text:
Celebrity Extra: What was it about the show and the character of Ashley that made you want to be a part of it?
Laura Leighton: The script came out when pilot season was casting, and it was definitely one of those scripts that everybody wanted to be a part of. It was talked about. The casting process was exciting. The script was a terrific read. The thing with the show, it makes you want to see what's going to happen next. You want to have the next episode. It's the same in the script. You just kept turning the page. It's fun; it's suspenseful; it's a huge cast of characters. I feel lucky that there was a part in there for me. I think the character of Ashley, it's always fun to play somebody who's got a bit of, two sides. On one hand she's trying to do her best, and on the other she's got a little something a dark going on. It's fun to play those characters. I'm just so lucky that it worked out.
CE: This is being called a cross between Desperate Housewives and I Know What You Did Last Summer. Would you agree? If not, how would you describe it?
LL: I do agree with that. I think it's an apt description. I also think there's a little bit of The Sisterhood of the Traveling Pants in there. These four girls are sort of brought together by this common mystery they share. They've all got secrets, and one of them is a big secret that they all share together. Their friendship, I think, is a very important element of the show. One other description that I heard that I think is a really great is "teen noir." It's got a little bit of a dark quality to it. At the same time it's every bit of a teen show with beautiful girls and the storyline is about that time in their lives. So I like the idea of calling it teen noir.
CE: That is great! I wish I had thought of that.
LL: I wish I had too, but I didn't. I stole it from somebody. One of our directors called it that and now I'm using it.
CE: This is a pretty risky teen drama, dealing with murder, affairs with teachers, blackmail, etc. — how has the reaction been to it so far from people who've seen the first episode?
LL: My experience of people's reactions is it's unanimous. They are excited to see it and they are excited by the possibility of the next episodes as the storyline keep building. Who doesn't like a good story with colorful drama, with mystery and suspense and all of that fun stuff? I just think it's going to be a really fun show for ABC Family.
(Photo: ABC Family)
CE: You are working with an excellent cast — all of the teen girls are excellent (Troian Bellisario, Ashley Benson, Lucy Hale, Shay Mitchell), as well as the parents: you, Chad Lowe, Holly Marie Combs, Nia Peeples. How is the cast to work with, and in later episodes will we get to see Ashley interact more with the other parents?
LL: So far I think they are really establishing the girls together and their relationship, their friendship with one another and then catching a glimpse of their home life with their parents. So far we've mostly focused on developing those stories. But I'm sure in the community of Rosewood, every now and then the parents are going to cross paths. There's a reason for the people in the neighborhood to interact with each other. We haven't developed those story lines yet where the families meet and cross paths. I look forward to having scenes with Chad and Holly and Nia, and I'm sure that that will happen in the future. But right now they are just getting their relationships launched and really established.
CE: I am excited to see a Melrose Place reunion of sorts wih you and Chad together onscreen again.
LL: We are definitely excited to work together again.
CE: On a side note, I always liked Sydney (Laura's character on Melrose) with Carter (Chad's character) best, and was sad when it didn't work out with them.
LL: I did too. It's funny — I remember back then in interviews, people would ask, "Who do you think was the real love of Sydney's life?" And I always thought that there was maybe a part of her that felt it was probably Carter. He was kind of eccentric and perfect for her. He left her on the tarmac, and that was just so romantic.
CE: Back to Pretty Little Liars, Ashley is different from the other moms in that now she is a single mom. How has her husband leaving her affected her as a wife and as a mom?
LL: I think that it's left her pretty vulnerable and slightly desperate to make it work. I think the idea of a failed marriage, feeling like she has failed as a wife — she doesn't want to fail as a mother. So she's making choices to just try to make everything OK. She's not necessarily seeing clearly what the right thing to do is. But I think in her vulnerability and sort of desperation, what's driving her is to make everything OK for her daughter. It's definitely, I think, going to keep unfolding what's going on in that family as a result of being a single-parent household.
(Photo: ABC Family)
CE: She has a line where she tells her daughter, "I buy you everything you need to be popular, so why do you have to steal?" And she sleeps with the detective to get her daughter off for shoplifting. Is there nothing Ashley wouldn't do for Hanna?
LL: Well, we'll see. I think that right now we are seeing that she's making a pretty drastic choice to try to cover up Hanna's misdemeanor. We'll see what sort of repercussions that has. I think it's going to unravel in a way that I don't know yet. It hasn't unfolded yet. The stories are still developing. But I definitely think that there will be some sort of either conclusion or unraveling to that choice.
And it's interesting, I think, with the relationship with a teenage girl and especially a single mother. I think that there's a really interesting color: They have things in common. Sometimes the parent is just as vulnerable as the child, and sometimes the child can relate to the parent in ways that she was never able to when she was younger. I think they become a lot closer when the children are that age. You find that you have a lot more in common. You can see your parent as human. You can see your parent as flawed. And sometimes the parent can even look to the child for advice or support. So I think it's a really interesting relationship to have a single mother and a teenage daughter just kind of going at it together. Trying to find their way and figure it out.
CE: The look on Ashley's face when she looked back her daughter while going up to bed with the detective was just devastating. What was going through her mind at that point?
LL: Well, I think the idea is that nobody wanted it to come to this. She wants her daughter to be clear that I'm doing this for you— for us. This is because of what you did, but I'm doing this for you. It wasn't a decision made without regret or remorse. So there is definitely anger and sort of desperate love for her daughter too.
CE: I like that this is based on a book series — anything to get kids to read! Have you read the books to prepare for the role?
LL: Well, the pilot script was the first I'd read, and after we shot the pilot I was interested. I was intrigued. I wanted to peek ahead a bit, so I did read a couple of the books. But I stopped because I decided I wanted to be surprised. As much as it was fun to see where it was going, I also wanted to be surprised. So much of the first book is in the pilot, so there is so much yet to come. Sara Shepard's eighth book in the series, Wanted, is coming out at the same time we are premiering the television show (today). In a perfect world, the show will go for a long time and they'll run out of books for material, and they'll go off in their own direction. I think that's really exciting. There's a lot of material left to explore and then there's a lot left for the imagination. The series is going to be appealing to people who have read the books and are dying to see it come to life. And then for people who have never read the books, it's a brand new series that has exciting episodes.
CE: I don't know if this a question you can answer, but this first season has 10 episodes to air throughout the summer. If it does well, is this the kind of things that can transfer to the fall, or is this meant to be a summer series?
LL: I'm not sure if I'm able to answer that; if I really know. I know that ABC Family runs their programming a little bit different than some of the other networks. I think that's a good thing. They structure 10 episodes and then a bit of a break. It's great for the crew and for the writers. I think that they are like any other network where they are going to wait to see how it does on the air before speculating on how to proceed.
CE: It's great with the new trend of having original shows airing in the summertime — now we always have something new to watch and entertain us!
LL: And I think ABC Family definitely has their finger on that pulse. People don't just want to watch television at certain times of the year. There is definitely a year-round appetite for it. I think it's a perfect summer series, but it does take place in a school year beginning in the fall, so why shouldn't it air in the fall as well?
CE: I was so excited when they announced the Melrose revamp, but angry that Sydney was killed off in the first episode (yet relieved that she still showed up in flashbacks). What were your thoughts when they presented this idea to you?
LL: You take a bit of a leap of faith and think, "Well, they must know what they are doing!" You do sort of wonder whether or not you entirely agree with that vision you think, "Well, somebody must know what they are doing." You just try to go with it. I certainly knew what I was signing on for, and I thought it was an interesting way to approach it and have to tell the story in flashbacks, approaching it from the end and looking back. You never know how things are going to end up. It was just one of those things where I was excited to go back and play the character and completely willing to try it.
(Heather Locklear as Amanda, Laura Leighton as Sydney in Melrose Place on The CW. Photo: Michael Desmond/The CW ©2010 The CW Network, LLC. All Rights Reserved.)
CE: Was it fun to work with and see your old Melrose Place castmates again?
LL: It was great fun. As much as I love the new cast and it was really great to get to know them and work with them and stuff, it was also particularly rewarding to get to catch up with the old cast members. That was our favorite part.
(Pictured: Daphne Zuniga as Jo Reynolds, Heather Locklear as Amanda Woodward, Thomas Calabro as Dr. Michael Mancini, Josie Bissett as Jane Andrews. Photo Credit: ©The CW/ Michael Desmond (C) 2010 The CW Network, LLC. All rights reserved.)
CE: I absolutely loved that scene, even though Sydney didn't get to be in it, where they were all at a party at the apartments, and there was Jane, Jo, Michael and Amanda all hanging out together, remarking on how weirdly familiar it felt. That was a great treat for us old-school Melrose fans.
LL: I do think that what's the old Melrose audience was interested in. I think that was a great thing to do, to get them all together, and it was a bummer to not get to be there that day.


text:
In This Section
- Our Practice
- Our Team
- Your Appointment
- Making a Request
- Emergencies
- Maps & Directions
Patient Portal
Access my Health Records. click here »
Educational Resources
Review information here. view now »
Our Team
Allan L. Crimm, MD
What I like most about caring for patients is the satisfaction I gain from helping them achieve better health. I enjoy long-term relationships with many, many of my patients and often care for several generations within one family. I see patient care as a partnership that requires me to bring my extensive hospital and outpatient experience to the table. This allows me to maintain involvement in all aspects of a patient's care, while being an effective advocate on their behalf. I ask my patients, in return, to set personal health care goals and to participate actively in their own care.
I chose medicine as a career after teaching elementary school & working with emotionally disturbed children for several years. In this path I followed my grandfather, who was one of the first physicians in this country to be trained after medical education was changed to have a more scientific basis in the second decade of the 20th century. I vividly remember accompanying him when he made hospital rounds and home visits.
In addition to seeing patients I have also been organizing a network of physician practices in the Greater Philadelphia area that are all organized on the principles of the Patient-Centered Medical Home. We are working together to improve the quality of patient care in this area.
In my spare time I enjoy being with my wife Sara and 3 children (now all teenagers!), whose artwork has been on display in my exam rooms. Together we love outdoor activities such as skiing, hiking, canoeing and camping. I also enjoy weekend barbecues and jazz and blues music.
Medical School
Internship & Residency
Board Certification
Special Procedures & Techniques
Undergraduate School

subset: commoncrawl


text:
Welcome to The Dub Pages...just a plain ol' website with info on my life. I'll provide everything vital to my life. Links, Editorials, Contact Info, Pictures (coming soon), Daily Diaries, and much more. And what's with the name "Dub"? Only a select few know. Search the page...maybe you'll find out! Keep in mind, this page is always "under construction," so check back soon for updates. And be sure to visit the "Contact Me" link above, I'd love to hear from you!
The Ballpark Project!
The Ballpark Project was just an idea I had at a Rangers game a couple years ago. I decided to put the idea on paper the other day. After reading it, I realized I should share the idea on the Internet. Check out the site and let me know what you think!
Click here to visit The Ballpark Project. Or click the picture above.
This is our logo that we'll use for our new merchandise. We'll soon have an on-line store selling killerfrogs.com and TCU merchandise. Special thanks to our buddies at LogoCenter.com for creating this sew-on design.


text:
Events for June 23, 2018 › Other Events
Events Search and Views Navigation
10:00 am
The Mega Market
The Mega Market presented by 3Run Entertainment A Short Term Shop For Long Term Goods! Do you have a craft that you love? Maybe you're a collector of an interesting item? Do you have gently worn clothes, household goods, decorations,Find out more »


text:
Automakers are scrambling to develop models with consumer appeal. "We believe the future of driving will be autonomous driving and that is true for China as well", said Daimler's China CEO Hubertus Troska, adding the firm would push to bring the next-generation functions to the Asian giant. Read More »
McDonald's staff tried to stall Facebook murder suspect, says owner
In other Facebook videos posted on the day of the killing, Stephens said he had "just snapped", blaming gambling debts and a broken-down relationship among other things. Maggie Green said of Lane: "It's not her fault". "She's the reason that this is about to happen to you", Stephens told the elderly father of nine in the video. Read More »
Microsoft buys startup to boost its cloud
For Azure customers, this acquisition should make Microsoft's container technologies much more attractive and versatile going forward. In particular, Deis provides a slew of tools that are essential for building and managing applications on Google's Kubernetes container service. Read More »
Samsung Galaxy S8 and Galaxy S8+ set to launch in India…
Galaxy S8 is a crucial launch for the company given its Note 7 never went on sale in India. Samsung Galaxy S8, S8+ feature infinity display, which has no bezels at the sides. Comparing the prices, both the devices may cost above Rs. 50,000 in India. Samsung has already said the English-version of its new voice-assistant "Bixby" will be missing from the devices sold in the US. Read More »
Adnan Sami hits back after being trolled by Pakistanis for slamming Snapchat
After Snapchat CEO Evan Spiegel allegedly termed India as a "poor country" earlier this week, patriotism invoked in all the Indian Snapchat users. Here's how Twitterati has reacted to Snapchat CEO Evan Spiegel's comment calling India poor. Read More »
Samsung Takes on Surface With $629 Galaxy Book
There is also an LTE version that only comes in a 12-inch model and will cost $1,299.99. Creators who are getting exhausted of waiting on Microsoft to revise its Surface Pro lineup will have an interesting new alternative to consider starting tomorrow: That's when Samsung begins launching its Galaxy Book 2-in-1 in the United States. Read More »
Facebook killer's mother says he 'snapped'
Incredibly, two of the victim's daughters said they forgive their father's killer. "I honestly can say right now that I hold no animosity in my heart against this man. He took his nuggets and drove off. "I couldn't be prouder of my employees, the restaurant, the Pennsylvania State Police, everyone came together and did what we had to do", DuCharme said. Read More »
Facebook CEO comments on tragic death of Robert Godwin Sr
The McDonald's employee called police as Stephens proceeded down the drive-thru, DuCharme said. Stephens fled after he killed Godwin, triggering a manhunt that extended to several states and ended when he shot himself on Tuesday in Pennsylvania. Read More »
Airtel Extends 30 GB Free Data Offer, Brings Plan For International Holidayers
The fact that users will have to pay only fordata charges makes Jio the cheapest network . The validity of this plan is 28 days. While the Rs 408 plan has daily-FUP limit of 1GB, it is 2GB in the Rs 608 plan. Moreover, there is no restriction of timing regarding the data consumption; this is a major incentive for users to buy the pack as they were restricted to 500MB for 12am and 6am, and the remaining 500MB for the rest of the day. Read More »
Verizon Q1 Report Disappoints With Lower Than Expected Wireless Sign-Ups
The telecom giant's much-talked-about acquisition of Marissa Mayer's Yahoo is still pending, complicated by the two data breaches that Yahoo announced past year, which affected up to 1.5 billion users throughout 2013 and 2014. For its Fios service, Verizon added 35,000 internet subscribers, lost 13,000 cable subscribers and lost 8,000 voice subscribers. For the second quarter, analysts are looking for EPS of $0.99 and revenues of $30.35 billion, compared with EPS of $0.94 and revenues of ... Read More »
Ivanka Trump And Kushner's Roles Inappropriate, Poll
Painter added that under the Criminal Conflict of Interest statute, she should not be allowed to advise on governmental issues that could affect her company, like trade policies with China. Painter and others have sued President Trump over his business conflicts. Sweet argued that the Chinese trademarks carry "the appearance of impropriety" because "it is possible that she knew about these pending business deals" after taking a role in the White House. Read More »
Console Repairmen Explain Why Cockroaches Love PS4s
I heard about the PS4's roach problem at an independent console fix shop on the Lower East Side of Manhattan. But why do roaches love the PS4 more than, say, the XBox One or various Nintendo products? The report said that the only thing PS4 users can do is to go to third-party fix store like Che's XCubicle to get their consoles fixed. Read More »
Mario Kart 8 Deluxe review
You can play as six new characters: Inkling Boy and Inkling Girl from Splatoon , and classic Mario Kart racers Dry Bones, King Boo, Bowser Jr., and Gold Mario. Instead of racing, you would gather items and try to take out as many racers as possible, either hoping to get the most points or be the last one standing. Read More »
Hundreds of jobs in danger as Debenhams boss announces warehouse closures
Debenhams , which operates a flagship store on London's Oxford Street, said it will close as many as 10 United Kingdom outlets over the next five years and begin consultations on shutting one of three central distribution centers. He said he wanted to de-clutter stores after customers complained it was like a "treasure hunt" shopping in some branches. Liberum also highlighted its average length of shop leases at around 20 years, which would "limit the scope for action" and doubted ... Read More »
Manhunt for Facebook Killing Suspect Ends When He Fatally Shoots Himself
Restaurant owner Thomas DuCharme Jr and a supervisor told him his fries were delayed, but he eventually drove off and was chased by police before he stopped and shot himself dead. "We have a lot of work and we will keep doing all we can to prevent tragedies like this from happening", Zuckerberg said. The killing was the latest in a string of disturbing crimes captured on Facebook video, including the alleged gang rape of a 15-year-old girl, two fatal shootings, and the kidnapping and torture ... Read More »
Mark Zuckerberg Doubles Down on AR Future for Facebook
During his keynote kickoff for F8 yesterday, Zuckerberg (pictured above) once again emphasized his belief that Facebook in all its incarnations will help "bring people closer together" and overcome the current polarization in society. Based on the company's demo, these photo effects will be able to move and adjust based on what the camera is pointing at. Messenger will have much more to offer: Messenger is also going to get an Update. Read More »
Turkey's opposition CHP to demand annulation of referendum
Erdogan's supporters celebrated through the night, while his opponents stayed home in front of television sets, reflecting on the future of a nation that seems to be moving steadily away from its secular, democratic ideals of the past. While AKP supporters stressed the need for Erdogan and other party members to connect with the "no" voters to make them feel included, President Erdogan had already overstepped boundaries of any other president, and with the new constitution in place, there ... Read More »
Microsoft is killing off the password
Then, when you need to sign in , an app alert will pop up on your phone asking you to approve the action. Alex Simons, director of program management at Microsoft's Identity Division argues the new feature is superior to standard two-step verification and much better than relying on exclusively on passwords, which can be forgotten, phished, or compromised. Read More »
Facebook CEO Mark Zuckerberg Addresses Cleveland Murder
Augmented reality involves the project of computer-generated images into real-world surroundings. Facebook has been adding technology very similar to that of Snapchat for months, since Instagram and Facebook added stories features that copied Snapchat, as ground work for the augmented reality they would eventually launch, he said. Read More »
Hyundai Xcent Facelift launched at Rs.5.38 lakh
The Xcent was the third most selling compact sedan in the first quarter of 2017 after the Maruti Suzuki Swift DZire and the Honda Amaze. The current-gen model gets the trademark clamshell bonnet, sweptback headlamps and the chrome garnished front grille. Read More »
Xiaomi Mi 6 With 6GB RAM, Snapdragon 835 SoC Launched
After all, the handset features the latest Qualcomm Snapdragon 835 chipset , also found in the Samsung Galaxy 8, and it includes no less than 6GB of RAM . The Mi 6 with 64GB of storage will go for around $360, while a 128GB model will be priced at around $420. For connectivity , the Xiaomi Mi 6 supports 2X2 MIMO Wi-Fi, and 4G+ networks with download speed of up to 600Mbps and up to 100Mbps upload speed. Read More »
Apple Pledges to Stop Mining and Use Only Recycled Materials
The report shows the company hopes to do this with a closed supply chain, which would likely use recycling schemes and robots - such as Apple's Liam project - to identify the reusable materials. "We are committing as a company to not necessarily having to source from the earth for everything that we need", Jackson noted. Other environmental milestones are also outlined in Apple's report. Read More »
Sharp Aquos R launched with Snapdragon 835 and AI Assistant
After not hearing about a major smartphone release from Sharp for a while, today the company is announcing a new high-end smartphone which carries the Aquos moniker with it. We have received exclusive news that a 4GB RAM is on the cards which will be paired with a Snapdragon 835 processor. The smartphone runs on Android Nougat 7.1. Read More »
Huntington Bancshares Incorporated (NASDAQ:HBAN) from Regional - Midwest Banks - Todays Top Gains
BlackRock Advisors LLC now owns 1,497,878 shares of the company's stock valued at $14,769,000 after buying an additional 745,402 shares during the last quarter. Huntington Bancshares Incorporated's P/B is valued at 1.48 exhibiting the investor sentiment on the value of the stock to its actual value. HBAN has been the topic of several research reports. Read More »
Fujifilm goes Instax Square with the SQ10
On the digital side, the SQ10 is also the first Instax camera to feature a built-in digital camera. Because of its digital insides, the camera, which arrives in May for $280 (approximately £218 and AU$370 in the United Kingdom and Australia, respectively), can do much more such as take close-ups as close as 4 inches (10 cm), crop in on your subject and shoot with 10 different filters, vignette control and brightness adjustment. Read More »
Samsung's Galaxy S8 is super hot in South Korea
Samsung Galaxy S8 and S8 Plus is running on Android 7.0 Nougat right out of the box. Comparing the newly launched Galaxy S8 with its predecessor will show you some major changes this time. The smartphones have an advanced 8MP F1.7 Smart autofocus front camera and 12MP F1.7 dual pixel rear camera for the best low-light, zoom and anti-blur photos with enhanced image processing. Read More »
Samsung heir denies bribery charges at first hearing
Samsung said its first-quarter sales rose only modestly, to 50 trillion won. The major cause for this growth is understood to be the company's component divisions, which have thrived as demand for the company's memory chips and OLED panels has grown. Read More »
Switch is Nintendo's fastest-selling console ever in the US
This is not very surprising, as the Legend of Zelda Breath of the Wild is one of the biggest games that is available for the Nintendo Switch at the time of launch and the attach rate between the console and this game is nearly one to one. Read More »
Facebook F8 Conference: What to Expect, Where to Watch It
Speaking on stage at Facebook's F8 developer conference , CEO Mark Zuckerberg said that AR would be the next major platform for computing. Zuckerberg elaborated about future technological advances such as 3D effects, simultaneous location and mapping to determine precise location, and more. Read More »
The hunt is on: 1000 flock to Lehi's Easter Egg Hunt
Besides the goodies to be found in the eggs during the hunt there will also be over 100 Easter baskets given away as door prizes to the lucky attendees. There will be Easter egg hunts , crafts, face painting and live music. "We're going to have a bounce house, egg toss, egg relay and other egg-related activities". Read More »
Meet Galaxy Note 8 - Samsung's first dual camera phone
The company launched these phones in India at an event in New Delhi. Such fact could suggest the possibility of experiencing a color balance issue because the Samsung Galaxy S8's panels have two greens. Their internal storage out of the box capacity is 64 GB. They also both feature Super AMOLED display , allowing for clearer videos and images with a resolution of 2960 x 1440 (Quad HD). Read More »
Cleveland Murder Intensifies Scrutiny of Facebook Live
After a brief pursuit, he pulled over, and as officers approached the vehicle, he took his own life, authorities said. "We were just trying to make sure [the employee] got in contact with the state police". Authorities said a cell phone tower east of Erie detected a signal from Stephens' cell phone at 4:30 p.m. Sunday. A strong faith in God is helping the family through this ordeal, Godwin-Baines said. Read More »
Facebook CEO Mark Zuckerberg addresses shooting controversy during San Jose F8 Conference
The conference is a chance for Zuckerberg and his executives to wax poetic about all they ways they're changing the world, while also getting brands excited to sell things on Facebook . Zuckerberg envisions the marriage of augmented reality and Facebook's camera feature enabling people to make even mundane chores, like doing the dishes, look entertaining with digital effects. Read More »
HTC U Official Launch Date has been Confirmed
HTC 11, or HTC Ocean, is the new highly awaited flagship device that is surrounded with rumors of unofficial updates. The HTC U Ultra's super shiny facade wasn't also enough to fend off criticism of its less than impressive features. The U, which gets deformed into a wavy curve, could hint at this being the HTC "Ocean" that has been previously leaked. Further, it will support connectivity options including, Wi-Fi 802.11 a/b/g/n/ac, Bluetooth 4.2 and a fingerprint sensor integrated into the ... Read More »
Wolff sees Mercedes and Ferrari 'equal'
Hamilton lost a position to Vettel at the start, but his race really started to unravel when he received a five-second penalty for holding up Daniel Ricciardo in the pit lane under the Safety Car. "I know that gap that was there doesn't just come from driving". "I completely understand the decision from the team to request that, and of course I did it". Read More »


text:
- Research
- Open Access
Involvement of HTLV-I Tax and CREB in aneuploidy: a bioinformatics approach
© de la Fuente et al; licensee BioMed Central Ltd. 2006
- Received: 10 January 2006
- Accepted: 05 July 2006
- Published: 05 July 2006
Adult T-cell leukemia (ATL) is a complex and multifaceted disease associated with human T-cell leukemia virus type 1 (HTLV-I) infection. Tax, the viral oncoprotein, is considered a major contributor to cell cycle deregulation in HTLV-I transformed cells by either directly disrupting cellular factors (protein-protein interactions) or altering their transcription profile. Tax transactivates these cellular promoters by interacting with transcription factors such as CREB/ATF, NF-κB, and SRF. Therefore by examining which factors upregulate a particular set of promoters we may begin to understand how Tax orchestrates leukemia development.
We observed that CTLL cells stably expressing wild-type Tax (CTLL/WT) exhibited aneuploidy as compared to a Tax clone deficient for CREB transactivation (CTLL/703). To better understand the contribution of Tax transactivation through the CREB/ATF pathway to the aneuploid phenotype, we performed microarray analysis comparing CTLL/WT to CTLL/703 cells. Promoter analysis of altered genes revealed that a subset of these genes contain CREB/ATF consensus sequences. While these genes had diverse functions, smaller subsets of genes were found to be involved in G2/M phase regulation, in particular kinetochore assembly. Furthermore, we confirmed the presence of CREB, Tax and RNA Polymerase II at the p97Vcp and Sgt1 promoters in vivo through chromatin immunoprecipitation in CTLL/WT cells.
These results indicate that the development of aneuploidy in Tax-expressing cells may occur in response to an alteration in the transcription profile, in addition to direct protein interactions.
- Chromosomal Missegregation
- Kinetochore Assembly
- Mitotic Spindle Checkpoint
- CREB Binding Site
- Nuclear Envelope Assembly
Human T-cell leukemia virus type 1 (HTLV-I) is a complex retrovirus that causes adult T-cell leukemia/lymphoma (ATLL), a CD4 lymphoproliferative disease [1, 2]. While endemic in Japan, South America, Africa, part of the Middle East and the Carribean, there is an increasing prevalence of HTLV-I seropositivity world wide [1–3]. ATL develops in 2–5% of HTLV-I-infected individuals after a long latency period of about 20–30 years [4–6]. Different clinical features have resulted in the division of this disease into four clinical subtypes characterized by increasing aggressiveness: smoldering, chronic, lymphoma, and acute ATL [7].
One important marker for the risk of ATL within patients is the percentage of abnormal T lymphocytes versus normal T lymphocytes within the peripheral blood [8]. Binucleated lymphocytes or lymphocytes containing cleaved/cerebriform nuclei (also termed "flower" cells) have been observed in blood smears of HTLV-I infected individuals and in ATLL cells [7, 9–12]. These cells are representative of aneuploidy or abnormal chromosomal content which develops due to aberrant mitotic divisions [13]. Since aneuploidy has been suggested to contribute to tumorigenesis, there is a growing interest in deciphering the events in late G2/mitosis phase and defects therein that lead to aneuploidy. Additionally, aneuploidy may be associated with an acquired resistance to chemotherapeutic agents such as imatinib or 5-fluorouracil [14]; therefore, therapeutics disrupting aneuploidy development may improve upon current therapies for ATLL patients.
There is also a growing body of evidence to suggest that Tax, a 40 kDa viral oncoprotein encoded by HTLV-I, controls various aspects of cell cycle check points needed for aneuploidy. In fact, we were one of the first groups to show that Tax controls the G1/S check point [15], which was later confirmed by others [16], resulting in failure of G1 checkpoint and NER deficiency [17]. For a more comprehensive review of the cell cycle and check point controls by Tax, we recommend some of the more relevant reviews published recently [18–21]. In addition to disrupting checkpoints at the G1/S resulting in continuous cellular proliferation, Tax also directly targets a number of G2 and mitotic regulators. One of the first indication of Tax's involvement in the G2 and M phases was shown by Jin and colleagues [22] who discovered that Tax binds to hsMAD1. MAD1 and MAD2 are two of several genes that are involved in the activation of the mitotic spindle checkpoint function (MSC) following chromosomal missegregation. Tax interaction hindered the binding of MAD1/MAD2 complex to kinetochores by inducing translocation of these factors from the nucleus to the cytoplasm [23]. Furthermore, recent reports have demonstrated that Tax promotes activation of the anaphase promoting complex (APC)- APCCdc20p leading to a reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent and human cells [6, 24]. Overall, the degradation of these critical check point proteins results in a delay or failure in mitotic entry and progression, and is accompanied by a loss of cellular viability, resulting in aberrant anaphase progression, chromosomal instability and severe DNA aneuploidy [25–27].
Concurrently, Tax has been shown to repress cellular DNA repair by binding to Chk2 [24, 28] and Chk1, thus impairing kinase activities in vitro and in vivo [25]. Moreover, Tax silences cellular checkpoints, which guard against DNA structural damage and chromosomal missegregation, thereby favoring the manifestation of a mutator phenotype in cells [18]. In such cells, rapidly induced cytogenetic damage can be measured by a significant increase in the number of micronuclei (MN) in cells knocked-out for DNAPKcs [29, 30]. Therefore, it is possible that Tax perturbs many dynamic complexes that coordinate the processes of cell cycle regulation and DNA repair.
Here we present evidence that cytotoxic T cells (CTLL) stably expressing wild type Tax (CTLL/WT) exhibited a higher incidence of aneuploidy when compared to a Tax clone deficient for CREB transactivation (CTLL/703) [31]. Given the role of Tax as a strong activator of both viral and cellular transcription, we address the role of Tax-dependent transcription through the CREB/ATF pathway in the possible development of aneuploidy. We performed gene expression microarray analysis comparing CTLL/WT to CTLL/703 cells. Those genes that were either up or down-regulated in CTLL/WT cells were functionally annotated using the NIH's Database for Annotation, Visualization, and Integrated Discovery (NIH-DAVID). Next, we used an online database – PromoSer – to extract promoters of annotated genes to determine which of these genes contained CREB binding sites. Finally, chromatin immunoprecipitation was used to determine if DNA binding proteins such as Tax, Pol II and CREB were present at the promoters of the few selected genes. Our results clearly indicate that Tax/CREB binds to promoters of many genes, including Sgt1 and p97 (Vcp), which have functions in spindle formation and disassembly, respectively. The consequences of their over-expression and involvement in aneuploidy will be discussed.
Results and discussion
Aneuploidy prevalence in CTLL/WT cells
Tax deregulates the expression of genes that encode interleukin-2 (IL-2) and the multisubunit (IL-2R alpha, IL-2R beta, and IL-2R gamma) IL-2 receptor (IL-2R) in the early phases of HTLV-I induced ATL [32]. In the later phases of ATL, cells no longer produce IL-2 but still continue to express the IL-2R [32]. Fujii and colleagues developed an IL-2 independent system where Tax was stably expressed in a mouse IL-2-dependent T-cell line, CTLL-2, and examined the growth property of these cells in the absence of IL-2 [31]. While the Tax M47 (703) mutant activates NF-κB-dependent transcription but not CRE-dependent transcription, the reverse is true for the Tax M22 mutant [31]. They also noted that in addition to Tax's role in cAMP-responsive element (CRE) and NFκB activation pathways, Tax also increases expression of mRNAs coding for various AP-1 transcription factor family members including c-Jun, JunB, JunD, c-Fos and Fra-1. Genes encoding AP-1 are immediate-early genes whose products play important roles in cell activation, proliferation and transformation. Thus, an alternate pathway, i.e., AP-1, may be involved in the dysregulated phenotypes of T cells expressing Tax (CTLL) or infected with HTLV-1 [33].
Initially, these mutants were used to investigate the involvement of the transcription pathways in the transformation of CTLL-2 cells by Tax. Wild type Tax expression in CTLL-2 cells resulted in IL-2 independent growth. The Tax M47 mutant still activated the NF-κB-dependent transcription, and was able to support the growth of CTLL-2 in the absence of exogenously added IL-2. Therefore, the CREB dependent activity of Tax may not be as critical for IL-2 dependent growth, but may be needed for Tax induced transformation of cells. Tax M47 induced transformation may be accomplished through deregulation of cellular oncogenes, tumor suppressor genes, or checkpoint genes for DNA damage.
Our previous work utilizing these cell lines have shown that depending on which phase of the cell cycle DNA damage occurred, two different phenotypes were observed. Using centrifugal elutriation, we were able to fractionate cells at G1, S, or G2/M based on differences in cell volume at these distinct phases [34]. These cell fractions were gamma irradiated and harvested 24 hrs later for cell cycle analysis by propidium iodide staining and FACS. We observed that the CTLL/WT cells exhibited a distinct phenotype; at G1, these cells were able to induce a G1/S checkpoint, while at S or G2/M phase, these cells apoptosed after gamma irradiation. Conversely, CTLL/703 cells continued to proliferate without any apoptosis. We believed these differences were due, at least in part, to the differing gene expression profile of these cells and the induction of DNA damage at a particular point in the cell cycle. Interestingly, it was also observed that unirradiated CTLL/WT cells displayed a higher prevalence of aneuploidy than CTLL/703. The appearance of aneuploidy occurred in the later fractions (G2/M phase) which represented the largest cell populations. Consistent with our findings, previous reports have also indicated that centrifugal elutriation was capable of separating mixed populations of diploid and aneuploid cells [35, 36].
Since both of these cells are transformed, i.e. IL-2 independent [31], differences in transformation status cannot explain the presence of aneuploidy in one cell line and not the other. While it is possible that mutation of the Tax protein, resulting in a CREB transactivation deficient Tax (CTLL/703 cells), may disrupt interactions of Tax with late cell cycle checkpoint proteins whose dysregulation contributes to aneuploidy, however, this seems to be an unlikely event. For instance, Tax interaction with hMAD1 (also known as Txbp181) appears to be dependent on the zinc finger motif located within the N-terminus of Tax [22], and not the N-terminal domain as seen in the M47 mutant. Therefore, it appears probable that Tax may contribute to aneuploidy development, at least in part, through transcriptional activation of cellular genes critical for aneuploidy. This result would not be without precedence, since the involvement of Tax in immortalization has been shown to be mediated both at the transcriptional level and by direct protein:protein interactions [18]. Furthermore, since Tax-dependent CREB transactivation was deficient in CTLL/703 and not in CTLL/WT cells (see below), it would appear that those genes involved in aneuploidy development may be CREB-dependent.
Gene expression profiling and promoter analysis
Cellular genes containing CREB response element activated by Tax
Genbank Accession Number
Fold Change
Transcription/RNA Processing
Cytoskeletal Protein Binding
M etabolism
Transm em brane Receptor
Cell Cycle
Stress Response
a po pto tic protease activating factor 1
ADP-ribosylation factor 1
ATP synthase, H+ transporting, m itochondrial F1 com plex, gam m a polypeptide 1
ATPase, H+ transporting, V0 subunit C
CCR4 carbon catabolite repression 4-like (S. cerevisiae)
centrin 3
checkpoint kinase 1 hom olog (S. pom be)
cytochrom e c oxidase, subunit Vb
carboxypeptidase E
dihydrolipoam ide dehydrogenase
early B-cell factor 1
ferredoxin 1
Sm c2l1
SM C2 structural m aintenance of chrom osom es 2-like 1 (yeast)
lipin 1
ferritin heavy chain
gam m a-am inobutyric acid (GABA-C) receptor, subunit rho 1
gap junction m em brane channel protein alpha 1
guanine nucleotide binding protein, alpha inhibiting 3
gene rich cluster, C10 gene
heat shock 70kD protein 5 (glucose-regulated protein)
guanylate kinase 1
histocom patibility 2, class II antigen A, alpha
histocom patibility 2, class II antigen A, beta 1
histocom patibility 2, D region locus 1
H2-DM b1
histocom patibility 2, class II, locus M b1
hexokinase 2
heat shock protein, A
im m ediate early response 2
Jun-B oncogene
Psm b8
proteosom e (prosom e, m acropain) subunit, beta type 8 (large m ultifunctional protea
M dh2
m alate dehydrogenase 2, NAD (m itochondrial)
nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, beta
phospholipase D3
protein phosphatase 1, catalytic subunit, beta isoform
Psm b5
proteasom e (prosom e, m acropain) subunit, beta type 5
Psm e3
proteasom e (prosom e, m acropain) 28 subunit, 3
dual specificity phosphatase 1
purine rich elem ent binding protein A
RAB6, m em ber RAS oncogene fam ily
regulator of G-protein signaling 2
chem okine (C-C m otif) ligand 1
golgi apparatus protein 1
solute carrier fam ily 31, m em ber 1
signal recognition particle 14
tum or necrosis factor receptor superfam ily, m em ber 9
phospholipid scram blase 1
ubiquitin-like 1
Von W illebrand factor hom olog
tyrosine 3-m onooxygenase/tryptophan 5-m onooxygenase activation protein, eta po
zeta-chain (TCR) associated protein kinase
craniofacial developm ent protein 1
Psm b2
proteasom e (prosom e, m acropain) subunit, beta type 2
caspase 8 associated protein 2
H2A histone fam ily, m em ber Y
ATP synthase, H+ transporting, m itochondrial F1 com plex, O subunit
Tax1 (hum an T-cell leukem ia virus type I) binding protein 1
heterogeneous nuclear ribonucleoprotein A2/B1
dynactin 3
zinc finger protein 265
Ras-related GTP binding C
ubiquitin carboxyl-term inal esterase L5
gam m a-am inobutyric acid receptor associated protein
arsA (bacterial) arsenite transporter, ATP-binding, hom olog 1
Parkinson disease (autosom al recessive, early onset) 7
interferon-stim ulated protein
vacuolar protein sorting 35
cytochrom e c-1
Usm g5
upregulated during skeletal m uscle growth 5
succinate dehydrogenase com plex, subunit B, iron sulfur (Ip)
SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae)
NADH dehydrogenase (ubiquinone) 1 beta subcom plex, 2
ubiquitin-associated protein 2
suppression of tum origenicity 13
casein kinase 1, alpha 1
Kruppel-like factor 7 (ubiquitous)
arginyl-tRNA synthetase
protein phosphatase 1, regulatory (inhibitor) subunit 15b
ATPase, H+ transporting, V0 subunit B
lactam ase, beta 2
am yloid beta precursor protein binding protein 1
alanyl-tRNA synthetase
eukaryotic translation initiation factor 2C, 2
valosin containing protein
filam in, beta
9 Unknowns: BC021353, BC037019, NM_026452, BC016544, AL023058, BC004013, BC032165BC032165, BC030844, and BC027100
Tax activated genes involved in aneuploidy
Genbank Accession Number
Fold change
ADP-ribosylation factor 1
Membrane vesicle formation, inactivation nec. for mitotic Golgi breakdown, chromosome segregation, and cytokinesis [95–97]
centrin 3
spindle cell body duplication need for bipolarity in cytokinesis [98–103]
checkpoint kinase 1 homolog (S. pombe)
needed for S and G2 checkpoints after DNA damage; Tax has been shown to disrupt its function [24,25,104–106]
dynactin 3
light-chain subunit of dynactin complex (p24); dynactin complex involved in vesicle movement; dynactin complex interacts with NuMA and dynein in order to tether microtubules at the spindle poles and that are essential for mitotic spindle pole assembly and stabilization; recruitment by ZW10 protein to the kinetichore [107–110]
valosin containing protein
aka cdc48; needed for spindle disassembly after segregation; Golgi disassembly-assembly [111–113]
protein phosphatase 1, catalytic subunit, beta isoform
antagonist of Aurora B kinase during mitosis, i.e. regulating the protein interface between the centromeres and the mitotic spindle; possible downstream targets include kinetochore protein Ndc10, CENP-A, which substitutes for histone H3 in centromeric nucleosomes, and inner centromere protein INCENP; mutation of PP1 or inactivation was shown to result in cytokinesis defects. [114]
purine rich element binding protein A
involved in DNA replication and transcription; overexpression results in G2 blockage [115,116]
SMC2 structural maintenance of chromosomes 2-like 1 (yeast)
aka CAP-E protein, that is part of the condensin protein complex originally identified in Xenopus; protein complex is needed for chromosome condensation; CAP-E has been found to interact with DNA ligase IV (DNA double-strand break repair protein) possible interaction important for genome stability [117–120]
SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae)
recent study shows that SGT1 is needed for kinetechore assembly [66,76]
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide
binds to CDC25B in a phosphorylation independent mechanism [121,122]
ZW10 interactor
specifies the kinetochore association of ZW10 which may act as part of, or immediately downstream of, the wait anaphase tension-sensing checkpoint [123,124]
While depletion of these particular factors have been shown to cause aneuploidy, over-expression of these factors may prove to be equally problematic. There are instances where over-expression of kinetochore or associated proteins such as dynamitin (p50; [58]), CENP-H [59], and CENP-A [60] led to disruption of the dynactin or kinetochore complex. In the case of CENP-H, over-expression contributed to the appearance ofaberrant micronuclei, a sign of aneuploidy. Therefore we envision a similar scenario where over-expression of dynactin 3 (p24), whichacts as a light-chain subunit of the dynactin complex that tethers microtubules to the kinetochore and was found to bepresent stoichiometrically at one molecule per complex, leads to sequesteration of other components of the dynactin complex away from microtubules leading to aneuploidy.
In vivoconfirmation of CREB binding sites
Chromatin immunoprecipitation (ChIP) has been used to determine if specific proteins bind to regions of a genome in vivo [61], to identify transcription factor binding to promoters [62, 63], and to identify the binding of modified proteins to DNA in vivo [64, 65]. To confirm that genes regulating aneuploidy were in fact transcriptionally activated in a CREB-dependent manner, a ChIP assay was performed on a few of the Tax regulated genes in CTLL cells. Sgt1 and p97 were amongst the list of the genes identified based on the presence of potential binding sites for CREB. Sgt1 has been shown to be an essential protein and a critical assembly factor for kinetochore assembly [66]. Experiments have been carried out in the past to demonstrate the functional significance of Sgt1. RNAi mediated Sgt1 depletion in HeLa cells leads to mitotic delay due to activation of the spindle checkpoint. Sgt1 depletion also led to the reduction in kinetochore levels of three MSC components – Mad1, Mad2 and BubR1 [66].
Direct effect of Tax on gene promoters
Finally, given our results in Table 2, we wondered how over-expression of some of these genes by Tax could contribute to aneuploidy? A careful examination of the p97 and Sgt1 literature indicates that when these proteins are over-expressed, they deregulate control of few cellular pathways including apoptosis, cell division, and cell cycle. For instance, over-expression of p97 in Tax expressing cells could exhibit multiple functions including a role in anti-apoptosis and metastasis by activation of the NFκB signaling pathway [70], as well as its binding to Cdc48 and its adaptors, Ufd1-Npl4. Cdc48 and Ufd1-NPl4 regulate membrane related functions and mitotic spindle disassembly by directly binding to membrane-associated proteins or spindle assembly factors, modulating their interactions with membranes or spindles, respectively. The AAA ATPase CDC48 was first identified in Saccharomyces cerevisiae as a cell cycle division gene. Cdc48, as well as its homolog p97 in vertebrates, forms a homohexameric ring that functions as an active ATPase. Together with its adaptor proteins, the Ufd1-Npl4 heterodimer, the Cdc48/p97-Ufd1-Npl4 complex participates in a variety of membrane-related functions, including morphological transformations in cell division, particularly in establishing a proper G1 phase after mitosis. Consistent with this idea, Cdc48/p97-Ufd1-Npl4 is required for post-mitotic nuclear envelope reassembly [71], one of the major morphological transformations occurring at the end of cell division.
Over-expression of the Cdc48/p97-Ufd1-Npl4 complex could also assist in modification of chromatin by removing proteins involved in chromosome condensation from the highly condensed mitotic chromatin. In addition, this complex may directly regulate nuclear envelope assembly by assisting proper assembly of nuclear envelope proteins on the chromatin, which puts the proteins at the right place to coordinate post-mitotic chromosome decondensation with nuclear envelope assembly [72]. A third function of over-expressed p97 could be its role with Cdc48 and the execution of "Start" in G1 by mediating the proteolysis of the G1-CDK inhibitors i.e., Far1 [73]. This would be consistent with the idea that Cdc48/p97-Ufd1-Npl4 has a general role in regulating proper M-G1 transition [72]. Finally, over-expressed p97 ATPase could extract class I MHC from ER membranes [74]. Polyubiquitinated substrates preferentially bind by p97 ATPase in complex with two adaptor proteins, Ufd1 and Npl4. This association triggers retrotranslocation of the substrates from the ER membrane, followed by proteasome-mediated degradation [75].
Increased expression of Sgt1 induced by Tax could also affect the dynamics of its many protein partners resulting in efficient substoichiometric complexes. Sgt1 (TPR+CS domain) binds to Skp1 and this interaction might play a role in activation of multiprotein complexes CBF3 and SCF [76], assembly of the CBF3 complex, its turnover, and influence proper kinetochore function [77]. Sgt1 (TPR+CS domain) binds to Ctf13 and this binding might influence proper CBF3 assembly [78]. Sgt1 also binds Rad6, which is involved in DNA repair and protein degradation as well as cell cycle progression [79]. Furthermore, Sgt1p contributes to the activity of the cyclic AMP (cAMP) pathway and physically interacts with the adenylyl cyclase Cyr1p/Cdc35p, where a Gα subunit-type protein called Gpa2p and the GTP binding Ras proteins both of which are implicated in adenylyl cyclase activation [80–83]. The major effector of cAMP in eukaryotes is the cAMP dependent protein kinase, or protein kinase A (PKA) [84]. In the absence of cAMP, the catalytic subunit of PKA is found in an inactive complex with the regulatory subunit. The binding of cAMP by the regulatory subunit leads to dissociation of the complex and activation of the catalytic subunit [85]. In budding yeast, the adenylyl cyclase pathway is notably involved in cell growth control and stress responses [80], and also regulates the cell cycle by modulating G1 cyclin expression [86–88] and the activities of the anaphase-promoting complex/cyclosome and the SCF pathway [89].
Therefore, given the role of Sgt1 in several different types of multimeric protein complexes, it is possible that it acts as some sort of protein chaperone, assembly factor, or allosteric activator. This hypothesis is consistent with structure predictions suggesting that the Sgt1 N-terminal region is similar to the TPR regions of the Sti1/Hop co-chaperone and the central CS domain adopts a fold similar to that of the p23 co-chaperone. Sti1/Hop facilitates protein substrate transfer from Hsp70 to Hsp90, whereas p23 stimulates the ATPase activity of Hsp90 and the release of bound substrate [90]. Finally, several experimental observations are also consistent with a role for Sgt1 as a co-chaperone or assembly factor. First, Sgt1 is required to functionally activate the Skp1 and Ctf13 subunits of the CBF3 kinetochore complex but is not itself a subunit of this complex [76]. Second, Sgt1 may be present in substoichiometric quantities in SCF [76] and Cyr1 complexes regulating the M to G1 transition, and thirdly Sgt1, like SKP1, is required for both the G1/S and G2/M transitions during the cell cycle [79].
The Tax oncoprotein is considered the cause for mitotic aberrations in HTLV-I infected cells. In addition to binding checkpoint factors and interfering with the MSC function, we propose a mechanism by which Tax induces these aberrations through over-expression of factors associated with kinetochore assembly. Wild-type Tax-expressing cells displayed higher incidence of aneuploidy than the CREB transactivation deficient mutant. Through microarray and promoter analyses, we have identified 95 genes that were over-expressed in CTLL/WT cells which may be regulated through CREB activity. Furthermore, 11 of these genes are involved in G2/M phase regulation, in particular kinetochore regulation. We have confirmed the regulation of Sgt1 and p97/Vcp by CREB and Tax in vivo by performing ChIP analysis. The over-expression of these factors may contribute to the observed aneuploidy phenotype and disrupt proper targeting of their binding partners. The mitotic aberrations generated may be in the form of improper spindle attachment and disassembly process that results in chromosomal mis-segregation and hence aneuploidy.
Cell culture
Chronic T Lymphocytic Leukemia (CTLL) cells stably tranfected with either wild-type Tax or the M47 Tax mutant (319LL → AS) have previously been described [31]. For the purpose of this study, they are designated as CTLL/WT (CTLL-2 cells transfected with wild type Tax) and CTLL/703 (CTLL-2 cells transfected with a CREB Tax mutant, M47). A comparison of Tax expression in WT and 703 cells has been published previously [15, 34]. Cells were grown in RPMI-1640 with 10% FBS, 1% streptomycin, penicillin antibiotics, and 1% L-glutamine, and at 5% CO2, 37°C.
Metaphase chromosome spread
CTLL/WT and CTLL/703 cells were incubated in 5 ml of complete media for 48 hrs. Both cultures were treated with 50 μg colcemid for 45 mins. Next, samples were centrifuged at 160 g for 10 mins. Cells were incubated at room temperature in 0.075 M KCl for 30 minutes and then fixed in 1 ml of methanol:glacial acetic acid (3:1). Cells were then centrifuged and washed again with the fixative solution for a total of three times. Finally, cells were resuspended in 250 μl of fixative and dropped onto prechilled slides at -20°C with a water vapor film formed on them just before dropping the cell suspension to induce better chromosome spread. Three to five drops of 70% glacial acetic acid was then added to remove cytoplasm. The slides were then allowed to dry overnight at room temperature. Slides were processed in Dulbecco's Phosphate Buffered Saline (D-PBS) for 1 minute, Giemsa stain working solution (2 ml Giemsa stain diluted with 25 ml D-PBS) for 10 mins, and in distilled water for 30 secs. After being dried at room temperature, cover slips were mounted and allowed to dry. Slides were then imaged using the Olympus BX-60 microscope (100x, oil immersion) and Image-Pro Plus 5.1 software. Spreads with clearly visible and distinct chromosomes were then selected and counted.
Cytoplasmic RNA isolation
Cells were centrifuged at 4°C, 3000 rpm for 10 min., quickly washed with D-PBS without Ca2+/Mg2+, and centrifuged twice. Pelleted cells were immediately frozen at -80°C until all time points were collected. Cytoplasmic RNA was isolated utilizing the RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer's directions with the addition of 1 mM dithiothreitol in Buffer RLN. Isolated RNA was quantified by UV spectrophotometric analysis and 3 μg of RNA was visualized on a non-denaturing 1% agarose TAE gel for quality control.
Expression profiling
Six micrograms of cytoplasmic RNA from each sample was used to synthesize double-stranded cDNA using the Superscript Choice System kit and T7-(dT) 24 primer (100 pmol/μL) (Invitrogen). The cDNA was purified using phenol/chloroform extraction and ethanol precipitation. The cDNA was then used to perform in vitro transcription using the BioArray HighYield RNA Transcript Labeling Kit (T7) (Enzo, Farmingdale, NY). The biotin-labeled cRNA was purified using the RNeasy Mini Kit (Qiagen) and quantified by spectrophotometric analysis and analyzed on a 1% agarose TAE gel. The biotin-labeled cRNA was then randomly fragmented to ~s35–200 base pairs by metal-induced hydrolysis using a fragmentation buffer according to the Affymetrix Eukaryotic Target Hybridization protocol. Two Murine Genome U74A GeneChips (Affymetrix) were used per sample. This array contained 6,000 functionally characterized sequences from the Mouse UniGene database (Build 74) and 6,000 Expressed Sequence Tag (EST) clusters. The array was washed, primed, and stained on the Affymetrix Fluidics Station 400 following manufacturer's recommendations. cRNA was detected through a primary scan with phycoerythrin-streptavidin staining and then amplified with a second stain using biotin-labeled anti-streptavidin antibody and a subsequent phycoerythrin-streptavidin stain. The emitted fluorescence was scanned using the Hewlett-Packard G2500A Gene Array Scanner, and the intensities were extracted from the chips using Microarray Suite 4.0 (MAS4.0) software. All raw chip data was scaled in MAS4.0 to 800 to normalize signal intensities for inter-array comparisons.
Data analysis
Comparative analyses were performed by normalizing the fluorescence intensity of each probe using robust multi-array average (RMA), as previously described [91]. The mean of the signal intensities from duplicate experiments was calculated. To filter for differentially expressed genes, those genes that were absent in both experiments were excluded from further analysis. Those genes that were either up-or down-regulated in CTLL/WT cells by a magnitude of two-fold were compiled and functionally annotated utilizing the Database for Annotation, Visualization, and Integrated Discovery program (DAVID;[39]), as detailed in Figure 2.
Utilizing Genbank accession IDs, promoter sequences [2100 bp surrounding the predicted transcription start site (TSS)] for our annotated genes were retrieved from PromoSer [47, 48], a large-scale database containing the promoters of human, mouse, and rat genes. The following criteria were used for the retrieval: 1) promoter sequence encompassing 2000 bp upstream and 100 bp downstream of the TSS, 2) sequences were required to contain a quality score of at least 2 and support score of 2, i.e. TSS prediction is supported by one or more mRNAs, 3) in the case of alternative promoters only the one that is best supported and 5' was included, and 4) promoters that mapped to more than one loci or did not align well were excluded. At least one third of the promoter sequences retrieved were checked for proper alignment against the mouse genome using Blastn [92] and MapViewer [93] tools through NCBI. CREB (TGACGT/C), CREB/p300 (A/GGGAGT), NFκB (GGGAA/CTTC/TCC), and SRF (CCCATATATGG) consensus sequences obtained through the TRANSFAC database [94] were searched within the promoters obtained. Factors that contained the CREB or CREB/p300 sequences within their promoters were further probed for genes that contribute directly to mitosis, cytokinesis, and microtubule organization.
Chromatin immunoprecipitation
Primers used for PCR
Accession #
Gene ID
Correctly mapped
This work was supported by grants from the George Washington University REF funds to A. Vertes and F. Kashanchi, and NIH grants AI44357, AI43894 and 13969 to F.K.
Authors' Affiliations
- Proietti FA, Carneiro-Proietti AB, Catalan-Soares BC, Murphy EL: Global epidemiology of HTLV-I infection and associated diseases. Oncogene. 2005, 24: 6058-6068. 10.1038/sj.onc.1208968.PubMedGoogle Scholar
- Taylor GP, Matsuoka M: Natural history of adult T-cell leukemia/lymphoma and approaches to therapy. Oncogene. 2005, 24: 6047-6057. 10.1038/sj.onc.1208979.PubMedGoogle Scholar
- Mahieux R, Gessain A: HTLV-1 and associated adult T-cell leukemia/lymphoma. Rev Clin Exp Hematol. 2003, 7: 336-361.PubMedGoogle Scholar
- Murphy EL, Hanchard B, Figueroa JP, Gibbs WN, Lofters WS, Campbell M, Goedert JJ, Blattner WA: Modelling the risk of adult T-cell leukemia/lymphoma in persons infected with human T-lymphotropic virus type I. Int J Cancer. 1989, 43: 250-253.PubMedGoogle Scholar
- Gatza ML, Watt JC, Marriott SJ: Cellular transformation by the HTLV-I Tax protein, a jack-of-all-trades. Oncogene. 2003, 22: 5141-5149. 10.1038/sj.onc.1206549.PubMedGoogle Scholar
- Liu B, Liang MH, Kuo YL, Liao W, Boros I, Kleinberger T, Blancato J, Giam CZ: Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability. Mol Cell Biol. 2003, 23: 5269-5281. 10.1128/MCB.23.15.5269-5281.2003.PubMed CentralPubMedGoogle Scholar
- Shimoyama M: Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma. A report from the Lymphoma Study Group (1984-87). Br J Haematol. 1991, 79: 428-437.PubMedGoogle Scholar
- Hisada M, Okayama A, Shioiri S, Spiegelman DL, Stuver SO, Mueller NE: Risk factors for adult T-cell leukemia among carriers of human T-lymphotropic virus type I. Blood. 1998, 92: 3557-3561.PubMedGoogle Scholar
- Fujimoto T, Hata T, Itoyama T, Nakamura H, Tsukasaki K, Yamada Y, Ikeda S, Sadamori N, Tomonaga M: High rate of chromosomal abnormalities in HTLV-I-infected T-cell colonies derived from prodromal phase of adult T-cell leukemia: a study of IL-2-stimulated colony formation in methylcellulose. Cancer Genet Cytogenet. 1999, 109: 1-13. 10.1016/S0165-4608(98)00141-1.PubMedGoogle Scholar
- Taguchi H, Miyoshi I: Three cases of pre-adult T-cell leukemia. Jpn J Clin Oncol. 1983, 13 Suppl 2: 209-214.PubMedGoogle Scholar
- Kinoshita K, Amagasaki T, Ikeda S, Suzuyama J, Toriya K, Nishino K, Tagawa M, Ichimaru M, Kamihira S, Yamada Y, et al: Preleukemic state of adult T cell leukemia: abnormal T lymphocytosis induced by human adult T cell leukemia-lymphoma virus. Blood. 1985, 66: 120-127.PubMedGoogle Scholar
- Sacher RA, Luban NL, Ameti DI, Friend S, Schreiber GB, Murphy EL: Low prevalence of flower cells in U.S.A. blood donors infected with human T-lymphotrophic virus types I and II. Br J Haematol. 1999, 105: 758-763. 10.1046/j.1365-2141.1999.01408.x.PubMedGoogle Scholar
- Kops GJ, Weaver BA, Cleveland DW: On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer. 2005, 5: 773-785. 10.1038/nrc1714.PubMedGoogle Scholar
- Lazaris AC, Kavantzas NG, Zorzos HS, Tsavaris NV, Davaris PS: Markers of drug resistance in relapsing colon cancer. J Cancer Res Clin Oncol. 2002, 128: 114-118. 10.1007/s00432-001-0310-5.PubMedGoogle Scholar
- Santiago F, Clark E, Chong S, Molina C, Mozafari F, Mahieux R, Fujii M, Azimi N, Kashanchi F: Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells. J Virol. 1999, 73: 9917-9927.PubMed CentralPubMedGoogle Scholar
- Lemoine FJ, Marriott SJ: Accelerated G(1) phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein. J Biol Chem. 2001, 276: 31851-31857. 10.1074/jbc.M105195200.PubMedGoogle Scholar
- Haoudi A, Semmes OJ: The HTLV-1 tax oncoprotein attenuates DNA damage induced G1 arrest and enhances apoptosis in p53 null cells. Virology. 2003, 305: 229-239. 10.1006/viro.2002.1642.PubMedGoogle Scholar
- Grassmann R, Aboud M, Jeang KT: Molecular mechanisms of cellular transformation by HTLV-1 Tax. Oncogene. 2005, 24: 5976-5985. 10.1038/sj.onc.1208978.PubMedGoogle Scholar
- Zhao RY, Elder RT: Viral infections and cell cycle G2/M regulation. Cell Res. 2005, 15: 143-149. 10.1038/sj.cr.7290279.PubMedGoogle Scholar
- Azran I, Schavinsky-Khrapunsky Y, Aboud M: Role of Tax protein in human T-cell leukemia virus type-I leukemogenicity. Retrovirology. 2004, 1: 20-10.1186/1742-4690-1-20.PubMed CentralPubMedGoogle Scholar
- Jeang KT, Giam CZ, Majone F, Aboud M: Life, Death and Tax: role of HTLV-I oncoprotein in genetic instability and cellular transformation. J Biol Chem. 2004Google Scholar
- Jin DY, Spencer F, Jeang KT: Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell. 1998, 93: 81-91. 10.1016/S0092-8674(00)81148-4.PubMedGoogle Scholar
- Kasai T, Iwanaga Y, Iha H, Jeang KT: Prevalent loss of mitotic spindle checkpoint in adult T-cell leukemia confers resistance to microtubule inhibitors. J Biol Chem. 2002, 277: 5187-5193. 10.1074/jbc.M110295200.PubMedGoogle Scholar
- Haoudi A, Daniels RC, Wong E, Kupfer G, Semmes OJ: Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response. J Biol Chem. 2003, 278: 37736-37744. 10.1074/jbc.M301649200.PubMedGoogle Scholar
- Park HU, Jeong JH, Chung JH, Brady JN: Human T-cell leukemia virus type 1 Tax interacts with Chk1 and attenuates DNA-damage induced G2 arrest mediated by Chk1. Oncogene. 2004, 23: 4966-4974. 10.1038/sj.onc.1207644.PubMedGoogle Scholar
- Liu B, Hong S, Tang Z, Yu H, Giam CZ: HTLV-I Tax directly binds the Cdc20-associated anaphase-promoting complex and activates it ahead of schedule. Proc Natl Acad Sci U S A. 2005, 102: 63-68. 10.1073/pnas.0406424101.PubMed CentralPubMedGoogle Scholar
- Hagting A, Den Elzen N, Vodermaier HC, Waizenegger IC, Peters JM, Pines J: Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J Cell Biol. 2002, 157: 1125-1137. 10.1083/jcb.200111001.PubMed CentralPubMedGoogle Scholar
- Park HU, Jeong SJ, Jeong JH, Chung JH, Brady JN: Human T-cell leukemia virus type 1 Tax attenuates gamma-irradiation-induced apoptosis through physical interaction with Chk2. Oncogene. 2006, 25: 438-447.PubMedGoogle Scholar
- Majone F, Semmes OJ, Jeang KT: Induction of micronuclei by HTLV-I Tax: a cellular assay for function. Virology. 1993, 193: 456-459. 10.1006/viro.1993.1145.PubMedGoogle Scholar
- Majone F, Luisetto R, Zamboni D, Iwanaga Y, Jeang KT: Ku protein as a potential human T-cell leukemia virus type 1 (HTLV-1) Tax target in clastogenic chromosomal instability of mammalian cells. Retrovirology. 2005, 2: 45-10.1186/1742-4690-2-45.PubMed CentralPubMedGoogle Scholar
- Peloponese JMJ, Iha H, Yedavalli VR, Miyazato A, Li Y, Haller K, Benkirane M, Jeang KT: Ubiquitination of human T-cell leukemia virus type 1 tax modulates its activity. J Virol. 2004, 78: 11686-11695. 10.1128/JVI.78.21.11686-11695.2004.PubMed CentralPubMedGoogle Scholar
- Yoshida M, Inoue J, Fujisawa J, Seiki M: Molecular mechanisms of regulation of HTLV-1 gene expression and its association with leukemogenesis. Genome. 1989, 31: 662-667.PubMedGoogle Scholar
- Wu K, Bottazzi ME, de la Fuente C, Deng L, Gitlin SD, Maddukuri A, Dadgar S, Li H, Vertes A, Pumfery A, Kashanchi F: Protein profile of tax-associated complexes. J Biol Chem. 2004, 279: 495-508. 10.1074/jbc.M310069200.PubMedGoogle Scholar
- Chen L, Ma S, Li B, Fink T, Zachar V, Takahashi M, Cuttichia J, Tsui LC, Ebbesen P, Liu X: Transcriptional activation of immediate-early gene ETR101 by human T-cell leukaemia virus type I Tax. J Gen Virol. 2003, 84: 3203-3214. 10.1099/vir.0.19283-0.PubMedGoogle Scholar
- Alexandre C, Charnay P, Verrier B: Transactivation of Krox-20 and Krox-24 promoters by the HTLV-1 Tax protein through common regulatory elements. Oncogene. 1991, 6: 1851-1857.PubMedGoogle Scholar
- Marriott SJ, Lemoine FJ, Jeang KT: Damaged DNA and miscounted chromosomes: human T cell leukemia virus type I tax oncoprotein and genetic lesions in transformed cells. J Biomed Sci. 2002, 9: 292-298.PubMedGoogle Scholar
- Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher MR, Peters JM, Kinzler KW, Vogelstein B, Lengauer C: Securin is required for chromosomal stability in human cells. Cell. 2001, 105: 445-457. 10.1016/S0092-8674(01)00340-3.PubMedGoogle Scholar
- Wang Z, Yu R, Melmed S: Mice lacking pituitary tumor transforming gene show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, and premature centromere division. Mol Endocrinol. 2001, 15: 1870-1879. 10.1210/me.15.11.1870.PubMedGoogle Scholar
- Neuveut C, Low KG, Maldarelli F, Schmitt I, Majone F, Grassmann R, Jeang KT: Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb. Mol Cell Biol. 1998, 18: 3620-3632.PubMed CentralPubMedGoogle Scholar
- Iwanaga Y, Tsukahara T, Ohashi T, Tanaka Y, Arai M, Nakamura M, Ohtani K, Koya Y, Kannagi M, Yamamoto N, Fujii M: Human T-cell leukemia virus type 1 tax protein abrogates interleukin-2 dependence in a mouse T-cell line. J Virol. 1999, 73: 1271-1277.PubMed CentralPubMedGoogle Scholar
- Waldmann TA: The promiscuous IL-2/IL-15 receptor: a target for immunotherapy of HTLV-I-associated disorders. J Acquir Immune Defic Syndr Hum Retrovirol. 1996, 13 Suppl 1: S179-85. 10.1097/00042560-199600001-00027.PubMedGoogle Scholar
- Iwai K, Mori N, Oie M, Yamamoto N, Fujii M: Human T-cell leukemia virus type 1 tax protein activates transcription through AP-1 site by inducing DNA binding activity in T cells. Virology. 2001, 279: 38-46. 10.1006/viro.2000.0669.PubMedGoogle Scholar
- de la Fuente C, Wang L, Wang D, Deng L, Wu K, Li H, Stein LD, Denny T, Coffman F, Kehn K, Baylor S, Maddukuri A, Pumfery A, Kashanchi F: Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in HTLV-1 Tax expressing cells. Mol Cell Biochem. 2003, 245: 99-113. 10.1023/A:1022866027585.PubMedGoogle Scholar
- Distelhorst CW, Benutto BM, Bergamini RA: Centrifugal elutriation of human lymphoid cells: synchronization and separation of aneuploid cells into diploid and tetraploid populations. Cell Immunol. 1983, 79: 68-80. 10.1016/0008-8749(83)90051-5.PubMedGoogle Scholar
- Davies R, Cain K, Edwards RE, Snowden RT, Legg RF, Neal GE: The preparation of highly enriched fractions of binucleated rat hepatocytes by centrifugal elutriation and flow cytometry. Anal Biochem. 1990, 190: 266-270. 10.1016/0003-2697(90)90191-B.PubMedGoogle Scholar
- Mann MB, Hodges CA, Barnes E, Vogel H, Hassold TJ, Luo G: Defective sister-chromatid cohesion, aneuploidy and cancer predisposition in a mouse model of type II Rothmund-Thomson syndrome. Hum Mol Genet. 2005, 14: 813-825. 10.1093/hmg/ddi075.PubMedGoogle Scholar
- Guenet JL: The mouse genome. Genome Res. 2005, 15: 1729-1740. 10.1101/gr.3728305.PubMedGoogle Scholar
- Dennis GJ, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA: DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003, 4: P3-10.1186/gb-2003-4-5-p3.PubMedGoogle Scholar
- de la Fuente C, Santiago F, Chong SY, Deng L, Mayhood T, Fu P, Stein D, Denny T, Coffman F, Azimi N, Mahieux R, Kashanchi F: Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2. J Virol. 2000, 74: 7270-7283. 10.1128/JVI.74.16.7270-7283.2000.PubMed CentralPubMedGoogle Scholar
- Fujii M, Niki T, Mori T, Matsuda T, Matsui M, Nomura N, Seiki M: HTLV-1 Tax induces expression of various immediate early serum responsive genes. Oncogene. 1991, 6: 1023-1029.PubMedGoogle Scholar
- Sekkai D, Gruel G, Herry M, Moucadel V, Constantinescu SN, Albagli O, Tronik-Le Roux D, Vainchenker W, Bennaceur-Griscelli A: Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells. Stem Cells. 2005, 23: 1634-1642. 10.1634/stemcells.2005-0182.PubMedGoogle Scholar
- Vandepoele K, Vlieghe K, Florquin K, Hennig L, Beemster GT, Gruissem W, Van de Peer Y, Inze D, De Veylder L: Genome-wide identification of potential plant E2F target genes. Plant Physiol. 2005, 139: 316-328. 10.1104/pp.105.066290.PubMed CentralPubMedGoogle Scholar
- Worden B, Yang XP, Lee TL, Bagain L, Yeh NT, Cohen JG, Van Waes C, Chen Z: Hepatocyte growth factor/scatter factor differentially regulates expression of proangiogenic factors through Egr-1 in head and neck squamous cell carcinoma. Cancer Res. 2005, 65: 7071-7080. 10.1158/0008-5472.CAN-04-0989.PubMedGoogle Scholar
- Allocco DJ, Kohane IS, Butte AJ: Quantifying the relationship between co-expression, co-regulation and gene function. BMC Bioinformatics. 2004, 5: 18-10.1186/1471-2105-5-18.PubMed CentralPubMedGoogle Scholar
- Argyropoulos C, Nikiforidis GC, Theodoropoulou M, Adamopoulos P, Boubali S, Georgakopoulos TN, Paliogianni F, Papavassiliou AG, Mouzaki A: Mining microarray data to identify transcription factors expressed in naive resting but not activated T lymphocytes. Genes Immun. 2004, 5: 16-25. 10.1038/sj.gene.6364034.PubMedGoogle Scholar
- Halees AS, Leyfer D, Weng Z: PromoSer: A large-scale mammalian promoter and transcription start site identification service. Nucleic Acids Res. 2003, 31: 3554-3559. 10.1093/nar/gkg549.PubMed CentralPubMedGoogle Scholar
- Halees AS, Weng Z: PromoSer: improvements to the algorithm, visualization and accessibility. Nucleic Acids Res. 2004, 32: W191-4.PubMed CentralPubMedGoogle Scholar
- Quinn PG, Wong TW, Magnuson MA, Shabb JB, Granner DK: Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol. 1988, 8: 3467-3475.PubMed CentralPubMedGoogle Scholar
- Jameson JL, Jaffe RC, Deutsch PJ, Albanese C, Habener JF: The gonadotropin alpha-gene contains multiple protein binding domains that interact to modulate basal and cAMP-responsive transcription. J Biol Chem. 1988, 263: 9879-9886.PubMedGoogle Scholar
- Rikitake Y, Moran E: DNA-binding properties of the E1A-associated 300-kilodalton protein. Mol Cell Biol. 1992, 12: 2826-2836.PubMed CentralPubMedGoogle Scholar
- Chen H, Hung MC: Involvement of co-activator p300 in the transcriptional regulation of the HER-2/neu gene. J Biol Chem. 1997, 272: 6101-6104. 10.1074/jbc.272.10.6101.PubMedGoogle Scholar
- Kashanchi F, Brady JN: Transcriptional and post-transcriptional gene regulation of HTLV-1. Oncogene. 2005, 24: 5938-5951. 10.1038/sj.onc.1208973.PubMedGoogle Scholar
- Westermann S, Cheeseman IM, Anderson S, Yates JR, Drubin DG, Barnes G: Architecture of the budding yeast kinetochore reveals a conserved molecular core. J Cell Biol. 2003, 163: 215-222. 10.1083/jcb.200305100.PubMed CentralPubMedGoogle Scholar
- De Wulf P, McAinsh AD, Sorger PK: Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes. Genes Dev. 2003, 17: 2902-2921. 10.1101/gad.1144403.PubMed CentralPubMedGoogle Scholar
- Rieder CL, Salmon ED: The vertebrate cell kinetochore and its roles during mitosis. Trends Cell Biol. 1998, 8: 310-318. 10.1016/S0962-8924(98)01299-9.PubMedGoogle Scholar
- Zhou J, Yao J, Joshi HC: Attachment and tension in the spindle assembly checkpoint. J Cell Sci. 2002, 115: 3547-3555. 10.1242/jcs.00029.PubMedGoogle Scholar
- Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB: Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol. 1996, 132: 617-633. 10.1083/jcb.132.4.617.PubMedGoogle Scholar
- Tomonaga T, Matsushita K, Ishibashi M, Nezu M, Shimada H, Ochiai T, Yoda K, Nomura F: Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res. 2005, 65: 4683-4689. 10.1158/0008-5472.CAN-04-3613.PubMedGoogle Scholar
- Tomonaga T, Matsushita K, Yamaguchi S, Oohashi T, Shimada H, Ochiai T, Yoda K, Nomura F: Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res. 2003, 63: 3511-3516.PubMedGoogle Scholar
- Kuo MH, Allis CD: In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment. Methods. 1999, 19: 425-433. 10.1006/meth.1999.0879.PubMedGoogle Scholar
- Spiller MP, Kambadur R, Jeanplong F, Thomas M, Martyn JK, Bass JJ, Sharma M: The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD. Mol Cell Biol. 2002, 22: 7066-7082. 10.1128/MCB.22.20.7066-7082.2002.PubMed CentralPubMedGoogle Scholar
- Holmes MC, Tjian R: Promoter-selective properties of the TBP-related factor TRF1. Science. 2000, 288: 867-870. 10.1126/science.288.5467.867.PubMedGoogle Scholar
- de la Fuente C, Deng L, Santiago F, Arce L, Wang L, Kashanchi F: Gene expression array of HTLV type 1-infected T cells: Up-regulation of transcription factors and cell cycle genes. AIDS Res Hum Retroviruses. 2000, 16: 1695-1700. 10.1089/08892220050193164.PubMedGoogle Scholar
- Ashburner BP, Westerheide SD, Baldwin ASJ: The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol Cell Biol. 2001, 21: 7065-7077. 10.1128/MCB.21.20.7065-7077.2001.PubMed CentralPubMedGoogle Scholar
- Steensgaard P, Garre M, Muradore I, Transidico P, Nigg EA, Kitagawa K, Earnshaw WC, Faretta M, Musacchio A: Sgt1 is required for human kinetochore assembly. EMBO Rep. 2004, 5: 626-631. 10.1038/sj.embor.7400154.PubMed CentralPubMedGoogle Scholar
- Andrisani OM: CREB-mediated transcriptional control. Crit Rev Eukaryot Gene Expr. 1999, 9: 19-32.PubMedGoogle Scholar
- Niikura Y, Kitagawa K: Identification of a novel splice variant: human SGT1B (SUGT1B). DNA Seq. 2003, 14: 436-441.PubMedGoogle Scholar
- Yamamoto S, Tomita Y, Hoshida Y, Nagano H, Dono K, Umeshita K, Sakon M, Ishikawa O, Ohigashi H, Nakamori S, Monden M, Aozasa K: Increased expression of valosin-containing protein (p97) is associated with lymph node metastasis and prognosis of pancreatic ductal adenocarcinoma. Ann Surg Oncol. 2004, 11: 165-172. 10.1245/ASO.2004.05.012.PubMedGoogle Scholar
- Hetzer M, Meyer HH, Walther TC, Bilbao-Cortes D, Warren G, Mattaj IW: Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat Cell Biol. 2001, 3: 1086-1091. 10.1038/ncb1201-1086.PubMedGoogle Scholar
- Cao K, Zheng Y: The Cdc48/p97-Ufd1-Npl4 complex: its potential role in coordinating cellular morphogenesis during the M-G1 transition. Cell Cycle. 2004, 3: 422-424.PubMedGoogle Scholar
- Fu X, Ng C, Feng D, Liang C: Cdc48p is required for the cell cycle commitment point at Start via degradation of the G1-CDK inhibitor Far1p. J Cell Biol. 2003, 163: 21-26. 10.1083/jcb.200307025.PubMed CentralPubMedGoogle Scholar
- Chevalier MS, Johnson DC: Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties. J Virol. 2003, 77: 4731-4738. 10.1128/JVI.77.8.4731-4738.2003.PubMed CentralPubMedGoogle Scholar
- Rabinovich E, Kerem A, Frohlich KU, Diamant N, Bar-Nun S: AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol. 2002, 22: 626-634. 10.1128/MCB.22.2.626-634.2002.PubMed CentralPubMedGoogle Scholar
- Kitagawa K, Skowyra D, Elledge SJ, Harper JW, Hieter P: SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol Cell. 1999, 4: 21-33. 10.1016/S1097-2765(00)80184-7.PubMedGoogle Scholar
- Rodrigo-Brenni MC, Thomas S, Bouck DC, Kaplan KB: Sgt1p and Skp1p modulate the assembly and turnover of CBF3 complexes required for proper kinetochore function. Mol Biol Cell. 2004, 15: 3366-3378. 10.1091/mbc.E03-12-0887.PubMed CentralPubMedGoogle Scholar
- Lingelbach LB, Kaplan KB: The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly. Mol Cell Biol. 2004, 24: 8938-8950. 10.1128/MCB.24.20.8938-8950.2004.PubMed CentralPubMedGoogle Scholar
- Yamamoto T, Mori Y, Ishibashi T, Uchiyama Y, Sakaguchi N, Furukawa T, Hashimoto J, Kimura S, Sakaguchi K: Characterization of Rad6 from a higher plant, rice (Oryza sativa L.) and its interaction with Sgt1, a subunit of the SCF ubiquitin ligase complex. Biochem Biophys Res Commun. 2004, 314: 434-439. 10.1016/j.bbrc.2003.12.144.PubMedGoogle Scholar
- Thevelein JM, de Winde JH: Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1999, 33: 904-918. 10.1046/j.1365-2958.1999.01538.x.PubMedGoogle Scholar
- Field J, Xu HP, Michaeli T, Ballester R, Sass P, Wigler M, Colicelli J: Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae. Science. 1990, 247: 464-467.PubMedGoogle Scholar
- Shima F, Okada T, Kido M, Sen H, Tanaka Y, Tamada M, Hu CD, Yamawaki-Kataoka Y, Kariya K, Kataoka T: Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation. Mol Cell Biol. 2000, 20: 26-33.PubMed CentralPubMedGoogle Scholar
- Suzuki N, Choe HR, Nishida Y, Yamawaki-Kataoka Y, Ohnishi S, Tamaoki T, Kataoka T: Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins. Proc Natl Acad Sci U S A. 1990, 87: 8711-8715. 10.1073/pnas.87.22.8711.PubMed CentralPubMedGoogle Scholar
- Beebe SJ: The cAMP-dependent protein kinases and cAMP signal transduction. Semin Cancer Biol. 1994, 5: 285-294.PubMedGoogle Scholar
- Tasken K, Skalhegg BS, Tasken KA, Solberg R, Knutsen HK, Levy FO, Sandberg M, Orstavik S, Larsen T, Johansen AK, Vang T, Schrader HP, Reinton NT, Torgersen KM, Hansson V, Jahnsen T: Structure, function, and regulation of human cAMP-dependent protein kinases. Adv Second Messenger Phosphoprotein Res. 1997, 31: 191-204.PubMedGoogle Scholar
- Baroni MD, Monti P, Alberghina L: Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature. 1994, 371: 339-342. 10.1038/371339a0.PubMedGoogle Scholar
- Hall DD, Markwardt DD, Parviz F, Heideman W: Regulation of the Cln3-Cdc28 kinase by cAMP in Saccharomyces cerevisiae. Embo J. 1998, 17: 4370-4378. 10.1093/emboj/17.15.4370.PubMed CentralPubMedGoogle Scholar
- Tokiwa G, Tyers M, Volpe T, Futcher B: Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature. 1994, 371: 342-345. 10.1038/371342a0.PubMedGoogle Scholar
- Irniger S, Baumer M, Braus GH: Glucose and ras activity influence the ubiquitin ligases APC/C and SCF in Saccharomyces cerevisiae. Genetics. 2000, 154: 1509-1521.PubMed CentralPubMedGoogle Scholar
- Young JC, Moarefi I, Hartl FU: Hsp90: a specialized but essential protein-folding tool. J Cell Biol. 2001, 154: 267-273. 10.1083/jcb.200104079.PubMed CentralPubMedGoogle Scholar
- Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003, 19: 185-193. 10.1093/bioinformatics/19.2.185.PubMedGoogle Scholar
- Basic Local Alignment Search Tool (nucleotide-nucleotide BLAST- BLASTn) [http://www.ncbi.nlm.nih.gov/BLAST/].Google Scholar
- NCBI Map Viewer [http://www.ncbi.nlm.nih.gov/mapview/].Google Scholar
- TRANSFAC database [http://www.gene-regulation.com/pub/databases.html#transfac].Google Scholar
- Altan-Bonnet N, Phair RD, Polishchuk RS, Weigert R, Lippincott-Schwartz J: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis. Proc Natl Acad Sci U S A. 2003, 100: 13314-13319. 10.1073/pnas.2234055100.PubMed CentralPubMedGoogle Scholar
- Gant TM, Wilson KL: ARF is not required for nuclear vesicle fusion or mitotic membrane disassembly in vitro: evidence for a non-ARF GTPase in fusion. Eur J Cell Biol. 1997, 74: 10-19.PubMedGoogle Scholar
- Boman AL, Taylor TC, Melancon P, Wilson KL: A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature. 1992, 358: 512-514. 10.1038/358512a0.PubMedGoogle Scholar
- Middendorp S, Kuntziger T, Abraham Y, Holmes S, Bordes N, Paintrand M, Paoletti A, Bornens M: A role for centrin 3 in centrosome reproduction. J Cell Biol. 2000, 148: 405-416. 10.1083/jcb.148.3.405.PubMed CentralPubMedGoogle Scholar
- Middendorp S, Paoletti A, Schiebel E, Bornens M: Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene. Proc Natl Acad Sci U S A. 1997, 94: 9141-9146. 10.1073/pnas.94.17.9141.PubMed CentralPubMedGoogle Scholar
- Antoniacci LM, Kenna MA, Uetz P, Fields S, Skibbens RV: The spindle pole body assembly component mps3p/nep98p functions in sister chromatid cohesion. J Biol Chem. 2004, 279: 49542-49550. 10.1074/jbc.M404324200.PubMedGoogle Scholar
- Paoletti A, Bordes N, Haddad R, Schwartz CL, Chang F, Bornens M: Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication. Mol Biol Cell. 2003, 14: 2793-2808. 10.1091/mbc.E02-10-0661.PubMed CentralPubMedGoogle Scholar
- Errabolu R, Sanders MA, Salisbury JL: Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles. J Cell Sci. 1994, 107 ( Pt 1): 9-16.Google Scholar
- Baum P, Furlong C, Byers B: Yeast gene required for spindle pole body duplication: homology of its product with Ca2+-binding proteins. Proc Natl Acad Sci U S A. 1986, 83: 5512-5516. 10.1073/pnas.83.15.5512.PubMed CentralPubMedGoogle Scholar
- Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J: Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol. 2005, 25: 3553-3562. 10.1128/MCB.25.9.3553-3562.2005.PubMed CentralPubMedGoogle Scholar
- Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J, Helleday T: The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol. 2005, 7: 195-201. 10.1038/ncb1212.PubMedGoogle Scholar
- Zachos G, Rainey MD, Gillespie DA: Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures. Mol Cell Biol. 2005, 25: 563-574. 10.1128/MCB.25.2.563-574.2005.PubMed CentralPubMedGoogle Scholar
- Pfister KK, Benashski SE, Dillman JF, Patel-King RS, King SM: Identification and molecular characterization of the p24 dynactin light chain. Cell Motil Cytoskeleton. 1998, 41: 154-167. 10.1002/(SICI)1097-0169(1998)41:2<154::AID-CM6>3.0.CO;2-E.PubMedGoogle Scholar
- Merdes A, Ramyar K, Vechio JD, Cleveland DW: A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. Cell. 1996, 87: 447-458. 10.1016/S0092-8674(00)81365-3.PubMedGoogle Scholar
- Starr DA, Williams BC, Hays TS, Goldberg ML: ZW10 helps recruit dynactin and dynein to the kinetochore. J Cell Biol. 1998, 142: 763-774. 10.1083/jcb.142.3.763.PubMed CentralPubMedGoogle Scholar
- Vallee RB, Vaughan KT, Echeverri CJ: Targeting of cytoplasmic dynein to membranous organelles and kinetochores via dynactin. Cold Spring Harb Symp Quant Biol. 1995, 60: 803-811.PubMedGoogle Scholar
- Cao K, Nakajima R, Meyer HH, Zheng Y: The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis. Cell. 2003, 115: 355-367. 10.1016/S0092-8674(03)00815-8.PubMedGoogle Scholar
- Cheeseman IM, Desai A: Cell division: AAAtacking the mitotic spindle. Curr Biol. 2004, 14: R70-2. 10.1016/j.cub.2003.12.048.PubMedGoogle Scholar
- Uchiyama K, Jokitalo E, Lindman M, Jackman M, Kano F, Murata M, Zhang X, Kondo H: The localization and phosphorylation of p47 are important for Golgi disassembly-assembly during the cell cycle. J Cell Biol. 2003, 161: 1067-1079. 10.1083/jcb.200303048.PubMed CentralPubMedGoogle Scholar
- Ceulemans H, Bollen M: Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol Rev. 2004, 84: 1-39. 10.1152/physrev.00013.2003.PubMedGoogle Scholar
- Itoh H, Wortman MJ, Kanovsky M, Uson RR, Gordon RE, Alfano N, Johnson EM: Alterations in Pur(alpha) levels and intracellular localization in the CV-1 cell cycle. Cell Growth Differ. 1998, 9: 651-665.PubMedGoogle Scholar
- Stacey DW, Hitomi M, Kanovsky M, Gan L, Johnson EM: Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Pur alpha. Oncogene. 1999, 18: 4254-4261. 10.1038/sj.onc.1202795.PubMedGoogle Scholar
- Hirano T: SMC-mediated chromosome mechanics: a conserved scheme from bacteria to vertebrates?. Genes Dev. 1999, 13: 11-19.PubMedGoogle Scholar
- Schmiesing JA, Ball ARJ, Gregson HC, Alderton JM, Zhou S, Yokomori K: Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics. Proc Natl Acad Sci U S A. 1998, 95: 12906-12911. 10.1073/pnas.95.22.12906.PubMed CentralPubMedGoogle Scholar
- Schmiesing JA, Gregson HC, Zhou S, Yokomori K: A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation. Mol Cell Biol. 2000, 20: 6996-7006. 10.1128/MCB.20.18.6996-7006.2000.PubMed CentralPubMedGoogle Scholar
- Przewloka MR, Pardington PE, Yannone SM, Chen DJ, Cary RB: In vitro and in vivo interactions of DNA ligase IV with a subunit of the condensin complex. Mol Biol Cell. 2003, 14: 685-697. 10.1091/mbc.E01-11-0117.PubMed CentralPubMedGoogle Scholar
- Mils V, Baldin V, Goubin F, Pinta I, Papin C, Waye M, Eychene A, Ducommun B: Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase. Oncogene. 2000, 19: 1257-1265. 10.1038/sj.onc.1203419.PubMedGoogle Scholar
- Uchida S, Kuma A, Ohtsubo M, Shimura M, Hirata M, Nakagama H, Matsunaga T, Ishizaka Y, Yamashita K: Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B. J Cell Sci. 2004, 117: 3011-3020. 10.1242/jcs.01086.PubMedGoogle Scholar
- Wang H, Hu X, Ding X, Dou Z, Yang Z, Shaw AW, Teng M, Cleveland DW, Goldberg ML, Niu L, Yao X: Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling. J Biol Chem. 2004, 279: 54590-54598. 10.1074/jbc.M407588200.PubMedGoogle Scholar
- Kops GJ, Kim Y, Weaver BA, Mao Y, McLeod I, Yates JR, Tagaya M, Cleveland DW: ZW10 links mitotic checkpoint signaling to the structural kinetochore. J Cell Biol. 2005, 169: 49-60. 10.1083/jcb.200411118.PubMed CentralPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.