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thanks "Vase" for all the wangotango pix!! ya saved me some time!
am i the only one who thinks tyler posey is 100% gay?
thanks "Vase" for all the wangotango pix!! ya saved me some time!
am i the only one who thinks tyler posey is 100% gay?
"Everyone wants to be adored."
This is the top ten reasons to love Colton Haynes. He currently plays Roy Harper on the CW show "Arrow".
1. His jaw line is actually literally perfect.
2. Like, if scientists had to build a perfect human face from scratch, they would probably just recreate this face exactly.
3. His hair and eyebrows and jaw are actually scientifically without flaw.
4. At any given moment, if hes not moving, he looks like a perfect golden statue.
5. He is, anatomically speaking, without flaw.
6. His face looks like it was chiseled from actual Grecian marble.
7. His body looks like it was actually just made to be in a suit and to look perfect and beautiful.
8. He is, according to science and gods and men, actually perfect.
9. He is, basically, the most beautiful and perfect man alive.
10. And he is actually just a flawless human being.
Last edited by MJEH89; May 13th, 2013 at 10:07 PM.
"Everyone wants to be adored."
^ Be still my fluttering heart....
Perfect 10 reasons
Add me on Kik Messenger for a chat - danny.mo.89
you forgot to mention that his ass and cock taste like pure heaven.....dont even get me started on the deliciousness of his cock-nectar
lol....ok mate......loved the finale last night
Colton at CW Upfronts this morning:
With Austin Butler:
Last edited by VaseFace808; May 16th, 2013 at 02:40 PM.
god damn yes
All the agencies/PR firms/talent companies/studios are in bed with each other. It's the system. And they're also in bed with a great deal of the press, particularly all the celeb press sites (Perez, justjared, etc.). In fact, someone like Perez who used to be on the outside is now on the inside. He's just another part of the system, hence, why he doesn't out or target people anymore...and in fact, I've seen write stuff about how straight so-and-so is when years ago these would be people he would be openly questioning.
And not only can press not ask certain things (all questions are scripted before hand...the celebs have their 'answers' usually rehearsed)... they are often bought off. Yes, payouts (large sums) can be dolled out to keep a certain "narrative" going ("so-and-so is a devoted husband, a good Christian", "the man is a playboy/manwhore" (like the Harry Styles narrative), etc....
That's usually how PR folks I've heard phrase it - 'you are "selling the narrative"...not the truth'. They create and package an image.
Most clients have NO say in it whatsoever... but some do, and take a very active hand in how they're marketed.
It all depends on your particular contract you've signed (some have more autonomy or control then others)...but a lot of the young Hollywood types that are building their careers usually have NO say.
They've signed their name on the dotted line...and they own them. Especially considering what you are 'selling' (the 'heartthrob' image, the 'playboy', the devoted good boy, etc.) and to what demographic (young women, teens, CW demo...like Colton Haynes and Matt Lanter).
Say hello to your new friend...the closet.
Last edited by paintact; May 16th, 2013 at 04:09 PM.
Colton was MADE to be a model.
His body shape is made to fit perfectly tailored clothing.
I love Colton, but he seldom looks genuinely happy in the photos that are taken of him.
I agree he's hot, has fans and everything so it shouldn't be hard for him to smile. I personally think that it's the fact that he has to be in the closet for a long long time that puts his frown upside down or inside out. Compared to the pictures that he was taken during his bf / non-famous days.
There's literally tons of pictures of Colton smiling and showcasing his perfect teeth & lips. There just so happens to be more pics here that are publicity stills, screen shots, modeling pics, etc showing him shirtless or less with solemn/stern look on his face. A simple Google search of 'Colton Haynes smile' or checking out his Instagram/Twitter pics will show you that he smiles every bit as much - if not more than he did before he was gracing our TV screens every week...
And for the hell of it:
no one has mentioned that this is CH in Hugo Boss.
it's called being a lost soul.
ty, but i actually got the idea from another site.
and also, i freakin' love the suit he's wearing here...and just for fun, a pic at the Versace Runway Show w/Darren Criss...
"Everyone wants to be adored."
^oooh girlfriend....snap
Where did that batman tee pic come from, awesome. Not many men can do it for me completely clothed but Colton in a suit is incredible
Country boy makes him even hotter
Vacancy, my love would you please convince him to send Dylan my way? You know once you come up for some air...
He can't Sinestro. Vacancy (with Colton's help) already led Dylan my way.
I now have him tied up to my bed. His little cute face turns to worry when I walk in....
Add me on Kik Messenger for a chat - danny.mo.89
You and I are going to be having problems then easyman. Everyone knows that Dylan is my man!
On topic though, Colton is always hot but I don't quite believe he's perfect XD (Please don't kill me)
On set with Nylon Magazine and DKNY
lol....i miss ya mate ....i hope you me and sincereisdamon can get it hot hard and wet this season when teen wolf 3 starts....ive been so busy with a new job.....love you guys so much,,,,,hope youve been gettin hot cock on a regular basis
I was hoping that Colton being good friends with Bryan Singer means that Bryan will cast Colton in "X Men: Days of Future Past".........oh well
Because Bryan Singer is gay and I heard that he has many toy boys/ twinks working for him....casting couch?
^ Colton could easily play Angel/Archangel I think!
"Your life is yours alone. Rise up and live it."
Richard Rahl
not my fave CH pic. he looks like a cheap, trashy, slutty NYC gay crack whore who will do anything for money.
speaking of which, where IS "sinestro", anyway...???
lol...you're funny!
funny that someone should say this because i kind of feel the same way. the Zefron is good-looking, no doubt, but CH seems sexier.
i've read a lot of stories about Singer, too. and yeah, "twink" is his fave flavor. if you're under 20, thin, cute, and have a penis, you're probably his type. i thought TL was supposed to be in the latest "X-Men" movie, too. don't know exactly what happened there.
"Everyone wants to be adored."
fyi....the crack sinestro is addicted to is anatomical not chemical.....he gives a hell of a rim-job
Thanks Dear, it's nice to know one of my men knows what he's talking about I forgive Mjeh though, what with poor Taytay being away so frequently... (Love ya Mjeh)
On topic a bit though: he does look great in that white ensemble. But I think the cap is somewhat out of place, it looks wrong on his head like that...
"My best bud Hoechlin! Had a blast last night"
Guesses on what went down last night with these two?
He's a good looking guy, but he doesn't come off like the nicest guy.


text:
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My Profile
Washington, District of Columbia
Member since Mar 2011
Flag This ProfileFlag as Inappropriate
Reviewed overnight oatmeal
Jul 29, 2014 on FoodNetwork.com
"Love, love, love this. I have a 7-quart Hamilton Beach Stay-n-Go slow cooker. I made a single batch and kept all of the liquid measurements/types the same. I don't like figs at all, so I decided to go with a cup of golden raisins and a cup of Granny Smith apple. I also added cinnamon and some nutmeg. I cooked it on high for 30 minutes, then left it..."
Reviewed Strawberry Tartlets
Mar 22, 2011 in Food Network Community Toolbox on FoodNetwork.com
"Baking is my specialty and I followed this recipe to the letter, but the shells were a sticky disaster - they were glued to the baking sheet despite the cooking spray. I was so frustrated I almost threw out the pan. The strawberries were delicious, however. I wound up serving them up shortcake style with some pound cake and whipped cream I had on..."
Reviewed Whole-Wheat Penne with Turkey and Asparagus
Mar 22, 2011 in Food Network Community Toolbox on FoodNetwork.com
"This recipe was great. It works with ground turkey too, which was all I had on hand. I just sauteed some garlic and added balsamic in with the turkey, then added the olive oil and more balsamic to the pasta once it was done cooking. I also offered parmesan for everyone to sprinkle over the top. Delicious!""


text:
On Sat, 2008-10-25 at 11:40 +0200, Robin Gareus wrote:
> Ivica Ico Bukvic wrote : "We need to start thinking about the function
Seems that belongs on the consortium list and shouldn't be on LAD and
> * http://wiki.linuxaudio.org
> We would like to support maintained and moderated namespaces adopted by
I'm not sure what you are talking about here and highly suspect others
will be even more puzzled. Balancing?
Regarding namespaces (nice example of coder speak not suitable for a
general audience), I see it as a problem that linuxaudio.org web
presence is so fractured already.
> ..and we're not going to stop there.
Consider conference.linuxaudio.org or conf.linuxaudio.org. Redundant
redundancy department / cryptic abbrev. ;)
> * http://portal.linuxaudio.org, http://linuxaudio.org,
I actually think portal shouldn't exist. Should be just a list of links
reachable from linuxaudio.org . Like it is, but the link name "Portal"
doesn't cut it. Maybe "Ressources"?
> We're urgently looking for 2-5 Content Maintainers (and Guitarists) so
I suspect writing a long and partly confusing email while assuming
everyone already knows what this is all about and what you want to
accomplish is not the best way to invite contributors.
> Besides in the longer term we're looking for volunteers to summarize or
One of the URLs will be dropped? Which one? I though one is an open wiki
for content that is not necessarily tied to single applications and the
other a directory of linux audio applications?
> * http://lau.linuxaudio.org and http://quicktoots.linuxaudio.org/
Ah yes, the fragmentation. _Hidden_ resources are great for Indiana
Jones style explorers, I guess? ;)
I would rather drop old material to have a chance of introducing
something structured, where a newcomer would know where to start and had
a chance of finding stuff without using a pit lamp.
Thorsten Wilms
thorwil's design for free software:
Consortium mailing list
LINUX® is a registered trademark of Linus Torvalds in the USA and other countries.
Linuxaudio.org logo copyright Thorsten Wilms © 2006.
Hosting provided by the Virginia Tech Department of Music and DISIS.


text:
Select () or exclude () categories to narrow your recipe search.
As you select categories, the number of matching recipes will update.
Find exactly what you're looking for with the web's most powerful recipe filtering tool.
Married mother of 3. American now living in Italy. I like to make healthy yummy meals for my family. Though I enjoy italian food I want to make our favorites from America too so im finding myself looking for recipes since i cant find the dishes here that we enjoyed in the US
Seafood, pasta, chocolate!, salads, rice


text:
1. allow members to create own accounts, adding details which can be used for newsletter and also regular snal mail distributions.
2. give users a calendar they can add events to and receive automatic email reminders/notifications of. similar to this: <sorry,no example urls>
3. includes a real time stats package, nothing too fancy but good basic info about visitors and individual page hits
thanks in advance for any help / recommendations..
- spacerace -
[edited by: tedster at 8:36 pm (utc) on June 28, 2006]
I personally use Joomla!. It's open source and it's based on PHP, MySQL and Apache. It has all the features you mentioned and many more. Very easy too.


text:
Teen Volleyball Tournament on the shores of West Grand Traverse Bay. Teams are limited to six members. There are two divisions ages 12-15 and 16-19. Players under 18 will need a parent signature.
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Corporate Members |


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recipe reviews for:
"There's nothing like a zesty, smooth dip filled with shrimp for enjoying on your favorite cracker!"
My Profile | Cooks I Like Reviews | Photos | Recipes | Menus | Blog
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Search Result
Search Tags: workforce
'Fired up' feds celebrate their achievements
Federal employees celebrated their achievements Wednesday at the Feds Feed Families closing ceremony. This year, feds collectively donated nearly 9 million pounds of food.
Tags: USDA , Feds Feed Families , Doug Keeler , Shefali Kapadia
Health insurance: Pay a little now or a lot later
You may be the healthiest person in the IRS or the most organic couple with the EPA, but that doesn't let you off the hook when it comes time to hunt for a health plan, Senior Correspondent Mike Causey says.
Tags: Mike Causey , Federal Report , pay and benefits , open season , Walt Francis , FEHBP
AFGE proposes TSA reforms after LAX shooting - November 8, 2013 and November 15, 2013
This week on AFGE's "Inside Government" National President J. David Cox Sr. proposes reforms at the Transportation Security Administration after last week's shooting at the Los Angeles International Airport. Rep. Donna Edwards (Md.) discusses upcoming budget battles on Capitol Hill, AFGE Federal Protective Service Local 918 President David Wright addresses training concerns with the FPS contractor workforce and DoD Local 2024 Chief Steward Donald Hands talks about ways to engage retired union members.
Tags: AFGE , Inside Government , J. David Cox Sr. , David Wright , Donald Hands , Donna Edwards
Archuleta looks to 'forge a new pathway' to better engage federal workforce
Just a week into the job and confronted with signs of the sagging morale of the federal workforce, new Office of Personnel Management Director Katherine Archuleta said she wants to take steps to make sure federal employees feel engaged in their work. Tuesday's annual public meeting of the Chief Human Capital Officers Council focused on ways to improve employee engagement and morale. OPM released its annual Employee Viewpoint Survey last week, revealing continuing declines in federal employees' overall job satisfaction and a sharp drop in satisfaction with their pay.
Tags: Katherine Archuleta , CHCO Council , management , OPM , Employee Viewpoint Survey , Justin Johnson , Heidi Sheaffer , Angela Bailey , Jack Moore
In wake of LAX shooting, real talk needed about TSA
The Nov. 1 shooting at Los Angeles International Airport sheds light on the public's negative perception of transportation security officers. Former DHS CHCO Jeff Neal says its time to reevaluate how those federal employees are treated.
Tags: Jeff Neal , commentary , TSA , TSOs , DHS ,
OPM Survey: Oldest federal employees most satisfied, engaged workers
The oldest federal employees are also the most satisfied and engaged workers, according to the Office of Personnel Management's annual Employee Viewpoint Survey. According to the survey, the pre-Baby Boom generation of federal workers is more likely to believe they are recognized for their service, believe they have sufficient resources and are satisfied with training opportunities. Knowing how satisfaction and engagement shake out across age barriers can be helpful as managers attempt to build back up the battered morale of the federal workforce, OPM said.
Tags: OPM , Employee Viewpoint Survey , Jack Moore
Pay freeze, sequestration take bite out of employee satisfaction, OPM survey reveals
Frustrations over federal pay, budget cuts and uncertain agency funding have weakened federal-employee satisfaction, according to the Office of Personnel Management's Federal Employee Viewpoint survey released Friday. For the second year in a row, overall employee satisfaction scores fell, dipping below 60 percent this year. Meanwhile, less than half of federal employees said they believe they have sufficient resources such as material, staff and funding to do their jobs effectively.
Tags: OPM , pay and benefits , employee satisfaction , morale , Katherine Archuleta , Colleen Kelley , NTEU , Employee Viewpoint Survey , Jack Moore
Army budget, tech, workforce issues take center stage
Federal News Radio Defense Reporter Jared Serbu was on-site at the 2013 AUSA Conference. While there, Jared had the chance to speak with Army officials on some of the biggest topics and issues facing the service.
Tags: Army , DoD , AUSA confrence , galleries , cybersecurity , technology ,
2013 Federal Technology Survey
Federal News Radio wants to know how the technology being used by federal agencies today is affecting federal employees, agencies and government contractors. Take our brief, anonymous survey. is conducting a survey on the use of technology at government agencies. as part of our research and measurement of the impact of technology on federal employees, agencies and government contractors. We will present our findings starting Nov. 19.
Tags: survey , technology ,
Young leaders find new avenues to help advance their careers
Young Government Leaders helps young professionals find classes and mentors that can help them to learn new skills and advance in their careers.
Tags: Miguel Joey Aviles , Young Government Leaders , leadership , mentoring , Senior Executives Association , Senior Executive Service , managing , Michael OConnell


text:
Hanson Ferry is a populated place located in Asotin County at latitude 46.027 and longitude -117.336.
The elevation is 1,601 feet. Hanson Ferry appears on the Mountain View U.S. Geological Survey Map. Asotin County is in the Pacific Time Zone (UTC -8 hours).
Asotin County: Cities & Towns | ZIP Codes | Schools | Features (airports, churches, hospitals, etc.)
BIG Map |
Driving Directions |
Data Source - USGS GNIS FID: 1514673
BOUNDARY MAPS: Asotin Division | Asotin County
Mountain View, WA
(1.4 miles N) Bartlett, OR (6.6 miles WSW) Troy, OR (7.9 miles SW) |
Paradise, OR
(8.2 miles SE) Flora, OR (8.9 miles S) Anatone, WA (12.3 miles NE) |
Hanson Ferry, WA
(< 0.1 mile) Grouse, WA (4.3 miles WSW) Grouse, OR (5.1 miles WSW) |
Eden, OR
(13.5 miles SW) |
The Demographic Data for the 1,481 Washington cities, towns, counties, ZIP Codes and Census Designated Places (CDPs) profiled in this gazetteer is updated annually. Last Update: July 1, 2014.
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The School Attendance Zone Maps for 2,201 Washington public schools are updated four times per year. Last Update: October 6, 2014.


text:
2012-05-23 / Front Page
Despite Great 'Love' For The Sport, Mike & Barbara Spinks Announce Retirement As OV Tennis Coaches
After Seven Years At The Helm, Couple To Hand Over Reigns Of Up & Coming Program
Monday morning, I sat down to my computer at the Spencer Evening World, checking my email as I do multiple times a day, when I ran across an email from Spencer residents and Owen Valley tennis coaches, Michael and Barbara Spinks. This was nothing new to me as the pair graciously sends me their OV boys' and girls' tennis recaps without fail, each night via email following a match.
The full version of this story is only available to online subscribers.
© 2009-2015 Spencer Evening World, Inc.
No commercial reproduction without written consent.
Electronic reproduction of any kind forbidden without written consent.


text:
Phone: (315) 866-8562
School Type: Regular
School Level:
Primary School
School Status: Operational
School District: Herkimer Central School District
County: Herkimer
Location: Rural, Fringe
Title I School: Yes
Students: 665
Teachers: 48
Student-Teacher Ratio: 13.9 to 1
Lowest Grade in School: Pre-Kindergarten
Highest Grade in School: 06
Students by Grade
Pre-K: 28
Kindergarten: 97
Grade 1: 98
Grade 2: 98
Grade 3: 87
Grade 4: 89
Grade 5: 86
Grade 6: 82
Corrections to the school database are encouraged! School Edit Form.
Herkimer Elementary School, NY
(< 0.1 mile) Herkimer-Fulton-Hamilton-Otsego Boces, NY (0.2 miles NW) Herkimer High School, NY (2.3 miles WSW) Gregory B Jarvis Junior-Senior Hs, NY (3.1 miles SW) Harry M Fisher Elementary School, NY (3.2 miles SW) |
Remington Elementary School, NY
(3.9 miles WSW)
Little Falls High School, NY (4.4 miles ENE) Little Falls Middle School, NY (4.4 miles ENE) Ilion Junior-Senior High School, NY (4.4 miles WSW) Barringer Road Elementary School, NY (5.2 miles WSW) |
St. Francis de Sales Regional, NY
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Mohawk Valley Christian Academy, NY (4.7 miles E) |
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text:
Fri, Jul 27, 12 at 6:45
I didn't think the "tipping" question would be so confusing.
Made sense to me. Dinner/tip Terilyn was the first one to get yesterday's answer right. it takes the average person 7 minutes to do this. what is it?
This weekend's question is:
Follow-Up Postings:
cheat on their spouse |
Have weekly dates Separate beds/bedrooms |
Do NOT cheat on their spouses. |
Maybe stay married to their first spouses. |
Eat dinner together |
Speak to each other? :) Kiss goodbye when leaving the house. |
File separate income tax returns |
Hide bills or money. |
Get a divorce. |
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GOD'S WORD Translation (GW) | New International Version (NIV) |
23 Like the laughter of a fool when he carries out an evil plan, so is wisdom to a person who has understanding. | 23 A fool finds pleasure in evil conduct, but a man of understanding delights in wisdom. |
GOD'S WORD® is a copyrighted work of God's Word to the Nations. Copyright © 1995 by God's Word to the Nations. All rights reserved. Used by permission. (God's Word Translation -Online Bible) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |


text:
The Youngest Doll - Latin American woman writers (Paperback)
by Rosario Ferre, Jean Franco, Rosario Ferre
Format: | Paperback 170 pages |
Not in stock
Usually despatched in 4-7 days
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in the UK
"...a collection that displays such talent and power." (New York Times Book Review). "Rosario Ferre shines, and it is high time for English-speaking readers to bask in her light." (The Nation). "The 14 stories in The Youngest Doll, as radiant as they are disturbing, are animated by ferocious river prawns, trees that weep and a 'town with beaches of white gunpowder which thundered at dusk when the tide began to rush in.' In masterly prose, the Puerto Rican-born poet, essayist and fiction writer Rosario Ferre conveys a world in which stories hide within stories, the personal is always political, mystery is as common and sudden as tenderness and endings are violent. Ms. Ferre (who has deftly translated the stories, occasionally with a collaborator, from the original Spanish) writes with an irony that cloaks anger about the oppression and danger inherent in being either a protected upper-class woman or a marginalized working-class woman in Puerto Rico's patriarchal society." (New York Times Book Review). In two closing essays, Ferre considers how her experiences as a Latin American woman with ties to the United States has brought to her writing a dual cultural perspective. Rosario Ferre's works include Sweet Diamond Dust and The House on the Lagoon.
Book details
University of Nebraska Press
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text:
The Xpc gene markedly affects cell survival in mouse bone marrow.
ABSTRACT The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc-/- mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc-/- and strain-matched wild-type mice. Using several independent methods, Xpc-/- bone marrow was approximately 10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc-/- mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.
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ABSTRACT: Organic selenium compounds are known to prevent certain cancers although mechanisms may be complex. A widely-held view is that selenium compounds can induce apoptosis in cancer cells, or more precisely, in aberrant cells that are undergoing clonal evolution somewhere along the carcinogenesis process. There are at least 20 different selenium compounds, inorganic as well as organic, that have been used in various published studies. Extrapolation between studies should therefore be undertaken with caution. Similarly, it will be important to ascertain the physiological relevance of the selenium concentrations used in some studies. While cancer prevention by selenium is well-established, recently, organic selenium in the form of pure seleno-L-methionine (SeMet) has been used in combination with cancer chemotherapy drugs. SeMet can induce a DNA repair response in some cell types including bone marrow. Cancer cells generally lack a SeMet-inducible DNA repair response. Thus, SeMet appears to selectively regulate a DNA repair pathway and thereby potentially alter responses to cancer chemotherapy drugs. The specific pathway implicated, nucleotide excision DNA repair (NER) is required for repair of cisplatin or carboplatin DNA damage relevant to chemotherapy. Moreover, some studies have implicated NER as a factor in carcinogenesis processes. Thus, the capacity of SeMet to selectively regulate NER may prove useful in both therapeutic and preventive contexts.Molecular and Cellular Pharmacology 01/2009; 1(4):218-221. - SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: About 15 years ago, several groups including ours had used matched pairs of cell lines carrying wild type or mutant p53 genes to ascertain a role for p53 in cell survival. These were isogenic cell lines differing only by p53 status. The trend at that time was to support p53-mediated apoptosis. Accordingly, p53-wildtype cells were sensitive to DNA damage compared to p53-mutant cells which were thought to evade apoptosis. However, this finding was not universal. In particular, after UV-radiation, p53-mutant cells were more sensitive than their wild type p53 counterparts in several studies. The finding that p53 controlled a major DNA repair pathway, nucleotide excision repair (NER) which repairs UV-damage, provided a mechanism for the observations. We coined the term "the two faces of tumor suppressor p53" to illustrate that p53 can on one hand induce apoptosis leading to cell sensitivity, but p53 can also enhance the rate of DNA repair thereby protecting cells from DNA damage. This concept has gained acceptance and has been expanded to other DNA-damaging agents. New insights into how p53 is "switched" from a protective function to an apoptotic function are reviewed.Molecular and Cellular Pharmacology 01/2010; 2(3):117-119. - SourceAvailable from: iupui.edu[Show abstract] [Hide abstract]
ABSTRACT: DNA is subject to various types of damage that can impair cellular function or cause cell death. DNA damage blocks normal cellular processes such as replication and transcription and can have catastrophic consequences for the cell and for the organism. It has long been thought that the G1/S cell cycle checkpoint allows time for DNA repair by delaying S-phase entry. The p53 tumor suppressor pathway regulates the G1/S checkpoint by regulating the cyclin-dependent kinase inhibitor p21Waf1/Cip1, but p53 also regulates the nucleotide excision DNA repair protein XPC. Here, using p53-null cell lines we show that additional mechanisms stabilize XPC protein and promote NER in concert with the G1/S checkpoint. At least one mechanism to stabilize and destabilize XPC involves ubiquitin-mediated degradation of XPC, as the ubiquitin ligase inhibitor MG-132 blocked XPC degradation. The retinoblastoma protein, RB, in its unphosphorylated form actually stabilized XPC and promoted NER as measured by host-cell reactivation experiments. The data suggest that XPC protein and XPC-mediated NER is tightly linked to the G1/S checkpoint even in cells lacking functional p53. Indiana University-Purdue University Indianapolis (IUPUI)
Page 1
Mutagenesis vol. 24 no. 4 pp. 309–316, 2009
Advance Access Publication 16 April 2009
The Xpc gene markedly affects cell survival in mouse bone marrow
Joshua L. Fischer, M.A. Suresh Kumar, Travis W. Day1,
Tabitha M. Hardy, Shari Hamilton2, Cynthia Besch-
Williford2, Ahmad R. Safa1, Karen E. Pollok3and Martin
L. Smith*
Department of Microbiology and Walther Oncology Center, Indiana University
Simon Cancer Center and Walther Cancer Institute,1Department of
Pharmacology, Indiana University Simon Cancer Center, Indiana University
School of Medicine, Indianapolis, IN 46202, USA,2Department of Veterinary
Pathology and Research Animal Diagnostic Laboratory, University of
Missouri-Columbia, Columbia, MO 65211, USA and3Herman B. Wells Center
for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN
The XPC protein (encoded by the xeroderma pigmentosum
Xpc gene) is a key DNA damage recognition factor that is
required for global genomic nucleotide excision repair (G-
NER). In contrast to transcription-coupled nucleotide
excision repair (TC-NER), XPC and G-NER have been
reported to contribute only modestly to cell survival after
DNA damage. Previous studies were conducted using
fibroblasts of human or mouse origin. Since the advent of
Xpc2/2 mice, no study has focused on the bone marrow of
these mice. We used carboplatin to induce DNA damage in
Xpc2/2 and strain-matched wild-type mice. Using several
independent methods, Xpc2/2 bone marrow was ?10-fold
more sensitive to carboplatin than the wild type. Impor-
tantly, 12/20 Xpc2/2 mice died while 0/20 wild-type mice
died. We conclude that G-NER, and XPC specifically, can
contribute substantially to cell survival. The data are
important in the context of cancer chemotherapy, where
Xpc gene status and G-NER may be determinants of
response to DNA-damaging agents including carboplatin.
Additionally, altered cell cycles and altered DNA damage
signalling may contribute to the cell survival end point.
The xeroderma pigmentosum Xpc gene is defective in a subset
of human patients exhibiting the cancer-prone disease xero-
derma pigmentosum which results from defective nucleotide
excision DNA repair (NER). XPC patients are sensitive to
sunlight and ultraviolet (UV) radiation-induced DNA damage
and skin cancers. They also exhibit internal cancers with
advanced age. The gene products encoded by Xpc and other
XP genes A-G have been characterized biochemically (1).
Specifically, the XPC protein (encoded by the xeroderma
pigmentosum Xpc gene) is required and is rate limiting for
global genomic nucleotide excision repair (G-NER). XPC is
neither required nor apparently involved in transcription-
coupled nucleotide excision repair (TC-NER). Thus, studies
of Xpc are good models for G-NER separate from TC-NER (1).
Mice lacking Xpc genes (Xpc?/? mice) were generated
some 10 years ago (2). A number of studies of carcinogenesis
and mutagenesis have been conducted, consistent with the
cancer-prone human genetic disease discussed above (2–4).
This is the first study to examine bone marrow in Xpc?/? mice.
Bone marrow is often dose limiting in response to cancer
chemotherapy drugs including carboplatin. While cell survival
is a complex end point, the goal of chemotherapy is to sensitize
cancercellswhile retainingbonemarrowcellularity.Agentsthat
can protect bone marrow are important adjuncts to chemother-
apy. Presently, the cytokine granulocyte macrophage colony
stimulating factor is administered which causes proliferation of
bone marrow myeloid stem/progenitor cells and thus repopu-
lates bone marrow after chemotherapy (5). The idea of G-NER
as a protective mechanism in bone marrow is novel. Xpc?/?
mouse bone marrow was highly sensitive to carboplatin.
Patients carrying mutant XPC genes may exhibit an adverse
bone marrow response to carboplatin.
Materials and methods
Mice originated from Sands et al. (2) and were purchased from Taconic Farms
and bred at Indiana University under licence agreement as B6;129s7-XPCtml/Brd
mice. Female mice were 10 weeks old at the time of initiating the experiments.
Bone marrow was directly harvested and cultured for 24–72 h for indicated in
vitro experiments (Figures 1–3). For in vivo experiments (Figures 4 and 5),
carboplatin (Sigma, St Louis, MI, USA) was dissolved in sterile saline and
administered intra-peritoneally at weekly intervals over 6 weeks as indicated in
the figure (0.5-ml injections, 60 or 100 mg/kg body weight). White blood cell
(WBC) counts were by weekly tail vein blood collection in ethylenediaminete-
traacetic acid-treated haematocrit tubes (6). Counting was done using a Hemavet
950 (Drew Scientific, Dallas, TX). Statistical analysis was conducted using a t-
test using Microsoft Office 2003 or GraphPad Prism software. All mouse
treatments were in accord with protocols approved by the Institutional Animal
Care and Use Committee at Indiana University. Mice were sacrificed after 41
days using approved methods. Mice that appeared moribund before 41 days
were sacrificed by veterinary staff.
DNA repair assay
To confirm the DNA repair defect in Xpc?/? bone marrow, we used an
immunoassay in which genomic DNA from UV-irradiated bone marrow cells,
at 0, 4, 8 or 16 h after UV irradiation, was fixed to 96-well microtiter plates at
10 ng per well. Cells were washed and re-suspended in 2-ml phosphate-
buffered saline and irradiated as a thin monolayer in 60-cm2dishes, then placed
back in culture medium. An antibody to 6-4 photoproducts (Trevigen Inc.,
Gaithersburg, MD) was used to detect removal of lesions. Immunoassays were
developed with peroxidase secondary antibody and 2,2#-azino-bis 3-ethyl-
benzthiazoline-6-sulphonic acid chromogenic substrate and read at 405 nm in
a Tecan Spectra plate reader. Immunoblotting of Xpc was with a rabbit
polyclonal antibody (Santa Cruz Biotech, Santa Curz, CA).
*To whom correspondence should be addressed. Indiana University Simon Cancer Center, 1044 West Walnut Street, Room 155, Indiana University School of
Medicine, Indianapolis, IN 46202, USA. Tel: þ1-317-278-0225; Fax: þ1-317-278-3331; Email: email@example.com
? The Author 2009. Published by Oxford University Press. All rights reserved.
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Page 2
Cell cycle analysis
Bone marrow of wild-type and Xpc?/? genotypes were cultured in Iscove's
modified Dulbecco medium (IMDM)-containing cytokines as above for 48 h to
stimulate proliferation and then treated with carboplatin for an additional 15 h.
Untreated cultures served as controls for each genotype. Cells were fixed in 70%
(PI) content. To analyse only proliferating cells, 10-lM bromodeoxyuridine
(BrdU) was added to the cultures during the 48-h growth period. Cycling cells
were first gated for BrdU using a fluorescein isothiocyanate-conjugated mouse
monoclonal antibody to BrdU (Becton-Dickinson, Franklin Lakes, NJ, USA)
and then with PI. The data represent ?15 000 cells per data point.
For lineage marker analysis, antibodies to CD4, CD8, B220, Gr-1, lin, c-kit
and Sca-1 were used in accord with manufacturer protocols (Becton-
Dickinson). Samples were analysed on a Becton-Dickinson FACScalibur
using CellQuest software, scoring at least 10 000 events per sample.
Immunoblotting of cell cycle proteins
Initially whole-cell lysates were used to probe for differences in cell cycle
proteins between wild-type and Xpc?/? bone marrow. Given apparent
differences in Cdt1 and Cul4a, we purified total ubiquitinated proteins on
a ubiquitin-binding resin (Pierce Chemical, Rockford, IL). Cell lysates
corresponding to 107viable cells per sample were prepared in RIPA lysis
buffer to which 50 ll of resin was added and rotated overnight at 4?C. The resin
was collected by centrifugation and then boiled 15 min in sodium dodecyl
sulphate/gel-loading buffer and subject to electrophoresis in 4–20% poly-
acrylamide gels (Invitrogen, Carlsbad, CA). Immunodetection was on
nitrocellulose membranes using rabbit anti-Cul4a (34897; Abcam, Cambridge,
MA) or rabbit anti-Cdt1 (sc-28262; Santa Cruz Biotech).
Human H1299 lung cancer cell line was transfected using a FuGene
(Boehringer-Mannheim, Indianapolis, IN) and a short hairpin RNA (shRNA)
served as a control (Origene). Cells were analysed by immunoblotting after 72 h.
Histochemical staining
Femurs were fixed in 10% buffered formalin overnight, then paraffin embedded
and thin sectioned for histological examination by a veterinary pathologist.
Slides were stained with haematoxylin and eosin for morphological analysis or
4#,6-diamidino-2-phenylindole to visualize intact nuclear DNA. Slides were
visualized by a Nikon HB-10101AF fluorescence microscope using a ?100
objective. Digital photographs were captured.
Colony-forming assays
Bone marrow was collected from femurs, fibulas and iliac crests. Total bone
marrow cells were then added to complete methylcellulose medium consisting
of IMDM liquid medium (Sigma), 20% foetal bovine serum, interleukin-6 (200
U/ml) and stem-cell factor 100 ng/ml (Stem Cell Technologies, Vancouver,
Canada). Triplicate 35-mm culture dishes were seeded with 105cells and
placed in a humidified incubator at 37?C for 10 days at which time colonies
were counted manually as in ref. (7).
MTS assay of cell viability
Bone marrow of wild-type and Xpc?/? genotypes was cultured in 96-well
plates in IMDM medium with cytokines as above and then treated in vitro with
increasing concentrations of carboplatin for 2 h. Cell yield was determined after
72 h in culture by adding MTS (Promega, Madison, WI) to the wells and
reading the absorbance at 490 nm in a Tecan Spectra plate reader. Cells that did
not receive carboplatin served as controls for each genotype representing 100%
cell yield.
The data show a pronounced effect on mouse bone marrow
owing to the presence or absence of a functional XPC gene
product. The first observation was that the number of viable
cells recovered from untreated Xpc?/? bone marrow was
always 2- to 3-fold lower than wild type. To remedy this
problem, we normalized the number of viable cells for both
genotypes for the cell culture experiments. Cell numbers were
normalized typically to 107cells representing pooled cells of
three mice of each genotype. The normalized cell populations
then were used as a source of cells for tissue culture
experiments. Flow cytometric analysis was conducted both
immediately after harvest and after tissue culture experiments,
to ascertain if any one cell lineage was affected in the Xpc?/?
mice. As shown in Figure 1A, no single lineage was uniquely
affected in Xpc?/? mice. Populations representing lineage
markers Gr-1þ, B220þ, Lin?, CD4þ, CD8þ and the triply
gated Lin?/Sca1þ/c-kitþ population were not significantly
different between wild-type and Xpc?/? mice when compar-
ing equal numbers of cells of both genotypes (Figure 1A). The
relative distribution of the lineage markers did not change over
the course of cell culture experiments (results not shown),
although the cell culture experiments were typically conducted
within 48 h and were therefore short-term cultures.
Mice of wild type and Xpc?/? genotypes were assayed for
XPC expression and G-NER activity. Bone marrow was used
directly for western blotting using an XPC antibody. Bone
Fig. 1. G-NER defect in cultured Xpc?/? bone marrow. (A) For cell culture
experiments (Figures 1–3) bone marrow was harvested from untreated wild-
type or Xpc?/? mice. Cell yields were lower in Xpc?/? compared to wild
type; therefore, cell numbers were normalized to 107viable cells per genotype.
Flow cytometry was conducted using six different lineage markers. No
significant differences were observed in the lineage markers in comparing 107
cells of each genotype. As shown in the pie graph, lineage markers tested were
Gr1þ, B220þ, Lin?, CD4þ, CD8þ and the small subpopulation Lin?/
Sca1þ/c-kitþ. The pie segment labelled 'neg' was unreactive for any of the
lineage markers tested. (B) XPC protein was undetectable in Xpc?/? bone
marrow (inset). Removal of DNA lesions was markedly slow in Xpc?/? bone
marrow, consistent with the known rate-limiting role of XPC in G-NER.
Pooled bone marrow of three or more untreated 10-week-old mice of
each genotype was used. Cells were cultured in cytokine-containing
medium for 15 h, irradiated with 20 J/m2254 nm UV radiation in phosphate-
buffered saline and returned to tissue culture. Time points were taken at 0, 4, 8
and 16 h and assayed using an antibody to 6-4 photoproducts (P , 0.02
by t-test).
J. L. Fischer et al.
Page 3
marrow was additionally cultured 24 h, UV irradiated and then
subjected to an immunoassay for removal of DNA damage.
Removal of DNA lesions was 80% complete in wild-type mice
after 16 h, while Xpc?/? mice were defective in removal of
the lesions (Figure 1B). Thus, the Xpc?/? mice exhibit the
expected defective NER phenotype.
We reasoned that the Xpc?/? DNA repair defect might
affect the cell cycle as a secondary consequence of lack of
lesion removal. A comparison was made between wild type
and Xpc?/? for cell cycle distribution in the presence or
absence of carboplatin. The majority (80%) of bone marrow
cells when freshly harvested were in the G1 phase of the cell
cycle irrespective of genotype (Figure 2A). By PI staining
alone, no cell cycle defect of Xpc?/? bone marrow or
difference from wild type was observed in any cell cycle phase
(Figure 2A). To further analyse the cell cycle, we grew the cells
for 48 h in the presence of BrdU, thereby labelling proliferating
cells. Cells were then treated with 10-lM carboplatin for 15 h,
fixed and stained for PI. Cells were gated for BrdU positivity
using a fluorescein-labelled antibody and then for PI staining.
A modest but significant decrease in the G1 phase was
observed in Xpc?/? compared to wild type (Figure 2B). A G2
arrest by carboplatin was observed in both genotypes, which
was more pronounced in Xpc?/? than in wild type (Figure 2B).
The flow cytometric profiles corresponding to the bar graph in
Figure 2B are shown in Figure 2C. The intent of the cell cycle
experiments was to reveal cell cycle differences between wild-
type and Xpc?/? bone marrow, while minimally causing
apoptosis or other cell death. However, use of a higher
concentration of carboplatin revealed a G2 population of 8% in
the wild type and 26% in the Xpc?/?. Moreover, the sub-G1
apoptotic populations were 21% for the wild type and 38% for
Xpc?/?. Thus, G2 arrest may precede apoptosis, both of
which were more pronounced in the Xpc?/? mutant bone
marrow compared to wild type (Figure 2C).
We examined several key cell cycle regulatory proteins for
differences between Xpc?/? and wild type, including Rb,
cyclin D1 and cyclin E, which were unremarkable (results not
shown). Our analyses by western blotting would reveal not
only protein steady-state levels but also phosphorylation and/or
Fig. 2. Evidence of G1 and G2 cell cycle checkpoint alterations in cultured Xpc?/? bone marrow. (A) Bone marrow harvested from untreated 10-week-old mice
was cultured in cytokine-containing medium for 15 h and then treated with 10-lM carboplatin for an additional 24 h. Cells were fixed in 70% ethanol and analysed
by PI staining. By PI staining alone, cell cycle differences were not significant and 68–80% of cells were in G1. (B) Bone marrow harvested from untreated 10-
week-old mice was stimulated with cytokines and cultured for 48 h in the presence of 10-lM BrdU to label proliferating cells, then treated with 10-lM carboplatin
for 15 h and then fixed and stained with PI. A fluorescein isothiocyanate-conjugated antibody to BrdU was used to gate the BrdU-labelled population, which were
then assayed for PI content. Values for carboplatin-treated bone marrow were divided by values for untreated bone marrow conducted side by side. The data
represent three pooled mice of each genotype. Two separate experiments yielded similar results. A modest but significant decrease in G1 population was observed in
Xpc?/? mice compared to wild-type mice (P , 0.05 by t-test). A significant increase was observed in the G2 population in Xpc?/? mice compared to wild-type
mice (P , 0.02 by t-test). The plot shows relative cell cycle distribution after carboplatin treatment. (C) Raw flow cytometric data corresponding to the bar graph
shown in panel (B). Cell number is plotted on the y-axis, at least 15 000 events per sample; PI staining is shown on the x-axis. Use of a higher concentration of
carboplatin (40 lM) revealed a sub-G1 apoptotic population which was more pronounced in the Xpc?/? mutant bone marrow. The G2 population was also more
pronounced in the mutant at the higher carboplatin concentration.
Xpc and cell survival
Page 4
Fig. 3. (A) CUL4A and CDT1 cell cycle checkpoint proteins in cultured wild-type and Xpc?/? bone marrow. We wanted to determine if the presence or absence of
XPC would alter DNA damage signalling. Immunoblots were conducted using 50 lg of total cell lysates (lanes 1 and 2, upper and lower panels). Equal amounts of
cellular proteins, 5 mg, were then affinity purified on a ubiquitin-binding resin and immunoblotting of bound proteins was conducted. The higher molecular weight
ubiquitinated forms of CUL4A (upper panel) and CDT1 (lower panel) clearly differ between the two genotypes (lanes 3 and 4 of upper and lower panels). The data
suggest a defect in CUL4A and CDT1 ubiquitin modification in Xpc?/? mice. To help identify ubiquitinated CUL4A and CDT1, we used in vitro ubiquitin-
conjugated proteins. Omission of ubiquitin from the reaction shows that mainly ubiquitinated CUL4A is detected by the CUL4A antibody (lanes 5 and 6, upper
panel). Plasmid-encoded CDT1 protein was also used as a marker (lanes 5 and 6, lower panel). (B) Knocking down XPC in H1299 cells alters the ubiquitin
modification of CDT1. We used transiently transfected H1299 cells expressing an shRNA to XPC to test if the results shown in panel (A) were due directly to loss of
XPC. Although Xpc was not completely silenced, it was clearly decreased by the shRNA. Higher molecular weight ubiquitinated forms of CDT1 were largely
absent where XPC was knocked down, consistent with a role for XPC in DNA damage signalling to the CDT1 cell cycle checkpoint protein.
Fig. 4. Effect of carboplatin administration in vivo in bone marrow of wild-type and Xpc?/? mice. (A) Kaplan–Meier plots of mouse survival. Twenty mice of each
genotype received carboplatin or saline only as controls. Saline-only did not affect mouse survival nor alter WBC counts; saline-only control groups are not shown
in order to simplify the figure. Mice received carboplatin at weekly intervals up until day 27. By day 41, 12/20 Xpc?/? mice died, while 0/10 wild-type mice died.
(B) WBC counts were monitored during the course of the experiments. WBC counts were not significantly different prior to day 27, but were significantly different
after day 27 (P , 0.05, lower panel). Each circle represents an individual mouse, black, wild type; red, Xpc?/?. The horizontal bars represent the mean of each
group. By day 41, the few surviving Xpc?/? mice had significantly lower WBC counts (P , 0.05 by t-test). The carboplatin dosing schedule is shown in black,
wild type; red, Xpc?/?. Note that wild-type mice received an additional dose on day 27 that was not administered to Xpc?/? mice. (C) Histological evaluation of
wild-type and xpc?/? mice in carboplatin-treated and saline-control groups, day 41. Haematoxylin–eosin staining of formalin-fixed femur sections. Marked
hypocellularity was observed in Xpc?/? mice receiving carboplatin (upper panel); 4#,6-diamidino-2-phenylindole staining (lower panel). The data are
representative of at least three mice of each treatment group and genotype.
J. L. Fischer et al.
Page 5
other modifications that might be affected as a secondary
consequence of lack of lesion removal in the Xpc?/? mutant.
Interesting results were obtained for CUL4A and CDT1 cell
cycle checkpoint proteins, which are involved in DNA damage
signalling. The rationale for studying CUL4A and CDT1 was
that XPC is a key substrate for CUL4A after DNA damage;
thus, in the absence of XPC, other downstream targets
including CDT1 might be either exaggerated or attenuated.
Higher molecular weight forms of CUL4A and CDT1 were
largely absent in Xpc?/? bone marrow suggesting that Xpc
may play a role in the DNA damage signalling mechanism
(Figure 3A). The differences in higher molecular weight
CUL4a and CDT1 were observed in untreated, freshly
harvested bone marrow. Both CUL4A and its downstream
substrate CDT1 are ubiquitinated as evidenced by binding to
a ubiquitin-binding resin (Figure 3A), although we do not
exclude other possible post-translational modifications that
may occur as well. The differences are not merely reflective of
cell cycle differences, as cell cycle differences in untreated
cells are not significant (Figure 2A). The differences are not
merely reflective of bone marrow cell subpopulations, which
could theoretically differ between wild type and Xpc?/?. No
significant differences were observed between wild type and
Xpc?/? for any of the six lineage markers shown in Figure 1A.
Fig. 4. (Continued)
Xpc and cell survival
Page 6
Thus, the apparent defective ubiquitination of CUL4A and its
downstream substrate CDT1 appears to be a characteristic of
Xpc?/? bone marrow. We used transiently transfected H1299
cells to test if knocking down Xpc would mirror the mouse
bone marrow findings. H1299 cells in which Xpc was knocked
down by an shRNA plasmid showed a decrease in ubiquiti-
nated CDT1, similar to that observed in the bone marrow
(Figure 3B). XPC may therefore affect DNA damage signalling
to the cell cycle checkpoint protein CDT1, although further
studies are needed.
Having conducted cell culture experiments to characterize
Xpc?/? bone marrow (Figures 1–3), we conducted experi-
ments to address carboplatin sensitivity in vivo. Twenty mice of
each genotype were divided into four treatment groups and
administered carboplatin at weekly intervals. Mice were 10
weeks old at the time of initiating the experiment. By day 41 of
carboplatin regimen, 12/20 Xpc?/? mice had died unexpect-
edly. No deaths were observed in wild-type mice receiving
carboplatin (Figure 4A). No deaths were observed in saline-
only control groups of either genotype (results not shown).
During the course of carboplatin treatments, peripheral WBC
counts were measured weekly (6). Peripheral WBC counts in
Xpc?/? mice were significantly lower than in wild-type mice
(Figure 4B). Remaining mice were sacrificed on or after day 41
using approved protocols. Mice were evaluated by a veterinary
pathologist. The most significant pathology was marked
hypocellularity in bone marrow in 10/10 Xpc?/? mice
examined by the pathologist (Figure 4C). Brain lesions were
observed in ?50% of the mice which, however, occurred with
equal frequency in wild type and Xpc?/? (results not shown).
No significant lesions were found in cecum, colon, duodenum,
heart, ileum, jejunum, kidney, liver, lymph nodes, spleen,
stomach or thymus. Perivascular lymphoid infiltrates were
observed in lung in both genotypes (results not shown).
To quantify and further characterize the hypocellularity in
Xpc?/? bone marrow, we conducted colony-forming assays
and cell viability assays using a vital dye. A hallmark of
myeloid stem/progenitor cell populations is their colony-
forming ability when placed in culture and stimulated with
cytokines. Colony-forming assays of bone marrow were
conducted as in ref. (7). The data were plotted as total colonies
per femur. In the absence of carboplatin, Xpc?/? mice
exhibited a 3-fold decrease in colony-forming units compared
to wild type (Figure 5A). With carboplatin, the difference was
10- to 12-fold (Figure 5A). We also used the vital stain
thiazolyl blue to measure cell viability after 4 days in culture.
Using this method, carboplatin concentrations corresponding to
50% cell yield (known as IC50dose) was approximately 10-
fold lower in Xpc?/? compared to wild type (Figure 5B). It is
likely that a higher fraction of apoptosis contributed to
decreased cell yield in Xpc?/? bone marrow, evidenced by
the sub-G1 (apoptotic) fractions shown in Figure 2C. We also
conducted experiments utilizing annexin V, an indicator of
apoptosis that is assayed by flow cytometry. The Xpc?/? bone
marrow showed a higher apoptotic fraction by annexin V
staining (results not shown), although neither of these findings
of apoptosis can exclude other mechanisms such as irreversible
cell cycle arrests that may also contribute to overall cell yield.
Untreated Xpc?/? mice always yielded 2- to 3-fold fewer
cells per femur compared to wild type. We used equal numbers
of viable cells for cell culture experiments shown in Figures
1–3, while Figures 4 and 5 reflect the difference in overall bone
marrow yield between wild type and Xpc?/?. All six lineage
markers were decreased in proportion to total cell yield in
Xpc?/? as compared to wild type, i.e. no single lineage was
uniquely affected. Table I shows raw cell numbers of distinct
lineages, per femur, recovered from bone marrow of wild type
and Xpc?/? genotypes after 41 days receiving either saline
only or carboplatin. The Gr-1þ, B220þ, Lin?, CD4þ and
CD8þ lineages differed significantly between wild type and
Xpc?/?, as indicated by P values shown in Table I.
It has long been thought that, in contrast to TC-NER, G-NER
mediated by XPC contributes only modestly to cell survival
after DNA damage (8). Essentially all studies were conducted
in human or mouse fibroblasts which showed 2- to 3-fold
differences in cell survival after DNA damage (8–12). The
present study is the first to examine bone marrow in Xpc?/?
mice. Xpc?/? bone marrow exhibited 10-fold greater
sensitivity to carboplatin compared to strain-matched wild-
type bone marrow (Figures 4 and 5 and Table I). The data
Fig. 5. Quantification of bone marrow hypocellularity in Xpc?/? mice
compared to wild type. (A) Colony-forming assays of bone marrow harvested
from the respective genotypes and carboplatin treatment groups shown in
Figure 4. Bone marrow was harvested and grown in complete methylcellulose
medium containing interleukin-6 and stem cell factor for 10 days. Total
colonies per femur are shown. Xpc?/? bone marrow in the carboplatin-treated
groupwas decreased10- to 12-fold comparedto wild type(P, 0.008by t-test).
Xpc?/? bone marrow in the untreated group was decreased 3-fold compared to
wild type (P , 0.02 by t-test). Each set of experiments utilized bone marrow
from three or more mice of each genotype. (B) Assay of cell yield in bone
marrow cells treated with carboplatin in vitro. Bone marrow of untreated
10-week-old wild-type and Xpc?/? mice was cultured for 24 h and then treated
with indicated concentrations of carboplatin for 2 h. Cell yield after 72 h in
culture is shown (P , 0.006 by t-test). The data shown were averaged from
three experiments. The dose modification factor for 50% cell survival in the
presence of carboplatin is ?10-fold, consistent with the other data.
J. L. Fischer et al.
Page 7
indicate that the Xpc gene can contribute substantially (10-fold)
to cell survival in bone marrow. The cell survival end point was
biologically relevant as bone marrow myelosuppression was
dose limiting for carboplatin, consistent with other studies (13)
and the majority of Xpc?/? mice died during the course of the
experiments (Figures 4 and 5). No deaths were observed in
wild-type mice irrespective of the carboplatin regimen and/or
duration (Figure 4 and results not shown).
Carboplatin and cisplatin produce a similar DNA damage
spectrum, mainly 1,2 and 1,3 platinum diadducts that are
substrates for NER in vitro (14,15). However, our data do not
exclude that minor lesions such as platinum monoadducts,
interstrand cross-links and/or base damage by reactive oxygen
species may also contribute to the cell survival end point.
Recent studies point to a role for XPC in repair of oxidative
DNA damage (12). A catalytic role for XPC in base-excision
DNA repair (BER) was suggested by the finding that XPC
enhanced the activity of the DNA glycosylase 8-oxoguanine
DNA glycosylate (OGG1) (12). Colony-forming ability of
Xpc?/? bone marrow was decreased 3-fold compared to wild
type even in the absence of carboplatin (Figure 5A). As endo-
genous base damage is removed by the separate BER pathway,
the decreased cell yield in Xpc?/? mice may be due to the XPC
role in BER (12). Interestingly, Xpc?/? fibroblasts exhibited
sensitivity to a 20% oxygen environment compared to wild-type
fibroblasts consistent with a role in base damage repair (11).
Cell survival though is a complex end point that involves
cell cycle effects. We found that Xpc?/? bone marrow cells
were defective in a G1 checkpoints and showed increased
accumulation in G2 compared to wild type (Figure 2). One
possibility is that Xpc?/? bone marrow cells exit the cell cycle
after a prolonged G2 or may be irreversibly arrested in G2
(Figure 2). We show that CUL4A and CDT1 cell cycle proteins
exhibit defective ubiquitination even in untreated Xpc?/?
mice. The data suggest an alteration in DNA damage signalling
in Xpc?/? bone marrow (Figure 3), although further studies
are warranted. Given that these data were obtained in the
absence of carboplatin, it is possible that XPC signalling of
oxidative base damage involves the CUL4A/CDT1 mechanism
(16–18). Thus, XPC may contribute to DNA damage
sensitivity including base damage by (i) controlling the BER
protein OGG (12), (ii) altering cell cycles and (iii) affecting
DNA damage signalling to downstream effectors including
CUL4A and CDT1. Our data pertain to bone marrow; thus, it is
not known if absence of functional XPC would exhibit these
same features in other cell types.
Human patients with xeroderma pigmentosum XPC mutant
alleles typically develop skin cancers well prior to age 20 and
develop internal cancers with ageing. Similarly, Xpc?/? mice
mice as young as possible for the current study. Perhaps human
XPC patients would exhibit myelosuppression were they to live
toadvanced age.Ontheotherhand,themicearecompletelynull
for Xpc genes, while the human mutant XPC alleles probably
exhibit variable penetrance. One study did show myelosuppres-
sion in an XPC patient (19). Thus, cancer therapeutic regimens
may require modification in the case of XPC patients.
In summary, the Xpc gene product protects mouse bone
marrow from exogenous and probably endogenous DNA
damage with cell survival as an end point. The biological
relevance is indicated by the fact that 12/20 Xpc?/? mice
receiving carboplatin died while 0/20 wild-type mice died. No
deaths were observed in saline-only controls of either
genotype. The proximal cause of death appeared to be bone
marrow failure, although other tissues may have contributed.
The XPC protein may be linked to the CUL4A/CDT1 cell
cycle checkpoint, probably relevant to DNA damage signalling
and may contribute to the cell death mechanism.
National Institutes of Health (R01 HL086978) to M.L.S.;
American Institute for Cancer Research (04B010) to M.L.S.;
US Department of Defense (BC051172) to J.L.F. T.M.H. was
supported by 1R25 GM079657 training grant to Dr. Hal E.
We thank Mark R. Kelley, Laura Haneline and D. Wade Clapp for comments
on the figures. We thank four anonymous reviewers for their helpful comments.
Conflict of interest statement: None declared.
1. Friedberg, E. C., Aguilera, A. M., Gellert, M. et al. (2006) DNA repair, from
molecular mechanism to human disease. DNA Repair (Amst.), 5, 986–996.
2. Sands, A. T., Abuin, A., Sanchez, A., Conti, C. J. and Bradley, A. (1995)
High susceptibility to ultraviolet-induced carcinogenesis in mice lacking
XPC. Nature, 377, 162–165.
3. Berg, R. J., Ruven, H. J., Sands, A. T., deGruiji, F. R. and
Mullenders, L. H. (1998) Defective global genome repair in XPC mice is
associated with skin cancer susceptibility but not with sensitivity to UVB
induced erythema and edema. J. Invest. Dermatol., 110, 405–409.
4. Hollander, M. C., Philburn, R. T., Patterson, A. D., Velasco-Miguel, S.,
Friedberg, E. C., Linnoila, R. I. and Fornace, A. J., Jr (2006) Deletion of
XPC leads to lung tumors in mice and is associated with early events in
human lung carcinogenesis. Proc. Natl Acad. Sci USA, 102, 13200–13205.
5. Wolf, T. and Densmore, J. J. (2004) Pegfilgrastim use during chemother-
apy: current and future applications. Curr. Hematol. Rep., 3, 419–423.
Table I. Lineage marker distribution after six weeks carboplatin administration
Genotype WT Xpc?/?
Treatment SalineCarboplatinSaline Carboplatin
aIndicates statistical significance for the comparison of wild type and Xpc?/? for carboplatin treatment groups; values represent the mean and standard deviation of
three mice per group.
P , 0.005a
P , 0.005a
P , 0.008a
P , 0.05a
P , 0.05a
Not significant
5.7 (?0.3) ? 106
1.5 (?0.2) ? 106
3.0 (?0.5) ? 105
2.1 (?0.4) ? 105
3.3 (?0.5) ? 105
2.0 (?0.4) ? 104
1.6 (?0.1) ? 106
6.0 (?1.5) ? 105
0.8 (?0.2) ? 105
1.6 (?0.2) ? 105
1.3 (?0.1) ? 105
2.1 (?0.5) ? 104
3.5 (?0.5) ? 106
7.4 (?0.3) ? 105
1.8 (?0.3) ? 105
1.4 (?0.2) ? 105
1.9 (?0.2) ? 105
3.0 (?0.5) ? 104
0.3 (?0.1) ? 106
1.5 (?0.1) ? 105
0.3 (?0.1) ? 105
1.2 (?0.7) ? 105
1.0 (?0.1) ? 105
0.3 (?0.1) ? 104
Xpc and cell survival
Page 8
6. Cai, S., Ernstberger, A., Wang, H. et al. (2008) In vivo selection of
hematopoietic stem cells transduced at a low multiplicity-of-infection with
a foamy viral MGMT(P140K) vector. Exp. Hematol., 36, 283–292.
7. Haneline, L. S., Broxmeyer, H. E., Cooper, S., Hangoc, G., Carreau, M.,
Buchwald, M. and Clapp, D. W. (1998) Multiple inhibitory cytokines
induce deregulated progenitor growth and apoptosis in hematopoietic cells
from Fac?/? mice. Blood, 91, 4092–4098.
8. Hanawalt, P. C. (2002) Subpathways of nucleotide excision repair and their
regulation. Oncogene, 21, 8949–8956.
9. Muotri, A. R., Marchetto, M. C., Zerbini, L. F., Libermann, T. A.,
Ventura, A. M., Sarasin, A. and Menck, C. F. (2002) Complementation of
the DNA repair deficiency in human xeroderma pigmentosum group A and
C cells by recombinant adenovirus-mediated gene transfer. Hum. Gene
Ther., 13, 1833–1844.
10. Anaudeau-Begard, C., Brellier, F., Chevallier-Lagente, O., Hoeijmakers, J.,
Bernerd, F., Sarasin, A. and Magnaldo, T. (2003) Genetic correction of
DNA repair-deficient/cancer-prone xeroderma pigmentosum group C
keratinocytes. Hum. Gene Ther., 14, 983–996.
11. Melis, J. P., Wijnhoven, S. W. W., Beems, R. B. et al. (2008) Mouse
models for xeroderma pigmentosum group A and group C show divergent
cancer phenotypes. Cancer Res., 68, 1347–1353.
12. D'Errico, M., Parlanti, E., Teson, M., Bernardes de Jesus, B. M. et al.
(2006) New functions of Xpc in the protection of human skin cells from
oxidative damage. EMBO J., 25, 4305–4315.
13. Colby, C., Koziol, S., McAfee, S. L., Yeap, B. and Spitzer, T. R. (2002)
High-dose carboplatin and regimen-related toxicity following autologous
bone marrow transplant. Bone Marrow Transplant., 29, 467–472.
14. Shivji, M. K., Moggs, J. G., Kuraoka, I. and Wood, R. D. (2006) Assaying
for the dual incisions of nucleotide excision repair using DNA with a lesion
at a specific site. Methods Mol. Biol., 314, 435–456.
15. Trego, K. S. and Turchi, J. J. (2006) Pre-steady state binding of damaged
DNA by XPC-hHR23B reveals a kinetic mechanism for damage
discrimination. Biochemistry, 45, 1961–1969.
16. Hu, J. and Xiong, Y. (2006) Targeted ubiquitination of Cdt1 by the DDB1-
Cul4a-Roc1 ligase in response to DNA damage. J. Biol. Chem., 281,
17. Arias, E. E. and Walter, J. C. (2007) Strength in numbers: preventing
rereplication via multiple mechanisms in eukaryotic cells. Genes Dev., 21,
18. Sugasawa, K., Okuda, Y., Saijo, M. et al. (2005) UV-induced
ubiquitinylation of Xpc protein mediated by UV-DDB-ubiquitin ligase
complex. Cell, 121, 387–400.
19. Salob, S. P., Webb, D. K. H. and Atherton, D. J. (1992) A child with
xeroderma pigmentosum and bone marrow failure. Br. J. Dermatol., 126,
Received on January 27, 2009; revised on March 6, 2009;
accepted on March 21, 2009
J. L. Fischer et al.


text:
Brussels Bar du Matin
This photo was shot on film. Taken by elvismartinezsmith with a Canon AE-1 loaded with Lomography X Tungsten 64 film (available in our Online Shop) in Brussels, Belgium. These tags describe this photo: fisheye, 15mm, lens, music, smith, freres, brothers, afrobeat, afro, funk, afrofunk, touring, tour, live, summer, road, tungsten, xpro, x-pro, cross, processed, double, multiple, and exposure. Date: 2012-07. Time: Afternoon. This photo can also be found in the album Experience Tour 2012: Life on tour.Please login to get more out of this site
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U.S. State Department
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text:
The classic Mary Jane gets a sexy update with this patent platform style. Slip it on with wide-leg trousers during the day and change it up with a formfitting dress for a stunning nighttime look.
• Peep-toe Mary Jane pump. T-strap detail at vamp.
• Perforated detail at platform and upper
• Adjustable buckle closure at ankle
• 1 ½" platform
• 5" stiletto heel
• Leather
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Orders placed during regular business hours (Monday through Friday, 8:00 a.m. to 4:00 p.m. EST) are typically processed and shipped within one business day.
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For complete details, click here.
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Take it back to your local GUESS store for FREE or ship it back to us.
GUESS gladly accepts unworn, unwashed or defective full-priced merchandise for up to thirty days after purchase date. Original receipt, hygiene strip and tags must be attached. Unfortunately, pierced jewelry, innerwear, fragrances, lotions and items marked "final sale" are not returnable. Sale merchandise may only be returned to a store for an exchange. Shipping charges are not refundable.
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text:
Traffic 360 is the latest version in Just Flight's best-selling Traffic series of AI traffic expansions and is the new benchmark for Artificial Intelligence traffic add-ons.
Traffic 360 features the most realistic worldwide aircraft schedules ever, using data from real-world flight schedules covering the entire globe, and adds over half a million flight plans and over 2,400 individual aircraft variants to your flight simulator environment.
New and updated aircraft include commercial airliners, regional jets and turboprops, business aircraft, GA aircraft, helicopters, military jets and props and - for the first time in the Traffic series - custom airport ground vehicles. Airliners include the mighty A380, 787 and the yet-to-fly A350XWB, and Traffic 360 also includes all the content from Just Flight's three Traffic X PlusPaks.
With a host of custom utilities and innovative functionality to control and monitor your AI traffic around the globe, this is THE essential addition to your FS world!
Traffic Control Centre (TCC) lets you easily customise and expand your AI traffic.
Traffic Editor has improved flight planning and greatly enhanced traffic generation features to allow you to easily create new traffic, whether randomly generated traffic or detailed scheduled connections as in the real world. The Editor lets you set up one-way flights and compile flights by region.
Correct identifiers - all flights use their correct aircraft/airline identifiers and tail numbers according to the country of origin.
ChronoTraffic - AI time period selection feature. Every Traffic 360 AI aircraft and flight can now be set to be only operational during a user-defined period of years. In the traffic database you can easily select the year or range of years in which you want the aircraft to appear. This flexible feature means that you can easily see older aircraft such as Concorde or out-of-service airlines, or up-to-date traffic or even future aircraft chosen from the included Traffic 360 aircraft fleets.
Flyable aircraft tool – allows you to make the Traffic 360 AI aircraft flyable (generic cockpits and flight models employed).
Driveable Follow Me car and pushback truck - you can explore the airport from the ground.
Spotty, the plane spotting feature, and the unique Control Tower view let you watch aircraft as they are land, taxi, park and depart as well as listen to their ATC communications.
Control Tower comes in four different sizes from 'Airfield' to 'Large International Airport' and can be placed anywhere in the world. A 2D panel and a complete Tower interior provide everything you need to observe all AI traffic in your area:
Custom sounds to replace the generic FSX default sounds - a total of 29 different sound sets, recorded from real aircraft, provide more variety and more realism thanks to special take-off, landing, thrust reverser sounds and many more. Hear the typical take-off sound of an Airbus 321 or the noisy thrust reversers of a landing Tornado – Traffic 360 is an immersive experience for all the senses!
A complete list of the Traffic 360 aircraft and liveries is available as a PDF - click here to download.
Traffic 360 includes many new and improved custom aircraft models featuring advanced and improved animations, providing high quality models while reducing the impact on frame rates.
Aircraft take advantage of the advanced FSX animation features at airports such as baggage handlers, docking jetways and pushback tugs. These service vehicles will service AI aircraft when they park and, when they are ready to depart, the pushback tug will push the aircraft back off the stand (only operates at stands enabled for this feature by default in FSX). AI aircraft have numerous detailed animations including animated control surfaces, landing gear, propellers, control surfaces, jet engine fans and doors. All aircraft are painted in real-world airline liveries, with updated and improved reflective textures and real aircraft registrations. 500 liveries are included for 123 different civil and military aircraft types, giving a total of more than 2,400 aircraft variants:
General Aviation
Traffic 360 also uses the default GA aircraft in Flight Simulator X:
Military aircraft


text:
Douay-Rheims (RHE) | New International Version (NIV) |
9 Behold, the day of the Lord shall come, a cruel day, and full of indignation, and of wrath, and fury, to lay the land desolate, and to destroy the sinners thereof out of it. | 9 See, the day of the LORD is coming --a cruel day, with wrath and fierce anger-- to make the land desolate and destroy the sinners within it. |
The Douay-Rheims Bible is in the public domain. (Douay Rheims Catholic Bible Translation Online) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |


text:
Facepalm Momments
Everyone pretty much has them sooner or later. So what yours? Mine is when I played a dominion game with a guy who bought Alistar with the IP instead of getting it for free. While ingame he just ignores the control points. Double *facepalm*
You can get alistar for free? o-o
I have a "WTF" moment, like i'm dumbfounded. Playing summoner's rift normal mode, this akali says in all caps: "I'M THE BEST EFFING MID THERE IS *****EZZ" in game goes 0/5 to a xin that decided to go mid then rage quits because were noobs.
When I use Mordekaiser, my ult is up, and I see an extremely low hp AP carry running from a team fight. And upon using my ult... I accidentally hit the full HP tank who walked RIGHT infront of my intended target, with no idea that he had just saved his team from getting DPS'd down by their own carry. It's happened more than once, lol.
accidently igniting the minion next to a 100 hp running teemo, faceflashing into a wall while trying to escape, noxian guillotine being screwed by zilean or GA, i have a lot a facepalm moments .___.
The other day I dodged two ranked games BC of obvious trolls.....then hour and a half later I was in ranked picked then went to get something to drink...when I got back I saw in chat that an Alistair was a troll BUT it was too late to Dodge.timer was up.....ended up with 2 trolls and 2 Afks. FACEPALM.
Sorry so long |
Fail flashes. That is all.
I've recently been messing up my Ahri ulti by for some unexplained reason, continuously dashing into walls. I've done it twice now and it so funny and depressing.
Funny, because I still manage to kill my intended target no matter what Depressing, because I have no idea what I am doing! "ah! WHY! WHY WALL! WHY!" |
dont forget running through jungle at super low hp to get finished off by your sunfire cape .-.
All times are GMT -8. The time now is 09:54 AM. |
(c) 2008 Riot Games Inc


text:
Who We Treat
Consultation information for individuals with suspected or diagnosed chest malignancies.
Your First Visit
A list of materials to bring with and information about what to expect at your initial appointment.
Forms and Paperwork
Demographic and medical history forms for NYU Clinical Cancer Center patients.
Location of Clinics
Contact information and directions to the division's clinics.
Directions to the division and affiliated hospitals using various modes of transportation.
Where to park when driving to your appointment.
Places to stay when visiting NYU Medical Center.


text:
U.S. Sen. Pat Roberts was able to introduce a high-profile visitor he called "one of my heroes" to an enthusiastic crowd of GOP supporters Wednesday. Sen. John McCain, a Republican and the senior senator from Arizona, joined a handful of famous Republicans visiting Kansas to support Roberts in his campaign against Independent candidate and businessman ...Continue Reading
Arts Upload VIDEO
Is Kansas City a theatre town, an incubator for music-makers, a haven for visual artists? Does it blaze trails in animation, the spoken word and creative ways to preserve the past? KCPT's ARTS UPLOAD says "yes" to all of the above, and we're building the show to prove it! It's a half-hour exploration of the ...Continue Reading
Lunch time at Harris Bilingual Elementary School in Fort Collins, Colo., displays all the usual trappings of a public school cafeteria: Star Wars lunch boxes, light up tennis shoes, hard plastic trays and chocolate milk cartons with little cartoon cows. It's pizza day, the most popular of the week, and kids line up at a ...Continue Reading
Emperor dads stand watch as penguin hatchlings peck their way free. Penguins: Spy in the Huddle, A Nature Special Presentation Emperor penguins cross a treacherous frozen sea to reach their breeding grounds. Rockhoppers brave the world's stormiest seas only to come ashore and face a daunting assault up a 300-foot cliff, hopping most of the ...Continue Reading
Consumers in Missouri and Kansas should see more companies offering coverage through the health insurance marketplaces established by the Affordable Care Act, according to preliminary estimates released Tuesday by the U.S. Department of Health and Human Services. A report issued by the department, released in advance of the open enrollment period scheduled to run from ...Continue Reading
Grocery stores and restaurants serve up more than 400 million pounds of food each year, but nearly a third of it never makes it to a stomach. With consumers demanding large displays of un-blemished, fresh produce or massive portion sizes, many grocery stores and restaurants end up tossing a mountain of perfectly edible food. Despite ...Continue Reading


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Public notice is given that I, Sebastien Kinkela Mbanunu will file a Petition in the Circuit Court of Champaign County, IL on March 13, 2015 to change the name of my minor daughter from Muaka Mbanunu to Remercia Muaka Mbanunu pursuant to the statute in such case made and provided.
Sebastien Kinkela
Mbanunu on behalf of
Muaka Mbanunu
1262577 1/30,6,13
The Mahomet-Seymour Community Unit School District #3 Board of Education is soliciting a request for proposals (RFP) for mowing services. For specifications, please contact Trent Nuxoll, Chief School Business Official, at tnuxoll@ ms.k12.il.us. Proposals are due by February 11, 2015 at 2:00pm, at which time they will be opened. The successful bidder will be selected at the Board meeting on February 17, 2015.
Cheryl Melchi, Secretary Board of Education Mahomet Seymour CUSD #3
1262591 1/30
SBA Towers V, LLC (SBA) proposes to construct a 195' monopole (199' overall) along Highway 12, approx 875' N of Dunlap Woods N in Sidney, Champaign County, IL (Project 28143).
In accordance with the National Historic Preservation Act of 1966 and the 2005 Nationwide Programmatic Agreement, SBA is hereby notifying the public of the proposed undertaking and soliciting comments on Historic Properties which may be affected by the proposed undertaking. If you would like to provide specific information regarding potential effects that the proposed undertaking might have to properties that are listed on or eligible for listing in the National Register of Historic Places and located within 1/2 mile of the site, please submit the comments (with project number) to: RAMAKER, Contractor for SBA, 1120 Dallas St, Sauk City, WI 53583 or via e-mail to history@
ramaker.com within 30 days of this notice.
1262569 1/30