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- Officer's trial exposes fault lines over police shootings
- Black-footed ferrets return to where they held out in wild
- Senator: Is Medicare drug plan vulnerable to exploitation?
- As Fed ends meeting, few expect much clarity on next hike
- Washington scientist launches effort to digitize all fish
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Officer's trial exposes fault lines over police shootings
PORTSMOUTH, Va. (AP) - It's a familiar story: an unarmed black male killed at the hands of a white law enforcement officer. But it didn't take place in Baton Rouge, Minneapolis, or any of the other cities most recently at the center of discussions about police use of force and race. | 3:01 a.m.
Black-footed ferrets return to where they held out in wild
MEETEETSE, Wyo. (AP) - A nocturnal species of weasel with a robber-mask-like marking across its eyes has returned to the remote ranchlands of western Wyoming where the critter almost went extinct more than 30 years ago. | 2:24 a.m.
Senator: Is Medicare drug plan vulnerable to exploitation?
WASHINGTON (AP) - A senior senator is examining whether Medicare's prescription drug benefit is vulnerable to manipulation by pharmaceutical companies that set very high prices for medications. | 2:17 a.m.
As Fed ends meeting, few expect much clarity on next hike
WASHINGTON (AP) - With consumers spending and employers hiring more freely, the U.S. economy has fared better of late. But when the Federal Reserve ends its latest policy meeting Wednesday, most analysts think it will signal that it wants to see further gains before raising interest rates again. | 2:06 a.m.
Washington scientist launches effort to digitize all fish
SEATTLE (AP) - University of Washington biology professor Adam Summers no longer has to coax hospital staff to use their CT scanners so he can visualize the inner structures of sting ray and other fish. | Updated: 1:36 a.m.
47 people arrested at Guns N' Roses concerts in New Jersey
EAST RUTHERFORD, N.J. (AP) - Police say more than 40 people were arrested at two Guns N' Roses concerts in New Jersey over the weekend on a variety of charges including assaulting police and selling items without a permit. | 1:15 a.m.
Coast Guard: 46 people rescued from sinking boat off Alaska
ANCHORAGE, Alaska (AP) - Two Good Samaritan vessels rescued 46 people Tuesday night who abandoned their sinking fishing boat in the Bering Sea off Alaska's Aleutian Islands, the Coast Guard said. | Updated: 1:04 a.m.
US Navy sued over live-fire training plan in Pacific
HONOLULU (AP) - Community members and an environmental group on Wednesday sued the U.S. Navy, the Department of Defense and the secretary of defense over a plan to turn two Pacific islands into live-fire testing sites. | 12:40 a.m.
The Latest: 46 people rescued from sinking vessel off Alaska
ANCHORAGE, Alaska (AP) - The Latest on the rescue of 46 crew members who abandoned a fishing vessel in Alaska's Aleutian Islands on Tuesday (all times local): | 12:15 a.m.
Boston boy found dead after wandering away from summer camp
BOSTON (AP) - Police are trying to determine how an 8-year-old boy wandered away unnoticed from a city-run summer camp at a Boston beach and ended up dead, his body found five hours later in the water. | Yesterday 11:59 p.m.
Black-footed ferrets return to where they held out in wild
MEETEETSE, Wyo. (AP) - A nocturnal species of weasel with a robber-mask-like marking across its eyes has returned to the remote ranchlands of western Wyoming where the critter almost went extinct more than 30 years ago. | Updated: Yesterday 11:00 p.m.
The Latest: Black-footed ferrets released near Yellowstone
MEETEETSE, Wyo. (AP) - The Latest on the release of black-footed ferrets in an area of Wyoming where they almost went extinct. (all times local): | Yesterday 10:29 p.m.
California wildfire forces shutdown of famed Big Sur parks
BIG SUR, Calif. (AP) - California's signature parks along the Big Sur coastline that draw thousands of daily visitors were closed Tuesday as one of the state's two major wildfires threatened the scenic region at the height of the summer tourism season. | Updated: Yesterday 10:28 p.m.
The Latest: Clinton sees 'biggest crack' in glass ceiling
PHILADELPHIA (AP) - The Latest on the Democratic National Convention (all times EDT): | Updated: Yesterday 10:24 p.m.
Environmentalists take aim at TransCanada pipeline project
BOSTON (AP) - Environmentalists are again taking aim at the company that proposed the Keystone XL pipeline - this time for another of its projects they fear would send hundreds of supertankers laden with crude oil down the Atlantic coast to refineries in Texas and Louisiana. | Updated: Yesterday 9:50 p.m.
Sanders loyalists bash Clinton nomination, clash with police
PHILADELPHIA (AP) - Bernie Sanders loyalists protested inside and outside the Democratic National Convention site and clashed with police on Tuesday after Hillary Clinton won the party's presidential nomination. | Updated: Yesterday 9:38 p.m.
Arizona sheriff: 3 slain in possible murder-suicide
PHOENIX (AP) - Authorities found the bodies of a mother and her two children inside a home in suburban Phoenix in what could be a case of murder-suicide, authorities said Tuesday. | Yesterday 9:06 p.m.
Young boy who went missing from Boston beach found dead
BOSTON (AP) - An 8-year-old boy who went missing from a summer day camp at a Boston beach has been found dead in the water. | Yesterday 8:17 p.m.
Authorities ID remains as those of missing treasure hunter
ALBUQUERQUE, N.M. (AP) - A treasure hunter who disappeared this year while searching for an author's cache of gold and jewels in the New Mexico wilderness was confirmed dead by authorities Tuesday after his remains were discovered west of Santa Fe. | Updated: Yesterday 8:03 p.m.
Indiana GOP panel backs Gov. Pence's preferred successor
INDIANAPOLIS (AP) - Indiana Republican leaders selected Lt. Gov. Eric Holcomb on Tuesday to replace Gov. Mike Pence on the November ballot, backing the governor's preferred successor over two members of Congress who also sought the nomination when Pence withdrew to become Donald Trump's running mate. | Updated: Yesterday 7:37 p.m.
Last charges dropped in case over Planned Parenthood videos
"Regardless of however it came, we're happy today this matter is over." | Updated: Yesterday 7:30 p.m.
Wish granted: Six-year-old boy is garbage man for a day
SACRAMENTO, Calif. (AP) - Ethan Dean has always dreamed of being a garbage man. He never tires of playing with toy garbage trucks and loves to watch the real ones drive past his house. | Updated: Yesterday 7:01 p.m.
Los Angeles officer shot; suspect dies in running gun battle
LOS ANGELES (AP) - A man fleeing police was killed after he wounded an officer during a running gun battle, authorities said. | Updated: Yesterday 7:01 p.m.
St. Louis police add ice cream truck as outreach tool
ST. LOUIS (AP) - St. Louis police have a question to ask as they take their new truck into neighborhoods: One scoop or two? | Yesterday 6:38 p.m.
Feds: NYC bank burglars stole $5 million in cash, diamonds
NEW YORK (AP) - Bank burglars reminded New York's top police official of a Hollywood movie as they pulled off two nighttime heists, snatching about $5 million in cash, diamonds, jewelry, coins and baseball cards from safe deposit boxes before they were caught, authorities said Tuesday as they announced the arrest of three men. | Updated: Yesterday 6:35 p.m.
Recent deaths of kids in hot cars prompt pleas to parents
DALLAS (AP) - The deaths of four children in hot cars in recent days has brought the number across the U.S. this year to at least 23, nearly matching the total for all of last year and prompting experts to plead for vigilance and warn parents that it can happen to anyone. | Updated: Yesterday 6:30 p.m.
Why Vermont delegates want to go last in roll call vote
PHILADELPHIA — Sen. Bernie Sanders has waited 15 months to watch his delegates cast ballots for him at the Democratic National Convention. On Tuesday night, his wait will end when nearly 1,900 fervent Sanders supporters will vote for his nomination as the party's presidential nominee. | Yesterday 5:03 p.m.
Suspect in student's killing was convicted of similar attack
TOLEDO, Ohio (AP) - The circumstances, separated by 26 years, are chilling: Two women are attacked while bicycling alone down lonely Ohio country roads on warm July days. | Updated: Yesterday 4:59 p.m.
Black women take over top spots for Democratic convention
PHILADELPHIA — Three black women are running the Democratic National Convention this week, taking charge of the top jobs that keep the massive gathering on schedule and, as much as possible, on message. | Yesterday 4:52 p.m.
Delegate, 102, shares a tender moment with congressman
Jerry Emmett, the 102-year-old honorary chair of the Arizona Democratic delegation, shared an unexpected and tender moment with U.S. Rep. John Lewis of Georgia on Tuesday. | Yesterday 4:44 p.m.
Violence at club signals changing city in southwest Florida
FORT MYERS, Fla. (AP) - A couple of doors down from Club Blu, where a young man and a 14-year-old boy were killed while attending a teen party, shop owner Idis Edouard hoisted a rod above his head to gently move a girl's blue dress aside. | Updated: Yesterday 4:28 p.m.
Black Lives Matter protesters want to send message to Clinton
Several hundred demonstrators aligned with the Black Lives Matter movement and other social justice groups took to the streets of North Philadelphia Tuesday afternoon to raise concerns about police brutality and speak out about presumptive Democratic nominee Hillary Clinton. | Yesterday 4:12 p.m.
Powerball jackpot $422 million after months without winner
DES MOINES, Iowa (AP) - The Powerball jackpot has soared to $422 million thanks to nearly three months without a winner of the big prize. | Yesterday 3:55 p.m.
Dems make Clinton official today as dissent lingers
PHILADELPHIA — The protracted, arduous struggle for control of the heart of the Democratic Party comes to a close tonight when delegates to the national convention formally select Hillary Clinton as their candidate for president -- much to the chagrin of Bernie Sanders' faithful. | Yesterday 3:54 p.m.
Condemned home invasion killer is resentenced to life term
NEW HAVEN, Conn. (AP) - A man condemned to die for killing a woman and her two daughters during a 2007 home invasion was resentenced Tuesday to life in prison without the possibility of release because the state abolished the death penalty. | Updated: Yesterday 3:33 p.m.
Dr. Dre handcuffed after driveway encounter
The Los Angeles County Sheriff's Department says it cited Dr. Dre after a man accused him of pointing a gun at him because he was blocking the rapper's driveway in Malibu, Calif., on Monday. | Yesterday 2:41 p.m.
WATCH LIVE: Democratic National Convention Day 2
On Tuesday, delegates hear from former President Bill Clinton. His eighth convention speech takes place on the night delegates formally select his wife as their candidate for president. | Yesterday 2:38 p.m.
Jinger Duggar engaged to ex-soccer player Jeremy Vuolo
For once, Jinger Duggar won't have to play her family's engagement and wedding photographer — because this time, she'll be the bride. | Yesterday 2:37 p.m.
NYPD bee squad ready for sting operations on urban swarms
NEW YORK (AP) - Daniel Higgins is one cop who knows a thing or two about sting operations. | Updated: Yesterday 2:32 p.m.
Sanders touts Clinton in surprise visit to N.Y. delegates
Fresh off his rousing speech Monday night on the convention floor, Bernie Sanders visited New York's delegation Tuesday morning — urging delegates to back Hillary Clinton for president. | Yesterday 2:31 p.m.
Waiters at this Arkansas Olive Garden go the extra mile for parents
A mom found comfort in more than just salad and breadsticks last week at an Arkansas Olive Garden. | Yesterday 2:26 p.m.
What is a convention roll call vote, and what does it mean for Clinton?
Tonight, Hillary Clinton will officially secure the number of delegates (regular delegates and superdelegates combined) to become the Democratic Party's nominee during a process called the "roll call." | Yesterday 2:25 p.m.
Conquering Kilimanjaro: 'You felt like you wanted to die'
It was minus-30 degrees halfway between the final ridge of Tanzania's Mount Kilimanjaro and its 19,341-foot-tall peak and Max Crider was stuck. | Yesterday 2:22 p.m.
Ohio teens trick Google into honoring Harambe
CINCINNATI — If a teenage boy was given the chance to rename the street his high school was on, his mouth would likely salivate at the opportunity for mischief. | Yesterday 2:13 p.m.
The Latest: Pence praises GOP replacement choice
INDIANAPOLIS (AP) - The Latest on replacing Republican vice presidential nominee Mike Pence as the GOP candidate for Indiana governor (all times local): | Updated: Yesterday 2:10 p.m.
'Pokémon Go' downloads top 75 million
The viral mobile hit Pokémon Go just keeps on going. | Yesterday 2:07 p.m.
Beyoncé, Adele lead MTV's VMA nominations
Lemonade is the drink of choice at this year's MTV Video Music Awards. | Yesterday 2:00 p.m.
'Magnificent Seven' to open Toronto Film Festival
Director Antoine Fuqua's Western, The Magnificent Seven, will open the Toronto International Film Festival, organizers announced Tuesday. | Yesterday 1:52 p.m.
$15 billion Volkswagen emissions deal clears 1st hurdle
SAN FRANCISCO (AP) - A nearly $15 billion settlement over Volkswagen's emissions cheating scandal cleared a key hurdle Tuesday, with a federal judge giving preliminary approval to the deal that includes an option for owners to have the carmaker buy back their vehicles. | Updated: Yesterday 1:50 p.m.
Roger Federer announces he's done for season
Roger Federer announced he's out for the rest of the season and will miss the upcoming Rio Olympics. | Yesterday 1:48 p.m.
Texas dentist shares brutal pictures of shark attack
A Texas dentist is recovering after being attacked by a bull shark while spearfishing in the Bahamas over the weekend. | Yesterday 1:39 p.m.
Warrant: Mom chained kids to bed, locked them in rooms in North Carolina home
CHARLOTTE, N.C. — A mother has been arrested after police say she chained her four children around their wrists and waists inside their North Carolina home. | Yesterday 1:36 p.m.
Bus driver admits to raping student, walks free
A 35-year-old former school bus driver won't serve time in jail after pleading guilty to raping a 15-year-old student in Hamilton County, Tenn. | Yesterday 12:58 p.m.
Booker responds to Trump attack: 'I love Donald Trump' | Video
"Let me tell you right now: I love Donald Trump. I don't want to answer his hate with hate. I'm going to answer it with love. I'm not going to answer his darkness with darkness," Cory Booker said. | Yesterday 12:22 p.m.
Some Sanders backers spurn Clinton, say they'll go Green Party | Video
Hundreds of pro-Sanders demonstrators marched through Center City on the first day of the Democratic National Convention on Monday, chanting "Bernie or Jill, No Hill." | Yesterday 12:14 p.m.
Study: More Colo. children hospitalized for mistaking pot brownies, cookies for treats
As more Colorado adults become comfortable with marijuana in their homes, more young children are ingesting the drug, a new study reports. | Yesterday 12:10 p.m.
Big Ten commissioner Jim Delany sets window on retirement
CHICAGO – One of the most powerful men in college sports is retiring – someday. | Yesterday 12:03 p.m.
Obama expanding refugee program for Central Americans
WASHINGTON (AP) - The Obama administration will soon expand efforts to help Central American families and children legally immigrate to the United States amid another surge of migrants caught crossing the border illegally. | Updated: Yesterday 12:02 p.m.
Review: 'Cursed Child' has that Harry Potter magic
LONDON — "Apparently, wizardry's moved on since we were kids," notes Harry Potter to Hermione Granger, in Harry Potter and the Cursed Child: Parts One and Two (* * * ½ our of four stars). | Yesterday 11:52 a.m.
PHOTOS: Wild and wacky sights from the convention
The spectacle of the Democratic National Convention wasn't confined to Philadelphia's Wells Fargo Center. Enjoy some of the sillier sights.
Pokémon Go isn't a defense, lawyers say
SAN FRANCISCO — "The Pikachu made me do it," isn't going to cut it in court, say lawyers.
| Yesterday 11:38 a.m.
Family member holding gun shoots teen in face, kills her
TEMPE, Ariz. — A 16-year-old girl died after being shot in the face when a handgun accidentally fired as a relative was handling it, police said. | Yesterday 11:34 a.m.
Day 1: Democrats prove they can be dysfunctional, too | Video
Here are some takeaways from the first day of the Democratic convention, which began only after host Debbie Wasserman Schultz was essentially disinvited. | Yesterday 11:26 a.m.
Guanabara Bay water still overrun by sewage as Rio Olympics loom
RIO DE JANEIRO – Luiz Goldfeld was excited in 2009 when he heard the Olympics were coming to Rio de Janeiro and the sailing events would take place on Guanabara Bay. | Yesterday 11:04 a.m.
Shootings feed fears, anguish and preparation
MELBOURNE, Fla. — Not long ago, movie theaters, churches, shopping malls and even hospitals carried an unspoken air of security and civility. | Yesterday 10:51 a.m.
Fact check: Night 1 of Democratic convention
The factual inaccuracies on the first night focused on income, college tuition and something the Republican ticket had said or done. | Yesterday 10:50 a.m.
Police: Wanted man used dead man's name for decades
INDIANAPOLIS — An Indiana man who walked out on his wife and children 23 years ago has been living in Florida under a name he stole from a dead man, police said. | Yesterday 10:44 a.m.
Missouri man accused of seeking out cow for sex
FAIR GROVE, Mo. — Authorities spent six weeks investigating a case of cow rape and decided last week to charge a 68-year-old man with unlawful sex with an animal. | Yesterday 10:38 a.m.
Japan knife attack suspect: 'It's better that the disabled disappear'
An apparently disturbed man arrested in a stabbing rampage at a care facility for mentally disabled people near Tokyo on Tuesday had earlier warned authorities that he might carry out the attack, according to police and local news media. At least 19 are dead and 26 injured after the man, a former employee, allegedly broke into the facility early Tuesday and went room to room stabbing patients with a knife. | Yesterday 10:21 a.m.
ISIL claims two of its 'soldiers' killed French priest, seized four hostages
The Islamic State claimed responsibility for an attack on a French church Tuesday in which an elderly priest was killed and four hostages seized. The two attackers, whom the terror group described as "soldiers," according to the Associated Press, were later shot and killed by police. | Yesterday 10:10 a.m.
Personal attacks shadow Hillary Clinton's big moment | Video, Podcast
PHILADELPHIA — Feminists here to see Hillary Clinton become the first major-party female presidential nominee worry that the historic sound of her breaking what she calls "the hardest glass ceiling'' is being drowned out by insults — "Liar!" "Crook!'' and worse. | Yesterday 10:05 a.m.
Clinton and Sanders: Newfound allies as Democrats divide | Video
PHILADELPHIA — It's a sign of how divided the Democrats are that Hillary Clinton was counting on Bernie Sanders to put it back together. | Yesterday 9:58 a.m.
Michelle Obama, Bernie Sanders own Twitter and Google | Video
The most retweeted tweet of Day 1 of the convention was President Obama praising his wife. | Yesterday 9:51 a.m.
Inmates with hepatitis C sue Tennessee prison officials for treatment
The lawsuit says Tennessee Department of Correction officials knowingly denying inmates care for their hepatitis C constitutes cruel and unusual punishment. | Yesterday 9:43 a.m.
Party of Clinton looks different than in 1992
The question tonight is whether Bill Clinton can resonate with a more progressive party than the one he led in the 1990s. | Yesterday 9:36 a.m.
Gender, race, Trump: Michelle Obama went there | Video
"Don't let anyone ever tell you that this country isn't great, that somehow we need to make it great again, because this right now is the greatest country on earth," she said. | Yesterday 9:30 a.m.
Remains of lost Spanish fort found on South Carolina coast
PARRIS ISLAND, S.C. (AP) - Archaeologists have found the location of a long-sought Spanish fort on the South Carolina coast near Beaufort. | Yesterday 9:25 a.m.
Shutting down 'shark finning': US fishermen promise a fight
PORTLAND, Maine (AP) - American fishermen are digging in for a fight over a congressional proposal to send a message to the rest of the world by shutting down the vestiges of the U.S. harvest of shark fins, prized for soup and traditional medicine in Asia. | Updated: Yesterday 8:34 a.m.
Mom of slain Florida teen warned son of nightclub shootings
A shooting at a Florida nightclub early Monday morning killed two people and wounded as many as 17, police said. The attack apparently occurred at a teen party, billed as a "Swimsuit Glow Party," at Club Blu in Fort Myers, according to local media. | July 25, 2016
The Latest: Russian diplomat scoffs at DNC hack allegations
The Latest on the Democratic National Convention in Philadelphia. | July 25, 2016
More than 50 pro-Sanders demonstrators cited by police
Police briefly detained more than 50 people after they tried to storm the barricades outside the Democratic convention Monday. | July 25, 2016
Most evacuations canceled for Southern California wildfire
An unpredictable wildfire that destroyed more than a dozen houses has surged back to life. | July 25, 2016
Groups seek to help veteran held as motel hostage for years
ALBANY, N.Y. (AP) - Veterans' organizations have reached out to help a Korean War-era veteran who authorities say was held hostage in a motel room for four years by a man who stole his benefit checks. | July 25, 2016
The Latest: 2 shootings took place outside club last year
The Latest on a shooting a nightclub in Fort Myers, Florida. | July 25, 2016
APNewsBreak: Pfizer: Arkansas execution would 'misuse' drug
LITTLE ROCK, Ark. (AP) - An execution drug obtained by the Arkansas prison system this month appears to have been made by a subsidiary of Pfizer, even though the pharmaceutical giant has said it doesn't want its drugs to be used in executions. | July 25, 2016
Up there: Netherlands, Latvia lead world for people's height
NEW YORK (AP) - If you want to see a tall population of men, go to the Netherlands. Tall women? Latvia. | July 25, 2016
Boston Marathon bombing amputee scales mountain in Ecuador
BOSTON (AP) - Boston Marathon bombing survivor Adrianne Haslet can check mountaineering off her bucket list. | July 25, 2016
Michael Jordan donating $2 million to help address police-related shootings
Current NBA stars Carmelo Anthony, LeBron James, Chris Paul and Dwyane Wade are among the many professional athletes who have spoken out about the recent police-related shootings and the importance of social change in America, using their celebrity and the professional sports platform to do so. | July 25, 2016
Prince Harry 'regrets' not talking about Princess Diana's death
Princess Diana's sons almost never talked about their grief after their mother's 1997 death, at least not publicly; Prince Harry now says he regrets he didn't talk to anyone about the trauma. | July 25, 2016
For the first time, hotter than normal everywhere in the USA
Literally: It has never been this hot, everywhere. | July 25, 2016
Heckled offstage, Wasserman Schultz now seeks re-election
TALLAHASSEE, Fla. (AP) - The furor over leaked emails not only got U.S. Rep. Debbie Wasserman Schultz heckled out of her speaking role Monday at the Democratic National Convention; it's also providing fodder for her congressional primary opponent. | July 25, 2016
Tim LaHaye, author of "Left Behind" end-times novels, dies
The Rev. Tim LaHaye, co-author of the "Left Behind" series, a multimillion-selling literary juggernaut that brought end-times prophecy into mainstream bookstores, died Monday. He was 90. | July 25, 2016
Acquiring Aroldis Chapman gives Cubs a championship look
CHICAGO -- Go ahead, Chicago, make those parade route plans. Time to start hiring more bartenders and waitresses in Wrigleyville. Better make sure that liquor supply is locked and loaded. | July 25, 2016
Gabby Douglas back on track for Rio Olympics
Something about being at the Karolyi ranch brings out the best in Gabby Douglas. | July 25, 2016
Van in wreck with Dallas Cowboys staff bus was turning left
KINGMAN, Ariz. (AP) - Four people died when their van turned left at a highway intersection in northwestern Arizona and collided with a bus carrying staffers but no players of the Dallas Cowboys football team, authorities said Monday. | July 25, 2016
Investors learn Nintendo doesn't own Pokémon, stock tanks
Nintendo stock nose-dived Monday, falling close to 18% in Tokyo and opening more than 8% lower in the U.S., following the company issuing a press release Friday saying the hit mobile game Pokémon Go will not greatly affect its upcoming earnings. | July 25, 2016
Kabul bombing raises worries about Islamic State threat
The weekend suicide bombing in Kabul that killed 80 people has raised worries that the Islamic State may be gaining a foothold in Afghanistan, even as the U.S.-backed government makes headway in its fight against the Taliban. | July 25, 2016
Starbucks CEO Howard Schultz outlines plan to court premium coffee drinkers
Starbucks may be known for frilly frappuccinos, but CEO Howard Schultz has a new priority: premium coffee drinkers. | July 25, 2016
Busy New York subway tunnel to close for 18 months
The New York subway system will close one of its busiest tunnels for 18 months to make repairs, a move that will cause one of the largest commuter disruptions in the transit system's history. | July 25, 2016
Chevrolet could be seeing end of era in NASCAR
INDIANAPOLIS – Alba Colon doesn't take pictures of race cars. As Chevrolet's Sprint Cup program manager, she's involved in the monotonous, science-and-sweat daily business of helping crew chiefs and engineers put her manufacturer in victory lane. | July 25, 2016
Baby panda alert! Toronto Zoo has 9-month-old twins
If the kids — or you — have panda fever after watching Po and his pals in the Kung Fu Panda 3 movie, now's the time to head to the Toronto Zoo, where 9-month-old cubs Jio Panpan and Jia Yueyue and their parents are delighting visitors. | July 25, 2016
Al Gore, convention no-show, endorses Clinton
Former Vice President Al Gore pledged his support for presumptive Democratic presidential nominee Hillary Clinton on Monday despite having no plans to attend the Democratic National Committee's convention, which kicked off the same day. | July 25, 2016
Hundreds rally in Philly on opening day of convention
PHILADELPHIA -- About 100 people gathered outside City Hall Monday morning to rally on behalf of the Equality Coalition, many carrying placards supporting former Democratic presidential candidate Bernie Sanders or slamming the Democratic National Committee. | July 25, 2016
Experts: Hard to prove Russians behind DNC hack
SAN FRANCISCO – Computer security researchers say it's difficult to definitively say the cyber theft of files from the Democratic National Committee was perpetrated by Russian hackers, as some media outlets have reported. | July 25, 2016
Bernie Sanders' call to elect Hillary Clinton booed by his delegates
PHILADELPHIA – Bernie Sanders drew loud boos from his delegates Monday afternoon when he told the crowd of nearly 1,900 "We have got to elect Hillary Clinton." | July 25, 2016
Wasserman Schultz says she won't open convention
PHILADELPHIA — Outgoing party chairwoman Debbie Wasserman Schultz has told a Florida newspaper she will no longer open the Democratic National Convention on Monday. | July 25, 2016
Who's to blame for massive traffic jam on I-95 around Philly?
PHILADELPHIA -- I-95 outside the city was jammed more than most days Monday morning. | July 25, 2016
Bernie Sanders, turmoil kick off Democrats' fest
PHILADELPHIA — A lineup of progressive speakers featuring vanquished presidential hopeful Bernie Sanders on Monday highlight opening day of a Democratic National Convention already struggling to stay on message amid another email scandal and the ensuing resignation of the party's chairwoman. | July 25, 2016
Debbie Wasserman Schultz draws boos, cheers at Florida delegation breakfast
PHILADELPHIA – A breakfast for Florida delegates to the Democratic National Convention turned raucous on Monday when supporters of Sen. Bernie Sanders booed outgoing party chairwoman Debbie Wasserman Schultz off the stage. | July 25, 2016
NFL clears Peyton Manning after 7-month HGH investigation
The NFL has cleared Peyton Manning after a seven-month investigation found no credible evidence he used human growth hormone or violated the league's performance-enhancing drug policy, the league said Monday. | July 25, 2016
Paralympic swimmer Mallory Weggemann to attend Democratic convention
American Paralympic swimmer Mallory Weggemann was raised in a household where she was told she could make a difference. Every night her father would tell her that she could change the world if she wanted to. | July 25, 2016
Live coverage: Democratic National Convention 2016
Get live updates from the 2016 Democratic National Convention in Philadelphia. | July 25, 2016
NFL clears Peyton Manning after 7-month HGH investigation | Video
Peyton Manning and his wife, Ashley, "were fully cooperative with the investigation and provided both interviews and access to all records sought by the investigators," the league said in a statement. | July 25, 2016
Poll: Trump gets bump following GOP convention
Donald Trump got a 6-point bump and has passed Hillary Clinton coming out of the Republican convention, according to a new CNN/ORC poll. | July 25, 2016
Economist experiences life on a food-stamp budget
An expert's opinion on poverty is only as good as their experience. That's the reason Janet Harrah, a Northern Kentucky University economist, decided to live for one year as if she used only food stamps, also known as SNAP benefits. | July 25, 2016
One dead, one wounded in tiger attack at China safari park | Video
In surveillance video captured of the incident, the woman is seen getting out of the vehicle and walking to the driver's side. As she stands by the door, a tiger runs out of the woods and drags her away. | July 25, 2016
Cops: Man busted in decadeslong secret life that fooled his wife
He's accused of spending more than 20 years in Florida disguised as another man. | July 25, 2016
Watch Live: Democratic convention Day 1
A lineup of progressive speakers featuring vanquished presidential hopeful Bernie Sanders on Monday highlight opening day of the Democratic National Convention. | July 25, 2016
Miami judge: Bitcoin isn't money
A Miami judge ruled Monday that Bitcoin is not really money in a closely-watched decision that dismissed charges against a local man for illegally selling and laundering the virtual currency. | July 25, 2016
Simple surrogacy case turns into legal nightmare for gay couple
MILWAUKEE — Jay Timmons and Rick Olson expected little legal trouble when they filed papers in Dane County Circuit Court last year to become the fathers of a son through a surrogate mother in Wisconsin. | July 25, 2016
Up to 16 shot, 2 dead in Florida club shooting
Authorities received reports shortly before 1 a.m. Monday of shots fired near the Club Blu Bar and Grill in Fort Myers, Fla. | July 25, 2016
Cowboys tour bus involved in fatal crash in Arizona
Four people were killed when a Dallas Cowboys tour bus carrying staffers but no players and a van were involved in an accident just outside Dolan Springs in northwest Arizona on Sunday afternoon, officials said. | July 25, 2016
One dead, one wounded in tiger attack at China safari park
A woman was killed by a tiger and another injured after the pair got out of their vehicle at a China safari park over the weekend. | July 25, 2016
Sneak peek: 'Moana' is 'beautiful' quest of courage
Auli'i Carvalho hopes that fans of her adventurous teen in the animated Moana will call her a heroine rather than a Disney princess. | July 25, 2016
Bomber who blew himself up in Germany pledged allegiance to Islamic State
BERLIN — Germany is on edge following a string of attacks by refugees, including a 27-year-old Syrian man who pledged allegiance to the Islamic State before blowing himself up outside an open-air music festival in the southern part of the country. | July 25, 2016
Sofia Boutella is in the captain's seat with 'Trek'
Sofia Boutella is sitting in a prime position after her breakout performance as the alien Jaylah in Star Trek Beyond, which topped the weekend box office. | July 25, 2016
Last of slain Baton Rouge officers to be buried Monday
BATON ROUGE, La. (AP) - Hundreds of law enforcement officers from around the country streamed past the casket Monday of a slain Baton Rouge police officer, some solemnly saluting and others making the sign of the cross as they paid their respects and mourned one of their own. | July 25, 2016
Seven Russian swimmers ruled ineligible for Rio
Seven Russian swimmers were ruled ineligible to compete in Rio by their international federation and the Russian Olympic Committee. | July 25, 2016
USA Gymnastics buys Karolyi ranch
There will still be a Karolyi connection to the women's national team even after Martha Karolyi retires. | July 25, 2016
'Tampon tax' eliminated in N.Y.
ALBANY - Gov. Andrew Cuomo signed a bill into law Thursday that will eliminate sales tax on feminine hygiene products. | July 25, 2016
Texas police officer killed in apparent burglary attempt
ROUND ROCK, Texas (AP) - A Texas sheriff's deputy was shot and killed at his home north of Austin before dawn on Monday in what authorities said appeared to be an attempted burglary. | July 25, 2016
'Hillary's America' documentary cracks top 10 at box office
Politics isn't just center stage after last week's Republican National Convention in Cleveland and the upcoming Democratic National Convention in Philadelphia. | July 25, 2016
The casual viewer's guide to the Democratic convention
Everyone is talking about the conventions, and I know I'm supposed to know this — but what are they again?
| July 25, 2016
One dead, at least 10 hurt in Bavarian bar explosion
One person is dead and at least 10 hurt after an explosion at a restaurant/bar Sunday night in Ansbach, Germany, according to various media reports. | July 25, 2016
Clinton's history of hiring women includes mentoring, office crib
By any office standard, and especially for Bill Clinton's 1990s-era, male-dominated West Wing, Patti Solis Doyle had an unusual work arrangement: a baby crib in her office. | July 25, 2016
Trump's trade policies worry economists
"Our country is getting ripped off." That's the sub-headline on Donald Trump's campaign website for his position on trade. A vote for Trump is a vote for standing up to trade manipulators. | July 25, 2016
Best friends, bound by the law, bonded by gift of life
City and suburb. Black and white. A 45-year-old man built like an offensive lineman and a 58-year-old woman who is lean like a dancer. | July 25, 2016
News anchor dies in plunge over falls in Gorges State Park
TRANSYLVANIA COUNTY, N.C. — A Georgia morning news anchor plunged approximately 160 feet to her death Thursday after being swept over the top of Rainbow Falls just a day before her 25th birthday, according to authorities. | July 25, 2016
The long, troubled history of Yahoo's top execs
On Tuesday, Yahoo CEO Marissa Mayer unveiled a cost-cutting initiative that includes trimming 15% of its workforce. It's the latest chapter in Yahoo's recent drama, as the company continues to weigh how to handle its Internet businesses amid shareholder scrutiny. | July 25, 2016
Fact check: Hillary Clinton's false and misleading claims
In advance of the Democratic National Convention, which begins July 25 in Philadelphia, we present a wrap-up of some of the more egregious falsehoods from Hillary Clinton, who is set to accept her party's nomination for president later this week. | July 25, 2016
#ClintonNation: This is why Americans are voting for Hillary Clinton
Many supporters of the candidate who'll be America's first female presidential nominee don't care that she's a woman any more than they care her opponent's a man. | July 25, 2016
Verizon is winner in Yahoo sale with $4.8B bid
With Verizon's $4.8 billion acquisition of Yahoo, the telecommunications giant hopes to merge it with fellow Web portal pioneer AOL to create a new mobile and online powerhouse. | July 25, 2016
Bernie Sanders to address convention amid drama over leaked emails
WASHINGTON — Democrats hope to project an image of unity Monday as Bernie Sanders delivers opening remarks at the party's national convention. But an email scandal and the subsequent resignation of the national party's top official threaten to undermine that theme. | July 25, 2016
A guide to Day 1 of the Democratic convention
PHILADELPHIA — Donald Trump and Hillary Clinton have one thing in common: Their conventions got off to rocky starts. | July 25, 2016
Californian who tried joining Islamic State faces sentencing
SANTA ANA, Calif. (AP) - A California man who aspired to join the Islamic State group in Syria and lied to get a replacement American passport to head overseas could face two decades in prison Monday when he is set to be sentenced. | July 25, 2016
Crash kills Nebraska punter, former Michigan State punter
WAUKESHA, Wis. (AP) - Nebraska punter Sam Foltz and former Michigan State punter Mike Sadler died in a car crash in Wisconsin after working at a kicking clinic, a sheriff's department official said Sunday. LSU kicker Colby Delahoussaye was injured in the crash. | July 24, 2016
'Like a freight train': California wildfire guts 18 homes
LOS ANGELES (AP) - Flames raced down a steep hillside "like a freight train," leaving smoldering remains of homes and forcing thousands to flee the wildfire churning through tinder-dry canyons in Southern California, authorities said Sunday. | July 24, 2016
Dallas Cowboys bus involved in fatal crash in Arizona
PHOENIX (AP) - A bus carrying Dallas Cowboys staffers but no players and a van collided Sunday on an Arizona highway, killing four people in the van, authorities said. | July 24, 2016
28 injured in crash involving bus, Jeep and tractor-trailer
YEADON, Pa. (AP) - More than two dozen people have been injured in a crash involving a bus, a Jeep and a tractor-trailer. | July 24, 2016
Thousands of pro-Sanders, anti-fracking marchers hit streets
Thousands of demonstrators are taking to Philadelphia's sweltering streets Sunday. | July 24, 2016
Heat wave, drought showing no signs of slowing down
PHILADELPHIA (AP) - The heat wave gripping parts of the country including Philadelphia, where tens of thousands are descending upon the city for the Democratic National Convention this week, is not going away anytime soon and will hit a peak Monday with temperatures in the city feeling like 108 degrees. | July 24, 2016
2 years after Ferguson, recriminations roil governor's race
JEFFERSON CITY, Mo. (AP) - It has been two years since a white police officer fatally shot black teenager Michael Brown in Ferguson, Missouri, touching off days of rioting, but the political repercussions from the incident have only intensified, fanned by a governor's race in which all four Republican candidates are pledging an aggressive law-and-order approach. | July 24, 2016
Ken Griffey Jr. and Mike Piazza inducted into Hall of Fame
Two players who began their careers at opposite ends of the spectrum nearly three decades ago ended up in the same place on Sunday - with their names etched on plaques at the Baseball Hall of Fame. | July 24, 2016
Behavior changes offer clues that dementia could be brewing
Memory loss may not always be the first warning sign that dementia is brewing - changes in behavior or personality might be an early clue. | July 24, 2016
Airbnb sues hometown San Francisco over rental regulation
SAN FRANCISCO (AP) - San Francisco wants people who rent out their homes through Airbnb and other online platforms to follow some rules, and it wants the platforms to advertise only those rule-abiding listings - or face steep fines. | July 24, 2016
In Dallas, burgeoning movement overshadowed by shooting
DALLAS (AP) - The leadership of the Next Generation Action Network drove all night from Baton Rouge, Louisiana, arriving in Dallas early on July 7, just hours before the start of their hastily arranged march that ended in the worst attack on law enforcement since 9/11. | July 24, 2016
The Latest: 3 hurt in Virginia shooting, suspect sought
RICHMOND, Va. (AP) - The Latest on a shooting in Virginia that left three people injured early Sunday (all times local): | July 24, 2016
Burned body found as wildfire burns near Los Angeles
LOS ANGELES (AP) - Thousands of homes remained evacuated Sunday as two massive wildfires raged in tinder-dry California hills and canyons, and authorities said a burned body was found in one neighborhood swept by the flames. | July 24, 2016
Baton Rouge to continue to mourn officers slain in ambush
BATON ROUGE, La. (AP) - The city of Baton Rouge will continue to memorialize officers slain in a shoot-out outside a convenience store just a week ago with funeral services planned Monday for police officer Montrell Jackson. | July 24, 2016
Burned body found as wildfire burns near Los Angeles
LOS ANGELES (AP) - A burned body was found Saturday at the scene of a brushfire north of Los Angeles that has scorched 31 square miles and prompted the evacuation of 1,500 homes, authorities said. | July 24, 2016
The Latest: Burned body found at California fire scene
SANTA CLARITA, Calif. (AP) - The Latest on California wildfires (all times local): | July 24, 2016
As Democratic convention nears, excessive heat settles in
PHILADELPHIA (AP) - As thousands of delegates arrive in Philadelphia for the Democratic National Convention, it's not just politics they have to contend with - it's also the hot and sticky weather. | July 23, 2016
Lines form ahead of Garafola's funeral service
BATON ROUGE, La. (AP) - The Baton Rouge sheriff's deputy killed last Sunday ran to help another officer when he could have stayed safe in the convenience store where he was working off-duty, a minister said at his funeral Saturday. | July 23, 2016
Slain Kansas police captain remembered as hero at funeral
KANSAS CITY, Kan. (AP) - Just before the funeral of a detective who was killed by a fleeing suspect in May, the police chief of Kansas City, Kansas, asked one of his trusted veterans, Capt. Robert "Dave" Melton, to craft a how-to manual for holding a slain officer's funeral in case the department ever needed to do so again. | July 23, 2016
Weary of protest, Baltimore activists seek change elsewhere
BALTIMORE (AP) - Under the beating summer sun, retired steelworker Arthur B. Johnson Jr. stood outside the Clarence Mitchell Courthouse in Baltimore, clutching the fraying wooden handle of a homemade sign. | July 23, 2016
Philadelphia primps, activists prepare for DNC convention
PHILADELPHIA (AP) - Protesters, party leaders and city officials were making final plans as Philadelphia prepared to host the four-day Democratic National Convention that starts Monday. | July 23, 2016
A poll on American attitudes about guns
Nearly two-thirds of Americans say they believe the nation's gun laws should be stricter. | July 23, 2016
AP Poll: Support grows among Americans for stricter gun laws
Americans increasingly favor tougher gun laws by margins that have grown wider after a steady drumbeat of shootings in recent months, but they also are pessimistic that change will happen anytime soon, according to a new Associated Press-GfK poll. | July 23, 2016
The Latest: Democratic delegates mostly positive about Kaine
WASHINGTON (AP) - The Latest on Campaign 2016 between the Republican and Democratic nominating conventions (all times local): | July 22, 2016
California governor denies parole for Manson follower
SACRAMENTO, Calif. (AP) - California Gov. Jerry Brown denied parole Friday for Leslie Van Houten, the youngest follower of murderous cult leader Charles Manson who is serving a life sentence for killing a wealthy grocer and his wife more than 40 years ago. | July 22, 2016
Girl, 14, killed by lightning while on personal watercraft
SALT LAKE CITY (AP) - A 14-year-old girl has died and a woman was hospitalized in critical condition after lightning struck the personal watercraft they were riding across a reservoir straddling the Utah-Wyoming border, authorities said. | July 22, 2016
Virginia court nixes order restoring felons' voting rights
RICHMOND, Va. (AP) - Virginia Gov. Terry McAuliffe's sweeping executive order restoring the voting rights of more than 200,000 felons is unconstitutional, the state's highest court ruled Friday, siding with Republican lawmakers who said the governor overstepped his authority. | July 22, 2016
At slain officer's funeral, calls for respect and unity
BATON ROUGE, La. (AP) - When protests erupted here over a black man's killing during a struggle with two white police officers, Baton Rouge police officer Matthew Gerald made a promise to an old friend who urged him to be careful. | July 22, 2016
OSU officials: Coger died after 40-minute outdoor workout
Oklahoma State says junior college transfer Tyrek Coger has died after collapsing following a basketball team workout. | July 22, 2016
Toxic algae bloom swarms Utah Lake, seeps into farm supply
SPANISH FORK, Utah (AP) - A huge toxic algae bloom in Utah has closed one of the largest freshwater lakes west of the Mississippi River, sickening more than 100 people and leaving farmers scrambling for clean water for days during the hottest part of the year. | July 22, 2016
Katey Sagal, Jack McBrayer join Penny's 'Big Bang' family
We're about to meet two more members of the extended Big BangTheory family: Penny's mom and brother, both of whom will appear in the 10th-season opener, Sept. 19 on CBS. | July 22, 2016
'NeverEnding Story' is returning to the big screen
The classic coming-of-age film The NeverEnding Story will hit theaters again for the first time in more than 30 years this September. | July 22, 2016
Trump convention speech draws 32.2 million TV viewers
LOS ANGELES (AP) - The Nielsen company says an estimated 32.2 million people watched Donald Trump deliver his GOP nomination acceptance speech. | July 22, 2016
Cruz is right. In politics, family should be off-limits: Robert Robb
Trump suggested Cruz's wife was ugly and that his father was tied to JFK's death. And Donald expected an endorsement?
| July 22, 2016
3 Florida men charged with aiding Islamic State
MIAMI — A Florida man was arrested as he tried to fly out of
Medical examiner: Oklahoma State's Tyrek Coger died from enlarged heart
The Oklahoma State Medical Examiner's Office says
Top Massachusetts lawmaker makes play for NBA All-Star Game
BOSTON (AP) - Boston is apparently out of the running for the 2017 NBA All-Star game, which became available after the league decided to move the event out of Charlotte, North Carolina. | July 22, 2016
The Latest: Officer who shot therapist has clean record
NORTH MIAMI, Fla. (AP) - The Latest on the police shooting of an unarmed black mental health worker in North Miami (all times local): | July 22, 2016
Obama rejects Trump depiction of US in crisis
Obama fiercely rejected Donald Trump's depiction of an America in crisis on Friday. | July 22, 2016
Kansas court upholds death sentence for sheriff's killing
TOPEKA, Kan. (AP) - The Kansas Supreme Court on Friday upheld the death sentence imposed against a man who fatally shot a sheriff during a 2005 drug raid. | July 22, 2016
'Merciless' heat, humidity sticks to nearly all of US
WASHINGTON (AP) - Call it the United Sweats of America. A heat wave spreading across the United States is leaving few places to hide from the relentless mugginess of high humidity. Not the north. Not the cool of night. | July 22, 2016
Turkey will work with U.S. on Gulen extradition
WASHINGTON — Turkey's ambassador said Friday that his country will accept a U.S. offer to work with the State and Justice departments on an extradition request for a Turkish cleric living in Pennsylvania who Ankara accuses of orchestrating last week's failed coup. | July 22, 2016
Cowboys' Ezekiel Elliott denies domestic assault accusation
Police shootings study sparks debate on role of racial bias
HOUSTON (AP) - A Harvard University economist found that although police nationwide are more likely to use nonfatal force against blacks and Hispanics, that racial bias didn't translate to the use of deadly force by officers in Houston, one of the country's largest and most racially diverse cities. | July 22, 2016
FBI offers up to $10K reward for stolen weapons
WASHINGTON — The FBI is offering a reward of up to $10,000 for a handgun, a rifle and other equipment stolen from an agent's vehicle earlier this month. | July 22, 2016
Early ratings: 30 million watch Trump speech
Preliminary ratings for
'I vote Hillary', other fake Wi-Fi hotspots lure RNC attendees
Many of the Republican party faithful gathered in Cleveland this week are as sloppy about Internet security as Democratic candidate
Facebook says it's not negating 'great talent'
SAN FRANCISCO — Under fire for remarks that insinuated it could not hire more women and minorities because of a dearth of qualified candidates, Facebook says it's not negating "the great talent that exists in the software engineering space." | July 22, 2016
Black Texas woman seeks unity as white officers investigated
AUSTIN, Texas (AP) - A black Texas teacher thrown to the ground by a white officer during a traffic stop, and then told by another white officer on the way to jail that blacks have "violent tendencies," said Friday she is grateful the police chief has publicly apologized. | July 22, 2016
Ex-KKK leader David Duke runs for Senate: 'My time has come'
Former Ku Klux Klan leader David Duke announced Friday on his website that he plans to run for U.S. Senate in Louisiana. | July 22, 2016
Edward Snowden appears at secret screening | Video, Podcast
SAN DIEGO — From a world away, Edward Snowden critiqued the guy playing him in the upcoming Snowden as well as his own Hollywood performance. | July 22, 2016
Back home in Japan, Pokemon Go finds success is unreal
TOKYO — The wildly popular
Indiana court tosses woman's feticide conviction
INDIANAPOLIS (AP) - The Indiana Court of Appeals overturned the feticide conviction of a woman found guilty of killing her premature infant by taking abortion-inducing drugs, saying Friday the state's law wasn't intended to be used "to prosecute women for their own abortions." | July 22, 2016
Roger Ailes' exit may help all sexual harassment victims
The resignation of
Fans applaud Fox's new 'Rocky Horror' | Video, Podcast
Comic-Con fans responded enthusiastically Thursday to a screening of the first 25 minutes of the TV revival of the 1975 camp film classic, The Rocky Horror Picture Show. | July 22, 2016


text:
"If you establish a democracy, you must in due time reap the fruits of a democracy. You will in due season have great impatience of the public burdens, combined in due season with great increase of the public expenditure. You will in due season have wars entered into from passion and not from reason; and you will in due season submit to peace ignominiously sought and ignominiously obtained, which will diminish your authority and perhaps endanger your independence. You will in due season find your property is less valueable, and your freedom less complete." ~ Benjamin Disraeli
City Stops Residents From Backing Out of Driveways Safely
"Glendaleofficials are forcing residents to take down mirrors they've attached to trees in the public right-of-way so they can better see oncoming traffic while exiting their driveways. Residents say in the absence of any speed-reduction enhancements, such as speed humps, the round convex mirrors are one of the few safety measures they have. City officials, though, say it's about more than tree damage. City codes don't allow anything to be attached to city-owned trees or street-light poles. Also, nothing can be placed in the public right-of-way because of liability concerns."
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"...city-owned trees..."?????????
Hmmmm, I thought if they were on the "public right-of-way", i.e. the "common right-of-way", that they are common property, i.e. everyone owns them. Damaging the bark. [laughing] Then I suppose woodpeckers and squirrels will have to be notified as well.
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Driveway safety is comprised of two categories. Preventing your child from being run over as a car is backing out of the driveway. The other is preventing your child from running out into the street while playing in the yard with a driveway safety net. We will now cover each topic individually. Thanks.
cell phone directory
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subset: commoncrawl


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Do I have ANY Other Recourse if I Want to Use YouTube?
Started Mar 24, 2014 | Discussions thread
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Monicakm • Senior Member • Posts: 2,352
Do I have ANY Other Recourse if I Want to Use YouTube?
Mar 24, 2014
Sometime over the last few months, YouTube/Google has conspired once again to chip away at our right to privacy (like that even exists!). After not attempting to manage my YouTube Videos for quite sometime, I find that I can not even access them because I don't have a Google+ Acct. I already knew I couldn't leave comments without a G+ account but to deny me access to the videos that have been up for years....that's just wrong. I've been sly enough to not get tricked into signing up for a G+ acct TILL TONIGHT. They got me! I'll be decommissioning that acct asap.
QUESTION: Does anyone know how to get around this stupid forced Gmail+ acct to use (upload and comment) YouTube?
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The tact the author takes to organize the information contained in this book is masterful. He relies on first person sources as well as naval insight. Read morePublished 17 days ago by JOHN SR
Perhaps the worst navy book I've ever tried to finish, with the exception of Danger's Hour by M. T. Kennedy. Read morePublished 26 days ago by Coz
Wow. Read the whole Book on a flight to China. Well done. See if you can spot the Capt. Queeg character. He's real.Published 1 month ago by Amazon Customer
Well written, it holds the reader's interest throughout. There are some minor errors, (for instance the USS CANBERRA is mistakenly identified as Australian) but none of these... Read morePublished 2 months ago by 1250man
It is the winter of 1944 in the Pacific Ocean. This is the true story of a typhoon which takes the lives of almost 800 men, sinks 3 destroyers, renders 12 ships inoperable, and... Read morePublished 4 months ago by Bibliophile
Fascinating read, I highly recommend this book for any amateur Naval Historian. A great back story is presented as well as personal experiences before, during, and after the... Read morePublished 4 months ago by Michael L. irvin


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Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCβII was activated during increased angiogenesis and that PKCβII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCβII-selective inhibitor, βIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCβII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCβII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be upregulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is upregulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCβII inhibitor such as βIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.
In the US, prostate cancer is the third leading cause of cancer-related deaths in males (1). Although early detection and new therapies have increased survival rates, many men develop androgen-independent prostate cancers against which chemotherapeutic drugs have been generally ineffective (2). Furthermore, increases in microvessel density and expression of pro-angiogenic factors are associated with negative outcomes in patients with prostate cancer (3). Targeting cells that support tumor growth in addition to using cytotoxic agents to induce cancer cell death has therapeutic advantages (4–7). However, rather than targeting a single pro-angiogenic factor, there is a strong rationale for the development of new pharmacological treatments that target both tumor angiogenesis and tumor cell proliferation for the treatment of prostate cancer (8).
The protein kinase C (PKC) family of serine/threonine kinases plays an important role in angiogenesis both in vitro and in vivo (9–12). Also, PKC is activated by tumor-promoting phorbol esters and its involvement in carcinogenesis was proposed many years ago (13). Its role has since been substantiated in many human cancers, including prostate cancer (14–17). However, the role of PKC in prostate cancer angiogenesis has not been explored explicitly. Currently, a PKCβ inhibitor, Enzastaurine (a novel macrocyclic bisindolylmaleimide), is being tested in clinical trials for its anti-angiogenic and anti-cancer effects with promising phase II studies of high-grade gliomal tumors (18). However, although the initial reports suggested that Enzastaurine is selective for PKCβ (15), subsequent studies showed that it also inhibits PKCγ, δ, and ε to a similar degree at the same concentration (14).
PKC family members are known to mediate cytokinesis and cell proliferation by microtubule regulation (19-21). Functional studies have shown a key role for pericentrin, a centrosomal protein, in microtubule organization, spindle assembly, and chromosome segregation (22, 23). Chen et al. showed that endogenous PKCβII and pericentrin interact in K562 cells (19) and that PKCβII co-localizes with pericentrin in G2 and mitotic cells, i.e. dividing cells in culture. In addition, overexpression of a fragment of pericentrin that binds PKCβII leads to mislocalization of PKCβII away from the centrosome and a loss of microtubule anchoring at the centrosome resulting in cytokinesis failure and aneuploidy. Also, overexpression of a PKCβII fragment that binds pericentrin induces the same phenotype, suggesting that increased levels of PKCβII could also disrupt interaction with pericentrin. Therefore, there is strong evidence that PKCβII and pericentrin regulate cytokinesis in cells, but the role of PKCβII and pericentrin in prostate cancer progression, both in endothelial and tumor cells in vivo, has not been established.
Here, we set out to determine how PKC activity affects angiogenesis and tumor cell proliferation during different stages of prostate tumor growth in a xenograft model. Our data from xenografts and patients suggest PKCβII as a target in anti-cancer treatment for prostate cancer against tumor-induced angiogenesis and tumor growth.
PC-3 human prostate cancer cells and mouse tumor endothelial cells (2H-11) were obtained from the American Type Culture Collection (ATCC, Manassas,VA) and cultured in DMEM media with 10% fetal bovine serum (FBS, Gibco, NY) with 1% antibiotics (penicillin and streptomycin, Gibco, NY). Primary cultures of normal human epithelial cells were established from the peripheral zone of a radical prostatectomy specimen according to established techniques (24). The tissue of origin was confirmed to be normal by histopathological analyses. Cells were cultured in a serum-free medium, "Complete PFMR-4A" (24). For in vitro tumor endothelial cell culture, 5000 cells were seeded into each well of chamber slides in DMEM with 10% FBS and grown for 2 days in DMEM or conditioned medium from PC-3, i.e. 2 day old medium from PC-3 cell cultures. Tumor endothelial cells were then treated with TAT (carrier peptide) or βIIV5-3-TAT at a final concentration of 1 μM, 3 times per day for 2 days.
For Western blot analyses, rabbit antibodies directed against Gαi-3 (C-10) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and anti-GAPDH antibody, clone 6C5, was from Advanced Immunochemical (Long Beach, CA). For immunofluorescence, α-tubulin and γ-tubulin Cy3 antibodies were from Sigma (St. Louis, MO). Pericentrin antibodies used for immunofluorescence were from Abcam (4448, Cambridge, MA). Pericentrin antibodies used for Western blot analyses (M1, 4b and UM225) were from Dr. Stephen Doxsey (University of Massachusetts, Worcester, MA). Paraffin-embedded prostate tissues were from the Urology Department at Stanford Medical School (IRB # 348).
The PKCβII-selective inhibitor (βIIV5-3) was derived from the PKCβII V5 region (amino acids 645–650 [QEVIRN]) (25). For intracellular delivery, peptides were synthesized and conjugated to a membrane-permeable TAT carrier peptide as previously described (26). TAT carrier peptide or saline was used as a control. Peptides were delivered in vivo using Alzet osmotic mini-pumps (Alzet model 2001) as described (27). The peptides were dissolved in saline and administered at a constant rate (0.5μl/hr) corresponding to 2.4 or 24 mg/day/kg (3mM or 30mM of TAT) and 3.6 or 36 mg/day/kg (3mM or 30mM of βIIV5-3-TAT). Pumps were replaced every 2 weeks because of the t1/2 (about 2 weeks) of the peptides in the pump (27). Peptides were delivered for up to 5 weeks.
Six week old male nude mice were purchased from Harlan (Indianapolis, IN) and housed at the animal care facility at Stanford University Medical Center (Stanford, CA). All mice were kept under standard temperature, humidity, and timed lighting conditions and provided mouse chow and water ad libitum. All animal experimentation was conducted in accordance with the Guide for Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (revised 1996) and was approved by the Stanford University Animal Care and Use Committee. Five million PC-3 tumor cells were injected subcutaneously in the flank of male, 7–8 week old, athymic nude mice in sterile PBS (Figure 2) or in a mixture of 1:1 serum-free medium and Matrigel (Figure 3, Beckton Dickinson, Bedford, MA). Peptide treatment began when the tumors reached a group average of 100mm3 after about 1 week. Tumor volume (mm3) was calculated using the equation 0.52× (width (cm))2 × (length (cm)).
Animals were administered 4% deuterated water and tumor endothelial cells and tumor cells were isolated using flow cytometric sorting (refer to supplemental Figure 1 for cell isolation) and prepared for GC-MS analyses as previously described (28, 29).
Dual-color immunofluorescence was performed on fresh-frozen sections fixed in O.T.C. compound (Torrance, CA) using PKC and biotin linked rat-anti mouse CD31 antibodies (Santa Cruz Biotech Inc, Santa Cruz, CA and BD Pharmingen, San Diego, CA, respectively). For pericentrin and PKC detection, sections were stained with rabbit anti-pericentrin (ab4448, Abcam, Cambridge, MA) followed by PKC antibodies. TUNEL staining was carried out using an in situ cell death detection kit (TMR red) according to manufacturer's instructions (Roche Applied Science, Indianapolis, IN). Cleaved caspase-3 antibody was from Cell Signaling (Danvers, MA). CD31- and TUNEL-positive areas were measured using Photoshop (Version 9.0.1). Hoechst 333242 was from Molecular Probes (Carlsbad, CA). The apparatus for immunofluorescence experiments consisted of a Leica DMI 6000B microscope with 350FX camera (JH Technologies, San Jose, CA).
Frozen tumors and livers were weighed and two volumes of homogenization buffer [20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 250 mM sucrose, 1:300 protease inhibitor cocktail (Sigma) and 1:300 phosphatase inhibitor cocktail (Sigma)] were added. The tissue was homogenized and were fractionated by spinning at 100,000g for 30 min at 4°C. The supernatants correspond to the cytosolic fractions. The particulate fractions correspond to the rest of the intracellular organelles including nuclear and plasma membrane. The particulates were resuspended in homogenization buffer with 1% Triton X-100 and both detergent soluble and insoluble fractions were analyzed together. Translocation of PKCα, βI, βII and ε was determined in cytosolic and particulate fractions from tumor and liver samples as described (26). Whole cell lysates refer to total homogenates without fractionation. For all PKC detections, 10 μg of whole cell lysates, cytosolic and particulate fractions were used. Antibodies against GAPDH (1:10,000) and Gαi-3 (1:1000) were used as loading controls for cytosolic and particulate fraction, respectively.
Tumor lysates were subjected for immunoprecipitation using PKCβ II according to Chen et al. (19) and the immunoprecipitate was assayed for kinase activity in the absence of PKC activators (30).
Tissue sections in the slides were deparaffinized and with xylene, hydrated by using a diluted alcohol series, and immersed in 3% H2O2 in distilled water for 15 minutes to quench endogenous peroxidase activity. The sections were then microwaved in a pressure cooker for 30 minutes in distilled water containing 1mM EDTA. To avoid non-specific staining, each section was incubated with 4% bovine serum albumin (Qbiogene, Irvine, CA) in PBS with 0.1% Tween 20 (PBST) for 30 minutes at room temperature. The sections were then incubated with rabbit anti-pericentrin polyclonal antibody (4b, dilution: 1:250) in TBST [50mM Tris (pH7.5), 150mM NaCl, and 0.5% Tween 20] containing 4% Tryptone casein (Amresco, Solon, OH) for 1 hour at room temperature. HRP-conjugated secondary antibody against rabbit immunoglobulins (DAKO, Carpinteria, CA) was applied for 20 min at room temperature. Signals were amplified by catalyzed reporter deposition tyramine signal amplification (CSA II kit, DAKO, Carpinteria, CA), following manufacturer's instructions. Each section was incubated with fluorescyl-conjugated tyramide for 15 minutes and protected from light. Sections were then incubated with HRP-conjugated anti-fluorescein antibody for 15 minutes at room temperature. Each step was followed by three successive rinses with TBST for 5 min. The chromogen used was 3,3′-diaminobenzidine (DAKO, Carpinteria, CA). Sections were counterstained in Meyer's hematoxylin.
Data are expressed as mean±SE. Paired t test and repeated ANOVA were used to assess significance (p<0.05).
Because PKC activation has been implicated during growth of various tumors (31, 32), we first determined which PKC isozyme is present in growing PC-3 human prostate cancer cells in a xenograft model, in vivo. PKCα, βI, βII and ε were all found in the PC-3 tumors (Figure 1A). We next compared the cellular distribution of the PKC isozymes in the PC-3 xenografts with that in a primary culture of normal human prostate epithelial cells (PEC). We used the cellular distribution of the isozymes in PEC as a measure of basal levels of PKC activation (Figure 1A, left; cytosolic enzyme represents inactive PKC (33)). All the PKC isozymes were more active in the PC-3 xenografts relative to the primary PEC and PKCβII appeared more active relative to the other isozymes, as evidenced by high levels of this isozyme in the particulate fraction relative to the cytosolic fraction. This was also apparent when comparing PKCβII and its alternatively spliced form, PKCβI (n=4 each, p=0.01, Figure 1A, right). Immunofluorescence studies demonstrated that PKCβII was more localized to endothelial cells relative to PKCα, βI or ε (Figure 1B, arrow heads). Based on these results, we focused our study on determining the role of PKCβII in angiogenesis and in tumor growth.
To examine the proliferation kinetics of tumor cells and endothelial cells in the growing tumor, we used a new method that measures directly the proliferation rates of these cells, in vivo (Figure 2A, B, C). PC-3 cancer cells were injected subcutaneously (5×106 cells) into the flank area of male nude mice and the resulting solid tumors were isolated each week, for up to 6 weeks after tumor cell injection (Figure 2A, B). For each time point, deuterated water was administered in drinking water for 1 week before sacrificing and the tumor endothelial cells and tumor cells were isolated by fluorescence activated cell sorting (FACS). The proliferation rate of each cell type was calculated by measuring the amount of deuterium in the DNA of these cells, as we previously described (28, 29).
Interestingly, the rise in tumor endothelial cell (TEC) proliferation rate preceded the rise in the proliferation rate of the tumor cells (TC) during the first 4 weeks (Figure 2C); rates of proliferation of these two cell types continued to change in an oscillating pattern for about 4 weeks. These data support the predicted coordination between tumor growth and angiogenesis with TEC proliferation and angiogenesis rising to meet the metabolic demand of the growing tumor (4, 34). After week 4, the tumor endothelial cell and tumor cell proliferation rates appeared to reach a steady-state, suggesting that the rate of angiogenesis had matched the metabolic demand of the growing tumor (4, 34). We further examined the kinetics of cell proliferation during the days 0–7 of post-tumor injection (n=6–10 animals each, insert). The proliferation rate of tumor endothelial cells was several-fold higher than that of tumor cells during days 0–4 and days 4–7 (Figure 2C, insert), indicating active tumor angiogenesis during the early period of tumor growth with only moderate tumor cell proliferation at that period. This confirms that angiogenesis is particularly active in the early period of tumor growth and suggests a window of treatment for anti-angiogenesis.
Because we found PKCβII to localize mainly in endothelial cells, we next determined its role in tumor growth and angiogenesis, in vivo. We implanted osmotic pumps with saline, control peptide (TAT47–57 carrier peptide (35, 36)) or βIIV5-3 (PKCβII-selective inhibitor peptide) conjugated to TAT47–57 to deliver the PKCβII inhibitor (25) into the cells. Specifically, one week after injection of the PC-3 cells, mice were implanted with osmotic pumps with saline/TAT or βIIV5-3 at 3.6 mg/kg/day for 2 weeks followed by 36 mg/kg/day for the following 3 weeks. Already after two weeks of treatment, there was a trend towards decreased tumor size in the βIIV5-3-treated animals (Figure 3A). When the βIIV5-3 concentration was increased from 3.6 mg/kg/day to 36 mg/kg/day for the next 3 weeks [a dose that was well tolerated (37)], tumor volume was found to be significantly smaller in the βIIV5-3-treated group over time (Figure 3A, repeated ANOVA, *; p<0.05, n=4–5 each). Previous in vivo studies demonstrated that inhibition of PKC translocation by systemic peptide delivery was observed in all tissues (26). Similarly, we found here that βIIV5-3 treatment reduced the level of PKCβII in the particulate fraction relative to the cytosolic fraction in tumor as well as in other tissues e.g., in liver (βIIV5-3 treatment decreased PKCβII translocation by 25–35%; Figure 3B, right, p<0.05, n=3 each). We have previously shown that such a decrease in PKC translocation is sufficient to inhibit its pathological activity [e.g., (38, 39)]. We further confirmed the selectively of the PKCβII inhibitor; sustained treatment of βIIV5-3 did not affect translocation of the closely related PKCμI in the tumor nor PKCε in the liver (supplemental Figure 3). Using kinase assay in vitro in the absence of added PKC activators, we found that βIIV5-3 treatment resulted in an ~85% reduction in the catalytic activity of PKCβII immunoprecipitated from total tumor lysates containing equal amounts of protein (Figure 3C), confirming sustained inhibition of PKCβII in the treated tumors.
We next set out to confirm the tumor growth inhibitory effect in vivo by administering βIIV5-3 at the higher dose of 36 mg/kg/day from week 1 - week 5 (i.e., when we observed the most active angiogenesis, see Figure 2C). This treatment decreased overall tumor growth rate by 60% compared to 16% with the lower dose of βIIV5-3, calculated as the change in tumor volume over time (Figure 3D; p<0.05, n=8–9 each vs. Figure 3A).
Next, we determined whether βIIV5-3 affected cell division of tumor endothelial cells by directly measuring in vivo cell proliferation rates using deuterated water, as in Figure 2. At week 3 with two weeks of sustained treatment with βIIV5-3 (3.6 mg/kg/day), tumor endothelial cell (TEC) proliferation rates were reduced by 40%, as compared to control mice (Figure 4A, p=0.008, n=8–9 each). However, there was no difference in the proliferation rates five weeks after sustained treatment (at week 6) of βIIV5-3 (3.6 mg/kg/day followed by 36 mg/kg/day). These data suggest that the anti-angiogenic effect of β IIV5-3 is more pronounced at the early stage of tumor growth, even at a lower dose. To confirm the anti-angiogenic effect of βIIV5-3, tumor sections at week 3 and 6 were stained with anti-CD31 antibody, a marker of endothelial cells (Figure 4B). There was a significantly lower number of CD31-positive tumor vessels in the βIIV5-3-treated samples compared to controls at week 3 (28±11 vs. 6±3 %, p<0.05, Figure 4B), but not at week 6 (not significant, data not shown), quantified using Photoshop program (Ver. 9.0.1, San Jose, CA).
βIIV5-3 treatment also decreased tumor cell (TC) proliferation rates at week 3 (Figure 4C, p<0.001) and showed a trend for inhibition at week 6 (p=0.065, Figure 4C). Moreover, there was a stronger tendency of increased TUNEL-positive cells in the βIIV5-3-treated tumors at week 6 relative to TAT or saline controls (Figure 4D, ,22±1 vs. 8±2 %, p=0.06), compared to week 3 (not significant). We found that TUNEL-positive cells overlapped with staining for cleaved caspase 3 (Figure 4D, insert), further confirming an increase in apoptosis. This suggests that at an earlier tumor stage, βIIV5-3 treatment decreases cell proliferation of both the tumor endothelial cells and tumor cells rather than inducing apoptosis.
We next set out to determine the molecular basis for inhibition of tumor growth by β IIV5-3. An unbiased two hybrid screen by Newton and collaborators demonstrated that a centrosomal protein, pericentrin, which is involved in controlling cytokinesis, microtubule organization and spindle formation, binds PKCβII (19).
Pericentrin levels in human prostate cancer have been shown to be elevated with increasing Gleason grade (23, 40). Furthermore, pericentrin overexpression is associated with centrosomal defects leading to chromosomal instability, microtubule mis-segregation, larger nuclei and increased cell proliferation in human prostate and other types of cancer cells (23, 40–43). We therefore hypothesized that regulation of interaction of PKCβ II with pericentrin may play a role in the phenotype that we observed. Pericentrin staining appears as a dot or two in normal cells (19, 23, 40 and in supplemental Figure 2). However, staining of the PC-3 tumor xenografts showed an abnormal pattern of pericentrin staining with elongated filamentous structures (Figure 5A, left panel). Treatment with 36 mg/kg/day of βIIV5-3 for 4 weeks significantly reduced the abnormal filamentous pericentrin staining, reduced the overall staining intensity and resulted in the return of a dotted staining pattern with anti-pericentrin antibodies (Figure 5A, right panel). This was confirmed by Western blot analyses of pericentrin (~220kDa) and its cleaved form (~150kDa) in the total tumor lysate (Figure 5B). The amount of cleaved form of pericentrin was reduced by ~90% with βIIV5-3 treatment compared to TAT controls (Figure 5B, lower arrow). We also found that enlongated and filamentous pericentrin did not co-localize with PKCβII in TAT-treated tumors (asterisk, Figure 5C, ,2,2, ,3,3, ,4),4), whereas dotted pericentrin co-localized with PKCβII in βIIV5-3-treated tumors (arrows, Figure 5C, ,6,6, 7, 8). This was confirmed by determining the interaction between pericentrin and PKCβII in vivo by co-immunoprecipitation assay followed by Western blot analysis of the immunoprecipitate. PKCβII was immunoprecipitated from the total tumor lysate using anti-PKCβII antibodies and detected with antibodies against pericentrin (Figure 5D, upper panel, lanes 2 and 3). We detected pericentrin (~220kDa) and its cleaved form (~150kDa) in the immunoprecipitate (44, 45). Even though the total level of pericentrin was lower in the βIIV5-3-treated group as compared with the TAT-treated group (Figure 5B), there was 10-fold more pericentrin associated with PKCβII as compared with that associated in immunoprecipitates from TAT-treated tumors (Figure 5D, upper panel, lanes 2 and 3). Immunoprecipitate from the lysate of primary culture of PECs that were not treated with βIIV5-3 was also used to show interaction of PKCβII and pericentrin in normal human cells. The interaction between PKCβII and pericentrin was stronger than that in untreated PC-3 tumors (Lane 5).
Because we found that filamentous pericentrin was present in structures similar to microvessels (Figure 5A, left panel), we co-stained tumor sections for CD31 (a marker of vessels) and pericentrin. In TAT-treated tumors, enlongated and filamentous pericentrin was seen in tumor vessels (Figure 6A, left panel, arrows). In βIIV5-3-treated tumors, pericentrin staining was significantly reduced in tumor microvessels and dotted staining was apparent in both tumor cells (arrow heads) and tumor endothelium (arrows, Figure 6A, right panel).
Since tumor endothelial cells are contributed by the mice, we used a mouse tumor endothelial cell line cultured in medium from PC-3 human cancer cells to simulate in vivo system (46). Medium from PC-3 cells increased the level of pericentrin staining in the endothelial cells (Figure 6B, left panel, middle row) relative to those cultured in normal DMEM (Figure 6B, left panel, top row). βIIV5-3 treatment reduced this effect (Figure 6B, left panel, bottom row). To confirm that the pericentrin abnormality correlates with centrosomal defect, endothelial cells were also stained for γ-tubulin (Figure 6B, middle panel). PC-3-conditioned medium increased the centrosomal γ-tubulin staining in the endothelial cells (Figure 6B, second panel, middle row) whereas βIIV5-3 treatment normalized it similarly to TAT-treated cells (Figure 6B, second panel, bottom and top rows). Also, PC-3-conditioned medium resulted in disorganized forms of α-tubulin, representative of microtubule organization (Fig. 6C, second panel, middle row) in the endothelial cells, whereas βIIV5-3 treatment resulted in an organized form of microtubules, similar to TAT - treated cells in DMEM (Fig. 6C, second column, bottom and top rows).
To determine the clinical relevance of our findings, we assessed location and levels of pericentrin in prostate tissue from patients with Gleason grades 3, 4 and 5 cancers. Similar to our data in the xenograft model, in some patients, we found higher levels of pericentrin in the cytoplasm of endothelial cells adjacent to tumor glands (Figure 6D, middle, 100X) compared to those among benign prostatic hyperplasia (Figure 6D, left, 100X). The figure on the right is showing a magnified view of the middle figure (right, 400X). In tumors of other patients, some tumor endothelial cells were strongly stained for pericentrin, whereas others were stained at similar levels to those seen in endothelial cells among normal glands. These data suggest that, at least in some patients with Gleason grades 3 and up prostate cancers, there is upregulation of pericentrin levels and localization in tumor endothelium.
Determining isozyme-specific roles of PKC in tumors has been hampered by a lack of `isozyme-specific regulators for each PKC isozyme. Here, we show that isozyme-specific inhibition of PKCβII by βIIV5-3 reduces PC-3 tumor growth in a xenograft model by decreasing angiogenesis, tumor cell proliferation and normalizing pericentrin levels and subcellular localization.
First, we found an oscillatory pattern of increase in the proliferation rates of tumor endothelial and tumor cells. This in vivo interplay between the tumor endothelial cells and tumor cell proliferation has not been reported and provides a new insight into the relationship between the tumor and microenvironment during prostate cancer progression. These findings also identified a possible time window for drug treatment to reduce angiogenesis and tumor growth. Our findings may relate to the alternate apoptotic waves of these two cells types in Lewis lung carcinoma xenografts with chemotherapy as evidenced by TUNEL staining (6, 34); both are likely reflecting the tight regulation of angiogenesis by the tumor, to match metabolic demand of the tumor mass.
Microvessel density, the most frequently used method to measure angiogenesis, is not without limitations (4); vessel density does not represent the angiogenic activity of the tumor. Rather, it represents local tumor metabolic burden expressed as vessel to tumor ratio (4). Also, this measurement is laborious and quite subjective (47). We therefore used 2H2O to label DNA in vivo. Our method accurately measures net in vivo proliferation (i.e. turnover) rates of the tumor cells and the endothelial cells separately during the 2H2O administration period (28, 29) by analyzing isolated endothelial cells and tumor cells from the tissue (see supplemental Figure 1).
PKC family members are known to mediate cytokinesis and cell proliferation by regulation of microtubule organization (19–21). Expression studies of pericentrin fragments in cultured cells demonstrated a key role for pericentrin as a scaffold protein for PKCβII in microtubule organization, spindle assembly, and chromosome segregation (19). Here, βIIV5-3 appeared to normalize centrosome defects and microtubule misalignment seen in tumor endothelial cells. Knockdown of pericentrin in TEC and PC-3 cells using siRNA resulted in decreased γ-tubulin staining and reduction in the number of cells, supporting our data (supplemental Figure 4B and C). Because centrosome aberration and microtubule misorganization are thought to be possible causes of aneuploidy and chromosomal instability in some types of cancer, including prostate cancer (40, 41), the role of PKCβII/pericentrin interaction in the molecular events leading to aberration in cytokinesis and chromosomal mis-segregation needs to be determined. The cleaved form of pericentrin was suggested to be involved in malignant transformation (44, 45). Increased binding of PKCβII to pericentrin, especially the cleaved form, may inhibit further carcinogenesis. The effect of βIIV5-3, which inhibits the binding of PKCβII to its RACK, a receptor for activated C kinase (25, 48), may leave more PKCβII available for binding with pericentrin at the centrosome. We also found increased staining of pericentrin in endothelial cells can be induced by PKCβII-activating factor(s) secreted from the tumor cells. Our data suggest that the secreted factor is unlikely to be VEGF (see supplemental Figure 5); the role of other secreted factors from prostate cancer cells (e.g. TGF-α, basic fibroblast growth factor and insulin-like growth factor (3)) remains to be determined.
The findings that pericentrin levels are greatly elevated in human prostate tumors relative to normal prostate tissue, that pericentrin levels correlate with the Gleason grade (23, 40) and our immunohistochemistry data of high levels of pericentrin, specifically in the tumor endothelium (Figure 6D) of some patients suggest that correction of pericentrin abnormalities with a PKCβII inhibitor, such as βIIV5-3, may improve both anti-angiogenic and anti-tumor therapy. It remains to be determined whether the catalytic activity of PKCβII plays a role in pericentrin regulation or whether its role is confined to simply anchoring pericentrin. Our data also suggest that a larger study of humans with prostate cancer is warranted, to assess the correlation between the levels of pericentrin in tumor endothelium and the Gleason grade of the cancer.
In conclusion, we show that an isozyme-specific inhibitor of PKCβII localization and function reduces tumor growth by reducing angiogenesis and tumor cell proliferation in a human prostate cancer xenograft model. We determined the appropriate window of treatment by analyzing proliferation kinetics of tumor endothelial cells and tumor cells in vivo using a direct measurement of cell proliferation. PKCβII inhibition corrected pericentrin localization and reduced other abnormalities, especially in the tumor vessels. Overall, our results suggest that a PKCβII inhibitor may provide a useful adjuvant treatment to the current therapy for patients with prostate cancer (and perhaps for patients with other solid tumors) by inhibiting proliferation of both tumor endothelial cells as well as tumor cells in the early phase.
Isolation of tumor endothelial cells and tumor cells.
Tumors were cut into fine pieces and rinsed with PBS on ice. Cell suspensions were obtained by treatment with collagenase [1mg/ml Collagenase A (Roche, 1088785) in Hank's buffer, 10ml] at 37°C for 10 minutes, shaking vigorously. After 10 minutes, digested tissue was centrifuged at 1100 rpm for 2 minutes and the supernatant was decanted to remove dead cells. The pellet was resuspended in 10ml of fresh collagenase solution and incubated for an additional 10 minutes. The cell suspension was filtered using a cell strainer (BD Falcon, 100μm Nylon, Cat# 352360), pelleted at 800g for 5 minutes and the pellet was resuspended in 1ml of Hank's buffer. Cells were washed twice with PBS containing 0.1% BSA and 2mM EDTA in 1ml buffer, spun at 6000 rpm (tabletop Eppendorf centrifuge) for 1 minute and stained with appropriate antibodies for the fluorescence activated cell sorter (FACS) [e.g. 1 μl of ant-CD31 FITC (1:100) for endothelial cells, 0.1 μl of anti-CD45-Cy5 phycoerythrine (PE) (1:1000) for leukocytes and 1 μl of anti-CD14-PE for macrophages (1:100, all three antibodies were from BD Biosciences) per 100 μl]. Appropriate control antibodies were used for each fluorophore at the same concentrations. One to five million cells/100 μl were used for FACS analysis. Cells were analyzed and sorted with a FACStar flow cytometer. Tumor endothelial cells were collected from CD31-positive, CD14-negative and CD45-negative cell populations (to prevent macrophage and leukocyte contamination in the tumor endothelial cells) and tumor cells were collected from CD31-negative, CD14-negative and CD45-negative cell populations. Contamination from CD14-positive cells was negligible, so the representative FACS plot shows analysis with CD31 and CD45 antibodies.
Supplemental Figure 2. Pericentrin staining in normal mouse spleen appears as dots.
Frozen section of normal mouse spleen was stained for nuclei (blue) and pericentrin (Cy3) to show normal pattern of pericentrin (arrows, n=3). Scale bar: 10 μm.
Supplemental Figure 3. Translocation of PKCβI and ε to the particulate fraction. (A) Supplementary for Figure 3B, left. Five-week continuous βIIV5-3-treatment did not alter PKCβI levels in the cytosolic (C) and particulate (P) fractions of tumors. The active level of PKCβI was analyzed by Western blot after fractionation and probed with antibodies against PKCβI. (B) Supplementary for Figure 3B, right. Five-week continuous βIIV5-3-treatment did not alter PKCε levels in the cytosolic and the particulate fraction of livers. Loading controls (GAPDH and Gαi) are shown.
Supplemental Figure 4. Treatment of TEC with PC-3 media on cell number and RNA interference of pericentrin and PKCβII in TEC and PC-3 cells. (A) Number of TEC after treatment with TAT, medium from PC-3 cells and media from PC-3 cells plus βIIV5-3 as analysed by Hoechst staining. *, p<0.05, unpaired t-test (TEC treated with TAT vs. TEC treated with medium from PC-3 cells). TEC treated with medium from PC-3 showed significantly higher number of cells compared with TAT-treated group. βIIV5-3 treatment together with PC-3 medium reduced the number of cells compared with PC-3 medium-treated group but did not reach statistical significance (p=0.06). (B) siRNA knockdown of pericentrin in TEC. In supplemental Figure 4B, Western blot analyses after siRNA of pericentrin in TEC is shown (upper panel). A mixture of rabbit anti-pericentrin antibodies (Gift from Dr. Doxsey, U. Mass) that recognize both mouse and human pericentrin were used to detect pericentrin. For loading control, blots were probed with mouse anti-GAPDH antibody (Advanced Immunochemical (Long Beach, CA)). siRNA (1μg/1ml) were added in each well in 6-well plate. Cells were stained with Hoechst 33342 and γ-tubulin (Sigma) after fixation (supplementary Figure 4B, lower panels). (C) siRNA knockdown of pericentrin and PKCβII in PC-3 cells. In supplemental Figure 4C, Western blot analyses after siRNA of pericentrin and PKCβII in PC-3 cells are shown (supplemental Figure 4C, upper panels). A mixture of rabbit anti-pericentrin antibodies (Gift from Dr. Doxsey, U. Mass) and anti-PKCβII antibodies (Santa Cruz Biotech) were used to detect pericentrin and PKCβII. For loading control, anti-GAPDH antibody (Advanced Immunochemical (Long Beach, CA)) was used. The concentration of siRNA was the same as in (B). Cells were stained with DAPI and γ-tubulin (Sigma) after fixation (supplemental Figure 4C, lower panels).
Supplemental Figure 5. VEGF levels from the serum of TAT vs. βIIV5-3 treated mice.
Mice treated with TAT or βIIV5-3 at 36 mg/kg/day for 4 weeks were sacrificed at the end of the treatment at week 5 and the serum was collected. VEGF concentration was determined using an ELISA kit (Quantikine, R &D Systems, San Diego, CA).
Supplemental Figure 6. Qunatitation of pulled-down PKCβII in the kinase assay
Membrane used for kinase assay was re-probed with anti-PKCβII antibody and the bands were quantitated (n=3, each).
Supplemental Figure 7. Interaction of pericentrin with PKCβII in PECs
PKCβII was immunoprecipitated from lysates of primary culture of PEC and PC-3 tumor, respectively (left and middle). Then, the precipitates were probed for pericentrin and PKCβII (arrows). Whole cell lysate of heart tissue was used as a control to show low level of pericentrin in non-proliferating cells (right).
This study was supported in part by PHS Grant Number CA09151 awarded by the National Cancer Institute, DHHS to J Kim.
DMR is the founder of KAI Pharmaceuticals, Inc., a company that plans to bring PKC regulators to the clinic. However, none of the work described in this study is based on or is supported by the company.
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