening rate due to defective telomere lagging strand synthesis, suggesting that WRN
is necessary for efficient replication of telomeric DNA (Crabbe et al., 2004). Data from
our lab also suggests that WRN may influence telomerase activity (Li et al., 2008).
Alternative lengthening of telomeres (ALT)
Telomeres are nucleoprotein complexes located at the very end of human chromosomes
and consist of long tandem repetitive G rich sequences with telomere binding and
associated proteins, the shelterin complex, attached to them (Greenberg, 2005). The 3’
end of the DNA is longer than the paired 5’ DNA and it forms a single strand overhang
that invades the duplex DNA of the telomeric repeats to form a telomeric loop (T-loop).
This unique structure protects the telomere being recognized by the DNA double strand
break repair system and being fused with the ends of other chromosomes (de Lange,
2005). Besides, telomeres solve the end replication problem of semi-consecutive DNA
replication. When DNA polymerase reaches the end of the chromosome, the end portion
of the lagging strand is lost due to lack of space to produce the primer that is needed to
3

start the last Okazaki fragment in the 3’ end (Levy et al., 1992). The chromosome is
shortened after every cell division because of the telomere attrition. The most widely
believed model involving cell aging is that the shortening of telomere length is a
molecular counting mechanism to trigger cellular senescence or apoptosis. The telomere
length is usually maintained by telomerase, a reverse transcriptase that utilizes a
telomeric specific RNA as templates to add the TTAGGG repeats to the ends of the
chromosome (Shay and Bacchetti, 1997). However, the level of telomerase is insufficient
in human somatic cells to prevent telomere attrition, so cells have a limited capacity of
cell division and eventually undergo replicative senescence. Most cancer cells overcome
this problem by overexpressing telomerase or by elevating telomerase activity. Thus, low
level of telomerase is known to play a tumor-suppressor role in normal cells by restricting
the capacity a cell can divide. High level of telomerase activity is accepted as a
characteristic of cancer cells. However, about 15% of cancer cells maintain their telomere
with telomerase-independent pathways referred to as alternative lengthening of telomere
(ALT).
ALT is an aberrant situation of cell regulation that is not seen in normal cells but only in
cancer cells or genetically modified organisms. The telomeric structure of ALT is similar
to that of other cells with the presence of TTAGGG tandem repeats, the binding of
sheltering complex and the existence of T-loop. Nevertheless, ALT cells have some
4

unique attributes, including the presence of extrachromosomal telomeric DNA (t-circles),
which are linear or circular telomeric repeat of DNA fragments outside of the
chromosome. These DNA structures, some telomere-specific proteins and some
recombination proteins, are normally found in promyelocytic leukemia nuclear bodies
(PML nuclear bodies). These PML bodies are normally in association with ALT cells and
are recognized as the phenotypic characteristic of ALT cells (Yeager et al., 1999). Besides,
highly heterogeneous and rapidly changing telomere lengths as well as elevated level of
telomeric recombination are other characteristics of ALT cells. (Cesare and Reddel, 2010).
Accumulating evidence indicates that telomere length maintenance in ALT cell involves
recombination of telomeric DNA, but the molecular mechanism of this process is not
very well understood. (Lundblad and Blackburn, 1993).
The relationship between WRN and ALT
There is evidence suggesting that WRN participates in the maintenance of telomere and
plays an important role in telomeric recombination (Rossi et al., 2010). Indeed, in
telomerase-negative fission yeast, increased level of a WRN homolog protein is
expressed in response to the type II ALT phenotype (Rossi et al., 2010). In addition,
studies show that WRN localize to ALT associated PML bodies and interacts with
proteins in these bodies, including PML, RAD52 and NBS1. Besides, WRN has
5

functional connections with TRF1, TRF2, and FEN1, proteins which are important in
ALT cell telomere maintenance (Bhattacharyya et al., 2010). Studies from our lab
demonstrate that WRN is required for telomere dysfunction and telomeric circle (t-circle)
formation induced by expression of a TRF2 mutant missing the GAR domain (Li et al.,
2008). T-circles are one of the key features of ALT cancer cells, suggesting that WRN is
linked to ALT by t-circle formation in pathological conditions. Moreover, WRN may play
a role in recombination at telomeres in telomerase positive cells. In our lab, we show that
WRN strongly interacts with Ku70/80, PARP1 and DNA-PKcs proteins in Hela cell.
These proteins are involved in DNA repair and telomere maintenance, indicating that
WRN may influence telomere maintenance by forming protein complex with these
proteins in telomerase-positive cells. Since ALT cells maintain their telomere using
recombination processes, WRN may also play a role during the process of alternative
lengthening of telomere. Thus, it is possible for WRN to take part in telomeric
recombination in telomerase-negative cells. The rapid telomere deletions and elongations
in ALT cells are believed to occur through high rate of interchromosomal recombination,
and it is generally agreed that ALT cells depend on recombination mechanisms to
maintain telomere length. These studies, collectively, indicate that WRN, together with its
specific binding partners, potentially plays a role in the regulation of telomere
metabolism in cancer cells that use alternative telomere lengthening pathway.
6

The relationship between WRN and its molecular partners are also regulated in a cell
cycle dependent manner. Cells from Werner Syndrome patients display a prolonged S
phase, suggesting the dominant functional interactions between WRN and associated
proteins happen during S phase (Poot et al., 1992). There is also evidence that XPG
interacts directly with WRN in mid- to late S phase (Trego et al., 2011).
Immunoprecipitation experiments from Hela cells synchronized to G1, S and G2/M phase
has shown that Ku70 is bound to WRN throughout the cell cycle, while the associations
of WRN with TRF2, DNA-PKcs, PARP1 and hnRNP-C are observed during S phase (Jog
et al., 2011). In telomerase positive cells, WRN is likely to bind to specific proteins and
forms diverse megacomplex to carry out its distinct functions in different phase of cell
cycles.
It is not entirely certain how cellular factors interact with each other to elongate telomere
in ALT positive cells. Based on what we know about WRN and the characteristic of ALT
cells, I hypothesize that WRN regulate the activity of ALT by binding to different
proteins and form distinct functional complexes during specific stages of cell cycle. To
test this hypothesis experimentally, I therefore want to identify the component of WRN
complex and its relationship with different stages of cell cycles in ALT cells. In this study,
I utilized double thymidine block to synchronize CCL75.1 cells (ALT-positive) to S phase
and performed immunoprecipitation of WRN protein followed by Western Blot analysis
7

to study the interaction between WRN and Ku70, PARP1 and TRF2. In ALT cells, my
results indicate that WRN complex of CCL75.1 cell is similar to that of Hela cell during
S phase, suggesting the difference between telomerase positive cell and ALT cell is
probably not due to variation of WRN complex composition in S phase. Thus, the
influence of WRN on ALT pathway is yet to be defined.
8

Chapter 1: Materials and Methods
Cell Culture, synchronization and transfection
Hela and CCL75.1 cells were purchased from ATCC and were grown in DMEM medium
containing 10% FBS and 1% Pen-Strep at 37 ℃ in an atmosphere containing 5% CO
2
.
For Hela cell synchronization, cells were cultured on 10 cm plates for FACS analysis and
15 cm plates for immunoprecipitation. Cells were arrested by double thymidine block at
70% confluency. 1
st
blocking was 18 h incubating in media containing 2 mM thymidine,
and 1
st
release was incubating in regular media for 9 h after washing with PBS. Then the
2
nd
blocking was 17 h with media containing 2 mM thymidine to arrest the cells at the
beginning of G0/G1 phase. The 2
nd
release was 0 h (G1 phase), 3 h (S phase) or 7 h
(G2/M phase) after culturing in regular media.
In general, the condition for CCL75.1 synchronization was similar to that of Hela cell. In
the process of optimizing CCL75.1 synchronization condition, the time of the 1
st
blocking
and release as well as the time for the 2
nd
blocking and release was indicated in specific
cases. Briefly, a 3-factor-3-level orthogonal test was performed and the time of 1
st

blocking, release and the concentration of thymidine were the 3 factors. The levels of the
factors were as follows: the time of 1
st
blocking was 18, 21 or 24 hours; the time of 1
st

release was 9, 11 or 13 hours and the concentration of thymidine was 1, 2, or 4 mM. At 0
9

hour or 3 hours time point culturing in regular media after 2
nd
blocking, cells were fixed
and were ready for flow cytometry analysis.
For lentiviral transfection, Hela cells were transfected with pSlik/Neo-FLAG-hTERT
(hTERT) and pSlik/Puro-hTR (hTR) plasmids using method described in (Shin et al.,
2006).
Flow cytometry Analysis
Fix the cells: Wash with PBS twice followed by adding 1.2 ml of PBS. Scrape cells with
cell lifter. Adjust volume to 1.5 ml and add 3.5 ml of 100% ethanol to a final
concentration of 70%. Cells were kept at 4 ℃ overnight. Pellet the cells by centrifuging
at 1,500 rpm for 10 min. Resuspend the cells in 1 ml PBS and add RNase A to a final
concentration of 100 μg/ml. Incubate in 37 ℃ for 25 min. Pellet the cells by centrifuging
at 1,500 rpm for 10 min. Resuspend pellet in 1 ml of PBS with 50 μg/ml of Propidium
Iodide (PI). Incubate at room temperature for 1 hour. Cells were ready for Fluorescence
Induced Cell Sorting (FACS) analysis, or may be kept at 4 ℃ in dark for less than two
weeks before the FACS analysis.
The stained cells were then analyzed by FACSCanto II flow cytometer (BD Biosciences).
PI (PI-A and PI-H), FSC and SSC were chose as inspector channel and the voltage was
10

set at 320V, 150V and 280V, respectively. For each sample, 10,000 events were recorded.
First, events were plotted against PI-A and PI-H to gate single cells that didn’t form
aggregates with other cells. Then the histogram of the percentage of these cells in each
stage of cell cycle was analyzed using Watson (Pragmatic) model in Flowjo 7.6.1
software (Tree Star, Inc.).
Preparation of protein extract
All the protein extraction steps were carried out on ice or in cold room. Hela and
CCL75.1 cells were washed with PBS, scraped with cell lifter and pellet with 1,500 rpm
for 10 min. The resulting cell pellet was resuspended in 5 packed cell volumes (PCV) of
Hypo Buffer (10 mM Hepes pH=7.5, 1 mM EDTA, 0.5% NP-40, 1 mM PMSF, 1 mM
DTT, 1.7 μg/ml aprotinin and 1 μg/ml leupeptin) and incubated for 10 min. Cells were
pellet by 3,000 rpm for 5 min and resuspended in 3 PCV of high salt buffer (20 mM
Hepes pH=7.5, 420 mM NaCl, 1 mM EDTA, 10% Glycerol, 1 mM PMSF, 1 mM DTT,
1.7 μg/ml aprotinin and 1 μg/ml leupeptin) for 15 min and subsequently centrifuged at
13,000 rpm for 15 min. The supernatant was dialyzed at 4 ℃ overnight with 2 L of
dialysis buffer (20 mM Tris-HCl pH=8.0, 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 1
mM DTT, 1 mM PMSF), and was centrifuged at 12,000 rpm for 20 min at 4 ℃. The
resulting supernatant was collected and protein concentration was determined by Bio-Rad
11

BSA standard Curve Assay. The protein extract was saved for immunoprecipitation
experiments.
Coimmunoprecipitation analysis and immunoblotting
Nuclear extract (1.0 mg) was incubated with 5 μg of anti-WRN antibody (BL 1308;
Bethyl Inc.) and 5 μg of control rabbit IgG (Promega) on nutator in cold room for
overnight. 30 μl of Protein-A and Protein-G beads (1:1) equilibrated with dialysis buffer
were added to the extract and further incubated for 2 more hours. The beads were washed
5 times with dialysis buffer to wash out nonspecific antibody binding. Each wash was 10
min nutating at 4 ℃ followed by centrifugation at 1,000 rpm for 2 min. The beads were
resuspended in 30 μl of SDS sample buffer. 25 μl of the suspension was resolved by 8%
SDS-PAGE gel and subsequently transferred to PVDF membrane. Western blot analysis
was performed with a mouse antibody against PARP1 (F-2; sc-8007), a goat antibody
against Ku70 (C-19; sc-1486, Santa Cruz Biotechnology, Inc.), a rabbit antibody against
WRN (BL1309; A300-239A, Bethyl Inc.), and a mouse antibody against TRF2 (4A794;
OP129, Calbiochem). The anti-mouse, anti-goat, and anti-rabbit immunoglobulin G
horseradish peroxidase-coupled antibodies were purchased from Promega and Santa Cruz
Biotechnology Inc. The results were visualized by EPL PLUS from GE.
12

Telomere Repeats Amplification Protocol (TRAP)
TRAP was performed using TRAPeze Telomerase Detection Kit (CHEMICON). Pellet
the cells, wash once with PBS and carefully remove all PBS. The cells were stored at -80 ℃
or resuspended in 200 μl CHAPS Lysis Buffer/10
6
cells. 200 units/ml of RNase inhibitor
were added to the CHAPS Lysis Buffer (10 mM Tris-HCl, pH=7.5, 1 mM MgCl
2
, 1 mM
EGTA, 0.1 mM Benzamidine, 5 mM β-mercaptoethanol, 0.5% CHAPS, 10% glycerol)
prior to the extraction. The suspension was incubated on ice for 30 min. Pellet the sample
at 12,000 rpm for 20 min at 4 . Transfer 160 μ ℃ l of the supernatant into a fresh tube and
determine the protein concentration. Aliquot and quick-freeze the remaining extract at
-80 . Dilute the stock aliquots 1:20 with 1 ℃ ×CHAPS Lysis Buffer and dispense 2 μl per
assay (2 μl ≈500 cells). PCR were performed in the following condition. 10× TRAP
reaction buffer 5.0 μl, 10 mM dNTP 0.25 μl, TS primer 1.0 μl, TRAP Primer Mix 1.0 μl,
Taq polymerase 2 units, cell extract 2.0 μl. Add H
2
O to a final volume of 50 μl. HEK
293T cell extract was used as positive control and CCL75.1 cell extract was used as
negative control. PCR was performed in 3 steps: 94 /30 ℃ s, 59 /30 ℃ s and 72 /1 ℃ min
for 30 cycles. 5 μl of loading dye (0.25% bromophenol blue and 0.25% xylene cyanol in
50% glycerol/ 50 mM EDTA) was added and 25 μl was loaded to 12.5% non-denaturing
PAGE in 0.5× TBE buffer. Gel was stained with 1 μg/ml Ethidium Bromide in ddH
2
O for
30 min and destained for 25 min in ddH
2
O.
13

Chapter 2: Results
Optimize conditions for S phase CCL75.1 cell synchronization using double
thymidine block
CCL75.1 cell enters S phase 3 hours after double thymidine block.
To investigate the components of WRN complex in S phase CCL75.1 cells, I decided to
synchronize the cells to S phase with double thymidine block, because this was an
established method for synchronization of Hela cells (Jog et al., 2011). Hela Cells were
blocked by culturing in media with 2 mM thymidine for 18 hrs (1
st
blocking), released in
regular media for 9 hrs (1
st
release), blocked again for 17 hrs (2
nd
blocking) and then
released in regular media (2
nd
release). When cells were fixed at 0h, 3h and 7h after 2
nd

blocking and analyzed by flow cytometry, as shown in Figure 1A and Table 1, three
distinct cell profiles were observed. Cells were synchronized to G0/G1, S and G2/M
phase, respectively. (In this work, I characterized synchronization as more than 70% of
cells were in the same stage of the cell cycle). When I tried to find out the optimal time
point for collecting S phase CCL75.1 cells, however, I observed that CCL75.1 cells were
not synchronized to any single stage of G1, S or G2/M at the time points of 0, 2.5, 5, 7
and 10 hrs after the 2
nd
blocking (Figure 2B and Table 1). Nevertheless, CCL75.1 cells
fixed at 2.5 hours showed the highest percentage of cells in S phase, indicating the
optimal time for harvesting S phase CCL75.1 cells was around 2.5 hour. 3 hour time
14

point was preferred for further study because this was the time point for harvesting S
phase Hela cells after double thymidine block.
15

Figure 1: The different outcome of cell cycle synchronization of Hela cell and CCL75.1 cell after double thymidine
block. (A)Histogram of flow cytometry analysis of Hela cell untreated (a) and collected 0, 3 or 7 hours (b, c, and d,
respectively) after double thymidine block, indicating asynchronous, G1, S and G2/M phase, respectively. (B)
Untreated CCL75.1 cells (a) and cells harvested at 0, 2.5, 5, 7 and 10 hours (b, c, d, e, and f, respectively) after double
thymidine block were analyzed by Fluorescence Induced Cell Sorting (FACS). Green, brown and blue indicate cells in
G1, S and G2/M phase, respectively.
Table 1: Profile of FACS analysis results of Hela and CCL75.1 cells from Figure 1, showing percentage of cells in
different stages of cell cycle.
Panel in Fig. 1 Property Time after 2
nd
Blocking/ h G1% S% G2/M %
A-a Asynchronous -- 68.2 18.7 12.8
A-b G1 0 83.7 14.8 1.1
A-c S 3 12.8 83.6 4.1
A-d G2/M 7 8.4 18.6 73.0
B-a Asynchronous -- 45.5 37.9 17.1
B-b -- 0 40.8 50.2 9.1
B-c -- 2.5 23.1 67.7 10.6
B-d -- 5 21.9 45.2 33.5
B-e -- 7 23.7 32.8 43.3
B-f -- 10 34.3 31.8 34.7

16

4 mM of thymidine treatment in double thymidine block increases percentage of S phase CCL75.1 cells collected
at 3 h
Next, considering there are many differences between Hela and CCL75.1 cell lines, I
hypothesized that CCL75.1 cells might require different conditions of double thymidine
block to be synchronized to S phase. In other words, the original blocking parameters (the
blocking time, release time or the concentration of thymidine) may not be ideal for
harvesting CCL75.1 cells. In order to find out the optimal combination of these
parameters, I carried out 3-factor-3-level orthogonal test using the model L
9
(3
4
). As
shown in Table 2, the first factor was the time for 1
st
blocking, and the levels were 18, 21
and 24 hrs. The time for 1
st
release was the second factor, and the 3 levels were 9, 11, and
13 hrs. The concentration of thymidine was the third factor, and the 3 levels were 1, 2,
and 4 mM. Cells were fixed at 0 h (G1) or 3 h (S) after 2
nd
blocking. After FACS analysis,
the influence of each factor was determined by one-way analysis of variance (Table 3 and
4). According to the data below, the concentration of thymidine was more important to
yield more cells entering S phase at 3 h than the difference of time interval of blocking
and release, but the importance was not significant at α=0.10 level. In addition, more
cells were in S phase when treated with 4 mM of thymidine than that of 1 mM thymidine
treated (Figure2 A, right panel, p<0.05). I then looked at the difference of percentage of
G1 cells between 0 h and 3 h. This number represented the portion of cells that were
17

initially blocked by thymidine and were moving towards S phase after 2
nd
blocking and
thus indicated the potential of the cells for entering S phase. Thymidine concentration had
stronger influence on the results and it was significant at α=0.10 level. I further tested the
results using t-test and found out that 4 mM of thymidine increased the potential of
CCL75.1 cells entering S phase than 2 mM did (Figure2 B, right panel, p<0.05).
Table 2: The experimental design and results of L
9
(3
4
) orthogonal test

1
st
Blocking
Time/ h
1
st
Release
Time/h
Thymidine
Conc./mM
G1%
0h
G1%
3h
S%
3h
Exp. 1 18 9 1 35.04 27.69 56.40
Exp. 2 18 11 2 39.26 24.79 63.77
Exp. 3 18 13 4 54.65 21.29 70.67
Exp. 4 21 9 2 35.17 20.43 64.57
Exp. 5 21 11 4 39.11 16.52 68.94
Exp. 6 21 13 1 30.81 28.36 57.25
Exp. 7 24 9 4 50.52 16.89 68.72
Exp. 8 24 11 1 35.84 27.61 51.83
Exp. 9 24 13 2 26.21 23.42 50.62

Table 3: One way ANOV A result of the influence of 3 factors on the percentage of S phase cells at 3 h point
Factors
Sum of
Squares
Degree of
Freedom
F Value
F critical
α=0.1
1
st
Blocking 85.63 2 2.405
9.000
1
st
Release 20.76 2 0.583
Thymidine
Conc.
320.05 2 8.989

18

Table 4: One way ANOVA result of the influence of 3 factors on the percentage of cells moving from G1 phase to S
phase between 0 h and 3 h
Factors
Sum of
Squares
Degree of
Freedom
F Value
F critical
α=0.1
1
st
Blocking 41.31 2 0.409
9.000
1
st
Release 49.63 2 0.492
Thymidine
Conc.
958.90 2
9.505*
(p<0.10)

Figure 2: T-test results of orthogonal test. (A) The percentage of cells in S phase at 3 h after 2
nd
blocking with different
double thymidine blocking conditions. 4 mM of thymidine treatment is significantly better than 1 mM treatment (*
indicates p<0.05) in terms of percentage of cells in S phase at 3 h. (B) The potential of CCL75.1 cells entering S phase
after double thymidine block. The graph shows the difference of percentage of G1 cells between 0 h and 3 h, which
indicates the proportion of cells that were moving towards S phase after 2
nd
blocking. The percentage yield of 4 mM
thymidine treatment is significantly better than that of 2 mM (*, p<0.05) and 1 mM (**, p<0.01)

T
re
hr
Fi
th
15
in
fo
U
in
o
p
b
C
he optimal cond
elease for 9 hrs,
rs.
igure 3: Histogra
he X-axis, CCL7
5, 17 or 19 h. Af
n specific stage o
or 19 h during 2
n
Using previou
nstead of 2 m
f the duratio
ercentage of
locking was
CCL75.1 cel
0
10
20
30
40
50
60
70
80
Percentage/%
dition for harve
, 4 mM thymidi
am of CCL75.1
75.1 cells were tr
fter collecting ce
of cell cycle was
nd
blocking.
usly identifi
mM in tradit
on of the 2
n
f cell in S ph
s preferred.
ls were as f
27.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1
Perce
esting S phase C
ine block again
cells synchroniz
reated with 4 mM
ells at 3 h after 2
s shown in the g
ied condition
tional doubl
nd
blocking.
hase increase
To conclude
follows: inc
0
59.1
14.3
18-9-15
entage of C
CCL75.1 cells is
for 19 hrs and
zed to S phase w
M thymidine for
2
nd
blocking, FAC
graph. There we
ns in orthogo
e thymidine
When I inc
ed (Figure 3)
e, the optim
ubate cells
19.9
68.
18-9-
Cell in S ph
s 4 mM thymidi
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20

media for 9 hrs, 4 mM thymidine for 19 hrs and harvest cells after regular media
incubation for 3 hrs.
WRN associated proteins in S phase ALT cell
In both Hela and CCL75.1 cell lines, the interactions of WRN with Ku70 and PARP1 can be detected in
asynchronous cells, but the interaction with TRF2 can only be detected during S phase.
To investigate whether the interactions between WRN and its molecular partners were
cell cycle regulated in CCL75.1 cell line, and whether the components of this complex
was different from that of Hela cell, I performed WRN immunoprecipitation from protein
extracts prepared from asynchronous and S phase Hela and CCL75.1 cells. I used regular
double thymidine block to synchronize Hela cells and the optimized method for CCL75.1
cells. From the Western Blot analysis, I found out that in both Hela and CCL75.1 cell
lines, WRN exhibit similar distributions in terms of interactions with Ku70, PARP1 and
TRF2. The associations of WRN with Ku70 and PARP1 were observed in asynchronous
and S phase samples and the binding of TRF2 was only present in cells synchronized to S
phase (Figure 4). These results indicate that WRN probably binds Ku70 and PARP1
throughout the cell cycle, while TRF2 interacts with WRN only during S phase.
21

Figure 4: WRN complex in asynchronous and S phase Hela and CCL75.1 cells. (A) Regular and optimized double
thymidine block were used to synchronize Hela and CCL75.1 cells, respectively. A small portion of the cells used for
WRN-IP were analyzed by flow cytometry and the histogram of cells in different stages of cell cycle was shown. From
left to right: asynchronous Hela (a), S phase Hela (73.1%, b), asynchronous CCL75.1 (c) and S phase CCL75.1 (74.3%,
d). (B) Western blot results of immunoprecipitation of WRN complex from asynchronous and S phase Hela and
CCL75.1 cells. Nuclear protein extracts (1.0 mg) from these cells were incubated with WRN1308 antibody or control
rabbit antibody (IgG) and the immunoprecipitated materials were resuspended in sample buffer and subjected to
Western Blot with antibodies against WRN, PARP1, Ku70 and TRF2.
22

The function of WRN complex in telomerase activity and processivity
To test directly that the WRN complex influences the activity and processivity of
telomerase, I decided to set up a cell-free system using purified or recombinant WRN and
its associated proteins. First I needed to generate telomerase with high activity. I
transfected Hela cell with pSlik/Neo-FLAG-hTERT (hTERT) and pSlik/Puro-hTR (hTR)
plasmids (Figure 5A) using lentiviral transfection system described in (Shin et al., 2006).
Then I tested telomerase activity of Hela cells and Hela cells transfected with these two
plasmids (Hela-hTERT+hTR) using Telomere Repeats Amplification Protocol (TRAP).
The activity of telomerase in cell extracts from 500 cells of Hela and Hela-hTERT+hTR
were first examined. The result shows that there isn’t any significant difference of the
telomerase activity between Hela and Hela-hTERT+hTR, according to the distribution
and intensity of the PCR ladder product on the gel (Figure 5B). It is likely that the signal
was saturated after the PCR amplification. I then diluted the protein extracts from 500
cells to 100, 30 and 10 cells and applied TRAP analysis again. As shown in Figure 5C,
Hela cells show a ladder signal when diluted to 10 cells while Hela-hTERT+hTR don’t,
demonstrating that the telomerase activity of Hela-hTERT+hTR is not better than that of
Hela cell.

23

Figure 5: Telomerase activity of Hela cells transfected with hTERT and hTR. (A) Map of pSlik/Neo-FLAG-hTERT
(hTERT) and pSlik/Puro-hTR (hTR) plasmids. hTERT encoded the protein part of telomerase and was tagged by FLAG
epitope. The expression of FLAG-hTERT was induced by doxycycline. hTR encoded the RNA template of telomerase.
(B) Activities of telomerase from Hela cells and Hela-hTERT+hTR cells were determined by TRAP assay with 500 cell
lysate. HEK 293T (293T) cells were used as positive control and CCL75.1 cells and CHAPS Lysis Buffer (CHAPS LB)
were used as negative control. The wider distribution of the ladder and the stronger intensity of the bands indicate
higher activity of telomerase. HI means heat inactivation which was used as another negative control. Hela-TT:
Hela-hTERT+hTR. (C) Activities of telomerase from Hela cell and Hela-hTERT+hTR cell determined by TRAP assay
from a series of diluted protein extracts from 500 cell lysate. The numbers indicate the corresponding cell number of
protein extracts used in the assay. HI: Heat inactivation. CHAPS LB: CHAPS lysis buffer.

24

Chapter 3: Discussion
Werner protein and its molecular partners in ALT cell
About 15% of cancer cells maintain telomere length by a telomerase-negative pathway
termed alternative lengthening of telomere (Shay and Bacchetti, 1997), suggesting high
level of telomerase activity is not essential for unlimited proliferation in cancer cells. It is
widely believed that the ALT process requires telomeric recombination, but the exact
molecular mechanism is not well understood. Since WRN plays important roles in
telomere maintenance and DNA recombination, I am interested in investigating the role
WRN plays in regulating telomere length in ALT-positive cancer cells.
Hela cells were easily synchronized to S phase using an established double thymidine
block, but the synchronization of CCL75.1 cells was different. The excess of thymidine
causes an imbalanced nucleotide pool which sends feedback signal to inhibit nucleotide
synthesis. Cells are arrested at G1/S transition and S phase due to lack of nucleotide
synthesis. The 1
st
blocking will yield a population throughout G1 and S phase. The 1
st

release makes sure that all cells are out of S phase and will only be blocked to G1/S
transition upon next thymidine treatment. The reason that I was not able to synchronize
CCL75.1 cells in the first place was probably because the cell cycle time or the sensitivity
to thymidine were different in CCL75.1 cell compared to that of Hela cells. To solve
25

these issues, I performed a 3-factor-3-level orthogonal test. The test is an experimental
technique designed to minimize experimental practice needed to find out optimal
conditions using statistical analysis of the results. When I performed one-way ANOV A on
the results of the potential of cells entering S phase from G1 phase after 2
nd
blocking,
only thymidine concentration differed the outcome significantly on α=0.10 level. The
influence of the duration of 1
st
blocking and 1
st
release was not significant. Also, 4 mM
thymidine treatment showed an improvement in terms of cells in S phase at 3 h, but the
difference was not significant from that of 2 mM treatment. Although 4 mM thymidine
treatment seemed better in this orthogonal test analysis, further validations were required
to confirm the condition. So I performed additional tests and finally confirmed that 4 mM
thymidine treatment could yield more than 70% of CCL75.1 cells in S phase. Besides,
from these results I can tell that CCL75.1 cells were less sensitive to thymidine than Hela
cells were.
The double thymidine block may not be the ideal method for synchronizing CCL75.1 cell.
CCL75.1 cells were derived from WI-38 human diploid fibroblasts. Study showed that
WI-38 human diploid fibroblasts and the SV40 virus-transformed subline WI38/VA13
could be synchronized at the G1-S boundary by treatment with 1 mM hydroxyurea for 24
hrs and at M phase by treatment with 0.4 μg/ml nocodazole for 16 h (Matranga and
Shapiro, 2002). As I was unable to achieve more than 70% of CCL75.1 cells in G1 phase
26

or G2/M phase, these methods might be an alternative way to synchronize the cells in G1
or G2 phase. Thus I would be able to investigate the formation of WRN complex
throughout the cell cycle in CCL75.1 cells.
Previous work from our lab demonstrated that WRN and its associations with DNA-PKcs,
PARP1 and TRF2 were cell cycle regulated in telomerase positive Hela cells. I
hypothesized that this regulation may be different in ALT cells and this difference in
WRN complex formation may play a role in ALT telomere maintenance. In this study, I
used CCL75.1 cell as a representative for ALT cell lines and identified interactions
between WRN with Ku70, PARP1 and TRF2 by immunoprecipitation of cell extracts
from CCL75.1 cells synchronized to S phase. I observed that the interactions of WRN
and its molecular partners in CCL75.1 cell were similar to that of Hela cell, indicating the
cell cycle related regulation of WRN complex is not different in ALT cells.
We would be more confident to say that WRN interacts with Ku70, PARP1 and TRF2 if
the reverse immunoprecipitation experiments were done. But when our lab did this
experiment with Ku70, the amount of Ku70 protein was so high that we were unable to
detect the association of endogenous WRN with Ku70. The fact that we didn’t detect the
presence of WRN from immunoprecipitation of Ku70 does not mean the interaction is
artificial, because Ku70 interacts with many proteins and WRN may not be an important
27

partner to Ku70 while Ku70 can be a very important partner to WRN. Also, the
interaction could be detectable when we over-express WRN in the cell (data not shown).
On the other hand, we can’t rule out the possibility that WRN complex formation of
CCL75.1 cells may still be different from that of Hela cells. Although I didn’t detect the
difference in the formation of Ku70, PARP1 and TRF2 between these two cell lines, there
might be other WRN associated proteins that were present in one but absent in the other
cell line. Indeed, mass spectrometry results of silver stain of IP WRN from asynchronous
CCL75.1 cells from our lab revealed that a protein named DPYSL-3 was associated to
WRN (data not shown). This interaction was not seen in IP experiments with Hela cells,
indicating further detailed analysis are needed to identify whether the formation of WRN
complex is the same between CCL75.1 cell and Hela cell.
My results are consistent with previous studies on WRN interacting proteins. For
example, Ku70/80 complex has been shown to interact with and stimulate WRN
exonuclease activity (Cooper et al., 2000). And WRN possesses a functional interaction
with PARP1 in the process of DNA repair (von Kobbe et al., 2003). In addition, the
binding of WRN to TRF2 in ALT cell is not surprising, as study shows that WRN
colocalizes with TRF2 in U-2 OS (telomerase-negative) cell line (Opresko et al., 2002).
Although the method I was using could not identify new interaction partners of WRN, I
28

demonstrated that some of the interactions between WRN and its partners were not
always present during the entire cell cycle. Indeed, the binding between TRF2 and WRN
was not strong enough to be seen in cell cycle stages other than S phase, suggesting WRN
functions differently during different stages of cell cycle. For example, the helicase
activity of WRN increases upon binding to TRF2, leading to more efficient unwinding of
double strand DNA in S phase (Opresko et al., 2002). Study from cells derived from WS
patients showed a prolonged S phase, which was evidence that WRN functions more
active in S phase. DNA replication takes place in S phase, and WRN is believed to
recover replication fork stalling and resolving secondary structure that block polymerase
elongation. WRN probably functions as a complex to fulfill these tasks and it’s plausible
that the protein complex responsible for replication is only assembled during S phase.
Considering that WRN forms various complexes to perform distinct functions during
different stages of cell cycle, the investigation of WRN partners should take account of
cell cycle status. For Hela cell, the percentage of cells in S phase or G2/M phase is as low
as 20% in asynchronous cells. Therefore, it would be very difficult to identify WRN
partners that present only in S or G2/M phase with just asynchronous Hela cell
population. Besides, the interactions identified in asynchronous cells, which consists
mainly G0/G1 cells, may not take part in the process of DNA replication due to change of
WRN complex profile during cell cycle progression. The reason TRF2 binds to WRN
29

only during S phase could be that the binding potential of the interaction might be
hindered by post-translational modifications of TRF2 or WRN. There is evidence that
WRN enzyme activity can be modulated by post-translational modifications (Kusumoto
et al., 2007).
Although people have been studying WRN for many years, the distinct physiological
function of WRN is not very well defined. This might because there are still some
important molecular partners of WRN yet to be discovered. Recently, a proteome-wide
identification of WRN-interacting proteins using immunoprecipitation and mass
spectrometry has identified some new WRN interactors like TMPO, HNRNPU, etc.
(Lachapelle et al.), indicating new techniques and methods can discover novel
underrepresented interactions. Given that these proteins may suggest novel WRN
functions, we have to be aware the limitations of every method. For example, the number
of interaction proteins dropped considerably when they treated the protein extract with
nuclease before performing immunoprecipitation. WRN is a DNA binding protein, so it
can be found associated with other proteins indirectly through interactions with DNA.
It has been suggested that WRN is involved in regulation of the ALT process through
associations with proteins like flap endonuclease 1 (FEN1) (Brosh et al., 2002; Saharia
and Stewart, 2009). FEN1 is required for maintenance of telomeres in ALT cells and also
30

likely functions in the same mechanism as WRN (Saharia et al., 2008). On the other hand,
WRN may not be essential for ALT pathway, as indicated by the fact that WRN represses
telomere sister chromatids exchange (T-SCE) and thus prevents ALT process. And cells
with WRN-deficient and telomerase dysfunctional showed elevated recombination rates
which facilitate the activation of ALT pathway (Laud et al., 2005).
Although the connection between WRN and ALT is somewhat unclear, the study of the
relationship between WRN and ALT would promote our understanding of WRN
functions or ALT mechanisms. Further research in WRN-depleted ALT cells would reveal
more information on the role of WRN in ALT cell. Understanding the specific functions
of proteins involved in the recombination processes of ALT telomere maintenance will be
necessary to address the role of WRN in telomerase-negative cells. This knowledge
would surely take us a step forward in identifying new therapeutic targets for
telomerase-negative cancer cells.
The function of WRN complex in telomerase activity and processivity
WRN is suggested to be involved in telomere maintenance. Cells from WS patients have
accelerated telomere shortening (Crabbe et al., 2004). And data from our lab suggests that
WRN may influence telomerase activity. To test the idea directly, I proposed to set up a
cell-free system to investigate the influence of WRN complex on telomerase activity and
31

processivity using purified or recombinant WRN complex proteins. First I had to generate
telomerase with high activity. The endogenous telomerase of Hela cell is not sufficient for
the in vitro direct telomerase assay. As a result, I decided to over express telomerase in
Hela cells. I transfected pSlik/Neo-FLAG-hTERT and pSlik/Puro-hTR into Hela cell
using lentivirus transfection system. After selection with puromycin and G418, protein
extracts of 500 cells were prepared and the activity of telomerase was tested by Telomere
Repeat Amplification Protocol (TRAP). This assay required the telomerase to elongate an
18 nucleotide single strand DNA primer whose sequence resembled that of telomere. The
products of telomerase were then amplified by PCR with a reverse primer attached to
random places on the telomerase products leading to the formation of a ladder of DNA
fragments on the gel. Because of the PCR amplification, the signal intensity could be
saturated and thus could not be allowed to evaluate differences in activity and
processivity of telomerase. I diluted the protein extracts from Hela and
Hela-hTERT+hTR and carried out the TRAP analysis again. The less the activity of
telomerase, the diminishing of the ladder would be more sensitive to the dilution. It
turned out that the telomerase activity in Hela cells transfected with both hTERT and hTR
was not better than the endogenous telomerase activity in non-transfected Hela cells. It is
likely that the hTERT protein was not over-expressed in the transfected cell. This was
confirmed by Western Blot against FLAG epitope tag antibodies using the whole extract
32

of these cells (data not shown). Studies have been using HEK 293 cell in contract to Hela
cell when preparing telomerase with high activity. So I plan to transfect HEK 293 cell
with these two plasmids and hopefully be able to generate high expression of telomerase
in this cell line.
FLAG-tag affinity purification will be used to purify the over-expressed telomerase and it
will be used in direct telomerase assay. Different combinations of purified recombinant
WRN complex proteins (WRN, Ku70, PARP1 and TRF2 et al.) from Baculovirus
Expression System will be added to the telomerase assay to determine how these proteins
influence the activity and processivity of telomerase.

33

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Abstract (if available)

Abstract The Werner protein (WRN) is encoded by the WRN gene. Mutations in this gene are the causal factor for the outset of an autosomal recessive progerid disorder known as Werner Syndrome. WRN has been proposed to function in ALT, a pathway that maintains telomere length independent of telomerase and involves high level of recombination processes observed in about 15% of cancer cells. These functions are probably accomplished by the interactions between WRN and many other proteins. Thus I studied the WRN complex formation in ALT cells to further investigate the potential role of WRN in ALT pathway. In this study, I used CCL75.1 cell as a representative for ALT cell lines and synchronized CCL75.1 cells to S phase using optimized double thymidine block. I found out that the optimal condition for harvesting S phase CCL75.1 cells was as follows: cells were blocked with media containing 4 mM thymidine for 18 hours, released in regular media for 9 hours, blocked again in 4 mM thymidine media for 19 hours and harvested after regular media incubation for 3 hours. Protein extracts from asynchronous and S phase CCL75.1 and Hela cells were subjected to immunoprecipitation of WRN protein followed by Western Blot analysis to determine the interaction between WRN and Ku70, PARP1 and TRF2. In both Hela and CCL75.1 cell lines, the interactions of WRN with Ku70 and PARP1 can be detected in asynchronous cells, but the association with TRF2 can only be detected during S phase. My results suggest that WRN complex in CCL75.1 cell is similar to that of Hela cell during S phase, indicating that the cell cycle related regulation of WRN complex is not different in ALT cells and that the difference between telomerase positive cell and ALT cell is probably not due to the variation of WRN complex composition in S phase. It is likely that WRN influences the activity of ALT cells in other ways.

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Asset Metadata

Creator Huang, Fangjin (author)

Core Title Identify Werner protein molecular partners in S phase alt cell

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 05/01/2012

Defense Date 03/14/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alternative lengthening of telomere,cell cycle synchronization,immunoprecipitation,OAI-PMH Harvest,Werner protein

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Comai, Lucio (committee chair), Reddy, Sita (committee member), Schonthal, Axel H. (committee member)

Creator Email fangjinh@usc.edu,huangfangjin@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-21674

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