text:
Get to Know the Rodin Duo
Make sure to catch the Rodin Duo this Sunday at the Columbus Museum of Art at 3:00 PM. This is truly a duo of lovely musicians with a firm grasp on music both new and classic.
Listen to the Story
127 Hours
Starring: James Franco, Kate Mara, Amber Tamblyn
Director: Danny Boyle
Genre: Drama
Rating: R for for language and some disturbing violent content/bloody images
Runtime: 1 hour, 33 minutes
Rico: 127 Hours is only 1 hour and 33 minutes long, but it seems to last a lifetime. Not than it's long or drags; it isn't and it doesn't.
Joyce: This is a biopic about a catastrophe, unanticipated by the experienced, featured rock-climber.
Rico: 127 hours is the length of time Aaron Ralston (James Franco) is trapped in a remote canyon before he desperately cuts his arm off, as the only way to find release.
Joyce: One should keep in mind, as you suffer along with Aron that in spite of his expertise as a rock-climber and survivalist, he made almost fatal mistakes. No.1: he's alone. No.2: he didn't tell anyone where he was going.
Rico: James Franco pulls off the one-note plot throughout the film. He is a good enough actor to make it work. Danny Boyle, the genius behind Slumdog Millionaire, is the director, and he brings enough camera magic and pacing to make this an exciting experience.
Joyce: Franco has proved to be a very strong actor and he carries off this role successfully. And Danny Boyle knows how to keep a largely one-set location interesting.
Rico: The scene of his release is graphic and a number of people who have seen it have become quite ill. If you prepare yourself or have a strong constitution, you'll be fine. It's an excellent film about the desire to survive in a really bad situation. ***1/2
Joyce: I was geared up for the worst but I found that the graphic scenes were so well done, they weren't nearly as hard to watch or bloody as I expected. I thought it was terrific. ****


text:
Retired IAS officer's wife refused to lend Rs 5000 to ex-servant, what happens next will shock you
Devoid of Blessing: Do you know why this bus fell from over bridge?
SHOCKER: Girl faints while writing board exam, dies on way to nursing home
SHOCKER: Man wakes up in night only to find wife in objectionable position with friend, beats them to death
HYGIENE FACTOR MISSING: Food being made in pantry cars of trains from dirty waterBhopal (MP): Passengers not only on the platform but also in the trains, having pantry, are being served with unhygienic food and what is more interesting is that they are overcharged for it. From tea-coffee to lunch and dinner, everything is provided but three times of the fixed rate. When the...Friday, March 20, 2015 03:36 PM [IST]
OMG! Boy, playing cricket, falls into 60 feet deep well while trying to take a catchDevas: A child fell in a well filled with slit while trying to take a catch during a Cricket match in Devas. Shubham was severely injured after he fell in the 60 feet deep well. In the meanwhile, a man who saw it happen came forward and went down inside the well and pulled Shubham to the...Wednesday, March 18, 2015 03:05 PM [IST]
MOB JUSTICE: Road Romeo taken to task by crowd, allegedly pestered married woman with WhatsApp messages!Burhanpur: In a shocking incident a man, who allegedly pestered a mother of two children with messages on mobile phone app WhatsApp, was roughed up by the public on Monday at 11 in the morning near Iqbal Chowk. Soon Nirbhaya police, a team constituted for women protection, too reached the spot....Wednesday, March 18, 2015 10:24 AM [IST]
SHOCKING: 3 youths burnt alive in the car of DEATHRatlam/Indore: A car caught fire on Tuesday morning near Dodar in Ratlam district, 190km from Indore, resulting in the death of three youths who burnt alive inside it. On Tuesday, at around 2 am, a Santro car (M-43-C-2566) caught fire in which four young boys were travelling....Tuesday, March 17, 2015 01:52 PM [IST]
Disturbing images: Bus crashes into railing of a bridge near Korba, 14 dieKorba/Bilaspur (Chhattisgarh): In a road accident, 10 persons lost their life, including three women and a six-year-old girl, when a passenger bus crashed into railing of a small over bridge near Korba. The accident took place at village Madanpur on Badni nullah main road,...Tuesday, March 17, 2015 12:10 PM [IST]
Bravo! They taught law to three youths undergoing civil judge training; forced police to actBhopal: A minor girl who has won gold and silver medals at National games was travelling with her family by train. The family was on its way back to Bhopal from Jabalpur. Near Itarsi three youth allegedly started misbehaving with the minor girl. What is more shocking is the fact that the trio is...Monday, March 16, 2015 07:07 PM [IST]
In pics: When a truck trailer got stuck in the middle of the roadDewas/Indore: In a one of its kind incident, a truck trailer got stuck in the middle of the road at Ujjain bypass. Due to very little space on both sides, the highway witnessed five hours traffic jam. In the evening, with the help of two cranes, it was removed from the road. ...Monday, March 16, 2015 10:06 AM [IST]
SHOCKER: Engineer tests HIV-positive; burns alive wife, daughters in Madhya PradeshMultai (Bhopal): In a shocking incident, an engineer working with a public sector undertaking in Madhya Pradesh burnt to death his wife and two daughters, aged nine and two, after finding out that he was HIV-positive. The 39-year-old Praveen Manvar, who was brought by police to...Wednesday, March 11, 2015 03:00 PM [IST]
WOW! These 2-year-old & 6-year-old kids earn up to Rs 8 lakh per year through modellingIndore: The little children are earning fame at national and international level through modelling these days. The 2-year-old Ekam and 6-year-old Oshan earn up to Rs 8 lakh per annum through this profession. They both are models for 11 huge brands in the country. Several producers are fan of...Tuesday, March 10, 2015 08:26 PM [IST]
A HOLE in Modi's security! Unmanned spots like this can be used by terrorists to target PMIndore: The media was not allowed to cover Prime Minister Narendra Modi's visit when he landed at Indore airport on Thursday. Citing security reasons all efforts were made to keep media at bay, but these pictures reveal that there was a serious lapse in PM's security. Video journalists...Thursday, March 5, 2015 06:01 PM [IST]
Top News

subset: commoncrawl


text:
'Tweet Like Dame Dash' Hashtag Erupts on Twitter
The "Tweet Like Dame Dash" hashtag erupts on Twitter after the music mogul's controversial interview on Power 105.1's 'The Breakfast Club.'
The "Tweet Like Dame Dash" hashtag erupts on Twitter after the music mogul's controversial interview on Power 105.1's 'The Breakfast Club.'
Brandy will be making her Broadway debut in the hit musical 'Chicago' next month.
When it comes to Hustle Simmons defending himself against an attack, the guy is no slouch at all. He proves this in newly released footage that showcases men in rapper Bleu Davinci's crew attempting to beat him up.
When an artist is in the spotlight 24 hours a day, seven days a week, sometimes all of the public scrutiny can take its toll on their self-esteem. Iggy Azalea may be a four-time Grammy Award-nominated rapper but she's just as human as the rest of us.
Now that XXL's 10th Spot has ended, we thought it'd be a good time to make our own predictions on which rappers -- and singers -- are deserving to be included in the 2015 XXL Freshmen class. Did your favorite new jack make the list? Check out our selections.
In The Boombox Battle this week, songs from Kid Cudi and T-Pain compete for the win.
Unfortunately, Charles Hamilton's new song "New York Raining" didn't beat Kid Cudi's "Love" in The Boombox Battle this week, but that doesn't mean he isn't winning in other areas. The rapper stopped by 'Late Night with Seth Meyers' to perform the track with Rita Ora.
T.I. gives details on his forthcoming album, 'Trap's Open,' which is the follow-up to 'Paperwork: The Motion Picture.'
Rich Homie Quan recently had a James Bond moment. The Atlanta rhymer is accused of punching a security guard at a Miami nightclub and then making a quick getaway in a speedboat.
'Love & Hip Hop New York' is coming to an end with the season finale airing next week, but that hasn't stopped Mona Scott-Young -- the mastermind behind the realty TV series -- from continuing to keep the ball rolling. VH1 released a teaser of the upcoming fourth season of 'Love & Hip Hop Atlanta,' which has gone on to the become the most popular spin-off of the 'L&HH' franchise to date.


text:
Shop now and get Free Value Shipping on most orders over $49 to the contiguous 48 states, DC and to all U.S. Military APO/FPO/DPO addresses.
We want to ensure that making a return is as easy and hassle-free as possible! If for any reason you decide that your purchase just isn't for you, simply return the item within 30 days of receipt and we'll cover the cost of return shipping. Learn more about our Return Policy.
If you're searching for a great bargain on the BenchMaster Rifle Rest Replacement Front Bag, then you have found yourself in the right spot. These Shooting Accessories through the professional product experts at BenchMaster Rifle Rest are designed by using long lasting and dependable components, as a way to provide you with product which is going to be beneficial for quite some time. For a number of years, BenchMaster Rifle Rest has been making high-end products, and the BenchMaster Rifle Rest Replacement Front Bag is proof of their persistence for making sure that you've got the products you'll want to succeed in virtually any situation. Right here at OpticsPlanet, we ensure that it is our responsibility to make sure you obtain the most suitable product for all your specifications, and showcasing the BenchMaster Rifle Rest Replacement Front Bag is one thing that we're pleased to do. And because you won't be charged for delivering on purchases of $49 or more, there has not ever really been an even better moment to purchase the unbelievable BenchMaster Rifle Rest Replacement Front Bag. For a good way to make sure you are utilizing some of the finest solutions obtainable, buy the BenchMaster Rifle Rest Replacement Front Bag.
The Benchmaster Cordura Nylon Front Site Bag. Made from DuPont Cordura, sewn by American Artisans.
Connect with OpticsPlanet
Become our fan, follow us, & subscribe for the latest updates and deals
Subscribe to our email list
Sign up and receive $5 Off and get exclusive access to promotions, sales
events, pre-order sales & more!
Contact Information
Toll-Free: 800-504-5897
Intl.: 847-513-6201
Fax: 847-919-3003
Email: email@example.com
Hours of Operation
7AM-8PM CST Mon-Fri9AM-5PM CST Sat-Sun
Our Guarantees


text:
Used Hearthstone Heritage with catalytic converter
newlanderSeptember 19, 2011
Hi all,
I'm looking at buying a 15-20 year old Hearthstone Heritage stove, from when the company used catalytic converters. It's a huge savings from a new one, but I'm not sure if it's too out of date.
What do you think, do I go for the good deal and end up paying for a new cat or do I hold out for a newer model?


text:
Las Vegas, NV 89109
Get Directions:
- Hours
Monday: 11:00 AM - 11:00 PMTuesday: 11:00 AM - 11:00 PMWednesday: 11:00 AM - 11:00 PMThursday: 11:00 AM - 11:00 PMFriday: 11:00 AM - 12:00 AMSaturday: 11:00 AM - 12:00 AMSunday: 11:00 AM - 11:00 PM
- Atmosphere Outdoor Dining, Scenic View, Family / Children, Bar Scene
- Amenities TV, Private Party Room
- Special Needs Smoking Section, Booster/High Chairs, Wheelchair Access, Child Menu
- Bar Full Bar
- Banquet Facilities Over 100 People
- Reservation Information Not Required
- Attire Casual
- Parking Availability Valet, Parking on Site
- Meal Times Served Lunch, Dinner, Late Night, Happy Hour
- Payment Methods
Stripburger is an all-outdoor restaurant and bar serving 100 all natural beef burgers, hand cut fries creamy milk shakes and delicious baby cakes on the sunny Las Vegas Strip. Stripburgers are not made from typical hamburger meat, they are made from100 all natural selected cuts, never frozen and specially chosen for flavor, juiciness, and leanness. Fries are made from hand cut, skin on Idaho potatoes, specially seasoned and cooked in trans fat free oil. Host your next special event, BBQ, or party on the patio at Stripburger. We can accommodate groups ranging from 12 to 150 and have both indoor and outdoor accommodations. Enjoy our energetic vibe, strip views, and the best burgers in town.
- Best Known For
- Blue Cheese Burger
- Awards
- Bite of Las Vegas "Best Burger"
People who viewed this restaurant also bought:
Craving for Savings?Sign up today to receive special offers for Stripburger and other restaurants in your area!
Subscribe Now


text:
Question Guidelines:
Is your question about beauty?
Please help us by staying on-topic. Unrelated questions will be deleted.
Is your question a question?
A good general rule when asking a question is to start with the words What, Where or How. You should also include as much detail as possible - the more detailed your question, the easier it will be for people to give you good answers.
Please be nice!
When answering, asking or commenting, please always be respectful of others in the community.
Need advice on multiple makeovers?
Attach multiple makeovers to allow the community to vote on the best look and provide valuable feedback.
Good example questions:
- What kind of foundation is best for oily skin?
- Which hair color suits me best?
- How do I apply a gel eyeliner?
- Which hair color suits me best?
- How do I apply a gel eyeliner?
Give Beauty & Fashion Advice
When it comes to pretty, you're the pro. TAAZ users have submitted their questions below, and are now awaiting your beauty expertise!
Sort by Most Recent
Show All
Gostaria de saber como fica meu cabelo ruivo
Ele está com mechas loiras e eu queria mudar para ruivo.
Submitted by JoyceA1
0 Answers
What kind of bob hair suits me?
I have very long brown hair and I want to cut it and have a long bob. But I am afraid that my hair will look like…
Submitted by anna11a
0 Answers
I want blonde hair but...
Hi. I want to achieve a nice blonde color, but... the Orange takes over pretty much everything. I can see some blonde in…
Submitted by tifditif
1 Answer
what are the best haircut styles for short, thick hair?
I need to cut off all my damaged hair, my hair is really think and needs to be cut really short so I'm just seeking…
Submitted by shellykrj382007
5 Answers
Will baby shampoo work to eliminate semi or demi permanent dye quicker?
I have heard baby shampoo works for dading color quickly. My question is, one, is this true and two, if it is true HOW does…
Submitted by KarahMC
1 Answer
Would it be okay to die my hair the same color brown twice
Okay I died my hair Sunday with Revlon 33 dark soft brown I am naturally a dirty blonde and I had wanted to darken my…
Submitted by CodyEverettMiller
0 Answers
Get Beauty & Fashion Advice
Ask a question and get real-time advice from the TAAZ community.
Advice Type *
What's Your Question? *
Explain Your Question
Does your question follow the Question Guidelines?
Category *
Attach an Image(Limit 1)
Attach Images & Captions (Limit 12)


text:
Chinese and Japanese Department
Want to engage deeply and knowledgeably with China and Japan? Grinnell takes you there, with a full curriculum in Chinese language and literature and two years of Japanese language and literature.
Want to engage deeply and knowledgeably with China and Japan? Grinnell takes you there, with a full curriculum in Chinese language and literature and two years of Japanese language and literature.
This program reflects the assumption that educated persons will wish to extend their experience of literature beyond what has been written in their native tongue and beyond what they can read in the foreign languages they have mastered. The education of any student can be enriched by exploring literature in translation. Courses in General Literary Studies make possible the discovery of a variety of illuminating relationships among works in different languages.
The ALSO program offers highly motivated and independent students the opportunity to learn a language through a combination of self-instruction and peer tutoring. Languages offered have included Czech, Modern Greek, Hindi, Hebrew, Hungarian, Italian, Korean, Polish, Portuguese, and Swahili.
Students interested in any language may fill out an online application form. The Program Director will contact you about your application and desired course.
The Grinnell writer dropped her forehead onto her arms in abject despair. She lay there, limp and hopeless, like a corn doll abandoned in the rain. The husks of her notebooks lay about her, fluttering idly in the Iowa wind that whistled through the partially open window. A page of one particularly kind and caring notebook draped itself across her shoulder in a reassuring way. It's all right, sweetie, that touch seemed to say. It'll all work out.
Mark Baechtel
When Grinnell's English department brought Ralph Savarese to Iowa six years ago from Florida, he saw it as a chance to nourish a range of interests that -- to an outsider, at least -- looks not only exhaustive, but downright exhausting.
Grinnell, IA - Writers@Grinnell will open its fall semester program with a reading from novelist and essayist Natalie Bakopoulos on Thurs., Sept. 13. All readings will be in Room 101 of the Joe Rosenfield '25 Center on the Grinnell College campus at 8 p.m. unless noted.
"We have an incredible group of writers coming this year," said Dean Bakopoulos, assistant professor of English, who is directing this year's program. "Some of the most entertaining readers and engaging teachers of writing in the country are coming to Grinnell this year. I'm excited for our students."
Natalie Bakopoulos will hold a roundtable discussion in Room 226 of the Joe Rosenfield '25 Center at 4:15 p.m. Bakopoulos recently released her first novel "The Green Shore," which paints a finely-etched portrait of one family, whose heartbreaking stories of love and resistance play out against the backdrop of the late 1960s Greek military dictatorship. She has also written essays for "Granta," "Salon," and "The New York Times" about Greece's current economic crisis. She is the sister of Dean Bakopoulos, who will join her for the roundtable discussion entitled "Sibling Rivalry: Writing About Family Without Getting Disowned."
Historian, biographer, and journalist Sam Tanenhaus, a 1977 Grinnell College graduate and visiting faculty in English, will give a presentation on Thurs., Sept. 27. Tanenhaus is the editor of "The New York Times Book Review" and the author of "The Death of Conservatism" and "Whittaker Chambers: A Biography."
On Oct. 4, author Charles Baxter, acclaimed fiction writer, critic, and one of the nation's most beloved creative writing teachers, will read from his most recent story collection, "Gryphon: New and Selected Stories," recently published by Pantheon/Random House. Baxter is also the author of a dozen other books, including the National Book Award finalist "The Feast of Love."
May-lee Chai, a writer, educator and 1989 graduate of Grinnell College, has participated in past Writers@Grinnell series and will be back on Thurs., Oct. 11 to lead a roundtable discussion on "Writing after Grinnell" at 4:15 p.m. in Room 226 of the Joe Rosenfield '25 Center. Following the roundtable, Chai will read from her new memoir "Hapa Girl." This event will be held at the college's Faulconer Gallery at 8 p.m.
On Thurs., Nov. 1, Ronald Wallace, will read from his poetry, including selections from "Long for this World" and "For a Limited Time Only." Wallace will also lead a roundtable presentation on formal poetry at 4:15 p.m. in Room 209 of the Joe Rosenfield '25 Center. Praised as "one of our liveliest, most readable poets" by Charles Harper Webb, Wallace's trademark warmth and wit makes poetry lovers out of the form's sworn enemies.
Brothers Davy and Peter Rothbart began FOUND Magazine in 2001. The magazine spawned the 2004 bestseller "Found: The Best Lost, Tossed, and Forgotten Items from Around the World," as well as two other collections in 2006 and 2009. On Sun., Nov. 11, Davy will read selections from his new essay collection "My Heart is an Idiot" and will be accompanied by musical selections composed and performed by Peter. True performers, the Rothbart brothers have sat on David Letterman's interview couch and have been featured on the radio show "This American Life."
Students of the Grinnell Review will round off the fall semester readings on Thurs., Dec. 6, with readings from the campus's literary journal, which is edited and designed entirely by Grinnell students.
The Joe Rosenfield '25 Center is located at 1115 8th Ave. on the Grinnell College campus. Faulconer Gallery is located in the Bucksbaum Center for the Arts at 1108 Park Street, also on the college's campus. Grinnell welcomes the participation of people with disabilities. Information on parking and accessibility is available on the college website. Accommodation requests may be made to Conference Operations at 641-269-3235 or firstname.lastname@example.com.
Grinnell, IA - Writers@Grinnell will open its fall semester program with a reading by novelist and faculty member Dean Bakopoulos on Thurs., Sept. 8 at 8 p.m. in Room 101 of the Joe Rosenfield '25 Center on the Grinnell College campus.
Bakopoulos' latest novel, "My American Unhappiness," was reviewed this summer by the Los Angeles Times and Wall St. Journal and listed as the "#1 Title to Pick Up Now." He is currently at work on a television series based on his first novel, "Please Don't Come Back from the Moon," set in his hometown of Detroit. Bakopoulos is a member of the Grinnell English faculty and also teaches in the creative writing and environment program at Iowa State University.
Poet Nell Regan will read on Sept. 13 from her debut collection, "Preparing for Spring," reviewed as a "stingingly clear outdoor light." Regan has also published historical biography in "Female Activists: Irish Women and Change." Regan will read in Room 102 of Alumni Recitation Hall.
On Sept. 29, author Ed Pavlic will read from his most recent books, "But Here Are Small Clear Refractions," "Winners Have Yet to be Announced," and "Labors Lost Left Unfinished." Pavlic's works have drawn recognition from the African American Review, the American Poetry Review, and the Georgia Writers Association.
Non-fiction writer Susanne Antonetta will discuss her work in the fields of environment, mental health, diversity, spirituality, and the sciences during a reading on Oct. 6 co-sponsored by the Environmental Studies concentration. Antonetta is the author of "Body Toxic," a New York Times Notable Book, as well as four books of poetry under her legal name Suzanne Paola and a soon-to-be released book on adoption titled "Inventing Family."
All Writers@Grinnell events will be held at 8 p.m. in Room 101 of the Joe Rosenfield '25 Center, 1115 8th Ave. on the Grinnell College campus, unless otherwise noted. Grinnell welcomes the participation of people with disabilities. Information on parking and accessibility is available on the college website. Accommodation requests may be made to Conference Operations at 641-269-3235 or email@example.com.
GRINNELL, IA—Writers@Grinnell will open its fall semester program with a reading by fiction writer and faculty member Jeremy Jackson on Thurs., Sept. 9 at 8 p.m. in Room 101 of the Joe Rosenfield '25 Center on the Grinnell College campus.
Jackson, who teaches fiction writing at Grinnell, has written two novels, "Life at These Speeds" and "In Summer," plus three cookbooks, one of which was nominated for the James Beard award. He also writes young adult novels under the pseudonym of Alex Bradley.


text:
Octavia's Bistro is known for its delicious seafood, steaks & spirits and lively atmosphere. Enjoy a glass of wine with fresh Northwestern cuisine while looking out over the beautiful English gardens and harbor. Call and ask about Pub Night, tasty specials and live music every Friday night.
Reserve the entire Orcas Hotel for your group, conference or unique retreat or wedding. Whatever your occasion, we invite you to create your own story and good times at The Orcas Hotel.
Octavia's Bistro is open from 4:00pm till 8:30pm every night for dinner and drinks and Sunday mornings from 7:00am till 12:00pm for a full service breakfast.
Seahawks are going to the Super Bowl! February 1st we will have the game here on the big screen at Octavia's Bistro. Happy hour from 4-5pm
Join us Saturday February 14th for a romantic four course meal in Octavia's Bistro. Call (360) 376-4300 for more details and view the menu below. Reservations requested.
Prime Rib special every thursday night! Starting November 11th we are bringing back prime rib night. Full menu is still available and we have vegetarian options as well. Dinner starts at 5:00pm and goes till 8:30.


text:
Page 1
Identification of ASF/SF2 as a critical, allele-specific effector of the
cyclin D1b oncogene
Nicholas A. Olshavskya,c, Clay E.S. Comstockb,c, Matthew J. Schiewerb,c, Michael A.
Augellob,c, Terry Hyslopb,e, Claudio Settef, Jinsong Zhanga, Linda M. Paryseka, and Karen
E. Knudsenb,c,d,*
aDepartment of Cancer and Cell Biology, University of Cincinnati, Cincinnati, OH 45267
bKimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107
cDepartment of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107
dDepartment of Urology, Thomas Jefferson University, Philadelphia, PA 19107
eDepartment of Pharmacology and Experimental Therapeutics, Thomas Jefferson University,
Philadelphia, PA 19107
fDepartment of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata,
00133 Rome, Italy
The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors
markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent
studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of
tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b
induction remain unsolved. Here, we identify the RNA binding protein SF2/ASF as a critical, allele-
specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2
associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous
transcript in prostate cancer (PCa) cells. SF2-association was altered by the CCND1 G/A870
polymorphism, which resides in the splice donor site controlling transcript-b production. This finding
was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary
PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this
disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation
of G870-derived transcript-b. Finally, relevance of SF2 function was established, as SF2 strongly
correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a
novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a
factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production.
Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies
designed to suppress the oncogenic alternative splicing event.
The cyclin D1b variant, produced via alternative splicing of the CCND1 transcript (1,2), is a
potent oncogene that harbors distinct functions from full-length cyclin D1 (cyclin D1a) (3–
5). Unlike cyclin D1a, cyclin D1b independently confers cellular transformation (4,5). In
addition, only the cyclin D1b isoform has the capacity to promote anchorage-independent
*To whom correspondence should be addressed email@example.com, Phone: 215-503-8574.
NIH Public Access
Author Manuscript
Cancer Res. Author manuscript; available in PMC 2011 May 15.
Published in final edited form as:
Cancer Res. 2010 May 15; 70(10): 3975–3984. doi:10.1158/0008-5472.CAN-09-3468.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 2
growth and cell invasiveness (6). Further confirmation of novel oncogenic capabilities was
identified in mouse models, wherein animals expressing human cyclin D1b under the bovine
K5 promoter demonstrated increased papilloma multiplicity (3). Given the enhanced oncogenic
function of cyclin D1b, it is imperative to define the mechanisms that regulate cyclin D1b
production in systems of clinical relevance.
The alternative splicing event that produces the cyclin D1b transcript (referred to as transcript-
b) arises from failure to splice at the CCND1 exon 4-intron 4 boundary. Due to intronic
transcriptional termination, cyclin D1b lacks exon 5 encoded sequences and contains a novel
C-terminal domain of unknown function (1,2). Previous studies showed that transcript-b/a
ratios are enhanced in selected tumor types, thus providing evidence that the alternative splicing
event may be altered as a function of tumorigenesis or tumor progression (6–10). Lending
support to this posit, recent analyses of a large cohort of prostate cancer (PCa) specimens
revealed that cyclin D1b (but not cyclin D1a) is induced in PCa as compared to non-neoplastic
tissue (11). These findings were of interest, as cyclin D1b has specialized functions in this
tumor type that are hypothesized to promote tumor progression (7).
Despite the compelling evidence identifying cyclin D1b as a potent, novel oncogene, the tumor-
associated factor(s) that promote the alternative splicing event remain poorly defined. It has
been long suggested that a polymorphism within the exon 4 splice donor site (G/A870) may
contribute to transcript-b production, wherein the A-allele was suggested to favor the
alternative splicing event (1,12,13). Recent analysis using minigenes supported this contention,
and analysis of non-neoplastic prostate tissue demonstrated that the presence of the A-allele
predicted for higher transcript-b production; however, the impact of the A-allele was lost in
tumor tissue, thus indicating that tumor-associated factor(s) likely bypass or modify the impact
of the G/A870 polymorphism with regard to transcript-b production (11). Here, the present
study identifies the SF2 (also known as ASF or SRp30a) RNA binding protein, as a critical,
allele-selective factor that associates directly with transcript-b and modulates cyclin D1b
production in model systems of cancer relevance. Importantly, analyses of tumor tissue further
support a model wherein tumor-associated elevation of SF2 specifically enhances cyclin D1b
expression in human disease. Together, these findings provide a mechanism by which
CCND1 alternative splicing is controlled in tumorigenesis, and identify SF2 as a critical
regulator of cyclin D1b oncogene production.
Materials and Methods
Cell Culture, Transfections, Generation of Stables
LNCaP, C33A, and LAPC4 cell lines were obtained, cultured and transfected as previously
described (7,14–17). The DT40-ASF cell line was a generous gift from James Manley
(Columbia University) and maintained as previously described (18). DT40-ASF cells were
transfected using the Amaxa Nucleofector protocol. C33A stable cell lines were generated by
transfecting the indicated expression constructs encoding empty vector, o r individual cyclin
D1 minigenes and selected with 400 µg/ml G418 (MP Biomedicals). Clonal isolates were
screened for expression by immunoblot. Isolates utilized herein are denoted as C33A-Vec,
C33A-G1 (containing the G870 allele minigene) and C33A-A1 (containing the A870 allele
The pCGT7, pCGT7-ASF/SF2 and pCGT7-SRp40 expression constructs were generous gifts
from Adrian Krainer (Cold Spring Harbor) and previously described (19). pCEP4-ASF/SF2
was a generous gift from Woan Yuh-Tarn (Institute of Biomedical Sciences, Taiwan) and was
previously described (20). The cyclin D1 minigenes were previously described (11).
Olshavsky et al. Page 2
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 3
Cells were harvested, lysed in NETN, and subjected to SDS-PAGE and immunoblotting as
previously described (14,15). Immunoblots were performed using antisera against ASF/SF2
(Invitrogen), β-tubulin (Sigma), cyclin D1a (Neomarker, Ab3), cyclin D1b (7), GAPDH
(Invitrogen), or T7 (Novagen), as indicated.
RNA Isolation, PCR, PCR-RFLP and Real Time PCR
RNA was isolated using TRIzol per manufacturer's protocol. PCR analyses and primer
sequences for transcript-a, transcript-b and GAPDH were previously described (7,15,21). The
PCR-RFLP was performed as previously described (7,22); briefly, RIP recovered products
were subjected to a second amplification using primers designed for RFLP analyses. The
product was then digested and visualized after electrophoresis by ethidium bromide. Real-time
PCR for transcript-b was performed using the Syber Green protocol from Applied Biosystems
and a 7900F Real Time PCR machine.
Immunohistochemistry and AQUA system analysis
IHC and AQUA analyses were performed as previously described (11) using a human prostate
cancer array (SuperBio Chips Labs) and antisera indicated above. Comparison of relative SF2
levels from microarrays were analyzed using one way analysis of variance. Correlations of
SF2 with cyclin D1a and D1b AQUA expression levels was completed using Spearman
correlation coefficients, with Bonferroni correction of p-values for multiple comparisons.
RNA Immunoprecipitation (RIP)
RIPs were performed as previously described (23). Briefly, cells (LNCaP, LAPC4, C33A and
the C33A-derived stables cell lines) were harvested and nuclear extracts prepared by
resuspending in isotonic buffer. After a 7 min incubation on ice, samples were centrifuged at
700g for 7 min and nuclei were isolated, resuspended in buffer supplemented with 90 mM
NaCl and 0.5% Triton X-100, and sonicated. After centrifugation at 5,000g for 15 min, nuclear
extracts were precleared for 1h and immunoprecipitated with ASF/SF2 antibody or mouse IgG
antibody. The antibody-antigen complex was precipitated by the addition of protein G-
sepharose beads (Invitrogen) for 3h at 4° with rotation. Beads were washed thrice with lysis
buffer and an aliquot was eluted in SDS sample buffer for immunoblot analysis. The remaining
beads were incubated with lysis buffer in the presence of (RNase-free) DNase (Ambion) for
15 min at 37°C and washed thrice with lysis buffer before incubation with 50 µg proteinase K
(Roche) for 15 min at 37°C. Co-precipitated RNA was then extracted by the TRIzol procedure
and used for RT-PCR analyses.
G/A870 Polymorphism alters cyclin D1b production and SF2 binding
Given the enhanced oncogenic function of the cyclin D1b variant (3–5), and the established
observation that cyclin D1b levels are enhanced as a function of prostate tumorigenesis (7,
11), it is imperative to discern the mechanism(s) underpinning the alternative splicing event.
As was recently reported, the CCND1 G/A870 polymorphism plays a context-specific role in
the alternative splicing event, wherein the A-allele favors cyclin D1b production in minigene
expression studies in which the alleles were individually examined (11). These studies were
validated here, wherein minigenes harboring either the G- or A870 allele (Figure 1A) were
independently introduced by stable transfection into C33A cells, which have been previously
shown to harbor low to undetectable levels of endogenous cyclin D1 isoforms (24).
Introduction of the G-allele minigene (stable line C33A-G1), resulted in detectable expression
of both CCND1 transcripts (a and b), but a preference towards transcript-a and cyclin D1a
Olshavsky et al.Page 3
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 4
protein was observed (lanes 2). Transcript-b levels were comparatively increased in the
presence of the A-allele minigene (lanes 3), consistent with previous studies wherein the
minigenes were individually analyzed in parallel (11). Subsequent analyses of individual
minigene studies in spontaneously immortalized CV1 cells or viral oncoprotein immortalized
RWPE-1 prostate epithelial cells (25,26) were also carried out. Similar findings were observed
in both non-tumorigenic model systems, wherein the A-allele predisposed to transcript-b and
cyclin D1b production (Supplemental Figure S1). These studies further support the premise
that in isolated comparison, the G/A870 polymorphism influences CCND1 alternative splicing.
As G/A870 lies within the splice donor site, it was reasoned that the polymorphism may
influence RNA binding protein recognition or activity. The splicing factor binding resource
ESEfinder3.1 (27,28) was therefore utilized to predict distinctions in the profile of associated
splicing factors between the G- and A-870 allele splice donor sites. As shown, binding of SF2
was suggested to be altered in both position and strength by changes in the polymorphic site
(Figure 1C). To initially assess whether SF2 could play a role in CCND1 alternative splicing,
an RNA immunoprecipitation (RIP) was performed using the same stable cell lines described
in Figure 1B. Using standard techniques, RNA associated with either SF2-specific or control
antisera was isolated, reverse transcribed, and detected by PCR amplification. As illustrated
in Figure 1D (top panel), transcript-b was only weakly detected in control cells and
immunoprecipitates thereof (lanes 1–3). In cells containing the G-allele minigene (C33A-G1),
SF2 readily associated with transcript-b, providing the first evidence that SF2 associates with
this mRNA species (lanes 4–6). SF2 association with transcript-b was reduced in cells
containing the A-allele minigene (lanes 7–9), as was confirmed by quantitative-RT-PCR (Fig.
1D, bottom panel). For these analyses, signal observed in C33A-Vec cells was set to "1" for
ease of comparison. The finding that SF2 binding is influenced by the polymorphism was
subsequently confirmed in additional stable isolates of minigene integration (Supplemental
Figure S2, panel A) as well through transient analyses (panel B). Together, these data strongly
indicate for the first time that the G/A870 polymorphism influences recognition by the SF2
splicing factor.
SF2 specifically correlates with cyclin D1b in prostate cancer
The observation that the G/A870 polymorphism may alter SF2 association with transcript-b
was of interest, given reports that cyclin D1b, not cyclin D1a, is induced as a function of prostate
tumorigenesis (7,11), and that cyclin D1b has oncogenic activity (3–5). Intriguingly, analyses
of gene microarray data from human PCa demonstrated that SF2 expression increases with
tumor progression (Figure 2A) (29–31). This observation was further validated upon screening
a panel of PCa cell lines, wherein it was revealed that SF2 expression is lowest in non-
transformed RWPE-1, and as compared to cells derived from primary and distant metastases
of prostate cancer (Supplemental Figure S3). Among the cancer cell lines representative of
hormone-therapy sensitive disease, SF2 was relatively low in cells known to express lower
levels of cyclin D1b (LNCaP) and higher in cells of this subtype known to express high levels
of cyclin D1 (LAPC4) (7,11). For the androgen-receptor negative cells, unexpected high levels
of expression were observed in PC3 cells, which exhibit the most aggressive phenotype in
vivo with regard to metastases (32). To probe the impact of SF2 on cyclin D1b production in
human tumors, comparative analyses of nuclear SF2 and cyclin D1 (a and b isoform) expression
was objectively analyzed using serial sections of PCa specimens and quantitative AQUA
analyses. Representative images of tumors scoring with low SF2 (Figure 2B, top panels) or
high SF2 (lower panels) are shown. Signal was quantified across each specimen within the
nuclear compartment (DAPI positive, blue) of epithelial-derived carcinoma cells (cytokeratin-
positive; green), and resultant data are plotted in Figure 2C. As shown, tumors expressing low
SF2 exhibited low cyclin D1b expression; conversely, tumors with high SF2 scored high for
cyclin D1b. Quantification of all tumors examined revealed a significant correlation between
Olshavsky et al. Page 4
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 5
SF2 and cyclin D1b (p=0.023), but not cyclin D1a (p=0.19). Co-expression of cyclin D1b and
SF2, in serial sections, was observed in glandular epithelial cells (data not shown). Together,
these data demonstrate that SF2 expression is strongly correlated with only the cyclin D1b
isoform in human disease.
SF2 modulates cyclin D1b production in multiple model systems
Since SF2 predicted for high cyclin D1b in PCa, the effect of SF2 on cyclin D1b production
was examined in model systems of disease relevance. Initially, LNCaP cells were utilized,
which express low endogenous levels of cyclin D1b (7). Epitope tagged SR proteins (SRp40
or SF2) were individually introduced (Supplemental Figure S4) by transient assays with high
transfection efficiency (Supplemental Figure S5), and the impact on CCND1 mRNA isoform
expression determined (Figure 3A). Consistent with previous results, these cells express low
levels of endogenous transcript-b (lane 1), and SRp40 failed to elicit changes in either
CCND1 transcript (lane 2). Significantly, SF2 promoted a dramatic increase in transcript-b
expression without altering transcript-a (lane 3). Quantification revealed an approximate 2.7-
fold increase in transcript-b following SF2 introduction (lower panel), thus providing the first
functional evidence for SF2-mediated regulation of cyclin D1b. Increased expression of SF2
in LNCaP cells also resulted in an increase in cyclin D1b protein expression (Supplemental
Figure S4). Unfortunately, depletion of endogenous SF2 in PCa models using siRNA were
unsuccessful (data not shown), consistent with previous observations that knockdown of SF2
induces cell death (33). As such, a previously developed DT40-ASF/SF2 cell line was utilized
(DT40-ASF), in which endogenous SF2 was replaced with a tet-repressible SF2 construct
(Figure 3B) (18). In this system, SF2 expression is undetectable approximately 24 h following
addition of Dox. Previous reports indicated that after 48 h post-knockdown, the DT40-ASF
cell line begins to undergo apoptosis (33). Thus, experiments analyzed endpoints after 24 hrs,
whereupon no loss of cell viability was noted (data not shown). As expected, no detectable
cyclin D1 isoforms were recognized (Figure 3B, left panel lanes 1, 2). Stable introduction of
the G870 minigene revealed that although both isoforms were produced (left panel, lanes 4,
5), suppression of endogenous SF2 (left panel, lane 5) diminished cyclin D1b (but not cyclin
D1a) production. Similar results were observed at the level of the transcripts following
depletion of SF2 (Figure 3B, right panel). Collectively, these results identify SF2 as an effector
of cyclin D1b production in multiple model systems.
Endogenous SF2 associates with both alleles in PCa cells
As it was observed that elevated SF2 induces cyclin D1b in PCa (Figure 2), and that SF2
differentially binds the transcript dependent on G/A870 (Figure 1), the impact of the
polymorphism on SF2 association was investigated in PCa cells. First, LAPC4 cells were
utilized, which are homozygous for the A870 allele (7). Consistent with the individual minigene
analyses in Fig. 1, endogenous SF2 associates with the endogenous transcript-b in these cells
(Figure 4A). RFLP (Restriction Fragment Length Polymorphism) analyses of the SF2-
associated transcript was also performed (Figure 4B). These observations show that in PCa,
SF2 can associate with transcript-b generated by the A870 allele; however, as individual
minigene analyses in Fig. 1 indicated a preference for SF2 to associate with transcript-b from
the G870 allele (when compared in isolation), the most critical analyses were generated in
LNCaP cells, which are heterozygous for the polymorphism (shown as genotype control in
Figure 4B). This model system afforded the first opportunity to dissect the impact of SF2 on
G870- versus A870-derived transcripts in the same model system. As expected, SF2 was
associated with total transcript-b in this model system (Figure 4C). Using the SF2 bound and
recovered transcript, two key determinations were made. First, RFLP analyses of the total
transcript (Figure 4D, lane 1) revealed that approximately 61% of the endogenous transcript-
b was derived from the G-allele, and 39% from the A-allele, thus indicating that in the
endogenous setting, both alleles significantly contribute to transcript-b production.
Olshavsky et al.Page 5
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 6
Intriguingly, RFLP analyses and quantification of the SF2 bound fraction (Figure 4D, lane 2)
resulted in a similar ratio of transcript generated from the G- and A-alleles, based on at least
five independent analyses and quantification (Figure 4D, lower panel). Thus, these data show
that endogenous SF2 can associate with both transcripts when present, but demonstrates a slight
preference for the G870-derived transcript.
SF2 predominantly associates with and produces transcript-b from the G-allele
Given the observation that elevated SF2 correlates with high cyclin D1b in human PCa and is
sufficient in PCa model systems to induce cyclin D1b production, the impact of the G/A870
polymorphism for this event was determined. As shown, SF2 levels were elevated though
ectopic expression of an HA-tagged allele in cells heterozygous for the polymorphism and that
express low endogenous cyclin D1b (Supplemental Figure S6), so as to mimic tumor-
associated SF2 induction. Subsequent analyses (Figure 5A) confirmed SF2-mediated
enhancement of overall transcript-b (compare lanes 1, 4). In addition, SF2 association with
transcript-b was markedly enhanced in the SF2-transfected cells (compare lanes 2, 5),
demonstrating that elevated SF2 results not only in enhanced transcript-b production, but also
enhanced SF2-associated transcript.
Furthermore, these findings also allowed determination of: 1) the allele composition of
transcript-b levels elevated by SF2; and 2) whether elevated SF2 altered the likelihood of either
allele to directly associate with SF2. To answer both questions, RFLP analyses were performed
with the SF2-associated transcripts after SF2 induction or in control cells. First, it was observed
that SF2 predominantly resulted in an induction of transcript-b from the G870 allele (panel
5B, lanes 1 vs. 3; quantified in panel C). These data implicate SF2 as an allele-selective modifier
of the alternative splicing event. Second, to determine whether this action of SF2 was direct,
RFLP analyses of the SF2-bound fraction was compared in cells with steady-state SF2 (control)
vs. those with elevated SF2 expression (Figure 5B, compare lanes 2, 4). For ease of comparison,
the percent of SF2 bound to the A870-derived transcript (relative to input) in control cells was
set to "1", and relative association determined for each (Figure 5D). As shown, SF2 association
with the G-allele was statistically unchanged under conditions mimicking tumor-associated
SF2 elevation. By contrast, SF2 association with the A-allele was enhanced by approximately
2-fold under these conditions.
Collectively, these data suggest a model (summarized in Figure 6) wherein SF2 elicits allele-
selective effects on cyclin D1b production in prostate cancer that are dose dependent. Under
steady state conditions, SF2 binds with slight preference to transcript generated from the G870
allele; concordantly, the G-allele accounts for 61% of transcript-b production with the A-allele
accounting for the remaining 39%. These data are consistent with the supposition that SF2 acts
(potentially directly) to suppress splicing at the intron-4/exon-4 boundary, and that this event
is facilitated by the G870 allele. Elevated SF2, such as observed in human disease, can modestly
enhance SF2 association with the A-allele transcript, but induced SF2 remains predominantly
associated with and produces cyclin D1b from the G-allele transcript.
Here, we identify SF2 as a dose-dependent effector of cyclin D1b production, and provide
evidence of a tumor-associated mechanism that alters the influence of the G/A870
polymorphism. While minigene analyses of individual alleles (Figure 1) and evaluation of non-
neoplastic human tissue support a role for the A870 allele in promoting transcript-b and
resultant cyclin D1b production, the influence of the A-allele is thought to be relieved in PCa
(11). The present data demonstrate that the RNA binding protein SF2, which is induced as a
function of PCa progression, predicts for cyclin D1b (not cyclin D1a) elevation in human
disease (Figure 2). By contrast, cyclin D1b production was attenuated in model systems of SF2
Olshavsky et al.Page 6
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 7
depletion (Figure 3). Functional studies in PCa cells heterozygous for the polymorphism
unexpectedly demonstrated that SF2 predominantly associates with and induces transcript-b
derived from the G870-allele, however, association with and production of transcript-b from
the A-allele can still occur (Figures 4 and 5). Together, these studies provide a novel mechanism
by which cyclin D1b oncogene production induced in human disease, and identify tumor-
associated SF2 as an allele selective effector of cyclin D1b.
Despite the potent oncogenic activity of cyclin D1b, knowledge of the factor(s) that regulate
the CCND1 alternative splicing event are poorly defined. The present identification of SF2 as
an effector of transcript-b and cyclin D1b production using both in vitro models and analyses
of human tumors provides strong evidence linking splicing factor deregulation to oncogene
activation. It is tempting to speculate as to whether SF2 can cooperate with few effectors of
cyclin D1b production that were identified in other tissue types. For example, in colorectal
cells it was observed that knockdown of the SWI/SNF chromatin remodeling complex subunit
Brahma (BRM) increased transcript-b production without altering transcript-a (34). These
findings are of note, as BRM was recently shown to be significantly downregulated in human
PCa (35), and it has yet to be determined whether BRM loss might also impact SF2 levels. In
a separate study, a chromosomal translocation-derived transcription factor known to be
upregulated in Ewing's sarcoma (EWS-FLI1) was found to enhance cyclin D1b production by
diminishing the rate of transcriptional elongation (10). More recently, it was demonstrated that
Sam68 promotes cyclin D1b production through a splicing repressive mechanism by blocking
U1-70k association, a constitutive spliceosome accessory factor of the U1 snRNP that is
necessary for 5' splice site recognition (36,37). It is well established that chromatin remodeling
complexes can alter RNA Pol II accessibility by alteration of the native chromatin structure
(38); moreover, pre-mRNA splicing occurs co-transcriptionally, and is aided by the function
of SR proteins (including SF2) which can bind to Pol II and selected SWI/SNF subunits
(BAF155 and BAF53A) (39). Therefore, an attractive hypothesis is that SF2 could act in
concert with either BRM or EWS-FLI1 to modulate the CCND1 splicing event. Given the
marked pro-tumorigenic activity of cyclin D1b, these collective observations further
underscore the importance of delineating the mechanisms that regulate or influence the SF2-
mediated alternative splicing event in models of disease relevance.
With regard to clinical relevance, it is notable that in PCa cells, SF2 exhibited an allele selective
effect on the alternative splicing event. Previous studies showed that in non-neoplastic tissue,
the A870 allele is associated with higher transcript-b production but that the influence of the
A-allele was lost in PCa specimens (11); these findings suggested tumor-associated factors
may either bypass the impact of the polymorphism or bolster production of transcript-b from
the G-allele. The present data support the hypothesis that SF2 may serve as such a factor, since
SF2 induction (such as occurs in human disease) predominantly binds to transcript-b derived
from the G-allele and promotes accumulation of this transcript. Production of transcript-b still
occurs from the A-allele (about 40%), however, appears to remain unaltered following
modulation of SF2 expression, thus it will be of interest to examine the functional relationship
between SF2 and the A-allele. Examination of how SF2 influences allele-specific cyclin D1b
production in other tumor types in which cyclin D1b levels are elevated as a function of
tumorigenesis (e.g. colon, bladder or breast carcinoma) will be critical (6,9,40,41). Given the
propensity of SF2 to preferentially bind to and induce cyclin D1b from the G-allele, the present
data indicate that SF2 may promote intron inclusion at the CCND1 exon4-intron4 boundary,
and precedence for a splicing repressor function of SF2 was previously established for the
MNK2 kinase (42). It cannot be presently ruled out that the impact of SF2 could be manifest
through other means, given the ability of SF2 to affect mRNA metabolism, mRNA transport
and/or stability, and mTOR-mediated translation (43,44). Future analyses will be directed at
defining the action of SF2 at the exon4-intron4 boundary.
Olshavsky et al. Page 7
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 8
Finally, the present findings provide new insight into a potential means through which SF2
promotes cellular transformation. It is noteworthy that SF2 can independently induce
transformation and induce tumor growth in vivo (42), thus demonstrating phenotypes similar
to those observed with cyclin D1b (3,5). As SF2 levels correlated with cyclin D1b in human
PCa, these data implicate cyclin D1b as a possible downstream effector of SF2-mediated
cellular transformation in the prostate. Additional mechanisms are predicted to contribute to
this event, as perturbations in spliceosome function and RNA processing proteins have been
recently identified as major contributors to genomic instability (45). Importantly, dysregulation
of splicing factors has also been shown to accelerate PCa progression and metastases, such as
observed by deregulated expression of SRp40, or in the presence of specific, cancer-risk
associated polymorphisms in the intronic region of the KLF6 tumor suppressor that create
novel SRp40 binding sites (46). Given the disease relevance of this event, it will be imperative
to determine not only the consequence of SR protein dysregulation, but whether these events
act in concert to promote tumor progression. Ongoing studies suggest that tumor-associated
SF2 induction may induce and enhanced migration and invasion phenotype in prostate cancer
cells (data not shown), providing the impetus to discern the impact of SF2 on development of
tumor metastases.
In summary, our study identifies SF2 as a novel, clinically relevant effector of CCND1
alternative splicing, capable of promoting allele-specific induction of cyclin D1b in prostate
cancer. The findings presented are among the first to determine how the cyclin D1b oncogene
is enhanced in human disease, and provide the foundation for future studies directed at
developing mechanisms to target oncogene induction.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
We thank the K.Knudsen lab for ongoing discussions, Dr. E. Knudsen for critical commentary, Drs. J. Bruzik, C.
Chalfant, and R. Carstens for valuable discussions, and J. Tulenko for assistance with figure presentation. This study
was supported by NIH grants R01 CA09996 and CA116777 (to K.E.K.), Associazione Italiana Ricerca sul Cancro
and Association for International Cancer Research (to C.S.), a DOD New Investigator Award (to C.E.S.C.). M.J.S.
and M.A.A. are supported by pre-doctoral fellowships from the Department of Defense, and N.A.O. is supported by
NIEHS training grant 2T32ES007250-21A1.
1. Betticher DC, Thatcher N, Altermatt HJ, Hoban P, Ryder WD, Heighway J. Alternate splicing produces
a novel cyclin D1 transcript. Oncogene 1995;11:1005–1011. [PubMed: 7675441]
2. Hosokawa Y, Tu T, Tahara H, Smith AP, Arnold A. Absence of cyclin D1/PRAD1 point mutations in
human breast cancers and parathyroid adenomas and identification of a new cyclin D1 gene
polymorphism. Cancer letters 1995;93:165–170. [PubMed: 7621424]
3. Rojas P, Benavides F, Blando J, et al. Enhanced skin carcinogenesis and lack of thymus hyperplasia
in transgenic mice expressing human cyclin D1b (CCND1b). Molecular carcinogenesis 2009;48:508–
516. [PubMed: 18942117]
4. Solomon DA, Wang Y, Fox SR, et al. Cyclin D1 splice variants. Differential effects on localization,
RB phosphorylation, and cellular transformation. The Journal of biological chemistry
2003;278:30339–30347. [PubMed: 12746453]
5. Lu F, Gladden AB, Diehl JA. An alternatively spliced cyclin D1 isoform, cyclin D1b, is a nuclear
oncogene. Cancer research 2003;63:7056–7061. [PubMed: 14612495]
6. Kim CJ, Nishi K, Isono T, et al. Cyclin D1b variant promotes cell invasiveness independent of binding
to CDK4 in human bladder cancer cells. Molecular carcinogenesis 2009;10:953–964. [PubMed:
Olshavsky et al.Page 8
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 9
7. Burd CJ, Petre CE, Morey LM, et al. Cyclin D1b variant influences prostate cancer growth through
aberrant androgen receptor regulation. Proceedings of the National Academy of Sciences of the United
States of America 2006;103:2190–2195. [PubMed: 16461912]
8. Li R, An SJ, Chen ZH, et al. Expression of cyclin D1 splice variants is differentially associated with
outcome in non-small cell lung cancer patients. Human pathology 2008;39:1792–1801. [PubMed:
9. Millar EK, Dean JL, McNeil CM, et al. Cyclin D1b protein expression in breast cancer is independent
of cyclin D1a and associated with poor disease outcome. Oncogene 2009;28:1812–1820. [PubMed:
10. Sanchez G, Bittencourt D, Laud K, et al. Alteration of cyclin D1 transcript elongation by a mutated
transcription factor up-regulates the oncogenic D1b splice isoform in cancer. Proceedings of the
National Academy of Sciences of the United States of America 2008;105:6004–6009. [PubMed:
11. Comstock CE, Augello MA, Benito RP, et al. Cyclin D1 Splice Variants: Polymorphism, Risk, and
Isoform-Specific Regulation in Prostate Cancer. Clin Cancer Res 2009;17:5338–5349. [PubMed:
12. Holley SL, Heighway J, Hoban PR. Induced expression of human CCND1 alternative transcripts in
mouse Cyl-1 knockout fibroblasts highlights functional differences. International journal of cancer
13. Howe D, Lynas C. The cyclin D1 alternative transcripts [a] and [b] are expressed in normal and
malignant lymphocytes and their relative levels are influenced by the polymorphism at codon 241.
Haematologica 2001;86:563–569. [PubMed: 11418364]
14. Petre CE, Wetherill YB, Danielsen M, Knudsen KE. Cyclin D1: mechanism and consequence of
androgen receptor co-repressor activity. The Journal of biological chemistry 2002;277:2207–2215.
[PubMed: 11714699]
15. Petre-Draviam CE, Cook SL, Burd CJ, Marshall TW, Wetherill YB, Knudsen KE. Specificity of
cyclin D1 for androgen receptor regulation. Cancer research 2003;63:4903–4913. [PubMed:
16. Petre-Draviam CE, Williams EB, Burd CJ, et al. A central domain of cyclin D1 mediates nuclear
receptor corepressor activity. Oncogene 2005;24:431–444. [PubMed: 15558026]
17. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Molecular
and cellular biology 1987;7:2745–2752. [PubMed: 3670292]
18. Wang J, Takagaki Y, Manley JL. Targeted disruption of an essential vertebrate gene: ASF/SF2 is
required for cell viability. Genes & development 1996;10:2588–2599. [PubMed: 8895660]
19. Caceres JF, Misteli T, Screaton GR, Spector DL, Krainer AR. Role of the modular domains of SR
proteins in subnuclear localization and alternative splicing specificity. The Journal of cell biology
1997;138:225–238. [PubMed: 9230067]
20. Lai MC, Tarn WY. Hypophosphorylated ASF/SF2 binds TAP and is present in messenger
ribonucleoproteins. The Journal of biological chemistry 2004;279:31745–31749. [PubMed:
21. Burd, CJ.; Petre, CE.; Moghadam, H.; Wilson, EM.; Knudsen, KE. Molecular endocrinology. Vol.
19. Baltimore, Md: 2005. Cyclin D1 binding to the androgen receptor (AR) NH2-terminal domain
inhibits activation function 2 association and reveals dual roles for AR corepression; p. 607-620.
22. Sawa H, Ohshima TA, Ukita H, et al. Alternatively spliced forms of cyclin D1 modulate entry into
the cell cycle in an inverse manner. Oncogene 1998;16:1701–1712. [PubMed: 9582018]
23. Paronetto MP, Achsel T, Massiello A, Chalfant CE, Sette C. The RNA-binding protein Sam68
modulates the alternative splicing of Bcl-x. The Journal of cell biology 2007;176:929–939. [PubMed:
24. Wang Y, Dean JL, Millar EK, et al. Cyclin D1b is aberrantly regulated in response to therapeutic
challenge and promotes resistance to estrogen antagonists. Cancer research 2008;68:5628–5638.
[PubMed: 18632615]
25. Jensen FC, Girardi AJ, Gilden RV, Koprowski H. Infection of Human and Simian Tissue Cultures
with Rous Sarcoma Virus. Proceedings of the National Academy of Sciences of the United States of
America 1964;52:53–59. [PubMed: 14192657]
Olshavsky et al. Page 9
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 10
26. Webber MM, Bello D, Kleinman HK, Hoffman MP. Acinar differentiation by non-malignant
immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis
1997;18:1225–1231. [PubMed: 9214606]
27. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: A web resource to identify exonic
splicing enhancers. Nucleic acids research 2003;31:3568–3571. [PubMed: 12824367]
28. Smith PJ, Zhang C, Wang J, Chew SL, Zhang MQ, Krainer AR. An increased specificity score matrix
for the prediction of SF2/ASF-specific exonic splicing enhancers. Human molecular genetics
2006;15:2490–2508. [PubMed: 16825284]
29. Lapointe J, Li C, Higgins JP, et al. Gene expression profiling identifies clinically relevant subtypes
of prostate cancer. Proceedings of the National Academy of Sciences of the United States of America
2004;101:811–816. [PubMed: 14711987]
30. Vanaja DK, Cheville JC, Iturria SJ, Young CY. Transcriptional silencing of zinc finger protein 185
identified by expression profiling is associated with prostate cancer progression. Cancer research
2003;63:3877–3882. [PubMed: 12873976]
31. Welsh JB, Sapinoso LM, Su AI, et al. Analysis of gene expression identifies candidate markers and
pharmacological targets in prostate cancer. Cancer research 2001;61:5974–5978. [PubMed:
32. Crowley CW, Cohen RL, Lucas BK, Liu G, Shuman MA, Levinson AD. Prevention of metastasis by
inhibition of the urokinase receptor. Proceedings of the National Academy of Sciences of the United
States of America 1993;90:5021–5025. [PubMed: 8389464]
33. Li X, Wang J, Manley JL. Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis,
but inhibits internucleosomal DNA fragmentation. Genes & development 2005;19:2705–2714.
[PubMed: 16260492]
34. Batsche E, Yaniv M, Muchardt C. The human SWI/SNF subunit Brm is a regulator of alternative
splicing. Nature structural & molecular biology 2006;13:22–29.
35. Shen H, Powers N, Saini N, et al. The SWI/SNF ATPase Brm is a gatekeeper of proliferative control
in prostate cancer. Cancer research 2008;68:10154–10162. [PubMed: 19074882]
36. Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annual review of biochemistry
37. Paronetto MP, Cappellari M, Busa R, et al. Alternative splicing of the cyclin D1 proto-oncogene is
regulated by the RNA-binding protein Sam68. Cancer research 70:229–239. [PubMed: 20028857]
38. Clapier CR, Cairns BR. The biology of chromatin remodeling complexes. Annual review of
biochemistry 2009;78:273–304.
39. Das R, Yu J, Zhang Z, et al. SR proteins function in coupling RNAP II transcription to pre-mRNA
splicing. Molecular cell 2007;26:867–881. [PubMed: 17588520]
40. Bala S, Peltomaki P. CYCLIN D1 as a genetic modifier in hereditary nonpolyposis colorectal cancer.
Cancer research 2001;61:6042–6045. [PubMed: 11507050]
41. Hong Y, Eu KW, Seow-Choen F, Fook-Chong S, Cheah PY. GG genotype of cyclin D1 G870A
polymorphism is associated with increased risk and advanced colorectal cancer in patients in
Singapore. Eur J Cancer 2005;41:1037–1044. [PubMed: 15862753]
42. Karni R, de Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR. The gene encoding the splicing
factor SF2/ASF is a proto-oncogene. Nature structural & molecular biology 2007;14:185–193.
43. Michlewski G, Sanford JR, Caceres JF. The splicing factor SF2/ASF regulates translation initiation
by enhancing phosphorylation of 4E-BP1. Molecular cell 2008;30:179–189. [PubMed: 18439897]
44. Zhong XY, Wang P, Han J, Rosenfeld MG, Fu XD. SR proteins in vertical integration of gene
expression from transcription to RNA processing to translation. Molecular cell 2009;35:1–10.
[PubMed: 19595711]
45. Paulsen RD, Soni DV, Wollman R, et al. A genome-wide siRNA screen reveals diverse cellular
processes and pathways that mediate genome stability. Molecular cell 2009;35:228–239. [PubMed:
46. Narla G, Difeo A, Reeves HL, et al. A germline DNA polymorphism enhances alternative splicing
of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer
research 2005;65:1213–1222. [PubMed: 15735005]
Olshavsky et al.Page 10
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 11
Fig. 1. SF2 associates with transcript-b and shows allele preference in minigene analyses
(A) Schematic of the pcDNA3.1-cyclin D1-Intron 4 minigenes. Primers used to amplify
transcript-a (bars; exon 1 and exon 5) and transcript-b (arrows; exon 4 and intron 4) are
indicated. Clonal isolates C33A-Vec, C33A-G1 and C33A-A1 are as described in the Materials
and Methods section. (B) Representative mRNA (left panel) and protein (right panel) analyses
of cell lines engineered to individually express the G870 or A870-containing minigenes. Non-
template (NT) is the negative control. (C) Analyses of predicted SF2 binding to the CCND1
splice donor site as determined using the ESEfinder3.1 resource. Exonic (upper case) and
intronic (lower case) sequence is shown, and the G/A870 polymorphic site underlined. (D)
C33A-derived stable cell lines shown in panel B were subjected to a RIP. Representative RT-
PCR reaction of input and SF2 or IgG-associated transcript is shown (top panel), and
quantification by qPCR of at least 3 independent experiments provided (bottom panel).
Statistical significance (p<0.05) was determine by ANOVA.
Olshavsky et al. Page 11
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 12
Fig. 2. SF2 is enhanced in PCa and positively correlates with cyclin D1b
(A) Analyses of independent microarray analyses were determined using the Oncomine
resource. Relative SF2 expression in non-neoplastic (N), primary PCa (PCa) and metastatic
PCa (Met) are shown and p-values given. (B) Representative immunostaining of tissue
microarrays using the AQUA platform. As previously described, cells of epithelia origin were
identified using a pan-cytokeratin antisera (green), nuclei were stained with DAPI (blue), and
cyclin D1 isoforms detected in serial sections using well characterized, isoform specific
antisera (red). (C) Quantification of all samples analyzed (n=49) across each core was used to
determine the relevance of SF2 for cyclin D1a (left) and cyclin D1b (right) levels. P-values
were determined via Spearman Rank Correlation.
Olshavsky et al.Page 12
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 13
Fig. 3. Cyclin D1b production is induced in multiple models systems by SF2
(A) LNCaP cells were transfected with constructs encoding empty vector, T7-SRp40 or T7-
SF2, harvested after 48 h, and examined for expression of indicated transcripts. Representative
PCR (upper panel) and quantification by qPCR of at least 3 independent experiments (bottom
panel) are shown. (B) DT40-ASF/SF2 cells were transfected with plasmids encoding either
empty vector or the G870 minigene. Transfected cells were split after 24 h into populations
treated with vehicle or doxycycline for 24 h. Cells were harvested, lysed and subjected to
immunoblot analyses with the indicated antisera (left panel) or RNA was isolated and subjected
to RT-PCR analyses for the indicated transcripts (right panel).
Olshavsky et al.Page 13
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 14
Fig. 4. Endogenous SF2 can associate with transcript generated from both G/A870 alleles in PCa
(A) Asynchronous LAPC4 cells were harvested and subjected to a RIP. An aliquot of
immunoprecipitated complexes was subjected to SDS-PAGE and immunoblot to validate SF2
recovery (left panel). From the remaining sample, RNA was isolated, converted to cDNA, and
analyzed for the presence of transcript-b by PCR (representative sample, right panel). qPCR
quantification of at least 4 independent experiments is shown (lower left). P-value determined
by ANOVA. (B) Input and SF2-recovered transcripts from Fig. 4A were amplified with primers
required for RFLP analyses of the CCND1 polymorphism, which have been previously
described (38). Following amplification, the PCR product was ScrFI digested and visualized
on an agarose gel. Genotype control is from amplification of genomic DNA from LNCaP cells.
(C) Asynchronous LNCaP cells were treated as per panel A. Relative SF2 pulldown and
transcript-b recovery are shown. Quantification was from at least 5 independent RIP
experiments. (D) Analyses of input and SF2-associated transcripts by RFLP from panel C is
shown (top panel). Relative band intensities of endogenous, overall transcript-b levels as a
Olshavsky et al.Page 14
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 15
function of the G/A870 allele (generated from the input band) or SF2-associated transcript
(generated from the SF2-RIP studies) were quantified from at least five independent
experiments (lower panel). Averages and SEM are shown. The ratio of transcript-b as A to G
(A/G) is shown for the representative image.
Olshavsky et al. Page 15
Cancer Res. Author manuscript; available in PMC 2011 May 15.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript


text:
His brother is Thomas Everett same company.
There is more information about Green W. Everett in Thomas records.
Discovered by
More Spotlights by cmikeb (See all)
Source Information
Popular Titles
Missing Air Crew Reports, WWII
WWII US Air Force Photos Free
WWII "Old Man's Draft" Registration Cards
Civil War "Widows' Pensions"
Civil War and Later Veterans Pension Index
War of 1812 Pension Files Free Updated
Revolutionary War Pensions
Vietnam Veterans Memorial Free
Brady Civil War Photos Free
Revolutionary War Service Records


text:
Gifting note: This item ships in the manufacturer's original packaging. If intended as a gift, the packaging may reveal the contents.
About this item
Get in shape with the Gold's Gym Trainer 315 Treadmill! Gold's Gym gives you the best home workout options. This treadmill is equipped with everything you need to lose weight in comfort! With six weight-loss workouts built-in to the console, you can quickly select a program and get going! Each workout is designed by a certified personal trainer to maximize your calorie burn! Plus, with this Gold's Gym treadmill, AirSoft cushioning reduces impact on your joints by up to 15 percent as compared to running on the road! This absorption system creates a comfortable workout surface! Other features include digital speed control, power incline control, a 2.5 CHP velocity motor and a dual-grip precision response heart rate monitor.
Gold's Gym Trainer 315 Treadmill:
- 2.5 hp velocity motor
- 16" x 50" treadbelt
- Accommodates up to 250 lbs
- 6 weight-loss workouts designed by a certified personal trainer
- Oversized LCD with priority display track your speed, time, incline, distance, calories burned and pulse
- Select between 0-10 mph with the speed control arrows
- Select between 0-10 percent with the incline control arrows
- Heart rate monitor built-in to the handlebars
- Deck folds vertically for storage
- 5-year motor warranty; 90-day parts and labor warranty
See all treadmills on Walmart.com.
|Battery Type:
|Does Not Contain a Battery
|Multi Pack Indicator:
|Model No.:
|Shipping Weight (in pounds):
|Product in Inches (L x W x H):
|64.5 x 28.5 x 55.0
|Assembled in Country of Origin:
|Origin of Components:
|Walmart No.:
Customer Q&A
Ask a question
If you would like to share feedback with us about pricing, delivery or other customer service issues, please contact customer service directly.
Preview Question
Walmart does not sponsor, recommend or endorse any third party product or service, or any customer ideas or advice.
Thank you.
Questions will be checked against our question guidelines and posted within five to seven business days.


text:
Jan 08 09:19:55
..... SUPpin through a rainbow...
Date: 01/08/2011
Owner: Movie Upload User
Winter on the North Shore means waves on the North Shore and boy did we get them this past week. We had an incredible eight day run of perfect, then way too big, then back to perfect waves up and down the north shore. Read more...


text:
Dec 24, 2012 at 08:23AM EST
What happened to the episodes? We haven't gotten one for like 2 months now.
Cheezburger Blog Advertising Developers Jobs Contact Us
Legal Information: Know Your Meme ® is a trademark of Cheezburger, Inc. By using this site, you are agreeing by the site's terms of use and privacy policy. © 2007-2015 Cheezburger, Inc.
Login Now!
Sign up Now!


text:
Start Training for the 2015 Mount Wilson Trail Race
Sierra Madre, CA. - January 30, 2015 - The Mt. Wilson Trail Race Committee is again offering a comprehensive 17-week program to get runners ready for the challenging Mt. Wilson Trail Race, scheduled for Saturday, May 23, in Sierra Madre. This program is free and the public is invited to participate.


text:
Super 8 Bismarck
No availability found through our partners. Please contact the business directly or check some of our recommended alternatives.
How has this motel rated in the past?
- Continental Breakfast
- Free High-Speed Internet
- Business Services
- Free parking
- Pets allowed
Photos of Super 8 Bismarck
Travelers also recommend:
TripAdvisor Reviews Super 8 Bismarck
Travel Blogs from Bismarck
A chillaxed day in and around Bismarck
After a fantastic breakfast we left for Fort Lincoln State Park. Happy to get out of the car we followed some trails. Terrorized by mosquitoes we speeded up the hike to get back to the car ;-). The rest of the day we were on the lookout for the Missouri, fascinated by it's majesty and beauty. Dinner at the local pub ended the day. We have not decided which way we are driving tomorrow, but we're heading for the Black Hills. Pictures will follow and you'll find out which way we ...
A long drive
... drove on and crossed the border and entered North Dakota. The landscape was filled with oil drilling facilities and trailer parks for the workers. We were happy to arrive in Bismarck where we're staying at Kay's B&B. Now it's time for Apéro and a big ...
North Dakota State Capitol
... outside pictures of the capital. But it was not impressive, it was reminded us of just a large medical office building. The pictures I've included inside were more interesting. They did offer a 45 minute tour, but due to repair work being done the tour was shortened and therefore so were the photo ...
Wet finale...
Today was a cool and very wet day in the Bismarck area. We toured the State Capitol building including the Roughrider Gallery...portraits of famous North Dakotans. We also toured the beautiful North Dakota Heritage Center. Due to the rain we passed on our planned Lewis & Clark Riverboat Cruise and headed to Fort Abraham Lincoln State Park where we toured in the rain then happily stopped to camp for the night. Tomorrow we leave for ...
In Bismarck, North Dakota
... and do a pretend trip and it was called Trip Across America Tick tock
8:59 pm: that's how I'm going to bug her Tick tock
Linda 9:01 pm: Consider it done. I'm sure the teacher will enjoy the bugging. NOT! (But that's why ...


text:
Sort by:
- Shoots spring 2015 (mainly weekends) in L.A.
- Seeking submissions from: Los Angeles, CA
Casting "Transference," a film. Synopsis: Six teens have fun at a house while the mother of the household is out of town. They discover an ancient game in her antique collection and decide to play, not realizing what they are in for. Director states: "We are casting underaged teens to ...Mar 19
Nashville Shakespeare Festival 2015
- Season runs Nashville, TN.
- Auditions: Apr 24, 25 in Nashville, TN and Apr 24, 25 in Nashville, TN
Seeking actors for the Nashville Shakespeare Festival 2015 Season. Season includes "Henry V" (Nat McIntyre, dir. Rehearsals begin July 13; runs Aug. 13-Sept. 13); "King Lear" (Denice Hicks, dir. Rehearsals begin Dec. 7; runs Jan. 7-31).Mar 19
- Rehearses early to mid April; shoots late April in L.A.
- Auditions: Apr 3, 4 in Los Angeles, CA and Apr 3, 4 in Los Angeles, CA
Casting "Thumb and Toe," a film about a woman who goes to extreme measures to make her body her own. Director states: "This film is a co-production between the CalArts Film Directing Program and the German Film and Television Academy in Berlin." Synopsis: Sienna is a high achieving graphic designer ...Mar 19
- Shoots Apr. 3 in Malibu, CA.
- Seeking submissions from: Los Angeles, CA
Casting "Meat," the story and adventure of French tourists trying to talk to an American girl, with an unexpected twist.Mar 19
- Rehearsal begins July 7; runs July 30-Aug. 22 in Colorado Springs, CO.
- Auditions: Apr 4 in Colorado Springs, CO
Casting "A Midsummer Night's Dream," by William Shakespeare.Mar 19
- Rehearsed May 31-July 15, in Seattle, WA. Performances run July 16-August 1, at the Richard Hugo House, in Seattle, WA.
- Seeking submissions from: Seattle, WA
Casting the northwest premier of the play, "Sidewinders." Project description: "An absurdly existential wild western written in the style of waiting for Godot, this play advocates the acceptance of a gender spectrum and is ultimately about love."Mar 19
- Rehearsals early-mid April; shoots late April in L.A.
- Auditions: Apr 3, 4 in Los Angeles, CA and Apr 3, 4 in Los Angeles, CA
Casting "The Feeling," a film about making a mess and inviting death into a world where cleanliness is God. Director states: "A co-production between the film directing program at CalArts and the DFFB German Film and Television Academy in Berlin. Set in the future, Anna (40) and Jona (45) live ...Mar 19
- Rehearses this April in NYC; shoots May 1-3 in Brooklyn.
- Auditions: Apr 4, 5, 11, 12 in New York, NY and Apr 4, 5, 11, 12 in New York, NY and Apr 4, 5, 11, 12 in New York, NY and Apr 4, 5, 11, 12 in New York, NY
Casting a short for NYU, "Mortsan," in which a 70-year-old veteran fearing an asynchronous death between his mind and body, struggles to find a suitable caretaker for his turtle so that he may euthanize himself.Mar 19
- Performances run July 10-August 16, at Lakewood Center for the Arts in Lake Oswego, OR.
- Auditions: Apr 4, 6 in Lake Oswego, OR and Apr 4, 6 in Lake Oswego, OR
Casting the play, "Unnecessary Farce." Project description: "In a cheap motel room, an embezzling mayor is supposed to meet with his female accountant, while in the room next door, two undercover cops wait to catch the meeting on videotape. But there's some confusion as to who's in which room."Mar 19
- Rehearsal and production will occur mid-April in the L.A.
- Seeking submissions from: Los Angeles, CA
Casting a short, "Allison's Impasse," in which a film school student struggles with getting her student film completed on time.Mar 19
- Shoots June-July in Austin, TX.
- Seeking submissions from: Austin, TX; Dallas, TX; Houston, TX; San Antonio, TX; Lubbock, TX
Casting "It's Cold in Texas," in which a womanizer has an identity crisis.Mar 19
- Shoots Apr. 25 in NYC.
- Seeking submissions from: Long Island, NY; Queens, NY; Brooklyn, NY; Manhattan, NY
Seeking models and actors for a music video that will be broadcast on Vevo, and later picked up by MTV. The Artist is 'S-Quire', and his hit single is entitled "The One I Love." For more info, visit www.mtv.com/artists/s-quire.Mar 19
- Shoots in early-April in Vancouver, BC.
- Seeking submissions from: Vancouver, BC
Casting a lifestyle video shoot. Project description: "A couple take in a tranquil area in this lifestyle piece for web usage."Mar 19


text:
Category Archives: Uncategorized
Change is hard. Change is good…sometimes. Each of the Big Three sports is considering major changes in order to make their games more appealing. Apparently "that's the way it has always been done" isn't good enough. Minor League Baseball, … Continue reading
Even at the end of his career, when Hot Rod Hundley's eyes and memory weren't what they once were, he was still a big part of the show. He never showed up at EnergySolutions Arena, a.k.a. the Delta … Continue reading
Talking with an Oregon sportswriter in Portland, last week, he told me he has enjoyed his interactions with Larry Krystkowiak. Like me, he also covered the Ute coach when he was an NBA player. So it came as no surprise … Continue reading
In the department of strange NCAA occurrences, I present last Wednesday's press conference, preceding the Utah-Stephen F. Austin game. All reporters are required to state a name and affiliation before proceeding with questions, but when Larry Krystkowiak took the podium, … Continue reading
Last time I was in the Moda Center, it was still the Rose Garden. Jeff Hornacek was standing outside the locker room talking about retirement. The Jazz had been easily dispatched by the Trail Blazers in spring of 2000. … Continue reading
At one point, the Utes were expecting to be a No. 2 seed in this weeks NCAA tournament. Then No. 3. They ended up No. 5 in Thursday's game against No. 12—seeded Stephen F. Austin. Whether that's a huge disappointment … Continue reading
Great work by Deseret News staffer Roy Burton on Damian Lillard forgetting to tell his mother he had graduated from college. The Weber State alumnus told a writer at ForwardCenter.net, a Blazers' blog, that he mentioned his graduation during a … Continue reading
Much has been said about Michele Roberts comments regarding media access to the locker room. It's safe to say the executive director of the NBA Player Association thinks reporters are hanging out just to hang out. Among her observations are … Continue reading
One of the most appealing things about ex-Jazz forward Andrei Kirilenko was that he did things with his heart. He stayed in Salt Lake, in part because he and his wife loved it. He cried when he had problems figuring … Continue reading
Going into tonight's game, the Jazz are 19-34, the identical record they had last year at this time. So are Jazz fans being sold a load of goods by team management? The complicated thing is that the Jazz do seem … Continue reading


text:
Winner South Dakota: We want you to be a part of our future. We invite you to take a moment to acquaint y... more
Official site of Holiday Inn Express & Suites Winner. Stay Smart, rest, and recharge at Holiday Inn Expre... more
The best selection of winter clothes accessories in Southern South Dakota! Whether you're looking to keep... more
Our mission is to help people manage the risks of everyday life, recover from the unexpected and realize ... more
Edward Jones is an investment firm that believes your financial goals deserve a face to face conversation... more
For more than 80 years, we've made it our business to give our customers peace of mind by making their in... more
Affordable living at it's best in Winner visit our website for more info at http://frontierapts.net more
Top Trending Articles
Which Camping Style Fits Your Personality?
Many different types of camping are popular with people... more
How to Plan a Camping Trip to the Rocky ...
Get last-minute camping tips to make your next trip to ... more
How to Pack For Your Next Camping Trip
So you're off on holiday with the Family, you lucky thi... more
Top 5 Places to Camp With Your Family in the ...
Camping is a wonderful experience everyone can enjoy. L... more
How to Safely Take Your Dog With You When You...
Camping with your dog can be fun, but you need to keep ... more
© 2015 Local Corporation - US Patent 7,231,405
Some data provided by Acxiom. Questions and Comments?


text:
retro fitting non grounded receptacles
have a simple question: i would like to replace the old non ground receps in my 1955 house with grounded, three prong receps. each existing outlet box is metal and has a white and black entering the box but no green or bare wire. how would you suggest i install the new receps with 3 prongs? Should i use those "green clips" that attached to the side of metal boxes and that hold the bare wire which is then screwed onto the green ground screw of the new recep? what is the right way to go? thanks!! larry
I assume you want a ground for the added safety. If so, your "ground" needs to be connected to an actual ground. It is not safe to assume that all metal boxes are grounded. If it is just a metal box in the wall, then that won't help at all.
If those boxes aren't connected to ground, you would need to run new cable to each outlet you want grounded.
You can obtain nearly the same level of protection by using GFCI receptacles.
This is a code approved way to put 3-hole receptacles on a circuit without a grounding conductor.
Every GFCI receptacle comes with the required stickers.
The 'No equipment ground sticker' goes on the GFCI receptacle, while any downstream (load side of the GFCI) 3-hole duplex receptacles get the 'GFCI protected' AND a 'No equipment ground' stickers.


text:
Date: 3 years ago
Runtime: 8:56
Views: 107,760
Adam Comes Back For More! (Part 2 - CUM)
http://www.xtube.com as he stands up for a couple minutes and has his way with my throat. Once again, i lightly lick his balls while he gets himself close and then blasts his usual thick, gooey, rich load of baby batter all over my mouth and face. And although the camera gets cut off at the very end, you can BET that I swallowed every last drop. Fucking delicious! 8===D
cum, cumshot, suck, blowjob, swallow, balls, uncut, throat, str8, straight, ginger, skater, bro, tongue, lips, buddy, prep, suckjob
Categories: General Gay, Blow Jobs, Amateur
Top Comments
All Comments
Kaden Smith of Houston, Texas y'all
damn what a beautiful face too
Wish you'd let one of these hot young guys get up your arse
GEEZ Kaden, that was the hottest of the three with Adam! lol He has progressed - from abrupt edits to gay porn on the laptop and even a look at his handsome face - also, he can`t keep his hands off you! You`re the best!!!
Oh, yeah!!! Fucking delicius!!!
he has a hot ass uncut cock i love to swallow him too
Mmmmm ... delicious foreskin!!!
very hot, and inspiring. ok, i'm gonna get a hot straight boy now haha : ]
Well that was hot
My favourite position is on my back with a guy fucking my throat so that they can control getting the full length of their cock into my throat and man what a beautiful cock this guy has
fucking hot and like your written background descriptions for each video
We want more Adam vids! He is adorable :P
Fuck man you are good! I'm dripping with precum and now one to suck me off. Thanks, lol
Does Adam have a gf? Did he ever have anal sex?..It'd be great if u tell us more about him
Cocksuckers like you make uncut men like myself feel appreciated.
You two are so hot! Really! Which you were in my country
Foooooooock, this is good!
Wow, what a beautiful piece of uncut and fat meat, yum!
best videos on here!!!
it is truly amazing how much you love cum. I LOVE IT
ver yhot
great sucking
i love your vids....thanks for posting
very hot
Your videos are work of art!
Damn, you're the hottest cumswallower on the net! Love to see you taste and swallow that cum, and I loooooove your hot video detail desriptions! Good job!!


text:
Latest Commission on Accreditation of Rehabilitation Facilities Stories
Regional healthcare leader successfully attains the highest industry recognized standards for acute inpatient medical rehabilitation care AMARILLO, Texas, Dec.
CARF International announced that NSIGHT Psychology & Addiction has earned preliminary accreditation for its Intensive Outpatient Treatment: Integrated: AOD/MH (Adults) program.
In its latest blog post, Best Drug Rehabilitation, which offers treatment programs and believes that having family close by during a stay in rehab can make a big difference in whether or not the
The multi-location organization has been providing superior treatment and support services throughout Southern Maryland for over 40 years.
Regional healthcare leader setting course for meeting the highest industry recognized standards for acute inpatient medical rehabilitation care AMARILLO, Texas, June 30, 2014 /PRNewswire/
The Commission on Accreditation of Rehabilitation Facilities (CARF) is an independent non-profit accreditor of health and human services.
Regional leader in acute inpatient medical rehabilitation voted best rehab for 2nd consecutive year MARLTON, N.J., June 16, 2014 /PRNewswire/ -- Marlton Rehabilitation Hospital ("Marlton
NE Delta HSA, which directs the operation of community-based programs to improve the quality of life of people with major mental illness, developmental disabilities and addictive disorders, receives
The Narconon Arrowhead drug rehab facility has just launched a monthly newsletter with updates on the facility, reviews, and addiction and recovery information. Canadian,
Narconon Arrowhead holds a 48th Anniversary event to celebrate 48 years of effective rehabilitation and the first Narconon program. Canadian, Oklahoma (PRWEB)
- One who brings meat to the table; hence, in some countries, the official title of the grand master or steward of the king's or a nobleman's household.


text:
Subscribe to TESOL Blog
TESOL English Language Bulletin
- Expansion, more funds needed for English language program 31 March 2015
- The impact of Moroccan students' attitudes towards English language on speaking skill 31 March 2015
- After learning new words, brain sees them as pictures 31 March 2015
- How to measure English learners' development more accurately 31 March 2015
- Cebuano? Here are all the languages Jersey City students speak 31 March 2015
TESOL Videos
TESOL president, speaks about TESOL.
Tag Archives: two-word verbs
Figuring out the frequency of two word verbs
The December 2011 TESOL Quarterly contains a wonderful study by Dilin Liu analyzing the frequency of phrasal verbs across several corpora. This is a really useful article for teachers and materials writers, with lots of great information in it and … Continue reading
Posted in TESOL Blog Tagged as analysis, corpus, Dilin Liu, ELT, ESL, frequency, idioms, Joe McVeigh, phrasal verbs, teaching, TESOL Quarterly, two-word verbs 2 Comments


text:
Center for Entrepreneurship
Entrepreneurship in Chico goes back to its founding by the visionary pioneer John Bidwell in 1860. The Chico State Center for Entrepreneurship is dedicated to keeping that same entrepreneurial spirit alive in Chico and throughout the North State. Since its founding in 2005, the Center has strived to connect students with the community and a wide range of entrepreneurs from around the country.
Today, the Center works hand-in-hand with the College of Business and our thriving academic program in Entrepreneurship to help introduce students to a place beyond "getting a job" and into a world of creating their own future. Our growing number of success stories, to which you'll find reference throughout this web site, are but the tip of the iceberg. We believe entrepreneurship is a gateway for young people to better understand themselves for whatever they end up doing.
We are under no illusions that the entrepreneurial skills learned here at CSUC will immediately result in a new business; it's hard enough for anyone to start up a new business, let alone a newly minted graduate. Nevertheless, we are convinced the skills learned in our entrepreneurship program and in the Center will carry forward in a student's life and can be used in a wide variety of ways.
In the meantime, of course, there is, as previously stated, a growing number of students and recent graduates who have started businesses. Some are already thriving. Most are wonderful learning experiences. All are exciting. So, what are you waiting for?
We invite you to dive in.
Peter Straus
BCC Spring 2015
Get your business concepts ready! On April 15th the Chico State Business Concept Competition finals will be held. Submit your 1-2 page abstract by Thursday April 2nd, 2015 at 5:00pm. Submit via email to: email@example.com
Access the BCC Rules and Abstract Guidelines (.pdf).
Chico State eWeek has turned into eMonth 2015 - April 2nd - 30th
Mark your calendars for a whole month devoted to entrepreneurship–with 24 speakers, movie screenings, entrepreneurs-in-residence and a multi-school business concept competition. All events are open to the public and are FREE!
View the Speaker Biographies (.pdf) and Schedule of Events (.pdf)
Chico State Alum Achieving Inc. 500
Alumni Andrew Gazdecki started a small company called Bizness Apps while attending Chico State. It is now the world's leading mobile app development platform for small businesses, experiencing a growth rate of more than 5,000% in four short years, was ranked as the 58th fastest growing private company by Inc. magazine.
The Center for Entrepreneurship and 3CORE Forge Partnership
Chico, CA – The Center for Entrepreneurship (CFE) at CSU, Chico and 3CORE, Inc. are pleased to announce its formal partnership between the two organizations with the goal of creating and fostering an entrepreneurial ecosystem for new and emerging businesses. For more information on the partnership, click here.
Student Success
Upper Park Designs
Disc golf anyone?
Often it seems student entrepreneurs are concentrating exclusively on building web sites and apps, but it's simply not so. Case in point, the winners of the Center for Entrepreneurship's Spring 2011 Business Concept Competition (Hobie Jensen, Jon Richardson and Pat Edelman, aka Upper Park Designs) have just released their product line of backpacks for the casual disc golfer.


text:
Moms & BabiesThe Stefani-Rossdales
Gwen Stefani, Gavin Rossdale and their two boys, Kingston and Zuma, are a fab family of four. Take a look at their rockin' life, both on and off the road
Credit: Axelle/Bauer-Griffin
Updated: Friday Sep 06, 2013 | 07:00 PM EDT By: Shanelle Rein-Olowokere and Karen J. Quan
More from PEOPLE
Latest News!
- Sean Hayes & Husband Lip Sync Iggy Azalea & Jennifer Hudson's 'Trouble'
- Read the Cover Story: At Home with Britney Spears and Her Boys!
- FROM TIME: Police Say Germanwings Pilot 'Hid' Mental Illness Before Crash
- What Does Dolly Parton Think of Taylor Swift?
- What 'Rite of Passage' Is Nick Jonas Looking Forward To?


text:
Montreal is in a snit because someone in Ottawa suggested honouring Maurice Richard. Fine: Toronto should take the lead in establishing the Rocket Richard Bridge. Just for a start.
Mostly, the anger stems from the sheer Ottawa-ness of it all, a bright pre-electoral bauble aimed at currying support among nostalgic voters without consulting local voters
The NDP's stand against tolls suggests Mr. Mulcair realizes his dream of besting both the Conservatives and Liberals is just that – wishful thinking – and is instead reinforcing his defences in his home province.
For once, are we reading too little into byelection results? Is the new Champlain Bridge design too boring? Should religious doctors be able to deny patients birth control? These are the questions that preoccupy is this Canada Day week.
Michel Fournier was at the helm of the the Federal Bridge Corp. when it chose SNC-Lavalin to carry out a $125M refurbishing of the Jacques Cartier Bridge
When the bridge was completed in 1962, it was recognized by all as a tremendous achievement and a wonderful reflection of the energy and drive.
Public-private partnerships are a better model for building public infrastructure.
Ensuring the announced service life of 100 years would require a design life of about 150 years
A modern bridge is expected to last more than a century. Yet barely five decades after it opened in 1962, the Champlain Bridge is entering its twilight years
The briefing notes said the government would face 'significant pressure' to build a new bridge — a promise the government made less than six months later